Pediatric Hematopoietic Stem Cell Transplantation

DK3062_half 5/9/06 3:51 PM Page 1

Pediatric Hematopoietic Stem Cell Transplantation

DK3062_title 5/16/06 8:33 AM Page 1

Pediatric Hematopoietic Stem Cell Transplantation

edited by

Ronald M. Kline Pediatric Division Comprehensive Cancer Centers of Nevada Las Vegas, Nevada, U.S.A.

New York London

Informa Healthcare USA, Inc. 270 Madison Avenue New York, NY 10016 © 2006 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8247-2445-3 (Hardcover) International Standard Book Number-13: 978-0-8247-2445-0 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Informa Web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com

T&F_LOC_B_Master.indd 1

6/14/06 8:55:05 AM

I dedicate this book to the memory of my father: He triumphed over adversity from boyhood onwards and instilled in me the values that I live by today and will pass on to my children. Ronald Kline

Preface

“Children are not little adults.” This much-overused expression characterizes the pride that pediatricians take in their specialized knowledge of the growing and developing human being. The quote is also an admonition to our adult medicine colleagues that specialized knowledge is required for the care of children with complex diseases and therapies, not simply a dosage adjustment for size. However, because of the general good health of most children, the pediatric specialties are inherently small. As a result, practitioners of such niche subspecialties as pediatric blood and marrow transplantation have in the past been required to depend on general texts written primarily for the care of adult transplant patients. Yet as all pediatric transplanters know, “children are not little adults.” The diseases they encounter, their responses to treatment, and the types and relative risks of various complications are markedly different than in adults. Thus the time has come for a comprehensive text focused exclusively on the care of the pediatric hematopoietic stem-cell transplant patient. The purpose of this work is to provide a focused, comprehensive and up-to-date reference work for those of us caring for pediatric BMT patients. Chapter topics have been chosen with particular respect to their relevance to pediatric hematopoietic stemcell transplantation and direction has been given to the authors to focus primarily on the pediatric aspects of disease and treatment, rather than to generate a broader treatment that includes adult issues. I have been most fortunate in assembling a group of distinguished and dedicated authors to contribute to this work. I am grateful for their hard work and hope that readers will find this text both enjoyable and informative. Ronald M. Kline

v

Contents Preface : : : : v Contributors : : : : xvii

SECTION I: GENERAL PRINCIPLES 1. Supportive Care of the Pediatric Hematopoietic Stem-Cell Transplant Patient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Victor M. Aquino and Eric S. Sandler Introduction : : : : 1 Prevention and Management of Mucositis : : : : 1 Hepatic Veno-occlusive Disease : : : : 2 Nutrition Support : : : : 6 Hemorrhagic Cystitis : : : : 7 Prevention and Management of Renal Disease : : : : 7 Neurologic Complications of Hematopoietic Stem-Cell Transplant : : : : 9 Transfusion Support : : : : 11 Hematopoietic Growth Factor Support : : : : 16 Conclusion : : : : 19 References : : : : 19 2. Prevention and Treatment of Infectious Disease . . . . . . . . . . . . . . . 27 Scott M. Bradfield, Steven Neudorf, Elyssa Rubin, and Eric S. Sandler Bacterial Infections : : : : 27 Invasive Fungal Infections : : : : 35 Viral Infections : : : : 43 References : : : : 55 3. Acute Graft-Versus-Host Disease . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Theodore B. Moore and Stephen A. Feig Pathophysiology : : : : 65 Staging/Clinical Description : : : : 66 vii

viii

Contents

Risk Factors : : : : 68 Prophylaxis/Therapy : : : : 71 Treatment of Acute Graft-Versus-Host Disease : : : : 76 References : : : : 77 4. Chronic Graft-Versus-Host Disease in Children . . . . . . . . . . . . . . . 85 David A. Jacobsohn, Georgia B. Vogelsang, and Kirk R. Schultz Overview of the Biology of Chronic Graft-Versus-Host Disease : : : : 85 Chronic Graft-Versus-Host Disease—Incidence and Risk Factors in Children : : : : 91 Classification of Chronic Graft-Versus-Host Disease : : : : 93 Treatment of Chronic Graft-Versus-Host Disease : : : : 98 References : : : : 100 5. Cellular Engineering of the Hematopoietic Graft . . . . . . . . . . . . . 111 Ralph Quinones Introduction : : : : 111 Definitions : : : : 111 Minimally Manipulated Products : : : : 117 Extensively Manipulated Products : : : : 118 T-Cell Depletion to Prevent Graft-Versus-Host Disease : : : : 119 Purging of Tumor Cells from Autologous Hematopoietic Stem Cells : : : : 122 Translational Research and the Future: Gene Therapy and Stem-Cell Expansion : : : : 126 Regulation : : : : 127 References : : : : 128 6. Issues in Pediatric Peripheral Blood Stem-Cell Collection . . . . . . 137 Stephan A. Grupp Introduction : : : : 137 Pheresis and Vascular Access : : : : 137 Collection : : : : 139 Techniques for Stem-Cell Mobilization : : : : 139 Target Dose for PBSC Infusion : : : : 140 Processing and Storage of Peripheral Blood Stem Cells : : : : 142 Tumor Cell Purging : : : : 142 Storage : : : : 143 References : : : : 143 7. Pediatric Unrelated Donor Stem-Cell Transplantation . . . . . . . . . 147 Monica Bhatia and Naynesh R. Kamani History : : : : 147 Unrelated Stem Cell Donor Registries : : : : 148 Histocompatibility : : : : 148

Contents

ix

HLA Typing : : : : 149 Registries and Donor Selection : : : : 149 Preparative Regimens : : : : 151 Complications : : : : 151 The Role of Unrelated Donor Transplants in Specific Diseases : : : : 152 References : : : : 155 8. Umbilical Cord Blood Transplantation . . . . . . . . . . . . . . . . . . . . . 161 Satkiran S. Grewal and John E. Wagner Introduction : : : : 161 Biological Features of Umbilical Cord Blood Grafts : : : : 162 Umbilical Cord Blood Transplantation Clinical Experience : : : : 163 Practical Considerations When Selecting a Donor Graft : : : : 179 Umbilical Cord Blood Transplantation for Larger Sized Recipients : : : : 181 Summary and Future Considerations : : : : 181 References : : : : 181 9. Cellular Immunotherapeutic Approaches to the Hematopoietic Stem-Cell Transplant Patient . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 Dennis Hughes and John Levine Introduction : : : : 189 Immunotherapy and Loss of Immunologic Control of Leukemia : : : : 190 Donor Leukocyte Infusion : : : : 192 Nonmyeloablative Transplant : : : : 194 Summary : : : : 196 References : : : : 196 10. Partially Mismatched Related Donor Transplantation . . . . . . . . . 201 Kuang-Yueh Chiang, P. Jean Henslee-Downey, and Kamar T. Godder Background : : : : 201 Terminology of Partially Mismatched Related Donor (Haplo-Identical) Transplant : : : : 202 Alloreactivity : : : : 202 Donor Selection Criteria : : : : 204 Methods to Cross Major-Human Leukocyte Antigen Barriers : : : : 205 Granulocyte-Colony Stimulating Factor Primed Bone Marrow Cells and Peripheral Blood Stem Cells : : : : 207 Conditioning of the Recipient : : : : 208 Engraftment : : : : 208 Graft-Versus-Host Disease and Outcomes : : : : 209 Infection and Immune Reconstitution : : : : 211

x

Contents

Comparison Between Alternative Donor Transplants : : : : 212 Summary : : : : 215 References : : : : 215 11. Nursing Care of the Pediatric Blood and Marrow Transplant Patient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 Linda Z. Abramovitz and Vicki L. Fisher Introduction : : : : 223 Nursing Roles and Practice Settings : : : : 223 Nursing Education : : : : 225 Professional Nursing Organizations : : : : 226 Patient Education : : : : 227 Special Programs : : : : 228 References : : : : 232 12. Psychological Dimensions of Pediatric Hematopoietic Stem-Cell Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Bryan D. Carter, William G. Kronenberger, Tanya F. Stockhammer, and Christi Bartolomucci Process-Related Stressors of Pediatric Hematopoietic Stem-Cell Transplantation on the Child and Family : : : : 235 Psychosocial Outcomes of Pediatric HSCT : : : : 237 Influences on Adjustment to Bone Marrow Transplantation : : : : 241 Psychosocial Intervention : : : : 244 Grief and Loss : : : : 246 Caring for the Professional and Psychosocial Team : : : : 247 References : : : : 247 13. Ethical Considerations in Pediatric Hematopoietic Stem-Cell Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Raymond Barfield and Eric Kodish Introduction : : : : 251 Foundational Concepts : : : : 252 Consent for Stem-Cell Transplantation : : : : 253 The Second Patient : : : : 258 Research, Therapy and Human Rights : : : : 262 Quality of Life and End-of-Life Issues : : : : 266 Conclusion : : : : 267 References : : : : 267 14. Immune Reconstitution in Pediatric Patients Following Hematopoietic Stem-Cell Transplantation . . . . . . . . . . . . . . . . . . 271 Trudy N. Small Introduction : : : : 271

Contents

xi

Natural Killer Cell Reconstitution Post-hematopoietic Stem-Cell Transplantation : : : : 272 T-Cell Reconstitution : : : : 273 B-Cell Reconstitution : : : : 277 Antigen-Specific Responses : : : : 278 Adoptive Immunotherapy : : : : 280 Immunomodulatory Factors : : : : 281 Conclusion : : : : 281 References : : : : 281 15. Endocrine Complications of Childhood Hematopoietic Stem-Cell Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Wassim Chemaitilly, Farid Boulad, and Charles Sklar Introduction : : : : 287 Impaired Linear Growth and Growth Hormone Deficiency : : : : 287 Disturbances of the Other Hypothalamic-Pituitary Axes : : : : 290 Thyroid Dysfunction : : : : 290 Gonadal and Reproductive Dysfunction : : : : 291 Osteoporosis : : : : 293 Disorders of Glucose Homeostasis : : : : 294 Conclusion : : : : 294 References : : : : 295 16. Future Directions in Pediatric Stem-Cell Transplantation . . . . . . 299 Edwin M. Horwitz Principles of Bone Marrow Cell Therapy : : : : 299 Principles of Gene Therapy : : : : 301 Mesenchymal Stem Cells : : : : 304 Fundamental Steps in the Development of Bone Marrow Cell Therapy : : : : 305 Patient-Based Research of Bone Marrow Cell Therapy and Gene Therapy : : : : 305 Bone Marrow Cell Therapy for Genetic Disorders of Bone : : : : 306 Clinical Trials of Gene Therapy of Severe Combined Immunodeficiency Disorders : : : : 310 Marrow Mesenchymal Stem Cells as Cell Therapy for Inborn Errors of Metabolism : : : : 311 Mesenchymal Stem Cells as Modulators of Immune Function and the Treatment of Graft-Versus-Host Disease : : : : 312 Mesenchymal Stem Cells to Facilitate Hematopoietic Stem-Cell Engraftment : : : : 312 Preclinical Models and Clinical Trials of Blood and Marrow Transplantation as Cell Therapy for Nonhematopoietic Disorders : : : : 313

xii

Contents

Parting Thoughts : : : : 315 References : : : : 315 SECTION II:

NONMALIGNANT DISEASES

17. Primary Immunodeficiencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 Brett J. Loechelt and Naynesh R. Kamani Introduction : : : : 321 Special Considerations : : : : 321 Clinical Results : : : : 325 Future Directions : : : : 331 References : : : : 332 18. Hematopoietic Stem-Cell Transplantation for the Inherited Bone Marrow Failure Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . 337 Adrianna Vlachos, Carole Paley, and Jeffrey Michael Lipton Introduction : : : : 337 The Syndromes : : : : 339 Perspectives on the Future : : : : 359 References : : : : 360 19. Hematopoietic Stem-Cell Transplantation for Acquired Aplastic Anemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 Carole Paley, Adrianna Vlachos, and Jeffrey Michael Lipton Introduction : : : : 369 Pathophysiology : : : : 370 Epidemiology : : : : 371 Clinical Features : : : : 371 Immunosuppressive (Immunomodulatory) Therapy for Severe Aplastic Anemia : : : : 371 Other Immunologic Therapies : : : : 372 Hematopoietic Stem-Cell Transplantation for Severe Aplastic Anemia : : : : 372 Graft-Versus-Host Disease : : : : 374 Survival : : : : 375 Hematopoietic Stem-Cell Transplantation Compared with Immunosuppressive Treatment : : : : 375 Alternative Donor Transplant : : : : 377 Unrelated Donor Transplant : : : : 377 HLA-Nonidentical Related Donors : : : : 378 Conclusions and Future Perspectives : : : : 378 References : : : : 379 20. Hematopoietic Stem-Cell Transplantation for the Treatment of Beta Thalassemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383 Farid Boulad Introduction : : : : 383

Contents

xiii

Hematopoietic Stem-Cell Transplantation from HLA-Matched Siblings : : : : 384 Hematopoietic Stem-Cell Transplantation Using Alternative Donors : : : : 386 Hematopoietic Stem-Cell Transplantation Using Alternative Sources of Stem Cells : : : : 388 Mixed Chimerism Posttransplant for Thalassemia : : : : 388 Alternative Approaches for Allogeneic Stem-Cell Transplantation for Thalassemia : : : : 389 Late Effects Posttransplantation—The “Ex-thalassemic” Patient : : : : 390 Summary : : : : 392 References : : : : 393 21. Hematopoietic Stem-Cell Transplantation for Sickle Cell Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397 Paul Woodard Introduction : : : : 397 Bone Marrow Transplantation for Sickle Cell Disease : : : : 397 Umbilical Cord Blood Transplantation for Sickle Cell Disease : : : : 399 Acute Toxicities of Hematopoietic Stem-Cell Transplantation : : : : 399 Effects on Organs : : : : 400 Challenges to Transplantation : : : : 403 Mixed Chimerism After Transplantation : : : : 404 Alternate Sources of Allogeneic Hematopoietic Stem Cells : : : : 407 Use of Peripheral Blood Stem Cells : : : : 408 Future Directions : : : : 409 Summary : : : : 409 References : : : : 409 22. Metabolic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413 Charles Peters Introduction : : : : 413 Hematopoietic-Cell Transplantation : : : : 416 Mucopolysaccharidoses : : : : 417 Leukodystrophies : : : : 427 Glycoprotein Metabolic Disorders : : : : 433 Miscellaneous Disorders : : : : 434 Developing Therapies and Future Directions : : : : 437 References : : : : 438

xiv

Contents

23. Hematopoietic Stem-Cell Transplantation for Autoimmune Diseases in Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449 Richard K. Burt, Larissa Verda, I. M. de Kleer, and Nico Wulffraat Introduction : : : : 449 Rationale : : : : 449 Animal Models : : : : 450 Stem-Cell Mobilization in Patients with Autoimmune Diseases : : : : 452 Ex Vivo Stem-Cell Selection : : : : 452 Rationale for Design of Autologous Autoimmune Hematopoietic Stem-Cell Transplantation (HSCT) Regimens : : : : 453 Juvenile Idiopathic Arthritis : : : : 454 Crohn’s Disease : : : : 458 Systemic Lupus Erythematosus : : : : 461 Type I Diabetes : : : : 465 Juvenile Dermatomyositis : : : : 466 Immunologic Mechanisms of Hematopoietic Stem-Cell Transplantation : : : : 467 Allogeneic Hematopoietic Stem-Cell Transplantation : : : : 468 References : : : : 468 SECTION III:

MALIGNANT DISEASES

24. Transplantation for Childhood Acute Lymphoblastic Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477 Donna A. Wall, Kirk R. Schultz, and Gregor S. D. Reid Introduction : : : : 477 Biology of Transplantation for Acute Lymphoblastic Leukemia : : : : 477 Importance of Disease Control Pretransplant : : : : 478 When to Transplant in Childhood Acute Lymphoblastic Leukemia : : : : 478 Preparative Regimens : : : : 482 Hematopoietic Stem Cell Source : : : : 483 Graft-Versus-Leukemia in Acute Lymphoblastic Leukemia: Fact or Fiction : : : : 486 Strategies to Augment Posttransplant Immune Activity : : : : 488 References : : : : 489 25. Bone Marrow Transplantation for Acute Myeloid Leukemia in Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497 Allen R. Chen and Robert J. Arceci Historical Background : : : : 497

Contents

xv

Risk Groups and Prognostic Factors : : : : 498 Preparative Regimens : : : : 501 Allogeneic Transplantation : : : : 506 Autologous Transplantation : : : : 510 QOL/Late Effects : : : : 513 Future Directions and Controversies : : : : 514 References : : : : 518 26. Hematopoietic Stem-Cell Transplantation for Children with Hodgkin’s and Non-Hodgkin’s Lymphoma . . . . . . . . . . . . . . 529 Bruce Gordon and K. Scott Baker Non-Hodgkin’s Lymphoma : : : : 529 Hodgkin’s Disease : : : : 539 References : : : : 549 27. Hematopoietic Stem-Cell Transplantation for Pediatric Malignant Brain Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555 Sharon L. Gardner and Ira J. Dunkel Introduction : : : : 555 Gliomas : : : : 555 Brainstem Gliomas : : : : 558 Ependymoma : : : : 558 Medulloblastoma : : : : 559 Other Primitive Neuroectodermal Tumors : : : : 561 Germ Cell Tumors : : : : 561 Infants : : : : 562 Future Directions : : : : 563 References : : : : 565 28. Hematopoietic Stem-Cell Transplantation for Pediatric Solid Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569 ˝ zkaynak and Marcio H. Malogolowkin M. Fevzi O Ewing’s Sarcoma/Peripheral Primitive Neuroectodermal Tumors : : : : 569 Wilms Tumor : : : : 576 Rhabdomyosarcoma (RMS) : : : : 578 Hepatoblastoma : : : : 579 Osteosarcoma : : : : 580 Extracranial Germ Cell Tumors : : : : 580 References : : : : 583 29. Stem-Cell Transplantation in Neuroblastoma . . . . . . . . . . . . . . . . 589 Stephan A. Grupp Introduction : : : : 589 Autologous Transplant in Neuroblastoma : : : : 590 References : : : : 598

xvi

Contents

Appendix: A Brief Overview of Hematopoietic Stem-Cell Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601 Vicki L. Fisher and Linda Z. Abramovitz Introduction : : : : 601 Pretransplant Considerations : : : : 603 The Transplant Process : : : : 606 Learning Disabilities : : : : 623 Conclusion : : : : 624 Index : : : : 625

Contributors

Linda Z. Abramovitz Pediatric Bone Marrow Transplant, Children’s Hospital at the University of California, San Francisco, California, U.S.A. Victor M. Aquino Texas, U.S.A.

University of Texas Southwestern Medical Center at Dallas, Dallas,

Robert J. Arceci Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins University, Baltimore, Maryland, U.S.A. K. Scott Baker Pediatric Blood and Marrow Transplant Program, University of Minnesota, Minneapolis, Minnesota, U.S.A. Raymond Barfield Division of Stem Cell Transplantation, St. Jude Children’s Research Hospital, Memphis, Tennessee, U.S.A. Christi Bartolomucci

Kids on the Move, Atlanta, Georgia, U.S.A.

Monica Bhatia Division of Pediatric Hematology and Blood and Marrow Transplantation, Columbia University, New York, New York, U.S.A. Farid Boulad Department of Pediatrics, Bone Marrow Transplant Service, Memorial Sloan-Kettering Cancer Center, New York, New York, U.S.A. Scott M. Bradfield Division of Hematology/Oncology, Mayo Clinic College of Medicine, Nemours Children’s Clinic-Jacksonville, Jacksonville, Florida, U.S.A. Richard K. Burt Division of Immunotherapy, Feinberg School of Medicine, Northwestern University Medical Center, Chicago, Illinois, U.S.A. Bryan D. Carter Kosair Children’s Hospital and University of Louisville School of Medicine, Louisville, Kentucky, U.S.A. Wassim Chemaitilly Department of Pediatrics, New York Presbyterian Hospital-Cornell Weill Medical Center, New York, New York, U.S.A.

xvii

xviii

Contributors

Allen R. Chen Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins University, Baltimore, Maryland, U.S.A. Kuang-Yueh Chiang Pediatric Blood and Marrow Transplant Program, Aflac Cancer Center and Blood Disorders Service, Children’s Healthcare of Atlanta, Emory University, Atlanta, Georgia, U.S.A. I. M. de Kleer Pediatric BMT Unit, University Medical Center Utrecht, Utrecht, The Netherlands Ira J. Dunkel The Steven D. Hassenfeld Center for Children with Cancer and Other Blood Disorders, NYU Medical Center and Memorial Sloan-Kettering Cancer Center, New York, New York, U.S.A. Stephen A. Feig Mattel Children’s Hospital at UCLA, David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A. Vicki L. Fisher Pediatric BMT Program, Rainbow Babies and Children’s Hospital, Cleveland, Ohio, U.S.A. Sharon L. Gardner The Steven D. Hassenfeld Center for Children with Cancer and Other Blood Disorders, NYU Medical Center, New York, New York, U.S.A. Kamar T. Godder Division of Pediatric Hematology Oncology, Virginia Commonwealth University, Medical College of Virginia Campus, Children’s Medical Center, and Stem Cell Transplantation, Richmond, Virginia, U.S.A. Bruce Gordon Pediatric Hematology/Oncology and Stem Cell Transplantation, University of Nebraska Medical Center, Omaha, Nebraska, U.S.A. Satkiran S. Grewal Department of Pediatrics, Division of Hematology/Oncology, Tufts University School of Medicine, Baystate Medical Center, Springfield, Massachusetts, U.S.A. Stephan A. Grupp Division of Oncology and Department of Pathology, Stem Cell Biology, Children’s Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A. P. Jean Henslee-Downey EMD Pharmaceuticals, Inc., An Affiliate of Merck KGaA, Darmstadt, Germany and Durham, North Carolina, U.S.A. Edwin M. Horwitz Department of Hematology-Oncology, Divisions of Stem Cell Transplantation and Experimental Hematology, St. Jude Children’s Research Hospital, Memphis, Tennessee, U.S.A. Dennis Hughes The Children’s Cancer Hospital, MD Anderson Cancer Center, Houston, Texas, U.S.A. David A. Jacobsohn Stem Cell Transplant Program, Children’s Memorial Hospital, Northwestern University Feinberg School of Medicine, Chicago, Illinois, U.S.A.

Contributors

xix

Naynesh R. Kamani Division of Stem Cell Transplantation and Immunology, Children’s National Medical Center and The George Washington University School of Medicine, Washington, D.C., U.S.A. Eric Kodish Department of Bioethics, Cleveland Clinic Foundation, Lerner College of Medicine at Case, Cleveland, Ohio, U.S.A. William G. Kronenberger Riley Hospital for Children and Indiana University School of Medicine, Indianapolis, Indiana, U.S.A. John Levine Department of Pediatrics and Communicable Diseases, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan, U.S.A. Jeffrey Michael Lipton Division of Pediatric Hematology, Oncology and Stem Cell Transplantation, Schneider Children’s Hospital, New Hyde Park, New York, U.S.A. Brett J. Loechelt Clinical Immunology, Division of Stem Cell Transplantation and Immunology, Children’s National Medical Center, The George Washington University School of Medicine, Washington, D.C., U.S.A. Marcio H. Malogolowkin Bone and Soft Tissue Tumor Program, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A. Theodore B. Moore Mattel Children’s Hospital at UCLA, David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A. Steven Neudorf Blood and Marrow Transplant Program, Children’s Hospital of Orange County, Orange, California, U.S.A. ˝ zkaynak Pediatric Blood and Marrow Transplantation, Division of M. Fevzi O Hematology/Oncology, Department of Pediatrics, New York Medical College, Valhalla, New York, U.S.A. Carole Paley

Novartis Pharmaceuticals, East Hanover, New Jersey, U.S.A.

Charles Peters Hematopoietic Stem Cell Transplantation, Division of Hematology/Oncology, Children’s Mercy Hospital, Kansas City, Missouri, U.S.A. Ralph Quinones Pediatric Bone Marrow Transplantation, Center for Cancer and Blood Disorders, The Children’s Hospital and Department of Pediatrics, University of Colorado School of Medicine, Denver, Colorado, U.S.A. Gregor S. D. Reid Department of Oncology, The Children’s Hospital of Philadelphia, Joseph Stokes, Jr. Research Institute, Abramson Research Center, Philadelphia, Pennsylvania, U.S.A. Elyssa Rubin Pediatric Hematology/Oncology, Children’s Hospital of Orange County, Orange, California, U.S.A.

xx

Contributors

Eric S. Sandler Hematology/Oncology, Mayo School of Medicine, Nemours Children’s Clinic-Jacksonville, Jacksonville, Florida, U.S.A. Kirk R. Schultz Division of Hematology/Oncology, Blood and Marrow Transplantation Program, British Columbia Children’s Hospital, University of British Columbia, Vancouver, British Columbia, Canada Charles Sklar Department of Pediatrics, Bone Marrow Transplant Service, Memorial Sloan-Kettering Cancer Center, New York, New York, U.S.A. Trudy N. Small Department of Pediatrics and Clinical Laboratories, Memorial SloanKettering Cancer Center, New York, New York, U.S.A. Tanya F. Stockhammer Kosair Children’s Hospital and University of Louisville School of Medicine, Louisville, Kentucky, U.S.A. Larissa Verda Division of Immunotherapy, Feinberg School of Medicine, Northwestern University Medical Center, Chicago, Illinois, U.S.A. Adrianna Vlachos Division of Pediatric Hematology, Oncology and Stem Cell Transplantation, Schneider Children’s Hospital, New Hyde Park, New York, U.S.A. Georgia B. Vogelsang Kimmel Comprehensive Cancer Center of Johns Hopkins, Johns Hopkins University, Baltimore, Maryland, U.S.A. John E. Wagner Pediatric Hematology/Oncology/Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, U.S.A. Donna A. Wall Pediatric Blood and Marrow Transplant Program, Methodist Children’s Hospital/Texas Transplant Institute, San Antonio, Texas, U.S.A. Paul Woodard Hematology/Oncology, Division of Stem Cell Transplantation, St. Jude Children’s Research Hospital, Memphis, Tennessee, U.S.A. Nico Wulffraat Pediatric BMT Unit, University Medical Center Utrecht, Utrecht, The Netherlands

SECTION I:

GENERAL PRINCIPLES

1

Supportive Care of the Pediatric Hematopoietic Stem-Cell Transplant Patient Victor M. Aquino University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, U.S.A.

Eric S. Sandler Hematology/Oncology, Mayo School of Medicine, Nemours Children’s Clinic-Jacksonville, Jacksonville, Florida, U.S.A.

INTRODUCTION Hematopoietic stem-cell transplant (HSCT) is an effective therapy for a variety of malignant and nonmalignant conditions. Morbidity and mortality after HSCT are often due to toxicity of the conditioning regimen and resulting pancytopenia, acute and chronic graft versus host disease (GVHD), and the complications of drug therapy. Early recognition and treatment of the complications of HSCT will keep morbidity and mortality to a minimum. The following chapter summarizes the pathophysiology, prevention and management of the noninfectious complications encountered by HSCT recipients.

PREVENTION AND MANAGEMENT OF MUCOSITIS Mucositis is a common complication of HSCT (1,2), occurring in 99% of HSCT recipients in one series with 67.4% having severe mucositis (i.e., grade III and IV) (3). Not only is its occurrence frequent, but mucositis was rated by 42% of patients as the most debilitating side effect of HSCT (4). The etiology of mucositis is multifactorial. The inclusion of melphalan, etoposide, and total body irradiation (TBI) in the conditioning regimen is highly associated with the occurrence and severity of mucositis. Other contributing factors include the use of methotrexate for GVHD prophylaxis, the development of infection, especially herpes simplex and candida, and GVHD. The pathophysiology of mucositis is complex and includes direct cytotoxic effects of therapy on the mucosa, local inflammatory responses, lack of neutrophils for healing, and changes in the oral microflora (5). The morbidity associated with mucositis in patients is significant and includes significant pain, inability to take in adequate calories, predisposition to systemic infection, particularly with oral flora including Streptococcus viridans and anaerobes, and respiratory failure secondary to upper airway damage (6). Although mucositis is typically observed in the mouth 1

2

Aquino and Sandler

and throat, it often extends throughout the gastrointestinal tract and may result in further infection risk with gram-negative bacilli, diarrhea, nausea and vomiting, ileus, and bowel wall injury. The consequences of mucositis include an increase in the number of days of fever, greater use of parenteral antibiotics, greater need for total parenteral nutrition (TPN), and increased use of narcotic therapy. In one study, severe mucositis was associated with a 3.9-fold increase in 100-day mortality, an increase of 2.6 days of narcotic use, an average increase of 2.6 days of hospitalization, and $43,000 in additional hospital charges (7). One problem in defining effective agents for the prevention and treatment of mucositis has been the difficulty in objectively defining the severity of mucositis. There are now multiple studies utilizing different assessment instruments in studying mucositis severity (8). Mucositis may be graded by the National Cancer Institute common toxicity criteria (NCI-CTC) (9), which are summarized in Table 1. Although the treatment of established mucositis has been primarily supportive, many interventions have been studied to avoid this complication. One primary preventative therapy has been infection prophylaxis (10). Acyclovir is used commonly to prevent herpes infection and antifungals to prevent candidal infections. There has been significant controversy about the role of antibacterial prophylaxis of mucositis with topical agents, such as chlorhexidine or nonabsorbable antibiotics. Several studies have failed to clearly demonstrate the efficacy of these agents (11,12). A study by the Pediatric Blood and Marrow Transplant Consortium (PBMTC) evaluated the use of a vancomycin paste for the prevention of mucositis and, particularly, secondary systemic infection (12). Although vancomycin paste was found to significantly decrease the severity of mucositis, use of narcotics, and positive blood cultures, the agent has not been recommended for further study because of concerns for the emergence of vancomycin resistant organisms. A more recent study by this group, as well as several others, has demonstrated the efficacy of glutamine, given either intravenously or orally, in decreasing the frequency and severity of mucositis, with secondary improvement in the duration of narcotic use and hospital days (13), especially in patients undergoing allogeneic HSCT. Other studies have resulted in conflicting outcomes with several showing that glutamine given either orally or intravenously does improve outcomes and others failing to show a difference in outcome (14–16). Different results may be explained by patient selection, dosing of glutamine, or the formulation used in the study. Recently, palifermin (recombinant keratinocyte growth factor) was shown to reduce the incidence and duration of mucositis in patients undergoing HSCT (17). In addition to the improvement in clinical mucositis, there was a statistically significant reduction in the number of days of morphine and TPN use. Other agents that have shown efficacy in nonrandomized studies include topical lidocaine or “magic mouthwash preparations,” sucralfate, clarithromycin, granulocyte-macrophage colony-stimulating factor (GM-CSF) oral rinses, topical tretinoin, cryotherapy, and propantheline, an anticholinergic agent (18–22). Several other studies have attempted to decrease the severity of mucositis by decreasing the dose or number of doses of methotrexate given as GVHD prophylaxis. Another study added leucovorin after each dose of methotrexate to prevent side effects, including mucositis (23). Newer agents under study currently include Traumeel, interleukin (IL) 11, and EN3247 (2). In conclusion, mucositis is a common and significant complication of HSCT. Preventive efforts include avoidance of specific conditioning agents when possible, avoidance of high doses of methotrexate, and prophylaxis of infection. The best treatment is not currently known, and the mainstay of treatment remains supportive care with nutritional support, pain management, and treatment of infection. The efficacy of glutamine and other new agents remains to be studied.

HEPATIC VENO-OCCLUSIVE DISEASE Veno-occlusive disease (VOD) of the liver is a clinical syndrome that occurs as a result of damage to the liver by pretransplant radiation and chemotherapy. The incidence of VOD varies

None

None

Radiationinduced

HSCT

Grade 2 (moderate)

Grade 3 (severe)

Painless ulcers, erythema, Painful erythema, edema, Painful erythema, edema, or ulcers requiring IV hydration or ulcers, but can eat or or mild soreness in the swallow absence of lesions Confluent pseudomembranous Erythema of the mucosa Patchy pseudomemreaction (contiguous patches branous reaction generally O 1.5 cm in (patches generally diameter) R1.5 cm in diameter and noncontiguous) Painless ulcers, erythema, Painful erythema, edema, Painful erythema, edema, or ulcers preventing swallowing or or ulcers, but can or mild soreness in the requiring hydration or swallow absence of lesions parenteral (or enteral) nutritional support

Grade 1 (mild)

Abbreviation: HSCT, hematopoietic stem-cell transplant. Source: From Ref. 9.

None

Grade 0

Grading of Oral Mucositis According to National Cancer Institute Common Toxicity Criteria

Chemotherapyinduced

Table 1

Severe ulceration requiring prophylactic intubation or resulting in documented aspiration pneumonia

Severe ulceration or requires parenteral or enteral nutritional support or prophylactic intubation Necrosis or deep ulceration; may include bleeding not induced by minor trauma or abrasion

Grade 4 (life threatening)

Supportive Care of the Pediatric HSCT Patient 3

4

Aquino and Sandler

but appears to be more common in adult stem cell transplant recipients (7–54%) (24) than in children (11%) (25). VOD is caused by endothelial damage to the hepatic sinusoids and small hepatic venules by radiation and chemotherapy administered as part of the conditioning regimen. This damage appears to induce a local hypercoagulable state by activating the coagulation cascade, leading to occlusion of the hepatic venous outflow tract, which then leads to intrahepatic portal hypertension. As the syndrome progresses, progressive sinusoidal fibrosis appears, exacerbating the portal hypertension (26). The differential diagnosis of liver dysfunction in HSCT recipients is summarized in Table 2. VOD may occur with conventional doses of chemotherapeutic agents, such as Ara-C, 6-thioguanine, and actinomycin-D, but is much more common after high dose therapy. Chemotherapeutic agents used in HSCT preparative regimens associated with the development of VOD include busulfan, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), and mitomycin-C. Risk features associated with the development of VOD include age greater than 15 years at the time of transplant, recipients of a second transplant, active hepatitis, a history of prior liver disease (viral or drug induced hepatitis), and an elevated AST prior to transplant. Children with a diagnosis of acute lymphoblastic leukemia also had a higher incidence of VOD, perhaps due to exposure of methotrexate as part of their chemotherapeutic regimen (27). Pathologic changes of VOD are centered in the terminal hepatic venules and the central lobular hepatocytes in zone 3 of the liver acinus (28). Complete or partial hepatic venular occlusion by thrombosis or endophlebitis is seen. Histologically, VOD leads to nonthrombotic obstruction of centrolobular veins by subendothelial connective tissue and centrolobular hepatocyte necrosis (29). Other pathologic changes in zone 3 include eccentric venular luminal narrowing, phlebosclerosis, sinusoidal fibrosis, and necrosis of hepatocytes (30). VOD usually occurs within the first thirty days after transplant (27). The diagnosis of VOD is made by the presence of clinical symptoms and laboratory tests. The diagnostic criteria for VOD are summarized in Table 3. The earliest clinical sign of VOD is progressive weight gain and peripheral edema due to renal sodium retention. Patients often become thrombocytopenic and may be refractory to platelet transfusion. Patients with severe VOD may develop renal dysfunction, which can progress to renal failure, pleural effusions, and hepatic encephalopathy. Multiorgan failure may develop and is associated with 90% mortality in patients with VOD. The severity of VOD, scored according to mild, moderate, and severe disease, is defined retrospectively according to outcome (33). Mild disease is defined by no apparent adverse effect with complete resolution of symptoms. Moderate disease is characterized by liver dysfunction requiring therapy, such as diuresis for fluid retention and analgesia for pain, but with complete resolution. Severe disease is defined as disease causing the death of the patient. Although histologic examination of the liver parenchyma is required for definitive diagnosis of VOD, it is rarely performed. Percutaneous liver biopsy may be helpful in differentiating VOD from other disorders of the liver, but it is difficult to perform in the patient Table 2

Causes of Liver Dysfunction After Hematopoietic Stem-Cell Transplant (HSCT)

Acute graft-versus-host disease Chronic graft-versus-host disease Veno-occlusive disease Hepatitis Viral: CMV, adenovirus, hepatitis A, B and C, Epstein-Barr virus Bacterial: Septicemia Fungal: Candida, aspergillus, cryptococcus Drug Toxicity: cyclosporine, erythromycin, metotrexate, ketoconazole, fluconazole Total parenteral nutrition Abbreviation: CMV, cytomegalovirus.

Supportive Care of the Pediatric HSCT Patient Table 3

5

Criteria for the Diagnosis of Hepatic Veno-occlusive Disease

Jones criteria (31) Hyperbilirubinemia R2 mg/dL before day 21 after transplant and at least two of the following: Hepatomegaly, usually painful Ascites Weight gain O5% from pretransplant baseline Berman scale (32) Presence of two of the following within 2 weeks of transplantation: Bilirubin R2.0 mg/dL Weight gain R2.5% from pretransplant baseline Hepatomegaly and/or right upper quadrant pain

with thrombocytopenia who responds poorly to platelet transfusion. The transjugular route can be used for both biopsy and measurement of the hepatic vein pressure gradient (34), which has been shown to correlate with histologic features of VOD (30). In patients without prior liver disease, a hepatic venous pressure gradient of more than 10 mmHg as measured by the transjugular route is highly specific (O90%) and moderately sensitive (60%) for VOD (35). Ultrasonography can detect ascites, hepatomegaly, and hepatic vein dilatation. Gall bladder wall thickening of greater than 4 mm has been described in patients with VOD, and a correlation with hepatic venous pressure gradient has been noted (36). Ultrasonography has been used to detect reversal of flow through the para-umbilical vein, but this is often a late finding in VOD (31). Fortunately, in most pediatric patients VOD is reversible. However, more than 25% of patients have irreversible disease, which usually leads to multiorgan failure and death (27,32). In a series of 335 patients with VOD, outcome was predicted by the serum bilirubin and the amount of weight gain (37). Reversal of hepatic flow on Doppler ultrasound is predictive of mortality but is often a late finding. Several drugs have been studied as prophylactic agents against the development of VOD, although the efficacy of such interventions has been disappointing. Several randomized studies have been performed using a continuous infusion of low-dose heparin, the majority of which did not show a reduction in the development of VOD (26). A recent study has shown a lower incidence of VOD in patients receiving low molecular weight heparin (38). Prophylactic use of ursodiol has been shown to reduce liver toxicity but not reduce the incidence or severity of VOD (39). Studies have failed to show that prostaglandin-E1 or pentoxifylline are effective prophylactic agents (26). The mainstay of treatment of VOD is supportive care with fluid restriction, maintenance of sodium balance, and administration of pain medication. The efficacy of low-dose dopamine to maintain renal blood flow is controversial (40). Drugs, such as heparin (41), tissue plasminogen-activator (42), and streptokinase, have been utilized with varying results. Unfortunately, the use of these agents is associated with potentially fatal bleeding, especially in patients with thrombocytopenia due to VOD. Difibrotide (43,44) is a new agent that stimulates the synthesis of thrombomodulin, increases endogenous tissue plasminogen activator, and decreases plasminogen activator inhibitor type I with very little anticoagulant activity. In one study, complete resolution of severe VOD was seen in 40% of cases without significant toxicity (45). Defibrotide has also been shown to be effective in the prevention of VOD. In one study (46), use of prophylactic defibrotide was found to reduce the maximum total bilirubin levels and to lead to an improvement in survival. Large prospective randomized trials are required to determine the efficacy of defibrotide in the management and prevention of VOD. Transjugular intrahepatic portosystemic shunting (47) and liver transplantation can be considered in patients who do not respond to conventional therapy.

6

Aquino and Sandler

In summary, VOD is a commonly seen complication of HSCT that may progress to multiorgan failure and death. Fortunately, VOD spontaneously reverses in most pediatric patients. The mainstay of therapy is early recognition, with careful fluid management and pain control. Interventions to prevent and/or treat the disorder have not been successful. Newer agents, such as defibrotide, appear promising but require further study.

NUTRITION SUPPORT Nutritional problems are extremely common after HSCT, with more than 90% of patients requiring nutritional support in some studies (48,49). These children are prone to the development of severe gastrointestinal complications as well as metabolic derangements. The results are decreased intake, increased metabolic needs, and wasting. Decreased intake is a result of: (1) mucositis secondary to conditioning and infection, (2) nausea and vomiting, (3) anorexia, (4) changes in taste, making it hard to find pleasing foods, (5) restrictions in food choices, (6) development of gastroparesis, and (7) acute and chronic GHVD (50). Wasting in the form of diarrhea and protein losing enteropathy is also common. Patients will often have increased metabolic needs as a consequence of GVHD, infection, or other complications that must be met. The end result is often protein calorie malnutrition. Nutrition support has historically been centered on TPN (51). The majority of HSCT patients start on TPN around the day of transplant, and most require several weeks of support. Often ongoing support is necessary even after recovery from other acute manifestations and hospital discharge. In those patients with acute and chronic GVHD, nutrition supplementation may continue for weeks to months. Recently, there has been an increased interest in the use of enteral support in the HSCT patient (52,53). Such support has taken the form of nasogastric feedings or placement of gastrostomy tubes (54,55). There are several advantages to the use of enteral support. Its use is associated with lower costs and less need for laboratory monitoring when compared with TPN. Enteral feeding may theoretically protect the GI enterocyte from damage caused by conditioning, especially when such agents as glutamine are added to the formula. The use of enteral feedings has also been associated with a decreased risk of infection when compared with TPN administered via a central venous catheter (56). In general, the few studies comparing parenteral and enteral support have found similar efficacy in most outcome measures but decreased complications with enteral feeding. In one study by Pietsch et al. 17 children undergoing intensive chemotherapy or HSCT received glutamine supplemented NG feeds and tolerated it well (57). They compared the costs to a similar group of patients receiving TPN and found that for the same number of days, the cost differential was $25,348 versus $112,299 for NG feeds and TPN, respectively. Another study compared 12 patients receiving NG feedings to 22 receiving TPN. Both groups achieved 85% of their nutritional needs, GI symptoms were equally frequent, but again the cost associated with the use of NG feedings was significantly less than that of TPN (14). Although several groups are currently advocating the use of enteral feeds, these are not without potential complications, including the problems of NG insertion in a patient with mucositis and the risk of vomiting and aspiration. The most important changes resulting in a decreased need for nutrition support have been in the area of general supportive care. These include the use of conditioning regimens associated with development of less severe mucositis, prevention of GVHD and infections, use of newer more effective antiemetics, and newer agents that may prevent severe mucositis and wasting. The issue of when to discontinue parenteral and enteral nutrition support is controversial. Some groups will continue support after discharge from the hospital, whereas others make every attempt to stop support at the time of discharge. One study specifically addresses this issue by randomizing patients to continued TPN or hydration fluids after discharge from the hospital.

Supportive Care of the Pediatric HSCT Patient

7

They found that patients who were discharged with fluids only had a more rapid resumption of oral intake (58). In summary, malnutrition is a frequent complication of HSCT. It is important that nutritional needs be addressed in all patients undergoing HSCT. Further studies are ongoing to define the best methods of nutritional support.

HEMORRHAGIC CYSTITIS Hemorrhagic cystitis (HC), defined as greater then 100 RBC/high power field persisting for longer then 2 days, is a common complication of HSCT, occurring in 10–50% of HSCT recipients (59). Patients may develop dysuria and urinary obstruction. Early onset HC, occurring within 48 hours of conditioning treatment, is typically secondary to cyclophosphamide or ifosfamide use (60). With current preventive therapies including MESNA and aggressive hydration, it is now rarely seen. Late onset HC, occurring days to several months after transplant, predominates. The etiology of late onset disease is likely secondary to reactivation of viruses during a time of immunosuppression (61,62). The use of pretransplant cyclophosphamide and irradiation, as well as thrombocytopenia, during the time of myeloablation may increase the severity of early HC. Recent literature supports the reactivation of latent virus, including BK virus, a human polyoma virus, adenovirus, and Cytomegalovirus (CMV) as etiologic factors in the development of late HC (59,63). These viruses are ubiquitous, and once infection occurs, they remain dormant in the kidney with clinically relevant infection developing during times of immunosuppression (64,65). As a result, treatment for GVHD has been associated with an increased risk of HC. In one study of 63 patients undergoing HSCT, 11 patients developed 19 episodes of HC, with 89% having documented viruria or bacteruria (12 BK, 2 adenovirus, 1 CMV, and 3 bacteria) (59). Patients with GVHD had a significantly increased risk of HC. In another study of adult patients, 52% of patients were BK positive (66). HC occurred in 50% of those who were BK positive and in none of those who were BK negative. In a more recent study of more than 800 urine samples from 50 patients, the BK viral load in the urine was significantly higher in patients who developed HC (67). In the past, treatment of severe HC was largely supportive, including correction of coagulopathy and thrombocytopenia, hyperhydration or continuous bladder irrigation, localized treatment, including cystoscopy to evacuate clots, and local instillation of Prostaglandin-E, alum, silver nitrate, or formaldehyde (68,69). Other supportive therapies reported include the instillation of GM-CSF (70), aminocaproic acid (71), and the use of hyperbaric oxygen (72). More recently, new antiviral agents have been shown to be very effective, including ganciclovir for CMV, intravenous ribavirin for adenovirus, and cidofovir for treatment of both BK virus and adenovirus (73,74). In summary, HC is a common complication of HSCT. The early onset disease seen in the past and associated with the conditioning chemotherapy is now rarely observed. However, late onset disease, frequently seen, is associated with the level of immunosuppression and viral reactivation. New antiviral agents especially cidofovir have been extremely effective in treatment of HC.

PREVENTION AND MANAGEMENT OF RENAL DISEASE Renal dysfunction after bone marrow transplantation is a relatively frequent event, occurring in approximately 30–50% of children undergoing HSCT (75,76). Early renal injury (within the first 100 days after transplant) most often results from infection, its prophylaxis, and treatment. The causes of early renal dysfunction in HSCT recipients are summarized in Table 4. Acute tubular necrosis may arise as a direct consequence of sepsis with or without hypotension or from therapy with a variety of nephrotoxic drugs (77). Other causes of early renal injury include tumor lysis syndrome, marrow infusion-associated acute renal failure, and VOD.

8 Table 4

Aquino and Sandler Causes of Early Renal Dysfunction After Hematopoietic Stem-Cell Transplant

Septicemia Hypotension: hemorrhage, cardiac failure Veno-occlusive disease Drug induced: amphotericin, cyclosporine, aminoglycosides, vancomycin, foscarnet, acyclovir, previous chemotherapy with cisplatin or ifosfamide Total body irradiation Hypertension: cyclosoporine, steroids

Late renal injury (after 100 days post transplantation) may be caused by a syndrome similar to hemolytic-uremic syndrome and is thought to evolve from the late effects of radiation therapy and cytotoxic chemotherapy.

Nephrotoxic Drugs A variety of nephrotoxic drugs are administered to children and adolescents undergoing HSCT, as well as drugs that may potentiate the nephrotoxicity of other agents. Cyclosporine and tacrolimus, commonly used for prophylaxis of acute GVHD, are nephrotoxic. The dose of cyclosporine and tacrolimus should be reduced if patients are receiving voriconazole (78). Antibiotics, such as the aminoglycosides that are routinely administered to prevent and treat infections, are associated with renal toxicity. Drug levels should be carefully monitored in patients receiving these agents to minimize renal damage. Amphotericin-B is nephrotoxic and causes electrolyte wasting. Fluid boluses prior to infusion may reduce the nephrotoxicity of this agent. The use of liposomal amphotericin products and other new antifungal agents may reduce the risk of nephrotoxicity when compared with conventional amphotericin.

Tumor Lysis Syndrome In patients with tumor lysis syndrome, renal failure occurs after induction of massive tumor cell death, results from the rapid death of tumor cells that results in the release of tumor cell products, and the development of hyperuricemia, hyperkalemia, and hyperphosphatemia. Acute renal failure results from the accumulation of uric acid and phosphate in the renal tubules, which causes obstruction and filtration failure. Because most HSCT recipients who come to transplant receive prior antineoplastic therapy and are usually in complete or partial remission, this syndrome rarely complicates HSCT [approximately 1% of transplant patients (79)]. Prevention of tumor lysis syndrome includes the use of aggressive volume expansion, urine alkalinization, and allopurinol. Recombinant uricase (80) has been shown to be effective in reducing the amount of uric acid and the risk of renal failure. Renal dialysis may be employed if patients develop severe hyperphosphatemia and/or renal failure develops.

Marrow Infusion-Associated Acute Renal Failure Red blood cell (RBC) hemolysis with release of free hemoglobin can occur during bone marrow collection or if cold storage (freezing and thawing) is performed. Infusion of such marrow can be associated with heme protein–induced nephrotoxicity, which potentially can lead to renal failure. Heme protein cast formation, which can occur in acidic urine, renal vasoconstriction, and proximal tubular cell heme loading are the main pathogenic mechanisms that lead to this form of renal damage.

Supportive Care of the Pediatric HSCT Patient

9

Heme protein associated nephrotoxicity may be prevented by vigorous hydration and intravascular volume expansion before and during marrow infusion. This limits cast formation, renal vasoconstriction, and proximal tubular heme uptake. Potential therapeutic interventions include iron chelation therapy, the use of endothelial antagonists, and nitric oxide supplementation by L-arginine therapy (79).

Hemolytic Uremic Syndrome The most common cause of renal failure beyond the period of marrow engraftment is hemolytic uremic syndrome , which can be documented in approximately 5% to 25% of posttransplant patients (81,82). Hemolytic Uremic Syndrome (HUS) is characterized by the development of microangiopathy with end organ damage (especially the kidney and central nervous system), microangiopathic hemolytic anemia, and thrombocytopenia. HUS occurs between 3 and 12 months post–bone marrow transplant. TBI is the most likely cause of HUS, although infectious agents (e.g., CMV), immunologic reactions (e.g., GVHD), and nephrotoxic drugs (cyclosporine, tacrolimus) have also been implicated (83). The management of HUS has largely been supportive with the use of RBC and platelet transfusions and hemodialysis if renal failure develops. Although a variety of specific treatments have been studied, their efficacy in patients with HUS has been limited. Patients with severe HUS are often treated with plasmapheresis, although no compelling evidence of its efficacy has been documented to date (83). Other interventions have included discontinuation of cyclosporine, hemoperfusion over staphylococcal protein A column, and intravenous gamma-globulin therapy. The prognosis is predicted by the severity of the disease. The disease appears to undergo spontaneous resolution, although the organ damage that occurs may be permanent.

Long-Term Renal Complications of Hematopoietic Stem-Cell Transplant Late renal toxicity (O100 days) has been reported in 11–54% of patients who have received HSCT (84). It appears that the single most important risk factor in the development of late nephropathy is the use of radiation therapy as a part of the conditioning regimen (81,85). Radiation-induced nephritis usually presents 6–12 months posttransplant and is thought to be due to damage to the renal vascular epithelium (86,87), the latent period being attributed to slow endothelial turnover and progressive tissue damage (88). Radiation-induced nephritis may present acutely, as seen in hemolytic-uremic syndrome, and presents with severe anemia, microscopic hematuria, proteinuria, elevation of BUN and creatinine, hypertension and evidence of microangiopathic intravascular hemolysis (81,85). The chronic form presents with isolated renal impairment with or without hemolysis (89). A variety of other nephrotoxins may play a role in the development of late nephrotoxicity. Chemotherapeutic agents, such as cisplatin, appear to predispose patients to the development of late nephrotoxicity (85,90). Therapy with cyclosporine has been shown to cause microangiopathy, hypertension, and potentially HUS (91). Drug therapy with amphotericin and aminoglycoside antibiotics may also predispose patients to late nephrotoxicity. In summary, early and late renal dysfunction are an important cause of morbidity and mortality in children undergoing HSCT. Careful monitoring of drug levels and renal function are required to prevent long-term complications in the survivors of HSCT.

NEUROLOGIC COMPLICATIONS OF HEMATOPOIETIC STEM-CELL TRANSPLANT Neurologic complications of HSCT can be generally divided into five areas: encephalopathies, infectious complications, chemotherapy, radiation therapy, and cerebrovascular disorders (92).

10 Table 5

Aquino and Sandler Neurologic Complications After Hematopoietic Stem-Cell Transplant (HSCT)

Seizures Drug induced: busulfan, cyclosporine, imipenem Hyponatremia or H2O intoxication: cyclophosphamide, inappropriate antidiuretic hormone secretion Hypomagnesemia: cyclosporine, fanconi syndrome Hypocalcemia Hypoglycemia: inadequate infusion, pentamidine Dystonic reactions: metoclopramide or phenothiazines Encephalopathy: uremia, liver failure (e. g., veno-occlusive disease), drug induced Intracranial hemorrhage Infection: aspergillus, cryptococcus, herpesvirus encephalitis, pneumococcal meningitis, toxoplasmosis Polymyositis Thrombotic thrombocytopenia purpura

The various causes are summarized in Table 5. Significant neurologic events have been reported to occur in from 14% to O50% of HSCT recipients in various series and are associated with a mortality of up to 10% of patients (93). Risk factors for neurologic events include alternative donor transplants, development of severe GVHD, and the use of TBI (94). The most common complications are in those patients who develop encephalopathy. These encephalopathies may occur with or without seizures. The most common etiology of encephalopathy is medication related. Both busulfan and BCNU have been associated with seizures and altered mental status (95). The incidence of seizures in children receiving busulfan seems to be higher then that seen in adults. Most pediatric centers routinely prophylaxis these patients with anticonvulsants during the period of drug administration. Other medications commonly associated with seizures and/or encephalopathies are the immunosuppressant medications cyclosporine and tacrolimus. Typically patients will develop severe hypertension and headaches prior to the onset of seizures and/or encephalopathy. Neurologic complications are more likely when these patients are also treated with steroids and have hypertension or hypomagnesemia. In most cases, drug levels are elevated at the time of neurologic dysfunction. Seizures may be focal in nature, and MRI will typically show white matter changes, often in the posterior circulation. Transient cortical blindness is occasionally seen as well. Fortunately, all neurologic complications usually resolve with discontinuation of the medication and the use of antiseizure medication. Long-term sequelae are rare (96). Other drugs frequently used in the transplant setting are occasionally associated with seizures and encephalopathy as well, including antimicrobials (imipenem), narcotics, and chemotherapy agents. Depressed mental status can also be seen in patients with multiorgan failure or those with isolated liver disease who develop hyperammonemia. The development of depressed mental status in these situations is considered a poor prognostic sign. Inciting factors for depressed neurologic function may include injury due to cytokines, organ dysfunction (i.e., hypoxia secondary to pulmonary failure), or microthrombotic disease causing multiorgan function (97). CNS infection is also an ominous complication of HSCT (98). Whenever CNS dysfunction occurs in these severely immunocompromised patients, infection must be considered. Bacterial and viral meningitis/encephalitis may occur without the usual clinical manifestations of meningitis. A diagnostic lumbar puncture must be strongly considered as part of the evaluation in these circumstances. Until results of the lumbar puncture and radiographic examination are available, empiric coverage with broad-spectrum antibiotics and antiviral therapy should be strongly considered. More concerning is the high risk of opportunistic infections of the CNS, particularly in the alternative donor patient receiving significant immunosuppressive therapy. The herpes family of viruses, including HHV6, cryptococcus,

Supportive Care of the Pediatric HSCT Patient

11

fungal disease with abscess, toxoplasmosis, and nocardia infections, have all been reported in posttransplant patients. Acute changes in neurologic function, particularly those associated with focal signs, are very suggestive for cerebrovascular disease. The incidence of intracranial hemorrhage ranges from 1–30% depending on the series reviewed (99,100). Risk factors for intracranial hemorrhage include the sometimes severe thrombocytopenia commonly seen prior to engraftment, coagulation defects secondary to infection or organ dysfunction, and CNS infections. Ischemic lesions are much less common but have also been reported. This may be due to underlying infection, embolic disease, or an underlying hypercoagulability, which has now been well described in the HSCT setting (101). Although not proven, some investigators have described a CNS angitis that may be associated with both acute and chronic GVHD (102). Particularly in leukemia patients, recurrence of leukemia in the CNS may also present with neurologic signs and symptoms. In addition, the development of a somnolence syndrome approximately one month after treatment with TBI has been well described. Late CNS complications reported include not only recurrent disease but also leukoencephalopathy and secondary CNS malignancies. Other rare neurologic complications seen after HSCT include encephalopathy associated with thrombotic thrombocytopenic purpura and unusual immune mediated peripheral neuropathies associated with chronic GVHD, such as polymyositis and Guillain Barre´ Syndrome (103,104). TTP is a systemic microvascular disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia, and ischemic manifestations (105) TTP and HUS were initially described as distinct disorders but are now considered different expressions of the same disease process characterize by the nonimmune destruction of platelets. TTP shares many features with HUS, including consumptive thrombocytopenia, microangiopathic hemolytic anemia, and renal dysfunction. TTP occurs in 10–15% of allogeneic patients and up to 7% of autologous patients. Risk factors include extensive prior therapy, the occurrence of GVHD and VOD, and the use of cyclosporine. A majority of patients will develop renal and neurologic symptoms. Treatment is controversial in the posttransplant setting. Studies have reported mixed results with plasmapheresis and immune suppression. Recently defibrotide has been suggested as a successful treatment of TTP (83,106,107). Any patient undergoing HSCT who develops neurologic dysfunction should have immediate assessment with a careful physical exam, CT scan, and empiric coverage for possible infection as well as correction of any coagulopathy. Strong consideration should be given to LP with CSF analysis and MRI. Prognostic implications of neurologic dysfunction are dependent on the underlying etiology.

TRANSFUSION SUPPORT Transfusion of blood products is critical to support the HSCT patient prior to engraftment. All blood products administered to HSCT recipients should be leukoreduced and irradiated prior to infusion.

Components Red Blood Cells RBCs are transfused in patients with anemia to improve oxygen carrying capacity. In general, patients with a hemoglobin greater than 10 gm/dl are asymptomatic and do not require transfusion. Although centers have different criteria for transfusion, most centers transfuse patients when their hemoglobin is less than 7–8 gm/dl. RBCs are removed from whole blood by centrifugation. The volume of the average unit of RBCs is about 350 mL, which contains 200 mL of RBCs with a hematocrit of 60%. Citrate phosphate dextrose (CPD) and CP2D are preservative solutions approved by the

12

Aquino and Sandler

Food and Drug Administration (FDA) for 21-day storage of RBCs. Blood collected in adenine-fortified CPD may be stored for 35 days. Adsol (AS-1), Nutricel (AS-3), and Optisol (AS-5) are newer preservatives that can extend the storage time to a maximum of 42 days (108). Washed RBCs are rarely used and are available for patients who have severe reactions to plasma, such as those with IgA deficiency.

Platelets Platelets are transfused in order to prevent or treat bleeding complications related to thrombocytopenia. There are two types of platelet concentrates: those prepared from centrifugation of whole blood (“random-donor”) and those collected by apheresis. Platelets are then stored in anticoagulant containing CPD or citrate-phosphate-dextrose-adenosine and then stored on elliptical, circular, or flat-bed agitators. Platelets can be stored for up to five days at temperatures of 20 to 248C, after which the increased risk of bacterial contamination mandates the discarding of the unit. In general, one random unit of platelets per 5 kilograms of patient body weight will raise the platelet count by 40,000–50,000/mm3. A single apheresis unit of platelets will raise the platelet count of an adolescent or adult to 50,000/mm3. The decision as to whether to administer platelet transfusions depends on the cause of the thrombocytopenia, the anticipated duration of the thrombocytopenia, the patient’s platelet count, and the clinical condition of the patient. The risk of serious spontaneous bleeding when the platelet count is above 20,000/mm3 is small but increases with lower platelet counts. Most centers administer platelet counts on a prophylactic basis when the patient’s platelet count decreases to less than 20,000/mm3 in an otherwise asymptomatic patient. The trigger for platelet transfusion may be higher in patients at increased risk of bleeding such as mucositis. Patients who receive multiple platelet transfusions are at risk of developing refractoriness to platelet transfusions. Platelet refractoriness should be suspected in patients who do not get the expected increase in platelet count after a platelet transfusion. There are two general causes of platelet refractoriness. Patients with immune mediated thrombocytopenia develop antibodies to HLA class I molecules, ABO blood group antigens, or platelet membrane specific antigens. The platelet refractory state may be prevented by the use of single donor platelets, and leukoreduced platelet products. These patients can be managed with the use of HLA-matched or ABO compatible platelet transfusions if available. Nonimmune platelet refractoriness is caused by sequestration of platelets (such as in patients with splenomegaly) or due to increased platelet destruction [such as disseminated intravascular coagulation (DIC) or fever]. The use of a one-hour posttransfusion platelet count may help to differentiate these two conditions. Patients with no increase in the one-hour posttransfusion platelet count are likely to have immune-mediated platelet destruction. Granulocytes Infections that occur prior to engraftment and in the immediate posttransplant period are a major cause of morbidity and mortality in children and adolescents undergoing stem cell transplant. Such patients even after recovery from postconditioning neutropenia exhibit neutrophil dysfunction and may manifest defective cellular and humoral immunity for months following HSCT. It appears that granulocyte infusions are effective for the treatment of gramnegative bacteremia in neutropenic neonates (109). However, the efficacy of granulocyte infusion in patients undergoing HSCT is less clear. Early studies were limited by the administration of low doses of granulocytes and the unavailability of hematopoietic growth factors (CSFs) to increase the number of peripheral blood neutrophils. The use of granulocytes harvested from cytokine-stimulated donors has not been well studied. Recent data also suggests that compatibility testing of granulocytes may contribute to prolonged granulocyte survival and may improve their efficacy (110).

Supportive Care of the Pediatric HSCT Patient

13

Granulocytes are collected by apheresis and are separated based upon density. Granulocyte collections taken by centrifugation from healthy, unstimulated donors usually yield approximately 0.5–1.0!1010 granulocytes per liter of donor blood. Donor granulocyte counts may be increased by the administration of agents, such as steroids or CSFs, that mobilize granulocytes from the marginal pool into the circulating pool or increase their production. Granulocytes are then irradiated prior to infusion to prevent the development of transfusion related graft-versus-host-disease. Reactions after granulocyte transfusions are common. Fever, chills, dyspnea, chest tightness, acrocyanosis, hypoxia, and pulmonary infiltrates on chest X-ray may be seen. These episodes may take up to 12 hours to resolve. The concurrent administration of amphotericin B and granulocyte concentrates has been reported to cause pulmonary reactions, the mechanism of interaction is unknown (111). However, separating the administration of granulocytes and amphotericin B by several hours may avoid this adverse effect.

Fresh Frozen Plasma Plasma is removed from whole blood by centrifugation and frozen at K188C or lower within 8 hours of collection and may be stored for up to seven years. The volume of a unit of fresh frozen plasma is 200–250 mL. FFP is indicated for the correction of documented deficiencies of coagulation factors, after massive RBC transfusion or for the correction of the coagulopathy associated with DIC. The usual dose of FFP is 10–15 ml/kg administered over one to two hours. Each unit of FFP contains an average of 1 unit/ml of factors II, V, VIII, IX, and X. FFP does not require irradiation prior to infusion in immunocompromised patients. Cryoprecipitate Cryoprecipitate is the cold insoluble portion of FFP thawed at 1–68C and is stored at K188C. Cryoprecipitate contains Factor VIII, fibrinogen, and von Willebrand factor. The main indication for cryoprecipitate is for the replacement of fibrinogen in patients with DIC. A bag of cryoprecipitate contains approximately 200 to 250 mg of fibrinogen. An appropriate dose of cryoprecipitate is one bag for each 5–10 kilograms of body weight.

Processing of Blood Products Prior to Infusion Leukocyte Depletion Leukocytes may be removed from blood products by centrifugation or by filtration. The current filters in use are able to remove 99.9% of leukocytes (112,113). The filters may be used either in the blood bank or at the bedside as the RBCs are being transfused. Leukocyte depletion leads to a decrease in febrile transfusion reactions, prevention of CMV transmission, prevention of alloimmunization, and prevention of the immunomodulatory effects of transfusion.

Irradiation of Blood Products All blood products are irradiated prior to administration to HSCT recipients to prevent transfusion associated GVHD. Blood units are irradiated with either a cesium (137Cs) or cobalt (60Co) radiation source. Most blood banks use radiation doses between 1500 and 3500 cGy. Irradiation damages the lymphocytes by forming electrically charged particles or ions that alter the DNA, rendering the lymphocytes unable to proliferate. After irradiation, there is no reduction in the life span of the cells transfused, and granulocytes appear to maintain their function.

14

Aquino and Sandler

Management of ABO Incompatibility Between Donor and Recipient ABO incompatibility between donor and recipient is encountered in approximately 25% of all allogeneic HSCT. A major incompatibility exists in which the recipient plasma contains isohemagglutinins directed against the donor’s ABO antigens (example O recipient and A, B, or AB donor). This situation can lead to immediate transfusion reaction (see above). In this situation, the donor’s RBCs must be removed by centrifugation prior to infusion. A minor incompatibility exists when the donor plasma contains isohemagglutinins directed against the recipient’s RBCs (example A, B, or AB recipient and O donor). In this situation, the donor’s plasma must be removed by centrifugation prior to infusion. If the donor and recipient have a bidirectional mismatch (donor A and recipient B or vice versa), then both RBCs and plasma must be removed prior to infusion. Studies have shown no significant effect of major or minor ABO mismatch on the incidence of graft rejection, GVHD, or survival, although in one cohort analysis bidirectional mismatch was associated with a poorer survival post-HSCT (114). The ABO and Rh type of the stem cell donor and recipient must be considered in RBC transfusion of the HSCT recipient. Acute and delayed transfusion reactions may result secondary to major and minor incompatibilities. Table 6 summarizes the guidelines for RBC transfusion of recipients of ABO-incompatible stem cell grafts.

Complications of Transfusion Therapy Transfusions may be associated with either febrile or allergic reactions. Febrile reactions are caused by cytotoxic or agglutinating antibodies in the recipient’s plasma that react with antigens on transfused white blood cells or cytokines. The frequency of febrile reactions is roughly 0.5% to 1.0% per unit of RBCs infused and 20% of all platelet infusions. An allergic transfusion reaction occurs when the patient has been previously sensitized to a plasma protein in the platelet concentrate. The reaction may be mild, with erythema, urticaria, and pruritus, or severe, progressing to anaphylaxis. Such reactions occur in 1% to 2% of blood product infusions. These reactions can be managed by stopping the infusion and administering intravenous diphenhydramine and/or methylprednisolone.

Hemolytic Transfusion Reactions Immediate hemolytic transfusion reactions are most often due to ABO incompatibility between the donor and the recipient. These events are usually due to a “clerical error,” such as mislabeling the unit to be transfused or a unit being transfused into the wrong patient. These reactions are characterized by fever, chills, abdominal and lower back pain, tachycardia, Table 6 Transfusion Support in Hematopoietic Stem-Cell Transplant Recipients Transplanted from ABO Incompatible Donors Recipient type A A A B B B AB AB AB Source: From Ref. 115.

Donor type

Transplant incompatibility

Type of RBCs to transfuse

Type of plasma to transfuse

O B AB O A AB O A B

Minor Major Major Minor Major Major Minor Minor Minor

O O A, O O O B, O O A, O B, O

A, AB AB A, AB B, AB AB B, AB AB AB AB

Supportive Care of the Pediatric HSCT Patient

15

hypotension, nausea, and hemoglobinuria, leading to oliguria and anuria. If a hemolytic transfusion reaction is suspected, the blood transfusion should be stopped immediately, and a blood sample should be sent to the laboratory for a Coombs test to confirm the diagnosis. Therapy consists of intravenous hydration and maintenance of urinary output. Diuretic therapy with furosemide or mannitol may be required to maintain urine output. Delayed hemolytic transfusion reactions may also occur. Primary immunization is mild and can occur weeks after transfusion. This rarely causes significant hemolysis and should be suspected when an unexplained decrease in hemoglobin occurs two to three weeks after transplant. An anamnestic response occurs 3 to 10 days after transfusion and is related to sensitivity to minor blood groups. These reactions can result in profound anemia. The diagnosis is confirmed with a Coombs test and the development of antibodies to one of the minor blood groups.

Transfusion Associated Lung Injury Transfusion associated lung injury (TRALI) is the third leading cause of death related to transfusions (116). Clinically, TRALI is similar to acute respiratory distress syndrome. TRALI may occur after transfusion with whole blood components, FFP, cryoprecipitate, or intravenous gamma-globulin. Within six hours of receiving a blood transfusion containing plasma, the patient develops fever, tachypnea, and dyspnea. Chest X-ray shows pulmonary edema. The mortality associated with TRALI is approximately 10%. Patients are managed with oxygen and supportive care, with some patients requiring mechanical ventilation. Bacterial Contamination of Blood Units Bacterial contamination of blood units can be seen and potentially lead to the development of shock and ultimately death. Bacterial contamination is most often due to inadequate cleaning of the skin before the venous puncture is performed prior to blood collection and is occasionally due to a transient bacteremia in the donor. As expected, the most common cause of blood product contamination is due to bacterial skin flora. Platelets are more commonly contaminated than RBCs because platelets are stored at 20–248 C. In one study, one in 4300 platelet units were contaminated, and the risk of contamination was higher in pooled platelets when compared to apheresis platelets (117). Transfusion Transmission of Viral Infection Transfusions may also be associated with the transmission of certain infectious agents, including viruses, parasites, and potentially prions, although the risk of such infections is small. The aggregate risk of infection from a blood transfusion in a unit of blood that has passed both donor and viral testing is one in 34,000 units transfused, with hepatitis B and C accounting for 88% of the infections (118). Approximately 300 cases of virally transmitted infection occur each year in transfusion recipients. Hepatitis A is usually transmitted by the fecal-oral route, and its transmission in blood is rare. Symptoms of infection occur early in the illness, and the duration of viremia is short. Currently blood products are not routinely screened for hepatitis A. Hepatitis D and E are also rarely transmitted by transfusion. Hepatitis B and C virus are the most common viral pathogens transmitted by blood transfusions (118). Donors with viremia from these infections can be healthy without a history of symptoms. Hepatitis B virus (HBV) can be identified in one in 63,000 units. Infection with HBV can be associated with acute fulminant hepatitis. Hepatitis C (HCV) can be seen one in 103,000 units. Eighty percent of those infected are asymptomatic (119). Hepatitis C can be associated with relapsing hepatitis. Infection with HBV and HCV can be associated with both chronic persistent and chronic active hepatitis, and both are associated with an increased risk of developing hepatocellular carcinoma.

16

Aquino and Sandler

Hepatitis G is a new RNA virus that has been identified in some patients with non-A, non-B, non-C hepatitis. From 1% to 4% of otherwise normal blood donors are carriers of hepatitis G. Currently, donors are not screened routinely for hepatitis G. Human immune deficiency virus (HIV) can be transmitted by blood transfusion, although only a very small number of new cases of acquired immunodeficiency syndrome (AIDS) are caused by infection via blood transfusion. Currently, antibody testing can detect antibodies in 98% to 100% of HIV C donors, and testing and donor screening has greatly reduced the risk of HIV infection from a blood transfusion. The risk of HIV infection from a screened unit is estimated to be one in 493,000 units transfused (118). Human T-lymphocyte virus (HTLV) is a retrovirus that has been associated with the development of T-cell leukemia in adults and is associated with the development of a form of myelopathy in humans. The incidence of HTLV-1 antibodies in blood donors is 0.025%. Currently blood units are screened for this infection. CMV is a herpes virus that causes infections in all age groups. CMV is likely harbored in neutrophils and can be transmitted via blood transfusion. The risk of primary infection from a blood transfusion ranges from 2.5% to 12%. It appears that the risk of infection is directly related to the number of leukocytes transfused. Although the issue of whether to provide blood products at low risk of transmitting CMV is controversial, it appears that the use of leukofiltration is adequate in preventing primary CMV infection in patients with malignancy or those undergoing HSCT (120). Transfusion of parvovirus B19 and infection is rare and may be transmitted by plasma products and albumin (121). It is estimated that one in 3300–50,000 units are contaminated with parvovirus B19 and can transmit the virus. The risk of transfusion of Creutzfeldt-Jakob disease (CJD) is unknown. The disease is thought to be caused by a prion, though the exact etiologic agent is unknown. CJD is known to be transmitted by the transplantation of dura or cornea and by the administration of pituitary derived growth hormone. Although no evidence of blood transmission has been identified, the fact that a blood donor subsequently developed CJD has raised fears about the potential for transfusion-borne infection. In summary, the use of blood products is critical to the success of HSCT. The risks of transfusion can be reduced by improving donor selection, improving testing for potential infectious agents, and reducing the number of blood units transfused.

HEMATOPOIETIC GROWTH FACTOR SUPPORT Infectious complications after HSCT are due to prolonged neutropenia induced by myeloablation and neutrophil dysfunction in the early phase of recovery after transplantation. Therefore, shortening of the period of neutropenia should reduce the risk of life-threatening infection and improve survival after transplant. Both recombinant granulocyte CSF (G-CSF) and GM-CSF have been shown to increase the number of stem cells harvested from the peripheral blood or bone marrow. Hematopoietic CSFs have also been shown to shorten the duration of neutropenia and decrease the incidence of infectious complications in HSCT patients. The optimal dose, schedule and method of administration of growth factors remain to be standardized. Guidelines for the use of CSFs in HSCT recipients have been published by the American Society of Clinical Oncology (122,123).

Growth Factors in the Priming of Donors Prior to the Collection of Bone Marrow Administration of G-CSF before harvesting autologous or allogeneic bone marrow may be of benefit to both the donor and the recipient. However, the optimal dose and duration of G-CSF to be used for bone marrow priming prior to harvest is unknown. In one study, administration of

Supportive Care of the Pediatric HSCT Patient

17

2 mg/kg/day of G-CSF for five days increased the number of harvested CD34C cells and granulocyte macrophage colony-forming (GM-CFU) in the donor’s bone marrow exceeding baseline values by 4.2-fold and 1.6-fold, respectively, when compared with unprimed donors (124). The use of G-CSF priming allowed the collection of an average of 180 ml less bone marrow in the primed group when compared to the unprimed group. However, G-CSF primed bone marrow did not shorten the time to trilineage engraftment or the duration of hospitalization compared with unprimed bone marrow. Morton et al. (125) randomized patients undergoing allogeneic transplant to G-CSF primed bone marrow or G-CSF primed PBSCs. The median time to neutrophil and platelet engraftment was similar in the two groups. The use of the G-CSF primed peripheral blood stem cells (PBSCs) was associated with steroid-refractory acute GVHD, chronic GHVD, and prolonged requirements for immunosuppressive therapy. Survival was similar for the two groups. However, a study by Couban et al. (126) demonstrated an acceleration in neutrophil and platelet engraftment in patients receiving G-CSF primed bone marrow when compared to PBSCs.

Growth Factors in the Mobilization and Collection of Peripheral Blood Stem Cells PBSCs from autologous donors have replaced bone marrow as the preferred source of hematopoietic stem cells, based upon the ease of collection and the rapidity of engraftment. G-CSF mobilized PBSCs have also been used in allogeneic HSCT. Although the incidences of grade II-IV acute GVHD have been similar for patients receiving allogeneic PBSC when compared with bone marrow (127,128), the incidence of chronic GVHD has been higher in PBSC recipients (127,129). It also appears that the chronic GHVD that has been experienced has been more difficult to treat, requiring a greater number of immunosuppressive regimens (130). PBSCs are collected by leukapheresis, stored, and then infused after the administration of myeloablative chemotherapy and radiation. Harvesting PBSCs without the use of priming with either chemotherapy and/or CSFs requires multiple pheresis procedures and yields a relatively small number of stem cells (131). Although administration of non-myeloablative doses of chemotherapy, such as cyclophosphamide, can be used to increase the number of circulating stem cells, the use of G-CSF and GM-CSF either alone or in combination with chemotherapy allows for the collection of larger numbers of PBSCs. G-CSF stimulated donors have a 2.5- to 5.5-fold increase in the number of mononuclear cells recovered during PBSC collection when compared with collection from unstimulated donors (132). The peak number of stem cells is seen 4–8 days after CSF treatment alone or shortly after recovery from neutropenia when CSFs are given after recovery from chemotherapy. It appears that higher doses of G-CSF (10 mcg/kg per day or higher) may yield a higher content of CD34C cells during PBSC collection (133).

Use of Colony-Stimulating Factors During Autologous Transplantation Both G-CSF and GM-CSF have been studied in randomized, placebo controlled studies in adult patients undergoing autologous bone marrow transplantation (134,135). Recovery from severe neutropenia was reduced from 20 to 13 days in patients receiving CSFs in one randomized study (136). Use of G-CSF or GM-CSF prophylaxis after autologous BMT demonstrated reduction in the duration of fever, days of antibiotic use and number of hospital days (137). The benefit of post-transplant CSFs in recipients of autologous PBSC HSCT is less clear. Several studies have shown no significant benefit for post infusion G-CSF in terms of neutrophil recovery, raising the possibility that the larger number of progenitor cells infused obviates the beneficial effects of CSFs therapy. In one randomized trial, although faster neutrophil recovery was associated with the use of G-CSF (mean of 10 vs. 12 days), there was no difference in

18

Aquino and Sandler

the incidence and severity of infection, days of fever or days of antibiotic use (138). No financial benefit was associated with the use of G-CSF in this study.

Use of Colony-Stimulating Factors During Allogeneic Transplantation The benefits of the use of CSFs in the allogeneic setting have been less pronounced than those seen in patients undergoing autologous transplant. In randomized trials which evaluated the use of CSFs after allogeneic bone marrow transplant the time to neutrophil recovery has usually been shortened (135); however this has not translated into a reduction in the number of infections or the number of hospital days. Several studies have reported potential negative side effects of CSF use. Several authors have noted that the time to platelet engraftment has been delayed in patients receiving CSFs (139,140). CSFs have been noted to increase the levels of soluble IL-2 receptor-a, which can potentially aggravate GVHD (141). However, there have been conflicting reports of whether the use of growth factors results in a clinically detectable increase in the incidence of acute and chronic GVHD (115,136,140,142–145).

Use of Colony-Stimulating Factors in Umbilical Cord Blood Transplantation As more experience has been gained with the use of umbilical cord blood units for transplant, engraftment appears delayed when compared with bone marrow and peripheral stem cells, likely due to the lower cell doses transplanted (146,147). Growth factors have been used in umbilical cord blood transplant to hasten engraftment although the efficacy of this approach is controversial. Ex vivo expansion of cord blood using stem cell growth factors may improve engraftment time and allow their use in adult donors (148).

Use of Colony-Stimulating Factors for the Management of Engraftment Failure After Hematopoietic Stem-Cell Transplant G-CSF and GM-CSF have been studied in patients in whom engraftment does not occur, is delayed, or is lost after the return of granulopoiesis. Recipients of T cell depleted grafts, patients who received grafts purged with chemotherapy in vitro, patients whose donors are HLA-C mismatched, and patients who receive a relatively low cell dose are at increased risk of graft failure. Most studies have evaluated the use of GM-CSF in patients who failed to show evidence of engraftment by 3–4 weeks after autologous bone marrow transplant. Neutrophil responses are seen in approximately one-half to two-thirds of patients (133). These trials are limited by the fact that results are compared with historical controls, and the response rates are difficult to compare to the variable incidence of spontaneous neutrophil recovery. The role of CSFs in this patient population requires further study.

Role of Erythropoietin in Hematopoietic Stem-Cell Transplantation In order to reduce the RBC transfusions in stem cell transplant recipients, interest has arisen in the use of human recombinant erythropoietin (EPO). During the time of bone marrow hypoplasia during the first two weeks following myeloablative therapy, the levels of EPO increase and are disproportionately high relative to the degree of anemia (149). EPO has been shown to be efficacious in decreasing the number of RBC transfusions and in improving the quality of life of patients receiving conventional chemotherapy (150,151). A reduction in the number of PRBC transfusions has been demonstrated in small numbers of patients undergoing allogeneic HSCT (152,153). In one study, the number of RBCs transfusions was reduced from an average of 10 to 5 units (152). However, in several trials

Supportive Care of the Pediatric HSCT Patient

19

involving autologous HSCT recipients, there appears to be no reduction in the number of RBC transfusions associated with the use of EPO (153). In autologous HSCT recipients with delayed engraftment and prolonged anemia (hemoglobin less than 9 g/dL at more than 30 days from transplant), a response rate of 83% was seen (154). EPO has also been studied in patients undergoing hematopoietic progenitor cell mobilization and harvest. In one retrospective study of 34 patients, EPO increased the number of colony-forming unit-granulocyte macrophage progenitors and CD34C cells in patients who received G-CSF plus EPO versus those who received G-CSF plus placebo (154). However, there was no associated decrease in RBC transfusion or time to engraftment in the patients who received G-CSF/EPO stimulated stem cells.

Use of Thrombopoietic Agents in Hematopoietic Stem-Cell Transplant Recipients Due to the expense and complications associated with the use of platelet transfusions, the development of agents with thrombopoietic activity would be an important advance in the management of patients undergoing HSCTs. A variety of molecules, including IL-3, GMCSF/IL-3 fusion product (PIXY), IL-11 and recombinant thrombopoietin, have been developed and evaluated in preliminary studies and have demonstrated only a modest decrease in platelet transfusion requirements (155). Currently, only IL-11 is commercially available. In a randomized trial of patients undergoing autologous transplant for breast cancer, there was no statistically significant difference in the number of platelet transfusions associated with the use of IL-11 (156). A pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) has recently been tested in phase II trials in women with breast cancer undergoing autologous transplant (157). Although well tolerated, no significant differences in the kinetics of early thrombopoiesis or number of platelet transfusions after autologous HSCT were observed. In summary, the use of G-CSF and GM-CSF has been shown to be of benefit to patients undergoing HSCT when used in peripheral stem cell collection and in patients undergoing autologous HSCT. Their benefit in allogeneic transplant is not as pronounced and the use of growth factors in this patient group remains controversial. The efficacy of EPO in HSCT patients is not well studied. Although the reduction in RBC transfusions may be modest at best, the improvement in quality of life associated with the use of EPO in patients receiving conventional chemotherapy may be present in HSCT recipients and requires further study. The efficacy of thrombopoietic agents to date has not been demonstrated, although newer molecules currently in phase II or III testing may be shown to be of benefit.

CONCLUSION The use of supportive care is critical to successful HSCT. A variety of preventive and interventional strategies have been developed to optimize the prevention and treatment of the noninfectious complications associated with HSCT. Further studies are necessary to refine preexisting regimens and to develop new strategies to reduce the morbidity and mortality of HSCT recipients.

REFERENCES 1. Demarosi F, Bez C, Carrassi A. Prevention and treatment of chemo- and radiotherapy-induced oral mucositis. Minerva Stomatol 2002; 51:173–186. 2. Stiff P. Mucositis associated with stem cell transplantation: current status and innovative approaches to management. Bone Marrow Transplant 2000; 27:S3–S11.

20

Aquino and Sandler

3. Wardley AM, Jayson GC, Swindell R, et al. Prospective evaluation of oral mucositis in patients receiving myeloablative conditioning regimens and haemopoietic progenitor rescue. Br J Haematol 2000; 110:292–299. 4. Bellm LA, Cunningham G, Durnell L, et al. Defining clinically meaningful outcomes in the evaluation of new treatments for oral mucositis: oral mucositis patient provider advisory board. Cancer Invest 2002; 20:793–800. 5. Blijlevens NM, Donnelly JP, DePaw BE. Mucosal barrier injury: biology, pathology, clinical counterparts and consequences of intensive treatment for haematological malignancy: an overview. Bone Marrow Transplant 2000; 25:1269–1278. 6. Ruescher TJ, Sodeifi A, Scrivani SJ, et al. The impact of mucositis on alpha-hemolytic streptococcal infection in patients undergoing autologous bone marrow transplantation for haematologic malignancies. Cancer 1998; 82:2275–2281. 7. Sonis ST, Oster G, Fuchs H, et al. Oral mucositis and the clinical and economic outcomes of hematopoietic stem-cell transplantation. J Clin Oncol 2001; 19:2201–2205. 8. McGuire DB, Peterson DE, Muller S, et al. The 20 item oral mucositis index: reliability and validity in bone marrow and stem cell transplant patients. Cancer Invest 2002; 20:893–903. 9. DCTD, NCI, NIH et al. Cancer therapy evaluation program: common toxicity criteria version 1998; 2.0. 10. Gomez RS, Carneiro MA, Souza LN, et al. Oral recurrent human herpes virus infection and bone marrow transplantation survival. Oral Surg Oral Med Pathol Oral Radiol Endod 2001; 91:552–556. 11. Bondi E, Baroni C, Prete A, et al. Local antimicrobial therapy of oral mucositis in paediatric patients undergoing bone marrow transplantation. Oral Oncol 1997; 33:322–326. 12. Gamis A, Personal communication. 2003. 13. Aquino VM, Harvey A, Garvin JH, et al. The use of supplemental glutamine to decrease morbidity in children undergoing stem cell transplantation: A pediatric blood and marrow transplant consortium study. Bone Marrow Transplant 2005. In press. 14. Coghlin-Dickson TM, Wong RM, Offrin RS, et al. Effect of oral glutamine supplementation during bone marrow transplantation. JPEN J Parenter Enteral Nutr 2000; 24:61–66. 15. Cockerham MB, Weinberger BB, Lerchie SB. Oral glutamine for the prevention of oral mucositis associated with high-dose paclitaxel and melphalan for autologous bone marrow transplantation. Ann Pharmacother 2000; 34:300–303. 16. Schloerb PR, Skikne BS. Oral and parenteral glutamine in bone marrow transplantation: a randomized, double-blind study. JPEN J Parenter Nutr 1999; 23:117–122. 17. Spielberger R, Stiff P, Bensinger W, et al. Palifermin for oral mucositis after intensive therapy for hematologic cancers. N Engl J Med 2004; 351:2590–2598. 18. Bez C, Demarosi F, Sardella A, et al. GM-CSF mouth rinses in the treatment of severe oral mucositis: a pilot study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999; 88:311–315. 19. Castagna L, Benhamou E, Pedraza E, et al. Prevention of mucositis in bone marrow transplantation: a double blind randomised controlled trial of sucralfate. Ann Oncol 2001; 12:953–955. 20. Cohen G, Elad S, Or R, et al. The use of tretinoin as oral mucositis prophylaxis in bone marrow transplantation patients: a preliminary study. Oral Dis 1997; 3:243–246. 21. Yuen KY, Woo PC, Tai JW, et al. Effects of clarithromycin on oral mucositis in bone marrow transplant recipients. Haematologica Budap 2001; 86:554–555. 22. Elad S, Cohen G, Zylber-Katz E, et al. Systemic absorption of lidocaine after topical application for the treatment of oral mucositis in bone marrow transplantation patients. J Oral Pathol 1999; 28:170–172. 23. Nevill TJ, Tirgan MH, Deeg HJ, et al. Influence of post-methotrexate folinic acid rescue on regimen-related toxicity and graft-versus-host disease after allogeneic bone marrow transplantation. Bone Marrow Transplant 1992; 9:349–354. 24. Carreras E, Bertz H, Arcese W, et al. Incidence and outcome of hepatic veno-occlusive disease after blood or marrow transplantation: a prospective cohort study of the European Group for Blood and Marrow Transplantation Chronic Leukemia Working Party. Blood 1998; 92:3599–3604. 25. Meresse V, Hartmann O, Vassal G, et al. Risk factors for hepatic veno-occlusive disease after highdose busulfan-containing regimens followed by autologous bone marrow transplantation: a study in 136 children. Bone Marrow Transplant 1992; 10:135–141. 26. Carreras E. Veno-occlusive disease of the liver after hemopoietic cell transplantation. Eur J Haematol 2000; 64:281–291.

Supportive Care of the Pediatric HSCT Patient

21

27. McDonald GB, Sharma P, Matthews DE, et al. Venoocclusive disease of the liver after bone marrow transplantation: diagnosis, incidence, and predisposing factors. Hepatology 1984; 4:116–122. 28. Allen JR, Carstens LA, Katagiri GJ. Hepatic veins of monkeys with veno-occlusive disease. Sequential ultrastructural changes. Arch Pathol 1969; 87:279–289. 29. Shulman HM, Gown AM, Nugent DJ. Hepatic veno-occlusive disease after bone marrow transplantation. Am J Pathol 1987; 127:549–558. 30. Shulman H, Fisher LB, Schoch HG, et al. Venoocclusive disease of the liver after marrow transplantation: histological correlates of clinical signs and symptoms. Hepatology 1994; 19:1171–1181. 31. Yoshimoto K, Yakushiji K, Ijuin H, et al. Colour Doppler ultrasonography of a segmental branch of the portal vein is useful for early diagnosis and monitoring of the therapeutic course of venoocclusive disease after allogeneic haematopoietic stem cell transplantation. Br J Haematol 2001; 115:945–948. 32. Jones RJ, Lee KSK, Beschorner WE, et al. Venoocclusive disease of the liver following bone marrow transplantation. Transplantation 1987; 44:778–783. 33. McDonald GB, Hinds MS, Fisher LD, et al. Veno-occlusive disease of the liver and multiorgan failure after bone marrow transplantation: a cohort study of 355 patients. Ann Intern Med 1993; 118:255–267. 34. Azoulay D, Castaing D, Lemoine A, et al. Transjugular intrahepatic portosystemic shunt (TIPS) for severe veno-occlusive disease of the liver following bone marrow transplantation. Bone Marrow Transplant 2000; 25:987–992. 35. Shulman H, Gooley T, Dudley MD, et al. Utility of transvenous liver biopsies and wedged hepatic venous pressure measurements in sixty marrow recipients. Transplantation 1995; 59:1015–1022. 36. Nicolau C, Bru C, Carreras E, et al. Sonographic diagnosis and hemodynamic correlation in venoocclusive disease of the liver. J Ultrasound Med 1993; 12:437–440. 37. Bearman SI, Anderson GL, Mori M, et al. Venoocclusive disease of the liver: development of a model for predicting fatal outcome after marrow transplantation. Blood 1993; 11:1729–1736. 38. Or R, Nagler A, Shpilberg O, et al. Low molecular weight heparin for the prevention of venoocclusive disease of the liver in bone marrow transplantation patients. Transplantation 1996; 61:1067–1071. 39. Ruutu T, Eriksson B, Remes K, et al. Ursodeoxycholic acid for the prevention of hepatic complications in allogeneic stem cell transplantation. Blood 2002; 100:1977–1983. 40. Bacq Y, Gaudin C, Hadengue A, et al. Systemic, splanchnic and renal hemodynamic effects of a dopaminergic dose of dopamine in patients with cirrhosis. Hepatology 1991; 14:483–487. 41. Simon M, Hahn T, Ford LA, et al. Retrospective multivariate analysis of hepatic veno-occlusive disease after blood or marrow transplantation: possible beneficial use of low molecular weight heparin. Bone Marrow Transplant 2002; 27:627–633. 42. Kulkarni S, Rodriguez M, Lafuente A, et al. Recombinant tissue plasminogen activator (rtPA) for the treatment of hepatic veno-occlusive disease (VOD). Bone Marrow Transplant 1999; 23:803–807. 43. Chopra R, Eaton JD, Grassi A, et al. Defibrotide for the treatment of hepatic veno-occlusive disease: results of the European compassionate-use study. Br J Haematol 2000; 111:1122–1129. 44. Abescasis MM, Conceicao S, Ferreira I, et al. Defibrotide as salvage therapy for refractory venoocclusive disease of the liver complicating allogeneic bone marrow transplantation. Bone Marrow Transplant 1999; 23:843–846. 45. Richardson P, Elias AD, Krishnan A, et al. Treatment of severe veno-occlusive disease with defibrotide: compassionate use results in response without significant toxicity in a high-risk population. Blood 1998; 92:737–744. 46. Chalandon Y, Roosnek E, Mermillod B, et al. Prevention of veno-occlusive disease with defibrotide after allogeneic stem cell transplantation. Biol Blood Marrow Transplant 2004; 10:347–354. 47. Fried MW, Connaghan DG, Sharma S, et al. Transjugular intrahepatic portosystemic shunt for the management of severe venoocclusive disease following bone marrow transplantation. Hepatology 1996; 24:588–591. 48. Papadopoulou A, Lloyd DR, Williams MD, et al. Gastrointestinal and nutritional sequelae of bone marrow transplantation. Arch Dis Child 1996; 75:208–213. 49. Papadopoulou A, MacDonald A, Williams MD, et al. Enteral nutrition after bone marrow transplantation. Arch Dis Child 1997; 77:131–136.

22

Aquino and Sandler

50. Hermann VM, Petruska PJ. Nutrition support in bone marrow transplant patients. Nutr Clin Pract 1993; 8:19–27. 51. Lipman TO. Clinical trials of nutritional support in cancer. Parenteral and enteral therapy. Hematol Oncol Clin North Am 1991; 5:91–102. 52. Langdana A, Tully N, Molloy E, et al. Intensive enteral nutrition support in paediatric bone marrow transplantation. Bone Marrow Transplant 2001; 27:741–746. 53. Mercadante S. Parenteral versus enteral nutrition in cancer patients: indication and practice. Support Care Cancer 1998; 6:85–93. 54. Barron MA, Duncan DS, Green GJ, et al. Efficacy and safety of radiologically placed gastrostomy tubes in paediatric haematology/oncology patients. Med Pediatr Oncol 2000; 34:177–182. 55. Sefcick A, Anderton D, Byrne JL, et al. Naso-jejunal feeding in allogeneic bone marrow transplant recipients: results of a pilot study. Bone Marrow Transplant 2001; 28:1135–1139. 56. Pencharz PB. Aggressive oral, enteral or parenteral nutrition: prescriptive decisions in children with cancer. Int J Cancer Suppl 1998; 11:73–75. 57. Pietsch JB, Ford C, Whitlock JA. Nasogastric tube feedings in children with high-risk cancer: a pilot study. J Pediatr Hematol Oncol 1999; 21:111–114. 58. Charuhas PM, Fosberg KL, Bruemmer B, et al. A double-blind randomized trial comparing outpatient parenteral nutrition with intravenous hydration: effect on resumption of oral intake after marrow transplantation. JPEN J Parenter Enteral Nutr 1997; 21:157–161. 59. Russell SJ, Vowels MR, Vale T. Haemorrhagic cystitis in paediatric bone marrow transplant patients: an association with infective agents, GVHD and prior cyclophosphamide. Bone Marrow Transplant 1994; 13:533–539. 60. Greene JN, Sandin RL, Fields KK, et al. Hemorrhagic cystitis in bone marrow transplant patients: is it an infection or chemotherapy toxicity? Cancer Control 1994; 1:411–415. 61. Vogeli TA, Peinemann F, Burdach S, et al. Urological treatment and clinical course of BK polyomavirus-associated hemorrhagic cystitis in children after bone marrow transplantation. Eur Urol 1999; 36:252–257. 62. Bogdanovic G, Priftakis P, Taemmeraes B, et al. Primary BK virus (BKV) infection due to possible BKV transmission during bone marrow transplantation is not the major cause of hemorrhagic cystitis in transplanted children. Pediatr Transplant 1998; 2:288–293. 63. Akiyama H, Kurosu T, Sakashita C, et al. Adenovirus is a key pathogen in hemorrhagic cystitis associated with bone marrow transplantation. Clin Infect Dis 2001; 32:1325–1330. 64. Pahari A, Rees L. BK virus-associated renal problems—clinical implications. Pediatr Nephrol 2003. In press. 65. Holt DA, Sinnott JT, IV, Oehler RL, et al. BK virus. Infect Control Hosp Epidemiol 1992; 13:738–741. 66. Bedi A, Miller CB, Hanson JL, et al. Association of BK Virus with failure of prophylaxis against hemorrhagic cystitis following bone marrow transplantation. J Clin Oncol 1995; 13:1103. 67. Leung AY, Suen CK, Lie AK, et al. Quantification of polyoma BK viruria in hemorrhagic cystitis complicating bone marrow transplantation. Blood 2001; 98:1971–1978. 68. Trigg ME, O’Reilly J, Rumelhart S, et al. Prostaglandin E1 bladder instillations to control severe hemorrhagic cystitis. J Urol 1990; 143:92–94. 69. deVries CR, Freiha FS. Hemorrhagic cystitis: a review. J Urol 1990; 143:1–9. 70. Vela-Ojeda J, Tripp-Villanueva F, Sanchez-Cortes E, et al. Intravesical rhGM-CSF for the treatment of late onset hemorrhagic cystitis after bone marrow transplant. Bone Marrow Transplant 1999; 24:1307–1310. 71. Lakhani A, Raptis A, Frame D, et al. Intravesicular instillation of E-aminocaproic acid for patients with adenovirus-induced hemorrhagic cystitis. Bone Marrow Transplant 1999; 24:1259–1260. 72. Hattori K, Yabe M, Matsumoto M, et al. Successful hyperbaric oxygen treatment of life-threatening hemorrhagic cystitis after allogeneic bone marrow transplantation. Bone Marrow Transplant 2001; 27:1315–1317. 73. Gavin PJ, Katz BZ. Intravenous ribavirin treatment for severe adenovirus disease in immunocompromised children. Pediatrics 2002; 110:e9. 74. Miyamura K, Hamaguchi M, Taji H, et al. Successful ribavirin therapy for severe adenovirus hemorrhagic cystitis after allogeneic marrow transplant from close HLA donors rather than distant donors. Bone Marrow Transplant 2000; 25:545–548. 75. Kist-van Holte J, van-Zwet JM, Brand R, et al. Bone marrow transplantation in children: consequences for renal failure shortly after and one year post-BMT. Bone Marrow Transplant 1998; 22:559–564.

Supportive Care of the Pediatric HSCT Patient

23

76. Van Why SK, Friedman AL, Wei LJ, et al. Renal insufficiency after bone marrow transplantation in children. Bone Marrow Transplant 1991; 7:383–388. 77. Zager RA, O’Quigley J, Zager BK, et al. Acute renal failure following bone marrow transplantation: a retrospective study of 272 patients. Am J Kidney Dis 1989; 13:210–216. 78. Romero AJ, Pogamp PL, Nilsson LG, et al. Effect of voriconazole on the pharmacokinetics of cyclosporine in renal transplant patients. Clin Pharmacol Ther 2002; 71:226–234. 79. Zager RA. Acute renal failure syndromes after bone marrow transplantation. Adv Nephrol Necker Hosp 1997; 27:263–280. 80. Pui CH, Jeha S, Irwin D, et al. Recombinant urate oxidase (rasburicase) in the prevention and treatment of malignancy-associated hyperuricemia in pediatric and adult patients: results of a compassionate-use trial. Leukemia 2001; 15:1505–1509. 81. Tarbell NJ, Guinan EC, Chin L, et al. Renal insufficiency after total body irradiation for pediatric bone marrow transplantation. Radiother Oncol 1990; 18:139S–142S. 82. Loomis LJ, Aronson AJ, Rudinsky R. Hemolytic uremic syndrome following bone marrow transplantation: a case report and review of the literature. Am J Kidney Dis 1989; 14:324–328. 83. Pettitt AR, Clark RE. Thrombotic microangiopathy following bone marrow transplantation. Bone Marrow Transplant 1994; 14:495–504. 84. Lieper AD. Non-endocrine late complications of bone marrow transplantation in childhood: part I. Br J Haematol 2002; 118:3–22. 85. Guinan E, Tarbell NJ, Niemeyer CM, et al. Intravascular hemolysis and renal insufficiency after bone marrow transplantation. Blood 1988; 72:451–455. 86. Keane WF, Crossan JT, Staley NA, et al. Radiation-induced renal disease. a clinicopathologic study. Am J Med 1996; 60:127–137. 87. Luxton RW, Kunkler PB. Radiation nephritis. Acta Radiol Ther Phys Biol 1997; 2:169–178. 88. Baker DG, Krochak RJ. The response of the microvascular system to radiation: a review. Cancer Invest 1989; 7:287–294. 89. Lonnerholm G, Carlson K, Bratteby LE, et al. Renal function after autologous bone marrow transplantation. Bone Marrow Transplant 1991; 8:129–134. 90. Tarbell NJ, Guinan E, Miemeyer C, et al. Late onset of renal dysfunction in survivors of bone marrow transplantation. Int J Radiat Oncol Biol Phys 1988; 15:99–104. 91. Shulman H, Striker G, Deeg HJ, et al. Nephrotoxicity of cyclosporin A after allogeneic marrow transplantation: glomerular thromboses and tubular injury. N Engl J Med 1981; 305:1392–1395. 92. Krouwer HGJ, Wijdicks EFM. Neurologic complications of bone marrow transplantation. Neurol Clin N Am 2003; 21:319–352. 93. Antonini G, Ceschin V, Morino S, et al. Early neurologic complications following allogeneic bone marrow transplant for leukemia: a prospective study. Neurology 1998; 50:1441. 94. Crenshaw H, Slatkin NE. Neurological complications. In: Hematopoietic Cell Transplantation, 1999:45–54. 95. Shah AK. Cyclosporine. A neurotoxicity among bone marrow recipients. Clin Neuropharmacol 1999; 22:67–73. 96. Gordon B, Spadinger A, Hodges E, et al. Effect of granulocyte-macrophage colony-stimulating factor on oral mucositis after hematopoietic stem-cell transplantation. J Clin Oncol 1994; 12:1917. 97. Faraci M, Lanino E, Dini G, et al. Severe neurologic complications after hematopoietic stem cell transplantation in children. Neurology 2002; 59:1895–1904. 98. Bleggi-Torres LF, deMedeiros BC, Werner B, et al. Neuropathologic findings after bone marrow transplantation: an autopsy study of 180 cases. Bone Marrow Transplant 2000; 25:301–307. 99. Bleggi-Torres LF, Werner B, Gasparetto EL, et al. Intracranial hemorrhage following bone marrow transplantation: an autopsy study of 58 patients. Bone Marrow Transplant 2002; 29:29–32. 100. Kaufman PA, Jones RB, Greenberg CS, et al. Autologous bone marrow transplantation and factor XII, factor VII, and protein C deficiencies. Cancer 1990; 66:512–521. 101. Gallardo D, Ferra C, Berlanga JJ, et al. Neurologic complications after allogeneic bone marrow transplantation. Bone Marrow Transplant 1996; 18:1135–1139. 102. Ma M, Barnes G, Pullilam J, et al. CNS angiitis in graft vs host disease. Neurology 2002; 59:1994–1997. 103. Anderson B, Young V, Kean WF, et al. Polymyositis in chronic graft versus host disease. Arch Neurol 1982; 39:188–190. 104. Greenspan A, Deeg HJ, Cottler-Fox M, et al. Incapacitating peripheral neuropathy as a manifestation of chronic-graft-versus-host disease. Bone Marrow Transplant 1990; 5:349–352.

24

Aquino and Sandler

105. Tsai HM. Molecular mechanisms in thrombotic thrombocytopenic purpura. Semin Thromb Hemost 2004; 30:549–557. 106. Fuge R, Bird JM, Fraser A, et al. The clinical features, risk factors and outcome of thrombotic thrombocytopenic purpura occurring after bone marrow transplantation. Br J Haematol 2001; 113:56–64. 107. Elliot MA, Nichols WLJ, Plumhoff EA, et al. Posttransplantation thrombotic thrombocytopenic purpura: a single-center experience and a contemporary review. Mayo Clin Proc 2003; 78:421–430. 108. Quirolo KC. Transfusion medicine for the pediatrician. Pediatr Clin North Am 2002; 49:1211–1238. 109. Strauss RG. Current status of granulocyte transfusions to treat neonatal sepsis. J Clin Apheresis 1989; 5:25–29. 110. Adkins D, Goodnough LT, Shenoy S, et al. Effect of leukocyte compatibility on neutrophil increment after transfusion of granulocyte colony-stimulating factor-mobilized prophylactic granulocyte transfusions and on clinical outcomes after stem cell transplantation. Blood 2000; 95:3605–3612. 111. Wright DG, Robichaud KJ, Pizzo PA, et al. Lethal pulmonary reactions associated with the combined use of amphotericin B and leukocyte transfusions. N Engl J Med 1981; 304:1185–1189. 112. Sirchia G, Rebulla P, Parravicini A. Leukocyte depletion of red cells. Curr Stud Hematol Blood Transfus 1994; 60:6–17. 113. Sirchia G, Rebulla P, Parravicini A, et al. Quality control of red cell filtration at the patient’s bedside. Transfusion (Paris) 1994; 34:26–30. 114. Stussi G, Muntwyler J, Passweg JR, et al. Consequences of ABO incompatibility in allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant 2003; 30:87–93. 115. Powles R, Smith C, Milan S, et al. Human recombinant GM-CSF in allogeneic bone-marrow transplantation for leukaemia: double-blind, placebo-controlled trial. Lancet 1990; 336:1417. 116. Silliman CC. Transfusion-related lung injury. Transfus Med Rev 1999; 13:177–186. 117. Ness P, Braine H, King K, et al. Single-donor platelets reduce the risk of septic platelet transfusion reactions. Transfusion (Paris) 2001; 41:857–861. 118. Schreiber GB, Busch MP, Kleinman SH. The risk of transfusion-transmitted viral infections. N Engl J Med 1996; 334:1685–1690. 119. Weiland O, Schvarcz R. Hepatitis C: virology, epidemiology, clinical course, and treatment. Scand J Gastroenterol 1992; 27:337–342. 120. Verdonck LF, de Graan-Hentzen YC, Dekker AW, et al. Cytomegalovirus seronegative platelets and leukocyte poor red blood cells can prevent primary cytomegalovirus infection after bone marrow transplantation. Bone Marrow Transplant 1987; 2:73–78. 121. Lefrere JJ, Mariotti M, de la Croix I, et al. Albumin batches and B19 parvovirus DNA. Transfusion (Paris) 1995; 35:389–391. 122. American Society of Clinical Oncology. Recommendations for the use of hematopoietic colonystimulating factors: evidence-based, clinical practice guidelines. J Clin Oncol 1996; 11:2471–2508. 123. Ozer H, Armitage J, Bennett CL. 2000 update of recommendations for the use of hematopoietic colony-stimulating factors: evidence based, clinical practice guidelines. J Clin Oncol 2000; 22:227–241. 124. MacHida U, Tojo A, Takahashi S, et al. The effect of granulocyte colony-stimulating factor administration in healthy donors before bone marrow harvesting. Br J Haematol 2000; 108:747–753. 125. Morton J, Hutchins C, Durrant ST. Granulocyte-colony-stimulating factor (G-CSF)-primed allogeneic bone marrow: significantly less graft-versus-host-disease and comparable engraftment to G-CSF-mobilized peripheral blood stem cells. Blood 2001; 98:3186–3191. 126. Couban S, Messner HA, Andreous P, et al. Bone marrow mobilized with granulocyte colonystimulating factor in related allogeneic transplant recipients: a study of 29 patients. Biol Blood Marrow Transplant 2000; 6:422–427. 127. Couban S, Simpson DR, Barnett MJ, et al. A randomized multicenter comparison of bone marrow and peripheral blood in recipients of matched sibling allogeneic transplants for myeloid malignancies. Blood 2002; 100:1525–1531. 128. Vigorito AC, Marques Junior JF, Aranha FJ, et al. A randomized, prospective comparison of allogeneic bone marrow and peripheral blood progenitor cell transplantation in the treatment of hematologic malignancies: an update. Haematologia (Budap) 2001; 86:665–666.

Supportive Care of the Pediatric HSCT Patient

25

129. Schmitz N, Beksac M, Hasenclever D, et al. Transplantation of mobilized peripheral blood cells to HLA-identical siblings with standard-risk leukemia. Blood 2002; 100:761–767. 130. Mohty M, Kuentz M, Michallet M, et al. Chronic graft-versus-host disease after allogeneic blood stem cell transplantation: long-term results of a randomized study. Blood 2002; 100:3128–3134. 131. Kessinger A, Armitage J. The evolving role of autologous peripheral stem cell transplantation following high-dose therapy for malignancies. Blood 1991; 77:211. 132. Teshima T, Harada M, Takamatsu Y, et al. Granulocyte colony-stimulating factor (G-CSF)-induced mobilization of circulating haemopoietic stem cells. Br J Cancer 1993; 84:570. 133. Nademanee A, Sniecinsk iI, Schmidt GM, et al. High-dose therapy followed by autologous peripheral-blood stem-cell transplantation for patients with Hodgkin’s disease and non-Hodgkin’s lymphoma using unprimed and granulocyte colony-stimulating factor-mobilized peripheral-blood stem cells. J Clin Oncol 1994; 12:2176–2186. 134. Armatage JO. Emerging applications of recombinant human granulocyte-macrophage colonystimulating factor. Blood 1998; 92:4491–4508. 135. Lieschke G, Burgess A. Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor (first of two parts). N Engl J Med 1992; 327:28. 136. Gisselbrecht C, Haioun C, Lepage E, et al. Placebo-controlled phase III trial of lentogastrim (glycosylated recombinant human granulocyte colony-stimulating factor) in aggressive nonHodgkin’s lymphoma: factors influencing chemotherapy administration. Leuk Lymphoma 1997; 25:289–300. 137. Schmitz N, Dreger P, Zander AR, et al. Results of a randomised, controlled, multicentre study of recombinant human granulocyte colony-stimulating factor (filgrastim) in patients with Hodgkin’s disease and non-Hodgkin’s lymphoma undergoing autologous bone marrow transplantation. Bone Marrow Transplant 1995; 15:261. 138. Ojeda E, Garcia-Bustos J, Aguado MJ, et al. A prospective randomized trial of granulocyte colonystimulating factor therapy after autologous blood stem cell transplantation. Bone Marrow Transplant 1999; 24:601–607. 139. Ringde´n O, Barrett AJ, Zhang M, et al. Decreased treatment failure in recipients of HLA-identical bone marrow or peripheral blood stem cell transplants with high CD34 cell doses. Br J Haematol 2003; 121:874–885. 140. Ringden O, Labopin M, Gorin N-C, et al. Treatment with granulocyte colony-stimulating factor after allogeneic bone marrow transplantation for acute leukemia increases the risk of graft-versushost disease and death: a study from the acute leukemia working party of the European group for blood and marrow transplantation. J Clin Oncol 2004; 22:416–423. 141. Kobayashi S, Imamura M, Hashino S, et al. Possible role of granulocyte colony-stimulating factor in increased serum soluble interleukin-2 receptor-alpha levels after allogeneic bone marrow transplantation. Leuk Lymphoma 1999; 33:559–566. 142. Hiraoka A, Masaoka T, Mizoguchi H, et al. Recombinant human non-glycosylated granulocytemacrophage colony-stimulating factor in allogeneic bone marrow transplantation: double-blind placebo-controlled phase III clinical trial. Jpn J Clin Oncol 1994; 24:205. 143. De Witte T, Vreugdenhil G, Shattenberg A. Prolonged administration of recombinant granulocytemacrophage colony-stimulating factor (GM-CSF) after T-cell-depleted allogeneic bone marrow transplantation. Transplant Proc 1993; 25:37. 144. Eapen M, Horowitz MM, Klein JP, et al. Higher mortality after allogeneic peripheral-blood transplantation compared with bone marrow in children and adolescents: the histocompatibility and alternate stem cell source working committee of the international bone marrow transplant registry. J Clin Oncol 2004; 22:4872–4880. 145. Ho VT, Mirza NQ, del Junco D, et al. The effect of hematopoietic growth factors on the risk of graftvs-host disease after allogeneic hematopoietic stem cell transplantation: a meta-analysis. Bone Marrow Transplant 2003; 32:771–775. 146. Cairo MS, Wagner JE. Placental and/or umbilical cord blood: an alternative source of hematopoietic stem cells for transplantation. Blood 1997; 90:4665–4678. 147. Locatelli F, Rocha V, Chastang C, et al. Factors associated with outcome after cord blood transplantation in children with acute leukemia. Blood 1999; 93:3662–3671. 148. Shpall EJ, Quinones R, Giller R, et al. Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant 2002; 8:368–376. 149. Ireland RM, Atkinson K, Concannon A, et al. Serum erythropoietin changes in autologous and allogeneic bone marrow transplant patients. Br J Haematol 1990; 76:128–134.

26

Aquino and Sandler

150. Henry DH. Epoetin alfa and high-dose chemotherapy. Semin Oncol 1998; 25:54–57. 151. Demetri GD, Kris M, Wade J, et al. Quality-of-life benefit in chemotherapy patients treated with epoetin alfa is independent of disease response or tumor type: results from a prospective community oncology study. Procrit Study Group. J Clin Oncol 1998; 16:3412–3425. 152. Klaesson S, Ringden O, Ljungman P, et al. Reduced blood transfusions requirements after allogeneic bone marrow transplantation: results of a randomised, double-blind study with highdose erythropoietin. Bone Marrow Transplant 1994; 13:397–402. 153. Locatelli F, Zecca M, Pedrazzoli P, et al. Use of recombinant human erythropoietin after bone marrow transplantation in pediatric patients with acute leukemia: effect on erythroid repopulation in autologous versus allogeneic transplants. Bone Marrow Transplant 1994; 13:403–410. 154. Olivieri A, Offidani M, Cantori I, et al. Addition of erythropoietin to granulocyte colony-stimulating factor after priming chemotherapy enhances hemopoietic progenitor mobilization. Bone Marrow Transplant 1995; 16:765–770. 155. Maslak P, Nimer SD. The efficacy of IL-3, SCF, IL-6, and IL-11 in treating thrombocytopenia. Semin Hematol 1998; 35:253–260. 156. Vredenburgh JJ, Hussein A, Fisher D, et al. A randomized trial of recombinant human interleukin11 following autologous bone marrow transplantation with peripheral blood stem cell support in patients with breast cancer. Biol Blood Marrow Transplant 1998; 4:134–141. 157. Schuster MW, Beveridge R, Frei-Lahr D, et al. The effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet recovery in breast cancer patients undergoing autologous bone marrow transplantation. Exp Hematol 2002; 30:1040–1044.

2 Prevention and Treatment of Infectious Disease Scott M. Bradfield Division of Hematology/Oncology, Mayo Clinic College of Medicine, Nemours Children’s Clinic-Jacksonville, Jacksonville, Florida, U.S.A.

Steven Neudorf Blood and Marrow Transplant Program, Children’s Hospital of Orange County, Orange, California, U.S.A.

Elyssa Rubin Pediatric Hematology/Oncology, Children’s Hospital of Orange County, Orange, California, U.S.A.

Eric S. Sandler Hematology/Oncology, Mayo School of Medicine, Nemours Children’s Clinic-Jacksonville, Jacksonville, Florida, U.S.A.

Infection and disease recurrence are the two major causes of death in the stem-cell transplant recipient. Although current advances in hematopoietic stem cell transplant (HSCT) have attempted to improve overall survival by limiting these complications, few have been able to simultaneously decrease both. More intensive therapies reduce the risk of relapse at the cost of increasing infectious risk, whereas efforts to reduce infectious death often require less intensive conditioning regimens with a resultant increase in relapse rate. Infection continues to be a major obstacle to the goal of successful cure of the pediatric stem-cell transplant patient. This section will address the issues surrounding bacterial infection in this population, including risk factors, as well as prevention and treatment options.

BACTERIAL INFECTIONS Epidemiology The pediatric patient undergoing HSCT is at risk for a number of opportunistic bacterial pathogens, in addition to the “normal” infections seen in same-aged healthy children. Due to the immunosuppressive therapy involved in HSCT, opportunistic pathogens account for the majority of culture isolates. These bacteria are often found colonizing human body surfaces (e.g., respiratory tract, skin, gastrointestinal system) or commonly encountered environmental objects (e.g., plants, tap water sources). In the clinical context of transplant, they may become 27

28

Bradfield et al.

pathogenic. In the early years of bone marrow transplantation, gram-negative bacteria accounted for most bloodstream infections, and had a mortality rate approaching 40% (1). Changes in antibiotic therapy, transplant regimens, and supportive care techniques (e.g., surgically implanted central venous catheter use) have significantly impacted these statistics, such that gram-positive organisms now account for the majority of culture-positive infections in epidemiological studies of both adult and pediatric transplant populations (1–5). In a large pediatric study, gram-positive cocci seen included coagulase-negative Staphylococcus (16%), Enterococcus (15%), and Staphylococcus aureus (8%). Less common organisms were alphahemolytic Streptococcus and Streptococcus viridans, Streptococcus pneumoniae and Streptococcus sanguis. Gram-negative organisms cultured included Klebsiella (11%), Pseudomonas (8%), Enterobacter (6%), and Escherichia coli (5%). Acinetobacter, Stenotrophomonas, Citrobacter, and Serratia were also significant findings. Clostridium difficile, through isolation of its toxin in stool, accounted for 7% of bacterial infections (3). The fact remains, however, that in the neutropenic patient with fever, only an estimated 25% will have confirmed bacteremia (1). With viral and fungal isolates much less common, this leaves a substantial percentage of patients with unexplained fevers, representing possible culture-negative bacterial infections. They are regularly treated as if they do have such an infection, and the epidemiology of these “possible” organisms is unknown. The risk of antibiotic resistance must be considered. The tremendous genetic variability of bacteria allows the continued emergence of new mechanisms of resistance to antibacterial therapy. Thorough monitoring of resistance patterns at a local level is required, and this data must be factored into any recommendations or analysis of published literature. The concern for emerging antibiotic resistance should affect choices for antibiotic prophylaxis, empiric treatment, and definitive treatment choices made after identifying a bacterial organism. In choosing antibacterial medications, the transplant physician has a duty to both current and future patients to use thoughtful, rational decision-making, with attention to potential future antibacterial resistance.

Patient Risk Factors The risk of fever in the posttransplant period approaches 100% (6). Assessment of the febrile patient’s true risk for serious infection depends on a variety of factors. The transplant course is often divided into three phases [early or preengraftment, postengraftment through day [(D)C100, and late or after DC100] for evaluating infectious risk factors (Fig. 1). The epidemiology of infectious organisms has been shown to vary according to the phase following stem-cell infusion. This variation reflects the patient’s count recovery and immune status during each of these stages. The early, or preengraftment stage, characteristically represents the period of maximal neutropenia and the period of recovery from conditioning regimen toxicities. Of course, in the patient with relapse of a hematologic malignancy or severe aplastic anemia, this period may actually predate the stem-cell infusion. The type of transplant affects the length of this stage postinfusion. Unrelated volunteer donor and umbilical cord blood transplants typically have delayed engraftment, whereas peripheral blood stem cell donors often provide more rapid engraftment. Nonmyeloablative conditioning regimens may avoid neutropenia and significant conditioning toxicity altogether. When a conditioning regimen is received as an outpatient, the patient may avoid the risk of nosocomial infections, which often have increased antibiotic resistance. Growth factor use may shorten the preengraftment period. The toxicities inherent to the conditioning regimen give potential clues as to the organisms responsible for infection in this early stage. Severe mucositis is a result of total body irradiation, many chemotherapeutic regimens and methotrexate as graft-versus-host disease (GVHD) prophylaxis. It results in a breakdown of the mucosal barrier immune function and creates a ripe situation for transmigration of gastrointestinal or oral bacteria into the bloodstream. The gram-negative Enterobacteriaciae are, therefore, a common isolate during this stage, and gastrointestinal

Prevention and Treatment of Infectious Disease Phase I, Pre-engraftment, < 30 days

Phase II, Postengraftment, 30-100 days

Neutropenia, mucositis, and acute graft-versushost disease

Host immune system defect

29 Phase III, Late phase, < 100 days

Impaired cellular immunity and acute and chronic graftversus-host disease

Impaired cellular and humoral immunity and chronic graft-versus-host disease

Central line

Device risk

Respiratory and enteric viruses

Allogeneic patients

Herpes simplex virus* Cytomegalovirus* Varicella-zoster virus Epstein-Barr virus lymphoproliferative disease Facilitate gram-negative bacilli

Staphylococcus epidermidis Encapsulated bacteria (e.g., Pneumococcus)

Gastrointestinal tract Streptococci species All Candida species

Aspergillus species

Aspergillus species Pneumocystis carini Toxoplasma gondii Strongyloides stercoralis

0

30

100

360

Days after transplant *Without standard prophylaxis Primarily among persons who are seropositive before transplant

High incidence (> _10%) Low incidence (<10%) Episodic and endemic Continuous risk

Figure 1 Phases of opportunistic infections after hematopoietic stem-cell transplantation. Source: Adapted from Ref. 7.

anaerobes are also a possibility, especially in the setting of a perineal/perianal abscess. Alphahemolytic Streptococci infections increase in the setting of oral mucositis. The need for frequent accessing of central venous catheters during this stage, however, makes infection with gram-positive cocci such as the skin commensal, coagulase-negative Staphylococcus, even more likely. Severe skin breakdown from radiation burns contributes to bacterial bloodstream access as well. Antibiotic prophylaxis using fluoroquinolones has been shown to increase the percentage of gram-positive isolates (8). Upon engraftment, the risk from absolute neutropenia resolves, but the patient remains at risk due to impaired humoral and cellular immunity (9,10). Autografts tend to be at a lower risk than allografts because acute GVHD has a major impact during this stage. Adequate neutrophil numbers decrease the risk of gram-negative organisms, but gut GVHD increases the risk due to continued gastrointestinal bacterial transmigration. Posttransplant immunosuppression for GVHD prophylaxis can extend mucositis and decrease immunity, while GVHD itself is immunosuppressive, as are the medications needed to treat it. Diarrheal symptoms attributed to acute gut GVHD should be cultured for C. difficile, an important part of the differential diagnosis. Gram-positive organisms continue to be a factor during this stage as long as central

30

Bradfield et al.

venous catheters remain in place. Importantly, viral and mold pathogens also need to be considered when evaluating infections at this stage. Patient immunity has generally improved by DC100 and continues to improve as the patient moves closer to one year posttransplant (9,10). The major risk factor for delayed postengraftment infections is chronic GVHD. In allogeneic transplants, patients with chronic GVHD have continued immunosuppression both from this disorder as well as from the various medications used for its treatment. There may continue to be oral and gastrointestinal mucosal breakdown as well. Patients with chronic GVHD have a high risk for encapsulated organisms. This requires antibiotic prophylaxis (11,12). The most common encapsulated organisms are S. pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. As immunosuppression is increased, patients are at increasing risk for viral, and mold pathogens as well, especially with corticosteroid use.

Prevention and Treatment Environmental Protection Protection from bacterial exposure is a first step in controlling patient infections. Good hand washing with soap and water or antibacterial hand gels is required for all people entering the patient’s room, as well as for the patient after using the bathroom. Other proposed techniques to manipulate the environment for infection control are less well supported. Much of the difficulty lies in the nature of the bacteria. Many are endogenous organisms to which exposure has already occurred. Regular use of masks and/or gowns has not been shown to reduce bacterial infections, although standard contact precautions should be used when appropriate (e.g., C. difficile infection). A low microbial (immunosuppressed) diet is recommended during all periods of immunosuppression and care should be taken in handling raw and undercooked meats, seafood, and eggs, as well as unwashed fruits and vegetables (7). Two controversial areas of infection control are isolation/ventilation techniques and gut decontamination. Studies have suggested that laminar airflow (LAF) reduces sepsis and major local infections but does not affect overall survival (13). High-efficiency particulate air (HEPA) filtration is more commonly used and is felt by many to be equally protective. The major benefit of these techniques is to reduce spore exposure and fungal disease, but bacterial infection is often simultaneously mentioned, especially when concomitant prophylactic antibiotics are given. One recent study has suggested that allogeneic stem-cell transplantation can be safely performed without confining patients to isolation or even hospitalization (14). This certainly is not the standard of care today. The current Centers for Disease Control (CDC) recommendation is that LAF rooms are optional, but HEPA filtration should be used when LAF is not in place (7). Gut decontamination includes attempts to sterilize the oral and gastrointestinal flora in order to prevent infection from endogenous bacteria. Antibacterial medications used have been absorbable and nonabsorbable antibiotics, such as colistin, penicillin, vancomycin, gentamicin, and the fluoroquinolones. Complete sterilization has been difficult by this method. Furthermore, it has not been shown to improve survival, although studies have reported decreased documented bacteremias (15). One study showed the use of ciprofloxacin decontamination to be associated with increased leukemic relapse (16). At this time, the CDC guidelines for prevention of opportunistic infections in stem cell transplant recipients do not recommend gut decontamination (7). Antibiotic Prophylaxis The role of antibiotic prophylaxis remains controversial. With fever incidence approaching 100% posttransplant and the significant risk of infectious death during neutropenia, many researchers have evaluated the potential efficacy of upfront systemic prophylactic antibiotics to improve overall survival. The fluoroquinolones are one well-studied option (2,17). They are

Prevention and Treatment of Infectious Disease

31

active against the most dangerous gram-negative organisms but tend to leave the anaerobic gut flora relatively undisturbed. A large meta-analysis of trials using ciprofloxacin as oral prophylaxis showed a reduction in gram-negative bacteremias and no concomitant increase in gram-positive bacteremia. Unfortunately, this did not have any significant effect on number of patient days with fever or overall mortality (17,18). Vancomycin is another antibiotic that has been studied due to its excellent gram-positive coverage. Initial data suggested its benefit in reducing gram-positive bacteremias and decreasing infectious morbidity (19). Since then, the emergence of vancomycin-resistant enterococci has only strengthened the argument for limiting overuse of the most potent antibiotics. A 1998 study examined the possibility of limiting vancomycin use in this prophylactic setting (20). This paper compared vancomycin prophylaxis to penicillin/cefazolin to no prophylaxis (although all patients received trimethoprim-sulfamethoxasole prophylaxis). They found vancomycin reduced gram-positive bacteremias without any significant reduction in mortality over the other two groups. This study showed vancomycin could be safely withheld until specifically needed. Another study examined rifampin along with ciprofloxacin for gram-positive coverage and, again, could not demonstrate improved survival (21). Currently, CDC guidelines do not recommend prophylactic antibiotics and specifically recommend not using vancomycin (7). The decision rests on concerns about bacterial resistance. No prophylaxis regimen during early stem cell transplant has been shown to alter survival (17). Until a survival benefit is proven, antibacterial prophylaxis during the early transplant period is not recommended. The situation is completely different in the late postengraftment period. Patients who have undergone allogeneic transplant and have chronic GVHD are at significant risk for infection with encapsulated organisms, especially with S. pneumoniae (12). For this reason, the CDC recommendation is to treat these patients prophylactically while on immunosuppressive therapies (7). Many patients are receiving Pneumocystis jiroveci (formerly P. carinii) pneumonia (PCP) prophylaxis during this time. If given daily, trimethoprim-sulfamethoxazole can be used for both indications. If this agent is not tolerated daily due to allergy, cytopenias, or gastrointestinal upset, penicillin has been used for bacterial prophylaxis with an alternate medication to prevent PCP disease. Penicillin has been associated with decreased fatal pneumococcal infections in the chronic GVHD setting (12). Unfortunately, penicillinresistance is present and increasing, so local bacterial sensitivities must be known in choosing the proper prophylactic agent.

Fever and Neutropenia Fever and neutropenia is an almost universal occurrence in the early posttransplant stage. The issues involved in choosing antibacterial therapy are similar to those involved in choosing prophylactic antibiotics (i.e., duration of neutropenia, type of transplant, conditioning regimen, presence of mucositis). However, there is consensus that once fever is present during neutropenia, antibiotics are required without waiting for identification of an organism on blood culture. Overall survival is decreased if antibiotic therapy is delayed. A variety of studies have examined the best empirical use of antibiotics at the onset of fever and neutropenia. Traditional regimens used a combination of an anti-pseudomonal penicillin (e.g., Ticarcillin-clavulanic acid) and an aminoglycoside (e.g., gentamicin or tobramycin). This combination has excellent gram-negative coverage, including for Pseudomonas aeruginosa. In the past decade, a number of single agent regimens have been shown to be equally effective in children, including ceftazadime, cefipime, and meropenem (22–24). In adults, piperacillintazobactam has also been shown to be effective monotherapy (25). Fluoroquinolones are used as single agent treatment as well. These results all suggest that the use of a nephrotoxic, ototoxic aminoglycoside can be avoided in fever and neutropenia. A number of antibacterial options are available for initial choice and further decision-making should be based on knowledge of institutional sensitivity patterns.

32

Bradfield et al.

No particular regimen should be used as a standard approach for all patients. At this time, initial antibiotic regimens tend to have poor gram-positive coverage because these organisms generally have been less virulent. Consideration should be given to empirically covering for gram-positive bacteria, commonly with vancomycin or clindamycin, in certain settings where the incidence of these bacteria is much higher. With severe mucositis and following cytosine arabinoside, an increased incidence of alpha-hemolytic streptococci has been documented (26). This particular organism is known to cause an acute toxic shock syndrome with respiratory failure and hypotension. Clinically evident line infections or tunnel infections also suggest a gram-positive bacteria as the source of fever and should be covered appropriately. If such prophylactic antibiotics as fluoroquinolones are used, bacteremias may shift towards grampositive organisms (6). The clinical situation must be considered. For example, in the context of hypotension, consideration should be given to double-coverage for gram-negative organisms since the lipopolysaccharide cell membrane acts as an endotoxin affecting vascular tone. An aminoglycoside is commonly used as an additional antibiotic. Coverage for anaerobic bacteria, not well covered in most initial regimens, might be instituted for such findings as severe gastrointestinal pain or perianal tenderness. Anaerobic bacterial flora can often be cultured from abdominal or perianal abscesses. For patients who have no confirmed bacteremia, duration of therapy is not entirely clear. Many transplant physicians would continue antibacterial coverage until the patient is neither febrile nor neutropenic. Some advocate stopping only vancomycin after the initial 48 hours, if cultures do not suggest its continued need, to prevent the emergence of drugresistant species (27). Others permit drug discontinuation in the afebrile patient after a full fourteen-day treatment course, even if the patient remains neutropenic. This decision rests on the theory that an undocumented bacteremia would have been fully treated in that time.

Antibacterial Treatment Data suggest bacteremia is only documented in approximately 25% of febrile neutropenic episodes (1,2). Every attempt should be made to identify an infectious organism in order to focus treatment appropriately. Blood cultures are usually obtained in the continuously febrile patient on a daily basis to help identify an organism. However, there is a recent study suggesting that blood cultures beyond the first day of fever are not helpful and do not alter therapy. In this study, patients were mainly autograft recipients on vancomycin and ciprofloxacin prophylactic therapy. These results may not generalize to other institutions (28). Any other sites of clinical suspicion should be investigated further. Skin lesions should be biopsied. Central venous catheter exit sites should be examined and cultured if signs of infection exist. Significant pulmonary disease should also be aggressively explored. Bronchoalveolar lavage (BAL) is often the first diagnostic step for newly developing pulmonary infiltrates, although lung biopsy must be considered if this procedure is nondiagnostic (29). A persistently febrile patient may be observed on empiric broad-spectrum antibiotics for a short period. After three to five days, antifungal therapy should be initiated, as discussed further in the section on Fungal Infections. At that point, a computed tomographic (CT) scan of the sinuses, chest, abdomen, and pelvis may not only identify fungal disease but also perhaps identify asymptomatic areas of sinusitis or abscess formation. Once a bacterial infection is confirmed, therapy should be tailored specifically to that species, based first on known institutional patterns of resistance, and then on laboratory-tested sensitivities of the organisms. Generally speaking, gram-positive species can be treated with vancomycin before tailoring to sensitivities. Gram-negative rods are typically double-covered pending their sensitivities, because these organisms historically have a worse outcome and a number of them have inducible beta-lactamases when treated solely with a penicillin or cephalosporin. Suspected central venous catheter infections do not require line removal in most cases, although this should always be a consideration in the patient who cannot achieve clearance of the bloodstream (30,31). Antibiotics are commonly given for 10–14 days following the last positive blood culture. Nontuberculous mycobacteria are an infrequent cause

Prevention and Treatment of Infectious Disease

33

of infection in transplant patients but can be successfully treated with prolonged therapy with two or three antimicrobial agents (32). C. difficile is a frequently encountered bacterial pathogen in the stem cell transplant population (3). This bacterium causes pseudomembranous colitis and is diagnosed by isolating either a common antigen or one of its toxins from a stool sample (33). It frequently causes diarrhea and can be a complicating factor in acute gut GVHD, where in severe cases it can be fatal. Upon isolation, treatment should be instituted with metronidazole, orally or intravenously, for ten days. Following this, repeat toxin assay of the stool may be performed, and retreatment with metronidazole can be used if it remains positive. This may happen due to the bacterium’s ability to form spores to noxious exposures. The common antigen assay does not differentiate between toxigenic and nontoxigenic species and does not need to be repeated. If repeat toxin testing shows positivity again at the end of a second course or if a patient has many recurrences, vancomycin taken orally, and therefore without systemic absorption, can be administered. This should be avoided any earlier in therapy to prevent the emergence of antibiotic resistance, but it is effective when necessary.

Supplementary Therapies Other treatments have been instituted in an attempt to reduce the impact of bacterial infections on the transplant patient. These may act as preventive measures or as adjunctive forms of treatment. Colony-stimulating factors have been looked at as a way to reduce the duration of the neutropenic preengraftment period and, therefore, shorten the greatest risk period for infection. To date, studies on this subject have been inconclusive. A number of adult and pediatric studies have shown both recombinant human granulocyte colony stimulating factor (G-CSF) and recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) may shorten the duration of the neutropenic period. However, they have not demonstrated a clear benefit in terms of decreased antibiotic use, decreased severe infections or improved overall survival. A recent manuscript found G-CSF in pediatric transplant patients to improve neutrophil recovery, but this only translated to clinical advantage in the bone marrow (as opposed to peripheral blood stem cell) recipients (34). Another study evaluating GM-CSF use in pediatric allogeneic transplant recipients found enhanced neutrophil recovery without shortening the duration of systemic antibiotics given for documented infections or as prophylaxis to prevent infections, and without decreasing the risk of significant infections (35). No significant evidence has shown that initiating colony-stimulating factors at the time of a fever and neutropenia episode, or at the time of documentation of infection, improves survival outcomes. For now, use of colony-stimulating factors should be assumed to have potential benefit on duration of hospitalization and neutropenia in individually tested transplantation protocols, but this may change with differing conditioning regimens, donor stem cell products and donor sources. Generalizable effects on overall infectious outcome have not been proven. Infusions of intravenous immunoglobulins (IVIG) posttransplant have been touted as a method to reduce bacterial, viral, and fungal infections as well as to reduce GVHD in the allogeneic transplant setting. Clearly, there is delayed humoral and cellular immune reconstitution following transplantation. This is true even in the presence of a noninvoluted thymus in the pediatric population (10). However, the benefit of regular infusions of immunoglobulin without documented hypogammaglobulinemia has not reliably been shown to decrease bacterial infection risk. This does not exclude possible efficacy in preventing other complications. There is some concern that these infusions may impair the recovery of humoral immunity even longer (36). On the other hand, patients with documented hypogammaglobulinemia (especially IgG subclass 2), which may last an extended time in the setting of chronic GVHD, are at higher risk of infection and are good candidates for immunoglobulin replacement to keep them at serum levels greater than 400 mg/dl. Granulocyte transfusions are occasionally used to treat neutropenic patients during severe, life-threatening infections. Evidence clearly does not support routine transfusions

34

Bradfield et al.

during neutropenia without infection due to the risk of acquired infections [e.g., cytomegalovirus (CMV)] and the known side effects (e.g., pulmonary reactions, fever, chills, alloimmunization). The support for such transfusions in neutropenic patients with lifethreatening infection has also been marginal. However, most previous reports use “unstimulated” granulocyte donors. A recent Phase I/II trial found that by stimulating donors with G-CSF (600 mcg) and dexamethasone (8 mg) at 12 hours prior to collection, substantially higher quantities of neutrophils could be harvested (37). Transfusion of these cells restored peripheral neutrophil counts to the normal range in 17 of 19 patients and the capacity of these neutrophils to migrate to tissue sites in vivo was restored to normal in the majority of patients. Side effects were minimal and infections resolved in 8 of 11 patients. This method of granulocyte collection requires further study, in larger patient populations. It does hold promise for providing the neutropenic patient with some passive restoration of immunity during the preengraftment period.

Vaccinations Maintaining up-to-date immunizations is perhaps the greatest contribution pediatricians make to their patients’ health. The process of stem cell transplantation, as well as any preceding chemotherapy or subsequent GVHD, significantly affects recipient host immunity. This makes vaccination an important part of the transplant physician’s care-giving too. Antibody levels are known to wane over the years following transplantation if reimmunization is not performed. Unfortunately, the best way to achieve this in patients who may have reduced vaccine responses is not clear. Individual transplant centers have developed a variety of vaccination schedules without standardization (38). Based on the best data available, the CDC’s Advisory Committee on Immunization Practices has published recommendations regarding vaccination for prevention of bacterial disease. Both Haemophilus influenzae b (Hib) conjugate vaccine and diphtheria toxoid-tetanus toxoid-pertussis vaccine (DTaP) are given to pediatric patients under seven years of age at 12, 14, and 24 months posttransplantation. For patients greater than seven years, DTaP is substituted with Tetanus-diphtheria toxoid (Td) for all three doses. Hib vaccine is given as previously stated, regardless of age. Td should then be given at a minimum of every 10 years as in normal, healthy adults (7). The recent introduction of pneumococcal conjugate vaccine (PCV7) complicates the recommendations regarding the prevention of posttransplant pneumococcal disease, a significant pathogen in late postengraftment infections in children (39). With the older 23-valent-polysaccharide vaccine, protective levels of antibody were difficult to achieve within the first two years following transplant, the period of greatest risk for this organism. This was true whether the vaccine was given at 12 and 24 months posttransplant or just at 24 months (38). The introduction of the new PCV7 will hopefully change this. The CDC has made no specific recommendations regarding this vaccine due to limited data (7). Since then, a recent publication showed that a 3-dose schedule of PCV7 is immunogenic when given at 3, 6, and 12 months following transplantation. This study found that immunization of the donor with PCV7 one week prior to harvesting did not improve overall recipient immunity by the end of the evaluation but did increase early antibody responses (40). This method of “donor immunization” needs further evaluation for prevention of all types of infection. Conclusion It is rewarding to see the progress that has been made over the past few decades in preventing bacterial infectious morbidity and mortality in pediatric stem-cell transplant patients. Supportive care has been greatly improved, even in the face of higher risk donor and patient selection. Novel antibacterial agents have been steadily introduced to help combat infections and save lives. Nevertheless, bacteria’s genetic diversity will likely continue to find a way to defeat our best defenses. Only careful and conservative use of our antibacterial armamentarium will allow today’s outstanding advances in therapeutics to be tomorrow’s medications of

Prevention and Treatment of Infectious Disease

35

choice. Resistance is inevitable, but with thoughtful antibiotic choices we can hope to slow the process and continue to improve cure rates.

INVASIVE FUNGAL INFECTIONS As a result of increasing indications for transplant and the increasing use of alternate donors, a greater number of pediatric patients have become eligible for stem-cell transplantation. Fungal disease has become a major factor impacting the success of these therapies. There is now some encouraging news for the transplant physician, who may have a number of options for more successfully treating invasive fungal infections (IFI) and, potentially, with fewer toxic effects. Historically IFI have represented a virtual death sentence to the unfortunate patient given this diagnosis following HSCT. The frequent combination of disease and treatment-related factors, including poor marrow function, regimen-related neutropenia, severe T-cell deficits, immunosuppressive treatments, coincident bacterial, and viral infections, and often accompanying organ damage have, until recently, produced survival rates for patients diagnosed with IFI in the posttransplant setting at less than 20% (41). However, the last 10 years represent an exciting period in the care of the immunosuppressed patient, with the introduction of novel pharmacologic agents to the antifungal armamentarium. Recently published randomized clinical trials have demonstrated their efficacy.

Epidemiology of Posttransplant Invasive Fungal Infections Fungal Epidemiology Fungi are eukaryotic organisms ubiquitously present in nature and divided morphologically between yeast and molds. There are an estimated 250,000 fungal species, of which fewer than 150 have been documented as human pathogens (42). Fungal pathogens identified in pediatric transplant patients are listed in. Candida species are by far the most common pathogenic yeast, and are frequent commensal organisms colonizing the gastrointestinal tract in the normal healthy human host. They are also commonly found on plant material. Infection with Candida spp is commonly felt to occur through direct exposure at the time of infection or by initial colonization of the patient’s body at some time prior to infection. Human-to-human transmission is not thought to contribute to spread, although some evidence suggests that such spread is possible (43–45). In human IFI, C. albicans historically has accounted for more than 50% of candidal infections with C. tropicalis, C. parapsilosis, C. glabrata, and C. krusei much less common (42,46). C. parapsilosis has been associated with central venous catheters and has been linked to infected parenteral nutrition (47,48). C. lusitaniae and C. guilliermondi are uncommon. This rank order may be changing, however, following institution of effective fluconazole fungal prophylaxis in the early 1990s. Such a preventive strategy creates selective pressure supporting fluconazole-resistant organisms, such as C. krusei and C. glabrata, over the more sensitive C. albicans and C. tropicalis. Whether or not this shift is currently occurring is controversial, but will nevertheless need to be followed over time (46,49,50). Mold infection is overwhelmingly caused by Aspergillus species (A. fumigatus, most common, but also A. niger, A. flavus, A. terreus, and others) (51). However, a significant quantity of less common opportunistic fungi (e.g., Fusarium, Scedosporium, Rhizopus) have emerged as organisms causing infection in the severely immunosuppressed, pediatric stem-cell transplant population (41,52–56). Unlike yeasts, molds are not thought to be colonizers of the human host, and infection is felt to arise from environmental exposure to aerosolized fungal spores. Construction sites are known to be risky areas, both in and out of the hospital, but aerosolized water can also be a dangerous source of exposure. Recent evidence points to hospital water systems and patient showers as a potentially contaminated source that may require changes in supportive care recommendations (57).

36

Bradfield et al.

Patient Risk Factors A variety of factors inherent to the patient, his/her disease and condition, as well as his/her treatment regimen contribute to the risk of acquiring IFI (58). The incidence of Aspergillus infection appears to be increasing. This increase may reflect the institution of fluconazole prophylaxis (to which Aspergillus is resistant). However, a recent epidemiological study from the Fred Hutchinson Cancer Research Center found other factors in the pediatric transplant recipient that play a role. Such factors include delayed CMV infections due to better monitoring and early ganciclovir use, T-cell depletion or CD34-selection of stem cell products, corticosteroid use, neutropenia, lymphopenia, GVHD, and respiratory viral infections (53). Immunosuppression has been and continues to be the overwhelming risk factor in IFI. This typically occurs during the preengraftment period of neutropenia. Indeed, this is a significant risk period in patients with delayed engraftment kinetics, particularly, with unrelated, and umbilical cord blood donors. However, advances in stem cell transplantation, such as more frequent use of peripheral blood stem cell donors, nonmyeloablative conditioning regimens, and improved HLA matching, have reduced the duration of neutropenia, and shifted the majority of IFI to the postengraftment phase (59). Immunosuppression categorically remains the largest contributing factor, but it is manifested through means other than neutropenia. The use of T-cell-depleted and CD34-selected stem cell products produce prolonged deficits in adequate lymphocyte function. GVHD itself is immunosuppressive, and its prevention and treatment, often including corticosteroid medications, decreases cellmediated immunity. Lastly, viral infections make a significant contribution to risk, although the mechanism for this is not clearly understood. Marr et al. suggest the known inhibitory effects of viral infections on antimicrobial effector mechanisms likely account for a portion of the risk. They also speculate on the probable minor contribution of ganciclovir treatment for CMV (and its associated neutropenia), as well as the possibility of a confounding factor missing from the immune system that creates a predisposition to both fungal and viral infections (53). Nevertheless, patients require an exposure to the organisms to acquire disease. For this reason, patients with prior fungal disease are at increased risk of reactivation of their disease during the immunosuppressive phase of their transplant course (42). These patients, with proper treatment during their transplant, may still remain eligible transplant candidates (60). Patients with fungal colonization are at risk for invasive disease with the specific species detected and are usually treated to remove known colonization. Protective isolation is of minimal help. LAF hospital rooms during the transplant procedure do reduce the inpatient risk of IFI but cannot prevent exposures in the hospital outside of the room or once the patient becomes an outpatient. Antibacterial medication use is a major risk factor as well. This intervention allows the overgrowth of resistant organisms, such as Candida spp. previously existing as noninvasive commensals. Finally, mucositis, as a regimen-related toxicity, contributes to patient risk by providing a portal of entry to the bloodstream, most commonly for yeast organisms, and therefore should be prevented when possible.

Diagnosis and Detection Clinical Presentation Colonization with Candida spp. is commonly detected in the stem cell transplant population with up to 80% of patients colonized at the start of transplant (61). Candida will often be picked up on routine surveillance cultures, if performed, yet it generally responds to the institution of standard fluconazole prophylaxis. Conservative thought dictates treatment of any fluconazoleresistant organisms picked up on surveillance cultures or found as superficial infection on oral examination (62). This “thrush” appears as white patches on an erythematous base often located on the buccal mucosa, palate, or tongue. Candida esophagitis often presents as dysphagia and retrosternal burning, but, in severe cases, it may also be a cause of hematemesis in the thrombocytopenic stem cell transplant patient. The most common presentation of Candidemia

Prevention and Treatment of Infectious Disease

37

or invasive Candidal infection is persistent fever without explanation. Common invasive sites of disease are the bloodstream, liver, spleen, kidney, brain, heart, and retina. The prevalence of pretransplant detection of molds is low but may be underestimated. Detection of mold on surveillance cultures in a pediatric stem cell transplant patient should be treated, although the data supporting routine screening is limited. As with the yeasts, invasive disease most frequently presents as unexplained fever. Specific sites of disease may be associated with particular signs and symptoms related to that body site. Aspergillus spp. with exposure through inhalation of aerosolized spores, present with signs, and symptoms from the respiratory tree. Pulmonary disease, most common with Aspergillus species, may present with cough, pleural chest pain, dyspnea, or pneumothorax. Massive hemoptysis may be the presenting sign in a patient with disease near the pulmonary hilum and great vessels. Rhinosinusitis is another relatively frequent site of disease and presents as nasal or sinus pain and swelling, nasal obstruction, and mucosal crusting or discoloration of the nares. Bleeding is uncommon, even in the thrombocytopenic patient (63,64). The Zygomycoses (e.g., Rhizomucor and Rhizopus) are organisms with a propensity to infiltrate at this site (65). Signs and symptoms of a brain abscess, such as headache, and increased intracranial pressure, may be noted if intracranial extension has occurred. Although the skin is a potential site of involvement with any of the molds, Fusarium has an extreme predilection for the skin, with 91% of patients having primary or metastatic skin lesions in one study (56). In children, Scedosporium species generally present with pulmonary or central nervous system lesions (55).

Diagnostic Studies Establishing a certain diagnosis of IFI is difficult in the pediatric stem cell transplant recipient. In many cases, treatment must be initiated immediately based solely on presumptive evidence, particularly because awaiting a definitive diagnosis may delay initiation of therapy and result in a poor outcome. Reflecting this uncertainty, scientific studies concerning IFI often divide infections by the level of evidence for infection. “Proven” infections have histopathologic or microbiologic documentation of disease from biopsied tissues, whereas “probable” infections are defined as BAL specimens that culture fungus in the appropriate clinical setting (53). “Possible” infection is a much less stringent category, encompassing clinical and radiographic findings consistent with fungal infection but without any laboratory confirmation of disease. Blood cultures identifying yeast or a number of the molds are considered evidence of fungemia, but due to the rare occurrence of identification of Aspergillus species in the blood, a positive culture with this organism should be carefully considered for the possibility of contamination in the laboratory. In the pediatric HSCT population, the identification of fungus in the bloodstream may be the only localizing diagnostic information in a persistently febrile patient. After starting treatment, this finding should prompt a more extensive work-up for hematogenously disseminated disease. This work-up is also indicated any time “proven” disease is identified at another site. CT scans of the head, sinuses, chest, and abdomen will identify most visceral involvement. An ophthalmologic examination assessing retinal involvement is indicated, and many transplant physicians also recommend an echocardiogram to identify fungal vegetations. The skin should be examined as well for subcutaneous nodular lesions or ulcerative lesions. Radiographic evidence of disease differs according to site. The characteristic finding on CT scan of the chest is nodular disease, singular or multiple, and often peripheral, with a “halo sign.” This term describes the surrounding hazy, low attenuation shadow around a dense nodule that reflects adjacent fungal invasion and tissue necrosis. Similar to this is the “air crescent” sign, a radiologic finding caused by disease progression, and cavitation around the nodules. Both of these findings are considered highly suggestive of pulmonary IFI (51). Larger hilar nodules may also be seen and represent a greater risk for massive, potentially fatal hemoptysis. However, pulmonary disease can also present as free air due to spontaneous pneumothorax (51). Neutropenic patients with overwhelming disease may show extensive, diffuse bilateral consolidation on X-ray or CT scan. This finding is associated with an extremely poor prognosis.

38

Figure 2

Bradfield et al.

Computed tomographic scan showing hepatosplenic candidiasis.

Cerebral lesions most often resemble the brain abscesses seen with other infectious organisms. Fungal rhinosinusitis appears as air-fluid levels, orbital involvement, adjacent abscesses, and/or bony erosion, but, as in other body sites, it may not present radiographically during the early stages of infection (63,64). Hepatosplenic candidiasis characteristically shows up on CT scans as multiple parenchymal “bulls-eye” lesions consistent with fungal nodules (Fig. 2). Retinal involvement is seen as patchy lesions on direct examination. Attempts should be made in almost all cases to obtain material from suspected sites of disease for histopathologic and microbiologic identification, due to differing patterns of susceptibility amongst the fungi and the possibility of incorrect diagnoses. In any patient who can tolerate the procedure, a biopsy should be obtained of intracranial, sinus, skin, or abdominal parenchymal disease, although positive blood cultures will often limit the diagnostic aggressiveness of many transplant physicians to these areas. Nodular pulmonary disease may be amenable to open or CT-guided biopsy, whereas diffuse disease may require bronchoscopic biopsies. BAL is a much less convincing diagnostic tool, although positive fluid in a patient with clinically consistent pulmonary disease would be assumed “probable” disease and treated as such. Candida species in this situation are most often the result of oral contamination.

New Methods Better techniques of early fungal detection might greatly improve survival by allowing early detection and treatment. Much active research is ongoing in this area, but at this time, no technique is used routinely. Experimental studies are looking at fungal antigens, such as Aspergillus’ galactomannan or Candidal DNA as novel markers in blood and/or urine, measuring them by antigen/antibody detection or by amplification using the polymerase chain reaction (66–69). This research holds much promise for the future diagnostic assessment of IFI. Prevention and Treatment Prevention Adherence to proper supportive care techniques to prevent IFI is crucial in the stem cell transplant setting, but clearly environmental protection measures do not completely eliminate the risk of acquiring invasive disease. Proper central venous line care is important, as well as avoidance of known high-risk areas (e.g., construction sites) outside of the hospital. Within the hospital setting, nosocomial transmission can be reduced using air filtration, whether through HEPA filtration or laminar air flow protective isolation, to reduce mold spore exposure.

Prevention and Treatment of Infectious Disease

39

The wearing of masks by patients or hospital personnel has not been shown to reduce infection. Candidal infection is often due to endogenous flora, which makes environmental protection more difficult, but limitation of antibacterial use to prevent yeast overgrowth should be followed. Good hand washing is always recommended and should only lessen the risk of handto-hand human transmission of yeast (70,71). Finally, there have been suggestions that, in the highest-risk period, patients may be better served by substituting sponge baths for showers to avoid aerosolized mold spores found in hospital water distribution systems (57). This has not become standard-of-care as of yet, pending further investigation.

Prophylaxis for Yeast Organisms The standard institution of antifungal prophylaxis has been a major improvement in supportive care for pediatric stem cell transplantation. In the early 1990s, the results from two randomized controlled trials were published showing that fluconazole prophylaxis until engraftment or until D C75 is effective in preventing IFI when administered to patients at risk (61,72). The best dose and duration of antifungal therapy continues to be debated in the adult literature (52,73). Available data has been presented to suggest that 400 mg fluconazole given once daily significantly reduces fatal Candidal infections in allogeneic recipients during the engraftment phase and the period of acute GVHD (until D C75). There continues to be a reduction in candidiasis and candidiasis-related death even after discontinuation of the medication. There may also be an associated decrease in severe gut GVHD (52). The prolonged protective effect most likely relates to a decreased level of colonization during the period of immunosuppressive GVHD treatments. Without similar risks for GVHD, autograft recipients can have their fluconazole discontinued following engraftment and resolution of mucositis, as neutropenia and loss of barrier function represent their major risk factors. The major success of fluconazole prophylaxis has been related to a decrease in infections due to C. albicans and C. tropicalis, those generally fluconazole-susceptible, but a reduction in C. glabrata and C. krusei infections has also been noted (62). Pediatric patients should receive 5 mg/kg/day (maximum 400 mg) from the start of their conditioning therapy until engraftment and resolution of mucositis. In autograft recipients, the medication may be discontinued at that time unless they require continued significant corticosteroid medications. However, in allogeneic recipients, fluconazole should be continued until D C75. The known side effects of fluconazole include nausea, rash, and liver function abnormalities. Liver function tests should be monitored at least weekly during the prophylaxis period. The drug is renally excreted and dosage modifications may be needed in cases of renal impairment. Fluconazole does have a significant interaction with cyclosporine, which necessitates following cyclosporine levels while on therapy. Prophylaxis for Mold Infections Because colonization is less common with mold infections, one might assume prevention of invasive mold infection would be greatest by avoiding exposures rather than by prophylactic medication. Unfortunately, as stated earlier, the ubiquitous nature of Aspergillus species, and the other molds has made prevention quite difficult. No studies convincingly support the use of prophylactic intravenous amphotericin B in stem-cell transplant recipients. This therapy has much inherent toxicity and in previously uninfected patients, the toxicity of the drug probably outweighs any benefits that may be seen, even with low-dose therapy in the prophylactic setting. Many transplant centers, however, make an exception to this in the highest-risk patients, and administer a low-dose intravenous amphotericin B product in those expected to have markedly delayed engraftment. In the pediatric stem cell transplantation, this generally occurs in patients receiving umbilical cord blood stem cell products. Patients with invasive fungal infection prior to coming to HSCT represent a different risk category altogether. These patients historically had survivals reported at less than 5–10% during HSCT and were often declared ineligible for transplant based solely on this prior

40

Bradfield et al.

diagnosis, due to the risk of reactivation. In modern therapy, patients are generally considered high-risk, yet eligible, if their fungal infection is well-controlled going into the conditioning therapy. Patients with uncontrolled, active infection are still poor candidates for surviving stem cell transplantation. With well-controlled IFI, patients can be transplanted, but they require persistent prophylactic treatment from conditioning through D C365 or longer, if still requiring immunosuppressive medications. Intravenous amphotericin B lipid complex (ABLC) (5 mg/kg/day) or liposomal amphotericin B (3–5 mg/kg/day) is best used during conditioning therapy to reduce the drug interactions of the newer azoles and the nephrotoxic effects of conventional amphotericin B. Amphotericin B was for many years the single option available for antifungal treatment. This intravenous medication has a multitude of dreadful infusional side effects, including fever, rigors, dyspnea, hypotension, headache, and nausea. Sodium loading of the kidney with a normal saline bolus prior to drug infusion has been demonstrated to decrease its nephrotoxic effects. Premedication with analgesics, antipyretics, meperidine and/or hydrocortisone is often needed to prevent the above infusional side effects. In addition, continued use of this medication for more than a few doses generally results in renally mediated hypokalemia, hypophosphatemia, hypomagnesaemia, and azotemia. The nephrotoxic risk of HSCT overall makes conventional amphotericin B a poor choice for today’s transplant recipient. Newer lipidassociated products, such as ABLC and liposomal amphotericin B, have been shown to reduce nephrotoxicity and reduced infusional-related events. The kidneys excrete these products slowly, so they do have some advantage over the azoles for their ability to be used in patients with hepatotoxicity. Following conditioning therapy, voriconazole can be administered. Voriconazole is one of a new class of second-generation triazoles with an extended mold spectrum of coverage compared to fluconazole (although not for the Zygomycoses) and greatly reduced toxicity when compared to amphotericin B formulations. Voriconazole has excellent bioavailablity and can be given orally when possible. The pediatric intravenous dose requires initial loading at 6 mg/kg every 12 hours for two doses and then 4 mg/kg every 12 hours. Suggested oral dosing is 10 mg/kg/day divided in two doses up to the adult dose of 200 mg every 12 hours. Known toxic side effects consist mainly of nausea, liver function abnormalities, which necessitate monitoring, and photopsia or visual spots and color changes, which do not generally require drug discontinuation. Oral administration does not need to be adjusted for renal or hepatic impairment, except in extreme cirrhosis. Intravenous administration is contraindicated in renal impairment due to accumulation of the vehicle component needed for administration by this route. Voriconazole inhibits cytochrome P450, creating a number of drug–drug interactions. Specifically important to transplantation, it increases blood levels of cyclosporine and tacrolimus and is contraindicated with sirolimus. Itraconazole, another azole showing promise prior to voriconazole’s FDA approval, has similar drug interactions, causes nausea, and can also create significant problems maintaining stable blood levels. Intranasal administration of amphotericin B has been used by some investigators to prevent Aspergillus infection. To date, its efficacy has not been adequately proven. This prophylactic therapy is based on the fact that exposure occurs through the respiratory tree. Side effects of this daily therapy are thought to be mainly local, but nasal steroids are often needed to reduce nasal stinging and irritation (74).

Empirical Therapy During Fever and Neutropenia For reasons previously mentioned, the suspicion of fungal infection should increase in patients with unexplained fever that continues on broad-spectrum antibacterial coverage. Following 3–5 days of fever or recurrent fever while on antibacterial therapy, the institution of antifungal therapy is indicated. In 1999, Walsh et al. published the results of a randomized, double-blind, multicenter trial comparing liposomal amphotericin B with conventional amphotericin B for empiric therapy during fever and neutropenia (75). This study concluded that liposomal amphotericin was overall equivalent to conventional amphotericin B, by using the scoring

Prevention and Treatment of Infectious Disease

41

system in the study but more effective in reducing the frequency of proven breakthrough infections (3.2% vs. 7.8%), infusion-related toxic reactions (38% vs. 74%), and nephrotoxic effects (19% vs. 34%). Since then, voriconazole has been FDA approved and Walsh et al. have produced another comprehensive randomized study comparing, in an open-label format, the use of voriconazole versus liposomal amphotericin B for empiric therapy of fever and neutropenia (76). Although the study found voriconazole to have significantly fewer breakthrough fungal infections (1.9% vs. 5%), voriconazole did not meet criteria for noninferiority according to the composite end points defined in the study. Despite the statistical findings, the authors and other experts in mycology believe the study was successful in demonstrating that voriconazole can be used successfully in this situation with fewer adverse effects than amphotericin B products (76,77). Decision-making for fever and neutropenia requires that a patient’s risk of mold infection is assessed. Only those patients who are autograft recipients without prior fungal infection, current corticosteroid use, or prolonged neutropenia should be considered low-risk. Assuming they are already receiving antifungal prophylaxis, these patients can be treated with an amphotericin B product with product choice based on nephrotoxic risk and renal status. Conversely, they may be treated with voriconazole at the doses previously discussed. Proof of this drug’s benefits and efficacy in low-risk patients has not been assessed. In all other patients, those in the high-risk group, voriconazole or a lipid-based amphotericin product (ABLC 5 mg/kg/day or liposomal amphotericin B 3–5 mg/kg/day) should be given. Decision-making in this situation should be based on liver and renal status, drug interactions, and infection knowledge. Amphotericin B products are suggested in those with prior known Zygomycosis (Mucor, Rhizopus), whereas voriconazole is preferred for a history of Fusarium and Scedosporium. Once started, patients without documented fungal infection should remain on antifungal treatment regardless of continued fever until their neutropenia resolves. The suspicion that instigated initial therapy should not change because the risk of IFI remains while the patient continues in such a markedly immunosuppressed state.

Invasive Yeast Once a diagnosis of Candidal infection is made in the pediatric HSCT recipient, proper treatment is of primary concern. The great majority of these patients will be on fluconazole prophylaxis at the time of documented infection and are assumed to have a fluconazoleresistant organism. However, non-neutropenic patients who have not had recent fungal prophylaxis are eligible to have C. albicans treated with fluconazole, once susceptibilities are known. In general, Candida species require alternative treatment. Until recently, an amphotericin B product would be used at higher doses (conventional amphotericin 1.0 mg/kg/day, ABLC 5 mg/kg/day, or liposomal amphotericin 3–5 mg/kg/day) due to some resistance seen in C. glabrata or C. krusei. This still may be the optimal choice for some but nevertheless comes with the known side effects previously discussed for amphotericin B products. A recent randomized double blind clinical trial conducted from 1997–2000 assessed the noninferiority of a new medication, caspofungin, compared to conventional amphotericin B for invasive candidiasis (78). Caspofungin is a new fungicidal agent from the class of medications known as echinocandins. These drugs target fungal cell wall synthesis by a different mechanism than previous antifungal medications and, therefore, may be active in azole or amphotericin B resistant organisms, having activity against both Candida and Aspergillus species. Either medication was given for 14 days following the last positive blood culture. In this study, caspofungin was at least as effective as amphotericin B, with successful outcome in 73% versus 62% of patients and, specifically for candidemia, caspofungin was at least as effective (72% vs. 63%) (78). Caspofungin also has the benefit of causing significantly fewer adverse events. For invasive candidiasis, caspofungin is an excellent alternative to amphotericin B therapy, although it has not been extensively tested in children. The medication is only available

42

Bradfield et al.

in IV formulation and is dosed the same as in adults, one initial 70 mg loading dose followed by 50 mg given once daily. Side effects include fever, chills, nausea, increased liver function abnormalities, and hypokalemia, all observed less frequently than with amphotericin B. This drug should be dose-reduced when there is moderate hepatic damage but does not require renal status adjustments. Significant drug–drug interactions are unknown. Only further study will inform practitioners of the best method of using this drug; however, it holds promise for the future treatment of fungal disease either alone or in combination therapy. Consideration should be given to treating for two weeks following the last positive blood culture and, in hepatosplenic disease, for at least three weeks to prevent recurrence. Practice guidelines for the treatment of candidiasis also find reasonable support in the literature for the practice of removing central venous catheters with Candidal infections, especially in C. parapsilosis where the evidence is even stronger (79). For now, infectious disease consultation is warranted in all of these patients.

Invasive Mold Documented mold infection in the pediatric transplant setting is extremely worrisome. Patients are at risk for overwhelming fatal sepsis, sudden massive hemoptysis in pulmonary disease, intracranial bleeding with brain abscesses, or, at best, a subacute infection requiring intensive antifungal therapy and probable alteration of any subsequent treatment for the patient’s underlying disease or GVHD. Fortunately, the options have expanded recently so that effective, less toxic medications are now available. Following biopsy, surgical options must be explored. Investigations have suggested that surgical removal of high-risk mediastinal or hilar pulmonary fungal disease should be performed, especially in patients who have demonstrated hemoptysis (80,81). This can be done, using proper antibacterial coverage and continuous platelet infusions, even in the context of absolute neutropenia and thrombocytopenia. The invasive nature of mold infections makes this the preferred plan due to the extreme risk of sudden and massive hemoptysis. Surgery has also been advocated in invasive fungal sinus disease (63,64). Extensive surgical debridement, in conjunction with prolonged antifungal medications, should be attempted for any patient for whom prolonged survival is being pursued, although the potentially disfiguring, painful nature of this procedure must be assessed in the context of the patient’s overall condition. Antifungal drug therapy is the area that has seen the greatest development in successfully treating IFI. Historically, an amphotericin B product would be used at high doses (conventional 1.0–1.5 mg/kg/day, ABLC 5 mg/kg/day or liposomal amphotericin B 5 mg/kg/day) in the setting of documented mold infection. This has all the severe toxicity discussed previously. Fortunately, voriconazole has become an excellent alternative in this setting. A large, randomized, unblinded study published in 2002 compared voriconazole to conventional amphotericin B as treatment for invasive aspergillosis (82). This trial found initial therapy with voriconazole led to better responses and improved survival, and it resulted in fewer severe side effects than the standard approach of initial therapy with amphotericin B. Successful complete or partial response at 12 weeks from start of therapy was 53% in the voriconazole group compared to 32% in the amphotericin group. Survival was also improved (71% vs. 58%) in the voriconazole group. Other publications have confirmed this beneficial effect. Voriconazole was used to treat IFI refractory to conventional therapy or in patients who could not tolerate their antifungal therapy. Efficacy was 44% in Aspergillosis, 58% for candidiasis, 46% for fusariosis, and 30% for scedosporiosis. These numbers are quite acceptable for patients who would otherwise have no treatment for their less-common, emerging, or refractory fungal infections (54). Walsh et al. even examined the results of voriconazole in children through the National Cancer Institute’s compassionate release program (55). Again, voriconazole was reasonably effective (45% with complete or partial response) with only 7% discontinuing due to intolerance. More recent studies have suggested it may be beneficial to combine antifungal therapies. Due to the differing modes of action, voriconazole can be used effectively in combination

Prevention and Treatment of Infectious Disease

43

therapy with the echinocandin, caspofungin. Neither medication has the adverse side effects characteristically seen with amphotericin B. The dose of voriconazole is 6 mg/kg IV every 12 hours for two doses, followed by 4 mg/kg IV every 12 hours thereafter. When tolerated, patients can be switched to 100 or 200 mg by oral formulation twice daily (!40 kg, O40 kg, respectively). Caspofungin, which is only available intravenously, is dosed as previously stated. ABLC or liposomal amphotericin would still be indicated for patients who should not receive voriconazole due to allergy, hepatotoxicity, or infection with Zygomycoses (resistant organisms). The proper duration of therapy is not well known and infectious disease consultation is recommended. In general, treatment needs to be continued until the infection is well under control before switching to monotherapy or maintenance medication. Then, an assessment of continued risk factors (further neutropenia, corticosteroid use) should dictate the full length of antifungal therapy to prevent recurrence.

Other Treatments Additional treatments for supporting the neutropenic patient with IFI have been reported. These modalities include colony-stimulating factor administration and granulocyte transfusions. Little data has been presented to support either method as a standard of care in IFI. However, either may be instituted in selected patients. Colony-stimulating factors may be used to shorten the time to engraftment but have not been shown to decrease fungal infections in pediatric stem cell transplant recipients. Granulocyte transfusions have characteristically been used for the neutropenic patient with life-threatening infection, when no immediate recovery of neutrophil count is expected. In patients following HSCT, this has not been shown to be significantly helpful. One retrospective study found no benefit in resolution of infection (29% vs. 23% with and without transfusion, respectively) in noncandidal infections or in candidal sepsis (56% vs. 50%) (83). Side effects were tolerable consisting of fever, chills, and/or respiratory distress. In light of present knowledge, this treatment remains an unsupported last resort. Hyperbaric oxygen has also been suggested as a fungicidal treatment for invasive mold infections, especially for Zygomycotic rhinosinusitis (84). In the literature, there are many cases reporting its use, but no clinical trials have demonstrated its efficacy. This therapeutic modality requires further study before it can be recommended. New Agents and Conclusion Several new antifungal agents are expected to be available in the near future. These include the triazole, Posaconazole, shown to have particular efficacy in the zygomycoses, as well as ravuconazole. Another echinocandin, micafungin, is also recently approved and has demonstrated activity against both yeast and molds (85). In addition, development is ongoing for liposomal nystatin; alternate echinocandins, as well as novel antifungal therapy classes known as nikkomycins, pradimicins, and sordarins (86). The optimal use of these newer antifungal agents for prophylaxis, empirical use, and fungal treatment will continue to be studied in the coming years (Table 1). Clearly, long-held standard recommendations for IFI as presented in the text are changing. There are great reasons to remain hopeful for the pediatric stem cell transplant recipient with an IFI. These medicines, along with methods of detecting disease earlier, have the potential to greatly reduce the morbidity and mortality of fungal disease.

VIRAL INFECTIONS Viral infections are a major cause of morbidity and mortality following stem-cell transplantation. Recipients of allogeneic transplants are particularly at risk for complications from viral infections due to prolonged immunosuppression from the treatment of GVHD and/or or

Antifungal Spectrum

Hypersensitivity to micafungin.

Aspergillus species, Candida species, Fusarium species, Scedosporium apiospermum, Cryptococcus neoformans, Histoplasma capsulatum, (not Rhizopus arrhizus, not zygomycosis).

Mechanism of Action

Hypersensitivity to caspofungin.

Candida species, Aspergillus species, (not Cryptococcus neoformans, Fusarium solani, Rhizopus, zygomycetes, or Trichosporum cutaneum).

Aspergillus species, Candida species (Rhizopus, Scedosporium and Fusarium are less susceptible) (not Cryptococcus neoformans).

A second-generation triazole agent; inhibits fungal P450-dependent ergosterol synthesis, which disrupts fungal cell membrane and halts fungal growth.

Approved Indications

Contraindications

Candida species (albicans, glabrata, krusei, tropicalis, parapsilosis, and others), Aspergillus species (fumigatus, flavus, niger and others), Coccidioides, Fusarium, Histoplasma, Zygomycetes, Cryptococcus.

An echinocandin lipopeptide agent; inhibits b-(1,3)-D-glucan synthase which interferes with fungal cell wall formation.

An echinocandin lipopeptide agent; inhibits b-(1,3)-D-glucan synthase which interferes with fungal cell wall formation.

Treatment of invasive aspergillosis; treatment of various other Candida infections, included esophageal candidiasis, and other Scedosporium and Fusarium infections.

Coadministration with terfenadine, astemizole, cisapride, pimozide (Orap, Teva), quinidine, rifampin, carbamazepine, barbiturates, ritonavir, efavirenz, rifabutin (Mycobutin, Pharmacia and Upjohn), ergot alkaloids, or sirolimus (Rapamune, Wyeth). Pregnancy.

A second-generation triazole agent; inhibits fungal P450-dependent ergosterol synthesis, which disrupts fungal cell membrane and halts fungal growth.

Treatment of esophageal candidiasis; prophylaxis of candida infections in highrisk hematopoietic stem cell transplant patients.

Empiric therapy for presumed fungal infections in febrile, neutropenic patients; treatment of Candida esophagitis, candidemia, and Candida-caused intra-abdominal abscesses, peritonitis, and pleural space infection; treatment of refractory invasive aspergillosis.

Hypersensitivity to posaconazole.

Not yet approved (under investigation for treatment and prophylaxis of invasive fungal infections in patients intolerant or refractory to other antifungal therapies. Potential indications include invasive aspergillus infections, neutropenic fevers, and azole-refractory esophageal candidiasis)

Schering-Plough

Fugisawa

Merck

Noxafil

Posaconazole

Pfizer

Mycamine

Micafungin

Manufacturer

Cancidas

Caspofungin

Vfend

Voriconazole

Comparison of New Antifungal Agents

Brand Name

Agent

Table 1

44 Bradfield et al.

Primarily hepatic metabolism, some elimination unchanged in stool and urine.

For patients with moderate liver disease (Child-Pugh score 7-9), dose modification may or may not be needed. There is no data regarding the use in patients with severe liver disease.

Nausea, vomiting, diarrhea, hyperbilirubinemia, phlebitis, rash.

Largely metabolism in liver (n-acetylation) and in plasma (hydrolysis).

For patients with moderate hepatic impairment, use normal loading dose, then 35 mg IV daily. For patients with severe hepatic impairment, further reduce dosage or withhold therapy.

Fever, infusion-related complications, phlebitis, nausea, vomiting, rash, flushing, headache, increased transaminases, diarrhea.

Primarily hepatic metabolism.

For patients with mild to moderate hepatic cirrhosis (Child-Pugh Class A & B), use normal loading dose, then reduce to 2 mg/kg IV q12h, then 100 mg PO Q12h (for patients >40 kg) or 50 mg PO Q12h (for patients <40 kg).

Transient visual disturbances, rash, fever, nausea, vomiting, diarrhea, headache, sepsis, edema, abnormal liver function tests.

Elimination.

Dose Modifications

Common Adverse Effects

(Continued)

Nausea, vomiting, diarrhea, abdominal pain, headache, fatigue, rash, increased transaminases.

None defined yet. May be needed with liver dysfunction.

Primarily eliminated unchanged in the stool, some hepatic metabolism, some excreted unchanged in urine.

400 mg PO BID or 200 mg PO QID, taken with food.

70 mg IV once, then 50 mg IV daily.

Usual Dosage

Prophylaxis in HSCT patients: 50 mg IV daily Treatment of esophageal candidiasis: 100150 mg IV daily. Treatment of invasive aspergillosis: 75100 mg IV daily. Treatment of Candida albicans infections: 50-100 mg IV daily. Treatment of nonalbicans Candida infections: 100 mg IV daily.

6 mg/kg IV Q12h for two doses, then 4 mg/kg IV Q12h. Can be changed to 200 mg PO Q12h (for patients >40 kg) or 100 mg PO Q12h (for patients <40 kg.) Dose can be increased if inadequate response. Oral doses are preferably taken on an empty stomach.

Hypersensitivity to other azole antifungal agents. Coadministration with tacrolimus or cyclosporine.

Hypersensitivity to caspofungin. Liver disease or impairment, myelosuppression.

Liver disease or impairment, myelosuppression, renal insufficiency.

Hypersensitivity to azoles, severe hepatic cirrhosis (Child-Pugh Class C), moderate-severe renal dysfunction (accumulation of IV vehicle − avoid use).

Precautions

Prevention and Treatment of Infectious Disease 45

Can interact with carbamazepine, cyclosporine, dexamethasone, efavirenz, nelfinavir, nevirapine, phenytoin, rifampin, tacrolimus. None reported.

Abbreviations: HSCT, hematopoietic stem cell transplant; PO, per os (orally); QID, quater in die (four times a day). Source: From Ref. 86. Reprinted with permission from HemOnc Today and SLACK Incorporated.

Interactions

(Continued )

Inhibits CYP2C19, 2C9, 3A4. Can interact with amprenavir, astemizole, atazanavir (Reyataz, Bristol-Meyers Squibb), atorvastatin, barbiturates, benzodiazepines, calcium channel blockers, carbamazepine, cisapride, cyclosporine, delavirdine (Rescriptor, Agouron), efavirenz, ergot alkaloids, erlotinib (Tarceva, OSI), erythromycin, fosphenytoin, imatinib (Gleevec, Novartis), lovastatin, nelfinavir (Viracept, Agouron), nevirapine, omeprazole, phenytoin, pimozide, quinidine, rifabutin, rifampin, ritonavir, saquinavir, simvastatin, sirolimus, tacrolimus, terfenadine, vinca alkaloids, warfarin.

Table 1 Comparison of New Antifungal Agents

Can interact with tacrolimus, cyclosporine, rifabutin, rifampin. More may be defined later.

46 Bradfield et al.

Prevention and Treatment of Infectious Disease

47

delayed immune reconstitution. There have been numerous advances in the methods to diagnose and treat viral infections, which will be reviewed in this chapter.

Cytomegalovirus Prior to the availability of effective methods to detect and treat early CMV infections, CMV disease occurred in 20–30% of recipients of allogeneic grafts. Using strategies that define patients at risk for developing CMV infections and disease, and implementing antiviral therapy to at-risk patients has dramatically reduced the incidence of CMV disease to !5% of patients (87). Clinical manifestations of CMV disease include pneumonia, enteritis, hepatitis, retinitis, and encephalitis. CMV is typically associated with allogeneic BMT but has been reported to occur in 5% of autologous transplant recipients with a higher incidence reported in recipients of autologous CD34 selected grafts (88). Among allogeneic BMT recipients, patients who receive highly immunosuppressive preparative therapies, such as those containing alemtuzumab or fludarabine, or who are recipients of haploidentical grafts and are at risk for severe GVHD and/or delayed immune reconstitution, the risks for CMV infection may be higher (89,90).

Epidemiology CMV infection is defined as the detection of CMV by culture, PCR or antigenemia. CMV disease is defined as the histologic demonstration of CMV in biopsy specimens or in culture from visceral sites or demonstration of CMV in BAL fluid in the presence of new or changing pulmonary infiltrates. CMV infection can arise due to a de novo infection or reactivation of latent virus. De novo infections can be acquired from infusions of leukocytes from CMV seropositive donors and from the seropositive stem cell donors. It is possible to prevent transfusion associated CMV by using either blood products from CMV seronegative donors or leukodepletion prior to transfusion. In randomized trials comparing the effectiveness of using blood product support from only seronegative donors versus leukodepletion, the incidence of CMV disease was slightly higher in patients who received only blood from seronegative donors (2.4% vs. 0%), but those differences were not statistically significant (91). CMV infections can often be detected prior to the development of clinical manifestations using the detection of pp65 antigenemia in leucocytes or PCR assays (92). Both pp65 antigenemia and PCR assays are easily performed and have the rapid turn around times necessary to implement prompt therapy. Although reactivation usually occurs between 30 and 50 day post transplant, reactivation of CMV can be seen as early as the first week post BMT (89,90,93). Cases of CMV reactivation occurring beyond day 100 post transplant have been reported (89,94). In one series, CMV disease developed in 17.8% of CMV seropositive patients a median of 169 (range 96–784) days post transplantation. Significant risk factors for late reactivation included lymphopenia (!100/mm3), lack of a cellular immune response to CMV, a history of prior reactivation of CMV, having received donor leukocyte infusions, and acute or chronic GVHD (94,95). The mortality rate in patients who developed late CMV infections has been reported to be as high as 46% (94). Prevention of Cytomegalovirus Disease Following Transplant There are two strategies for prevention of CMV disease following HSCT; the use of prophylactic antiviral agents and preemptive antiviral therapy at the first sign of reactivation. Acyclovir has low potency against CMV in vitro and yet has been shown to have activity in preventing CMV disease. Winston et al. compared the efficacy of oral valacylovir to intravenous ganciclovir for the prevention of CMV disease. CMV seropositive patients received acyclovir until engraftment and then were randomized to receive either valacyclovir (2 grams qid) or intravenous ganciclovir (5 mg/kg q12 hour for 1 week then 6 mg/kg/day for

48

Bradfield et al.

5 days/wk). Patients received valacyclovir or ganciclovir until day 100 post HSCT. The incidence of CMV disease in the valacyclovir treated patients was 12% and 19% in patients treated with ganciclovir. These differences were not statistically significant (96).

Preemptive Therapy The initiation of antiviral therapy at the onset of reactivation has reduced the incidence of CMV disease following HSCT. Both ganciclovir and foscarnet have successfully been used in preemptive therapy. In one randomized trial, patients with reactivation determined by antigenemia or PCR were randomized between ganciclovir (5 mg/kg/dose given every 12 hours intravenously for two weeks) and foscarnet (60 mg/kg every 12 hours intravenously for two weeks). Patients who had persistent antigenemia or a positive PCR two weeks after starting therapy were crossed over to receive either foscarnet or ganciclovir at the above-mentioned doses for an additional two weeks. They reported no difference in survival and no difference in the incidence of CMV disease. Both arms were associated with a 5% incidence of CMV disease (pneumonia, gastrointestinal, and retinitis) that developed at a median of 30–40 days after the initial randomization. CMV disease developed as early as 3 days and as late as 128 days following randomization (97). Patients who received ganciclovir had a higher incidence of severe neutropenia, while patients who received foscarnet had a significantly higher incidence of hypomagnesaemia, hypokalemia, and hypophosphatemia. The incidence of renal impairment was not different in recipients of ganciclovir compared to foscarnet; however, the investigators stated that recipients of foscarnet required additional hydration and the dose of foscarnet was modified for renal dysfunction. This indicates that both ganciclovir and foscarnet are effective in preventing CMV in the majority of patients who show reactivation. The choice of which drug to use should depend on the stability of engraftment and whether patients are at risk for renal dysfunction. Cidofovir has also been shown to be effective treatment for CMV (98). Cidofovir is associated with a high incidence of nephrotoxicity that can be minimized using aggressive hydration and probenecid. A potential advantage of cidofovir over ganciclovir or foscarnet is that it is administered weekly. In that retrospective review, cidofovir was given for multiple indications, including failure of previous preemptive therapy, treatment of CMV pneumonia, and as primary preemptive therapy. The outcome of patients with CMV pneumonia treated with cidofovir is similar to those reported using ganciclovir (98–100). An alternative approach is to initiate anti-CMV therapy when viral cultures performed as part of routine surveillance become positive. Schmidt et al. showed that as many as 40% of patients who underwent a routine BAL at day 35 post transplant isolated CMV and that initiation of ganciclovir therapy for culture positive patients reduced the likelihood of developing CMV pneumonia (101). In that study, patients who had CMV isolated from BAL fluid were randomized to receive ganciclovir or no treatment. The incidence of CMV pneumonia was 25% in the ganciclovir treated arm compared to 70% in the control arm and those differences were statistically significant. Of note was that 21% of patients where CMV was not isolated from the BAL eventually developed CMV pneumonia. Goodrich reported a lower incidence of CMV disease and transplant related mortality when ganciclovir was initiated when throat, urine or blood cultures became positive (102). In that study, culture positive patients were randomized to receive ganciclovir or placebo. The ganciclovir treated cohort had a significantly lower incidence of CMV disease compared to the placebo treated group (3% vs. 43%). Similar to the findings from BAL, a significant proportion of patients (12%) who were culture negative eventually developed CMV disease. The Role of Intravenous Immunoglobulins in the Prevention and Treatment of Cytomegalovirus Disease IVIG has been used to prevent CMV infection with conflicting results. A meta-analysis of these results showed that patients treated with IVIG had a slight reduction in the incidence of CMV disease (103). These trials were carried out prior to the use of preemptive antiviral therapy.

Prevention and Treatment of Infectious Disease

49

Because of the high cost of IVIG infusions and small benefit in preventing CMV disease, most centers have adopted the use of preemptive antiviral therapy instead of IVIG for the prevention of CMV disease. IVIG may have a role in the treatment of CMV pneumonia. Several nonrandomized trials using IVIG plus ganciclovir showed survival rates of 65–85% in patients with CMV pneumonia (99,100). Although preemptive therapy with ganciclovir or foscarnet is effective in preventing CMV disease in the majority of cases, there are a proportion of patients who develop CMV disease or who demonstrate rising antigenemia or PCR titers despite preemptive antiviral therapy (92,97,104). Nichols reported that as many as 39% of patients on ganciclovir for reactivation had rising antigenemia titers (104). Progressive reactivation was confirmed using PCR. Ninety percent of cases of rising antigenemia occurred after 1–2 weeks of ganciclovir therapy. Concurrent corticosteroid therapy was the main risk factor associated with rising antigenemia. CMV isolates obtained from patients with rising antigenemia did not demonstrate resistance to ganciclovir. Patients who had rising antigenemia did not have a higher incidence of CMV disease compared to patients without rising antigenemia. Initial antigenemia level or the maximal antigenemia did not correlate with the development of CMV disease (104).

Resistance Infections with ganciclovir-resistant CMV have been reported in BMT recipients (105–108), and the issue of resistance is raised when patients with reactivation of CMV do not respond to ganciclovir. Resistance has been most extensively studied in patients with AIDS and is usually seen in patients who have had prolonged exposure to ganciclovir. Seven percent of CMV isolates obtained from patients with AIDS who received three months of ganciclovir were resistant and the incidence of ganciclovir resistant CMV increased to 27% after 12 months of ganciclovir treatment (109). In contrast, patients with AIDS who received !3 months of therapy demonstrated no ganciclovir resistance (110). In HSCT recipients, resistance has usually been seen in patients who had prior antiviral therapy either either for prophylaxis or for treatment of reactivation. Ganciclovir resistance has not been shown to be a common cause of progressive antigenemia that can occur once antiviral therapy has been initiated (104). Some of the mechanisms for resistance have been defined. Ganciclovir, foscarnet, and cidofovir are all prodrugs that require phosphorylation to become active. A common mechanism for resistance is the hypophosphorylation of ganciclovir, foscarnet, or cidofovir caused by mutations in the viral UL97 kinase or UL54 DNA polymerase (111). Mutations in UL97 confer resistance to ganciclovir, but not foscarnet or cidofovir, whereas UL54 mutations result in resistance to ganciclovir, foscarnet, and cidofovir. UL97 mutations account for O90% of the mutations seen in ganciclovir-resistant CMV (112). Resistance can also be detected using plaque reduction assays where CMV is grown in the presence of various concentrations of anti-viral drugs. After several days, the number of plaques is compared to a standard culture, and the concentration necessary to inhibit 50% growth (IC50) is determined. Plaque assays can detect mutations not detected using DNA-based methods (107) but are not routinely performed by most hospitals, and they also suffer from interassay variabilities and interlaboratory variability in the cutoff used to define resistance (111). Because of the time required to perform resistance assays, it may not be practical to depend on the results of these assays to document resistance and change antiviral therapy. Instead, when ganciclovir resistance is suspected, patients should be empirically changed to foscarnet or cidofovir. Another approach to managing resistant CMV has been to add lefluomide to an antiviral therapy regimen. Lefluomide is an immunosuppressive drug approved for the treatment of rheumatoid arthritis and has been successfully used in solid organ transplant recipients with CMV infections (113,114). Lefluomide has also been shown to inhibit virion assembly (115), although the mechanism of lefluomide’s antiviral activity is still under investigation.

50

Bradfield et al.

Epstein-Barr Virus Infections Severe infections by Epstein-Barr virus (EBV) are relatively uncommon following HSCT. The most serious EBV related complication is post transplant lymphoproliferative disease (PTLD). PTLD is the result of the uncontrolled proliferation of donor derived EBVCB cells due to the lack of T cell immunity. The most significant risk factors for developing PTLD include the use of unrelated donor grafts, T cell depletion of donor grafts, the use of OKT3 for prophylaxis, or treatment of acute GVHD, and having received antithymocyte globulin (116). T-cell depletion methods that selectively deplete T cells such as E rosette depletion are more likely to result in PTLD (116,117). This suggests that selective T-cell depletion results in depletion of immunoregulatory T cells while leaving behind EBVCB cells that are susceptible to uncontrolled proliferation. PTLD occurs in !2% of allogeneic transplants (116) and typically occurs during the first six months after transplant. PTLD occurring beyond the first year post HSCT have been reported and occur mainly in patients with chronic GVHD (116). Clinical manifestations of PTLD include fever, anorexia, lymphadenopathy, lethargy or confusion, hepatitis, or hepatosplenomegaly (118). The diagnosis of PTLD is made by biopsy of affected organs. EBV serology is not helpful in establishing a diagnosis of PTLD, but monitoring changes in viral load may be useful. Unlike PCR screening for CMV, EBV DNA can be detected in O60% of allogeneic BMT recipients (119). However, patients with PTLD have a significantly higher viral load compared to patients without PTLD (119). Viral load alone, however, is not sufficient to predict the development of PTLD because as many as 30% of allogeneic transplant recipients will have a transient rise in EBV viral load on one or more occasions and not develop PTLD. Even in patients with sustained increases in EBV viral load, only approximately 50% will develop PTLD (120). In addition, the predictive value of threshold viral load may vary from center to center (119,120), and the patients’ individual risk for PTLD must be taken into account when acting on PCR results. The interval between first demonstrating a rise in viral load and clinical signs of PTLD is often short. In one series, patients who developed PTLD often had a viral load that did not differ from patients without PTLD up to one week prior to developing symptoms of PTLD (119). Successful treatments for PTLD have included donor leukocyte infusions (121), EBV specific cytotoxic T lymphocytes (122), chemotherapy (123), and anti-CD20 monoclonal antibodies (119,124,125). Anti-CD20 antibodies (rituximab) are commercially available, thus making them attractive choices for therapy. Preemptive treatment with rituximab based on rising EBV DNA titers has prevented the development of PTLD in one series (124). Patients whose viral load exceeded the institutional threshold of 1000 genome equivalents/ml of blood received a single dose of rituximab (375 mg/m2). Fifteen patients were treated and 14/15 patients had a clearance of viral load in a median of 8 days (range 1–46 days). One patient progressed to PTLD and was successfully treated with rituximab and donor lymphocytes. Another approach is to initiate treatment as soon as patients develop symptoms of PTLD. In the report by Wagner et al. 85 patients at high risk for developing PTLD based on having received T cell depleted grafts were monitored for reactivation of EBV. Nine patients showed a single elevation in viral load above the institutional threshold of 4000 copies/mcg peripheral blood DNA, and 16 patients had an elevation in viral load on two or more occasions. Despite having persistent elevation in EBV DNA, only 8/16 patients developed clinical signs of PTLD. All were successfully treated with rituximab and/or EBV specific cytotoxic lymphocyte infusions (120). Both preemptive rituximab treatment based on viral load, and treatment at the first clinical signs of PTLD can be effective therapy, although initiating treatment only when patients demonstrate a high viral load may result in the unnecessary treatment of some patients.

Adenovirus Adenoviruses are DNA viruses that can cause lytic and latent infections, which can persist from days to years. There are about 51 human types of adenovirus serotypes, all of which share

Prevention and Treatment of Infectious Disease

51

a common group specific complement-fixing antigen. However, less than 40% of these cause human disease (126). Most people have been affected by one or more serotypes by age five. Transmission is by respiratory droplets or via the fecal-oral route. Because adenovirus becomes latent, and chronic shedding can occur in an asymptomatic host, it is likely that many of the infections observed in stem cell transplant patients represent reactivation rather than acute infection. This hypothesis is supported by the nonseasonal pattern of virus isolation after HSCT and occurrence of virus shedding at a time similar to that of CMV infection (median time 2–3 months after HSCT) (127). The most common clinical presentations in immunocompetent hosts are pharyngitis, tracheitis, bronchitis, and pneumonitis. In fact, adenovirus accounts for approximately 5–10% of bronchitis and viral pneumonias in children. In immunocompetent hosts, it does not commonly cause disseminated infections after the newborn period, but in immunocompromised patients, adenovirus infections can be severe and life threatening (128). Adenovirus has been found to infect an average of one-fifth of pediatric stem-cell transplant recipients (126). Clinical manifestations of adenovirus infection in immunocompromised patients include hepatitis, hemorrhagic cystitis, and gastroenteritis. The severity of infection increases with the degree of immunosuppression (127). Risk factors for adenovirus infections include receiving T-cell depleted grafts, having received alemtuzumab as part of conditioning, GVHD, and lymphopenia (129–131). Adenovirus can be identified by viral culture, as well as by immunofluorescence testing of blood, urine, throat, or stool (127). Because diarrhea and fever are the most common presenting symptoms, it is not surprising that stool is a common site of virus isolation (126). Culture results can usually be obtained about 5–7 days after inoculation. Isolation of adenovirus from multiple sites, especially viremia, has been associated with a poorer outcome (126). Studies have shown that adenovirus can be detected in peripheral blood more than three weeks prior to the onset of clinical symptoms (129,132). Serologies are unreliable, especially early posttransplant, because of the variable ability of patients to mount an antibody response. Also, the results are difficult to interpret because one may get a positive serological result secondary to passively acquired antibody from IVIG or other blood products (127). There is little data supporting the use of antivirals in the treatment of adenovirus infections. A recent study using Cidofovir in ten severely ill HSCT patients with adenovirus showed promising results, with virologic resolution in 9/10 patients and only one death. Cidofovir was given intravenously at 5 mg/kg once weekly for up to six weeks and then once every two weeks for up to three additional doses. Probenecid and vigorous hydration was given with the Cidofovir and renal function was monitored closely (133).

Human Herpes Virus Type 6 Human Herpes Virus Type 6 (HHV-6) has been divided into two groups based on molecular characteristics, variant A, and variant B (134). In immunocompetent hosts, HHV-6 variant B causes exanthem subitum, better known as roseola infantum. HHV-6 is thought to persist in lymphocytes and salivary glands after a primary infection and may become reactivated in the immunosuppressed host (135,136). HHV-6 infections typically occur 2–4 weeks after transplant. Recipients of allografts and those patients undergoing transplant for leukemia or lymphoma are at higher risk for developing HHV-6 infections (135). Reactivation of HHV-6 is common. One report showed that 38% of transplanted patients showed reactivation of HHV-6 (137). Rash was the most common clinical manifestation but was seen in as many as 54% of patients, suggesting that many patients with HHV-6 infection after transplant are asymptomatic (137–139). HHV-6 has also been shown to cause encephalitis (136), pneumonia (134) and bone marrow suppression following transplantation. HHV-6 can be detected by several methods. It can be cultured from body fluids, detected in cells using ELISA or immunofluroescence assays, and detected using PCR (140). PCR has

52

Bradfield et al.

the advantage of rapid turn-around time and sensitivity compared to culture or immunologic methods. Ganciclovir, foscarnet, or cidofovir all have in vitro activity against HHV-6 and have been reported to be effective treatment (140–142). Yoshihara et al. reported that donor leukocyte infusions were effective treatment in a patient with HHV-6 induced encephalitis that failed ganciclovir and foscarnet treatment (143).

BK Virus BK virus is a polyomavirus, which are a genus of the family Papovaviridae and are nonenveloped double-stranded DNA viruses. BK virus is acquired in early childhood, and most children are seropositive by the age of five. By adulthood, 70–90% of people have antibodies to BK virus. The respiratory tract is thought to be the site of primary infection in many individuals, but most primary infections with these viruses are asymptomatic or produce only a mild nonspecific respiratory illness (144). After primary infection, BK virus remains latent in the body within the kidney and, as is common with other DNA viruses, is prone to reactivation. Reactivation typically occurs during pregnancy or in immunosuppressed states. BK virus has been found to shed in the urine of patients after renal transplantation, with a recent report suggesting that up to 10% of kidney transplants fail as a result of BK virus infection (145). Hemorrhagic cystitis secondary to BK virus has been described after HSCT (146), and BK virus has been associated with late-onset hemorrhagic cystitis, defined as occurring greater than two weeks after stem cell transplantation (147–149). It has been found that patients with hemorrhagic cystitis excrete a significantly higher BK viral load in the urine than post-HSCT patients without hemorrhagic cystitis (150–152). However, the incidence of BK viruria is higher than that of hemorrhagic cystitis, suggesting that there are other factors involved in the development of hemorrhagic cystitis after HSCT. One study conducted by Bogdanovic et al. found that a high BK viral load in the urine (O1 million copies/microliter) in combination with acute GVHD put HSCT patients at greatest risk for developing hemorrhagic cystitis (153). In a report by Erard et al. BK viremia was found to be associated with late-onset hemorrhagic cystitis (154). There is no specific treatment for BK virus infection. The quinolones have been shown to have activity against the Polyoma BK virus, inhibiting replication (155,156), and a promising study by Leung et al. demonstrated the successful use of Ciprofloxacin to prevent and treat BK virus reactivation after HSCT (157). Studies with Levofloxacin are underway, and there is in vitro evidence that suggests Levofloxacin may be even more efficacious than Ciprofloxacin in treating BK virus (157). Vidarabine (158) and Cidofovir (159,160) have also been reported as being beneficial in the treatment of BK virus-associated cystitis.

Respiratory Syncytial Virus Respiratory syncytial virus (RSV) is an enveloped RNA virus from the family of paramyxoviruses. The envelope contains the G protein, which shows antigenic diversity and is responsible for attachment to the cell and the F protein, which is more conserved and is responsible for cell entry and fusion formation. There are two subgroups of RSV, termed A and B. Both cause disease in humans. Humans are the only source of RSV infection. In immunocompetent hosts, RSV causes bronchiolitis and pneumonia, especially in infants and young children. Most children have been infected by the age of 2, and reinfection throughout one’s lifetime is common (161). This suggests that the immunity developed following RSV infection may be temporary. The observations that immunocompromised patients can develop life-threatening RSV infections suggests that cell-mediated immunity may also play a role in controlling RSV. Transmission of RSV is via respiratory droplets or fomites. The incubation time of RSV is four to six days, and viral shedding may subsequently occur for up to three to four weeks. Identification of RSV is performed using immunofluorescence and enzyme immunoassay

Prevention and Treatment of Infectious Disease

53

techniques that detect viral antigen in nasopharyngeal washings (161). RSV outbreaks usually occur during the winter season, but isolates have been recovered from nasopharyngeal secretions throughout the year (162). Among 190 adults with multiple myeloma undergoing chemotherapy or HSCT who were prospectively screened, RSV was isolated in 37% of cases. Twenty one percent of cases occurred between May and September, the rest between October and April. RSV can cause severe, sometimes fatal lower respiratory tract infections in HSCT patients (163,164). Among adult patients with multiple myeloma who had documented RSV infection, 29% developed severe respiratory complicatings defined as hypoxemia, pneumonia, transfer to the intensive care unit, intubation, tracheobronchitis, or death within 30 days from the start of chemotherapy (162). Multivariate analysis showed that myeloma patients with mucositis, renal failure, or elevated LDH were independent variables predicting the development of severe respiratory complications. Other factors that may predict the development of RSV pneumonia are increasing age and receiving a graft from a mismatched or unrelated donor (165).

Prevention of Respiratory Syncytial Virus Disease Avoidance of exposure and frequent hand washing are the most effective methods to prevent RSV infection. For patients who develop RSV infection prior to starting conditioning, therapy should be delayed until symptoms resolve. Peck et al. showed that, in patients who had RSV upper respiratory tract symptoms immediately prior to conditioning and who had their preparative therapy delayed until there was resolution of symptoms, there was a significantly lower incidence of severe RSV disease compared to patients who proceeded with conditioning (166). Preemptive treatment with inhaled ribavirin may be helpful in preventing RSV pneumonia. Adams et al. performed a study in which patients had nasopharyngeal washings performed on a weekly basis during RSV season. One hundred and forty-five nasal aspirations were performed on 25 patients. Seven patients had positive RSV nasal aspirates and received aerosolized ribavirin (2 grams in 33 ml sterile water) three times per day for five days. Repeat nasopharyngeal testing was performed at 48 and 72 hours after five days of treatment. If either of the tests were positive, then the patient was treated with another 5-day course of ribavirin along with RSV-IVIG (Respigamw), which has a fivefold higher concentration of RSV neutralizing antibodies compared to standard IVIG. Six of the seven patients cleared the RSV after one 5-day course of ribavirin; the seventh patient required two 5-day courses of treatment. In this study, all positive RSV events were treated successfully and no patients became symptomatic, suggesting that ribavirin is effective in clearing asymptomatic RSV infections in pediatric transplant patients (164).

Treatment of Respiratory Syncytial Virus Disease Since RSV pneumonia occurs in only a minority of patients who present with upper respiratory tract symptoms, the optimal treatment of RSV pneumonia remains to be determined. Anecdotal reports have demonstrated some success using ribavirin alone as treatment of RSV pneumonia (164,167). IVIG may have a role in the treatment of RSV disease. Hemming et al. reported results of a trial where 35 infants and children (not with malignancies or undergoing HSCT) with RSV pneumonia or bronchiolitis were randomized to receive IVIG or placebo. Ribavirin was not used in that study. They reported that recipients of IVIG had significant improvements in oxygenation and significantly faster clearing of RSV. There was no difference in length of hospital stay, no difference in supplemental oxygen requirements, and there were no deaths. They concluded that IVIG treatment was safe and had some clinical benefit (168). Nine patients with RSV pneumonia or tracheobronchitis, who received a combination of aerosolized ribavirin and standard preparations of IVIG (500 mg/kg every other day for 12 days) prior to the development of respiratory failure, had a 22% mortality (169). In contrast,

54

Bradfield et al.

3 patients with respiratory failure at the time when treatment was initiated died from RSV pneumonia, as did 4 patients who did not receive antiviral therapy. A high titer preparation of IVIG (Respigamw) is commercially available. RSV-IVIG/ Respigam contains approximately a fivefold increase in RSV neutralizing antibodies compared to standard IVIG preparations. A compassionate use trial of RSV-IVIG was performed in 11 pediatric patients with RSV pneumonia defined radiographically or clinically. All patients had hypoxia or respiratory distress. One patient was on mechanical ventilation at the onset of therapy. The majority of patients (10/11) were receiving or had received aerosolized ribavirin prior to starting RSV-IVIG. Patients received a single dose (1500 mg/kg) of RSV-IVIG. Ten patients (including the patient on mechanical ventilation) survived and one patient died from RSV pneumonia (163). Palivizumab (Synagisw) is a humanized mouse monoclonal antibody that is administered intramuscularly and is approved (along with Respigamw) for the prophylaxis of RSV disease in selected patient populations. Palivizumab is directed at the RSV’s highly conserved F protein. Palivizumab is 50–100 times more active than RSV specific immunoglobulin and has been shown to lower the incidence of RSV-related hospitalizations in children with chronic lung disease. Results of a phase I study of Palivizumab in adults who developed RSV pneumonia post HSCT showed that it was well tolerated. Eighty-three percent (10/12) of patients with RSV pneumonia survived. Two patients died from RSV pneumonia. All had received concomitant aerosolized ribavirin (2 grams over two hours three times/day). There have not been prospective trials evaluating the efficacy of standard IVIG, high titer IVIG, and monoclonal anti-RSV preparations in the treatment of RSV pneumonia. One report suggested there was no difference in the outcomes based on immunoglobulin preparation used (170). Another report suggested that outcomes were similar regardless of whether patients received ribavirin alone or ribarivin C IVIG and suggested that a more important predictor of survival was the initiation of therapy prior to the development of respiratory failure (166). Given the high mortality rate associated with RSV pneumonia, treatment with aerosolized ribavirin with or without IVIG should be initiated as soon as possible. Although the addition of IVIG to ribavirin has not been shown to be superior to ribavirin alone, IVIG has relatively few side effects that would limit its use. A more difficult scenario is the management of patients with RSV upper respiratory tract infection. For patients who develop RSV infection prior to the start of conditioning, therapy should be delayed until there is resolution of symptoms. For patients who develop RSV upper respiratory tract infections early posttransplant, it is reasonable to consider treatment of patients who have mucositis, are recipients of mismatched/unrelated donor grafts, or have other organ dysfunction since limited data suggests that these patients are at higher risk for developing RSV pneumonia. Likewise, antiviral therapy should be considered for patients with RSV upper respiratory tract infections who have progression in symptoms.

Herpes Simplex Viruses Herpes simplex viruses (HSV) are part of the large family of herpesviruses with humans as the only reservoir. There are two widely recognized subtypes of HSV, type 1 and type 2. Type 1 HSV generally causes infections involving the face, oropharynx, and skin above the waist, and establishes latency in the trigeminal ganglia. Type 2 HSV most often leads to infection in the genital region and skin below the waist, and establishes latency in the lumbosacral sensory ganglia. However, both type 1 and type 2 HSV can cause infections in any area (127,161). Pulmonary, ocular, CNS, and disseminated disease may occur as well. Control of HSV infections is through T-cell mediated immunity, and those patients with impaired T-cell function are at most risk for serious HSV infections. It is therefore not surprising that 70–80% of HSCT patients who are seropositive for HSV will have a clinical reactivation if not treated with antiviral prophylaxis. The median time until reactivation has

Prevention and Treatment of Infectious Disease

55

been reported as 17 days after transplant. About 85% of HSV infections involve the oral cavity and the other 15% involve the genitalia (127). In the early 1980s, the first study to use acyclovir as prophylaxis to prevent HSV infection was performed and published and has influenced HSCT practice to this day (171). This landmark study and subsequent confirmatory studies (172,173) showed that, not only was acyclovir very effective prophylaxis for preventing HSV reactivation and infection in the HSCT setting but also that acyclovir had little toxicity and did not delay engraftment. HSV prophylaxis is now a universal practice in the transplant setting. There are also newer prodrugs available today, including Ganciclovir, Penciclovir, Famiciclovir, and Valacyclovir. In addition to expanding the physician’s choices, some of these newer agents, such as Valacyclovir, are better absorbed and are have increased bioavailability after hepatic metabolism as compared to acyclovir (127). The new challenge is the emergence of acyclovir-resistant strains of HSV. These strains are often resistant to the other drugs in the acyclovir family as well. Resistance to acyclovir arises from spontaneous mutations in one of the two HSV genes, TK, or DNA polymerase. Studies have shown that most resistant isolates have TK mutations (174,175). Treatment of acyclovir-resistant HSV with foscarnet can be effective (174,176).

REFERENCES 1. Collin BA, Leather HL, Wingard JR, Ramphal R. Evolution, incidence, and susceptibility of bacterial bloodstream isolates from 519 bone marrow transplant patients. Clin Infect Dis 2001; 33:947–953. 2. Kru¨ger W, Ru¨ssmann B, Kro¨ger N, et al. Early infections in patients undergoing bone marrow or blood stem cell transplantation—a 7 year single center investigation of 409 cases. Bone Marrow Transplant 1999; 23:589–597. 3. Benjamin DK, Miller WC, Bayliff S, Martel L, Alexander K, Martin PL. Infections diagnosed in the first year after pediatric stem cell transplantation. Pediatr Infect Dis J 2002; 21:227–234. 4. Mullen CA, Nair J, Sandesh S, Chan KW. Fever and neutropenia in pediatric hematopoietic stem cell transplant patients. Bone Marrow Transplant 2000; 25:59–65. 5. Dini G, Castagnola E, Comoli P, van Tol MJD, Vossen JM. Infections after stem cell transplantation in children: state of the art and recommendations. Bone Marrow Transplant 2001; 28:S18–S21. 6. Mullen CA, Petropoulos D, Roberts WM, et al. Outpatient treatment of fever and neutropenia for low risk pediatric cancer patients. Cancer 1999; 86:126–134. 7. Centers for Disease Control and Prevention. Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients: recommendations of CDC, the infectious disease society of America, and the American society of blood and marrow transplantation. MMWR 2000; 49:1–128. 8. de Pauw BE, Donnelly JP, De Witte T, Novakova IR, Schattenberg A. Options and limitations of long-term oral ciprofloxacin as antibacterial prophylaxis in allogeneic bone marrow transplant recipients. Bone Marrow Transplant 1990; 5:179–182. 9. Hammarstro¨m V, Pauksen K, Svensson H, et al. Serum immunoglobulin levels in relation to levels of specific antibodies in allogeneic and autologous bone marrow transplant recipients. Transplantation 2000; 69:1582–1586. 10. de Vries E, van Tol MJD, van den Bergh RL, et al. Reconstitution of lymphocyte subpopulations after pediatric bone marrow transplantation. Bone Marrow Transplant 2000; 25:267–275. 11. Leather HL, Wingard JR. Infections following hematopoietic stem cell transplantation. Infect Dis Clin North Am 2001; 15:483–520. 12. Kulkani S, Powles R, Treleaven J, et al. Chronic graft versus host disease is associated with longterm risk for pneumococcal infections in recipients of bone marrow transplants. Blood 2000; 95:3683–3686. 13. Buckner CD, Clift RA, Sanders JE, et al. Protective environment for marrow transplant recipients: a prospective study. Ann Intern Med 1978; 89:893–901. 14. Russell JA, Chaudhry A, Booth K, et al. Early outcomes after allogeneic stem cell transplantation for leukemia and myelodysplasia without protective isolation: A 10-year experience. Biol Blood Marrow Transplant 2000; 6:109–114.

56

Bradfield et al.

15. Gluckman E, Cavazzana M, Devergie A, et al. Prevention of bacterial infections after bone marrow graft by broad-spectrum oral antibiotics, absorbable (pefloxacin, penicillin) and non absorbable (cephalosporin, gentamycin, bacitracin). Pathol Biol 1988; 36:902–906. 16. Carlens S, Ringden O, Aschan J, et al. Risk factors in bone marrow transplant recipients with leukemia. Increased relapse risk in patients treated with ciprofloxacin for gut decontamination. Clin Transplant 1998; 12:84–92. 17. Sepkowitz KA. Antibiotic prophylaxis in patients receiving hematopoietic stem cell transplant. Bone Marrow Transplant 2002; 29:367–371. 18. Cruciani M, Rampazzo R, Malena M, et al. Prophylaxis with fluoroquinolones for bacterial infections in neutropenic patients: a meta-analysis. Clin Infect Dis 1996; 23:795–805. 19. Attal M, Schlaifer D, Rubie H, et al. Prevention of gram-positive infections after bone marrow transplantation by systemic vancomycin: a prospective, randomized trial. J Clin Oncol 1991; 9:865–870. 20. Arns da Cunha C, Weisdorf D, Shu XO, DeFor T, Pastor JD, Johnson JR. Early gram-positive bacteremia in BMT recipients: impact of three different approaches to antimicrobial prophylaxis. Bone Marrow Transplant 1998; 21:173–180. 21. Gomez-Martin C, Sola C, Hornedo J, et al. Rifampin does not improve the efficacy of quinolone antibacterial prophylaxis in neutropenic cancer patients: results of a randomized clinical trail. J Clin Oncol 2000; 18:2126–2134. 22. Chuang Y-Y, Hung I-J, Yang C-P, Jaing T-H, Lin T-Y, Huang Y-C. Cefipime versus ceftazidime as empiric monotherapy for fever and neutropenia in children with cancer. Pediatr Infect Dis J 2002; 21:203–209. 23. Mustafa MM, Carlson L, Tkaczewski I, McCracken GH, Jr., Buchanan GR. Comparative study of cefepime versus ceftazidime in the empiric treatment of pediatric cancer patients with fever and neutropenia. Pediatr Infect Dis J 2001; 20:362–369. 24. Fleischhack G, Hartmann C, Simon A, et al. Meropenem versus ceftazidime as empirical monotherapy in febrile neutropenia of pediatric patients with cancer. J Antimicrob Chemother 2001; 47:841–853. 25. Viscoli C, Castagnola E. Treatment of febrile neutropenia: what is new? Curr Opin Infect Dis 2002; 15:377–382. 26. Engelhard D, Elishoov H, Or R, et al. Cytosine arabinoside as a major risk factor for Streptococcus viridans septicemia following bone marrow transplantation: a 5-year prospective study. Bone Marrow Transplant 1995; 16:565–570. 27. Wingard JR. Bacterial infections. In: Thomas ED, Blume KG, Forman SJ, eds. Hematopoietic Cell Transplantation. Malden, MA: Blackwell Science 1998:537–549. 28. Serody JS, Berrey MM, Albritton K, et al. Utility of obtaining blood cultures in febrile neutropenic patients undergoing bone marrow transplantation. Bone Marrow Transplant 2000; 26:533–538. 29. Hayes-Jordan A, Benaim E, Richardson S, et al. Open lung biopsy in pediatric bone marrow transplant patients. J Pediatr Surg 2002; 37:446–452. 30. Engelhard D, Elishoov H, Strauss N, et al. Nosocomial coagulase-negative staphylococcal infections in bone marrow transplantation recipients with central vein catheter: a 5-year prospective study. Transplantation 1996; 61:430–434. 31. Wang CC, Mattson D, Wald A. Corynebacterium jeikeium bacteremia in bone marrow transplant patients with Hickman catheters. Bone Marrow Transplant 2001; 27:45–449. 32. Gaviria JM, Garcia PJ, Garrido SM, Corey L, Boeckh M. Nontuberculous mycobacterial infections in hematopoietic stem cell transplant recipients: characteristics of respiratory and catheter-related infections. Biol Blood Marrow Transplant 2000; 6:361–369. 33. Lee SD, Turgeon DK, Ko CW, Fritsche TR, Surawicz CM. Clinical correlation of toxin and common antigen enzyme immunoassay testing in patients with clostridium difficile disease. Am J Gastroenterol 2003; 98:1569–1572. 34. Dallorso S, Rondelli R, Messina C, et al. Clinical benefits of granulocyte colony-stimulating factor therapy after hematopoietic stem cell transplant I children: results of a prospective randomized trial. Haematology 2002; 87:1274–1280. 35. Trigg ME, Peters C, Zimmerman MB. Administration of recombinant human granulocytemacrophage colony-stimulating factor to children undergoing allogeneic marrow transplantation: a prospective, randomized, double-masked, placebo-controlled trial. Pediatr Transplant 2000; 4:123–131.

Prevention and Treatment of Infectious Disease

57

36. Sullivan KM, Storek J, Kopecky KJ, et al. A controlled trial of long-term administration of intravenous immunoglobulin to prevent late infection and chronic graft-vs.-host disease after marrow transplantation: clinical outcome and effect on subsequent immune recovery. Biol Blood Marrow Transplant 1996; 2:44–53. 37. Price TH, Bowden RA, Boeckh M, et al. Phase I/II trial of neutrophil transfusions from donors stimulated with G-CSF and dexamethasone for treatment of patients with infections in hematopoietic stem cell transplantation. Blood 2000; 95:3302–3309. 38. Avignan D, Pirofski L-A, Lazarus HM. Vaccination against infectious disease following hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2001; 7:171–183. 39. Schutze GE, Mason EO, Wald ER, et al. Pneumococcal infections in children after transplantation. Clin Infect Dis 2001; 33:16–21. 40. Molrine DC, Antin JH, Guinan EC, et al. Donor immunization with pneumococcal conjugate vaccine and early protective antibody responses following allogeneic hematopoietic cell transplantation. Blood 2003; 101:831–836. 41. Marr KA, Carter RA, Crippa F, Wald A, Corey L. Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients. Clin Infect Dis 2002; 34:909–917. 42. Wingard JR. Fungal infections after bone marrow transplant. Biol Blood Marrow Transplant 1999; 5:55–68. 43. Schmid J, Tay YP, Wan L, Carr M, Parr D, McKinney W. Evidence for nosocomial transmission of Candida albicans obtained by Ca3 fingerprinting. J Clin Microbiol 1995; 33:1223–1230. 44. Fowler SL, Rhoton B, Springer SC, Messer SA, Hollis RJ, Pfaller MA. Evidence for person-toperson transmission of Candida lusitaniae in a neonatal intensive-care unit. Infect Control Hosp Epidemiol 1998; 19:343–345. 45. Fanello S, Bouchara JP, Jousset N, Delbos V, LeFlohic AM. Nosocomial Candida albicans acquisition in a geriatric unit: epidemiology and evidence for person-to-person transmission. J Hosp Infect 2001; 47:46–52. 46. Abi-Said D, Anaissie E, Uzun O, Raad I, Pinzcowski H, Vartivarian S. The epidemiology of hematogenous candidiasis caused by different candida species. Clin Infect Dis 1997; 24:1122–1128. 47. Karabinis A, Hill C, Leclerq B, Tancrede C, Baume D, Andremont A. Risk factors for candidemia in cancer patients: a case-control study. J Clin Microbiol 1988; 26:429–432. 48. Martino P, Girmenia C, Venditti M, et al. Candida colonization and subsequent infections in neutropenic patients: a retrospective study. Cancer 1989; 64:2030–2034. 49. Kunova A, Trupl J, Dluholucky S, Galova G, Krcmery VJ. Use of fluconazole is not associated with a higher incidence of Candida Krusei and other non-albicans candida species. Clin Infect Dis 1995; 21:226–227. 50. Wingard JR, Merz WG, Rinaldi MG, Johnson TR, Karp JE, Saral R. Increase in Candida Krusei infection among patients with bone marrow transplantation and neutropenia treated prophylactically with fluconazole. N Engl J Med 1991; 325:1274–1277. 51. Denning DW. Invasive Aspergillosis. Clin Infect Dis 1998; 26:781–805. 52. Marr KA, Seidel K, Slavin MA, et al. Prolonged fluconazole prophylaxis is associated with persistent protection against candidiasis-related death in allogeneic marrow transplant recipients: long-term follow-up of a randomized, placebo-controlled trial. Blood 2000; 96:2055–2061. 53. Marr KA, Carter RA, Boeckh M, Martin P, Corey L. Invasive aspergillosis in allogeneic stem cell transplant recipients: changes in epidemiology and risk factors. Blood 2002; 100:4358–4366. 54. Perfect JR, Marr KA, Walsh TJ, et al. Voriconazole treatment for less-common, emerging, or refractory fungal infections. Clin Infect Dis 2003; 36:1122–1131. 55. Walsh TJ, Lutsar I, Driscoll T, et al. Voriconazole in the treatment of aspergillosis, scedosporiosis, and other invasive fungal infections in children. Pediatr Infect Dis J 2002; 21:240–248. 56. Boutati EI, Anaissie EJ. Fusarium, a significant emerging pathogen in patients with hematologic malignancy: ten years’ experience at a cancer center and implications for management. Blood 1997; 90:999–1008. 57. Anaissie EJ, Stratton SL, Dignani MC, et al. Pathogenic molds (including Aspergillus species) in hospital water distribution systems: a 3-year prospective study and clinical implications for patients with hematologic malignancies. Blood 2003; 101:2542–2546. 58. Wald A, Leisenring W, van Burik JA, Bowden RA. Epidemiology of aspergillus infections in a large cohort of patients undergoing bone marrow transplantation. J Infect Dis 1997; 175:1459–1466.

58

Bradfield et al.

59. Fukuda T, Boeckh M, Carter RA, et al. Risks and outcomes of invasive fungal infections in recipients of allogeneic hematopoietic stem cell transplants after nonmyeloablative conditioning. Blood 2003; 102:827–833. 60. Wright JA, Bradfield SM, Park JR, Hawkins DS. Prolonged survival after invasive aspergillosis: a single institution review of eleven cases. J Pediatr Hematol Oncol 2003; 25:286–291. 61. Slavin MA, Osborne B, Adams R, et al. Efficacy and safety of fluconazole for fungal infections after marrow transplant-a prospective, randomized, double blind study. J Infect Dis 1995; 171:1545–1552. 62. Marr KA, Seidel K, White TC, Bowden RA. Candidemia in allogeneic blood and marrow transplant recipients: evolution of risk factors after the adoption of prophylactic fluconazole. J Infect Dis 2000; 181:309–316. 63. Gillespie MB, O’Malley BW. An algorithmic approach to the diagnosis and management of invasive fungal rhinosinusitis in the immunocompromised patient. Otolaryngol Clin North Am 2000; 33:323–334. 64. Choi SS, Milmoe GJ, Dinndorf PA, Quinones RR. Invasive aspergillus sinusitis in pediatric bone marrow transplant patients. evaluation and management. Arch Otolaryngol Head Neck Surg 1995; 121:1188–1192. 65. Sugar AM. Mucormycosis. Clin Infect Dis 1992; 14:S126–S129. 66. Sulahian A, Boutboul F, Ribaud P, Leblanc T, Lacroix C, Derouin F. Value of antigen detection using an enzyme immunoassay in the diagnosis and prediction of invasive aspergillosis in two adult and pediatric hematology units during a 4-year prospective study. Cancer 2001; 91:311–318. 67. Bialek R, Moshous D, Casanova JL, Blanche S, Hennequin C. Aspergillus antigen and PCR assays in bone marrow transplanted children. Eur J Med Res 2002; 7:177–180. 68. Fujita SI, Lasker BA, Lott TJ. Microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species in blood. J Clin Microbiol 1995; 33:962. 69. Einsele H, Hebart H, Roller G, et al. Detection and identification of fungal pathogens in blood by using molecular probes. J Clin Microbiol 1997; 35:1353–1360. 70. Trick WE, Vernon MO, Hayes RA, et al. Impact of ring wearing on hand contamination and comparison of hand hygiene agents in a hospital. Clin Infect Dis 2003; 36:1383–1390. 71. Strausbaugh LJ, Sewell DL, Ward TT, Pfaller MA, Heitzman T, Tjoelker R. High frequency of yeast carriage on hands of hospital personnel. J Clin Microbiol 1994; 32:2299–2300. 72. Goodman JL, Winston DJ, Greenfield RA, et al. A controlled trial of fluconazole to prevent fungal infections in patients undergoing bone marrow transplantation. N Engl J Med 1992; 326:845–851. 73. MacMillan ML, Goodman JL, DeFor TE, Weisdorf DJ. Fluconazole to prevent yeast infections in bone marrow transplantation patients: a randomized trial of high versus reduced dose, and determination of the value of maintenance therapy. Am J Med 2002; 112:369–379. 74. Trigg ME, Morgan D, Burns TL, et al. Successful program to prevent aspergillus infections in children undergoing marrow transplantation: use of nasal amphotericin. Bone Marrow Transplant 1997; 19:43–47. 75. Walsh TJ, Finberg RW, Arndt C, et al. Liposomal Amphotericin B for empirical therapy in patients with persistent fever and neutropenia. N Engl J Med 1999; 340:764–771. 76. Walsh TJ, Pappas P, Winston DJ, et al. Voriconazole compared with liposomal Amphotericin B for empirical antifungal therapy in patients with neutropenia and persistent fever. N Engl J Med 2002; 346:225–234. 77. Marr KA. Empirical antifungal therapy—new options, new tradeoffs. N Engl J Med 2002; 346:278–280. 78. Mora-Duarte J, Betts R, Rotstein C, et al. Comparison of caspofungin and amphotericin B for invasive candidiasis. N Engl J Med 2002; 347:2020–2029. 79. Rex JH, Walsh TJ, Sobel JD, et al. Practice guidelines for the treatment of candidiasis. Infectious diseases society of America. Clin Infect Dis 2000; 30:662–678. 80. Caillot D, Casasnovas O, Bernard A, et al. Improved management of invasive pulmonary aspergillosis in neutropenic patients using early thoracic computed tomographic scan and surgery. J Clin Oncol 1997; 15:139–147. 81. Stevens DA, Kan VL, Judson MA, et al. Practice guidelines for diseases caused by aspergillus. Infectious diseases society of America. Clin Infect Dis 2000; 30:696–709. 82. Herbrecht R, Denning DW, Patterson TF, et al. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J Med 2002; 347:408–415.

Prevention and Treatment of Infectious Disease

59

83. Bhatia S, McCullough J, Perry EH, Clay M, Ramsay NK, Neglia JP. Granulocyte transfusions: efficacy in treating fungal infections in neutropenic patients following bone marrow transplantation. Transfusion 1994; 34:226–232. 84. Ferguson BJ, Mitchell TG, Moon R, Camporesi EM, Farmer J. Adjunctive hyperbaric oxygen for treatment of rhinocerebral mucormycosis. Rev Infect Dis 1988; 10:551–559. 85. van Burik JH, Ratanatharathorn V, Stepan DE, et al. Micafungin versus fluconazole for prophylaxis against invasive fungal infections during neutropenia in patients undergoing hematopoietic stem cell transplantation. Clin Infect Dis 2004; 39:1407–1416. 86. Chiou CC, Groll AH, Walsh TJ. New drugs and novel targets for treatment of invasive fungal infections in patients with cancer. Oncologist 2000; 5:120–135. 87. Ljungman P, Aschan J, Lewensohn-Fuchs I, et al. Results of different strategies for reducing cytomegalovirus-associated mortality in allogeneic stem cell transplant recipients. Transplantation 1998; 66:1330–1334. 88. Holmberg LA, Boeckh M, Hooper H, et al. Increased incidence of cytomegalovirus disease after autologous CD34-selected peripheral blood stem cell transplantation. Blood 1999; 94:4029–4035. 89. Chakrabarti S, Mackinnon S, Chopra R, et al. High incidence of cytomegalovirus infection after nonmyeloablative stem cell transplantation: potential role of campath-1H in delaying immune reconstitution. Blood 2002; 99:4357–4363. Chakrabarti S, Mackinnon S, Chopra R, et al. High incidence of cytomegalovirus infection after nonmyeloablative stem cell transplantation using campath. Blood 2002; 100:3843–3844. 90. Bainton RD, Byrne JL, Davy BJ, Russell NH. CMV infection following nonmyeloablative allogeneic stem cell transplantation using campath. Blood 2002; 100:3843–3844. 91. Bowden RA, Slichter SJ, Sayers M, et al. A comparison of filtered leukocyte-reduced and cytomegalovirus (CMV) seronegative blood products for the prevention of transfusion-associated CMV infection after marrow transplant. Blood 1995; 86:3598–3603. 92. Einsele H, Hebart H, Kauffmann-Schneider C, et al. Risk factors for treatment failures in patients receiving PCR-based preemptive therapy for CMV infection. Bone Marrow Transplant 2000; 25:757–763. 93. Einsele H, Ehninger G, Hebart H, et al. Polymerase chain reaction monitoring reduces the incidence of cytomegalovirus disease and the duration and side effects of antiviral therapy after bone marrow transplantation. Blood 1995; 86:2815–2820. 94. Boeckh M, Leisenring W, Riddell SR, et al. Late cytomegalovirus disease and mortality in recipients of allogeneic hematopoietic stem cell transplants: importance of viral load and T-cell immunity. Blood 2003; 101:407–414. 95. Kim DH, Kim JG, Lee NY, et al. Risk factors for late cytomegalovirus infection after allogeneic stem cell transplantation using HLA-matched sibling donor: donor lymphocyte infusion and previous history of early CMV infection. Bone Marrow Transplant 2004; 34:21–27. 96. Winston DJ, Yeager AM, Chandrasekar PH, Snydman DR, Petersen FB, Territo MC. Valacyclovir cytomegalovirus study group. Randomized comparison of oral valacyclovir and intravenous ganciclovir for prevention of cytomegalovirus disease after allogeneic bone marrow transplantation. Clin Infect Dis 2003; 36:749–758. 97. Reusser P, Einsele H, Lee J, et al. Infectious diseases working party of the European group for blood and marrow transplantation. Randomized multicenter trial of foscarnet versus ganciclovir for preemptive therapy of cytomegalovirus infection after allogeneic stem cell transplantation. Blood 2002; 99:1159–1164. 98. Ljungman P, Deliliers GL, Platzbecker U, et al. Cidofovir for cytomegalovirus infection and disease in allogeneic stem cell transplant recipients. Blood 2001; 97:388–392. 99. Schmidt GM, Horak DA, Niland JC, Duncan SR, Forman SJ, Zaia JA. A randomized, controlled trial of prophylactic ganciclovir for cytomegalovirus pulmonary infection in recipients of allogeneic bone marrow transplants; the city of hope-stanford-syntex CMV study group. N Engl J Med 1991; 324:1005–1011. 100. Emanuel D, Cunningham I, Jules-Elysee K, et al. Cytomegalovirus pneumonia after bone marrow transplantation successfully treated with the combination of ganciclovir and high-dose intravenous immune globulin. Ann Intern Med 1988; 109:777–782. 101. Schmidt GM, Kovacs A, Zaia JA, et al. Ganciclovir/immunoglobulin combination therapy for the treatment of human cytomegalovirus-associated interstitial pneumonia in bone marrow allograft recipients. Transplantation 1988; 46:905–907.

60

Bradfield et al.

102. Goodrich JM, Mori M, Gleaves CA, et al. Early treatment with ganciclovir to prevent cytomegalovirus disease after allogeneic bone marrow transplantation. N Engl J Med 1991; 325:1601–1607. 103. Bass EB, Powe NR, Goodman SN, et al. Efficacy of immune globulin in preventing complications of bone marrow transplantation: a meta-analysis. Bone Marrow Transplant 1993; 12:273–282. 104. Nichols WG, Corey L, Gooley T, et al. Rising pp65 antigenemia during preemptive anticytomegalovirus therapy after allogeneic hematopoietic stem cell transplantation: risk factors, correlation with DNA load, and outcomes. Blood 2001; 97:867–874. 105. Julin JE, van Burik JH, Krivit W, et al. Ganciclovir-resistant cytomegalovirus encephalitis in a bone marrow transplant recipient. Transplant Infect Dis 2002; 4:201–206. 106. Wolf DG, Lurain NS, Zuckerman T, et al. Emergence of late cytomegalovirus central nervous system disease in hematopoietic stem cell transplant recipients. Blood 2003; 101:463–465. 107. Avery RK, Bolwell BJ, Yen-Lieberman B, et al. Use of leflunomide in an allogeneic bone marrow transplant recipient with refractory cytomegalovirus infection. Bone Marrow Transplant 2004; 34:1071–1075. 108. Eckle T, Lang P, Prix L, et al. Rapid development of ganciclovir-resistant cytomegalovirus infection in children after allogeneic stem cell transplantation in the early phase of immune cell recovery. Bone Marrow Transplant 2002; 30:433–439. 109. Jabs DA, Enger C, Dunn JP, et al. Cytomegalovirus retinitis and viral resistance: ganciclovir resistance. J Infect Dis 1998; 177:770–773. 110. Drew WL, Miner RC, Busch DF, et al. Prevalence of resistance of in patients receiving ganciclovir for serious cytomegalovirus infection. J Infect Dis 1991; 163:716–719. 111. Gilbert C, Boivin G. Human cytomegalovirus resistance to antiviral drugs. Antimicrob Agents and Chemother 2005; 49:873–883. 112. Erice A, Gil-Roda C, Perez JL, et al. Antiviral susceptibilities and analysis of UL97 and DNA polymeriase sequences of clinical cytomegalovirus isolates from immunocompromised patients. J Infect Dis 1997; 175:1087–1092. 113. Williams JW, Mital D, Chong A, et al. Experiences with leflunomide in solid organ transplantation. Transplantation 2002; 73:358–366. 114. John GT, Manivannan J, Chandy S, Peter S, Jacob CK. Leflunomide therapy for cytomegalovirus disease in renal allograft recepients. Transplantation 2004; 77:1460–1461. 115. Waldman WJ, Knight DA, Lurain NS, et al. Novel mechanism of inhibition of cytomegalovirus by the experimental immunosuppressive agent leflunomide. Transplantation 1999; 68:814–825. 116. Curtis RE, Travis LB, Rowlings PA, et al. Risk of lymphoproliferative disorders after bone marrow transplantation: a multi-institutional study. Blood 1999; 94:2208–2216. 117. Hale G, Waldmann H. Risks of developing Epstein-Barr virus-related lymphoproliferative disorders after T-cell-depleted marrow transplants. Campath users. Blood 1998; 91:3079–3083. 118. Shapiro RS, McClain K, Frizzera G, et al. Epstein-Barr virus associated B cell lymphoproliferative disorders following bone marrow transplantation. Blood 1988; 71:1234–1243. 119. Gartner BC, Schafer H, Marggraff K, et al. Evaluation of use of Epstein-Barr viral load in patients after allogeneic stem cell transplantation to diagnose and monitor posttransplant lymphoproliferative disease. J Clin Microbiol 2002; 40:351–358. 120. Wagner HJ, Cheng YC, Huls MH, et al. Prompt versus preemptive intervention for EBV lymphoproliferative disease. Blood 2004; 103:3979–3981. 121. Papadopoulos EB, Ladanyi M, Emanuel D, et al. Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation. N Engl J Med 1994; 330:1185–1191. 122. Rooney CM, Smith CA, Ng CY, et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 1998; 92:1549–1555. 123. Gross TG, Hinrichs SH, Winner J, et al. Treatment of post-transplant lymphoproliferative disease (PTLD) following solid organ transplantation with low-dose chemotherapy. Ann Oncol 1998; 9:339–340. 124. van Esser JW, Niesters HG, van der Holt B, et al. Prevention of Epstein-Barr viruslymphoproliferative disease by molecular monitoring and preemptive rituximab in high-risk patients after allogeneic stem cell transplantation. Blood 2002; 99:4364–4369.

Prevention and Treatment of Infectious Disease

61

125. Kuehnle I, Huls MH, Liu Z, et al. CD20 monoclonal antibody (rituximab) for therapy of EpsteinBarr virus lymphoma after hemopoietic stem-cell transplantation. Blood 2000; 95:1502–1505. 126. Baldwin A, Kingman H, Darville M, et al. Outcome and clinical course of 100 patients with adenovirus infection following bone marrow transplantation. Bone Marrow Transplant 2000; 26:1333–1338. 127. Thomas ED, Blume KG, Forman SJ. Hematopoietic Cell Transplantation. Second edition. Boston. Blackwell Science, 1999: Chapters 51–55. 128. Hierholzer JC. Adenoviruses in the immunocompromised host. Clin Microbiol Rev 1992; 5:262–274. 129. Suparno C, Mautner V, Osman H, et al. Adenovirus infections following allogeneic stem cell transplantation: incidence and outcome in relation to graft manipulation, immunosuppression, and immune recovery. Blood 2002; 100:1619–1627. 130. Avivi I, Chakrabarti S, Milligan DW, et al. Incidence and outcome of adenovirus disease in transplant recipients after reduced-intensity conditioning with alemtuzumab. Biol Blood Marrow Transplant 2004; 10:186–194. 131. Chakrabarti S, Mautner V, Osman H, et al. Adenovirus infections following allogeneic stem cell transplantation: the incidence and outcome in relation to graft manipulation, immunosuppression, and immune recovery. Blood 2002; 100:1619–1627. 132. Lion T, Baumgartinger R, Watzinger F, et al. Molecular monitoring of adenovirus in peripheral blood after allogeneic bone marrow transplantation permits early diagnosis of disseminated disease. Blood 2003; 102:1114–1120. 133. Muller WJ, Levin MJ, Shin YK, et al. Clinical and in vitro evaluation of cidofovir for treatment of adenovirus infection in pediatric hematopoietic stem cell transplant recipients. Clin Infect Dis 2005; 41:1812–1816. 134. Cone RW, Huang MLW, Corey JZ, Ashley R, Bowden R. human herpesvirus 6 infections after bone marrow transplantation: clinical and virologic manifestations. J Infect Dis 1999; 179:311–318. 135. Yoshikawa T, Asano Y, Ihira M, et al. Human herpesvirus 6 viremia in bone marrow transplant recipients: clinical features and risk factors. J Infect Dis 2002; 185:847–853. 136. Zerr DM, Gooley TA, Leung Y, et al. Human herpesvirus 6 reactivation and encephalitis in allogeneic bone marrow transplant recipients. CID 2001; 33:763–771. 137. Yoshikawa T, Asano Y, Ihira M, et al. Human herpesvirus 6 viremia in bone marrow transplant recipients: clinical features and risk factors. J Infect Dis 2002; 185:847–853. 138. Cone RW, Huang ML, Corey L, Zeh J, Ashley R, Bowden R. Human herpesvirus 6 infections after bone marrow transplantation: clinical and virologic manifestations. J Infect Dis 1999; 179:311–318. 139. Kadakia MP, Rybka WB, Stewart JA, et al. Human herpesvirus 6: infection and disease following autologous and allogeneic bone marrow transplantation. Blood 1996; 87:5341–5354. 140. Dockrell DH. Human herpesvirus 6: molecular biology and clinical features. J Med Microbiol 2003; 52:5–18. 141. Singh N, Paterson DL. Encephalitis caused by human herpesvirus-6 in transplant recipients: relevance of a novel neurotropic virus. Transplantation 2000; 69:2474–2479. 142. Zerr DM, Gupta D, Huang ML, Carter R, Corey L. Effect of antivirals on human herpesvirus 6 replication in hematopoietic stem cell transplant recipients. Clin Infect Dis 2002; 34:309–317. 143. Yoshihara S, Kato R, Inoue T, et al. Successful treatment of life-threatening human herpesvirus-6 encephalitis with donor lymphocyte infusion in a patient who had undergone human leukocyte antigen-haploidentical nonmyeloablative stem cell transplantation. Transplantation 2004; 77:835–838. 144. Hirsch HH, Steiger J. BK Polymavirus. Lancet 2003;611–623. 145. Hirsch HH, Brennan DC, Drachenberg CB, et al. Polyomavirus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations. Transplantation 2005; 79:1277–1286. 146. Leung AY, Mak R, Lie AK, et al. Clinicopathological features and risk factors of clinically overt haemorrhagic cystitis complicating bone marrow transplantation. Bone Marrow Transplant 2002; 29:509–513. 147. Apperley JF, Rice SJ, Bishop JA, et al. Late-onset hemorrhagic cystitis associated with urinary excretion of polyomaviruses after bone marrow transplantation. Transplantation 1987; 43:108–112. 148. Arthur RR, Shah KV, Baust SJ, et al. Association of BK viruria with hemorrhagic cystitis in recipients of bone marrow transplants. N Engl J Med 1986; 315:230–234.

62

Bradfield et al.

149. Azzi A, Fanci R, Bosi S, et al. Monitoring of polyomavirus BK viruria in bone marrow transplantation patients by DNA hybridization assays and polymerase chain reaction: an approach to assess the relationship between BK viruria and hemorrhagic cystitis. Bone Marrow Transplant 1994; 14:235–240. 150. Azzi A, Cesaro S, Laszio D, et al. Human polyomavirus BK load and haemorrhagic cystitis in bone marrow transplantation patients. J Clin Virol 1999; 14:79–86. 151. Biel SS, Held TK, Landt O, et al. Rapid quantification and differentiation of human polyomavirus DNA in undiluted urine from patients after bone marrow transplantation. J Clin Microbiol 2000; 38:3689–3695. 152. Leung AY, Suen CK, Lie AK, et al. Quantification of polyoma BK virus in hemorrhagic cystitis complicating bone marrow transplantation. Blood 2001; 98:1971–1978. 153. Bogdanovic G, Priftakis P, Giraud G, et al. Association between a high BK virus load in urine samples of patients with graft-versus-host disease and development of hemorrhagic cystitis after hematopoietic stem cell transplantation. J Clin Microbiol 2004; 42:5394–5396. 154. Erard V, Storer B, Corey L, et al. BK virus infection in hematopoietic stem cell transplant recipients: frequency, risk factors, and association with postengraftment hemorrhagic cystitis. Clin Infect Dis 2004; 39:1861–1865. 155. Portolani M, Pietrosemoli P, Cermelli C, et al. Suppression of BK virus replication and cytopathic effect by inhibitors of prokaryotic DNA gyrase. Antiviral Res 1988; 9:205–218. 156. Ferrazzi E, Peracchi M, Biasolo MA, et al. Antiviral activity of gyrase inhibitors norfloxacin, coumermycin A1 and nalidixic acid. Biochem Pharmacol 1988; 37:1885–1886. 157. Leung AYH, Chan MTL, Yuen KY, et al. Ciprofloxacin decreased polyoma BK virus load in patients who underwent allogeneic hematopoietic stem cell Transplantation. Clin Infect Dis 2005; 40:528–537. 158. Kawakami M, Ueda S, Maeda T, et al. Vidarabine therapy for virus-associated cystitis after allogeneic bone marrow transplantation. Bone Marrow Transplant 1997; 20:485–490. 159. Held TK, Biel SS, Nitsche A, et al. Treatment of BK virus-associated hemorrhagic cystitis and simultaneous cytomegalovirus reactivation with cidofovir. Bone Marrow Transplant 2000; 26:347–350. 160. Wong CL, Williams C, Chopra R, Mutton K. Haemorrhagic cystitis and BK virus: association and treatment. Annu Sci Meet Br Soc Haemotol 2004; April:000 . Abstract. 161. Pickering LK. 26th ed. Red Book 2003 Report of the Committee on Infectious Diseases. American Academy of Pediatrics; 344–353, see also 523–528. 162. Anaissie EJ, Mahfouz TH, Asian T, et al. The natural history of respiratory syncytial virus infection in cancer and transplant patients: implications for management. Blood 2004; 103:1611–1617. 163. DeVincenzo JP, Hirsch RL, Fuentes RJ, Top FH, Jr. Respiratory syncytial virus immune globulin treatment of lower respiratory tract infection in pediatric patients undergoing bone marrow transplantation-a compassionate use experience. Bone Marrow Transplant 2000; 25:161–165. 164. Adams R, Christenson J, Petersen F, Beatty P. Pre-emptive use of aerosolized ribavirin in the treatment of asymptomatic pediatric marrow transplant patients testing positive for RSV. Bone Marrow Transplant 1999; 24:661–664. 165. Peck A, Corey L, Boeckh M. Pretransplantation respiratory syncitial virus infection: impact of a strategy to delay transplantation. Clin Infect Dis 2004; 39:673–680. 166. Nichols G, Gooley T, Boeckh M. Community acquired respiratory syncitial virus and parainfluenza virus infections after hematopoietic stem cell transplantation: the Fred Hutchinson cancer research center experience. Biol Blood Marrow Transplant 2001; 7:11s–15s. 167. Harrington RD, Hooton RD, Hackman RC, et al. An outbreak of respiratory syncytial virus in a bone marrow transplant center. J Infect Dis 1992; 165:987–993. 168. Hemming V, Rodriguez W, Kim H, et al. Intravenous immunoglobulin treatment of respiratory syncitial virus infections in infants and young children. Antimicrob Agents Chemother 1987; 31:1882–1886. 169. Whimbey E, Champlin RE, Englund JA, et al. Combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients. Bone Marrow Transplant 1995; 16:393–399. 170. Champlin R, Whimbey E. Community respiratory virus infections in bone marrow transplant recipients: the MD Anderson cancer center experience. Biol Blood Marrow Transplant 2001; 7:8s–10s. 171. Saral R, Burns WH, Laskin OL, et al. Acyclovir prophylaxis of herpes-simple-virus infections. N Engl J Med 1981; 305:63–67.

Prevention and Treatment of Infectious Disease

63

172. Hann IM, Prentice HG, Blacklock HA, et al. Acyclovir prophylaxis against herpes virus infections in severely immunocompromised patients: randomized double blind trial. Br Med J 1983; 287:384–388. 173. Gluckman E, Lotsberg J, Devergie A, et al. Prophylaxis of herpes infections after bone marrow transplantation by oral acyclovir. Lancet 1983; 2:706–708. 174. Stranska R, van Loon AM, Polman M, et al. Genotypic and phenotypic characterization of acyclovir-resistant herpes simplex viruses isolated from haemotopoietic stem cell transplant recipients. Antivir Ther 2004; 4:565–575. 175. McLaren C, Chen MS, Ghazzouli I, et al. Drug resistance patterns of herpes simplex virus isolates from patients treated with acyclovir. Antimicrob Agents Chemother 1985; 28:740–744. 176. Safrin S, Crumpacker C, Chatis P, et al. A controlled trial comparing foscarnet with vidarabine for acyclovir-resistant mucocutaneous herpes simplex in the acquired immunodeficiency syndrome. N Engl J Med 1991; 325:551–555.

3 Acute Graft-Versus-Host Disease Theodore B. Moore and Stephen A. Feig Mattel Children’s Hospital at UCLA, David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A.

Since the first successful allogeneic marrow transplant (1), prevention and management of acute graft-versus-host disease (aGVHD) have remained two of the major problems of stem cell transplantation. A better understanding of the pathophysiology of aGVHD and the human leukocyte antigen (HLA) barrier, as well as the development of more effective and selective immunosuppressive agents, are crucial to reducing the morbidity and mortality of aGVHD.

PATHOPHYSIOLOGY The development of aGVHD is complex and dependent on many factors with several levels of interaction. A commonly accepted simplified model summarizes it in a three step process with afferent and efferent phases (2–6), including a “conditioning-induced tissue damage phase,” “donor lymphocyte activation phase,” and “a cellular and inflammatory effector phase.” During the first phase (Conditioning-Induced Tissue Damage), tissue damage occurs as a result of the transplant conditioning regimen or infection, which leads to cellular activation, release of inflammatory cytokines, and host tissue factors, including IL-1, IL-6, tumor necrosis factor-a (TNF-a), and others (7), which, in turn, leads to increased expression of host antigens and adhesion molecules. This leads to increased maturation/activation of host dendritic cells with subsequent recognition of host major and minor histocompatibility antigens by mature donor T cells. Conditioning causes damage to the intestinal mucosa lining, compromising its integrity, allowing bacteria and their endotoxins to be released into the circulation, further causing a surge in the release of inflammatory cytokines from macrophages. Early intervention or the introduction of preventative measures may reduce the impact of these factors on aGVHD. Reduced intensity conditioning regimens are one strategy, but they may not be appropriate for all patients. Blockade of lipopolysaccharide (endotoxin) can result in a reduction of aGVHD without compromise of the graft-versus-leukemia effect (8). In experimental models both keratinocyte growth factor (KGF) and IL-11 have shown a protective effect on the gastrointestinal (GI) mucosa, as well as a reduction in aGVHD (9–11). In clinical use, IL-11 has prohibitive toxicity (12), whereas KGF has demonstrated efficacy in reducing the duration and severity of mucositis after intensive chemotherapy and radiotherapy in adult trials (7). Pediatric trials with KGF are in process. More intensive conditioning regimens cause increased release of inflammatory cytokines (13–15) and significant damage to the thymus, where reeducation and elimination of antiself T cells occur (16,17). 65

66

Moore and Feig

During the second phase (Donor Lymphocyte Activation), host antigen presenting cells (APC) present recipient antigens to donor T cells. APC process proteins and then present peptides bound to HLA molecules on the cell surface. Following infusion of donor T cells, these antigens are recognized by T cells via specific T-cell receptors. Despite matching of major HLA antigens, minor antigens that are not matched (such as the H–Y antigen and others) may be recognized as foreign. Following recognition, there is activation of donor T cells and cytokine production. CD4C helper T cells can differentiate into two populations: Th1 and Th2 cells. Th1 cells secrete IL-2, IL-12, and Interferon (IFN)-g and appear to play a role in potentiating aGVHD and the inflammatory response, whereas Th2 cells producing IL-4, IL-5, and IL-13 appear to suppress it. Inflammatory cytokines may cause additional donor T-cell activation against recipient antigens (18–20). Host APC play a critical role in aGVHD. Immature dendritic cells are present in significant numbers in barrier organs, including the skin and bowel (21). Tissue damage can lead to dendritic cell maturation within those tissues, making the tissue a specific target for aGVHD. In some models, elimination of APC in specific organs resulted in elimination of GVHD in those organs, but not others (22,23). T cells that interact with APC require a second costimulatory signal before becoming completely activated. Identification of these costimulatory pathways and their blockade is an area of significant interest for potential therapeutic intervention (24). In the third phase (Cellular and Inflammatory Effector), IL-2 and IFN-g secreted by Th1 cells are responsible for activation of natural killer (NK) cells, and cytotoxic T lymphocytes (CTLs), as well as stimulating host and donor mononuclear phagocytes to produce additional IL-1, TNF-a, and nitric oxide. There is a clonal expansion of specific T cells, which then differentiate. Finally, CTLs as well as monocytes and NK cells cause further tissue injury. NK cells can be activated by Th1 cells and are negatively regulated by MHC class I-specific inhibitory receptors. Thus, donor NK cell alloreactivity may be seen in HLA mismatched transplants (25). However, there is evidence that NK cells may play a significant role in graft versus leukemia (GVL), while actually decreasing aGVHD (20,26–28). Mouse models have provided evidence that donor NK cells may directly eliminate host APC. In addition, either NK cells or NK-induced cells produce TGF-b, which has immunosuppressive effects. Blockade of TGF-b reduces the protective effects of NK cells on aGVHD (29). Regulatory T cells play an important role in aGVHD as well. Recently, there has been significant evidence that a T-cell subpopulation consisting of CD4C CD25CT cells may suppress the development of aGVHD (30,31), as do CD3CCD4-CD8- T cells (32). Results from in vitro studies suggest that there may be a differential effect of various immunosuppressive agents on regulatory T cell subsets (see section on Sirolimus). There is an increasing interest in differentiating between cells that are responsible for aGVHD and those that play a role in GVL (33–35). The complete role of each lymphocyte subset remains unclear, although data continues to emerge (36). As a result of increasing knowledge of the pathophysiology of aGVHD, therapy may be designed to target either the responsible T-cell subtype or inhibiting specific cytokines through the use of monoclonal antibodies or small molecules (see below).

STAGING/CLINICAL DESCRIPTION Acute GVHD is characterized by manifestations in the skin, liver, and GI tract. Grading of the severity of aGVHD is based on evaluation of the degree of involvement in each of these organs. The most commonly used grading system was proposed in 1974 (Tables 1 and 2) (37–40). Symptoms of aGVHD may appear prior to signs of marrow recovery. Initial presentation often involves the skin and may seem like a flush of the face, ears, palms, and soles. Upon closer examination, there is a fine maculopapular rash that may become generalized (Fig. 1A). When severe, the rash may progress to bulla formation and widespread desquamation. The aGVHD rash must be distinguished from other rashes that may be secondary to bacterial, fungal, or viral infections or drug reactions. A skin biopsy may be useful

Acute Graft-Versus-Host Disease Table 1

67

Clinical Staging Stage I

Stage II

Stage III

Stage IV

Skin

Rash !25% body surface

Rash 25–50% body surface

Rash 50–100% body surface

Desquamation and bulla formation

Gastrointestinal

Diarrhea O5 K%10 ml/kg/day or persistent nausea

Diarrhea O10 –%15 ml/kg/day

Diarrhea O15 ml/kg/day

Pain C/K Ileus

Liver

Bilirubin 2–3 mg/dL

Bilirubin 3–6 mg/dL

Bilirubin 6 –15 mg/dL

Bilirubin O15 mg/dL

Source: Adapted from Refs. 37–40.

in distinguishing these since aGVHD is characterized by eosinophilic bodies, apoptotic bodies, and lymphocytic infiltration of the dermis (Fig. 1B). Obstructive hyperbilirubinemia is the primary hepatic manifestation, but transaminase elevation may also occur. Measurement of the severity of liver involvement is based on the total bilirubin level (Table 1). The jaundice of aGVHD must be differentiated from that of venoocclusive disease, biliary stones, drug toxicity (especially cyclosporine and tacrolimus), and infection. A liver biopsy is often helpful in discerning the underlying etiology and shows bile duct damage and lymphocytic infiltration (Fig. 2). GI manifestations of aGVHD can be highly variable and often lack specificity. The most common symptom is diarrhea. Stool volume will determine the severity (grade) of GVHD (Table 1). Other manifestations may include anorexia, nausea, food intolerance, cramping abdominal pain, bloody stools, mucosal sloughing, and ileus. The differential diagnosis of GI aGVHD includes infection, malabsorption, and mucosal toxicity of the conditioning regimen. An endoscopic biopsy may help to determine the cause of symptoms; if aGVHD is present, the biopsy may show lymphocytic infiltration with crypt cell necrosis (Fig. 3). Involvement of other organs has been described, including ocular aGVHD. Ocular aGVHD may be manifested by a hemorrhagic conjunctivitis and pseudomembrane formation.

Graft-Versus-Host Disease After Autologous Stem-Cell Transplantation Although aGVHD is typically seen in the context of allogeneic transplantation, it has been observed in recipients of autologous and syngeneic transplants (43). This has been reproduced in several animal studies (44). Sudden withdrawal of immunosuppression may precipitate aGVHD, as may the exogenous administration of such cytokines as IL-2 (45). This strategy has been employed in an attempt to induce a “graft-versus-malignancy” effect for some recipients of autologous transplants in both children (e.g., Children’s Oncology Group study AHOD0121 “A Phase II/III Study of Immunomodulation after High Dose Myeloablative Therapy with Table 2

Clinical Grading

Overall grade 0 I (mild) II (moderate) III (severe) IV (life threatening) Source: Adapted from Refs. 37–40.

Skin

Stage (Table 1) Gastrointestinal

Liver

Functional impairment

0 I to II I to III II to III II to IV

0 0 I II to III II to IV

0 0 I II to III II to IV

0 0 I II III

68

Moore and Feig

Figure 1 Skin graft-versus-host disease (GVHD). (A) maculopapular rash consistent with acute graft-versus-host disease. (B) Skin biopsy. This high power photo shows dying keratinocytes in association with lymphocytes (arrow) as well as sparse dermal lymphocytic infiltrate. Note also the epithelial separation from the dermis that is seen in severe (grade IV) GVHD. Source: Photos courtesy of Sarah Dry, M.D.

Autologous Stem Cell Rescue for Refractory/Relapsed Hodgkin Disease”) and adults (46). Several trials in adults have demonstrated that the development of autologous GVHD is associated with an increased production of IL-10 and may impact antitumor response (46). Polymorphisms within the IL-10 promoter region significantly impact the degree of IL-10 production. Inheritance of specific alleles that affect IL-10 and g IFN production may be responsible for the variable effects seen among autologous transplant patients in whom immune-based strategies have been employed (46).

RISK FACTORS Source of Stem Cells The stem cell source used for transplantation may impact the degree of aGVHD manifested. Differences between marrow, peripheral blood and cord blood stem cells are outlined below.

Figure 2 Liver graft-versus-host disease (GVHD). This photograph shows a portal tract with a bile duct (arrow), artery (above the bile duct), and vein (to the right of the bile duct). The bile duct shows epithelial cell loss (arrow) consistent with acute GVHD. Source: Photos courtesy of Sarah Dry, M.D.

Acute Graft-Versus-Host Disease

69

Figure 3 Gastrointestinal graft-versus-host disease (GVHD). (A) Colon, high power. This photograph demonstrates apoptotic bodies in crypts (arrows). Previous treatment of this patient with steroids reduced the lymphocyte infiltration that might otherwise be present. (B) Small bowel, low power. This case of acute GVHD shows extensive crypt loss and injury on the right side of the photograph; crypt injury can be seen as epithelial attenuation and atrophy (arrowheads). Source: Photos courtesy of Sarah Dry, M.D.

Bone Marrow The greatest experience in stem cell transplantation is with bone marrow. Bone marrow can be administered fresh or thawed (following previous cryopreservation). Although most observers have reported no differences between outcomes using cryopreserved and fresh marrow, there has been at least one report that has suggested that aGVHD is reduced in marrow that has been cryopreserved prior to use (47).

Peripheral Blood Stem Cells Over the last five to ten years, the use of allogeneic peripheral blood stem cells (PBSC) has been gaining in popularity due to the ease of harvesting and the more rapid hematopoietic engraftment seen. PBSC contain ten times as many T lymphocytes in the specimen compared to bone marrow (48). Nevertheless, the majority of both retrospective and prospective studies have shown little if any statistically significant increase in aGVHD (49,50), although an increase in the frequency and severity of chronic GVHD (cGVHD) has been reported (51). There are conflicting data in this regard (52–57). Although the literature is rich in publications comparing bone marrow and PBSC’s for the risk of aGVHD and cGVHD in the adult population, there is very little published pediatric experience (58–64). Nagatoshi et al. reported no increase in acute GVHD but observed a significant increase in cGVHD after PBSC transplant (58).

Cord Blood Umbilical cord blood has been used with increasing frequency as a stem cell source for transplantation. Several groups, including the Cord Blood Transplantation trial, have reported a reduced incidence of aGVHD in patients receiving cord blood mismatched at 0–2 HLA loci, in comparison to historical BMT controls (65–69). Even in these significantly mismatched donorrecipient pairs the incidence of grade III–IV aGVHD at day 100 was only 25% (67). This may be due to the younger age of recipients of cord blood stem cells or the immunologic immaturity of the fetal lymphocyte. There are reduced numbers of alloreactive CTLs in cord blood. Important differences in the pattern of immune reconstitution compared to other stem cell sources have been reported (70–72). Cytokine production and responsiveness also appear to be decreased as well (71,73).

70

Moore and Feig

Degree of Match/Human Leukocyte Antigen Typing One of the most crucial factors in the potential for the development of aGVHD is the degree of HLA disparity between donor and recipient. There is a strong correlation between the incidence and severity of aGVHD and the degree of HLA mismatch (74–80). HLA typing may be performed by either serologic or molecular analyses. Molecular typing is crucial in diminishing the risk of GVHD in unrelated donor transplants and in related transplants where serologic homozygosity is found. Although an HLA mismatch at any locus increases overall risk of aGVHD, there are conflicting reports as to the relative importance of a mismatch at class I or class II loci. Initial observations stressed the importance of matching at HLA-DRB (60,75,76). Increases in grade III/grade IV aGVHD and mortality were demonstrated when donor and recipient were mismatched at the DRB locus. Other studies have shown equivalency between class I and class II single antigen mismatches, whether detected by DNA or serologic methods (77,78). Leung et al. (79) performed a retrospective analysis of 248 consecutive pediatric allogeneic BMT from related (nZ119) or unrelated donors (nZ129). Class I HLA antigens were determined by serology and class II HLA antigens by DNA typing. By these methods, 69% were completely matched on the A, B, and DR loci. There was a decrease in survival in all patients who had a one-antigen mismatch, regardless of the location. There was an increase in the occurrence of severe aGVHD in patients with mismatch at one or more antigens at the class I loci, although no significant increase was associated with a class II mismatch. In addition to the major HLA loci considered, several additional “minor antigens” have been described and appear to play a role in aGVHD (81,82). Matched related donors appear to have less difficulty with aGVHD than similarly matched unrelated donors. This suggests the existence of other factors or minor antigens that may play a significant role in the development of aGVHD. It is important to optimize the match chosen for the recipient by using the best possible typing methodology with the inclusion of the most important compatibility antigens. As technology improves and our understanding of histocompatibility increases, we will need to continue the process of optimizing donor searches and procurement processes to achieve the best possible match while maintaining efficiency of time and cost.

Donor Characteristics (Gender Differences and Age Differences) Although the importance of optimizing HLA matching of donors for the recipient remains one of the most crucial aspects for reducing aGVHD, there are other factors, such as age and gender, that may have significant impact on the development of aGVHD (83–85). It is difficult to demonstrate an impact of age on related donor stem cell transplants because siblings are often of similar age. Even an examination of the impact of donor age in the unrelated setting can be fraught with confounding influences and careful multivariate analyses must be performed. Nademanee (86) showed an increased risk of developing aGVHD that correlated with increasing donor age. Thus, choice of a younger donor is preferable when all other factors are similar. Increased recipient age is associated with an increase in the incidence and severity of aGVHD (83–85). Gender differences between donor and recipient also can increase the risk of aGVHD, specifically the use of a female donor for a male recipient (83–85). The exact mechanism for this increased risk of aGVHD is not clear. It has been postulated that this may be due to female donor T cells that recognize the H–Y antigens on male recipient cells, as a result of prior sensitization due to pregnancy, sexual activity or both. Certain acquired characteristics, such as CMV and HSV seropositivity of the donor, have been reported to be associated with an increase in the risk of developing aGVHD. The development of active CMV infection may lead to an increase in aGVHD (87,88). Other investigators have not found an association with increased aGVHD in the scenario of either CMV positive or HSV positive status in the donor or recipient (85).

Acute Graft-Versus-Host Disease

71

Conditioning Regimen More intensive conditioning regimens and more advanced disease have also been associated with increased risk of aGVHD (85,89,90). Higher doses of total body irradiation were especially associated with increased incidence and severity of aGVHD. The reason for this is not defined, although it has been postulated that more intensive conditioning causes increased tissue damage that, in turn, results in exposed host antigens and an increase in cytokine release. Damage to host organs by the conditioning regimen may prevent the administration of full doses of immunosuppressive medication. Maintenance of full doses of cyclosporine in one study (85) or of target levels (91) has been associated with a lower incidence and severity of aGVHD. Although granulocyte colony stimulating factor (G-CSF) is often used to shorten the period of neutropenia, one study suggests it may increase the risk of aGVHD (92).

PROPHYLAXIS/THERAPY Several strategies have developed over the years for both the prevention and treatment of aGVHD. After optimizing the choice of donor, the aGVHD prophylaxis regimen is the next important step to minimize aGVHD. A variety of agents have been used over the years, as single agents or in combination. These agents are primarily aimed at reducing the number or function of T cells or at modulating cytokines. The following section reviews a number of different agents and examines their role and efficacy in prevention or treatment of aGVHD.

Agents Used Nonselective Agents (Cytotoxic Agents) Methotrexate. Methotrexate, a well-established antimetabolite, has been used for more than 30 years in prophylaxis of aGVHD. When administered as a single agent, it is generally given at an initial dose of 15 mg/M2 on day C1 post transplant. Subsequent dosing is at 10 mg/M2 and is administered on days C3,C6,C11 and weekly thereafter until day C102. It may also be used in a “short course” in combination with other agents. It may be administered by oral or parenteral routes. The toxicities of this and other agents used in the management or prevention of aGVHD are listed in Table 3. Pentostatin. Pentostatin is a nucleoside analog that is a potent immunosuppressive and is currently being studied in Phase II trials for both acute and cGVHD. In the Phase I study two thirds of the patients showed complete or partial responses, although a significant increase in infection was observed at the 2 mg/M2 dosing level (121). Thus, data suggest dosing for efficacy trials at 1.5 mg/M2 level, although the optimal dosing regimen has not been established at this time. Steroids: (Prednisone, Methylprednisolone, Dexamethasone, and Others). Steroids play a role in both the prophylaxis as well as the treatment of aGVHD (see below). Treatment doses of methylprednisolone typically begin at 2 mg/kg/day in divided dosing, although some have advocated higher dosing (122). Corticosteroids can suppress both the number and function of lymphocytes. Preparations can be administered in oral, parenteral, inhaled, and topical forms. T-Cell Inhibitory Agents Cyclosporine. Cyclosporine can be used either as a single agent or in combination. It is a calcineurin inhibitor that interferes with T-lymphocyte function. It is frequently used for aGVHD prophylaxis. Prophylactic dosing typically begins on day-2

Yes Yes

T-cell inhibition

T-cell inhibition

T-cell inhibition

Pan anti-T cell

Cyclosporine

Tacrolimus

Sirolimus

Mycophenolate mofetil Antithymocyte globulin

Yes

No No No Yes

Anticytokine

T-cell cytotoxic

T-cell removal preinfusion

No

Yes

Yes

Yes

Yes

Yes

Yes

No

No

No

Yes

Yes

No

Therapeutic use

Anti-T cell CD52 specific Anticytokine

Abbreviations: TNF, tumor necrosis factor; PUVA, photopheresis ultraviolet A.

Alemtuzumab campath-1H Daclizumab (anti-IL-2Ra) Infliximab (anti-TNF) Photopheresis (PUVA) T cell depletion (ex vivo)

Yes

Yes

Yes

Yes

No

Nonselective cytotoxic T-cell inhibition

Pentostatin

Yes

Prophylactic use

Steroids

Class of agent (mechanism)

Nonselective cytotoxic Nonselective cytotoxic

Agents/Drugs

Methotrexate

Agent

Table 3

Infection, headache, tremor, GI bleeding, seizures, stroke, nausea, vomiting (112,113) Anemia, thrombocytopenia/leukopenia (transient), hypotension, abdominal pain (114–118) Non-engraftment, infection (119,120)

Nausea, vomiting, myelosuppression, mucositis, skin rash, pulmonary fibrosis, hepatotoxicity, renal toxicity (93) Nausea, vomiting, infection, fever, rash, pruritis, cough, lymphopenia, neutropenia, thrombocytopenia, elevated transaminases (93) Hypertension, hyperglycemia, mood disturbances, gastric ulcerations, elevated triglycerides, osteoporosis (93) Hypertension, nephrotoxicity, hypomagnesemia, seizures, demyelination leukoencephalopathy, hepatotoxicity (94) Hypertension, nephrotoxicity, hypomagnesemia, seizures, hepatotoxicity, myelotoxicity (94) Hypertension, seizures, hepatotoxicity, elevated triglycerides and cholesterol, hemolytic uremic syndrome, myelotoxicity (95,96) Myelotoxicity, gastrointestinal toxicity, diarrhea, nausea, hepatotoxicity (97,98) Rash, urticaria, hypotension, anaphylaxis, infection, headache, dizziness, tremor, nausea, vomiting, posttransplant lymphoproliferative disease (99) Rash, urticaria, hypotension, infection, headache, dizziness, tremor, nausea, vomiting, diarrhea (100–105) Infection, hemorrhagic cystitis, interstitial pneumonia (106–111)

Common toxicities

72 Moore and Feig

Acute Graft-Versus-Host Disease

73

prior to stem cell infusion with a loading dose of 3 mg/kg IV over 12 hours, followed by a dosing of 1.5 mg/kg IV every 12 hours. Dose adjustments are subsequently made based on trough levels and individual laboratory standards. It may be given by oral or parenteral routes. When oral administration is indicated, the starting dose is approximately three times the parenteral dose. Tacrolimus (FK506). Tacrolimus is an immunosuppressive agent used both in solid organ and stem cell transplantation. It is a macrolide lactone that inhibits T-cell activation by down regulation of IL-2 gene expression (123). It forms a complex with an FK-binding protein, which inhibits calcineurin. This inhibition prevents a transcription factor from translocating to the nucleus, and thus, prevents transcription of the IL-2 gene. Tacrolimus is primarily used for aGVHD prophylaxis (124). Prophylactic dosing typically begins on day -2 prior to stem cell infusion at a dose of 0.015 mg/kg IV every 12 hours. It may be given by oral or parenteral routes. When oral administration is indicated, the starting dose is 0.07 to 0.15 mg/kg every 12 hours. Dose adjustments for oral and parenteral administration are subsequently made based on trough levels and individual laboratory standards. Sirolimus. Sirolimus (rapamycin) is a macrocyclic lactone with structure similar structure to the calcineurin inhibitors cyclosporine and tacrolimus. It primarily inhibits signal transduction and cell cycle progression by binding to FK-binding proteins (124). Animal studies and solid organ transplant studies have shown potential efficacy and safety of the drug (95,96,124–126). In fact recent data suggest that the use of rapamicin may be more effective than cyclosporine in the induction of tolerance as rapamicin preserved the dominance of the potent suppressor CD4CCD25CCD27C over the CD27K Treg subset displaying stronger suppressor capacity in vitro (127). In addition, there is some evidence that it may have an anti-leukemic effect in Precursor B acute lymphoblastic leukemia (128). Its use in bone marrow transplant patients is still under investigation, with optimal dosing for prophylaxis (129,130) and treatment (131,132) yet to be determined. Early clinical evidence suggests a synergistic effect when used in combination with tacrolimus (133,134). Randomized trials comparing the combination to standard therapy have yet to be completed. Interestingly, there are in vitro data that demonstrate a differential effect of sirolimus and cyclosporine on the CD27C Treg cell (CD4CCD25C) subset dominance, which may favor the use of sirolimus over cyclosporine in inducing tolerance (127). Sirolimus can be administered by oral or parenteral routes. Mycophenolate Mofetil. Mycophenolate mofetil (MMF) is a prodrug that is converted to Mycophenolic acid. It acts as a potent competitive inhibitor of purine synthesis, particularly the synthesis of guanine nucleotides (97,135,136). It has been used for both prophylaxis (98,137,138) and treatment of aGVHD (139,140). It may be given by the oral or parenteral route. Oral dosing typically begins at 30–45 mg/kg/day, administered three times per day; maintenance dosing seeks target trough levels in the 1.0 to 3.5 mcg/mL range. There is some preliminary evidence, however, that peak levels may better represent AUC (area under the curve) (141,142). The pharmacokinetics in stem cell transplant patients are significantly different from those observed in solid organ transplant patients (143). Significantly higher doses are required to achieve similar levels and AUC’s for the unbound molecule (141,142). Bioavailability may be better after oral administration. Antibody Therapy Antithymocyte Globulin (Thymoglobulin). Antithymocyte globulin (ATG, Thymoglobulin) is a xenogenic serum (horse or rabbit) that contains pan anti-T-cell

74

Moore and Feig

antibodies. It has been used in the conditioning regimen to prevent rejection (especially with cord blood transplants) as well as a second line treatment of aGVHD (144). The dose and timing of ATG is highly variable between protocols for both prophylaxis and treatment. ATG is administered intravenously and has a high frequency of significant allergic reactions. Alemtuzumab (Campath-1H). Alemtuzumab (Campath-1H) is recombinant humanized monoclonal antibody directed against CD52 positive cells. CD52 is found on lymphocytes, NK cells, macrophages, monocytes, and also on some granulocytes (100). The optimal dose and role of Alemtuzumab has yet to be established, but it has been used for both prophylaxis and treatment of aGVHD (101). In addition, it has been used as a component of conditioning regimens for non-myeloablative or reduced intensity transplants (100–105). Alemtuzumab is administered intravenously. Daclizumab (Anti-IL2Ra). Daclizumab (Anti-IL2Ra) is a recombinant humanized monoclonal antibody directed against IL-2Ra (106,135). Daclizumab acts as an anticytokine, with the intended purpose of countering the effects of IL-2Ra. It is currently being investigated for its role in the treatment of aGVHD (see below) (106–111). In a large multi-institutional randomized study treating acute GVHD with either steroids alone or in combination with daclizumab GVHD, responses were found to be similar but with a significantly worse 100-day survival in the latter group, leading to early cessation of the study (108). In another study in steroid refractory patients with aGVHD improved 100-day survival was demonstrated when Daclizumab was used in combination with preemptive antimicrobial therapy (111). Daclizumab is administered by vein. Infliximab (Anti-Tumor Necrosis Factor). Infliximab (Anti-TNF) a recombinant humanized monoclonal antibody directed against TNF. It is currently being investigated for its role in the treatment of aGVHD (105,112,113,145–147). Activity has been demonstrated in the context of treating steroid resistant aGVHD with response in two thirds of the patients, especially those with GI aGVHD. There is concern over a possible increase in the number of invasive fungal infections and the use of preemptive antifungal therapy should seriously be considered with its use. It is administered by vein.

Prophylaxis for Acute Graft-Versus-Host Disease No Prophylaxis Prophylaxis to prevent aGVHD is routinely used in unmanipulated stem cell transplants. There have been several initial reports that have compared prophylaxis versus no prophylaxis (148–150). All of these studies contained small numbers of patients. Fifty-nine to one hundred percent of the patients had aGVHD Ograde II. Subsequent studies have shown differences in survival as well (149). Except in the situations where significant T-cell depletion occurs prior to transplant, pharmacologic prophylaxis is required for allogeneic transplant recipients.

Single-Agent Prophylaxis Initial studies relied primarily on a single drug for prophylaxis, utilizing additional drugs for treatment of clinical aGVHD. The use of cytotoxic agents, such as methotrexate, azathioprine, and cyclophosphamide, were mainstays of therapy in the late 60s and early 70s (151). The introduction of cyclosporine in the 1970s provided an alternative to methotrexate and became a foundation upon which to combine additional agents (152,153). A significant decrease in aGVHD was observed when aGVHD prophylaxis with cyclosporine and methotrexate in combination was compared to cyclosporine alone (33–54% vs. 56–70%) (154–156).

Acute Graft-Versus-Host Disease

75

More recently, Tacrolimus has been used as a single agent, especially after transplantation from a matched sibling donor (157). Primary prophylaxis using a single agent generally uses cyclosporine (1.5 mg/kg q 12 hours initial dosing) or tacrolimus (0.015 mg/kg q 12 hours initial dosing), with dose adjustment based on trough levels and the reported normal therapeutic range for the individual laboratory and method.

Combined-Agent Prophylaxis Most potential hematopoietic transplant recipients will not have a matched related donor. Thus, the use of unrelated and less-than-ideally matched related donors has been attempted. In this situation, single agent aGVHD prophylaxis is inadequate and combination prophylaxis regimens have been used. These have typically been built on a foundation of immunosuppression utilizing a T-cell inhibiting agent, such as Tacrolimus or cyclosporine, with the addition of various agents, such as methotrexate, steroids, MMF (98,137,138), Sirolimus (127–130), ATG, and others. Most of the combinations have shown a relative equivalence in the efficacy and overall survival in pediatric patients. Tacrolimus has been used for more than decade for the prophylaxis and treatment of aGVHD in the bone marrow transplant setting (158,159). Although initially used as salvage therapy for steroid- and cyclosporine-resistant cGVHD (160), several large multi-institutional studies have been performed in adults comparing it to cyclosporine in combination with short course methotrexate for aGVHD prophylaxis (161–164). These and other studies showed that tacrolimus prophylaxis was as good as or superior to cyclosporine in reducing the risk of aGVHD in both related and unrelated donor allogeneic transplants. No difference in survival has been clearly demonstrated when cyclosporine or tacrolimus containing regimens are compared. Despite the decrease of GVHD, there was no increase in infection, lymphoproliferative disease, or relapse. Recommended dosing for beginning tacrolimus prophylaxis is 0.03 mg/kg/day (lean body mass) as a continuous infusion by vein beginning one or two days prior to transplant. Serum levels between 5 and 15 ng/ml are targeted to optimize control and minimize toxicity. Toxicity of tacrolimus is similar to that of cyclosporine (94), although hypertension is less common in patients on tacrolimus than cyclosporine. The use of tacrolimus instead of cyclosporine for aGVHD prophylaxis is common in adult transplant programs. Despite the increased use of tacrolimus in the adult population, cyclosporine is still predominantly used as frontline prophylaxis in children, although experience with tacrolimus in this population is increasing. There is a paucity of large pediatric studies comparing cyclosporine and tacrolimus (159,165,166). T-Cell Depletion/CD34D Cell Selection. The use of mismatched donors carries a higher risk of aGVHD. Some investigators have turned to T-cell depletion as a means of preventing aGVHD in the mismatched transplant recipient. Handgretinger et al. (119) reported the use of purified CD34C cells obtained from G-CSF mobilized PBSC’s from mismatched related donors in thirty-nine children with leukemia who lacked an alternate donor. No other aGVHD prophylaxis was used. Patients achieved neutrophil engraftment (ANC O500) at a median of 11 days and three of the patients did not engraft. Two of these patients engrafted after a second transplant when monoclonal anti-CD3 antibody was added to the conditioning regimen. Acute GVHD was minimal in the evaluable patients. Many of the patients subsequently received T-cell “add backs” or donor leukocyte infusions (DLI). Following DLI, aGVHD grade I occurred in two patients, grade II in three, and grade IV in one patient. Fifteen patients were alive and disease-free at a median of two years (1–4.5 years); thirteen patients died of relapsed disease, five of viral infection, two of fungal infection, two from VOD, one patient from nonengraftment and one from nontransplant related causes. Similar experience has been reported by others (120,167,168). Thus, T-cell depletion is effective in preventing aGVHD but is associated with some increased risk of graft failure, disease relapse, and prolonged immunocompromise (which may predispose to opportunistic infection).

76

Moore and Feig

TREATMENT OF ACUTE GRAFT-VERSUS-HOST DISEASE Although aGVHD prophylaxis is geared to minimizing or eliminating aGVHD, 30% to 50% of patients with a matched sibling donor and 50–80% of recipients with unrelated donors may still develop aGVHD of sufficient severity as to require treatment (169–171). Grade I aGVHD requires careful monitoring and adjustment/optimization of the dosing of prophylactic immunosuppression. Appearance of O Grade II aGVHD requires prompt and aggressive therapy. First line treatment of aGVHD is typically with methylprednisolone, although MMF, ATG, and monoclonal antibody therapy may be used (35).

Steroids Primary treatment of aGVHD is started when the grade of disease is OGrade 2. The immunoprophylaxis is usually continued and methylprednisolone therapy is added. A wide range of doses (up to 60 mg/kg/day) have been used, but most practitioners begin with a pulse of 2 mg/kg/day which is subsequently tapered if the aGVHD responds (35,36,112,172,173). The duration of steroid taper has not been found to impact the risk of infection, recurrent aGVHD, or survival (172).

Mycophenolate Mofetil MMF is a potent inhibitor of T-lymphocyte function and has been used for prophylaxis and treatment of aGVHD (139,140). Dosing is generally started at 30–45 mg/kg/day, with the best pharmacokinetics achieved with every 8 hours dosing (141,142).

Sirolimus (Rapamycin) There is a paucity of published information in regards to the use of sirolimus to treat aGVHD. Benito used sirolimus to treat 21 patients with steroid-resistant aGVHD (132). The first four patients were given an initial loading dose of sirolimus (15 mg/M2), with subsequent daily dosing of 5 mg/M2/day for the remainder of a two-week course. Subsequent patients were treated without a loading dose at either 5 (nZ7 patients) or 4 (nZ10 patients) mg/M2/day for a full two weeks. Eighteen of the patients were able to receive greater than 6 doses and 12 patients responded (5 CR and 7 PR). The ultimate role for sirolimus in the management of aGVHD has not yet been established.

Antibody Therapy The use of antibodies for the treatment of aGVHD was designed to provide specificity of therapy and reduction of nonspecific side effects. ATG has been incorporated into conditioning regimens to prevent rejection of the transplanted marrow and has been used as second line therapy for the treatment of steroid resistant aGVHD. Although very effective in its prevention and treatment of aGVHD (99), it may cause a significant serum sickness and profound immunosuppression. With the use of humanized monoclonal antibodies to more specific targets, the potential exists to both improve efficacy and reduce toxicity. Four major classes of monoclonals are currently being developed and tested, including antipan T-lymphocyte antibodies (such as anti-CD2, anti-CD3, anti-CD5, anti-CD8, and anti-CD52) (100–104,144), antiactivated T-lymphocyte antibodies (including anti-CD25 HAT, anti-CD25 BT563 and antiCD25 2A3), antiadhesion molecule antibodies (such as anti-CD11a-antiLFA1), and anticytokine antibodies, such as Infliximab (anti-TNF) and Daclizumab (anti-IL-2Ra) (106).

Acute Graft-Versus-Host Disease

77

The role of these agents in the treatment of aGVHD is under study. Major limitations to their use include the increased risk of infection, low sustained response rates, and a potential increase in the risk of posttransplant lymphoproliferative disease.

Photopheresis In the last decade extracorporeal photochemotherapy (ECP) has been evaluated as a means to treat resistant acute and cGVHD (114–118). This is an in vivo method to inactivate T cells while attempting to minimize the systemic complications that can be experienced with other agents. Essentially, lymphocytes are removed from the patient by leukapheresis. UVA (ultraviolet A) and 8-MOP (8-methoxypsoralen) are used to treat the cells that are in the extracorporeal circuit. UVA activates the 8-MOP, which in turn inactivates T lymphocytes. The half-life of photoactivated 8-MOP is short, so there are few systemic side effects upon reinfusion of the pheresed cells. In a review of 31 studies (147) that explored the use of ECP for the treatment of GVHD, there were 76 patients treated for steroid resistant acute and cGVHD. Of note, only a third of these patients were female. There were 59 patients with skin involvement, 28 with GI involvement, and 47 with liver involvement. Patients were treated from 1 to 24 months with mixed results. A complete response of the skin manifestations of GVHD was observed in two thirds of the patients, with 83% showing at least partial regression. Half of the patients with GI involvement demonstrated a complete response of their symptoms. Approximately 38% of patients with liver GVHD had complete regression. Patient survival was 53% overall.

REFERENCES 1. Gatti RA, Meuwissen HF, Allen HD, Hong R, Good RA. Immunological reconstitution of sex-linked lymphopenic immunological deficiency. Lancet 1968; 2:1366–1369. 2. Iwasaki T. Recent advances in the treatment of GVHD. Clin Med Res 2004; 2:243–252. 3. Jaksch M, Mattson J. The pathophysiology of acute GVHD. Scand J Immunol 2005; 61:398–409. 4. Antin JH, Ferrara JL. Cytokine dysregulation and acute graft-versus-host disease. Blood 1992; 80:2964–2968. 5. Ferrara JL, Cooke KR, Teshima T. The pathophysiology of acute GVHD. Int J Hematol 2003; 78:181–187. 6. Krenger W, Hill GR, Ferrara JL. Cytokine cascades in acute graft-versus-host disease. Transplantation 1997; 64:553–558. 7. Xun CQ, Thompson JS, Jennings CD, Brown SA, Widmer MB. Effect of total body irradiation, busulfan-cyclophosphamide, or cyclophosphamide conditioning on inflammatory cytokine release and development of acute graft-versus-host disease in H-2-incompatible transplanted SCID mice. Blood 1994; 83:2360–2367. 8. Cooke KR, Gerbitz A, Crawford JM. LPS antagonism reduces graft-versus-host disease and preserves graft-versus-leukemia activity after experimental bone marrow transplantation. J Clin Invest 2001; 107:1581–1589. 9. Hill GR, Cooke KR, Teshima T. Interleukin-11 promotes T cell polarization and prevents acute graft-versus-host disease after allogeneic bone marrow transplantation. J Clin Invest 1998; 2:115–123. 10. Panoskaltsis-Mortari A, Lacey DL, Vallera DA, Blazar BR. Keratinocyte growth factor administered before conditioning ameliorates graft-versus-host disease after allogeneic bone marrow transplantation in mice. Blood 1998; 92:3960–3967. 11. Clouthier SG, Cooke KR, Teshima T. Repifermin (keratinocyte growth factor-2) reduces the severity of graft-versus-host disease while preserving a graft versus leukemia effect. Biol Blood Marrow Transplant 2003; 9:592–603. 12. Antin JH, Lee SJ, Neuberg D. A phase I/II double blind, placebo controlled study of recombinant human interleukin-11 for mucositis and acute graft-versus-host disease prevention in allogeneic stem cell transplantation. Bone Marrow Transplant 2002; 29:373–377.

78

Moore and Feig

13. Spielberger R, Stiff P, Bensinger W, et al. Palifermin for oral mucositis after intensive therapy for hematologic cancers. N Engl J Med 2004; 351:2590–2598. 14. Krenger W, Ferrara JLM. Dysregulation of cytokines during graft-versus-host disease. J Hematother 1996; 5:3–14. 15. Shenoy S, Mohanakumar T, Todd G, et al. Immune reconstitution following allogeneic peripheral blood stem cell transplants. Bone Marrow Transplant 1999; 23:335–346. 16. Fukushi N, Arase H, Wang B, et al. Thymus: a direct target tissue in graft-versus-host reaction after allogeneic bone marrow transplantation that results in abrogation of induction of self-tolerance. Proc Natl Acad Sci USA 1990; 87:6301–6305. 17. Weinberg K, Blazar BR, Wagner JE, et al. Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation. Blood 2001; 97:1458–1466. 18. Teshima T, Ferrara JL. Understanding the alloresponse: new approaches to graft-versus-host disease prevention. Semin Hematol 2002; 39:15–22. 19. Nikolic B, Lee S, Bronson RT, Grunsby SJ, Sykes M. Th1 and Th2 mediate acute graft-versus-host disease, each with distinct end-organ targets. J Clin Invest 2000; 105:1289–1298. 20. Hashimoto D, Aakura S, Miyake A, et al. Stimulation of host NKT cells by synthetic glycolipid regulates acute graft-versus-host disease ny inducing Th2 polarization of donor T cells. J Immunol 2005; 174:551–556. 21. Janeway CA, Jr., Medzhitov R. Innate immune recognition. Ann Rev Immunol 2002; 20:197–216. 22. Zhang Y, Shlomchik WD, Joe G. APC’s in the liver and spleen recruit activated allogeneic CD8CT cells to elicit hepatic graft-versus-host disease. J Immunol 2002; 169:7111–7118. 23. Murai M, Yoneyama H, Ezaki T. Peyer’s patch is the essential site in initiating murine acute and lethal graft-versus-host reaction. Nat Immunol 2003; 4:154–160. 24. Sayegh MH, Turka LA. The role of T-cell costimulatory activation pathways in transplant rejection. N Engl J Med 1998; 338:1813–1821. 25. Murphy WJ, Koh CY, Raziuddin A, Bennett M, Longo DL. Immunobiology of natural killer cells and bone marrow transplantation: merging of basic and preclinical studies. Immunol Rev 2001; 181:279–289. 26. Klingemann HG. Relevance and potential of natural killer cells in stem cell transplantation. Biol Blood Marrow Transplant 2000; 6:90–99. 27. Ruggeri L, Capanni M, Martelli MF, Velardi A. Cellular therapy: exploiting NK cell alloreactivity in transplantation. Curr Opin Hematol 2001; 8:355–359. 28. Ruggeri L, Capanni M, Urbani E. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295:2097–2100. 29. Asai O, Longo DL, Tian ZG. Suppression of graft-versus-host disease and amplification of graftversus-tumor effects by activated natural killer cells after allogeneic bone marrow transplantation. J Clin Invest 1998; 101:1835–1842. 30. Taylor PA, Lees CJ, Blazar BR. The infusion of ex vivo activated and expanded CD4CCD25C immune regulatory cells inhibits graft-versus-host disease lethality. Blood 2002; 99:3493–3499. 31. Ermann J, Hoffmannn P, Edinger M. Only the CD62LC subpopulation of CD4CCD25C regulatory T cells protects from lethal acute GVHD. Blood 2005; 105:2220–2226. 32. Young KJ, DuTemple B, Phillips MJ, Zhang L. Inhibition of graft-versus-host disease by double negative regulatory T cells. J Immunol 2003; 171:134–141. 33. Weiden PL, Fluornoy N, Thomas ED, et al. Antileukemic effect of graft-versus-host disease in human recipients of allogeneic marrow grafts. NEJM 1979; 300:1068–1073. 34. Chao NJ. 2nd ed. Graft-versus-Host Disease. Austin: R.G. Landes Company, 1999. 35. Goker H, Hazenedaroglu IC, Chao NJ. Acute graft-versus-host disease: pathobiology and management. Exp Hematol 2001; 29:259–277. 36. Ferrara JLM, Levy R, Chao NJ. Pathophysiologic mechanisms of acute graft-vs.-host disease. Biol Blood Marrow Transplant 1999; 5:347–356. 37. Blume KG, Forman SJ, Appelbaum FR, Eds. 3rd ed. Thomas’ Hematopoietic Cell Transplantation. Malden, MA: Blackwell Science Ltd, 2004. 38. Glucksberg H, Storb R, Fefer A, et al. Clinical manifestation of graft-versus-host disease in human recipients of marrow from HLA-matched sibling donors. Transplantation 1974; 18:295–304. 39. Przepiorka D, Weisdorf D, Klingemann HG, Beatty P, Hows J, Thomas ED. 1994 Consensus conference on acute GVHD grading. Bone Marrow Transplant 1995; 15:825–828. 40. National Institutes of Health, National Cancer Institute, Common Toxicity Criteria. Version 3.0, 2003; pages 15, 20, 34.

Acute Graft-Versus-Host Disease

79

41. Thomas ED, Storb R, Clift RA, et al. Bone marrow transplantation. N Engl J Med 1975; 292:832–843. 42. Jack MK, Jack GM, Sale GE, Shulman HM, Sullivan KM. Ocular manifestations of graft-vs-host disease. Arch Ophthalmol 1983; 101:1080–1084. 43. Thein SL, Goldman JM, Galton DAG. Acute “graft-versus-host disease” after autografting for chronic granulocytic leukemia in transformation. Ann Intern Med 1981; 94:210–211. 44. Hess AD, Horowitz L, Beschorner WE, Santos GW. Development of graft-versus-host disease-like syndrome in cyclosporine-treated rats after syngeneic bone marrow transplantation I. Development of cytotoxic T-lymphocytes with apparent polyclonal anti-Ia specificity, including autoreactivity. J Exp Med 1985; 161:718–730. 45. Fischer AC, Beschorner WE, Hess AD. Requirements for the induction and adoptive transfer of cyclosporine-induced syngeneic graft-versus-host disease. J Exp Med 1989; 169:1031–1041. 46. Wu JM, Bensen-Kennedy D, Miura Y, et al. The effects of interleukin 10 and interferon g cytokine gene polymorphisms on survival after autologous bone marrow transplantation for patients with breast cancer. Biol Blood Marrow Transplant 2005; 11:455–464. 47. Eckardt JR, Roodman GD, Boldt DH, et al. Comparison of engraftment and acute GVHD in patients undergoing cryopreserved or fresh allogeneic BMT. Bone Marrow Transplant 1993; 11:125–131. 48. To LB, Haylock DN, Simmons PJ, Juttner CA. The biology and clinical use of blood stem cells. Blood 1997; 89:2233–2258. 49. Powles R, Mehta J, Kulkarni S, et al. Allogeneic blood and bone-marrow stem-cell transplantation in haematological malignant diseases: a randomized trial. Lancet 2000; 355:1231–1237. 50. Blaise D, Kuentz M, Fortanier C, et al. Randomized trial of bone marrow vs lenograstim-primed blood cell allogeneic transplantation in patients with early-stage leukemia: a report from the societe Francaise de Greffe de Moelle. J Clin Oncol 2000; 18:537–546. 51. Storek J, Gooley T, Siadek M, et al. Allogeneic peripheral blood stem cell transplantation may be associated with a high risk of chronic graft-vs-host disease. Blood 1997; 90:4705–4709. 52. Bensinger WI, Weaver CH, Appelbaum FR, et al. Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor. Blood 1995; 85:1655–1658. 53. Korbling M, Prezpiorka D, Huh YO, et al. Allogeneic blood stem call transplantation for refractory leukemia and lymphoma: potential advantage of blood over marrow allografts. Blood 1995; 85:1659–1665. 54. Vigorito AC, Azvedo WM, Marques JFC, et al. A randomized, prospective comparison of allogeneic bone marrow and peripheral blood progenitor cell transplantation in the treatment of haematological malignancies. Bone Marrow Transplant 1998; 22:1145–1151. 55. Mahmoud HK, Fahmy OA, Kamel A, Kemel M, El-Haddad A, El-Kadi D. Peripheral blood vs. bone marrow as a source for allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant 1999; 24:355–358. 56. Khoury HJ, Loberiza FR, Ringde´n O, et al. Impact of posttransplantation G-CSF on outcomes of allogeneic hematopoietic stem cell transplantation. Blood 2006; 107:1712–1716. 57. Stem Cell Collaborative Group. Allogeneic peripheral blood stem-cell compared with bone marrow transplantation in the management of hematologic malignancies: an individual patient data metaanalysis of nine randomized trials. J Clin Oncol 2005; 23:5074–5087. 58. Nagatoshi Y, Kawano Y, Watanabe T, et al. Hematopoietic and immune recovery after allogeneic peripheral blood stem cell transplantation and bone marrow transplantation in a pediatric population. Pediatr Transplant 2002; 6:319–326. 59. Benito AI, Gonzalez-Vicent M, Garcia F, et al. Allogeneic peripheral blood stem cell transplantation (PBSCT) from HLA-identical sibling donors in children with hematological diseases: a single center pilot study. Bone Marrow Transplant 2001; 28:537–543. 60. Matsubara H, Makimoto A, Takayama J, et al. Possible clinical benefits of the use of peripheral blood stem cells over bone marrow in the allogeneic transplantation setting for the treatment of childhood leukemia. Jpn J Clin Oncol 2001; 31:30–38. 61. Diaz MA, Kanold J, Vicent MG, Halle P, Madero L, Demeocq F. Using peripheral blood progenitor cells (PBPC) for transplantation in pediatric patients: a state of the art review. Bone Marrow Transplant 2000; 26:1291–1298. 62. Watanabe T, Kajiume T, Abe T, et al. Allogeneic peripheral blood stem cell transplantation in children with hematological malignancies from HLA-matched siblings. Med Pediatr Oncol 2000; 34:171–176.

80

Moore and Feig

63. Li CK, Yuen PM, Chik KW, et al. Allogeneic peripheral blood stem cell transplant in children. Med Pediatr Oncol 1998; 30:147–151. 64. Korbling M, Chan KW, Anderlini P, et al. Allogeneic peripheral blood stem cell transplantation using normal patient-related pediatric donors. Bone Marrow Transplant 1996; 18:885–890. 65. Gluckman E, Rocha V, Boyer-Chammard A, et al. Outcome of cord-blood transplantation from related and unrelated donors. Eurocord transplant group and the european blood and marrow transplantation group. N Engl J Med 1997; 337:373–381. 66. Rocha V, Wagner JE, Jr., Sobocinski KA, et al. Graft-versus-host disease in children who have received a cord blood or bone marrow transplant from an HLA-identical sibling. N Engl J Med 2000; 342:1846–1854. 67. Wall DA, Carter SL, Kernan NA, et al. COBLT steering committee. Busulfan/melphalan/antithymocyte globulin followed by unrelated donor COBLT for treatment of infant leukemia and leukemia in young children: the COBLT study experience. Biol Blood Marrow Transplant 2005; 1:637–646. 68. Barker JN, Davies SM, DeFor T, Ramsay NKC, Weisdorf DJ, Wagner JE. Survival after transplantation of unrelated donor cord blood is comparable to that of human leukocyte antigenmatched unrelated donor bone marrow: results of a matched pair analysis. Blood 2001; 97:2957–2961. 69. Wagner JE, Barker JN, DeFor TE, et al. Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34C cell dose and HLA disparity on treatment-related mortality and survival. Blood 2002; 100:1611–1618. 70. Wang XN, Sviland L, Ademokun AJ, et al. Cellular alloreactivity of human cord blood cells detected by T-cell frequency analysis and a human skin explant model. Transplantation 1998; 66:903–909. 71. Macardle PJ, Wheatland L, Zola H. Analysis of the cord blood T lymphocyte response to superantigen. Hum Immunol 1999; 60:127–139. 72. Hromadnikova I, Sedlacek P, Stary J, et al. An in vitro skin explant assay as a predictive assay for graft-versus-host disease in a cohort of pediatric transplants. Pediatr Transplant 2001; 5:258–265. 73. Cohen SBA, Madrigal JA. Immunological and functional differences between cord and peripheral blood. Bone Marrow Transplant 1998; 21:s9–s12. 74. Beatty PG, Clift RA, Mickelson EM, et al. Marrow transplantation from related donors other than HLA-identical siblings. N Engl J Med 1985; 313:765–771. 75. Servida P, Gooley T, Hansen JA, et al. Improved survival of haploidentical related donor marrow transplants mismatched for HLA-A or B versus HLA-DR. Blood 1996; 86:484a. 76. Hansen JA, Gooley TA, Martin PJ, et al. Bone marrow transplants from unrelated donors for patients with chronic myeloid leukemia. N Eng J Med 1998; 338:962–968. 77. Petersdorf EW, Gooley TA, Anasetti C, et al. Optimizing outcome after unrelated marrow transplantation by comprehensive matching of HLA class I and II alleles in the donor and recipient. Blood 1998; 92:3515–3520. 78. Sasazuki T, Juji T, Morishima Y. Effect of matching of class I HLA alleles on clinical outcome after transplantation of hematopoietic stem cells from unrelated donor. N Engl J Med 1998; 339:1177–1185. 79. Leung WH, Turner V, Richardson SL, et al. Effect of class I or class II incompatibility in pediatric marrow transplantation from unrelated and related donors. Hum Immunol 2001; 62:399–407. 80. Petersdorf EW, Longton GM, Anasetti C, et al. The significance of HLA-DRB1 matching on clinical outcome after HLA-A , B, DR identical unrelated donor marrow transplantation. Blood 1995; 86:1606–1613. 81. Goulmy E, Schipper R, Pool J, et al. Mismatches of minor histocompatibility antigens between HLA-identical donors and recipients and the development of graft-versus-host disease after bone marrow transplantation. N Engl J Med 1996; 334:281–285. 82. Martin PJ. How much benefit can be expected from matching for minor antigens in allogeneic marrow transplantation? Bone Marrow Transplant 1997; 20:97–100. 83. Bross DS, Tutschka PJ, Farmer ER, et al. Predictive factors for acute graft-versus-host disease in patients transplanted with HLA-identical bone marrow. Blood 1984; 63:1265–1270. 84. Weisdorf D, Hakke R, Blazer B, et al. Risk factors for acute graft-versus-host disease in histocompatible donor bone marrow transplantation. Transplantation 1991; 51:1197–1203. 85. Nash RA, Pepe MS, Storb R, et al. Acute graft-versus-host disease: analysis of risk factors after allogeneic marrow transplantation and prophylaxis with cyclosporine and methotrexate. Blood 1992; 80:1838–1845.

Acute Graft-Versus-Host Disease

81

86. Nademanee A, Schmidt GM, Parker P, et al. The outcome of matched unrelated donor bone marrow transplantation in patients with hematologic malignancies using molecular typing for donor selection and graft-versus-host disease prophylaxis regimen of cyclosporine, methotrexate and prednisone. Blood 1995; 86:1228–1234. 87. Grundy JE, Shanley JD, Shearer GM. Augmentation of graft-versus-host reaction by cytomegalovirus infection resulting in interstitial pneumonitis. Transplantation 1985; 39:548–553. 88. Gratama JW, Zwaan FE, Stijnen T, et al. Herpes-virus immunity and acute graft-versus-host disease. Lancet 1987; 1:471–474. 89. Petz LD, Yam P, Wallace RB, et al. Mixed hematopoietic chimerism following bone marrow transplantation for hematologic malignancies. Blood 1987; 70:1331–1337. 90. Clift RA, Buckner CD, Appelbaum FR, et al. Allogeneic marrow transplantation in patients with acute myeloid leukemia in first remission: a randomized trial of two irradiation regimens. Blood 1990; 76:1867–1871. 91. Yee G, Slef SG, McGuire TR, Carlin JC, Sanders JE, Deeg HJ. Serum cyclosporine concentration and risk of acute graft-versus-host disease after allogeneic marrow transplantation. N Engl J Med 1988; 319:65–70. 92. Przepiorka D, Kernan NA, Ippoliti C, et al. Daclizumab, a humanized anti-interleukin-2 receptor alpha chain antibody, for treatment of acute graft-versus-host disease. Blood 2000; 95:83–89. 93. Murray L. Physician’s Desk Reference. 60th ed. Montvale, NJ: Thomson PDR, 2006; pp. 2110, 3216, 3319. 94. Woo MH, Przepiorka D, Ippoliti C, et al. Toxicities of tacrolimus and cyclosporine after allogeneic blood cell transplantation. Bone Marrow Transplant 1997; 20:1095–1098. 95. Davidson RJ, Marley SB, Gordon MY. Rapamycin for GVHD prophylaxis-potential for severe myelotoxicity. Bone Marrow Transplant 2001; 27:115. 96. Groth CG, Backman L, Morales JM, et al. Sirolimus (rapamycin)-based therapy in human renal transplantation. Transplantation 1999; 67:1036–1042. 97. European Mycophenolate Mofetil Cooperative Study Group. Placebo-controlled study of mycophenolate mofetil combined with cyclosporin and corticosteroids for prevention of acute rejection. Lancet 1995; 345:1321–1325. 98. Basara N, Blau WI, Kiehl MG, et al. Mycophenolate mofetil for the prophylaxis of acute GVHD in HLA-mismatched bone marrow transplant patients. Clin Transplant 2000; 14:121–126. 99. Deeg HJ, Loughran TP, Storb R, et al. Treatment of human acute graft-versus-host disease with antithymocyte globulin and cyclosporine with or without methylprednisolone. Transplantation 1985; 40:162–166. 100. Jani CR, Walsh W, Emmons R, Alzoubi A. Review of alemtuzumab (campath-1H) related adverse events and a case of gas gangrene after its use. Blood 2002; 100:362b. 101. Carrella AM, Beltrami G, Scalzulli PR. Altemtuzumab can successfully treat steroid-refractory acute graft-versus-host disease. Bone Marrow Transplant 2004; 33:131–132. 102. Claxton DF, Kopp M, Mierski J, Ehmann WC, Rybka W. Campath-1H allows non-myeloablative conditioning for allogeneic stem cell transplantation. Blood 2002; 100:430a. 103. Tauro S, Piper K, Cobbold M, et al. The use of in vivo CAMPATH-1H is associated with persistent defects in humoral immunity after reduced intensity conditioned sibling allogeneic stem cell transplantation despite enhanced reconstitution of marrow CD20CB-cells. Blood 2002; 100:422a. 104. Khouri IF, Keating MJ, Saliba RM, et al. Mini-dose Campath-1H in-vivo induces significant reduction of GVHD associated with high dose chemotherapy and allogeneic transplantation (ASCT). Blood 2002; 100:423a. 105. Rai KR, Keating MJ, Coutre S, Rizzeri DA. Patients with refractory B-CLL and T-PLL treated with alemtuzumab (campath) on a compassionate basis. A report on efficacy and safety of CAM 511 trial. Blood 2002; 100:802a. 106. Wolff D, Roessler V, Steiner B, et al. Treatment of steroid resistant acute GVHD with daclizumab and etanercept. Bone Marrow Transplant 2005; 35:1003–1010. 107. Jacobsohn DA, Vogelsang GB. Anticytokine therapy for the treatment of graft-versus-host disease. Curr Pharm Des 2004; 10:1195–1205. 108. Lee SJ, Zhrich D, Agura E, et al. Effect of up front daclizumab when combined with steroids for the treatment of acute graft-versus-host disease: results of a randomized trial. Blood 2004; 104:155901564. 109. Soumelis V, Delarue R, Benettaib B, Hirsch I, Varet B, Buzyn A. Prolonged administration of anti-interleukin-2 receptor monoclonal antibody therapy for severe steroid-refractory acute graft-versus-host disease: preliminary report of a pilot study. Blood 2002; 100:444b.

82

Moore and Feig

110. Maciejewski JP, Nunez O, Sloand EM, Young NS. Recombinant-humanized-anti-IL2-receptor antibody (daclizumab) produces responses inpatients with moderate aplastic anemia. Blood 2002; 100:233a. 111. Srinivasan R, Chakrabarti S, Walsh T, et al. Improved survival in steroid-refractory aGVHD after non-myeloablative allogeneic transplant using a daclizumab based strategy with comprehensive infectious prophylaxis. Br J Hematol 2004; 124:777–786. 112. Srinivasan R, Geller N, Chakrabarti S, et al. High response rate and improved survival in patients with steroid-refractory aGVHD treated with daclizumab with or without infliximab. Blood 2002; 100:173a–174a. 113. Marty FM, Lee S, Fahey M, et al. Infliximab use in patients with severe GVHD and other emerging risk factors of non-candida invasive fungal infections in allogeneic hematopoietic stem cell transplant recipients: a cohort study. Blood 2003; 102:2768–2776. 114. Dall’Amico R, Messina C. Extracorporeal Photochemotherapy for the treatment of graft-versushost disease. Ther Apher 2002;296–304. 115. Salvaneschi L, Perotti C, Zecca M, Locatelli F, et al. Extra-corporeal photochemotherapy for treatment of acute and chronic graft-versus-host disease in childhood. Transfusion 2001; 1:1299–1305. 116. Dall’Amico R, Rossetti F, Zulian F, et al. Photopheresis in pediatric patients with drug-resistant chronic graft-versus-host disease. Br J Haematol 1997; 97:848–854. 117. Greinix TH, Volc-Platzer B, Kalhs P, et al. Extracorporeal photochemotherapy in the treatment of severe steroid refractory acute graft-versus-host disease: a pilot study. Blood 2000; 96:2426–2431. 118. Smith EP, Sniecinski I, Dagis AC, et al. Extracorporeal photochemotherapy for the treatment of drug resistant graft-versus-host disease. Biol Blood Marrow Transplant 1998;27–37. 119. Handgretinger R, Klingbiel T, Lang P, et al. Megadose transplantation of purified peripheral blood CD34C progenitor cells from HLA-mismatched parental donors in children. Bone Marrow Transplant 2001; 27:777–783. 120. Lang P, Handgretinger R, Niethammer D, et al. Transplantation of highly purified CD34C progenitor cells from unrelated donors in pediatric leukemia. Blood 2003; 101:1630–1636. 121. Bolamos-Meade J, Jacobsohn D, Margolis J. Pentostatin in steroid resistant acute graft-versus-host disease. Blood 2002; 100:420A. 122. Kendra J, Barrett AJ, Lucas C, et al. Response of graft versus host disease to high doses of methylprednisolone. Clin Lab Haematol 1981; 3:19–26. 123. Yoshimura N, Matsui S, Hamashima T, Oka T. Effect of a new immunosuppressive agent, FK506, on human lymphocyte responses in vitro. Inhibition of the production of IL-2 and gamma-IFN, but not B cell-stimulating factor 2. Transplantation 1989; 47:356–359. 124. Blazer BR, Taylor PA, Panoskaltsis-Mortari A, Vallera DA. Rapamycin inhibits the generation of graft-versus-host disease- and graft-versus-leukemia-causing T cells by interfering with the production of Th1 or Th2 cytotoxic cytokines. J Immunol 1998; 160:5355–5365. 125. Homma M, Damoiseaux JGMC, van Breda Vriesman PJC. Differential effects of cyclosporin-A and rapamycin on in vivo thymocyte maturation. Transplant Proc 1997; 29:1743–1744. 126. Bachar-Lustig E, Reich-Zeliger S, Reisner Y. Synergism between rapamycin and host-non-reactive veto CTLs in murine models for BM allograft rejection: a new approach to facilitate engraftment of T-cell depleted allogeneic BMT under reduced intensity conditioning. Blood 2002; 100:211a–212a. 127. Coenen JJA, Koenen HJPM, van Rijssen E, Hilbrands LB, Joosten I. Rapamycin, and not cyclosporine A, preserves the highly suppressive CD27C subset of human CD4CCD25C regulatory T cells. Blood 2006; 107:1018–1023. 128. Brown VI, Fang J, Alcorn K, et al. Rapamycin is active against B precursor leukemia in vitro and in vivo, an effect that is modulated by IL-7 mediated signaling. PNAS 2003; 100:15113–15118. 129. Antin JH, Edwin AP, Kim H, et al. Combination sirolimus, tacrolimus and methotrexate to prevent GVHD after unrelated donor or mismatched family member marrow transplantation. Blood 2002; 100:174a–175a. 130. Popescu D, Kopp M, Ehmann C, Rybka W, Claxton DF. Engraftment of haploidentical or alternative donor stem cells after non-myeloablative conditioning with combination sirolimus and tacrolimus immunoprophylaxis in elderly and poor prognosis patients. Blood 2002; 100:637a. 131. Couriel DR, Hicks K, Saliba R, et al. Sirolimus (rapamycin) for the treatment of steroid refractory chronic graft versus host disease. Blood 2002; 100:847a. 132. Benito AI, Furlong T, Martin PJ, et al. Sirolimus (rapamycin) for the treatment of steroid-refractory acute graft-versus-host disease. Transplantation 2001; 72:1924–1929.

Acute Graft-Versus-Host Disease

83

133. Cutler C, Kim HT, Hochberg E, et al. Sirolimus and tacrolimus without methotrexate as graftversus-host disease prophylaxis after matched related donor peripheral blood stem cell transplantation. Biol Blood Marrow Transplant 2004; 10:328–336. 134. Antin JH, Kim HT, Cutler C, et al. Sirolimus, tacrolimus, and low-dose methotrexate for graftversus-host disease prophylaxis in mismatched related donor or unrelated donor transplantation. Blood 2003; 102:1601–1605. 135. Ettinger RB. New immunosuppresive agents in pediatric renal transplantation. Transplant Proc 1998; 30:1956–1958. 136. Allison AC, Eugui EM. Purine metabolism and immunosuppressive effects of mycophenolate mofetil (MMF). Clin Transplant 1996; 10:77–84. 137. McSweeney PA, Abhyankar S, Becker C, et al. Low incidence of early transplant mortaltity using tacrolimus and mycophenolate mofetil for GVHD prevention after conventional allografting. Blood 2002; 100:419a. 138. Pavletic SZ, Abhyankar S, Foran JC, et al. Tacrolimus and mycophenolate mofetil are superior to cyclosporine and methotrexate for GVHD prevention after HLA-matched related donor hematopoietic cell transplantation. Blood 2002; 100:445b. 139. Basara N, Blau W, Romer E. Mycophenolate mofetil for the treatment of acute and cGVHD in bone marrow transplant patients. Blood 1997; 90:105a. 140. Basara N, Blau WI, Kiehl MG, et al. Efficacy and safety of mycophenolate mofetil for the treatment of acute and cGVHD in bone marrow transplant recipient. Transplant Proc 1998; 30:4087–4089. 141. Giaconne L, Maris MB, Sandmeier BM, et al. Mycophenolic acid (MPA) plasma levels in patients following non-myeloablative HLA-matched unrelated donor hematopoietic cell transplantation (NMURD-HCT). Blood 2002; 100:840a–841a. 142. Jacobson PA, Mifek J, Rogoshske J, et al. Highly variable mycophenolate pharmacokinetics in hematopoietic stem cell transplantation (HSCT): potential need for clinical drug monitoring. Blood 2002; 100:411a. 143. Bessmertny O, Harrison L, Osunkwo I, et al. Significant alterations of mycophenolate mofetil (MMF) pharmacokinetics in pediatric allogeneic stem cell transplant (AlloSCT) recipients: alternate dosing is required in AlloSCT compared to solid organ autograft recipients. Blood 2002; 100:418a. 144. Tse JC, Moore TB. Monoclonal antibodies in the treatment of steroid resistant acute graft-vs-host disease. Pharmacotherapy 1998; 18:988–1000. 145. Trenschel R, Ditchskowski M, Biersack H, et al. Infliximab as treatment for steroid refractory acute and chronic graft-versus-host disease. Blood 2002; 100:450b. 146. Jacobsohn DA, Hallick J, Andres V, McMillan S, Morris L, Vogelsang GB. Infliximab for steroid refractory acute GVHD: a case series. Am J Hematol 2003; 74:119–124. 147. Couriel D, Saliba R, Hicks K, et al. Tumor necrosis factor-alpha blockade for the treatment of acute GVHD. Blood 2004; 104:649–654. 148. Lazarus H, Coccia PF, Herzig RH, et al. Incidence of acute graft-versus-host disease with and without methotrexate prophylaxis in allogeneic bone marrow transplantation. Blood 1984; 64:215–220. 149. Sullivan K, Deeg HJ, Sanders J, et al. Hyperacute graft-v-host disease in patients not given immunosuppression after allogeneic marrow transplantation. Blood 1986; 67:1172–1175. 150. Elfenbein G, Goedect T, Graham-Poole J, Skoda-Smith S, Gross S, Weiner R. Is prophylaxis against acute graft-versus-host disease necessary if treatment is effective and survival is not impaired? Proc Am Soc Clin Oncol 1986; 5:643. 151. Storb R, Epstein RB, Graham TC, Thomas ED. Methotrexate regimens for control of graft-versushost disease in dogs with allogeneic marrow grafts. Transplantation 1970; 9:240–246. 152. Powles RL, Clink HM, Spence D, et al. Cyclosporine A to prevent graft-versus-host disease in man after allogeneic bone marrow transplantation. Lancet 1980; 1:327–329. 153. Tutschka P, Beschorner WE, Hess AD, Santos GW. Cyclosporine to prevent graft-versus-host disease: a pilot study in 22 patients receiving allogeneic marrow transplant. Blood 1983; 61:318–325. 154. Deeg HJ, Storb R, Thomas ED, et al. Cyclosporine as prophylaxis for graft-versus-host disease: a randomized study in patients undergoing marrow transplantation for acute nonlymphocytic leukemia. Blood 1985; 65:1325–1334. 155. Santos G, Broxmeyer R, Saral R, Tutschka PJ. Cyclosporine (CsA) versus cyclophosphamide: prevention of graft-versus-host disease (GVHD). Exp Hematol 1985; 13:427.

84

Moore and Feig

156. Storb R, Deeg HJ, Fisher LD, et al. Cyclosporine versus methotrexate for graft-versus-host disease prevention in patients given marrow grafts for leukemia: long-term follow-up of three controlled trials. Blood 1988; 71:293–298. 157. Nash RA, Etzioni R, Storb RF, et al. Tacrolimus (FK-506) alone or in combination with methotrexate or methylprednisolone for the prevention of acute graft-versus-host disease after marrow transplantation from HLA-matched siblings: a single-center study. Blood 1995; 85:3514–3519. 158. Przepiorka D, Devine SM, Fay JW, Uberti J, Wingard J. Practical considerations in the use of tacrolimus for allogeneic marrow transplantation. Bone Marrow Transplant 1999; 24:1053–1056. 159. Przepiorka D, Petropoulos D, Mullen CA, Danielson M, Mattewada V, Chan KW. Tacrolimus for the prevention of graft-versus-host disease after mismatched cord blood transplantation. Bone Marrow Transplant 1999; 23:1291–1295. 160. Fung JJ, Tzakis A, Przepiorka D. FK506 for the treatment of resistant liver involvement in chronic graft-versus-host disease: a pilot study. Blood 1990; 76:539a. 161. Ratanatharathorn V, Nash RA, Przepiorka D. Phase III sstudy comparing methotrexate and tacrolimus (prograf, FK506) with methotrexate and cyclosporine for graft-versus-host disease prophylaxis after sibling bone marrow transplantation. Blood 1997; 92:2303–2314. 162. Nash RA, Antin JH, Karanes C. A phase III study comparing methotrexate and tacrolimus with methotrexate and cyclosporine for prophylaxis of acute graft-versus-host disease after marrow transplantation from unrelated donors. Blood 1997; 90:561a. 163. Japanese FK506 BMT Study Group. Hiraoka AF. Results of a phase III study on prophylactic use of FK506 for acute GVHD compared with cyclosporine in allogeneic bone marrow transplantation. Blood 1997; 90:561a. 164. Fay JW, Wingard JR, Antin JH, et al. FK-506 (tacrolimus) monotherapy for prevention of graftversus-host disease after histocompatible sibling allogeneic bone marrow transplantation. Blood 1996; 87:3514–3519. 165. Koehler MT, Howrie D, Mirro J, et al. FK-506 (tacrolimus) in the treatment of steroid-resistant acute graft-versus-host disease in children undergoing bone marrow transplantation. Bone Marrow Transplant 1995; 15:895–899. 166. Yanik G, Levine JE, Ratanatharathorn V, Dunn R, Ferrara J, Hutchinson RJ. Tacrolimus (FK506) and methotrexate as prophylaxis for acute graft-versus-host disease in pediatric allogeneic stem cell transplantation. Bone Marrow Transplant 2000; 26:161–167. 167. Aversa F, Terenzi A, Felicini R. Mismatched T cell depleted hematopoietic stem cell transplantation for children with high risk leukemia. Bone Marrow Transplant 1998; 5:S29–S32. 168. Schlegel PG, Eyrich M, Bader P, et al. OKT-3-based reconditioning regimen for early graft failure in HLA-non-identical stem cell transplants. Br J Haematol 2000; 111:668–673. 169. Gale RP, Bortin MM, van Bekkum DW, et al. Risk factors for acute graft-versus-host disease. Br J Haematol 1987; 67:397–406. 170. Beatty PG, Hansen JA, Longton GM, et al. Marrow transplantation from HLA-matched unrelated donors for treatment of hematologic malignancies. Transplantation 1991; 51:443–447. 171. Beatty PG. Results of allogeneic bone marrow transplantation with unrelated or mismatched donors. Semin Oncol 1992; 19:13–19. 172. Hings IM, Filipovich AH, Miller WJ, et al. Prednisone therapy for acute graft-versus-host disease: short-versus long-term treatment. A prospective randomized trial. Transplantation 1993; 56:577–580. 173. Deeg HJ, Henslee-Downey PJ. Management of acute graft-versus-host disease. Bone Marrow Transplant 1990; 6:1–8.

4 Chronic Graft-Versus-Host Disease in Children David A. Jacobsohn Stem Cell Transplant Program, Children’s Memorial Hospital, Northwestern University Feinberg School of Medicine, Chicago, Illinois, U.S.A.

Georgia B. Vogelsang Kimmel Comprehensive Cancer Center of Johns Hopkins, Johns Hopkins University, Baltimore, Maryland, U.S.A.

Kirk R. Schultz Division of Hematology/Oncology, Blood and Marrow Transplantation Program, British Columbia Children’s Hospital, University of British Columbia, Vancouver, British Columbia, Canada

OVERVIEW OF THE BIOLOGY OF CHRONIC GRAFT-VERSUS-HOST DISEASE Chronic graft-versus-host disease (GVHD) is a complex disease, probably most similar in biology to the autoimmune disease Systemic Lupus Erythematosus with the major exception that alloreactive donor-derived T cells induce the disease. GVHD results from alloreactivity of donor T lymphocytes against recipient cells. T lymphocytes recognize peptide antigens in the context of human leukocyte antigen (HLA) molecules. Exogenous proteins are endocytosed by antigen-presenting cells (APC) and transported to lysosomes where acid hydrolases cleave the protein into peptides. These peptides are then loaded into HLA class II molecules and transported to the cell surface for presentation to CD4CT lymphocytes. Endogenous antigens are processed and presented using similar end major histocompatibility complex (MHC) class II pathways (1). Endogenous peptides associate with class I HLA molecules in the Golgi apparatus of APC and are presented to CD8CT lymphocytes (2–6). Alloreactivity also requires costimulatory signals from APC, including the secretion of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a) (7).

Role of T Cells in Chronic Graft-Versus-Host Disease Helper CD4CT cells play a major role in induction and maintenance of chronic GVHD. In contrast to the acute GVHD mouse models, which have correlated relatively closely with human acute GVHD, the chronic GVHD mouse models have many times been less enlightening. This may be due to the higher level of complexity and heterogeneity of chronic GVHD. Even in humans, the presence of alloreactive T cells post-BMT do not necessarily 85

86

Jacobsohn et al.

correlate with chronic GVHD but do with acute GVHD (8). Severe chronic GVHD is associated with the presence of preponderance of CD4C effector memory cells compared to central memory cells. Effector memory CD4CT cells are defined as being CCR7K/CD62Llow with CD4CT cells divided into three groups: (a) naı¨ve (CD45RAC/CCR7C), (b) central memory (CD45RAK/CCR7C) and (c) effector memory (CD45RAK/CCR7K). Whereas central memory CD4CT cells are not polarized [can be either type 1 helper T cells (Th1) or type 2 helper T cells (Th2)], effector memory CD4CT cells produce high levels of IFN-g, IL-4, IL-5 and moderate levels of IL-2 (9). This is in contrast to that seen in chronic GVHD mouse models in which a naı¨ve subset of alloreactive CD4CT cells (CD45RChigh) are able to induce chronic GVHD but in which memory (CD45RClow) cannot (10). Other adhesion molecules expressed on CD4CT cells have recently been described in patients with GVHD: CD156b, a transmembrane protein, and CD167a, an epithelial tyrosine kinase receptor (11). CD8CT cells appear to be an important effector in chronic GVHD. CD8CT cells infiltrates correlate with microvessel loss in skin of patients with chronic GVHD (12) and increased cytolytic CD8CT cells that are found in intestinal biopsies of patients with chronic GVHD (13). Response to extra corporeal photopheresis as therapy for chronic GVDH has been associated with a decrease in CD8CT cells and an increase in CD4CT cells but the changes were not dramatic (14). The activation marker for both CD4C and CD8CT cells, OX40, (15), is associated as an early marker in both populations for development of chronic GVHD at the time of onset and with clinical response. The role of regulatory T cells is not clear in chronic GVHD. Regulatory T cells are associated with control of autoimmune reactive T cells and murine GVHD models have suggested that they may result in less GVHD post BMT (16). Recently, it has been observed in humans that chronic GVHD is not due to a lack of CD4C/CD25C regulatory T cells (17). Surprisingly, patients with chronic GVHD appeared to have an increased number of CD4C/ CD25CT cells with low levels of CD62L compared to patients without. Murine models also support the conclusion that regulatory T cells do not appear to play a role in chronic GVHD in that infusion of donor cells selected to contain the regulatory T cell fraction could not prevent chronic GVHD (10). Some groups have also evaluated for T cell clonality as a marker for chronic GVHD. Clonal T cells have been isolated out of chronic GVHD myositis patients post-transplantation (18). Others have shown that chronic GVHD patients that have the presence of clonal T cell receptor gamma (TCR-g) rearrangements were better responders to photopheresis than those that were not (19). Such interpretation are complicated by the fact that patients without chronic GVHD have clonal expansion of T cells post BMT that is part of the normal immune reconstitution (20).

Cytokine Profiles of T Cells in Chronic Graft-Versus-Host Disease The cytokine secretion profiles of T cells have been used as the basis for dividing T cells into two distinct populations, known as type 1 and type 2. According to this model, Th1 secrete primarily IL-2, IFN-g, TNF-a, and TNF-b, while Th2 produce IL-4, IL-5, IL-6, IL-10 and IL-13. Both of these cell types are thought to develop from a common precursor T cell, the direction of the differentiation being influenced by the conditions present at the time of activation (21). Cytokine-producing CD8CT cells can be classified as Tc1 and Tc2 and will expand at the same time as CD4CT cell populations (22). The phenotype of the T cell that predominates, Th1 or Th2, dictates the nature of the subsequent immune response. Th1 cells mediate primarily cytotoxic and inflammatory responses, and Th2 cells promote humoral and anti-inflammatory responses. Factors implicated in influencing the type of T cell response that develops include ligand density (23), the type of costimulatory molecules involved (24) and the presence of particular cytokines (IL-4/5 for Th2 induction and IL-12 and IFN-g for Th1 induction) (25). Chronic GVHD involves alloreactive helper and cytotoxic T cells, nonspecific suppressor cells, TNF-a-secreting macrophages, and autoreactive T cells (26,27). Chronic

Chronic Graft-Versus-Host Disease in Children

87

GVHD in humans may involve an alteration in the balance of Th1/Tc1 and Th2/Tc2 populations, with a predominant Th1/Tc1 response that is aberrant, with IFN-g production but not IL-2. Chronic GVHD has been associated with immunophenotypic changes including a relative increase in the number of CD8C cells lacking CD28 (28) and a decrease in NK (CD3K/ CD16C/CD56C) cells (28,29). Altered T cell regulation and reduced numbers of T cells may be due to NKT cells (30). T cells from patients with chronic GVHD are poorly responsive to mitogenic and alloantigenic challenge, suggesting these cells may be naive or have a defect in T cell activation pathway (25,27). Some investigators have suggested that alloreactive effector cells can be identified by constitutive expression of “activation” markers, including CD69, IL-2R, Fas receptor (CD95), and Fas ligand (31–34). Autoimmunity, responsible for at least some of the clinical manifestations of chronic GVHD, may be due to impaired thymic deletion of autoreactive T cells and to altered presentation of cryptic antigens secondary to increased IL-6 production. Cytokines also play a role in disease manifestations, with sclerosis associated with TGF-b, fatigue and wasting associated with TNF-a, and immunodeficiency associated with IL-10 and TGF-b (35).

Th1/Tc1 Versus Th2/Tc2 Murine Graft-Versus-Host Disease Extensive studies of mouse models of GVHD have revealed that the type of helper T cell response correlates with the manifestations of GVHD—Th1 cytokines predominate in acute GVHD models (36,37), whereas Th2 cytokines are prevalent in a chronic GVHD model (38). The two forms of the disease share common beginnings, with recognition of host alloantigens by donor T cells causing IL-2 production (39). This is followed, in both cases, by an increase in Th2 cytokine production, and resultant B cell activation. In cases where acute GVHD develops, by day 7 there is an expansion of CD8C cells and increased IFN-g production (40). The shift to a Th1 cytokine pattern results in the activation of macrophages, with the resultant release of the inflammatory cytokines TNF-a, IL-1b, and IL-6, which are thought to be responsible for the tissue destruction seen in acute GVHD (41). LPS released by bacteria that transgress injured mucosa also stimulate macrophages to a hyperactive response with excessive cytokine release (42). In mice that develop chronic GVHD, there is continued lymphoproliferation, B cell activation, and autoantibody production, in contrast to the reduction in host lymphocyte number and reduced serum antibody levels seen in acute forms of the disease (40). As yet, it is unclear what signals are responsible for dictating the nature of the helper T cell response. One candidate for this role is IL-12, a cytokine produced by antigen-presenting cells, that has been shown to mediate the growth and differentiation of Th1 cells in a variety of disease models. Evidence supporting such a role for IL-12 has been obtained from experiments in which the neutralization of IL-12 by antibody was sufficient to prevent the development of acute GVHD without promoting chronic disease (43). It has recently been shown that IL-12 can act synergistically with the IFN-g-inducing cytokine IL-18 to reduce immunoglobulin production by B cells from chronic GVHD mice, indicating that perhaps more than one cytokine is required to produce the necessary conditions for the development of GVHD (44). A reciprocal role for IL-10 has been postulated, whereby the production of this cytokine inhibits the generation of a Th1 response, and thus inhibits development of Th1-mediated acute GVHD. Unfortunately, despite a clear correlation between cytokine profile and disease, IL-10 therapy was unsuccessful in modulating acute GVHD in mice (40).

Th1/Tc1 Versus Th2/Tc2 in Human Graft-Versus-Host Disease While fewer studies of cytokine expression profiles have been performed in human GVHD patients than in mice, those that have been carried out suggest that dysregulation of cytokine expression may be central to disease progression. In contrast to the murine models, it is not clear that human acute and chronic GVHD correlate as closely with the distinct Th1/Th2mediated responses. The small numbers of patients that have been studied hampers

88

Jacobsohn et al.

interpretation of the data from all of these studies. In the case of acute GVHD, suppression of gene expression of the Th2 cytokines IL-4, IL-10 and IL-13 has been seen in human PBMC. In addition, expression of IL-12, an inducer of Th1 responses, was elevated (45) However, in contrast to the mouse Th1/acute GVHD relationship, IL-2 expression was not increased in the PBMC, suggesting that IL-12 may be mediating effects distinct from its upregulation of the Th1 response. In chronic GVHD in humans, reduced expression of IL-10 and increased IFN-g production has been described in patients with chronic GVHD as compared to healthy individuals and patients without GVHD, indicating a greater role for Th1 cytokines in human chronic GVHD than in mouse (46). This finding is further supported by the detection of increased IFN-g expression in skin biopsies taken from patients with cutaneous chronic GVHD (45). Transcription of the Th2 cytokines IL-4 and IL-5 was not detected in these skin samples and IL-10 expression was not significantly different from that seen in controls. However, in a separate study Con A-stimulated T cell production of IFN-g was decreased in patients who developed extensive chronic GVHD (47). Some indirect support for a role of Th2 donor cells in the manifestations of human chronic GVHD is demonstrated by the fact that autoantibodies against cytoskeletal proteins (tubulin, actin, myosin) appear to be consistently present in chronic GVHD (48). Overall, it appears that chronic GVHD can present with either Th1/Tc1 or Th2/Tc2 predominance and it is likely that predominance of one or the other may result in different manifestations of chronic GVHD. Thus, intestinal chronic GVHD may present with a Th1/Tc1 profile where as Myasthenia gravis secondary to chronic GVHD may preset with a Th2/Tc2. Studies to elucidate such differences are imperative and critical for improved therapy.

Role of Cytokine Polymorphisms in Chronic Graft-Versus-Host Disease Multiple roles of cytokines in chronic GVHD have been described. Polymorphisms of IL-6 and IFN-g in sibling donor BMTs (49) may play a role in the incidence and severity of chronic GVHD. Patients with the donor IL-6-174GG homozygous genotype that correlates with increased IL-6 production have increased chronic GVHD severity and incidence. In sibling donor transplants, TNF receptor type II receptor 169R allele if homozygous in the donor is associated with an increase in chronic GVHD (50). Increased chronic GVHD may also be associated with recipient IL-10 GG homozygotes and with the recipient IL-1Ra polymorphism, IL-1RN*2 (51).

Role of Antigen-Presenting Cells in Chronic Graft-Versus-Host Disease To develop adequate activation of alloreactive T cell that induce chronic GVHD, antigenpresenting cells are needed to present self-antigens. All three professional antigen-presenting cells: dendritic cells (DCs), B cells and macrophages have been evaluated in their role in chronic GVHD. Although macrophages clearly have a role in acute GVHD and probably play a role in chronic GVHD, there is no definitive evidence that macrophages play a role in chronic GVHD. DCs are the most potent antigens-presenting cell for T cell activation and most investigations have focused on the role of DCs in induction of chronic GVHD. The role of plasmacytoid (interferon alpha producing) DCs in chronic GVHD is mixed. Plasmacytoid DCs have been associated with the presence of chronic GVHD in humans and are of donor origin (52). Some patients without GVHD continued to have recipient DCs. There is good evidence that decreased chronic GVHD is associated with a predominance of plasmacytoid DCs. This conclusion is supported by the fact that extracoporeal photopheresis results in a shift from myeloid DCs to plasmacytoid DCs (53). Others have shown that a lower incidence of chronic GVHD and higher leukemia relapse rate was associated with higher numbers of infused plasmacytoid DCs. This may be due to the ability of plasmacytoid DCs to induce a shift to a Th2/Tc2 response post BMT (54) and increased chronic GVHD after G-CSF stimulated PBPC transplantation decreased numbers of plasmacytoid DCs (55). Some of the conflicting results regarding the role of DCs in chronic GVHD may be due to whether the DCs are of donor or host

Chronic Graft-Versus-Host Disease in Children

89

origin. Unlike in acute GVHD, where host DCs appear to be a major antigen-presenting, both donor plasmacytoid DCs (56) and host DCs (57) have been associated with increased chronic GVHD in humans. Another potential factor that may impact of DCs function is the fact that increased recipient age is associated with higher chronic GVHD (58). DCs from older individuals may be more potent stimulators of alloreactive T cells as seen in an acute GVHD model in mice (59). DCs also play a role in the development of skin GVHD. Whereas CD1aC DCs are not found in skin chronic GVHD (60,61) there is an increase in the number of factor XIIIa positive DCs in the dermis (59). B cells appear to play a role in the development of chronic GVHD as both producers of auto reactive immunoglobulin and antigen-presenting cells. Depletion of recipient B cells as well as depletion of donor B cells play a role in the development of chronic GVHD in mice (62). Anti-CD20 mAb also works as a therapy in human chronic GVHD (63). Interestingly, chronic GVHD in humans is associated with decreased B cell lymphopoiesis and decreased B cell precursors (64). B cell production of auto reactive antibodies is important in the murine chronic GVHD models. A limited CD4C alloreactive T cell repertoire is associated with induction of anergic B cells that could produce IgM but a diverse CD4CT cell repertoire is required for IgG switching. Thus, a diverse CD4CT cell response is needed to maintain a GVHD like response (65). In such models IL-12 plus IL-18 can inhibit both auto reactive antibody production in mice with chronic GVHD and appears to do so by increasing IFN-g production (66). Chronic GVHD, in humans, is associated with diseases due to auto-immune antibody production such as Myasthenia Gravis, but the understanding of the mechanisms behind development of recipient reactive immunoglobulin production, in humans, is limited. Activation of donor-derived alloreactive T cells is dependent on T cell antigenpresenting interactions of co-stimulatory molecules including CD40, CD80 and CD86. Blockade of these molecules have been shown to play a role in acute GVHD in mice and can be used to induce tolerance in alloreactive T cells (67–72). Since lack of adequate co-stimulatory interaction between donor T cells and antigen-presenting cells is critical for development of alloreactive T cells, this has been a major area of research for development of therapy of human GVHD. Adequate interaction of the T cell receptor and MHC molecule are also critical for development of an alloreactive T cells response. Blockade of that interaction has also been shown to be effective in preventing chronic GVHD in mice (73–74). Another set of molecules that may play in important role in induction GVHD are the Toll-like receptors (TLRs) expressed on antigen-presenting cells. Lipopolysaccharide binding to TLR4 on DCs or macrophages appears to be an important mechanism for acute GVHD (75). Interestingly, one of the major mechanisms of action of the 4-aminoquinoline, hydroxychloroquine (HCQ), may be inhibition of TLR9 signaling (76–78). TLR9 signaling appears to be important in chronic GVDH as well (79). Numerous myeloid cell populations including neutrophils, mast cells, and eosinophils also are important effector cells in chronic GVHD. Eosinophils are increased in the gut of patients with chronic intestinal GVHD and correlate with severity (80). It has been suggested that mast cells are important in T cell trafficking and activation in the micro vasculature of patients with chronic GVHD (81).

Antigens Associated with the Development of Chronic Graft-Versus-Host Disease Although the role of T cell in development of chronic GVHD is well established, the antigens recognized by these donor-derived recipient reactive T cells populations are poorly characterized. The T cell response appears to be very heterogeneous and polyclonal. Except in the situation of HLA mismatching of donor and recipient, the minor histocompatibility antigens recognized by donor T cells appear to be highly expressed polymorphisms of normal cellular proteins that differ between the donor and recipient. This is supported by the fact that differences in donor and recipient ethnicity is associated with a higher rate of chronic GVHD

90

Jacobsohn et al.

(57). The HLA molecules may play a role in that a particular HLA molecule will preferentially present a selected repertoire of minor histocompatibility antigens. As an example, HLA-B27 has been correlated with lower incidence of chronic GVHD (82). The best-characterized minor histocompatibility antigens associated with MHC class I and have focused on the H–Y antigen. The increased rate of chronic GVHD associated with female donor into a male recipient appears to predominately be due to donor T cell recognition of the male specific antigen, H–Y, located at the Y chromosome (83–84). Evaluation of T cell recognizing H–Y associated with HLA-A2 and HLA-B7 correlates with the presence of chronic GVHD and the number of H–Y reactive T cells decreases after response to GVHD therapy (85). Increase antibodies reactive with donor and H–Y antigen have been shown to be present in patients with chronic GVHD (85–87). A second antigen that appears to be recognized as part of chronic GVHD is HA-1. T cells reactive with this antigen are present in patients with GVHD and decrease when the GVHD is improved after therapy (86). Subsequent studies have only shown a correlation of HA-1 mismatch with acute GVHD and not chronic GVHD (88). One of the problems in demonstrating this correlation may be the fact that the frequency of HA-1 disparity is so rare that larger studies are required before it can be concluded that it does not play a role in chronic GVHD (89). T cell responses post transplants against other minor histocompatibility antigens such as ACC1 restricted to HLA–A*2402 have been measured but are not associated with chronic GVHD (90). CD31 nonidentity with all three codons 125, 563 and 670 is associated with acute GVHD and there is a trend toward chronic GVHD with out conclusive correlation (91). Both need to be confirmed with larger studies. Only one minor histocompatibility antigen has been shown to be restricted to MHC class II. The DBY gene restricted to HLA-DQ5 presentation is recognized by a cytolytic CD4CT cells (92) but has only been isolated out of one patient.

Immunological Differences Between Bone Marrow and Peripheral Blood as Donor Cell Sources When an individual is treated with G–CSF, there is a general expansion in a number of hematopoietic cell populations. It is now well established that there is a higher rate of chronic GVHD after PBPC transplantation (93), possibly due to higher numbers of CD45RO (memory CD4CT cells although there are also higher numbers of CD45RA (naive) ones as well. Others have shown that T cells have a lower activation level after G–CSF stimulated peripheral blood (G–PB) is used as the donor source compared to bone marrow (94). G–PB cells are derived from bone marrow after G–CSF treatment, and one of the major differences between the two sources appears to be that the G–PB are cells that have different adhesion molecule expression, resulting in release from the bone marrow, whereas the remaining bone marrow cells, which have not been released, have not developed this property. Decreased adherence by G–CSF mobilized CD34C PB has been associated with decreased expression of the cell surface lectins, LFA-1 and VLA-4 (95) as well as CXCR4 and SDF-1 (96). These effects on CD34C cells do not directly suggest a mechanistic reason for a difference in the ability of G–CSF stimulated bone marrow (G–BM) to induce GVHD, compared to G–PB. Other effects of G–CSF on G–PB immune populations reveal that, although G–PB have increased numbers of T cells, CD4CT cell precursor evaluations that usually predict the onset of GVHD are not correlated with GVHD in patients receiving G–PB (97). Evaluation of the DC populations in G–PB showed that there is an increased number of the plasmacytoid DC2 that are associated with induction of Th2 type T cell response and tolerance (98,99). An increase in plasmacytoid DCs in G–PB could be associated with an increased rate of chronic GVHD but it is difficult to assign such a relationship since G–CSF stimulated bone marrow has even higher concentrations of plasmacytoid DCs (100) and is associated with a lower rate of chronic GVHD. Mature B cell reconstitution at three months after transplantation from PBPC compared to BM appears to be more rapid (93,101,102), with an increased number of activated B cell post

Chronic Graft-Versus-Host Disease in Children

91

PBPC and increased number of anti-HLA antibodies (103). Further support for a potential role of increased mature B cell reconstitution paying a role in chronic GVHD after G–PB is the fact that G–BM, a donor source that has a lower rate of chronic GVHD, has increased naive B cells compared to G–PB (100). The number of CD34C cells infused has been associated with the rate of chronic GVHD in patients receiving G–PB (104) whereas higher numbers of CD4C or CD8CT cells are not (105). This suggests that immature CD34C DCs may play a significant role in whether patients develop GVHD after G–PB transplantation. More recently, G–CSF treatment has been shown to induce GATA-3, an important transcription factor in induction of Th2 T cells (106); this activation was associated with IL-4 secretion. Interestingly, G–CSF given shortly after BMT increases the incidence of chronic GVHD (107).

Impact of Donor and Recipient Age on the Biology of Chronic Graft-Versus-Host Disease It is well established that a lower age of either the donor or recipient can result in a lower incidence of chronic GVHD. These differences have been hypothesized to be due to a lower exposure of the donor or recipient to viral infections particularly CMV. In fact, clinical correlations have shown that donors or recipient with positivity to CMV are associated with a higher rate of chronic GVHD (108,109). More recently, it has been shown in murine models that recipient age may be related to antigen-presenting cell function with increased function associated with higher recipient or donor age (58). One of the best examples of the impact of donor age on the development of chronic GVDH is the lower rate of chronic GVHD with umbilical cord blood (UCB) transplants. UCB over all has an immature T cell phenotype with increased numbers of naı¨ve T cells that are shifted toward a Th2 phenotype (110). As part of this Th2 bias, neonatal CD8CT cells require exogenous IL-4 to develop into Tc2 cells and, upon activation with anti-CD3/B7, cord blood CD8CT cells co-express CD4 as well as CCR5 and CXCR4. The decreased ability of UCB T cells to activate is associated with a physical linkage of CD26 with CD45RA outside lipid rafts that may attenuate T-cell activation signaling through CD26. This attenuation of CD26 signaling may be the reason for the decreased incidence of GVHD in cord blood transplantation (111). There is good evidence that the Th2 bias of UCB cells is probably not due to an intrinsic T cell defect but rather to particular conditions of Ag presentation at priming. UCB DCs are characteristically immature and as a result inefficient in presentation antigen (112,113). Because of this, UCB DCs appear to bias toward a Th2 response after transplantation (114). B cells are immature in their response with lower expression of CD62L and CCR7 on cord blood B cells than on adult B cells, suggesting possible homing defects (115). Cord blood B cells have empty HLA–DR molecules suggesting that they are inefficient antigen-presenting cells (116). Overall, the decreased incidence of chronic GVHD seen with UCB transplantation is probably due to both ineffective T cell activation and a shift to a Th2/Tc2 response due to shifted antigenpresentation by DCs and B cells resulting in increased T cell tolerance.

CHRONIC GRAFT-VERSUS-HOST DISEASE—INCIDENCE AND RISK FACTORS IN CHILDREN The incidence of chronic GVHD is generally lower in children than adults. Results of different stem cell sources are always compared to matched-sibling bone marrow, which was the only source available for much of the history of transplantation. In a recent publication of registry data from the IBMTR, the three-year cumulative incidence of chronic GVHD in 2052 children that received HLA-matched sibling bone marrow transplants was 15%. All of these transplants were myeloablative and roughly one third of these children had received a total-body irradiation (TBI-based) regimen and the rest a chemotherapy-only regimen. Two-thirds of the

92

Jacobsohn et al.

patients had malignant disease and the vast majority received cyclosporine-based GVHD prophylaxis. Factors associated with less chronic GVHD were younger age, nonmalignant disease, and the use of methotrexate with cyclosporine (117). These results are very consistent with previous reports. Given that few patients have the luxury of having an HLA-matched sibling, centers then turned to using HLA-matched unrelated donor marrow as a stem cell source. The use of unrelated donor transplants is still associated with significant morbidity from chronic GVHD in children. Seattle recently published their results on unrelated-donor marrow transplantation for ALL. All patients received the same preparative regimen with cyclophosphamide and TBI and the vast majority received cyclosporine with methotrexate for GVHD prophylaxis. Of 88 patients, chronic GVHD was evaluable in 62 who survived past day 80. Extensive chronic GVHD developed in 29 patients (47%) and limited chronic GVHD was observed in 7 patients (11%) (118). No factors could be identified to be associated with the risk of chronic extensive GVHD, likely due to the similarity of the patients and how they were treated. The use of peripheral blood stem cells (PBSC) as opposed to bone marrow is becoming more common for adults, patients receiving either HLA-matched sibling or unrelated donor stem cells. Studies in adults have demonstrated either a higher incidence of chronic GVHD or a higher incidence of refractory chronic GVHD when PBSC is used as opposed to bone marrow (119,120). The data are limited in pediatrics due to the small number of pediatric centers, which have adopted the use of PBSC. No randomized studies have been done in children comparing PBSC to standard bone marrow, so it is impossible to be certain of the difference in chronic GVHD. A few series have shown that it is feasible to do PBSC in children. It is likely that the rate of chronic GVHD using HLA-matched-sibling PBSC as the stem cell source is higher than standard bone marrow (121,122). However, this cannot be confirmed until a randomized study takes place. Also, whether the possible increase in chronic GVHD confers a better (due to more graft-versus-leukemia) or a worse survival (due to chronic GVHD complications) is unknown. Given the toxicities, cost, potential risk to the donor, and long wait time to obtain unrelated marrow (or PBSC), there is a great deal of interest in the use of cord blood as a source of stem cells. HLA-matched sibling cord blood provides adequate engraftment with very low rates of GVHD. In a recent IBMTR publication, it was shown that the three-year cumulative incidence of chronic GVHD in 113 children that received matched-sibling cord blood transplant was 6%. Patients whose indication for HSCT was a non-malignant diagnosis had even lower rates of chronic GVHD (117). Given the low incidence of acute and chronic GVHD using matched-sibling cord blood transplants, the use of unrelated cord blood transplants (UCBT) has become increasingly popular. Obtaining HLA-matched sibling cord blood is obviously difficult and rare given that most patients do not have the luxury of waiting for an HLA-matched sibling to be born in order to use the cord blood for transplantation. As discussed in the cord blood chapter, the rates of GVHD are fairly low even with 1 or 2 HLA allele mismatches. In 99 UCBT transplants reviewed through the Eurocord registry, the two-year incidence of chronic GVHD in survivors after day 100 with sustained engraftment was 12%. Half of these patients had 1 or more HLA antigen mismatch (es). These patients were compared to recipients of HLA-matched unrelated bone marrow (nZ262) where the cumulative incidence of chronic GVHD was 46% and to recipients of T-cell depleted HLA-matched unrelated bone marrow (nZ180) where the incidence of chronic GVHD was 11% (123). The indication for transplant for all of these patients was leukemia, although preparative regimens and GVHD prophylaxis varied by center. Another trend in transplantation is the use of non-myeloablative preparative regimens, with the goal of decreasing both short and long-term toxicity while allowing the graft-versustumor effect to dominate. The incidence of GVHD appears to be similar in adults as compared to standard myeloablative SCT, although other toxicities are reduced. While data are limited in pediatrics, it appears the rates of chronic GVHD may be lower than in adults. Slavin et al. reported 9 patients aged 20 or younger receiving fludarabine, busulfan, and ATG followed by matched sibling allografts for leukemia and nonmalignant disorders (Fanconi anemia, b-thalassemia, and Gaucher disease). Chronic GVHD developed in 4 patients (124).

Chronic Graft-Versus-Host Disease in Children

93

At Children’s Memorial Hospital in Chicago, there is a growing experience with the same regimen. Of 12 patients transplanted for nonmalignant disorders (five immunodeficiencies, three metabolic disorders, four hemoglobinopathies), eight are evaluable for chronic GVHD (survivors past day 100 with donor chimerism). Of these, one of five patients that underwent HLA-matched related transplants had chronic GVHD (extensive) and two of three matchedunrelated donor transplant patients developed chronic GVHD (both limited) (125). Among pediatric patients with malignancies receiving the same regimen, the rates of chronic GVHD are higher. In the matched-related group, three of four evaluable patients developed chronic GVHD (two limited, one extensive), and in the matched-unrelated donor group, five of six developed chronic GVHD (three limited, two extensive) (126). Horwitz et al. attempted to decrease GVHD in young patients with chronic granulomatous disease by giving T celldepleted grafts after fludarabine, cyclophosphamide, and ATG followed by DLI beginning on day 30. Out of six patients under age 21 in this report, two had graft failure (one of them died). Of the other four patients, all remain alive and well, and only one patient has limited chronic GVHD (127). Only by accumulating more patients in organized clinical trials will we be able to answer whether long-term toxicities and rates of chronic GVHD are lowered by using reduced intensity conditioning in children. The largest series documenting the incidence and risk factors of chronic GVHD in children comes from the Italian group led by Zecca. In this analysis, 696 consecutive patients that underwent HSCT in Italy between 1991 and 1999 were analyzed. The vast majority of patients received bone marrow as stem cell sources and 66% were from HLA-matched sibling donors. Two-thirds of diseases leading to HSCT were malignant. The overall incidence of chronic GVHD in this population was 25%, with 16% being limited and 9% extensive. Median time to diagnosis was 116 days after transplantation. In multivariate analysis, the following factors were associated with a higher incidence of chronic GVHD: patient age greater than 15 years, donor age greater than five years, female donor into male recipient, TBI use during conditioning, malignant diagnosis as transplant indication, and previous grade II–IV acute GVHD. The latter risk factor had the highest relative risk for chronic GVHD of all the factors. Use of cord blood was associated with significantly less chronic GVHD. A graft-versusleukemia was demonstrated for ALL with a lower relapse rate for patients with chronic GVHD. Overall, in malignant diseases, the six years disease-free survival was 68% in the presence of chronic GVHD and 54% in the absence of chronic GVHD. Presence of chronic GVHD did not affect survival in non-malignant diseases (128). This very important analysis confirms that in children, chronic GVHD confers a graft-versus-leukemia effect and that limited chronic GVHD is probably beneficial. Future directions need to focus on limiting GVHD to a degree the child can tolerate without leading to infections or increased morbidity. What remains to be determined in children is whether there are risk factors at diagnosis of chronic GVHD that are predictive for increased mortality. For adults, extensive skin involvement, thrombocytopenia, and progressive onset chronic GVHD at diagnosis of chronic GVHD confer a poor survival (129,130). These data will be useful in pediatrics as well as allowing us to stratify patients and direct them to experimental therapy for chronic GVHD if we can a priori predict a poor outcome with standard therapy.

CLASSIFICATION OF CHRONIC GRAFT-VERSUS-HOST DISEASE The most commonly employed classification is the one originally proposed by Seattle in 1980, which was devised to define a group needing therapy. Patients with limited disease have localized skin involvement and/or hepatic dysfunction due to chronic GVHD, and were felt not to require treatment. Those with extensive disease (i.e., those needing therapy) have either generalized skin involvement (O50% BSA) or localized skin involvement and/or hepatic dysfunction plus an additional organ involved (131). Criteria have been slightly modified in

94

Jacobsohn et al.

order to classify patients with severe disease (i.e., with contractures or scleroderma) that may have fallen into the limited category to the clinically extensive category (132). Another method of classification relies on type of onset. The majority of patients with chronic GVHD have had acute GVHD. If chronic GVHD evolves from acute GVHD, it is termed progressive and is associated with poor outcome (133). Those that have resolution after acute but later have chronic GVHD have quiescent GVHD and have an intermediate prognosis. The best outcome for chronic GVHD patients is for those who have never had acute GVHD—this type of chronic GVHD is termed de novo. More recently, investigators have tried to develop prognostic grading scales based on survival as the primary endpoint. The two main grading scales recently proposed essentially show that at diagnosis of chronic GVHD, the following findings clearly decrease survival: thrombocytopenia, progressive onset, extensive skin involvement, GI involvement, and low Karnofsky performance status (134,135). The grading scale from Johns Hopkins, in which the first three findings mentioned were associated with poor survival, has been validated at four other centers (136). The population used in the Johns Hopkins study was adult and pediatric; however, there were not enough pediatric patients to look at separately. Nevertheless, there was no effect of age on the whole model suggesting there is a good chance the same model would apply in pediatrics. Unfortunately, most of these studies have been done in adult populations so the same risk factors may not exactly apply to pediatrics.

Clinical Manifestations Cutaneous Cutaneous involvement in chronic GVHD is usually manifested by patchy erythema with scaling and erythematous or violaceous papules. This is known as lichen-planus or a lichenoid rash (137). It is not unusual to find, within the lichenoid rash, areas with hyperkeratotic papules and desquamation (138). Patients can also present in a very subtle fashion with hypoor hyper-pigmented areas (132). Patients usually progress, if treatment is ineffective, to sclerodermatous GVHD. Sclerodermatous GVHD may involve the dermis or the muscular fascia and clinically resembles systemic sclerosis. Diffuse induration with morphea-like plaques is seen. Furthermore, contractures can develop at this point (135). Contractures are usually a sign of progressive disease and damage and are difficult to reverse, although physical therapy is recommended. With advanced scleroderma, the skin is thickened, tight, and fragile with very poor wound healing. This usually leads to hair loss and sweat gland destruction. Poor lymphatic drainage can lead to blistering and ulceration. The skin is especially sensitive to trauma at this point as well (132). In addition, viral skin infections can worsen or activate chronic GVHD (136). Some alteration in pigmentation, either hypo- or hyper-pigmentation, is very common at this stage. In very unusual cases, patients can remain with partial or total leucoderma (similar to albinism) which is thought to be from damage to melanocytes (137,138). Finally, chronic GVHD is often a difficult diagnosis that can simulate other syndromes, for example, dyskeratosis congenita in children (139,140). Obtaining histology and doing careful follow-up to observe the evolution of skin findings are therefore imperative. Dermal Appendages All dermal appendages can be affected by chronic GVHD. Hair can become brittle and alopecia can ensue. The association between alopecia and chronic GVHD has been well-documented in children (141,142). Premature graying of the hair is common, even in children. The mechanism of this premature graying is not clear, but may affect hair and eye brows. The scalp can develop changes similar to seborrhea. At our center, steroid mousses have been helpful in managing these symptoms. Nails can also become brittle, crack, and develop ridging (143). These changes can be permanent in many cases. Sweat glands may be destroyed in sclerodermatous

Chronic Graft-Versus-Host Disease in Children

95

disease, leading to an inability to sweat in warm temperatures and serious hyperthermia. Biopsies of chronic GVHD often show complete sweat gland destruction with fibrosis and occasionally squamous metaplasia and dilation of the sweat glands can be identified (144).

Musculoskeletal System While skin changes are usually seen overlying fascial involvement, fasciitis may develop with normal but fixed overlying skin (145). Therefore, a very careful examination is required as patients’ skin may look normal but on exam be very fixed underneath. Fasciitis may cause limitations in range of motion if joint areas are involved. Fascial biopsies can be technically difficult but can be useful. They would often show dense fibrous connective tissue with focal perivascular chronic inflammation (146). Furthermore, muscle cramps are a common complaint in patients with chronic GVHD, although the pathophysiology is not understood. Children tend to complain of cramping after repeated use of an extremity. Cramping is not explained by alteration in electrolytes or by myositis. The diagnosis of myositis can occur with chronic GVHD, albeit it is very rare. Chronic GVHD myositis can be debilitating and should be considered when a patient, after stem cell transplant, has diffuse weakness (147). The work-up of myositis would involve evaluating muscle enzymes (CK–MB, aldolase, LDH) and muscle biopsy. The group from Children’s Hospital of Philadelphia recently reviewed their experience with orthopedic complications of chronic GVHD in children. All fourteen patients reviewed positive skin biopsies. A number of patients had biopsies of other musculoskeletal sites and they were able to show chronic inflammation or lymphocytic infiltrates in muscle, joint capsules, fascia, nerve, and fat. Three patients underwent surgical procedures, all of which involved joint capsular releases of various joints for contractures. In all cases, contractures recurred within six months after the surgical procedure (146). Therefore, it is difficult to suggest surgical management of orthopedic complications and a medical approach with physical and occupational therapy is recommended. Other orthopedic complications that are common are osteoporosis and avascular necrosis (148,149). These are overwhelmingly the result of steroid therapy for chronic GVHD. Careful follow-up with bone density studies and use of biphosphonates in select patients are therefore warranted. Eyes Ocular GVHD usually presents with irritation or dry eyes. The probability of developing ocular involvement in patients with chronic GVHD is estimated to be 35–40% at 15 years (150). Irreversible destruction of the lacrimal glands results in dryness, photophobia, and burning. Interestingly, symptoms can begin with excessive tearing (151). Conjunctival GVHD is a rare manifestation of severe chronic GVHD and is more difficult to treat. Lacrimal gland specimens from patients with dry eyes show prominent fibrosis and an increase in CD34C stromal fibroblasts in the glandular interstitium in addition to infiltration of T cells into the periductal areas (152). Local therapy involves preservative-free tears and ointment and placement of punctal plugs by an experienced ophthalmologist. It is important to follow these patients with serial Schirmer’s tests to assess degree of wetting. Mouth Children complain of food sensitivity with oral GVHD. Typically acidic or spicy foods exacerbate the sensitivity. Patients can also have severe dryness. A more advanced stage of disease may cause odynophagia due to extension of damage down the throat, although rarely patients will have esophageal involvement without oral disease. At first, mild disease shows only erythema with a few white plaques that may be confused with thrush or herpetic infections (153). Lichenoid changes of more advanced disease cause more extensive plaques. Patients can progress to pseudomembranes with large ulcers with salivary gland destruction (154).

96

Jacobsohn et al.

Pseudomembranes may be covered by fibrin exudates and surrounded by atrophic, erythematous areas (155). Labial salivary gland biopsies can be useful as they can demonstrate atrophy and/or destruction in association with diffusely infiltrating lymphocytes (mostly CD3C cells) (156). Secondary infections with viruses (especially herpes simplex) and yeasts are almost universal. Patients can also have difficulty chewing secondary to fibrosis causing decreased range of motion. Finally, it is not unusual to have extremely dry lips causing sensitivity in that area. In general, children with isolated oral chronic GVHD can be treated with steroid rinses. Since the rate of infection with fungus is so high, using a local antifungal preparation in combination with the steroid rinse is recommended.

Gastrointestinal Tract Children with chronic GVHD can have GI complaints consisting of anorexia, nausea, lower abdominal pain, cramping and diarrhea. While these symptoms may be directly related to chronic GVHD, very often these symptoms are attributable to other disease states including acute GVHD, infection, dysmotility, lactose intolerance, pancreatic insufficiency (157), and drug-related side effects (for example, mycophenolate mofetil can lead to diarrhea with colitis on biopsy that actually resembles acute GVHD (158). As many of these problems are very correctable, full evaluation of these symptoms is important before adding or continuing immunosuppressive medication, as this tactic may worsen the child’s symptom (159). In a retrospective review of the intestinal biopsies of 40 patients at Hopkins with chronic GVHD and persistent GI symptoms, histopathological evidence of chronic GVHD was found in only 11 patients. The majority of these patients had evidence of both acute and chronic GVHD, with only three patients (7%) found to have isolated chronic GVHD (160). This study illustrated that although chronic GVHD alone may involve the gastrointestinal (GI) tract, it may be difficult to diagnose and is seldom seen without concurrent acute GVHD. It is generally accepted that while acute GVHD can involve the whole GI tract, chronic GI GVHD mostly affects the esophagus, sparing the lower GI tract (131,161), leading to desquamation of the esophageal mucosa with submucosal fibrosis (162). Damage to the esophageal mucosa eventually leads to symptoms such as dysphagia and odynophagia. Webs and strictures are sometimes identified on upper endoscopies in these patients. Patients with strictures may benefit from endoscopic dilatations and antacid medications. Weight loss is a major problem in patients with chronic GVHD and is often not explained by the actual symptoms of chronic GVHD that may prevent adequate caloric intake. Of 93 patients with chronic GVHD reviewed at Johns Hopkins, 43% were considered malnourished and 14% had severe malnutrition, defined by a body mass index less than 18.5 (163). It does appear that patients with chronic GVHD have elevated resting energy expenditures (164), which probably is at least partially responsible for their weight loss. Liver Hepatic chronic GVHD usually presents with obstructive jaundice, with elevations of alkaline phosphatase, gamma-glutamyl transferase, and serum bilirubin (direct). Isolated hepatic chronic GVHD is being seen with increased frequency with the use of donor-lymphocyte infusions (165). Furthermore, while cholestasis is the norm, the concept of hepatitic chronic GVHD is being identified more often recently, with some patients presenting mostly with elevations of serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) (166,167). Viral studies including hepatitis A, B, C, and EBV, CMV, and adenovirus are recommended to exclude infection with these agents. Also, certain agents prescribed to these children, including fluconazole and calcineurin inhibitors can cause elevations of bilirubin and liver enzymes. Liver biopsy is required to confirm the diagnosis, especially important in patients with no other symptoms of chronic GVHD. It is well known that other factors, such as viral infections, drug toxicity, or total-parenteral nutrition-related cholestasis may mimic

Chronic Graft-Versus-Host Disease in Children

97

GVHD. Liver biopsies can show portal fibrosis and bile duct dropout but may progress to cirrhosis and bridging necrosis (168).

Respiratory Tract Bronchiolitis obliterans (BO) is usually a late and serious manifestation of chronic GVHD. The pathophysiology is not completely understood but we know, since its first description in 1982, that it is an obstructive disease of small airways (169) and damage is mediated by donor-lymphocytes targeting host epithelial cells (170), as in other forms of GVHD. This disorder has also shown in other reports to be associated with the use of methotrexate and hypogammaglobulinemia. The overwhelming majority of patients following HSCT with BO, though, have it concurrently with chronic GVHD (171–173). The survival of patients with BO is dismal despite aggressive therapy. In a recent retrospective study from Minnesota of 2859 SCT recipients, the overall Three-year incidence of BO was 3% and the Five-year survival of patients with BO was 10%. While not a strictly pediatric study, there were a large number of pediatric patients included (median age, 24.7 years; range, 0.1–67.4 years) (174). Patients with BO typically present with a cough or dyspnea (175). Pulmonary function studies show FEV1 and FVC !80% predicted with rapid declines thereafter. As other clinical diagnoses can be associated with these findings, a full work-up is recommended. Recommendation for work-up include high-resolution computer-assisted tomography scan of the chest which would confirm air trapping and lung biopsy showing granulation tissue, fibrosis, and obliteration of the small airways. Finally, aggressive therapy of concurrent diagnoses such as infection is important. Patients with chronic GVHD are also at risk for chronic sinopulmonary disease, chronic cough, and bronchospasm. Sinus infections are more common in patients with GVHD and symptoms may be minimal (176). Therefore, computer-assisted tomography of the sinuses can often be helpful in this group of patients. Hematopoietic System While platelets are the most common hematopoietic cells affected during chronic GVHD, any cytopenia can be found. This may be a result of stromal damage, but autoimmune neutropenia (177), anemia (178), and/or thrombocytopenia (179) are also seen. A number of studies, mostly in adults, have shown that thrombocytopenia at the time of chronic GVHD diagnosis confers a poor prognosis (180,181). However, thrombocytopenia post transplant may be a poor prognostic factor, independent of GVHD (182). Eosinophilia is often seen in children with chronic GVHD and sometimes precedes the occurrence of chronic GVHD (183). While the hypothesis is that the eosinophilia is secondary to activation of the Th-two arm of the immune system, the exact etiology is unknown. Eosinophilia usually resolves with adequate systemic treatment of GVHD. Immune System Chronic GVHD patients are severely immunosuppressed, both from the disease and its therapy. Most deaths in patients with active chronic GVHD are related to infection. Patients with chronic GVHD have reduced number and function of lymphocytes making them at risk for fungal, viral, and bacterial infections. In addition, mucosal barriers are compromised in children with GVHD, increasing the risk for infections (184–186). Another complication of chronic GVHD is functional asplenia, causing increased risk of infections with encapsulated organisms such as pneumococcus. Functional asplenia can remain for life despite resolution of chronic GVHD. This is evidenced by persistence of Howell-Jolly bodies in some patients who have had chronic GVHD and a higher incidence of pneumococcal sepsis in these patients (187,188).

98

Jacobsohn et al.

Given the compromised immune state of these children, we recommend lifelong prophylaxis against encapsulated organisms as well as prophylaxis against Pneumocystis until complete resolution of chronic GVHD, and at least six months after discontinuation of immunosuppressive therapy. It is also recommended that patients’ IgG level be kept above 500 mg/dL with supplemental intravenous IgG. Patients at risk for CMV should be monitored closely with CMV PCR or antigenemia. Patients receiving steroid rinses for oral GVHD are at high risk for local Candidal infections so an antifungal such as nystatin swishes or clotrimazole troche should be used. Consideration should also be given to antifungal and antiviral prophylaxis, although this can be dependent on each patient and the intensity of their therapy. Vaccinations are delayed until there is no active disease and at least one year after completion of immunosuppressive therapy. At that point, the usual vaccinations recommended for children following HSCT are recommended (189).

TREATMENT OF CHRONIC GRAFT-VERSUS-HOST DISEASE Standard Therapy While there is no specific “standard therapy,” it seems that most centers start with prednisone and CsA for the treatment of chronic GVHD. One regimen that appears to improve survival in patients with high-risk features such as thrombocytopenia and extensive skin involvement is alternate-day prednisone and CsA (133). Most of the data on this regimen exists in adults but has been extrapolated to pediatrics. At our institution, patients are evaluated every three months and therapy is continued for three months after maximal response. The three-month time frame for evaluation of response to a given therapy is based on our own observation that 90% of patients who are ultimately going to respond to therapy will show signs of response at that point (190).

Salvage Regimens An agent initially shown by Vogelsang to have a role in chronic GVHD is thalidomide (191). The response of high-risk chronic GVHD to thalidomide appears to be 20–30% (192). Unfortunately, the side effects (particularly sedation and constipation) are intolerable to many patients and the drug has therefore fallen out of favor to other newer agents. Two reports of thalidomide use in pediatrics do suggest that this drug has activity in steroid refractory chronic GVHD and is actually well-tolerated by children (193,194). Another agent sometimes used in steroid-refractory chronic GVHD is azathioprine. However, given its myelosuppressive effects and high incidence of infections, it is best avoided (195,196). The use of MMF combined with FK506 is being studied in chronic GVHD. At Johns Hopkins, a retrospective review of 26 patients with refractory chronic GVHD treated with this steroid-sparing combination showed that it was well tolerated, and nearly half the patients showed an objective response (197). The Seattle group reported a complete response rate of 65% in 26 pediatric patients who had progressed on therapy with prednisone and CSA. MMF was added to this combination but had to be maintained for upto three years in some patients to see resolution. This highlights what many investigators are noting—that MMF appears to have efficacy in chronic GVHD but can take many months to start seeing an effect. The drug was remarkably well tolerated, as toxicity was only transient leucopenia in one patient (198). Finally, a randomized, placebo-controlled phase III multi-center study recently started where the addition of MMF to standard therapy at initial diagnosis of chronic GVHD is being evaluated. Another agent that has shown efficacy in acute GVHD and is therefore being studied in chronic GVHD is pentostatin. Pentostatin is a nucleoside analog that irreversibly inhibits adenosine deaminase by blocking the metabolism of 2 0 -deoxyadenosine (199). Patients with adenosine deaminase deficiency have few T cells and have a form of severe combined

Chronic Graft-Versus-Host Disease in Children

99

immunodeficiency (200). Pentostatin inhibition of adenosine deaminase creates a state similar to severe combined immunodeficiency. Lymphocytes have a high ratio of deoxycytidine kinase to Five-nucleotidase, favoring the formation of 2 0 -deoxyadenosine 5 0 -triphosphate (dATP) from 2 0 -deoxyadenosine (201). A study using pentostatin for chronic GVHD is under way at Johns Hopkins and Children’s Memorial Hospital. Patients are treated with 4 mg/m2 IV every two weeks for six months and can continue receiving therapy beyond that point if they are partial responders. Of 19 pediatric and young adult patients (received HSCT under age of 21) treated so far, there have been objective responses in 15. The drug is well tolerated in this group. Response usually is seen starting at six weeks into therapy. This response is very encouraging given that all of these patients were refractory to at least two different previous regimens and the majority had very severe scleroderma (202). Given the encouraging data, a phase II study in pediatric chronic GVHD, where the drug will be incorporated immediately after failing a steroid-based regimen, is starting through the Pediatric Blood and Marrow Transplant Consortium. Hydroxychloroquine (HCQ), a lysosomotropic four-aminoquinoline antimalarial drug but has been used to treat autoimmune disorders for more than 30 years (203). HCQ is particularly effective for mucocutaneous manifestations of autoimmune disease and is also useful for decreasing the dose of prednisone needed to control autoimmune diseases (204). The most common (but infrequent) side effects are GI symptoms. The most worrisome toxicity of HCQ is retinopathy resulting in visual field deficits. Retinal toxicity is very uncommon and is related to the daily and cumulative dose and to the duration of therapy, rarely occurring before two years of treatment (205,206). HCQ affects several steps of the immune response involved in chronic GVHD. HCQ is a weak base that interferes with antigen processing and presentation through the major histocompatibility complex (MHC) class II pathway by raising lysosomal and endosomal pH (207). HCQ also reduces production of the proinflammatory cytokines IL-1, IL-6 and TNF-a (208,209). Finally, synergism between HCQ and CSA or FK506 has been demonstrated in vitro (210,211). Clinically, a phase II trial in children and adults studied the addition of HCQ to patients with steroid-refractory chronic GVHD. Of 32 evaluable patients, 17 had objective responses. Also crucial is that all patients with response tolerated a O50% wean in their steroid dose (212). Because of this encouraging data, a phase III randomized study evaluating the addition of HCQ versus placebo to standard therapy in initially diagnosed chronic GVHD pediatric patients is currently in progress through the Children’s Oncology Group. Extracorporeal photopheresis (ECP) is currently used for the treatment of cutaneous T-cell lymphoma and certain autoimmune diseases (213). ECP is based on the infusion of autologous peripheral blood mononuclear cells collected by apheresis, incubated with the photoactivatable drug 8-methoxypsoralen (8-MOP) and UV–A irradiation. 8-MOP is a naturally occurring photoactive substance found in the seeds of the Ammi majus (Umbelliferae) plant. It belongs to a group of compounds known as psoralens or furocoumarins. The exact mechanism of action of 8-MOP is not known. The best-known biochemical reaction of 8-MOP is with DNA. 8-MOP, upon photoactivation, conjugates and forms covalent bonds with DNA which leads to the formation of both monofunctional (addition to a single strand of DNA) and bifunctional adducts (crosslinking of psoralen to both strands of DNA) (UVADEX package insert). ECP was approved in 1988 by the FDA as a medical device for the treatment of cutaneous T cell lymphoma (CTCL) after a landmark study showed significant response in 27 of 37 refractory patients (214). Since then, more than 300 patients with CTCL have been treated with ECP (214). ECP is well tolerated with few complications or adverse effects (214,216–218). Extremely rare adverse reactions in adults have included exacerbation of congestive heart failure or arrhythmias (215), superficial thrombophlebitis (215), catheterrelated sepsis (218), disseminated fungal infection (215), and a single episode of mechanically induced hemolysis (216). ECP has recently shown efficacy in children with steroid-refractory chronic and acute GVHD. A number of promising results, mostly from Europe, have been reported in children. Salvaneschi et al. has treated 35 children with steroid-refractory GVHD. 17 had acute grade

100

Jacobsohn et al.

II–IV GVHD with a 58% response rate and ability to taper steroids in 53%. Eighteen had extensive chronic GVHD with a 78% response rate ability to taper steroids in 67%. ECP was safe and well tolerated without increase in infections in this study (219). In another recent study of ECP for children with steroid-refractory chronic GVHD, out of 44 children, 15 (44%) showed a complete response and 10 (29%) a significant improvement after ECP. As a result of treatment with ECP, it was possible to discontinue immunosuppressive therapy in 15 (44%) and to reduce it in 10 (29%) of these patients. Patients as small as 15 kg were able to undergo the procedure. ECP was performed on two consecutive days weekly for one month, followed by every two weeks over the next two months, followed by monthly for at least three more months. The procedure was well-tolerated, even in the smaller children (220). Clearly this modality will likely be adopted by more centers given these encouraging data in children. Recent data from Tufts University suggest that in chronic GVHD, maximal improvement in chronic GVHD correlates with a dramatic shift in the balance of DCs. ECP causes an increase in plasmacytoid DC2 cells and a decrease in monocytoid DC1 cells. Essentially this leads to an activation of Th2 cells (and production of IL-4 and IL-10) and a deactivation of Th1 cells (causing less production of inflammatory cytokines such as TNF-a and IFN-g) (221). These data would suggest a Th-1 mediation of chronic GVHD (87,157).

REFERENCES 1. Aichinger G, Lechler RI. Antigen processing and presentation. Endogenous pathway of class II presentation. Biochem Soc Proc 1995; 23:657–661. 2. Eckels DD. Alloreactivity: allogeneic presentation off endogenous peptide or direct recognition of MHC polymorphism? a review. Tissue Antigens 1990; 35:49–55. 3. Lechler RI, Lombardi G, Batchelor JR, Reinsmoen N, Bach FH. The molecular basis of alloreactivity. Immunol Today 1990; 11:83–88. 4. Germain RN. The ins and outs of antigen processing and presentation. Nature 1986; 322:687–689. 5. Brodsky FM, Guagliardi LE. The cell biology of antigen processing and presentation. Annu Rev Immunol 1991; 9:707–744. 6. Weaver CT, Unanue ER. The costimulatory function of antigen-presenting cells. Immunol Today 1990; 11:49–55. 7. Quentmeier H, Klaucke J, Muhlradt PF, Drexler HG. Role of IL-6, IL-2, and IL-4 in the in vitro induction of cytotoxic T cells. J Immunol 1992;3316–3320. 8. Russell CA. Alloreactivity and the predictive value of anti-recipient specific interleukin 2 producing helper T lymphocyte precursor frequencies for alloreactivity after bone marrow transplantation. Dan Med Bull 2002; 49:89–108. 9. Yamashita K, Choi U, Woltz PC, et al. Severe chronic graft-versus-host disease is characterized by a preponderance of CD4C effector memory cells relative to central memory cells. Blood 2004; 103:3986–3988. 10. Xystrakis E, Bernard I, Dejean AS, Alsaati T, Druet P, Saoudi A. Alloreactive CD4 T lymphocytes responsible for acute and chronic graft-versus-host disease are contained within the CD45RChigh but not the CD45RClow subset. Eur J Immunol 2004; 34:408–417. 11. Regeczy N, Kormos L, Szigetvari CM, et al. Reactivity of new adhesion molecules on lymphocytes from patients with chronic graft versus host disease. Acta Microbiol Immunol Hung 2003; 50:55–65. 12. Biedermann BC, Sahner S, Gregor M, et al. Endothelial injury mediated by cytotoxic T lymphocytes and loss of microvessels in chronic graft versus host disease. Lancet 2002; 359:2078–2083. 13. Patey-Mariaud de Serre N, Reijasse D, Verkarre V, et al. Chronic intestinal graft-versus-host disease: clinical, histological and immunohistochemical analysis of 17 children. Bone Marrow Transplant 2002; 29:223–230. 14. Seaton ED, Szydlo RM, Kanfer E, Apperley JF, Russell-Jones R. Influence of extracorporeal photopheresis on clinical and laboratory parameters in chronic graft-versus-host disease and analysis of predictors of response. Blood 2003; 102:1217–1223.

Chronic Graft-Versus-Host Disease in Children

101

15. Kotani A, Ishikawa T, Matsumura Y, et al. Correlation of peripheral blood OX40C(CD134C) T cells with chronic graft-versus-host disease in patients who underwent allogeneic hematopoietic stem cell transplantation. Blood 2001; 98:3162–3164. 16. Johnson BD, Konkol MC, Truitt RL. CD25C immunoregulatory T-cells of donor origin suppress alloreactivity after BMT. Biol Blood Marrow Transplant 2002; 8:525–535. 17. Clark FJ, Gregg R, Piper K, et al. Chronic graft-versus-host disease is associated with increased numbers of peripheral blood CD4CCD25high regulatory T cells. Blood 2004; 103:2410–2416. 18. Kojima K, Kurokawa MS, Tanimoto K, et al. Clonal expansion of limited T cell clonotypes in affected muscle from a patient with post-transplant polymyositis. Bone Marrow Transplant 2002; 30:467–470. 19. French LE, Alcindor T, Shapiro M, et al. Identification of amplified clonal T cell populations in the blood of patients with chronic graft-versus-host disease: positive correlation with response to photopheresis. Bone Marrow Transplant 2002; 30:509–515. 20. Matsutani T, Yoshioka T, Tsuruta Y, et al. Restricted usage of T-cell receptor alpha-chain variable region (TCRAV) and T-cell receptor beta-chain variable region (TCRBV) repertoires after human allogeneic haematopoietic transplantation. Br J Haematol 2000; 109:759–769. 21. Rocken M, Racke M, Shevach EM. IL-4-induced immune deviation as antigen-specific therapy for inflammatory autoimmune disease. Immunol Today 1996; 17:225–231. 22. Halverson DC, Schwartz GN, Carter C, Gress RE, Fowler DH. In vitro generation of allospecific human CD8CT cells of Tc1 and Tc2 phenotype. Blood 1997; 90:2089–2096. 23. Murray JS. How the MHC selects Th1/Th2 immunity. Immunol Today 1998; 4:157–163. 24. Thompson CG. Distinct roles for the costimulatory ligands 137-1 and 137-2 in T helper differentiation? Cell 1998; 81:979–982. 25. Seder RA, Pau WE. Acquisition of lymphokine-producing phenotype by CD4CT cells. Annu Rev Immunol 1994; 12:635–7673. 26. Ferrara JLM, Deeg HJ. GVHD—review article. N Engl Med J 1991; 324:667–674. 27. Facon T, Jouet JP, Noel-Walter, Bloget F, Bauters F, Janin A. Involvement of TNF-alpha secreting macrophages in lethal forms of human graft-vs.-host disease. Bone Marrow Transplant 1997; 20:511–515. 28. Atkinson K. Chronic graft-versus-host disease. Bone Marrow Transplant 1990; 5:69–82. 29. Garin L, Rigal D, Souillet G, et al. Allogeneic BMT in children: differential lymphocyte subset reconstitution according to the occurrence of acute GVHD. Clin Immunol Immunopathol 1995; 77:139–148. 30. Zeng D, Lewis D, Dejbakhsh-Jones S, et al. Bone marrow NK1.1(K) and NK1.1(C) T cells reciprocally regulate acute graft versus host disease. J Exp Med 1999; 189:1073–1081. 31. Kook H, Goldman F, Padley D, et al. Reconstruction of the immune system after unrelated or partially matched T-cell-depleted bone marrow transplantation in children: immunophenotypic analysis and factors affecting the speed of recovery. Blood 1996; 88:1089–1097. 32. Cranston R, Koh M, McDermott A, Ray N, Prentice HG, Lowdell MW. Temporal dynamics of CD69 expression on lymphoid cells. J Immunol Methods 1997;209. 33. Foley R, Couban S, Walker I, et al. Monitoring soluble interleukin-2 receptor levels in related and unrelated donor allogeneic bone marrow transplantation. Bone Marrow Transplant 1998; 21:769–773. 34. Lee S, Chong SY, Lee JW, et al. Difference in the expression of Fas/Fas ligand and the lymphocyte subset reconstitution according to the occurrence of acute GVHD. Bone Marrow Transplant 1997; 20:883. 35. Liem LM, Fibbe WE, van Houwelingen HC, Goulmy E. Serum transforming growth factor-beta1 levels in bone marrow transplant recipients correlate with blood cell counts and chronic graftversus-host disease. Transplantation 1999; 67:59–65. 36. Shustov A, Nguyen P, Finkelman F, Elkon KB, Via CS. Differential expression of fas and fas ligand in acute and chronic graft-versus-host disease: up-regulation of fas and fas ligand requires CD8CT cell activation and INF-gamma production. J Immunol 1998; 161:2848–2855. 37. Garside P, Reid S, Steel M, Mowat A, Mel. Differential cytokine production associated with distinct phases of murine graft-vs.-host disease. Eur J Immunol 1993; 23:333–341. 38. De Wit D, Van Mechelen M, Zanin C, et al. Preferential activation of Th2 cells in chronic graft-vs. host reaction. J Immunol 1993; ISO:361–371. 39. Via CS, Finkelman FD. Critical role of intedeukin-2 in the development of acute graft-vs. host disease. Int Immunol 1993;S565–S572.

102

Jacobsohn et al.

40. Via CS. Kinetics of T cell activation in acute and chronic forms of murine graft-vs. host disease. J Immunol 1991; 146:2603–2612. 41. Rus V, Svetic A, Nguyen P, Gause WC, Via CS. Kinetics of Th1 and Th2 cytokine production during the early course of acute and chronic murine graft-vs. host disease. J Immunol 1995; 155:2396–2405. 42. Price KS, Nestel FP, Lapp WS. Progressive accumulation of bacterial lipopolysaccharide in vivo during murine acute graft-vs.-host disease. Scand J Immunol 1997; 45:294–300. 43. Williamson E, Garside P, Bradley JA, More IAR, Mowat A, Mcl. Neutralizing IL 12 during induction of murine acute graft-vs. host disease polarizes the cytokine profile toward a Th2-type alloimmune response and confers long term protection from disease. J Immunol 1997; 159:1208–1215. 44. Lauwerys BR, Renauld JC, Houssiau FA. Inhibition of in vitro immunoglobulin production by IL-12 in murine chronic graft-vs.-host disease, synergism with IL-18. Eur J Immunol 1998; 28:2017–2024. 45. Tanaka J, Imamura M, Kasai M, et al. The important balance between cytokines derived from type 1 and type 2 helper T cells in the control of graft-vs.-host disease. Bone Marrow Transplant 1997; 19:571–576. 46. Ochs LA, Blazar SR, Roy J, Rest EB, Weisdorf DJ. Cytokine expression in human cutaneous chronic graft-vs.-host disease. Bone Marrow Transplant 1996; 17:1085–1092. 47. Tanaka J, Imamura M, Kasai M, et al. Cytokine gene expression by concanavalin A-stimulated peripheral mononuclear cell after bone marrow transplantation, an indicator of immunological abnormality due to chronic graft-vs. host disease. Bone Marrow Transplant 1994; 14:695–701. 48. Dighiero G, Intrator L, Cordonnier C, Tortevoye P, Vernant JP. High levels of anticytoskeleton autoantibodies are frequently associated with chronic GVHD. Br J Haematol 1987; 67:301–305. 49. Cavet J, Dickinson AM, Norden J, Taylor PR, Jackson GH, Middleton PG. Interferon-gamma and interleukin-6 gene polymorphisms associate with graft-versus-host disease in HLA-matched sibling bone marrow transplantation. Blood 2001; 98:1594–1600. 50. Stark GL, Dickinson AM, Jackson GH, Taylor PR, Proctor SJ, Middleton PG. Tumour necrosis factor receptor type II 196M/R genotype correlates with circulating soluble receptor levels in normal subjects and with graft-versus-host disease after sibling allogeneic bone marrow transplantation. Transplantation 2003; 76:1742–1749. 51. Rocha V, Franco RF, Porcher R, et al. Host defense and inflammatory gene polymorphisms are associated with outcomes after HLA-identical sibling bone marrow transplantation. Blood 2002; 100:3908–3918. 52. Clark FJ, Freeman L, Dzionek A, et al. Origin and subset distribution of peripheral blood dendritic cells in patients with chronic graft-versus-host disease. Transplantation 2003; 75:221–225. 53. Gorgun G, Miller KB, Foss FM. Immunologic mechanisms of extracorporeal photochemotherapy in chronic graft-versus-host disease. Blood 2002; 100:941–947. 54. Waller EK, Rosenthal H, Sagar L. DC2 effect on survival following allogeneic bone marrow transplantation. Oncology (Huntingt) 2002; 16:19–26. 55. Arpinati M, Chirumbolo G, Urbini B, et al. Acute graft-versus-host disease and steroid treatment impair CD11cC and CD123C dendritic cell reconstitution after allogeneic peripheral blood stem cell transplantation. Biol Blood Marrow Transplant 2004; 10:106–115. 56. Clark FJ, Freeman L, Dzionek A, et al. Origin and subset distribution of peripheral blood dendritic cells in patients with chronic graft-versus-host disease. Transplantation 2003; 75:221–225. 57. Chan GW, Gorgun G, Miller KB, Foss FM. Persistence of host dendritic cells after transplantation is associated with graft-versus-host disease. Biol Blood Marrow Transplant 2003; 9:170–176. 58. Remberger M, Aschan J, Lonnqvist B, et al. An ethnic role for chronic, but not acute, graft-versushost disease after HLA-identical sibling stem cell transplantation. Eur J Haematol 2001; 66:50–56. 59. Ordemann R, Hutchinson R, Friedman J, et al. Enhanced allostimulatory activity of host antigenpresenting cells in old mice intensifies acute graft-versus-host disease. J Clin Invest 2002; 109:1249–1256. 60. Deguchi M, Ohtani H, Sato E, et al. Proliferative activity of CD8(C) T cells as an important clue to analyze T cell-mediated inflammatory dermatoses. Arch Dermatol Res 2001; 293:442–447. 61. Hasseus B, Jontell M, Brune M, Johansson P, Dahlgren UI. Langerhans cells and T cells in oral graft versus host disease and oral lichen planus. Scand J Immunol 2001; 54:516–524. 62. Schultz KR, Bader S, Paquet J, Li W. Chloroquine treatment affects T-cell priming to minor histocompatibility antigens and graft-versus-host disease. Blood 1995; 86:4344–4352.

Chronic Graft-Versus-Host Disease in Children

103

63. Ratanatharathorn V, Ayash L, Reynolds C, et al. Treatment of chronic graft-versus-host disease with anti-CD20 chimeric monoclonal antibody. Biol Blood Marrow Transplant 2003; 9:505–511. 64. Storek J, Wells D, Dawson MA, Storer B, Maloney DG. Factors influencing B lymphopoiesis after allogeneic hematopoietic cell transplantation. Blood 2001; 98:489–491. 65. Busser BW, Adair BS, Erikson J, Laufer TM. Activation of diverse repertoires of autoreactive T cells enhances the loss of anti-dsDNA B cell tolerance. J Clin Invest 2003; 112:1361–1371. 66. Lauwerys BR, Renauld JC, Houssiau FA. Inhibition of in vitro immunoglobulin production by IL-12 in murine chronic graft-vs.-host disease: synergism with IL-18. Eur J Immunol 1998; 28:2017–2024. 67. Seung E, Iwakoshi N, Woda BA, et al. Allogeneic hematopoietic chimerism in mice treated with sublethal myeloablation and anti-CD154 antibody: absence of graft-versus-host disease, induction of skin allograft tolerance, and prevention of recurrent autoimmunity in islet-allografted NOD/Lt mice. Blood 2000; 95:2175–2182. 68. Liu H, Mao N, Hou C, Li X, Shen B, Tang PH. Protective effect of human CD40-Ig fusion protein in a murine model of acute graft-versus-host disease. Chin Med J (Engl) 2001; 114:685–689. 69. Via CS, Rus V, Nguyen P, Linsley P, Gause WC. Differential effect of CTLA4Ig on murine graftversus-host disease (GVHD) development: CTLA4Ig prevents both acute and chronic GVHD development but reverses only chronic GVHD. J Immunol 1996; 157:4258–4267. 70. Saito K, Yagita H, Hashimoto H, Okumura K, Azuma M. Effect of CD80 and CD86 blockade and anti-interleukin-12 treatment on mouse acute graft-versus-host disease. Eur J Immunol 1996; 26:3098–3106. 71. Blazar BR, Taylor PA, Panoskaltsis-Mortari A, Sharpe AH, Vallera DA. Opposing roles of CD28:B7 and CTLA-4:B7 pathways in regulating in vivo alloresponses in murine recipients of MHC disparate T cells. J Immunol 1999; 162:6368–6377. 72. Lang TJ, Nguyen P, Peach R, Gause WC, Via CS. Vivo CD86 blockade inhibits CD4CT cell activation, whereas CD80 blockade potentiates CD8CT cell activation and CTL effector function. J Immunol 2002; 168:3786–3792. 73. Schlegel PG, Aharoni R, Smilek DE, et al. Prevention of graft-versus-host disease by peptides binding to class II major histocompatibility complex molecules. Blood 1994; 84:2802–2810. 74. Schlegel PG, Aharoni R, Chen Y, et al. A synthetic random basic copolymer with promiscuous binding to class II major histocompatibility complex molecules inhibits T-cell proliferative responses to major and minor histocompatibility antigens in vitro and confers the capacity to prevent murine graft-versus-host disease in vivo. Proc Natl Acad Sci USA 1996; 93:5061–5066. 75. Cooke KR, Hill GR, Gerbitz A, et al. Hyporesponsiveness of donor cells to lipopolysaccharide stimulation reduces the severity of experimental idiopathic pneumonia syndrome: potential role for a gut-lung axis of inflammation. J Immunol 2000; 165:6612–6619. 76. Macfarlane DE, Manzel L. Antagonism of immunostimulatory CpG-oligodeoxynucleotides by quinacrine, chloroquine, and structurally related compounds. J Immunol 1998; 160:1122–1131. 77. Lund J, Sato A, Akira S, Medzhitov R, Iwasaki A. Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells. J Exp Med 2003; 198:513–520. 78. Schultz KR, Su WN, Hsiao CC, et al. Chloroquine prevention of murine MHC-disparate acute graftversus-host disease correlates with inhibition of splenic response to CpG oligodeoxynucleotides and alterations in T-cell cytokine production. Biol Blood Marrow Transplant 2002; 8:648–655. 79. She K, Schultz KR. CpG responses in human chronic graft-versus-host disease CBMTG meeting abstract. June. 2004 2. 80. Daneshpouy M, Socie G, Lemann M, Rivet J, Gluckman E, Janin A. Activated eosinophils in upper gastrointestinal tract of patients with graft-versus-host disease. Blood 2002; 99:3033–3040. 81. Zhao ZZ, Savage NW, Sugerman PB, Walsh LJ. Mast cell/T cell interactions in oral lichen planus. J Oral Pathol Med 2002; 31:189–195. 82. Remberger M, Persson U, Hauzenberger D, Ringden O. An association between human leucocyte antigen alleles and acute and chronic graft-versus-host disease after allogeneic haematopoietic stem cell transplantation. Br J Haematol 2002; 119:751–759. 83. Gratwohl A, Hermans J, Niederwieser D, van Biezen A, van Houwelingen HC, Apperley J. Chronic leukemia working party of the European Group for blood and marrow transplantation EBMT. Female donors influence transplant-related mortality and relapse incidence in male recipients of sibling blood and marrow transplants. Hematol J 2001; 2:363–370.

104

Jacobsohn et al.

84. Rufer N, Wolpert E, Helg C, et al. HA-1 and the SMCY-derived peptide FIDSYICQV (H-Y) are immunodominant minor histocompatibility antigens after bone marrow transplantation. Transplantation 1998; 66:910–916. 85. Mutis T, Gillespie G, Schrama E, Falkenburg JH, Moss P, Goulmy E. Tetrameric HLA class I-minor histocompatibility antigen peptide complexes demonstrate minor histocompatibility antigenspecific cytotoxic T lymphocytes in patients with graft-versus-host disease. Nat Med 1999; 5:839–842. 86. Randolph SS, Gooley TA, Warren EH, Appelbaum FR, Riddell SR. Female donors contribute to a selective graft-versus-leukemia effect in male recipients of HLA-matched, related hematopoietic stem cell transplants. Blood 2004; 103:347–352. 87. Miklos DB, Kim HT, Zorn E, et al. Antibody response to DBY minor histocompatibility antigen is induced after allogeneic stem cell transplantation and in healthy female donors. Blood 2004; 103:353–359. 88. Gallardo D, Arostegui JI, Balas A, et al. GvHD subcommittee of the grupo espanol de trasplante hemapoyetico (GETH). Disparity for the minor histocompatibility antigen HA-1 is associated with an increased risk of acute graft-versus-host disease (GvHD) but it does not affect chronic GvHD incidence, disease-free survival or overall survival after allogeneic human leucocyte antigenidentical sibling donor transplantation. Br J Haematol 2001; 114:931–936. 89. Lin MT, Gooley T, Hansen JA, et al. Absence of statistically significant correlation between disparity for the minor histocompatibility antigen-HA-1 and outcome after allogeneic hematopoietic cell transplantation. Blood 2001; 98:3172–3173. 90. Nishida T, Akatsuka Y, Morishima Y, et al. Clinical relevance of a newly identified HLA-A24restricted minor histocompatibility antigen epitope derived from BCL2A1, ACC-1, in patients receiving HLA genotypically matched unrelated bone marrow transplant. Br J Haematol, 2004; 124: 629–35. 91. Grumet FC, Hiraki DD, Brown BWM, et al. CD31 mismatching affects marrow transplantation outcome. Biol Blood Marrow Transplant 2001; 7:503–512. 92. Vogt MH, van den Muijsenberg JW, Goulmy E, et al. The DBY gene codes for an HLA-DQ5restricted human male-specific minor histocompatibility antigen involved in graft-versus-host disease. Blood 2002; 99:3027–3032. 93. Korbling M, Anderlini P. Peripheral blood stem cell versus bone marrow allotransplantation: does the source of hematopoietic stem cells matter? Blood 2001; 98:2900–2908. 94. Tayebi H, Lapierre V, Saas P, et al. Societe francaise de greffe de moelle et de therapie cellulaire. Enhanced activation of B cells in a granulocyte colony-stimulating factor-mobilized peripheral blood stem cell graft. Br J Haematol 2001; 114:698–700. 95. Carion A, Domenech J, Herault O, et al. Decreased stroma adhesion capacity of CD34C progenitor cells from mobilized peripheral blood is not lineage- or stage-specific and is associated with low beta 1 and beta 2 integrin expression. J Hematother Stem Cell Res 2002; 11:491–500. 96. Lapidot T, Petit I. Current understanding of stem cell mobilization. The roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells. Exp Hematol 2002; 30:973. 97. Healey G, Schwarer AP. The helper T lymphocyte precursor (HTLp) frequency does not predict outcome after HLA-identical sibling donor G-CSF-mobilised peripheral blood stem cell transplantation. Bone Marrow Transplant 2002; 30:341–346. 98. Arpinati M, Green CL, Heimfeld S, Heuser JE, Anasetti C. Granulocyte-colony stimulating factor mobilizes T helper 2-inducing dendritic cells. Blood 2000; 95:2484–2490. 99. Klangsinsirikul P, Russell NH. Peripheral blood stem cell harvests from G-CSF-stimulated donors contain a skewed Th2 CD4 phenotype and a predominance of type 2 dendritic cells. Exp Hematol 2002; 30:495–501. 100. Shier LR, Schultz KR, Imren S, et al. Differential effects of granulocyte colony-stimulating factor on marrow- and blood-derived hematopoietic and immune cell populations in healthy human donors. Biol Blood Marrow Transplant 2004; 10:624–634. 101. Storek J, Dawson MA, Storer B, et al. Immune reconstitution after allogeneic marrow transplantation compared with blood stem cell transplantation. Blood 2001; 97:3380–3389. 102. Tayebi H, Tiberghien P, Ferrand C, et al. Societe francaise de greffe de moelle allogeneic peripheral blood stem cell transplantation results in less alteration of early T cell compartment homeostasis than bone marrow transplantation. Bone Marrow Transplant 2001; 27:167–175.

Chronic Graft-Versus-Host Disease in Children

105

103. Lapierre V, Auperin A, Tayebi H, et al. Societe francaise de greffe de moelle et de therapiue cellulaire. Increased presence of anti-HLA antibodies early after allogeneic granulocyte colonystimulating factor-mobilized peripheral blood hematopoietic stem cell transplantation compared with bone marrow transplantation. Blood 2002; 100:1484–1489. 104. Zaucha JM, Gooley T, Bensinger WI, et al. CD34 cell dose in granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cell grafts affects engraftment kinetics and development of extensive chronic graft-versus-host disease after human leukocyte antigen-identical sibling transplantation. Blood 2001; 98:3221–3227. 105. Rocha V, Carmagnat MV, Chevret S, et al. Influence of bone marrow graft lymphocyte subsets on outcome after HLA-identical sibling transplants. Exp Hematol 2001; 29:1347–1352. 106. Franzke A, Piao W, Lauber J, et al. G-CSF as immune regulator in T cells expressing the G-CSF receptor: implications for transplantation and autoimmune diseases. Blood 2003; 102:734–739. 107. Ringden O, Labopin M, Gorin NC, et al. Treatment with granulocyte colony-stimulating factor after allogeneic bone marrow transplantation for acute leukemia increases the risk of graft-versus-host disease and death: a study from the Acute leukemia working party of the European Group for blood and marrow transplantation. J Clin Oncol 2004; 22:416–423. 108. Jacobsen N, Badsberg JH, Lonnqvist B, et al. Graft-versus-leukaemia activity associated with CMV-seropositive donor, post-transplant CMV infection, young donor age and chronic graftversus-host disease in bone marrow allograft recipients. The Nordic bone marrow transplantation group. Bone Marrow Transplant 1990; 5:413–418. 109. Bostrom L, Ringden O, Jacobsen N, Zwaan F, Nilsson B. A European multicenter study of chronic graft-versus-host disease. The role of cytomegalovirus serology in recipients and donors–acute graft-versus-host disease, and splenectomy. Transplantation 1990; 49:1100–1105. 110. Delespesse G, Yang LP, Ohshima Y, et al. Maturation of human neonatal CD4C and CD8CT lymphocytes into Th1/Th2 effectors. Vaccine 1998; 16:1415–1419. 111. Kobayashi S, Ohnuma K, Uchiyama M, et al. Association of CD26 with CD45RA outside lipid rafts attenuates cord blood T-cell activation. Blood 2004; 103:1002–1010. 112. Hunt DW. Blood 1994; 84:4333–4343. Sorg RV, Kogler G, Wernet P. Identification of cord blood dendritic cells as an immature CD11c- population. Blood 1999; 93:2302–2307. 113. Sorg Ru¨diger V, Ko¨gler Gesine, Wernet Peter. Identification of Cord blood dendritic cells as an immature CD11c population. Blood 1999; 93:2302–2307. 114. Langrish CL, Buddle JC, Thrasher AJ, Goldblatt D. Neonatal dendritic cells are intrinsically biased against Th-1 immune responses. Clin Exp Immunol 2002; 128:118–123. 115. Tasker L, Marshall-Clarke S. Functional responses of human neonatal B lymphocytes to antigen receptor cross-linking and CpG DNA. Clin Exp Immunol 2003; 134:409–419. 116. Garban F, Ericson M, Roucard C, et al. Detection of empty HLA class II molecules on cord blood B cells. Blood 1996; 87:3970–3976. 117. Rocha V, Wagner JE, Jr., Sobocinski KA, et al. Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. Eurocord and international bone marrow transplant registry working committee on alternative donor and stem cell sources. N Engl J Med 2000; 342:1846–1854. 118. Woolfrey AE, Anasetti C, Storer B, et al. Factors associated with outcome after unrelated marrow transplantation for treatment of acute lymphoblastic leukemia in children. Blood 2002; 99:2002–2008. 119. Cutler C, Giri S, Jeyapalan S, et al. Acute and chronic graft-versus-host disease after allogeneic peripheral—blood stem-cell and bone marrow transplantation: a meta-analysis. J Clin Oncol 2001; 19:3685–3691. 120. Flowers ME, Parker PM, Johnston LJ, et al. Comparison of chronic graft-versus-host disease after transplantation of peripheral blood stem cells versus bone marrow in allogeneic recipients: longterm follow-up of a randomized trial. Blood 2002; 100:415–419. 121. Watanabe T, Kajiume T, Abe T, et al. Allogeneic peripheral blood stem cell transplantation in children with hematologic malignancies from HLA-matched siblings. Med Pediatr Oncol 2000; 34:171–176. 122. Benito AI, Gonzalez-Vicent M, Garcia F, et al. Allogeneic peripheral blood stem cell transplantation (PBSCT) from HLA-identical sibling donors in children with hematological diseases: a single center pilot study. Bone Marrow Transplant 2001; 28:537–543. 123. Rocha V, Cornish J, Sievers EL, et al. Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood 2001; 97:2962–2971.

106

Jacobsohn et al.

124. Slavin S, Nagler A, Naparstek E, et al. Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood 1998; 91:756–763. 125. Jacobsohn DA, Duerst R, Tse W, Kletzel M. Reduced intensity haemopoietic stem-cell transplantation for treatment of non-malignant diseases in children. Lancet 2004; 364:156–162. 126. Duerst R, Jacobsohn DA, Kletzel M. Reduced intensity conditioning (RIC) and allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric patients [abstract]. Blood 2003; 102:435b. 127. Horwitz ME, Barrett AJ, Brown MR, et al. Treatment of chronic granulomatous disease with nonmyeloablative conditioning and a T-cell-depleted hematopoietic allograft. N Engl J Med 2001; 344:881–888. 128. Zecca M, Prete A, Rondelli R, et al. Chronic graft-versus-host disease in children: incidence, risk factors, and impact on outcome. Blood 2002; 100:1192–1200. 129. Akpek G, Zahurak ML, Piantadosi S, et al. Development of a prognostic model for grading chronic graft-versus-host disease. Blood 2001; 97:1219–1226. 130. Akpek G, Lee SJ, Flowers ME, et al. Performance of a new clinical grading system for chronic graft-versus-host disease: a multicenter study. Blood 2003; 102:802–809. 131. Shulman HM, Sullivan KM, Weiden PL, et al. Chronic graft-versus-host syndrome in man. A longterm clinicopathologic study of 20 Seattle patients. Am J Med 1980; 69:204–217. 132. Lee SJ, Vogelsang G, Flowers ME. Chronic graft-versus-host disease. Biol Blood Marrow Transplant 2003; 9:215–233. 133. Sullivan KM, Witherspoon RP, Storb R, et al. Alternating-day cyclosporine and prednisone for treatment of high-risk chronic graft-v-host disease. Blood 1988; 72:555–561. 134. Lee SJ, Klein JP, Barrett AJ, et al. Severity of chronic graft-versus-host disease: association with treatment-related mortality and relapse. Blood 2002; 100:406–414. 135. Sullivan KM, Shulman HM, Storb R, et al. Chronic graft-versus-host disease in 52 patients: adverse natural course and successful treatment with combination immunosuppression. Blood 1981; 57:267–276. 136. Kawano N, Gondo H, Kamimura T, et al. Chronic graft-versus-host disease following varicellazoster virus infection in allogeneic stem cell transplant recipients. Int J Hematol 2003; 78:370–373. 137. Nagler A, Goldenhersh MA, Levi-Schaffer F, Bystryn JC, Klaus SN. Total leucoderma: a rare manifestation of cutaneous chronic graft- versus-host disease. Br J Dermatol 1996; 134:780–783. 138. Jacobsohn DA, Ruble K, Moresi JM, Vogelsang GB. Rapid-onset leucodrema associated with graftversus-host disease. Bone Marrow Transplant 2002; 30:705–706. 139. Ivker RA, Woosley J, Resnick SD. Dyskeratosis congenita or chronic graft-versus-host disease? A diagnostic dilemma in a child eight years after bone marrow transplantation for aplastic anemia Pediatr Dermatol 1993; 10:362–365. 140. Ling NS, Fenske NA, Julius RL, Espinoza CG, Drake LA. Dyskeratosis congenita in a girl simulating chronic graft-vs.-host disease. Arch Dermatol 1985; 121:1424–1428. 141. Vowels M, Chan LL, Giri N, Russell S, Lam-Po-Tang R. Factors affecting hair regrowth after bone marrow transplantation. Bone Marrow Transplant 1993; 12:347–350. 142. Locatelli F, Giorgiani G, Pession A, Bozzola M. Late effects in children after bone marrow transplantation: a review. Haematologica 1993; 78:319–328. 143. Andrews ML, Robertson I, Weedon D. Cutaneous manifestations of chronic graft-versus-host disease. Australas J Dermatol 1997; 38:53–62. 144. Akosa AB, Lampert IA. The sweat gland in graft versus host disease. J Pathol 1990; 161:261–266. 145. Janin A, Socie G, Devergie A, et al. Fasciitis in chronic graft-versus-host disease. A clinicopathologic study of 14 cases. Ann Intern Med 1994; 120:993–998. 146. Beredjiklian PK, Drummond DS, Dormans JP, et al. Orthopaedic manifestations of chronic graftversus-host disease. J Pediatr Orthop 1998; 18:572–575. 147. Stevens AM, Sullivan KM, Nelson JL. Polymyositis as a manifestation of chronic graft-versus-host disease. Rheumatology (Oxford) 2003; 42:34–39. 148. Stern JM, Chesnut CH, Bruemmer B, et al. Bone density loss during treatment of chronic GVHD. Bone Marrow Transplant 1996; 17:395–400. 149. Tauchmanova L, De Rosa G, Serio B, et al. A vascular necrosis in long-term survivors after allogeneic or autologous stem cell transplantation: a single center experience and a review. Cancer 2003; 97:2453–2461.

Chronic Graft-Versus-Host Disease in Children

107

150. Tichelli A, Duell T, Weiss M, et al. Late-onset keratoconjunctivitis sicca syndrome after bone marrow transplantation: incidence and risk factors. European Group or blood and marrow transplantation (EBMT) Working party on late effects. Bone Marrow Transplant 1996; 17:1105–1111. 151. Johnson DA, Jabs DA. The ocular manifestations of graft-versus-host disease. Int Ophthalmol Clin 1997; 37:119–133. 152. Ogawa Y, Kuwana M. Dry eye as a major complication associated with chronic graft-versus-host disease after hematopoietic stem cell transplantation. Cornea 2003; 22:S19–S27. 153. Schubert MM, Sullivan KM, Morton TH, et al. Oral manifestations of chronic graft-v-host disease. Arch Intern Med 1984; 144:1591–1595. 154. Nagler R, Marmary Y, Krausz Y, et al. Major salivary gland dysfunction in human acute and chronic graft-versus-host disease (GVHD). Bone Marrow Transplant 1996; 17:219–224. 155. Schubert MM, Sullivan KM. Recognition, incidence, and management of oral graft-versus-host disease. NCI Monographs 1990;135–143. 156. Hiroki A, Nakamura S, Shinohara M, Oka M. Significance of oral examination in chronic graftversus-host disease. J Oral Pathol Med 1994; 23:209–215. 157. Akpek G, Valladares JL, Lee L, Margolis J, Vogelsang GB. Pancreatic insufficiency in patients with chronic graft-versus-host disease. Bone Marrow Transplant 2001; 27:163–166. 158. Papadimitriou JC, Cangro CB, Lustberg A, et al. Histologic features of mycophenolate mofetilrelated colitis: a graft-versus-host disease-like pattern. Int J Surg Pathol 2003; 11:295–302. 159. Jacobsohn DA, Montross S, Anders V, Vogelsang GB. Clinical importance of confirming or excluding the diagnosis of chronic graft-versus-host disease. Bone Marrow Transplant 2001; 28:1047–1051. 160. Akpek G, Chinratanalab W, Lee LA, et al. Gastrointestinal involvement in chronic graft-versus-host disease: a clinicopathologic study. Biol Blood Marrow Transplant 2003; 9:46–51. 161. McDonald GB, Shulman HM, Sullivan KM, Spencer GD. Intestinal and hepatic complications of human bone marrow transplantation Part I. Gastroenterology 1986; 90:460–477. 162. Iqbal N, Salzman D, Lazenby AJ, Wilcox CM. Diagnosis of gastrointestinal graft-versus-host disease. Am J Gastroenterol 2000; 95:3034–3038. 163. Jacobsohn DA, Margolis J, Doherty J, Anders V, Vogelsang GB. Weight loss and malnutrition in patients with chronic graft-versus-host disease. Bone Marrow Transplant 2002; 29:231–236. 164. Zauner C, Rabitsch W, Schneeweiss B, et al. Energy and substrate metabolism in patients with chronic extensive graft-versus-host disease. Transplantation 2001; 71:524–528. 165. Arai S, Lee L, Vogelsang G. A systematic approach to hepatic complications in hematopoietic stem cell transplantation. J Hematother Stem Cell Res 2002; 11:215–230. 166. Strasser SI, Shulman HM, Flowers ME, et al. Chronic graft-versus-host disease of the liver: presentation as an acute hepatitis. Hepatology 2000; 32:1265–1271. 167. Akpek G, Boitnott JK, Lee LA, et al. Hepatitic variant of graft-versus-host disease after donor lymphocyte infusion. Blood 2002; 100:3903–3907. 168. Shulman HM, Sharma P, Amos D, Fenster LF, McDonald GB. A coded histologic study of hepatic graft-versus-host disease after human bone marrow transplantation. Hepatology 1988; 8:463–470. 169. Roca J, Granena A, Rodriguez-Roisin R, et al. Fatal airway disease in an adult with chronic graftversus-host disease. Thorax 1982; 37:77–78. 170. Crawford SW, Clark JG. Bronchiolitis associated with bone marrow transplantation. Clin Chest Med 1993; 14:741–749. 171. Clark JG, Schwartz DA, Flournoy N, et al. Risk factors for airflow obstruction in recipients of bone marrow transplants. Ann Intern Med 1987; 107:648–656. 172. Schultz KR, Green GJ, Wensley D, et al. Obstructive lung disease in children after allogeneic bone marrow transplantation. Blood 1994; 84:3212–3220. 173. Holland HK, Wingard JR, Beschorner WE, Saral R, Santos GW. Bronchiolitis obliterans in bone marrow transplantation and its relationship to chronic graft-v-host disease and low serum IgG. Blood 1988; 72:621–627. 174. Dudek AZ, Mahaseth H, Defor TE, Weisdorf DJ. Bronchiolitis obliterans in chronic graft-versushost disease: analysis of risk factors and treatment outcomes. Biol Blood Marrow Transplant 2003; 9:657–666. 175. Ratanatharathorn V, Ayash L, Lazarus HM, Fu J, Uberti JP. Chronic graft-versus-host disease: clinical manifestation and therapy. Bone Marrow Transplant 2001; 28:121–129. 176. Thompson AM, Couch M, Zahurak ML, Johnson C, Vogelsang GB. Risk factors for post-stem cell transplant sinusitis. Bone Marrow Transplant 2002; 29:257–261.

108

Jacobsohn et al.

177. Khouri IF, Ippoliti C, Gajewski J, Przepiorka D, Champlin RE. Neutropenias following allogeneic bone marrow transplantation: response to therapy with high-dose intravenous immunoglobulin. Am J Hematol 1996; 52:313–315. 178. Au WY, Lo CM, Hawkins BR, et al. Evans’ syndrome complicating chronic graft versus host disease after cadaveric liver transplantation. Transplantation 2001; 72:527–528. 179. Tomonari A, Tojo A, Lseki T, et al. Severe autoimmune thrombocytopenia after allogeneic bone marrow transplantation for aplastic anemia. Int J Hematol 2001; 74:228–232. 180. Sullivan KM, Witherspoon RP, Storb R, et al. Prednisone and azathioprine compared with prednisone and placebo for treatment of chronic graft-v-host disease: prognostic influence of prolonged thrombocytopenia after allogeneic marrow transplantation. Blood 1988; 72:546–554. 181. Akpek G, Zahurak ML, Piantadosi S, et al. Development of a prognostic model for grading chronic graft-versus-host disease. Blood 2001; 97:1219–1226. 182. Nevo S, Enger C, Hartley E, et al. Acute bleeding and thrombocytopenia after bone marrow transplantation. Bone Marrow Transplant 2001; 27:65–72. 183. Jacobsohn DA, Schechter T, Seshadri R, Thormann K, Duerst R, Kletzel M. Eosinophilia correlates with the presence or development of chronic graft-versus-host disease in children. Transplantation 2004; 77:1096–1100. 184. Siadak M, Sullivan KM. The management of chronic graft-versus-host disease. Blood Rev 1994; 8:154–160. 185. Storek J, Witherspoon RP, Webb D, Storb R. Lack of B cells precursors in marrow transplant recipients with chronic graft-versus-host disease. Am J Hematol 1996; 52:82–89. 186. Maury S, Mary JY, Rabian C, et al. Prolonged immune deficiency following allogeneic stem cell transplantation: risk factors and complications in adult patients. Br J Haematol 2001; 115:630–641. 187. Rege K, Mehta J, Treleaven J, et al. Fatal pneumococcal infections following allogeneic bone marrow transplant. Bone Marrow Transplant 1994; 14:903–906. 188. Kulkarni S, Powles R, Treleaven J, et al. Chronic graft versus host disease is associated with longterm risk for pneumococcal infections in recipients of bone marrow transplants. Blood 2000; 95:3683–3686. 189. Vogelsang GB. How I treat chronic graft-versus-host disease. Blood 2001; 97:1196–1201. 190. Wingard JR, Piantadosi S, Vogelsang GB, et al. Predictors of death from chronic graft-versus-host disease after bone marrow transplantation. Blood 1989; 74:1428–1435. 191. Vogelsang GB, Farmer ER, Hess AD, et al. Thalidomide for the treatment of chronic graft-versushost disease. N Engl J Med 1992; 326:1055–1058. 192. Parker PM, Chao N, Nademanee A, et al. Thalidomide as salvage therapy for chronic graft-versushost disease. Blood 1995; 86:3604–3609. 193. Cole CH, Rogers PC, Pritchard S, Phillips G, Chan KW. Thalidomide in the management of chronic graft-versus-host disease in children following bone marrow transplantation. Bone Marrow Transplant 1994; 14:937–942. 194. Rovelli A, Arrigo C, Nesi F, et al. The role of thalidomide in the treatment of refractory chronic graft-versus-host disease following bone marrow transplantation in children. Bone Marrow Transplant 1998; 21:577–581. 195. Sullivan KM. Acute and chronic graft-versus-host disease in man. Int J Cell Cloning 1986; 4:42–93. 196. Sullivan KM, Witherspoon RP, Storb R, et al. Prednisone and azathioprine compared with prednisone and placebo for treatment of chronic graft-v-host disease: prognostic influence of prolonged thrombocytopenia after allogeneic marrow transplantation. Blood 1988; 72:546–554. 197. Mookerjee B, Altomonte V, Vogelsang G. Salvage therapy for refractory chronic graft-versushost disease with mycophenolate mofetil and tacrolimus. Bone Marrow Transplant 1999; 24:517–520. 198. Yusuf U, Sanders JE, Stephan V. Mycophenolate Mofetil (MMF) as salvage treatment for steroidrefractory chronic graft-versus-host-disease (GVHD) in children [abstract]. Blood 2001; 98:398a. 199. Saven A, Piro L. Newer purine analogues for the treatment of hairy-cell leukemia. N Engl J Med 1994; 330:691–697. 200. Giblett ER, Anderson JE, Cohen F, Pollara B, Meuwissen HJ. Adenosine-deaminase deficiency in two patients with severely impaired cellular immunity. Lancet 1972; 2:1067–1069. 201. Saven A, Piro L. Newer purine analogues for the treatment of hairy-cell leukemia. N Engl J Med 1994; 330:691–697. 202. Jacobsohn DA, Margolis J, Chen AR, et al. Pentostatin: a promising treatment for refractory chronic GVHD [abstract]. Blood 2001; 98:399a. 203. Mackenzie AH. Pharmacologic actions of 4-aminoquinoline compounds. Am J Med 1983; 75:5–10.

Chronic Graft-Versus-Host Disease in Children

109

204. Olson NY, Lindsley CB. Adjunctive use of hydroxychloroquine in childhood dermatomyositis. J Rheumatol 1989; 16:1545–1547. 205. Easterbrook M. Ocular effects and safety of antimalarial agents. Am J Med 1988; 85:23–29. 206. Mackenzie AH. Dose refinements in long-term therapy of rheumatoid arthritis with antimalarials. Am J Med 1983; 75:40–45. 207. Ziegler HK, Unanue ER. Decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of antigen presentation to T cells. Proc Natl Acad Sci USA 1982; 79:175–178. 208. Salmeron G, Lipsky PE. Immunosuppressive potential of antimalarials. Am J Med 1983; 75:19–24. 209. Ertel W, Morrison MH, Ayala A, Chaudry IH. Chloroquine attenuates hemorrhagic shock-induced suppression of kupffer cell antigen presentation and major histocompatibility complex class II antigen expression through blockade of tumor necrosis factor and prostaglandin release. Blood 1991; 78:1781–1788. 210. Schultz KR, Bader S, Nelson D, Wang MD. Hay Glass KT. Immune suppression by lysosomotropic amines and cyclosporine on T-cell responses to minor and major histocompatibility antigens: does synergy exist? Transplantation 1997; 64:1055–1065. 211. Schultz KR, Nelson D, Bader S. Synergy between lysosomotropic amines and cyclosporin A on human T cell responses to an exogenous protein antigen, tetanus toxoid. Bone Marrow Transplant 1996; 18:625–631. 212. Gilman AL, Chan KW, Mogul A, et al. Hydroxychloroquine for the treatment of chronic graftversus-host disease. Biol Blood Marrow Transplant 2000; 6:327–334. 213. Heald P, Perez M, McKiernan G, Christiensen I, Edelson R. Extracorporeal photochemotherapy: indications, methodology, safety aspects, side effects and long-term results. Photodermatol 1989; 6:171–176. 214. Edelson R, Berger C, Gasparro F, et al. Treatment of cutaneous T-cell lymphoma by extracorporeal photochemotherapy Preliminary results. N Engl J Med 1987; 316:297–303. 215. Zic J, Arzubiaga C, Salhany KE, et al. Extracorporeal photopheresis for the treatment of cutaneous T-cell lymphoma. J Am Acad Dermatol 1992; 27:729–736. 216. Heald PW, Perez MI, Christensen I, et al. Photopheresis therapy of cutaneous T-cell lymphoma: the yale-new haven hospital experience. Yale J Biol Med 1989; 62:629–638. 217. Duvic M, Hester JP, Lemak NA. Photopheresis therapy for cutaneous T-cell lymphoma. J Am Acad Dermatol 1996; 35:573–579. 218. Lim HW, Edelson RL. Photopheresis for the treatment of cutaneous T-cell lymphoma. Hematol Oncol Clin North Am 1995; 9:1117–1126. 219. Salvaneschi L, Perotti C, Zecca M, et al. Extracorporeal photochemotherapy for treatment of acute and chronic GVHD in childhood. Transfusion 2001; 41:1299–1305. 220. Messina C, Locatelli F, Lanino E, et al. Extracorporeal photochemotherapy for paediatric patients with graft-versus-host disease after haematopoietic stem cell transplantation. Br J Haematol 2003; 122:118–127. 221. Gorgun G, Miller KB, Foss FM. Immunologic mechanisms of extracorporeal photochemotherapy in chronic graft-versus-host disease. Blood 2002; 100:941–947.

5 Cellular Engineering of the Hematopoietic Graft Ralph Quinones Pediatric Bone Marrow Transplantation, Center for Cancer and Blood Disorders, The Children’s Hospital and Department of Pediatrics, University of Colorado School of Medicine, Denver, Colorado, U.S.A.

INTRODUCTION The goal of all hematopoietic stem cell (HSC) processing is to provide a safe, high-quality HSC product that can support rapid and sustained in vivo hematologic recovery in the transplant patient. Processing of human HSC for clinical transplantation includes any manipulation of the cells between harvest and infusion. All HSC are processed to some extent, if solely to determine the product’s characteristics, quality, and safety. HSC may then be further processed prior to infusion, using procedures that involve minimal manipulation or those that involve extensive manipulations, which are defined as altering the infused cells compared to the harvested cells. Minimal manipulation processes are generally performed in order to avoid infusionrelated complications. Minimal manipulation procedures include: red blood cells (RBC) depletion to avoid major transfusion reactions or hemolysis after cryopreservation; plasma depletion to avoid minor transfusion reactions or allergic reactions; volume reduction due to patient size or cardiovascular status; reducing levels of anticoagulant or preservative; and the cryopreservation and thawing of HSC products for use in future hematopoietic stem cell transplantation (HSCT). Extensive manipulations are those that fundamentally alter the HSC product compared to its state at the time of harvest. Extensive manipulations usually involve selecting or depleting the HSC product of a defined cell population, such as T cells or tumor cells. More recently, extensive manipulations have focused on biologically altering the cell populations to be infused.

DEFINITIONS HSCs, also called hematopoietic progenitor cells, are cells that are capable of further differentiation and expansion into mature cells capable of hematologic function. HSC originally differentiate from mesodermal cells and can be in various states of differentiation. They have varying potential for further differentiation, both in their ability for self-replication and the types of cells into which they can differentiate. In vivo, the process of HSC differentiation is thought to be terminal and not usually reversible. There are recent data, from in vitro studies, in vivo studies in laboratory animals (1,2), and in human histologic samples, after transplantation (3) that challenge this hypothesis. Such de-differentiation or transdifferentiation of HSC into 111

112

Quinones

non-hematopoietic cells are active fields of basic research that could have wide-sweeping clinical implications (4). There is abundant evidence that in an individual, at any point in time, the process of hematopoiesis is stochastic, resulting from a finite number of active pluripotent HSC. Other pluripotent HSC remain cycling until they are recruited to differentiate and expand. The biologic signals that result in an individual pluripotent HSC either remaining in quiescent cycling versus committing to expansion and terminal differentiation are incompletely understood. In animal studies, it has been shown that although more terminally differentiated HSC can support hematopoiesis, this is for a finite time (i.e., limited potential for self-renewal), (5) as compared to the long-term repopulating HSC that can be transplanted through multiple generations. HSC can be obtained from different tissues. This has led to a number of terms for HSC and clinical HSCT (Table 1). Clinically, HSCT is the transplantation of HSC into a patient to support hematopoietic function. Regardless of the source of HSC, a major goal of clinical HSCT is the recovery of adequate, sustained in vivo hematopoietic or blood-making function. This is defined by the sustained production of functional cells of the myeloid, monocytoid, lymphoid, erythroid, and megakarycytoid lineages. In the setting of allogeneic HSCT, the goal is that hematopoiesis is of donor origin. Bone marrow, peripheral blood and cord blood (CB) are clinically proven HSC sources for HSCT. In the following discussions, HSC will be identified by their source (e.g., marrow HSC). HSC can also be obtained from various fetal tissues that developmentally support hematopoiesis in utero, such as fetal liver HSC that has been used for clinical HSCT, (6) but is not a common clinical source. The engrafting potential of a given HSC graft is often discussed in terms of cell doses per kilogram of recipient body weight. The doses that are usually of concern clinically are the total nucleated cell dose (TNC/kg), total mononuclear cell dose (TMNC/kg), the HSC-containing CD34C cells/kg (CD34C cells/kg), and graft-versus-host disease (GVHD)-causing T cells (T cells/kg). The HSC products collected from different sources vary widely in these cell doses (Table 2). In the setting of providing additional donor T cells as potential immune therapy against tumor or infection, a donor lymphocyte infusion (DLI) can be used and the dose is determined as the number of T cells infused (7–10). In HSC processing there are clear definitions for the level of processing (11). Minimally manipulated HSC are HSC processed in such a way that their biologic properties are not altered in comparison to their state at harvest. Such manipulations are usually performed to improve the safety of the HSC product for infusion or store them until needed for clinical HSCT.

Table 1 Common Names and Abbreviations in Clinical Hematopoietic Stem-Cell Transplantation Term used in this chapter

Abbreviation

Hematopoietic stem cells

HSC

Bone marrow hematopoietic stem cells Peripheral blood hematopoietic stem cells

BM HSC

Cord blood hematopoietic stem cells Hematopoietic stem-cell transplantation a

PBSC

CB HSC HSCT

Alternate term Hematopoietic progenitor cellsa Hematopoietic progenitor cells—marrowa Peripheral blood progenitor cells Hematopoietic progenitor cells—apheresisa Hematopoietic progenitor cells—cord blooda Blood and marrow transplantation Bone marrow transplantation

Foundation for Accreditation of Cellular Therapy (FACT) approved term and abbreviation.

Abbreviation HPCa HPC-Ma PBPC HPC-Aa HPC-Ca BMT BMT

Cellular Engineering of the Hematopoietic Graft

113

Table 2 Cell Doses in Clinical Allogeneic Hematopoietic Stem-Cell Transplantation by Hematopoietic Stem Cell Source Total nucleated cells/kg 2–5!108 2–5!109 2–10!107 –

Marrow HSC PBSC Cord blood HSC Donor lymphocyte infusion

CD34C cells/kg

T cells/kg

1–5!106 2–5!106 0.5–2C!105 –

0.5–3!108 1–3!109 0.5–3!107 0.1–5!108

Abbreviations: HSC, hematopoietic stem cell; PBSC, peripheral blood stem cells.

Extensively manipulated HSC are HSC that have been processed in such a way as to alter the biologic properties of the HSC graft as compared to their harvested state. The type of transplantation is defined by not only the source of the HSC, but the type of donor, which determines the risks of a given HSCT (Table 3). The use of patient’s own HSC is an autologous HSCT, regardless of the HSC source. If the HSC are from a monozygotic twin it is a syngeneic HSCT. Using HSC from another individual, other than a monozygotic twin is an allogeneic HSCT. Use of HSC from another species is xenogeneic HSCT, which is not a clinical practice at this time. Allogeneic HSCT is further defined as coming from an HLA identical sibling, a genotypically HLA matched allogeneic HSCT, a closely HLA matched relative, a partially HLA matched relative, or an unrelated HLA closely matched donor; unrelated allogeneic HSCT. The most significant barrier to allogeneic HSCT has been and is GVHD, which is an attack on host tissues by donor T cells sensitized to unshared host major histocompatibility antigens or minor histocompatibility antigens. GHVD is often accompanied by a graft-versus leukemia (GVL) effect that confers a significant therapeutic advantage in certain leukemias. In order to improve the quality and comparability between HSC products and in anticipation of Food and Drug Administration (FDA) regulatory oversight, clinicians, and scientists in the field of clinical HSCT and laboratory processing of HSC for clinical use, under the auspices of the International Society for Hematotherapy and Graft Engineering and the American Society of Blood and Marrow Transplantation, created the Foundation for Accreditation of Cellular Therapy (FACT) in 1996. FACT provides guidelines and a voluntary inspection and certification process for clinical HSCT programs, HSC collection sites and HSC processing laboratories, including CB banks (11,12). In parallel, and in collaboration with FACT, the American Association of Blood Banks (AABB) has developed standards for the collection and clinical processing of human HSC, including CB banks (13). The FACT and AABB standards have resulted in improved consistency and quality of HSC collected and processed for clinical use. A unique facet of the FACT accreditation is that the clinical HSCT program is an integral part of the process that must meet specific FACT standards. In the spring of 2005, the initial FDA regulations concerning Human Cells, Tissues, and Cellular and Tissue Based Products (HCT/P) were released (14). These include HSC from Table 3 Types of Hematopoietic Stem-Cell Transplantation as Defined in Clinical Allogeneic Hematopoietic Stem-Cell Transplantation by Hematopoietic Stem Cell Donor Source Type of donor Autologous Syngeneic Allogeneic

Source Self Identical twin Another person

Rejection risk None None Risk varies with HLA match

Abbreviation: GVHD, graft-versus-host disease.

GVHD risk

Immune deficiency

None None Risk varies with HLA match

3–6 months 3 months 6 to 24 months

114

Quinones

peripheral blood apheresis and CB sources but do not include marrow HSC. These regulations have as their primary focus the FDA’s mandate of preventing transmissible disease through the transplantation of a cellular or tissue products. The FDA regulations divide HSC processing based on the donor source. Minimally manipulated autologous or allogeneic grafts from first degree relatives are regulated less stringently, as compared to an unrelated donor or an extensively manipulated product. This provides a relatively greater degree of freedom in reassessing the risk of a first degree related or autologous donor deemed ineligible. For example, a positive serologic screen for a blood-borne pathogen that makes a donor ineligible can be further investigated to determine the risk of transmission of an infectious disease. In such as case, a very sensitive, negative assay (i.e., viral genome study) could provide sufficient data to justify the use HSC from that ineligible donor, where the total risk conferred by the donor is considerably less than the risks with an unrelated donor. In contrast, the identical ineligible seropositive screening test with a negative follow-up evaluation in an unrelated donor, who is the best available donor for the patient, presents a different scenario. Choosing an ineligible unrelated donor using the same reasoning for an exception could require justification to an FDA inspector. The Joint Commission on Accreditation of Healthcare Organizations now also has guidelines that closely parallel those of the FDA. The advances in clinical medicine and quality control and improvement continue to improve HSCT outcomes. The gold standard for all aspects of the HSCT process, starting with patient selection, the type of transplant chosen, the preparative regimen, graft harvesting, processing, and infusion, as well as supportive care, is the probability of eventfree survival (EFS).

Collection The collection of HSC for clinical transplantation requires specific expertise in donor selection, screening, and evaluation, as well as in the methods of HSC collection to ensure maximal safety of the donor, the HSC product and the HSCT recipient. Bone marrow was the initial source of human HSC successfully used for clinical HSCT (15–17). In steady-state human marrow there are sufficient primitive HSC to result in sustained hematopoiesis after HSC. Bone marrow can be harvested from a donor by multiple percutaneous aspirations under regional or general anesthesia, the latter being almost universal with pediatric donors. If there are no ABO incompatibilities, then the marrow can be infused without any further processing. The cell dose necessary for successful engraftment is 2–3!108 nucleated marrow cells/kg of the recipient in allogeneic HSCT, although there are data demonstrating improved clinical outcome with higher doses (18,19). In autologous HSCT the desired minimal marrow cell dose is 1!108 nucleated marrow cells/kg of the patient. Although larger autologous cell doses may promote more rapid engraftment, this advantage must be weighed against the risk of also harvesting contaminating tumor cells. Peripheral blood can also be a source HSC that can be efficiently obtained using apheresis devices that allow the processing of several blood volumes to collect a specific blood cell fraction. Apheresis can be used to collect stem cells, lymphocytes, platelets, and RBC. However the number of circulating HSC at rest are insufficient to be able to collect enough for HSCT, therefore different strategies have been used to “mobilize” peripheral blood HSC or Peripheral Blood Stem Cells (PBSC). The most frequently used strategy involves giving the donor recombinant hematopoietic growth factors, usually granulocyte-colony stimulating factor (G-CSF; Filgrastim) or granulocyte-monocyte colony-stimulating factor (GM-CSF; Sargramostim), usually for five days prior to harvest. In autologous PBSC, clinicians have been able to exploit a “rebound” effect of elevated circulating PBSC in the early recovery phase from intensive chemotherapy. This is usually enhanced by the use of G-CSF or GM-CSF. The HSC donor is then connected to an apheresis device using either a central venous catheter or large caliber percutaneous needles. Several blood volumes (3–6C) are processed through sterile, single-use disposable plastic tubing and containers (20–22). The apheresis device is set

Cellular Engineering of the Hematopoietic Graft

115

to harvest the mononuclear WBC fraction, which contains the HSC. In our center, the collection of PBSC from pediatric donors is an outpatient procedure using double lumen external central venous (Broviac) catheters in small children or larger short-term apheresis catheters placed by interventional radiology (23). The HSC content of a PBSC product is determined by the number of CD34C cells. CD34C cells are believed to contain most HSC and have been clinically correlated with the ability to engraft a recipient in both the autologous and allogeneic settings. In the setting of an allogeneic HSCT, the desired CD34C cell dose is at 5!106 CD34C cells/kg recipient (24). In a healthy, G-CSF mobilized allogeneic donor, sufficient PBSC can usually be harvested in one to two days of apheresis sessions. Most autologous donors are patients who have serious medical conditions, usually malignancies. Their HSC have often been compromised, either by the primary disease or by therapy used to control the primary disease. Both chemotherapy and irradiation result in significant HSC damage, which increases with both the repeated exposures and cumulative dose. As a result, the harvesting of autologous PBSC can require multiple daily apheresis sessions and potentially multiple rounds of mobilization and collection to obtain sufficient stem cells for HSCT, especially in the heavily pretreated cancer patient. In autologous PBSC, the minimal acceptable dose is 1–2!106 CD34C cells/kg of the patient, (25–27) although many transplant physicians prefer a higher dose (often O5!106 CD34C cells/kg) to hasten engraftment and decrease complications. There are patients in whom adequate PBSC can not be harvested. Such patients may require either autologous marrow HSC or an allogeneic HSCT. Umbilical CB was demonstrated in the 1980s to contain circulating HSC (28). CB HSC were found to be more potent on a per cell basis in in vitro colony-forming assays and were predicted to be able to result in sustained engraftment despite relatively small numbers (29). CB HSC are harvested immediately following a healthy infant’s birth by collecting the infant’s blood that remains in the cord and placenta after the cord is clamped and transected. CB HSC can achieve durable clinical engraftment across allogeneic barriers with a log lower cell numbers (O2!107 nucleated cells/kg and O1!105 CD34C cells/kg) compared with either BM HSC or PB HSC (Table 2). Successful clinical outcomes are correlated with nucleated cell/kg dose, with the optimal outcomes being with O3.7!107 nucleated cells/kg (30–34). The collection of autologous or sibling donor CB HSC requires advanced planning and consultation with an experienced CB bank. The dose of CB HSC is fixed by the amount of blood harvested at birth and may need to be supplemented by additional cells from a related donor. In unrelated CB donor banking, the mandated anonymity of the unrelated infant donor makes supplementation of HSC from that donor impossible. Recently, the infusion of either expanded CB or two CB units has been used to obtain a higher cell dose/kg with promising early clinical results (35,36). The use of two CB units to attain an adequate cell dose for adolescent and adults is currently being evaluated in multi-institutional trials. The inability to access a CB donor must also be considered and contrasted with the potential of requesting additional donor lymphocytes, BM or peripheral blood HSC from an unrelated volunteer adult donor if there is a high likelihood of needing DLI for a patient at very high-risk of relapse.

Processing General Principles of Hematopoietic Stem-Cell Processing and Specific Concerns in Pediatrics The primary goal of any HSC processing is to insure the safety and potential engrafting efficacy of the HSC product. In HSCT in adults and larger children, the primary concerns are preserving cell dose and viability, decreasing the potential of infectious complications, and avoiding significant complications when using an ABO mismatched graft. For this reason, every HSC donor should be the subject of appropriate screening and testing procedures for blood-borne pathogens prior to collection (Table 4). In allogeneic HSCT, HLA typing will determine the risk of GVHD and rejection risks for the transplant. The donor data are reviewed by the transplant physician to determine donor eligibility prior to collection. All HSC intended for

116 Table 4

Quinones Evaluation of a Prospective Hematopoietic Stem Cell Donor

Parameter History Past medical Infections HIV, CJD, West Nile virus, SARS Transfusions Transplants Pregnancy(s) Immunizations Travel Allergies Medications Family history Malignancy Donor risk history Examination General health Infection Malignancy AIDS Serologies Hep B (cAb; sAG) Hep Cb (Ab) HIVb HTLV Syphilis West Nile virusb CMV HLA type (allo only) ABO/Rh

Labs Pregnancy test LFTs a b

c

Eligible

Ineligible

Testing to allow an exception

None

Any

May need further testing to determine if infection is present &/or active (Further ID testing)

Nonea Nonea Nota

Any Any Pregnant

No recenta Nonea Nonea Nonea No riska

Recent Recent Present Medications Potential risk

None No risk

Yes Potential risk

Further ID testing Further ID testing Determine risk of donation on mom, fetus, & recipient Further ID testing Further ID testing Make recipient & physician aware Make recipient & physician aware Make recipient & physician aware. Possible further specific testing Further testing, inform donor & patient Make recipient & physician aware Further ID testing

Yes No No Noa

No Yes Yes Yes

Assess risk to donor & recipient Further testing, inform donor & patient Further testing, inform donor & patientc Further testing, inform donor & patientc

No No No No No No Noa Full match Compatible

Yes Yes Yes Yes Yes Yes Yes Partial match Incompatible

Futher ID testing, inform Futher ID testing, informc Futher ID testing, informc Futher ID testing, inform Futher ID testing, inform Futher ID testing, inform Futher ID testing, inform Assess risk to recipient and alternatives Assess infusion risk. Process HSC accordingly. Inform blood bank of special needs. Assess risk to engraftment

Negative

Positive

Normal

Abnormal

Determine risk of donation on mom, fetus, & recipient, inform Further ID testing, inform

Not an absolute criterion to determine a donor is ineligible. FDA-mandated nucleic acid testing to detect at risk individuals prior to the production of a specific antibody response. HIV, Hep C and Malignancy exclude all but autologous donors.

transplantation are qualified based on quantification of clinically relevant cell numbers, cell viability, and appropriate microbiologic testing upon arrival in the HSC processing lab, after significant manipulations and prior to cryopreservation and at the time of infusion. The cell numbers are calculated as doses per kilogram of recipient body weight and communicated to the transplant physician prior to proceeding with high-dose therapy. Additionally, in the setting

Cellular Engineering of the Hematopoietic Graft

117

of an ABO mismatched allogeneic HSC, depending on the disparities, the HSC graft may require specific processing to improve safety and efficacy of the infusion. Smaller pediatric patients require additional attention to increase the safety of infusing the HSC product. The collected or thawed volume may represent a significant volume challenge (O10 ml/kg) for very small patients or for those with cardiac or respiratory compromise. Simple volume reduction of the product allows for a recipient-specific infusion volume. Similarly, the volume of anticoagulant remaining in the graft may result in clinical anticoagulation. In our program, we further process an HSC graft to keep the infused dose of heparin less than 10 units/kg infused dose. This can be accomplished by volume reduction and washing of the HSC product. In cryopreserved HSC products, the dose of the cryoprotectant, DMSO, can lead to significant clinical complications including hypertension, headache, respiratory difficulties, and anaphylaxis (37–39). The half-life of DMSO is 24 hours (38), and this is prolonged when there is significant renal compromise. Our program’s policy is to infuse less than 1g DMSO/kg/24 hours, which occasionally necessitates infusing HSC over several days. Alternately, DMSO may be removed by washing the cryopreserved products, which is required when the patient has an anaphylactic reaction to, or is at high risk for, severe complications from DMSO.

MINIMALLY MANIPULATED PRODUCTS Processes that do not fundamentally alter the HSC graft with regard to TNCs, CD34C cells or other cell population’s content or function are considered minimal manipulation. Procedures to cryopreserve and then thaw viable HSC are minimal manipulation procedures as they do not fundamentally alter the biologic properties of HSC products. Minimal processing procedures also include volume reduction in the smaller patient or a patient with cardiac, pulmonary, or renal compromise; the reduction of heparin, DMSO content, RBC (major ABO incompatibility; patient antidonor RBC) or plasma (minor ABO incompatibility; donor antipatient RBC). These processes increase the safety of infusing these products. All HSC processing should be performed using clearly written standard operating procedures with clearly defined assessments and goals for the final product. Any deviations from these predetermined processes requires documentation, and if goal parameters are not met, discussion with the HSC laboratory director and the HSCT physician. In the setting of a major ABO incompatibility there is a significant risk of a lifethreatening hemolytic transfusion reaction in which recipient anti-ABO antibodies would lyse infused donor RBC (i.e., an A donor into an O recipient with anti-A antibodies). This can trigger DIC and renal insufficiency (40,41). It is important to help define the risk by assessing the patient’s titer(s) against the donor’s ABO antigen(s). In most major ABO incompatible cases, it is safer and simpler to deplete the graft of the target donor RBC, using either density gradients or semiautomated differential centrifugation, often using a clinical apheresis device (42,43). Although both types of RBC depletion procedures are effective, density gradients become necessary with very small grafts for which there is inadequate volume and RBC mass to effect a good separation with semiautomated processes. In exceptional cases where a very high antidonor ABO/Rh titer may affect engraftment, reducing the recipient’s titer with plasmapheresis can also decrease the risk of hemolysis of the infused donor RBC (44,45). There is not a consensus of the maximal volume of incompatible RBC that can be safely infused. In our center the goal is to infuse less than 0.15 ml RBC/kg. When the donor and recipient are of differing ABO types, there needs to be collaboration with the transfusion medicine service and a predetermined hierarchy for the use of specific blood components to avoid hemolytic reactions and/or delayed engraftment (Table 5). In the setting of ABO minor incompatibility, where donor anti-ABO antibodies can trigger a hemolytic reaction by lysing recipient RBC, reducing the plasma volume is an effective intervention. This is usually accomplished by centrifugation in a sealed blood container (i.e., a blood bag), with the plasma “expressed” off in a closed system. If there is

118

Quinones

Table 5 Transfusion Guidelines in ABO Incompatible Allogeneic Hematopoietic Stem Cell Transplantation Recipients Donor A A B B AB AB AB O O O A B ABO-compatible Donor O A B AB

Recipient

Packed RBCs

B O A O A B O A B AB AB AB

O O O O A or O B or O O O O O A or O B or O

Recipient

Packed RBCs

O A B AB

O A, O B, O AB, A, B, O

Platelets (plasma)a AB, B, A, O A, AB, B, O AB, A, B, O B, AB, A, O AB, A, B, O AB, B, A, O AB, A, B, O A, AB, B, O B, AB, A, O AB, A, B, O AB, A, B, O AB, B, A, O Platelets (plasma)a O, A, B, AB A, AB, B, O B, AB, A, O AB, A, B, O

Note that special issues in product selection and processing need to be discussed and resolved between transfusion medicine and BMT attending physician (e.g., washing). a Plasma products in order of preference of use in order to decrease the risk of hemolysis. Abbreviation: RBC, red blood cells.

a very high titer ABO antibody, there may be the need for the addition of saline and a repeat centrifugation and plasma/saline removal, often referred to as “washing.” Cryopreservation of human HSC is possible using cryoprotectant chemicals, such as DMSO, that prevent macroscopic crystal formation with the ensuing rupture of cell membranes and consequent cell death. HSC undergo controlled-rate freezing in specially designed chambers that use computer-controlled injection of liquid nitrogen to lower the product slowly through the point of fusion at K98C, and then more rapidly to solid state at K75–908C, after which the product is transferred to liquid nitrogen storage in either a vapor or liquid phase. The length of time an HSC product can be cryopreserved and still support engraftment is unknown, however, patients have been reconstituted with cryopreserved stem cells stored for ten years. Thawing of cryopreserved cells is often performed on the transplant unit or at the bedside. The product is rapidly warmed in a body-temperature bath and then infused over a short period of time. If a product exceeds acceptable parameters for infusion (i.e., volume, DMSO, anticoagulant) or the patient has additional risks, then the product may be processed to reduce volume or remove additives. Freshly thawed autologous products are routinely infused without further processing; however, a patient can have a history of a severe DMSO reaction, often severe hypertension and/or bradycardia, or an anaphylactic reaction to DMSO. In this setting, a washing procedure can be used to remove DMSO without significant loss of thawed cells.

EXTENSIVELY MANIPULATED PRODUCTS Extensive manipulation of HSC products results in a fundamental alteration in the HSC product, either by altering the numbers of a specific cell population or by changing a cell

Cellular Engineering of the Hematopoietic Graft

119

population’s inherent biology. Three primary areas of extensive HSC product manipulation used in multiple centers include depletion of GVHD-causing T cells, purging of residual tumor cells from autografts, and in vitro HSC expansion. Although this is not a comprehensive list of extensive manipulations, these examples serve as a focus for the process of implementing such processing procedures and the difficulties in determining efficacy.

T-CELL DEPLETION TO PREVENT GRAFT-VERSUS-HOST DISEASE GVHD remains one of the major challenges in allogeneic HSCT, especially in older patients or when using unrelated or non-HLA identical family member donors (46,47). It has been well established using murine models that eliminating the immunocompetent T lymphocytes from the infused graft can prevent GVHD even across MHC barriers (48–50). This has also been demonstrated clinically by using a variety of methods to remove mature T cells from the graft and by using fetal tissue, at a stage prior to the development of mature T cells (6). The development of monoclonal antibodies (MAb) allowed confirmation of the pivotal role of T cells, which are necessary and sufficient to initiate the GVHD process (51,52). Such studies also elucidated the role of CD8CT cells that recognize antigen in the context of MHC class I molecules and are the primary effector cells in both MHC-matched, minor histocompatibility antigen directed GVHD and class I disparate GVHD. Similarly, it has been shown that CD4C T cells, which recognize antigens in the context of MHC class II molecules, are the primary effectors in class II disparate GVHD and a few minor histocompatibility antigens (52–55). These studies also emphasized that, as with all T cell mediated immunity, the synergy between CD4C and CD8CT cells results in a more vigorous and effective in vivo GVHD response, (52,53) making the depletion of both T-cell subsets desirable in many clinical settings. Observations in murine systems formed the rationale and basis for the initial clinical trials of T-cell depletion (TCD) to prevent GVHD. The initial clinical proof of principle came from Reisner et al., who used a combination of sequential agglutination with soybean lectin and sheep erythrocytes to remove T cells from HLA incompatible marrow to successfully transplant children with severe combined immunodeficiency and leukemia (56). TCD exploiting the differential binding to cell surface molecules expressed on T cells, but not by HSC using soybean agglutination or sheep erythrocyte rosetting, has been a clinically effective strategy for the last two decades (57); however, it is a complex and cumbersome process that relies on a variable biologic agent, the sheep RBC. The use of MAb specific for T cells has been explored as a more practical means of clinical TCD. Trials with multiple different TCD methodologies have identified many variables in clinical TCD (58). These variables include the T-cell population that express the molecule; the MAb’s affinity; the targeting strategy; the extent of TCD (or the infused T-cell dose); the donor type and HLA match; and the differing clinical settings, as defined by the patient’s disease and infection history. The success of TCD in preventing or significantly decreasing GVHD was also offset by the identification of significant new concerns, including graft failure, increase leukemic relapse, prolonged immunodeficiency, and opportunistic infections. Currently, there has not been a universally embraced clinical TCD strategy (Table 6). There are clinical data using MAb with specificities for CD2, CD3, CD4, CD5, CD6 CD7, CD8, the T-cell receptor complex, and CD52, alone, and in combinations; however, the choice of MAb specificity has not been clear. The most widely used MAb is the anti-CD52 MAb, Campath-1 (59), which targets T cells, B cells, natural killer (NK) cells, and monocytes. Campath-1H (humanized) also efficiently fixes human complement, eliminating the need for heterologous complement. With the notable exception of Campath-1H, most MAb are not effective for TCD when used without an exogenous second step (targeting strategy) to eliminate the T cells, such as heterologous complement or immunomagnetic beads. One of the most commonly used types of complement is rabbit serum prescreened for lytic efficacy and lack of toxicity to hematopoietic progenitors. Frequently, multiple cycles of complement-mediated lysis result in a greater log reduction. The use of a variable biologic agent, such as rabbit complement, necessitates additional screening for safety and efficacy that is cumbersome and

120 Table 6

Quinones T-Cell Depletion Methodologies

Targeting T-cell surface molecule Monoclonal antibodies CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD52, TcRab Complement-mediated lysis Immunotoxin Solid phase fixation Immunomagnetic beads Biotin-avidin columns Polyclonal antibodies (ATG, ALG) Soybean agglutinin E-rosetting (sheep erythrocytes) Soybean agglutinin CE-rosetting Physical separation Counterflow centrifugation elutriation Density gradients (albumin, percoll) Combined monoclonal antibody C pharmacological purging CD34 selection

expensive. Also there is the potential of variability between complement lots that can alter depletion efficiency, thereby impacting clinical results. Alternate MAb targeting strategies have focused on conjugating the MAb(s) either to a solid-phase that can be used to separate the T cells from the hematopoietic progenitors or to potent toxins. MAb have been conjugated to different solid-phase components, often polystyrene beads, that are then removed with immunomagnetic beads, columns, or differential density centrifugation (58). The use of MAb conjugated to toxins or immunotoxins uses the exquisite specificity of the monoclonal antibody to deliver a potent cellular toxin to the targeted cell. The most often used toxins—ricin, pseudomonas exotoxin, diphtheria toxin or their derivatives—are all very potent inhibitors of protein synthesis, resulting in complete inhibition and eventual cell death (60,61). Positive selection techniques can result in TCD by using MAb to select a CD34C HSC rich fraction (62–64). Finally, there are TCD methods that do not rely on MAb but on separating T cells based on their physical characteristics. Counter flow elutriation uses centripetal force against a high fluid flow to effectively separate T cells from HSC based on their size and density (65–67). This method separates T cells from HSC but does not lyse, poison, or functionally alter the T cells, allowing for the potential of recovering the T cells for add-back at the end of the depletion. The T cells are also available for cryopreservation for future donor T-cell infusions or to augment immune recovery, provide a GVL effect or a specific anti-infection effect, such as against EBV (68–70). The degree of TCD (determining the infused T-cell dose) was initially a challenge and has remained an issue as the donor source varies. This has been most effectively addressed using limiting dilution analysis, which allowed for the very sensitive quantification of residual functional T cells after a two to four log reduction (71,72). In TCD methods that physically deplete the graft of T cells at the time of processing, highly sensitive, rare event flow cytometry has also been useful (67). Using limiting dilution analyses, a dose of !1!105 T cells/kg was determined to be the GVHD threshold in recipients of TCD HLA identical marrow grafts who did not receive postgrafting immune suppression (73). This T-cell dose may also be protective in the setting of HLA disparity or the multiple minor histocompatibility mismatches seen in unrelated donor transplantation (74). Although GVHD was reduced in these clinical settings (with a T-cell dose of less than 1!105 T cells/kg), it was not completely eliminated. GVHD can be completely eliminated, however, in HLA mismatched and unrelated donor transplants with

Cellular Engineering of the Hematopoietic Graft

121

a greater degree of TCD (2.5–3 log depletions), often with the addition of postgrafting immune suppression (75). The use of G-CSF-mobilized PBSC from allogeneic donors results in durable allogeneic grafts and has become a focus for TCD. The significantly higher numbers of CD34C PBSC HSC that can be harvested, as compared to BM HSC, have allowed the clinical studies of extensively T-cell depleted, very high HSC dose allogeneic transplants across HLA barriers (62). Although TCD was effective in preventing or markedly decreasing life-threatening acute GVHD, not all TCD resulted in the elimination of chronic GVHD. In HLA identical HSCT, the incidence of chronic GVHD varied with the method of TCD used (58). In the setting of HLA disparity, the challenges of both acute and chronic GVHD are greater. That TCD can result in GVHD-free engraftment with long-term disease-free survival was demonstrated using extensively TCD (R3 logs) haploidentical parental marrow HSC in SCID patients (76). SCID patients are unable to reject the grafts and therefore need less immunoablative therapy, the risk of leukemic relapse. These variables all became significant clinical issues when extensive TCD in the HLA disparate setting was applied to patients with other non-SCID genetic diseases or leukemia, where increased graft rejection, reduced but still present GVHD, and increased leukemic relapse were all observed (77,78). Many of the clinical issues that resulted from broader applications of TCD allogeneic HSC technologies were, in retrospect, predicted by studies of murine immunobiology. Graft failure, either primary immunologic graft rejection or late graft failure with preserved mixed chimerism, has been observed after TCD allografting. This was unexpected in the setting of unmanipulated HLA identical sibling HSCT for leukemia, where the observed graft failure was w1% but occurred in up to 25% of recipients of TCD HLA identical grafts (79,80). Graft failure has been reported to be as high as 50% in the clinical setting of HLA disparate TCD grafts (81,82). In murine studies designed to study graft failure, engraftment varied with the degree of TCD and MHC disparity, with the frequency of graft failure increasing with either increasing TCD or greater genetic disparity or combinations of both (83,84). In these models graft failure could be overcome by either increasing the immune suppression or the infused HSC dose, or both. Similarly, the increased incidence of graft failure in the clinical setting has been overcome by additional immune suppression (higher TBI doses, adding Thiotepa or Cytarabine to TBI and Cyclophosphamide regimens and/or adding anti-T-cell serologic agents, such as ATG, Campath or anti-T cell MAb) (85–88), adding postgrafting immune suppression or increasing the CD34C HSC dose (89) or combining multiple approaches. The mechanism of late graft failure is poorly understood as there is often sustained mixed chimerism arguing against classic host immune-mediated graft rejection. A potential etiology could be damage to the host’s marrow microenvironment, from the primary disease, it’s therapies or immune or virally mediated damage. When the observations of successful TCD in SCID patients were applied to leukemic patients, there was a reported increase in leukemic relapse, which was most marked in CML and AML patients (77,78,90). These data are now viewed as part of the proof of the importance of GVL, which was initially observed in murine models as an antileukemic effect of allogeneic cells (91). In a few prospective and multiple retrospective analyses, the use of TCD is associated with a significant increase in the risk for leukemic relapse, especially in myeloid leukemias (77,78). A significant exception to these data are the results in adult AML patients receiving a soybean agglutination and sheep erythrocyte TCD graft after aggressive myeloablation and without postgrafting immune suppression in whom the DFS was O70% with a relapse rate !5% (92). The experience with CML is instructive, with effective TCD preventing GVHD-related mortality, but being associated with an unacceptably high incidence of relapse in HLA identical sibling HSCT (77). There was not a significant effect on survival or DFS compared to the non-TCD patients secondary to their GVHD-related mortality. Now, such relapsed patients would receive infusions of DLI to elicit a GVL effect as salvage therapy (7,69,93,94). In the setting of HLA identical HSCT for CML, depletion of selective T-cell subsets, such as CD8 depletion, are being investigated (95,96).

122

Quinones

Finally, extensive TCD has the potential to alter the pace of immune reconstitution, resulting in increased opportunistic infectious complications. All recipients of myeloablative allogeneic HSCT experience a period of profound lymphoid immunodeficiency. There is the potential that TCD can further impair this recovery by removing any potential for the transfer of immunocompetent T cells. Conversely, TCD can improve immune recovery by eliminating GVHD and its therapies, both of which are very immunosuppressive. To date studies have shown rapid immune recovery in young children in the absence of GVHD (97–99). In contrast, immune recovery is more delayed with increasing age, possibly due to relative thymic insufficiency. One often fatal, opportunistic infection that became apparent with the extensive use of TCD HLA nonidentical allografts have been B-cell lymphoproliferative disorders (LPD), which invariably involve EBV, usually in donor B cells (100). EBV-LPD is believed to occur due to the lack of EBV-specific CTL in the months following grafting. Adoptive immunotherapy with either donor lymphocytes or donor EBV-specific CTL can effectively treat EBV-LPD (101–103). More recently, the in vivo use of B cell specific MAb has been effective (104), without creating a risk for GVHD (as the infusion of donor lymphocytes does). Clinicians who use TCD allografts have recognized the risk of opportunistic infections due to the prolonged and profound T-cell immunodeficiency. This often has necessitated prolonged prophylaxis for PCP, surveillance for viral pathogens (such as EBV, CMV, and HHV6) and a high index of concern with rapid diagnostic and treatment responses to symptoms until there is documentation of adequate immune recovery using in vitro assessments of immune function. In some centers, DLI are being studied to support immune recovery (97,105). More recently, marrow-resident immunoregulatory T cells have been described in both mouse and humans, which when increased in frequency appear to down-regulate GVHD but not abrogate GVL (106). Based on in vitro data, techniques to augment such immunoregulatory cells are being implemented. These studies require the selection of this rare T-cell population using MAb, followed by stimulation with MAb and cytokines, and the expansion of these cells in culture using strict, reproducible clinical isolation conditions. As the field of allogeneic HSCT focuses more on the graft-versus-tumor effect, the use of selected, activated, and cultured products are being investigated. Examples include the use of dendritic cells in studies of antitumor vaccines or antigen-specific T cells to treat viral infections, EBV, CMV, or cancer (107–109).

PURGING OF TUMOR CELLS FROM AUTOLOGOUS HEMATOPOIETIC STEM CELLS In patients with malignancies that are refractory to conventional dose chemotherapy there have been demonstrated benefits to high-dose combination chemotherapy regimens that require HSC support to reduce treatment-related mortality from prolonged marrow aplasia (25). For most patients without a primary hematologic malignancy, autologous HSC provide the optimal rescue of hematopoietic function without risk of GVHD. In autologous HSCT, the most common reason for treatment failure is relapse of the malignancy. Two major concerns in autologous HSCT are the potential of reinfusing tumor cells contaminating the graft into a patient rendered potentially tumor-free by the high-dose chemotherapy and the inability to mount an effective anticancer immune response to their residual endogenous tumor cells. The latter is the subject of significant ongoing research in immunotherapy. The concern of tumor cells contaminating the graft has been long-standing in autologous HSCT. Initially, the hypothesis that was widely embraced was that tumor cells potentially contaminating the graft would contribute to an increased incidence of relapse, and purging the HSC would improve DFS. Although intuitively appealing, this hypothesis required a series of proofs, some of which are still incomplete. First, it had to be demonstrated that autologous HSC products contained viable tumor cells. Next, technologies needed to be developed to purge tumor cells while sparing HSC required for hematopoietic recovery. Parallel to the development of purging technologies, sensitive assays for residual tumor cells

Cellular Engineering of the Hematopoietic Graft

123

were required to detect the degree of tumor cell depletion, usually measured as a log10 reduction. Although these were considerable challenges, they were met with a variety of creative and effective solutions that have increased both our understanding of the tumor and our technologic armamentarium. The purging strategy that is optimal remains elusive and appears to vary with the malignancy and the targeting strategy. However, the greatest challenges have been in the clinical setting, where it had to be demonstrated that reinfused tumor cells could contribute to relapse and that tumor cell purging improved DFS. Inherent in these are the questions of how effective tumor cell depletion needs to be, or stated differently, how many tumor cells need to be infused to result in relapse. Clinically, the paramount barrier in most cases of autologous HSCT still remains relapse due to insufficiently effective in vivo tumor eradication in the patient. The concern of infusing viable tumor cells into a patient rendered tumor-free after highdose therapy is a logical one. Indeed studies of autologous HSCT in leukemia have show very high rates of relapse compared to allogeneic HSCT (110,111). This was especially true in ALL, where the allogeneic GVL effect is the least potent; implying that increased relapse following identical myeloablative therapy was due to reinfused tumor. Indeed purged autologous HSCT has shown benefit in only ALL patients that were highly selected by clinical criteria to have a low risk of a significant marrow leukemic burden (110,112). In some instances, it was possible to demonstrate that autologous HSC products contained viable tumor cells present at levels of 1–5% by simple careful histologic examination (a 1–2 log detection) (113–115). However, for many tumor types more sensitive techniques had to be developed. MAb specific for tumorassociated antigens provided these sensitive techniques, and these assays have served the dual purpose of assessing the HSC products at harvest and after purging. For many solid tumors this has been demonstrated primarily by immunohistochemistry and detailed histologic examination, which has provided a three to six log detection in neuroblastoma and breast cancer (116–118). In leukemia and some solid tumors, multicolor, rare event flow cytometry has been able to achieve leukemic detection with a sensitivity of two to four logs (119–121). Parallel advances in both the cytogenetics and the molecular biology of the leukemias and solid tumors have also contributed to ability to detect low levels of contaminating tumor cells. Although standard cytogenetics rarely contributed more than confirmation of histologic concern of rare leukemic cells (both one to two log detection), fluorescent in situ hybridization can provide an additional log of detection (122–124). Similarly, southern blot analysis, although sufficient to detect as low as 1–5% contaminating tumor cells, is inadequate to detect lower levels of contaminating cells before or after purging. The use of carefully controlled polymerase chain reaction (PCR) assays with oligonucleotide primers to specific or novel tumor genes has allowed for a six-log detection ability (1 in 106 cells). This is especially true when the PCR is for a novel gene product resulting from a tumor specific gene or chromosomal translocation, such as bcr/abl in CML, MLL/MF4 in infant leukemia, bcl-2/IgH in B cell lymphoma, EWS/FLI1 in Ewing’s sarcoma, or PAX3/FKHR in alveolar rhabdomyosarcoma (115,118,122,125–128). Although the above assays have provided exquisite sensitivity in detecting tumor cells, they could not address whether these cells were viable, which is especially important in tumor purging strategies that do not result in immediate elimination of tumor cells from the graft, such as immunotoxins or chemotherapeutic purging. It would be ideal to monitor tumor purging using sensitive clonogenic assays, either ex vivo or in vivo in immunodeficient rodent that will allow the growth of xenogeneic tumors; Although there has been considerable effort to establish such assays, these have remained elusive for most tumor types. For the few tumors where clonogenic assays have been developed, they demonstrated the ability to achieve multiple log kills of tumor cells while sparing HSC. Such assays, although useful for assessing the potential of a purging technology, are often impractical for routine prospective assessment of clinical grafts and have been difficult to reproduce, as well as being very labor and cost intensive (129,130). In summary, the results of refinements of many investigational methodologies has resulted in sensitive assays being developed that both convincingly demonstrate tumor cell contamination of autologous HSC, effective methods to eliminate many logs of tumor cells

124

Quinones

while preserving HSC, while also providing accurate assessments of the effectiveness of various tumor purging strategies. As many solid tumors had been know to metastasize to the marrow, marrow contamination with residual tumor cells below the level of detection by standard histologic examination was predicted and subsequently confirmed (114,117,131,132). There was initial hope that this could be circumvented by the use of autologous mobilized PBSC. However, using the same assays to assess autologous PBSC, there has been the consistent demonstration of tumor contamination, although usually at a lower level when compared to marrow in neuroblastoma, lymphoma, and Ewing’s and breast cancer (116,117,121,127). As the number of PBSC/kg collected is often an order of magnitude greater than marrow cells/kg, there is a concern that the infused tumor burden can be significant with PBSC and therefore may be improved by purging. The consistent demonstration of contaminating tumor cells in autologous HSC products provided justification to develop technologies to purge tumor cells. The primary principle of purging is to eliminate the greatest amount of tumor possible, while preserving HSC required for hematopoietic recovery. This can be approached by either negatively selecting or purging the graft of tumor cells or by the positive selection of HSC from the graft. Two major purging approaches have been extensively investigated. Immunologic purging, using the exquisite selectivity of MAb to target tumor cells, and pharmacologic purging, where an adequate therapeutic index between tumor kill and HSC toxicity can be achieved. As observed with the uses of MAb for TCD, there are a number of targeting strategies that have been used for purging (Table 7). MAb-mediated purging strategies have resulted in direct tumor cell elimination from the graft by complement-mediated lysis or fixation to beads, followed by immunomagnetic depletion or with column filtration (133–137). In the case of immunotoxins, and with some chemotherapeutic purges, the tumor cells initially remain viable, then progressively die (67,138). This creates unique challenges, which have primarily been solved by the use of clonogenic tumor cell line add-backs to normal HSC as a model to optimize the purging strategy. Limitations of such assessments are that tumor cell lines may differ from actual tumor cells in the level and/or homogeneity of targeted antigen expression, as well as their susceptibility to the specific targeting strategy (e.g., sensitivity to a toxin). Nevertheless, this has had to suffice when purged clinical grafts cannot be prospectively or retrospectively assessed. Multiple studies of purging in various tumors have shown that although a single MAb may result in excellent purging of a tumor cell line while preserving HSC, relapses can still occur. Relapse can be due to the outgrowth of a malignant clone that does not express the target Table 7

Tumor Purging Methodologies

Negative selection Immunologic-mediated by monoclonal antibodies Complement-mediated lysis Immunotoxin Solid phase fixation Immunomagnetic beads Biotin-avidin columns Pharmacologic 4-Hyoperoxicyclophophamide (4-HC) Mafosfamide Etoposide Methylprednisolone Combined monoclonal antibody C pharmacologic purging Positive selection CD34 selected Combined positive HSC selection C negative tumor purging Abbreviation: HSC, hematopoietic stem cell.

Cellular Engineering of the Hematopoietic Graft

125

antigen, expresses the antigen at a very low level, or modulates expression of the antigen by shedding or endocytosis. As with TCD, the use of multiple antibodies with varying specificities has improved purging efficiency. Similarly, multiple cycles of purging have consistently demonstrated greater log depletions of tumor cells, although there is the ever-present potential for the nonspecific loss of HSC. In pediatric HSCT, there have been and are ongoing trials to assess purging of autologous HSC in neuroblastoma, AML, T, and pre-B ALL and NHL (110,112,134,137). The use of chemotherapeutic purging of HSC requires careful selection of the agent, as the therapeutic index between HSC and tumor cells is usually not sufficient to have a welldepleted product without significant impairment of engraftment potential. Observations from studies by the Hopkins group were seminal to these efforts. They observed that although Cyclophosphamide was an effective in vivo agent against many tumors, its effects on hematopoietic cells were less profound and more transient than other chemotherapeutic drugs. Upon further investigation, they defined the cyclophosphamide metabolites that possessed these properties (139). One metabolite, 4-hydroxycyclophosphamide was identified as being spontaneously formed in vitro from its pro-drug, 4 hydroperoxycyclophosphamide (4-HC), and was highly active against tumor cells while seeming to spare HSC due to their high content of aldehyde dehydrogenase (139–141). When assessed for in vitro purging effectiveness, 4-HC could effectively purge AML cells from a graft, and although it also affected HSC, delaying engraftment, most patients nevertheless engrafted (142,143). There was initial hope that using technologies to positively select HSC from a tumorcontaminated graft could provide a universally applicable approach. There have been a number of strategies to use CD34 selection that have resulted in two plus log depletions of tumor cells (117,136,144). The combination of positive CD34 selection and negative tumor cell purging has resulted in significant log depletions (136,145). The use of a CD34CThy1C HSC subpopulation yielded tumor reduction below the limits of detection and has resulted in rapid autologous engraftment in a limited number of adults (146). Additional strategies to select HSC by in vitro culture techniques that do not support tumor cells have also been investigated (147). The greatest challenges have been in the clinical setting, where it needed to be demonstrated that reinfused tumor cells could contribute to relapse and that tumor cell purging could contribute to improved DFS. In order to determine how effective tumor cell depletion needs to be, one has to know how many tumor cells need to be infused to result in relapse, which remains unanswered to date. However, two seminal studies provided significant insights. Brenner and colleagues used gene-marking technologies in autografts for neuroblastoma and AML, which, demonstrated that in vivo relapse in patients could come from marked tumor cells re-infused in the graft (148,149). In other studies, Gribben et al. retrospectively analyzed autografts purged of B cell lymphoma and demonstrated superior survival in those patients receiving grafts that were purged to negativity as assessed by a very sensitive PCR technique (131). The logical interpretation of this study is that relapse came from the low level of tumor cells in the positive grafts. An alternate interpretation, however, is that the inability to completely purge a graft was a surrogate marker for a greater tumor burden in the patient that could not be eliminated in vivo by the high dose chemotherapy. This is a critique that remains in all studies of graft purging in autologous HSCT for high-risk malignancies. Although the results of syngeneic HSCT, which can be considered a “tumor-free” autograft (without the confusing effects of an allogeneic GVL) are superior to autologous HSCT (111), there are insufficient numbers of such studies to make definitive conclusions. Despite these caveats, the most recent published multi-institutional trial in neuroblastoma demonstrated superior survival for high dose chemotherapy with a purged autologous marrow graft (no nonpurged arm) and post-HSCT biotherapy with cisretinoic acid (134). The DFS for patients who received an autologous HSCT and cis-retinoic acid was 55% at three years compared to patients who received chemotherapy with cisretinoic acid with a DFS of 32% or only chemotherapy (DFS 18%). This has led to the current study that will compare purged PBSC to unpurged PBSC, which will also be

126

Quinones

analyzed by using very sensitive detection techniques for minimal residual tumor in all grafts (COG 3973). Despite significant laboratory and clinical research, the role of purging for a specific tumor type remains undecided with strong proponents on either side of the issue, and will only be resolved by well-designed, adequately powered, disease-specific, multiinstitutional studies.

TRANSLATIONAL RESEARCH AND THE FUTURE: GENE THERAPY AND STEM-CELL EXPANSION There have been great advances in the ability to genetically manipulate hematopoietic cells to study their biology and to modify function. At the same time there has also been progress in the ability to expand HSC able to provide long-term in vivo hematopoiesis. These advances have led to promising and ongoing clinical trials designed to provide the data to initially understand and then translate these experimental finding into changes in clinical HSCT practice. Initially, stable gene insertion was used to study the biology of engraftment and relapse after HSCT (150,151). However the goal has always been the stable insertion of functioning genetic material. The ability to correct a life-threatening inborn metabolic deficiency by inserting genes into autologous HSC offers the promise of curing these diseases without the risks of allogeneic HSCT. Genetic alteration of murine HSC is now a routine procedure in labs studying HSC biology to either model the correction of genetic deficiencies or to alter cell function (152–155). Clinical studies of genetic manipulation of HSC are more limited (149,156–159). Stable expression of the inserted gene in HSC can clinically correct defects in lympho-hematopoietic cells, specifically X-linked common gamma chain deficiency SCID or adenosine deaminase deficiency SCID (149,157–161). This technology could also be effective in metabolic diseases where, currently, allogeneic HSC can correct the disease be providing a body-wide cell population containing normal enzyme. There remain many issues in making gene therapy using HSC clinically practical. Most were anticipated and focus on optimizing the establishment of a long-term stable gene expression in vivo. However, unexpected events have already occurred in SCID patients where there was a recurring insertion of the common gamma chain receptor into LMO2, a known site of gene rearrangement in T-cell ALL. Several of the patients have developed T-cell ALL involving the LMO2 site (162,163). This resulted in the temporary cessation of trials of gene therapy in HSC. Knowledge of HSC biology has provided the ability to generate models where highly enriched or in vitro expanded pluripotent HSC resulted in sustained in vivo hematopoiesis (164–167). The benefits of clinical translation of HSC expansion could offer methodologies to provide tumor-free autografts, GVHD-free allogeneic grafts, or the ability to augment cell dose in CB HSCT. In addition, HSC expansion is often part of the gene therapy process. Pioneering clinical expansion studies in autologous HSCT (35,168,169) showed engraftment without toxicities. Clinical experiences with expanded allogeneic HSC are limited. In order to potentially augment the cell dose and improve engraftment, there have been efforts to expand CD34C unrelated CB cells (35,170). Other promising areas include the selection, expansion, and differentiation of functional hematopoietic cellular subpopulations. These are often able to promote a specific immunologic response using in vitro derived CTL or using antigen-pulsed dendritic cells to generate antitumor responses or immunoregulatory cells to down-regulate GVHD (103,106,171). The observations in experimental models that may transform the clinical practice of HSCT are those showing the potential for the transdifferentiation of HSCs into somatic cells, providing regeneration of cardiac muscle, pulmonary epithelium, neurons, and other cells once believed to have little regenerative potential (1,2,172). Assessing recipients of allogeneic HSCT has confirmed the existence of human donor-derived somatic cells (3,173). Although there are preliminary clinical trials in progress, clinical applications will require greater

Cellular Engineering of the Hematopoietic Graft

127

understanding of the basic biology of these cells, as well as the practical aspects of scaling up these models and translating them into practical clinical applications.

REGULATION As the field of HSCT has grown in the types of HSCT performed, the number of patients treated, and the number of centers performing HSCT; it became apparent that it was necessary to develop uniform standards to improve quality and comparability between HSC products processed at different institutions. HSCT clinicians, HSC processing labs, scientists, and experts in transfusion medicine collaborated to establish standards and implement an external oversight process. These collaborative efforts resulted in the creation of the FACT. The initial FACT Standards were published in 1996 and updated in 2000 (11). FACT collaborated with the AABB (13) in designing these standards, and although each group has its own standards, they concur in most of the fundamental aspects. Most recently, FACT collaborated with the European-based Netcord group to establish comprehensive standards for CB banking (12). FACT standards address: (1) Terminology and definitions; (2) Clinical Program Standards; (3) HSC Collection Standards; (4) HSC Processing Standards; and (5) Quality Management of the above processes. FACT offers voluntary inspection for clinical HSCT programs, HSC collection, and HSC processing laboratories, including CB banks, and can grant accreditation to programs and laboratories meeting the rigorous FACT standards. Accreditation is time-limited and institutions may apply for reaccreditation. These standards have resulted in improved consistency and quality and are continually monitored to ensure that HSC products result in consistent patient engraftment. Although not mandating specific techniques, many of the standards move processing toward GMP (good manufacturing process), which due to the variability of individually harvested biologic products, is inherently more difficult to fully standardize in comparison, to the pharmaceutical industry. The FACT Standards and process were also created in anticipation of FDA oversight and FACT has been instrumental in providing expert guidance to the process of establishing Federal regulations. In the spring of 2005, the FDA regulations pertaining to HCT/P went into effect. These regulations (21 CFR part 1271) (14) are different for allogeneic and autologous HSC, especially allografts from other than immediate family members. These regulations pertain to HSC from PBSC and CB products but not marrow HSC. The regulations have as their primary focus the FDA’s mandate of preventing transmissible disease through the transplantation of a cellular or tissue product. These regulations include the widely used, FDA-mandated donor serologic testing, donor screening for risk of blood transmissible diseases, and microbiologic cultures (Table 4), and also mandate an extensive in depth process control of essentially all aspects of HSC collection, processing, and distribution. Under the FDA regulations, the distributing HSC processing lab will continue to ensure that HSC are acceptable for clinical HSCT. The laboratory will track any reagents and devices (syringes, blood bags, needles, etc.) that are in direct contact with the HSC product and that all testing done to determine HSC acceptability for clinical transplantation were preformed using FDA-approved reagents. HSC laboratories must also be able to backtrack to individual reagent or component lot numbers. In summary, HSCT has evolved from an experimental therapy to a well accepted clinical therapy that is continually changing and evolving, based on ongoing research and understanding of tumor and transplantation biology and supportive care. Similarly, the processing of HSC grafts has also evolved and moved from the research laboratory setting to a clinical laboratory setting. It is in this context that the quality of the HSC product needs to be as assured so as to offer patients undergoing these life-threatening procedures and the physicians caring for them the confidence in the product’s ability to result in safe engraftment with the minimal possible risk. HSC processing is at the cutting edge of biomedical knowledge and innovation, which remains the cornerstone of our ability to offer more patients safer HSCT.

128

Quinones

Innovative HSC processing will still originate in the research laboratory and a prominent role for most HSC processing laboratories will be to translate these advances into reproducible, clinical-scale products.

REFERENCES 1. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001; 410:701–705. 2. Jiang Y, Vaessen B, Lenvik T, et al. Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp Hematol 2002; 30:896–904. 3. Quaini F, Urbanek K, Beltrami AP, et al. Chimerism of the transplanted heart. N Engl J Med 2002; 346:5–15. 4. Orlic D. BM stem cells and cardiac repair: where do we stand in 2004? Cytotherapy 2005; 7:3–15. 5. Jones RJ, Wagner JK, Celano P, Zicha MS, Sharkis SJ. Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells. Nature 1990; 347:188–189. 6. Touraine JL, Raudrant D, Laplace S. Transplantation of hemopoietic cells from the fetal liver to treat patients with congenital diseases postnatally or prenatally. Transplant Proc 1997; 29:712–713. 7. Mackinnon S, Papadopoulos EB, Carabasi MH, et al. Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia responses from graft-versus-host disease. Blood 1995; 86:1261–1268. 8. Slavin S. Donor lymphocyte infusions for hematopoietic malignancy. Cancer Chemother Biol Response Modif 2002; 20:291–299. 9. Giralt S, Hester J, Huh Y, et al. CD8-depleted donor lymphocyte infusion as treatment for relapsed chronic myelogenous leukemia after allogeneic bone marrow transplantation. Blood 1995; 86:4337–4343. 10. Aker M, Kapelushnik J, Pugatsch T, et al. Donor lymphocyte infusions to displace residual host hematopoietic cells after allogeneic bone marrow transplantation for beta-thalassemia major. J Pediatr Hematol Oncol 1998; 20:145–148. 11. Standards for Hematopoietic Progenitor Cell Collection, Processing and Transplantation. 2nd ed. North America: FACT, 2002. 12. International Standards for Cord Blood Collection, Processsing, Testing, Banking, Selection and Release. 2nd ed. U.S.A.: Fahct Netcord: NB, 2001. 13. Standards for Cellular Therapy Product Services. 1st ed. U.S.A.: American Association of Blood Banks, 2004. 14. FDA. 21 CFR Part 1271 Human Cells, Tissues, and Cellular and Tissue Based Products. 2004. 15. Bach FM, Albertini RJ, Anderson JL, Joo P, Bortin MM. Bone marrow transplantation in a patient with the Wiskott-Aldrich syndrome. Lancet 1968; 2:1364. 16. Gatti RA, Meuwissen JJ, Allen HD, Hong R, Good RA. Immunologic reconstitution of sex-linked lymphopenic immunologic deficiency. Lancet 1968; 1:1366–1369. 17. Thomas ED, Storb R, Clift RA, et al. Bone marrow transplantation. N Engl J Med 1975; 292:832. 18. Deeg HJ, Self S, Storb R, et al. Decreased incidence of marrow graft rejection in patients with severe aplastic anemia: changing impact of risk factors. Blood 1986; 68:1363–1368. 19. Niederwieser D, Pepe M, Storb R, Loughran TP, Jr., Longton G. Improvement in rejection, engraftment rate and survival without increase in graft-versus-host disease by high marrow cell dose in patients transplanted for aplastic anemia. Br J Haematol 1988; 69:23–28. 20. Watanabe T, Kawano Y, Watanabe A, Takaue Y. Autologous and allogeneic transplantation with peripheral blood CD34C cells: a pediatric experience. Haematologica 1999; 84:167–176. 21. Rowley SD, Yu J, Gooley T, et al. Trafficking of CD34C cells into the peripheral circulation during collection of peripheral blood stem cells by apheresis. Bone Marrow Transplant 2001; 28:649–656. 22. Diaz MA, Alegre A, Benito A, Villa M, Madero L. Peripheral blood progenitor cell collection by large-volume leukapheresis in low-weight children. J Hematother 1998; 7:63–68. 23. Harned RK, Kelly SS, Foreman NK, Giller RH. Peripheral placement of apheresis catheters in children: feasibility, safety, and efficacy in the collection of blood stem cells—initial experience. Radiology 2001; 218:294–298.

Cellular Engineering of the Hematopoietic Graft

129

24. Kawano Y, Watanabe T, Takaue Y. Mobilization/harvest and transplantation with blood stem cells, manipulated or unmanipulated. Pediatr Transplant 1999; 3:65–71. 25. Anderson KC. Autologous peripheral blood progenitor cell transplantation. J Clin Apher 1995; 10:131–138. 26. Bensinger WI, Longin K, Appelbaum F, et al. Peripheral blood stem cells (PBSCs) collected after recombinant granulocyte colony stimulating factor (rhG-CSF): an analysis of factors correlating with the tempo of engraftment after transplantation. Br J Haematol 1994; 87:825–831. 27. Shpall EJ, Champlin R, Glaspy JA. Effect of CD34C peripheral blood progenitor cell dose on hematopoietic recovery. Biol Blood Marrow Transplant 1998; 4:84–92. 28. Broxmeyer HE, Douglas GW, Hangoc G, et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA 1989; 86:3828–3832. 29. Broxmeyer HE, Hangoc G, Cooper S, et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci USA 1992; 89:4109–4113. 30. Gluckman E. Current status of umbilical cord blood hematopoietic stem cell transplantation. Exp Hematol 2000; 28:1197–1205. 31. Laughlin MJ, Barker J, Bambach B, et al. Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors. N Engl J Med 2001; 344:1815–1822. 32. Migliaccio AR, Adamson JW, Stevens CE, et al. Cell dose and speed of engraftment in placental/umbilical cord blood transplantation: graft progenitor cell content is a better predictor than nucleated cell quantity. Blood 2000; 96:2717–2722. 33. Rocha V, Labopin M, Sanz G, et al. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 2004; 351:2276–2285. 34. Rubinstein P, Carrier C, Scaradavou A, et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 1998; 339:1565–1577. 35. Shpall EJ, Quinones R, Giller R, et al. Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant 2002; 8:368–376. 36. Barker JN, Weisdorf DJ, DeFor TE, et al. Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 2005; 105:1343–1347. 37. Davis J, Rowley SD, Santos GW. Toxicity of autologous bone marrow graft infusion. Prog Clin Biol Res 1990; 333:531–540. 38. Egorin MJ, Rosen DM, Sridhara R, Sensenbrenner L, Cottler-Fox M. Plasma concentrations and pharmacokinetics of dimethylsulfoxide and its metabolites in patients undergoing peripheral-blood stem-cell transplants. J Clin Oncol 1998; 16:610–615. 39. Rowley SD. Hematopoietic stem cell cryopreservation: a review of current techniques. J Hematother 1992; 1:233–250. 40. Petz LD. Immunohematologic problems associated with bone marrow transplantation. Transfus Med Rev 1987; 1:85–100. 41. Warkentin PI. Transfusion of patients undergoing bone marrow transplantation. Hum Pathol 1983; 14:261–266. 42. Koristek Z, Mayer J. Bone marrow processing for transplantation using the COBE spectra cell separator. J Hematother Stem Cell Res 1999; 8:443–448. 43. Karandish S, Luban N, Gutierrez R, et al. Automated marrow mononuclear cell enrichment using the Fenwall CS3000 for pediatric bone marrow transplantation [abstract]. ISBT/AABB joint congress 1990; S714:179. 44. Lasky LC, Warkentin PI, Kersey JH, et al. Hemotherapy in patients undergoing blood group incompatible bone marrow transplantation. Transfusion 1983; 23:277–285. 45. Warkentin PI, Hilden JM, Kersey JH, Ramsay NK, McCullough J. Transplantation of major ABOincompatible bone marrow depleted of red cells by hydroxyethyl starch. Vox Sang 1985; 48:89–104. 46. Ferrara JL, Yanik G. Acute graft versus host disease: pathophysiology, risk factors, and prevention strategies. Clin Adv Hematol Oncol 2005; 3:415–419 see also 428. 47. Bolanos-Meade J, Vogelsang GB. Acute graft-versus-host disease. Clin Adv Hematol Oncol 2004; 2:672–682. 48. Tyan ME. Modification of severe graft-versus-host-disease with anti-sera to the theta antigen or to whole serum. Transplantation 1973; 15:601.

130

Quinones

49. Korngold R, Sprent J. Lethal graft-versus-host disease after bone marrow transplantation across minor histocompatibility barriers in mice. Prevention by removing mature T cells from marrow. J Exp Med 1978; 148:1687–1698. 50. Vallera DA, Youle RJ, Neville DM, Kersey JH. Bone marrow transplantation across major histocompatibility barriers. V. Protection of mice from lethal GVHD by pretreatment of donor cells with monoclonal anti-Thy-1.2 coupled to the toxic lectin, ricin. J Exp Med 1982; 155:949–954. 51. Blazar BR, Taylor PA, Vallera DA. CD4C and CD8CT cells each can utilize a perforin-dependent pathway to mediate lethal graft-versus-host disease in major histocompatibility complex-disparate recipients. Transplantation 1997; 64:571–576. 52. Pietryga DW, Blazar BR, Soderling CCB, Vallera DA. The effect of T-subset depletion on the incidence of lethal graft versus host disease in a murine major histocompatibility complex mismatched transplantation system. Transplantation 1987; 43:442. 53. Korngold R, Sprent J. T cell subsets and graft-versus-host disease. Transplantation 1987; 44:335–339. 54. Korngold R, Sprent J. Features of T cells causing H-2-restricted lethal graft-versus-host disease across minor histocompatibility barriers. J Exp Med 1982; 155:872–883. 55. Meuer SC, Schlossman SF, Reinherz EL. Clonal analysis of human cytotoxic T lymphocytes: T4C and T8C effector T cells recognize products of different major histocompatibility complex regions. Proc Nat Acad Sci USA 1982; 79:4395–4399. 56. Reisner Y, Kapoor N, Kirkpatrick D, et al. Transplantation for severe combined immunodeficiency with HLA-A, B, D, DR incompatible parental marrow cells fractionated by soybean agglutinin and sheep red blood cells. Blood 1983; 61:341–348. 57. O’Reilly RJ. Immunologic aspects of hematopoietic stem-cell transplantation. Cytotherapy 2002; 4:431–432. 58. Collins NH, Fernandez JM. T-cell depletion and manipulation in allogeneic hematopoietic cell transplantation. Immunomethods 1994; 5:189–196. 59. Waldmann H, Hale G, Cividalli G, et al. Elimination of graft-versus-host disease by in-vitro depletion of alloreactive lymphocytes with a monoclonal rat anti-human lymphocyte antibody (CAMPATH-1). Lancet 1984;1984. 60. Vallera DA, Quinones RR, Azemove SM, Soderling CCB. Monoclonal antibody-toxin conjugates reactive against human T lymphocytes. A comparison of antibody linked to intact ricin toxin with antibody linked to ricin A chain. Transplantation 1984; 37:387–392. 61. Filipovich AH, Ramsay NKC, Arthur DC, et al. Allogeneic bone marrow transplantation with related donors other than HLA MLC-matched siblings, and the use of antithymocyte globulin, prednisone, and methotrexate for prophylaxis of graft-versus-host disease. Transplantation 1985; 39:282–285. 62. Tabilio A, Falzetti F, Zei T, et al. Graft engineering for allogeneic haploidentical stem cell transplantation. Blood Cells Mol Dis 2004; 33:274–280. 63. Aversa F, Martelli MF. Transplantation of haploidentically mismatched stem cells for the treatment of malignant diseases. Springer Semin Immunopathol 2004; 26:155–168. 64. Cornelissen JJ, van der HB, Petersen EJ, et al. A randomized multicenter comparison of CD34(C)selected progenitor cells from blood versus from bone marrow in recipients of HLA-identical allogeneic transplants for hematological malignancies. Exp Hematol 2003; 31:855–864. 65. DeWitte T, Hoogenhout J, DePauw B, et al. Depletion of donor lymphocytes by counterflow centrifugation successfully prevents acute graft-versus-host disease in matched allogeneic marrow transplantation. Blood 1986; 67:1302–1308. 66. Wagner JE, Santos GW, Noga SJ, et al. Bone marrow graft engineering by counterflow centrifugal elutriation: results of a phase I-II clinical trial. Blood 1990; 75:1370–1377. 67. Quinones RR, Gutierrez RH, Dinndorf PA, et al. Extended cycle elutriation to adjust T cell content in HLA disparate bone marrow transplantation. Blood 1993; 82:307–317. 68. Schaap N, Schattenberg A, Bar B, et al. Induction of graft-versus-leukemia to prevent relapse after partially lymphocyte-depleted allogeneic bone marrow transplantation by pre-emptive donor leukocyte infusions. Leukemia 2001; 15:1339–1346. 69. Drobyski WR, Hessner MJ, Klein JP, et al. T-cell depletion plus salvage immunotherapy with donor leukocyte infusions as a strategy to treat chronic-phase chronic myelogenous leukemia patients undergoing HLA-identical sibling marrow transplantation. Blood 1999; 94:434–441. 70. Ferra C, Rodriguez-Luaces M, Gallardo D, et al. Individually adjusted prophylactic donor lymphocyte infusions after CD34-selected allogeneic peripheral blood stem cell transplantation. Bone Marrow Transplant 2001; 28:963–968.

Cellular Engineering of the Hematopoietic Graft

131

71. Moretta A, Pantaleo G, Moretta L, Cerottini JC, Mingari MC. Direct demonstration of the clonogenic potential of every human peripheral blood T cell. Clonal analysis of HLA-DR expression and cytolytic activity. J Exp Med 1985; 157:743–754. 72. Kernan NA, Flomenberg N, Collins NH, O’Reilly RJ, Dupont B. Quantitation of T lymphocytes in human bone marrow by a limiting dilution assay. Transplantation 1985; 40:317–322. 73. Kernan NA, Collins NH, Juliano L, et al. Clonable T lymphocytes in T cell-depleted bone marrow transplants correlate with development of graft-versus-host disease. Blood 1986; 68:770–773. 74. Muller S, Schulz A, Reiss U, et al. Definition of a critical T cell threshold for prevention of GVHD after HLA non-identical PBPC transplantation in children. Bone Marrow Transplant 1999; 24:575–581. 75. Cornelissen JJ, Fibbe WE, Schattenberg AV, et al. A retrospective Dutch study comparing T celldepleted allogeneic blood stem cell transplantation versus T cell-depleted allogeneic bone marrow transplantation. Bone Marrow Transplant 1998; 21:S66–S70. 76. Antoine C, Muller S, Cant A, et al. Long-term survival and transplantation of haemopoietic stem cells for immunodeficiencies: report of the European experience 1968–1999. Lancet 2003; 361:553–560. 77. Goldman JM, Gale RP, Horowitz MM, et al. Bone marrow transplantation for chronic myelogenous leukemia in chronic phase: increased risk for relapse associated with T-cell depletion. Ann Int Med 1988; 108:806–814. 78. Mitsuyasu RT, Champlin RE, Gale RP, et al. Treatment of donor bone marrow with monoclonal anti-T-cell antibody and complement for the prevention of graft-versus-host disease—a prospective, randomized, double-blind trial [abstract]. Ann Int Med 1986; 105:20–26. 79. Kernan NA, Bordignon C, Heller G, et al. Graft failure after T-cell-depleted human leukocyte antigen identical marrow transplants for leukemia:I. Analysis of risk factors and results of secondary transplants. Blood 1989; 74:2227–2236. 80. Martin PJ, Hansen JA, Torok-Storb B, et al. Graft failure in patients receiving T cell depleted HLAidentical allogeneic marrow transplants. Bone Marrow Transpl 1988; 3:445–456. 81. Kernan NA, Bordignon C, Keever CA, et al. Graft failure after T cell depleted marrow transplants for leukemia. Transpl Proc 1987; 19:29–32. 82. Kernan NA, Flomenberg N, Dupont B, O’Reilly RJ. Graft rejection in recipients of T-cell-depleted HLA-nonidentical marrow transplants for leukemia. Transplantation 1987; 43:842–847. 83. Gale RP, Reisner Y. Graft rejection and graft-versus-host disease: mirror images. Lancet 1986; 1:1468–1470. 84. Soderling CCB, Song CW, Blazar BR, Vallera DA. A correlation between conditioning and engraftment in recipients of MHC-mismatched T cell-depleted murine bone marrow transplants. J Immunol 1985; 135:941–946. 85. O’Reilly RJ, Shank B, Collins NH. Increased total body irradiation (TBI) abrogates resistance to HLA-matched marrow grafts depleted of T cells by lectin agglutination and E-rosette depletion (SBA-E-BMT). Exp Hematol 1985; 13:406. 86. Sondel PM, Bozdech MJ, Trigg ME. Additional immunosuppression allows engraftment following HLA-mismatched T cell-depletion bone marrow transplantation for leukemia. Transpl Proc 1985; 17:460–461. 87. Cavazzana-Calvo M, Jabado N, Bordigoni P, et al. In vivo infusion of anti-LFA-1 and anti-CD2 antibodies prevents graft failure after HLA partially incompatible bone marrow transplantation in children with high risk acute lymphoblastic leukaemia. Leuk Lymphoma 1997; 28:103–112. 88. Friend PJ, Waldmann H, Hale G, et al. Reversal of allograft rejection using the monoclonal antibody Campath-1G. Transpl Proc 1991; 23:2253–2254. 89. Aversa F, Tabilio A, Velardi A, et al. Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype. N Engl J Med 1998; 339:1186–1193. 90. Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990; 75:555–562. 91. Fefer A. Adoptive tumor immunotherapy in mice as an adjunct to whole-body x-irradiation and chemotherapy. A review. Isr J Med Sci 1973; 9:350–365. 92. Papadopoulos EB, Carabasi MH, Castro-Malaspina H, et al. T-cell depleted allogeneic bone marrow transplantation as post-remission therapy for acute myelogenous leukemia: freedom from relapse in the absence of graft-versus-host disease. Blood 1998; 91:1083–1090.

132

Quinones

93. Dazzi F, Szydlo RM, Cross NC, et al. Durability of responses following donor lymphocyte infusions for patients who relapse after allogeneic stem cell transplantation for chronic myeloid leukemia. Blood 2000; 96:2712–2716. 94. Guglielmi C, Arcese W, Dazzi F, et al. Donor lymphocyte infusion for relapsed chronic myelogenous leukemia: prognostic relevance of the initial cell dose. Blood 2002; 100:397–405. 95. Shimoni A, Gajewski JA, Donato M, et al. Long-term follow-up of recipients of CD8depleted donor lymphocyte infusions for the treatment of chronic myelogenous leukemia relapsing after allogeneic progenitor cell transplantation. Biol Blood Marrow Transplant 2001; 7:568–575. 96. Zorn E, Wang KS, Hochberg EP, et al. Infusion of CD4C donor lymphocytes induces the expansion of CD8C donor T cells with cytolytic activity directed against recipient hematopoietic cells. Clin Cancer Res 2002; 8:2052–2060. 97. Small TN, Papadopoulos EB, Boulad F, et al. Comparison of immune reconstitution after unrelated and related T-cell-depleted bone marrow transplantation: effect of patient age and donor leukocyte infusions. Blood 1999; 93:467–480. 98. Dror Y, Gallagher R, Wara DW, et al. Immune reconstitution in severe combined immunodeficiency disease after lectin-treated, T-cell-depleted haplocompatible bone marrow transplantation. Blood 1993; 81:2021–2030. 99. Haddad E, Landais P, Friedrich W, et al. Long-term immune reconstitution and outcome after HLAnonidentical T-cell-depleted bone marrow transplantation for severe combined immunodeficiency: a European retrospective study of 116 patients. Blood 1998; 91:3646–3653. 100. Gross TG, Steinbuch M, DeFor T, et al. B cell lymphoproliferative disorders following hematopoietic stem cell transplantation: risk factors, treatment and outcome. Bone Marrow Transplant 1999; 23:251–258. 101. Papadopoulos EB, Ladanyi M, Emanuel D, et al. Infusions of donor leukocytes to treat Epstein-Barr virus- associated lymphoproliferative disorders after allogeneic bone marrow transplantation [see comments]. N Engl J Med 1994; 330:1185–1191. 102. Papadopoulos EB, Ladanyi M, Emanuel D, et al. Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation. N Engl J Med 1994; 330:1185–1191. 103. Rooney CM, Smith CA, Ng CY, et al. Infusion of cytotoxic T cells for the prevention and treatment of epstein-barr virus-induced lymphoma in allogeneic transplant recipients. Blood 1998; 92:1549–1555. 104. van Esser JW, Niesters HG, van der HB, et al. Prevention of Epstein-Barr virus-lymphoproliferative disease by molecular monitoring and preemptive rituximab in high-risk patients after allogeneic stem cell transplantation. Blood 2002; 99:4364–4369. 105. Kishi Y, Kami M, Oki Y, et al. Donor lymphocyte infusion for treatment of life-threatening respiratory syncytial virus infection following bone marrow transplantation. Bone Marrow Transplant 2000; 26:573–576. 106. Blazar BR, Taylor PA. Regulatory T cells. Biol Blood Marrow Transplant 2005; 11:46–49. 107. Fujii S, Fujimoto K, Shimizu K, et al. Presentation of tumor antigens by phagocytic dendritic cell clusters generated from human CD34C hematopoietic progenitor cells: induction of autologous cytotoxic T lymphocytes against leukemic cells in acute myelogenous leukemia patients. Cancer Res 1999; 59:2150–2158. 108. Peggs KS, Mackinnon S. Augmentation of virus-specific immunity after hematopoietic stem cell transplantation by adoptive T-cell therapy. Hum Immunol 2004; 65:550–557. 109. Trivedi D, Williams RY, O’Reilly RJ, Koehne G. Generation of CMV-specific T lymphocytes using protein-spanning pools of pp65-derived overlapping pentadecapeptides for adoptive immunotherapy. Blood 2005; 105:2793–2801. 110. Uckun FM, Kersey JH, Haake R, Weisdorf D, Ramsay NK. Autologous bone marrow transplantation in high-risk remission B-lineage acute lymphoblastic leukemia using a cocktail of three monoclonal antibodies (BA-1/CD24, BA-2/CD9, and BA-3/CD10) plus complement and 4-hydroperoxycyclophosphamide for ex vivo bone marrow purging. Blood 1992; 79:1094–1104. 111. Bierman PJ, Sweetenham JW, Loberiza FR, Jr., et al. Syngeneic hematopoietic stem-cell transplantation for non-Hodgkin’s lymphoma: a comparison with allogeneic and autologous transplantation—the lymphoma working committee of the international bone marrow transplant registry and the European group for blood and marrow transplantation. J Clin Oncol 2003; 21:3744–3753.

Cellular Engineering of the Hematopoietic Graft

133

112. Billett AL, Kornmehl E, Tarbell NJ, et al. Autologous bone marrow transplantation after a long first remission for children with recurrent acute lymphoblastic leukemia. Blood 1993; 81:1651–1657. 113. Franklin WA, Glaspy J, Pflaumer SM, et al. Incidence of tumor-cell contamination in leukapheresis products of breast cancer patients mobilized with stem cell factor and granulocyte colonystimulating factor (G-CSF) or with G-CSF alone. Blood 1999; 94:340–347. 114. Cheung NK, Heller G, Kushner BH, Liu C, Cheung IY. Detection of metastatic neuroblastoma in bone marrow: when is routine marrow histology insensitive? J Clin Oncol 1997; 15:2807–2817. 115. Zwicky CS, Maddocks AB, Andersen N, Gribben JG. Eradication of polymerase chain reaction detectable immunoglobulin gene rearrangement in non-Hodgkin’s lymphoma is associated with decreased relapse after autologous bone marrow transplantation. Blood 1996; 88:3314–3322. 116. Moss TJ, Sanders DG, Lasky LC, Bostrom B. Contamination of peripheral blood stem cell harvests by circulating neuroblastoma cells. Blood 1990; 76:1879–1883. 117. Franklin WA, Shpall EJ, Archer P, et al. Immunocytochemical detection of breast cancer cells in marrow and peripheral blood of patients undergoing high dose chemotherapy with autologous stem cell support. Breast Cancer Res Treat 1996; 41:1–13. 118. Cheung IY, Barber D, Cheung NK. Detection of microscopic neuroblastoma in marrow by histology, immunocytology, and reverse transcription-PCR of multiple molecular markers. Clin Cancer Res 1998; 4:2801–2805. 119. Borowitz MJ, Pullen DJ, Winick N, et al. Comparison of diagnostic and relapse flow cytometry phenotypes in childhood acute lymphoblastic leukemia: implications for residual disease detection: a report from the children’s oncology group. Cytometry B Clin Cytom 2005; 68:18–24. 120. Robillard N, Cave H, Mechinaud F, et al. Four-color flow cytometry bypasses limitations of IG/TCR polymerase chain reaction for minimal residual disease detection in certain subsets of children with acute lymphoblastic leukemia. Haematologica 2005; 90:1516–1523. 121. Tsang KS, Li CK, Tsoi WC, et al. Detection of micrometastasis of neuroblastoma to bone marrow and tumor dissemination to hematopoietic autografts using flow cytometry and reverse transcriptase-polymerase chain reaction. Cancer 2003; 97:2887–2897. 122. Tbakhi A, Pettay J, Sreenan JJ, et al. Comparative analysis of interphase FISH and RT-PCR to detect bcr/abl translocation in chronic myelogenous leukemia and related disorders. Am J Clin Pathol 1998; 109:16–23. 123. Rubnitz JE, Downing JR, Crist WM. Pathobiology and clinical significance of molecular genetic findings in childhood tumors. Adv Pediatr 1998; 45:315–336. 124. Shannon K. Genetic predispositions and childhood cancer. Environ Health Perspect 1998; 106:801–806. 125. Kuroda T, Saeki M, Nakano M, Mizutani S. Clinical application of minimal residual neuroblastoma cell detection by reverse transcriptase-polymerase chain reaction. J Pediatr Surg 1997; 32:69–72. 126. Freedman AS, Neuberg D, Mauch P, et al. Long-term follow-up of autologous bone marrow transplantation in patients with relapsed follicular lymphoma. Blood 1999; 94:3325–3333. 127. de AE, Lozano MD, Patino A, Sierrasesumaga L, Pardo-Mindan FJ. Ewing family tumors: potential prognostic value of reverse-transcriptase polymerase chain reaction detection of minimal residual disease in peripheral blood samples. Diagn Mol Pathol 1998; 7:152–157. 128. Kelly KM, Womer RB, Barr FG. Minimal disease detection in patients with alveolar rhabdomyosarcoma using a reverse transcriptase-polymerase chain reaction method. Cancer 1996; 78:1320–1327. 129. Mach-Pascual S, Legare RD, Lu D, et al. Predictive value of clonality assays in patients with nonHodgkin’s lymphoma undergoing autologous bone marrow transplant: a single institution study. Blood 1998; 91:4496–4503. 130. Uckun FM, Kersey JH, Vallera DA, et al. Autologous bone marrow transplantation in high-risk T-lineage acute lymphoblastic leukemia using immunotoxins plus 4-hydroxycyclophosphamide for marrow purging. Blood 1990; 76:1723–1733. 131. Gribben JG, Neuberg D, Freedman AS, et al. Detection by polymerase chain reaction of residual cells with the bcl-2 translocation is associated with increased risk of relapse after autologous bone marrow transplantation for B-cell lymphoma. Blood 1993; 81:3449–3457. 132. Faulkner LB, Tintori V, Tamburini A, et al. High-sensitivity immunocytologic analysis of neuroblastoma cells in paired blood and marrow samples. J Hematother 1998; 7:361–366.

134

Quinones

133. Webb IJ, Friedberg W, Gribben JG, et al. Effective purging of autologous hematopoietic stem cells using anti-B-cell monoclonal antibody-coated high-density microparticles prior to high-dose therapy for patients with non-Hodgkin’s lymphoma. Biol Blood Marrow Transplant 2002; 8:429–434. 134. Matthay KK, Villablanca JG, Seeger RC, et al. Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children’s cancer group. N Engl J Med 1999; 341:1165–1173. 135. Negrin RS, Kusnierz-Glaz CR, Still BJ, et al. Transplantation of enriched and purged peripheral blood progenitor cells from a single apheresis product in patients with non-Hodgkin’s lymphoma. Blood 1995; 85:3334–3341. 136. Mohr M, Hilgenfeld E, Fietz T, et al. Efficacy and safety of simultaneous immunomagnetic CD34C cell selection and breast cancer cell purging in peripheral blood progenitor cell samples used for hematopoietic rescue after high-dose therapy. Clin Cancer Res 1999; 5:1035–1040. 137. Weisdorf DJ, Haake R, Miller WJ, et al. Autologous bone marrow transplantation for progressive non-Hodgkin’s lymphoma: clinical impact of immunophenotype and in vitro purging. Bone Marrow Transplant 1991; 8:135–142. 138. Vallera DA. Immunotoxins for ex vivo bone marrow purging in human bone marrow transplantation. Cancer Treat Res 1988; 37:515–535. 139. Sensenbrenner LL, Marini JJ, Colvin M. Comparative effects of cyclophosphamide, isophosphamide, 4-methylcyclophosphamide, and phosphoramide mustard on murine hematopoietic and immunocompetent cells. J Natl Cancer Inst 1979; 62:975–981. 140. Sharkis SJ, Santos GW, Colvin M. Elimination of acute myelogenous leukemic cells from marrow and tumor suspensions in the rat with 4-hydroperoxycyclophosphamide. Blood 1980; 55:521–523. 141. Kohn FR, Landkamer GJ, Manthey CL, Ramsay NK, Sladek NE. Effect of aldehyde dehydrogenase inhibitors on the ex vivo sensitivity of human multipotent and committed hematopoietic progenitor cells and malignant blood cells to oxazaphosphorines. Cancer Res 1987; 47:3180–3185. 142. Rowley SD, Zueshlsdorf M, Braine HG, et al. CFU-GM content of bone marrow graft correlates with time to hematologic reconstitution following autologous bone marrow transplantation with 4-hydroperoxycyclophosphamide-purged bone marrow. Blood 1987; 70:271–275. 143. Yeager AM, Kaizer H, Santos G, et al. Autologous bone marrow transplantation in patients with acute non-lymphocytic leukemia. Using ex vivo marrow treatment with 4-hydroperoxycyclophosphamide. N Eng J Med 1986; 315:141–147. 144. Benedetti F. [CD34C cells: biological aspects]. Tumori 1996; 82:S3–S13. 145. Preti RA, Lazarus HM, Winter J, et al. Tumor cell depletion of peripheral blood progenitor cells using positive and positive/negative selection in metastatic breast cancer. Cytotherapy 2001; 3:85–95. 146. Negrin RS, Atkinson K, Leemhuis T, et al. Transplantation of highly purified CD34CThy-1C hematopoietic stem cells in patients with metastatic breast cancer. Biol Blood Marrow Transplant 2000; 6:262–271. 147. Chang J, Morgenstern GR, Coutinho LH, et al. The use of bone marrow cells grown in long-term culture for autologous bone marrow transplantation in acute myeloid leukaemia: an update. Bone Marrow Transplant 1989; 4:5–9. 148. Philip T, Zucker JM, Bernard JL, et al. Improved survival at 2 and 5 years in the LMCE1 unselected group of 72 children with stage IV neuroblastoma older than 1 year of age at diagnosis: is cure possible in a small subgroup? J Clin Oncol 1991; 9:1037–1044. 149. Heslop HE, Rooney CM, Rill DR, Krance RA, Brenner MK. Use of gene marking in bone marrow transplantation. Cancer Detect Prev 1996; 20:108–113. 150. Keller G, Paige C, Gilboa E, Wagner EF. Expression of a foreign gene in myeloid and lymphoid cells derived from pluripotential stem hematopoietic precursors. Nature 1985; 318:149. 151. Rill DR, Santana VM, Roberts WM, et al. Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells. Blood 1994; 84:380–383. 152. Halene S, Wang L, Cooper RM, et al. Improved expression in hematopoietic and lymphoid cells in mice after transplantation of bone marrow transduced with a modified retroviral vector. Blood 1999; 94:3349–3357. 153. Li AL, Li C, Feng YG, et al. Antileukemic effect of interleukin-7-transduced bone marrow stromal cells in mice following allogeneic T-cell-depleted bone marrow transplantation. Transplant Proc 2005; 37:2297–2299.

Cellular Engineering of the Hematopoietic Graft

135

154. Ellinwood NM, Vite CH, Haskins ME. Gene therapy for lysosomal storage diseases: the lessons and promise of animal models. J Gene Med 2004; 6:481–506. 155. Kiem HP, Sellers S, Thomasson B, et al. Long-term clinical and molecular follow-up of large animals receiving retrovirally transduced stem and progenitor cells: no progression to clonal hematopoiesis or leukemia. Mol Ther 2004; 9:389–395. 156. Kohn DB, Hershfield MS, Carbonaro D, et al. T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34C cells in ADA-deficient SCID neonates. Nat Med 1998; 4:775–780. 157. Aiuti A, Slavin S, Aker M, et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 2002; 296:2410–2413. 158. Aiuti A, Vai S, Mortellaro A, et al. Immune reconstitution in ADA-SCID after PBL gene therapy and discontinuation of enzyme replacement. Nat Med 2002; 8:423–425. 159. Cavazzana-Calvo M, Fischer A. Efficacy of gene therapy for SCID is being confirmed. Lancet 2004; 364:2155–2156. 160. Gaspar HB, Parsley KL, Howe S, et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gamma retroviral vector. Lancet 2004; 364:2181–2187. 161. Carlucci F, Tabucchi A, Aiuti A, et al. Evaluation of ADA gene expression and transduction efficiency in ADA/SCID patients undergoing gene therapy. Nucleosides Nucleotides Nucleic Acids 2004; 23:1245–1248. 162. Hacein-Bey-Abina S, von KC, Schmidt M, et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003; 348:255–256. 163. Kohn DB, Sadelain M, Glorioso JC. Occurrence of leukaemia following gene therapy of X-linked SCID. Nat Rev Cancer 2003; 3:477–488. 164. Dick JE. Stem cells: self-renewal writ in blood. Nature 2003; 423:231–233. 165. Sprangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopietic stem stem cells. Science 1988; 241:58–62. 166. Aglietta M, Bertolini F, Carlo-Stella C, et al. Ex vivo expansion of hematopoietic cells and their clinical use. Haematologica 1998; 83:824–848. 167. Devine SM, Lazarus HM, Emerson SG. Clinical application of hematopoietic progenitor cell expansion: current status and future prospects. Bone Marrow Transplant 2003; 31:241–252. 168. Bachier CR, Gokmen E, Teale J, et al. Ex-vivo expansion of bone marrow progenitor cells for hematopoietic reconstitution following high-dose chemotherapy for breast cancer. Exp Hematol 1999; 27:615–623. 169. Chabannon C, Olivero S, Blaise D, Maraninchi D, Viens P. Ex vivo expansion of human hematopoietic progenitors and cells to support high-dose chemoradiation therapy: five years of clinical experience. Cytokines Cell Mol Ther 2000; 6:97–108. 170. Jaroscak J, Goltry K, Smith A, et al. Augmentation of umbilical cord blood (UCB) transplantation with ex vivo-expanded UCB cells: results of a phase 1 trial using the AastromReplicell system. Blood 2003; 101:5061–5067. 171. Riddell SR, Warren EH, Gavin MA, et al. Immunotherapy of human viral and malignant diseases with genetically modified T-cell clones. Cancer J 2000; 6:S250–S258. 172. Anversa P, Kajstura J, Leri A. Circulating progenitor cells: search for an identity. Circulation 2004; 110:3158–3160. 173. Albera C, Polak JM, Janes S, et al. Repopulation of human pulmonary epithelium by bone marrow cells: a potential means to promote repair. Tissue Eng 2005; 11:1115–1121.

6 Issues in Pediatric Peripheral Blood Stem-Cell Collection Stephan A. Grupp Division of Oncology and Department of Pathology, Stem Cell Biology, Children’s Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

INTRODUCTION The primary source of hematopoietic stem and progenitor cells for use in autologous and allogeneic transplantation had been bone marrow, but this has changed over the past decade. During this period, there has been increasing use of peripheral blood containing mobilized stem and progenitor cells as a source of these cells for transplantation (Table 1) (1,2). This product is variously referred to as peripheral blood stem cells (PBSC), peripheral blood progenitor cells, or given the shorthand designation “stem cells.” Although each cell source used for hematopoietic transplantation contains stem cells, when the term “stem cells” is used without a qualifier, it is usually referring to PBSC. Even in pediatrics, there has been increasing and wide use of PBSC for transplant. As in adult transplant, PBSC have become the standard of care in autologous transplant, even for diseases such as neuroblastoma, where the mean age of the patient population is under 3 years (3–9). The pros and cons of the use of PBSC in pediatric allogeneic transplant are beyond the scope of this chapter and remain controversial (10). Issues include the use of minor siblings as stem-cell donors (11), well established in the case of bone marrow donation but somewhat less well characterized in the setting of PBSC collection (12), use of G-CSF to mobilize cells in the minor sibling donor (11,13), and the challenges of PBSC collection in the small child (detailed below). The use of PBSC has advantages and disadvantages, and the relative balance between these differs depending on the type of transplant being performed, the disease state being treated, and the age and physiological status of both donor and recipient. This chapter will explore issues and challenges in the collection of PBSC, focusing on practical approaches to the collection and processing of these cells, with particular reference to special issues in pediatric transplantation.

PHERESIS AND VASCULAR ACCESS There are a variety of techniques to increase the number of stem and progenitor cells circulating in the peripheral blood. This process is called mobilization and is discussed below. After mobilization, the cells are then collected using an apheresis device, which can separate specific 137

138 Table 1

Grupp Cellular Characteristics of Various Stem Cell Sources

Stem cell source Stem cell content Progenitor cell content T cell content Risk of tumor cell contamination in autologous transplant

Bone marrow

PBSC

G-CSF primed bone marrow

Umbilical cord blood

CC CC C

CC CCCC CCCC

CC CCC C

CCC

C

CCC

C C C/functionally immature Not applicable

Abbreviation: PBSC, peripheral blood stem cells.

components from whole blood continuously and in real time by centrifugation. Any component can be targeted for relatively specific removal—plasma, red cells, platelets, or white cells. After venous blood is collected and processed through the apheresis machine, the targeted product is retained for collection and the nontargeted components are returned to the patient. In order to allow continuous blood processing for PBSC collection, two ports of vascular access are necessary. In most adults, this can be accomplished using two antecubital lines. In 5–10% of adults and most children, percutaneous antecubital large-bore access is not possible and a pheresis catheter is used instead, although a veno-arterial approach, utilizing an arterial line to draw blood and a conventional venous catheter to return it to the patient, has been described. Although there are many configurations, a pheresis catheter is generally a double lumen catheter with offset proximal and distal ports and side holes along the tip of the catheter. This offset configuration minimizes mixing of processed and unprocessed blood and maximizes the efficiency of the collection. Conventional Broviac-type catheters are difficult to use for pheresis, especially in pediatric sizes, because the lumen collapses under the negative pressure used to draw blood at a useful rate for apheresis (generally1–2 ml/kg/min). Thus, a pheresis catheter is designed to allow faster draw rates using a combination of larger lumen size, shorter catheter length, and stiffer walls. This general design is similar to dialysis catheters and these catheters can and are used for pheresis purposes. However, as the flow rates required for pheresis are lower than those required for dialysis, a dialysis-type catheter is generally larger and stiffer than a catheter specifically designed for pheresis. Pheresis catheters are available both for temporary and tunneled insertion. For smaller patients, we have generally used an 8 Fr cuffed tunneled pheresis catheter (MedCompw). We have routinely placed these catheters in neuroblastoma patients as small as 12 kg, and in selected smaller patients. Smaller patients generally require femoral line placement. The concern in smaller patients is threefold: (1) the risk of partial or complete vessel occlusion with the catheter, (2) the risk of vessel erosion and perforation, which may be greater with stiffer catheters in small vessels, and (3) the difficulty in placing an offset catheter in a short vessel where, if the proximal port is in adequate position, the distal port may be too far advanced. Femoral catheters are short, allowing faster collect rates for a given diameter. However, a patient with a percutaneous nontunneled femoral catheter cannot walk, necessitating admission to the hospital for what otherwise would be an outpatient procedure. Also, the perceived risk of complications (especially infection) with a femoral catheter is higher. For both of these reasons, femoral catheters are generally only placed temporarily, except in unusual circumstances. Another approach used at some institutions is to place a single lumen 7 Fr Broviac-type central venous catheter on the opposite side of the patient’s existing double lumen catheter. The single lumen line is then used as the draw line and the smaller double lumen catheter is used as the return line.

Peripheral Blood Stem-Cell Collection

139

COLLECTION Even in a mobilized patient, the number of stem cells circulating in the entire blood volume may be inadequate to provide engraftment. Thus, processing of multiple blood volumes, often over more than one day, is required for many patients to collect adequate numbers of stem cells. The minimum required for most patients is one large volume leukopheresis (LVL), which represents approximately 20 liters in an adult or 3–4 blood volumes in a child. This volume is the typical goal for a single apheresis session, although some physicians will pherese for a total of six or more blood volumes. Depending on the collection goal and effectiveness of mobilization, patients will often undergo two or more days of apheresis. This is most often true in patients serving as autologous donors, who have been extensively pretreated with chemotherapy. On the other hand, an untreated and healthy allogeneic donor, equal or larger in size than the recipient, will often only require one session. There are two issues in PBSC collection that require special consideration in children. First is the issue of priming. Even using devices that minimize extracorporeal volume, smaller children will require priming of the apheresis machine with red cells. This prevents unacceptable dilutional anemia. Second is the issue of anticoagulation. In older patients, anticoagulation required for the apheresis procedure is accomplished using ACD (anticoagulant citrate dextrose). Although rapidly reversible, ACD creates a higher risk of symptomatic hypocalcemia in young patients. These patients may be managed with a combination of ACD and heparin to achieve anticoagulation, or they may receive a calcium infusion in the apheresis return line.

TECHNIQUES FOR STEM-CELL MOBILIZATION Although early attempts to collect PBSC used steady-state peripheral blood as the source, it soon became apparent that large increases in the number of circulating stem and progenitor cells occur at the point of recovery from myelosuppressive chemotherapy, at which time large numbers of these cells are mobilized from the bone marrow. This typically occurs at a point when the absolute neutrophil count (ANC) has reached 1000 after the nadir and is increasing rapidly. The exact point of maximal stem-cell mobilization is difficult to predict and highly patient dependent (see Target Dose below). Chemotherapy-induced mobilization is possible using a large variety of myelosuppressive regimens. The most commonly used approach is to give cyclophosphamide (often at a total dose of 4 g/m2 over 2 days; Table 2). Multiple-drug regimens that are used in the treatment of the primary disease can also be used to induce a nadir, after which PBSC collection is possible. PBSC can be collected successfully after most chemotherapy regimens, including topoisomerase inhibitors such as etoposide. However, there is a concern that the DNA damage that occurs after treatment with etoposide may occur in stem cells, and this damage may increase the risk of secondary (treatment-related) leukemia in a patient who is reconstituted with these stem cells. In one study, use of PBSC collected after etoposide resulted in a 7–12-fold relative risk of secondary leukemia (14), with other studies also demonstrating an increased risk Table 2

Regimens for Peripheral Blood Stem-Cell Mobilization

Cyclophosphamide 2000 mg/m2/day over 2 days, followed by G-CSF 5 mg/kg/d from day 3 to the end of pheresis G-CSF 5 mg/kg/d for 3–4 days followed by pheresis on days 4–5 and following GM-CSF 250 mg/m2/d for 3–4 days followed by pheresis on days 4–5 and following Combination of G-CSF 5–10 mg/kg/d (in AM) and GM-CSF 250 mg/m2/d (in PM) for 4 days, pheresis starting on d 5 Other chemotherapy/HGF combinations

140

Grupp

attributable more to the prior chemotherapy than to the preparative regimen used for the stem-cell transplant (15). This effect, together with the observation that multiple cycles of chemotherapy reduce yields of PBSC collection (16), argue that PBSC should be collected as early in treatment as possible but after sufficient therapy (usually two to three cycles of chemotherapy) to clear circulating tumor (17). The use of chemotherapy to mobilize PBSC may not be possible or desirable in every patient, has a risk of toxicity during the nadir, and is clearly not appropriate for normal allogeneic donors in whom the chemotherapy has no potential benefit. An alternative approach of using hematopoietic growth factors (HGF) is in widespread use. Donors are placed on a daily regimen of HGF injections, followed by initiation of PBSC collection on day 4–5 of treatment. HGF treatment continues until the apheresis is complete. There are several choices of HGF doses and regimens. Filgrastim (rhuG-CSF) is the most common HGF used for this purpose. Doses given vary widely. There is a modest dose-response effect between 2 and 16 mg/kg of G-CSF. Although doses as high as 24 mg/kg/d have been used for mobilization, there is little evidence that these very high doses are more efficacious and they have the disadvantage of greater cost and a higher incidence of side effects, especially bone pain. Sargramostim (rhuGM-CSF) is an alternative. Comparisons of G-CSF and GM-CSF as single agents reveal either no significant advantage of one HGF over the other in terms of PBSC collection efficiency or extent of progenitor cell mobilization (18), or a modest advantage for G-CSF (19). Laboratory studies have suggested that PBSC collected after G-CSF mobilization may have a polarization in T-cell response toward the more suppressive T-helper lymphocyte type 2 (Th2) response (20). This may in turn have an impact (1) on the recovery of cellular immunity after stem cell transplant (SCT) and (2) the risk of graft-versus-host disease (GVHD) after allogeneic SCT, but this has not been proven in a clinical trial. The extent to which GM-CSF may not cause this Th2 polarization has not been well studied. The combination of G-CSF and GM-CSF may be superior to either alone, although one pediatric study failed to show an advantage for the combination (21). In any case, most donors will mobilize well with either HGF alone and will not require this combination. The one setting in which the combination of G-CSF plus GM-CSF may be superior is when a patient has had inadequate numbers of stem cells collected over several apheresis. In these so-called “poor mobilizers,” combination HGF regimens may improve the likelihood that adequate PBSC can be collected (22). Other HGF have been tested as PBSC mobilizers and work as well, including stem-cell factor and thrombopoietin, but there is no evidence to suggest superiority in terms of clinical outcome during transplant over the standard use of G-CSF, even when higher numbers of CD34C cells are collected. On the other hand, collection of higher numbers of CD34C cells has the potential to reduce the number of LVL a donor must undergo, which is a benefit in terms of cost, convenience, and potential donor exposure, especially in pediatrics. Balanced against this is the high cost of HGF, and the fact that adding a second HGF doubles this cost. In patients who are receiving myelosuppressive chemotherapy, HGF such as filgrastim are often used to improve recovery. The concurrent use of chemotherapy and an HGF improves PBSC mobilization as well (23,24), although a randomized trial did not show this improved mobilization to have an impact on survival or engraftment (25). Thus, any patient receiving chemotherapy after which PBSC collection is planned should be placed on an HGF, even if similar courses during the treatment are not supported by an HGF.

TARGET DOSE FOR PBSC INFUSION When bone marrow is collected, most operators target a final volume, or, more commonly, a volume and a nucleated cell dose. Because of the high variability in stem- and progenitor-cell content in PBSC, a more direct assay is needed to assure that adequate numbers for reliable engraftment have been collected. There is no well-established assay for human stem cells, although stem-cell activity is likely to be found in a portion of cells that are detected by the long-term culture initiating cell assay or an immunodeficient mouse repopulating cell assay.

Peripheral Blood Stem-Cell Collection

141

Progenitor cell content can be assessed by the colony forming unit-granulocyte/monocyte (CFU-GM) assay. The presence of 2–10!104 CFU-GM/kg of recipient weight is predictive of engraftment, but the assay is laborious, expensive, and difficult to standardize. It also takes 14 days to complete, making it useless to assess PBSC collections in real time. For all these reasons, most centers have moved away from CFU-GM assays. A major advance in the use of PBSC was the recognition that most [although perhaps not all (26)] of the cells in the hematopoietic stem and progenitor cell compartment bear the antigen CD34, regardless of lineage. Enumeration of CD34C cells allows for more accurate assessment of engraftment potential provided by a given number of mononuclear cells. There is a threshold for reliable engraftment and a rough correlation between numbers of CD34C cells above the threshold of engraftment and time to engraftment (Table 3). The threshold for reliable engraftment is generally thought to be 1!106 CD34C cells/kg (27). Below this threshold, the likelihood of delayed engraftment of neutrophils and especially platelets increases (28). Increasing the minimum acceptable number to 2–2.5!106 CD34C cells/kg decreases this likelihood somewhat further, and this is the threshold that most transplant centers attempt to achieve. Some authors have advocated a goal (rather than a minimum) of five to up to 15!106 CD34C cells/kg (22). This higher goal may be unrealistic in some patients, especially patients who have been treated with multiple cycles of chemotherapy prior to collection, and has the potential to increase the cost and length of apheresis. CD34C cells in the bone marrow can range from 1–4%, while CD34C cells in mobilized pheresis products can range from 0.1% (in a poor mobilizer) to O1%. The assay for CD34C cells is a flow cytometric assay (29,30), and this technique is inherently inaccurate at low percentages. This means that the number of CD34C cells based on a measured frequency of 0.1% could easily be off by twofold in either the direction of more or fewer cells and this must be borne in mind when assessing when to stop apheresis in patients with poor collections. In collections that have undergone CD34 selection (see below), the CD34 purity is generally O60% and these determinations are extremely accurate. Another consideration is that low CD34 PBSC collections may often have a higher granulocyte content, which can complicate freezing/ thawing and therefore have an impact on the yield of cells actually infused after storage. There are many different approaches to determining when a donor will be most successfully pheresed for the highest number of PBSC. The goals are to collect adequate numbers of PSBC as defined above, preferably in a single collection procedure. When HGF regimens alone are used, timing is simple: the donor is pheresed on either the fourth or fifth day of HGF administration. When a patient is recovering marrow function after chemotherapy, the point at which the best collection can be obtained is more difficult to predict. Peripheral WBC count is a poor predictor of stem-cell mobilization (31), and there is some theoretical concern for donor safety and possible hyperleukocytosis at WBC counts of more than 70!109/L. Some advocate G-CSF dose reductions for donors whose WBC reaches this level. Many centers use some variation on the following algorithm: 1–3 days after the ANC reaches 1000 after the nadir, at a point where there is some evidence of platelet recovery, stem-cell collection begins. Rather Table 3 Dose level Minimum

Optimum Ideal

Choosing Doses of Peripheral Blood Stem Cells for Stem-Cell Transplantation CD34C cells/kg of recipient weight 6

1!10

2–2.5!106 5!106

Notes At this dose, there is a risk of prolonged neutropenia and extended platelet transfusion requirements Threshold dose for many centers There is a limited dose-response effect at doses O2.5!106 cells, and this target may increase number of phereses needed and cost

142

Grupp

than using a rising neutrophil count to trigger apheresis, some centers with access to rapidturnaround, quantitative (or “absolute”) CD34C cell counts use the rise in peripheral CD34C cells to time initiation of collection. Detection of !5 CD34C cells/mL of blood is highly predictive of poor PBSC collection, whereas O10–20 CD34C cells/mL correlates well with the likelihood of collecting O2.5!106 CD34 cell/kg in a single LVL procedure (31).

PROCESSING AND STORAGE OF PERIPHERAL BLOOD STEM CELLS Many PBSC products collected to support transplant procedures are autologous, and must therefore be cryopreserved for later use. In the allogeneic setting, products can be collected prior to starting pretransplant conditioning in the recipient or they can be collected on the day of the intended infusion. In addition to cryopreservation, other processing options exist, depending on the purpose for which the PBSC will be used. Specific engineering of the graft is possible to remove or expand desired cell populations. For patients undergoing allogeneic PBSC transplantation, cells are collected from normal donors (see below). Since the donor does not undergo conditioning for the transplant, these cells can be collected at the time of use and do not need to be cryopreserved. The advantage of this “just-in-time” strategy is that there is no cell loss due to freezing and thawing. The disadvantage is that, if there is trouble collecting PBSC either because of access issues or because the donor is a poor mobilizer, the recipient has already been conditioned and there is considerable time pressure to accomplish any back-up measures (line placement, alternative mobilization strategies, or harvest of bone marrow). After collection, the PBSC product is taken to the stem-cell processing lab. This is where procedures to ensure quality of the product take place, including determination of CD34C cell content (see Target Dose), viability determinations, mononuclear cell counts, and confirmation of sterility. Stem-cell practise has attracted more regulatory attention recently. The Foundation for the Accreditation of Cell Therapy has been established to provide uniform standards for collection and processing of stem cell products, as well as the clinical care of both donors and recipients (32). The various procedures involved in stem-cell processing have also attracted more scrutiny from the Food and Drug Administration. Options for stem-cell processing include (1) depletion of granulocytes by density gradient centrifugation, (2) depletion of potential tumor cells by a direct purging technique or CD34 selection (33), and (3) depletion of T cells in an allogeneic product to decrease the risk of GVHD. Many of the stem-cell processing steps described here and below, with the notable exception of CD34 selection, were developed using marrow products and all are made somewhat more complicated by the considerably higher number of cells found in PBSC compared with bone marrow.

TUMOR CELL PURGING All of these processing procedures either depend on negative selection (removal of the cell type that is unwanted) or positive selection (selection of stem/progenitor cells, leaving all other cells behind). CD34 selection is the primary positive selection technique available to stem-cell labs. CD34 is a cellular antigen that is expressed on stem cells, as well as progenitor cells of all hematopoietic lineages. Automated processes are available that select the CD34C cell population (34) away from the 99% of PBSC that are irrelevant for engraftment, and one of these technologies, the Isolex 300i device, is FDA approved. An alternative device, the Miltenyi CliniMACS device (35), is approved in Europe and may become available in the United States. In general, CD34 selection will result in a product that is 60–95% CD34C, removing more than 99% of T cells (36) and tumor cells (37–39), providing that the tumor cells do not express the CD34 antigen. Hematopoietic tumors such as acute leukemias often express CD34 and are therefore not depleted by CD34 selection. CD34 selection has been used to purge

Peripheral Blood Stem-Cell Collection

143

stem-cell products in patients with neuroblastoma, but concerns have been raised that some neuroblastoma cells or cell lines may express CD34 or express surface epitopes that cross-react with monoclonal antibodies that recognize CD34 (40,41). Our data have not confirmed expression of CD34 on neuroblastoma (37), and we and others have shown purging of neuroblastoma cells from PBSC products in the clinical setting (42). These data suggest that CD34 selection may be a purging alternative for PBSC products obtained from neuroblastoma patients. Negative selection procedures, by contrast, are tumor- or cell-type specific. For example, many techniques have been developed to negatively select T cells or T-cell subsets away from stem and progenitor cells in bone marrow and PBSC (43). These include: (1) a variety of monoclonal antibodies directed against T cells; (2) counterflow centrifugal elutriation (44), which separates out lymphocytes based on physical characteristics; (4) sheep red blood cell rosetting (45); and (5) immunomagnetic removal of T cells. Some of these procedures allow for more specific graft engineering by removing specific T-cell subsets such as CD8CT cells or T cells expressing activation markers such as CD25 or CD69 (46). It is also possible to deplete tumor cells using specific antitumor monoclonal antibodies, often followed by a magnetic depletion step. This approach has been proven to purge tumor cells from stem-cell products collected from patients with B-cell lymphomas (47). This technique has been the mostly widely used method to purge neuroblastoma (48,49). That tumor contaminating stem-cell products may contribute to relapse was shown in gene marking studies in neuroblastoma patients undergoing autologous bone marrow transplant. In these studies, a bone marrow aliquot was transfected with a marker gene and infused after transplant. Tumor cells at sites of relapse were found to contain the marker gene, suggesting that clonogenic tumor had been infused with the graft (50). In follicular lymphoma, inability to detect tumor cells in the stem-cell product after purging is associated with improved outcome after autologous transplant (47), but no study has shown that purging itself improves outcome.

STORAGE After processing, PBSC are then cryopreserved for later infusion. Controlled-rate freezing with temperature curve monitoring is required. Products are stored in the vapor phase of liquid nitrogen until they are required for infusion. Usually the storage period is weeks to months, but stem-cell products have provided adequate engraftment when infused 8–10 years after cryopreservation (51). After thawing, PBSC are again checked for viability. Granulocytes do not survive cryopreservation, so loss of this cell fraction from the collection is expected. In order to allow cells to survive freezing and thawing, they are placed in a medium containing 7.5–10% dimethyl sulfoxide. Stem and progenitor cells lose viability over time in this medium, so it is important to infuse the cells immediately after thawing. Some institutions mandate thawing of products at the bedside for this reason.

REFERENCES 1. Korbling M, Dorken B, Ho AD, Pezzutto A, Hunstein W, Fliedner TM. Autologous transplantation of blood-derived hemopoietic stem cells after myeloablative therapy in a patient with Burkitt’s lymphoma. Blood 1986; 67:529–532. 2. Kessinger A, Armitage JO, Landmark JD, Weisenburger DD. Reconstitution of human hematopoietic function with autologous cryopreserved circulating stem cells. Exp Hematol 1986; 14:192–196. 3. Kletzel M, Abella EM, Sandler ES, et al. Thiotepa and cyclophosphamide with stem cell rescue for consolidation therapy for children with high-risk neuroblastoma: a phase I/II study of the pediatric blood and marrow transplant consortium. J Pediatr Hematol Oncol 1998; 20:49–54.

144

Grupp

4. Kletzel M, Katzenstein HM, Haut PR, et al. Treatment of high-risk neuroblastoma with triple-tandem high-dose therapy and stem-cell rescue: results of the Chicago pilot II study. J Clin Oncol 2002; 20:2284–2292. 5. Grupp SA, Stern JW, Bunin N, et al. Rapid-sequence tandem transplant for children with high-risk neuroblastoma. Med Pediatr Oncol 2000; 35:696–700. 6. Grupp SA, Stern JW, Bunin N, et al. Tandem high-dose therapy in rapid sequence for children with high-risk neuroblastoma. J Clin Oncol 2000; 18:2567–2575. 7. Cairo MS, Davenport V, Bessmertny O, et al. Phase I/II dose escalation study of recombinant human interleukin-11 following ifosfamide, carboplatin and etoposide in children, adolescents and young adults with solid tumors or lymphoma: a clinical, haematological and biological study. Br J Haematol 2005; 128:49–58. 8. Pradhan KR, Johnson CS, Vik TA, Sender LS, Kreissman SG. A novel intensive induction therapy for high-risk neuroblastoma utilizing sequential peripheral blood stem cell collection and infusion as hematopoietic support. Pediatr Blood Cancer 2005. 9. Grupp SA, Cohn SL, Wall D, Reynolds CP. Collection, storage, and infusion of stem cells in children with high-risk neuroblastoma: saving for a rainy day. Pediatr Blood Cancer 2006. 10. Eapen M, Horowitz MM, Klein JP, et al. Higher mortality after allogeneic peripheral-blood transplantation compared with bone marrow in children and adolescents: the histocompatibility and alternate stem cell source working committee of the international bone marrow transplant registry. J Clin Oncol 2004; 22:4872–4880. 11. Pulsipher MA, Nagler A, Iannone R, Nelson RM. Ethical and safety considerations regarding risks for normal pediatric bone marrow donors: the use of G-CSF prior to stem cell harvest. Pediatric Blood Cancer 2006; 46. 12. Pulsipher MA, Levine JE, Hayashi RJ, et al. Safety and efficacy of allogeneic PBSC collection in normal pediatric donors: the pediatric blood and marrow transplant consortium experience (PBMTC) 1996–2003. Bone Marrow Transplant 2005; 35:361–367. 13. Grupp SA, Frangoul H, Wall D, Pulsipher MA, Schultz KR. Use of G-CSF in matched sibling donor pediatric allogeneic transplantation: a consensus statement from the children’s oncology group (COG) transplant discipline committee and pediatric blood and marrow transplant consortium (PBMTC) executive committee. Pediatr Blood Cancer 2006; 46:414–421. 14. Krishnan A, Bhatia S, Slovak ML, et al. Predictors of therapy-related leukemia and myelodysplasia following autologous transplantation for lymphoma: an assessment of risk factors. Blood 2000; 95:1588–1593. 15. Kollmannsberger C, Beyer J, Droz JP, et al. Secondary leukemia following high cumulative doses of etoposide in patients treated for advanced germ cell tumors. J Clin Oncol 1998; 16:3386–3391. 16. Jerjis S, Croockewit S, Preijers F, Schaap N, De Witte T. Impact of chemotherapy on the mobilization, harvest and economic costs of autologous peripheral stem cell transplantation in patients with multiple myeloma. Leuk Lymphoma 2000; 37:551–560. 17. Moss TJ, Sanders DG, Lasky LC, Bostrom B. Contamination of peripheral blood stem cell harvests by circulating neuroblastoma cells. Blood 1990; 76:1879–1883. 18. Gazitt Y. Comparison between granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the mobilization of peripheral blood stem cells. Curr Opin Hematol 2002; 9:190–198. 19. Weaver CH, Schulman KA, Buckner CD. Mobilization of peripheral blood stem cells following myelosuppressive chemotherapy: a randomized comparison of filgrastim, sargramostim, or sequential sargramostim and filgrastim. Bone Marrow Transplant 2001; 27:S23–S29. 20. Sloand EM, Kim S, Maciejewski JP, et al. Pharmacologic doses of granulocyte colony-stimulating factor affect cytokine production by lymphocytes in vitro and in vivo. Blood 2000; 95:2269–2274. 21. Madero L, Gonzalez-Vicent M, Molina J, Madero R, Quintero V, Diaz MA. Use of concurrent G-CSF C GM-CSF versus G-CSF alone for mobilization of peripheral blood stem cells in children with malignant disease. Bone Marrow Transplant 2000; 26:365–369. 22. Stiff PJ. Management strategies for the hard-to-mobilize patient. Bone Marrow Transplant 1999; 23:S29–S33. 23. Knudsen LM, Gaarsdal E, Jensen L, Nielsen KJ, Nikolaisen K, Johnsen HE. Improved priming for mobilization of and optimal timing for harvest of peripheral blood stem cells. J Hematother 1996; 5:399–406. 24. Levine JE, Boxer LA. Clinical applications of hematopoietic growth factors in pediatric oncology. Curr Opin Hematol 2002; 9:222–227.

Peripheral Blood Stem-Cell Collection

145

25. Narayanasami U, Kanteti R, Morelli J, et al. Randomized trial of filgrastim versus chemotherapy and filgrastim mobilization of hematopoietic progenitor cells for rescue in autologous transplantation. Blood 2001; 98:2059–2064. 26. Goodell MA, Rosenzweig M, Kim H, et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 1997; 3:1337–1345. 27. Shpall EJ, Champlin R, Glaspy JA. Effect of CD34C peripheral blood progenitor cell dose on hematopoietic recovery. Biol Blood Marrow Transplant 1998; 4:84–92. 28. Weaver CH, Potz J, Redmond J, et al. Engraftment and outcomes of patients receiving myeloablative therapy followed by autologous peripheral blood stem cells with a low CD34C cell content. Bone Marrow Transplant 1997; 19:1103–1110. 29. Trischmann TM, Schepers KG, Civin CI. Measurement of CD34C cells in bone marrow by flow cytometry. J Hematother 1993; 2:305–313. 30. Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. The ISHAGE guidelines for CD34C cell determination by flow cytometry. International society of hematotherapy and graft engineering. J Hematother 1996; 5:213–226. 31. Yu J, Leisenring W, Bensinger WI, Holmberg LA, Rowley SD. The predictive value of white cell or CD34C cell count in the peripheral blood for timing apheresis and maximizing yield. Transfusion 1999; 39:442–450. 32. Rowley SD. Regulation of hematopoietic stem cell processing and transplantation. Int J Hematol 2002; 75:237–245. 33. Civin CI, Strauss LC, Fackler MJ, Trischmann TM, Wiley JM, Loken MR. Positive stem cell selection—basic science. Prog Clin Biol Res 1990; 333:387–401. 34. Strauss LC, Trischmann TM, Rowley SD, Wiley JM, Civin CI. Selection of normal human hematopoietic stem cells for bone marrow transplantation using immunomagnetic microspheres and CD34 antibody. Am J Pediatr Hematol Oncol 1991; 13:217–221. 35. Schumm M, Lang P, Taylor G, et al. Isolation of highly purified autologous and allogeneic peripheral CD34C cells using the cliniMACS device. J Hematother 1999; 8:209–218. 36. Beelen DW, Peceny R, Elmaagacli A, et al. Transplantation of highly purified HLA-identical sibling donor peripheral blood CD34C cells without prophylactic post-transplant immunosuppression in adult patients with first chronic phase chronic myeloid leukemia: results of a phase II study. Bone Marrow Transplant 2000; 26:823–829. 37. Donovan J, Temel J, Zuckerman A, et al. CD34 selection as a stem cell purging strategy for neuroblastoma: pre-clinical and clinical studies. Med Ped Oncol 2000; 35:677–682. 38. Klein JL, Hamm C, Dansey RD, et al. High-dose chemotherapy and CD34-selected peripheral blood progenitor cell transplantation for patients with breast cancer metastatic to bone and/or bone marrow. Bone Marrow Transplant 2001; 28:1023–1029. 39. Mohr M, Dalmis F, Hilgenfeld E, et al. Simultaneous immunomagnetic CD34C cell selection and B-cell depletion in peripheral blood progenitor cell samples of patients suffering from B-cell nonHodgkin’s lymphoma. Clin Cancer Res 2001; 7:51–57. 40. Hafer R, Voigt A, Gruhn B, Zintl F. Neuroblastoma cells can express the hematopoietic progenitor cell antigen CD34 as detected at surface protein and mRNA level. J Neuroimmunol 1999; 96:201–206. 41. Voigt A, Hafer R, Gruhn B, Zintl F. Expression of CD34 and other haematopoietic antigens on neuroblastoma cells: consequences for autologous bone marrow and peripheral blood stem cell transplantation. J Neuroimmunol 1997; 78:117–126. 42. Kanold J, Yakouben K, Tchirkov A, et al. Long-term results of CD34(C) cell transplantation in children with neuroblastoma. Med Pediatr Oncol 2000; 35:1–7. 43. Ho VT, Soiffer RJ. The history and future of T cell depletion as a graft-versus-host disease prophylaxis for allogeneic hematopoietic stem cell transplantation. Blood 2002; 98:3192–3204. 44. Wagner JE, Santos GW, Noga SJ, et al. Bone marrow graft engineering by counterflow centrifugal elutriation: results of a phase I-II clinical trial. Blood 1990; 75:1370–1377. 45. Reisner Y, Kapoor N, Kirkpatrick D, et al. Transplantation for acute leukaemia with HLA-A and B nonidentical parental marrow cells fractionated with soybean agglutinin and sheep red blood cells. Lancet 1981; 2:327–331. 46. Fehse B, Goldmann M, Frerk O, Bulduk M, Zander AR. Depletion of alloreactive donor T cells using immunomagnetic cell selection. Bone Marrow Transplant 2000; 25:S39–S42. 47. Freedman AS, Neuberg D, Mauch P, et al. Long-term follow-up of autologous bone marrow transplantation in patients with relapsed follicular lymphoma. Blood 1999; 94:3325–3333.

146

Grupp

48. Reynolds CP, Seeger RC, Vo DD, Black AT, Wells J, Ugelstad J. Model system for removing neuroblastoma cells from bone marrow using monoclonal antibodies and magnetic immunobeads. Cancer Res 1986; 46:5882–5886. 49. Seeger RC, Vo DD, Ugelstad J, Reynolds CP. Removal of neuroblastoma cells from bone marrow with monoclonal antibodies and magnetic immunobeads. Prog Clin Biol Res 1986; 211:285–293. 50. Rill DR, Santana VM, Roberts WM, et al. Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells. Blood 1994; 84:380–383. 51. Attarian H, Feng Z, Buckner CD, MacLeod B, Rowley SD. Long-term cryopreservation of bone marrow for autologous transplantation. Bone Marrow Transplant 1996; 17:425–430.

7 Pediatric Unrelated Donor Stem-Cell Transplantation Monica Bhatia Division of Pediatric Hematology and Blood and Marrow Transplantation, Columbia University, New York, New York, U.S.A.

Naynesh R. Kamani Division of Stem Cell Transplantation and Immunology, Children’s National Medical Center and The George Washington University School of Medicine, Washington, D.C., U.S.A.

HISTORY The era of hematopoietic stem cell transplantation (HSCT) was launched following work from the late 1940s and early 1950s that showed that mice could be protected from the lethal effects of ionizing radiation on bone marrow by shielding their spleens with lead (1). Shortly thereafter, Lorenz and colleagues showed that protection against radiation could also be conferred by intravenous infusion of bone marrow (2). Initially, this was thought to be due to some humoral factor in the spleen or bone marrow that stimulated recovery (1). In the 1950s, Thomas and others reported on outcomes following infusions of syngeneic marrow into twins with leukemia who had received supralethal doses of total body irradiation. Their prompt hematologic and clinical recovery attested to the protective cellular effects of bone marrow (3–5). Progress in the field over the next four decades has resulted in allogeneic stem cell transplantation being recognized as curative therapy for several malignant and nonmalignant disorders. Before the mid-1980s, almost all allogeneic HSCT performed utilized marrow from HLA-identical siblings as the stem cell source. Because only 30% of patients have HLAmatched siblings, the use of alternative donors who were less than perfectly matched was explored (6). The first group of donors to be used were family members who were closely matched or haploidentical, such as parents or siblings (6,7). Although the use of related donor haploidentical T-cell depleted marrow as a stem cell source for infants with SCID became commonplace at a few select transplant institutions, the widespread use of this approach for patients with hematologic malignancies did not ensue because of the tedious methodologies for T-cell depletion and the increased risk of graft failure, relapse, and posttransplant lymphoproliferative syndromes following this approach. The search for alternative donors led to investigations focused on typing large numbers of individuals who volunteered to register with a number of private marrow donor banks. When necessary, they agreed to be called upon to donate marrow to a tissue type matched recipient in need of a donor. 147

148

Bhatia and Kamani

Speck et al. reported the first unrelated donor bone marrow transplant in a patient with aplastic anemia in which the donor was HLA-A and HLA-B matched but engraftment was not documented (8). Subsequent to this, Horowitz et al. reported a 10-year-old female with acute lymphoblastic leukemia (ALL) in second remission who received an HLA-A, HLA-B, and HLA-D matched unrelated marrow. A complete remission lasting 2 years with full donor engraftment was observed without any evidence of graft versus host disease (GVHD) (9). Other reports of successful unrelated donor transplants followed (10,11). However, this was more the exception than the rule as most unrelated donor transplants were associated with severe GVHD or with morbidity and mortality from the immunosuppression needed to treat the GVHD.

UNRELATED STEM CELL DONOR REGISTRIES As the numbers of transplants using unrelated donors increased, an effort ensued to bring together the private donor banks to set up a single international registry to provide all allogeneic hematopoietic stem cells for transplant recipients. The Anthony Nolan Bone Marrow Transplant Registry, established in 1975, was the first registry established to identify unrelated donors for unrelated stem cell transplantation (12). Similar initiatives were started in the U.S.A. in Milwaukee, St. Paul, Seattle, and Iowa City by approaching previously HLA-typed platelet donors (13). The National Marrow Donor Program (NMDP) was set up in 1986 through a contract from the United States Navy to the American Red Cross. With increasing recruitment efforts through the 1980s and into the present, well over five and a half million volunteer donors are registered and listed in the NMDP registry database. Currently the operations of the NMDP are jointly funded by the Health Resources and Services Administration and the U.S. Office of Naval Research. To further expand the pool, the NMDP registry, as well as other national donor registries, are connected through Bone Marrow Donors Worldwide (BMDW), which is sponsored by the Europdonor Foundation in the Netherlands. The BMDW database was developed to help facilitate donor searches internationally (14). To date, this program includes 49 bone marrow donor registries from 37 countries, and 24 cord blood registries from 16 countries. The registry currently lists close to ten million donors, and the likelihood of finding a suitably HLA-matched unrelated stem cell graft donor varies by race. For Caucasian patients, the probability of finding at least one potential HLA-A, B, DR match is more than 85% whereas in African Americans, this decreases to 60%. The problem of finding matches for racial/ethnic minorities, especially African Americans, is secondary to a multiplicity of factors, including higher HLA polymorphism (15), smaller numbers of minority volunteer donors, and the higher rates at which these donors become unavailable to searching patients. A recent study assessing the optimal size and composition of the U.S. national registry suggests that although the recruitment of additional new donors to the registry may improve the likelihood of finding matches, it may be more cost effective to improve donor availability and patient access to the NMDP registry (16).

HISTOCOMPATIBILITY 1. Histocompatibility antigens are cell surface determinants that help to mediate immune reactions and graft rejection after stem cell transplantation between the donor and recipient. They are widely expressed and encoded by genes within a chromosomal region termed the HLA complex (17,18). It is located on chromosome 6 and contains more than 200 genes. The major loci are grouped into class I, II, and III genes, although the role of class III genes in transplantation has yet to be fully determined. HLA genes are usually inherited as a unit within chromosome 6, also known as the haplotype. The combination of haplotypes inherited from each parent is known as the genotype.

Unrelated Donor Stem-Cell Transplantation

149

2. Class I antigens contain an a-polypeptide chain, encoded by class I genes, and a b chain, encoded by the b2-microglobulin gene on chromosome 15. HLA-A, HLA-B, and HLA-C are the class I genes shown to be of utmost importance in transplantation (19,20). 3. Class II molecules have an a and b polypeptide chain encoded by class II genes, designated by the class, family, and chain, respectively (21,22). Individual genes are identified by numbers and alleles preceded by an asterisk (19–22). Class II genes are known as HLA-D region genes and code for antigens responsible for the activation of T cells (23). There are five subtypes of class II genes: DR, DQ, DO, DN, and DP, which make up nine loci (DRA, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPB1, and DPB2) (17). HLA-DRB1, and DQB1 are the most important transplant related antigens, with HLA-DRB1 being the most diverse (24).

HLA TYPING Historically, class I antigens have been serologically defined on peripheral blood mononuclear cells (PBMC) by a complement dependent microcytotoxicity assay using panels of alloantisera containing HLA-antibodies, or by the use of monoclonal antibodies. Class II antigens have been described by serology for DR and DQ on PBMC enriched for B lymphocytes. Mixed lymphocyte culture (MLC) may also be used to evaluate differences in class II HLA-D antigens (25,26). The antigens involved in the MLC are combinations of HLA-DR, DP, and DQ. Serologic methods have several limitations. The initial lack of specificity seen with antisera can now be improved by the use of monoclonal antibodies. Another problem is the lack of antisera recognizing antigens expressed by non-Caucasians (13). Lymphocytopenia may sometimes interfere with class II testing as viable B cells are necessary for class II serologic testing. Serologic HLA typing methods do not detect the polymorphisms determined by molecular methods which define class I and II alleles (13). Because of all these limitations, molecular techniques are now widely used in searching for unrelated donors. DNA typing characterizes the genotype coding of the unique sequences in a molecule. Polymerase chain reaction based methods may directly establish a coding region of a sequence (27,28). Low resolution DNA typing provides the same level of HLA characterization as serologic typing. High resolution DNA or molecular typing denotes allele level identification. Class I molecular typing is more challenging than class II typing, owing to the higher number of polymorphisms and the presence of pseudogenes (29,30).

REGISTRIES AND DONOR SELECTION Durable engraftment of donor cells is an essential step in successful stem cell transplantation. Several studies have shown that in recipients of marrow from non-HLA-identical family members, the incidence of graft failure increases with increasing HLA disparity (31,32). Selecting an unrelated donor for HSCT is primarily based on HLA matching. Usually, HLA-A, HLA-B, and HLA-DR types are assigned for volunteer donors and patients seeking an unrelated donor. These assignments are submitted by many different laboratories testing on behalf of transplant centers or donor recruitment centers (33). Search criteria are often based only on the HLA-A, -B, and -DR loci, although information on other loci, such as HLA-C or -DQ, may also be provided. Although these secondary assignments for donors are stored by registries and sometimes may appear on search reports, they are usually not included in matching algorithms. More data attesting to the importance of these secondary loci on transplant outcome is emerging and may become a factor in matching algorithms in the future. For example, a report by Flomenberg et al. suggested that HLA-C matching should be given the same priority as HLA-A, HLA-B, and HLA-DR matching (34).

150

Bhatia and Kamani

An NMDP analysis showed that younger donor age was associated with better survival. Five year overall survival rates for recipients were 33%, 29%, and 25%, respectively, with donors aged 18 to 30 years, 31 to 45 years, and greater than 45 years (pZ0.0002). There were no significant associations between donor cytomegalovirus (CMV) serology for CMV-negative patients, sex, parity, race, or ABO matching and survival (35). In an analysis of 481 recipients of matched related, mismatched related and unrelated donor partially T-cell depleted transplants, Keever-Taylor et al. identified ABO disparity, the method of T-cell depletion, and an HLA mismatch of R2 Ag as factors contributing to higher rates of GVHD (36). In an analysis of 469 matched sibling donor transplants, Weisdorf et al. found that in univariate analysis, patient or donor age greater than 18 years was associated with an increased GVHD risk (37). This suggests that donor age may contribute to higher rates of GVHD in transplants utilizing volunteer unrelated adult donors. In those patients with multiple highly matched suitable donors, there have been reports of additional benefits with matching at the HLA-DQB1, -DPB1, and -DRB3/4/5 loci. However, the association between HLA-DQ and -DP mismatching and mortality remains unproven, and the association of HLA-DRB3/4/5 mismatching and survival has not been studied (33). At the time a search is being initiated, DNA based testing methods should be used to identify the patient’s HLA alleles. Although it is paramount to type HLA-A, -B, and -DRB1, other loci, including HLA-C and DQ, should also be characterized. Some are important in matching while others assist in designing an efficient search strategy for the patient. It is now commonplace to search for 10/10 allele level matched unrelated donors. HLA typing of members of the patient’s immediate family is necessary to identify a potential related donor, to confirm patient HLA assignments, and to define haplotypes. In the absence of a related donor, an unrelated donor search can be initiated. Once donors are selected from the NMDP search report as potential matches, they should have higher resolution testing done to select the best match. The optimal number of potential donors to select from the search report should be customized for each patient, because multiple factors influence the likelihood of finding a donor. These factors include the patient’s alleles, haplotypes, and clinical urgency. In a clinically urgent situation, multiple donors should be simultaneously evaluated. The success in finding a DRB1-matched donor is directly related to the allele prevalence in the population, and the combination of class I and II antigens inherited by the subject. The probability of identifying a DRB1-matched donor among serologically matched HLA-A, HLA-B, and HLA-DR donors is about 40% if only one serologically matched donor is available, but 90% if five donors are available (30). For those patients with highly conserved haplotypes, further typing of a small number of donors is usually sufficient. More than one donor should always be selected, as some donors may be unavailable, mistyped, or not matched at the allele level. For patients with rare alleles and haplotypes, O10 donors may be needed to find the best match. Thorough HLA typing at the allele level is the basis of the entire search process. Although allele level typing is more time consuming than lower resolution testing, it ultimately speeds donor selection and enables better matching. This leads to fewer posttransplant complications. In the event that a well matched donor is not available, it allows the process to proceed more rapidly to the search for a partially matched donor (related or unrelated) (33). Once confirmatory typing has been completed, prospective donors are contacted and prepared for transplant. They are interviewed, medically evaluated, and checked for transmissible diseases. Once clinical clearance has been obtained by the NMDP-approved donor or collection center, the patient’s transplant team is alerted and a transplant date is scheduled. The marrow donation harvest or peripheral blood stem cell collection is performed and the graft is shipped to the recipient’s hospital within 24–48 hours. The search process and preparation of the donor can take between 2 and 6 months from initiation to completion. Patients needing urgent transplants or those with advanced or aggressive disease may develop complications or disease progression while awaiting donor identification. They may benefit from the use of unrelated cord blood as a stem cell source.

Unrelated Donor Stem-Cell Transplantation

151

PREPARATIVE REGIMENS Multiple different preparative (or pre-transplant) conditioning regimens have been used in stem cell transplantation which include combinations of chemotherapy, total body radiation (TBI), and biologic agents. Traditionally, myeloablative regimens that are also intensely immunosuppressive have been recommended in unrelated donor HSCT. These generally include TBI and high dose cyclophosphamide or purine analogs, with or without ATG (13). Patients receiving autologous BMT or matched sibling SCT may be able to tolerate more intensive conditioning regimens because of a lower incidence of GVHD (38,39). In those situations where a graft-versus-tumor or graft-versus-leukemia effect is desired, a shift towards more immunosuppression,without ablation, to ensure engraftment may be favored. Nonmyeloablative regimens are based on the premise that using a less ablative regimen with enough immunosuppression will result in donor engraftment. It allows for a graft versus tumor effect while at the same time significantly reducing both short and long term toxicities. Slavin et al. recently showed that reduced intensity conditioning with busulfan, fludarabine, and ATG followed by matched unrelated donor bone marrow transplantation can result in complete donor chimerism (40). Several studies have since shown that a fludarabine based reduced intensity regimen with or without cyclophosphamide, busulfan, melphalan, low dose TBI (200 cGy), thymic irradiation, or ATG in patients with malignant or nonmalignant diseases, followed by HLA matched unrelated donor transplantation can result in stable mixed or complete donor chimerism (41–47). The use of nonmyeloablative regimens in patients with nonmalignant diseases can be expected to result in higher rates of graft rejection and mixed donor cell chimerism, especially in matched unrelated donor transplantation.

COMPLICATIONS The complications seen following unrelated donor transplantation are similar to those seen after matched related HSCT. Opportunistic infections, acute, and chronic GVHD, and disease relapse comprise the major complications. Although the risk of GVHD in unrelated donor transplants remains high and survival currently is not as favorable as in HLA-identical sibling transplants, better supportive care and GVHD prophylaxis are providing a gradual improvement in the long term disease free survival of URD transplants (48).

Infections The pediatric SCT patient, much like other transplant hosts, is at highest risk of infection in the first 30 days post-transplantation. The cumulative incidence of infection was evenly distributed by the type of transplant in those first 30 days. After day 30, patients receiving matched unrelated donor transplants and unrelated cord blood transplants were at much higher risk than autologous or matched sibling transplant recipients (49). Disseminated Aspergillus infection after unrelated transplants has an incidence of approximately 15% with more than a 90% mortality rate (50). These patients were also at much higher risk for Candida and Adenovirus with a case fatality rate of 50–90% (49). These infections generally occur after Day 30 posttransplantation and are related to prolonged neutropenia and GVHD-conditions more strongly associated with unrelated cord blood transplants and matched unrelated donor transplantation. The advent of prophylactic and preemptive therapy with gancyclovir has significantly reduced the morbidity and mortality associated with CMV infection.

Graft-Versus-Host Disease GVHD is due to T lymphocytes from the donor that recognize the recipient tissue as foreign.

152

Bhatia and Kamani

Acute Graft-Versus-Host Disease A detailed discussion of acute GVHD is beyond the scope of this chapter. Although minor antigen disparity is believed to play an important role in GVHD following matched sibling donor HSCT, this along with disparity at major HLA antigens may contribute to the significantly higher GVHD risk than that seen following matched sibling donor transplantation. In an analysis of 363 children who had undergone unrelated donor BMT for ALL in second remission, 71% of patients developed acute GVHD with 29% developing grades III-IV acute GVHD (51). This is markedly higher than the incidence reported by Al-Kasim et al. Their work reported an incidence of grade II-IV acute GVHD of 24% in children with ALL undergoing a matched related transplant (52). Because volunteer unrelated donors are adults and because donor age plays an important role in the causation of GVHD, it can be expected that pediatric recipients of very well matched unrelated donor marrows will experience a higher risk of GVHD compared to those receiving sibling donor transplants. This may result in a decreased risk of leukemia relapse and a consequent survival benefit in patients with certain hematologic malignancies. Chronic Graft-Versus-Host Disease This is the most common nonrelapse complication affecting long-term survivors of allogeneic stem cell transplantation (53). The incidence of chronic GVHD is lower in children but still remains significant. In retrospective analyses of unrelated donor BMT, extensive chronic GVHD incidence rates of 25–30% have been reported (51,54). Chronic GVHD is becoming a more frequent problem due to the increasing use of alternative donors, including haploidentical family members, increasing age limits of transplantation, the use of peripheral blood stem cells (PBSC) instead of bone marrow as the source of the graft, and the use of donor lymphocyte infusions (DLI) for treatment or prophylaxis to prevent relapse in those patients at high risk (55). The most important risk factor for the development of chronic GVHD is the development of clinically significant acute GVHD. Other risk factors include older age, use of mismatched or unrelated donors, infusion of DLI, use of PBSC versus BM, lack of T-cell depletion and the combination of female donors and male recipients (56–62). Graft Failure Durable engraftment of donor hematopoietic stem cells depends on multiple factors, including underlying patient diagnosis, disease status at the time of transplant, allosensitization, intensity of the pretransplant conditioning regimen, and posttransplant immunosuppression. The dose of stem cells infused is crucial for successful engraftment and lower cell doses are known to increase the likelihood of graft failure. Other causes of graft rejection include T-cell depletion, infections, and medications. Late graft failures have been reported in 2–15% of unrelated donor transplant recipients (31,63). Causes include HLA-incompatibility, GVHD, graft T-cell depletion, low numbers of progenitor cells, and infectious etiologies such as CMV, EBV, HSV, hepatitis, parvovirus B19, and human herpesvirus-6. Prognosis after graft failure is usually poor (11,31). Depending on the general condition of the patient, a second transplant may be considered. The outcome of a second transplant for primary graft failure is worse than that for secondary graft failure (64).

THE ROLE OF UNRELATED DONOR TRANSPLANTS IN SPECIFIC DISEASES Acute Lymphoblastic Leukemia ALL is the most common indication for marrow transplantation in children. Because chemotherapy provides excellent control of the disease in newly diagnosed patients, transplantation in first remission is limited to those with very high risk ALL. For those

Unrelated Donor Stem-Cell Transplantation

153

children who relapse within 36 months of diagnosis, there is a general consensus that related or unrelated donor allogeneic transplantation offers the best chance for long-term disease free survival. In those relapsing later, the role of allogeneic transplantation is more controversial. There are no randomized therapeutic trials to determine the role of allogeneic stem cell transplantation in these children, and the decision to pursue this option may rely on the availability of a matched related donor, the duration of first remission, and institutional bias. The development of donor registries has increased the availability of HLA-matched or closely matched donors for the 70% of patients without an HLA-matched family donor. Compared with historical controls, results with unrelated donor transplants have approached those with transplants from HLA-matched siblings. Woolfrey et al. identified factors associated with outcomes of unrelated donor marrow transplantation in childhood ALL. Leukemia free survival (LFS) at 3 years, according to phase of disease at transplantation, was 70%, 46%, 20%, and 9%, respectively, for patients in CR1, CR2, CR3, and relapse. Other factors identified as important prognostic variables included younger age and a CR1 duration of longer than 24 months (65). In an NMDP analysis of 363 children with ALL in CR2 who underwent unrelated donor BMT, Bunin et al. reported a 5 year LFS of 36% and identified recipient age less than 15 years and HLA matching as important factors in improved LFS (51).

Acute Myelogenous Leukemia Children with acute myelogenous leukemia (AML) who are treated with standard chemotherapeutic regimens have a long term disease free survival of 35–46%. Those who have an HLA-matched sibling usually receive BMT in first complete remission with a disease free survival of 50–70%, and a relapse rate of 20–30%. Although BMT appears to offer a better therapeutic outcome for AML in first remission, only 30 percent of patients have an HLAmatched sibling donor. In 2000, a report from Seattle studied 161 patients with primary AML between 1985 and 1998 who received T-replete BMT from unrelated donors. The median age was 30 years ranging from 1–55 years. The median post transplant follow-up was 2.9 years, with a LFS at 5 years of 50C/K 12% for transplants during first complete remission. The incidence of relapse was 19% after 5 years. Transplantation during remission, a marrow cell dose above 3.5!108/kg, and CMV seronegative status before BMT in both patient and donor were favorable prognostic factors. Although this data was not limited to the pediatric population, it does support the use of unrelated donor BMT for the treatment of patients with high risk AML when a family match is not available (66).

Myelodysplastic Syndrome The only curative therapy available for patients with myelodysplastic syndrome is allogeneic HSCT. Previous reports have clearly shown that BMT from HLA-matched siblings can cure 40–50% of patients with MDS. The main obstacles limiting the success of BMT in these patients have been disease relapse and treatment related mortality (TRM) (67–75). Outcomes after related donor HSCT have shown that disease free survival is strongly correlated with patient age, MDS subtype, prognostic score, and chromosomal abnormalities with relapse being associated with MDS subtype, chromosomal abnormalities, and disease duration. Transplant related mortality is strongly correlated with disease duration, patient age, and HLA matching. The largest study to date is a retrospective analysis of the first 510 patients with MDS who underwent unrelated donor BMT facilitated through the NMDP (54). Results were comparable to those seen with related HSCT with a disease free survival (DFS) of 29% at 2 years and 26% at 4 years. The 2-year cumulative incidence of relapse was 14%. The most common cause of death was treatment related complications. Infections, acute and chronic GVHD, and regimen related toxicity were the most frequent complications leading to death. In this study, which included 144 children, recipient age was not a factor independently associated with any difference in DFS. This study showed that HSCT is a valid option for those with MDS

154

Bhatia and Kamani

and that the best results are achieved in the early stages of disease and within the first year of diagnosis (54).

Chronic Myelogenous Leukemia Chronic myelogenous leukemia (CML) occurs in children in two forms: the more common adult type characterized by the presence of the Philadelphia chromosome and the much rarer juvenile form. Children undergoing allogeneic BMT for adult type CML have a disease free survival rate of 50–85% with a relapse rate of 15–30%. Patients who have progressed beyond the chronic phase to an accelerated or blast phase of CML have a much lower response rate. In an analysis of more than 1400 patients with CML who underwent URD-BMT, graft failure and acute and chronic GVHD were identified as the major obstacles, whereas relapses occurred in only 6% of the patients. By contrast, transplant in chronic phase or within 1 year of diagnosis, younger recipient age, a CMV seronegative recipient and the lack of severe acute GVHD were independent factors for improved DFS. Despite promising advances in the therapy of CML with interferon and imatinib mesylate, allogeneic stem cell transplantation currently remains the only curative therapy for CML. Children with adult-type CML should be strongly considered for allogeneic BMT at or soon after diagnosis if a matched sibling donor is available. With the advent of imatinib, those lacking donors are placed on imatinib therapy and only considered for unrelated donor BMT if they are refractory to imatinib. Juvenile chronic myelogenous leukemia (JCML), more appropriately referred to as Juvenile myelomonocytic leukemia (JMML) is much less common than CML. It occurs in children under 5 years of age and is characterized by an elevated fetal hemoglobin and an aggressive course. Because conventional chemotherapy offers no long-term benefits, the recommended therapy for JCML is allogeneic BMT after high dose chemoradiotherapy as soon as a suitable related or unrelated donor is identified.

Severe Aplastic Anemia In children with severe aplastic anemia, bone marrow transplantation from a matched related donor is the therapy of choice at diagnosis with large cohort studies showing overall survival rates between 90–95%. For the 70% of patients who lack matched related donors, immunosuppressive therapy is the recommended approach because acute toxicity is moderate and response rates as high as 70% have been reported. However, recurrence of aplasia can occur, some patients have incomplete hematologic responses and 20% to 45% of patients receiving immunosuppressive therapy have been reported to develop a clonal hematopoietic disorder 5 to 7 years after treatment. Patients who fail one or more courses of immune suppressive therapy can be considered for unrelated donor HSCT. Transplant related mortality with an unrelated donor historically has been high. Causes of transplantation failure include increased incidence of graft rejection, regimen related toxicity due to intensified conditioning regimens aimed at preventing graft rejection, and a high incidence of GVHD. In a retrospective analysis of NMDP data, Deeg et al. reported outcomes following unrelated donor transplantation in 141 patients with severe aplastic anemia who had failed immune suppressive therapy (76). Slightly more than half of the cohort consisted of children. This analysis showed a sustained engraftment rate of 89% with a 52% incidence of grade II-IV acute GVHD and 31% of evaluable patients developing extensive chronic GVHD. At the time of analysis, 36% of patients were surviving at 11–94 months post transplant. Patients with donors matched by both serology and allele-level DRB1 typing had significantly better survival that DRB1 mismatched patients with 56% versus 15% surviving at 3 years (pZ0.001). Outcomes in patients transplanted within 3 years of diagnosis were superior to those patients transplanted with greater delay. Major causes of death included GVHD, graft failure, and infections. An ongoing NMDP analysis of more than 200 pediatric patients who underwent

Unrelated Donor Stem-Cell Transplantation

155

unrelated donor HSCT shows an overall 5-year survival of 45% with acute and chronic GVHD incidence rates comparable to those from the Deeg study (77).

Hurler Syndrome The mucopolysaccharidoses (MPS) are a group of heritable disorders caused by a deficiency of the lysosomal enzymes needed to degrade glycosaminoglycans. Hurler syndrome (MPS I H) is a progressive autosomal recessive inborn error of metabolism that leads to premature death usually by 5 years of age. By providing a renewable source of lysosomal enzymes, bone marrow transplantation has provided a method for correcting enzymatic deficiencies in several lysosomal and peroxisomal disorders (78). After HSCT for Hurler syndrome, recipients attain donor levels of serum and leukocyte a-L-iduronidase, normalization of urinary excretion of glycosaminoglycans, and decreased hepatosplenomegaly and obstructive sleep apnea (79,80). Despite stable engraftment, skeletal and corneal abnormalities progress necessitating surgical interventions. Peters et al. recently reported the retrospective experience using unrelated donor BMT for 40 children with MPS I H (81). All patients received high dose chemotherapy with or without radiation followed by BMT. Twenty-five of the 40 patients initially engrafted. An estimated 49% of patients were alive at 2 years, 63% alloengrafted and 37% autoengrafted. The probability of grade II to IV acute GVHD was 30%, and the probability of extensive chronic GVHD was 18%. Eleven patients received a second URD-BMT because of graft rejection or failure. Of the 20 survivors, 13 had complete donor engraftment, two children had mixed chimeric grafts, and five children had autologous bone marrow recovery. Although these results show that unrelated donor BMT can provide an alternative for those patients without a matched sibling donor, future endeavors are being targeted to address the high risk of graft rejection or failure and the impact of GVHD in this patient population.

Wiskott-Aldrich Syndrome Wiskott-Aldrich syndrome (WAS) is an X-linked, prematurely lethal inherited disorder primarily affecting the morphology and function of platelets and lymphocytes. It results from mutations in the WASP gene at Xp11.22 (82,83). Matched sibling donor BMT is the therapy of choice and curative. In a combined IBMTR/NMDP analysis of allogeneic transplants for WAS, Filipovich et al. recently reported a 5-year probability of survival for all subjects of 70% (84). Probabilities for survival differed by donor type: 87% with HLA-identical sibling donors, 52% with other related donors, and 71% with unrelated donors. Analyses indicated significantly lower survival using related donors other than HLA-identical siblings or unrelated donors. Most importantly, boys receiving an unrelated donor transplant before age five had survival rates similar to those receiving HLA-identical sibling transplants.

REFERENCES 1. Jacobson L, Simmons EL, Marks EK, et al. Recovery from radiation injury. Science 1949; 102:400–402. 2. Lorenz E, Uphoff D, Congdon CC, et al. Modification of irradiation injury in mice and guinea pigs by bone marrow injections. J Natl Cancer Inst 1951; 12:197–201. 3. Thomas ED, Lochte HL, Jr., Cannon J, et al. Supralethal whole body irradiation and isologous marrow transplantation in man. J Clin Invest 1959; 38:1709–1716. 4. Thomas ED, Weiden PL. Marrow transplantation of aplastic anemia and acute leukemia. World J Surg 1977; 2:197–206. 5. Thomas ED, Blume KG. Historical markers in the development of allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 1999; 5:341–346.

156

Bhatia and Kamani

6. Trigg ME, Gingrich R, Goeken N, et al. Low rejection rate when using unrelated or haploidentical donors for children with leukemia undergoing marrow transplantation. Bone Marrow Transplant 1989; 4:431–437. 7. Trigg ME. Bone marrow transplantation using alternative donors. Mismatched related donors or closely matched unrelated donors. Am J Pediatr Hematol Oncol 1993; 15:141–149. 8. Speck B, Zwaan FE, van Rood JJ, et al. Allogeneic bone marrow transplantation in a patient with aplastic anemia using a phenotypically HL-A-identical unrelated donor. Transplantation 1973; 16:24–28. 9. Horowitz SD, Bach FH, Groshong T, et al. Treatment of severe combined immunodeficiency with bone-marrow from an unrelated, mixed-leucocyte-culture-non-reactive donor. Lancet 1975; 2:431–433. 10. Lohrmann HP, Dietrich M, Goldman SF, et al. Bone marrow transplantation for aplastic anaemia from a HL-A and MLC-identical unrelated donor. Blut 1975; 31:347–354. 11. Hansen JA, Clift RA, Thomas ED, et al. Transplantation of marrow from an unrelated donor to a patient with acute leukemia. N Engl J Med 1980; 303:565–567. 12. Cleaver S. The anthony nolan research centre and other matching registries. In: Treleaven J, Barrett J, eds. Bone Marrow Transplantation in Practice. Edinburgh: Churchill Livingstone, 1992:361–366. 13. de Lima M, Champlin R. Unrelated donor hematopoietic transplantation. Rev Clin Exp Hematol 2001; 5:100–134. 14. Oudshoorn M, van Leeuwen A, vd Zanden HG, et al. Bone marrow donors worldwide: a successful exercise in international cooperation. Bone Marrow Transplant 1994; 14:3–8. 15. Beatty P, Mori M, Milford E, et al. Impact of racial genetic polymorphism upon the probability of finding an HLA-matched donor. Transplantation 1995; 60:778. 16. Kollman C, Abella E, Baitty RL, et al. Assessment of optimal size and composition of the U.S. National registry of hematopoietic stem cell donors. Transplantation 2004; 78:89–95. 17. Dausset J, Nenna A. Presence of leuko-agglutinin in the serum of a case of chronic agranulocytosis. C R Seances Soc Biol Fil 1952; 146:1539–1541. 18. Payne R, Tripp M, Weigle J, et al. A new leukocyte isoantigen system in man. Cold Spring Harb Symp Quant Biol 1964; 29:285–295. 19. Albert E, Amos DB, Bodmer WF, et al. Nomenclature for factors of the HLA-system. Z Immunitatsforsch Immunobiol 1977; 153:373–379. 20. Bodmer WF. HLA: a super supergene. Harvey Lect 1978; 72:91–138. 21. Klein J, Sato A. The HLA system. First of two parts. N Engl J Med 2000; 343:702–709. 22. Klein J, Sato A. The HLA system. Second of two parts. N Engl J Med 2000; 343:782–786. 23. DuPont B, Hansen JA. Human mixed-lymphocyte culture reaction: genetics, specificity, and biological implications. Adv Immunol 1976; 23:107–202. 24. Schreuder GM, Hurley CK, Marsh SG, et al. The HLA dictionary 1999: a summary of HLA-A, -B, C, -DRB1/3/4/5, -DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR, and -DQ antigens. Hum Immunol 1999; 60:1157–1181. 25. Bach F, Hirschhorn K. Lymphocyte interaction: a potential histocompatibility test in vitro. Science 1964; 143:813–814. 26. Petersdorf E, Anasetti C, Servida P, et al. Effect of HLA matching on outcome of related and unrelated donor transplantation therapy for chronic myelogenous leukemia. Hematol Oncol Clin North Am 1998; 12:107–121. 27. Allen M, Liu L, Gyllensten U, et al. A comprehensive polymerase chain reaction-oligonucleotide typing system for the HLA class I A locus. Hum Immunol 1994; 40:25–32. 28. Gao X, Jakobsen IB, Serjeantson SW, et al. Characterization of the HLA-A polymorphism by locusspecific polymerase chain reaction amplification and oligonucleotide hybridization. Hum Immunol 1994; 41:267–279. 29. Santamaria P, Lindstrom AL, Boyce-Jacino MT, et al. HLA class I sequence-based typing. Hum Immunol 1993; 37:39–50. 30. Petersdorf EW, Hansen JA. A comprehensive approach for typing the alleles of the HLA-B locus by automated sequencing. Tissue Antigens 1995; 46:73–85. 31. Davies SM, Ramsay NK, Haake RJ, et al. Comparison of engraftment in recipients of matched sibling of unrelated donor marrow allografts. Bone Marrow Transplant 1994; 13:51–57. 32. Karanes C. Unrelated donor stem cell transplant: donor selection and search process. Pediatr Transplant 2003; 7:59–64.

Unrelated Donor Stem-Cell Transplantation

157

33. Hurley CK, Baxter-Lowe LA, Logan B, et al. National marrow donor program HLA-matching guidelines for unrelated marrow transplants. Biol Blood Marrow Transplant 2003; 9:610–615. 34. Flomenberg N, Baxter-Lowe LA, Confer D, et al. Impact of HLA class I and class II high-resolution matching on outcomes of unrelated donor bone marrow transplantation: HLA-C mismatching is associated with a strong adverse effect on transplantation outcome. Blood 2004; 104:1923–1930. 35. Kollman C, Howe CW, Anasetti C, et al. Donor characteristics as risk factors in recipients after transplantation of bone marrow from unrelated donors: the effect of donor age. Blood 2001; 98:2043–2051. 36. Keever-Taylor CA, Bredeson C, Loberiza FR, et al. Analysis of risk factors for the development of GVHD after T cell-depleted allogeneic BMT: effect of HLA disparity, ABO incompatibility, and method of T-cell depletion. Biol Blood Marrow Transplant 2001; 7:620–630. 37. Weisdorf D, Hakke R, Blazar B, et al. Risk factors for acute graft-versus-host disease in histocompatible donor bone marrow transplantation. Transplantation 1991; 51:1197–1203. 38. Bearman SI, Mori M, Beatty PG, et al. Comparison of morbidity and mortality after marrow transplantation from HLA-genotypically identical siblings and HLA-phenotypically identical unrelated donors. Bone Marrow Transplant 1994; 13:31–35. 39. Appelbaum FR. Is there a best transplant conditioning regimen for acute myeloid leukemia? Leukemia 2000; 14:497–501. 40. Slavin S, Aker M, Shapira MY, et al. Reduced-intensity conditioning for the treatment of malignant and life-threatening non-malignant disorders. Clin Transpl 2003;275–282. 41. Khouri IF, Keating M, Korbling M, et al. Transplant-lite: induction of graft-versus-malignancy using fludarabine-based nonablative chemotherapy and allogeneic blood progenitor-cell transplantation as treatment for lymphoid malignancies. J Clin Oncol 1998; 16:2817–2824. 42. Amrolia P, Gaspar HB, Hassan A, et al. Nonmyeloablative stem cell transplantation for congenital immunodeficiencies. Blood 2000; 96:1239–1246. 43. Bornhauser M, Thiede C, Schuler U, et al. Dose-reduced conditioning for allogeneic blood stem cell transplantation: durable engraftment without antithymocyte globulin. Bone Marrow Transplant 2000; 26:119–125. 44. Childs R, Chernoff A, Contentin N, et al. Regression of metastatic renal-cell carcinoma after nonmyeloablative allogeneic peripheral-blood stem-cell transplantation. N Engl J Med 2000; 343:750–758. 45. Nagler A, Slavin S, Varadi G, et al. Allogeneic peripheral blood stem cell transplantation using a fludarabine-based low intensity conditioning regimen for malignant lymphoma. Bone Marrow Transplant 2000; 25:1021–1028. 46. Giralt S, Thall PF, Khouri I, et al. Melphalan and purine analog-containing preparative regimens: reduced-intensity conditioning for patients with hematologic malignancies undergoing allogeneic progenitor cell transplantation. Blood 2001; 97:631–637. 47. Khouri IF, Saliba RM, Giralt SA, et al. Nonablative allogeneic hematopoietic transplantation as adoptive immunotherapy for indolent lymphoma: low incidence of toxicity, acute graft-versus-host disease, and treatment-related mortality. Blood 2001; 98:3595–3599. 48. Hansen JM, Abildgaard U, Fogh-Andersen N, et al. The transplanted human kidney does not achieve functional reinnervation. Clin Sci (Lond) 1994; 87:13–20. 49. Benjamin DK, Jr., Miller WC, Bayliff S, et al. Infections diagnosed in the first year after pediatric stem cell transplantation. Pediatr Infect Dis J 2002; 21:227–234. 50. Anasetti C, Etzioni R, Petersdorf EW, et al. Marrow transplantation from unrelated volunteer donors. Annu Rev Med 1995; 46:169–179. 51. Bunin N, Carston M, Wall D, et al. Unrelated marrow transplantation for children with acute lymphoblastic leukemia in second remission. Blood 2002; 99:3151–3157. 52. Al-Kasim FA, Thornley I, Rolland M, et al. Single-centre experience with allogeneic bone marrow transplantation for acute lymphoblastic leukaemia in childhood: similar survival after matchedrelated and matched-unrelated donor transplants. Br J Haematol 2002; 116:483–490. 53. Socie G, Stone JV, Wingard JR, et al. Long-term survival and late deaths after allogeneic bone marrow transplantation. Late effects working committee of the international bone marrow transplant registry. N Engl J Med 1999; 341:14–21. 54. Castro-Malaspina H, Harris RE, Gajewski J, et al. Unrelated donor marrow transplantation for myelodysplastic syndromes: outcome analysis in 510 transplants facilitated by the national marrow donor program. Blood 2002; 99:1943–1951. 55. Vogelsang GB. How I treat chronic graft-versus-host disease. Blood 2001; 97:1196–1201.

158

Bhatia and Kamani

56. Storb R, Prentice RL, Sullivan KM, et al. Predictive factors in chronic graft-versus-host disease in patients with aplastic anemia treated by marrow transplantation from HLA-identical siblings. Ann Intern Med 1983; 98:461–466. 57. Ringden O, Paulin T, Lonnqvist B, et al. An analysis of factors predisposing to chronic graft-versushost disease. Exp Hematol 1985; 13:1062–1067. 58. Atkinson K, Farewell V, Storb R, et al. Analysis of late infections after human bone marrow transplantation: role of genotypic nonidentity between marrow donor and recipient and of nonspecific suppressor cells in patients with chronic graft-versus-host disease. Blood 1982; 60:714–720. 59. Bostrom L, Ringden O, Jacobsen N, et al. A European multicenter study of chronic graft-versus-host disease. The role of cytomegalovirus serology in recipients and donors—acute graft-versus-host disease, and splenectomy. Transplantation 1990; 49:1100–1105. 60. Ochs LA, Miller WJ, Fillipovich AH, et al. Predictive factors for chronic graft-versus-host disease after histocompatible sibling donor bone marrow transplantation. Bone Marrow Transplant 1994; 13:455–460. 61. Carlens S, Ringden O, Remberger M, et al. Risk factors for chronic graft-versus-host disease after bone marrow transplantation: a retrospective single centre analysis. Bone Marrow Transplant 1998; 22:755–761. 62. Prezepiorka D, Anderlini P, Saliba R, et al. Chronic graft-versus-host disease after allogeneic blood stem cell transplantation. Blood 2001; 98:1695–1700. 63. Hansen JA, Gooley TA, Martin PJ, et al. Bone marrow transplants from unrelated donors for patients with chronic myeloid leukemia. N Engl J Med 1998; 338:962–968. 64. Champlin RE, Horowitz MM, van Bekkum DW, et al. Graft failure following bone marrow transplantation for severe aplastic anemia: risk factors and treatment results. Blood 1989; 73:606–613. 65. Woolfrey AE, Anasetti C, Storer B, et al. Factors associated with outcome after unrelated marrow transplantation for treatment of acute lymphoblastic leukemia in children. Blood 2002; 99:2002–2008. 66. Sierra J, Storer B, Hansen JA, et al. Unrelated donor marrow transplantation for acute myeloid leukemia: an update of the Seattle experience. Bone Marrow Transplant 2000; 26:397–404. 67. Anderson JE, Appelbaum FR, Fisher LD, et al. Allogeneic bone marrow transplantation for 93 patients with myelodysplastic syndrome. Blood 1993; 82:677–681. 68. Ratanatharathorn V, Karanes C, Uberti J, et al. Busulfan-based regimens and allogeneic bone marrow transplantation in patients with myelodysplastic syndromes. Blood 1993; 81:2194–2199. 69. O’Donnell MR, Long GD, Parker PM, et al. Busulfan/cyclophosphamide as conditioning regimen for allogeneic bone marrow transplantation for myelodysplasia. J Clin Oncol 1995; 13:2973–2979. 70. Demuynck H, Verhoef GE, Zachee P, et al. Treatment of patients with myelodysplastic syndromes with allogeneic bone marrow transplantation from genotypically HLA-identical sibling and alternative donors. Bone Marrow Transplant 1996; 17:745–751. 71. Sutton L, Chastang C, Ribaud P, et al. Factors influencing outcome in de novo myelodysplastic syndromes treated by allogeneic bone marrow transplantation: a long-term study of 71 patients societe francaise de greffe de moelle. Blood 1996; 88:358–365. 72. Ballen KK, Gilliland DG, Guinan EC, et al. Bone marrow transplantation for therapy-related myelodysplasia: comparison with primary myelodysplasia. Bone Marrow Transplant 1997; 20:737–743. 73. Appelbaum FR, Anderson J. Allogeneic bone marrow transplantation for myelodysplastic syndrome: outcomes analysis according to IPSS score. Leukemia 1998; 12:S25–S29. 74. Nevill TJ, Fung HC, Shepherd JD, et al. Cytogenetic abnormalities in primary myelodysplastic syndrome are highly predictive of outcome after allogeneic bone marrow transplantation. Blood 1998; 92:1910–1917. 75. Runde V, de Witte T, Arnold R, et al. Bone marrow transplantation from HLA-identical siblings as first-line treatment in patients with myelodysplastic syndromes: early transplantation is associated with improved outcome. Chronic leukemia working party of the european group for blood and marrow transplantation. Bone Marrow Transplant 1998; 21:255–261. 76. Deeg HJ, Seidel K, Casper J, et al. Marrow transplantation from unrelated donors for patients with severe aplastic anemia who have failed immunosuppressive therapy. Biol Blood Marrow Transplant 1999; 5:243–252. 77. Perez E, Kamani N, Sinner P, et al. Outcome of unrelated donor stem cell transplantation for children with severe aplastic anemia. Blood 2001; 98:675a.

Unrelated Donor Stem-Cell Transplantation

159

78. Krivit W, Lockman LA, Watkins PA, et al. The future for treatment by bone marrow transplantation for adrenoleukodystrophy, metachromatic leukodystrophy, globoid cell leukodystrophy and Hurler syndrome. J Inherit Metab Dis 1995; 18:398–412. 79. Hobbs JR, Hugh-Jones K, Barrett AJ, et al. Reversal of clinical features of Hurler’s disease and biochemical improvement after treatment by bone-marrow transplantation. Lancet 1981; 2:709–712. 80. Whitley CB, Belani KG, Chang PN, et al. Long-term outcome of Hurler syndrome following bone marrow transplantation. Am J Med Genet 1993; 46:209–218. 81. Peters C, Balthazor M, Shapiro EG, et al. Outcome of unrelated donor bone marrow transplantation in 40 children with Hurler syndrome. Blood 1996; 87:4894–4902. 82. Derry JM, Ochs HD, Francke U, et al. Isolation of a novel gene mutated in Wiskott-Aldrich syndrome. Cell 1994; 78:635–644. 83. Derry JM, Ochs HD, Francke U, et al. Isolation of a novel gene mutated in Wiskott-Aldrich syndrome. Cell 1994; 79:922. 84. Filipovich AH, Stone JV, Tomany SC, et al. Impact of donor type on outcome of bone marrow transplantation for Wiskott-Aldrich syndrome: collaborative study of the international bone marrow transplant registry and the national marrow donor program. Blood 2001; 97:1598–1603.

8 Umbilical Cord Blood Transplantation Satkiran S. Grewal Department of Pediatrics, Division of Hematology/Oncology, Tufts University School of Medicine, Baystate Medical Center, Springfield, Massachusetts, U.S.A.

John E. Wagner Pediatric Hematology/Oncology/Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, U.S.A.

INTRODUCTION The first successful human allogeneic hematopoietic stem cell transplantations (HSCT) were performed in children with congenital immunodeficiencies in 1968 (1–3). Since then, bone marrow transplantation (BMT) has been proven to cure or provide survival advantage for several malignant and nonmalignant diseases. However, several challenges limit the success rate and ability to utilize BMT. Only about 30% of potential HSCT recipients have a suitably human leukocyte antigen (HLA)–matched related donor. Unrelated donor (URD) transplants are associated with higher transplant-related mortality (TRM) due to graft failure, opportunistic infections, and graft-versus-host disease (GVHD). URD searches are time consuming with median search times reported in the range of four months (4–8). Donor attrition is another challenge: about 30% of suitably matched URD, even when identified, are not available when needed for a variety of reasons (8). As a result, only about 50% of the initiated URD searches are successful in terms of identifying an HLA-matched donor who is still motivated and available (8). Hence, an unknown number of patients succumb to their primary disease while awaiting a suitable graft (9). These limitations led to incentives for newer strategies, including the search for alternative sources of hematopoietic stem cells (HSC). The first umbilical cord blood transplantation (UCBT) was performed in 1988, in a child with Fanconi anemia, using the cryopreserved umbilical cord blood (UCB) of a HLA-identical sibling (10). Since then it is estimated that more than 2500 UCBT have been performed worldwide (11–27). With increasing experience, UCBT is now established as a viable alternative to marrow transplants, particularly in pediatric recipients, and in some settings even as a preferable option. This chapter will focus on current experience with related and URD UCBT. Also, guidelines for choice of marrow versus UCB graft in the URD setting are provided based on what we understand today. 161

162

Grewal and Wagner

BIOLOGICAL FEATURES OF UMBILICAL CORD BLOOD GRAFTS Progenitor Cell Function Various in-vitro and animal studies demonstrated the presence of sufficient numbers of primitive hematopoietic progenitor cells in UCB, their viability after cryopreservation for extended periods of time, as well as their proliferative potential compared to adult bone marrow (BM) derived progenitors (results summarized in Table 1) (28–43). Whereas the original question was whether a banked UCB collection would contain sufficient number of HSC for reconstitution of donor hematopoiesis in a human recipient, the question now is what is the minimum number of nucleated cells or CD34C cells in a banked UCB collection required for a successful HSCT? In an analysis of 65 UCB collections, Broxmeyer et al. reported total nucleated cell (TNC) content of 0.13–58.3!108 cells (mean, 14.3!108 cells) (44). With the exception of young pediatric recipients, this cell content would be estimated to provide approximately a one-log lower graft TNC dose to the recipient in comparison to a typical marrow graft (O2!108/kg). However, comparative colony-forming assays have shown that the percentage and proliferative potential of primitive hematopoietic cells in UCB (as compared to marrow) is higher (Table 1).

Table 1 Reference (28,29) (30,31)

(32)

(33–38)

(39)

(40) (32,41,42)

Umbilical Cord Blood (UCB) Progenitor Cell Biological Characteristics Result UCB contains primitive progenitors Granulocyte-macrophage (CFU-GM), erythroid (BFU-E) and multipotent (CFU-GEMM) progenitor cells are present in large numbers, and remain viable in an anticoagulated collection for 3 days, showing that processing and cryopreservation of UCB is possible after a remote obstetrical collection High efficiency recovery of immature and mature UCB progenitors after 10–15 years of freezing in liquid nitrogen. High yeild of nucleated cells form the thawed product and that of granulocytemacrophage (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM) progenitors. Self renewal of CFU-GEMMs generated from the cryopreserved UCB is maintained as demonstrated by efficient replating of CFU-GEMMs to yield secondary colonies of CFU-GEMM, CFU-E, and CFU-GM Higher in-vitro proliferative potential of UCB primitive progenitors (as compared to marrow derived progenitors) and UCB LTC-IC can be maintained in culture for longer periods of time as compared to adult marrow CD34CCD38- immunophenotype defines a highly primitive hematopoietic cell population in UCB, similar to adult marrow; however, CD34CCD38- UCB cells have a higher cloning efficiency, proliferate more rapidly in response to cytokine stimulation, and develop about sevenfold more progeny than do their counterparts in bone marrow CD34C, CD33-, CD38-, Rho-, c-kitC fraction of UCB cells have highest frequency of ML-IC, LTC-IC and NK-IC Presence of SCID-repopulating cells (SRC) in UCB; in contrast to studies using human bone marrow grafts, human UCB derived hematopoiesis in SCID mice can proliferate and differentiate in the absence of human cytokines

Abbreviations: ML-IC, myeloid-lymphoid initiating cell; LTC-IC, long-term culture initiating cell; NK-IC, natural killer-initiating cells; SCID, severe combined immunodeficiency; UCB, umbilical cord blood.

Umbilical Cord Blood Transplantation

163

Both, the number of colonies formed per cells plated and the size of the colonies formed are greater for UCB (28,31,37,45). Also, transplantation of human UCB cells into sublethally irradiated combined immunodeficiency mice has been reported to result in more efficient engraftment compared to similar transplants using human marrow (41). This difference in frequency of progenitors, and the proliferative characteristics of UCB HSC, may explain its potential to successfully engraft in human HSCT despite a lower TNC dose provided by the graft.

Immunological Phenotype The content and function of UCB lymphocytes and their subsets have been studied with interest, due to the possibility of a lower incidence of GVHD and better immune tolerance of HLA-mismatch with the use of UCB grafts (43,46–53). The absolute numbers of T and B lymphocytes and natural killer (NK) cells in UCB are comparable or even higher than that seen in adult peripheral blood. However, UCB T-lymphocytes show an increased proportion of cells with naı¨ve phenotype, decreased reactivity to alloantigens, and increased apoptosis following stimulation. Specifically, UCB T cells have a significantly higher proportion of CD45RAC cells as compared to adult peripheral blood. This isoform of CD45, phenotypically defines T cells that are antigen naı¨ve, in contrast to the CD45ROC isoform, which phenotypically defines a memory T-cell population (54). In addition, the CD4CCD45RAC T cells in UCB are less reactive as compared to cells with this phenotype in adult peripheral blood; IL-2 production in response to stimulation via CD2 monoclonal antibodies is decreased. Although UCB T-lymphocytes respond to primary allostimulation, they do not proliferate upon rechallenge with alloantigens. The B-lymphocytes in UCB similarly demonstrate a more naı¨ve phenotype with a higher proportion of CD19C/CD23K cells and CD5 “B1” cells, as compared to adult peripheral blood. The levels of NK cells in UCB are comparable or higher to those in adult peripheral blood. Although no NK-like cytotoxic activity is observed in fresh UCB, short-term cultures of UCB cells with IL-2 results in the appearance of significant NK-like lytic activity. Also, preferential activation of NK cells, rather than T lymphocytes, in response to viral infections or allogeneic stimuli has been observed with UCB.

UMBILICAL CORD BLOOD TRANSPLANTATION CLINICAL EXPERIENCE After the first successful UCBT in a sibling with Fanconi anemia, considerable interest was generated in its use as an alternative to BM grafts, due to the limitations of URD marrow grafts detailed above. Transplants using UCB grafts have now been performed in both the related and URD settings, in pediatric as well as adult recipients. However, a larger proportion have been performed in pediatric recipients utilizing URD grafts.

Related Donor Umbilical Cord Blood Transplantation The experience with related donor UCBT has been relatively less, as the availability of a related UCB graft when needed is not frequent. Situations in which related UCBT have been possible include 1. Fortuitous birth of a suitably matched sibling when another child in the family needs HSCT 2. When a directed donor UCBT has been stored in a cord blood bank and is acceptably HLA-matched 3. Deliberate conception, with or without the use of preimplantation genetic diagnosis (PGD)

164

Grewal and Wagner

Directed-Donor Umbilical Cord Blood Transplantation There is a paucity of data regarding directed donor UCB collection and banking. Prior to the development of UCB banks, transplant centers coordinated to collect UCB during delivery of a newborn, when the couple had another child (with a malignant or nonmalignant disorder) in need of HSCT. This is evident in isolated case reports of related UCBT (55–59). Recently, the Sibling Donor Cord Blood Program, Oakland, CA (U.S.A.), reported its experience with sibling donor UCB collection and described its procedures and policies (60). Starting in November 1997, 540 families were enrolled, from which 504 (93%) units were successfully stored after collection at remote sites. Failure was most commonly the result of inadequate collections but was also attributed to missed collections resulting from maternal health emergencies, attrition of enrolled families, death of recipient, and one stillbirth. Factors associated with low volume collection included late enrollment, birth outside the hospital, emergency caesarian section, multiple gestation, and precipitous third stage of labor. The mean TNC count was 8.9!108 cells, and in 90% of the units the TNC dose was more than 1.5!107/kg for the intended recipient at the time of collection. The center also reported outcomes of attempts to augment the UCB collection volume by placing the newborn on the mother’s abdomen prior to cord clamping, which did not result in an increase in the mean volume of collection. It should be noted that the policies for routine UCB collection and donation in the URD setting allow only for collection of placental UCB from an otherwise unmanipulated birth. Seventeen of the 504 collections (3.4%) had been utilized at the time of the report (60). The use was higher for nonmalignant disorders. However, the majority of sibling patients with malignancy were either in remission or on chemotherapy without yet an indication for HSCT. In this study, the direct cost per family was approximately US$3000. Approximately 75% of the banked UCB units were not HLA-identical to the intended recipient. In addition to the sibling donor cord blood program at Oakland, CA, some other private cord blood banks are involved in directed donor cryopreservation of UCB. A recent report from the University of Minnesota involved 44 collections followed by on-site banking (61). The cost of collection, processing, and banking was approximately US$1600. Eighteen of these units have been utilized for transplants in malignant and nonmalignant disorders, with a median recipient age of 5.5 years. The donor and recipient were HLA-identical in 14 of the 18 transplants, and the median CD34C cell dose was 2.6!105/kg. With a median follow-up of 3.5 years, only one patient failed to engraft (2-antigen mismatched graft), and no patient developed grade III–IV GVHD. Survival was particularly encouraging for Fanconi anemia patients [as compared to historical results (62)] with six of seven patients alive at a median of 16 months. With these reports, the feasibility of long-distance directed donor collections has been demonstrated; however, at present there is still a paucity of standardization or clear policy guidelines for directed donor UCB collection and banking. Preimplantation Genetic Diagnosis in Related Donor Umbilical Cord Blood Transplantation In certain hereditary genetic disorders involving single gene mutations or structural chromosomal rearrangements, PGD (63–65) in association with in vitro fertilization has been utilized to select disease unaffected embryos prior to uterine transfer. This technique ensures pregnancy with embryo(s) free of the particular genetic disorder. It has now been successfully, and with high accuracy, utilized in several hundred cases for such disorders as cystic fibrosis, sickle cell disease (SCD), and b-Thalassemia. A major advantage of PGD over prenatal diagnosis is the establishment of diagnosis prior to implantation, averting the need to consider the termination of an established pregnancy. More recently, in families with a child with a hereditary genetic disorder where HSCT is indicated, PGD was extended to select embryos that are not only free of the particular genetic disease but also HLA-identical to the affected sibling (66,67). This allows the birth of a disease

Umbilical Cord Blood Transplantation

165

unaffected baby that is also HLA-identical to the affected sibling. The UCB collected and banked at birth can be subsequently used as the source of HLA-identical HSCs for HSCT. In the first case of its kind, for a child with Fanconi anemia associated BM failure and myelodysplasia, the UCB collected from a disease unaffected HLA-identical newborn, who was created using PGD to select for both variables (disease unaffected and HLA-identical), was successfully utilized to restore normal hematopoiesis after UCBT in the affected sibling (67). Although there is ongoing debate concerning medical, legal, and ethical issues associated with such use of this technology (67–70), there has also been considerable interest in its use. The major attraction is predicted superior HSCT outcomes following an HLA-identical sibling donor HSCT, particularly for nonmalignant diseases. As the procedure requires several months and even years for success, the primary potential for use of this technology in HSCT may be in diseases that are slowly progressive. These are typically nonmalignant disorders, such as Fanconi anemia, SCD, and certain hemoglobinopathies. For these conditions, it is frequently possible to perform HSCT in a relatively elective manner, allowing time to pursue this route. Also, if the parents desire to have another child, PGD is independently indicated to prevent pregnancy with a disease-affected embryo. However, the most common indication for pediatric HSCT is in the setting of acute leukemia, where the time span is too short to pursue the PGD route to create a donor once HSCT is definitely indicated (such as in the event of a relapse). For these and other situations, several couples have requested and are pursuing PGD with in vitro fertilization to “create” an HLA-identical stem cell donor for a family member. As this does not involve testing for a lethal disease trait, such use raises ethical concerns as to testing and selection for which embryo trait or combination of traits should be permissible. Other challenges that have been faced include fertility of the couple; financial limitations (as the technology is expensive); frequent requirement for multiple IVF attempts; reluctance of third-party payers to compensate; who follows the clinical status and course of the intended transplant recipient during the incubation period of this process (i.e., when should the medical decision be taken that the patient should no longer wait for the creation of a HLA-identical donor and proceed for an URD transplant); and ongoing ethical issues facing embryo-related research and therapeutics. The interested reader is referred to other articles for a detailed discussion on ethical issues and other challenges related to use of this technology (67–69).

Outcomes Following Related Donor Umbilical Cord Blood Transplantation Several series have described results after related UCBT (14,17,18,23,71,72). In a multiinstitutional study from Eurocord and other centers, Gluckman et al. (14) reported outcomes of 78 related cord blood transplants in recipients under 20 years of age (median, five years) with malignant and nonmalignant disorders. Sixty of the 78 grafts were HLA-identical with the recipient, while the others were 1–3 antigen mismatched (1 graft was 4-antigen mismatched); the median TNC dose was 3.7!107/kg. The probability of neutrophil and platelet engraftment by day 60 was 79% and 62%, respectively (Table 2). Moderate to severe acute GVHD (Table 2) occurred at estimated rates of 9% in the 60 HLA-matched transplants and 50% in the 18 mismatched transplants (p!0.001). The overall survival (OS) at 1 year was 63% (as compared to 29% for URD UCBT in the same study); the OS was 73% in recipients of HLA-matched UCBT and 33% in the HLA-mismatched group (pZ0.006). Variables favorably associated with survival were young age, weight !20 kg, CMV seronegativity of the recipient, and HLAidentity. In an earlier report (71) of 44 related UCBT (34, HLA-identical) for malignant (nZ25) and nonmalignant (nZ19) diseases, the probability of hematopoietic recovery by day 50 was 82%; graft failure was more frequent in patients with a nonmalignant disease. All patients with a malignant disease who received an HLA-identical or HLA 1-antigen mismatched graft showed donor engraftment; five of 18 evaluable patients with a nonmalignant disease and 0–1 antigen-matched grafts did not demonstrate donor-derived hematopoietic recovery. The probability of grades II—IV acute GVHD was low (3%), and no patient with a 0–1

(18)

(14)

Ref

HLA-identical sibling UCBT (nZ113)

!1–15 (5) years

0.3–45 (9) years

Unrelated UCBT (nZ65)

Age (median)

0.2–20 (5) years

General characteristics

HLAidentical sibling

85% of grafts:1–3 HLA-mm

At day 60a 62%

Platelet engraftment (median days)

Favorable factors associated with engraftment

Grade II–IV (III–IV) acute GVHD

At day 60 89% (26)

At day 180a 86% (44)

1. The relative risk of 14% (2%) neutrophil recovery in the first month after UCBT was 0.4 as compared with BMT (95% CI, 0.32–0.51; p! 0.001)

2. Platelet recovery: HLA match

1. Age !6 years 18% (5%) associated with reaching ANC O 500 by day 60 (median, 30 days (median, 56 days 2. For platelet for related for related recovery: HLA C unrelated) C unrelated) identity was most important 87% 39% 1. Neutophil recovery: 32% (20%) donor nucleated cells/Kg

Myeloid engraftment (median days)

At day 60 23% of 79% grafts: 1–4 HLA-mm

Donorrecipient HLAmismatch

Engraftment and Acute Graft-Versus-Host Disease After Umbilical Cord Blood Transplantation

Related UCBT (nZ78)

Table 2

1. The relative risk of grade II-IV GVHD after UCBT compared with BMT was 0.41 (95% CI, 0.24–0.70; pZ0.001)

1. Only CMV serostatus of host 2. No relation to HLAmm

Only HLA-mm

Factors associated with acute GVHD

166 Grewal and Wagner

(15)

K39% of grafts: 1 HLA-mm

18% patients: !18 years

HLAidentical sibling

For whole cohort: K7% of grafts: 6/6 HLAmatch

!1–15 (8) years

82% patients: Analysis of 562 %17 years patients receiving UCB grafts from New York blood center between 1992–1998

HLA-identical sibling BMT (nZ2052)

1. Older age (%12 vs. R12)

At day 60 At day 180b 6/6 HLA1. Higher nucleated 91% (28 days) match 85% (90 days) cell dose, HLAUCBT: match, US center, 27% (9%) and diagnosis other than chronic myeloid leukemia & aplastic anemia were associated with successful myeloid engraftment 1 HLA-mm 2. Younger age, UCBT: absence of infection 48% after HSCT, and (22%) absence of acute GVHD were associated with platelet engraftment

(Continued)

2. Non-US center location

2. Significantly lower rates of chronic GVHD were also seen following UCBT

96% (24)

24% (11%) 2. Time to platelet recovery in the early period after transplant was significantly longer after UCBT as compared with BMT (p!0.001)

98% (18) (p!0.001)

Umbilical Cord Blood Transplantation 167

(21)

Ref

18%: 1 HLA-mm

96% (18 days)

5–12 (8) years

UBMT: 262

K54% of grafts: 2–3 HLA-mm

At day 60

Age (median)

Myeloid engraftment (median days)

Type of HSCT: patient numbers

General characteristics

Donorrecipient HLAmismatch

85% (29 days)

At day 180a

Platelet engraftment (median days) 2–3 HLAmm UCBT: 49% (25%)

Grade II–IV (III–IV) acute GVHD

2. Neutrophil and T-UBMT: platelet recovery 19% (8%) correlated with nucleated cell dose (!3.7 vs. O3.7x107 cell/kg) but not with HLA disparity for UCBT

1. Significant delay of UBMT: 56% neutrophil and (29%) platelet recovery reported in UCBT group

Favorable factors associated with engraftment

Table 2 Engraftment and Acute Graft-Versus-Host Disease After Umbilical Cord Blood Transplantation (Continued)

Decreased acute GVHD in the UCBT and T-UBMT groups as compared to UBMT group was seen. No other association reported

3. HLA mismatch (0 vs. R1) approached significance (pZ0.06), but no correlation with number of mismatches was seen

Factors associated with acute GVHD

168 Grewal and Wagner

40%: 1–2 HLA-mm 89%: 1–3 HLA-mm At day 180b

At day 42

65% engrafted (86 days)

90% (81 days)

85% (29 days)

80% (32 days)

90% (16 days)

88% engrafted 84% of (23 days) grafts: 1–2 HLA-mm

102 UCBT at single 0.2–56.9 (7.4) 14% of years grafts: No center between HLA-mm 1994–2001

6–12 (8) years 2.5–10 (6 years) 39% (11%) 1. Neutrophil recovery: higher CD34C cell dose infused (O1.7x105/kg) 2. Platelet recovery: higher CD34C cell dose infused and absence of severe acute GVHD 3. No association of either with HLA match was observed

UCBT: 33% (21%)

No association with CD3 cell dose, HLA disparity or class of HLA-mismatch

Results of five large peer-reviewed published studies are shown. [14] Eurocord and other centers with malignant and non-malignant diagnoses; [18] Data from Eurocord and International Bone Marrow Transplant Registry; [15] Malignant and non-malignant diagnoses; [21] Eurocord and other centers, with pediatric acute leukemia patients only; [22] Malignant and nonmalignant diagnoses. Myeloid engraftment: neutrophil count R0.5!109/L, first of 3 consecutive days. a Platelet engraftment: R50!109 (untransfused) platelets/L, first of 7 days. b Platelet engraftment: R20!109 (untransfused) platelets/L, first of 7 days. Abbreviations: HLA-mm, HLA-mismatch; UBMT, unmanipulated (unrelated) bone marrow transplantation; T-UBMT, T-cell depleted unrelated bone marrow transplantation; GVHD, graft-versus-host disease; UCBT, umbilical cord blood transplantation; ANC, absolute neutrophil count; HLA, human leukocyte antigen; BMT, bone marrow transplantation; UCB, umbilical cord blood; HSCT, hematopoietic stem cell transplantation. Source: From Refs. 14, 15, 18, 21, 22.

(22)

UCBT: 99

T-UBMT: 180

Umbilical Cord Blood Transplantation 169

170

Grewal and Wagner

HLA-mismatched graft developed severe acute GVHD. The probability of limited chronic GVHD was 6%, and no patient developed extensive chronic GVHD. The first study (Table 2) comparing outcomes between HLA-identical sibling UCBT (nZ113) and BMT (nZ2052) verified decreased incidence of acute and chronic GVHD in UCB recipients (18). Although OS was similar in the two cohorts (three years probability of survival: UCBT, 64%; BMT, 66%), the risk of acute and chronic GVHD was significantly lower following UCBT (Fig. 1 and

100

Acute GVHD (%)

(A)

80 60 40 Bone marrow transplantation P=0.02

20

Cord blood transplantation 0 0

10

20

30

40

50

70

60

80

90

100

Days since Transplantation No. at Risk Bone marrow 2012 1810 1597 1489 1422 1378 1350 1328 1311 1293 1260 transplantation 107 97 85 84 83 81 79 78 77 76 71 Cord blood transplantation 100

Chronic GVHD (%)

(B)

80

60

40 P=0.02 Bone marrow transplantation

20

Cord blood transplantation 0 0

1

2

3

4

5

Years since Transplatation No. at Risk Bone marrow 1779 transplantation 93 Cord blood transplantation

1141

883

651

479

338

61

36

18

8

5

Figure 1 (A) Risk of graft-versus-host disease after human leukocyte antigen (HLA)-identical sibling umbilical cord blood versus (B) HLA-identical sibling bone marrow transplantation. Abbreviation: GVHD, graft-versus-host disease. Source: With permission from Ref. 18.

Umbilical Cord Blood Transplantation

171

Table 2). However UCBT was associated with longer intervals to neutrophil and platelet ecovery compared with BMT. Despite reduced GVHD, relapse as a cause of death did not differ between the two groups. The risk of GVHD has unique implications in the setting of HSCT for nonmalignant diseases; in these disorders, there is no known benefit of GVHD following HSCT, and GVHD is only associated with increased morbidity and mortality. Hence a decreased risk of GVHD is particularly important in nonmalignant diseases. Specific outcomes following related UCBT for leukemia (17) and hemoglobinopathies (23) have also been reported. In a multicenter report (23) of related UCBT for thalassemia and SCD, there was no treatment related mortality. Graft failure was more common with thalassemia with 1 of 11 SCD patients and 7 of 33 thalassemia recipients not showing sustained donor engraftment. Use of methotrexate for GVHD prophylaxis was associated with lower rate of engraftment. In summary, these results suggest that in the related-donor setting, UCB is a viable alternative to a BM graft. HLA-identical UCBT has comparable overall outcomes as compared to HLA-identical BMT (18). Although the risk of acute and chronic GVHD is lower with UCBT, hematopoietic recovery is faster following BMT. In comparison to matched-sibling UCBT, HLA-mismatched related UCBT is associated with a higher risk of GVHD and inferior survival. Lower recipient weight and age was associated with superior outcomes, possibly due to a higher cell dose in this group (14).

Unrelated Donor Umbilical Cord Blood Transplantation Currently, the majority of UCBT are performed in the URD setting. When HSCT is indicated, typically there is no stored related donor UCB unit available and insufficient time to plan a pregnancy. Even if the patient’s disease allows time, the parents may not be successful in having another child or the conceived child may not be suitably matched. For these reasons it became necessary to develop and maintain large repositories of UCB for public use. In 1993 the New York Blood Center developed a Placental Blood Program. Shortly thereafter, banks developed in Milan and Dusseldorf. At present, cord blood banks are established in several countries in the Americas, Europe, Asia, and Australia. A listing of 16 large cord blood banks registered with Netcord (73), and the growth of 29 cord blood registries with BM Donors Worldwide (74), are shown in Figure 2. Another organization coordinating cord blood registries is the National Marrow Donor Program (75). The Netcord repository alone exceeded 70,000 cryopreserved units as of September 2003, from which approximately 2,600 transplants had been performed. For latest updated data please refer to websites in references (73–75). Outcomes following URD UCBT are described below.

Engraftment UCBT series have evaluated the influence of the UCB graft cell dose (TNC and CD34C cell dose) and donor-recipient HLA-match on engraftment (Table 2). Data indicate that the single most important factor affecting speed of myeloid recovery and probability of donor engraftment is the cell dose provided by the UCB unit (Table 2) (12,14,15,17,21,22). In the Eurocord UCBT dataset (14,17) (in which approximately half of the patients received TNC dose of O3.7!107/kg), neutrophil engraftment by day 60 occurred in 76% of patients who received TNC dose of !3.7!107/kg and 94% of patients who received a TNC dose of O3.7!107/kg (pZ0.008) (14). Infused TNC dose above 3.7!107/kg was the most important factor influencing neutrophil recovery (14,17). Platelet engraftment was associated with HLA-identity (14). In two other large series of URD UCBT, increasing quartiles of TNC or CD34C cell dose were associated with shortened time to myeloid recovery (Fig. 3 and Table 2) (15,22). In the study by Wagner et al. (22), inferior speed and probability of myeloid recovery was observed in patients receiving a thawed CD34C cell dose less than 1.7!105/kg. The incidence of neutrophil

172 (A)

Grewal and Wagner NETCORD Inventory and Use March 2006 Inventory Transplanted Children Adults CB Bank 14836 170 125 39 AusCord 6435 245 123 122 Barcelona Düesseldorf 9702 303 184 112 5329 368 173 195 France Cord Helsinki 2504 8 3 5 723 12 12 0 Jerusalem 3286 35 18 17 Leiden Leuven 6457 52 38 14 Liege 4749 115 52 63 7352 116 82 34 London Milan (Grace) 10795 420 241 179 New York 26676 1951 1319 632 Prague 1962 9 5 4 Santiago de Compostela 3785 16 12 4 Tel Hashomer 1320 4 4 0 Tokyo 3860 416 139 277 109771 4240 2530 1697 TOTAL

140 120 100 80 60 40 20 0 19 93 19 94 19 95 19 96 19 97 19 98 19 99 20 00 20 01 20 02

Number (× 1000)

(B)

Figure 2 Netcord and Bone Marrow Donor Worldwide (BMDW) repositories of unrelated umbilical cord blood (UCB) units.

recovery by day 42 for all 102 patients was 88% at a median of 23 days, whereas the 29 patients infused with less than 1.7!105 CD34C cells/kg required a median of 34 days for neutrophil recovery. Similar results were observed in 562 patients receiving URD UCBT by Rubinstein et al. (15). The cumulative rate of neutrophil engraftment was 81% by day 42 and 91% by day 60; the speed of myeloid engraftment was associated primarily with the TNC dose. Interestingly, although the cumulative probability of engraftment was inferior at the lowest graft cell doses, it was similar for patients who received any TNC dose more than 2.5!107/kg (15,76). With marrow HSCT, histoincompatability (URD transplants or HLA-mismatch) is a major factor that adversely influences myeloid engraftment (4,77–80). With UCBT, the influence of HLA-mismatch on engraftment is less clear. Several studies have failed to observe an association between HLA-mismatch and myeloid engraftment kinetics (14,17,22). However, a large series of unrelated UCBT (15), and its more recent update with 861 unrelated UCBT (81), revealed an adverse correlation between HLA-mismatch and myeloid engraftment. The median time to neutrophil recovery with six-antigen matched grafts was 23 days, in comparison to 28 days with mismatched grafts (pZ.0027) (81). Importantly, the effect appears to be only between HLAmatch versus HLA-mismatch, i.e., no association between engraftment and number of HLA-mismatches (1 vs. O1 HLA-mismatch) was observed (81). However, the data needs to be interpreted with the knowledge that the numbers of HLA-matched UCBT (w6%) were a much smaller subset than the mismatched transplants in this study. Engraftment characteristics after UCBT in comparison to BMT have been analyzed in retrospective studies (18,21,82). In the HLA-identical sibling donor setting (18), the speed of myeloid and platelet recovery is slower, and the cumulative incidence of donor engraftment is

Umbilical Cord Blood Transplantation

173

1.0 Cumulative Proportion with ANC > 500/mm3

(A)

0.8

0.6

> 100 million/kg (n=65) 50 million-99 million/kg (n=121) 25 million-49 million/kg (n=198) 7 million-24 million/kg (n=162)

0.4

0.2 P<0.001

0.0 0

7

14

21

28

35

42

49

56

63

70

77

Days after Transplantation 2.8–5.4

(B)

1.0

P<0.001

Probability

>5.4

0.8

1.7–2.7

0.6

<1.7

0.4 0.2 0.0 0

7

14

21 Days

28

35

42

Figure 3 (A) Association of umbilical cord blood nucleated cell dose and (B) CD34C cell dose with speed of myeloid engraftment. Abbreviation: ANC, absolute neutrophil count. Source: From Refs. 15, 22.

inferior with UCBT. Similar results have been observed in the URD transplant setting. In a registry study, a matched pair analysis was conducted comparing 0–3 antigen mismatched UCBT with HLA-A, -B, and -DRB1 matched BM transplants. The patients who received UCBT were matched for age, disease, and disease status with patients undergoing BMT using either methotrexate (BM-MTX) or T-cell depletion (BM-TCD) for GVHD prophylaxis (82). The median time for UCBT to achieve neutrophil recovery was significantly longer than either of the BMT groups, regardless of the type of GVHD prophylaxis used with the BMT. It was 29 versus 22 days when matched to the methotrexate (BM-MTX) group and 27 versus 14 days when compared to the BM-TCD group (82). However, by day 45, the overall probability of donorderived myeloid engraftment was comparable in all groups. The time to achieve platelet independence was similar in the UCB versus BM-TCD groups (61 vs. 59 days); although the BM-MTX group recovered platelet count faster than its matched UCB group (30 vs. 66 days), the difference was not found to be statistically significant (pZ.12).

174

Grewal and Wagner

Also, in another registry study of pediatric patients with acute leukemia, Rocha et al. (21) compared similar groups of transplants as shown in Table 2. This study also observed significant delay in both neutrophil and platelet engraftment after UCBT when compared to URD marrow transplant. In summary, current data on engraftment after unrelated UCBT suggest that graft cell dose (either TNC or CD34C) is a critical predictor of engraftment. Overall, speed of recovery is inferior as compared to BMT; however, this can be overcome at least partly with use of UCB units with higher cell doses. Also, with increasing realization of the role of cell dose on UCBT outcomes, and consequently routine utilization of grafts with higher cell doses, future engraftment results may be superior to current outcomes. HLA-match also appears to influence engraftment; however, at present its role is less clear.

Graft-Versus-Host Disease Risk of GVHD is an important factor that has limited utilization of BMT, particularly in the URD setting. Grade II–IV GVHD is reported in 45% to 70% of phenotypically matched URD marrow transplants and in 63–95% of one-antigen mismatched URD transplants (4,83–87). Chronic GVHD affects approximately 60% of matched URD transplants and maybe as high as 80% after one-antigen mismatched URD HSCT, with high morbidity and mortality in patients suffering extensive chronic GVHD (88,89). URD and histoincompatible grafts have been the strongest risk factors for GVHD with marrow transplants (78,90–94). This risk has been successfully reduced by the use of T-cell-depleted marrow grafts; however, no survival benefit has been observed in part due to increased complications associated with graft failure and poor immune reconstitution resulting in opportunistic infections, posttransplant lymphoproliferative disease, and relapse (95–99). In hematologic malignancies, donor-recipient genetic diversity and GVHD may contribute to the development of a graft-versus-leukemia (GVL) effect, reducing risk of relapse (100–102). However, despite this potential benefit the development of severe acute GVHD or extensive chronic GVHD is not desirable as it is associated with diminished quality of life and OS. GVHD outcomes following URD UCBT are summarized in Table 2. The risk of GVHD with URD UCBT is difficult to accurately compare with BMT data, as it is fraught with the usual limitations of historical comparisons over different eras. No randomized prospective studies comparing the two stem cell sources have been performed (and may never be possible). However, the existing data suggest that UCB grafts may have better tolerance for histoincompatability and lower risk of GVHD: (1) in the HLA-identical sibling donor setting, the risk of acute and chronic GVHD is lower with UCBT in comparison to BMT (18). This study perhaps allows the most reliable comparison for risk of GVHD between the two stem cell sources, as other studies are complicated by heterogeneity of recipient characteristics or differences in degree of donor-recipient HLA-mismatch, (2) the observed risk of acute and chronic GVHD with unrelated UCBT (12,14,15,20–22,82) is lower than that reported following HLA-matched URD BMT (4,90,91,93,103). Larger URD UCBT series have observed 32–44% and 11–22% incidence of grades II–IV and III–IV GVHD respectively (14,17,21,22). Similar comparatively lower risk of GVHD is observed from dataset of URD UCBT limited to adult recipients (104), (3) studies that have retrospectively analyzed comparable cohorts of patients receiving URD UCBT and URD BMT have observed similar or even lower risk of acute GVHD with (predominantly 1–2 antigen mismatched) UCBT when compared to HLA-matched unmanipulated URD BMT (Table 2) (21,82,105). Unlike outcomes following use of BM grafts, the association of histoincompatability with GVHD in the UCBT setting is less clear (Table 2). In the URD UCBT setting, no association of HLA mismatch with risk of grade II–IV GVHD has been observed in the Eurocord dataset (14,17). Similarly, another single institution U.S. study of 102 URD UCBT (22) did not observe any influence of HLA-mismatch on probability of acute GVHD (Table 2). However, the New York Blood Center URD UCBT dataset (15,76), similar to its observations on the impact of HLA-mismatch on engraftment, noted significantly higher rates of severe acute GVHD in the

Umbilical Cord Blood Transplantation

175

HLA-mismatched group as compared to the HLA 6-antigen matched group. Again, there was no association of the degree of HLA-mismatch (1 vs. O1) on probability of GVHD. The small number of patients who received HLA 6-antigen matched UCBT in this study may explain these results. The apparent lower risk of GVHD after UCBT, particularly in the HLA-mismatched setting, is hypothesized to result from a decreased alloreactivity of cord blood T cells rather than from the lower T cell dose infused. In an unmanipulated marrow graft, the recipient typically receives 1–5!107 T-cells/kg body weight (106). However, even when only 0.5!106 T-cells/kg are infused with HLA-identical sibling donor grafts, the risk of acute GVHD reported is 22% and with 1!106 T-cells/kg, it increases to 45% (107,108). The median T-cell dose infused with UCB grafts is in the order of 8!106 per kg weight of recipient (22). This level of reduced T-cell content seems unlikely to explain the observed reduced frequency of acute GVHD with UCB grafts. Also, the progressive benefit of increasing UCB nucleated cell dose/kg of recipient (presumably with concomitantly increasing T-cell numbers) on engraftment, TRM, and survival is not associated with any increase in the risk of developing GVHD. Mature T cells present in the donor graft are the chief contributor to repopulating T cells in the first year after HSCT (109). The functional and immunophenotypic profile of UCB lymphocytes reflects a population of cells that are naı¨ve (with a T-cell repertoire characterized by a lack of prior antigenic stimulation) with reduced immunoreactivity as previously described (46,47,50,110,111). Also, although UCB cells can respond to alloantigens with a strong initial proliferative response (similar to that of adult T cells), restimulation is shown to induce a state of unresponsiveness (46,47). These and other factors may contribute to reduced clonal expansion of UCB derived lymphocytes when exposed to alloantigens (50,111). Hence, it is postulated that in the allogeneic setting, large numbers of mature, antigen specific, alloreactive T cells infused in an unmanipulated marrow graft (as compared with UCB graft), are more likely to initiate GVHD due to recognition of cross-reactive alloantigens (50).

Immune Reconstitution and Relapse Studies of immune reconstitution after marrow HSCT show that the reconstitution of T cells occurs in two phases (52,112–116). The initial wave is proliferation of mature T cells that accompany the donor graft. The second phase is the appearance of thymic derived T cells, which are probably derived from HSC, colonize the lymphoid tissues, and are responsible for later, long-term immune function. It has been hypothesized that with its lower and previously unstimulated T cell content, the early thymus-independent (peripheral) expansion of UCB-derived T cells may be considerably less than that seen with marrow graft. Poor initial T-cell expansion could potentially predispose to infectious risks in the early posttransplant period. However, clinical experience after UCBT with adequate cell doses shows that it is not associated with an increased rate of infectious deaths relative to BMT (117). Also, several investigators have demonstrated that the recovery and function of immune-cells after UCBT is comparable to that with unmanipulated marrow HSCT (13,20,118–121). NK-cell numbers are reconstituted promptly, and the recovery of B lymphocytes and CD4CT cells may actually be faster after UCBT. Interestingly, the typical inversion of CD4C/CD8C cells seen after HSCT using marrow grafts, including after T-cell-depleted grafts (113,122,123), is not observed with UCBT (20,118,120). The observed pattern after UCBT is of rapid CD4C recovery but a slower CD8C recovery. Delayed CD8CT-cell recovery may be related to the paucity of mature T cells in the UCB graft, but it is unclear why this selectively affects CD8C cells. Although recovery of lymphocyte numbers into the normal range occurs weeks to months after HSCT, full reconstitution of immune function takes longer (52,124,125). Longterm antigen specific immune function correlates with host thymopoietic activity and the ability to generate a phenotypically naı¨ve diverse T-cell repertoire. T-cell diversity analysis after HSCT has been evaluated by assessment of naı¨ve phenotype (CD45RAC) and by the size of

176

Grewal and Wagner

the b-chain complimentary determining region 3 (CDR3). A newer, and perhaps more reliable technique to assay thymopoietic capacity is measurement of the frequency of T-cell receptor excision circles (TRECs) in peripheral blood T cells (52,124,125). TRECs measure the formation of a thymic dependant, donor-derived reservoir of T cells with a diverse repertoire. TRECs are increased when the recipient is younger and when there is the absence of GVHD (52,124). Quantification of TRECs after UCBT and non-T-cell-depleted BMT have shown that T-cell repertoire normalizes onwards of two years after transplant. Interestingly, frequency of TRECs after UCBT is comparable or even superior to that following non-T-cell-depleted BMT (125). The functional naı¨vete´ of UCB lymphocytes and comparatively lower risk of GVHD has also raised concern for a reduced GVL effect after UCBT (126). Current results of UCBT for malignant diagnoses do not show an increased risk of relapse after URD UCBT as compared to historical URD BMT. However, in the absence of randomized prospective studies, such comparison of historical data need to be interpreted with caution as they can be complicated by variable patient selection (such as age and disease status at HSCT) in different studies, which can independently affect outcome. Among pediatric reports using marrow grafts, Balduzzi et al. (91) described outcomes including 84 children with hematological malignancies (0.5–17 years; median 9.1 years) after URD transplant (46, HLA-matched; 42, one minor antigenmismatched). Thirty of 84 (36%) of patients relapsed; the relapse rate was 36% for standard-risk patients and approximately 60% in patients with high-risk disease. Young age and advanced disease status were associated with risk of relapse. Similar adverse effect of advanced leukemia on risk of relapse was observed by Casper et al. in pediatric patients (127). UCBT studies have reported incidences and risk factors for relapse similar to those in pediatric BMT series (Table 3). In addition, a retrospective comparison of URD BMT with UCBT in pediatric patients with acute leukemia does not reveal a higher risk of relapse with UCBT (21). In this study (Table 2) Rocha et al. retrospectively compared outcome after URD unmanipulated (UBMT) marrow, URD T-cell depleted (T-UBMT) marrow, and UCBT. Recipients of UCBT were younger [median age six vs. eight years (pZ0.0004) with a higher percentage of children under two years (pZ0.0001)], more likely to have a diagnosis of AML, and had a higher frequency of previous HSCT. A larger proportion of recipients in the UBMT and UCBT groups (18–20%) had advanced stage leukemia compared to the T-UBMT (9%) group. Also, a significantly higher percentage of UCBT than marrow transplants used mismatched grafts. In all groups the risk of relapse was higher for advanced leukemia at HSCT. Interestingly, although in both T-UBMT and UCBT groups a lower incidence of acute and chronic GVHD was noted, the T-UBMT group (but not the UCBT group) had a higher risk of early relapse when compared to UBMT group (pZ0.02). The outcome of the three groups was comparable for long-term relapse.

Survival: Influence of Age, Disease Characteristics, Cell Dose, and Human Leukocyte Antigen Match UCB graft leukocyte content relative to recipient weight has consistently been observed to be an important predictor of survival after UCBT (12,15,17,21,22,81). In the initial Eurocord dataset of related and URD UCBT (14,17), approximately half the patients received UCB with a TNC dose of O3.7!107/kg. In this dataset, cell dose of O3.7!107/kg was associated with superior survival. Subsequent larger studies have confirmed the critical role of cell dose on survival (Fig. 4). Particularly inferior survival has been reported in cohorts receiving less than 1–1.5!107/kg of TNC dose or !1.7!105/kg of infused CD34 cell dose (20,22,104). Although recognizing the importance of cell dose, there has been no uniform method to quantify cell content of an UCB collection. In the majority of UCBT series, graft “potency” has been assessed by measuring the TNC content only, as CFU-GM or CD34C content have either not been measured or the methods used have varied. Nonetheless, CFU-GM or CD34C cell content of the UCB grafts (with data typically available on thawed units) correlates well with

102, 54 with acute leukemia (URD UCBT)

Minnesota (22)

AML

ALL, 28 AML, 26

Acute leukemia

Disease

0.1–15 (4.8)

0.2–56.9 (7.4)

0.3–15

CR3, 20% Not in CR, 61%

ALL, 43% AML, 47%

77%

CR1, 10% CR2, 23%

ALL, 10% AML, 25%

31%

2-year relapse in 2-year relapse patients with high in standard risk risk group group

1. Relapse rate was similar for related and URD UCBT 2. The most important factors associated with risk of relapse were (respectively) advanced disease at transplant and weight !20 kg 1. The standard risk AML group had only 4 patients 2. Relapse was not associated with cell dose, HLA-match or history of GVHD 1. May have some common patients with earlier Eurocord study 2. Only 5 patients in study were in CR3 3. Features associated with increased risk of relapse were high risk disease and weight !21 kg 4. No association with history of GVHD

Comments

Abbreviations: URD, unrelated donor; UCBT, umbilical cord blood transplantation; HLA, human leukocyte antigen; GVHD, graft-versus-host disease AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia.

Multicenter 95 (URD UCBT) (129)

102 (Related C URD UCBT)

N

Age (median) in years

Risk of Leukemia Relapse Following Umbilical Cord Blood Transplantation

Eurocord (17)

Ref

Table 3

Umbilical Cord Blood Transplantation 177

178

Grewal and Wagner

(A) 1.0

P <.01

0.8 Probability

>5.4 0.6 1.7–2.7 2.8–5.4

0.4 0.2

<1.7

0.0 0

(B) 1.0

1

2 Years

3

4

P =.03

0.8 0.6

HLA 0–1 ag mm

0.4 HLA 2 ag mm 0.2 0.0 0

1

2 Years

3

4

Figure 4 (A) Influence of infused CD34C cell dose and (B) human leukocyte antigen (HLA)mismatch on survival. Abbreviation: HLA, human leukocyte antigen. Source: From Ref. 22.

survival and rate of myeloid recovery (22,104,128), and preliminary results suggest that these may be more reliable methods for assessing potency of UCB grafts (22,128). In addition to the striking effect of cell dose, there also appears to be an influence of HLAmismatch on OS after UCBT. The initial European series, interestingly, did not observe an adverse influence of 1–2 antigen mismatched UCB grafts on survival in the URD setting, although HLA-mismatch was associated with inferior survival in the related UCBT cohort (14,17). Also, in one of the Eurocord reports by Locatelli et al. (17) that involved pediatric acute leukemia patients only, survival among related and unrelated UCBT groups was similar. However, other larger series have subsequently reported a significant adverse effect of HLAmismatch on survival and treatment-related mortality after unrelated UCBT (Fig. 4) (15,22). Recipient age and disease-risk status have also been shown to be important predictors of survival after transplant, however, these cannot be modified (17,22,129). Within the limitations of retrospective comparisons, the probability of survival following UCBT appears to be similar to that following BMT. One study (but not others) observed higher early TRM (!100 days) in UCBT patients as compared to marrow transplants (21). The reasons for this observation are not clear and possibly reflect poor outcomes among the subset of UCBT patients receiving lower cell doses and not the stem cell source per se. In a registry study of HLA-identical sibling donor HSCT, the 3-year survival of 113 UCBT recipients (64%) was similar to 2052 BMT recipients (66%). Two reports have retrospectively compared outcomes of UCBT and BMT in the URD setting and reported comparable survival between HLA-mismatched UCBT and HLA-matched BMT recipients (21,82). In addition, preliminary results from an International BM Transplant Registry Study of URD HSCT that compared

Umbilical Cord Blood Transplantation

179

296 UCBT with 210 BMT recipients, observed similar OS between recipients of BMT and 0–1 antigen mismatched UCBT but inferior outcomes after two antigen mismatched UCBT (105).

PRACTICAL CONSIDERATIONS WHEN SELECTING A DONOR GRAFT When choosing an appropriate graft for a patient, the questions facing the transplant physician include: 1. Whether to opt for a UCB or marrow or PBSC graft 2. In the case of a UCB graft, how to balance cell dose with degree of HLA match

Unrelated Donor Transplants: Marrow or Umbilical Cord Blood In terms of logistics, UCB offers potential advantages, including shortened time to graft acquisition (11,130–132), 100% donor retention and availability (other than loss by use of the unit), absence of risk to the donor, and the possibility of more effectively targeting donation from minority populations. Median time for an URD marrow to be available is approximately four months, whereas, in one report, the median time to acquire URD UCB units was 13.5 days (130). Also, it is recommended that the birth process not be altered in any way for the purpose of UCB collection, so as to not pose any collection-associated risks to the newborn donor. Marrow donors face risks associated with anesthesia and a surgical procedure. UCB grafts are associated with a reduced risk of transmission of specific viral infections (133–135) particularly CMV (134). As limited HLA-mismatch is better tolerated with UCB grafts, it is available to most patients (including those from ethnic minorities) even with the size of current repositories (136). Also, as UCB units are cryopreserved and banked, there is complete flexibility of rescheduling the transplant date if required. However, an UCB unit has fixed nucleated cell content. As nucleated cell dose has proven to be a critical determinant of engraftment and survival with UCB grafts, it represents the major limiting factor, particularly for larger sized recipients. In addition, returning to the same donor for a second HSC graft or donor lymphocytes has not been an option, and there is a small but real possibility of the UCB graft transmitting a hereditary disease undetected at birth, such as Gaucher’s disease, hereditary spherocytosis, or even infant leukemia. Such an event has not yet been reported. With BMT, there has been sufficient experience regarding outcomes and efficacy for a variety of malignant and nonmalignant diseases. In comparison, experience with UCBT, although less, is growing and in the last decade sufficient experience has accumulated to make comparisons with BMT. In the related donor UCBT setting, HLA-identical grafts appear to be associated with comparable overall outcomes to HLA-identical BMT: similar survival, lower risk of acute and chronic GVHD, but slower speed and cumulative incidence of myeloid and platelet engraftment (18). In the URD setting, data indicate that the overall outcome after 0–1, and possibly 2-antigen mismatched UCBT is similar to that after phenotypically matched marrow HSCT, whereas neutrophil engraftment is slower and the probability of acute GVHD is less with UCBT. At present, sufficient experience exists to say that URD UCBT has comparable efficacy to URD BMT in patients with acute leukemia (17,22,129). Practically speaking, there is often an urgency to proceed to HSCT in children with acute leukemia and not uncommonly a need to reschedule the date of transplant. A particular advantage of UCB grafts in this setting is the rapid availability of an identified unit, and the ease of rescheduling the HSCT. Two studies (137,138) in children !2–2.5 years of age have reported encouraging results in nonmalignant and malignant diseases after UCBT. Notably, in this age group, a high graft TNC dose is typically delivered. In one of these studies (137), the median TNC dose was 9.5!107/kg. Hence, preliminary evidence also supports the use of UCBT for nonmalignant disorders in children, particularly in small sized recipients where high graft cell doses are typically infused.

180

Grewal and Wagner

Also, in other nonmalignant diseases where a long wait for URD marrow may seriously compromise ultimate outcomes (such as inherited metabolic storage disorders with potential for rapid neurological progression or severe combined immunodeficiency disease) (139–141), UCB offers the attraction of a rapid graft acquisition. Therefore, the urgency for HSCT or rate of disease progression has to be considered when choosing a UCB versus marrow graft. In diseases associated with a higher risk of graft failure (such as aplastic anemia, Fanconi anemia, chronic myeloid leukemia), the slower speed of engraftment observed with UCBT as compared to BMT has raised concerns for an increased risk of graft rejection. Also, a second HSC infusion or even donor-lymphocyte infusions from the same donor may be deemed necessary for the recipient after HSCT, which are not an option after UCBT. Conversely, URD BMT for certain disorders (such as Wiskott-Aldrich syndrome in children under five years of age) has been associated with excellent results (142,143). At present, in these conditions, UCBT should perhaps be considered only if a suitably matched URD marrow donor is not available.

Umbilical Cord Blood Graft Selection: Cell Dose and Human Leukocyte Antigen-Match Current experience with URD UCBT suggests that four of six (HLA-A, -B, -DRB1) antigenmatched UCB is an acceptable donor graft for HSCT and that the donor cell dose (nucleated cells/CD34C cells) can overcome the negative impact of HLA-disparity, at least in part. Outcomes are significantly inferior below an infused TNC dose of 2.0!107/kg or a CD34C cell dose of less than 1.7!105/kg. Hence, acceptable criteria for selection of a UCB graft should include TNC dose R2.0!107/kg of recipient and a %2 antigen HLA-mismatch. Although use of UCB grafts for HSCT is increasing, not only is there an increase in accumulated experience but also a possibility for changing standards of acceptable UCB grafts. With current HLA-matching criteria of 0–2 antigen disparities and minimum acceptable TNC dose of 2.0!107/kg or CD34C cell dose of 1.7!105/kg for URD UCBT, a majority of searches will find an acceptable donor within the present size of the cord blood repositories. However, studies thus far have shown that the optimal UCB graft is one with a higher cell dose (although the exact threshold needs further refinement), and preliminary evidence suggests that 0–1 antigen mismatched grafts is superior to two antigen mismatched grafts (105). As the number of UCB units being harvested and stored continues to expand, it can be foreseen that future standards of optimal UCB grafts will include 0–1 HLA-antigen mismatched units and, at least in pediatric recipients, an acceptable TNC or CD34C cell dose set higher than the current 2.0!107/kg or 1.7!105/kg, respectively.

Recommendations for Selection of Umbilical Cord Blood Units Based on current data, the authors recommend the following order of preference for selection of URD UCB grafts: 1. 2. 3. 4.

HLA 6 of 6 antigen match, TNC dose O2.0!107/kg HLA 5 of 6 match, TNC dose O2.0!107/kg HLA 4 of 6 match, TNC dose O3.0!107/kg Can consider: HLA 6 of 6 or 5 of 6 match, TNC dose 1.0–1.9!107/kg

If two UCB units have comparable TNC dose, CD34C cell content can be utilized for choosing the between the two grafts. If two units are similarly HLA-matched with the recipient, high resolution molecular typing should be performed to assess if one unit is better matched. If a six antigen and a five antigen HLA-matched graft are simultaneously available, preference should be given to the unit with higher cell content. However, at present it is not entirely clear how much of an increase in cell dose is required to compensate for a given degree of HLA-mismatch.

Umbilical Cord Blood Transplantation

181

With related donor grafts, it would be fair to presume that similar to the URD UCBT experience, 0–2 HLA-mismatches are acceptable. A choice of cell dose however does not exist in the related UCBT setting, although sibling UCB grafts with low cell contents can potentially be “topped-off” with supplemental marrow from the same donor (60). A unique issue with UCB grafts may be that a related donor graft may not always be superior to a URD graft. In particular, in comparison to a 2-antigen mismatched related UCB graft, a URD 0–1 antigen mismatched UCB graft with a higher cell dose may be predicted to be associated with superior outcomes.

UMBILICAL CORD BLOOD TRANSPLANTATION FOR LARGER SIZED RECIPIENTS As both age and cell dose have an impact on transplant outcomes, there is a greater need for larger UCB grafts in adults and larger sized pediatric recipients. Therefore, we and others have explored newer approaches to reduce toxicity and improve outcomes after UCBT in this group, including ex vivo expansion of primitive progenitors, infusion of two different partially matched UCB grafts in an attempt to augment cell dose, coinfusion of mesenchymal stem cells, and the use of reduced intensity conditioning (45,144–148). Longer follow-up and greater number of patients are required to determine the efficacy and role of these unique approaches and represent the next phase of UCBT.

SUMMARY AND FUTURE CONSIDERATIONS With more than a decade of experience, UCB has now been established as a viable and in some cases a preferable alternative to BM grafts for HSCT. Advantages of this stem cell source include speed of acquisition, ease of rearranging date of stem cell infusion, and low risk of transmission of viral diseases. The better tolerance of HLA-disparity with UCB grafts makes it possible to identify a graft for almost all recipients. As the UCB cell dose infused is an important prognostic marker of outcomes following UCBT, the fixed cell content of a UCB unit can be a limiting factor, particularly in larger sized recipients. However, newer strategies, such as coinfusion of a second unit of UCB, peripheral blood stem cells or mesenchymal stem cells, or the use of a nonmyeloablative conditioning therapy may overcome this limitation. At present, UCB is established as an alternative to marrow grafts for patients with acute leukemia. Preliminary studies also show equivalent efficacy and utility in certain nonmalignant diseases in children, particularly where the rate of disease progression is rapid and thereby rapid acquisition of the donor graft is important. However, in certain diseases where either the results with marrow HSCT have been associated with good outcomes, or where a second donor infusion may be anticipated, UCB should be recommended only when a suitably matched marrow graft is not available or until the time that results with UCB have been shown to be at least equivalent to BMT in these diseases.

REFERENCES 1. Bach FH, Albertini RJ, Joo P, Anderson JL, Bortin MM. Bone-marrow transplantation in a patient with the Wiskott-Aldrich syndrome. Lancet 1968; 2:1364–1366. 2. De Koning J, Van Bekkum DW, Dicke KA, et al. Transplantation of bone-marrow cells and fetal thymus in an infant with lymphopenic immunological deficiency. Lancet 1969; 1:1223–1227. 3. Gatti RA, Meuwissen HJ, Allen HD, Hong R, Good RA. Immunological reconstitution of sexlinked lymphopenic immunological deficiency. Lancet 1968; 2:1366–1369.

182

Grewal and Wagner

4. Kernan NA, Bartsch G, Ash RC, et al. Analysis of 462 transplantations from unrelated donors facilitated by the national marrow donor program. N Engl J Med 1993; 328:593–602. 5. Davies SM, Wagner JE, Shu XO, et al. Unrelated donor bone marrow transplantation for children with acute leukemia. J Clin Oncol 1997; 15:557–565. 6. McGlave P, Bartsch G, Anasetti C, et al. Unrelated donor marrow transplantation therapy for chronic myelogenous leukemia: initial experience of the national marrow donor program. Blood 1993; 81:543–550. 7. McGlave PB, Shu XO, Wen W, et al. Unrelated donor marrow transplantation for chronic myelogenous leukemia: 9 years’ experience of the national marrow donor program. Blood 2000; 95:2219–2225. 8. Confer DL. Unrelated marrow donor registries. Curr Opin Hematol 1997; 4:408–412. 9. Davies SM, Ramsay NK, Weisdorf DJ. Feasibility and timing of unrelated donor identification for patients with ALL. Bone Marrow Transplant 1996; 17:737–740. 10. Gluckman E, Broxmeyer HA, Auerbach AD, et al. Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 1989; 321:1174–1178. 11. Wagner JE, Rosenthal J, Sweetman R, et al. Successful transplantation of HLA-matched and HLAmismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graftversus-host disease. Blood 1996; 88:795–802. 12. Kurtzberg J, Laughlin M, Graham ML, et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996; 335:157–166. 13. Locatelli F, Maccario R, Comoli P, et al. Hematopoietic and immune recovery after transplantation of cord blood progenitor cells in children. Bone Marrow Transplant 1996; 18:1095–1101. 14. Gluckman E, Rocha V, Boyer-Chammard A, et al. Outcome of cord-blood transplantation from related and unrelated donors. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group. N Engl J Med 1997; 337:373–381. 15. Rubinstein P, Carrier C, Scaradavou A, et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 1998; 339:1565–1577. 16. Rocha V, Chastang C, Laporte JP, et al. Unrelated umbilical cord blood transplants in adults with hematologic malignancies [abstract]. Blood 1998; 92:144a. 17. Locatelli F, Rocha V, Chastang C, et al. Factors associated with outcome after cord blood transplantation in children with acute leukemia. Eurocord-Cord Blood Transplant Group. Blood 1999; 93:3662–3671. 18. Rocha V, Wagner JE, Jr, Sobocinski KA, et al. Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. Eurocord and International Bone Marrow Transplant Registry Working Committee on Alternative Donor and Stem Cell Sources. N Engl J Med 2000; 342:1846–1854. 19. Rubinstein P, Carrier C, Carpenter C, et al. Graft selection in unrelated placental/umbilical cord blood (PCB) transplantation: influence and weight of HLA match and cell dose on engraftment and survival [abstract]. Blood 2000; 96:588a. 20. Thomson BG, Robertson KA, Gowan D, et al. Analysis of engraftment, graft-versus-host disease, and immune recovery following unrelated donor cord blood transplantation. Blood 2000; 96:2703–2711. 21. Rocha V, Cornish J, Sievers EL, et al. Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood 2001; 97:2962–2971. 22. Wagner JE, Barker JN, DeFor TE, et al. Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival. Blood 2002; 100:1611–1618. 23. Locatelli F, Rocha V, Reed W, et al. Related umbilical cord blood transplantation in patients with thalassemia and sickle cell disease. Blood 2003; 101:2137–2143. 24. Ooi J, Iseki T, Takahashi S, et al. Unrelated cord blood transplantation for adult patients with de novo acute myeloid leukemia. Blood 2003. 25. Long GD, Laughlin M, Madan B, et al. Unrelated umbilical cord blood transplantation in adult patients. Biol Blood Marrow Transplant 2003; 9:772–780. 26. Isoyama K, Ohnuma K, Ikuta K, et al. Unrelated cord blood transplantation for second hemopoietic stem cell transplantation. Pediatr Int 2003; 45:268–274. 27. Isoyama K, Ohnuma K, Kato K, et al. Cord blood transplantation from unrelated donors: a preliminary report from the Japanese Cord Blood Bank Network. Leuk Lymphoma 2003; 44:429–438.

Umbilical Cord Blood Transplantation

183

28. Nakahata T, Ogawa M. Hemopoietic colony-forming cells in umbilical cord blood with extensive capability to generate mono- and multipotential hemopoietic progenitors. J Clin Invest 1982; 70:1324–1328. 29. Leary AG, Ogawa M, Strauss LC, Civin CI. Single cell origin of multilineage colonies in culture. Evidence that differentiation of multipotent progenitors and restriction of proliferative potential of monopotent progenitors are stochastic processes. J Clin Invest 1984; 74:2193–2197. 30. Koiki K. Cryopreservation of pluripotent and committed hematopoietic progenitor cells from human and marrow cord blood. Acta Paediatr Jpn 1983; 25:275–282. 31. Broxmeyer HE, Douglas GW, Hangoc G, et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA 1989; 86:3828–3832. 32. Broxmeyer HE, Srour EF, Hangoc G, et al. High-efficiency recovery of functional hematopoietic progenitor and stem cells from human cord blood cryopreserved for 15 years. Proc Natl Acad Sci USA 2003; 100:645–650. 33. Leary AG, Ogawa M. Blast cell colony assay for umbilical cord blood and adult bone marrow progenitors. Blood 1987; 69:953–956. 34. Carow CE, Hangoc G, Cooper SH, Williams DE, Broxmeyer HE. Mast cell growth factor (c-kit ligand) supports the growth of human multipotential progenitor cells with a high replating potential. Blood 1991; 78:2216–2221. 35. Lu L, Xiao M, Shen RN, Grigsby S, Broxmeyer HE. Enrichment, characterization, and responsiveness of single primitive CD34 human umbilical cord blood hematopoietic progenitors with high proliferative and replating potential. Blood 1993; 81:41–48. 36. Carow CE, Hangoc G, Broxmeyer HE. Human multipotential progenitor cells (CFU-GEMM) have extensive replating capacity for secondary CFU-GEMM: an effect enhanced by cord blood plasma. Blood 1993; 81:942–949. 37. Lansdorp PM, Dragowska W, Mayani H. Ontogeny-related changes in proliferative potential of human hematopoietic cells. J Exp Med 1993; 178:787–791. 38. Hows JM, Bradley BA, Marsh JC, et al. Growth of human umbilical-cord blood in long term haemopoietic cultures. Lancet 1992; 340:73–76. 39. Hao QL, Shah AJ, Thiemann FT, Smogorzewska EM, Crooks GM. A functional comparison of CD34C CD38K cells in cord blood and bone marrow. Blood 1995; 86:3745–3753. 40. Liu H, Verfaillie CM. Myeloid-lymphoid initiating cells (ML-IC) are highly enriched in the rhodamine-c-kit(C)CD33(K)CD38(K) fraction of umbilical cord CD34(C) cells. Exp Hematol 2002; 30:582–589. 41. Vormoor J, Lapidot T, Pflumio F, et al. Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice. Blood 1994; 83:2489–2497. 42. Hogan CJ, Shpall EJ, McNulty O, et al. Engraftment and development of human CD34(C)enriched cells from umbilical cord blood in NOD/LtSz-scid/scid mice. Blood 1997; 90:85–96. 43. Canto E, Rodriguez-Sanchez JL, Vidal S. Distinctive response of naive lymphocytes from cord blood to primary activation via TCR. J Leukoc Biol 2003; 74:998–1007. 44. Broxmeyer HE, Cooper S. High-efficiency recovery of immature haematopoietic progenitor cells with extensive proliferative capacity from human cord blood cryopreserved for 10 years. Clin Exp Immunol 1997; 107:45–53. 45. Broxmeyer HE, Hangoc G, Cooper S, et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci USA 1992; 89:4109–4113. 46. Risdon G, Gaddy J, Horie M, Broxmeyer HE. Alloantigen priming induces a state of unresponsiveness in human umbilical cord blood T cells. Proc Natl Acad Sci USA 1995; 92:2413–2417. 47. Takahashi N, Imanishi K, Nishi da H, Uchiyama T. Evidence for immunologic immaturity of cord blood T cells. Cord blood T cells are susceptible to tolerance induction to in vitro stimulation with a superantigen. J Immunol 1995; 155:5213–5219. 48. Roncarolo MG, Bigler M, Martino S, et al. Immune functions of cord blood cells before and after transplantation. J Hematother 1996; 5:157–160. 49. Hassan J, Reen DJ. Cord blood CD4C CD45RACT cells achieve a lower magnitude of activation when compared with their adult counterparts. Immunology 1997; 90:397–401. 50. Garderet L, Dulphy N, Douay C, et al. The umbilical cord blood alpha-beta T-cell repertoire: characteristics of a polyclonal and naive but completely formed repertoire. Blood 1998; 91:340–346.

184

Grewal and Wagner

51. Moretta A, Locatelli F, Mingrat G, et al. Characterisation of CTL directed towards non-inherited maternal alloantigens in human cord blood. Bone Marrow Transplant 1999; 24:1161–1166. 52. Klein AK, Patel DD, Gooding ME, et al. T-Cell recovery in adults and children following umbilical cord blood transplantation. Biol Blood Marrow Transplant 2001; 7:454–466. 53. Szabolcs P, Park KD, Reese M, et al. Coexistent naive phenotype and higher cycling rate of cord blood T cells as compared to adult peripheral blood. Exp Hematol 2003; 31:708–714. 54. Han P, Hodge G, Story C, Xu X. Phenotypic analysis of functional T-lymphocyte subtypes and natural killer cells in human cord blood: relevance to umbilical cord blood transplantation. Br J Haematol 1995; 89:733–740. 55. Issaragrisil S, Visuthisakchai S, Suvatte V, et al. Brief report: transplantation of cord-blood stem cells into a patient with severe thalassemia. N Engl J Med 1995; 332:367–369. 56. Issaragrisil S, Visuthisakchai S, Tangnaitrisorana Y, et al. Collection of cord blood stem cells for transplantation in thalassemic patients. Stem Cells 1995; 13:71–75. 57. Lau YL, Ma ES, Ha SY, et al. Sibling HLA-matched cord blood transplant for beta-thalassemia: report of two cases, expression of fetal hemoglobin, and review of the literature. J Pediatr Hematol Oncol 1998; 20:477–481. 58. Chik KW, Shing MM, Li CK, et al. Autologous marrow recovery in a multi-transfused betathalassemia major patient after umbilical cord blood transplantation. Blood 1996; 88:755. 59. Miniero R, Rocha V, Saracco P, et al. Cord blood transplantation (CBT) in hemoglobinopathies. Eurocord. Bone Marrow Transplant 1998; 22:S78–S79. 60. Reed W, Smith R, Dekovic F, et al. Comprehensive banking of sibling donor cord blood for children with malignant and nonmalignant disease. Blood 2003; 101:351–357. 61. O’Brien TA, McKenna DH, Tan Y, Ramsay NKC, Wagner JE. Directed donor umbilical cord blood: assessment of banking practices and transplant outcomes [abstract]. Blood 2003; 102:466b. 62. MacMillan ML, Auerbach AD, Davies SM, et al. Haematopoietic cell transplantation in patients with Fanconi anaemia using alternate donors: results of a total body irradiation dose escalation trial. Br J Haematol 2000; 109:121–129. 63. Handyside AH, Kontogianni EH, Hardy K, Winston RM. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990; 344:768–770. 64. Handyside AH. Clinical evaluation of preimplantation genetic diagnosis. Prenat Diagn 1998; 18:1345–1348. 65. Kanavakis E, Traeger-Synodinos J. Preimplantation genetic diagnosis in clinical practice. J Med Genet 2002; 39:6–11. 66. Verlinsky Y, Rechitsky S, Schoolcraft W, Strom C, Kuliev A. Preimplantation diagnosis for Fanconi anemia combined with HLA matching. JAMA 2001; 285:3130–3133. 67. Grewal SS, Kahn JP, MacMillan ML, Ramsay NK, Wagner JE. Successful hematopoietic stem cell transplantation for Fanconi anemia from an unaffected HLA genotypically-identical sibling selected using preimplantation genetic diagnosis. Blood 2004 Feb 1; 103(3):1147–1151. 68. Robertson JA, Kahn JP, Wagner JE. Conception to obtain hematopoietic stem cells. Hastings Cent Rep 2002; 32:34–40. 69. Burgio GR, Gluckman E, Locatelli F. Ethical reappraisal of 15 years of cord-blood transplantation. Lancet 2003; 361:250–252. 70. Wolf SM, Kahn JP, Wagner JE. Using preimplantation genetic diagnosis to create a stem cell donor: issues, guidelines & limits. J Law Med Ethics 2003; 31:327–339. 71. Wagner JE, Kernan NA, Steinbuch M, Broxmeyer HE, Gluckman E. Allogeneic sibling umbilicalcord-blood transplantation in children with malignant and non-malignant disease. Lancet 1995; 346:214–219. 72. Gluckman E. Current status of umbilical cord blood hematopoietic stem cell transplantation. Exp Hematol 2000; 28:1197–1205. 73. https://office.de.netcord.org. 74. http://www.bmdw.org. 75. http://www.marrow.org. 76. Rubinstein P, Stevens CE. The New York blood center’s placental/umbilical cord blood program. Experience with a “new” source of hematopoietic stem cells for transplantation. Ernst Schering Res Found Workshop 2001; 33:47–70. 77. Anasetti C, Amos D, Beatty PG, et al. Effect of HLA compatibility on engraftment of bone marrow transplants in patients with leukemia or lymphoma. N Engl J Med 1989; 320:197–204. 78. Beatty PG, Clift RA, Mickelson EM, et al. Marrow transplantation from related donors other than HLA-identical siblings. N Engl J Med 1985; 313:765–771.

Umbilical Cord Blood Transplantation

185

79. Davies SM, Ramsay NK, Haake RJ, et al. Comparison of engraftment in recipients of matched sibling of unrelated donor marrow allografts. Bone Marrow Transplant 1994; 13:51–57. 80. Szydlo R, Goldman JM, Klein JP, et al. Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings. J Clin Oncol 1997; 15:1767–1777. 81. Rubinstein P, Stevens CE. Placental blood for bone marrow replacement: the New York blood center’s program and clinical results. Best Pract Res Clin Haematol 2000; 13:565–584. 82. Barker JN, Davies SM, DeFor T, et al. Survival after transplantation of unrelated donor umbilical cord blood is comparable to that of human leukocyte antigen-matched unrelated donor bone marrow: results of a matched-pair analysis. Blood 2001; 97:2957–2961. 83. Schiller G, Feig SA, Territo M, et al. Treatment of advanced acute leukaemia with allogeneic bone marrow transplantation from unrelated donors. Br J Haematol 1994; 88:72–78. 84. Martin PJ, Schoch G, Fisher L, et al. A retrospective analysis of therapy for acute graft-versus-host disease: initial treatment. Blood 1990; 76:1464–1472. 85. Weisdorf D, Haake R, Blazar B, et al. Treatment of moderate/severe acute graft-versus-host disease after allogeneic bone marrow transplantation: an analysis of clinical risk features and outcome. Blood 1990; 75:1024–1030. 86. Ratanatharathorn V, Nash RA, Przepiorka D, et al. Phase III study comparing methotrexate and tacrolimus (prograf, FK506) with methotrexate and cyclosporine for graft-versus-host disease prophylaxis after HLA-identical sibling bone marrow transplantation. Blood 1998; 92:2303–2314. 87. Nash RA, Antin JH, Karanes C, et al. Phase 3 study comparing methotrexate and tacrolimus with methotrexate and cyclosporine for prophylaxis of acute graft-versus-host disease after marrow transplantation from unrelated donors. Blood 2000; 96:2062–2068. 88. Sullivan KM, Agura E, Anasetti C, et al. Chronic graft-versus-host disease and other late complications of bone marrow transplantation. Semin Hematol 1991; 28:250–259. 89. Goerner M, Gooley T, Flowers ME, et al. Morbidity and mortality of chronic GVHD after hematopoietic stem cell transplantation from HLA-identical siblings for patients with aplastic or refractory anemias. Biol Blood Marrow Transplant 2002; 8:47–56. 90. Anasetti C, Beatty PG, Storb R, et al. Effect of HLA incompatibility on graft-versus-host disease, relapse, and survival after marrow transplantation for patients with leukemia or lymphoma. Hum Immunol 1990; 29:79–91. 91. Balduzzi A, Gooley T, Anasetti C, et al. Unrelated donor marrow transplantation in children. Blood 1995; 86:3247–3256. 92. Beatty PG, Anasetti C, Hansen JA, et al. Marrow transplantation from unrelated donors for treatment of hematologic malignancies: effect of mismatching for one HLA locus. Blood 1993; 81:249–253. 93. Petersdorf EW, Gooley TA, Anasetti C, et al. Optimizing outcome after unrelated marrow transplantation by comprehensive matching of HLA class I and II alleles in the donor and recipient. Blood 1998; 92:3515–3520. 94. Petersdorf EW, Longton GM, Anasetti C, et al. The significance of HLA-DRB1 matching on clinical outcome after HLA-A, B, DR identical unrelated donor marrow transplantation. Blood 1995; 86:1606–1613. 95. Antin JH, Bierer BE, Smith BR, et al. Selective depletion of bone marrow T lymphocytes with antiCD5 monoclonal antibodies: effective prophylaxis for graft-versus-host disease in patients with hematologic malignancies. Blood 1991; 78:2139–2149. 96. Papadopoulos EB, Carabasi MH, Castro-Malaspina H, et al. T-cell-depleted allogeneic bone marrow transplantation as post-remission therapy for acute myelogenous leukemia: freedom from relapse in the absence of graft-versus-host disease. Blood 1998; 91:1083–1090. 97. Novitzky N, Thomas V, Hale G, Waldmann H. Ex vivo depletion of T cells from bone marrow grafts with CAMPATH-1 in acute leukemia: graft-versus-host disease and graft-versus-leukemia effect. Transplantation 1999; 67:620–626. 98. Hale G, Waldmann H. Control of graft-versus-host disease and graft rejection by T cell depletion of donor and recipient with Campath-1 antibodies. Results of matched sibling transplants for malignant diseases. Bone Marrow Transplant 1994; 13:597–611. 99. Wagner J, Thompson J, Carter S, Jensen L, Kernan N. Impact of graft-versus-host disease prophylaxis on 3 year disease free survival. Results of a multi-center, randomized phase II–III trial comparing T-cell depletion/cyclosporine and methotrexate/cyclosporine in recipients of unrelated donor bone marrow [abstract]. Blood 2002; 100:75a.

186

Grewal and Wagner

100. Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990; 75:555–562. 101. Weiden PL, Flournoy N, Thomas ED, et al. Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 1979; 300:1068–1073. 102. Weiden PL, Sullivan KM, Flournoy N, Storb R, Thomas ED. Antileukemic effect of chronic graftversus-host disease: contribution to improved survival after allogeneic marrow transplantation. N Engl J Med 1981; 304:1529–1533. 103. Hongeng S, Krance RA, Bowman LC, et al. Outcomes of transplantation with matched-sibling and unrelated-donor bone marrow in children with leukaemia. Lancet 1997; 350:767–771. 104. Laughlin MJ, Barker J, Bambach B, et al. Hematopoietic engraftment and survival in adult recipients of umbilical- cord blood from unrelated donors. N Engl J Med 2001; 344:1815–1822. 105. Rubinstein P, Kurtzberg J, Loberiza FR, et al. Comparison of unrelated cord blood and unrelated bone marrow transplants for leukemia in children: a collaborative study of the New York Blood Center and the International Bone Marrow Transplant Registry [abstract]. Blood 2001; 98:814a. 106. Ho VT, Soiffer RJ. The history and future of T-cell depletion as graft-versus-host disease prophylaxis for allogeneic hematopoietic stem cell transplantation. Blood 2001; 98:3192–3204. 107. Wagner JE, Donnenberg AD, Noga SJ, et al. Lymphocyte depletion of donor bone marrow by counterflow centrifugal elutriation: results of a phase I clinical trial. Blood 1988; 72:1168–1176. 108. Wagner JE, Santos GW, Noga SJ, et al. Bone marrow graft engineering by counterflow centrifugal elutriation: results of a phase I–II clinical trial. Blood 1990; 75:1370–1377. 109. Roux E, Helg C, Dumont-Girard F, et al. Analysis of T-cell repopulation after allogeneic bone marrow transplantation: significant differences between recipients of T-cell depleted and unmanipulated grafts. Blood 1996; 87:3984–3992. 110. Harris DT, Schumacher MJ, Locascio J, et al. Phenotypic and functional immaturity of human umbilical cord blood T lymphocytes. Proc Natl Acad Sci USA 1992; 89:10006–10010. 111. Madrigal JA, Cohen SB, Gluckman E, Charron DJ. Does cord blood transplantation result in lower graft-versus-host disease? It takes more than two to tango Hum Immunol 1997; 56:1–5. 112. Lum LG. The kinetics of immune reconstitution after human marrow transplantation. Blood 1987; 69:369–380. 113. Small TN, Papadopoulos EB, Boulad F, et al. Comparison of immune reconstitution after unrelated and related T-cell-depleted bone marrow transplantation: effect of patient age and donor leukocyte infusions. Blood 1999; 93:467–480. 114. Godthelp BC, van Tol MJ, Vossen JM, van Den Elsen PJ. T-Cell immune reconstitution in pediatric leukemia patients after allogeneic bone marrow transplantation with T-cell-depleted or unmanipulated grafts: evaluation of overall and antigen-specific T-cell repertoires. Blood 1999; 94:4358–4369. 115. Atkinson K. Reconstruction of the haemopoietic and immune systems after marrow transplantation. Bone Marrow Transplant 1990; 5:209–226. 116. Parkman R, Weinberg KI. Immunological reconstitution following bone marrow transplantation. Immunol Rev 1997; 157:73–78. 117. Barker J, Hough RE, van Burik JA, et al. Despite naive neonatal immune system, URD UCB transplantation is associated with comparable risk of serious infection early after transplant but lower risk beyond 6 months as compared to URD BM [abstract]. Blood 2003; 102:478a. 118. Giraud P, Thuret I, Reviron D, et al. Immune reconstitution and outcome after unrelated cord blood transplantation: a single paediatric institution experience. Bone Marrow Transplant 2000; 25:53–57. 119. Comoli P, Locatelli F, Moretta A, et al. Human alloantigen-specific anergic cells induced by a combination of CTLA4-Ig and CsA maintain anti-leukemia and anti-viral cytotoxic responses. Bone Marrow Transplant 2001; 27:1263–1273. 120. Niehues T, Rocha V, Filipovich AH, et al. Factors affecting lymphocyte subset reconstitution after either related or unrelated cord blood transplantation in children—a Eurocord analysis. Br J Haematol 2001; 114:42–48. 121. Moretta A, Maccario R, Fagioli F, et al. Analysis of immune reconstitution in children undergoing cord blood transplantation. Exp Hematol 2001; 29:371–379. 122. Ottinger HD, Beelen DW, Scheulen B, Schaefer UW, Grosse-Wilde H. Improved immune reconstitution after allo-transplantation of peripheral blood stem cells instead of bone marrow. Blood 1996; 88:2775–2779. 123. Martinez C, Urbano-Ispizua A, Rozman C, et al. Immune reconstitution following allogeneic peripheral blood progenitor cell transplantation: comparison of recipients of positive CD34C selected grafts with recipients of unmanipulated grafts. Exp Hematol 1999; 27:561–568.

Umbilical Cord Blood Transplantation

187

124. Weinberg K, Blazar BR, Wagner JE, et al. Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation. Blood 2001; 97:1458–1466. 125. Talvensaari K, Clave E, Douay C, et al. A broad T-cell repertoire diversity and an efficient thymic function indicate a favorable long-term immune reconstitution after cord blood stem cell transplantation. Blood 2002; 99:1458–1464. 126. Linch DC, Brent L. Marrow transplantation. Can cord blood be used? Nature 1989; 340:676. 127. Casper J, Camitta B, Truitt R, et al. Unrelated bone marrow donor transplants for children with leukemia or myelodysplasia. Blood 1995; 85:2354–2363. 128. Migliaccio AR, Adamson JW, Stevens CE, et al. Cell dose and speed of engraftment in placental/umbilical cord blood transplantation: graft progenitor cell content is a better predictor than nucleated cell quantity. Blood 2000; 96:2717–2722. 129. Michel G, Rocha V, Chevret S, et al. Unrelated cord blood transplantation for childhood acute myeloid leukemia: a Eurocord group analysis. Blood 2003; 102:4290–4297. 130. Barker J, Krepski T, De For TE, Wagner J, Weisdorf D. Searching for unrelated donor hematopoietic stem cell grafts: availability and speed of umbilical cord blood versus bone marrow. Biol Blood Marrow Transplant 2002; 8:257–260. 131. Rubinstein P. Placental blood-derived hematopoietic stem cells for unrelated bone marrow reconstitution. J Hematother 1993; 2:207–210. 132. Rubinstein P, Rosenfield RE, Adamson JW, Stevens CE. Stored placental blood for unrelated bone marrow reconstitution. Blood 1993; 81:1679–1690. 133. Stevens CE, Beasley RP, Tsui J, Lee WC. Vertical transmission of hepatitis B antigen in Taiwan. N Engl J Med 1975; 292:771–774. 134. Stagno S, Pass RF, Cloud G, et al. Primary cytomegalovirus infection in pregnancy. Incidence, transmission to fetus, and clinical outcome. JAMA 1986; 256:1904–1908. 135. Chang RS, Seto DS. Perinatal infection by Epstein-Barr virus [letter]. Lancet 1979; 2:201. 136. Baxter-Lowe LA, Kim Y, Carter S, et al. Ability of minority patients to find donors from an ethnically diverse cord blood bank [abstract]. Blood 2002; 100:640a. 137. Kurtzberg J, Scaradavou M, Wagner J, et al. Banked umbilical cord blood is an excellent source of donor hematopoietic stem cells for infants with malignant and non malignant conditions lacking a related donor [abstract]. Blood 2000; 96:587a. 138. Staba S, Martin P, Ciocci G, Allison-Thacker J, Kurtzberg J. Correction of Hurler syndrome with unrelated umbilical cord blood transplantation [abstract]. Blood 2001; 98:667a. 139. Krivit W, Aubourg P, Shapiro E, Peters C. Bone marrow transplantation for globoid cell leukodystrophy, adrenoleukodystrophy, metachromatic leukodystrophy, and Hurler syndrome. Curr Opin Hematol 1999; 6:377–382. 140. Peters C, Shapiro EG, Krivit W. Hurler syndrome: past, present, and future. J Pediatr 1998; 133:7–9. 141. Shapiro E, Krivit W, Lockman L, et al. Long-term effect of bone-marrow transplantation for childhood-onset cerebral X-linked adrenoleukodystrophy. Lancet 2000; 356:713–718. 142. Filipovich A. Stem cell transplantation from unrelated donors for correction of primary immunodeficiencies. Organ Bone Marrow Transplant 1996; 16:377–393. 143. Filipovich AH, Shapiro RS, Ramsay NK, et al. Unrelated donor bone marrow transplantation for correction of lethal congenital immunodeficiencies. Blood 1992; 80:270–276. 144. Hows JM, Bradley BA, Marsh JC, et al. Growth of human umbilical-cord blood in long term haemopoietic cultures. Lancet 1992; 340:73–76. 145. Ruggieri L, Heimfeld S, Broxmeyer HE. Cytokine-dependent ex vivo expansion of early subsets of CD34C cord blood myeloid progenitors is enhanced by cord blood plasma, but expansion of the more mature subsets of progenitors is favored. Blood Cells 1994; 20:436–454. 146. Barker JN, Weisdorf DJ, Wagner JE. Creation of a double chimera after the transplantation of umbilical-cord blood from two partially matched unrelated donors. N Engl J Med 2001; 344:1870–1871. 147. Barker JN, Weisdorf DJ, DeFor TE, et al. Rapid and complete donor chimerism in adult recipients of unrelated donor umbilical cord blood transplantation after reduced-intensity conditioning. Blood 2003; 102:1915–1919. 148. Barker J, Weisdorf D, DeFor T, McGlave P, Wagner J. Multiple unit unrelated donor umbilical cord blood transplantation in high risk adults with hematological malignancies: impact on engraftment and chimerism. Blood 2002; 100:40 [abstract].

9 Cellular Immunotherapeutic Approaches to the Hematopoietic Stem-Cell Transplant Patient Dennis Hughes The Children’s Cancer Hospital, MD Anderson Cancer Center, Houston, Texas, U.S.A.

John Levine Department of Pediatrics and Communicable Diseases, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan, U.S.A.

INTRODUCTION Allogeneic bone marrow transplantation (BMT) for relapsed or resistant leukemia was initially conceived as a mechanism for rescuing the patient from the toxicities of intensive chemotherapy, but studies from both the clinic and the laboratory soon demonstrated that the specific immune responses of transplanted, allogeneic immune cells—predominantly T cells—made a significant contribution to survival after BMT. Early observations of a correlation between development of graft versus host disease (GVHD) and relapse-free survival led to the identification of a graftversus-leukemia (GVL) effect (1–4) that was potentially separable from the GVH response (5,6). Yet precisely because BMT is used to treat those malignancies with the poorest outcomes, relapses are frequent. Because allo-immune responses contribute the efficacy of therapy, the loss of the GVL effect is an important part of relapse after BMT. When patients do relapse after allo-BMT, it is possible to reestablish the GVL response by using a fresh source of T cells from the original donor, termed donor leukocyte infusion (DLI) (7–9). The efficacy of this treatment is inversely related to the rate of growth of the leukemia (10), and can be improved with cytoreduction prior to DLI (11,12). DLI can also be combined with maintenance therapy, which may take the form of traditional chemotherapy, specific inhibitors, such as Gleevec (13,14), prodifferentiating agents, such as Arsenic trioxide (15–17) or all-trans-retinoic acid (18–21), or immune modulators, such as cytokines (22,23). In this chapter we will begin by discussing T-cell responses and their potential contribution to GVL and maintaining remission. The ways in which antileukemia responses can fail will also be discussed. We will then review the mechanisms of DLI and a history of its use, analyzing the timing of response and efficacy of therapy in each disease for which it has been used in children. Finally we will examine the use of nonmyeloablative transplants for pediatric 189

190

Hughes and Levine

disease. In this rapidly evolving field, we hope that the information in this chapter will give the reader a basic understanding of the mechanisms of action of adoptive immunotherapy after BMT and provide a foundation from which future studies can be understood.

IMMUNOTHERAPY AND LOSS OF IMMUNOLOGIC CONTROL OF LEUKEMIA To understand GVH and GVL, one must first understand the mechanism of antigen recognition by T cells. The T-cell receptor (TCR) achieves its antigenic specificity through binding by the unique CDR3 domain, as well as the V gene-encoded CDR1 and CDR2 domains, of specific antigenic peptides presented by self HLA (Fig. 1) (24–26). T cells bearing receptors that can mediate activating signals after engaging self HLA–presenting self peptides are deleted during thymic development (27), and it is estimated that such TCR rearrangements may outnumber useful rearrangements by 1000-fold (28). T cells surviving thymic selection are able to interact with self HLA, but each has specificity for nonself peptide being presented by that HLA. Most of these specificities remain irrelevant throughout the life of the person, but, for example, those TCRs with specificity for influenza peptides become important when the patient is infected with the virus.

Figure 1 Interaction of T-cell receptor (TCR) with human leukocyte antigen (HLA). The alpha and beta chains of the TCR pair such that the variable regions bind to the peptide–HLA complex, defining the antigenic specificity of the T cell. These chains then mediate signaling through the TCR/CD3 complex. The CDR3 domain, which is created through somatic rearrangement during T-cell development, provides most of the peptide/HLA complex recognition, whereas the CDR1 and CDR2 domains encoded by the V gene regions provide mostly HLA recognition. Abbreviations: HLA, human leukocyte antigen; TCR, T-cell receptor. Source: From Ref. 26.

Cellular Immunotherapeutic Approaches to HSCT

191

To understand GVL and GVH, it is essential to remember that the thymus only provides negative selection against T cells whose receptors can recognize self HLA and self peptide (29). T cells with specificity for nonself HLA, or for self HLA presenting a peptide from an allelic protein not found in the person, do not get removed by negative selection and remain a part of the TCR repertoire. For example, the allele that causes brown eye color, to a person with blue eyes, is a foreign protein. It is possible, at least in theory, for peptides from this allele to be recognized by a TCR, because the cell bearing such a receptor would not have been deleted during thymic development in a blue-eyed individual. Each person is made unique in part due to the expression of hundreds, if not thousands, of proteins that can have multiple alleles. Except for identical twins, no two people will have exactly the same complement of genetic alleles. All these varied proteins, when presented as peptide fragments by HLA, form the basis of minor antigen recognition and GVH in patients who receive an HLA-matched BMT. Leukemia clones may also express a distinct proteonome. Some of the proteins expressed, such as the BCR-ABL fusion protein created by the Philadelphia chromosome rearrangement (30–32), are tumor-specific antigens, whereas others are simply tumorassociated (33,34). In either case, T-cell recognition of antigenic peptides from these proteins can lead to destruction of tumor cells. In cases where such a peptide antigen is expressed by both normal and malignant tissue, the responses of a particular T-cell clone can mediate both GVH and GVL in the BMT patient. In those cases where the antigen is relatively tumor specific, it is possible to separate the GVL effect from GVH. Identifying the specific cellular and cytokine signals that help promote GVL and lessen GVH is an area of active investigation (5,35–38). Several mechanisms have been proposed for how tumors evade immune reactions. First, any of the proteins involved in peptide processing, loading of peptide into HLA, and trafficking of the HLA/peptide complex to the cell surface may be down-regulated, resulting in decreased expression of peptide antigen on the tumor cell surface (39). This reduction is termed “antigen escape.” Second, tumor cells may also specifically decrease expression of costimulatory molecules or of regulatory signals, such as cell-surface Fas (40), or increase of Th1-inhibiting cytokines, such as IL-4 or IL-10 (41,42). T-cell responses are not the only immune reactions responsible for antitumor effects. natural killer (NK) cells are lymphocytes that mediate cytotoxic responses against multiple targets, including leukemia cells (43,44). Similar to CD8C cytotoxic T lymphocytes (CTLs), NK cells utilize genes encoded within the class I HLA locus for their recognition, though the mechanism of recognition differs from that of CTLs. Early evidence led to a theory that the absence of cell-surface HLA renders a target susceptible to NK-mediated lysis (45,46). This model did not explain all NK activity, however, because NK cells may also preferentially target cells with non-self HLA, especially for myeloid lineages. More recent studies have identified a wide range of NK cell receptors, some of which mediate inhibiting signals and others that transmit activating signals (47). These include the killer-cell immunoglobulin receptors (KIRs) that specifically recognize HLA class I alleles (predominantly HLA-C), the C-type lectin family of receptors that recognize HLA-E molecules loaded with peptides derived from the leader sequences of HLA-A, -B, -C or -G, and the NKG2D C-type lectin receptor that transmits activating signals when it binds to proteins induced by “stress” or neoplastic transformation. Additionally, the natural cytotoxicity receptors (NCRs) NKp46, NKp30 and NKp44, the latter of which is only expressed after IL-2 exposure, can be directly responsible for the lysis of certain tumor types, including melanoma. The ligand for these is unknown. The activity of KIRs may be especially important for determining GVH and GVL. This family consists of matching inhibitory and activating receptors that share identical extracellular domains and ligand specificities but have different cytoplasmic domains. For example, KIR2DL2 is an inhibitory receptor specific for an epitope defined by 77serine; 80asparagine of group 1 HLA-C molecules, whereas KIR2DS2 is the activating receptor with the same specificity. The higher affinity of the inhibitory receptors helps to keep NK cells from reacting against autologous hematopoietic cells. Because NK cells interact with HLA in a peptide-independent manner at a specific, highly conserved locus (position 77 and 80 of HLA-C), all HLA-C alleles can be divided into two groups based upon which KIRs bind them: group 1 alleles are bound

192

Hughes and Levine

by KIR2DL2/KIR2DS2 and KIR2DL3/KIR2DS3, whereas group 2 alleles are bound by KIR2DL1/KIR2DS1. The KIRs expressed by an individual’s NK cells are those specific for that person’s HLA KIR epitope, i.e., group 1 or group 2. In the setting of an allogeneic transplant, when host cells lack this KIR epitope, the moderating signal of the inhibitory KIRs is absent, allowing activating signals from other NK receptors, such as the NCRs and C-type lectin receptors, to mediate donor NK cell activation. This response may mediate a GVL effect. In addition, it will tend to reduce the number of host antigen-presenting cells, reducing T cell mediated GVH responses. In a retrospective analysis by Ruggeri and colleagues, it was found that high-risk AML patients given a haploidentical, T-cell depleted, reduced-intensity transplant with KIR mismatched PBSCs had a 0% probability of relapse at five years, compared to 75% for the matched group (44). Graft rejection and severe acute GVHD were also absent in the mismatched group, whereas the matched group had a 15.5% rate of graft rejection and a 13.7% rate of grade 2 or greater GVHD. NK cells are clearly better at controlling myeloid disease than lymphoid malignancies. In one study alloreactive NK cell clones killed 100% of acute and chronic myeloid leukemia lines, while ALL lines were more resistant to NK-mediated lysis (48). ALL cells may be recognized by gd T cells in some circumstances (49). Both gd and NK cells are also subject to T-cell regulatory influences and may also become anergized in the presence of overwhelming antigen and dysfunctional T cells. If T-cell responses are important in maintaining remission, then relapses, when they occur, must include a loss of these effects. T-cell immunity may be lost either through the death of the responding clone, usually through activation-induced cell death (50), or through anergy, a state in which cells with antigen-specific receptors persist in the body but fail to make immune reactions, such as cytokine release, cytolysis of targets, or proliferation in response to antigen (51–54). The signals that mediate anergy are complex, but they include inadequate or inappropriate costimulatory signals during activation (51), overwhelming amounts of antigen (55–58) and the presence of inhibitory cytokines (59,60). All of these conditions may occur during leukemic relapse (61–63).

DONOR LEUKOCYTE INFUSION For the patient with relapse after BMT, it is possible to regain the GVL effect through DLI. By receiving unfractionated leukocytes from the original donor, patients are likely to receive a renewal of all donor cell–mediated activity, whether helpful or disadvantageous. If the GVHrelated toxicities in these patients can be controlled, this renewed GVL response can bring about renewed leukemia control. In 1995 Kolb and colleagues reported the first large series of patients treated for relapse after BMT with DLI (10). The best responses were seen with chronic myelogenous leukemia (CML) in cytogenetic or hematologic relapse, with 82% and 78%, respectively, achieving remission with DLI alone, and both groups having a 5-year survival of 67%. DLI was less effective against transformed phase CML, with only one patient out of 14 achieving a complete remission (CR). CML is rarely observed in pediatric patients, however, and those diseases that are seen in children had less promising results, perhaps because of their more rapid rates of growth. DLI provided some benefit to AML and MDS patients, with CR rates of 29% and 25%, respectively, but even those with a CR had a median survival of less than a year. ALL had the worst results in this study, with a median survival of less than six months and a 100% probability of relapse by 15 months after DLI. No ALL patients achieved a CR with DLI alone. Three years later this group issued a follow-up report confirming these findings (64), and others made the same observations about ALL (Table 1) (65–69). The timing of responses following DLI may explain, in part, its differing efficacy against CML, AML, and ALL. Baurmann and colleagues studied the hematopoietic engraftment in eight adults with relapsed CML after DLI (70). They found that donor granulocyte engraftment began between 5 and 13 weeks after infusion and was complete 9 to 17 weeks after infusion. The onset of GVHD occurred 12 to 21 weeks after DLI. Five of eight

Cellular Immunotherapeutic Approaches to HSCT Table 1

193

Summary of Donor Leukocyte Infusion Study Outcomes EFS (%)

OS (%)

2.4 (0.1–7.8) NR NR 5.7 (0.5–14) NR

30 – 62 48 19 15 15.4 NR 100 73

35 55 19 26 80

50 NR NR 22 NR

Y N

8.9 (6.3–13) 1.83 T cells infused

100 0

0 0

0 0

100 33

Collins ’97 (66) Collins ’00 (67)

22 26 10 15 28

N Y N N Y

2.9 (0.3–11) NR NR 2.8 (1–5.7) NR

0 50 10 18 25

– 14 10 – 7

22 14 10 27 7

40 NR NR 20 25

Porter ’99 (65) Mandanas (68) Slavin ’96 (69)

15 4 2 6

N N Y N

NR 13 NR 100 7.2 (6.9–7.4) 100 1.58 (0.5–3.2) 66 T cells infused

7 25 0 66

7 75 50 50

25 NBO 100 GVHD

MDS Kolb ’95 (10) Kolb ’98 (64) Collins ’97 (66) Porter ’99 (65)

4 9 6 3

N N N N

7.6 (4–15) NR 7.3 (5.5–10) NR

25 – – 66

25 – 33 66

NR NR 50 NBO

Study

n

CTx

Collins ’97 (66) Porter ’99 (65)

23 21 26 46 15

N Y N N Y

Mandanas (68) Slavin ’96 (69)

4 3

AML Kolb ’95 (10) Kolb ’98 (64)

ALL Kolb ’95 (10) Kolb ’98 (64)

Cell # (range)

CR (%)

25 33 40 100

GVHD (%)

Comments

CTx vs No CTx ave. 3 DLI Only CR patients

CTx vs No CTx GVHD for total group 1/4 given CTx GVHD pts were all survivors

ave 3 doses DLI

The outcomes of several recent reports of donor leukocyte infusion (DLI) are presented, groups by disease. The patients in all studies have received a prior allogeneic transplant with relapse of disease. Note: In the Slavin study, the number of T cells infused per patient was given, rather than the number of mononuclear cells. Abbreviations: n, number of patients with this disease in the report; CTx, indicates whether chemotherapy was used prior to DLI; Cell #, indicates the average dose of mononuclear cells infused, in units of 100M per kg, with the range of doses within the study listed in parentheses; CR, indicates the percentage of patients achieving a CR with the indicated treatment, whether achieved prior to DLI or following infusion; EFS, indicates the event-free survival at one year; OS, indicates the overall survival at one year; GVHD, indicates the percentage of patients developing acute GVHD of grade 2 or higher; NR, indicates that the data were not reported in that study; NBO, indicates that the data for GVHD were not broken out by individual patient or disease and thus cannot be summarized here. AML, acute myelogenous leukemia; MDS, myelodysplastic syndrome; ALL, acute lymphoblastic leukemia.

patients achieved a complete molecular remission between 6 and 13 weeks, with a sixth going into remission by week 21. It is interesting to note that, in Baurmann’s study, the majority of T cells at the time of relapse were still donor-derived, as were most B cells and erythrocytes. This residual hematopoiesis may be essential for good outcomes (71). This would suggest that leukemia-specific T cells are selectively depleted or anergized in the relapsed patient. Therapeutic responses are observed when DLI renews the leukemia-reactive cell population. DLI can have a fairly high rate of toxicity with treatment-related mortality ranging from 15–23% and graft-versus-host disease presenting in 42–56% of patients treated (12,65,67,72).

194

Hughes and Levine

One strategy that has been utilized to diminish GVHD associated with DLI has been escalating dose DLI. Mackinnon and colleagues at memorial Sloan Kettering Cancer Center in New York utilized this approach with adult patients with CML (73). Beginning with doses as low as 1!105 T cells per kilogram they escalated up to doses as high as 1!108 T cells per kilogram. With this approach, 19 of 22 patients treated achieved CR. Only 1 of those 19 developed acute graft-versus-host disease. Dazzi and colleagues in the United Kingdom reported a similar study using escalating dose DLI for relapsed CML in which they had similar response rates (72). In the Mackinnon study 9 of 19 patients developed chronic GVHD, whereas in Dazzi’s study only 10% of their patients developed chronic GVHD, compared to 44% treated with bulk dose GVHD. In the Dazzi study the rates of more severe grades of GVHD were also higher with bulk dose DLI. An approach for using escalating dose DLI for the acute leukemias has not yet been developed. One of the biggest predictors of efficacy for DLI is the particular disease being treated and CML has the best response rates for DLI. In Kolb’s 1995 report, CML patients in early stages of relapse, either cytogenetic or hematologic, had remarkable response rates using DLI alone (82% and 78% respectively), and the five-year survival in this population was 67% (10). However, patients with transformed phase CML rarely achieved a CR (only 1 of 14 patients). In 1997 Collins and colleagues reported the first large North American study of DLI (66). In their study, which included both children and adults CML patients, again had excellent response rates: a 60% CR rate at two years with much better responses for patients with a cytogenetic or hematologic relapse (100% and 73.5% CR rate), whereas accelerated phase or blast phase were much lower (33% and 16.7% CR rates). DLI was less efficacious against the acute leukemias. This technique achieved a CR rate of only 18.2% for ALL patients in relapse, whereas AML patients had a response rate of only 15.4%. Acute and chronic GVHD occurred at a rate 60% each, and pancytopenia occurred in nearly 19% of patients treated with DLI. These toxicities were manageable, however, as only 22% of patients experienced GVHD that was grade 3 or grade 4 in severity. In all cases both acute and chronic GVHD correlated with a better outcome. Because the median survival of acute leukemic relapse after BMT has already been shown to be less than one month (74), the toxicities did not seem so excessive as not to merit further exploration of DLI for the treatment of acute leukemias. It seemed evident at least from the CML data, that outcomes from DLI are best when there is a low overall disease burden. Thus trials were undertaken using cytotoxic therapy to induce a minimal residual disease state prior to DLI. Levine et al. undertook a prospective trial of chemotherapy followed by DLI for patients with advanced myeloid disease that had relapsed after transplant (12). The chemotherapy utilized was submyeloablative, and no immunosuppression was used after the cells were infused. The median T-cell dose in this study was 1!108 CD-3 positive cells per kilogram. The overall survival at two years in this study was 19% with a median follow-up of 871 days. In this trial 27 of 57 evaluable patients achieved a CR (47%). The overall survival of those 27 patients was 51% at one year and 41% at two years. By contrast the nonresponders had a one-year overall survival rate of 5%. Other reports have also shown a benefit for cytoreduction prior to DLI or second BMT for acute leukemia relapse (64,66,69,75,76).

NONMYELOABLATIVE TRANSPLANT The treatment of the acute leukemia patient who relapses after BMT requires a difficult balance to be achieved. Often these patients are not able to tolerate high intensity therapy and yet will not have a good outcome from such therapies as DLI unless the cells can be infused at a minimal residual disease state. A second myeloablative transplant is often deemed too toxic for the BMT patient in relapse, but it may be difficult to achieve effective cytoreduction while still allowing sufficient time for marrow recovery to take place prior to a DLI. For this reason several investigators have explored the option of a nonmyeloablative stem cell transplant, also

Cellular Immunotherapeutic Approaches to HSCT

195

termed reduced intensity stem cell transplantation (RIST). In these protocols patients receive a reduced intensity conditioning and then receive an infusion of stem cells and allogeneic T cells immediately after that conditioning. In some cases this conditioning follows induction and consolidation chemotherapy designed for the relapsed patient. Pawson and colleagues reported a trial of acute leukemia patients, relapsed after allogeneic BMT, in which fludarabine, cytarabine, and anthracyclines were used for conditioning for a second transplant (77). In this reduced intensity protocol there was rapid recovery of hematopoiesis with neutrophil recovery averaging 13.5 days and platelet recovery averaging 21 days. Though the number of patients was small (nZ14), there were no toxic deaths in their study. Actuarial survival was 60% nearly five years later, though chronic GVHD was frequent (eight patients). The Socie´te´ Franc¸aise de Greffe de Moelle Registry reported a large series of adults treated with reduced intensity stem cell transplants, which included 41 acute leukemia patients (14 ALL, 18 AML, and 10 MDS) (78). The findings were hard to interpret, because the prior therapies were not uniform—some patients had received a myeloablative allo-BMT, auto-BMT or both prior to the RIST—and the patient outcomes were not broken down by prior treatment to allow for detailed analysis. Nonetheless, the trends were similar to standard BMT and to DLI: younger age, lower disease burden at the time of treatment, and presence of GVHD were all associated with better outcome. Several groups have combined RIST with DLI in an attempt to maximize the GVL effect while limiting direct toxicities of therapy. Dey and colleagues treated a group of 13 patients with a variety of diseases (Hodgkin’s disease, non-Hodgkin’s lymphoma, MDS) with allogeneic RIST using cyclophosphamide (150–200 mg/kg) antithymocyte globulin C/K thymic irradiation as a preparative regimen (79). The seven patients who did not develop GVHD were given prophylactic DLI 5–6 weeks after transplant. Seven of 13 achieved a CR (4 DLI, 3 GVHD), and the two-year actuarial survival was 45%, with a disease-free survival of 37.5%. The same group reported the outcomes of 21 patients with relapsed hematologic malignancies treated with a similar conditioning regimen (80). In this study 10 of 21 patients required DLI to induce GVHD or full conversion to donor hematopoiesis, and a 40% CR rate was achieved. Treatment-related toxicities were felt to be low, given the patient population. Carella et al. reported the combination of autologous SCT followed by allogeneic RIST to treat 15 refractory lymphoma patients (81). The autologous transplant preparative regimen consisted of carmustine, etoposide, Ara-C, and melphalan. Approximately two months later, patients were reconditioned with fludarabine (90 mg/M2 total) and cyclophosphamide (900 mg/ M2 total) followed by donor peripheral blood stem cell infusion. Seven of 15 patients required a second DLI to achieve full engraftment. This approach led to a 73% CR rate, and 33% of patients had a durable remission. Depletion of CD8C cells from both the stem cell pool and the cells used for DLI may reduce GVHD without eliminating the benefit of GVL. Baron and colleagues reported a series of ten patients with high-risk malignancies treated with RIST using donor cells that were either CD34 selected (three patients) or CD8 depleted (seven patients), followed by DLI using CD8 depleted lymphocytes (82). The conditioning regimen utilized fludarabine with either total body irradiation or (for previously irradiated patients) cyclophosphamide. None of these patients developed grade II-IV GVHD, whereas the incidence was 75% in a control group undergoing the same treatment with unselected donor cells. The one-year actuarial survival of this group was 69%, but it is not clear from the data available whether this represents an improvement over standard BMT. All patient deaths were in the CD8-depleted group, although the number of patients evaluated was too small to draw any meaningful conclusions. Several reports have demonstrated that a reduced intensity transplant can be effective for a first allogeneic BMT, especially for patients who are not candidates for a traditional myeloablative approach (83–85). A RIST can also be an excellent option for nonmalignant conditions, and this treatment has been effective for patients with chronic granulomatous disease (86,87). Patients with severe combined immunodeficiency have traditionally been treated with this approach, because they are less likely to reject allografts and may achieve

196

Hughes and Levine

significant benefit from hematopoietic chimerism (88–90). It has also been shown that SCIDS patients develop a normal T cell repertoire following BMT, including RIST (91). A detailed analysis of the various conditioning regimens used for RIST is beyond the scope of this chapter, but the topic has been the subject of several recent reviews (92–95). For the present time, RIST in pediatrics should probably be reserved for those patients enrolled in a clinical trial or whose poor organ function or low performance score precludes their consideration for a more standard, myeloablative transplant.

SUMMARY The lost of GVL and immunoreactivity that comes with relapse after BMT need not be an insurmountable problem. The factors that led to the death or anergy of the initial responding cells—whether it be cytokine release or the presence of overwhelming antigen—are difficulties that can be corrected with a medical approach. Cytotoxic therapy in the form of a standard induction and consolidation for relapse are usually what is required. Once patients have a low disease burden they can receive an infusion of their original donor leukocytes to reinitiate the GVL affect. It is understood that this may also reinitiate the GVH effect and that patients may experience toxicities from GVHD. With improved understanding of the GVH and GVL effects and the development of new agents to control GVHD, it is hoped that the toxicities of GVHD will only be lower in the future. The use of DLI for treatment of leukemic relapse after transplant remains a promising area of investigation, and one can expect to see further exciting developments in this field over the next several years. This approach offers hope to a population of patients that previously would have had a very poor prognosis. As such it remains worthy of further attention and can be hoped to bring further developments to this arena.

REFERENCES 1. Horowitz M, Gale R, Sondel P, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990; 75:555–562. 2. Russell L, Jacobsen N, Heilmann C, Simonsen A, Christensen L, Vindelov L. Treatment of relapse after allogeneic BMT with donor leukocyte infusions in 16 patients. Bone Marrow Transplant 1996; 18:411–414. 3. Tringali S, Vasta S, Scime R, Catania P, Cavallaro A, Majolino I. Testicular relapse of AML during chronic graft-versus-host disease induced by donor leukocyte infusion. Haematologica 1996; 81:339–342. 4. Claret EJ, Alyea EP, Orsini E, et al. Characterization of T cell repertoire in patients with graft-versusleukemia after donor lymphocyte infusion. J Clin Invest 1997; 100:855. 5. Teshima T, Hill GR, Pan L, et al. IL-11 separates graft-versus-leukemia effects from graft-versushost disease after bone marrow transplantation. J Clin Invest 1999; 104:317–325. 6. Mehta J, Powles R, Kulkarni S, Treleaven J, Singhal S. Induction of graft-versus-host disease as immunotherapy of leukemia relapsing after allogeneic transplantation: single-center experience of 32 adult patients. Bone Marrow Transplant 1997; 20:129–135. 7. Ferster A, Bujan W, Mouraux T, Devalck C, Heimann P, Sariban E. Complete remission following donor leukocyte infusion in ALL relapsing after haploidentical bone marrow transplantation. Bone Marrow Transplant 1994; 14:331–332. 8. Garicochea B, van Rhee F, Spencer A, et al. Aplasia after donor lymphocyte infusion (DLI) for CML in relapse after sex-mismatched BMT: recovery of donor-type haemopoiesis predicted by nonisotopic in situ hybridization (ISH). Br J Haematol 1994; 88:400–402. 9. Kolb H, Mittermuller J, Clemm C, et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 1990; 76:2462–2465. 10. Kolb H, Schattenberg A, Goldman J, et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. European group for blood and marrow transplantation working party chronic leukemia [see comments]. Blood 1995; 86:2041–2050.

Cellular Immunotherapeutic Approaches to HSCT

197

11. Kolb HJ, Schmid C, Muth A, et al. Donor cell transfusion for the treatment of recurrence—adoptive immunotherapy using donor cells and GM-CSF for recurrent acute leukemia and acute phase CML after allogeneic marrow transplantation. American Society of Hematology Annual Meeting, Blood, 1998; 92/1:344b, A4481. 12. Levine JE, Braun T, Penza SL, et al. Prospective trial of chemotherapy and donor leukocyte infusions for relapse of advanced myeloid malignancies after allogeneic stem-cell transplantation. J Clin Oncol 2002; 20:405–3590. 13. Thiesing JT, Ohno-Jones S, Kolibaba KS, Druker BJ. Efficacy of STI571, an Abl tyrosine kinase inhibitor, in conjunction with other antileukemic agents against Bcr-Abl-positive cells. Blood 2000; 96:3195–3199. 14. Buchdunger E, Cioffi CL, Law N, et al. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther 2000; 295:139–3199. 15. Chen G, Zhu J, Shi X, et al. In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins. Blood 1996; 88:1052–3353. 16. Chen G-Q, Shi X-G, Tang W, et al. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic Leukemia (APL): I. As2O3 exerts dose-dependent dual effects on APL Cells. Blood 1997; 89:3345–3353. 17. Shen Z-X, Chen G-Q, Ni J-H, et al. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): II. clinical efficacy and pharmacokinetics in relapsed patients. Blood 1997; 89:3354–3360. 18. Chen Z, Tao R, Xia X. The present status in all-trans retinoic acid (ATRA) treatment for acute promyelocytic leukemia patients: further understanding and comprehensive strategy are required in the future. Leuk Lymphoma 1992; 8:247–252. 19. Degos L. All-trans retinoic acid (ATRA) therapeutical effect in acute promyelocytic leukemia. Biomed Pharmacother 1992; 46:201–209. 20. Degos L. All-trans-retinoic acid treatment and retinoic acid receptor alpha gene rearrangement in acute promyelocytic leukemia: a model for differentiation therapy. Int J Cell Cloning 1992; 10:63–69. 21. Dombret H, Sutton L, Duarte M, et al. Combined therapy with all-trans-retinoic acid and high-dose chemotherapy in patients with hyperleukocytic acute promyelocytic leukemia and severe visceral hemorrhage. Leukemia 1992; 6:1237–1242. 22. Grigg A, Kannan K, Schwarer A, Spencer A, Szer J. Chemotherapy and granulocyte colony stimulating factor-mobilized blood cell infusion followed by interferon-alpha for relapsed malignancy after allogeneic bone marrow transplantation. Intern Med J 2001; 31:15–22. 23. Mehta J, Powles R, Treleaven J, et al. Outcome of acute leukemia relapsing after bone marrow transplantation: utility of second transplants and adoptive immunotherapy. Bone Marrow Transplant 1997; 19:709–719. 24. Carbone FR, Moore MW, Sheil JM, Bevan MJ. Induction of cytotoxic T lymphocytes by primary in vitro stimulation with peptides. J Exp Med 1988; 167:1767–1779. 25. Townsend A, Ohlen C, Bastin J, Ljunggren H, Foster L, Karre K. Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature 1989; 340:443–448. 26. Krensky A, Weiss A, Crabtree G, Davis M, Parham P. T-lymphocyte-antigen interactions in transplant rejection. N Engl J Med 1990; 322:510–517. 27. Kisielow P, Bluthmann H, Staerz U, Steinmetz M, von Boehmer H. Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4C8C thymocytes. Nature 1988; 333:742–746. 28. Kisielow P, Teh H, Bluthmann H, von Boehmer H. Positive selection of antigen-specific T cells in thymus by restricting MHC molecules. Nature 1988; 335:730–733. 29. Palmer E. Negative selection–clearing out the bad apples from the T-cell repertoire. Nat Rev Immunol 2003; 3:383–391. 30. Tanaka Y, Takahashi T, Nieda M, et al. Generation of HLA-DRB1*1501-restricted p190 minor bcr-abl (e1a2)-specific CD4CT lymphocytes. Br J Haematol 2000; 109:435–437. 31. Osman Y, Takahashi M, Zheng Z, et al. Generation of bcr-abl specific cytotoxic T-lymphocytes by using dendritic cells pulsed with bcr-abl (b3a2) peptide: its applicability for donor leukocyte transfusions in marrow grafted CML patients. Leukemia 1999; 13:166–174. 32. Yotnda P, Firat H, Garcia-Pons F, et al. Cytotoxic T cell response against the chimeric p210 BCRABL protein in patients with chronic myelogenous leukemia. J Clin Invest 1998; 101:2290–2296.

198

Hughes and Levine

33. Li Q, Chang A. Adoptive T-cell immunotherapy of cancer. Cytokines Cell Mol Ther 1999; 5:105–117. 34. Barth RJ, Jr., Bock SN, Mule JJ, Rosenberg SA. Unique murine tumor-associated antigens identified by tumor infiltrating lymphocytes. J Immunol 1990; 144:1531–1537. 35. Hill GR, Teshima T, Gerbitz A, et al. Differential roles of IL-1 and TNF-{alpha} on graft-versus-host disease and graft versus leukemia. J Clin Invest 1999; 104:459–467. 36. Sefrioui H, Billiau A, Waer M. Graft-versus-leukemia effect in minor antigen mismatched chimeras given delayed donor leucocyte infusion: immunoregulatory aspects and role of donor T and ASGM1positive cells. Transplantation 2000; 70:348–353. 37. Boyer MW, Vallera DA, Taylor PA, et al. The role of B7 costimulation by murine acute myeloid leukemia in the generation and function of a CD8CT-cell line with potent in vivo graft-versusleukemia properties. Blood 1997; 89:3477–3485. 38. Billiau AD, Fevery S, Rutgeerts O, Landuyt W, Waer M. Crucial role of timing of donor lymphocyte infusion in generating dissociated graft-versus-host and graft-versus-leukemia responses in mice receiving allogeneic bone marrow transplants. Blood 2002; 100:1894. 39. Kourilsky P, Jaulin C, Ley V. The structure and function of MHC molecules. Possible implications for the control of tumor growth by MHC-restricted T cells. Semin Cancer Biol 1991; 2:275–282. 40. Bennett MW, O’Connell J, O’Sullivan GC, et al. The Fas counterattack in vivo: apoptotic depletion of tumor-infiltrating lymphocytes associated with Fas ligand expression by human esophageal carcinoma. J Immunol 1998; 160:5669–5675. 41. Shen L, Chiang A, Liu W, Li G, Liang R, Srivastava G. Expression of HLA class I, beta(2)microglobulin, TAP1 and IL-10 in epstein-barr virus-associated nasal NK/T-cell lymphoma: implications for tumor immune escape mechanism. Int J Cancer 2001; 92:692–696. 42. Fujii S-i, Shimizu K, Shimizu T, Lotze MT. Interleukin-10 promotes the maintenance of antitumor CD8CT-cell effector function in situ. Blood 2001; 98:2143. 43. Ruggeri L, Capanni M, Martelli M, Velardi A. Cellular therapy: exploiting NK cell alloreactivity in transplantation. Curr Opin Hematol 2001; 8:355–359. 44. Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295:2097–2100. 45. Ljunggren H, Karre K. In search of the “missing self”: MHC molecules and NK cell recognition. Immunol Today 1990; 11:237–244. 46. Lowdell M, Lamb L, Hoyle C, Velardi A, Prentice H. Non-MHC-restricted cytotoxic cells: their roles in the control and treatment of leukaemias. Br J Haematol 2001; 114:11–24. 47. Farag SS, Fehniger TA, Ruggeri L, Velardi A, Caligiuri MA. Natural killer cell receptors: new biology and insights into the graft versus leukemia effect. Blood 2002; 1935–1947. 48. Ruggeri L, Capanni M, Casucci M, et al. Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation. Blood 1999; 94:333–339. 49. Lamb L, Musk P, Ye Z, et al. Human gammadelta(C) T lymphocytes have in vitro graft vs. leukemia activity in the absence of an allogeneic response. Bone Marrow Transplant 2001; 27:601–606. 50. Hughes DP, Crispe IN. A naturally occurring soluble isoform of murine Fas generated by alternative splicing. J Exp Med 1995; 182:1395–1401. 51. Gimmi C, Freeman G, Gribben J, Gray G, Nadler L. Human T-cell clonal anergy is induced by antigen presentation in the absence of B7 costimulation. Proc Natl Acad Sci USA 1993; 90:6586–6590. 52. LaSalle J, Hafler D. T cell anergy. FASEB J 1994; 8:601–6590. 53. Lechler R, Chai J, Marelli-Berg F, Lombardi G. The contributions of T-cell anergy to peripheral T-cell tolerance. Immunology 2001; 103:262–269. 54. Tham EL, Shrikant P, Mescher MF. Activation-induced nonresponsiveness: a Th-dependent regulatory checkpoint in the CTL response. J Immunol 2002; 168:1190–1197. 55. Switzer S, Wallner B, Briner T, Sunshine G, Bourque C, Luqman M. Bolus injection of aqueous antigen leads to a high density of T-cell-receptor ligand in the spleen, transient T-cell activation and anergy induction. Immunology 1998; 94:513–522. 56. Korb LC, Mirshahidi S, Ramyar K, Sadighi Akha AA, Nasseri S. Induction of T cell anergy by low numbers of agonist ligands. J Immunol 1999; 162:6401–6409. 57. Falb D, Briner T, Sunshine G, et al. Peripheral tolerance in T cell receptor-transgenic mice: evidence for T cell anergy. Eur J Immunol 1996; 26:130–135. 58. Gao E, Lo D, Sprent J. Strong T cell tolerance in parent—F1 bone marrow chimeras prepared with supralethal irradiation. Evidence for clonal deletion and anergy. J Exp Med 1990; 171:1101.

Cellular Immunotherapeutic Approaches to HSCT

199

59. Shrikant P, Khoruts A, Mescher M. CTLA-4 blockade reverses CD8CT cell tolerance to tumor by a CD4CT cell- and IL-2-dependent mechanism. Immunity 1999; 11:483–493. 60. Perez V, Van Parijs L, Biuckians A, Zheng X, Strom T, Abbas A. Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity 1997; 6:411–417. 61. Cardoso A, Schultze J, Boussiotis V, et al. Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen. Blood 1996; 88:41–6590. 62. Gorin LJ, Jeha SC, Sullivan MP, Rosenblatt HM, Shearer WT. Burkitt’s lymphoma developing in a 7-year-old boy with hyper-IgE syndrome. J Allergy Clin Immunol 1989; 83:5–10. 63. Staveley-O’Carroll K, Sotomayor E, Montgomery J, et al. Induction of antigen-specific T cell anergy: an early event in the course of tumor progression. Proc Natl Acad Sci USA 1998; 95:1178–6590. 64. Kolb HJ. Donor leukocyte transfusions for treatment of leukemic relapse after bone marrow transplantation. EBMT immunology and chronic leukemia working parties. Vox Sang 1998; 74:321–329. 65. Porter DL, Collins RH, Jr., Shpilberg O, et al. Long-term follow-up of patients who achieved complete remission after donor leukocyte infusions. Biol Blood Marrow Transplant 1999; 5:253–261. 66. Collins RH, Jr., Shpilberg O, Drobyski WR, et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. [see comments]. J Clin Oncol 1997; 15:433–444. 67. Collins R, Goldstein S, Giralt S, et al. Donor leukocyte infusions in acute lymphocytic leukemia. Bone Marrow Transplant 2000; 26:511–516. 68. Mandanas R, Saez R, Selby G, Confer D. G-CSF-mobilized donor leukocyte infusions as immunotherapy in acute leukemia relapsing after allogeneic marrow transplantation. J Hematother 1998; 7:449–456. 69. Slavin S, Naparstek E, Nagler A, et al. Allogeneic cell therapy with donor peripheral blood cells and recombinant human interleukin-2 to treat leukemia relapse after allogeneic bone marrow transplantation. Blood 1996; 87:2195. 70. Baurmann H, Nagel S, Binder T, Neubauer A, Siegert W, Huhn D. Kinetics of the graft-versusleukemia response after donor leukocyte infusions for relapsed chronic myeloid leukemia after allogeneic bone marrow transplantation. Blood 1998; 92:3582–3590. 71. Keil F, Haas OA, Fritsch G, et al. Donor leukocyte infusion for leukemic relapse after allogeneic marrow transplantation: lack of residual donor hematopoiesis predicts aplasia. Blood 1997; 89:3113. 72. Dazzi F, Szydlo RM, Craddock C, et al. Comparison of single-dose and escalating-dose regimens of donor lymphocyte infusion for relapse after allografting for chronic myeloid leukemia. Blood 2000; 95:67–3590. 73. Mackinnon S, Papadopoulos E, Carabasi M, et al. Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia responses from graft-versus-host disease. Blood 1995; 86:1261–1268. 74. Keil F, Prinz E, Kalhs P, et al. Treatment of leukemic relapse after allogeneic stem cell transplantation with cytotoreductive chemotherapy and/or immunotherapy or second transplants. Leukemia 2001; 15:355–361. 75. Verdonck L, Petersen E, Lokhorst H, et al. Donor leukocyte infusions for recurrent hematologic malignancies after allogeneic bone marrow transplantation: impact of infused and residual donor T cells. Bone Marrow Transplant 1998; 22:1057–1063. 76. Mrsic M, Horowitz M, Atkinson K, et al. Second HLA-identical sibling transplants for leukemia recurrence. Bone Marrow Transplant 1992; 9:269–275. 77. Pawson R, Potter M, Theocharous P, et al. Treatment of relapse after allogeneic bone marrow transplantation with reduced intensity conditioning (FLAG C/- Ida) and second allogeneic stem cell transplant. Br J Haematol 2001; 115:622–629. 78. Michallet M, Bilger K, Garban F, et al. Allogeneic hematopoietic stem-cell transplantation after nonmyeloablative preparative regimens: impact of pretransplantation and posttransplantation factors on outcome. J Clin Oncol 2001; 19:3340. 79. Dey B, McAfee S, Sackstein R, et al. Successful allogeneic stem cell transplantation with nonmyeloablative conditioning in patients with relapsed hematologic malignancy following autologous stem cell transplantation. Biol Blood Marrow Transplant 2001; 7:604–612.

200

Hughes and Levine

80. Spitzer T, McAfee S, Sackstein R, et al. Intentional induction of mixed chimerism and achievement of antitumor responses after nonmyeloablative conditioning therapy and HLA-matched donor bone marrow transplantation for refractory hematologic malignancies. Biol Blood Marrow Transplant 2000; 6:309–320. 81. Carella AM, Cavaliere M, Lerma E, et al. Autografting followed by nonmyeloablative immunosuppressive chemotherapy and allogeneic peripheral-blood hematopoietic stem-cell transplantation as treatment of resistant Hodgkin’s disease and non-Hodgkin’s lymphoma. J Clin Oncol 2000; 18:3918–3924. 82. Baron F, Baudoux E, Frere P, et al. Nonmyeloablative stem cell transplantation with CD8-Depleted or CD34-selected peripheral blood stem cells. J Hematother Stem Cell Res 2002; 11:301–314. 83. Giralt S, Estey E, Albitar M, et al. Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: harnessing graft-versus-leukemia without myeloablative therapy. Blood 1997; 89:4531–4536. 84. Anderlini P, Giralt S, Andersson B, et al. Allogeneic stem cell transplantation with fludarabine-based, less intensive conditioning regimens as adoptive immunotherapy in advanced Hodgkin’s disease. Bone Marrow Transplant 2000; 26:615–620. 85. Childs R, Clave E, Contentin N, et al. Engraftment kinetics after nonmyeloablative allogeneic peripheral blood stem cell transplantation: full donor T-cell chimerism precedes alloimmune responses. Blood 1999; 94:3234. 86. Nagler A, Ackerstein A, Kapelushnik J, Or R, Naparstek E, Slavin S. Donor lymphocyte infusion post-non-myeloablative allogeneic peripheral blood stem cell transplantation for chronic granulomatous disease. Bone Marrow Transplant 1999; 24:339–342. 87. Horwitz ME, Barrett AJ, Brown MR, et al. Treatment of chronic granulomatous disease with nonmyeloablative conditioning and a T-cell-depleted hematopoietic allograft. N Engl J Med 2001; 344:881–888. 88. Buckley RH, Schiff SE, Schiff RI, et al. Hematopoietic stem-cell transplantation for the treatment of severe combined immunodeficiency. N Engl J Med 1999; 340:508–516. 89. Amrolia P, Gaspar HB, Hassan A, et al. Nonmyeloablative stem cell transplantation for congenital immunodeficiencies. Blood 2000; 96:1239–1246. 90. Woolfrey A, Pulsipher M, Storb R. Non-myeloablative hematopoietic cell transplant for treatment of nonmalignant disorders in children. Int J Hematol 2002; 76:271–277. 91. Patel DD, Gooding ME, Parrott RE, Curtis KM, Haynes BF, Buckley RH. Thymic function after hematopoietic stem-cell transplantation for the treatment of severe combined immunodeficiency. N Engl J Med 2000; 342:1325–1332. 92. McCarthy NJ, Bishop MR. Nonmyeloablative allogeneic stem cell transplantation: early promise and limitations. Oncologist 2000; 5:487. 93. Carella A, Champlin R, Slavin S, McSweeney P, Storb R. Mini-allografts: ongoing trials in humans. Bone Marrow Transplant 2000; 25:345–350. 94. Spitzer TR. Nonmyeloablative allogeneic stem cell transplant strategies and the role of mixed chimerism. Oncologist 2000; 5:215. 95. Storb RF, Champlin R, Riddell SR, Murata M, Bryant S, Warren EH. Non-myeloablative transplants for malignant disease. Hematology 2001; 2001:375.

10 Partially Mismatched Related Donor Transplantation Kuang-Yueh Chiang Pediatric Blood and Marrow Transplant Program, Aflac Cancer Center and Blood Disorders Service, Children’s Healthcare of Atlanta, Emory University, Atlanta, Georgia, U.S.A.

P. Jean Henslee-Downey EMD Pharmaceuticals, Inc., An Affiliate of Merck KGaA, Darmstadt, Germany and Durham, North Carolina, U.S.A.

Kamar T. Godder Division of Pediatric Hematology Oncology, Virginia Commonwealth University, Medical College of Virginia Campus, Children’s Medical Center, and Stem Cell Transplantation, Richmond, Virginia, U.S.A.

BACKGROUND Replacement of damaged, defective, or diseased hematopoietic and immune systems by hematopoietic progenitor cells, through a process of transplantation, has become the treatment of choice for many congenital and acquired diseases. Unfortunately, only 25% of patients who may benefit from hematopoietic stem cell transplantation (HSCT) have a genotypically identical sibling available. When a human leukocyte antigen (HLA)–matched related donor cannot be identified, an alternative donor becomes the only available option. Despite having more than eight million volunteers in the international registries network, and increasing numbers of umbilical cord blood banked units, many patients needing HSCT still cannot identify an appropriate donor within a reasonable period of time. In general, ethnic groups other than Caucasians are poorly represented in the registries. Moreover, in some nonCaucasian ethnic groups, the probability of finding a matched donor is even smaller due to greater degree of HLA polymorphism within these populations (1–4). Unrelated banked cord blood units represent similar ethnic groups as the adult unrelated donors regestrip but can be used with a lesser degree of HLA match. Nevertheless, because of the size of the graft it cannot be used in heavy or older children. Partially mismatched related donor transplantation (PMRD-HSCT) remains the only therapeutic option available to most recipients thus has been the focus of interest for many researchers. In this chapter we will review the challenges of decreasing the risk of graft-versus-host disease (GVHD), relapse, and transplant-related mortality (TRM) while avoiding graft rejection in partially mismatched related donor transplantation. 201

202

Chiang et al.

TERMINOLOGY OF PARTIALLY MISMATCHED RELATED DONOR (HAPLO-IDENTICAL) TRANSPLANT The terms PMRD or haplo-identical donor applies to any related donor who shares only one HLA-haplotype with the recipient. On the unshared haplotype, there may be dissimilarities in one, two or all of the three major HLA loci (A, B, and DR). Mismatch can either be in the donor (rejection) or the host (GVHD) direction. Mismatch in the rejection direction will occur when there are homozygous antigens (Ag) of one or several HLA loci found on host tissues. The donor will recognize both homozygous Ags and will not react against them, whereas the recipient will recognize one of the heterozygous Ags of the donor as nonself and potentially induce rejection. The reverse will happen when there are homozygous Ags on donor tissues (GVHD or host direction), where there will be a reduced risk of rejection but increased stimulation from the heterozygous Ag mismatch in the recipient, which may increase the risk of GVHD (Table 1). Approximately 5–10% of patients have a relative with only a single HLA-locus mismatched, another 25% have a two Ag mismatched (Ag MM) relative and almost everybody has a three Ag MM donor within the family; specifically children and parents are at least haploidentical. It is also more likely that a genetically haploidentical related donor who shares three of six major HLA Ags will share other chromosomes that may express minor histocompatible Ags, than a random unrelated donor. Because earlier studies using PMRD donors reported serological typing only, it is possible that some of the one Ag MM donors had a higher degree of HLA disparity that now could be detected using current high-resolution techniques. Nonetheless, the results of these studies may have been interpreted based on incomplete information that would have suggested that donor-recipient pairs were less mismatched than they were in reality. In the 1980s it became clear that mismatched related donor transplant was feasible and could result in long term disease-free-survival (DFS) in acute leukemia patients; however, regimen-related toxicity was felt to be greater than what was expected with sibling donor transplants (5–7). The first successful haplo-identical attempt in children was in patients with severe combined immune deficiency (SCID). Reisner and O’Reilly, using T-cell depletion (TCD) with soybean agglutinin and sheep red cell rosetting, successfully achieved engraftment and long-term survival with partial restoration of the immune system in those patients (8–10). Since their original work, this modality has been extended to other disease entities and other approaches to transplant have been developed.

ALLOREACTIVITY Two unique and complimentary networks are involved in the human immune system. The cells in the innate system [such as granulocytes, macrophages, natural killer (NK) cells] are predetermined and do not undergo rearrangement, unlike the adaptive system, such as T cells and B cells, which is capable of recognizing different Ags through rearrangement of its receptor Table 1 Examples of Human Leukocyte Antigen Mismatch in the Graft-Versus-Host Disease (Host) Direction and in the Rejection (Donor) Direction Antigen mismatch Antigen mismatch rejection GVHD (host) direction (donor) direction Host Donor

AxAy AxAz

BxBy BxBz

DRxDRy DRxDRz

3 Ag

Host Donor

AxAx AxAz

BxBy BxBz

DRxDRy DRxDRz

2 Ag

Host Donor

AxAy AxAx

BxBy BxBx

DRxDRy DRxDRx

3 Ag

Abbreviations: GVHD, graft-versus-host disease; Ag, antigen.

3 Ag 3 Ag 0 Ag

Mismatched Related Donor Transplantation

203

Table 2

Human Natural Killer Immunoglobulin-Like Receptors and Their Ligands

Receptor

Ligand

KIR3DL2 KIR3DL1 KIR2DL1 KIR2DL2 KIR2DL3

HLA-A (A3, A11) HLA-B (Bw4) HLA-Clys80 (Cw2,4,5,6)-group 2 HLA-Casn80 (Cw1,3,7,8)-group 1 HLA-Casn80 (Cw1,3,7,8)-group 1

Abbreviations: asn, asparagine; KIR, killer immunoglobulin-like receptors; HLA, human leukocyte antigen; lys, lysinc.

genes (11). In the allogeneic mismatched setting, donor and recipient Ag disparity increase the risk of developing GVHD and/or host-versus-graft alloreactivity mediated by T cells. In recent years, NK cell reactivity was also recognized as a major factor in HLA matching. This reactivity has additional relevance in HLA-mismatched transplantation because NK cells express inhibitory receptors that structurally mimic immunoglobulin receptors and are termed killer immunoglobulin-like receptors (KIR). The KIR react to specific ligands on target cells controlled by HLA class I Ags (12). The structure of the KIR molecule is organized by the number of extracellular domains (2D or 3D) and the length of the cytoplasmic tail (L for long, S for short) (13). There are two dominant ligands on HLA-C that are different by a single amino acid (asparagine vs. lysine) at position 80 (14). The different KIR and its ligand are illustrated in Table 2 (12,15,16). Ruggeri and coworkers first reported the effect of mismatched KIR between donors and recipients (in the GVHD direction) on avoiding leukemia relapse and protecting against GVHD (17). Since the initial observation, several analyses have yielded conflicting outcomes with respect to KIR mismatches (Table 3). The exact role KIR ligand incompatibility plays in transplantation remains an area of active investigation. Table 3

Outcomes of Killer Immunoglobulin-Like Receptors Mismatches in Clinical Studies

Donor type

T-cell depletion

ATG

Outcomes

No

Protected against rejection, GVHD and AML relapse; no effect on ALL No advantage observed

Mismatched related donor

Yes

Mismatched unrelated donor Unrelated donor

Some Yes No

Yes

Unrelated donor

No

Yes

HLA-matched related and unrelated donors

No

No

Unrelated donor

No

Yes

HLA-matched sibling

Yes

Better survival for myeloid malignancies Higher relapse rate Superior long-term antileukemia effect in myeloid malignancies Inferior survival, higher treatment related mortality Increased survival in highrisk AML and MDS; no effect on ALL, CML, or standard-risk AML

Reference Ruggeri L et al. 2002 (18) Davies SM et al. 2002 (19) Giebel S et al. 2003 (20) Bomha¨user M et al. 2004 (21) Beelen DW et al. 2005 (22) Malmberg KJ et al. 2005 (23) Hsu KC et al. 2005 (24)

Abbreviations: ATG, antithymocyte globulin; AML, acute myeloid leukemia, ALL, acute lymphoblastic leukemia; CML, chronic myelogenous leukemia; GVHD, graft-versus-host disease; HLA, human leukocyte antigen; MDS, myelodysplastic syndrome.

204

Chiang et al.

DONOR SELECTION CRITERIA In general, HLA matching between the donor and recipient is the major factor affecting HSCT donor choice. Although data have shown a correlation between donor-recipient HLA mismatch and transplant related complications, including graft failure, regimen related toxicity, and GVHD (5,6,25,26), the risk of these complications was not related to disparity in the class I versus class II loci mismatch (5,6,27). Current studies focus on whether the degree of mismatch correlates with the risk of complications, and on the identification of specific Ag pairs that are more likely to be associated with transplant complications (see KIR discussion). Based on earlier studies showing similar outcomes between transplants from completely matched versus one Ag MM disparate related donors, the practice adopted is to combine these two groups of transplant recipients (6/6 matched and 5/6 matched donor) together in outcome studies (5,25). When greater than 1 Ag mismatch was considered, Szydlo et al. in one of the largest studies using donors other than HLA-identical siblings, showed that TRM was related to the degree of HLA disparity; with fully matched sibling donor (MSD) transplant recipients having a better outcome than one Ag MM transplants, which in turn did better than a two Ag MM transplant recipients. Interestingly, in sub-analysis of patients with early acute leukemia, relapse was not significantly different between recipients of one Ag MM and matched donor transplants, or between recipients of one or two Ag MM donor transplants (26). In studies when three Ag MM PMRD-HSCT are included, there seem to be worse outcomes in patients who received transplants from donors with greater than 2 Ag MM (25,28). These findings were not confirmed in pediatric studies, possibly because small patient numbers did not have the statistical power to detect a true difference (29,30). In preparation for a PMRD transplant, when multiple donors may have the same degree of HLA match, other factors may play a role in donor selection. In a study from the University of South Carolina using PMRD-HSCT in pediatric patients with acute leukemia, favorable outcome was associated with donors who were younger than 30 years of age regardless of the degree of serological HLA matching or the consanguinity of the donor (parent vs. sibling) (29). Similar findings were observed in adult leukemia patients from the same institution as well as in a report from the IBMTR, where using younger donors was associated with a decreased risk of GVHD and transplant related complications (5,28,31). This factor cannot be objectively tested in studies utilizing peripheral blood stem cell (PBSC) transplants, where the logistics of cell collection and its yield favor an adult donor. Based on the hypothesis that long-term microchimerism of fetal or maternal cells in the donor may represent acquired immunological hyporesponsiveness to noninherited maternal HLA Ags (NIMA), investigators analyzed data from the Japanese society for hematopoietic cell Table 4 Donors

Recommended Criteria for Donor Selection When Using Partially Mismatched Related

Youngest possible donor as long as it does not compromise cell yield Least degree of major HLA mismatch in donor (rejection) direction Sibling preferred to parental donor Avoid recipient-donor cross-match positivity Same sex donor CMV negative donor for a CMV negative recipient Minimal donor exposure risk factors (per FACT guidelines) Minimal risk to the donor, physical or emotional Emerging criteria for consideration Donor sharing non-inherited maternal antigens Consider KIR matching Abbreviations: CMV, cytomegalovirus; FACT, Foundation for the Accreditation of Cellular Therapy; HLA, human leukocyte antigen; KIR, killer immunoglobulin-like receptors.

Mismatched Related Donor Transplantation

205

transplantation registry (32,33). They showed that in patients who received T-cell replete bone marrow or PBSC from an HLA haploidentical family donor, the most significant factor predicting a low risk of GVHD was NIMA mismatch in the recipient (GVHD direction). Similar findings were reported from a retrospective IBMTR analysis of 269 recipients of sibling T-replete PMRD-HSCT and in mismatched cord blood transplantation (31,34). In addition to other medical considerations, PMRD donors by virtue of being family members (often parents) are usually enthusiastic and willing. The choice of donor should consider immediate availability, without any compromise to the donor, either physically or emotionally, as well as future accessibility for cell collection procedures for donor-derived cell therapy. In this respect, PMRD donors are excellent potential donors. Based on current information, a recommended guideline for donor selection is shown in Table 4, which will undoubtedly be updated as new data become available (6,35,36).

METHODS TO CROSS MAJOR-HUMAN LEUKOCYTE ANTIGEN BARRIERS The optimal strategy to achieve engraftment across HLA barriers should include effective suppression of the host to allow engraftment, while minimizing transplant-related complications so that prompt immune reconstitution will protect the recipient from infection and relapse of the primary disease. Other considerations may be cost, complexity, and reproducibility of the transplant regimen. Multiple approaches have been developed to cross major HLA barriers (Table 5) and will be discussed below.

Graft Manipulation T-Cell Depletion (Negative Selection) The observation that there is an increased risk of GVHD in PMRD-HSCT and the recognition of the role of T cells in the pathophysiology of GVHD has led to the development of TCD methodologies (5,6,25). The first successful TCD in a PMRD transplant was achieved in patients with immunodeficiency, using soybean agglutinin and sheep red cell rosetting (8,9). This method alone was insufficient for the treatment of patients with acute leukemia (40) because of the need for additional therapy, resulting in the development of other TCD methods for these diseases (25,27,41–46). In an attempt to sustain engraftment and obtain greater antileukemic effect of the graft, researchers at the University of Kentucky developed a partial TCD methodology, with a specific monoclonal antibody directed against the ab heterodimer of the T-cell receptor (T10B9.1A-31) while sparing the gd receptor, followed by rabbit complement lysis (47,48). The choice of targeting this receptor was based on the finding that abCT cells but not the gdCT cells initiate the GVHD response (49,50). In the clinical setting, this observation was confirmed by the fact that gdCT-cell dose was not associated with the risk of acute or chronic GVHD (51). In order to be able to use a Food and Drug Administration (FDA)–approved product for marrow preparation, the next step in TCD was taken at the University of South Carolina using Orthoclone OKTw3 (Ortho Biotech Raritan, New Jersey). Several reports on the use of T10B9 and then OKT3 demonstrated excellent engraftment and a low risk of GVHD, specifically when pre and post transplant immunosuppression was utilized (29,43,51–54). Godder et al. published the pediatric experience at the University of South Carolina describing the characteristics of T10B9 or OKT3-manipulated grafts showing the median total nucleated cell dose was 1.68 and 1.06! 108/kg, respectively, and the T-cell dose was 10.1 and 4.27!104/kg, respectively (29). No differences were noted in the degree of TCD between OKT3 and published data using soybean agglutination. Other studies had shown that a better nucleated cell yield with less TCD was obtained when using T10B9 compared to OKT3 (28,55,56).

Acute leukemia

ATG cyclosporine steroids

Cyclosporine MMF

Cyclosporine methotrexate Tacrolimus based

Hem malignancy

Hem malignancy

Hem malignancy NMD Hem malignancy

SCID

None

none

Hem malignancy

Disease

Cyclosporine based

Posttransplant immune suppression

Good engraftment low incidence of acute and chronic GVHD Minimal acute and chronic GVHD Good engraftment minimal GVHD Good engraftment GVHD NIMA!IPA Good engraftment minimal GVHD

Engraftment-delayed high incidence of GVHD Engraftment-variable minimal GVHD

Results

Lu et al. (39)

Ichinohe et al. (33)

Guinan et al. (38)

Lang et al. (37)

Godder et al. (29)

O’Reilly et al. (8) Reisner et al. (9)

Beatty et al. (5), Anasetti et al. (6)

Reference

Abbreviations: ARA-C, cytosine arabinoside; Cytoxan, cyclophosphamide; ATG, antithymocyte globulin; PBSC, peripheral blood stem cells; GVHD, graft-versus-host disease; NIMA, noninherited maternal antigens; IPA, inherited paternal antigens; MMF, mycophenolate mofetil; NMD, nonmalignant disorders; G-CSF, granulocyte-colony stimulating factor; SCID, severe combined immune deficiency; TBI, total body irradiation; BM, bone marrow; MPD, methylprednisolone; CTLA-4-Ig, (Repligen, Cambridge, MA).

Busulfan cytoxan ATG

TBI Thiotepa fludarabine rabbit ATG TBIGARA-C cytoxan MPD Multiple regimens

BM and or PBSC G-CSF mobilized

BM soybean agglutination & sheep E rosetting BM partial T-cell depletion with T10B9 or OKT3 PBSC CD34C or CD133C selection BM cocultured & host cells with CTLA-4-Ig BM or PBSC replete

None

TBI Etoposide ARA-C cytoxan

BM replete

TBI Cytoxan

Graft and its manipulation

Published Methods for Crossing Major-Human Leukocyte Antigen Barriers in Pediatric Patients

Conditioning therapy

Table 5

206 Chiang et al.

Mismatched Related Donor Transplantation

207

Whether the TCD technique makes a difference in outcome is almost impossible to unravel because of differences in conditioning therapy, posttransplant GVHD prophylaxis regimens, and other supportive care standards between the studies (28,52,57). Other TCD methodologies have also been used in small numbers of patients.

CD34C Selection (Positive Selection) In the late 1990s, Reisner et al. showed in a mouse model that megadose TCD marrow might overcome HLA barriers (58). Using CD34C selected PBSC, initially with marrow and later without, Aversa et al. were the first to report on 43 adult and pediatric patients with acute leukemia who received large doses of cells from a haplo-identical donor (45). Marrow was TCD with soybean agglutination and e-rosetting, whereas mobilized PBSC were TCD by e-rosetting followed by CD34 selection by immunoadsorbtion using a biotin-avidin column. The entire process took nine hours, and was later changed to positive immune selection using the CliniMacs system. The mean final marrow-derived product contained 26G5.4!106, and the PBSC product contained 14.0G8.7!106 CD34C cells/kg. The use of megadose mobilized and selected PBSC in children first employed positive selection with a semi-automatic magnetic activated cell sorting technique and later was performed with a fully automated selection procedure using the CliniMacs device (59,60). Adult donors underwent mobilization by administration of 10 mcg/kg of granulocyte-colony stimulating factor (G-CSF) daily for five days, and were harvested in one to three procedures. The targeted progenitor cell count was 10!106/kg recipient weight (61,37). Similar to the Aversa study, patients received a mean of 19.5!106 CD34C cells/kg and a mean of 11!103 CD3CT cells/kg recipient weight. CD34C cell dose was shown to be associated with increased speed of neutrophil and platelet engraftment but the source of stem cells (bone marrow vs. PBSCs) was not (37,62,63).

Induction of Anergy An interesting alternative modality was developed by Guinan et al. who explored ex vivo induction of alloantigen-specific anergy in pediatric patients (38). Bone marrow from a mismatched donor was cocultured with irradiated cells from the recipient in the presence of an agent that inhibits B7:CD28 mediated co-stimulation. After the induction of anergy, the frequency of T cells recognizing alloantigens of the recipient decreased, while those which recognized a third party were preserved. Of the 11 evaluable recipients (10 of which were children), all engrafted and only three developed GVHD (64).

GRANULOCYTE-COLONY STIMULATING FACTOR PRIMED BONE MARROW CELLS AND PERIPHERAL BLOOD STEM CELLS This novel approach of marrow stimulation followed by PBSC mobilization, without ex vivo manipulation of the graft, was piloted in China in the treatment of leukemia patients receiving haplo-identical transplant. In a recent update on 135 leukemia patients (51 patients were younger than 20 years of age), the investigators showed comparable outcomes to similar patients who received matched sibling donor (MSD) transplants (39). Most patients received a combined marrow-PBSC graft. Similar results were reported with a small pediatric study using exclusively G-CSF primed marrow (65). The acceptable incidence of GVHD with this approach may be explained by the G-CSF stimulation, enriching the graft for Th2 cells that may modulate type 2 dendritic cells and protect against GVHD (66).

208

Chiang et al.

CONDITIONING OF THE RECIPIENT Any method of TCD using positive or negative selection, is associated with increased risk of engraftment failure (25), and has always required careful consideration of optimal host conditioning. Various methods have been applied, including intensification of pretransplant conditioning (67–70), addition of total nodal irradiation (71,72), adding immunosuppressive agents to the conditioning regimen, such as thiotepa or fludarabine (45,73,74), and immunosuppressive therapy with anti-thymocyte globulin (ATG) (10,29,39,43,64,74,75) or other monoclonal antibodies or immunocytokines (27,44,64,68,76). Unfortunately, any additional therapy may be associated with increased toxicity in this high-risk population. To decrease regimen related toxicity, nonmyeloablative preparation therapy has increasingly been used, mainly in adults, with the results still too early to evaluate.

ENGRAFTMENT The ultimate goal of transplant is to have a competent immuno-hematopoietic recovery. Partial engraftment of the donor cells resulting in a mixed donor/recipient chimerism may be adequate for correction of nonmalignant conditions, such as severe combined immunodeficiency or hemoglobinopathies; however, for malignant diseases, full donor engraftment is required. In mismatched transplantation, engraftment can be hindered by HLA disparity, suboptimal suppression of the recipient’s immune system, inadequate donor hematopoietic progenitor cell (HPC) dose, graft manipulation, or combinations of any of the above (52,63,64,72,76). To prevent graft rejection two therapeutic approaches have been studied; increasing the intensity of pretransplant conditioning and enriching the stem cell content of the graft. Increasing conditioning intensity improved engraftment in the majority of patients who received PMRD TCD grafts (67,77). To improve the probability of engraftment above the 88% achieved with previous studies (43), investigators at the University of South Carolina treated PMRD-HSCT recipients with TCD grafts, increased the total body irradiation (TBI) dose and added equine ATG, both during conditioning and early following graft infusion. These changes resulted in an engraftment rate of 91% and 97% when using T10B9 and OKT3, respectively (69). Similarly, the Perugia group observed a marked benefit when they added the more potent rabbit antithymocyte globulin and thiotepa to the conditioning regimen improving the primary engraftment rate to 94% (78,79). The use of stem cells to pave the way for engraftment has been pursued through the combined work of Reisner, Martelli, and Aversa et al. Using a different approach, researchers at Perugia University in Italy introduced the concept of megadose CD34C cells in PMRD-HSCT. In their initial approach, both bone marrow cells and G-CSF-stimulated PBSC were combined. Patients were prepared with a single-dose TBI—based conditioning, including rabbit ATG, resulting in 80% primary engraftment (45). In subsequent studies, several steps were modified, including replacing cyclophosphamide with fludarabine in the preparative regimen, and positive immunoselection of CD34C cells from peripheral blood stem cell product using the CliniMacs system. This has improved the primary engraftment rate to 94% with a minimal change in mean CD34C cell dose (from 10!106/kg to 12!106/kg) (79). The immunoregulatory effect of megadose CD34C cells (and their progeny) reduces the frequency of cytotoxic T-lymphocyte precursors against the stimulator cells of the same origin, but not from the cells of third-party origin. This was very similar to what has been observed for veto cells. These immunoregulatory activities require cell contact, are mediated by apoptosis, and can be blocked by irradiation (80,81). Experience with PMRD-HSCT in the pediatric population is often combined with the adult experience. Very few studies have reported on pediatric results separately. Peters and co-worker from Vienna reported their experience in 14 pediatric patients who received a large cell dose of CD34C cells purified from PBSC using two different magnetic bead devices.

Mismatched Related Donor Transplantation

209

Ten of the fourteen patients (71%) demonstrated primary engraftment (82). Researchers at the University of Tubingen updated their results on 63 pediatric patients who underwent 1-3 HLA loci mismatched transplants from parental donors. The graft source was PBSC followed by either CD34C or CD133C positive selection. Primary engraftment was achieved in 83% of patients and an additional 15% engrafted with a second transplant for a combined engraftment rate of 98% (37). A primary engraftment rate of 97% was reported by Godder et al. using monoclonal antibody (T10B9 or OKT3) and complement for TCD of bone marrow grafts, in 67 pediatric patients receiving PMRD-HSCT (29). Although results of the different approaches are similar, it is important to realize that condition regimens, graft source, manipulation methods and both stem cell and T-cell dose play a role in the success of establishing donor cell engraftment in HLA-mismatched donorrecipient pairs.

GRAFT-VERSUS-HOST DISEASE AND OUTCOMES The other major obstacle in HLA mismatched transplant is the development of GVHD induced by alloreactive T cells. Table 6 summarizes the incidence of GVHD and other outcomes from the two major approaches of TCD (i.e., CD34C selection with no posttransplant GVHD prophylaxis and partial TCD with sequential GVHD prophylaxis post transplant) (28,63). Table 7 presents the two largest published pediatric patient studies in PMRD-HSCT. Each study illustrates unique observations in these studies’ experiences. In their report, Lang and coworkers used ATG and/or steroids with OKT3 for four weeks in the peri-transplant period for rejection prophylaxis. They also reported a significant incidence of lethal viral or fungal infections in the first six months posttransplant. However, the incidence of infection declined markedly in the last two years using preemptive monitoring (37). Godder et al. exploring factors associated with improved outcomes; showed a relationship between younger donor age (!30 years), and the absence of peripheral blasts (in relapsed patients) with an improved survival rate (29). Disease status at the time of transplant has been shown to be a major factor predicting outcome, particularly in mismatched transplants, where most of the patients have an advanced disease state. Goldman reviewed the experience at the University of Iowa, concluding that transplant utilizing mismatched family member donors is a poor option for patients in relapse at the time of transplant (53). Lang et al. also observed that none of the ALL/NHL patients with active disease survived (37). However, a more promising outcome was reported by Godder et al. in a subset of patients transplanted in relapse who had no circulating blasts at the time of transplant (29). Moreover, her group showed that patients who received a second transplant or those with AML induction failure were salvageable with PMRD transplant, and had comparable survival rate with those who received transplants from other donors, including matched sibling or matched unrelated donors (MUD) (83,84). Researchers from Beijing, China (85) combined G-CSF stimulated bone marrow and PBSC without ex vivo TCD, from HLA 1–3 Ag MM family donors. The combination of cyclosporine, mycophenolate and ATG was used as GVHD prophylaxis. Grade II–IV acute GVHD developed in 38% of the patients and chronic GVHD in 65%. The two-year DFS was 75% and 69% for standard- and high-risk patients, respectively. G-CSF has been widely accepted as part of the supportive care regimen to hasten WBC recovery after chemotherapy. Its use in HSCT has been controversial. G-CSF has been shown to promote the development T-helper type 2 cells, which may theoretically result in a lower incidence of acute GVHD (86). A recent report from the acute leukemia working party of the European group for blood and marrow transplantation (87) examined transplants from HLA matched siblings using either bone marrow (1789 patients) or PBSC (434 patients). In contrast to the above data, the study concluded that G-CSF should not be used shortly after BMT

210 Table 6

Chiang et al. Clinical Outcomes of Mismatched Related Donor Transplants

# of patients Median age (years) % not in remission 2–3 HLA loci mismatches Conditioning regimen Graft source Graft processing Graft (median)/kg recipient Primary engraftment GVHD prophylaxis Acute GVHD (II–IV) Chronic GVHD (limited & extensive) Nonrelapse mortality

Leukemia relapse

Event-free survival

Aversa F et al. 2005 (63)

Mehta J et al. 2004 (28)

104 (Jan 1999 to April 2004) 33 37 100% TBICchemoCrabbit ATG PBSC CliniMacs (CD34C selection) CD34C13.8!106/kg; T cell 1!104/kg 91% None

201 (Feb 1993 to Dec 1999) 23 67 92% TBICchemoCequine ATG Bone marrow T-cell depletion (T10B9 or OKT3)

8% 7% 37% (remission patients) at 2 years; 44% (relapsed patients) at 2 years 16% (remission patients) at 2 years; 51% (relapsed patients) at 2 years 47% (CR patients) at 2 years; 4% (relapsed patients) at 2 years

Total nucleated cells 1!108/kg; T cell 5!104/kg 98% SteroidCATGCCSA, Cyclosporine 13% 15% 51% (mostly relapsed patients) at 5 years 31% (mostly relapsed patients) at 5 years 39% (CR patients with young donor & 2 loci mismatches); 18% (mostly relapsed patients) at 5 years

Abbreviations: HLA, human leukocyte antigen; TBI, total body irradiation; ATG, antithymocyte globulin; PBSC, peripheral stem cell; GVHD, graft-versus-host disease; CR, complete remission; CSA, cyclosporine.

because it delayed platelet engraftment, increased TRM and GVHD, and reduced overall survival and leukemia-free survival. Similar findings have been reported in PMRD-HSCT population. When G-CSF was not given posttransplant, CD4C recovery occurred significantly earlier without an adverse effect on engraftment or the incidence of GVHD (88). This has the potential to reduce TRM and morbidity from infections (88). To reduce the risk of complications associated with TCD in PMRD-HSCT, investigators explored the possibility of T-cell add-back, late after transplant, when the risk of GVHD due to cytokine storm was reduced. To avoid development of GVHD with the infusion of T cells, various techniques have been applied. Amrolia et al. used recipient Epstein-Barr Virus (EBV) transformed lymphoblastoid cell line (LCL) as stimulators of donor alloreactive T cells and have demonstrated the feasibility of adding back the allo-LCLdepleted haplo-identical donor T cells (89). They showed that a dose as low as 3!105 cells/kg was sufficient to provide useful antiviral immunity. Researchers in Perugia selected specific donor T-cell clones against Aspergillus or cytomegalovirus Ags and removed these clones with cross-reactivity against recipient mononuclear cells (90). These donor alloantigendepleted, pathogen-specific CD4C cells produced high levels of interferon-gamma and low levels of interleukin-10. Dose ranges of 105 to 106 cells/kg have been infused to patients receiving haplo-identical transplantation without causing acute GVHD. T-cell add back has also been used to prevent relapse, but no conclusive data on pre-emptive therapy has been published to date.

Mismatched Related Donor Transplantation Table 7

211

Clinical Outcomes of Mismatched Related Donor Transplants in Pediatric Patients Lang P et al. 2004 (37)

# of patients Median age (years) % not in remission 2–3 HLA loci mismatches Conditioning regimen Graft source Graft processing Graft (median)/kg recipient T-cell dose (median)/kg recipient Posttransplant immune suppression

Acute GVHD (II–IV) Chronic GVHD (limited & extensive) Event-free survival

63 (10 nonmalignant) (1995 to 2003) 9 20 89% Chemo C/KTBIC ATGC/KOKT3 PBSC CliniMacs (CD34C or CD133C selection) 19.5!106/kg (CD34C cell) 1.1!104/kg None, however; all patients received antibody preparations (ATG and/or OKT3) as rejection prophylaxis 7% 13% ALL, acute lymphoblastic leukemia 48% (remission patients); ALL/NHL, non-Hodgkin lymphoma 0% (relapsed patients) at 3 years; AML: 18% at 3 years; non-malignancy: 60% at 3 years

Godder K et al. 2000 (29) 67 (ALL & AML) (Feb 1993 to May 1997) 8.3 61.2 73% TBICchemoC/Kequine ATG Bone marrow T-cell depletion (T10B9 or OKT3) T10B9: 1.68!108/kg OKT3: 1.06!108/kg (Total nucleated cell) T10B9: 10.1!104/kg OKT3: 4.27!104/kg SteroidCATGCCSA

24% 37.8% ALL similar to AML: 26% (mostly relapsed patients) at 3 years; 45% (mostly relapsed patients), with donors !30 years, at 3 years

Abbreviations: AML, acute myeloid leukemia; HLA, human leukocyte antigen; TBI, total body irradiation; ATG, anti-thymocyte globulin; PBSC, peripheral stem cell; GVHD, graft-versus-host disease; ALL, acute lymphoblastic leukemia; NHL, non-Hodgkin lymphon.

INFECTION AND IMMUNE RECONSTITUTION HLA disparity, TCD, and posttransplant immune suppression; all contribute to delayed immune reconstitution and increase the risk of fatal infections after PMRD-HSCT, specifically viral infections and aspergillus (37,61,91). In the Lang report, the actual risk of lethal infections was 16% for viral and 6% for fungal infections, whereas in the Godder study, infection was a cause of death in 12% of patients (6% died of sepsis, 3% of viral infections and 3% of fungal infections). Immune reconstitution after PMRD-HSCT was studied by several groups that tracked lymphocyte subset recovery. Similar to recovery post HLA-matched sibling transplantation, the initial population of donor-derived lymphocytes (seen as early as 14 days post transplant) was comprised mainly of NK cells. T lymphocytes (CD3C) mostly consisted of CD8C cells, appeared around day C180, whereas CD4C numbers and the CD4:CD8 ratio remained depressed for up to two years post transplant. B cells (CD19C, CD20C) recovered after 6–12 months (92,93). To study the role of TCD in immune reconstitution, Keever et al. compared immune recovery between recipients of HLA-matched TCD transplants to those of HLA-matched T-cell replete transplants showing no qualitative differences between the two populations other than a delay in the recovery of T-cell proliferation responses in the TCD

212

Chiang et al.

group (94). Possible factors resulting in superior immune recovery include younger patient age, higher marrow cell or CD34C cells dose, the absence of GVHD and GVHD treatment, and the absence of CMV infection (37,92–96). The obvious way to prevent infectious complications in these high-risk patients is to use prophylaxis against the major infectious organisms (aspergillus and other fungi, viruses, specifically CMV, and Pneumocystis carinii) combined with other supportive measures for the management of HSCT recipients. Because of the high risk of opportunistic infections, patients should undergo a thorough surveillance for viruses (EBV, CMV, adenovirus, HHV6, etc.) and fungal infections, specifically aspergillus. Patients should also be monitored closely for subtle signs of infection and be started promptly on empiric anti-infectious therapy. Indeed, with the ready availability of PCR for viral detection, and with preemptive therapy, the incidence of infection has declined markedly over the last two years (60). Another approach to enhance immune reconstitution is the use of donor lymphocyte infusions (DLI). Treatment with DLI is limited by the increased risk of GVHD due to the relatively high numbers of alloreactive compared to antiviral T cells in the donor peripheral blood. A promising approach is TCD of DLI (with anti-CD25C, CD69C, CD71C and HLA-DR), which preserves cell populations capable of responding to adenovirus, CMV Ags, and candida (97,98). Other investigators have developed clones against Aspergillus and cytomegalovirus Ags (90). Epstein-Bar Virus associated lymphoproliferative disease (EBV-LPD) is a welldocumented complication of primary and secondary immune deficiency. HLA disparity, TCD, and posttransplant immune suppression contribute to the increased risk of EBV-LPD following PMRD transplants. In general, the intensity of TCD is correlated with the increased risk of EBV infection (99). With partial TCD, the estimated probability was 13%, increasing to 22% in patients with multiple risk factors (99,100). B-cell depletion when combined with TCD may prevent this complication (101).

COMPARISON BETWEEN ALTERNATIVE DONOR TRANSPLANTS Despite increasing numbers of transplant patients, and the increased use of alternative donors for HSCT, no study to date has randomized patients to receive a PMRD-HSCT versus other types of alternative donor transplants. Some of the difficulty is in the limited number of institutions that perform PMRD transplants, perhaps because of lack of the resources needed for graft manipulation, or the concern of increased transplant-related complications, despite evidence that the risk when using unrelated donors is as high or higher. To date, PMRD transplants have usually been used as the “last resort” for the sicker patients (29,37,53), a different patient population from the one that undergoes unrelated adult or cord blood transplant. This makes it difficult to objectively assess and compare alternative donors for allogeneic transplant. Other than the initial studies, which recognized the increased risk of GVHD, and the need for graft manipulation, very few studies have compared PMRD-HSCT to other donor transplants. Fleming et al. compared 32 pediatric acute lymphoblastic leukemia patients who received PMRD- HSCT to 16 receiving MSD transplants and showed no difference in disease free survival at 6 years (102). Similar findings were seen in the study from the Tubingen/St Jude’s transplant team, where DFS for ALL/NHL PMRD transplant recipients in remission was similar to historical controls who received matched unrelated donor (MUD) transplants. Moreover, the three-year probability of relapse in all patients with malignancy who were in remission at time of transplant was similar to that of MUD recipients (40% vs. 37%, respectively) (37). Drobyski et al. reporting on the Milwaukee experience, showed no difference in the incidence of GVHD or engraftment between patients who received phenotypically mismatched unrelated donor and PMRD HSCTs. In his study, which included pediatric and adult patients, overall survival was also equal between PMRD and mismatched unrelated donor transplants but inferior to matched MUD transplant, due to TRM (103).

Years of study

262 (61%) 31 (23%) 26 (58%)

R T R

U U

Eurocord PBMTC

1994–2001 1995–1996

U U

1994–1999

76 (17%) 52 (44%) 180 (74%)

T T T

1990–2001 1990–1996 1994–1998

60 (30%) 26 (58%) 31 (23%) 550 (43%) 44

28 (0)

R (T 3)

1990–1997

50 (64%) 59 (21%)

T R

137 (18%) 50 (44%) 88 (63%)

# Pts (% HR)

1986–1991 1988–1998

T R (T 8) R

GS

Unrelated cord blood Eurocord 1990–1997 Minnesota 1994–1999

Minnesota

Milwaukee Karolinska institute Nordic Soc of Peds Hem/Onc CHOP St. Jude Eurocord centers

Unrelated adult donor Bristol 1988–1997 Minnesota 1984–1994 Seattle 1985–1993

Center

All 9 19 478 29

17 12

All All All

All

All 38

All All 84

0 17 12 22 15

14 14

0 0 0

0

0 21

0 0 4

Malignant Nondisease malignant

33 days 29 days 27 days w30 days 21 days

18 days 14 days 22 days

18 days 20 days 16 days

n/a

18 days 16 days

15 days 24 days 21 days

Median time to WBC engraftment

37% 42% 36% 36% 44%

58% 35% 35%

34% 22% 20%

64%

33% 28%

20–23% 49–67% 83–98%

GVHD grade II–IV

34% 53%a 52%a 34%a 43%

43% 56%a 41%a

39% 73% 37%

54%

40–45% 30–33% 10–75% depending on Dx 44% 52%a

EFS (3 years in most studies)

(Continued)

Gluckman et al. (114) Yu et al. (115)

Locatelli et al. (113) Barker et al. (104)

Barker et al. (104)

Bunin et al. (54) Hongeng et al. (112) Rocha et al. (105)

Casper et al. (109) Gustafsson et al. (110) Saarinen-Pihkala et al. (111)

Green et al. (106) Davies et al. (107) Balduzzi et al. (108)

Reference

Table 8 Pediatric Studies on the Outcomes of Patients Receiving Transplants from Alternative Donors: Unrelated Adult Donor, Unrelated Cord Blood and Partially Mismatched Related Donor

Mismatched Related Donor Transplantation 213

Years of study

GS

1995–2003

T

All All

28 (14%) 67 (64%)

57

All

32b

62 (34%)

All

39 (59%)

# Pts (% HR)

5

0 0

0

0

Malignant Nondisease malignant

11 days w G-CSF

18 days 17 days

31 days

27 days

Median time to WBC engraftment

7%

35% 24%

46%

38%

GVHD grade II–IV

36% 26%; 45% when donor !30 years 0–48% Malignant; 60% Nonmalignant

28%

49%

EFS (3 years in most studies)

Lang et al. (37)

Bunin et al. (54) Godder et al. (29)

Wall et al. (117)

Ohnuma et al. (116)

Reference

b

Overall survival, when citation did not indicate risk group, high risk was defined as per IBMTR criteria (26). The authors’ high-risk group differs from the definition of IBMTR. Abbreviations: GS, graft source; RC, related cord blood; U, unrelated cord blood; R, unrelated donor T-cell replete; T, unrelated donor T-cell depleted; PMRD, partially mismatched related donor; GVHD, graft-versus-host disease; COBLT, cord blood transplantation; G-CSF, granulocyte colony stimulating factor; IBMTR, International Bone Marrow Transplant Registry; HR, high risk; WBC, white blood cell; EFS, event free survival; PBMTC, Pediatric Blood and Marrow Transplant Consortium; CHOP, Children’s Hospital of Philadelphia.

a

Tubingen/St Jude

1997–2000 U Kanagawa Cord Blood Bank COBLT 1999–2002 U Partially Mismatched Related Donor CHOP 1990–2001 T South 1993–1997 T Carolina

Center

Partially Mismatched Related Donor (Continued)

Table 8 Pediatric Studies on the Outcomes of Patients Receiving Transplants from Alternative Donors: Unrelated Adult Donor, Unrelated Cord Blood and

214 Chiang et al.

Mismatched Related Donor Transplantation

215

Age was a significant factor in outcome, with pediatric patients generally doing better, although the pediatric population was not analyzed separately. Equal survival with increased risk of TRM in patients receiving PMRD versus unrelated donor was also reported by Bunin et al. (54). The availability of unrelated umbilical cord blood units has expanded the alternative donor pool and has increasingly been used in children. However, the small nucleated cell dose in cord blood units still limits its use in older children. Moreover, when using unrelated cord blood, there is no access to the donor for additional cells that can be used for cellular therapy aimed against infection or relapse. Several pediatric reports have shown comparable outcomes in recipients of unrelated adult donor and unrelated cord transplants, vis-a`-vis the risk of GVHD, DFS, and probability of overall survival (104,105). Nevertheless, cord blood transplant was associated with an increased risk of graft failure, and longer time to engraftment in those who did engraft. No study has compared the outcome of PMRD and cord blood transplant. Table 8 lists the major trials using alternative donor transplant in children. The least-explored option for patients who receive PMRD-HSCT is autotransplant. Obviously this modality is reserved for patients with malignancies in remission only. In pediatric patients, the use of autotransplant for acute leukemia was abandoned despite promising results (118,119). Only one study, including pediatric and adult patients, has compared PMRD transplant outcomes with outcomes of patients who received autotransplants for AML. This study showed, as expected, increased mortality in the allogeneic transplant patients; however most patients in the PMRD group were in relapse, whereas most of the autotransplant patients were in remission (120). Taken together, for the pediatric patient in need of transplant today, high resolution typing of the patient and the immediate family should be performed. If no matched related donor is identified, searches for an unrelated donor and cord blood should be initiated. Depending on patient’s clinical status, resources and donor availability, a PMRD donor should be considered. A randomized multi-institution trial—including the three allogeneic sources of stem cells, banked unrelated cord blood units, unrelated donor, and PMRD—is much needed.

SUMMARY The availability of PMRD-HSCT offers hope to patients who require allogeneic HSCT to cure their underlying diseases. Significant advancements have been accomplished in the last 20 years, including achieving sustained donor engraftment, an acceptable risk of GVHD, and long-term DFS. With the improvement in controlling and managing transplant complications, pediatric patients should be referred for PMRD-HSCT earlier in their disease state, before they develop organ damage that may hamper the outcome of transplant. For the patient who does not have an HLA-MSD, the use of an alternative, haploidentical donor is a valid option. To assess the role of the different alternative donor transplants, a comparative trial should be pursued. New considerations in donor selection, such as the potential for microchimerism in the recipient and KIR mismatching, should be further explored. Manipulation of graft sources (such as G-CSF-stimulated bone marrow and/or PBSC), selective depletion of alloreactive T cells (such as photodynamic cell purging) from the graft, modification of preparative regimen (such as reduced-intensity transplants), and sequential add-back of specific T cells (such as alloantigen-depleted, pathogen-specific, or leukemia-specific) post graft infusions are important cornerstones to pave the future successes of PMRD-HSCT.

REFERENCES 1. Laver JH, Hulsey TC, Jones JP, et al. Assessment of barriers to bone marrow donation by unrelated African-American potential donors. Biol Blood Marrow Transplant 2001; 7:45–48.

216

Chiang et al.

2. Kollman C, Abella E, Baitty RL, et al. Assessment of optimal size and composition of the U.S. national registry of hematopoietic stem cell donors. Transplantation 2004; 78:89–95. 3. Beatty PG, Mori M, Milford E. Impact of racial genetic polymorphism on the probability of finding an HLA matched donor. Transplantation 1995; 60:778–783. 4. Ballen KK, Hicks J, Dharan B, et al. Racial and ethnic composition of volunteer cord blood donor; comparison with volunteer unrelated marrow donor. Transfusion 2002; 42:1279–1284. 5. Beatty PG, Reginald AC, Mickelson EM, et al. Marrow transplantation from related donors other than HLA-identical siblings. N Engl J Med 1985; 313:765–771. 6. Anasetti C, Amos D, Beatty PG, et al. Effect of HLA compatibility on engraftment of bone marrow transplants in patients with leukemia or lymphoma. N Engl J Med 1989; 320:197–204. 7. Powels SL, Kay HEM, Clink HM, et al. Mismatched family donors for bone-marrow transplantation as treatment for acute leukemia. Lancet 1983; 1:612–615. 8. O’Reilly RJ, Kapoor N, Kirkpatrick D, et al. Transplantation of hematopoietic cells for lethal congenital immunodeficiencies. Birth Defects Orig Artic Ser 1983; 19:129–137. 9. Reisner Y, Kapoor N, Kirkpatrick D, et al. Transplantation for severe combined immunodeficiency with HLA-A, B, DR incompatible parental marrow cells fractionated by soybean agglutinin and sheep red blood cells. Blood 1983; 61:341–348. 10. Friedrich W, Goldmann SF, Vetter U, et al. Immunoreconstitution in severe combined immunodeficiency after transplantation of HLA-haploidentical, T-cell-depleted bone marrow. Lancet 1984; 1:761–764. 11. Caligiuri MA, Velardi A, Scheinberg DA, et al. Immunotherapeutic approaches for hematologic malignancies. Hematology 2004:337–353. 12. Moretta A, Moretta L. HLA class I specific inhibitory receptors. Curr Opin Immunol 1997; 9:694–701. 13. Parham P, McQueen KL. Alloreactive killer cells: hindrance and help for hematopoietic transplants. Nat Rev Immunol 2003; 3:108–122. 14. Colonna M, Brooks EG, Falco M, et al. Generation of allospecific natural killer cells by stimulation across a polymorphism of HLA-C. Science 1993; 260:1121–1124. 15. Gumperz JE, Litwin V, Phillips JH, et al. The Bw4 public epitope of HLA-B molecules confers reactivity with natural killer-cell clones that express NKB1, a putative HLA receptor. J Exp Med 1995; 181:1133–1144. 16. Colonna M, Samaridis J. Cloning of immunoglobulin-superfamily members associated with HLAC and HLA-B recognition by human natural killer cells. Science 1995; 268:405–408. 17. Ruggeri L, Capanni M, Casucci M, et al. Role of natural killer cell alloreactivity in HLAmismatched hematopoietic stem cell transplantation. Blood 1999; 94:333–339. 18. Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295:2097–2100. 19. Davies SM, Ruggeri L, DeFor T, et al. Evaluation of KIR ligand incompatibility in mismatched unrelated donor hematopoietic transplants. Blood 2002; 100:3825–3827. 20. Giebel S, Locatelli F, Lamparelli T, et al. Survival advantage with KIR ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors. Blood 2003; 102:814–819. 21. Bomha¨user M, Schwerdtfeger R, Martin H, et al. Role of KIR ligand incompatibility in hematopoietic stem cell transplantation using unrelated donors. Blood 2004; 103:2860–2861. 22. Beelen DW, Ottinger HD, Ferencik S, et al. Genotypic inhibitory killer immunoglobulin-like receptor ligand incompatibility enhances the long-term anti-leukemic effect of unmodified allogeneic hematopoietic stem cell transplantation in patients with myeloid leukemias. Blood 2005; 105:2594–2600. 23. Malmberg KJ, Schaffer M, Ringde´n O, et al. KIR-ligand mismatch in allogeneic hematopoietic stem cell transplantation. Mol Immunol 2005; 42:531–534. 24. Hsu KC, Keever-Taylor CA, Wilton A, et al. Improved outcome in HLA-identical sibling hematopoietic stem cell transplantation for acute myelogenous leukemia (AML) predicted by KIR and HLA genotypes. Blood 2005; 105:4878–4884. 25. Ash RC, Horowitz MM, Gale RP, et al. Bone marrow transplantation from related donors other than HLA-identical siblings effect of T cell depletion. Bone Marrow Transplant 1991; 7:441–452. 26. Szydlo R, Goldman JM, Klein JP, et al. Results of allogeneic bone marrow transplants for leukemia using donors other that HLA identical donors. J Clin Oncol 1997; 15:1767–1777. 27. Soiffer RJ, Mauch P, Fairclough D, et al. CD6CT cell depleted allogeneic bone marrow transplantation from genotypically HLA nonidentical related donors. Biol Blood Marrow Transplant 1997; 1:11–17.

Mismatched Related Donor Transplantation

217

28. Mehta J, Singhal S, Gee AP, et al. Bone marrow transplantation from partially mismatched family donors for acute leukemia: single-center experience in 201 patients. Bone Marrow Transplant 2004; 33:222–226. 29. Godder KT, Hazlett LJ, Abhyankar SH, et al. Partially mismatched related donors allow for early allogeneic transplant intervention and improved disease free survival in young patients with poor prognosis acute leukemia. J Clin Oncol 2000; 18:1856–1866. 30. Klingebiel T, Handgretinger R, Lang P, et al. Haploidentical transplant for acute lymphoblastic leukemia in childhood. Boll Rev 2004; 18:181–192. 31. Van Rood JJ, Fausto R, Loberiza, Jr., et al. Effect of tolerance to non-inherited maternal antigens on the occurrence of graft-versus-host disease after bone marrow transplantation from a parent or an HLA haploidentical sibling. Blood 2002; 99:1572–1577. 32. Tamaki S, Ichinohe T, Matsue K, et al. Superior survival of blood and marrow stem cell recipients given maternal grafts over recipients given paternal graft. Bone Marrow Transplant 2001; 28:375–380. 33. Ichinohe T, Uchiyama T, Shimazaki C, et al. Feasibility of HLA-haploidentical stem cell transplantation between non-inherited maternal antigen (NIMA)-mismatched family members linked with long-term feto-maternal microchimerism. Blood 2004; 104:3821–3828. 34. Wagner JE. Allogeneic umbilical cord blood transplantation. Cancer Treat Res 1997; 277:1735–1736. 35. Henslee-Downey PJ, Gluckman E. Allogeneic transplantation from donors other than HLA identical siblings. Hematol Oncol Clin North Am 1999; 13:1017–1039. 36. Abhyankar SH, Geier SS, Parrish R, et al. Effect of crossmatch reactions (CMR) and HLA typed cell panel antibody response (PRA-AbR) on engraftment using plasmapheresis (PMP) and Prosorba column absorption (PCA) during partially mismatched related donor (PMRD) BMT. Blood 1995; 86:393a. 37. Lang P, Greil J, Bader P, et al. Long-term outcome after haploidentical stem cell transplantation in children. Blood Cells Mol Dis 2004; 33:281–287. 38. Guinan EC, Vassiliki A, Boussiotis, et al. Transplantation of anergic histocompatible bone marrow allograft. N Engl J Med 1999; 340:1704–1714. 39. Lu DP, Dong L, Wu T, et al. Conditioning including antithymocyte globulin followed by unmanipulated HLA-mismatched/haploidentical blood and marrow transplantation can achieve comparable outcomes to HLA-identical sibling transplantation. Blood pre-published on line. 2005-05-2146. 40. O’Reilly RJ, Collins NH, Kernan NA, et al. Transplantation of marrow depleted T cells by soybean lectin agglutination and E-rosette depletion: a major histocompatibility complex related graft resistance in leukemic transplant patients. Transplant Proc 1985; 17:455–459. 41. Sondel PM, Hank JA, Trigg ME, et al. Transplantation of HLA-haploidentical T-cell-depleted marrow for leukemia: autologous marrow recovery with specific immune sensitization to donor antigens. Exp Hematol 1986; 14:278–286. 42. Trigg ME, Sondel PM, Billing R, et al. Mismatched bone marrow transplantation in children with hematologic malignancy using T- lymphocyte depleted bone marrow. J Biol Response Mod 1985; 4:602–612. 43. Henslee-Downey PJ, Abhyankar SH, Parrish RS, et al. Use of partially mismatched related donors extends access to allogeneic marrow transplant. Blood 1997; 89:3864–3872. 44. Martin PJ, Rowley SD, Anasetti C, et al. A phase I-II clinical trial to evaluate removal of CD4 cells and partial depletion of CD8 cells from donor marrow for HLA-mismatched unrelated recipients. Blood 1999; 94:2192–2199. 45. Aversa F, Tabilio A, Terenzi A, et al. Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype. N Engl J Med 1998; 339:1186–1193. 46. Bacigalupo A, Mordini N, Pitto A, et al. Transplantation of HLA- mismatched CD34C selected cells in patients with advanced malignancies: severe immunodeficiency and related complications. Br J Hematol 1997; 98:760–766. 47. Marciniak E, Henslee-Downey PJ, Baily K, et al. Bone marrow purging of T-lymphocytes with T10B9 monoclonal antibodies and complement. In: Areman E, Deeg HJ, Sacher R, eds. Bone Marrow and Stem Cell Processing: A Manual of Current Techniques. Philadelphia: F.A. Davis Co., 1992; 208–210. 48. Lee C, Brouillette M, Lamb L, et al. Use of a closed system for Vab-positive T-cells depletion of marrow for use in partially mismatched related donor (PMRD) transplantation. Advances in bone marrow purging and processing. Fourth International Symposium. 1994; 389: 523–532

218

Chiang et al.

49. Drobyski WR, Majewski D, Hanson G. Graft-facilitating doses of ex vivo activated gamma-delta T cells do not cause lethal murine graft-versus-host disease. Biol Blood Marrow Transplant 1999; 5:222–230. 50. Lamb LS, Jr., Musk P, Ye Z, et al. Human gamma-delta (C) T lymphocytes have in vitro graft versus leukemia activity in the absence of an allogeneic response. Bone Marrow Transplant 2001; 27:601–606. 51. Kawanishi Y, Passweg J, Drobyski WR, et al. Effect of T cell subset dose on outcome of T celldepleted bone marrow transplantation. Bone Marrow Transplant 1997; 19:1069–1077. 52. Soderling CC, Song CW, Blazar BR, et al. A correlation between conditioning and engraftment in recipients of MHC-mismatched T cell-depleted murine bone marrow transplants. J Immunol 1985; 135:941–946. 53. Goldman FD, Rumelhart SL, De Alarcon P, et al. Poor outcome in children with refractory/relapsed leukemia undergoing bone marrow transplantation with mismatched family member donors. Bone Marrow Transplant 2000; 25:943–948. 54. Bunin N, Aplenc R, Leahey A, et al. Outcomes of transplantation with partial T-cell depletion of matched or mismatched unrelated or partially matched related donor bone marrow in children and adolescents with leukemia. Bone Marrow Transplant 2005; 35:151–158. 55. Blazar BR, Quinones RR, Heinitz KJ, et al. Comparison of three techniques for the ex vivo elimination of T cells from human bone marrow. Exp Hematol 1985; 13:123–128. 56. Lee C, Henslee-Downey PJ, Brouillette M, et al. Comparison of OKT3 and T10B9 for ex-vivo T-cell depleted partially mismatched related donor (PMRD) bone marrow transplants (BMT). Blood 1995; 86:625a abstract. 57. Lamb LS, Gee AO, Hazlett LJ, et al. Influence of T cell depletion method on circulating gammadelta T cell reconstitution and potential role in the graft-versus-leukemia effect. Cytotherapy 1999; 1:7–19. 58. Bachar-Lustig E, Rahamim N, Li H-W, et al. Megadose of T cell depleted bone marrow overcomes MHC barriers in sub-lethally irradiated mice. Nat Med 1995; 1:1268–1273. 59. Schumm M, Lang P, Taylor S, et al. Isolation of highly purified autologous and allogeneic peripheral CD34C cells using the cliniMACS device. J Hematother 1999; 8:209–218. 60. Lang P, Schumm M, Taylor G, et al. Clinical scale isolation of highly purified peripheral CD34C progenitors for autologous and allogeneic transplantation in children. Bone Marrow Transplant 1999; 24:583–589. 61. Handgretinger R, Klingebiel T, Lang P, et al. Megadose transplantation of purified peripheral cells from HLA-mismatched parental donors in children. Bone Marrow Transplant 2001; 27:777–783. 62. Kato S, Yabe H, Yasui M, et al. Allogeneic hematopoietic transplantation of CD34C selected cells from an HLA haplo-identical related donor. A long-term follow-up of 135 patients and a comparison of stem cell source between the bone marrow and the peripheral blood. Bone Marrow Transplant 2000; 26:1281–1290. 63. Aversa F, Terenzi A, Tabilio A, et al. Full haplotype-mismatched hematopoietic stem-cell transplantation: a phase II study in patients with acute leukemia at high risk of relapse. J Clin Oncol 2005; 23:3447–3454. 64. Passweg JR, Kuhne T, Gregor M, et al. Increased stem cell dose, as obtained using current available technology, may not be sufficient for engraftment of haploidentical stem cell transplants. Bone Marrow Transplant 2000; 26:1033–1036. 65. Ji S-Q, Chen H-R, Wang H-X, et al. G-CSF primed haploidentical marrow transplantation without ex-vivo T-cell depletion: an excellent alternative for high-risk leukemia. Bone Marrow Transplant 2002; 30:861–866. 66. Arpinati M, Green CL, Heimfeld S, et al. Granulocyte colony-stimulating factor mobilizes T helper 2- inducing dendritic cells. Blood 2000; 95:2484–2490. 67. Bozdech MJ, Sondel PM, Trig ME, et al. Transplantation of HLA- haploidentical T-cell depleted marrow for leukemia: addition of cytosine arabinoside to the pre-transplant conditioning prevents rejection. Exp Hematol 1985; 13:1201–1210. 68. Henslee-Downey PJ, Parrish RS, MacDonald JS, et al. Combined in vitro and in vivo T lymphocyte depletion for the control of graft-versus-host disease following haploidentical marrow transplant. Transplantation 1996; 61:738–745. 69. Henslee Downey PJ, Godder K, Abhyankar S, et al. Sequential immunomodulation to achieve engraftment and control graft-versus-host disease across mismatched MHC barriers. In: Schechter G, Hoffman R & Schrier S, eds. Hematology. Washington DC: American Society of Hematology, 1999:317–322.

Mismatched Related Donor Transplantation

219

70. Champlin RE, Ho WG, Mitsuyasu R, et al. Graft failure and leukemia relapse following T-cell depleted bone marrow transplant: effect of intensification of immunosuppressive conditioning. Transplant Proc 1987; 19:2616. 71. Slavin S, Naparstek E, Aker M, et al. The use of total lymphoid irradiation for prevention of rejection of T-lymphocyte depleted bone marrow allografts in non-malignant hematologic disorders. Transplant Proc 1989; 21:3053. 72. Soiffer RJ, Mauch P, Tarbell NJ, et al. Total lymphoid irradiation to prevent graft rejection in recipient of HLA non-identical T-cell depleted allogeneic marrow. Bone Marrow Transplant 1991; 7:460. 73. Terenzi A, Aristei C, Chionne F, et al. Preliminary results of fludarabine as an immunosupressor in bone marrow transplantation conditioning. Bone Marrow Transplant 1996; 17:89 abstract. 74. Ortin M, Raj R, Kinning E, et al. Partially matched related donor peripheral blood progenitor cell transplantation in paediatric patients adding fludarabine and anti-lymphocyte gamma globulin. Bone Marrow Transplant 2002; 30:359–366. 75. Filipovich AH, Ramsey NK, Arthur DC, et al. Allogeneic bone marrow transplantation with related donors other than HLA MLC-matched siblings, and the use of antithymocyte globulin, prednisone and methotrexate for prophylaxis of graft versus-host disease. Transplantation 1985; 39:282–285. 76. Kernan NA, Flomenberg N, Dupont B, et al. Graft rejection in recipients of T-cell-depleted HLAnonidentical marrow transplants for leukemia. Identification of host-derived anti-donor allocytotoxic T lymphocytes. Transplantation 1987; 43:842–847. 77. Trigg ME, Gingrich R, Goeken N, et al. Low rejection rate when using unrelated or haploidentical donors for children with leukemia undergoing marrow transplantation. Bone Marrow Transplant 1989; 4:431–437. 78. Reisner Y, Martelli MF. Bone Marrow Transplantation across HLA barriers by increasing the number of transplanted cells. Immunol Today 1995; 16:437–440. 79. Martelli MF, Aversa F, Bachar-Lustig E, et al. Transplants across human leukocyte antigen barriers. Semin Hematol 2002; 39:48–56. 80. Rachamim N, Gan J, Segall H, et al. Tolerance induction by “megadose” hematopoietic transplants: donor-type human CD34 stem cells induce potent specific reduction of host anti-donor cytotoxic T lymphocyte precursors in mixed lymphocyte culture. Transplantation 1998; 65:1386–1393. 81. Gur H, Krauthgamer R, Berrebi A, et al. Tolerance induction by megadose hematopoietic progenitor cells: expansion of veto cells by short-term culture of purified human CD34C cells. Blood 2002; 99:4174–4181. 82. Peters C, Matthes-Martin S, Fritsch G, et al. Transplantation of highly purified peripheral blood CD34C cells from HLA-mismatched parental donors in 14 children: evaluation of early monitoring of engraftment. Leukemia 1999; 13:2070–2078. 83. Chiang KY, Van Rhee F, Godder K, et al. Allogeneic bone marrow transplantation from partially mismatched related donors as therapy for primary induction failure acute myeloid leukemia. Bone Marrow Transplant 2001; 27:507–510. 84. Godder KT, Mehta J, Chiang KY, et al. Partially mismatched related donor bone marrow transplantation as salvage for patients with AML who failed autologous stem cell transplant. Bone Marrow Transplant 2001; 28:1031–1036. 85. Huang XJ, Han W, Xu LP, et al. A novel approach to human leukocyte antigen-mismatched transplantation in patients with malignant hematological disease. Chin Med J (Engl) 2004; 117:1778–1785. 86. Franzke A, Piao W, Lauber J, et al. G-CSF as immune regulator in T cells expressing the G-CSF receptor: implications for transplantation and autoimmune diseases. Blood 2003; 102:734–739. 87. Ringden O, Labopin M, Gorin NC, et al. Treatment with granulocyte colony-stimulating factor after allogeneic bone marrow transplantation for acute leukemia increases the risk of graft-versus-host disease and death: a study from the acute leukemia working party of the European group for blood and marrow transplantation. J Clin Oncol 2004; 22:416–423. 88. Volpi I, Perruccio K, Tosti A, et al. Post-grafting administration of granulocyte colony-stimulating factor impairs functional immune recovery in recipients of human leukocyte antigen haplotypemismatched hematopoietic transplants. Blood 2001; 97:2514–2521. 89. Amrolia PJ, Mucioli-Casadei G, Huls H, et al. Add-back of allo-depleted donor T cells to improve immune reconstitution after haplo-identical stem cell transplantation. Cytotherapy 2005; 7:116–125.

220

Chiang et al.

90. Perruccio K, Tosti A, Burchielli E, et al. Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation. Blood 2005; 106:4397–4406. 91. Skinner J, Finlay JL, Sondel PM, Trig ME. Infection complications in pediatric patients undergoing transplantation with T- lymphocyte depleted bone marrow. Pediatr Infect Dis 1986; 5:319–324. 92. Lamb LS, Gee AP, Henslee-Downey PJ, et al. Phenotypic and functional reconstitution of peripheral lymphocytes following T cell-depleted bone marrow transplantation from partially mismatched related donors. Bone Marrow Transplant 1998; 21:461–471. 93. Foot ABM, Potter MN, Donaldson C, et al. Immune reconstitution after BMT in children. Bone Marrow Transplant 1993; 11:7–13. 94. Keever CA, Small TN, Flomenberg N, et al. Immune reconstitution following bone marrow transplantation: comparison of recipients of T cell depleted marrow with recipient of conventional marrow grafts. Blood 1989; 73:1340–1350. 95. Small TN, Papadopoulos EB, Boulad F, et al. Comparison of immune reconstitution after unrelated and related T-cell-depleted bone marrow transplantation: effect of patient age and donor leukocyte infusion. Blood 1999; 93:467–480. 96. Kook H, Goldman F, Padley D, et al. Reconstitution of the immune system after unrelated or partially matched T-cell depleted bone marrow transplantation in children: immunophenotypic analysis and factors affecting the speed of recovery. Blood 1996; 88:1089–1097. 97. Valteau-Couanet D, Cavazzano-Calvo M, Le Deist F, et al. Functional study of residual T-lymphocytes after specific elimination of alloreactive T-cells by a specific anti-interleukin 2 receptor Bk chain immunotoxin. Transplantation 1993; 56:1574–1576. 98. Amorelia PJ, Muccioli-Casadei G, Yvon E, et al. Selective depletion of donor alloreactive T cells without loss of antiviral or anti-leukemia responses. Blood 2003; 102:2293–3399. 99. Chiang KY, Hazlett LJ, Godder KT, et al. Epstein-Barr virus associated B cell lymphoproliferative disorder following mismatched related T cell depleted marrow transplantation. Bone Marrow Transplant 2001; 28:1117–1123. 100. Curtis RE, Travis LB, Rowlings PA, et al. Risk of lymphoproliferative disorders after bone marrow transplantation: a multi institutional study. Blood 1999; 94:2208–2216. 101. Cavazzana-Calvo M, Bensoussan D, Jabado N, et al. Prevention of EBV induced B-lymphoproliferative disease disorder by ex-vivo marrow B cell depletion in HLA-phenoidentical or nonidentical T-depleted bone marrow transplantation. Br J Haematol 1998; 103:543–551. 102. Fleming DR, Henslee-Downey PJ, Romond EH, et al. Allogeneic bone marrow transplantation with T-cell depleted partially matched related donors for advanced acute lymphoblastic leukemia in children and adults: a comparative matched cohort study. Bone Marrow Transplant 1996; 17:917–922. 103. Drobyski WR, Klein J, Flomenberg N, et al. Superior survival associated with transplantation of matched unrelated versus one-antigen-mismatched unrelated or highly human leukocyte antigendisparate haploidentical family donor marrow grafts for the treatment of hematologic malignancies: establishing a treatment algorithm for recipients of alternative donor grafts. Blood 2002; 99:806–814. 104. Barker JN, Davis SM, DeFor T, et al. Survival after transplantation of unrelated donor umbilical cord blood is comparable to that of human leukocyte antigen-matched unrelated donor bone marrow: results of a matched pair analysis. Blood 2001; 97:2957–2961. 105. Rocha V, Cornish J, Sievers E, et al. Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood 2001; 97:2962–2971. 106. Green A, Clarke E, Hunt L, et al. Children with acute lymphoblastic leukemia who receive T-cell depleted HLA mismatched marrow allografts from unrelated donors have an increased incidence of primary graft failure but a similar overall transplant outcomes. Blood 1999; 94:2236–2246. 107. Davies SM, Wagner JE, Shu XO, et al. Unrelated donor bone marrow transplantation for children with acute leukemia. J Clin Oncol 1997; 15:557–565. 108. Balduzzi A, Gooley T, Anasetti C, et al. Unrelated donor transplantation in children. Blood 1995; 86:3247–3256. 109. Casper J, Camitta B, Truitt R, et al. Unrelated bone marrow transplants for children with acute leukemia or myelodysplasia. Blood 1995; 85:2354–2363. 110. Gustafasson A, Remberger M, Winiarski J, et al. Unrelated bone marrow transplantation in children: outcome and a comparison with sibling donor grafting. Bone Marrow Transplant 2000; 25:1059–1065.

Mismatched Related Donor Transplantation

221

111. Saarinen-Pihkala UM, Gustafasson G, Ringde´n O, et al. No disadvantage in outcome of using matched unrelated donors as compared with matched sibling donors for bone marrow transplantation in children with acute lymphoblastic leukemia in second remission. J Clin Oncol 2001; 19:3406–3414. 112. Hongeng S, Krance RA, Bowman LC, et al. Outcomes of transplantation with matched-sibling and unrelated-donor bone marrow in children with leukemia. Lancet 1997; 350:767–771. 113. Locatelli F, Rocha V, Chastang C, et al. Factor associated with outcome after cord blood transplantation in children with acute leukemia. Blood 1999; 93:3662–3671. 114. Gluckman E, Rocha V, Arcese W, et al. Factors associated with outcomes of unrelated cord blood transplant: guidelines for donor choice. Exp Haematol 2004; 32:397–407. 115. Yu LC, Wall DA, Sandler E, et al. Unrelated cord blood transplant experience by the pediatric blood and marrow transplant consortium. Pediatr Hematol Oncol 2001; 18:235–245. 116. Ohnuma K, Isoyama K, Ikuta K, et al. Cord blood transplantation form HLA-mismatched unrelated donors as treatment for children with hematological malignancies. Br J Haematol 2001; 112:981–987. 117. Wall DA, Carter SL, Kernan NA, et al. Busulfan/ Melphalan/ antithymocyte globulin followed by unrelated donor cord blood transplantation for treatment of infant leukemia and leukemia in young children: the cord blood transplantation study (COBLT) experience. Biol Bone Marrow Transplant 2005; 11:637–646. 118. Godder K, Eapen M, Laver J, et al. Autologous stem cell transplantation (SCT) for acute myeloid leukemia (AML) in 1st or 2nd complete remission (CR)—prognostic factor analysis. J Clin Oncol 2004; 22:3798–3804. 119. Messina C, Valsecchi MG, Arico M, et al. Autologous bone marrow transplantation for treatment of isolated central nervous system relapse of childhood acute lymphocytic leukemia. AIEOP/FONOP/ TMO group. Associzione Italiana emato-oncologia pediatrica. Bone Marrow Transplant 1998; 21:9–14. 120. Singhal S, Henslee-Downey PJ, Powles R, et al. Haploidentical versus autologous hematopoietic stem cell transplantation in patients with acute leukemia beyond first remission. Bone Marrow Transplant 2003; 31:889–895.

11 Nursing Care of the Pediatric Blood and Marrow Transplant Patient Linda Z. Abramovitz Pediatric Bone Marrow Transplant, Children’s Hospital at the University of California, San Francisco, California, U.S.A.

Vicki L. Fisher Pediatric BMT Program, Rainbow Babies and Children’s Hospital, Cleveland, Ohio, U.S.A.

INTRODUCTION Nursing has always played an integral role in the care of children undergoing hematopoietic stem cell transplantation (HSCT). As a vital member of the transplant team, nurses provide both direct and indirect care throughout all phases of a child’s transplant course. Working closely with physicians, social workers, nutritionists, pharmacists, physical therapists, and others, the nurse often serves as a link between the family unit and the medical team.

NURSING ROLES AND PRACTICE SETTINGS The transplant nurse interacts with the child and family, as well as other disciplines on the transplant team, in a variety of settings. As the field of HSCT has evolved over the last several decades, so has the scope and practice of nurses who work with these children. Opportunities for nurses in HSCT are quite broad and include both inpatient and outpatient settings encompassing a variety of roles (Table 1). Staff nurses provide direct patient care on the inpatient transplant unit and in other hospital settings, including the intensive care unit and pheresis unit. In surgical settings, such as the operating room and postanesthesia recovery room, nurses care for the child at the time of catheter placement, bone marrow harvest, and on other rare occasions when surgery is necessary as a therapeutic intervention or as a diagnostic tool for posttransplant complications. In the ambulatory practice setting, nurses provide direct care at the time of the clinic visit or may assume responsibility for children receiving treatments, such as venipuncture, chemotherapy, transfusions, and medication administration in a day hospital or infusion center. The intensity and scope of outpatient care has increased over the last decade as transplant centers have moved to earlier discharge and modified outpatient transplants (1). Some pediatric transplant centers have reorganized their programs to incorporate both inpatient and ambulatory care into adjacent physical settings (2). This has benefited patients and families, enabling them to move easily between inpatient hospital stays and ambulatory care. In addition, nurses have been 223

224 Table 1

Abramovitz and Fisher Nursing Roles in Inpatient and Outpatient Practice Settings

Staff nurse Pheresis nurse Nurse manager Surgical nurse Recovery room Clinical nurse specialist Nurse practitioner Nurse coordinator Case manager Nurse educator

Administrative nurse Donor search coordinator Research nurse Clinic nurse Day hospital/infusion nurse Home care nurse Palliative care nurse Private sector Pharmaceutical Insurance

able to rotate between these settings to provide continuity of care. This type of care environment is positive for the staff as well as the child and family. The formal education among nurses caring for transplant patients varies. Nurses who have a special interest in the field of HSCT may return to school for additional education (typically a 2-year program) in order to receive a master’s degree. These nurses assume advanced practice roles [clinical nurse specialist (CNS) and/or nurse practitioner (NP)] with a pediatric focus and often a specialization in the field of immunology/hematology/oncology/transplant nursing. Many graduate nursing programs have combined both the CNS and NP curriculum and, as a result, have broadened the scope and practice settings of the advance practice nurse (APN) (3). An NP can practice in both inpatient and outpatient settings, performing histories, and physicals and (in most states) prescribing medications. Typically working closely with one physician, the NP will have a caseload of patients (1). In some centers, the NP rotates between the inpatient and outpatient service. The components of the CNS role include clinical work, consultation, education, and research. Some CNS’s may also assume the role of nurse coordinator in their programs. Working with patients and families as well as the medical and nursing staff, the CNS provides clinical expertise and education. Many transplant programs also have nurse educators in addition to the CNS to develop and implement educational programs pertaining to HSCT. The CNS typically assumes responsibility for the development of policies, procedures, and guidelines. The Board of Registered Nursing in each state as well as the standardized procedures established in their individual work setting regulates APN practice. APN’s must be certified as CNS’s or NP’s at a state and national level. A certified APN demonstrates specialized knowledge and meets a standard that reflects nationally accepted values and practice standards (4). APN’s become certified by sitting for comprehensive examinations currently available through several national organizations (3). This credentialing is not to be confused with the certification offered by the oncology nursing society (ONS) and the association of pediatric oncology nurses (APON), where an advanced practice degree is not required. The certification examination offered from the ONS is for any nurse working in the field of oncology nursing (5). There is also a certification exam offered by the APON to recognize expertise in the subspecialty of pediatric oncology (6). At this time, there is no certification for the subspecialty of pediatric HSCT nursing. Some transplant nurses may pursue a master’s degree in public health or an advanced degree in administration with the intent to utilize their advanced education to explore other areas in the field of HSCT nursing. A doctorate degree in nursing or a PhD in a related field also attracts other nurses who want to further their education. Several transplant programs have employed doctorate prepared nurse researchers to facilitate and build nursing knowledge for expanding evidence-based nursing practice (7). There are many opportunities for nurses to engage in research related to the specialty of HSCT nursing. Transplant nurses in a variety of roles and practice settings collaborate with physicians and other individuals on the team to pursue clinical research. It is essential that transplant nurses continue to be involved in research to further define practice and improve patient outcomes and quality of life.

Nursing Care of the PBMT Patient

225

Nurse coordinators are instrumental to the success of many transplant programs. These nurses are responsible for the support and day-to-day management of patients through the different phases of the transplant process. Possessing up-to-date information, along with knowledge about other key issues, nurse coordinators communicate essential aspects of care to parents, who rely heavily on them. The responsibilities of the nurse coordinator vary from program to program and may include scheduling and coordinating tests and procedures, obtaining insurance approval, participation in informed consent and patient care conferences, patient education, case management, and discharge planning, as well as functioning as a liaison between the family and nursing staff (1). At some transplant centers, the APN assumes some aspects of the coordinator role. The formal education of the coordinator role varies greatly. Many transplant programs have more than one coordinator to support different aspects of care and to focus on different phases of the transplant process, including pretransplant evaluation, inpatient, and outpatient care. Unrelated donor search coordinators are responsible for the ongoing searches for viable marrow, peripheral blood stem cell, and cord blood donors. At some centers, a nurse may be the individual to assume this role. The search coordinator must become very knowledgeable about human leukocyte antigen (HLA) typing as well as be sufficiently detail oriented to effectively manage complex searches. In both inpatient and ambulatory settings, nurse managers and administrative nurses are vital to the overall success of a transplant program from both a financial and staff management perspective. Working closely with other members of the transplant team, the nurse manager possesses knowledge about HSCT as well as valuable information about the operations and management of the hospital. With an overall hospital perspective, the nurse manager is in a unique position to facilitate and support the HSCT mission, goals, and quality management programs (1). As patients are transitioned home earlier and move away from the medical center environment, home care nurses are critical members of the health care team and are responsible for aiding in the transition of the child and family back to their home. Pediatric nurses who are knowledgeable about the specific needs of children and their families should provide this home care. Exceptional assessment skills and the astute ability to recognize and then communicate changes to the medical/nursing team at the transplant or referring medical center are essential elements for pediatric home care nurses. Many home care nurses are skilled in palliative care. Some experienced transplant nurses have redirected their careers, moving away from the traditional hospital and clinic settings to pursue job opportunities with pharmaceutical companies and as consultants for other health care organizations with interests in transplant care. These skilled nurses are excellent representatives who are able to interface with and share knowledge between the hospital setting and private industry.

NURSING EDUCATION Nurses come to HSCT programs with a variety of educational backgrounds and clinical experiences. Many programs will accept new graduates, whereas others prefer nurses with varying degrees of experience. Transplant programs must have educational training programs in place to provide basic knowledge about transplant nursing (8). This must be coupled with a clinical component to allow the “hands on” experience essential to nursing education. Both formal and informal educational opportunities allow the nurse to gain the knowledge and skills necessary to meet the child’s and families’ needs. Topics typically covered in a basic training program include basic immunology and hematology; HLA typing and donor selection; indications for transplant, including disease information; overview of transplant; chemotherapy and conditioning protocols; medications; supportive care management (isolation, diet, mouth care, skin care); complications (mucositis, bleeding, infection, veno-occlusive disease, graft-versushost disease); immune reconstitution; and long-term complications. Pediatric transplant nurses

226 Table 2

Abramovitz and Fisher Topics in Core Curriculum

Basic immunology Basic hematology Rationale for transplant Indications for transplant Overview of transplant (including terminology) HLA typing and donor selection Chemotherapy, total body irradiation Conditioning protocols Administration of HSC Medications Supportive care Isolation, diet, mouth care, skin care, catheter care, blood product administration Complications Mucositis, bleeding, infections, veno-occlusive disease Graft versus host disease Normal growth and development Psychosocial aspects of care Quality of life issues Ethical issues Immune reconstitution Long-term complications Abbreviation: HSC, hematopoietic stem cell.

must be familiar with normal growth and development across the age spectrum, as well as possess knowledge about the psychosocial needs and ethical issues facing the child and family undergoing HSCT (Table 2) (9). The Pediatric Blood and Marrow Transplant Consortium and Association of Pediatric Oncology Nurses have developed a comprehensive core curriculum of pediatric BMT. The core curriculum is a review of the transplant process covering topics from initial intent to transplant through survivorship issues. When HSCT programs are associated with oncology and/or hematology services, the scope of the training program is extensive in order to cover information thoroughly enough to meet all three programs’ needs. Many centers have an extensive chemotherapy course that may include certification. Working closely with the APN and nurse educator, the nurse manager is in a key position to support the educational needs of the staff. Nurses, physicians, social workers, nutritionists, pharmacists, child life specialists, and other experienced members of the health care team should all be involved in teaching. This type of educational environment provides invaluable opportunities for the new nurse to establish a relationship with members of the transplant team. Nurses must be kept informed about the newest advances in the field of transplant, modifications in protocols, research, and supportive care management updates. This can be accomplished by formal lectures, readings, in-services, nursing rounds, and updates at staff meetings. The topics taught in the basic orientation course can be covered in greater depth and detail in an advanced curriculum after the nurse gains experience. The experienced HSCT nurse should be encouraged to attend regional and national conferences to increase exposure to new information and to establish a network of colleagues.

PROFESSIONAL NURSING ORGANIZATIONS There are several professional organizations that have supported nurses working in the field of HSCT. These organizations provide educational opportunities at an international, national, and

Nursing Care of the PBMT Patient

227

regional level. The ONS has been instrumental in offering conferences, workshops, and networking opportunities among nurses. In addition to a yearly international congress attended by more than 5000 participants, ONS has an extensive website, a professional journal, and plays an active role in advocacy activities (10). Within the ONS, the blood and marrow transplantation (BMT) Group is one of oldest and largest special interest groups (SIG). The BMT SIG has a Web site and online discussion forum and publishes a newsletter several times per year. The BMT SIG is responsible for planning and coordinating a three-day nursing conference at the Tandem BMT meetings. This is in conjunction with the joint annual meetings of the American Society for Blood and Marrow Transplantation (ASBMT) and the Center for International Blood and Marrow Transplant Research (CIBMTR—formerly the international bone marrow transplant registry and the autologous blood and marrow transplant registry). Outside the United States, the European bone marrow transplant nurses group was formed in 1985 based on the need to share information between countries. Currently a three-day conference is held annually in conjunction with the physician conference. The format of joint sessions and separate nursing sessions encourages networking and sharing of information through the presentation of abstracts and instructional sessions (11). Over the last 20 years, the Canadian association of nurses in oncology (CANO) has provided opportunities for educational growth of transplant nurses through annual conferences and networking as a special interest group (11). In 1974, APON was established to focus on the care of children with cancer. Their professional journal, educational workshops and conferences at a local and national level have included state of the art updates on HSCT nursing (6). Nurses have taken an active role with the children’s cancer group (CCG) and pediatric oncology group (POG) when these national cooperative groups were first established in the 1970s. In 2000, these two groups merged to form Children’s Oncology Group (12). Collaborating with transplant physicians and other health care providers, transplant nurses are active on committees and have assumed an integral role in the development of clinical protocols as well as developing educational programs related to the care of the child undergoing HSCT (13). With a focused interest in improving the care of children receiving HSCT, the Pediatric Blood and Marrow Transplant Consortium (PBMTC) currently has 75 institutional members. The nursing component, first established in 1999, has worked together with physicians on clinical and research protocols. The nursing group focuses on the care of the pediatric patient throughout all the phases of transplantation (14).

PATIENT EDUCATION Throughout the course of transplant, the nurse plays an important role in patient/family education. Patient education is an ongoing process and includes all members of the health care team. The information should be presented to the child and the family using a variety of forms (verbal, written, and visual). Information is provided verbally during clinic visits, meetings, and the informed consent process as well as in the form of written materials, such as booklets and handouts. Many transplant centers also have Web sites that contain information accessible to many patients and families. There are numerous organizations that have resources available to assist the families of children undergoing HSCT. Information is accessible on the Internet and/or in pamphlets, handouts, and books. Visual media in the form of videotapes and slides may also be available. There has been an increased interest using computer-assisted learning for patient education. Given adequate funding sources and expert knowledge, educational programs can be created and distributed via CD-ROM, DVD, or other digital media. With the advent of widespread broadband access it will also be possible to distribute these programs via the Internet. This educational technology has been shown to be an effective tool accepted across socioeconomic and educational levels to provide the user with a large amount of information without becoming

228

Abramovitz and Fisher

overwhelmed (15). In addition to international and national organizations there are many community resources and regional chapters available to families (Table 3). The Internet is another valuable resource providing older patients and their family access to health information, community support, and electronic patient-physician communication. Families need to have access to reliable and accurate information; therefore, the transplant team can play an important role in directing families to appropriate Internet sites and resources. Families who “surf” the Internet, however should use caution. Patients and families should be encouraged to discuss their findings with the health care team to ensure accurate interpretation of information (24). Internet online discussion groups have become extremely popular and provide another venue for families to gain knowledge and support about particular diseases and/or the transplant process. Written educational materials about transplantation must incorporate a broad scope of information covering the pretransplant, transplant, and posttransplant periods (9). The content should address issues and concerns facing both the child and the parents and other family members. Information to be covered in a transplant center handbook includes general information about the center, the types of diseases transplanted and types of transplants offered, the transplant process itself, conditioning regimens, complications, daily and supportive care, and psychosocial aspects and coping strategies. Table 4 outlines the specific elements of a transplant booklet for patient and families. Many centers have separate discharge handbooks and materials. The material covered must be detailed and structured in a format for easy reference for the patients and parents once they return home. Information should include important names and phone numbers, isolation precautions, and potential problems, such as signs and symptoms of infections and graft-versushost disease. Other topics include medications, nutrition, school, housekeeping, pets, dental care, skin precautions, and immunizations. The specific topics of a discharge handbook are provided in Table 5. The APN, nurse coordinator, and staff nurse, along with key members of the transplant team (social work, child life, physicians), are best suited to develop written information specific to their program. This information needs to be reviewed and updated frequently. Patient education materials should be available in languages appropriate to individual patients. Given the diverse patient population cared for at many transplant centers, this is not always possible. An interpreter is essential to transmit accurate information to families and provide the link for dialogue between the child/family and the health care team. According to the Joint Commission on the Accreditation of Healthcare Organizations (JCAHO), patient education materials should be written at a fifth to eighth grade reading level whereas the American Academy of Family Physicians’ Health Education Program suggest a reading level of seventh grade or lower (25). In the general population, an individual’s functional health literacy is lower than one’s reading literacy (26). Based on this, the HSCT team should assess the readability of materials presented to families to facilitate increased comprehension and informed decision making.

SPECIAL PROGRAMS Transplant centers have looked beyond the immediate scope of their clinical programs to provide care to the child and family. In well-established transplant programs, the range of services and activities may include patient/family reunions, camps, and palliative care and bereavement programs. Although difficult at times to implement, the transplant team can successfully incorporate the philosophy and goals the palliative care model into their practice. In addition, hospitalbased bereavement programs and retreats for families and significant others have become increasingly more common. Ongoing follow-up and support can be as basic as mailing condolence cards and bereavement literature to a more complex program one to two days in

Nursing Care of the PBMT Patient Table 3

229

Organizations, Programs, and Other Patient Education

Allogeneic Bone Marrow and Stem-Cell Transplantation: a medical and educational handbook. Developed by the Bone Marrow Foundation. (Written in both English and Spanish, this booklet provides an excellent overview of the transplant process, including critical questions to ask) American Brain Tumor Association (16) 1-800-886-2282 (An independent, global organization providing research funding and providing the information patients need to make educated decisions about their health care) American Bone Marrow Donor Registry c/o the Caitlin Raymond International Registry 1-800-7-A-MATCH American Cancer Society (17) 1-800-227-2545 (As a nationwide, community-based voluntary health organization; the ACS provides cancer information, community programs and service, advocacy, and public policy) Aplastic Anemia Foundation of America (18) 1-800-747-2820 Autologous Bone Marrow and Stem-Cell Transplantation: a medical and educational handbook. Developed by the bone marrow foundation. (Written in both English and Spanish, this booklet provides an excellent overview of the transplant process, including critical questions to ask) Autologous Stem Cell Transplants: A Handbook for Patients by Susan Stewart (A comprehensive resource covering a broad range of topics before, during, and following autologous transplant for patients and families) HSCT information network Blood and Marrow Stem-Cell Transplantation: Leukemia, Lymphoma, and Myeloma. Developed by the leukemia and lymphoma society (An information booklet for patients and their families about the use of blood or marrow stem cell transplantation for the treatment of leukemia, lymphoma and myeloma) HSCT information network (bmtinfonet) (19,20) 888-597-7674 (Provides publications, including a newsletter and a resource directory, emotional support, and links to other organizations for patients and families) HSCT Newsletter (20) 1-847-433-3313/1-888-597-7674 (A quarterly publication written for stem cell transplant patients, survivors, and their families) The Bone Marrow Foundation (21) 1-800-365-1336 (A not-for-profit organization to improve the quality of life for bone marrow and stem cell transplant patients and their families by providing financial aid, education, and emotional support) Bone Marrow Transplant Resource Guide: Friends Helping Friends Developed by the national bone marrow transplant link Cancercare.inc 1-800-813-HOPE (4673) (Provides telephone and online support to patients and families as well as financial assistance) Cancer Information Service (22) 1-800-4-CANCER (Provides information about clinical trials, diseases and standard treatments) Cancersourcekids.com (23) (Developed in partnership with APON this website is for kids (6–12), teens (13–15), and parents to learn about cancer) (24–26) Candlelighters Childhood Cancer Foundation (27) 1-800-366-2223 (Continued)

230 Table 3

Abramovitz and Fisher Organizations, Programs, and Other Patient Education (Continued)

(A national nonprofit membership organization whose mission is to educate, support, serve, and advocate for families of children with cancer, survivors of childhood cancer, and the professionals who care for them) Coping magazine 1-615-790-2400 Crowe, K. Me and My Marrow: A Kid’s Guide to Bone Marrow Transplantation. Fujisawa Healthcare, Inc. (A very practical but comprehensive booklet about hematopoietic stem cell transplantation written for children and teens) (28) Curesearch (Information for families supported by the national childhood cancer foundation and the children’s oncology group) Fanconi’s Anemia Research Fund, Inc. (29) 1-541-687-4658 Histiocytosis Association of America 1-800-548-2758 Immune Deficiency Foundation (30) 1-800-296-4433 International Bone Marrow Transplant Registry (IBMTR) 1-414-456-8325 Kids Cancer Network (31) 1-805-693-1017 (Publishes an activity letter called FUNLETTERS for children and families living with cancer) Leukemia and Lymphoma Society (32) 1-800-955-4572 (A large voluntary health organization dedicated to funding blood cancer research, education, and patient services) MPS Society (33) 1-610-942-0100 Make-a-Wish-Foundation (34) 1-800-722-9474 National Bone Marrow Transplant Link 1-800-546-5268 (Booklets about transplant and resource listings for support groups and BMT information) National Brain Tumor Foundation (35) 1-800-934-CURE (2873) National Childhood Cancer Foundation (36) 1-800-458-6223 1-314-241-1600 (Provides financial assistance) National Children’s Cancer Society (37) 1-800-5-FAMILY National Coalition for Cancer Survivorship 1-301-650-8868 National Institutes of Health (NIH) (38) 1-800-4-CANCER (Provides cancer research information and publications) National Marrow Donor Program (NMDP) (39) 1-800-627-7692 (The NMDP has patient education resources; written information is available in print and on the Internet, and videos pertaining to transplant using an unrelated donor are available. Support is offered through the office of patient advocacy. NMDP publishes information about member transplant programs.) (Continued)

Nursing Care of the PBMT Patient

231

Table 3 Organizations, Programs, and Other Patient Education (Continued) National Organization for Rare Disorders (NORD) (40) 1-800-999-NORD (6673) Oncology Nursing Society (10) BMT Special Interest Group 1-412-921-7373 (Publishes a directory of major BMT centers in the U.S. and Canada) Abbreviations: ACS, American cancer society; HSCT, hematopoietic stem cell transplantation; APON, association of pediatric oncology nurses; BMT, blood and marrow transplantation.

Table 4 Table of Contents for a Transplant Center Blood and Marrow Transplantation Booklet Overview The medical center The pediatric bone marrow transplant program and team General information Bone marrow and hematopoietic stem cells Rationale for HSCT Types of transplants Diseases treated with HSCTs Cancer Noncancerous diseases Genetic diseases Finding a donor The transplant process The pretransplant work-up and evaluation Admission to the transplant unit Beginning of conditioning The day of transplant The posttransplant period Side effects of conditioning Transplant related complications Discharge Follow-up care Short-term follow-up Long-term follow-up Psychosocial and emotional aspects of hematopoietic stem cell transplant Coping strategies for patient and family members Planning for your transplant Insurance Transportation, housing, parking Family members and friends Packing Definition of frequently used terms Appendix Chemotherapy information Total body irradiation (TBI) information Hospital and BMT information (visiting hours, phone numbers, cafeteria) BMT diet Maps/directions Abbreviations: HSCT, hematopoietic stem cell transplantation; BMT, blood and marrow transplantation. Source: From Refs. 41–43.

232 Table 5

Abramovitz and Fisher Table of Contents for a Blood and Marrow Transplantation Discharge Booklet

Introduction Important resources and telephone numbers Who and when to call Special precautions post transplant General guidelines and rationale Protective isolation Dietary restrictions School Housekeeping/Laundry Family pets Signs and symptoms of infections Other posttransplant complications, including GVHD General care issues Oral care and dental follow-up issues Skin (sun exposure) Immunizations Medic-alert bracelet Medications Frequently asked questions (FAQ) Follow-up care/quality of life issues Abbreviation: GVHD, graft-versus-host disease. Source: From Refs. 41–43.

length. Such an extensive retreat would include didactic lectures, multiple support groups covering a variety of topics, and other activities (art, writing, yoga) and a time of remembering. Offered concurrently, age-appropriate programs for siblings would be available using art, music, and other group activities. As the numbers of children who are long-term survivors of HSCT have increased, so has the need to provide the physical and psychosocial supports for these patients and families. Long-term follow-up programs are well established at most transplant centers. The manner in which each transplant program may follow patients varies greatly and is influenced by such factors as type of transplant and complications, travel distance, and the expertise of referring physician. Many centers have incorporated a transplant reunion into their program. This annual event is a celebration of life not only for patients and their families but also for the entire transplant team. Finally, summer camps and weekend retreats provide children, siblings, and parents an opportunity to meet others who have gone through a similar experience. These structured programs offer the necessary emotional support that patients and families need following a transplant. The transplant team nurses, social worker, and nurse manager are usually the key individuals responsible for developing and implementing programs for patients, their families, and staff. As the transplant team has maximized their ability to meet both physical and psychological demands of the child and family, there must be a program in place to meet the needs of the caregivers. Support groups, staff meetings, hospital, and unit-based memorial services and such programs as “Care for the Caregiver” are all ways to support the emotional well-being of the transplant team.

REFERENCES 1. Adams J, Clifford K, Adornettom D. Considerations in hematopoietic stem cell transplant program development and sites of delivery. In: Ezzone, ed. Hematopoietic Stem Cell Transplantation: A Manual for Nursing Practice. Pittsburgh, PA: Oncology Nursing Society Publishing Division, 2004:61–84.

Nursing Care of the PBMT Patient

233

2. Bushsel P, Yarbo C. Oncology Nursing in The Ambulatory Setting: Issues and Models of Care. Boston, MA: Jones and Bartlett, 1995. 3. Murphy-Ende K. Advanced practice nursing: reflections on the past, issues in the future. Oncol Nurs Forum 2002; 29:106–112. 4. McClain N, Richardson B, Wyatt J. A profile of certification for pediatric nurses. Pediatr Nurs 2004; 30:207–211. 5. Frank-Stromborg M, Ward S, Hughes L, et al. Does certification status of oncology nurses make a difference in patient outcome? Oncol Nurs Forum 2003; 29:665–672. 6. www.apon.org. 7. Haberman M. Nursing Research in Blood Cell and Marrow Transplantation. In: Whedon and Wujck eds. Blood and Marrow Stem Cell Transplantation. 2nd edition. Boston: Jones and Bartlett 1997: 497–505. 8. Campbell L, Foody M. In: Buchsel, Whedon, eds. Administrative Issues of an Inpatient BMT Unit. In: Bone Marrow Transplantation: Administrative and Clinical Strategies. Boston: Jones and Bartlett, 1995:39–68. 9. Abramovitz L. In: Whedon, ed. Perspectives on Pediatric Bone Marrow Transplant. Bone Marrow Transplantation: Principles, Practice, and Nursing Insights. Boston: Jones and Bartlett, 1991:70–104. 10. www.ons.org. 11. Ezzone S, Fliedner M. In: Whedon, Wujck, eds. Transplant Networks and Standards of Care: International Perspective. 2nd ed. Blood and Marrow Stem Cell Transplantation, Boston: Jones and Bartlett, 1997:474–496. 12. Hockenberry M. Editorial: pediatric oncology. J Pediatr Oncol Nurs 2003; 18:85–86. 13. www.childrensoncologygroup.org. 14. V. Fischer, personal communication, 2004. 15. Agre P, Dougherty J, Pirone J. Creating a CD-ROM program for cancer-related patient education. Oncol Nurs Forum 2002; 29:573–580. 16. www.abta.org. 17. www.cancer.org. 18. www.aplastic.org. 19. www.bmtinfonet.org. 20. www.bmtnews.org. 21. www.bonemarrow.org. 22. www.nci.hih.gov. 23. www.cancersourcekids.com. 24. Penson R, Benson R, Parles K, et al. Virtual connections: Internet health care. Oncologist 2002; 7:555–568. 25. Scown S. Cancer-patient reading levels in the real world. www.onconurse.com March 2002. 26. Boswell C, Cannon S, Aung K, Eldridge J. The application of health literacy research. Appl Nurs Res 2004; 17:61–64. 27. www.candlelighter.org. 28. www.curesearch.org. 29. www.fanconi.org. 30. www.primaryimmune.org. 31. www.kidscancernetwork.org. 32. www.leukemia-lymphoma.org. 33. www.mpssociety.org. 34. www.wish.org. 35. www.braintumor.org. 36. www.nccf.org. 37. www.children-cancer.org. 38. www.nci.nih.gov. 39. www.marrow.org. 40. www.rarediseases.org. 41. Abramovitz L, Link V. In: Buchsel, Whedon, eds. Administrative Issues of the Pediatric BMT Unit. Bone Marrow Transplantation: Administrative and Clinical Strategies. Boston: Jones and Bartlett, 1995:95–111. 42. Allogeneic Bone Marrow and Stem Cell Transplantation: A Medical and Educational Handbook. Developed by the Bone Marrow Foundation 2001. 43. Autologous Bone Marrow and Stem Cell Transplantation: A Medical and Educational Handbook. Developed by the Bone Marrow Foundation 2001.

12 Psychological Dimensions of Pediatric Hematopoietic Stem-Cell Transplantation Bryan D. Carter Kosair Children’s Hospital and University of Louisville School of Medicine, Louisville, Kentucky, U.S.A.

William G. Kronenberger Riley Hospital for Children and Indiana University School of Medicine, Indianapolis, Indiana, U.S.A.

Tanya F. Stockhammer Kosair Children’s Hospital and University of Louisville School of Medicine, Louisville, Kentucky, U.S.A.

Christi Bartolomucci Kids on the Move, Atlanta, Georgia, U.S.A.

PROCESS-RELATED STRESSORS OF PEDIATRIC HEMATOPOIETIC STEM-CELL TRANSPLANTATION ON THE CHILD AND FAMILY For pediatric patients with an increasing range of life-threatening and life-shortening medical conditions, hematopoietic stem cell transplantation (HSCT) is often a life-saving treatment option. Nevertheless, although HSCT may enhance chances of survival, it is often associated with numerous physical and psychological challenges that impact on multiple dimensions of quality of life for the child and their family. Although the impact of treatment for pediatric cancers on psychosocial functioning is fairly well established (1), the HSCT process adds additional and unique stressors that deserve specialized attention from the multidisciplinary pediatric HSCT treatment team. The primary stages involved in the HSCT process include the decision-making phase, preparation for admission, hospitalization (conditioning, transplantation, and engraftment), and posthospitalization (early and extended convalescence) phases (2). During the decision-making and preparation for admission phases, the HSCT team must determine the appropriateness of the procedure for the patient’s disease, including the child’s ability to physically and emotionally tolerate the rigorous HSCT treatment regimen and the family’s ability to handle the post-HSCT medical regimen and lifestyle changes. The results from these investigations are then shared with the parent(s)/guardian(s) and the patient (dependent on the child’s age and capacity to understand the complex information). 235

236

Carter et al.

This information can be quite complex and challenging for the patient and their family to fully understand. Dermatis and Leske (3) found that the patients and families in their study typically recalled less than half of the information they were given regarding side effects and risk factors, possibly due to the cognitive “overload” and anxiety they experienced. This difficulty with recall may not improve even with the use of focused interventions designed to reduce anxiety (4). When prognosis is poor, even with HSCT, it becomes a viable option to not pursue this dramatic and aggressive treatment and to instead direct efforts towards maximizing the quality of life surrounding the end-stage of the child’s disease. In cases of allogeneic transplantation, the search for a suitably matched donor may take weeks, or even months, adding to the anxiety of patient and family and risking further complications of their disease. Financial and insurance-related complications may add to these stressors. Pre-HSCT psychosocial evaluation of the patient and family is essential to determine coping resources and risk factors for adjustment and adherence difficulties during hospitalization and post-HSCT (5). Particular emphasis should be placed on the child’s preHSCT coping style, adjustment, and cognitive/academic functioning, due to their association with post-HSCT longitudinal outcome. Avoidant and emotion-focused coping have been associated with increased emotional distress, in contrast to those patients who employ active and information-gathering strategies in dealing with illness and hospitalization (6). Upon hospitalization for HSCT, children enter the conditioning phase of treatment, where they are administered potent chemotherapeutic drugs designed to eradicate their primary disease and to suppress their immune system so that the transplanted stem cells will not be rejected. Depending on the HSCT protocol for their specific disease, they may also receive total body irradiation. During the conditioning phase, which typically lasts from four to eight days, the patient may go from feeling relatively well to deathly ill. The patient and parents may also experience the extreme anxiety of realizing that the child is highly vulnerable to possibly lethal infections due to their immunosuppressed condition. The toxicity of the potent chemotherapeutic drugs can potentially result in a number of life-threatening organ system failures, including acute renal failure, pneumonitis, liver failure, hemorrhagic cystitis, etc. Additionally, multiple side effects of the treatment (e.g., nausea and vomiting, mouth sores, diarrhea, and hair loss) serve to exacerbate the patient’s misery and distress, often resulting in symptoms of anxiety, depression, and withdrawal (7). The actual process of the child receiving the marrow for transplantation, which is experientially not much different than receiving a blood transfusion, is rather anticlimactic for the patient and family. Once signs of engraftment begin to emerge, which may vary from days to weeks, indicating that the transplanted stem cells have successfully begun to function with the production of platelets and red and white blood cells, there is often a psychological boost for patient and family with hopeful anticipation of discharge from the strict confines of the HSCT unit, and eventually a return home. Nevertheless, departing from the security and isolation of the HSCT unit can also be a difficult time of adjustment for patient and family. Family members often experience mixed emotions of joy and relief of the fact that their child survived the HSCT, while experiencing fear and anxiety at leaving the support, sense of security, and the attachments formed with the medical staff that may have come to be associated with the HSCT setting (8). Parents may question their ability to assume care for their child and to provide for their immediate needs. The posthospitalization phase is associated with mixed feelings of relief and renewed uncertainty. Families must stay in close proximity and communication with the HSCT center for at least the first few weeks following transplant, for careful monitoring of signs of infection, graft rejection by the recipient’s immune system, and graft-versus-host disease (GVHD). GVHD is associated with considerable morbidity and can be potentially life threatening (9). During the early posthospitalization phase, bone marrow failure, GVHD, immunodeficiency, and relapse of the patient’s primary disease (in the case of childhood cancers) all pose a threat to survival. Immune function generally improves gradually over the first year post transplant, decreasing the risk of infection, and allowing the patient to hopefully resume a more normal life with return to school and fewer restrictions on social and physical activities. During the entire

Psychological Dimensions of Pediatric HSCT

237

HSCT process the child has been forced to become highly dependent on parents and family members, often at a time when, particularly for adolescents, they would be striving for increased independence from family. As a result, separation anxiety may arise with the return to more normal function, due to the forced increased dependency, social isolation from peers due to the increased risk of opportunistic infections, and, finally, to decreased confidence in the academic arena associated with both school absence and possible neurocognitive changes associated with treatment and/or disease. Although HSCT may indeed control or even cure their underlying disease, there is significant long-term physical and psychosocial morbidity associated with HSCT survival that poses challenges for patient and family, some which may occur and continue for years after transplant. Fatigue is a frequent “late effect” of HSCT, with patients often becoming discouraged at their slow pace of recovery and failure to return to feeling “normal” (9). Chronic GVHD, recurrent infections, respiratory problems, concerns about fertility and sexuality, growth hormone production problems, thyroid deficiency, sleep problems, learning and school performance difficulties, etc. (all of which can continue for years), may be demoralizing to the patient and family that expected a return to more normal and healthy function, and may adversely impact on their daily functioning and relationships. Finally, the possibility of relapse and death are concerns that patients and families face almost continually. These long-term challenges to the pediatric HSCT patient and their families are deserving of significantly more research attention and resource allocation.

PSYCHOSOCIAL OUTCOMES OF PEDIATRIC HSCT Neurocognitive Impact Concern about the impact of HSCT on the neurocognitive (intellectual, learning, perceptual, information processing, and related) abilities of children stems from two possible causes. First, the illness and treatment typically involve long periods of time during which the child is removed from the academic environment and from other environments (social, clubs, teams, family, etc.) in which children learn critical information, such as basic facts, problem-solving, vocabulary, and reasoning. Even during periods when the child is not being treated, the effects of the illness, such as weakness and nausea, and required isolation precautions cause some restriction from daily learning activities. In general, these effects involve deprivation of experience rather than a direct physiological effect of the illness or treatment involved in HSCT (i.e., they are not a direct effect of the HSCT). Nevertheless, the impact of deprivation of experience can be great and should not be ignored. For example, children are often returned to school following their illness and HSCT without any testing to determine their current achievement level; this lack of knowledge can contribute to inappropriate academic placement and teaching. Alternatively, interventions to minimize the effects of deprivation on learning, such as meetings with a teacher/tutor in the hospital, integration of academic activities into the child’s hospital schedule (as much as the child’s medical status allows), and exposure to social activities, whether in a playroom, over a video link, or on the computer may produce a significant beneficial effect for the child. A second area of concern about the impact of HSCT on neurocognitive functioning involves the potential effects of the illness or components of the HSCT treatment on the developing nervous system. Illness effects on neurocognitive functioning vary considerably with the type, location, onset age, and severity of the illness (10–12) and are not a result of the HSCT per se. In fact, the beneficial medical impact of HSCT may slow or stop some of the negative effects of illness on neurocognitive functioning (13). However, because HSCT is accompanied by high doses of chemotherapy, radiation, or both during the course of treatment, there is a need to monitor and evaluate the effects of these potentially damaging events on the nervous system and on neurocognitive functioning. CNS radiation therapy and intrathecal chemotherapy have received the most attention for potentially damaging neurocognitive effects. There is considerable evidence, for example, that

238

Carter et al.

both radiation and chemotherapy have damaging effects on the central nervous system (CNS) (12,14,15). Scientific advances in the type, method, and magnitude of delivery of radiation and chemotherapy continue to evolve, however, and therefore the effects on the CNS may be changing with advances in treatment. Most studies of CNS radiation or intrathecal chemotherapy treatment report some impact on neurocognitive functioning, consistent with findings of CNS damage. However, the type, magnitude, and duration of the neurocognitive changes vary with characteristics of the person, disease, and treatment (12,16). Although most of these studies do not involve HSCT, the literature can be applied to cases of HSCT in which there is some CNS-based treatment. Despite the variability in outcomes reported, some consistent findings have emerged. Compared to intrathecal chemotherapy, CNS radiation has shown more significant impact on both neurological and neuropsychological functioning (14). In fact, some studies have shown minimal or no impact of chemotherapy on neurocognitive functioning (17), although the consensus view is that intensive intrathecal chemotherapy is associated with risk of neurocognitive effects in a large proportion of children. (Butler and Mulhern (14) estimate this proportion at 30% or greater.) Short-term effects of CNS radiation, which may occur immediately to several months after treatment, include attention problems, fatigue, slowed processing speed, and difficulty focusing, which are frequently attributed to white matter disruption (12,14). Short-term neurocognitive effects of intensive intrathecal chemotherapy can range from mild (e.g., ataxia, somnolence, weakness) to severe (e.g., seizures, neuropathies, hemiplegia, hearing loss), with higher doses of more toxic medications generally associated with the more severe effects (15,16). Longer-term and delayed effects of CNS radiation and intrathecal chemotherapy on neurocognitive functions have also been identified. These effects may emerge months or years after treatment and can be persistent. In particular, complex psychomotor skills, attention and concentration, fluency/speed, working memory, arithmetic (computational) skills, and visualorganizational skills appear to be particularly vulnerable, whereas language, fund of information, and verbal academic skills appear to be minimally (if at all) affected (14,15,18). Picard and Rourke (15) attribute much of this discrepancy in affected neurocognitive functioning to the susceptibility of white matter to dysfunction following CNS treatment. This explanation is supported by research demonstrating a connection between white matter dysfunction, CNS treatments (both radiation and chemotherapy), and age at onset of treatment (12). Such functions as motor skills, attention, processing speed, and visual-organization tend to be more white matter-dependent, whereas language and other crystallized abilities are less dependent on white matter functioning (19,20). This pattern of strengths and weaknesses seen following CNS radiation and/or chemotherapy fits the description of a nonverbal learning disability (13,15). Although it is possible to see neurocognitive effects shortly after the treatment, the effects are frequently more apparent weeks or months later, reflecting a delayed effect that may be due to cumulative effects on white matter over time. Other studies support the view that complex psychomotor skills, attention/concentration, memory, and visual-organizational skills are especially at risk when HSCT involves CNS radiation or intrathecal chemotherapy treatment. Tasks that require both complex processing (e.g., choices, associations) and speed are impaired in children who have undergone HSCT with CNS treatment (21). This latter type of processing demands more integrative, attentional, and organizational processes. Additionally, children who had HSCT and cranial radiation treatment have been found to be more socially passive and withdrawn, which are adjunctive characteristics of a nonverbal learning disability (22). As noted earlier, neurocognitive outcomes vary somewhat by individual characteristics. Younger age at diagnosis (especially before 3 or 4 years of age) and initiation of treatment are often associated with poorer neurocognitive outcomes, particularly in the case of CNS radiation treatment. Girls may also be at higher risk for neurocognitive deficits. Some studies suggest that neurocognitive problems may worsen with time since CNS radiation treatment. These deficits sometimes do not emerge immediately after treatment but may be seen months or years later (12,14).

Psychological Dimensions of Pediatric HSCT

239

In summary, although a range of neurocognitive outcomes are seen in children who have undergone HSCT, those at greatest risk for neurocognitive impact are the ones for whom treatment involves CNS radiation or (to a lesser extent) intensive intrathecal chemotherapy. For those with intensive CNS-based treatments, short-term and long-term neurocognitive deficits are frequently seen, with attention, concentration, complex psychomotor skills, visual-organization, processing speed, and working memory skills at particular risk. However, these conclusions must be taken in the context of the changing nature of medical treatment and the need for more HSCT-focused studies (many studies focus on CNS-based treatments and not directly on HSCT per se).

Psychosocial Outcomes Individual The severity and uniqueness of the stresses experienced by the child and family from the preHSCT through the post-HSCT phases have been cited as primary reasons for the adjustment problems seen in children undergoing HSCT (23–25). Children and their families confront numerous stresses during each stage of “bone marrow transplantation,” with prominent stresses arising from the demands of the child’s illness and the rigorous nature of the HSCT protocol. Uncertainty about survival, pain, nausea, and isolation are stresses that may be experienced before, during, and after HSCT (24,26,27). McConville and colleagues (28) found that although many children appear to adjust positively following HSCT, particularly children with relatively few complications during and in the months following HSCT, most children experience mild to moderate levels of psychological symptoms during the HSCT process. Indeed, survivors of childhood cancers who have not been treated with HSCT have been found to experience mood and behavioral disturbances and psychological adjustment problems (29,30). The additional stressor of HSCT could be expected to significantly increase this risk of adjustment problems. In a one-year longitudinal follow-up of post-HSCT pediatric patients, Phipps and colleagues found significant declines in social competence, overall self-confidence, and general well-being (31,32). Prior to HSCT, perceptions of family conflict had a moderate inverse correlation with patient adjustment post-HSCT. Phipps and Mulhern (32) identified three general factors that predicted adjustment among post-HSCT pediatric patients: child disposition/temperament/personality/intelligence; family environment (closeness, warmth, support); and external support from at least one extra-family source. Children’s difficulties with psychological adjustment following treatment for cancer and post-HSCT, as well as that of their parents (33), have been described as consistent with the diagnostic criteria for post-traumatic stress disorder (PTSD). In a study by Stuber and colleagues (25), symptoms of PTSD were identified in pediatric HSCT patients up to 12 months post transplant. The PTSD symptoms seen in these pediatric HSCT patients were different than that seen in children who experienced PTSD resulting from a single violent threat or experience, i.e., PTSD symptoms of denial and avoidance were most commonly observed rather than symptoms of arousal and hypervigilence. In a study by Parsons (34), parental ratings of their child’s general adjustment and quality of life were significantly lower than the ratings made by the children themselves on selfassessment scales. Regardless of the child’s disease severity rating, the parents viewed their child as having more behavior and adjustment difficulties, whereas child self-report was more consistent with disease severity rating. Although Barrera and colleagues (35) found pediatric HSCT patients’ behavioral adjustment to generally remain in the normal range during the HSCT process, poor pre-HSCT family adjustment and child pre-HSCT adaptive functioning was associated with greater likelihood of behavioral difficulties during HSCT. Children’s overall quality of life appeared to improve six months following HSCT and was most significantly correlated with level of family cohesion prior to the HSCT.

240

Carter et al.

Family Adjustment The waiting period prior to the child’s admission for HSCT may be the most stressful time for families (36). Pre-HSCT family environment variables were found to be highly predictive of family and patient adjustment post-HSCT in a study by Phipps and Mulhern (35). Specifically, family expressiveness, cohesion, and perceptions of family conflict were important predictors of post-HSCT adjustment. Perceived family cohesion and expressiveness served as a resilience factor to protect the pediatric patient from the stresses associated with HSCT. Major negative life stressors experienced by the parent and the use of avoidant coping strategies significantly predicted pre-HSCT adjustment problems. Kronenberger, Carter, and colleagues (6) found that, in the period awaiting the child going for HSCT, up to 15–25% of the pediatric patients and parents reported experiencing significant levels of psychological distress. “Bone marrow transplantation” requires a major adjustment for the entire family. Frequently, in a matched related donor transplant, this medical procedure requires two family members to undergo a major medical procedure, i.e., bone marrow harvest for the donor and HSCT for the patient. Additionally, family life may become significantly altered for the siblings of the pediatric HSCT patient. Siblings often report feeling “overlooked” at a time when they are experiencing increased levels of stress and have to make frequent adjustments due to the demands of their seriously ill sibling’s condition. They may report a variety of emotional reactions, including isolation from their parents, feelings of jealousy towards their ill sibling, loneliness, and anxiety (8). Often the numerous needs of the patient require so much parental time and energy that the siblings feel neglected. These feelings may be intensified by the guilt and responsibility they feel for even having these reactions. The experience of having a sibling diagnosed with a potentially life-threatening illness and undergoing HSCT, along with feelings of isolation and misunderstanding, have been found to result in acute or chronic stress reactions in up to one-third of siblings (37). The risk for increased anxiety and adjustment problems may be enhanced for sibling donors (37–39). For a sibling donor in a matched related transplant, being eligible as a potential donor may have both positive and negative implications. Siblings who have been selected as the donor have been found to exhibit increased symptoms of anxiety and low self-esteem, while nonselected siblings have been noted to have increased behavioral and school difficulties (37). If the patient relapses or dies, the donor sibling may experience feelings of guilt or responsibility that their donated marrow did not save their sibling’s life. Despite these concerns about sibling adjustment, there has been little systematic study of the impact of sibling adjustment on the adjustment of the child undergoing HSCT (or vice-versa). In families with a chronically and/or seriously ill child, mothers have been shown to carry the majority of physical and emotional responsibility in caring for the ill child. In examining the psychological well-being of mothers, improvement in the mothers’ psychological adjustment was associated with their child’s physical improvement and quality of life (35). Approximately 8% of mothers whose child was undergoing HSCT experienced clinical levels of depression, which is consistent with the clinical rate of depression in the general adult population (35). The marital relationship can be strained while a child endures the HSCT process, as parents are often required to spend extended periods of time apart while one parent remains with a child in the hospital with the other attempting to maintain normalcy on the home front for the siblings. Heiney and colleagues (40) have characterized many parents of children undergoing HSCT as experiencing post-traumatic stress reactions. However, although the family of a pediatric HSCT patient must endure significant stress and challenges to coping and adjustment, many families appear to handle this complex and demanding medical procedure with strength, hope, and success, attesting to the resiliency of the human spirit in the most difficult of circumstances (2).

Peer Adjustment Social isolation from experiences with persons other than family members, so important for social development, is typically required following HSCT to assure the patient’s recovery while

Psychological Dimensions of Pediatric HSCT

241

in an immune compromised state. Experiencing a childhood cancer and enduring the treatment, particularly the HSCT, can lead to changes in appearance, as well as cognitive and physical abilities (41,42). Appearance changes can include hair loss, skin changes, and growth delays. Children who have survived HSCT and returned to school are often perceived by their peers as less able and frequently sick (22). Without education within the schools, peers may experience fear and misunderstanding about cancer and may select to distance themselves from the patient. Although pediatric HSCT patients may tend to be less aggressive or disruptive than healthy students, they face potentially significant isolation and the misunderstanding of their peers. Vannatta and colleagues (22) found that pediatric HSCT patients were frequently liked by their peers but were less likely to be chosen as close or best friends. Peers often reported that they found pediatric HSCT patients less attractive and athletically skilled than their peers. It appears that the peers of the child who has undergone HSCT may remain distant, further isolating the child. Vannatta and colleagues speculate that the initial reasons for diminished peer contact (i.e., hospitalization) and negative reactions of peers (to appearance changes and decreased energy and physical stamina) lead to altered social self-perceptions, attributions, and expectations on the part of the HSCT patient. This may then lead the child to decrease efforts at initiating social interactions and other social behaviors that serve to sustain relationships. A vicious cycle begins whereby the patients may feel isolated and unsure of themselves socially. In response, their peers begin to perceive them as wanting or needing to be alone, further confirming the patient’s feelings of isolation and encouraging greater social withdrawal.

INFLUENCES ON ADJUSTMENT TO BONE MARROW TRANSPLANTATION Although HSCT presents children and families with substantial stress, there is a wide range of psychological adjustment shown in this population (6,28,43). This variability in adjustment within populations of individuals experiencing stress in general, and chronic illness in particular, has been well established, leading to the development of many theoretical models explaining individual differences in stress responses (44). These models typically identify illness characteristics, other life stressors, cognitive appraisal of stressors, coping characteristics, family environment, social support, personality/temperament, cognitive abilities, and genetics as major contributors to how the stresses of illness and treatment eventually affect the adjustment of individuals (44). Rather than operating as static, unchanging influences, these characteristics change and interact with each other throughout the development of the individual who undergoes HSCT. For example, the illness and treatment associated with HSCT act as stressors that the child and family appraise as being threatening or harmful. They react by attempting to cope with the stressor in order to reduce negative emotional and behavioral responses. Coping requires the accessing of resources within the self, family, and community. Personal and genetic characteristics may provide strengths or vulnerabilities that influence which emotional and behavioral responses are most likely to occur under stress. Several studies have sought to apply this model of adaptation to pediatric HSCT, finding that differences in stressors, coping, family characteristics, and pretransplant personality characteristics of the child can have a significant impact on how the child and family adjust to HSCT.

Illness Stressors Objective indices of disease stress (type of transplant, objective measures of severity of disease, etc.) are rarely found to be related to child adjustment (6,45), probably because the individual’s appraisal of the stress is more important for adaptation than is the actual stress that is experienced (44). However, one major exception to this finding is a strong relationship between CNS radiation treatment and increased internalizing behavior problems, such as inhibition,

242

Carter et al.

anxiety, withdrawal, and social passivity (22,46). CNS radiation treatment (and other CNS based treatments) may exert an indirect influence on behavior through its neurological effects, as opposed to through stress appraisal per se; therefore, it may operate in a different fashion than do the other medical stressors associated with HSCT. For example, Thompson and Gustafson (44), in an extensive literature review, found that children with chronic illness conditions involving the CNS consistently showed more behavior problems (especially inhibition and internalizing problems) than children with other chronic illness conditions. This finding suggests that disease stressors that involve the brain may be qualitatively different (in relationship to child behavior and adjustment) than other disease stressors. Although objective disease stressors (other than CNS-related stressors) typically are minimally related to child adjustment, the child’s appraisal of these stressors as threatening or harmful may show a stronger relationship to adjustment. Child appraisal of stress has been the subject of very little research, but one study showed that children with lower self-esteem following HSCT had lower levels of social competence (46). The functional impact of stressors may also have an influence on adjustment, when stressors prevent the child from accessing pleasant or important social activities. Vannatta and colleagues (22) found that changes in appearance and athletic abilities were related to reduced acceptance and more isolation from the peer group.

Coping Coping describes the process by which the individual attempts to reduce the negative effects of stress by either changing characteristics of the stressful situation (problem-focused coping) or by changing the individual’s emotional response (emotion-focused coping). Coping is therefore critical in determining how the individual adjusts to stress and whether problems arise. Given the centrality of coping in the adjustment process, it is therefore surprising that relatively few studies have focused on the coping behaviors of children undergoing HSCT. Mirroring other illness conditions, the findings of one study indicated that avoidant coping (disengaging from the situation; using denial and avoidance of things related to the HSCT) is related to more emotional and behavioral problems in children during the immediate pretransplant phase of HSCT (47). Use of religious beliefs as a means to cope with the condition and with the HSCT was related to less withdrawal and possibly to less aggression and depression (6).

Family Influences Characteristics of the family environment, parent adjustment, and interactions between family members appear to be very important in the child’s psychological adjustment before, during, and after HSCT. Higher levels of family conflict are related to child adjustment problems during all phases of transplantation (28,32,47,48). For example, Carter and colleagues (47) found that family conflict prior to HSCT was related to more withdrawal and less positivesocial behavior in children hospitalized for HSCT, and McConville and colleagues (28) reported a relationship between family maladjustment and unexpected medical complications (including unexpected death) following HSCT. Family support (positive emotional interchanges, sense of cohesion, and team spirit), on the other hand, appear to exert a positive influence on adjustment. Children from more supportive families have been found to be less withdrawn during HSCT (47) and more welladjusted following HSCT (32). This impact of family support may occur only during the periods of the most intensive stress (e.g., during hospitalization for HSCT), with family support less strongly related to child adjustment during the somewhat less stressful period prior to transplant (32). Other studies have looked specifically at the impact of parent behavior and parent adjustment on child adjustment during HSCT. Distress in parents (typically mothers have been studied) has been found to be related to emotional problems in children before transplantation

Psychological Dimensions of Pediatric HSCT

243

(6), although one study found that parent distress prior to transplantation did not predict the child’s adjustment during transplantation (47). Levels of stress on mothers of children undergoing HSCT (particularly the presence of other major life stresses in addition to HSCT) predict child adjustment prior to and during HSCT (6,47). Because most studies of parent adjustment have focused on mothers, it is not known whether these results generalize to fathers. For example, one early study found that increased paternal psychopathology was related to the child’s having more serious unexpected medical complications during and following HSCT (including unexpected death); however, the relationship was in the opposite direction for maternal psychopathology. Decreased maternal psychopathology was related to unexpected medical complications (28). Because of the importance of family adaptation for the child’s adjustment to HSCT (as well as the desire to help all family members adjust as well as possible to the stress of HSCT), several studies have looked at factors influencing the adaptation of the family (often focusing on the parents) before, during, and after child HSCT. These studies have found high rates of parental distress, with approximately 30–50% of mothers meeting clinical criteria for clinical distress before, during, and after their child’s HSCT (43,45). Levels of stress (ranging from major life stresses to daily hassles, social stresses, and lower socioeconomic status) appear to have a very strong impact on parental adjustment (43,45). However, as with children, family support may exert a positive influence only at the times of greatest stress during and immediately following the child’s hospitalization for HSCT. Kronenberger and colleagues (43), for example, found minimal relationships between family support and maternal depression/worry prior to transplantation, and McConville (28) and colleagues found that better parent adjustment during and following transplantation was promoted by having “a reliable support person” (p. 771). Parent coping may also be strongly related to adjustment, with disengagement coping (avoiding, denying, refusing to actively engage the situation) correlated with poorer adjustment in mothers of children preparing to undergo HSCT (43). Conversely, strong beliefs and “a consistent philosophy of life” (28) may predict better adaptation in parents.

Premorbid Temperament/Personality Like all children, children undergoing HSCT have a variety of temperamental and personality traits that often predate their condition and that may reflect genetic/biological contributions. These traits may create vulnerabilities for maladjustment or strengths for adaptation. For example, a child who tends to be sensitive and hyperreactive is more likely to react to the stress of HSCT with anxiety than is a child who tends to be more calm and unreactive to stresses. Several studies have offered strong support for the hypothesis that personality, temperament, and preexisting behaviors have a significant impact on the child’s adaptation to HSCT. Carter and colleagues (47) found that behaviors shown at home prior to HSCT were strongly predictive of child adjustment during the hospitalization for HSCT. Specifically, children with more externalizing behaviors (aggression, oppositionality, rule-breaking) in the home environment prior to transplantation were more likely to be oppositional, noncompliant, and withdrawn when in the hospital for HSCT. Pretransplant anxiety, somatization, and other internalizing problems have also been found to be related to later adjustment problems during and following HSCT (46,48). Although studies find a relationship between preexisting behaviors and adaptation to HSCT, the reason for this relationship is unclear. Presumably, these behaviors reflect, in part, the influence of personality or temperament, but family environment and parental modeling are likely to influence them as well. Hence, more study is needed to determine whether genetic, temperamental, personality, and/or environmental factors are responsible for the observed association between preexisting behavior and adaptation to HSCT.

244

Carter et al.

Sociocultural Factors Maloney, Clay and Robinson (49) have proposed a “sociocultural transplant experience model” that addresses the influence of age, sex, residence, disability, language, ethnicity, race, spirituality, SES, and other cultural factors on the different phases of the pediatric transplantation process. Sociocultural factors are viewed as moderating the experience of the pediatric patient and family and potentially impacting health, medical, and psychosocial outcomes. Although much of the preliminary research on the influence of these factors has been on patients receiving solid organ transplants, there are obvious implications for pediatric bone marrow transplantation. For example, patients/families who live in very remote areas often experience limited access to family and community social support during the HSCT treatment process while in the hospital and increased anxiety post-HSCT due to limited access to specialized services in their local community. Patients in families of lower SES may lack adequate in-home support post hospitalization, due to the need for the parent(s) to work to meet financial demands, which may impact adversely on their opportunity for parental tutoring to catch up on missed school work, parental supervision of medication adherence, and enhance the child’s feelings of social isolation.

Summary of Influences on Adjustment The previous review suggests that stressors, coping, family influences, personality/temperament, and sociocultural factors are all probable influences on the child’s (and family’s) adjustment to HSCT. Family stressors and CNS treatment appear to be the most significant known stress-related influences on adjustment, whereas objective illness indices (other than CNS-related) are minimally related to adjustment. Avoidant coping is related to maladjustment in both child and parent, but using a strong belief/value system (such as that found in religion) has been related to better adaptation. Family conflict, parent stress, and parent psychopathology predict adjustment problems, whereas family support appears to act as a buffer protecting the child from the deleterious effects of stress. Finally, pretransplant behaviors and temperamental characteristics predict behavioral adaptation during and after transplant. Monitoring and interventions directed at these key areas may offer some potential for improving psychological adjustment and medical compliance.

PSYCHOSOCIAL INTERVENTION Increasing emphasis has been placed on the development of evidence-based interventions to facilitate coping, adherence and short- and long-term adjustment and functioning in the survivors of pediatric cancers (50). However, rather than focusing on patient/family deficits and the presence of psychopathology, there needs to be a priority given to prevention and enhancement of strengths, competencies and supports.

Coping/Adjustment Very little investigative research has been done on structured psychosocial interventions with pediatric HSCT patients to enhance coping and adjustment. Much of the psychosocial intervention research literature within the more general area of pediatric oncology has focused on behavioral interventions to reduce anxiety and problems with nausea and vomiting associated with the toxic effects of chemotherapy (51,52). Although such interventions as relaxation training, imagery with positive suggestions, and distraction techniques have proven effective in reducing anticipatory and postchemotherapy nausea with many pediatric cancer

Psychological Dimensions of Pediatric HSCT

245

patients (53), they have largely been replaced by a new generation of more effective antiemetic, antidepressant, and anxiolytic medications that have a low incidence of side effects. Kazak and colleagues (54) have developed the surviving cancer competently intervention program (SCCIP) for pediatric cancer survivors and their families, which likely has direct applicability to those patients who have undergone HSCT. This four-session, one-day program targets patient and family distressing beliefs about cancer, and the current and likely future impact of the disease on the child and family, including siblings.

Adherence A major area of interest and concern in psychosocial functioning of pediatric patients with serious and chronic health conditions is in adherence to complex treatment regimens (55). Even when the treatment regimen adherence during HSCT is fairly tightly regulated by the strict controls of the HSCT team and unit nurses, patients can still have problems with adhering to treatments, such as the oral administration of medications. Phipps and DeCuir-Whaley (23) found that more than half of pediatric patients undergoing HSCT had significant adherence problems with taking oral antibiotics, particularly among elementary school age and preschool children, which is medically necessary to maintain gastrointestinal and oral-nasal sterility during their immunocompromised status. Post-HSCT adherence is a dynamic process that often requires subsequent regimen modifications and complex interactions of patients, parents, and the HSCT team. Indeed, strict adherence has been shown to decrease over time in most chronic pediatric illnesses (56,57). Post-HSCT patients, their parents, and health care professionals must work out interactions to facilitate compliance with a host of factors: frequent posthospitalization follow-up appointments and specialist consultations; monitoring of immune function and avoidance of public settings due to danger of opportunistic infections; recovery/prevention diets; minimizing sun exposure; modified exercise routines; oral care routines; central line care; and involved medication regimens. Noncompliance by post-HSCT pediatric patients may stem from a number of factors, including misunderstanding, miscommunication, child/adolescent attempts to exert some semblance of control over their lives and fears, patient testing of the limits to see what impact it will have on their health, and a desire by the patient to be “normal” in the eyes of their peers (58). However, noncompliance can have life-threatening consequences for the patient and requires serious monitoring and intervention.

Family Increasingly, pediatric behavioral health professionals are realizing that family involvement to maximize social support, and active family intervention, are needed to address the multitude of psychosocial concerns facing the pediatric patient with serious life-threatening illness (59). Kazak (60) has described a pediatric oncology consultation model based on family systems and developmental theories and enumerated the systems barriers to such a program. This comprehensive model, employed at Children’s Hospital of Philadelphia, engages the patient’s family from point of referral to outcome, emphasizing a collaboration between family and the oncology team in the recurring process of problem definition, reframing of symptoms, empowering competence in the family, and facilitating healthy patterns of interaction among the “triad” of the family, child, and staff. A longitudinal, multidisciplinary, supportive-consultative approach is advocated by McConville and colleagues (28). In light of the findings of the importance of family variables (support, positive emotional interchanges, sense of cohesion, team spirit, etc.) on the child’s adjustment during and after HSCT (32,47), family-based interventions should be employed throughout the HSCT process and for at least the following year, if not longer.

246

Carter et al.

Educational/Academic Frequently the pediatric HSCT patient will require educational accommodations due to the extensive school absence the child undergoing HSCT must experience and, for some, the neurocognitive effects of intrathecal chemotherapy and/or cranial radiation. This may be particularly true for children who were transplanted at the three-year-or-younger age (61). These accommodations may range from retention in a grade in order to catch up to their peers, increased time on test and projects, and reduced workload to learning disabilities resource assistance, and placement. Formal psycho-educational and neurocognitive assessment pre- and post-HSCT are important to assure that the child receives the necessary services and accommodations to prevent discouragement, the development of a negative academic selfimage, and possible social maladjustment.

Neurocognitive Interventions Although considerable intervention efforts have been directed at emotional and social adjustment, an encouraging literature suggests that the neurocognitive status of some affected children may be improved with some treatments. These neurocognitive interventions fall into two categories: educational-behavioral rehabilitation treatments and medication treatments. Educational-behavioral rehabilitation treatments consist of intensive, repetitive practice of key neuropsychological behaviors, thought processes, functions, and strategies. These include attention, organization, self-monitoring, and use of external devices (62). Medication interventions are symptom focused, usually targeting attention problems with psychostimulant medication (most research has investigated methylphenidate). Promising results have been reported in clinical trials for both types of these interventions (14).

GRIEF AND LOSS “Bone marrow transplantation” provides the possibility of cure of many conditions that were previously untreatable or minimally treatable. As such, it offers hope to many individuals who otherwise would have little hope. Along with this hope, however, is the possibility of real or possible loss that may range from functional losses to death. The possibility of this loss is one of the most difficult and stressful parts of HSCT. Children show considerable variability in their adjustment to current or impending loss. In part, this is related to age and developmental stage. For example, at younger ages, children’s conceptualizations of death are based on more concrete thought; it is not until early adolescence that children begin to provide abstract explanations and to pose abstract questions about death. Concepts of death as irreversible, final, inevitable, and caused by a pathogenic process are much more rare at early school ages than at adolescence, with increasing awareness of these concepts as the child’s abstract/conceptual thinking grows. Hence, a preschool child may be coping with loss or fears of death as a reversible, changing (e.g., not inevitable), unpredictable event, leading to a very different reaction than that of an adult. In fact, many very young children react to death or loss as they would react to any temporary separation or loss. Despite extensive speculation, there has been relatively little empirical study of the family grief process during and following HSCT, other than the family and child adjustment studies reviewed earlier. Even less has been studied about family reaction, adjustment, and coping following the death of a child following treatment with HSCT. Inferring from other sources of information about family reactions to death of a child, it seems likely that families show a very wide range of long-term adjustment, ranging from post-traumatic stress disorder and severe depression to severe grief that remits (albeit slowly and incompletely) with time. However, the short-term adjustment of families to death of a child tends to be more uniform and

Psychological Dimensions of Pediatric HSCT

247

involves periods of denial, cycling of strong emotional states (anger, sadness) with numbness, dealing with existential questions (meaning of life, life after death), juggling ongoing daily life responsibilities (including caring for other children in the family) with the debilitating impact of grief, and coming to terms with a new life (including assumptions about the world and life) that does not include the child. The impact of these issues is staggering, and family members are often in great need of support from friends and from each other. Strong beliefs and coping strategies also often seem to be important to prevent significant long-term psychological deterioration.

CARING FOR THE PROFESSIONAL AND PSYCHOSOCIAL TEAM There are many potential sources of burnout for HSCT team members. These include the intensity of patient and family medical and psychosocial needs, high acuity of illness parameters, the complex continuing educational needs of HSCT health care providers due to the rapid rate of innovation in treatments, and the quick transition from aggressive therapy to caring for the dying patient, to name just a few (2,63). Burnout prevention requires a host of preventive policies and practices to be in place. The importance of regular multidisciplinary team meetings cannot be overemphasized. This is often the most important vehicle for avoiding miscommunication and misunderstanding between members of the HSCT team, the patient and family. This also necessitates clear communication among team members to avoid the “splitting” that may occur when patients and families, under the high levels of stress associated with HSCT, tend to elicit conflicting differential responses from team members pursuant to their need to experience some sense of control (64). This process can be highly destructive to both the HSCT team integrity and patient care during the HSCT and post-HSCT phases, and regular team meetings can serve to prevent this situation from developing. It is important that support services be available, both formal and informal, to team members due to the highly stressful nature of the work with pediatric HSCT patients and their families. This should include on-site and off-site continuing education classes to directly address stress management and bereavement concerns, as well as employee assistance services and professional referral. Staff participation in clinical unit decision making is also important to give team members a sense of control and fellowship with their HSCT team colleagues. Mentoring programs can serve to link less experienced staff with more seasoned professionals who have learned to manage the stress of this demanding setting. Finally, periodic formal and informal evaluation of staff stress levels can serve to alert senior HSCT staff to individuals who may be in need of specific assistance to manage the stress of this rewarding yet highly demanding work.

REFERENCES 1. Patenuade AF, Kupst MJ. Psychosocial functioning in pediatric cancer. J Pediatr Psychol 2005; 30:9–27. 2. Bishop M, Welsh H, Coons M, Wingard J. Blood and marrow transplantation. In: Rodrigue J, ed. Biopsychosicial Perspectives on Transplantation. New York: Kluwer Academic/Plenum, 1996:19–37. 3. Dermatis H, Leske LM. Psychosocial correlates of physician-patient communication at time of informed consent for bone marrow transplantation. Cancer Invest 1999; 9:621–628. 4. Fogarty LA, Curbow B, Wingard JR, McDonnell K, Somerfield MR. Can 40 seconds of compassion reduce patient anxiety? J Clin Oncol 1999; 17:371–379. 5. Streisand RM, Tercyak KP. Evaluating the pediatric transplant patient: general considerations. In: Rodrigue J, ed. Biopsychosocial Perspectives on Transplantation. New York: Kluwer Academic/Plenum, 2001:71–92.

248

Carter et al.

6. Kronenberger WG, Carter BD, Stewart J, et al. Psychological adjustment of children in the pretransplant phase of bone marrow transplantation: relationships with parent distress, parent stress, and child coping. J Clin Psychol Med Settings 1996; 3:319–335. 7. Walch S, Achles TA. Psychosocial effects: pretransplant and acute treatment phase. In: Whedon MB, Wujcik D, eds. Blood and Marrow Stem Cell Transplantation. Boston: Jones & Bartlett, 1997:135–147. 8. Freund BL, Siegel K. Problems in transition following bone marrow transplantation: psychosocial aspects. Am J Orthopsychiatry 1986; 56:244–252. 9. Bishop M, Welsh H, Coons M, Wingard J. Blood and marrow transplantation. In: Rodrigue J, ed. Biopsychosocial Perspectives on Transplantation. New York: Kluwer Academic/Plenum, 1996:19–37. 10. Kennedy CK. Brain Tumors. In: Maria BL, ed. Current Management in Child Neurology. 2nd ed. Hamilton, Ontario: BC Decker, 2002:288–304. 11. Kolodny EH. Degenerative disorders. In: Maria BL, ed. Current Mangement in Child Neurology. 2nd ed. Hamilton, Ontario: BC Decker, 2002:405–416. 12. Moore BD. Neurocognitive outcomes in survivors of childhood cancer. J Pediatr Psychol 2005; 30:51–63. 13. Shapiro EG, Lipton ME, Krivit W. White matter dysfunction and its neuropsychological correlates: A longitudinal study of a case of metachromatic leukodystrophy treated with bone marrow transplant. J Clin Exp Neuropsychol 1992; 14:610–624. 14. Butler RW, Mulhern RK. Neurocognitive interventions for children and adolescents with cancer. J Pediatr Psychol 2005; 30:65–78. 15. Picard EM, Rourke BP. Neuropsychological consequences of prophylactic treatment for acute lymphocytic leukemia. In: Rourke BP, ed. Syndrome of Nonverbal Learning Disabilities: Neurodevelopmental Manifestations. New York: Guilford, 1995:313–328. 16. Packer RJ, Meadows AT, Rorke LB, Goldwein J, D’Angio G. Long-term sequelae of cancer treatment on the central nervous system in childhood. Med Pediatr Oncol 1987; 15:241–253. 17. Copeland DR, Moore BD, Francis DJ, Jaffe N, Culbert SJ. Neuropsychologic effects of chemotherapy on children with cancer: a longitudinal study. J Clin Oncol 1996; 14:2826–2835. 18. Moleski M. Neuropsychological, neuroanatomical, and neurophysiological consequences of CNS chemotherapy for acute lymphoblastic leukemia. Arch Clin Neuropsychol 2000; 15:603–630. 19. Pennington BF. Diagnosing Learning Disorders: A Neuropsychological Framework. New York: Guilford, 1991. 20. Rourke BP. The NLD syndrome and the white matter model. In: Rourke BP, ed. Syndrome of Nonverbal Learning Disabilities: Neurodevelopmental Manifestations. New York: Guilford, 1995:35–49. 21. Brouwers P, Riccardi R, Poplack D, Fedio P. Attentional deficits in long-term survivors of childhood acute lymphoblastic leukemia. J Clin Neuropsychol 1984; 6:325–336. 22. Vanatta K, Zeller M, Noll RB, Koontz K. Social functioning of children surviving bone marrow transplantation. J Pediatr Psychol 1998; 23:169–178. 23. Phipps S, DeCuir-Whaley S. Adherence issues in pediatric bone marrow transplantation. J Pediatr Psychol 1990; 15:459–475. 24. Pot-Mees CC. The psychosocial effects of bone marrow transplantation. Delft, the Netherlands: Eburon, 1989. 25. Stuber ML, Nader K, Yasuda PL, Pynoos RS, Cohen S. Stress responses after pediatric bone marrow transplantation: preliminary results of a prospective longitudinal study. J Am Acad Child Adolesc Psychiatry 1991; 30:952–957. 26. Cogliano-Shutta N, Broda E, Geiss J. Bone marrow transplantation: an overview and comparison of autologous, syngeneic, and allogeneic treatment modalities. Nurs Clin North Am 1985; 20:49–66. 27. Phipps S. Bone marrow transplantation. In: Bearison DJ, Mulhern RK, eds. Pediatric Psychooncology: Psychological Perspectives on Children with Cancer. New York: Oxford University Press, 1994:97–113. 28. McConville BJ, Steichen-Asch P, Harris R, et al. Pediatric bone marrow transplants: psychological aspects. Can J Psychiatry 1990; 35:769–775. 29. Chen E, Zelter L, Bentler P, et al. J Health Psychol 1998; 3:23–38. 30. Kazak A. Implications of survival: pediatric oncology patients and their families. In: Bearson DJ, Mulhern RK, eds. Pediatric Psychooncology. New York: Oxford University Press, 1994:171–192.

Psychological Dimensions of Pediatric HSCT

249

31. Phipps S, Brenner M, Heslop H, Krance R, Jayawardene D, Mulhern R. Psychological effects of bone marrow transplantation on children and adolescents: preliminary report of a longitudinal study. Bone Marrow Transplant 1995; 15:829–835. 32. Phipps S, Mulhern RK. Family cohesion and expressiveness promote resilience to the stress of pediatric bone marrow transplant: a preliminary report. J Dev Behav Pediatr 1995; 16:257–263. 33. Best M, Streisand R, Catania L, Kazak AE. Parental distress during pediatric leukemia and posttraumatic stress symptoms (PTSS) after treatment ends. J Pediatr Psychol 2001; 26:299–308. 34. Parsons SK, Barlow SE, Levy SL, Supran SE, Kaplan SH. Health-related quality of life in pediatric bone marrow transplant survivors: according to whom? Int J Cancer Suppl 1999; 12:46–51. 35. Barrera M, Boyd Pringle LA, Sumbler K, Saunders F. Quality of life and behavioral adjustment after pediatric bone marrow transplantation. Bone Marrow Transplant 2000; 26:427–435. 36. Streisand RM, Rodrigue JR, Houck C, Graham-Pole J, Berlant N. Brief report: parents of children undergoing bone marrow transplantation: documenting stress and piloting a psychological intervention program. J Pediatr Psychol 2000; 25:331–337. 37. Packman WL, Crittenden MR, Schaeffer E, et al. Psychosocial consequences of bone marrow transplantation in donor and nondonor siblings. J Dev Behav Pediatr 1997; 18:244–253. 38. Kinrade LC. Preparation of sibling donors for bone marrow transplant harvest procedure. Cancer Nurs 1987; 10:77–81. 39. Pot-Mees CC, Zeitlin H. Psychosocial consequences of bone marrow transplantation in children: a preliminary communication. J Psychosoc Oncol 1987; 5:73–81. 40. Heiney SP, Nueberg RW, Myers D, Bergman LH. The aftermath of bone marrow transplant for parents of pediatric patients: a post-traumatic stress disorder. Oncol Nurs Forum 1994; 21:843–847. 41. Simms S, Kazak AE, Golomb V, Goldwein J, Bunin N. Cognitive, behavioral, and social outcome in survivors of childhood stem cell transplantation. J Pediatr Hematol Oncol 2002; 24:115–119. 42. Lerner RM, Lerner JV, Hess LE, et al. Physical attractiveness and psychosocial functioning among early adolescents. J Early Adolesc 1991; 11:300–320. 43. Kronenberger WG, Carter BD, Edwards J, Morrow C, Stewart J, Sender L. Psychological adjustment of mothers of children undergoing bone marrow transplantation: the role of stress, coping, and family factors. Children’s Health Care 1998; 27:77–95. 44. Thompson RJ, Gustafson KE. Adaptation to chronic childhood illness. Washington, DC: American Psychological Association, 1996. 45. Rodrigue JR, MacNaughton K, Hoffmann RG, et al. Transplantation in children: a longitudinal assessment of mothers’ stress, coping, and perceptions of family functioning. Psychosomatics 1997; 38:478–486. 46. Arvidson J, Larsson B, Lonnerholm G. A long-term follow-up study of psychosocial functioning after autologous bone marrow transplantation in childhood. Psychooncology 1999; 8:123–134. 47. Carter BD, Kronenberger WG, Edwards J, et al. Prediction of in-hospital adjustment of children undergoing bone marrow transplantation: the role of pre-HSCT child/parent adjustment and family environment. Poster presented at the Annual Convention of the American Psychological Association, Toronto, Ontario, Canada, 1996. 48. Gunter M, Karle M, Werning A, Klingebiel T. Emotional adaptation of children undergoing bone marrow transplantation. Can J Psychiatry 1999; 44:77–81. 49. Maloney R, Clay DL, Robinson J. Sociocultural issues in pediatric transplantation: a conceptual model. J Pediatr Psychol 2005; 30:235–246. 50. Kazak A. Evidenced-based interventions for survivors of childhood cancer and their families. J Pediatr Psychol 2005; 30:29–39. 51. Kaufman KL, Tarnowski KJ, Olson R. Self-regulation treatment to reduce the aversiveness of cancer chemotherapy. J Adolesc Health Care 1989; 10:323–327. 52. Zeltzer LK, Dolgin MJ, LeBaron S, LeBaron C. A randomized, controlled study of behavioral intervention for chemotherapy distress in children with cancer. Pediatrics 1991; 88:34–42. 53. McQuald EL, Nassau JH. Empirically supported treatments of disease-related symptoms in pediatric psychology: asthma, diabetes and cancer. J Pediatr Psychol 1999; 24:305–328. 54. Kazak A, Simms S, Barakat L, Hobbie W, Foley B, Golomb V. Surviving cancer competently intervention program (SCCIP): a cognitive-behavioral and family therapy intervention for adolescent survivors of childhood cancer and their families. Fam Process 1999; 38:175–191. 55. Lemanek KL, Kamps J, Chung NB. Empirically supported treatments in pediatric psychology. J Pediatr Psychol 2001; 26:253–276. 56. La Greca AM, Schumann WB. Adherence to prescribed medical regimens. In: Roberts MC, ed. Handbook of Pediatric Psychology. 2nd ed. NewYork: Guilford, 1995:55–83.

250

Carter et al.

57. Rapoff MA, Barnard MU. Compliance with pediatric medical regimens. In: Cramer JA, Spiker B, eds. Patient Compliance in Medical Practice and Clinical Trials. New York: Raven, 1991:73–98. 58. Andrykowski MA. Psychiatric and psychosocial aspects of bone marrow transplantation. Psychosomatics 1994; 35:13–24. 59. Seagull EA. Beyond mothers and children: finding the family in pediatric psychology. J Pediatr Psychol 2000; 25:161–170. 60. Kazak A. Family systems practice in pediatric psychology. J Pediatr Psychol 2002; 27:133–144. 61. Phipps S, Dunavant M, Srivastava DK, Bowman L, Mulhern R. Cognitive and academic functioning in survivors of pediatric bone marrow transplantation. J Clin Oncol 2000; 18:1004–1011. 62. Butler RW, Copeland DR. Attentional processes and their remediation in children treated for cancer: a literature review and the development of a therapeutic approach. J Int Neuropsychol Soc 2002; 8:113–124. 63. Wingard JR. Quality of life following bone marrow transplantation. Curr Opin Oncol 1998; 10:108–111. 64. Robertson JM, Robison BD, Carter BD. Splitting on a pediatric consultation-liaison service. Int J Psychiatry Med 1996; 26:93–104.

13 Ethical Considerations in Pediatric Hematopoietic Stem-Cell Transplantation Raymond Barfield Division of Stem Cell Transplantation, St. Jude Children’s Research Hospital, Memphis, Tennessee, U.S.A.

Eric Kodish Department of Bioethics, Cleveland Clinic Foundation, Lerner College of Medicine at Case, Cleveland, Ohio, U.S.A.

INTRODUCTION Hematopoietic stem cell transplantation (HSCT) is a relatively young discipline. The field has come far since the first pediatric transplants in 1968 in infants with SCID, but much remains to be done. In some cases, such as matched-sibling transplantation for AML, HSCT has become the standard of care. But often HSCT is done in the context of a research study. Therefore, issues in both clinical ethics and research ethics are important to the field. This is an area of medicine in which information learned in the laboratory setting is quickly translated into changes in clinical protocols for children. Translation from the laboratory to the clinic is always aimed at decreasing toxicity, improving efficacy, or both. Often the failure of stem cell transplant to improve survival is not due to recurrence of disease but rather to toxicity related to the procedure itself. Developing strategies for reducing toxicity-related deaths without abrogating the effectiveness of the therapy is high on the agenda for transplant research, but every change in protocol is an experiment. Many such experiments will have to occur for HSCT to offer its full potential for cure without negating this potential through the toxicity of the therapy. The fact that children are often the beneficiaries of the cure offered by this therapy means they are also often the victims of the toxic deaths and of the experimental failures. Because research in HSCT sometimes involves children who are not capable of making their own decisions, pediatric HSCT raises unique ethical issues. Bioethics, like any philosophical endeavor, is about questions and answers. Our goal in this chapter will be to raise some of the challenging questions and, when possible, to suggest answers. But there are several important points to be made at the outset. First, there are many questions that fit comfortably under the rubric of bioethics that can be answered using the same sort of research model used to answer questions about therapeutics. On the other hand, there are many questions of at least equal importance that do not lend themselves to such a method. Here are several questions of the former kind: What are the measurable psychological differences in children who donated stem cells to siblings who were cured versus those who donated stem cells to siblings who died? Do parents experience informed consent as more ‘informed’ with the use of educational video tapes? 251

252

Barfield and Kodish

Is there a difference between the conversations that take place between physicians and families at the initial diagnosis of cancer compared to the time of relapse? Questions of the latter kind are these: Should I proceed with transplant in this anxious child who expresses a wish to play at home and who has a 25% chance of cure with HSCT?; If I have great laboratory data to support an experimental change in therapy that promises to reduce toxicity while retaining efficacy, how much should I emphasize the experimental nature of the procedure in the process of consent? The latter group of questions will never be answered by research. If it is to be useful to practitioners, a chapter on the bioethics of stem cell transplantation must acknowledge the fact that most issues involving a moral judgment require wisdom beyond data for resolution. The clinical ethics of pediatric stem cell transplantation are discussed in this chapter. The starting point for this discussion is a brief look at the kinds of foundations that have developed for this sort of deliberation. After this, specific issues are addressed, some of which are translatable to almost any field in which children with severe illnesses are treated, and others of which are fairly unique to stem cell transplantation. Many pediatric HSCT patients are treated on research protocols or with treatment plans recently derived from research protocols. Therefore research ethics, as it relates to HSCT, is discussed. Because of continuing, rapid changes in the field of HSCT, content relevant to ethical questions, such as the specifics of risk and benefit, are rapidly changing as well. The premise of this chapter is that it is reasonable to expect surprising new issues to arise with the advent of new technologies that demand a caseby-case approach but that generalization of what is learned from these past encounters with difficult issues can in turn arm us to better approach future unknowns. This move back and forth between lessons learned from past experiences and unprecedented challenges offered by the evolution of the field demands that the practice of bioethics remain simultaneously flexible and strong and open to reform without losing the value of what we have learned in medicine’s long attempt to heal and help.

FOUNDATIONAL CONCEPTS The fundamental challenge to developing a cohesive and consistent approach to complex bioethical questions is that these questions are raised in a pluralistic context in which the participants enter the discussion with a variety of religious, ideological, philosophical, educational, and vocational backgrounds. These differences can serve to open new perspectives on a problem. The differences can also lead to impasses, because frequently the endpoint of a discussion in biomedical ethics is not an agreement to discuss an issue further but rather is a concrete action, one that perhaps must be undertaken with some urgency and that is often irrevocable. Such a practical conclusion to a discussion in which the stakes are high—often human life or well-being is at the center of the issue—lends great significance to the endeavor of biomedical ethics. Compelled by reports of human subject abuses, such as those uncovered in the Nuremberg trials, reviews of the U.S. public health service sponsored Tuskegee syphilis study, and President Clinton’s advisory committee on human radiation experiments, a number of attempts have been made to distill principles that are broadly applicable and comprehensive (1,2). Stemming in part from the Tuskegee study, the U.S. Congress became increasingly concerned about research ethics, and in 1974 it formed The National Commission for the Protection of Research Subjects of Biomedical and Behavioral Research. This resulted in a report called The Belmont Report, published in 1979. This report embraced three now-familiar principles as particularly important for research involving human subjects: respect for persons, beneficence, and justice (3). The concept of respect for persons comprises the two principles that individuals be treated as autonomous agents and that those with less autonomy are entitled to protection. This latter aspect of the concept is especially relevant in pediatric disciplines and is somewhat fluid in its definition because the degree of autonomy held by pediatric patients changes as they approach adulthood. Beneficence is a principle that acknowledges the

Ethical Considerations in Pediatric HSCT

253

Hippocratic maxim “do no harm” and extends it to include maximizing possible benefits and minimizing possible harms (often the minimizing of harms is formulated as a separate principle known as the principle of nonmaleficence) (4). The principle of beneficence is operative in any discussion that considers whether or not the benefit of research done with human subjects justifies the risk, especially when the subject is a child. Simply stated, the principle of justice is concerned with the right and fair distribution of the benefits of research as well as the burdens. These principles are general. A more detailed set of basic principles attending to such issues as scientific integrity, professional competence, risk assessment, and competing interests was developed by the world medical association in the Declaration of Helsinki (5). This document also explicitly examined the important distinction between clinical research in which an investigation is combined with professional care and nontherapeutic research involving human subjects. Principle-based deliberation and debate has done much to clarify the issues at stake and to attempt to provide common ground for discussion across pluralistic boundaries. It has also been criticized as being inadequate for addressing issues of such complexity and nuance. The first important camp from which helpful criticism has come is a camp that is by no means antithetical to the use of principles—casuistry, or “case-based ethics.” In the approach of casuistry, the principles always follow upon cases, instances, and concrete particulars. Bioethics properly proceeds from cases to categorical principles, not the other way around. Casuists argue (after Aristotle) that ethics is not and cannot be a science. It is rather a field of practical wisdom, a field that grows out of experience, which, in turn, yields a recognition of significant particulars and informed prudence (6). A second important source of criticism of principlism also derives from Aristotle, namely communitarianism. This approach argues that apart from agreement upon certain goods and goals, there is no rational way to reach moral agreement (7). For example, within one community individual autonomy might be identified as an important good, whereas another community deriving from a different cultural background might identify goods, such as family obligation or religious authority, as more weighty than that of autonomy. This weighing of goods might yield very different approaches to ethical decisionmaking. From inside of a community, such a weighing of goods makes sense and might function as a basis for decision-making. However, communitarians argue that there is no “view from nowhere” outside of a particular community that allows one to demonstrate the superiority of one community’s view over another’s view. Other criticisms have been leveled at principlism. The approaches are not, however mutually exclusive, and much work has been done to show how different vantage points can complement each other (8,9).

CONSENT FOR STEM-CELL TRANSPLANTATION General Issues What Is “Consent” in Pediatrics? Many formulations have been devised in the attempt to define what is meant by the concept of informed consent. Most definitions derive from the first principle of the Nuremberg Code, which states that “The voluntary consent of the human subject is absolutely essential,” a statement that is subsequently unpacked as comprising legal capacity, free power of choice, knowledge, and comprehension (10). Informed consent has come to be understood less as a single event and more as a process (11). In pediatric medical subspecialties, such as oncology and stem cell transplant, informed consent is made more complex: that the patients (subjects) are children complicates the notions of legal capacity and autonomy; that the medical care is so complex complicates the notions of parental and patient knowledge and comprehension. The model of autonomy used for adults simply does not fit the needs of those involved in pediatric decision-making, including the patient, the guardians, and the caregivers. Indeed, the very notion of one person consenting for another is problematic. Furthermore, both pediatric oncology and stem cell transplantation often work through investigational protocols and

254

Barfield and Kodish

therefore constitute not only treatment but also research. Consent to the one is not synonymous with consent for the other. The language of consent and assent in pediatric therapy and research is not settled at this time. But the injunction to approximate informed consent in these settings focusing above all on the best interests of the child-subject is obligatory (12). Though the development of an adequate model for consent and assent in pediatric clinical research trials and nontherapeutic research trials is a work in progress, much of the formal discussion follows upon the report and recommendations on research involving children of the national commission, as a report published in 1977, which became the foundation for federal regulations governing such research (13). In this report the important distinction was made between research that directly benefits a child and research that does not. Regarding the former the national commission recommended that the level of risk to be taken by a child undergoing research that might directly benefit the child be left at the parents’ discretion, as long as the risk is justified by the potential benefit to the child. Assessing the balance of such risks and benefits prior to making the research available to parental discretion is, in part, the task of the institutional review boards (IRB). IRBs are federally mandated boards meant to provide independent review of research, though in fact we do not know much about the actual quality of the reviews or how structural and functional variations among institutions affects this quality. This is one of many areas in bioethics relevant to HSCT that requires further study.

Enrollment of a Child in Nontherapeutic Research HSCT offered in the context of a clinical research study must have the potential for benefit to the child. But when pediatric patients are enrolled on HSCT clinical studies, they may also be recruited into nontherapeutic research that holds no prospect of direct benefit to the child. Federal regulations require that research without the prospect of direct benefit be evaluated in a different way. The concept known as “minimal risk” is operative in evaluation of such research. In the Belmont Report minimal risk is defined as “the probability and magnitude of physical or psychological harm that is normally encountered in the daily lives, or in the routine medical or psychological examination, of healthy children.” Such a definition clearly limits the sorts of research for which a parent can give permission, though questions still remain as to what constitutes daily life experience and whether it is ever acceptable for a parent to give permission for any risk higher than minimal risk in the absence of benefit to the child. Indeed, the national commission was willing to allow parental discretion in giving permission for research that exposed a child to “a minor increase” over minimal risk, given the fact that parents routinely give permission for their children to participate in activities, such as contact sports, that present more than a minimal risk, and given the potential benefit to others derived from such research. However, this position has come under significant criticism given the children for whom acceptable risk is being redefined are children who are already at higher risk given their situation as sick children (14). Of the four categories of pediatric research delineated in the federal regulations, permission of one parent is sufficient in two and permission of both parents is required for two (45CFR 46:408b). In the former group (45CFR 46:404 and 405) is research that presents no more than minimal risk to the child, and research that exposes children to more than minimal risk but holds the prospect of direct benefit (i.e., therapeutic pediatric HSCT). Both parents are required to permit research in the latter two categories (45CFR 46:406 and 407) that are defined as research that presents more than minimal risk with no prospect of direct benefit to the child. What Constitutes Adequate Consent in Pediatrics? With the development of guidelines and goals for consent, the process has become more intentional and more highly scrutinized than before publication of the national commission’s reports. Nonetheless, very little data has been produced since that time to shed light on the process of informed consent in stem cell transplant generally and, more particularly, pediatric transplant (15–20). But some lessons learned from the pediatric oncology setting are perhaps

Ethical Considerations in Pediatric HSCT

255

relevant to any complex pediatric medical therapy, including stem cell transplantation. As with pediatric transplantation patients, many children with cancer are research subjects, generally participating in protocols developed to answer specific unknowns (21). Generally, the new study compares a previously studied intervention with a new intervention that has a scientific rationale. The scientific merit and the acceptability of the risks and benefits of the study in a human population are assessed either by an IRB or in cooperation between an IRB and a scientific review board. The same is true for transplant. However, the changes in supportive therapy, antibiotics, stem cell graft engineering, immunomodulation, quickly change the procedure-associated morbidity, and mortality of both new interventions and tried interventions. Furthermore, because the new interventions—deletion of T-cell subsets with retention of alloreactive natural killer cells in transplant for acute leukemia, for example—are based on evidence from past experience, there is sometimes powerful theoretical reasoning that makes it far less than clear that the “standard” arm is safer than the “experimental” arm. Mice are not humans, and experimental data gathered in the laboratory and the animal facility cannot substitute for clinical trials. How to present the weight of the laboratory data that has been used to justify the development of the experimental arm is a difficulty not infrequently encountered in stem cell transplantation, a discipline that is changing rather quickly based upon new techniques developed in the laboratory (22,23). These questions are always accompanied by the everpresent fact of what is at stake. Often stem cell transplant is the last recourse for patients for whom other therapies have failed. The Children’s Cancer Group (CCG) has conducted studies that have shown that this sense of pressure is perceived by parents when making the consent decision for intervention in the context of childhood cancer (24). More recently we have reported results indicating that despite the fact that there is significant dissatisfaction with the consent process among CCG clinician-investigators, the majority of parents are satisfied with the informed consent process (25). The same study indicates that satisfaction on the part of the parents does not constitute adequate informed consent. This is true, but it is a point that raises the following question: what function does the process of informed consent play in scenarios in which the proffered intervention is perhaps the only option realistically available for curative therapy. Even among clinicians assessment of the process of informed consent varies. Simon et al. found differences that split across years of experience. Clinicians with 10 or fewer years of experience were more likely to say that explaining the disease and treatment is the most important goal of informed consent, and they were more likely to suggest to parents that other children might benefit from the current research (26). In the end the same study finds that when reports from clinicians and parents are compared, clinicians are dissatisfied with aspects of consent that parents seem far more satisfied with. For example, clinicians expressed concern regarding the timing of the consent discussion (which often occurs while the parents are still in a state of shock regarding the diagnosis) and the problem with information overload. The difference in clinician perspective versus patient and family perspective is a topic worth further study. Insights into this difference have been gained from studies addressing the perspectives of parents who have children with cancer. The first study involved three focus groups with twenty two parents of children with cancer (27). The goal of the study was to retrospectively examine the perceptions of parents regarding the informed consent process. High levels of stress were consistently reported as following upon the confluence of multiple demands, including assimilating their child’s diagnosis, nurturing and supporting the child, understanding the information offered about the diagnosis and treatment, getting to know an entirely new group of people involved in the care of the child, and participating in the child’s treatment. Interestingly the study found that parents did not consistently distinguish research from their child’s medical treatment. The dilemma this presents is that one goal of “informing” in the consent process is to make it clear that the research is optional. If no distinction is made between research and treatment, one must question whether or not the goal of consent has been reached. Physician-investigators often face the tension between being the

256

Barfield and Kodish

physician for a patient and being a researcher offering an as-yet-unproven alternative to standard therapy. This study suggests that overcoming this dilemma may include not only changes on the part of the professional involved in the consent process but also an awareness that the nature of research qua research and the difference between research and standard therapy may not be concepts adequately grasped by the parents. A second study in which 20 parents of newly diagnosed children were interviewed found similar results (28). Most participants (80%) recalled survival statistics, and 100% recalled the diagnosis and general plan. But only just over half knew that the treatment protocol involved research and understood the concept of randomization. This is rather striking when coupled with the fact that three-fourths of the parents thought that discussion of alternatives to enrolling their child on a randomized protocol was insufficiently discussed. Again the majority of participants were satisfied with the consent process. But because randomization is central to answering clinical research questions in any protocol, including those used in stem cell transplantation, this is an important finding that should preface future studies of improving the consent process. A study we have recently published in JAMA confirms the challenges of effective communication of randomization (29). In a multisite study in which informed consent conferences were observed and audiotaped, then compared to information acquired in interviews with parents shortly after the conference, the investigators found that while randomization was explained in 83% of the cases, 50% of the parents did not understand randomization. Furthermore parents who did not understand randomization were more likely to consent to the randomized study than those who did understand it, though this particular result did not reach statistical significance (pZ0.07). These points are useful to consider in any clinical research setting, including stem cell transplantation. Are there further issues that are unique to consent in stem cell transplantation?

Consent in Pediatric Hematopoietic Stem-Cell Transplantation Perhaps one salient difference between the process of consent for stem cell transplantation and the process of consent for most therapies in oncology is that transplant is usually a second medical procedure. Common indications for transplantation with malignant diseases include failure to respond to conventional therapy or known high risk for relapse after conventional therapy. In both cases, patients have been treated prior to transplant. Several elements follow from this fact that puts these patients and families in a different situation than when they first present with their condition. Going through conventional chemotherapy is itself an education. One of the most difficult parts of the consent process for therapy in, for example, acute leukemia when there is some urgency to begin therapy is achieving the “informed” part of “informed consent.” The entire language of oncology is foreign. Often there is little time to absorb the gravity of the situation before permission is granted to proceed with therapy. On the other hand, by the time an oncology patient needs stem cell transplantation, the family is generally acquainted with the language of chemotherapy, immunocompromise, fever and neutropenia, mucositis, and so forth. Much of this language is used in the discussions about transplantation and so the familiarity often facilitates more deliberate questioning on the part of the patient and family. Furthermore, stem cell transplant—whether for malignant or nonmalignant diseases—is generally not something that occurs with the kind of urgency inherent in the initial phases of treatment for many malignancies in which delay compounds the risk for adverse events. This fact in principle allows more time for thought, study, discussion, and decision-making. But these are primarily cognitive considerations, and it is by no means clear that cognitive considerations are the most important aspects of the informed consent process for stem cell transplantation. No studies have specifically looked at the noncognitive considerations in consent for pediatric stem cell transplantation. However, one study has addressed the issue in adult transplant patients, and the finding might be useful as a guide to future assessments of the unique characteristics of consent and assent in pediatric stem cell transplantation (30).

Ethical Considerations in Pediatric HSCT

257

The study begins with the unusual hypothesis that “in the context of a potentially life-saving procedure without any viable treatment alternatives for a potential cure, ‘informed consent’ has little significance to the patient in terms of his or her autonomous decision to proceed with treatment and that other factors influence the patient’s decision-making process.” Because these patients have life-threatening illnesses “forcing” them to consider transplant as an option, the notion of “voluntary” action becomes more complex. Of the four factors considered—(1) a full understanding of the treatment, (2) trust in the physician, (3) trust in the treatment team, and (4) best chance for a good outcome—the most important factor was “best chance for a good outcome,” and the least important factor was “a full understanding of the treatment.” Such an outcome has important implications for informed consent in transplant, as well as in other high-risk, complex interventions in which the patient’s life is at stake. As mentioned above, the very nature of the illness and the lack of alternate therapies place a limit on the notion of voluntariness. Furthermore, if patients (and parents) do not place a high value on thorough understanding of information about the procedure, this would seem to undermine the importance of “informed” consent, given the traditional centrality of understanding to that concept. In this case the principle of beneficence has priority. Trust in the physicianinvestigator and the health-care team may be more significant. In the case of complex procedures, such as stem cell transplantation, that may represent a final option for cure, careful attention to the therapeutic rationale of the treatment is crucial for responsibly dealing with the vulnerability of transplant patients. This is perhaps especially true in the context of a research study in which HSCT is not considered standard of care or in which there is no established standard of care.

Advances in the Process of Consent As we learn more about the process of consent and assent in pediatric clinical investigation generally, and stem cell transplantation particularly, several aspects might be appropriately emphasized. First, when stem cell transplantation is the only therapeutic option with potential for cure of an otherwise fatal illness, elements of consent, such as voluntariness, may be more complex than in some other arenas where consent is required but where more therapeutic choices exist. If there is a discrepancy between what parents and patients want and think they need from the consent process and what investigators, physicians, and regulatory boards think they need, a rather large issue is raised that requires negotiation. Anyone who has been through the consent process more than a few times knows that the process is by no means uniform from one family to the next. Some want a great deal of factual detail; others want to avoid discussion of detail altogether. Although there is no limit on how many factual questions ought to be answered, it is less clear what appropriately constitutes an informed process of consent when the family prefers not to talk about the details of risks inherent in the investigation/treatment. Second, where long and complex clinical trials are offered, novel approaches to consent might be considered. For example, one recent study looked at the possibility of staged consent (31). This was the first CCG study in which investigators had the option of obtaining consent over a 28-day period using a staged approach. This allows parents and patients more time to discuss and to absorb the facts about the disease, the purpose of the trial, the design of the study, and the potential toxicities associated with the proposed interventions. Several measures in this study suggested benefit from the staged approach. For example, trust scores were higher for this study when compared to those from other protocols. In addition, understanding of treatment choice and of the distinction between a randomized controlled trial and standard therapy was higher in the staged approach (80% understanding) than in other studies (62.5%: pZ0.05). Finally, as we learn more, new concepts might be introduced that better match the possibilities and realities of contemporary medicine. For example, some have argued that “informed consent” may be too restrictive a notion and have suggested that “valid consent” be substituted (32,33). The three aspects of this notion are personal competence (does the patient have the capacity to make the decision), procedural competence (is the consent given

258

Barfield and Kodish

correctly?), and material competence (is the procedure consented to appropriate for valid consent? There may be some procedures for which valid consent cannot be obtained. For example, if a parent consents to donating bone marrow for a child, this may be acceptable. But can the parent consent to donating his or her heart to a child?). The concept of valid consent has been explored in the pediatric context by the SIOP Working Committee on Psychosocial Issues in Pediatric Oncology (34). The value of this notion, they suggest, is that it emphasizes the patient’s or parents’ understanding of what is being consented to and underscores the fact that there are both rational and irrational aspects to the decision-making process that must be understood. Their concern is that “informed consent” has come to mean legally signed documentation rather than the achievement of real understanding, insofar as that is possible. Because parents come from different backgrounds and because children of different ages have different abilities to grasp complex concepts, the level of understanding that is attainable might vary from situation to situation. The notion of valid consent acknowledges this and attempts to resist the idea that a signed document is equivalent to informed consent.

THE SECOND PATIENT Certainly one of the most obvious differences between stem cell transplantation and most other clinical enterprises is that, with the exception of autologous transplant, HSCT involves two patients—the recipient and the donor. Different issues arise depending upon whether the donor is matched unrelated (MUDSCT), matched related (SIBSCT), or haploidentical (HAPSCT). These differences are in part based upon the fact that in MUDSCT the donor is a stranger, in SIBSCT the donor is a brother or sister (a situation in which age also impacts upon the discussion), and in HAPSCT the donor is usually the parent of the patient. Whether things go well with a patient receiving a transplant or do not, the relationship between the donor and the recipient may have impact on the donor, which is relevant to a discussion of the bioethical issues in HSCT.

Matched Unrelated Donors Overall, 70% of people who are candidates for HSCT do not have a matched-sibling donor. However, more than seven million people are HLA typed and registered as potential marrow or peripheral blood stem cell (PBSC) donors (35). These are the donors who make matched unrelated HSCT possible. The risk of being a marrow donor is small. Life-threatening complications occur in approximately 0.1% of healthy donors (36). In the case of PBSC donation, 95% of donors do well with no more that minor complaints, and approximately 3–4% of donors have more intense complaints or experience symptoms for a longer period of time but eventually have resolution of all complaints (37). Modern HSCT sometimes requires multiple donations from a single donor for optimal care of a patient. For example, in a T-cell depleted graft, if the patient’s chimerism begins to drop or if there is other evidence of relapse or rejection, donor T cells are often used to recover the graft or to treat the relapse. This fact has raised questions about the ethical implications of asking for a second donation from a donor recruited solely as a one-time bone marrow donor. If a donor agrees to join a registry as a donator of bone marrow, what are the implications of asking for PBSC donation, T cells, or other cell products on one or even multiple occasions, thereby forcing the donor to choose for or against the donation, knowing that a stranger may depend upon the decision for his or her life? In MUDSCT both the donor’s rights and the patient’s needs must be considered. In a review by the World Marrow Donor Association (WMDA) a number of issues were outlined as relevant to establishing the appropriate standards for donor commitment (38). Donor autonomy must always be honored. This means at a minimum that the donor is informed about consequences of each act in the process, both as they bear on the donor and on the patient. Written consent includes both the donor’s right to withdraw at any time, including during the

Ethical Considerations in Pediatric HSCT

259

patient’s pretransplant conditioning period, as well as explicit statement of the possible consequences of donor withdrawal. The donor must not be pressured or coerced in any way by the donor center/registry. At the same time the donor center/registry has the right to exclude a donor. That is, a donor does not have right to donate stem cells to a patient, and this exclusion process should be part of the initial counseling of the donor. The donor center/registry must make clear the expectations of the donor, including maintenance of altruism, avoidance of risk groups, and ongoing health and fitness for donation. The patient, of course, has high expectations that the donor will maintain these things. This factor has the potential to conflict with the obligation never to coerce a donor. No donor is obligated to join a registry. But once a donor has joined the registry in light of the clear expectations, it seems that some moral obligation is incurred by the donor, incurred voluntarily. Furthermore, it seems that this obligation grows as the process continues. That is, a person who has agreed to come to the operating room the next day to donate bone marrow for a patient who is aware that a matched donor has been found seems to be under moral obligation to follow through with the commitment in a way in which a person who is merely considering information about donor registry is not. Donor centers have an obligation to communicate clearly with the donor in order to avoid, as far as possible, any divergence from the plan. But here there are also limits to the sense of obligation. A gray area is the request for cell products besides bone marrow—the product for which most registries were originally set up to acquire and for which most donors have been registered. If a donor restricts his or her cell donation, that decision must be respected, and the transplant center should be informed. If a bone marrow donor agrees to donate to a patient and fulfills that initial commitment to donation, though the recipient at some stage may need other cell products, such as T cells or additional stem cells for a boost in, for example, the context of a failing graft, the donor does not have the same obligation to fulfill the second need that he or she may have had to follow through on a commitment to the first need. To do so would be an act of supererogation, that is, going beyond what is morally obligatory. Finally, donation of cells must never jeopardize a donor’s health beyond standard risks. Nor must the donation interfere with other standards the donor may hold including emotional, moral, religious, and economic standards. This is an intensely humane endeavor between strangers, and so requires the highest regard for humane principles and for a rich understanding of the range of human elements involved both in donating and in receiving bone marrow or other cell products. Therefore, detailed guidelines have been provided to ensure that the transplant community attends to every important aspect of the process (39). All of this said, there is still a problem with donor unavailability. This problem falls under the rubric of ethical issues in transplant because, as noted above, donor registration presumes the voluntary assumption of some obligation on the part of the donor, and patient need retains some claim, if not to an individual donor, at least to the institution set up to ensure as far as possible that donors who are registered are most likely to follow through. The National Marrow Donor Program (NMDP) is the largest registry of volunteer unrelated stem cell donors in the world. When donor unavailability rates over one year were examined in the NMDP, the largest registry of volunteer unrelated stem cell donors in the world, several interesting facts came to light (40). The initial activity at the time of donor search is usually a request for “confirmatory typing.” Over the course of the year studied (March 1, 1999, to February 29, 2000), at the time of confirmatory testing, 20% of all donors were permanently deferred and an additional 12% were temporarily unavailable (for such things as pregnancy, high-risk exposure, job change, and location change). There were large and consistent differences in donor unavailability rates between donor centers. The self-identified racial or ethnic group of the donor also affected the likelihood that the donor would be available when requested. Such findings suggest several areas of practical improvement, including better medical screening, better education about the details of the commitment, better collection of contact information, and improvements in producing a pressure-free environment. Important issues, such as route of contact and cultural sensitivity, must be studied further. The goal must be to meet the urgent needs of the patient

260

Barfield and Kodish

through a donor registry that is diverse and that is structured so as to maximize donor availability through better practice models for donor recruitment, retention and contact. Having asked the question why there might not be availability of donors who have been recruited or who have initiated the process of donation through registration, a next reasonable question is why patients with an apparent human leukocyte antigen (HLA) matched donor do not proceed to transplantation—the flip side of this issue. In a study that again focused on searches coming out of the NMDP, investigators addressed this question (41). Approximately one fourth of searches that find an HLA-matched donor encounter other barriers to transplantation. The most common barrier is deterioration of the patient’s condition—a barrier that might be improved through earlier searches and improved efficiency of the process. Another important barrier was financial, but interestingly, although 41% listed finances as a potential barrier at the time of initial search, in only 5% of cases was it listed as an actual barrier by U.S.A. coordinators. Whether this indicates an improvement in securing financial coverage or a lack of NMDP searches in cases in which the financial resources are lacking is uncertain. The pediatric population has not been studied as rigorously, and the result might turn out to be different for this population than for the adult population. At St. Jude Children’s Research Hospital, for example, no family with a child going through transplant is asked to pay anything beyond what their insurance provides for the procedure.

Sibling Donors A second important source of cell donation, which raises important ethical questions, is that of sibling donors, especially sibling donors who are minors. The issues center around the uncomfortable notions of coercion of an anxious child dependent upon the parents who provide surrogate consent for his or her donation, and the question what, if anything, constitutes a benefit to the donor. When a child becomes ill, he or she experiences psychological, spiritual, and physical stress without asking for it. Similarly, when the sibling of a child in need of HSCT is asked to be the donor, the child can experience psychological, spiritual, and physical stress without asking for it. The pain and anxiety among adult sibling donors who gave either bone marrow of PBSC has been described (42–45). In children the issues are more complex because the variable of developmental age is added, a variable that has significant impact on the perception of events, which in turn impacts stress. The stress can be further potentially compounded if the sibling recipient dies (46). In sibling donor interviews, some interesting themes arise (47). Irrespective of the outcome of the transplant, pediatric siblings report a perception of having “no choice” in becoming a donor, whether the sense of constraint is derived from outside pressure (as indicated by participants use of such language as “guilt,” “propaganda,” “bribed,” “privileged,” and “conned”) or from the donor’s own beliefs. Hesitancy arises not from a lack of desire to help but rather from fear of the procedure and of pain. Among donors whose recipient sibling died, several unique themes arose. Anger, guilt, and blame were common emotions expressed. These seemed to be most difficult in cases in which the death was directly related to graft failure or to graft-versus-host disease (GVHD). Though the numbers studied so far have been too small to do more than provide an impetus for further study and reflection, certain ethical tensions become clear from data gathered so far. These include such questions as competence to consent, the weight accorded to refusal, the limits of parental decision, the consequences to a child should he or she refuse to donate, and the worrisome specter of the notion of battery in the face of a child’s refusal. The adamant refusal of a 5-year-old to assent to donation might be overridden. Perhaps the 5-year-old simply cannot understand what is at stake. What about an 8-year-old? A 10-year-old? A 14-year-old? A 17-year-old? This chapter began with a statement that some questions will not submit to empirical investigation but rather require wisdom. These are the kinds of questions that prompt that observation. Coercion is, by contrast, minimized in all cases of MUDSCT. Prima facie one might think the same should be the case with sibling donors. But the forced issue in this case is not that

Ethical Considerations in Pediatric HSCT

261

a stranger might fail to benefit from the donation but that a sibling might fail to benefit and that from that decision point on the child-donor would have lost a sibling. Because in many areas of family life, parents are allowed to give more weight to the interests of one child over those of others or of the family in general, some have advocated always leaving the decision of whether a minor child should donate bone marrow to a sibling in the hands of the parents (48). However these issues are formally resolved, it seems clear that these sibling donors deserve all developmentally appropriate information at the start and full psychological support throughout the procedure and afterward. For the sake of completeness, it should be noted that siblings who are not chosen as donors may have important issues of their own. If sibling donors do not feel they have real “choice,” siblings who are not able to donate are susceptible to ambivalent feelings of relief, disappointment, and guilt (49). Here, by involving siblings in the screening process, issues of psychological well-being and respect for persons meet. A recent study compared sibling donors with sibling nondonors (50). Sibling donors report significantly more anxiety and lower selfesteem than nondonors. Nondonors had more school problems than the donor group. A third of siblings in both groups reported moderate-to-severe levels of post-traumatic stress. How much of this is due to the fact of being a screened sibling who is nonetheless not chosen as a donor requires further study. But in any case medical encounters with the nondonor sibling may well yield suffering or other experiences that would not have occurred apart from the process of being screened for stem cell donation. Insofar as this is the case, an obligation of respect is incurred by the care-givers toward this person as much as toward any patient.

Cord Blood Donation A third, increasingly prominent, source of cell donation is cord blood (CB). The first successful CB transplantation was performed in 1988 (51). Several issues have arisen in the years since then during which more than 2000 such transplants have been performed (52). The ethical issues primarily concern autonomy, privacy, and confidentiality. Some of the issues will be discussed separately in the section addressing the bioethical issues of conceiving a child to save a child, but there are more general issues related to CB transplantation. First, as protection of the donor is of paramount importance, initially there was concern that early clamping of the umbilical cord to obtain maximal stem cells would negatively affect the neonate (53,54). This seems not to be the case (55). But the point is important nonetheless as minors are not allowed to be bone marrow donors for unrelated recipients, and so any potential adverse consequences of harvesting CB or any changes in practice to maximize the number of stem cells harvested must be examined closely (56). Second is the question of when to obtain informed consent for the CB collection. Whether this is done in the first, second, or third trimester, consent should clearly be obtained prior to labor and delivery given the impact such events have on the ability of one to assimilate information and to make an informed decision (57). To do otherwise is to commit a family to decide an important issue in the middle of a separate physical and emotional period of stress, without such urgency being necessary. Once the cells have been obtained at least two questions arise. First, should the CB be linked to the identity of the donor? The primary argument for maintaining the link is that this allows unsafe units to be identified in the future (58). Arguments against such linkage generally center on concerns about loss of privacy for donors (59). Because the CB DNA holds genetic information, the implications of which are not clear at this time (and often one concern is that insurance companies will at some point use such genetic information to exclude clients from certain kinds of coverage), the donor is potentially vulnerable to inappropriate disclosures (60). A second question is related: what are the limits of testing of the CB sample, and what is to be done with the results? Clearly some testing is needed, including a screen for infectious diseases and perhaps some genetic diseases that are transmissible by CB transplantation (61). To do less is to put the recipient at unnecessary risk. However, in 1994 a review was issued from the Institute of Medicine recommending that children should not be tested for abnormal genes

262

Barfield and Kodish

unless the disease has an effective curative or preventative treatment that must be instituted early in life (62). This may constitute a reason to test samples broadly (for the sake of the recipient) but to do so in a blinded way (so as to protect the donor). The details of how this is done will be an on-going project as more is learned about the human genome and prediction of disease.

Haploidentical Donation The last source of stem cells to be considered is the haploidentical donor, usually a parent. The great promise of this sort of transplant is that, although many pediatric patients have neither a matched sibling nor a compatible HLA-matched unrelated donor from the marrow registries, most children have a living parent who is a motivated donor. New technologies in stem cell engineering are making this a safer, though still investigational, procedure (63). No research has been done on the impact this sort of transplant has on parents and families, but it would not be surprising if the complexities found in sibling donation were compounded in parental donation. For example, when a child dies of graft failure in haploidentical stem cell transplantation, one reasonable explanation for the event is that the graft is highly T-cell depleted. Many studies have suggested that T-cell depletion (required for haploidentical transplantation) impairs engraftment and that the HLA barrier is primarily overcome by the dose of CD34C stem cells per kilogram of recipient body weight. There is nothing a parent can do to affect this biological fact. And yet when a child dies of graft failure, the parent is susceptible to feelings of guilt. Therefore the consent process for the haploidentical parent donor might include discussions about the biology of the transplant as a way of mitigating feelings of guilt should the graft fail. There is another issue that will arise in haploidentical transplantation. In the future, when early organ (e.g., liver) damage is incurred by infants with genetic metabolic problems amenable to cure through HSCT, it is reasonable to expect that partial organ transplants might accompany haploidentical transplants of stem cells. Therefore, the question will arise: is there a limit to acceptable parental sacrifice? As one of the pioneers of haploidentical transplant has pointed out, “Obviously we would not take a parent’s heart.” But the question (which will not be answered here) remains, how much of a parent’s body will be accepted as haploidentical transplant advances?

RESEARCH, THERAPY AND HUMAN RIGHTS Research-Associated Conflicts of Interest Simultaneous Obligations to Provide Therapy and to Advance Medical Science A strong ethical argument can be made against physician-researchers having equity such that the success of a clinical trial will also yield financial gain for him or her (64). But financial conflicts are not the only potential conflicts of interest. Research involving human subjects and expensive experimental therapies with some unknown risks conducted in an environment in which professional advancement and promotion is in part based upon success in developing novel and successful therapies is bound to be vulnerable to conflicts of interest. Stem cell transplant by its very nature as a relatively young and quickly changing field is often experimental. The importance of this fact was emphasized by the recent experience of the Fred Hutchinson Center regarding transplantation with T-cell depleted marrow—a strategy that many think still holds great potential for reducing the toxicity of transplant and that has made such strategies as haploidentical transplantation possible (65). Fred Hutchinson was charged with exposing subjects to undue risks in bone marrow transplantation trials in the 1980s and 1990s involving stem cell products that were T-cell depleted. This approach turned out to yield poorer rates of engraftment than transplant with grafts that were not T-cell depleted. One claim

Ethical Considerations in Pediatric HSCT

263

against the Hutch was that researchers failed to inform subjects properly about alternative therapies and about prior failures regarding engraftment. The jury ruled in favor of the Fred Hutchinson Center. The questions raised in such debates underscore the importance of uncertainty in the design of a randomized control trial (66). That is, the design of a trial requires the condition of equipoise: there must be adequate uncertainty as to which arm of the study would benefit patients most in order to justify enrolling patients (67,68). This point will become important when we consider comparing better known therapies (which themselves be experimental) with experimental therapies that involve ethically contentious steps, such as preimplantation selection of an embryo. One must consider the related point that to proceed with a therapy off-protocol because of some level of assurance that it is in the patient’s best interest to pick that particular therapy is also a step that must be taken with caution. IRBs review experimental therapies that will be studied. If the same new therapy is not studied but is rather used because the physician thinks it is better, the IRB is generally not required to review the plan. Therefore adverse events are not necessarily scrutinized at the same level as events in a controlled study. Does this put the patients in harm’s way? The question should be asked in light of studies that have shown benefit to patients enrolled in randomized controlled trials versus those who were eligible for such trials but not enrolled (69). The view that participation in research is risky is being challenged in some ways by the view that such participation is beneficial to the patient (70). Therefore the corollary to the principle of adequate uncertainty in the development of randomized controlled trials is the possibility of the “inclusion benefit” for patients on controlled studies. Because all therapy in stem cell transplant is rapidly impacted by improvements, both in terms of betterengineered grafts and in terms of better support, one might argue that all pediatric transplants proceed best in the context of studies in which adverse events are formally analyzed, and settings in which demonstrated improvements are quickly implemented.

What Is Ethical Research? Having acknowledged the complexity of whether a patient is better off being treated as a participant in a study or not; at least four questions relevant to pediatric stem cell transplantation present themselves. First, a general question: what makes research involving children ethical? The Belmont Report makes it clear that innovative work in medicine is not necessarily research but rather that the collection of data and presentation in public or printed form constitutes research (71). The conflict between the obligations of the physician-researcher to deliver the best care to an individual patient and the obligations of the same to advance medical science so that optimal treatment options are available in the future is an ongoing topic of intense discussion (72). One important step in resolving this issue is to understand what constitutes ethical research. Informed consent in HSCT was discussed above, and one common answer to the question of what makes research ethical is informed consent. But Ezekiel Emanuel et al. have argued that informed consent is not sufficient for ethical clinical research (73). Drawing on the main sources of guidance for ethical clinical research from the past 50 years, they propose seven general requirements for ethical clinical research. First, the research must be valuable in the sense of enhancing health or knowledge. Second, it must be scientifically valid. In the clinical arena this means at least that there is controversy in the scientific community about whether the new therapy is better than standard therapy and that the method used to answer the question is valid and feasible. Third, the selection of subjects for the research must be fair. Fourth, the potential risks to subjects must be minimized, the benefits must be enhanced, and the balance between risk and benefit must be proportionate. Fifth, a committee with a range of expertise must review the study, and the committee must have the authority to approve, amend, or terminate the study. Sixth, informed consent must be obtained, and in the case of children unable to give consent, the research must be consistent with their interests and

264

Barfield and Kodish

values. Finally, subjects should have their privacy protected, should able to withdraw from the study, and should have their well-being monitored. Each of these requirements applies to research in children, though in the case of pediatric therapy and research a parent assesses the adequacy of the conformation of the study to the guidelines. The national institutes of health has made the participation of children in research studies a priority. Indeed, the exclusion of children from a research protocol must be justified (74). Insofar as the parent acts as the surrogate decision maker for the child, the goal of the parent must always be to protect the welfare of the child (75). Realization of this goal will be improved insofar as factors that influence parents’ decision making are identified and strategies devised to improve disclosure of each of these seven aspects of a clinical research study (76).

Transplantation for Conditions in Children That Are Slowly Fatal A second question in HSCT that is related to concerns about potential conflicts between therapy and research is this: what are the unique issues involved in HSCT for nonmalignant conditions that, though amenable to this therapy, do not present an immediate threat to life? These conditions include hematological diseases, such as sickle cell disease (SCD) and thalassemia, and nonhematological diseases, such as the mucopolysaccharidoses. Currently the event-free survival (EFS) rate after allogeneic matched-sibling HSCT for SCD is 82%, with a two-year EFS of 90% and 79% for patients undergoing related umbilical CB transplantation for SCD and thalassemia, respectively (77–79). Therefore the dilemma involves deciding which children derive most benefit from HSCT in light of the current 10–20% likelihood of death, autologous hematopoietic reconstitution or the need for infusion of cryopreserved recipient hematopoietic stem cells. What risk is acceptable in light of the potential benefit? As a start toward answering this question, two approaches have been taken. First, because parents must assess this balance for their child, one might ask parents about their perception of acceptable risk. In one study, parental attitudes toward the procedure were assessed (80). Parents were presented with a series of hypothetical situations beginning with HSCT as offering 100% survival and cure of SCD, and then increasing the mortality rate in subsequent situations by 5% increments. Among the 67 parents, 54% were willing to accept some risk of short-term mortality (STM), 37% were willing to accept current estimated risks of STM, 12% were willing to accept 50% or greater risk of STM, and 13% said they would accept current estimated risks of STM and an additional 15% risk of GVHD. These results suggested that a substantial minority of parents might consent to HSCT for their child with SCD and that this willingness should be factored into the decision about whether or not to offer HSCT to such patients. When a similar assessment was done in adults with SCD regarding acceptable treatment-related death rates, 63 of 100 patients were willing to accept some short-term risk of mortality in exchange for certainty of cure. Fifteen percent of patients were willing to accept more than 35% mortality risk, indicating that a substantial proportion of adults with SCD are willing to accept considerable risk of death in exchange for cure (81). One difficulty is that the study found no differences in patient or disease-related variables between those accepting the risk and those not accepting the risk. Nor was there agreement between the recommendations of health care providers and the risk accepted by patients. So the difficulty of assigning acceptable risk of treatment-related death for a child in exchange for cure remains complex. The complexity is compounded by the fact that for both nonmalignant hematological conditions and nonhematological conditions, longer delays in HSCT can result in increased disease-related morbidity (such as iron overload, lung damage, and, in the case of the mucopolysaccharidoses, accumulation of glycosaminoglycans that can lead to irreversible organ damage), which in turn places patients at higher risk of transplant-associated morbidity and mortality (82).

Ethical Considerations in Pediatric HSCT

265

Conflicts of Interest Between Living Children in Need of Transplantation and Unborn Children Conceiving Children for Sibling Transplantation If a child has a disease for which HSCT is the only option for possible cure and no sibling or matched unrelated donor is available, one option for parents is to conceive a child who might be able to donate CB or bone marrow. What are the salient issues involved in conceiving a child with the hope of a sibling transplant, when that child might not have been conceived in the absence of this need? Two things might be said here. First, most discussions of this issue acknowledge that conceiving a child for whatever reason is a decision to be left to the parents without coercion of any sort one way or the other. There is more work that might be done on the outcomes of such events, especially regarding the psychological well-being of the child conceived in these conditions. But this is an issue that abuts against the limits of what bioethics outside the context of specific social and religious contexts can usefully discuss. However, there is a second point to be made, and this point will be even more relevant in the following section. Even in the absence of a sibling or matched unrelated donor, if parents exist to produce another child for sibling transplant, then an alternative form of transplant exists without conception of a child, namely haploidentical transplant. Most children, for whom the option of conceiving a sibling for transplant is possible, have at least one and usually two donors (healthy parents) who can give bone marrow or PBSC. Therefore, the momentous event of bringing a child into the world can continue to be an event driven not by medical urgency but by independent desire to have another child.

Preimplantation Selection of Embryos for Sibling Transplantation Bringing a child into the world under the pressure of desiring a sibling stem cell donor is a complex issue, but one that is probably solvable only case by case in the context of individual families and their own social and religious communities. However, there is another related question that has even more demanding complexities: namely, what are the ethical implications of preimplantation genetic diagnosis (PGD) of embryos for the sake of subsequent sibling transplantation? The selection may be for known genetic diseases, such as Fanconi anemia (currently there are more than 100 different genetic diseases for which are putative indications for PGD), or, as more recently reported, for HLA testing to obtain a matched sibling for donation in the context of diseases without a known genetic cause (83,84). The effort of the former procedure is to discard “sick” embryos. The latter procedure, however, is more permissive in the sense that embryos are not rejected on the basis that they carry a burdensome disease but rather merely on the basis that they do not have the desired HLA typing. As Burgio and Locatelli have suggested, “Acceptance of this practice raises concerns about generating new individuals for whom full respect for their personal uniqueness and dignity is in some way watered down by their role as producer of cells for sick siblings” (85). Despite the risks of HSCT, the benefit of embryo selection to the ill patient who has a genetically defined and lethal disease is clear. However, important ethical issues are raised by this procedure. Broadly speaking it has been argued that conceiving a child to save a child is morally defensible if any procedure performed on the future child is acceptable to perform on an existing child (86). Whatever the merits of that argument, PGD introduces several other important issues that distinguish it from the general conception of a child in part to donate stem cells. Grewal et al. recently raised some of these points in a recent paper on PGD for Fanconi anemia (87). The cost of each clinical cycle with IVF and PGD is $15,000–$20,000. Multiple cycles are usually required for success. Because insurance rarely pays for such interventions, only the wealthy can afford it. Regarding the concept that using a person as a means to an end is

266

Barfield and Kodish

wrong, there are no policies in place sufficient to ensure that parents cannot put a donor child up for adoption after collection of UCB. Some parents may use PGD to select a disease-free embryo with the intent to abort the fetus and use the liver for collection of stem cells: according to the article some parents have already inquired about this illegal practice. To carry out this procedure more embryos must be made and tested, adding to the already 400,000C embryos frozen in the United States alone. The fact that such complex ethical issues are so new, and not even close to resolution, might suggest that the issues should be considered before proceeding with such a controversial procedure, rather than being noted at the end of a report on the successful use of the controversial technique. The impact of this criticism is increased in light of the fact mentioned above that with haploidentical transplantation the entire procedure may be unnecessary— especially as advances are made in haploidentical transplant such as improved T-cell depletion and reduced intensity conditioning regimens that make the transplant less toxic—so no new embryos need to be created and no new children need to be brought into the world to provide stem cells for a sibling. These are difficult issues. The difficulty is underscored by the fact that the authors of this chapter disagree with one another about the moral legitimacy of the procedure in light of other options.

QUALITY OF LIFE AND END-OF-LIFE ISSUES A recent review of top-selling textbooks from multiple specialties revealed that most textbooks and most disease-oriented chapters had no or minimal information on caring for patients at the end of life (88). In pediatric HSCT, many of children we treat will not be longterm survivors. Therefore we have an ethical obligation to provide better end-of-life care and diligently attend to the relief of suffering. There is almost no literature on quality of life (QOL) and end-of-life issues in pediatric HSCT. A study done in adults concluded that a third of patients undergoing HSCT report a poor QOL. Poor QOL was predicted by older age, longterm sequelae, chronic GVHD, and short follow-up. QOL was superior in long-term survivors (89). That such findings were so striking in the adult population mandates similar studies in the pediatric population. As with those who care for the 500,000 children in the United States with life-threatening conditions, those with children under their care who have received an HSCT should be taught to recognize a child’s need for management of suffering and a family’s need for bereavement care (90). We know that children who die of cancer—many of whom receive aggressive treatment at the end of life—often have substantial suffering in the last month of life (91). More studies are needed to understand the experiences of children who are treated with HSCT in whom survival entails chronic disorders or in whom survival is curtailed. One model for achieving improvements in pediatric HSCT is the discipline of pediatric oncology. But even in this discipline there is a stark lack of formal courses or training in caring for the dying patient, and there is a high reliance on trial and error as a means of learning how to deal with such situations (92). Children who die in the hospital with complex chronic conditions are more likely to experience longer periods of mechanical ventilation and hospitalization before death (87). End-of-life care for pediatric patients in any specialty will require integrated, coordinated service from specialty hospitals and home communities (93). In the case of HSCT, in which there may be high risk of toxicity and, depending upon the patient, a low chance of cure, the practice of palliative care must be further developed and implemented. Advances in this field will likely include a move away from the sequestering of palliative care to “end-of-life” and toward the notion of attention to QOL throughout the child’s experience whether the present goal is cure, prolongation of life or—once attainment of these goals no longer feasible—comfort and relief of suffering.

Ethical Considerations in Pediatric HSCT

267

CONCLUSION HSCT offers great hope for children with otherwise incurable diseases and, at the same time, presses many difficult ethical issues. Future research must address such issues, including the changing implications for consent in malignant and nonmalignant illness, as transplant morbidity and mortality decreases, the complexities involved in transplanting patients who have a slowly fatal disease, the challenges of haploidentical transplantation, and the limits of what can be asked of a parent, a sibling, or an unborn child. Whatever the future holds, no appraisal of HSCT is complete without acknowledging that many children have been cured and are thriving because of the courage of patients and their families who face risks for the sake of life, and caretakers willing to stay with patients in such difficult situations and to work through to the end what is practicable, effective and right.

ACKNOWLEDGMENTS From the St. Jude Children’s Research Hospital and the Cleveland Clinic Foundation, Lerner College of Medicine at Case. Dr. Kodish is supported by grants from the Cancer Treatment Research Foundation (CTRF G-02035) and the National Cancer Institute (RO1 83267).

REFERENCES 1. Brody BA. The Ethics of Biomedical Research: An International Perspective. New York: Oxford University Press, 1998. 2. Moreno JD. Undue Risk: Secret State Experiments on Humans. New York: Routledge, 2001. 3. National Commission, The Belmont Report, 1979. 4. Beauchamp TL, Childress JF. Principles of Biomedical Ethics. 4th ed. New York: Oxford University Press, 1994. 5. World Medical Association. Declaration of Helsinki. Adopted by the 18th World Medical Assembly, Helsinki, Finland, June 1964 and amended in Tokyo, 1975, in Venice, 1983, in Hong Kong, 1989, and in South Africa, October 1996. 6. Jonsen AR, Toulmin S. The Abuse of Casuistry: A History of Moral Reasoning. Berkeley: University of California Press, 1988. 7. MacIntyre A. After Virtue. Notre Dame: University of Notre Dame Press, 1984. 8. Kuczewski MG. Fragmentation and Consensus: Communitarian and Casuist Bioethics. Washington, DC: Georgetown University Press, 1997. 9. Childress JF. Narrative(s) versus norm(s). In: Nelson HL, ed. Stories and Their Limits. New York: Routledge, 1997. 10. Levine RJ. Ethics and Regulation of Clinical Research. 2nd ed. New Haven: Yale University Press, 1986. 11. Katz J. The Silent World of Doctor and Patient. New York: Free Press, 1984. 12. Kodish E. Informed consent for pediatric research: is it really possible? J Pediatr 2003; 142:89–90. 13. The National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research, Report and Recommendations: Research Involving Children. Washington, DC: DHEW Publication No. 77-0004, 1977. 14. Kopelman LM. When is the risk minimal enough for children to be research subjects? In: Kopelman LM, Moskop JC, eds. Children and Health Care: Moral and Social Issues. Boston: Kluwer Academic Publishers, 1989. 15. Patenaude AF, Rappeport JM, Smith BR. The physician’s influence on informed consent for bone marrow transplantation. Theor Med 1986; 7:165–179. 16. Carney B. Bone marrow transplantation: nurses’ and physicians’ perception of informed consent. Cancer Nurs 1987; 10:252–259. 17. Lesko LM, Dermatis H, Penman D, Holland JC. Patients’, parents’ and oncologists’ perceptions of informed consent for bone marrow transplantation. Med Pediatr Oncol 1989; 17:181–187.

268

Barfield and Kodish

18. Singer DA, Donnelly MB, Messerschmidt GL. Informed consent for bone marrow transplantation: identification of relevant information by referring physicians. Bone Marrow Transplant 1990; 6:431–437. 19. Dermatis H, Lesko LM. Psychosocial correlates of physician-patient communication at time of informed consent for bone marrow transplantation. Cancer Invest 1991; 9:621–628. 20. Andrykowski MA, Brady MJ, Greiner CB, et al. “Returning to normal” following bone marrow transplantation: outcomes, expectations and informed consent. Bone Marrow Transplant 1995; 15:573–581. 21. Ross JA, Severson RK, Pollock BH, Robinson LL. Childhood cancer in the United States. Cancer 1996; 77:201–207. 22. Gordon PR, Leimig T, Mueller I, et al. A large-scale method for T cell depletion: towards graft engineering of mobilized peripheral blood stem cells. Bone Marrow Transplant 2002; 30:69–74. 23. Barfield RC, Otto M, Houston J, et al. A one-step large-scale method for T- and B-cell depletion of mobilized PBSC for allogeneic transplantation. Cytotherapy 2004; 6:1–6. 24. Ruccione K, Kramer RF, Moore IK, Perin G. Informed consent for treatment of childhood cancer: factors affecting parents’ decision making. J Pediatr Oncol Nurs 1991; 8:112–121. 25. Kodish ED, Pentz RD, Noll RB, Ruccione K, Buckley J, Lange BJ. Informed consent in the Childrens Cancer Group. Cancer 1998; 82:2467–2481. 26. Simon S, Eder M, Raiz P, Syzanski S, Pentz R, Kodish ED. Informed consent for pediatric leukemia research. Cancer 2001; 92:691–700. 27. Levi RB, Marsick R, Drotar D, Kodish E. Diagnosis, disclosure, and informed consent: learning from parents of children with cancer. J Pediatr Hematol Oncol 2000; 22:3–12. 28. Kupst MJ, Patenaude AF, Walco GA, Sterling C. Clinical trials in pediatric cancer: parental perspectives on informed consent. J Pediatr Hematol Oncol 2003; 25:787–790. 29. Kodish E, Eder M, Noll RB, et al. Communication of randomization in childhood leukemia trials. JAMA 2004; 291:470–475. 30. Jacoby LH, Maloy B, Cirenza E, Shelton W, Goggins T, Balint J. The basis of informed consent for BMT patients. Bone Marrow Transplant 1999; 23:711–717. 31. Angiolillo AL, Simon C, Kodish E, et al. Staged informed consent for a randomized clinical trial in childhood leukemia: impact on the consent process. Pediatr Blood Cancer 2004; 42:433–437. 32. Syse A. Norway: valid (as opposed to informed) consent. Lancet 2000; 356:1347–1348. 33. Blustein J, Moreno JD. Valid consent to treatment and the unsupervised adolescent. In: Blustein J, Levine C, eds. The Adolescent Alone: Decision Making in Health Care in the United States. New York: Cambridge University Press, 1999:100–110. 34. Spinetta JJ, Masera G, Jankovic M, et al. Valid informed consent and participative decision-making in children with cancer and their parents: a report of the SIOP working committee on psychosocial issues in pediatric oncology. Med Pediatr Oncol 2003; 40:244–246. 35. Gahrton G, van Rood JJ, Oudshoorn M, The World Marrow Donor Association (WMDA). The world marrow donor association (WMDA): its goals and activities. Bone Marrow Transplant 2003; 32:121–124. 36. Buckner CD, Peterson FB, Bolonesi BA. Bone marrow donors. In: Forman SJ, Blume KG, Thomas ED, eds. Bone Marrow Transplantation. 1st ed. Boston: Blackwell Scientific Publications, 1994:259–269. 37. Egeland T, Lie J, Persson U, Raymond J, Muller C. Donor and liability insurance of donor registries, donor centers, and collection centers—recommendations. Bone Marrow Transplant 2004; 33:467–470. 38. Bakken R, van Walraven A-M, Egeland T. Donor commitment and patient needs. Bone Marrow Transplant 2004; 33:225–230. 39. Rosenmayr A, Hartwell L, Egeland T. Informed consent—suggested procedures for informed consent for unrelated haematopoietic stem cell donors at various stages of recruitment, donor evaluation, and donor workup. Bone Marrow Transplant 2003; 31:539–545. 40. Confer DL. The national marrow donor program: meeting the needs of the medically underserved. Cancer 2001; 91:274–278. 41. Kollman C, Weis T, Switzer GE, et al. Non-HLA barriers to unrelated donor stem cell transplantation. Bone Marrow Transplant 2001; 27:581–587. 42. Fortanier C, Kuentz M, Sutton L, et al. Healthy sibling donor anxiety and pain during bone marrow or peripheral blood stem cell harvesting for allogeneic transplantation: results of a randomized. Bone Marrow Transplant 2002; 29:145–149.

Ethical Considerations in Pediatric HSCT

269

43. Auquier P, Macquart-Moulin G, Moatti JP, et al. Comparison of anxiety, pain and discomfort in two procedures of hematopoietic stem cell collection: leukapheresis and bone marrow harvest. Bone Marrow Transplant 1995; 16:541–547. 44. Munzenberger N, Fortanier C, Macquart-Moulin G, et al. Psychosocial aspects of hematopoietic stem cell donation for allogeneic transplantation: how family donors cope with their experience. PsychoOncol 1999; 8:55–63. 45. Switzer GE, Dew MA, Magistro CA, Goycoolea JM, Twillman RK, Alter C. The effects of bereavement on adult sibling bone marrow donors’ psychological well-being and reactions to donation. Bone Marrow Transplant 1998; 21:181–188. 46. Weisz V, Robbennolt JK. Risks and benefits of pediatric bone marrow donation: a critical need for research. Behav Sci Law 1996; 14:375–391. 47. MacLeod KD, Whitsett SF, Mash EJ, Pelletier W. Pediatric sibling donors of successful and unsuccessful hematopoietic stem cell transplants (HSCT): a qualitative study of their psychosocial experience. J Pediatr Psychol 2003; 28:223–231. 48. Mumford SE. Donation without consent? Legal developments in bone marrow transplantation Br J Haematol 1998; 101:599–602. 49. Packman WL. Psychosocial impact of pediatric BMT on siblings. Bone Marrow Transplant 1999; 24:701–706. 50. Packman WL, Crittenden MR, Schaeffer E, Bongar B, Fischer JB, Cowan MJ. Psychological consequences of bone marrow transplantation in donor and nondonor siblings. J Dev Behav Pediatr 1997; 18:244–253. 51. Gluckman E, Broxmeyer HA, Auerbach AD, et al. Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 1989; 321:1174–1178. 52. Grewal SS, Barker JN, Davies SM, Wagner JE. Unrelated donor hematopoietic cell transplantation: marrow or umbilical cord blood? Blood 2003; 101:4233–4244. 53. Gluckman E. European organization for cord blood banking. Blood Cells 1994; 20:601–608. 54. Ende N. Cord blood collection: effects on newborns. Blood 1995; 85:3361 (letter). 55. Bertolini F, Battaglia M, De Iulio C, Sirchia G. Effects of newborns (Medical-legal). Blood 1995; 86:4700 (letter). 56. Burgia GR, Locatelli F. Transplant of bone marrow and cord blood hematopoietic stem cells in pediatric practice, revisited according to the fundamental principles of bioethics. Bone Marrow Transplant 1997; 19:1163–1168. 57. Sugarman J, Kaalund V, Kodish E, et al. Ethical issues in umbilical cord blood banking. JAMA 1997; 278:938–943. 58. National Heart, Lung, and Blood Institute. Collection and Storage Centers for Clinical Research on Transplantation of Umbilical Cord Stem and Progenitor Cells. Bethesda, MD: National Institutes of Health, 1995. June 20. 59. Sugarman J, Reisner EG, Kurtzberg J. Ethical aspects of banking placental blood for transplantation. JAMA 1995; 274:1783–1785. 60. Cassell C, Kulwicki LA, Kodish ED, Sugarman J. Spinning straw into gold: ethical concerns in umbilical cord blood banking. In: Macpherson CR, Domen RE, Perlin TM, eds. Ethical Issues in Transfusion Medicine. Bethesda, MD: AABB Press, 2001. 61. Burgio GR, Gluckman E, Locatelli F. Ethical reappraisal of 15 years of cord-blood transplantation. Lancet 2003; 361:250–252. 62. Marshal E. Clinical promise, ethical quandary. Science 1996; 271:586–588. 63. Handgretinger R, Klingebiel T, Lang P, et al. Megadose transplantation of purified peripheral blood CD34C progenitor cells from HLA-mismatched donors in children. Bone Marrow Transplant 2001; 27:777–783. 64. Drazen JM, Koski G. To protect those who serve. N Engl J Med 2000; 343:1643–1645. 65. Marshall E. Fred Hutchinson Center under fire. Science 2001; 292:25. 66. Marquis D. How to resolve an ethical dilemma concerning randomized clinical trials. N Engl J Med 1999; 341:691–693. 67. Djulbegovic B. Acknowledgment of uncertainty: a fundamental means to ensure scientific and ethical validity in clinical research. Curr Oncol Rep 2001; 3:389–395. 68. Freedman B. Equipoise and the ethics of clinical research. N Engl J Med 1987; 317:141–145. 69. Schmidt B, Gillie P, Caco C, Roberts J, Roberts R. Do sick newborn infants benefit from participation in a randomized clinical trial? J Pediatr 1999; 134:151–155. 70. Lantos JD. The “inclusion benefit” in clinical trials. J Pediatr 1999; 134:130–131.

270

Barfield and Kodish

71. Glatstein E. What is research? Oncologist 2002; 7:6–8. 72. Grunberg SM, Cefalu WT. The integral role of clinical research in clinical care. N Engl J Med 2003; 348:1386–1388. 73. Emanuel EJ, Wendler D, Grady C. What makes clinical research ethical? JAMA 2000; 283:2701–2711. 74. National Institutes of Health: Policy on the Inclusion of Children as Subjects in Clinical Research. Bethesda: Office of Extramural Research, 1997. 75. Denham EJ, Nelson RM. Self determination is not an appropriate model for understanding parental permission and child assent. Anesth Analg 2002; 94:1049–1052. 76. Tait AR, Voepel-Lewis T, Malviya S. Participation of children in clinical research. Anesthesiology 2003; 99:819–825. 77. Walters MC, Storb R, Patience M, et al. For the multicenter investigation of bone marrow transplantation for sickle cell disease. Impact of bone marrow transplantation for symptomatic sickle cell disease: an interim report. Blood 2000; 95:1918–1924. 78. Hoppe CC, Walters MC. Bone marrow transplantation in sickle cell anemia. Curr Opin Oncol 2001; 13:85–90. 79. Locatelli F, Rocha V, Reed W, et al. For the eurocord transplant group. Related umbilical cord blood transplantation in patients with thalassemia and sickle cell disease. Blood 2003; 101:2137–2143. 80. Kodish E, Lantos J, Stocking C, Singer PA, Siegler M, Johnson FL. Bone marrow transplantation for sickle cell disease: a study of parents’ decisions. N Engl J Med 1991; 325:1349–1353. 81. van Besien K, Koshy M, Anderson-Shaw L, et al. Allogeneic stem cell transplantation for sickle cell disease: a study of patients’ decisions. Bone Marrow Transplant 2001; 28:545–549. 82. Mullen CA, Chan KW. Ethical considerations in allogeneic hematopoietic cell transplantation for children with slowly fatal conditions. Bone Marrow Transplant 2000; 26:1030–1031. 83. Grewal SS, Kahn JP, MacMillan ML, Ramsay NKC, Wagner JE. Successful hematopoietic stem cell transplantation for Fanconi anemia from an unaffected HLA-genotype-identical sibling selected using preimplantation genetic diagnosis. Blood 2004; 103:1147–1151. 84. Verlinsky Y, Rechitsky S, Sharapova T, Morris R, Taranissi M, Kuliev A. Preimplantation HLA testing. JAMA 2004; 291:2079–2085. 85. Burgio GR, Locatelli F. Ethics of creating programmed stem-cell donors. Lancet 2000; 356:1868–1869. 86. Pennings G, Schots R, Liebaers I. Ethical considerations on preimplantation genetic diagnosis for HLA typing to match a future child as a donor of haematopoietic stem cells to a sibling. Hum Reprod 2002; 17:534–538. 87. Feudtner C, Christakis DA, Zimmerman FJ, Muldoon JH, Neff JM, Koepsell TD. Characteristics of deaths occurring in children’s hospitals: implications for supportive care services. Pediatrics 2002; 109:887–893. 88. Rabow MW, Hardie GE, Fair JM, McPhee SJ. End-of-life care content in 50 textbooks from multiple specialties. JAMA 2000; 283:771–778. 89. Chiodi A, Spinelli S, Ravera G, et al. Quality of life in 244 recipients of allogeneic bone marrow transplantation. Br J Haematol 2000; 110:614–619. 90. Himelstein BP, Hilden JM, Boldt AM, Weissman D. Pediatric palliative care. N Engl J Med 2004; 350:1752–1762. 91. Wolfe J, Grier HE, Klar N, et al. Symptoms and suffering at the end of life in children with cancer. N Engl J Med 2000; 342:326–333. 92. Hilden JM, Emanuel EJ, Fairclough DL, et al. Attitudes and practices among pediatric oncologists regarding end-of-life care: results of the 1998 American society of clinical oncology survey. JCO 2001; 19:205–212. 93. Feudtner C, DiGiuseppe DL, Neff JM. Hospital care for children and young adults in the last year of life: a population-based study. BMC Med 2003; 1:1–9.

14 Immune Reconstitution in Pediatric Patients Following Hematopoietic Stem-Cell Transplantation Trudy N. Small Department of Pediatrics and Clinical Laboratories, Memorial Sloan-Kettering Cancer Center, New York, New York, U.S.A.

INTRODUCTION The success of hematopoietic stem cell transplantation (HSCT) depends not only on the eradication of primary disease but also on the rapid restoration of durable hematopoiesis and immune competence. To date, multiple groups have evaluated the immune reconstitution of infants afflicted with severe combined immunodeficiency disease (SCID) (1–4), as well as children transplanted for metabolic diseases, cancer, and bone marrow failure states. The majority of these studies have assessed the reconstitution of lymphoid populations and nonspecific mitogen responses. Fewer studies have included the kinetics of recovery of antigenspecific T- and B-cell responses, the development of which is crucial to decrease death from infection and to develop protection following immunization against vaccine-preventable disease. Over the last decade, disease free survival following unrelated donor (URD) transplantation has continued to improve, with many children and young adults enjoying survival rates approximating those observed following HLA-matched related (MRD) bone marrow transplant (BMT) (5–9). In patients with chronic phase CML transplanted within 1 year of diagnosis, Weisdorf et al. reported a 61% and 68% 5-year disease-free survival following matched unmodified URD and MRD BMT, respectively (5). In children with acute lymphoblastic leukemia in 2nd remission (ALL CR2), 3-year event-free survival (EFS) was 60G11% following URD and 63G18% post MRD unmodified BMT, respectively (6). Following T cell-depleted (TCD) marrow transplantation; Kernan et al. reported that 19 of 23 children and 10 of 11 children with acute leukemia in CR1 or CR2 survive disease free (7), respectively. In good-risk patients, infection, not relapse, continues to be the most common cause of failure following unrelated HSCT. The primary cause of death in 5075 recipients of an unrelated transplant facilitated by the National Marrow Donor Program was infection, occurring in 29% and 30% recipients of a T-replete or TCD transplant, respectively (10). In most transplant series, infectious complications are lower in children than adults matched for transplant type and stage of disease. Following HLA-mismatched related or unrelated HSCT, the duration of risk for severe infections extends later than that observed post-HLA-matched related HSCT (11–13). This is unlikely to simply reflect a lower exposure of children to virulent 271

272

Small

pathogens but rather the more rapid reconstitution of antigen specific responses observed in younger patients due to residual thymic function (14). This chapter will focus on lymphoid reconstitution following pediatric HSCT, including responses to routine childhood vaccinations and potential therapies to reduce the period of immune deficiency post HSCT.

NATURAL KILLER CELL RECONSTITUTION POST-HEMATOPOIETIC STEM-CELL TRANSPLANTATION Regardless of stem cell or donor source, natural killer (NK) cells, characterized by expression of CD56 and CD16 and cytotoxicity against the K562 cell line (15,16), are the first functional lymphoid cells to recovery post transplant, including autologous HSCT (17). The one exception is the markedly impaired NK cell recovery observed in a subset of SCID patients (Fig. 1) not given cytoreduction prior to a TCD HLA mismatched related HSCT (4). These patients are often plagued with recalcitrant papilloma–induced skin lesions, not observed in SCID given cytoreduction who develop NK cell function post HSCT (18). Following transplantation, NK cells offer the first line of defense against viral infections (15,16). In addition, recent data has shown that inhibitory killer immunoglobulin-like receptors present on NK cells may play an important role in recognizing and destroying tumor cells that fail to express their cognate ligand (19,20), particularly in patients transplanted for AML and MDS. Following TCD PBSCT, NK cells represented up to 90% of circulating lymphocytes for the first three months post TCD PBSCT for metabolic storage diseases (21). Similarly, children achieved normal numbers of NK cells at one month following an unrelated or HLA-matched related BMT T-cell depleted by soybean agglutination, followed by rosetting with sheep red blood achieved normal NK cell numbers by one month post transplant (13,22). Rapid NK recovery has also been observed in children who received G-CSF mobilized CD34 positively selected peripheral blood stem cells from haplo-identical parental donors (23,24). Failure to achieve donor NK cells by three weeks post TCD HSCT is often the first sign of immunologically mediated graft failure (25). Comparison of NK cell recovery in 88 children following an unmodified BMT derived from an HLA matched related (nZ40), mismatched related (nZ8), or phenotypically matched unrelated (nZ10) donor with 30 recipients of a cord blood (nZ20) or CD34 positively selected TCD peripheral blood stem cell transplant (nZ10) revealed prompt NK recovery in all patient groups without any significant differences observed (26). Figure 2 demonstrates the median NK cells/ul

Months Post-TCD MM Related BMT for SCID Figure 1 10th–50th–90th CD56 cell counts/ul. Abbreviations: TCD, T-cell depleted; SCID, severe combined immunodeficiency disease.

Immune Reconstitution Post-HSCT

273

1000

CD56 + cells/ul

100 Adults, TCD BMT, Unrelated, n=58 Adults, TCD BMT, Related, n=168 Adults, TCD PBSCT, n=14 Adults, MM-related PBSCT, n=26

10

Children, Unrelated, n=50 Children, Rel, SBA-E-, n=34 1 0-2m

2-4m

4-6m

6-9m

9-12m

Months post HCT

Figure 2 Median CD56CNK cells/ul post TCD HLA-matched, mismatched or unrelated HCT, nZ350. Abbreviations: BMT, bone marrow transplantation; HLA, human leukocyte antigen; MM, mismatched; SBA, soybean agglutination; TCD, T-cell depleted.

recovery in children and adults following an unmodified or TCD BMT derived from an unrelated or HLA-matched related donor performed at Memorial Sloan Kettering Cancer Center (MSKCC). Similar to the results of the multiple studies cited above, there is very little difference in the time to achieve normal numbers of NK cells among the different transplant groups, with normal numbers reached by all groups within the first two months post HSCT.

T-CELL RECONSTITUTION In contrast to NK cell reconstitution, T-cell recovery post HSCT is influenced by the stem cell and donor source, patient age, and use and method of T-cell depletion. Storek et al. determined that advanced patient age (pZ0.01), graft type (marrow vs. blood stem cells), CD34 cell dose, inclusion of total body irradiation in the conditioning regimen, and acute or chronic graft-versus host-disease impaired T-cell reconstitution in adults following T-cell replete HSCT (27). Studies performed at MSKCC demonstrated that age O19 years and the use of antithymocyte globulin (ATG) for the prevention of graft rejection following an SBA-E- TCD related or unrelated BMT adversely impacted recovery of CD4CT cells and acquisition of normal T-cell function (13,22). These studies demonstrated that adult recipients of a TCD unrelated BMT experience prolonged and profound deficiencies of CD3C, CD4C, and CD8C T-cell populations when compared with pediatric recipients of unrelated BMT and adults after related BMT (P!0.01), that adults have a significantly increased risk of life-threatening opportunistic infections, and that the rate of recovery of CD4 T cells correlates with the risk of developing these infections. Recovery of normal numbers of CD3C, CD8C, and CD4C T-cell populations is similar in children after related or unrelated BMT (13,22). Comparison of CD4 recovery following unrelated TCD BMT or unmodified BMT demonstrates that regardless of the type of transplant, children reconstitute CD4 cells much faster than their adult counterparts following any of these transplant types (Fig. 3). In 1993, Foot et al. compared lymphoid reconstitution in children who received a TCD or unmodified BMT (28). Patients in both groups demonstrated low T-cell numbers for the first 12 months post HSCT, with a prolonged phase of CD4 lymphopenia, exacerbated by graftversus-host disease (GVHD). Mackall et al. described delayed CD4 T cell recovery in children and young adults following autologous HSCT, particularly helper cells expressing a naive (CD45RA) phenotype (29). A prospective study by Eyrich et al. (30), compared lymphoid

274

Small

Figure 3 10th–50th–90th CD3 cell counts/ul. Abbreviations: TCD, T-cell depleted; SCID, severe combined immunodeficiency disease.

reconstitution in 25 pediatric recipients of a TCD unrelated PBSCT (nZ13) or unmodified HLA-matched sibling BMT (nZ12). The time to develop O100 CD3C cells/ul was prolonged in unrelated PBSCT recipients compared to HLA-matched sibling BMT recipients, occurring at a median of 140 versus 39 days, respectively. Recovery of O100 CD4 cells/ul was also delayed in these patients, occurring at a median of 180 and 78 days following unrelated TCD PBSCT and unmodified HLA-matched related BMT, respectively. These authors demonstrated that early T-cell reconstitution following TCD unrelated PBSCT was typified by a limited T-cell repertoire and predominance of T cells expressing a memory phenotype (i.e., CD45ROC). Despite this, by 3 months post HSCT, recovery of naive CD4CCD45RAC cells and T-cell proliferative responses to PHA were similar in the two groups and by 12 months, both had a similarly complex T-cell repertoire (30). Another study performed in pediatric recipients of a TCD PBSCT (nZ33) showed that the mean time to recover O200 CD4 or CD8CT cells was O6 months post transplantation (31). T-cell recovery in 102 children following a TCD HLAmismatched related (nZ88) or unrelated (nZ14) BMT revealed an inverted CD4/CD8 ratio for at least 12 months post transplant and delayed reconstitution of CD8 and CD4CT cells for 18 and 24–36 months, respectively (32). In this study, GVHD, and CMV infection were associated with a significantly delayed reconstitution of CD3C and CD4CT cells. Lack of T cells for 5–6 months was observed by Ball et al. in 13 children who engrafted following a CD34positively selected PBSCT TCD HLA-mismatched parental PBSCT (33). This contrasts with the more rapid reconstitution of CD3 positive T cells in infants with SCID transplanted with SBA-E- HLA-mismatched parental BM, even when cytoreduction is not employed (Fig. 4). For patients lacking an HLA-matched sibling or a suitable adult unrelated stem cell donor, the use of cord blood has become increasingly more common. The decreased incidence of GVHD when compared to transplants from adult URDs, even in the face of increasing degrees of HLA disparity, makes unrelated cord blood transplant (CBT) an attractive choice in children in whom larger cell doses are possible. Moretta et al. (34) compared immune reconstitution in pediatric recipients of cord blood or unmodified bone marrow from an HLA matched sibling or URDs. Despite lower total nucleated, CD34, and CD3 cell doses/kg, recipients of cord blood had more rapid recovery of CD4CT cells compared to unmodified BMT recipients, and there were no significant differences between the time to recover normal numbers of T lymphocytes and CD8CT cells and mitogen responses between the two groups (34). In a single center study evaluating immune reconstitution following unrelated CBT, CD4 cell counts O200/ul were achieved at a median of five months, but total CD3 and

Immune Reconstitution Post-HSCT

275

1000 Children, n=90

Adults, n=158

CD4 cell/ul

800 600 400 200 0 0-2m 2-4m 4-6m 6-9m 9- 12- 18- 0-2m 2-4m 4-6m 6-9m 9- 12- 1812m 18m 24m 12m 18m 24m

Figure 4 Comparison of the median CD4C cell count/ul following unrelated BMT:SBA-E (circles), MoAb (T10B9), complement, triangles, or unmodified (squares).

CD3CCD8C lymphocyte recovery was depressed for at least one year post HSCT (35). A study by Niuhus et al. (36) analyzed the median time to develop normal numbers of CD8, CD4, and CD3CT cells in cord blood recipients. Factors associated with faster T-cell recovery included higher TNC dose/kg, receipt of an HLA matched sibling CBT, and a CMV seropositive host. Acute graft-versus-host disease delayed T-cell reconstitution (36). To date, the majority of studies have evaluated immune reconstitution following a nonmyeloablative conditioning regimen have been performed in adults. Busca et al. compared the kinetics of immune reconstitution in 15 adult patients receiving a nonmyeloablative transplant following a fludarabine based conditioning regimen compared to 30 adult recipients of a conventional HSCT (37). The majority of patients received G-CSF mobilized peripheral blood stem cells. There were no significant differences in the absolute numbers of helper (CD4C) T cells, naive (CD4CCD45RAC) and memory (CD4CCD45ROC) T cells, or in the numbers of suppressor (CD8C) T cells in the two groups. Both groups achieved a median value of 200 CD4CT cells/ul by two months post transplant. There were no significant differences in the incidence of bacteremia or CMV viremia between the two groups. Bacteremia and CMV infection occurring in 13%, 36%, 15%, and 39% of patients receiving a nonmyeloablative or ablative conditioning regimen, respectively. Slavin et al. (38) compared immune reconstitution in children following an HLA-matched sibling transplant utilizing either an ablative cytoreduction regimen and BMT (nZ27) or reduced intensity conditioning regimen (fludarabine, oral busulfan, ATG) followed by an unmodified PBST (nZ10). Both groups received cyclosporine alone for GVHD prevention. Compared to healthy children, patients in both groups demonstrated a lower percentage of CD3 and CD4 positive T cells and an increased percentage of CD8 positive cells. In contrast to recipients of an ablative conditioning regimen, recipients of nonmyeloablative cytoreduction demonstrated an almost normal stimulation index in response to PHA at 1, 3, and 12 months post HSCT. Despite this, there were no statistically significant differences in the incidence of invasive bacterial infections, fungal infections, or pneumonia secondary to P. carinii between the two groups. The immune reconstitution of six children and young adults who underwent an unrelated nonmyeloablative PBSCT at MSKCC following conditioning with fludarabine based regimen with Campath and Cyclosporine A for GVHD prophylaxis is compared to adult recipients transplanted with the same regimen is shown in Figure 5, demonstrating that even following a nonablative transplant the differences in kinetics of CD4 and particularly CD45RAC helper cells. Several methodologies have been utilized to differentiate T cell differentiation occurring via thymic dependent or independent pathways, including T-cell receptor diversity, naı¨ve versus memory phenotype, and expression of T-cell receptor rearrangement excision circles (TRECs), nonreplicating episomal DNA generated in thymic progenitors during generation of

276

Small

600 Children, n=6

Adults, n=8

500

cells/ul

400 300 200 100 0 0-2m 2-4m 4-6m 6-9m 9-12m 0-2m 2-4m 4-6m 6-9m 9-12m Months post PBSCT

Figure 5 Median CD4C (triangle) and CD45RACCD4C (circles) T cells post nonmyeloablative unmodified unrelated PBST.

a mature T-cell receptor (14,39–41). Lewin et al. compared the numbers of TREC positive T cells in the circulation of children and adults following T-cell replete or TCD BMT following a myeloablative-conditioning regimen (42). This study demonstrated that patients !19 years of age at the time of HSCT had significantly more rapid recovery and reached higher levels of circulating TREC positive T cells than patients O19 years of age. Although at early time points, recipients of a TCD BMT had lower TRECCT cells than unmodified transplant recipients (P!0.01), there were no significant differences by 9 months post HSCT. Talvensaari et al. compared the kinetics of TCR repertoire diversity, TREC levels, and immune phenotype in patients undergoing CB transplant (nZ10) compared to 19 age-matched recipients of a bone marrow transplant with similar degrees of GVHD (43). This study demonstrated that within the first year post HSCT, both groups had low TREC levels and limited TCR receptor diversity. By two years post HSCT, TREC values and TCR diversity were actually higher in CB recipients than BMT recipients, demonstrating the efficient thymic differentiation of cord blood derived stem cells despite lower CD34C cell doses per patient weight. The number of circulating TREC positive T cells at any one time point depends on complex balance between thymic output, lifespan of resting TREC positive T cells, and dilution due to proliferation following binding of cognate ligand. Eyrich et al. (44) investigated TREC levels in children following an autologous (nZ51), HLA-matched sibling (nZ47), unrelated (nZ45) or TCD haplo-identical parental (nZ21) HSCT and the influence of age, transplant type, donor source, and acute and chronic GVHD on these parameters. The relationship of TREC levels with T-cell turnover rates, measured by spontaneous Ki67 expression, was evaluated a subset of children (nZ81). The authors determined the earliest time to detect TREC positive cells in the circulation and the maximum level of TREC positive cells reached post HSCT (44). All patients received an ablative conditioning regimen and the majority (nZ104) received a rigorously TCD PBSCT. The number of TRECC cells/ul correlated with the total number of naı¨ve CD3C, CD4C, and CD8CT cells defined by expression of CD45RA (Fig. 6). Spontaneous expression of Ki67 was significantly higher in patients studied before 6 months post HSCT compared to later time points. Ki67 expression was positively correlated between all T-cell subsets evaluated suggesting a common factor driving proliferation. Neither T-cell depletion nor GVHD appeared to influence Ki67 expression in the majority of T-cell populations evaluated, with the exception of decreased Ki67 in naı¨ve CD8CT cells derived from patients with GVHD following an unmodified BMT but not a TCD HSCT. In this study, the median time to develop TREC positive cells was 83 days in children less than 1 year of age at the time of HSCT with an additional 14 days added for every year of life at the time of transplant. This study

Immune Reconstitution Post-HSCT

277

Figure 6 Correlation of TRECS with naı¨ve T cell populations. After logarithmic transformation, TREC levels correlated with the numbers of (A) naı¨ve CD4C and (B) naı¨ve CD8C cells and with total numbers of (C) naı¨ve CD3C cells. (D) Furthermore, TREC content, given as TRECs per 100,000 T cells, correlated well with the calculated number of TRECC T cells per microliter. Source: Reprinted with permission, Eyrich et al. Biology of Blood and Marrow Transplantation 11:194–205 (2005).

found no effect on time to develop TREC positive cells on the basis of graft or donor type, conditioning regimen, or number of stem cells transplanted. Age and presence of acute or chronic GVHD were the only factors that influenced time to develop TREC positive cells post HSCT and CD34C cell dose/kg was the only factor which influenced the maximum output of TREC positive cells. This study demonstrated that multiple factors affect TREC levels following HSCT, and emphasizes the importance of age, GVHD, and stem cell dose on T-cell development via thymic dependent pathways (44).

B-CELL RECONSTITUTION The B-cell reconstitution of 88 consecutive children and adults following an autologous, conventional, or T-cell depleted bone marrow transplantation (45) demonstrated that the earliest detectable B cells normally expressed HLA-DR, CD19, surface immunoglobulin, CD21, Leu-8, and lacked expression of CD10 (CALLA). In addition, B cells circulating during the first post transplant year coexpressed CD1c, CD38, CD5, and CD23, antigens normally expressed on a small percentage of circulating B cells in normal adults but highly expressed on cord blood B cells. The B cells isolated during the first year following transplant proliferated normally to Staphylococcus aureus Cowan strain I (SAC) and produced IgM, but minimal or no IgG, when stimulated with pokeweed mitogen and SAC, similar to cord blood B cells but unlike

278

Small

normal adult B cells produce both IgM and IgG. In this study as well as a 1993 report by Storek et al. (46), patients with chronic GVHD experienced a marked delay in B-cell reconstitution. The similar phenotype and function of post transplant and cord blood B cells, and their similar rate of decline in patients and normal children supported the hypothesis that B-cell differentiation post transplant, in the absence of GVHD, recapitulates normal B-cell ontogeny. A 2001 study (47) that analyzed factors implicated in B-cell recovery following allogeneic transplant in patients 18–61 years of age demonstrated that patients with extensive GVHD had significantly fewer bone marrow B-cell precursors at 1 year post HSCT than patients with limited or no cGVHD. Donor or patient age, use of bone marrow or PBSCs, or CD34C cell dose infused did not affect the number of B-cell precursors at 1 year post transplant. Eyrich et al. compared B-cell recovery (30) in 13 children following a TCD unrelated PBSCT and 12 children following an unmodified HLA matched sibling BMT. Acute grade II-IV GVHD occurred in two of 13 and 10 of 12 children in the TCD unrelated and unmodified related transplant patients, respectively. The time to recover O100 CD19C cells/ul was shorter in the unrelated group compared to the matched sibling transplant recipients taking a median of 69 days (range, 49–105 days) in the UD-HSCT group and 176 days (range, 66–462 days) in the MSD-BMT group (PO0.001). A similar analysis comparing B-cell recovery in cord blood or unmodified bone marrow demonstrated a more rapid normalization of circulating B cells in the cord blood group (34). Comparison of B-cell recovery in children and adults following TCD related or unrelated BMT showed rapid recovery in all groups, generally by 3 months post transplant (13,22). Despite the rapid recovery of B-cells in most pediatric transplant recipients, except those with chronic GVHD (45), Di Martino, utilizing CDR3 fingerprinting, showed delayed polyclonality in all children evaluated until at least 6 months post transplant (48).

ANTIGEN-SPECIFIC RESPONSES The ability to protect the host from invading pathogens and vaccine preventable disease is crucial in order to decrease nonrelapse mortality following transplantation. Several studies have evaluated the kinetics of antigen-specific responses to CMV (49,50), EBV (51,52), adenovirus (53), and influenza (54) following HSCT, pathogens comprising a significant portion of fatal opportunistic infections post transplant. Hakki et al. analyzed CMV-specific T-cell reconstitution in adults and children following unmodified allogeneic HSCT (49). These authors found that CD4 and CD8 cell counts less than 100/ul and 50/ul, use of bone marrow as the stem cell source and treatment with high dose steroids were associated with impaired CMV immunity at 3 months post HSCT. Lang et al. (50) studied the ability of mononuclear cells (MNC) derived from 11 children who received a CD34 positively selected PBSCT from an unrelated or mismatched related donor. MNC from seven of 11 patients were capable of lysing CMV-infected targets without cytokine stimulation. Antibody-dependent complement mediated cytotoxicity was significantly increased in these patients by the addition of IL-2 or IL-15, suggesting a potential benefit of low-dose IL-2 or IL-15 combined with hyperimmune anti-CMV gammaglobulin for the treatment of CMV disease. In 1996, Lucas (51) used limiting dilution to analyze the levels of anti-EBV cytotoxic T-lymphocyte precursor (CTLp) frequencies in 26 recipients of unmodified or TCD BMT from an EBV-seropositive donor. At 3 months post BMT, only 5 of 26 patients had EBV CTLp frequencies in the range of normal EBV seropositive controls. By 6 months post BMT, 9 of 13 patients evaluated had EBV CTLp frequencies within the normal range. Marshall et al. used tetramers to quantitate the level of EBV-specific T cells in patients following unmodified, cord blood, or T-cell depleted HSCT (52), showing a marked delay in recovery of EBV-specific T cells. In a 2005 study, Heemskerk et al. (53) correlated adenovirus DNA levels in the blood and lymphocyte reconstitution, including adenovirus specific B- and T- cell responses in 48 children, 21 of whom had evidence of an adenovirus infection following allogeneic transplantation. These authors found that low lymphocyte counts at the onset of infection were predictive of adenovirus viremia and that

Immune Reconstitution Post-HSCT

279

survival in patients with viremia was associated with an increase in lymphocyte counts during the first weeks after infection. In those patients capable of clearing adenovirus from the blood, antigen-specific CD4CT cell responses and neutralizing antibody were detected.

Vaccine Responses Recently, Machado (55) reviewed the current literature on responses of allogeneic and autologous hematopoietic transplant recipients to routine childhood vaccinations and summarized the current recommendations of the Centers for Disease Control and European Group for Blood and Bone Marrow Transplantation. This report emphasizes the heterogeneity of currently published papers on vaccine responses as well as the lack of evidence-based recommendations for vaccination of patients post HSCT. A number of investigators have shown that in the absence of revaccination post allogeneic HSCT, the level of preexisting antibody titers wanes with time post transplant (55,56). The majority of allogeneic transplant patients lack protection against tetanus, polio, measles, mumps, and rubella by 1–3 years post transplant (55,56). Whereas 50% of patients with positive tetanus titers at the time of transplant become seronegative at 1 year and 100% by 2 years post transplant (57), titers against measles, mumps, and rubella decrease more slowly, persisting for at least 2 years in approximately 50% of patients (58). This may unfortunately lull physicians into omitting MMR in patients capable of responding to live attenuated vaccines (58), most of whom will have lost protection to measles, mumps, and rubella by 3–5 years post HSCT (57–59). Several studies have evaluated the efficacy of immunization against tetanus, polio, H. influenzae, pneumococcus, and/or measles, mumps, and rubella post HSCT (54,55). Ljungman et al. (57) have shown the superiority of three doses of tetanus toxoid initiated at 12 months post HSCT to a single dose given at a year post BMT, increasing the rate of seroconversion from 64% to 100% and the proportion of patients with persistent titers at 2 years from !30% to 100%. A similar study by the same group demonstrated that of 19 patients immunized with a single dose of IPV at 1 year post HSCT, only 42%, 36%, and 21% of patients developed a fourfold increase in antibody titers against poliovirus type 1, 2, and 3, respectively, and only one-half of 21 patients vaccinated with a three-dose schedule responded to all 3 serotypes (60). This contrasts with a study by Parkkali et al. (61) in which 45 adult recipients of an HLA-matched sibling BMT were randomized to receive IPV at 6, 8, and 14 months (nZ23) or at 18, 20, and 26 months (nZ22). This study demonstrated that both regimens were equally immunogenic, inducing protective antibody responses in all patients and persistent protective titers in 44 of 45 patients 22 months after the third vaccine dose. In both studies, chronic GVHD did not affect response rates (60,61). Few studies have evaluated the use of recombinant hepatitis B vaccination post HSCT (55,62). In 1997, Volti and colleagues (62) evaluated the kinetics of hepatitis B titers in twenty patients with thalassemia who underwent an unmodified allogeneic transplant from an HLA-matched hepatitis B seronegative sibling donor. All twenty patients were Hepatitis B seropositive prior to transplant secondary to vaccination with plasma derived HBV vaccine given 3–5 years prior to transplant (nZ13) or natural infection in early childhood (nZ7). Ten patients, nine of whom had previously been vaccinated, had negative titers post transplant. Following two vaccinations with the DNA recombinant HBV vaccine, Engerix-B, all 20 patients redeveloped protective titers. Although Machado et al. reported a 100% seroconversion rate in 50 autologous or unmodified allogeneic transplant recipients immunized at least 1 year post transplant, 60% of patients failed to sustain titers for more than 1 year following vaccination (63). At this center, we have vaccinated 260 seronegative patients with a series of recombinant hepatitis B vaccine (64). Approximately 70% of patients developed positive titers following the third hep B vaccination. Advanced patient age and history of chronic GVHD predicted poor response to hepatitis B. Multiple studies have shown the inadequacy of immunization with pure polysaccharide vaccines post HSCT and the superiority of response following vaccination with proteinconjugated polysaccharide vaccines (55). Guinan et al. (65) reported that only 19% of

280

Small

allogeneic or autologous HSCT recipients (nZ35) vaccinated with a single dose of unconjugated Pnu-immune at 24 months, or two doses at 12 and 24 months, responded to all 6 serotypes tested. In contrast, 56%, and 80% of these same patients developed protective titers following vaccination with one or two doses of conjugated HibTITER, respectively. Parkkali et al. (66) showed a limited response in 45 adult allogeneic HSCT recipients immunized with a single dose of unconjugated pneumococcal vaccine at 8 or 20 months post transplant. In contrast, Avanzini et al. (67) demonstrated an effect of time post HSCT on the probability of response in children immunized with a pure polysaccharide pneumococcal vaccine. Whereas 100% of children seroconverted when immunized one year post autologous or unmodified allogeneic HSCT, the rates decreased to 50%, and 30% if immunized at 1–2 or 0.5–1 years, respectively. In a recent randomized study, Molrine et al. (68) evaluated the effect of pretransplant vaccination of donor and host with a heptavalent pneumococcal conjugate vaccine (PCV7, Prevnar) on the development of protective antibody responses in patients immunized at 3, 6, and 12 months post HSCT. Sixty percent of patients in both groups developed significant titers against all 7 serotypes after the 3rd vaccination post HSCT. However, 67% of patients immunized pretransplant who received marrow from immunized donors developed protective antibody titers after the first vaccination post HSCT compared to 36% of nonimmunized recipients of marrow derived from unimmunized donors, PZ0.05. This study demonstrates the efficacy of early vaccination post HSCT with PCV7 when a 3-dose schedule is utilized, as well as the successful transfer of augmented donor B cell immunity following unmodified BMT. Although these studies have furthered our understanding of the ability of patients post HSCT to respond to different classes of vaccines post transplant, they have been limited by the small number of heterogeneous patients evaluated and the lack of long-term follow-up. Data derived from these studies have shown conflicting results even in adult and pediatric recipients of an HLA-matched sibling BMT. In addition, there are very few studies that include unrelated or TCD transplant recipients. Many currently used T-cell depletion techniques remove not only mature T cells from the graft but B cells as well, preventing the transfer of memory B cells from the donor. Although all recipients of an allogeneic HSCT are subject to a varying period of T and B lymphocytopenia and subsequent functional incompetence despite return of normal numbers of T and B cells, the severity and duration of this immunoincompetence is not uniform across transplant types and in patients of all ages (13,22,27,44,46). The 1990 measles epidemic in the United States (69) and the recent outbreak of varicella in a daycare center (70), in which the index case and the majority of affected children had previously received Viravax, underscore the importance of systematic evaluation of the duration of protective antibody titers post vaccination, even in immunocompetent individuals, and particularly in patients post HSCT.

ADOPTIVE IMMUNOTHERAPY The ability to passively transfer viral immunity has been shown by multiple investigators, particularly for the treatment of EBV and CMV infections. Infusion of small numbers of unfractionated donor leukocytes containing as few as 0.1!106 CD3C cells/kg can rapidly restore EBV-specific immunity when derived from EBV-seropositive transplant donors, leading to permanent resolution of EBV lymphomas (71–73). Although effective, this strategy can cause GVHD, particularly when infused !3 months post transplant, during which time many life-threatening opportunistic infections occur (71,72). Adoptive transfer of in vitro propagated donor-derived virus specific T cells can restore immunity to specific pathogens (e.g., CMV, EBV, adenovirus), by providing memory donor T cells capable of peripheral expansion in the patient (71,73,74). Although this strategy carries a limited risk of GVHD, it still leaves the patient vulnerable to other opportunistic infections, is labor intensive, and requires time for generation. In addition, extended survival of in vitro propagated anti-viral T cells requires sufficient numbers of functional circulating CD4CT cells (74), often lacking in patients with the highest risk of recurrent infection. Newer methodologies (75–78), including priming with viral peptides presented by dendritic cells, are being developed to decrease time to

Immune Reconstitution Post-HSCT

281

development and expand adoptive therapy for a wider range of pathogens, including adenovirus (76) and aspergillus (79).

IMMUNOMODULATORY FACTORS Several cytokines and growth factors (79) have been shown to have potent effects on the immune system, including, but not limited to, growth hormone (GH) and insulin-like growth factor-1 (IGF-1) (80), keratinocyte-growth factor (81), and interleukin-7 (IL-7) (79,82,83). GH and/or IGF-1 has been shown to restore thymic architecture in aged, Cy A treated, or diabetic animals. It is capable of enhancing thymic and splenic growth, and augmenting mitogen and specific antigen responses, as well as potentiating pro-B-cell development (80). IGF-1 levels peak in adolescence and decline with advancing age, paralleling thymic development and involution seen with normal aging (81). Following allogeneic transplantation, normal agespecific levels of circulating IGF-1 are rapidly restored children and adolescents (84). In contrast, adults following allo HSCT have levels significantly lower than age matched controls for years post transplant (85). Given the contribution of GH/IGF-1 to lymphoid development, its deficiency may play a role in post HSCT immunodeficiency. IL-7 is a nonredundant growth factor for lymphopoiesis, notably for thymopoiesis as demonstrated by B- and T-cell deficiencies in IL7 K/K or IL-7 RK/K genes knockout mice (86,87). IL-7 fosters the activation and proliferation of mature and developing T cells (88,85) and plays a key role in the homeostatic expansion of peripheral T cells (89). In animal models, IL-7 given to cyclophosphamide-treated or irradiated mice improved T- and B-lymphocyte recovery (90). In a syngeneic murine transplant model Bolotin et al. (91) demonstrated that IL-7 administration resulted in increase thymic cellularity. Fry et al. (92) demonstrated that IL-7 could enhance extrathymic peripheral expansion and restore primary or recall immune responses through the inhibition of programmed cell death in athymic T-cell-depleted mice. Following syngeneic transplantation, Mackall et al. found that IL-7 had, by far, the most potent effect on peripheral T-cell expansion (93). In the setting of allogeneic BMT, IL-7 administered in the early post transplant period to young and middle aged mice accelerated reconstitution of major T- and B-cell populations at day 28 (94,95) and enhanced thymopoiesis. IL-7 treatment increased T-cell numbers and increased T-cell proliferation in response to mitogens. Currently, phase I studies are undergoing to assess the effect of this cytokine in autologous transplant patients, and are soon to be initiated in allogeneic TCD transplant recipients.

CONCLUSION Although children enjoy more rapid recovery of immune function following HSCT when compared to adults, as many as 20% of transplanted children who lack an HLA-matched sibling donor still die from an opportunistic infection. For physicians faced with choosing an alternative donor and stem cell source for these pediatric patients, knowledge of immune reconstitution infectious complications, and effectiveness of immunomodulatory agents following different transplant is crucial.

REFERENCES 1. Myers LA, Patel DD, Puck JM, Buckley RH. Hematopoietic stem cell transplantation for severe combined immunodeficiency in the neonatal period leads to superior thymic output and improved survival. Blood 2002; 99:872–878. 2. Haddad E, Le Deist F, Aucouturier P, et al. Long-term chimerism and B-cell function after bone marrow transplantation in patients with severe combined immunodeficiency with B cells: a single-center study of 22 patients. Blood 1999; 94:2923–2930.

282

Small

3. Antoine C, Mu¨ller S, Cant A, et al. Long-term survival and transplantation of haemopoietic stem cells for immunodeficiencies: report of the European experience 1968–99. Lancet 2003; 361:553–560. 4. Small TN, Friedrich W, O’Reilly RJ. Hematopoietic cell transplantation for immunodeficiency diseases. In: Appelbaum F, Forman S, Blume KG, Thomas ED, eds. Hematopoietic Cell Transplantation. 3rd ed. Malden, MA: Blackwell Science, 2004; 1430–1442. 5. Weisdorf DJ, Anasetti C, Antin JH, et al. Allogeneic bone marrow transplantation for chronic myelogenous leukemia: comparative analysis of unrelated versus matched sibling donor transplantation. Blood 2002; 99:1971–1977. 6. Al-Kasim FA, Thornley I, Rolland, et al. Single-centre experience with allogeneic bone marrow transplantation for acute lymphoblastic leukaemia in childhood: similar survival after matchedrelated and matched-unrelated donor transplants. Br J Haematol 2002; 116:483–490. 7. Kernan NA, Boulad F, Small TN, et al. Improved outcome with T-cell depleted (SBAKEK) marrow transplants from related and unrelated donors for children with acute leukemia transplanted in first or second remission. Blood 2001; 98:674a. 8. Alyea EP, Weller E, Fisher DC, et al. Comparable outcome with T-cell-depleted unrelated-donor versus related-donor allogeneic bone marrow transplantation. Biol Blood Marrow Transplant 2002; 8:601–607. 9. Balduzzi A, Gooley T, Anasetti C, et al. Unrelated donor marrow transplantation in children. Blood 1995; 86:3247. 10. Wagner JE, Anasetti C, Kollman C, et al. Unrelated donor bone marrow transplantation in 5075 patients with malignant and non-malignant disease: effect of graft-versus-host disease prophylaxis on treatment outcome. Blood 1998; 92:686a. 11. Ochs L, Shu XO, Miller J, et al. Late infections after allogeneic bone marrow transplantation: comparison of incidence in related and unrelated donor transplant recipients. Blood 1995; 86:3979. 12. Chen CS, Boeckh M, Seidel K, et al. Incidence, risk factors, and mortality from pneumonia developing late after hematopoietic stem cell transplantation. Bone Marrow Transplant 2003; 32:515–522. 13. Small TN, Papadopoulos EB, Boulad F, et al. Comparison of immune reconstitution following unrelated and related T cell depleted bone marrow transplantation: effect of patient age and donor leukocyte infusions. Blood 1999; 93:467–480. 14. Haynes BF, Markert ML, Sempowski GD, Patel DD, Hale L. The role of the thymus in immune reconstitution in aging, bone marrow transplantation, and HIV-1 infection. Annu Rev Immunol 2000; 18:529–560. 15. Biron CA, Nguyen KB, Pien GC, Cousens LP, Salazar-Mather TP. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Ann Rev Immunol 1999; 17:189–220. 16. Vivier R, Biron CA. Immunology. A pathogen receptor on natural killer cells. Science 2002; 296:1248–1249. 17. Kalwak K, Gorczynska E, Toporski J, et al. Immune reconstitution after haematopoietic cell transplantation in children: immunophenotype analysis with regard to factors affecting the speed of recovery. Br J Haematol 2002; 118:74–89. 18. Laffort C, Le Deist F, Favre M, et al. Severe cutaneous papillomavirus disease after haemopoietic stem-cell transplantation in patients with severe combined immune deficiency caused by common gamma c cytokine receptor subunit or JAK-3 deficiency. Lancet 2004; 363:2051–2054. 19. Hsu K, Keever-Taylor CA, Wilton A, et al. Improved outcome in HLA-identical sibling hematopoietic stem-cell transplantation for acute myelogenous leukemia predicted by KIR and HLA genotypes. Blood 2005; 105:4878–4884. 20. Ruggeri R, Capanni M, Mancusi A, Martelli MF, Velardi A. The impact of donor natural killer cell alloreactivity on allogeneic hematopoietic transplantation. Transpl Immunol 2005; 14:203–206. 21. Corti P, Peters C, Balduzzi A, et al. Reconstitution of lymphocyte subpopulations in children with inherited metabolic storage diseases after haematopoietic cell transplantation. Br J Haematol 2005; 130:249–255. 22. Small TN, Avigan D, Dupont B, et al. Immune reconstitution following T-cell depleted bone marrow transplantation: effect of age and post transplant graft rejection prophylaxis. Biol Blood Marrow Transplant 1997; 3:65–75. 23. Eyrich M, Lang P, Lal S, et al. A prospective analysis of the pattern of immune reconstitution in a paediatric cohort following transplantation of positively selected human leucocyte antigen-disparate haematopoietic stem cells from parental donors. Br J Haematol 2001; 114:422–432.

Immune Reconstitution Post-HSCT

283

24. Kalwak K, Moson I, Cwian J, et al. A prospective analysis of immune recovery in children following allogeneic transplantation of T-cell-depleted or non-T-cell-depleted hematopoietic cells from HLAdisparate family donors. Transplant Proc 2003; 35:1551–1555. 25. Bordignon C. Graft failure after T-cell-depleted human leukocyte antigen identical marrow transplants for leukemia: II. In vitro analyses of host effector mechanisms. Blood 1989; 74:2237–2243. 26. Inoue H, Yasuda Y, Hattori K, et al. The kinetics of immune reconstitution after cord blood transplantation and selected CD34C stem cell transplantation in children: comparison with bone marrow transplantation. Int J Hematol 2003; 77:399–407. 27. Storek J, Joseph A, Dawson MA, Douek DC, Storer B, Maloney DG. Factors influencing T-lymphopoiesis after allogeneic hematopoietic cell transplantation. Transplantation 2002; 73:1154–1158. 28. Foot AB, Potter MN, Donaldson C, et al. Immune reconstitution after BMT in children. Bone Marrow Transplant 1993; 11:7–13. 29. Mackall CL, Stein D, Fleisher TA, et al. Prolonged CD4 depletion after sequential autologous peripheral blood progenitor cell infusions in children and young adults. Blood 2000; 15:754–762. 30. Eyrich M, Leiler C, Lang P, et al. A prospective comparison of immune reconstitution in pediatric recipients of positively selected CD34C peripheral blood stem cells from unrelated donors versus recipients of unmanipulated bone marrow from related donors. Bone Marrow Transplant 2003; 32:379–390. 31. Lang P, Handgretinger R, Niethammer D, et al. Transplantation of highly purified CD34C progenitor cells from unrelated donors in pediatric leukemia. Blood 2003; 101:1630–1636. 32. Kook H, Goldman F, Padley D, et al. Reconstruction of the immune system after unrelated or partially matched T-cell-depleted bone marrow transplantation in children: immunophenotypic analysis and factors affecting the speed of recovery. Blood 1996; 88:1089–1097. 33. Ball LM, Lankester AC, Bredius RGM, Fibbe WE, van Tol MJD, Egeler RM. Graft dysfunction and delayed immune reconstitution following haploidentical peripheral blood hematopoietic stem cell transplantation. Bone Marrow Transplant 2005; 35:S35–S38. 34. Moretta A, Maccario R, Fagioli F, et al. Analysis of immune reconstitution in children undergoing cord blood transplantation. Exp Hematol 2001; 29:371–379. 35. Giraud P, Thuret I, Reviron D, et al. Immune reconstitution and outcome after unrelated cord blood transplantation: a single paediatric institution experience. Bone Marrow Transplant 2000; 25:53–57. 36. Niehues T, Rocha V, Filipovich AH, et al. Factors affecting lymphocyte subset reconstitution after either related or unrelated cord blood transplantation in children—a eurocord analysis. Br J Haematol 2001; 114:42–48. 37. Busca A, Lovisone E, Aliberti S, et al. Immune reconstitution and early infectious complications following nonmyeloablative hematopoietic stem cell transplantation. Hematology 2003; 8:303–311. 38. Morecki S, Gelfand Y, Nagler A, et al. Immune reconstitution following allogeneic stem cell transplantation in recipients conditioned by low intensity versus myeloablative regimen. Bone Marr Transp 2001; 28:243–249. 39. Naylor K, Li G, Vallejo AN Won-Woo Lee W-W, et al. The influence of age on T cell generation and TCR diversity. J Immunol 2005; 174:7446–7452. 40. Douek DC, McFarland RD, Keiser PH, et al. The role of the thymus in immune reconstitution in aging, bone marrow transplantation, and HIV-1 infection. Ann Rev Immunol 2000; 18:529–560. 41. De Rosa SC, Herzenberg LA, Herzenberg LA, Roederer M. 11-Color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity. Nat Med 2001; 7:245–248. 42. Lewin SR, Heller G, Zhang L, et al. Direct evidence for new T-cell generation by patients after either T-cell-depleted or unmodified allogeneic hematopoietic stem cell transplants. Blood 2002; 100:2235–2242. 43. Talvensaari, Clave E, Douay C, et al. Abroad T-cell repertoire diversity and an efficient thymic function indicate a favorable long-term immune reconstitution after cord blood stem cell transplantation. Blood 2002; 99:1458–1464. 44. Eyrich M, Wollny G, Tzaribaschev N, et al. Onset of thymic recovery and plateau of thymic output are differentially regulated after stem cell transplantation in children. Biol Blood Marrow Transplant 2005; 11:194–205. 45. Small TN, Knowles RW, Keever CA, et al. M241 (CD1c) expression on B lymphocytes. J Immunol 1987; 138:2864. 46. Storek J, Ferrara S, Ku N, Giorgi JV, Champlin RE, Saxon A. B cell reconstitution after human bone marrow transplantation: recapitulation of ontogeny? Bone Marrow Transplant 1993; 12:387–398.

284

Small

47. Storek J, Wells D, Dawson MA, Storer B, Maloney DG. Factors influencing B lymphopoiesis after allogeneic hematopoietic cell transplantation. Blood 2001; 98:489–491. 48. Di Martino D, Terranova MP, Scuderi F, et al. V(H)3 and V(H)6 immunoglobulin M repertoire reconstitution after hematopoietic stem-cell transplantation in children. Transplantation 2005;98–107. 49. Hakki M, Riddell SR, Storek J, et al. Immune reconstitution to cytomegalovirus after allogeneic hematopoietic stem cell transplantation: impact of host factors, drug therapy, and subclinical reactivation. Blood 2003; 102:3060–3067. 50. Lang P, Griesinger A, Hamprecht K, et al. Antiviral activity against CMV-infected fibroblasts in pediatric patients transplanted with CD34(C)-selected allografts from alternative donors. Hum Immunol 2004; 65:423–431. 51. Lucas KG, Small TN, Heller G, Dupont B, O’Reilly RJ. The development of cellular immunity to epstein-barr virus after allogeneic bone marrow transplantation. Blood 1996; 87:2594–2603. 52. Marshall NA, Howe JG, Formica R, et al. Rapid reconstitution of epstein-barr virus-specific T lymphocytes following allogeneic stem cell transplantation. Blood 2000; 96:2814–2821. 53. Heemskerk B, Lankester AC, van Vreeswijk T, et al. Immune reconstitution and clearance of human adenovirus viremia in pediatric stem-cell recipients. J Infect Dis 2005; 191:520–530. 54. Haining WN, Evans JW, Seth NP, et al. Measuring T cell immunity to influenza vaccination in children after haemopoietic stem cell transplantation. Br J Haematol 2004; 127:322–325. 55. Machado CM. Reimmunization after hematopoietic cell transplantation. Vaccine 2005. 56. Avigan D, Pirofski LA, Lazarus HM. Vaccination against infectious disease following hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2001; 7:171–183. 57. Ljungman P, Wiklund-Hammarsten M, Duraj V, et al. Response to tetanus toxoid immunization after allogeneic bone marrow transplantation. J Infect Dis 1990; 162:496–500. 58. Ljungman P, Lewensohn-Fuchs I, Hammarstrom V, et al. Long-term immunity to measles, mumps, and rubella after allogeneic bone marrow transplantation. Blood 1994; 84:657–663. 59. Ljungman P, Fridell E, Lonnqvist B, et al. Efficacy and safety of vaccination of marrow transplant recipients with a live attenuated measles, mumps, and rubella vaccine. J Infect Dis 1989; 159:610–615. 60. Ljungman P, Duraj V, Magnius L. Response to immunization against polio after allogeneic marrow transplantation. Bone Marrow Transplant 1991; 7:89–93. 61. Parkkali T, Stenvik M, Ruutu T, Hovi T, Volin L, Ruutu P. Randomized comparison of early and late vaccination with inactivated poliovirus vaccine after allogeneic BMT. Bone Marrow Transplant 1997; 20:663–668. 62. Li Volti S, Di Gregorio F, Romeo MA, et al. Immune status and the immune response to hepatitis B virus vaccine in thalassemic patients after allogeneic bone marrow transplantation. Bone Marrow Transplant 1997; 19:157–160. 63. Machado CM, Rocha IF, Diomede B, et al. Effectiveness of Hepatitis B vaccination and persistence of immunity after BMT. The Ninth International Symposium on Infections in the Immunocompromised Host, 1996. 64. Small TN, Papadopoulos EB, Aberoth H, et al. Response to Hepatitis B vaccination following allogeneic hematopoietic cell transplantation (HCT). Blood 2005; 106:28a. 65. Guinan EC, Molrine DC, Antin JH, et al. Polysaccharide conjugate vaccine responses in bone marrow transplant patients. Transplantation 1994; 57:677–684. 66. Parkkali T, Kayhty H, Ruutu T, Volin L, Eskola J, Ruutu PA. Comparison of early and late vaccination with haemophilus influenzae type b conjugate and pneumococcal polysaccharide vaccines after allogeneic BMT. Bone Marrow Transplant 1996; 18:961–967. 67. Avanzini MA, Carra AM, Maccario R, et al. Antibody response to pneumococcal vaccine in children receiving bone marrow transplantation. J Clin Immunol 1995; 15:137–144. 68. Molrine DC, Antin JH, Guinan EC, et al. Donor immunization with pneumococcal conjugate vaccine and early protective antibody responses following allogeneic hematopoietic cell transplantation. Blood 2003; 101:831–836. 69. Machado CM, Goncalves FB, Pannuti CS, Dulley FL, de Souza VA. Measles in bone marrow transplant recipients during an outbreak in Sao Paulo, Brazil. Blood 2002; 1:83–99. 70. Galil K, Lee B, Strine T, et al. Outbreak of varicella at a day-care center despite vaccination. N Engl J Med 2002; 347:1909–1915. 71. O’Reilly RJ, Small TN, Papadopoulos E, Lucas K, Lacerda J, Koulova L. Adoptive immunotherapy for epstein-barr virus-associated lymphoproliferative disorders complicating marrow allografts. Springer Semin Immunopathol 1998; 20:455–491.

Immune Reconstitution Post-HSCT

285

72. Papadopoulos EB, Ladanyi M, Emanuel D, et al. Infusions of donor leukocytes to treat epstein-barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation. N Engl J Med 1994; 330:1185–1191. 73. Gottschalk S, Heslop HE, Rooney CM. Adoptive immunotherapy for EBV-associated malignancies. Leuk Lymphoma 2005; 46:1–10. 74. Walter EA, Greenberg PD, Gilbert MJ, et al. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N Engl J Med 1995; 333:1038–1044. 75. Leen AM, Sili U, Savoldo B, et al. Fiber-modified adenoviruses generate subgroup cross-reactive, adenovirus-specific cytotoxic T lymphocytes for therapeutic applications. Blood 2004; 103:1011–1019. 76. Peggs KS, Verfuerth S, Pizzey A, et al. Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines. Lancet 2003; 362:1375–1377. 77. Trivedi D, Williams RY, O’Reilly RJ, Koehne G. Generation of CMV-specific T lymphocytes using protein-spanning pools of pp65-derived overlapping pentadecapeptides for adoptive immunotherapy. Blood 2005; 105:2793–2801. 78. Ramadan G, Davies B, Kurup VP, Keever-Taylor CA. Generation of cytotoxic T cell responses directed to human leucocyte antigen class I restricted epitopes from the aspergillus f16 allergen. Clin Exp Immunol 2005; 140:81–91. 79. van den Brink MR, Alpdogan O, Boyd RL. Strategies to enhance T-cell reconstitution in immunocompromised patients. Nat Rev Immunol 2004; 4:856–867. 80. Clark R. The somatogenic hormones and insulin-like growth factor-1: stimulators of lymphopoiesis and immune function. Endocr Rev 1997; 18:157–179. 81. Min D, Taylor PA, Panoskaltsis-Mortari A, et al. Protection from thymic epithelial cell injury by keratinocyte growth factor: a new approach to improve thymic and peripheral T-cell reconstitution after bone marrow transplantation. Blood 2002; 99:4592–4600. 82. Fry TJ, Mackall CL. The many faces of IL-7: from lymphopoiesis to peripheral T Cell maintenance. J Immunol 2005; 174:6571–6576. 83. Alpdogan O, van den Brink MR. IL-7 and IL-15: therapeutic cytokines for immunodeficiency. Trends Immunol 2005; 26:56–64. 84. Small TN, Wan W, Selva-Kumar E, et al. Longitudinal analysis of insulin-like growth factor levels post bone marrow transplant. Blood 1997; 90:541a. 85. Marrack P, Bender J, Hildeman D, et al. Homeostasis of abTCRCT cells. Nat Immunol 2000; 1:107. 86. Peschon JJ, Morrissey PJ, Grabstein KH, et al. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J Exp Med 1955; 180. 87. von Freeden-Jeffry U, Vieira P, Lucian LA, McNeil T, Burdach SE, Murray R. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J Exp Med 1995; 181:1519. 88. Hassan J, Reen DJ. IL-7 promotes the survival and maturation but not differentiation of human postthymic CD4CT cells. Eur J Immunol 1998; 28:3057. 89. Ku CC, Murakami M, Sakamoto A, Kappler J, Marrack P. Control of homeostasis of CD8C memory T cells by opposing cytokines. Science 2000; 288:675. 90. Morrissey PJ, Conlon P, Braddy S, Williams DE, Namen AE, Mochizuki DY. Administration of IL-7 to mice with cyclophosphamide-induced lymphopenia accelerates lymphocyte repopulation. J Immunol 1991; 146:1547. 91. Bolotin E, Smogorzewska M, Smith S, Widmer M, Weinberg K. Enhancement of thymopoiesis after bone marrow transplant by in vivo interleukin-7. Blood 1996; 88:1887. 92. Fry TJ, Christensen BL, Komschlies KL, Gress RE, Mackall CL. Interleukin-7 restores immunity in athymic T-cell-depleted hosts. Blood 2001; 97:1525. 93. Mackall CL, Fry TJ, Bare C, Morgan P, Galbraith A, Gress RE. IL-7 increases both thymicdependent and thymic-independent T-cell regeneration after bone marrow transplantation. Blood 2001; 97:1491. 94. Akashi K, Kondo M, von Freeden-Jeffry U, Murray R, Weissman IL. Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice. Cell 1997; 89:1033. 95. Broers AE, Posthumus-van Sluijs SJ, Spits H, et al. Interleukin-7 improves T-cell recovery after experimental T-cell-depleted bone marrow transplantation in T-cell-deficient mice by strong expansion of recent thymic emigrants. Blood 2003; 102:1534–1540.

15 Endocrine Complications of Childhood Hematopoietic Stem-Cell Transplantation Wassim Chemaitilly Department of Pediatrics, New York Presbyterian Hospital-Cornell Weill Medical Center, New York, New York, U.S.A.

Farid Boulad Department of Pediatrics, Bone Marrow Transplant Service, Memorial Sloan-Kettering Cancer Center, New York, New York, U.S.A.

Charles Sklar Department of Pediatrics, Bone Marrow Transplant Service, Memorial Sloan-Kettering Cancer Center, New York, New York, U.S.A.

INTRODUCTION The endocrine organs are especially prone to injury from radiotherapy and chemotherapy. Endocrine abnormalities are, in fact, the most prevalent late effects observed in survivors of hematopoietic stem cell transplantation (HSCT); approximately 50% of survivors followed long-term will develop one of several endocrinopathies. This overview will focus on the late endocrine disturbances that are most commonly observed following successful HSCT.

IMPAIRED LINEAR GROWTH AND GROWTH HORMONE DEFICIENCY Growth impairment and reduced final height are common following HSCT for haematologic malignancies. The growth failure following HSCT is multifactorial and involves both endocrine and nonendocrine risk factors (1–5).

Risk Factors The most important risk factors for growth failure following HSCT relate to patient characteristics (e.g., young age, the patient’s primary disease), treatment variables [e.g., total body irradiation (TBI), prior cranial radiation], and posttreatment complications, such as chronic graft-versus-host disease (GVHD) (5). The resultant growth failure may be due to poor nutrition, growth hormone (GH) deficiency, and/or skeletal dysplasia secondary to the exposure of the growth plate to drug toxicity and irradiation. Interpretation of the available data has been difficult due to the different preparative regimens employed at different centers, the heterogeneity of diagnostic groups, and the relatively small number of subjects studied at most centers. 287

288

Chemaitilly et al.

Chemotherapy Non-TBI containing cytoreductive regimens are being used increasingly in HSCT to reduce the exposure of patients to radiotherapy and its side effects. These regimens include the use of cyclophosphamide for aplastic anemia, as well as high doses of cyclophosphamide and busulfan. Children treated with cyclophosphamide for aplastic anemia appear to grow normally, so long as they do not develop a significant complication (e.g., GVHD, liver dysfunction) posttransplant (6–9). The consequences on growth of pretransplant conditioning regimens combining high doses of busulfan and cyclophosphamide remain controversial. Most authors have not found evidence of sustained growth failure in the patients who received such conditioning regimens in the absence of significant posttransplant complications (2,6–9). However, growth failure, possibly due to GH deficiency, has been reported by some (10,11). For instance, Bakker et al. recently suggested that a significant number of patients receiving high doses of busulfan and cyclophosphamide as cytoreduction for HSCT had abnormal growth patterns, but the growth deceleration the authors based their conclusions upon were not statistically significant (11). More final height data from patients who have received such conditioning regimens are needed before any definitive conclusions can be drawn (2). De Sanctis et al. recently reported on the final height of 29 thalassemic patients treated with HSCT following conditioning with high doses of cyclophosphamide and busulfan (12). Only one patient was diagnosed with GH deficiency and most of the patients reached a final height above -2SD (10 males and 13 females). It is important to note, however, that thalassemia itself can cause short stature by several mechanisms, including hypopituitarism due to iron toxicity and bone lesions secondary to chelating therapy (12). Radiotherapy Total Lymphoid Irradiation. Treatment of aplastic anemia with cyclophosphamide plus total lymphoid irradiation (TLI) is associated with some decrease in growth. One study that examined growth after single dose TLI (750 cGy) demonstrated a small loss in height, which was statistically significant only at year three post–bone marrow transplant (13). Overall, the consequences of TLI on growth are less important than those induced by TBI, and they do not appear to be secondary to GH deficiency (14). Total Body Irradiation. TBI when administered during childhood alters linear growth. In a large series reported from Seattle, Sanders et al. noted that following TBI for leukemia or lymphoma all subjects experienced a decrease in their growth rate (6). Growth was more impaired in those with chronic GVHD and in those treated with single dose (920–1000 cGy) TBI compared to those who received fractionated irradiation (200 cGy to 225 cGy once daily for six to seven days). Most subsequent reports on the growth of children after stem cell transplant indicate smaller losses in height in those treated with fractionated TBI compared to those treated with single dose irradiation, despite the fact that the total dose of irradiation is higher in patients treated with fractionated TBI (15,16). These results are in keeping with our understanding of the radio-biologic basis of normal tissue toxicity and underscore that both the total dose and dose per fraction are critical determinants of the height outcome following HSCT (17,18). Interestingly, some, but not all, workers have observed disproportionate growth following both single fraction and fractionated TBI, with spinal growth being more impaired than growth of the legs (16,19,20). The mechanisms by which TBI impair linear growth are multiple and the effects may be additive. Both GH deficiency and skeletal dysplasia caused by exposure of the growth plate to radiation are well-documented consequences of TBI, although their relative importance in terms of growth impairment remains unclear (21). Most studies show an increased incidence of GH deficiency after TBI in childhood (3,20,22–24). However, the permanence of GH deficiency after TBI has been challenged by some authors, based on the results of follow-up data from patients diagnosed as GH deficient in childhood but who turned out to have normal GH responses when retested in adulthood. It has been suggested that these

Endocrine Complications of Childhood HSCT

289

discrepant results may, at least in part, be explained by poor nutritional status, which could influence the interpretation of the insulin growth factor-1 (IGF-1) results (14,19). Prior cranial irradiation (CRT) has emerged as an extremely important determinant of poor growth after transplantation, regardless of the type of preparative regimen administered, as it can cause hypothalamic/ pituitary damage (16,19,24–26). In an analysis of 72 children treated at Memorial Sloan-Kettering Cancer Center for acute leukemia after conditioning with hyperfractionated TBI (125 cGy three times per day for four days), we observed that patients treated with previous cranial irradiation experienced more than twice the decrease in height as those who had not received CRT prior to transplantation (24). Similarly, Cohen et al. found the mean final height z-score of children treated with both CRT and fractionated TBI to be -1.69, compared to a z-score of -0.98 for those treated with fractionated TBI but no prior CRT (2). Central precocious puberty, which can cause bone age progression and further reduce the patients’ growth potential, has been reported in children treated at a young age with CRT, followed later by a transplant with TBI-based cytoreduction (27).

Graft-Versus-Host Disease High dose glucocorticoids used to treat chronic GVHD cause transient growth deceleration. Catch-up growth does not always occur after recovery from chronic GVHD, as the primary disease and the previous treatments may alter the patients’ growth potential (8,21). Management of Growth Hormone Deficiency Following Hematopoietic Stem-Cell Transplantation A variable but high incidence of GH deficiency (inadequate responses to pharmacologic stimuli as well as reduced endogenous GH secretion) has been observed in the posttransplant period (6,20,24,26).

Diagnosis Clinical Presentation. Growth deceleration, defined as a growth velocity below the fifth percentile for age, can be easily diagnosed during the patient’s follow-up by plotting the patient’s height at every visit, at least on a six monthly basis (28). Special attention should also be paid to the patient’s nutritional status, weight gain, and body mass index, all of which provide key information should the patient have signs of growth failure. Children who demonstrate significant declines in growth velocity should be referred to an experienced pediatric endocrinologist for further evaluation. Laboratory Assessment. Due to the pulsatile and irregular pattern of GH secretion, establishing GH deficiency remains a diagnostic challenge, especially in childhood cancer survivors. Most clinicians rely on measuring the peak plasma GH values in response to diverse stimuli (pharmacological or physiological) in order to assess the body’s ability to produce GH. The results of provocative testing can vary with the timing of the assessment after HSCT and the provocative agent used. Moreover, the reliability and reproducibility of these tests have been questioned (19,20,29–31). The plasma concentrations of the surrogate markers, IGF-1, and IGF binding protein-3 (IGFBP-3), are not reliable indices of GH status after radiation to the brain (19,20,31). The poor correlation between the results of GH testing and the growth of many of these children suggests that additional factors, such as radiationinduced skeletal dysplasia, play an important role in the disturbed growth that can be seen following transplant (32). Treatment. GH replacement therapy is indicated in patients with documented GH deficiency and who have been in remission for more than a year. It does not appear to increase the risk of disease recurrence or death in survivors of childhood cancer but there may be a small increased risk of developing second tumors (33,34). However, the response to GH treatment has been reported only in a limited number of HSCT survivors and the results have

290

Chemaitilly et al.

been variable. Although some authors have observed only stabilization in height percentiles without evidence of catch-up growth on GH treatment, we and others have noted catch-up growth in some subjects treated with GH (16,22,24,26,34). Some of these differences might be due to the fact that younger patients and those exposed to fractionated, as opposed to single dose TBI, are likely to respond better to GH therapy. Recently, Sanders and colleagues reported on final height outcomes in 42 survivors of HSCT and fractionated TBI who were treated with GH (34). Compared to 48 survivors who were not treated with GH, there was a significant improvement in final height in those who were !10 years at initiation of GH; those older than 10 years at start of GH did not appear to demonstrate an improved final height (34).

DISTURBANCES OF THE OTHER HYPOTHALAMIC-PITUITARY AXES Corticotropin (ACTH) Corticotropin deficiency is very rare after HSCT. In a study by Ogilvy-Stuart et al. cortisol levels were found to be low in only two out of 31 children after fractionated TBI (23). Kauppila et al. reported similar results in an adult population (35). These results are in contrast to a previous report by Sanders et al. where an incidence as high as 24% was suggested for ACTH deficiency following TBI (6). The authors of that report had based their conclusions on subnormal levels of 11-deoxycortisol (Compound S) after metyrapone stimulation, a method that is subject to a great deal of variability. The clinical relevance of these latter findings is questionable (16,23,35,36).

Thyrotropin (TSH) Deficiency Central hypothyroidism is a rare complication following HSCT (16,23). However, Rose et al. described a surprisingly high incidence of “hidden” TSH deficiency following TBI, reaching 20% four years after HSCT (37). The authors based their conclusions on abnormal responses to TRH stimulation tests and/or a loss of the nocturnal TSH surge despite normal baseline levels of free T4. The clinical relevance of these findings is unclear, and the benefit of thyroxine replacement therapy in such patients has not been established.

Disorders of Gonadotropin Secretion Hypogonadotropic hypogonadism is very rare in the context of HSCT (16,23). On the other hand, cases of central precocious puberty following HSCT in patients who received prior cranial irradiation have been reported (27). This premature activation of the hypothalamicgonadotropin axis is thought to be triggered by the disruption of the inhibitory influence of the cortex secondary to the brain’s exposure to radiation. Central precocious puberty can worsen the height prediction of these patients who often have GH deficiency as well (27).

THYROID DYSFUNCTION A spectrum of thyroid abnormalities has been described among long-term survivors of HSCT. Therapy-induced primary hypothyroidism, autoimmune thyroid disease, and differentiated thyroid carcinoma have all been reported following transplant.

Therapy-Induced Primary Hypothyroidism The incidence of primary thyroid dysfunction has varied greatly from series to series, owing to differences in treatments delivered and the duration of follow-up. The overall incidence of hypothyroidism has been greater following single dose irradiation (23–73%) compared to that

Endocrine Complications of Childhood HSCT

291

seen after fractionated radiotherapy (10–28%) (23,38–41). Moreover, the longer the follow-up time, the higher the incidence of impaired thyroid function. In an analysis of thyroid function in 139 patients who had received hyperfractionated irradiation for a HSCT at our center, 21 patients (15.1%) became hypothyroid after a median follow-up period of 6.2 years (40). Hypothyroidism developed a median of 49 months (11–88) after HSCT, considerably later than recorded following single dose irradiation. The majority of patients develop a mild, compensated primary hypothyroidism that is often transient and may resolve spontaneously. We and others have also observed a small number of patients who have developed mildly elevated TSH levels following cytoreduction with busulfan and cyclophosphamide (9,40,42). More recently, Slatter et al. reporting on a series of 83 survivors treated with HSCT for primary immunodeficiency, who received cyclophosphamide and busulfan as a conditioning regimen, described more severe cases of primary hypothyroidism in two patients who required treatment with thyroxine (43).

Autoimmune Thyroid Disease Several case reports have documented the occurrence of autoimmune thyroid disease in the recipients of HSCT (43,44). At our center, we have observed several cases of autoimmune hyperthyroidism that developed following transplantation (5). All patients had suppressed plasma concentrations of TSH, raised levels of T4 and/or T3, and evidence of thyroid autoimmunity. Two of the three had markedly elevated levels of antibodies to the TSH receptor, the antibody responsible for the development of Graves’ disease. Studies performed on the blood of the three donors (two donors were siblings, one was unrelated) revealed that all three had elevated levels of antibodies to the TSH receptor but normal TSH levels. These data are consistent with the hypothesis that the patients’ thyroid disorder was due to adoptive transfer of abnormal clones of T or B cells from donor to recipient (44,45). Two of our patients required treatment with radioactive iodine to correct their hyperthyroidism, whereas the third patient’s thyroid hyperfunction resolved spontaneously over a six-month period.

Thyroid Carcinoma Radiation to the thyroid gland is a known risk factor for the subsequent development of thyroid neoplasms, both benign and malignant (46–50). Reporting on a series of 113 childhood HSCT patients, Cohen et al. showed that the incidence of secondary thyroid carcinoma was higher than in the general population, with eight cases diagnosed between 3.1 and 15.7 years after transplant (49). Because the latency period between radiation and the appearance of thyroid neoplasms is often prolonged, the number of affected individuals will almost certainly increase over time.

GONADAL AND REPRODUCTIVE DYSFUNCTION Males Chemotherapy Leydig Cell Function. Young boys and adolescent males who receive standard dose cyclophosphamide alone (200 mg/kg) as therapy for aplastic anemia appear to retain normal Leydig cell function; the vast majority are reported to have normal plasma concentrations of LH and testosterone and to enter and progress normally through puberty. In a recent report from Seattle, all 28 boys who received that conditioning regimen had a normal pubertal development (51). In young males treated with the combination of busulfan and cyclophosphamide, Leydig cell function appears to be preserved in most adult males (9,11,42). Recently, Bakker et al.

292

Chemaitilly et al.

demonstrated that all eight boys receiving such regimens experienced normal pubertal development, confirming similar findings previously reported by Afify et al. (9,11). Germ Cell Function. Evidence of germ cell (i.e., cells that produce sperm) damage has been reported following standard dose cyclophosphamide alone (200 mg/kg) as therapy for aplastic anemia and may be more common in those males treated during or after puberty compared to males treated prior to the onset of puberty. Sanders and colleagues reported that the plasma concentrations of FSH remain normal in most boys who are now pubertal but were treated before puberty, whereas FSH levels are increased in nearly half the males who were treated during or after puberty (38). Nonetheless, semen analyses have been normal in approximately two-thirds of the males, and a sizable number of males, including eighteen who were prepubertal at transplant, have fathered normal children after treatment with high-dose cyclophosphamide (38,51,52). Most of the young men treated with the combination of busulfan and cyclophosphamide do appear to sustain damage to their germinal epithelium. Anserini et al. in a small cohort of patients post-HSCT, demonstrated that recovery of some spermatogenetic activity occurred in 50% of cases after such conditioning regimens, but semen quality was impaired (53). Grigg et al. had previously suggested that recovery of spermatogenetic activity occurred more frequently in patients receiving lower doses of cyclophosphamide (120 mg/kg) and busulfan (16 mg/kg) than in patients who received higher doses of cyclophosphamide (200 mg/kg) (54). Overall, the ultimate effect of these treatments on male fertility remains unclear.

Total Body Irradiation Leydig Cell Function. Males treated with TBI-based preparative regimens, both single dose and fractionated, generally retain their ability to produce testosterone regardless of their age at treatment (6,22,38). All fourteen evaluable males treated before puberty with 1375– 1500 cGy hyperfractionated TBI plus a 400 cGy testicular boost at our institution have entered puberty spontaneously and have manifested normal secondary sexual characteristics (27). Although the majority demonstrates age-appropriate plasma concentrations of testosterone, a third of the patients’ baseline levels of LH are raised. This finding suggests that subclinical injury to the Leydig cells is common after TBI and may progress with time (27). We have found very similar data in our males treated after the onset of puberty, in keeping with the previous observation that Leydig cell function is preserved when the total dose of radiotherapy to the testis is !2000 cGy (55). In contrast, many males who have received testicular irradiation (e.g., 2000–2400 cGy) before coming to transplant experience Leydig cell failure and require androgen replacement therapy. Therefore, boys treated with radiation to the testes who fail to demonstrate evidence of pubertal maturation by the age of 14 years should be evaluated for possible Leydig cell failure. Additionally, it is important to continue to monitor Leydig cell function as these individuals age. Germ Cell Function. Germ cell dysfunction is present in essentially all males treated with TBI (52). The vast majority of subjects will have increased plasma levels of FSH as well as reduced testicular volume, indices that correlate with impaired spermatogenesis. Azoospermia is the rule for patients studied in the first few years after treatment with TBI. Recovery of germ cell function has occurred rarely and primarily following single dose irradiation (56). A few men have been reported to father a child following TBI (52). A sperm analysis is the only test that can establish whether or not a young adult survivor has the capacity to produce viable sperm. Females Chemotherapy Following transplant for aplastic anemia with high-dose cyclophosphamide, ovarian function has remained normal in females treated both prior to as well as after the onset of puberty

Endocrine Complications of Childhood HSCT

293

(38,57). Sanders et al. (57) have observed a number of pregnancies and normal offspring in their cohort treated with cyclophosphamide alone. Data from patients treated with high-dose alkylating agents for other indications does suggest, however, that these subjects may be at increased risk of an early menopause as they reach the third decade of life (58). Females treated with busulfan and cyclophosphamide are at very high risk of developing ovarian failure (5,9,11,42). This has been observed in patients treated before and after pubertal development, is characterized by menopausal levels of LH and FSH, delayed or arrested puberty, and amenorrhea. Recovery of function has been recorded only rarely but the follow-up time has been relatively brief for most of the patients. The majority of young girls treated with the combination of busulfan and cyclophosphamide will require long-term hormonal replacement therapy.

Total Body Irradiation The outcome of ovarian function following TBI appears to be determined to a large extent by the age of the patient at the time of irradiation (59). Our data as well as the data of others indicate that approximately 50% of prepubertal girls given fractionated TBI will enter puberty spontaneously and achieve menarche at a normal age (27,41,60). Although plasma gonadotropins have been elevated in up to two-thirds of these patients early after transplant, normalization of the plasma concentrations of LH and FSH can occur over time (27). Ovarian failure is seen in essentially all patients who are greater than age 10 years at the time they are treated with TBI (27,57,59,60). Patients require hormonal support in order to achieve normal sexual development and to maintain normal menstruation. Recovery of ovarian function has been documented in a small number of women who have received TBI (52). In a series from Seattle, among the girls who were treated with TBI when they were prepubertal, all five pregnancies ended in spontaneous abortions (52). For women treated with TBI at an older age, the small numbers of pregnancies were associated with an increased risk of preterm deliveries and delivery of low birth weight infants (52). The infants did not demonstrate an excess of congenital anomalies, but follow-up over a longer period of time is necessary before final conclusions can be drawn regarding the health status of these children. The uterine consequences of TBI may contribute to the high incidence of miscarriages observed in this population. Holm et al. (61) and Bath et al. (62) demonstrated that the uterine volume of patients treated with TBI is smaller than in normal controls and in cancer survivors exclusively treated with chemotherapy. The blood supply to the uterus was significantly decreased after TBI, suggesting a vascular mechanism for the observed abnormalities with increased risks in younger patients (61,62). The uterine blood flow improved after hormone replacement therapy and the uterine volume subsequently increased but remained smaller than in controls (62). Overall, the full impact of HSCT on reproduction and on the health of future offspring will remain in question until we have data from large numbers of survivors, followed long-term.

OSTEOPOROSIS Survivors of pediatric HSCT appear to be at increased risk for the development of reduced bone density in later life (63–65). Both the therapies employed to treat these malignancies as well as a variety of treatment-related complications appear to interfere with normal bone accretion (66). Risk factors for reduced bone mineral density in survivors of HSCT include treatment with glucocorticoids for chronic GVHD, prior cranial irradiation (a surrogate for GH deficiency), and sex hormone insufficiency (63,67,68). The molecular mechanisms of post-HSCT osteoporosis remain unclear. Lee and colleagues suggested that impaired differentiation of bone marrow stromal cells into osteoblasts might partly explain post-HSCT osteoporosis (69). The same authors had previously suggested that the progressive increase in bone resorption during the immediate post-HSCT period was

294

Chemaitilly et al.

related to both the dose of glucocorticoids and the increase in the levels of interleukin-6 in bone marrow (70). The latter is a potent stimulator of bone resorption in vivo and is also known to be involved in postmenopausal osteoporosis (70). Subjects deemed at high risk for the development of osteoporosis should undergo periodic bone density studies. Although the dual-energy X-ray absorptiometry remains the most widely used tool for measuring bone mineral density, its results should be interpreted according to age, pubertal stage, and height in the pediatric population. Failure to take these elements into account may result in overdiagnosing osteoporosis (65). Preventive measures (e.g., supplementation with calcium and vitamin D, smoking cessation, weight-bearing exercise) should be encouraged in all individuals with low or borderline bone mineral density. Therapeutic interventions (e.g., sex hormone therapy, GH replacement, bisphosphonates) may prove beneficial for those with abnormally reduced bone density, but long-term follow-up data are currently not available.

DISORDERS OF GLUCOSE HOMEOSTASIS Several authors have reported an increased risk of insulin resistance and the metabolic syndrome (insulin resistance, glucose intolerance, dyslipidemia, hypertension) in patients who have undergone HSCT (71–75). Surprisingly, although prior exposure to glucocorticoids and current obesity were not associated with the observed metabolic abnormalities, prior exposure to TBI has emerged consistently as a significant risk factor for insulin resistance (72,74,75). Two recent series suggest that the incidence of both type-1 (i.e., insulinopenia) and type-2 (i.e., insulin resistance) diabetes mellitus may be increased following TBI (73,76). Whether TBI alters glucose homeostasis, and, if so, whether it does so by causing insulinopenia and/or by causing insulin resistance remain open questions. The existing studies assessing the risk factors for diabetes mellitus and glucose intolerance in childhood cancer survivors have yielded contradictory results, and it remains difficult to determine which disorders are predominantly associated with TBI (72,75,76). These studies, in fact, suffer from several shortcomings. The large population-based studies, which rely on questionnaires returned by patients reporting on their current status with regards to diabetes mellitus, do not permit us to distinguish between type-1 and type-2 diabetes mellitus (75,77). In general, the heterogeneity of the populations included in the published studies, the diversity of the treatments they received, and the differences in the time intervals since the completion of cancer treatments, all make the interpretation of the findings difficult (72,74). Additional limitations include the possibility of ascertainment and surveillance bias, as few studies included contemporaneous controls that were subjected to similar investigations. More studies are needed in order to investigate the true prevalence of glucose intolerance after HSCT, as a first step before elucidating the possible mechanisms of this association.

CONCLUSION Childhood HSCT survivors are at risk of developing multiple endocrine complications. Data on the final height of patients and on the safety and efficacy of GH therapy are limited. Thyroid dysfunction and osteoporosis are relatively frequent after HSCT, and survivors should therefore have periodic evaluations for these complications as part of their follow-up. Most childhood HSCT survivors will have fertility issues as adults, and new strategies aiming at the preservation of their reproductive potential need to be investigated. Glucose intolerance is increasingly reported following HSCT; more studies are required in order to investigate the true prevalence of this association, its long-term impact on HSCT survivors, and the underlying mechanisms. The prevention, early diagnosis and treatment of the endocrine complications of childhood HSCT are crucial in order to maximize the quality of life of survivors.

Endocrine Complications of Childhood HSCT

295

REFERENCES 1. Boulad F, Sands S, Sklar C. Late complications after BMT in children and adolescents. Curr Probl Pediatr 1998; 28:277–297. 2. Cohen A, Rovelli A, Bakker B, et al. Final height of patients who underwent bone marrow transplantation for hematological disorders during childhood: a study by the working party for late effects-EBMT. Blood 1999; 93:4109–4115. 3. Clement-De Boers A, Oostdijk W, Van Weel-Sipman MH, Van den Broeck J, Wit JM, Vossen JM. Final height and hormonal function after bone marrow transplantation in children. J Pediatr 1996; 129:544–550. 4. Frisk P, Arvidson J, Gustafsson J, Lonnerholm G. Pubertal development and final height after autologous bone marrow transplantation for acute lymphoblastic leukemia. Bone Marrow Transplant 2004; 33:205–210. 5. Sklar C, Boulad F, Small T, Kernan N. Endocrine complications of pediatric stem cell transplantation. Front Biosci 2001; 6:17–22. 6. Sanders JE, Pritchard S, Mahoney P, et al. Growth and development following marrow transplantation for leukemia. Blood 1986; 68:1129–1135. 7. Giorgiani G, Bozzola M, Locatelli F, et al. Role of busulfan and total body irradiation on growth of prepubertal children receiving bone marrow transplantation and results of treatment with recombinant human growth hormone. Blood 1995; 86:825–831. 8. Adan L, de Lanversin M-L, Thalassinos C, Souberbielle JC, Fischer A, Brauner R. Growth after bone marrow transplantation in young children conditioned with chemotherapy alone. Bone Marrow Transplant 1997; 19:253–256. 9. Afify A, Shaw PJ, Clavano-Harding A, Cowell CT. Growth and endocrine function in children with acute myeloid leukaemia after bone marrow transplantation using busulfan/cyclophosphamide. Bone Marrow Transplant 2000; 25:1087–1092. 10. Sanders JE. Growth and development after bone marrow transplantation. In: Forman SJ, Blume KG, Thomas ED, eds. Bone Marrow Transplantation. Boston: Blackwell Scientific Publications, 1994:527–537. 11. Bakker B, Oostdijk W, Bresters D, Walenkamp MJE, Vossen JM, Wit JM. Disturbances of growth and endocrine function after busulfan-based conditioning for haematopoietic stem cell transplantation during infancy and childhood. Bone Marrow Transplant 2004; 33:1049–1056. 12. De Sanctis V. Growth and puberty and its management in thalassemia. Horm Res 2002; 58:72–79. 13. Bushhouse S, Ramsay NKC, Pescovitz OH, Kim T, Robison LL. Growth in children following irradiation for bone marrow transplantation. Am J Pediatr Hem/Oncol 1989; 11:134–140. 14. Couto-Silva AC, Trivin C, Esperou H, Michon J, Fischer A, Brauner R. Changes in height, weight and plasma leptin after bone marrow transplantation. Bone Marrow Transplant 2000; 26:1205–1210. 15. Brauner R, Fontoura M, Zucker JM, et al. Growth and growth hormone secretion after bone marrow transplantation. Arch Dis Child 1993; 68:458–463. 16. Thomas BC, Stanhope R, Plowman PN, Leiper AD. Growth following single fraction and fractionated total body irradiation for bone marrow transplantation. Eur J Pediatr 1993; 152:888–892. 17. Wither HR. Biologic basis for altered fractionation schemes. Cancer 1985; 55:2086–2095. 18. Yahalom J, Fuks ZY. Strategies for the use of total body irradiation as a systemic therapy in leukemia and lymphoma. In: Armitage JO, Antman KH, eds. High Dose Chemotherapy. Baltimore: Williams and Wilkins, 1992:61–83. 19. Holm K, Nysom K, Rasmussen MH, et al. Growth, growth hormone and final height after BMT. Possible recovery of irradiation-induced growth hormone insufficiency. Bone Marrow Transplant 1996; 18:163–170. 20. Brauner R, Adan L, Souberbielle JC, et al. Contribution of growth hormone deficiency to the growth failure that follows bone marrow transplantation. J Pediatr 1997; 130:785–792. 21. Brennan BMD, Shalet MS. Endocrine late effects after bone marrow transplantation. Br J Haematol 2002; 118:56–66. 22. Papadimitrou A, Uruena M, Stanhope R, Leiper AD. Growth hormone treatment of growth failure secondary to total body irradiation and bone marrow transplantation. Arch Dis Child 1991; 66:689–692. 23. Ogilvy-Stuart AL, Clark DJ, Wallace WHB, et al. Endocrine deficit after fractionated total body irradiation. Arch Dis Child 1992; 67:1107–1110. 24. Huma Z, Boulad F, Black P, Heller G, Sklar C. Growth in children after bone marrow transplantation for acute leukemia. Blood 1995; 86:819–824.

296

Chemaitilly et al.

25. Wingard JR, Plotnick LP, Freemer CS, et al. Growth in children after bone marrow transplantation: busulfan plus cyclophosphamide versus cyclophosphamide plus total body irradiation. Blood 1992; 79:1068–1073. 26. Bozzola M, Giorgiani G, Locatelli F, et al. Growth in children after bone marrow transplantation. Horm Res 1993; 39:122–126. 27. Sarafoglou K, Boulad F, Gillio A, Sklar C. Gonadal function after bone marrow transplantation for acute leukemia during childhood. J Pediatr 1997; 130:210–216. 28. Reiter EO, Rosenfeld RG. Normal and abnormal growth. In: Larsen PR, Kronenberg HM, Melmed S et al. eds. Williams Textbook of Endocrinology. Philadelphia: WB Saunders, 2003:1003–1114. 29. Lissett CA, Saleem S, Rahim A, Brennan BMD, Shalet SM. The impact of irradiation on GH responsiveness to provocative agents is stimulus dependent: results in 161 individuals with radiation damage to the somatotropic axis. J Clin Endocrinol Metab 2001; 86:663–668. 30. Darzy KH, Aimaretti G, Wieringa G, Rao Gattamaneni H, Chigo E, Shalet SM. The usefulness of the combined growth hormone (GH) releasing hormone and arginine stimulation test in the diagnosis of radiation-induced GH deficiency is dependent on the post-irradiation time interval. J Clin Endocrinol Metab 2003; 88:95–102. 31. Sklar C, Sarafoglou K, Whittam E. Efficacy of insulin-like growth factor binding protein 3 in predicting the growth hormone response to provocative testing in children treated with cranial irradiation. Acta Endocrinol 1993; 129:511–515. 32. Fletcher BD, Crom DB, Krance RA, Kun L. Radiation-induced bone abnormalities after bone marrow transplantation for childhood leukemia. Radiology 1994; 191:231–235. 33. Sklar CA, Mertens AC, Mitsby P, et al. Risk of disease recurrence and second neoplasms in survivors of childhood cancer treated with growth hormone: a report from the childhood cancer survivor study. J Clin Endocrinol Metab 2002; 87:3136–3141. 34. Sanders JE, Guthrie KA, Hoffmeister PA, Woolfrey AE, Carpenter PA, Appelbaum FR. Final adult height of patients who received hematopoietic cell transpantation in childhood. Blood 2005; 105:1348–1354. 35. Kauppila M, Koskinen P, Irjala K, Remes K, Viikari J. Long-term effects of allogeneic bone marrow transplantation (BMT) on pituitary, gonad, thyroid and adrenal function in adults. Bone Marrow Transplant 1998; 22:331–337. 36. Littley MD, Shalet SM, Morgenstern GR, Deaken DP. Endocrine and reproductive dysfunction following fractionated total body irradiation in adults. QJM 1991; 78:265–274. 37. Rose SR, Lustig RH, Pituksheewanont P, et al. Diagnosis of hidden central hypothyroidism in survivors of childhood cancer. J Clin Endocrinol Metab 1999; 84:4472–4479. 38. Sanders JE. The Seattle marrow transplant team. The impact of marrow transplant preparative regimens on subsequent growth and development. Semin Hematol 1991; 28:244–249. 39. Thomas BC, Stanhope R, Plowman PN, Leiper AD. Endocrine function following single fraction and fractionated total body irradiation for bone marrow transplantation in childhood. Acta Endocrinol 1993; 128:508–512. 40. Boulad F, Bromley M, Black P, et al. Thyroid dysfunction following bone marrow transplantation using hyperfractionated radiation. Bone Marrow Transplant 1995; 15:71–76. 41. Sklar CA, Kim TH, Ramsay NKC. Thyroid dysfunction among long-term survivors of bone marrow transplantation. Am J Med 1982; 73:688–694. 42. Michel G, Socie G, Gebhard F, et al. Late effects of allogeneic bone marrow transplantation for children with acute myeloblastic leukemia in first complete remission: the impact of conditioning regimen without total-body irradiation—a report from the socie´te´ franc¸aise de greffe de moelle. J Clin Oncol 1997; 15:2238–2246. 43. Slatter MA, Gennery AR, Cheetham TD, et al. Thyroid dysfunction after bone marrow transplantation for primary immune deficiency without the use of total body irradiation. Bone Marrow Transplant 2004; 33:949–953. 44. Karthaus M, Gabrysiak T, Brabant G, et al. Immune thyroiditis after transplantation of allogeneic CD34C selected peripheral blood cells. Bone Marrow Transplant 1997; 20:697–699. 45. Holland FJ, McConnon JK, Volpe´ R, Saunders EF. Concordant Graves’ disease after bone marrow transplantation: implications for pathogenesis. J Clin Endocrinol Metab 1991; 72:837–840. 46. Ron E, Lubin JH, Shore RE, et al. Thyroid cancer after exposure to external radiation: a pooled analysis of seven studies. Radiation Res 1995; 141:259–277. 47. Tucker MA, Jones PH, Boice JD, Jr., et al. Therapeutic radiation at a young age is linked to secondary thyroid cancer. Cancer Res 1991; 51:2885–2888.

Endocrine Complications of Childhood HSCT

297

48. Acharya S, Sarafoglou K, LaQuaglia M, et al. Thyroid neoplasms after therapeutic radiation for malignancies during childhood and adolescence. Cancer 2003; 97:2397–2403. 49. Cohen A, Rovelli A, van Lint MT, et al. Secondary thyroid carcinoma after allogeneic bone marrow transplantation during childhood. Bone Marrow Transplant 2001; 28:1125–1128. 50. Uderzo C, van Lint MT, Rovelli A, et al. Papillary thyroid carcinoma after total body irradiation. Arch Dis Child 1994; 71:256–258. 51. Sanders JE. Endocrine complications of high-dose therapy with stem cell transplantation. Pediatr Transplant 2004; 8:39–50. 52. Sanders JE, Hawley J, Levy W, et al. Pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation. Blood 1996; 87:3045–3052. 53. Anserini P, Chiodi S, Costa M, Conte N, Copello F, Bacigalupo A. Semen analysis following allogeneic bone marrow transplantation. Additional data for evidence-based counselling. Bone Marrow Transplant 2002; 30:447–451. 54. Grigg AP, McLachlan R, Zajac J, Szer J. Reproductive status in long-term bone marrow transplant survivors receiving busulfan-cyclophosphamide (120 mg/kg). Bone Marrow Transplant 2000; 26:1089–1095. 55. Sklar CA, Robison LL, Nesbit ME, et al. Effects of radiation on testicular function in long-term survivors of childhood acute lymphoblastic leukemia: a report from the children’s cancer study group. J Clin Oncol 1990; 8:1981–1987. 56. Sklar CA, Kim TH, Ramsay NKC. Testicular function following bone marrow transplantation performed during or after puberty. Cancer 1984; 53:1498–1501. 57. Sanders JE, Buckner CD, Amos D, et al. Ovarian function following marrow transplantation for aplastic anemia or leukemia. J Clin Oncol 1988; 6:813–818. 58. Byrne J, Fears TR, Gail MH, et al. Early menopause in long-term survivors of cancer during adolescence. Am J Obstet Gynecol 1992; 166:788–793. 59. Mertens AC, Ramsay NKC, Kouris S, Neglia JP. Patterns of gonadal dysfunction following bone marrow transplantation. Bone Marrow Transplant 1998; 22:345–350. 60. Matsumoto M, Shinohara O, Ishiguro H, et al. Ovarian function after bone marrow transplantation performed before menarche. Arch Dis Child 1999; 80:452–454. 61. Holm K, Nysom K, Brocks V, Hertz H, Jacobsen N, Mu¨ller J. Ultrasound B-mode changes in the uterus and ovaries and doppler changes in the uterus after total body irradiation and allogeneic bone marrow transplantation in childhood. Bone Marrow Transplant 1999; 23:259–263. 62. Bath LE, Critchley HOD, Chambers SE, Anderson RA, Kelnar CJH, Wallace WHB. Ovarian and uterine characteristics after total body irradiation in childhood and adolescence: response to sex steroid replacement. Br J Obstet Gyneacol 1999; 106:1265–1272. 63. Aisenberg J, Hsieh K, Kalaitzoglou G, et al. Bone mineral density (BMD) in long-term survivors of childhood cancer. J Pediatr Hematol Oncol 1998; 20:241–245. 64. Bhatia S, Ramsay NKC, Weisdorf D, Griffiths H, Robison LL. Bone mineral density in patients undergoing bone marrow transplantation for myeloid malignancies. Bone Marrow Transplant 1998; 22:87–90. 65. Nysom K, Holm K, Michaelsen KF, et al. Bone mass after allogeneic bone marrow transplantation for childhood leukaemia or lymphoma. Bone Marrow Transplant 2000; 25:191–196. 66. Cohen A, Shane E. Osteoporosis after solid organ and bone marrow transplantation. Osteoporos Int 2003; 14:617–630. 67. Stern JM, Chesnut CH, III, Bruemmer B, et al. Bone density loss during treatment of chronic GVHD. Bone Marrow Transplant 1996; 17:395–400. 68. Gilsanz V, Carlson ME, Roe TF, Ortega JA. Osteoporosis after cranial irradiation for acute lymphoblastic leukemia. J Pediatr 1990; 117:238–244. 69. Lee WY, Cho SW, Oh ES, et al. The effect of bone marrow transplantation on the osteoblastic differentiation of human bone marrow stromal cells. J Clin Endocrinol Metab 2002; 87:329–335. 70. Lee WY, Kang MI, Oh ES, et al. The role of cytokines in the changes in bone turnover following bone marrow transplantation. Osteoporos Int 2002; 13:62–68. 71. Smedmyr B, Wilbell L, Simonsson B, Oberg G. Impaired glucose tolerance after autologous bone marrow transplantation. Bone Marrow Transplant 1990; 6:89–92. 72. Lorini R, Cortona L, Scaramuzza A, et al. Hyperinsulinemia in children and adolescents after bone marrow transplantation. Bone Marrow Transplant 1995; 15:873–877. 73. Taskinen M, Saarinen-Pihkala UM, Hovi L, Lipsanen-Nyman M. Impaired glucose tolerance and dyslipidaemia as late effects after bone marrow transplantation in childhood. Lancet 2000; 356:993–997.

298

Chemaitilly et al.

74. Traggiai C, Stanhope R, Nussey S, Leiper AD. Diabetes mellitus after bone marrow transplantation during childhood. Med Pediatr Oncol 2003; 40:128–129. 75. Hoffmeister PA, Storer BE, Sanders JE. Diabetes mellitus in long-term survivors of pediatric hematopoietic cell transplantation. J Pediatr Hematol Oncol 2004; 26:81–90. 76. Mohn A, Di Marzio A, Capanna R, Fioritoni G, Chiarelli F. Persistence of impaired pancreatic beta cell function in children treated for acute lymphoblastic leukaemia. Lancet 2004; 363:127–128. 77. Hawkins MM, Robertson CM, Edge JA, Neil HAW. Is risk of diabetes mellitus increased after abdominal radiotherapy? Lancet 1996; 347:539–540.

16 Future Directions in Pediatric Stem-Cell Transplantation Edwin M. Horwitz Department of Hematology-Oncology, Divisions of Stem Cell Transplantation and Experimental Hematology, St. Jude Children’s Research Hospital, Memphis, Tennessee, U.S.A.

The coming years, the early 21st century, promise to be an incredibly exciting era for bone marrow transplantation (BMT) and specifically pediatric BMT. The scientific advances in stem cell biology, transplantation immunology, and gene transfer technology will certainly lead to a wave of clinical trials that will boost our armamentarium to treat childhood disease. Of all physicians, these advances will be most relevant to specialists in pediatric BMT, physicians who focus on the processing and transplantation of stem cells, as well as the delivery of immune-mediated therapy. As biologists unlock the full potential of marrow stem cells, hematopoietic, mesenchymal, and other, as yet undescribed populations, as well understand the fundamental mechanisms of immune effector cells, the clinical transplant specialist will be called upon to develop therapy for a wide variety of disorders that will continue to expand the spectrum of diseases we treat beyond the traditional bounds of hematology-oncology. For example, we have cared for children with metabolic storage diseases as well as osteopetrosis over the last two decades, because we recognized that the defective cell in these diseases originates from the hematopoietic stem cell (HSC). It is an indisputable truth that many aspects of pediatric BMT are rapidly progressing. Investigators will continue to develop novel strategies for engineering grafts composed of mobilized peripheral blood, umbilical cord blood, or bone marrow cells. Moreover, new innovative immunotherapies using T cells, dendritic cells, natural killer (NK) cells, and potentially B cells, will likely play a crucial role in furthering treatment for both hematologic malignancies and solid tumors. However, the two arenas that will constitute the most far reaching, and perhaps the most challenging, new frontiers in pediatric stem cell transplantation in the coming years will be the application of BMT to nonmalignant, nonhematologic disorders and the use of gene therapy in place of allogeneic stem cell transplantation to treat monogenic disorders of the lymphohematopoietic stem cell.

PRINCIPLES OF BONE MARROW CELL THERAPY BMT is, effectively, bone marrow cell therapy. In fact, BMT is truly the first stem cell therapy to be used clinically. The HSCs, however, are rather distinct among stem cells in that they are 299

300

Horwitz

easily obtained from living donors and readily transplanted into recipients by intravenous infusion. HSCs have cell surface recognition molecules that mediate homing of the circulating stem cells into their proper niche, the marrow space within bones. In contrast to HSCs, most tissue specific stem cells that have been described, present greater challenges for harvesting and require direct transplantation into the target tissue. Because of this relative ease of acquisition and delivery, marrow stem cells are ideal candidates for cell therapy of all somatic tissues; however, this approach is completely dependent on the capacity of marrow stem cells to contribute to nonhematopoietic tissues. There are three primary mechanisms by which marrow stem cells can potentially correct diseased or damaged nonhematopoietic tissues. First, the marrow-derived cells could differentiate to mature tissue cells and replace the damaged cells. This hypothesis is complicated by the controversy as to whether HSCs give rise to nonhematopoietic tissue, so-called “stem cell plasticity,” or multiple classes of stem cells reside within marrow. Second, the marrow-derived cells could fuse with the damaged cells, correcting the deficits resulting in a fully functional cell product. Finally, missing enzymes could be transferred from donor derived hematopoietic cells into somatic tissues genetically lacking the essential enzyme. The latter mechanism is termed cross correction. The classic paradigm of stem cell biology is that rare cells within specific tissues have the capacity for self-renewal, differentiation into the various cell lineages within that tissue, and extensive proliferation (1). The tissue-specific, committed stem cells provide a supply of terminally differentiated cells for physiologic tissue turnover for the life of the individual. Possibly, these stem cells may be able to replace injured cells, as seen in the bone marrow after chemotherapy and radiation. Stem cell plasticity describes the notion that a stem cell, which is committed to give rise to the “expected tissues,” may differentiate to cells other than these expected tissues, the socalled “unexpected tissues” (Fig. 1). This idea, therefore, is also termed trans differentiation. An equally plausible explanation of much of the current data suggesting stem cell plasticity is that there exist uncommitted stem cells within tissues that have the capacity to differentiate into many, or perhaps all, tissues. Local environmental factors could direct the differentiation pathway (Fig. 2). Convincing data to support the latter theory may be difficult to develop because most stem cells within a pool may be committed to the expected tissue and a very rare, less mature, uncommitted stem cell may reside within this population, with the potential to differentiate into a multiple lineages giving the appearance of transdifferentiation of committed stem cells. Fusion refers to the merging of healthy donor cells with diseased or damaged host cells leading to a normal functioning combined cell. This concept was demonstrated in cell culture when Terada et al. (2) and Ying et al. (3) simultaneously demonstrated that murine bone marrow cells and brain cells, respectively, obtained from a green fluorescent protein transgenic mice can fuse spontaneously with murine embryonic stem cells in vitro. The resulting cell displayed an undifferentiated stem cell phenotype carrying the transgenic marker. However, both studies of cell fusion used embryonic stem cells, a unique cell population distinctly different from bone marrow or other somatic cells and employed prolonged in vitro cell culture, a cell processing procedure not commonly used in clinical stem cell processing. Stem cell biologists who do not accept the experimental evidence for transdifferentiation (stem cell plasticity) invoke the concept of cell fusion to discredit it. However, when the goal is to develop effective cell therapy, a fusion mechanism, in contrast to a differentiation mechanism, does not detract from treatment strategy if the resulting cells are fully functional and physiologic correction of the defective tissue is achieved. Currently, the importance of cell fusion for the development of clinical marrow cell therapy is unclear, but, as discussed below, animal experiments have demonstrated the “proof of concept” for cell fusion in disease models, so it must be considered as a potential mechanism for the correction of disease tissue by transplanted marrow cells. Cross correction is the process by which proteins, usually enzymes, are transferred from normal cells to genetically defective cells resulting in the correction of the diseased tissue. First recognized in 1981, investigators demonstrated the direct transfer of lysosomal enzymes from

Future Directions

Unexpected Tissues

Bone Progenitors

Neural Progenitors

301

Local Environmental Factors

Self-renewal

Transdifferentiation Committed Hematopoietic Stem Cell

Other Tissue Progenitors

Differentiation

Myeloid Progenitors

Lymphoid Progenitors Expected Tissues

Figure 1 Schematic representation of the differentiation pathway of a committed stem cell that transdifferentiates to nonhematopoietic tissues. This schema depicts true stem-cell plasticity.

lymphoid cell to enzyme deficient fibroblasts (4). Cross correction was soon shown to be a property of some, but not all, lysosomal enzymes (5), indicating that each new genetic disorder would require a specific study. The lysosomal enzyme alpha-mannosidase was then shown to be capable of transfer from lymphocytes to enzyme deficient fibroblasts obtained from a patient during in vitro culture (6). Subsequently, a feline model of alpha-mannosidosis was treated by BMT. Functional enzyme was found in the neurons, glial cells, and cells associated with blood vessels, providing direct evidence of the potential of bone marrow derived cells to cross-correct nonhematopoietic tissues (7).

PRINCIPLES OF GENE THERAPY Gene therapy refers to the introduction of a gene (typically, a cDNA construct) into autologous cells that will be expressed to produce a protein that will elicit the desired effect. Originally conceived primarily to correct monogenic disorders by replacing missing proteins due to null mutations of the encoding gene, clinical gene therapy has been most often used to express immunomodulatory molecules to enhance the immune effects in the treatment of cancer. Although, the scope of gene therapy encompasses virtually all somatic tissues and stem cells, this discussion will focus on gene therapy of the lymphohematopoietic stem cells. Gene therapy of autologous lymphohematopoietic stem cells can potentially be used only to treat disorders that are amenable to treatment with allogeneic lymphohematopoietic stem

302

Horwitz

Self-renewal Uncommitted Stem Cell Differentiation

Other Committed Tissue Stem Cells

Committed Neural Stem Cells

Other Tissue Progenitors

Neural Progenitors

Committed Osteopoietic Stem Cells

Bone Progenitors

Committed Hematopoietic Stem Cell

Myeloid Progenitor

Lymphoid Progenitor

Figure 2 Schematic representation of the differentiation pathway of an uncommitted stem cell that differentiates to multiple committed tissue stem cells in completely different lineages. This is not stem-cell plasticity because there is no “unexpected tissue differentiation” from a committed stem cell. In this scenario, the uncommitted stem cell may differentiate to many committed tissue stem cells, although only four pathways are shown.

cells, i.e., BMT. These two therapeutic strategies are different means to achieve the same goal; thus, the challenge is to determine which approach is more effective with less risk to the patient. Both approaches require the harvest and reinfusion of marrow cells with the stem cells expected to home to the hematopoietic microenvironment. BMT requires a healthy donor; whereas gene therapy uses autologous cells; hence, another person is at risk for adverse events with BMT. Furthermore, a healthy donor cannot be identified in all cases so that some patients in need of a BMT as potentially curative therapy may not have the option (due to a lack of a suitable donor). Moreover, using allogeneic cells usually requires some type of preparative regimen with its associated risks, e.g., drug toxicity, immunosuppression, risk of infection, and engenders a risk of graft-versus-host disease (GVHD). Gene therapy bypasses many of these risks, such as the need for a healthy donor and the risk of GVHD, but creates new, unique risks and presents several new technical challenges. First, the target cells must be transduced with a genetic vector. The vector is the DNA construct that contains the gene of interest with the capacity for expression. To date, viral vectors have been most commonly used because they efficiently transduce cells, but many nonviral systems are being explored and may replace viral-based transduction in the future. Clinically, oncoretroviral, adenoviral, and adeno-associated viral vectors have been used, but lentiviral vectors are being actively studied in the laboratory and are currently being used in a clinical trial to investigate

Future Directions

303

therapy for human immunodeficiency virus infection (HIV-I) in adults (8) (C.H. June, personal communication). Whichever vector is selected for a given application, the construct must contain sequences that will allow the long term expression of the encoded gene in a lineage and differentiation stagespecific fashion, i.e., the gene must be expressed in the proper hematopoietic lineage (e.g., granulocytes vs. lymphocytes vs. erythrocytes, etc.) and at the proper stage of cell differentiation (e.g., promyelocyte vs. segmented neutrophil). These issues may be obviated if there is no detrimental effect of unregulated expression of a gene in all lymphohematopoietic progeny. Then, the viral promoter sequences can be used to promote the expression of the gene; however, these viral sequences (termed long terminal repeats, or LTRs) are subject to silencing so that long-term expression may be problematic. Alternatively, eukaryotic promoter and regulatory sequences may be included in the vector that will limit expression to the proper cells; however, these sequences may also be silenced. Moreover, this latter strategy, using regulatory sequences within the vector, is a formidable task, and lineage specific expression remains one of the barriers to clinical gene therapy. Next, a substantial number of stem cells must be transduced for a clinically significant correction of the underlying disorder to ensue. Alternatively, if the progeny of the transduced stem cells have a selective growth or survival advantage, then a relatively few corrected stem cells can give rise to an abundance of corrected terminally differentiated cells. The problem of transducing a large number of lymphohematopoietic stem cells has not be solved; however, patients with severe combined immunodeficiency disorder (SCID) have been successfully treated with gene therapy (9), as discussed below, because of the enormous selective advantage of the gene-corrected T cells derived from the gene-corrected stem cells in this disorder. Oncoretroviral vectors, such as the vectors used for stem cell gene marking (10) and in the trial of gene therapy for SCID (9), stably integrate into the genomic DNA, becoming part of the cellular genetic makeup. However, these vectors cannot transverse an intact nuclear membrane, thus, the target cells must be undergoing cell division with the consequent breakdown of the nuclear membrane during mitosis for successful transduction. Because stem cells are quiescent, the need to stimulate the cells into mitosis presents technical challenges as well as possibly adversely affecting the long-term capacity of the stem cell to generate multilineage, differentiated cells. The need for cell division can be obviated by using lentiviral vectors, which can transverse an intact nuclear membrane. The vectors are able to transduce lymphohematopoietic stem cells and integrate into the stem cell genome. Expression of the encoded genes may be under the control of the viral promoters, or an internal promoter, which presents the same concerns as with oncoretroviral vectors. Importantly, the insertion of the vector can disrupt the host genome, an event termed insertional mutagenesis. There are two general mechanisms by which the integration of a vector can affect the genome. First, the vector may insert within a gene disrupting expression of that gene. This could result in the loss of a functional protein product, the effects of which are dependent on the specific protein. A resulting metabolic disorder would unlikely be of clinical significance because a small number of cells would be affected. However, disruption of a tumor suppressor gene could initiate the development of a vector-associated malignancy. This potential adverse event has not been reported in human trials. Second, the promoter sequences can interact with host genomic promoters to alter the expression of a gene. The up- or downregulation of genes may have profound effects on the phenotype of the cell. The now famous example of this adversity was reported in the trial of gene therapy for SCID (9) in which the oncoretroviral vector encoding the common gamma chain integrated into the genome in proximity to the LMO2 gene (11). The expression of this gene was increased sufficiently to induce T-cell leukemia in three children enrolled on this study. Although gene therapy investigators have long recognized the potential for such an adverse event, the events of the SCID trial were the first documented cases of insertional mutagenesis in human subjects. Another major risk to gene therapy is the development of recombination competent retroviruses (RCR) during vector production that would then transduce the stem cells or simply be infused with the stem cells giving rise to oncoretroviral induced malignancies. This potential

304

Horwitz

serious risk was demonstrated early in the development of gene therapy when T-cell lymphoma was induced in nonhuman primates after infusion of transduced CD34C cells with RCR (12). The bothersome detail with this study is that far more RCR was infused into these irradiated animals than would likely occur in a clinical trial. However, this study does prove the principle that RCR may lead to lymphoma and must be rigorously excluded in the clinical setting. RCR associated adverse events (malignancy or other) have not been reported in clinical gene therapy trials.

MESENCHYMAL STEM CELLS One of the most exciting recent advances in bone marrow cell therapy is the recognition of the therapeutic potential of marrow cells termed mesenchymal stem cells (MSCs) (13). Alexander Friedenstein first identified marrow stromal cells as supportive elements for the hematopoietic function of bone marrow and as precursors of osteogenic tissue (14). He showed that these spindle-shaped cells adhered to the plastic surface of a tissue culture flask (in contrast to hematopoietic cells from marrow) and could give rise to clonal colonies of spindle shaped cells, which we now designate CFU-F (15,16). Friedenstein showed that these cells could partially transfer the hematopoietic microenvironment in animals (17), which led Maureen Owen to propose the existence of a stromal stem cell (18–20) in analogy to the hematopoietic stem cell. Arnold Caplan popularized the term “MSCs” in the early 1990s, because these cells could ostensibly undergo extensive self-renewal and differentiate into at least three lineages: bone, fat, and cartilage (21). However, some investigators questioned the “stemness” of this heterogeneous population of cells and opted to omit any reference to a stem cell identity when publishing preclinical (22,23) or clinical (24–26) studies of the marrow stromal cells, which may also be designated MSCs. As early as 2000, leading mesenchymal cell therapy investigators gathered at a workshop at the Annual Meeting of the International Society for Cellular Therapy (ISCT) and concluded that convincing data to support a stem cell identity of the unfractionated plasticadherent cells was lacking (27), and quite recently the Mesenchymal and Tissue Stem Cell committee of the ISCT has recommended that these cells be termed multipotent mesenchymal stromal cells but continue to be identified by the acronym MSCs (28). MSCs can be isolated from multiple sources other than bone marrow, including adipose tissue (29,30), cord blood (31,32), placenta (33,34), and possibly mobilized peripheral blood (35). The prevailing opinion is that MSCs have the same characteristics regardless of the tissue source. However, there is much ongoing investigation; hence, differences may be reported in the future. Typically, MSCs are isolated by “adherence selection.” A mononuclear cell preparation derived from tissue source is placed into cell culture with standard media, and the MSCs will adhere to the plastic flask whereas the other cells (e.g., hematopoietic cells) will remain in suspension. Simply changing the culture media and rinsing the remaining adherent cells will isolate the MSCs. Although other selection methods have been described, none has generated a cell product that has proven more useful clinically, although, this, too, is an area of active research. Currently, MSCs are rather crudely identified by a combination of characteristics, because we do not have a specific marker that uniquely defines these cells, as CD3 defines a T cell. MSCs are plastic adherent cells (in vitro) that are generally spindle-shaped, although subsets of cells may be more rounded. Although the MSCs were originally by recognized by the monoclonal antibodies, SH2 and SH3 (36), the antigens have been identified as epitopes on CD105 and CD73, respectively (37,38). Hence, we now consider MSCs to express these markers, CD73 and CD105 in addition to CD90 and the adhesion molecules, CD29, CD44, and CD166 (36,39). They lack expression of hematopoietic surface antigens, notably CD45, CD34, CD14, and CD11b (39). Most importantly, MSCs can readily differentiate in vitro to osteoblasts (OB) giving rise to mineralized nodules, adipocytes, and chondroblasts, which give rise to cartilage rests. The in vitro trilineage differentiation potential is, to date, the most characteristic feature of MSCs.

Future Directions

305

MSCs also possess striking immunologic characteristics. The cells express intermediate levels of HLA class I molecules but not HLA class II molecules, although surface expression of HLA class II molecules can easily be induced by exposure to interferon g (40). Interestingly, MSCs do not elicit a proliferative response from allogeneic lymphocytes in a standard mixed lymphocyte reaction (MLR) assay (41) and are not lysed by lymphocytes in vitro (41,42), suggesting that these cells are not recognized by the immune system. Moreover, MSCs inhibit T cell proliferation in an MLR reaction of lymphocytes stimulated by other HLA mismatched lymphocytes, suggesting that they may be immunosuppressive (41–46). However, MSCs do not inhibit the activity of cytotoxic T-lymphocytes or natural killer (NK) cells (42) against irradiated lymphocyte target cells; although, as stated above, these effector cells do not lyse mismatched MSCs. Thus, there are two major potential applications of MSCs for cell therapy. First, they may be used as progenitor cells to replace damaged tissues as treatment for genetic disorders or traumatic injury (13). Second, MSCs may be used to suppress the immune system to facilitate engraftment of HSCs in BMT or to treat steroid-resistant GVHD (47), which is one of the great challenges in clinical BMT. Beyond the scope of this chapter are the potential of MSCs to secrete cytokines, which could foster engraftment of transplanted HSCs by a mechanism distinct from immunosuppression, and the ease of introducing foreign DNA into MSCs by viral or nonviral methods, suggesting an enormous potential for these cells in gene therapy.

FUNDAMENTAL STEPS IN THE DEVELOPMENT OF BONE MARROW CELL THERAPY There are three basic issues to be addressed when considering bone marrow cell therapy for a nonhematopoietic tissue. First, which cell within bone marrow is best suited to deliver the therapeutic effect? In many cases, this question will be initially approached in animal models. Cell populations enriched for HSCs, such as CD34C cells or CD133C cells, may be the ideal cell for many applications. MSCs may be the appropriate cell if the goal is to regenerate bone or cartilage (13) or to modulate the immune response (48,49). Myeloid cells have been reported to contribute to muscle (50) and liver (51) after transplantation in animal models. Second, the route of administration may be a critical determinant of the success of any cell therapy. As specialists in BMT, physicians generally consider systemic infusion as the route of choice. Indeed, if systemic disease is being treated, intravenous infusion may be best. However, for cell therapy of the brain, liver, heart, or any other localized organ, direct injection of marrow cells may lead to a greater delivery of cells to the target tissue, as homing of marrow cells to other tissues has not been demonstrated. Finally, the level of tissue correction required to achieve a clinically significant benefit must be considered. In most nonhematopoietic organs, complete donor chimerism, as we generally seek to obtain for hematopoietic disorders, is generally not feasible with our current knowledge. However, subtotal cellular biochemical correction is often sufficient to arrest the progression of a disease and, occasionally, render a patient functionally “cured.” For example, only 3–5% of neutrophils are thought to require enzymatic correction to ameliorate the symptoms of chronic granulomatous disease (52).

PATIENT-BASED RESEARCH OF BONE MARROW CELL THERAPY AND GENE THERAPY Translational research has become a trendy term that evokes a sense of clinical relevance to research bearing this label. However, in the strictest sense, translational research should involve patients and “translate” in real time. Moreover, translational research represents a bidirectional flow of knowledge. Investigators not only apply laboratory-discovered biology to the clinical trials, but they also learn human biology, which may foster the development of new and/or

306

Horwitz

improved therapies, from the trials. Thus to speed the pace of discovery, translational research should proceed to patient based trials as quickly as safety and ethical considerations permit. This is especially true in the development of bone marrow cell therapy and gene therapy. Although animal models are critically important tools, and should be vigorously pursued to define the biomedical principles, as required by the Belmont report, we now recognize that animal models often do not accurately reflect human disease. Hypothesis driven translational and clinical trials, then, are a key element to the successful development of novel bone marrow cell therapies.

BONE MARROW CELL THERAPY FOR GENETIC DISORDERS OF BONE Bone marrow cell therapy for bone seems quite logical. Bone and bone marrow are closely aligned physiological compartments, suggesting that these tissues may constitute a single functional unit (53). Indeed, osteopoietic progenitors are thought to reside in bone marrow and may be represented by MSCs. The increasing interest in MSCs, which began in the middle 1990s, lead Prockop and colleagues to investigate the fate of murine MSCs after intravenous infusion. They demonstrated that these cells can engraft in bone (22) and in a murine model of osteogenesis imperfecta (OI) lead to a small but appreciable improvement in the disease phenotype (23). OI is a genetic disorder of mesenchymal cells, characterized by bony fractures and deformities, short stature, and often a reduced life expectancy (54,55). The underlying defect is a mutation in one of the two genes, COL1A1 and COL1A2, that encode collagen type I. There is a wide variety in the severity of the phenotype of the affected children. In the mildest form, Type I OI according to the Sillence classification (54), mutations are typically found in regulatory sequences, resulting in decreased expression of one of the two genes and an overall decrease in the amount of normally structured collagen. These mutations may be transmitted by autosomal dominant or autosomal recessive inheritance patterns, and the affected children have minimal problems with fractures, no significant deformities, are generally of normal stature, and have a normal life expectancy. In the more severe forms, Type II and Type III, new, spontaneous autosomal dominant mutations are typically found within one of the exons so that a structurally altered protein is expressed at reasonably normal levels. The protein disrupts bone structure by a dominant negative mechanism. Children with Type II OI, exhibit 60% mortality in the first day of life, and 80% mortality in the first month of life. By one year, there is O99% mortality (however, a few do survive). Children with Type III OI, the most severe form to routinely survive infancy, have numerous painful fractures, severe bony deformities, and markedly shortened stature. The life expectancy of these patients, historically, was quite short; however, with improved awareness and improved supportive medical and surgical care, many such patients may live a relatively long life, although mild trauma may still be fatal. The severity of the phenotype of children with OI truly exists along a spectrum, without unequivocal criteria to categorize a given child. Although a genotype-phenotype correlation is approximate at best (56,57), the specific mutation clearly impacts the phenotype. However, the balance of normal and mutated collagen, in this autosomal dominant disorder, also seems to affect the phenotype (58). This latter observation is the key to our selection of OI as a model disorder to investigate BMT for disorders of bone. For a cell therapy strategy to benefit these children, only low levels of mesenchymal engraftment, sufficient to alter the balance of normal and mutated collagen, are required to reduce the severity of the phenotype. Additionally, the defective osteogenic environment, may promote engraftment of donor-derived cells. Based on this rationale, and with the animal data in mind, our research team undertook a pilot study to demonstrate the feasibility of transplanting bone marrow cells in children with severe OI. Five children with severe OI (Sillence Type II or III) underwent BMT with unmanipulated bone marrow freshly harvested from HLA-compatible sibling donors (25,59). Approximately three months after BMT, we obtained a bone biopsy from the iliac wing and harvested OB in the laboratory. After culture expansion of the cells, we verified the absence of hematopoietic

Future Directions

307

contamination (!0.1%) and demonstrated donor OB engraftment in three of five patients, ranging from 1.2% to 2.0% of the total number of OB isolated from the iliac wing biopsy (Fig. 3). Although the level of donor engraftment determined in our assay may seem low, we were only able to assess the fraction of donor OB. Bone is comprised of both OB and osteocytes, the latter being developmentally derived from the former. In typical bone specimens, there are tenfold more osteocytes than OB in bone (60). Additionally, bone is quite heterogeneous, and it is conceivable that the epiphysis of long bones would contain a greater fraction of donor cells than the iliac wing, which is the standard site and the most surgically accessible for biopsy in the evaluation of metabolic bone disease. Finally, subsequent studies in a murine transplant model indicated that analysis of cultured OB underestimates (about tenfold) the total donor-derived bone cells (61). Nonetheless, this data unequivocally demonstrates the presence of donor-derived OB. If mesenchymal engraftment were to affect the phenotype of the patient, we hypothesized that it should alter the microscopic structure of the bone. We therefore histomorphometrically assessed the bone of our patients before and after BMT (Fig. 4). Specimens of trabecular bone taken before transplant typically contain numerous disorganized osteocytes, enlarged lacunae, and relatively few OB. The bone had the characteristic appearance of high bone turnover, including woven bone, which is characteristic of OI and other metabolic bone disorders. Fluorescence microscopy with tetracycline labeling showed disorganized formation of new bone and poor mineralization. In contrast, specimens taken about six months after transplant showed a reduced number of osteocytes, linearly organized OB and evidence of lamellar bone formation. Thus, marrow mesenchymal cell engraftment in the bone is associated with an improvement in bone formation and mineralization. The clinical outcome of patients in a pilot trial is an important measure of the potential of bone marrow cell therapy for any disorder and dictates the need for ongoing research. In our trial of bone marrow cell therapy for OI (59), all five patients showed a decreasing growth (A)

(B)

335

495

655

815

975

1200 Donor

800 400 0 1200

Recipient (pretransplant)

800 400 0 4000

Recipient Bone (posttransplant)

2000 0

Figure 3 Engraftment of donor-derived cells in bone. (A) Fluorescence in situ hybridization analysis of interphase nuclei from cultured osteoblasts (OB) obtained from a bone biopsy specimen. Both X and Y chromosomes are present in one OB, the left nucleus, from this female patient. This approach can be utilized for sex mismatched donor-recipient transplants. (B) Electropherograms based on an analysis of DNA polymorphisms (variable number of tandem repeats) of the donor (top panel) and patient (middle panel) before transplantation, and of the OB from a bone specimen obtained from the patient after bone marrow transplantation (lower panel). This approach can be utilized for all patients but is the only readily available method to detect donor cells in sex matched donor-recipient transplants. Abbreviation: OB, osteoblasts. Source: Adapted with permission from Ref. 25.

308

Horwitz

Figure 4 Bone histology before (left column) and after (right column) bone marrow transplantation. (A) Pretransplantation biopsy specimen of trabecular bone stained with GoldnersMason trichrome. Numerous, randomly arranged osteocytes (OC) are citing in large lacunae. Note also the peritrabecular marrow fibrosis, the paucity of osteoblasts (OB) relative to the posttransplantation specimens, and the incompletely calcified area of bone matrix. (B) Similarly prepared posttransplantation specimen, taken near the site shown in Fig. 3(A). The number of OC is reduced, and there is a small section of lamellar bone (L), suggesting normalization of the remodeling process. Magnification, 88X. (C) Fluorescence photomicrograph of the tetracyclinelabeled trabecular bone specimen [same section as in Fig. 3(A)]. The labeling is poorly defined, indicating disorganized formation of new bone and abnormal mineralization. (D) Contrasting posttransplantation specimen with definitive, crisp single and double tetracycline labeling, indicative of markedly improved new bone formation and mineralization. Magnification, 56X. (E) Pretransplantation trabecular bone specimen stained with toluidine blue and photographed under polarized light to enhance the woven (w) texture of the bone, a characteristic feature of patients with osteogenesis imperfecta. (F) Similarly prepared posttransplantation bone specimen demonstrating L formation, and linearly arranged OB in areas of active bone formation along the calcified trabecular surface. Magnification, 88X. Abbreviations: OB, osteoblasts; OC, osteocytes; L, lamellar bone. Source: Adapted with permission from Ref. 25.

velocity over the first year as expected for children with this disorder. In contrast to the natural history of severe OI, all five patients showed an acute acceleration of their growth velocity during the first six months after BMT. Subsequently the growth rates slowed but remained greater than controls. The patients also showed an increase of total body bone mineral content (TBBMC) measured by dual energy X-ray absorptiometry. The rate of gain in TBBMC among these patients slightly exceeded that for weight-matched healthy children, and the last few measurements approached the lower limit of the normal range. OI is a disease of osteopenia;

Future Directions

309

hence, we interpret these findings as suggestive of clinical improvement of bone mineralization, consistent with the histormorphometric findings. Finally, the rate of radiographically documented fractures acutely decreased during the first six months after transplant compared with the six months before transplant. Although the rate of fractures gradually declines with age, an immediate reduction in the rate of fractures is inconsistent with the natural history of OI. This first trial of BMT for children with severe OI represented a proof of principle for bone marrow cell therapy for nonhematopoietic disorders. It also constituted a significant advancement in the development of cell therapy for this disease as well as other mesenchymal disorders. However, the children were not sufficiently improved to consider BMT, as it is currently practiced, to be a sole, complete therapeutic intervention. In an effort to enhance the benefits observed after BMT, we developed the first clinical study to infuse allogeneic MSCs after BMT (26). We sought to test the hypothesis that isolated, allogeneic marrow mesenchymal cells could be safely infused after allogeneic BMT and would benefit children with severe OI. To unequivocally identify the marrow mesenchymal cells infused in this trial (compared to cells that may persist after the original BMT) we “genemarked” the cells by transduction with a retroviral vector. Furthermore, to investigate whether marrow mesenchymal cells could be expanded ex vivo and retain their biologic potential, we used a double gene marking strategy in which minimally processed cells and substantially ex vivo expanded cells were each transduced with one of two unique retroviral vectors. One vector expressed neomycin phosphotransferase (neor), a bacterial protein, and the other encoded neor but did not express it (i.e., a silent vector). These two proviral sequences could be readily distinguished by a polymerase chain reaction (PCR) assay. After the mesenchymal cells were isolated from bone marrow by adherence to plastic, the cells were divided into two fractions, and each was transduced with one of the two retroviral vectors used for the two fractions of MSCs among the patients. One fraction was allowed to remain in culture for the minimal time required for isolation and transduction, while the other was expanded over three passages. The minimally maintained cell preparation was infused into the patients, without a chemotherapy conditioning regimen, at a dose of 1!106 cells/kg, and the expanded mesenchymal cells were infused at a dose of 5!106 cells/kg after about two to three weeks, again without a conditioning regimen. About six weeks after the cell infusions, we obtained a biopsy of bone and skin and an aspirate of bone marrow and isolated OB, skin fibroblasts, and marrow stromal cells. We then used our PCR assay to assess for engraftment of each cell population. In five of the six patients, we were able to identify marked mesenchymal cells in at least one of the tissues studied. Both minimally processed cells and expanded cells engrafted. A consistently less intense PCR signal from the OB DNA suggested that the ex vivo expansion may diminish the osteogenic engraftment and/or differentiation potential of marrow mesenchymal cells. However, the small sample (5 evaluable patients) precludes conclusive statements regarding the effect of cell expansion. All five children in whom we documented mesenchymal cell engraftment showed an acute acceleration of their growth velocity in the first six months after the cell infusions compared with the six months immediately preceding the infusions. The outcome was most significant for the two patients, who did not grow in the six months prior to the cell therapy but accelerated their growth velocity to 94% and 67% of the predicted growth velocity for age- and sex-matched children. We did not observe an unambiguous improvement of the TBBMC after the mesenchymal cell infusions. Because a chemotherapy based conditioning regimen was not given to the children prior to the cell infusions and the cells were relatively pure compared to unmanipulated marrow (although still quite heterogeneous), the growth velocity data, TBBMC data notwithstanding, formulates a compelling argument supporting the therapeutic potential of marrow mesenchymal cells. Although, these two trials do not collectively yield a definitive therapeutic strategy with the potential to completely ameliorate the symptoms of OI, a foundation has been established upon which future research can strive for this realistic goal. Based, in part, on our initial studies, a child with another genetic disorder of bone, hypophosphatasia underwent a haploidentical BMT from her unaffected sister (62).

310

Horwitz

Hypophosphatasia is a genetic disorder characterized by deficient activity of the tissuenonspecific isoenzyme of alkaline phosphatase and, in the most severe cases, is lethal. The child attained complete hematopoietic chimerism and showed clinical improvement of her disease with healing of rickets and skeletal remineralization. She then developed mixed hematopoietic chimerism coincident with a worsening of her skeletal disease. After a second infusion of marrow cells, expanded ex vivo to enrich for MSCs, she again showed significant skeletal improvement. Most recently, another child with hypophosphatasia underwent BMT but also had bone fragments, obtained form the marrow donor, surgically placed in the peritoneum and OB-like cells, obtained from donor bone, intravenously infused (63). This child has shown remarkable improvement beginning about three months after BMT and continues to maintain her height and weight with normal bone mineralization more than four years after BMT.

CLINICAL TRIALS OF GENE THERAPY OF SEVERE COMBINED IMMUNODEFICIENCY DISORDERS The initial prospects for gene therapy received much attention in the lay press fueling what, in retrospect, seems to have been unrealistic expectations. In 1995, the Orkin-Motulsky Report (64) stated that confidence in the future of gene transfer as a therapeutic strategy would be greater if just one disorder would have been cured. In 2000, this call was answered with the first report that children with a SCID due to a deficiency of the common gamma chain had been cured by gene therapy of the lymphohematopoietic stem cell (9,65). The first clinical gene transfer in lymphohematopoietic stem cells was the pioneering gene marking studies of Brenner and colleagues in 1993 (10,66). These investigators transduced marrow stem cells with an oncoretroviral vector encoding neor to identify the individual cell and all progeny of that cell. They showed that autografted marrow cells, as treatment for neuroblastoma or acute myeloid leukemia, contained tumor cells that contributed to recurrent disease. Additionally, they proved that the reinfused stem cells contribute to the long-term hematopoietic reconstitution in the patients. Perhaps the most important contribution of these seminal studies was that they laid the foundation for future clinical trials of gene therapy, the transfer of therapeutic genes. Based on meticulous preclinical studies in animal models (67,68), Cavazzana-Calvo, Fischer, and many collaborators reported the first successful clinical gene therapy (9,65). These investigators studied patients with SCID caused by common gamma chain deficiency. The common gamma chain, the gene for which resides on the X chromosome, is an essential molecular component of five cytokine receptors necessary for the development of T cells and NK cells. This molecular defect is responsible for about 40% of SCID patients (69). Marrow lymphohematopoietic stem cells were transduced with an oncoretroviral vector expressing the common gamma chain under the control of the modified viral promoter (LTR, see above). The autologous, genetically modified marrow cells were reinfused without a cytotoxic preparative regimen. Overall, 9 of 10 children showed reconstitution of both T cells and NK cells. The one child who failed to benefit from this therapy was 15 years of age at the time of the treatment, and we now recognize that older patients do not seem to benefit from gene therapy for SCID due to common gamma chain deficiency (70). A second trial of SCID gene therapy, conducted in London, using a similar oncoretroviral vector enrolled four patients; all showed similarly outstanding outcomes (71). Recently, threeyear follow-up of a single patient who underwent gene therapy for SCID in Australia was reported (72). However, this child obtained only partial immunologic reconstitution, presumably due to a low dose of gene-corrected CD34C cells, and underwent a BMT from a matched unrelated donor 26 months after gene therapy. Thus, 15 children who underwent gene therapy for SCID due to common chain deficiency have been reported, and all were either cured or the reason for failure presumably identified.

Future Directions

311

Adenosine deaminase deficiency is another molecular etiology of SCID and accounts for about 10–15% of diagnoses. Unlike common gamma deficiency, ADA deficiency results in metabolic defect, the accumulation of toxic metabolites, as well as an immune defect, and correction of both defects may be important to confer clinical benefit. Thus, stem cell transduction is essential for clinical improvement in contrast to transduction of differentiated lymphocytes, which is technically more readily accomplished. Two children with SCID due to ADA deficiency have undergone gene therapy using an oncoretroviral vector (73). To promote engraftment and impart an initial growth advantage to the genetically modified stem cells, these patients were treated with busulfan (4 mg/kg total dose) prior to the infusion of the genetically modified autologous cells. Both patients showed engraftment of the genetically modified cells with an increase of lymphocyte counts, improved immune function, and lower toxic metabolites, demonstrating the feasibility of gene therapy for SCID due to ADA deficiency as well as the use of a very low dose, nonmyeloablative cytotoxic conditioning regimen to foster the engraftment of genetically modified autologous lymphohematopoietic stem cells. Unfortunately, these clinical studies have brought our worst fears to bear. Three children who were treated in the trial for common gamma chain deficiency SCID subsequently developed T-cell leukemia (74,75). As discussed above, we now recognize that the vector integrated into the host genome in proximity to the LMO2 oncogene, up-regulated its expression, and initiated a series of genetic events that resulted in a T-cell leukemia phenotype (74,76). Current opinion is that the LMO2 insertional mutagenesis is unique to SCID, caused by common gamma chain deficiency and related molecular etiologies (69,77). Interestingly, none of the patients transplanted in the London trial have developed leukemia; however, the investigators acknowledge that having studied the same disease and having used a similar molecular strategy, a subset of those patients are expected to develop leukemia as well (71). Although these very serious adverse events are a temporary setback for the field of gene therapy, the events were only recognized through clinical trials and all investigators in this arena have demonstrated a remarkable effort to understand the molecular basis for the observed leukemogenesis to improve what seems to be a very promising therapeutic approach. Moreover, all investigators have kept the gene therapy community updated on the progress of their patients fostering trust between biomedical research community and the regulatory agencies. Thus, the future of gene therapy for immunodeficiencies disorders is very bright, and it is likely that scientific advances, the need for which were recognized through early clinical trials, will foster the development of broadly applicable gene therapy strategies for many, if not all, primary immunodeficiency disorders.

MARROW MESENCHYMAL STEM CELLS AS CELL THERAPY FOR INBORN ERRORS OF METABOLISM BMT is the standard of care for several inborn errors of metabolism (78), such as alphamannosidosis, as discussed later in this chapter. MSC therapy could potentially benefit these children if they engrafted in the appropriate tissues and differentiated to the cells expressing the genetic deficit. Alternatively, enzymes may be transferred from donor MSCs to host tissues, providing cross correction of the enzyme deficit, thereby benefiting the patient. Although such an approach has yet to be definitely demonstrated, MSCs express high level of arylsulfatase A and a-L-iduronidase, the missing enzymes in metachromatic leukodystrophy (MLD) and Hurler disease, respectively (79). Eleven patients with these disorders underwent conventional BMT (standard therapy) and were subsequently treated with an intravenous infusion of donor derived MSCs. Although these patients did not show improvement in their overall outcome, four of the five patients with MLD were reported to show improvement of in nerve conduction velocity (80).

312

Horwitz

MESENCHYMAL STEM CELLS AS MODULATORS OF IMMUNE FUNCTION AND THE TREATMENT OF GRAFT-VERSUS-HOST DISEASE Over the last several years, human bone marrow MSCs have increasingly been found to possess immunomodulatory properties (48,49). Indeed, immunomodulation may become of the principle applications of MSCs in BMT. Interpretation of the literature on the immunologic properties of MSCs requires caution as in vitro assays do not necessarily predict in vivo activity, and data from animal models seem to differ markedly from those using human MSCs. In vitro data discussed above suggests that human MSCs may escape recognition from the immune system and may suppress an unrelated immune response. If this data represents the in vivo biology of MSCs, then these cells should be capable of suppressing an inappropriate or undesired immune response, such as GVHD. A case report of an eight-year-old boy with steroid-resistant Stage IV GVHD beautifully illustrates this hypothesis (47). The patient was treated with an intravenous infusion of MSCs obtained from his haploidentical mother (third party, i.e., not his BMT donor). He rapidly showed a dramatic decline in his bilirubin and a normalization of his stools. After weaning of his pharmacologic immunosuppression, his GVHD recurred, and a second infusion of MSCs produced a similarly impressive improvement. Ultimately, another attempt to wean immunosuppression resulted in another recurrence of active GVHD, and the child died. Although the MSC therapy did not lead to a permanent resolution of his GVHD, this single patient case report indicates the potential of MSCs to treat steroid resistant GVHD, and further clinical studies are warranted. However, the capacity of MSCs to suppress GVHD activity does not indicate a lack of immune recognition in immunocompetent hosts. Our MSC trial in OI patients (26) illustrates this important issue. In our studies, we infused gene-marked MSCs into children with OI, using two retroviral vectors: one that expressed neor, a bacterial protein and a silent vector. We identified cells transduced with the silent vector in all evaluable patients, but we were unable to identify the cells transduced with the neor expressing vector, suggesting these cells were immunologically recognized and destroyed when they were infused into these immunocompetent patients. One of the patients in our trial developed antibodies against proteins in fetal bovine serum (common media supplement for ex vivo MSC expansion) and developed a clinical allergic-like reaction to the second infusion of MSCs. This child was the only study subject in whom we were unable to identify any gene marked cells, suggesting these antibodies destroyed the MSCs upon infusion, precluding engraftment. Thus, our studies of MSCs in OI provide two examples of apparent immune recognition of MSCs. Whether MSCs are immunoprivileged may be a matter of context. The cells may not elicit a simple allogeneic response against differing HLA alleles but still may elicit a response when presenting foreign antigens, such as bacterial or bovine antigens. Additionally, the cells may suppress an immune response against third party cells but still not escape immune recognition. The limited data currently available leaves many questions unanswered, indicating that much research remains, but MSCs certainly hold great promise to advance BMT.

MESENCHYMAL STEM CELLS TO FACILITATE HEMATOPOIETIC STEM-CELL ENGRAFTMENT The immune suppressive potential of MSCs suggests that coinfusion of MSCs with a marrow or blood stem cell graft could attenuate the host versus graft (rejection) response. Furthermore, in vivo MSCs may be able to secrete cytokines, as is characteristic of hematopoietic supportive stromal cells, thereby fostering the survival, proliferation and differentiation of HSCs. Both mechanisms would predict that MSCs could facilitate hematopoietic engraftment and reconstitution This hypothesis was first investigated in a study of breast cancer patients undergoing autologous peripheral blood progenitor cell transplantation who received a coinfusion of autologous MSCs with their hematopoietic graft and granulocyte colony stimulating factor

Future Directions

313

daily after the cell infusion (81). The median time to achieve a neutrophil count of 500/ml and platelet count of 20,000/ml was 8 and 8.5 days, respectively; however, this feasibility and safety study did not include a control group, and, therefore, efficacy could not be assessed. A second study, which was a multi-institutional collaborative study of MSC cotransplanted with either bone marrow or peripheral blood stem cell grafts in patients receiving a transplant from an HLA matched sibling donor, demonstrated a median time to neutrophil and platelet engraftment (defined as above) of 14 and 20 days, respectively (82). Again, a control group was not included. These studies show the safety of MSC cotransplantation and the potential of MSCs to foster engraftment; however, more rigorous efficacy trials are required to prove the capacity of MSCs to improve hematopoietic stem cell engraftment to a clinically significant degree. Additionally, adult patients often have a higher risk of graft failure and regimen related toxicity. Thus, whether the effort and expense of MSC processing will be justified by the incremental benefit in pediatric hematopoietic stem cell transplantation remains to be determined.

PRECLINICAL MODELS AND CLINICAL TRIALS OF BLOOD AND MARROW TRANSPLANTATION AS CELL THERAPY FOR NONHEMATOPOIETIC DISORDERS Metabolic Storage Diseases Although the pathology of many metabolic storage diseases rest in the accumulation of metabolic intermediates within macrophages, readily explaining successful treatment by BMT, other metabolic disorders are manifest primarily in nonhematopoietic tissues. Alphamannosidosis is a prototypic disorder that can be treated by BMT as cell therapy. It is a rare autosomal recessive disease characterized by a deficiency of lysosomal alpha-mannosidase. There is an accumulation of oligosaccharides in multiple tissues including the central nervous system, liver, and bone marrow. As previously discussed, this enzyme was shown to be directly transferred from normal lymphocytes to enzyme deficient fibroblasts in vitro (4–6). Based on this data and the lack of alternative therapies, a child underwent allogeneic BMT as therapy for his disorder in 1987 (83). The child died 18 weeks after successful hematopoietic engraftment. The enzyme level in his spleen and liver were near normal; however, enzyme activity in his brain tissue was only 7%, and the enzyme kinetic parameters were not consistent with normal protein. These investigators concluded that they had proved cross correction of the liver and reversed the somatic changes of alpha-mannosidosis but were not able to affect the CNS disease. Report of a feline model of alpha-mannosidosis that showed arrest of progression of the neurological deficits after BMT (7) led clinical investigators to further efforts to treat these children with marrow cells. To date, at least six children have undergone BMT for alphamannosidosis, and all are reported to be alive with at least stabilization of their neurological symptoms (78,84–86); thus, cross-correction of enzyme deficient neurons from donor lymphohematopoietic cells may, in fact, ensue after BMT. Such an approach is not universally successful. Glycogen storage diseases, which manifest as liver the pathology, are not corrected by BMT (87). Similarly, children with Hunter Syndrome (Mucopolysaccharidosis II) do not seem to benefit from BMT, and it is not recommended (88), although enzyme cross correction was demonstrated in vitro over a decade ago (89). Finally, GM1-gangliosidosis, a neurodegenerative disorder due to deficiency of lysosomal acid beta-galactosidase, has shown the potential for cross-correction in an animal model (90), despite evidence of low efficiency human enzyme transfer during in vitro culture (91). Two patients with GM1 gangliosidosis are known to have been treated with BMT; both children died of disease progression (92) (unpublished data).

Experimental Metabolic Liver Disease Severe metabolic diseases of the liver are often fatal unless the patient undergoes liver transplantation. Undoubtedly, a cell therapy for the liver obviating the need for liver transplantation

314

Horwitz

would represent a major advance for these disorders. Cross-correction of enzyme deficiencies in alpha-mannosidosis is an example of successful marrow cell therapy for the liver; however, the failure to correct glycogen storage diseases reminds us of the challenges that remain to be overcome. To investigate the potential of marrow cells to incorporate into the liver and correct enzyme deficiencies, fumarylacetoacetate hydrolase-deficient mice, an animal model of fatal hereditary tyrosinemia type I, were treated by BMT. Surprisingly, transplantation of highly purified murine HSCs gave rise to donor-derived hepatic regeneration and restored the biochemical function of the liver (93). The donor HSCs seem to fuse with the enzyme deficient hepatocytes to produce a functional liver (94,95). Further studies suggested myelomonocytic cells can also fuse with enzyme-deficient hepatocytes in vivo and correct the biochemical deficit (51), suggesting a hematopoietic cell therapy approach for liver disease. Although these studies provide proof of principle for novel bone marrow cell therapy for metabolic diseases of the liver, different diseases will require specific studies as the lack of successful clinical therapy for the glycogen storage diseases and GM1-gangliosidosis reminds us. Although these ostensibly promising murine results may prove to be unique to this animal model, future studies are assuredly warranted.

Muscular Dystrophy Bone marrow cells can give rise to skeletal muscle, although the mechanism is controversial: hematopoietic (96) or mesenchymal (97) stem cell differentiation to muscle or incorporation of donor cells into muscle through myeloid intermediates (50). Duchenne muscular dystrophy is a progressive muscle degenerative disorder caused by mutations in the dystrophin gene. Bone marrow stem cell transplantation in an mdx mouse, which is a murine model of Duchenne muscular dystrophy because it lacks a functional dystrophin gene, lead to incorporation of donorderived nuclei into the muscle and partial restoration of dystrophin expression (98). This validity of this model was challenged due to the rate of spontaneous reversion of muscle fibers to synthesize dystrophin (99). BMT was studied in a second model of Duchenne muscular dystrophy, which showed donor-derived cells, constituted less than 1% of all muscle fibers and did not ameliorate the muscle dystrophy phenotype (99). The markedly discrepant outcomes of these two studies underscore the challenges of using animal models of human disease to predict responses to therapy in clinical trials. Two children with Duchenne muscular dystrophy have undergone BMT. One child received a haploidentical BMT at 12 months of age for X-linked severe combined immunodeficiency syndrome and then was diagnosed with Duchenne muscular dystrophy at 12 years of age (100). This age is quite old to present with this disorder, suggesting a mild phenotype. Analysis of a muscle biopsy revealed donor-derived nuclei within 0.5–0.9% of the muscle fibers, demonstrating the capacity of allogeneic bone marrow cells to incorporate into skeletal muscle in humans. However, the majority of myofibers produced a truncated, partially functional, isoform of dystrophin, and the investigators hypothesized that production of normal dystrophin by the donor derived nuclei, which would indicate successful therapy, did not account for the mild phenotype; rather, the phenotype was due to production of the partially functional dystrophin isoform. Another child with Duchenne muscular dystrophy underwent BMT as therapy for his disorder on an investigational protocol (M.K. Brenner, personal communication). This patient received a reduced intensity conditioning regimen and did not attain durable hematopoietic engraftment. Although this child did not show measurable clinical improvement, the lack of hematopoietic engraftment renders interpretation of this single patient outcome inconclusive. Current data, although quite limited, suggests that conventional BMT, a conditioning regimen followed by the intravenous infusion of a donor stem cell graft, is not viable as therapy for Duchenne muscular dystrophy. On the other hand, proof of incorporation of donor-derived nuclei into myofibers and persistence for at least 12 years (100), suggests that marrow cells have the potential to contribute to skeletal muscle and research to overcome the barriers to high level incorporation of donor cells may lead to successful marrow cell therapy for this lethal disorder.

Future Directions

315

PARTING THOUGHTS The wide recognition that bone marrow cells, hematopoietic stem/progenitor cells, MSCs, undescribed bona fide MSCs, as well as other tissue stem/progenitor cells resident in marrow, can contribute to nonhematopoietic tissues suggests that the range of disorders that may be treated with BMT is far greater than what is currently practiced. Moreover, the development of gene therapy will extend the potential of cure to virtually every patient. The coming years will undoubtedly witness a tremendous growth in BMT research and practice. The application of BMT as marrow cell therapy will evolve new indications for BMT therapy with extraordinary speed. However, the great promise of bone marrow cell therapy and gene therapy will only be realized through carefully executed clinical trials and patient-based translational research. It has become trite for senior academicians to tell young investigators and practitioners that, “this is a very exciting time to be in our field.” Here and now, in pediatric BMT, this sentiment could not be more true.

REFERENCES 1. Lemischka IR, Jordan CT. The return of clonal marking sheds new light on human hematopoietic stem cells. Nat Immunol 2001; 2:11–12. 2. Terada N, Hamazaki T, Oka M, et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002; 416:542–545. 3. Ying QL, Nichols J, Evans EP, Smith AG. Changing potency by spontaneous fusion. Nature 2002; 416:545–548. 4. Olsen I, Dean MF, Harris G, Muir H. Direct transfer of a lysosomal enzyme from lymphoid cells to deficient fibroblasts. Nature 1981; 291:244–247. 5. Olsen I, Muir H, Smith R, Fensom A, Watt DJ. Direct enzyme transfer from lymphocytes is specific. Nature 1983; 306:75–77. 6. Abraham D, Muir H, Olsen I, Winchester B. Direct enzyme transfer from lymphocytes corrects a lysosomal storage disease. Biochem Biophys Res Commun 1985; 129:417–425. 7. Walkley SU, Thrall MA, Dobrenis K, et al. Bone marrow transplantation corrects the enzyme defect in neurons of the central nervous system in a lysosomal storage disease. Proc Natl Acad Sci USA 1994; 91:2970–2974. 8. Manilla P, Rebello T, Afable C, et al. Regulatory considerations for novel gene therapy products: a review of the process leading to the first clinical lentiviral vector. Hum Gene Ther 2005; 16:17–25. 9. Hacein-Bey-Abina S, Le Deist F, Carlier F, et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 2002; 346:1185–1193. 10. Brenner MK, Rill DR, Holladay MS, et al. Gene marking to determine whether autologous marrow infusion restores long-term hematopoiesis in cancer-patients. Lancet 1993; 342:1134–1137. 11. Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003; 302:415–419. 12. Donahue RE, Kessler SW, Bodine D, et al. Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer. J Exp Med 1992; 176:1125–1135. 13. Prockop DJ. Marrow stromal cells as stem cells for non-hematopoietic tissues. Science 1997; 276:71–74. 14. Friedenstein AJ, Petrakova KV, Kurolesova AI, Frolova GP. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 1968; 6:230–247. 15. Friedenstein AJ, Gorskaja UF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hemat 1976; 4:267–274. 16. Friedenstein AJ, Deriglasova UF, Kulagina NN, et al. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol 1974; 2:83–92. 17. Friedenstein AJ, Chailakhyan RK, Latsinik NV, Panasyuk AF, Keiliss-Borok IV. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and re-transplantation in vivo. Transplantation 1974; 17:331–340. 18. Owen M. Marrow stromal stem cells. J Cell Sci Suppl 1988; 10:63–76. 19. Owen M, Friedenstein AJ. Stromal stem cells: marrow-derived osteogenic precursors. Ciba Found Symp 1988; 136:42–60.

316

Horwitz

20. Owen M. Lineage of osteogenic cells and their relationship to the stromal system. In: Peck W, ed. Bone and Mineral Research. Amsterdam, New York: Elsevier, 1985:1–25. 21. Caplan AI. Mesenchymal stem cells. J Orthop Res 1991; 9:641–650. 22. Pereira RF, Halford KW, O’Hara MD, et al. Cultured adherent cells from marrow can serve as longlasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc Natl Acad Sci USA 1995; 92:4857–4861. 23. Pereira RF, O’Hara MD, Laptev A, et al. Marrow stromal cells as a source of progenitor cells for non-hematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta. Proc Natl Acad Sci USA 1998; 95:1142–1147. 24. Keating A, Berkahn L, Filshie RA. Phase I study of the transplantation of genetically marked autologous bone marrow stromal cells. Hum Gene Ther 1998; 9:591–600. 25. Horwitz EM, Prockop DJ, Fitzpatrick LA, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999; 5:309–313. 26. Horwitz EM, Gordon PL, Koo WKK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci USA 2002; 99:8932–8937. 27. Horwitz EM, Keating A. Non-hematopoietic mesenchymal stem cells: what are they? Cytotherapy 2000; 2:387–388. 28. Horwitz EM, Le Blanc K, Dominici M, et al. Clarification of the nomenclature for MSC: the international society for cellular therapy position statement. Cytotherapy 2005; 7:393–395. 29. Lee RH, Kim B, Choi I, et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem 2004; 14:311–324. 30. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell- based therapies. Tissue Eng 2001; 7:211–228. 31. Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 2000; 109:235–242. 32. Wang JF, Wang LJ, Wu YF, et al. Mesenchymal stem/progenitor cells in human umbilical cord blood as support for ex vivo expansion of CD34(C) hematopoietic stem cells and for chondrogenic differentiation. Haematologica 2004; 89:837–844. 33. Zhang Y, Li C, Jiang X, et al. Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34C cells. Exp Hematol 2004; 32:657–664. 34. Igura K, Zhang X, Takahashi K, Mitsuru A, Yamaguchi S, Takashi TA. Isolation and characterization of mesenchymal progenitor cells from chorionic villi of human placenta. Cytotherapy 2004; 6:543–553. 35. Fernandez M, Simon V, Herrera G, et al. Detection of stromal cells in peripheral blood progenitor cell collections from breast cancer patients. Bone Marrow Transplant 1997; 20:265–271. 36. Haynesworth SE, Baber MA, Caplan AI. Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies. Bone 1992; 13:69–80. 37. Barry FP, Boynton RE, Haynesworth S, Murphy JM, Zaia J. The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105). Biochem Biophys Res Commun 1999; 265:134–139. 38. Barry F, Boynton R, Murphy M, Zaia J. The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from Human Mesenchymal Stem Cells. Biochem Biophys Res Commun 2001; 289:519–524. 39. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284:143–147. 40. Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringden O. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol 2003; 31:890–896. 41. Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 2003; 57:11–20. 42. Rasmusson I, Ringden O, Sundberg B, Le BK. Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells. Transplantation 2003; 76:1208–1213. 43. Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 2002; 99:3838–3843.

Future Directions

317

44. Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002; 30:42–48. 45. Krampera M, Glennie S, Dyson J, et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood 2003; 101:3722–3729. 46. Maitra B, Szekely E, Gjini K, et al. Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation. Bone Marrow Transplant 2004; 33:597–604. 47. Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004; 363:1439–1441. 48. Le BK. Immunomodulatory effects of fetal and adult mesenchymal stem cells. Cytotherapy 2003; 5:485–489. 49. Le BK, Ringden O. Immunobiology of human mesenchymal stem cells and future use in hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2005; 11:321–334. 50. Camargo FD, Green R, Capetenaki Y, Jackson KA, Goodell MA. Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nat Med 2003. 51. Willenbring H, Bailey AS, Foster M, et al. Myelomonocytic cells are sufficient for therapeutic cell fusion in liver. Nat Med 2004; 10:744–748. 52. Malech HL, Maples PB, Whiting-Theobald N, et al. Prolonged production of NADPH oxidasecorrected granulocytes after gene therapy of chronic granulomatous disease. Proc Natl Acad Sci USA 1997; 94:12133–12138. 53. Compston JE. Bone marrow and bone: a functional unit. J Endocrinol 2002; 173:387–394. 54. Sillence DO. Disorders of bone density, volume, and mineralization. In: Rimoin DL, Connor JM, Pyeritz RE, eds. In: Emery and Rimoin’s Principles and Practice of Medical Genetics, 3rd ed, Vol. 2. New York: Churchill Livingstone, 1997. 55. Byers PH. Disorders of collagen biosynthesis and structure. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill, 1995:4029–4077. 56. Byers PH, Wallis GA, Willing MC. Osteogenesis imperfecta: translation of mutation to phenotype. J Med Genet 1991; 28:433–442. 57. Wang Q, Orrison BM, Marini JC. Two additional cases of osteogenesis imperfecta with substitutions for glycine in the a 2(I) collagen chain. A regional model relating mutation location with phenotype. J Biol Chem 1993; 268:25162–25167. 58. Constantinou CO, Pack M, Young S, Prockop DJ. Phenotypic heterogeneity in osteogenesis imperfecta: the mildly affected mother of a proband with a lethal variant has the same mutation substituting cysteine for a1-glycine 904 in a type I procollagen gene (COL1A1). Am J Hum Genet 1990; 47:670–679. 59. Horwitz EM, Prockop DJ, Gordon PL, et al. Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood 2001; 97:1227–1231. 60. van der Plas A, Aarden EM, Feijen JH, et al. Characteristics and properties of osteocytes in culture. J Bone Miner Res 1994; 9:1697–1704. 61. Dominici M, Pritchard C, Garlits JE, Hofmann T, Persons DA, Horwitz EM. Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplantation. Proc Natl Acad Sci USA 2004; 101:11761–11766. 62. Whyte MP, Kurtzberg J, McAlister WH, et al. Marrow cell transplantation for infantile hypophosphatasia. J Bone Miner Res 2003; 18:624–636. 63. Cahill RA, Jones OY, Klemperer M, et al. Replacement of recipient stromal/mesenchymal cells after bone marrow transplantation using bone fragments and cultured osteoblast-like cells. Biol Blood Marrow Transplant 2004; 10:709–717. 64. Orkin SH, Motulsky AG. Report and recommendations of the panel to assess the NIH investment in research on gene therapy. NIH Report, 1995. 65. Cavazzana-Calvo M, Hacein-Bey S, de Saint BG, et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000; 288:669–672. 66. Brenner MK, Rill DR, Moen RC, et al. Gene-marking to trace origin of relapse after autologous bone-marrow transplantation. Lancet 1993; 341:85–86. 67. Hacein-Bey S, Basile GDS, Lemerle J, Fischer A, Cavazzana-Calvo M. gamma c Gene transfer in the presence of stem cell factor, FLT-3L, Interleukin-7 (IL-7), IL-1 alpha, and IL-15 cytokines restores T-cell differentiation from gamma c(-) X-linked severe combined immunodeficiency hematopoietic progenitor cells in murine fetal thymic organ cultures. Blood 1998; 92:4090–4097.

318

Horwitz

68. Cavazzana-Calvo M, Hacein-Bey S, Saint Basile G, et al. Role of interleukin-2 (IL-2), IL-7, and IL-15 in natural killer cell differentiation from cord blood hematopoietic progenitor cells and from gamma c transduced severe combined immunodeficiency X1 bone marrow cells. Blood 1996; 88:3901–3909. 69. Cavazzana-Calvo M, Lagresle C, Hacein-Bey-Abina S, Fischer A. Gene therapy for severe combined immunodeficiency. Annu Rev Med 2005; 56:585–602. 70. Thrasher AJ, Hacein-Bey-Abina S, Gaspar HB, et al. Failure of SCID-X1 gene therapy in older patients. Blood 2005; 105:4255–4257. 71. Gaspar HB, Parsley KL, Howe S, et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gamma-retroviral vector. Lancet 2004; 364:2181–2187. 72. Ginn SL, Curtin JA, Kramer B, et al. Treatment of an infant with X-linked severe combined immunodeficiency (SCID-X1) by gene therapy in Australia. Med J Aust 2005; 182:458–463. 73. Aiuti A, Slavin S, Aker M, et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 2002; 296:2410–2413. 74. Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003; 302:415–419. 75. Baum C. A balanced decision? Regulatory reaction to the “third case” Mol Ther 2005; 11:819–820. 76. McCormack MP, Rabbitts TH. Activation of the T-cell oncogene LMO2 after gene therapy for X-Linked severe combined immunodeficiency. N Engl J Med 2004; 350:913–922. 77. Baum C, von Kalle C, Staal FJT, et al. Chance or necessity? Insertional mutagenesis in gene therapy and its consequences. Mol Ther 2004; 9:5–13. 78. Krivit W, Peters C, Shapiro EG. Bone marrow transplantation as effective treatment of central nervous system disease in globoid cell leukodystrophy, metachromatic leukodystrophy, adrenoleukodystrophy, mannosidosis, fucosidosis, aspartylglucosaminuria, hurler, maroteauxlamy, and sly syndromes, and gaucher disease type III. Curr Opin Neurol 1999; 12:167–176. 79. Koc ON, Peters C, Aubourg P, et al. Bone marrow-derived mesenchymal stem cells remain hostderived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases. Exp Hematol 1999; 27:1675–1681. 80. Koc ON, Day J, Nieder M, Gerson SL, Lazarus HM, Krivit W. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH). Bone Marrow Transplant 2002; 30:215–222. 81. Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after co-infusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 2000; 18:307–316. 82. Lazarus HM, Koc ON, Devine SM, et al. Co-transplantation of HLA-identical sibling cultureexpanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant 2005; 11:389–398. 83. Will A, Cooper A, Hatton C, et al. Bone marrow transplantation in the treatment of alphamannosidosis. Arch Dis Child 1987; 62:1044–1049. 84. Grewal SS, Shapiro EG, Krivit W, et al. Effective treatment of alpha-mannosidosis by allogeneic hematopoietic stem cell transplantation. J Pediatr 2004; 144:569–573. 85. Wall DA, Grange DK, Goulding P, Daines M, Luisiri A, Kotagal S. Bone marrow transplantation for the treatment of alpha-mannosidosis. J Pediatr 1998; 133:282–285. 86. Albert MH, Schuster F, Peters C, et al. T-cell-depleted peripheral blood stem cell transplantation for alpha-mannosidosis. Bone Marrow Transplant 2003; 32:443–446. 87. Watson JG, Gardner-Medwin D, Goldfinch ME, Pearson AD. Bone marrow transplantation for glycogen storage disease type II (Pompe’s disease). N Engl J Med 1986; 314:385. 88. Peters C, Steward CG. Hematopoietic cell transplantation for inherited metabolic diseases: an overview of outcomes and practice guidelines. Bone Marrow Transplant 2003; 31:229–239. 89. Braun SE, Aronovich EL, Anderson RA, Crotty PL, McIvor RS, Whitley CB. Metabolic correction and cross-correction of mucopolysaccharidosis type II (Hunter syndrome) by retroviral-mediated gene transfer and expression of human iduronate-2-sulfatase. Proc Natl Acad Sci USA 1993; 90:11830–11834. 90. Sano R, Tessitore A, Ingrassia A, d’Azzo A. Chemokine-induced recruitment of genetically modified bone marrow cells into the CNS of GM1-gangliosidosis mice corrects neuronal pathology. Blood 2005; 106:2259–2268.

Future Directions

319

91. Sena-Esteves M, Camp SM, Alroy J, Breakefield XO, Kaye EM. Correction of acid betagalactosidase deficiency in GM1 gangliosidosis human fibroblasts by retrovirus vector-mediated gene transfer: higher efficiency of release and cross-correction by the murine enzyme. Hum Gene Ther 2000; 11:715–727. 92. Shield JP, Stone J, Steward CG. Bone marrow transplantation correcting beta-galactosidase activity does not influence neurological outcome in juvenile GM1-gangliosidosis. J Inherit Metab Dis 2005; 28:797–798. 93. Lagasse E, Connors H, Al Dhalimy M, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000; 6:1229–1234. 94. Wang X, Willenbring H, Akkari Y, et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 2003; 422:897–901. 95. Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003; 422:901–904. 96. Corbel SY, Lee A, Yi L, et al. Contribution of hematopoietic stem cells to skeletal muscle. Nat Med 2003. 97. Ferrari G, Cusella-De Angelis G, Coletta M, et al. Muscle regeneration by bone marrow derived myogenic progenitors. Science 1998; 279:1528–1530. 98. Gussoni E, Soneoka Y, Strickland CD, et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 1999; 401:390–394. 99. Ferrari G, Stornaiuolo A, Mavilio F. Failure to correct murine muscular dystrophy. Nature 2001; 411:1014–1015. 100. Gussoni E, Bennett RR, Muskiewicz KR, et al. Long-term persistence of donor nuclei in a Duchenne muscular dystrophy patient receiving bone marrow transplantation. J Clin Invest 2002; 110:807–814.

SECTION II: NONMALIGNANT DISEASES

17 Primary Immunodeficiencies Brett J. Loechelt Clinical Immunology, Division of Stem Cell Transplantation and Immunology, Children’s National Medical Center, The George Washington University School of Medicine, Washington, D.C., U.S.A.

Naynesh R. Kamani Division of Stem Cell Transplantation and Immunology, Children’s National Medical Center and The George Washington University School of Medicine, Washington, D.C., U.S.A.

INTRODUCTION Bone marrow transplantation from a histocompatible matched sibling donor was established as a curative therapy for lethal congenital immunodeficiencies with the successful transplant of a patient with severe combined immunodeficiency (SCID) (1) and another child with WiskottAldrich syndrome (WAS) (2) in 1968. Over the ensuing 35 years, our knowledge of hematopoietic stem cell transplantation (SCT) has increased greatly. As our knowledge of transplantation and immunology has expanded, patients with many other primary immunodeficiencies (PIDs) (Table 1) have undergone SCT (4–7). Furthermore, the use of alternative donors and donor stem cell sources, when matched sibling donors are not available, has greatly increased the number of individuals who benefit from this procedure (8–10).

SPECIAL CONSIDERATIONS Goal of Stem-Cell Transplantation for Primary Immune Deficiency Diseases In contrast to patients with malignant disease, the overarching goal of SCT for patients with PID is recovery of normal immunologic function that will protect them from infections and other complications of chronic immunodeficiency, including autoimmune disease and malignancy. As a result, 100% donor cell engraftment may not always be required to “cure” the underlying immunodeficiency and ameliorate problems associated with the disease (11–13), though this remains a general goal of therapy. Haddad and colleagues investigated immune reconstitution of 22 patients who underwent SCT for X-linked SCID with B cells (where the patient’s B cells are capable of normal function in vitro in the presence of normal T cells) with specific investigation of B-cell function (14). Of 22 patients who survived greater than 2 years post-SCT, in 4 of 5 patients receiving HLA-matched related donor stem cells and in 14 of 17 receiving HLA-haploidentical 321

322

Loechelt and Kamani

Table 1 Estimates of Immunodeficiency Types Undergoing Stem-Cell Transplantation in North America and Europe Immunodeficiency type Lymphoid PID Severe combined immunodeficiency (including PNP deficiency, Omenn syndrome, combined immune deficiency, ZAP-70 deficiency) Wiskott-Aldrich syndrome MHC class II deficiency X-linked hyper-IgM syndrome X-linked lymphoproliferative syndrome Other lymphoid (DiGeorge, cartilage-hair hypoplasia, ataxia telangiectasia, common variable immunodeficiency, IL-2 deficiency) Myeloid PID Chediak-Higashi syndrome Chronic granulomatous disease Leukocyte adhesion deficiency type I Other myeloid (Griscelli syndrome, neutrophil disorders)

Percentage by PID type (%)

Percentage of total (%)

75.0

68.9

17.2 3.3 0.8 0.8 2.9

15.8 3.1 0.7 0.7 2.7

27.8 19.0 40.5 12.7

2.3 1.5 3.3 1.0

Abbreviations: MHC, major histocompatibility complex; PID, primary immunodeficiencies. Source: Adapted from Ref. 51.

related donor stem cells, B cells were found to be of host origin. Engraftment of donor B cells was found to be associated with normal B cell function. In contrast, 10 of 18 patients with B cells of host origin continued to require immunoglobulin supplementation, despite normal T-cell function and amelioration of the most serious consequences of the disease. By contrast, in other diseases, such as chronic granulomatous disease (CGD), stable donor chimerism of greater than 10–15% appears to be associated with normal host defenses (12,13).

Pretransplant Infections and Evaluation Recurrent infections are frequently the presenting problems in children with immune deficiencies. These infections should be identified and treated. Furthermore, many children with immune deficiency may be colonized with infectious agents that have the capacity to complicate the transplant procedure. Common occult infections include cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, rotavirus, parainfluenza virus, and respiratory syncytial virus (RSV) and may manifest as asymptomatic colonization or with minimal respiratory or gastrointestinal symptoms. Furthermore, reactivation of CMV, EBV, adenovirus, and RSV in the peri-transplant period can be especially problematic in children with PID and should be monitored for very carefully. Most children with PID should be evaluated aggressively for acute and occult infections prior to and during the transplant procedure. The presence of certain infectious agents, especially those for which effective therapy is lacking, may impact on the preparative regimen used. Lastly, standard antimicrobial therapy recommendations are based on responses in immunologically competent individuals. In the presence of a poorly functioning or absent immune system, regimens may need to be modified, and cure of the underlying infection may not be achievable. A detailed evaluation for occult infection may require screening cultures and diagnostic PCRs of various body fluids including blood, cerebrospinal fluid, naso- and oropharyngeal secretions, bronchoalveolar lavage fluid, urine, and feces. Where warranted, bronchoscopy

Primary Immunodeficiencies

323

and/or endoscopy may be required to obtain optimal specimens to identify potential dangerous pathogens. In addition, endoscopic biopsy specimens may help differentiate between infection and maternally derived graft-versus-host disease (GVHD) in the SCID infant with diarrhea. Ophthalmologic examination may be required in the setting of CMV or Candida infection. A dermatologic evaluation and a skin biopsy may be required to evaluate skin rashes that are common in children with immune deficiency. In addition to infection, children with primary immune deficiency, especially those with WAS, are at higher risk for malignancies and myelodysplastic syndrome. In these cases, evaluation of bone marrow aspirate and biopsy specimens, along with CT scans of the head, chest, and abdomen, may need to be performed. Other diseases have increased risk of autoimmune endocrinopathies and thus thyroid, adrenal, and islet cell function may need to be evaluated prior to SCT.

Maternal Engraftment Fetuses with SCID are unable to reject maternal lymphocytes that normally enter the fetal circulation during intrauterine life. This can result in maternal lymphocyte engraftment that can lead to significant pretransplant and peritransplant problems. These maternal cells are usually unable to respond normally to mitogens. However, they can cause clinically significant GVHD. There is also the additional risk of a graft-versus-graft reaction in the setting of HLA-matched related and unrelated donor transplant that may contribute to an increased risk of donor graft rejection (15). Evaluation of maternal engraftment can at times be challenging. In male infants, FISH analysis can be utilized to evaluate for the presence of XX- versus XY-bearing cells. However, this is not feasible in female patients. Detection of maternal engraftment requires analysis through one of the various “DNA fingerprinting” techniques. This requires obtaining DNA from the infant that is not potentially contaminated with maternal cells. Sources of DNA include hair follicles, fingernail clippings, and skin fibroblasts. This allows for the comparison of maternal DNA compared to that obtained from the infant’s peripheral blood. Furthermore, comparison of the maternal and paternal HLA types to that of the infant with SCID may also provide information regarding the presence of maternally derived cells.

Donor Selection and Stem Cell Source An HLA-identical matched sibling donor (MSD) is the donor of choice for PID disorders. This is due to the decreased risk of GVHD (which has no theoretical benefit to patients with PID) and graft refection associated with MSD transplants. When the patient is fortunate enough to have more than one HLA-identical sibling, a sex-matched donor may be preferred. Because advanced donor age is an important risk factor for the development of GVHD after SCT, younger donors are often preferred (16,17). However, sibling donors for most children with PID are minors where issues of marrow volume arise due to the limited blood volumes in small children. In these cases, an older minor sibling donor may be preferable from a technical standpoint. Less than 25% of children have an acceptable MSD, as children with severe PID may be the first or second child or there are other affected children in the family that reduces the pool of acceptable sibling donors. The introduction of T-lymphocyte depletion of bone marrow in 1981 to prevent GVHD (18) and development of unrelated donor registries has greatly expanded the donor pool to include “alternative” donors as sources of hematopoietic stem cells (HSC). As a result, significant numbers of children have undergone SCT using alternative donors, including haploidentical parental donors (particularly common in SCID but with very poor results in other diseases, such as WAS), and HLA-identical or 1 antigen-mismatched unrelated donors. Furthermore, the development of high resolution HLA typing and donor-recipient matching for all relevant HLA antigens at the allele level has the potential to reduce the impact of GVHD and

324

Loechelt and Kamani

rejection, which are significantly more common in unrelated donor transplants compared to matched related donors. Historically, haploidentical donors have been utilized more frequently than unrelated donors in patients with SCID, as parental donors were readily available and unrelated donor registries were either small or nonexistent. However, in the past decade increasing numbers of unrelated donor transplants have been performed with excellent results. In a recent analysis by Antoine and colleagues, reviewing the European experience, they found a significant difference in 3-year survival for patients with SCID who received an HLA-identical versus and HLA-mismatched related donor transplant (77% vs. 54%; pZ0.02) (19). Within the HLA-identical group, the 3-year survival after transplant from genotypically or phenotypically identical related or unrelated donors did not differ significantly. For non-SCID patients, the difference between HLA-identical and HLA-mismatched donors was more marked. Furthermore, when comparing transplants since 1996 only, 3-year survival in HLAmismatched related donor transplants remains unchanged at !40% compared to improving trends in the HLA-identical unrelated donor transplants at O60%. In addition to donor types, sources of HSC also must be considered. The traditional stem cell source for PID transplants has been bone marrow. Other potential sources include peripheral blood (PBSC) and umbilical cord blood (UCB) (10,20). Successful transplants have been achieved with all sources of stem cells. The advantages of PBSC include the ability to obtain larger number of CD34C stem cells and, in adults, obviating the need for general anesthesia and a surgical suite as is required for collection of bone marrow. In the HLAidentical related donor setting for PID, donors are usually children in whom placement of a pheresis catheter under deep sedation or general anesthesia is often required. Concern has also been raised regarding the use of growth factors in normal children to mobilize PBSC for a successful pheresis. Additionally, the increased incidence of chronic GVHD seen following PBSC transplantation is of concern in patients with PID in whom GVHD provides no theoretical advantage (21,22). As a result, the majority of PBSC transplants for patients with PID have been from HLA-mismatched related and HLA-identical unrelated donors, where megadoses of CD34C cells can be administered to the recipient to overcome the increased risk of rejection seen in the context of a T-cell depleted transplant (23,24).

Conditioning Regimens Despite the first successful allogeneic bone marrow transplant for SCID occurring more than 30 years ago, it is still not clear what the optimal pretransplant approach in these children is, especially if an HLA-matched sibling is not available. Although the ideal goal of a SCT is a permanent cure of the underlying immune deficiency with normalization of immune function, a number of investigators have settled for donor T-cell engraftment as an acceptable outcome if the patient can be spared preparative chemotherapy or chemoradiotherapy. Although donor T-cell engraftment usually results in freedom from opportunistic infections, the patients continue to require lifelong supplementation with intravenous immune globulin. In their analysis of non-conditioned SCID patients undergoing SCT, Buckley et al. report that 20 of 89 patients required booster transplants and almost two-thirds of the survivors continued to require IVIG supplementation (25). The rapid decline in T-cell receptor episome numbers (TRECs) in the circulation of transplanted SCID infants after the first 2 years may reflect a defect in longterm engraftment of donor stem cells (26). Whether these findings will translate into significant clinical consequences remains to be determined. There is a theoretical benefit to have engraftment of T cells, B cells, natural killer (NK) cells, and monocytes of donor origin because the quality of immune response can be maximized, although this has not been definitively proven in man. It is unclear whether the significant number of autoimmune problems seen in SCID patients following unconditioned transplantation is related to the presence of differential chimerism of T cells and other immune cells (27).

Primary Immunodeficiencies

325

There continues to be tremendous center-to-center variability in the approach to attaining complete immunologic engraftment in SCID patients. Progress has been hampered by the low incidence of these disorders, their diversity, and the many options available for donor HSC. There is general consensus that T-cell and usually B-cell function can be reliably expected in SCID patients with low NK activity when a matched sibling marrow is used as the HSCT source. However once a less perfectly matched donor or alternative stem cell source is used or if there is residual host immune function, the reliability of T-cell engraftment is less certain and B cell engraftment and recovery of NK cell function is the exception rather than the rule (5). This has led many centers to use standard chemotherapeutic myeloablation, with such agents as busulfan and thiotepa, to ensure complete engraftment of donor immunologic cells, accepting the accompanying risk of organ damage and infection in this group of children who are frequently infected coming into transplant. With this approach, patients not only engraft with T cells but also with B cells, NK cells, and monocytes of donor origin. Within the cohorts of SCID patients who have undergone nonconditioned SCT, there are children who have had documented full immune recovery. The likelihood of successful engraftment of a haplocompatible T-cell depleted parental graft is significantly reduced in children with TKBKNKC SCID compared to those with TKBCNKKSCID indicating the importance of NK cells in transplantation. Nonmyeloablative or reduced-intensity conditioning regimens have the potential for achieving engraftment without the inherent risk of morbidity, mortality, and late sequelae associated with standard myeloablative regimens. Gaspar and colleagues report their initial results using a nonmyeloablative approach for children with a variety of immune deficiencies (28). Patients with a variety of immune deficiencies were treated, primarily with fludarabinebased conditioning regimens. Immune reconstitution was equivalent to historic controls, and there was no evidence of organ toxicity. A clinical trial is underway at member institutions of the pediatric blood and marrow transplant consortium designed to assess whether nonmyeloablative regimens can result in full donor immune cell chimerism (29). The trial design is based on the premise that distinct immunophenotypes and or genotypes of SCIDs, and transplants utilizing different stem cell sources may require different pre-transplant preparative regimens. It is estimated that approximately 25% of all children with SCID present with the TKBKNKC immunophenotype (30), and of these, about a third have a mutation in RAG 1 or 2 genes (31). Mutations in a recently identified gene, Artemis, that is an essential component of V(D)J recombination and DNA repair also result in TKBKNKC SCID (32,33). Due to the role of Artemis in DNA repair, children with SCID due to Artemis gene defects are likely to be sensitive to radiation therapy and alkylating agents used during transplant conditioning. The clinical experience to date with transplants in these children provides no firm data to support these concerns. Myeloablative chemotherapy conditioning regimens have generally been utilized for patients with non-SCID primary immune deficiency diseases undergoing SCT. Total body or lymphoid irradiation containing regimens are generally to be avoided in children with primary immune deficiency diseases patients unless the patient has failed to engraft with chemotherapyonly containing regimens. The commonest preparative regimen used in WAS, LAD, and other non-SCID diseases has been myeloablative doses of busulfan and cyclophosphamide. More recently, Amrolia et al. have reported successful donor cell engraftment in children with CD40 ligand deficiency, SCID, and other combined immune deficiency diseases with a preparative regimen consisting of a submyeloablative dose of melphalan (140 mg/m2) and fludarabine 30 mg/m2/day !5 days and rabbit antilymphocyte globulin (11).

CLINICAL RESULTS The lymphoid disorders are the most common immunodeficiency for which patients have undergone SCT. In an informal worldwide survey of 978 blood or marrow transplants for PID

326

Loechelt and Kamani

diseases (Table 1) performed between 1968 and 1997, 92% were for lymphoid disorders compared to 8% for myeloid disorders (25). In this same survey, 68.9% of all PID transplants were for all forms of SCID disorders.

Lymphoid Disorders Severe Combined Immunodeficiency Syndrome Of all the primary immune deficiency syndromes, SCID syndrome is the most common indication for SCT. SCID is a heterogeneous group of disorders that includes IL-2 common gamma chain receptor gene defects (X-linked SCID, the most common form of SCID), JAK3 defects, adenosine deaminase deficiency (ADA), interleukin-7 receptor alpha gene defects and RAG-1 and/or -2 gene defects as well as others for whom the genetic defect remains unknown (Table 2). These disorders are characterized by variable degrees of lymphopenia, hypogammaglobulinemia, and aberrant T-cell function. Due to the severity of the immune defect associated with these diseases, SCT is currently the treatment of choice. In many of these children, SCT has been performed without a preparative regimen due to the severity of the immune deficiency and the presumed inability of the recipient to reject donor stem cells. Matched sibling donors are the donors of choice for children with SCID. However, only approximately 25% of patients undergoing SCT for SCID have a suitable sibling donor. Alternative sources of HSC for children with SCID have typically come from haploidentical donors. Buckley and colleagues reviewed the Duke University experience with SCT in SCID (25). Of 89 consecutive infants with SCID, 12 received HLA-identical marrow from a related donor, whereas 77 received T-cell-depleted, HLA-haploidentical parental marrow. GVHD developed in 28 of 77 recipients of T-cell-depleted haploidentical marrow compared to 6 of 12 recipients of HLA-identical marrow. All 12 recipients of HLA-identical related donor stem cells survived with adequate T-cell engraftment. Of the 77 infants who underwent SCT from haploidentical donors, 60 (78%) survived with 56 having adequate T-cell engraftment. B-cell engraftment in this group was variable with 5 of 12 recipients of HLA-identical related and 21 of 60 recipients of haploidentical stem cells having some donor B-cell engraftment (range 2–100%). The use of unrelated donors as a source of HSC has increased significantly in the past decade. In a report of the initial University of Minnesota experience with 8 patients with SCID (and its variants) who underwent unrelated SCT (9), Filipovich and colleagues reported that

Table 2

Phenotypes and Genotypes of Severe Combined Immune Deficiency Syndromes

SCID form X-linked SCID Auto recessive SCID

MHC class II def. Omenn syndrome CD3 deficiencies

Genetic defect/pathogenesis

Immunophenotypic features

IL-2 receptor common g chain JAK3 RAG-1/RAG-2 defect Artemis Adenosine deaminase deficiency Purine phosphorylase deficiency IL-7Ra-chain ZAP-70 defect Defective p56lck expression Defect in MHCII gene transcription ?partial RAG-1/RAG-2 defect CD3g or CD33 gene defect

TK, BC, NKK, lymphopenia TK, BC, NKK, lymphopenia TK, BK, NKC, lymphopenia TK, BK, NKC, lymphopenia TK, BK, NKC, lymphopenia TK, BC, NKC, lymphopenia TK, BC, NKC, lymphopenia CD8C deficiency, nl TLC CD4C deficiency, Y TLC CD4C deficiency, nl TLC Nl to [T cells,Y B, Y Igs YCD3C, variable CD4, CD8

Abbreviations: MHC, major histocompatibility complex; SCID, severe combined immune deficiency syndromes; TLC, total lymphocyte count.

Primary Immunodeficiencies

327

6 of 8 patients were long-term survivors. Acute GVHD occurred in one patient (which resolved with treatment), whereas chronic GVHD occurred in none. All survivors had stable donor engraftment, and none required supplemental IVIG following recovery from SCT for hypogammaglobulinemia. As previously noted, in the European experience to 1999 (19) the 3-year survival with evidence of sustained engraftment and improvement of the immunodeficiency disorder was significantly better for HLA-identical than for HLAmismatched transplantation (77% vs. 54%; pZ0.02). There was no significant difference between related and unrelated HLA-matched donors in this experience. It has been suggested that children with SCID transplanted in the neonatal period (i.e., in the first 28 days of life) have superior immune reconstitution (34). In an analysis of consecutive patients undergoing SCT in the first 28 days of life, these children showed higher phytohemagglutinin-stimulated T-cell responses, higher numbers of CD3C and CD45RAC T cells and higher values of T-cell antigen receptor excision circles (TRECs) at three years post SCT compared to children with SCID who had undergone SCT beyond the neonatal period.

Wiskott-Aldrich Syndrome WAS is a disorder characterized by eczema, thrombocytopenia with microthrombocytes, poor antibody responses to polysaccharide antigens, and progressive loss of T-cell function. Unlike individuals with SCID, individuals with WAS require preparative chemotherapy to achieve donor engraftment as determined by Parkman et al. in 1978 (35,36). Matched sibling donors are the donors of choice with survival ranging 80–88% at five years (25,37,38). In the early 1990s, successful transplant using matched unrelated donors was reported (9,39). This compares to 34–52% with other related donors (including haploidentical donors) and 69–71% with matched unrelated donors (25,37). In a review of all unrelated transplants reported to the International Bone Marrow Transplant Registry and/or National Marrow Donor Program, there was a distinct survival difference in individuals transplanted prior to 5 years of age with an overall survival of 84%, which was indistinguishable from matched sibling donor transplants (37). The most common causes of death in the unrelated donor setting were GVHD with/without interstitial pneumonitis and/or infection as compared to posttransplant lymphoproliferative disease in the mismatched related donor setting. Non-TBI containing conditioning regimen have been most commonly utilized for patients with WAS for both MSD and MUD transplant (37,39). Cyclosporine and methotrexate were also the most common GVHD prophylactic agents utilized. Successful SCT results in correction of manifestations of WAS including eczema, autoimmunity, and risk of lymphomas. Major Histocompatibility Complex Class II Deficiency Major histocompatibility complex (MHC) class II deficiency (bare lymphocyte syndrome) is characterized by impaired antigen presentation and combined immunodeficiency. Due to the severity of the clinical phenotype, transplant is recommended for these individuals. Successful transplants from HLA-identical related and unrelated donors as well as haploidentical parental donors have been reported (7,25). In general, survival and cure was higher in both HLAidentical related and unrelated donors, though the number of unrelated donor transplants is small, as compared to haploidentical donors. In the setting in which an HLA-identical related donor is not available, survival was optimal if SCT occurred in the first two years of life, before the acquisition of chronic viral carriage and sequelae of infections. X-linked Hyper IgM Syndrome X-linked hyper-IgM syndrome (XHIM) is caused by the failure to express CD40 ligand (CD154) on activated T cells. The absence of the interaction of CD40 with CD154 results in the absence of isotype switching in B cells (resulting in defective production of IgG, IgA, and IgE) and defective cellular immunity due to poor macrophage activation (resulting in increased

328

Loechelt and Kamani

susceptibility to opportunistic organisms). The likelihood of survival into adulthood in patients with XHIM is 25% (40). Bone marrow transplantation is the only known curative procedure for this immunodeficiency, though only a small number of patients have undergone this procedure (25). A recent retrospective analysis of the European experience by Gennery et al. analyzed the results of SCT in 38 patients with XHIM. Donor stem cell sources included 14 HLAidentical siblings, 22 unrelated donors, and 2 phenotypically matched parental donors (41). Sixty-eight percent were alive and well with a median follow-up of 3.4 years. The most important risk factor for an adverse outcome was preexisting lung damage. Their outcome data suggest that SCT during early childhood from an HLA-identical donor is associated with a better outcome.

X-linked Lymphoproliferative Syndrome X-linked lymphoproliferative syndrome (XLP) or Duncan’s syndrome is a heterogeneous immune deficiency syndrome presenting in childhood or adolescence with one or more of several clinical manifestations including fulminant infectious mononucleosis (50%), dysgammaglobulinemia (30%), or malignant lymphoma (20%). SAP (signaling lymphocytic activation molecule-associated protein) is a T- and NK-cell-specific protein containing a single SH2 domain encoded by a gene that is defective or absent in patients with SLP (42). The majority of reported transplants have been from matched sibling donors (43–45). A review of the XLP registry by Gross and colleagues found that six of seven individuals who underwent transplant were from matched sibling stem cell sources (44). Four were alive and well more than three years following SCT. In their analysis unsuccessful SCT was associated with age at SCT (O15 years), TBI-containing regimens, and a significant history of pre-SCT infections. There have also been reports of successful unrelated transplants from both marrow (46) and UCB (47) sources. Other Lymphoid Immunodeficiency Diseases Small numbers of patients with other lymphoid immunodeficiencies have undergone SCT. These reports generally consist of single case reports or small case series. Diseases for which transplant has been performed include DiGeorge Syndrome (5,48–50), Cartilage-Hair Hypoplasia (51), Common Variable Immunodeficiency (3), autoimmune lymphoproliferative disorders (52), and IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome (53). Transplant regimens for these groups of disorders have often been modeled after those used for the SCID variants or WAS. Therefore, transplants for these disorders have often consisted of myeloablative conditioning regimens with match-sibling donors. Recently, as the success rate has improved, transplants from both unrelated UCB and adult donor transplants have been performed for some of these disorders. Many of these immune deficiencies are associated with other nonimmune sequelae. Although correction of the immune disorder and associated autoimmune phenomena may be corrected with transplant, other sequelae will persist. This is particularly true in Cartilage-Hair Hypoplasia and the bone issues associated with this disorder. Myeloid Disorders Chediak-Higashi Syndrome Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disease characterized by recurrent pyogenic infections, partial oculocutaneous albinism, a progressive neuropathy, and large granules in all granule-containing cells (54). CHS is caused by mutations affecting the lysosomal transport protein LYST (55). Although the adverse manifestations of CHS are not limited to the hematopoietic system, the immunodeficiency that accompanies the “stable

Primary Immunodeficiencies

329

phase” and the lymphohistiocytic infiltration that characterizes the “accelerated phase” are the primary sources of morbidity and mortality (6). Allogeneic SCT has been shown to correct the hematopoietic cell defects and prevent progression to the accelerated phase (9,56–63). The preparative regimens employed in these cases were myeloablative and included HLA-matched related, HLA-matched unrelated, and HLA-nonidentical related donors. The largest series, reported by Haddad and colleagues, described their results of 10 children who underwent SCT. Of these, seven were from HLAmatched related donors and three from HLA-nonidentical related marrow donors. At the time of their report, six of seven HLA-matched, and one of three HLA-nonidentical marrow donor patients were alive 1.5–13 years post-SCT. The three deaths were due to a new accelerated phase in two following rejection of transplanted marrow and one from CMV pneumonia. It has also been noted that stable mixed chimerism is sufficient to ameliorate the hematopoietic manifestations of the disease (58,62).

Chronic Granulomatous Disease CGD is a genetic syndrome characterized by susceptibility to recurrent severe infections (S. aureus and Aspergillus), along with granulomatous inflammation that is associated with dysfunctional NADPH oxidase and defective microbicidal function of phagocytic cells but with normal B-cell and T-cell function (64,65). At least four genetic subtypes are now recognized with the X-linked subtype affecting the gp91phox component accounting for the majority (76%) of cases (66,67). Many individuals with CGD can survive to adulthood as prophylaxis with trimethoprim-sulfamethoxazole, itraconazole, and therapy with g-interferon has considerably reduced the incidence of life-threatening infections (68). However, despite these measures, the annual rate of death due complications of CGD in the United States is 2–5% (69). CGD is also amenable to cure with SCT, and there are multiple reports of successful allogeneic transplant (12,13,70–83). Most patients in these reports received myeloablative preparative regimens, typically busulfan (16 mg/kg) and cyclophosphamide (200 mg/kg) followed by transplant from an HLA-matched sibling donor, though successful unrelated donor transplants have also been documented. The European experience documented 27 transplants for CGD between 1985 and 2000. All received HSCT from unmanipulated marrow allografts from HLA-matched sibling donors in which 23 of 27 received a myeloablative conditioning regimen (82). Overall survival was 23 of 27 with 22 of 23 cured of CGD. It was noted that survival was especially good in patients without infection at the time of transplant with 18 of 18 surviving. There have also been reports of successful transplants in individuals receiving unrelated donor SCT (84). For many individuals with CGD, issues related to morbidity and mortality associated with myeloablative SCT have had a negative impact on perceptions regarding SCT as a definitive curative therapy. Studies are currently ongoing investigating the utility of nonmyeloablative conditioning regimens in this disease. Horwitz and colleagues reported preliminary results in 10 individuals with CGD who underwent CD34C selected, T-cell depleted peripheral blood SCT from an HLA-matched sibling after conditioning with cyclophosphamide, fludarabine, and antithymocyte globulin (12). Patients also received donor lymphocyte infusions at periodic intervals post SCT. 8 of 10 individuals had 33–100% donor neutrophils that is a level expected to provide normal host defenses. There were three deaths 8–14 months after transplant, none due to toxicity associated with the transplant procedure. Leukocyte Adhesion Deficiency Type I Leukocyte Adhesion Deficiency Type I (LAD 1) is a rare disease characterized by defective expression of the b2 integrin subunit shared by the leukocyte adhesion proteins, resulting in impaired cell adhesion (85). Its complete absence leads to the severe phenotype of LAD 1 that predisposes to severe bacterial infections, often leading to death within the first years of life, necrotic skin lesions, and delayed separation of the umbilical cord after birth. In addition, they

330

Loechelt and Kamani

experience severe gingivitis, periodontitis, and alveolar bone loss that lead to early loss of deciduous and permanent teeth (86). Successful SCT using grafts from HLA-identical related, HLA-nonidentical related, HLA-identical unrelated, and UCB have been reported (87–91). In the largest series reported to date, Thomas, and colleagues present a retrospective analysis of 14 patients with LAD 1. Donor types included HLA-identical (nZ5) and HLA-nonidentical (nZ9) related donors. All patients received a myeloablative preparative regimen that included TBI in one. Graft loss occurred in five (36%). Ten patients are alive and well 12 months to 12 years following SCT at the time of the report, six with full donor chimerism and four with mixed but stable donor chimerism. One patient with less than 15% donor leukocytes has mild gingivitis and leukocytosis. These results suggest that engraftment is a significant issue in patients with LAD 1 and that although mixed donor chimerism results in cure of this disease, it needs to be greater than 15% for complete amelioration (91).

Hemophagocytic Lymphohistiocytosis Hemophagocytic Lymphohistiocytosis (HLH) is a life-threatening immunoregulatory disorder resulting in infiltration of lymphocytes and non-Langerhans histiocytes, with extensive hemophagocytosis particularly involving the liver, spleen, bone marrow, and central nervous system (92). It is characterized by fever, massive hepatosplenomegaly, pancytopenia, hypertriglyceridemia, hypofibrinogenemia, decreased NK cell function (93), and frequently seizures (94). HLH frequently occurs in children younger than 2 years of age as a familial autosomal recessive disorder (95). Symptomatic disease can be unremitting or may follow a relapsing course, leading to death from infection and/or bleeding with a median survival after onset of only a few months (96). The use of immunosuppressive agents, such as corticosteroids and cyclosporine as well as etoposide and intrathecal methotrexate (the latter in patients with CNS disease), has resulted in marked improvements in survival during the first months after diagnosis (97–99). However, due to the relapsing nature of HLH, SCT is recommended as definitive therapy (100). In 1994 the Histiocyte Society initiated a prospective international collaborative therapeutic study (HLH-94), aimed at improved survival. This study utilized initiation of therapy with etoposide and corticosteroids plus intrathecal methotrexate in individuals with CNS disease. Cyclosporine was instituted at 8 weeks. For those individuals with the familial form of the disease, SCT is recommended as soon as a donor could be identified. SCT was recommended for those with nonfamilial forms that experienced relapse of their HLH. Successful SCT has been described in this disorder using multiple donor sources included HLA-matched related and unrelated donors (4,9,57,101,102). The results of SCT using HLA-nonidentical related donors have generally been poor. However, in a review of their own experience by Fischer and colleagues, using HLA-nonidentical related donors, seven of ten patients who received donor stem cells from a T-cell-depleted 3/6 haploidentical donor were alive and free of disease 8–69 months following SCT (103). They concluded that SCT from HLA genetically nonidentical related donors may be an alternative in patients with HLH who lack a suitable HLA genetically identical donor. Due to the early excellent results of the HLH-94 protocol, etoposide was commonly included in many conditioning regiments for SCT. A common preparative regimen included busulfan, cyclophosphamide, and etoposide. A recent analysis of the HLH-94 results by Henter and colleagues show a 3-year probability of survival after SCT was 62%. There was no difference in survival when comparing matched related versus matched unrelated donors (67% vs. 68% survival; nZ15 and 25, respectively). Haploidentical related donors fared less well with a 43% overall survival (nZ14). Other Myeloid Disorders Small numbers of transplants have been reported for other myeloid disorders, including Griscelli syndrome and other neutrophil disorders (104). Transplants have utilized

Primary Immunodeficiencies

331

myeloablative regimens with the majority of donors being HLA-matched siblings. However, in those patients lacking MSD, use of closely matched unrelated donors is certainly an option based on an assessment of patient-specific risk-benefit ratio.

FUTURE DIRECTIONS It is anticipated that several additional gene defects responsible for causing SCID and other PID will be identified over the next decade. In addition to advances in genetic diagnosis of these disorders, these advances will have an impact on new therapies for PID, such as gene therapy, in utero transplantation, and preimplantation genetic diagnosis (PGD). The SCID disorders and many of the PID are ideal diseases for application of somatic gene therapy for a number of reasons. First, disease associated single gene defects have been identified for many of these disorders. Secondly, correction of the gene defect should provide a survival advantage for gene-transduced cells in many SCID disorders. Delivery of the genetransduced cells can be accomplished easily by intravenous administration. Most importantly, although correction of the defect in the patient’s pluripotent HSC would result in a permanent “cure,” correction in hematopoietic progenitors or mature T cells can provide long-lasting benefit (105). In ADA and X-linked SCID due to a common-gamma chain gene defect, the pattern of gene expression in transduced cells is very similar to normal cells and results in stable expression of functional protein. Indeed, children with these two diseases have served as subjects for gene therapy clinical trials. Although there is some evidence to suggest that patients who have received ADA gene modified autologous peripheral T cells (106) or cord blood CD34C cells (107) have derived clinical benefit from the procedure and have a small percentage of long lasting circulating transduced T cells, much work needs to be done to improve efficiency of transduction and allow the use of vectors that are safe and target quiescent pluripotent HSC. One approach has been to use cytoreductive therapy prior to infusing autologous transduced CD34C progenitor cells. Bordignon et al. recently achieved engraftment of 10% transduced peripheral myeloid cells with approximately 100% genemodified peripheral lymphocytes in ADA deficient children who had been given 4 mg/kg busulfan prior to infusion of gene-transduced autologous cells. These children have demonstrated sufficient immune reconstitution to allow them to come off PEG-ADA supplementation (108). Hacein-Bey-Abina et al. recently reported sustained correction of X-SCID by ex vivo gene transduction of marrow derived autologous CD34C cells in 9 of 10 children with X-SCID (109). Unfortunately, two of these children developed T cell acute lymphocytic leukemia 30 and 34 months after gene transfer, secondary to the inadvertent proviral integration within the LMO-2 locus and subsequent aberrant expression of the LMO-2 proto-oncogene (110) and a third one. These serious adverse events resulted in a temporary moratorium of gene therapy clinical trials for these diseases. Recent efforts have focused on improving vector safety by exploring the use of lentiviral vectors, optimization of choice of promoter, and improvements in gene expression (108). An alternative approach being currently explored is the use of gene modification and repair by the use of zinc-finger nucleases designed against the X-SCID mutation in the IL-2R gamma gene (111). Clearly, a lot more basic research needs to be carried out before gene therapy can gain clinical acceptance and become universally available. As more gene therapy clinical trials are carried out, the results of these will need to be compared to those obtained with hematopoietic SCT or other therapies. In utero transplantation is also a consideration due to the availability of early prenatal diagnosis. Small numbers of procedures have been preformed on a research basis with mixed results (112). Two infants with gc-deficient SCID who were diagnosed in utero received CD34C paternal stem cells intraperitoneally during the first half of gestation (113,114). Both infants developed immune function postnatally. Although these latter cases were successful the

332

Loechelt and Kamani

risks to the fetus from graft versus graft or GVHD or fetal trauma must be weighed versus those of an early postnatal conventional transplant. Finally, recent advances in PGD have further impact for individuals and families who are at risk for genetic diseases. Following in vitro fertilization, zygotes can be evaluated, and those free of disease can be implanted, resulting in the birth of an unaffected infant. For families in whom there are affected children, similar techniques can be used for implantation of identified zygotes that are HLA-identical to the affected individual and free of genetic disease. Cord blood or marrow could then be used for a curative procedure for the affected individual (115).

REFERENCES 1. Gatti RA, Meuwissen HJ, Allen HD, et al. Immunological reconstitution of sex-linked lymphopenic immunological deficiency. Lancet 1968; 2:1366–1369. 2. Bach FH, Albertini RJ, Joo P, et al. Bone-marrow transplantation in a patient with the WiskottAldrich syndrome. Lancet 1968; 2:1364–1366. 3. Buckley RH, Fischer A. Bone marrow transplantation for primary immunodeficiency diseases. In: Ochs HD, Smith CIE, Puck JM, eds. Primary Immunodeficiency Disease: A Molecular and Genetic Approach. New York: Oxford University Press, 1999:459–475. 4. Baker KS, DeLaat CA, Steinbuch M, et al. Successful correction of hemophagocytic lymphohistiocytosis with related or unrelated bone marrow transplantation. Blood 1997; 89:3857–3863. 5. Goldsobel AB, Haas A, Stiehm ER. Bone marrow transplantation in DiGeorge syndrome. J Pediatr 1987; 111:40–44. 6. Horwitz ME. Stem-cell transplantation for inherited immunodeficiency disorders. Pediatr Clin North Am 2000; 47:1371–1387. 7. Klein C, Cavazzana-Calvo M, Le Deist F, et al. Bone marrow transplantation in major histocompatibility complex class II deficiency: a single-center study of 19 patients. Blood 1995; 85:580–587. 8. Buckley RH, Schiff SE, Schiff RI, et al. Haploidentical bone marrow stem cell transplantation in human severe combined immunodeficiency. Semin Hematol 1993; 30:92–101 discussion 2–4. 9. Filipovich AH, Shapiro RS, Ramsay NK, et al. Unrelated donor bone marrow transplantation for correction of lethal congenital immunodeficiencies. Blood 1992; 80:270–276. 10. Kurtzberg J, Laughlin M, Graham ML, et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996; 335:157–166. 11. Amrolia P, Gaspar HB, Hassan A, et al. Nonmyeloablative stem cell transplantation for congenital immunodeficiencies. Blood 2000; 96:1239–1246. 12. Horwitz ME, Barrett AJ, Brown MR, et al. Treatment of chronic granulomatous disease with nonmyeloablative conditioning and a T-cell-depleted hematopoietic allograft. N Engl J Med 2001; 344:881–888. 13. Kamani N, August CS, Campbell DE, et al. Marrow transplantation in chronic granulomatous disease: an update, with 6-year follow-up. J Pediatr 1988; 113:697–700. 14. Haddad E, Le Deist F, Aucouturier P, et al. Long-term chimerism and B-cell function after bone marrow transplantation in patients with severe combined immunodeficiency with B cells: a singlecenter study of 22 patients. Blood 1999; 94:2923–2930. 15. Friedman NJ, Schiff SE, Ward FE, et al. Graft-versus-graft and graft-versus-host reactions after HLA-identical bone marrow transplantation in a patient with severe combined immunodeficiency with transplacentally acquired lymphoid chimerism. Pediatr Allergy Immunol 1991; 2:111–116. 16. Bross DS, Tutschka PJ, Farmer ER, et al. Predictive factors for acute graft-versus-host disease in patients transplanted with HLA-identical bone marrow. Blood 1984; 63:1265–1270. 17. Weisdorf D, Hakke R, Blazar B, et al. Risk factors for acute graft-versus-host disease in histocompatible donor bone marrow transplantation. Transplantation 1991; 51:1197–1203. 18. Reisner Y, Kapoor N, Kirkpatrick D, et al. Transplantation for severe combined immunodeficiency with HLA-A,B,D,DR incompatible parental marrow cells fractionated by soybean agglutinin and sheep red blood cells. Blood 1983; 61:341–348. 19. Antoine C, Muller S, Cant A, et al. Long-term survival and transplantation of haemopoietic stem cells for immunodeficiencies: report of the European experience 1968-99. Lancet 2003; 361:553–560.

Primary Immunodeficiencies

333

20. Rubinstein P, Carrier C, Scaradavou A, et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 1998; 339:1565–1577. 21. Anderson D, DeFor T, Burns L, et al. A comparison of related donor peripheral blood and bone marrow transplants: importance of late-onset chronic graft-versus-host disease and infections. Biol Blood Marrow Transplant 2003; 9:52–59. 22. Watanabe T, Takaue Y, Kawano Y, et al. HLA-identical sibling peripheral blood stem cell transplantation in children and adolescents. Biol Blood Marrow Transplant 2002; 8:26–31. 23. Handgretinger R, Klingbiel T, Lang P, et al. Megadose transplantation of highly purified haploidentical stem cells: current results and future prospects. Pediatr Transplant 2003; 7:51–55. 24. Handgretinger R, Klingbiel T, Lang P, et al. Megadose transplantation of purified peripheral blood CD34 (C) progenitor cells from HLA-mismatched parental donors in children. Bone Marrow Transplant 2001; 27:777–783. 25. Buckley RH, Schiff SE, Schiff RI, et al. Hematopoietic stem-cell transplantation for the treatment of severe combined immunodeficiency. N Engl J Med 1999; 340:508–516. 26. Patel DD, Gooding ME, Parrott RE, et al. Thymic function after hematopoietic stem-cell transplantation for the treatment of severe combined immunodeficiency. N Engl J Med 2000; 342:1325–1332. 27. Horn B, Viele M, Mentzer W, et al. Autoimmune hemolytic anemia in patients with SCID after T cell-depleted BM and PBSC transplantation. Bone Marrow Transplant 1999; 24:1009–1013. 28. Gaspar HB, Amrolia P, Hassan A, et al. Non-myeloablative stem cell transplantation for congenital immunodeficiencies. Recent Results Cancer Res 2002; 159:134–142. 29. Kamani NR, Infante AJ, DeSantes K, et al. Fludarabine (FLU) containing preparative regimens for hematopoietic stem cell transplantation (HSCT) in severe combined immune deficiency syndromes (SCIDS): interim results of a multi-institutional trial of the pediatric blood and marrow transplant consortium (PBMTC). Blood 2003; 102:469a. 30. Stephan JL, Vlekova V, Le Deist F, et al. Severe combined immunodeficiency: a retrospective single-center study of clinical presentation and outcome in 117 patients. J Pediatr 1993; 123:564–572. 31. Schwarz K, Gauss GH, Ludwig L, et al. RAG mutations in human B cell-negative SCID. Science 1996; 274:97–99. 32. Li L, Moshous D, Zhou Y, et al. A founder mutation in artemis, an SNM1-like protein, causes SCID in Athabascan-speaking native Americans. J Immunol 2002; 168:6323–6329. 33. Moshous D, Callebaut I, de Chasseval R, et al. Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency. Cell 2001; 105:177–186. 34. Myers LA, Patel DD, Puck JM, et al. Hematopoietic stem cell transplantation for severe combined immunodeficiency in the neonatal period leads to superior thymic output and improved survival. Blood 2002; 99:872–878. 35. Parkman R, Rappeport J, Geha R, et al. Complete correction of the Wiskott-Aldrich syndrome by allogeneic bone-marrow transplantation. N Engl J Med 1978; 298:921–927. 36. Rappeport JM, Parkman R, Belli JA, et al. Correction of congenital bone marrow disorders by allogeneic bone marrow transplantation following preparation with anti-human thymocyte serum and total body irradiation. Transplant Proc 1981; 13:241–244. 37. Filipovich AH, Stone JV, Tomany SC, et al. Impact of donor type on outcome of bone marrow transplantation for Wiskott-Aldrich syndrome: collaborative study of the international bone marrow transplant registry and the national marrow donor program. Blood 2001; 97:1598–1603. 38. Ozsahin H, Le Deist F, Benkerrou M, et al. Bone marrow transplantation in 26 patients with Wiskott-Aldrich syndrome from a single center. J Pediatr 1996; 129:238–244. 39. Lenarsky C, Weinberg K, Kohn DB, et al. Unrelated donor BMT for Wiskott-Aldrich syndrome. Bone Marrow Transplant 1993; 12:145–147. 40. Notarangelo LD, Duse M, Ugazio AG. Immunodeficiency with hyper-IgM (HIM). Immunodef Rev 1992; 3:101–121. 41. Gennery AR, Khawaja K, Veys P, et al. Treatment of CD40 ligand deficiency by hematopoietic stem cell transplantation: a survey of the European experience, 1993–2002. Blood 2004; 103:1152–1157. 42. Engel P, Eck MJ, Terhorst C. The SAP and SLAM families in immune responses and X-linked lymphoproliferative disease. Nat Rev Immunol 2003; 3:813–821.

334

Loechelt and Kamani

43. Filipovich AH, Blazar BR, Ramsay NK, et al. Allogeneic bone marrow transplantation for X-linked lymphoproliferative syndrome. Transplantation 1986; 42:222–224. 44. Gross TG, Filipovich AH, Conley ME, et al. Cure of X-linked lymphoproliferative disease (XLP) with allogeneic hematopoietic stem cell transplantation (HSCT): report from the XLP registry. Bone Marrow Transplant 1996; 17:741–744. 45. Pracher E, Panzer-Grumayer ER, Zoubek A, et al. Successful bone marrow transplantation in a boy with X-linked lymphoproliferative syndrome and acute severe infectious mononucleosis. Bone Marrow Transplant 1994; 13:655–658. 46. Hoffmann T, Heilmann C, Madsen HO, et al. Matched unrelated allogeneic bone marrow transplantation for recurrent malignant lymphoma in a patient with X-linked lymphoproliferative disease (XLP). Bone Marrow Transplant 1998; 22:603–604. 47. Ziegner UH, Ochs HD, Schanen C, et al. Unrelated umbilical cord stem cell transplantation for X-linked immunodeficiencies. J Pediatr 2001; 138:570–573. 48. Bensoussan D, Le Deist F, Latger-Cannard V, et al. T-cell immune constitution after peripheral blood mononuclear cell transplantation in complete DiGeorge syndrome. Br J Haematol 2002; 117:899–906. 49. Borzy MS, Ridgway D, Noya FJ, et al. Successful bone marrow transplantation with split lymphoid chimerism in DiGeorge syndrome. J Clin Immunol 1989; 9:386–392. 50. Matsumoto T, Amamoto N, Kondoh T, et al. Complete-type DiGeorge syndrome treated by bone marrow transplantation. Bone Marrow Transplant 1998; 22:927–930. 51. Berthet F, Siegrist CA, Ozsahin H, et al. Bone marrow transplantation in cartilage-hair hypoplasia: correction of the immunodeficiency but not of the chondrodysplasia. Eur J Pediatr 1996; 155:286–290. 52. Sleight BJ, Prasad VS, DeLaat C, et al. Correction of autoimmune lymphoproliferative syndrome by bone marrow transplantation. Bone Marrow Transplant 1998; 22:375–380. 53. Baud O, Goulet O, Canioni D, et al. Treatment of the immune dysregulation polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) by allogeneic bone marrow transplantation. N Engl J Med 2001; 344:1758–1762. 54. Barak Y, Nir E. Chediak-Higashi syndrome. Am J Pediatr Hematol Oncol 1987; 9:42–55. 55. Huizing M, Anikster Y, Gahl WA. Hermansky-Pudlak syndrome and Chediak-Higashi syndrome: disorders of vesicle formation and trafficking. Thromb Haemost 2001; 86:233–245. 56. Demory JL, Senlecq-Tack S, Decoster A, et al. Maladie de Chediak-Higashi: un nouveau cas traite par allogreffe de moelle osseuse. Ann de Pediatr 1989; 36:387–389. 57. Fischer A, Griscelli C, Friedrich W, et al. Bone-marrow transplantation for immunodeficiencies and osteopetrosis: European survey, 1968–1985. Lancet 1986; 2:1080–1084. 58. Haddad E, Le Deist F, Blanche S, et al. Treatment of Chediak-Higashi syndrome by allogeneic bone marrow transplantation: report of 10 cases. Blood 1995; 85:3328–3333. 59. Liang JS, Lu MY, Tsai MJ, et al. Bone marrow transplantation from an HLA-matched unrelated donor for treatment of Chediak-Higashi syndrome. J Formos Med Assoc 2000; 99:499–502. 60. Mottonen M, Lanning M, Saarinen UM. Allogeneic bone marrow transplantation in ChediakHigashi syndrome. Pediatr Hematol Oncol 1995; 12:55–59. 61. O’Reilly RJ, Brochstein J, Dinsmore R, et al. Marrow transplantation for congenital disorders. Semin Hematol 1984; 21:188–221. 62. Trigg ME, Schugar R. Chediak-Higashi syndrome: hematopoietic chimerism corrects genetic defect. Bone Marrow Transplant 2001; 27:1211–1213. 63. Virelizier JL, Lagrue A, Durandy A, et al. Reversal of natural killer defect in a patient with ChediakHigashi syndrome after bone-marrow transplantation. N Engl J Med 1982; 306:1055–1056. 64. Dinauer MC, Orkin SH. Chronic granulomatous disease. Annu Rev Med 1992; 43:117–124. 65. Quie PG. Chronic granulomatous disease of childhood: a saga of discovery and understanding. Pediatr Infect Dis J 1993; 12:395–398. 66. Curnutte JT. Chronic granulomatous disease: the solving of a clinical riddle at the molecular level. Clin Immunol Immunopathol 1993; 67:S2–S15. 67. Johnston RB, Jr. Clinical aspects of chronic granulomatous disease. Curr Opin Hematol 2001; 8:17–22. 68. International Chronic Granulomatous Disease Cooperative Study Group. A controlled trial of interferon gamma to prevent infection in chronic granulomatous disease. The International Chronic Granulomatous Disease Cooperative Study Group. N Engl J Med 1991; 324:509–516.

Primary Immunodeficiencies

335

69. Winkelstein JA, Marino MC, Johnston RBJ, et al. Chronic granulomatous disease. Report on a national registry of 368 patients. Medicine 2000; 79:155–169. 70. Akioka S, Itoh H, Ueda I, et al. Donor lymphocyte infusion at unstable mixed chimerism in an allogeneic BMT recipient for chronic granulomatous disease. Bone Marrow Transplant 1998; 22:609–611. 71. Bhattacharya A, Slatter M, Curtis A, et al. Successful umbilical cord blood stem cell transplantation for chronic granulomatous disease. Bone Marrow Transplant 2003; 31:403–405. 72. Bielorai B, Toren A, Wolach B, et al. Successful treatment of invasive aspergillosis in chronic granulomatous disease by granulocyte transfusions followed by peripheral blood stem cell transplantation. Bone Marrow Transplant 2000; 26:1025–1028. 73. Calvino MC, Maldonado MS, Otheo E, et al. Bone marrow transplantation in chronic granulomatous disease. Eur J Pediatr 1996; 155:877–879. 74. Del Giudice I, Iori AP, Mengarelli A, et al. Allogeneic stem cell transplant from HLA-identical sibling for chronic granulomatous disease and review of the literature. Ann Hematol 2003; 82:189–192. 75. Di Bartolomeo P, Di Girolamo G, Angrilli F, et al. Reconstitution of normal neutrophil function in chronic granulomatous disease by bone marrow transplantation. Bone Marrow Transplant 1989; 4:695–700. 76. Ho CM, Vowels MR, Lockwood L, et al. Successful bone marrow transplantation in a child with X-linked chronic granulomatous disease. Bone Marrow Transplant 1996; 18:213–215. 77. Kamani N, August CS, Douglas SD, et al. Bone marrow transplantation in chronic granulomatous disease. J Pediatr 1984; 105:42–46. 78. Leung T, Chik K, Li C, et al. Bone marrow transplantation for chronic granulomatous disease: longterm follow-up and review of literature. Bone Marrow Transplant 1999; 24:567–570. 79. Nagler A, Ackerstein A, Kapelushnik J, et al. Donor lymphocyte infusion post-non-myeloablative allogeneic peripheral blood stem cell transplantation for chronic granulomatous disease. Bone Marrow Transplant 1999; 24:339–342. 80. Rappeport JM, Newburger PE, Goldblum RM, et al. Allogeneic bone marrow transplantation for chronic granulomatous disease. J Pediatr 1982; 101:952–955. 81. Savage WJ, Bleesing JJ, Douek D, et al. Lymphocyte reconstitution following non-myeloablative hematopoietic stem cell transplantation follows two patterns depending on age and donor/recipient chimerism. Bone Marrow Transplant 2001; 28:463–471. 82. Seger RA, Gungor T, Belohradsky BH, et al. Treatment of chronic granulomatous disease with myeloablative conditioning and an unmodified hemopoietic allograft: a survey of the European experience, 1985–2000. Blood 2002; 100:4344–4350. 83. Watanabe C, Yajima S, Taguchi T, et al. Successful unrelated bone marrow transplantation for a patient with chronic granulomatous disease and associated resistant pneumonitis and Aspergillus osteomyelitis. Bone Marrow Transplant 2001; 28:83–87. 84. Hobbs JR, Monteil M, McCluskey DR, et al. Chronic granulomatous disease 100% corrected by displacement bone marrow transplantation from a volunteer unrelated donor. Eur J Pediatr 1992; 151:806–810. 85. Marlin SD, Morton CC, Anderson DC, et al. LFA-1 immunodeficiency disease. Definition of the genetic defect and chromosomal mapping of alpha and beta subunits of the lymphocyte functionassociated antigen 1 (LFA-1) by complementation in hybrid cells. J Exp Med 1986; 164:855–867. 86. Anderson DC, Schmalsteig FC, Finegold MJ, et al. The severe and moderate phenotypes of heritable Mac-1, LFA-1 deficiency: their quantitative definition and relation to leukocyte dysfunction and clinical features. J Infect Dis 1985; 152:668–689. 87. Fischer A, Haddad E, Jabado N, et al. Stem cell transplantation for immunodeficiency. Springer Semin Immunopathol 1998; 19:479–492. 88. Hattori H, Tsuruta S, Horikoshi Y, et al. Successful human leukocyte antigen one antigenmismatched related bone marrow transplantation in a 6-year-old boy with leukocyte adhesion deficiency syndrome. Pediatr Int 2001; 43:306–309. 89. Mancias C, Infante AJ, Kamani NR. Matched unrelated donor bone marrow transplantation in leukocyte adhesion deficiency. Bone Marrow Transplant 1999; 24:1261–1263. 90. Stary J, Bartunkova J, Kobylka P, et al. Successful HLA-identical sibling cord blood transplantation in a 6-year-old boy with leukocyte adhesion deficiency syndrome. Bone Marrow Transplant 1996; 18:249–252. 91. Thomas C, Le Deist F, Cavazzana-Calvo M, et al. Results of allogeneic bone marrow transplantation in patients with leukocyte adhesion deficiency. Blood 1995; 86:1629–1635.

336

Loechelt and Kamani

92. Favara BE. Hemophagocytic lymphohistiocytosis: a hemophagocytic syndrome. Semin Diagn Pathol 1992; 9:63–74. 93. Egeler RM, Shapiro R, Loechelt B, et al. Characteristic immune abnormalities in hemophagocytic lymphohistiocytosis. J Pediatr Hematol Oncol 1996; 18:340–345. 94. Loy TS, Diaz-Arias AA, Perry MC. Familial erythrophagocytic lymphohistiocytosis. Semin Oncol 1991; 18:34–38. 95. Gencik A, Signer E, Muller H. Genetic analysis of familial erythrophagocytic lymphohistiocytosis. Eur J Pediatr 1984; 142:248–252. 96. Henter JI, Soder O, Ost A, et al. Incidence and clinical features of familial hemophagocytic lymphohistiocytosis in Sweden. Acta Paediatr 1991; 80:428–435. 97. Henter JI, Arico M, Egeler RM, et al. HLH-94: a treatment protocol for hemophagocytic lymphohistiocytosis. HLH study group of the histiocyte society. Med Pediatr Oncol 1997; 28:342–347. 98. Henter JI, Arico M, Elinder G, et al. Familial hemophagocytic lymphohistiocytosis. Primary hemophagocytic lymphohistiocytosis. Hematol Oncol Clin North Am 1998; 12:417–433. 99. Loechelt BJ, Egeler M, Filipovich AH, et al. Immunosuppression: preliminary results of alternative maintenance therapy for familial hemophagocytic lymphohistiocytosis (FHL). Med Pediatr Oncol 1994; 22:325–328. 100. Blanche S, Caniglia M, Girault D, et al. Treatment of hemophagocytic lymphohistiocytosis with chemotherapy and bone marrow transplantation: a single-center study of 22 cases. Blood 1991; 78:51–54. 101. Durken M, Horstmann M, Bieling P, et al. Improved outcome in haemophagocytic lymphohistiocytosis after bone marrow transplantation from related and unrelated donors: a single-centre experience of 12 patients. Br J Haematol 1999; 106:1052–1058. 102. Henter JI, Samuelsson-Horne A, Arico M, et al. Treatment of hemophagocytic lymphohistiocytosis with HLH-94 immunochemotherapy and bone marrow transplantation. Blood 2002; 100:2367–2373. 103. Jabado N, de Graeff-Meeder ER, Cavazzana-Calvo M, et al. Treatment of familial hemophagocytic lymphohistiocytosis with bone marrow transplantation from HLA genetically nonidentical donors. Blood 1997; 90:4743–4748. 104. Tezcan I, Sanal O, Ersoy F, et al. Successful bone marrow transplantation in a case of Griscelli disease which presented in accelerated phase with neurological involvement. Bone Marrow Transplant 1999; 24:931–933. 105. Fischer A. Gene therapy of lymphoid primary immunodeficiencies. Curr Opin Pediatr 2000; 12:557–562. 106. Blaese RM, Culver KW, Miller AD, et al. T lymphocyte-directed gene therapy for ADA-SCID: initial trial results after 4 years. Science 1995; 270:475–480. 107. Kohn DB, Hershfield MS, Carbonaro D, et al. T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34C cells in ADA-deficient SCID neonates. Nat Med 1998; 4:775–780. 108. Podsakoff GM, Engel BC, Kohn DB. Perspectives on gene therapy for immune deficiencies. Biol Blood Marrow Transplant 2005; 11:972–976. 109. Hacein-Bey-Abina S, Le Deist F, Carlier F, et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 2002; 346:1185–1193. 110. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al. LMO2-Associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003; 302:415–419. 111. Urnov FD, Miller JC, Lee YL, et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 2005; 435:577–579. 112. Touraine JL. Treatment of human fetuses and induction of immunological tolerance in humans by in utero transplantation of stem cells into fetal recipients. Acta Haematologica 1996; 96:115–119. 113. Flake AW, Roncarolo M-G, Puck JM, et al. Treatment of X-Linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow. N Engl J Med 1996; 335:1806–1810. 114. Wengler GS, Lanfranchi A, Frusca T, et al. In-utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with X-linked severe combined immunodeficiency (SCIDXI). Lancet 1996; 348:1484–1487. 115. Grewal SS, Kahn JP, MacMillan ML, et al. Successful hematopoietic stem cell transplantation for Fanconi anemia from an unaffected HLA-genotype-identical sibling selected using preimplantation genetic diagnosis. Blood 2004; 103:1147–1151.

18 Hematopoietic Stem-Cell Transplantation for the Inherited Bone Marrow Failure Syndromes Adrianna Vlachos Division of Pediatric Hematology, Oncology and Stem Cell Transplantation, Schneider Children’s Hospital, New Hyde Park, New York, U.S.A.

Carole Paley Novartis Pharmaceuticals, East Hanover, New Jersey, U.S.A.

Jeffrey Michael Lipton Division of Pediatric Hematology, Oncology and Stem Cell Transplantation, Schneider Children’s Hospital, New Hyde Park, New York, U.S.A.

INTRODUCTION The proliferation and differentiation of pluripotent stem cells give rise to progeny that can populate the entire immunologic and hematopoietic systems, the committed progenitors of both the lymphoid and myeloid lineages. The common myeloid progenitor, the CFU-GEMM, gives rise to committed myeloid progenitors. In turn each of these progenitors is programmed to provide the recognizable precursors of the granulocyte, erythrocyte, monocyte/macrophage, megakaryocyte, eosinophil, and basophil lineages. The marrow microenvironment, consisting of lymphocytes, macrophages, fibroblasts, endothelial elements and stroma, in which hematopoietic cells reside, creates a regulatory “niche” that determines a “local area network” (1). Thus the potential mechanisms for bone marrow failure include faulty stem/progenitor cells or a “network” breakdown, defective stroma, accessory cells, or abnormal growth factors, as well as deficient nonspecific nutrients, or, as in the case of acquired aplastic anemia, immune mediated abnormalities. It seems clear, however, that the inherited bone marrow failure syndromes result as the consequence of an intrinsic stem cell/progenitor defect. The precise pathophysiology of the inherited single cell and multilineage cytopenias has not yet been elucidated despite the identification of many of the genes mutated in these disorders (Table 1). These mutations seem to result in the predilection to apoptosis. Indeed, accelerated hematopoietic cell apoptosis has been demonstrated in virtually all of the inherited bone marrow failure syndromes studied thus far (2–5). The “failure” in the inherited bone marrow failure syndromes is usually not restricted to hematopoiesis. The pathology of many of these disorders includes congenital anomalies, multiorgan dysfunction, and, in some cases, extreme susceptibility to the toxicity of transplantation conditioning regimens (6–8). To achieve “bone marrow success” through hematopoietic stem cell transplantation (HSCT), the physician must be aware of these confounding circumstances lest they lead to extreme toxicity when traditional conditioning regimens are utilized. 337

Amegakaryocytic thrombocytopenia

Kostmann syndrome (SCN)

Familial aplastic anemia Shwachman Diamond syndrome Diamond Blackfan anemia

Dyskeratosis congenita

FANCA FANCB FANCC FANCD1 FANCD2 FANCE FANCF FANCG FANCI FANCJ FANCL FANCM DKC1 DKC2 (hTERC) DKC3 TERT SBDS DBA1 (RPS19) DBA2 ELA2 GFI1 ? c-mpl

Gene 16q24.3 Xp22.31 9q22.3 13q12.3 3p25.3 6p21.3 11p15 9p13 ? 17q23 2p16.1 14q21.3 Xq28 3q21–q28 ? 5p15.33 7q11 19q13.2 8p23.3–p22 19p13.3 1p22 ? 1p34

Locus

Inherited Bone Marrow Failure Syndrome Genes, Known and Presumed

Fanconi anemia

Disorder

Table 1

Autosomal recessive X-linked recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive ? Autosomal recessive Autosomal recessive Autosomal recessive X-linked recessive Autosomal dominant Autosomal recessive Autosomal dominant Autosomal recessive Autosomal dominant Autosomal dominant Autosomal dominant Autosomal dominant Autosomal recessive Autosomal recessive

Genetics

FANCA FANCB FANCC BRCA2 FANCD2 FANCE FANCF XRCC9 ? BRIP1/BACH1 PHF9 FANCM Dyskerin Telomerase RNA component ? Telomerase reverse transcriptase SBDS RPS19 ? Neutrophil elastase-2 ZNF163 ? Thrombopoietin receptor

Gene product

338 Vlachos et al.

Inherited Bone Marrow Failure Syndromes

339

The decision to perform a transplant must involve a careful risk-versus-benefit assessment. As well, a detailed understanding of the clinical presentation and genetics of these disorders is necessary in order to prevent the inadvertent selection of a sibling donor who is also affected. Classic serial transplant experiments in mice (9) have demonstrated the markedly reduced engraftment potential of “damaged” stem cells. It is obvious that a HSCT utilizing an affected donor would, at best, result in no change in the hematologic status of the recipient. However, the more likely outcome would be nonengraftment. Traditionally the inherited bone marrow failure syndromes have been divided into those resulting in pancytopenia [Fanconi anemia (FA) and dyskeratosis congenita (DC)] and those presumably restricted to a single hematopoietic lineage [Diamond Blackfan anemia (DBA), congenital neutropenia {Kostmann syndrome (KS), cyclic neutropenia, Shwachman Diamond syndrome (SDS)}, congenital amegakaryocytic thrombocytopenia (CAT) and thrombocytopenia absent radii (TAR) syndrome]. We will preserve these classifications; however, it has become evident that most of these “single cell cytopenias” may manifest abnormalities in other hematopoietic cell lines. Indeed in some, SDS and CAT, for example, pancytopenia is fairly common. Again, Young and Alter’s small masterpiece, Aplastic Anemia Acquired and Inherited (10), published in 1994, is recommended to those readers desiring a more complete discussion of the inherited bone marrow failure syndromes. In this chapter we will endeavor to provide a discussion of developments since 1994. Those disorders for which HSCT is rarely indicated, such as TAR syndrome and cyclic neutropenia, will be mentioned only in passing.

THE SYNDROMES Pancytopenia Fanconi Anemia Pathophysiology and Clinical Features. FA, first described in 1927 (11), is a rare, usually autosomal recessive (!1% X-linked) inherited bone marrow failure syndrome characterized by abnormal skin pigmentation (cafe´ au lait and hypopigmented spots), short stature, and congenital malformations. These malformations are most frequently skeletal (upper limb and classical thumb abnormalities), followed by renal anomalies. Other abnormalities, including cardiac and genital anomalies, have been catalogued in great detail (10). However, it has been estimated that as many as 40% of FA patients lack obvious physical abnormalities (12). The median age at hematologic presentation of patients reported to the International FA Registry (IFAR) database is approximately seven years (13). All racial and ethnic groups are affected. Hematologic dysfunction usually presents with thrombocytopenia, often leading to progressive pancytopenia and severe aplastic anemia (SAA), frequently terminating in myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). Thrombocytopenia is usually preceded by macrocytosis. In addition to the increased risk of hematologic malignancies, there is also an increased risk of solid tumors of the head and neck, esophagus, liver, and female reproductive organs. The actuarial risks of developing bone marrow failure and hematologic and nonhematologic neoplasms are 90%, 33%, and 28%, respectively, by 40 years of age (14). Rosenberg and colleagues suggest that the risk of solid tumors may increase in the face of improved HSCT outcomes, as a result of the toxicity of HSCT conditioning regimens or graft-versus-host disease (GVHD), or both (15). FA cells are hypersensitive to chromosomal breaks induced by DNA cross-linking agents. This observation is the basis for the commonly used chromosome breakage test for FA utilizing the clastogens diepoxybutane and mitomycin C (16). Clastogens also induce cell cycle

340

Vlachos et al.

arrest in G2/M; recently flow cytometry to detect this arrest has become another screening tool for FA (17). Genetics. FA, inherited as an autosomal recessive disorder in more than 99% of cases and rarely, as an X-linked recessive (18), is the most frequently inherited aplastic anemia. Somatic cell hybridization studies have thus far defined twelve FA complementation groups. Of these, 10 FA genes have been cloned (Table 1) (19–23). The gene products of these 10 genes have been shown to cooperate in a common pathway. Eight of the FA proteins (FANCA, B, C, E, F, G, L and M) are hypothesized to assemble in a nuclear protein “core complex” that is required to monoubiquinate and activate FANCD2. Ubiquinated FANCD2 is translocated to a nuclear focus containing BRCA1 (18,24), FANCD1 (identified as BRCA2) (25), and FANCJ. The exact mechanism of FANCD2 monoubiquitination and the role of FANCD2, BRCA2 (FANCD1), FANCJ, and BCRA1 in DNA repair are yet to be unraveled (26). FANCA is the most common complementation group and represents 70% of cases. An analysis from the International Fanconi Anemia Registry (IFAR) suggests an earlier onset of pancytopenia and shorter survival for patients with FANCC (14). However, genotype-phenotype correlation is complex and probably relates as much to the nature of the gene product and other factors as to the specific complementation group (27,28). Frontline Therapy. The art of FA patient management, in particular, the diagnostic workup, treatment of growth failure and other endocrine abnormalities, detection and management of cancer, as well as medical and psychosocial support, are beyond the scope of this chapter. Those who are interested should refer to the handbook, FA Standards for Clinical Care, available from the FA Research Fund, Inc. (29), published in 2003. Treatment of bone marrow failure is considered for an absolute neutrophil count (ANC) %1000/mm3, hemoglobin %8 g/dl or a platelet count %50,000/mm3. Hematologic manifestations of FA can be improved in 50–75% of FA patients with the use of androgens and hematopoietic growth factors. For neutropenia alone, G-CSF or GM-CSF is the first line of therapy. For anemia and neutropenia, oxymetholone and G-CSF, or GM-CSF, are recommended.

Hematopoietic Stem-Cell Transplantation Indications, Outcomes and the Approach to the Patient. Historically the majority of patients succumb to the complications of SAA, followed by leukemia and solid tumors. Recently, with improved supportive care, the median survival for FA has increased to approximately 30 years of age (30). Unquestionably, since the groundbreaking observations of Gluckman and colleagues (6), marrow failure has been cured and the risk of leukemia in FA ameliorated by the use of allogeneic stem cell transplantation. Excellent results have been obtained with human leukocyte antigen (HLA)-matched sibling donors. Thus, there is general agreement that otherwise healthy FA patients with significant cytopenias (ANC !1000/mm3, hemoglobin !8 g/dl or a platelet count !50,000/mm3) and an available HLA-matched sibling donor should undergo HSCT rather than proceed to androgen/growth factor therapy (31). Transplant Regimens. We will summarize the development of successful HSCT regimens for FA. A further in-depth presentation of this topic can be found in the excellent review by Harris (31). FA patients have an increased sensitivity to DNA cross-linking agents, in particular alkylating agents commonly used for HSCT conditioning, such as cyclophosphamide, as well as radiation therapy. In initial trials, toxicity from traditional transplant conditioning regimens was extreme. Severe mucositis and gastrointestinal toxicity, as well as severe cardiac, hepatic and cutaneous complications, lead to frequent early death following transplantation. These observations ultimately resulted in a modified regimen instituted by Gluckman and colleagues. The regimen consisted of low-dose cyclophosphamide (5 mg/kg/day!4 for a total 20 mg/kg, or one-tenth the standard dose) and 500 cGy of thoracoabdominal irradiation (TAI) (6,32). These reductions in dose resulted in diminished morbidity and mortality posttransplantation. From 1981 to 1996, 50 FA patients underwent transplant from HLA identical sibling donors using this regimen

Inherited Bone Marrow Failure Syndromes

341

(33). Cyclosporine A alone was used for GVHD prophylaxis. Four patients had MDS and were given a higher dose of cyclophosphamide (10 mg/kg!4). The five-year disease-free survival was 74G6%. The only factor associated with a decreased survival was the number of transfusions prior to HSCT. Grade II or higher acute GVHD developed in half of the patients. Twenty patients developed chronic GVHD. A unique and significant late complication was the development of squamous cell carcinoma of the tongue in five patients. No cases of secondary MDS or AML were seen following HSCT. The reduced toxicity of this regimen, however, was accompanied by a concomitant increase in graft failure. Further modifications, including the pretransplant and posttransplant administration of anti-thymocyte globulin (ATG) and a decrease in the dose of irradiation to 400 cGy, resulted in improved engraftment and reduced incidence and severity of GVHD (34). With this approach 16 of 17 patients engrafted and were alive, without any grade II to IV acute GVHD. In a review of the results of 151 HLA-matched sibling HSCT done between 1978 and 1994 and reported to the International Bone Marrow Transplant Registry (IBMTR) (35), there was a two-year probability of survival of 66%. Age at the time of transplant was a significant prognostic factor with a two-year probability of survival of approximately 90% for patients %10 years but only 50% for those O10 years of age. Those transplants done using a conditioning regimen of low-dose cyclophosphamide and limited field irradiation had superior outcomes with an overall survival of 82%. Patients receiving regimens containing ATG also did better, as well as those receiving cyclosporine as part of GVHD prophylaxis. Unfortunately, most patients with FA do not have an HLA-matched, unaffected related donor. Unrelated donors show some promise as a stem cell source; however, the risk of complications is increased. Significant morbidity and mortality has been associated with grade III–IV GVHD in FA patients undergoing HSCT using HLA-matched unrelated donors. Davies and colleagues reported the results from the University of Minnesota between 1990 and 1994 using cyclophosphamide 10 mg/kg/d!4 with total body irradiation (TBI) 400–450 cGy without T-cell depletion. Severe oropharyngeal mucositis was noted in all patients. Three of seven patients survived, one without GVHD and two with extensive chronic GVHD. Two patients succumbed to infection along with grade IV GVHD. There were two early deaths due to infection, one with concurrent veno-occlusive disease (36). The European Group for Blood and Marrow Transplantation (EBMT) analyzed the results of 69 patients with FA undergoing HSCT using an HLA-matched unrelated donor (37). The overall actuarial survival at 3 years was 33%. The incidence of grade III–IV GVHD was dramatically reduced by T-cell depletion. However with the reduction in mortality from GVHD came a concomitant increase in primary and secondary graft failure, resulting in no difference in survival for those patients receiving either the T-cell depleted or nondepleted grafts. There was a statistically significant improvement in neutrophil recovery in those patients receiving R2!106 CD34C cells. The authors point out that molecular HLA typing should improve upon these results. The next major step was developed to exploit nonmyeloablative, highly immunosuppressive regimens in FA to prevent graft rejection. Kapelushnik and colleagues used a conditioning regimen consisting of fludarabine (30 mg/M2!5), ATG (10 mg/kg!4) and cyclophosphamide (10 mg/kg!2) for HLA-matched related HSCT. They were able to obtain sustained engraftment and only Grade II mucositis, diarrhea, abdominal pain, and hyperbilirubinemia (38). The use of fludarabine appears to facilitate the omission of radiation therapy from the conditioning regimen. This is of particular importance in diminishing immediate toxicity and may be of significance in reducing the incidence of second malignancies. The results reported from the EBMT were comparable using HLA-identical relatives and HLA-identical sibling donors, a finding not terribly unexpected in a rare autosomal recessive disorder. Unrelated donor HSCT was performed in 76 FA patients through the EBMT. The conditioning regimen for these unrelated HSCT used cyclophosphamide (20 or 40 mg/kg total) and TAI or TBI. Most patients received ATG, pre and post HSCT. The two-year event-free

342

Vlachos et al.

survival (EFS) was 23G5%. The main causes of death were GVHD and infections. A male donor and T-cell depletion resulted in better outcomes. Unrelated donor transplants did significantly better than those using an HLA-mismatched relative. Higher dose cyclophosphamide and the use of ATG did not improve the 2-year EFS (33). The Italian Bone Marrow Donor Registry reported ten FA patients transplanted using unrelated donors with only three patients having successful outcomes (39). They concluded that the fewer number of transfusions received prior to transplantation, the better the outcome. Their newest protocol in use for FA patients undergoing unrelated HSCT consists of cyclophosphamide (40 mg/kg), TBI (450 cGy), and ATG with a CD34C selected graft. As a final note, a preliminary report from MacMillan et al. (40) has described a dramatic improvement in outcome for both HLA-matched sibling as well as unrelated donor HSCT in FA. All eleven patients with FA transplanted from allogeneic HLA-matched sibling donors using a fludarabine and cyclophosphamide based conditioning regimen with T-cell depleted CD34C cells are alive and well. Furthermore, the actuarial survival for 25 selected “standard risk” patients, utilizing T-cell depleted CD34C stem cells, who received a matched unrelated transplant is 76% at approximately two years. These investigators define “standard risk” as those patients with a 6/6 HLA-matched unrelated bone marrow or a 5/6 HLA-matched umbilical cord blood (UCB) donor who are !18 years of age, free of infection, and have either SAA or early MDS only, with no evidence of refractory anemia with excess blasts or AML. The results of this study are promising. Current recommendations for transplant in FA include serological HLA-A/B typing of the patient and the family at the time of diagnosis and HSCT from an HLA-matched sibling for patients who develop significant cytopenias. Such patients should move on to HSCT prior to the institution of androgen therapy and/or blood product transfusions. Based on the above experience, the Minnesota team is advocating either HLA-matched sibling or “matched” unrelated HSCT for all “standard risk” patients prior, if possible, to any attempt at nontransplant medical management. This recommendation is based on their excellent preliminary results, reports of poor HSCT outcomes in androgen-treated and heavily transfused patients, and the improved donor selection possible with extended molecular typing. They suggest that older and sicker patients and those without good donors should be transplanted only when more conservative measures have failed. Transplant results for patients with advanced MDS and AML are poor, but no other options exist at this time. It has been suggested that any patient with an HLA-matched sibling donor be considered for HSCT in the context of a clinical research study. However, more data are required before this can be considered a standard of care. Issues, such as the potential for nonhematologic cancers as a result of the conditioning regimen and hematopoietic malignancy as a consequence of mixed hematopoietic chimerism and residual host cells, need to be explored further. Matched-Related Donor Evaluation. The pretransplant donor evaluation should include a very careful physical exam and relevant hematologic laboratory evaluation. A chromosome breakage test for FA must be done. To rule out FA mosaicism, it has been proposed that all potential donors who have negative chromosome breakage assays using peripheral blood lymphocytes should also be tested using skin fibroblasts. In the future, mutation analysis may provide a means of screening donors for “silent” homozygosity. Recently the advent of preimplantation genetic diagnosis has provided the opportunity for families of FA and other inherited bone marrow failure syndromes to conceive unaffected HLA-matched children who could be UCB donors. A short discussion of this emerging technology is provided in the final section of this chapter.

Dyskeratosis Congenita Pathophysiology and Clinical Features. Ectodermal dysplasia and hematopoietic failure characterize DC. The classic triad of abnormal skin pigmentation, dystrophic nails, and

Inherited Bone Marrow Failure Syndromes

343

leukoplakia of mucous membranes defines DC. In addition to the classic triad, there are a number of other somatic findings. The most common of these are epiphora, developmental delay, pulmonary disease, short stature, esophageal webs, dental caries, tooth loss, premature gray hair, and hair loss. Other ocular, dental, skeletal, cutaneous, genitourinary, gastrointestinal, and central nervous system (CNS) abnormalities have also been reported (8,10). The Dyskeratosis Congenita Registry (DCR), established in 1995, has provided valuable data regarding epidemiology, pathophysiology, genetics, and treatment of DC. A review (8) reported 148 patients from 92 families emanating from 20 countries enrolled in the DCR. The median age for the onset of mucocutaneous abnormalities in patients enrolled in the DCR is 6–8 years. Nail changes occur first. Pancytopenia is the hematologic hallmark of DC. The median age for the onset of pancytopenia is ten years. Approximately 50% of patients reported in the literature develop SAA (10) and greater than 90% of individuals reported in the DCR have developed at least a single cytopenia by 40 years of age. In a number of cases aplastic anemia preceded the onset of abnormal skin, dystrophic nails or leukoplakia. As with FA, it is the nonhematologic manifestations of DC that are of particular concern when HSCT for bone marrow failure is contemplated. Recent evidence strongly supports deficient telomerase activity to be the cause of DC (41). Telomerase adds DNA sequence back to the ends of chromosomes that are eroded with each DNA replication. Telomerase activity is found only in tissues with rapid turnover, such as the basal layer of the epidermis, squamous epithelium of the oral cavity, and hematopoietic stem cells and progenitors. The lack of telomerase activity also gives rise to chromosome instability, resulting in the high rate of premature cancer observed in these tissues. Epithelial malignancies start to develop at the third decade of life. About one in five patients will develop progressive pulmonary disease, characterized by fibrosis, resulting in diminished diffusion capacity and/or restrictive lung disease. It is likely that more pulmonary disease would be evident if patients did not succumb earlier to the complications of SAA and cancer. Clearly the overall prognosis for DC is poor. Sixty-seven percent of the deaths in the DCR appear to be a consequence of bone marrow failure, whereas 9% died of lung disease with or without HSCT. Almost 9% (13/148) of patients developed cancer; four patients had MDS, one had Hodgkin disease, and eight had carcinoma. The degree of predisposition to leukemia is yet to be clearly defined. The DCR has, somewhat surprisingly, revealed the presence of significant progressive immunodeficiency in DC. Indeed the vast majority of patients (80%), with or without consequential neutropenia, who died did so from infection, some opportunistic, usually before 30 years of age. With more than half of the patients studied having a predominantly cellular immune defect, it is reasonable to assume that immunodeficiency as well as neutropenia plays a significant role in infectious morbidity and mortality in DC (8). Alter’s review of the literature reveals a median survival of approximately 35 years for both X-linked and autosomal recessive forms of DC (10). There are too few autosomal dominant cases for such an analysis. Genetics. DC is most commonly inherited as an X-linked recessive, with 86% of patients in the DCR being male, although some of these represent autosomal dominant or recessive inheritance (8). The gene responsible for the X-linked form was mapped to Xq28 (42) and subsequently identified as DKC1 (43). DKC1 codes for dyskerin, a nucleolar protein associated with nucleolar RNAs. Dyskerin is also associated with the telomerase complex. This latter function appears to be the one involved in the pathophysiology of DC (41), as the dominant form has recently been mapped to a gene that codes telomerase RNA (hTR or hTERC) (44). Furthermore the clinical heterogeneity of DC may be explained by different allelic mutations giving rise to differences in mutated proteins as well as by mutations leading to decreased amounts of normal protein. A more severe variant of X-linked DC, HoyerallHreidarsson syndrome, has been described (45). Autosomal recessive forms have been inferred from pedigrees, in particular those described with brother-sister pairs in consanguineous families (8,10). There are many features in common to all three genetic

344

Vlachos et al.

subtypes; however, autosomal recessive patients appear to have a more severe phenotype (8). Affected members within the same family may exhibit wide variability in clinical presentation, suggesting the influence of modifying genes and environmental factors. Recently, a third member of the complex, telomerase reverse transcriptase, has been found to be mutated in patients with familial aplastic anemia. However, these patients lack other clinical manifestations of DC (46). Frontline Therapy. Responses to androgens, G-CSF or GM-CSF, as well as erythropoietin, have been documented in DC (10). However, these responses have been transient. There have been temporary responses to splenectomy. Supportive care with blood products, antibiotics, and antifibrinolytics is similar to that used for idiopathic aplastic anemia. Once these measures are required, HSCT should be considered for those patients with an HLAmatched related donor or an acceptable alternative donor and no DC-related contraindications. Standard medical management of SAA using immunomodulatory therapy was, as would be expected, ineffective in a patient reported by Drachtman (47) and is not recommended.

Hematopoietic Stem-Cell Transplantation Indications, Outcomes, and the Approach to the Patient. The vast majority of patients with DC succumb to the complications of pancytopenia and/or immune deficiency. Thus it seems intuitive that HSCT be instituted at the earliest sign of significant hematopoietic or immunologic failure. Criteria, such as those recommended for FA, are reasonable (31) (i.e., an ANC %1000/mm3, hemoglobin %8 g/dl and a platelet count %50,000/mm3). Patients meeting these criteria should be considered for HSCT if a DCunaffected matched sibling donor is available. Unfortunately the results of HSCT in DC to date have been abysmal and indeed much worse than that for recent patients with FA undergoing HSCT. Patients without a matched sibling donor and those at high risk for transplant-related organ failure should be observed and managed medically for as long as is reasonable. Alter describes seven HSCT in patients with DC reported between 1982 and 1992 (10). All transplants for which the information is reported were from allogeneic HLA-matched relatives utilizing a variety of standard myeloablative conditioning regimens. Three HSCT were performed using a conditioning regimen of cyclophosphamide with TAI or TBI. Four HSCT utilized chemotherapy only. Only two patients were alive at five and six years post transplant. These patients received cyclophosphamide only and cyclophosphamide/TAI. Two patients died of complications of GVHD at 51 days and three months post HSCT, respectively. One patient died from interstitial pneumonitis eight years post transplant, one died from diffuse vasculitis two years post transplant, and one died of renal failure and veno-occlusive disease seven years post transplant. The two patients alive at the time of the report were the youngest, each two years of age at the time of transplant. The late deaths in this series indicate that those surviving patients remain at risk for serious complications related to HSCT. There have been no case control studies done to evaluate the impact of HSCT on the natural history of pulmonary disease, but the rapidity of the onset of pulmonary failure in some cases suggests an incremental relationship to HSCT. A report from the DCR briefly described eight patients with DC who underwent HSCT, seven new and one previously reported. At the time of the report three of the matched allogeneic sibling and the one unrelated HSCT were alive at four, five, seven, and three years post HSCT, respectively. Table 2 describes the cases reported in the literature (48–59). The largest available series, consisting of eight patients with aplastic anemia and DC, was reported by the group from the Fred Hutchinson Cancer Center (55). Six HSCT utilized HLA-matched sibling donors, and two were transplanted from unrelated alternative donors. Those patients who received an HLA-matched sibling transplant were conditioned with cyclophosphamide with or without ATG, whereas those receiving an alternative donor

Sibling

? Sibling

Sibling

? URD Sibling

Sibling Sibling

Sibling Sibling Sibling Sibling URD URD DP Mismatch Sibling Sibling Sibling Sibling Sibling

4/F

2/M 6/M

11/M

?/? 29/M 2/M

3/F 11/F

8/M 26/M 33/M 22/M 23/M 20/M 2/M 11/M ?/M ?/M 18/M 21/F

Cy 200 mg/kg Cy 200 mg/kg Cy 140 mg/kg ATG 90 mg/kg Cy 140 mg/kg ATG 90 mg/kg Cy 120 mg/kg TBI 12 Gy Cy 120 mg/kg TBI 12 Gy Cy 200 mg/kg TAI 3 Gy ? ? ? Cy 80 mg/kg Bu (IV) 0.8 mg/kg Cy 80 mg/kg Bu (IV) 0.8 mg/kg

Cy 200 mg/kg Cy 200 mg/kg

Cy 200 mg/kg Cy 200 mg/kg Cy 200 mg/kg

Cy 150 mg/kg TAI 6 Gy

Cy 200 mg/kg TAI 3 Gy Cy 120 mg/kg TBI 7 Gy

Bu 8 mg/kg Cy 240 mg/kg

Cy 200 mg/kg

Conditioning regimen

Died (7 days) aspergillosis Died (70 days) pulmonary fibrosis Alive (463 days) rectal carcinoma Died (44 days) acute GVHD Died (13 days) candidasis Died (14 days) candidasis Died (7 years) pulmonary fibrosis Died (4 years) VOD/liver failure Alive (8 years) pulmonary fibrosis Died (19 months) GVHD/invasive aspergillosis/CMV Alive (6 years) Alive (5 years)

Died (20 years) pulmonary fibrosis Died (8 years) pulmonary fibrosis

Alive (8 months) Rejection Alive (5 years)

Died (2 years) VOD/renal failure pulmonary fibrosis

Alive (6 years) Died (6 years) VOD/renal failure pulmonary fibrosis

Died (3 months) GVHD/GI bleeding

Died (51 days) liver failure

Outcome

Mahmoud (1985) (48) Young and Alter (1994) (10) Conter (1988) (49) Young and Alter (1994) (10) Young and Alter (1994) (10) Berthou (1991) (50) Rocha (1998) (51) Young and Alter (1994) (10) Berthou (1991) (50) Rocha (1998) (51) Young and Alter (1994) (10) Berthou (1991) (50) Dokal (1992) (52) Phillips (1992) (53) Chessells (1992) (54) Young and Alter (1994) (10) Langston (1996) (55) Langston (1996) (55) Ling (1985) (56) Young and Alter (1994) (10) Langston (1996) (55) Storb (1991) (57) Langston (1996) (55) Langston (1996) (55) Langston (1996) (55) Langston (1996) (55) Langston (1996) (55) Yabe (1996) (58) Rocha (1998) (51) Rocha (1998) (51) Rocha (1998) (51) Ghavamzadeh (1999) (59) Ghavamzadeh (1999) (59)

Reference

Abbreviations: Cy, cyclophosphamide; Bu, busulfan; GVHD, graft-versus-host disease; IV, intravenous; TAI, thoracoabdominal irradiation; TBI, total body irradiation; VOD, venoocclusive disease; ATG, antithymocye globulin; URD, unrelated donor transplant; IV, intravenous. Source: From Ref. 58.

Sibling

HLA match

Stem-Cell Transplants for Dyskeratosis Congenita

33/M

Age/sex

Table 2

Inherited Bone Marrow Failure Syndromes 345

346

Vlachos et al.

transplant were conditioned with cyclophosphamide and TBI. There were four early deaths. One patient died from refractory GVHD on day 44 post transplant, and three died of invasive fungal infection. Three of the four remaining patients developed pulmonary failure at 70 days, eight years, and 20 years post transplant, and one was alive at the time of the report but had developed Duke stage C rectal carcinoma. The three unique patients [two previously reported by Alter (10)] reported by Rocha et al. (51) also fared poorly. One patient succumbed to venoocclusive disease and hepatic failure at 17 years of age, six years post transplant. One patient developed pulmonary fibrosis, three years post transplant and was alive at the time of the report. The remaining patient died secondary to immune deficiency and an invasive aspergillus infection 19 months post transplant. An isolated case, terminating in chronic restrictive pulmonary disease at age nine, seven years post transplant from a partially matched sibling, was conditioned with cyclophosphamide and TAI (58). Two more recent cases reported by Ghavamzadeh and colleagues (59) received a milder busulfan/cyclophosphamide conditioning regimen and were alive and well at five and six years post transplant. In summary, of the 22 adequately documented cases of HSCT for DC, seven are alive, and 15 are dead. Six of the deaths are attributable to interstitial pulmonary disease, four patients had VOD and liver failure (two with concomitant pulmonary failure), and seven died from other transplant related causes. The median time to pulmonary death in these patients was 7.5 years post transplant (range, 70 days to 20 years). Thus, many of the patients remained at risk for pulmonary disease at the time they were reported. Indeed one of the living patients had pulmonary fibrosis eight years post transplant at the time of publication. A preliminary description of a recent series (60) describes five patients with DC who received HLA-matched sibling HSCT. All five were conditioned with cyclophosphamide 200 mg/kg and were alive at 20, 449, 1070, 1593, and 2949 days at the time of the abstract. These patients are too early to evaluate for pulmonary and hepatic toxicity; however, less aggressive conditioning regimens may provide better results in the modern transplant era. The number of documented cases is too few to determine the precise incremental risk of pulmonary and hepatic death as a consequence of HSCT. In conclusion, HSCT does have the potential to cure aplastic anemia in DC and should be utilized in selected patients with significant marrow failure. However the overall results have been sufficiently poor to date so as to warrant a study that would evaluate a potentially less toxic immunoablative rather than a myeloablative conditioning regimen, similar to the approach taken with FA. The cloning of both the X-linked recessive and the autosomal dominant genes for DC may allow, in the future, a predictive genotype-phenotype correlation for predisposition to pulmonary or hepatic disease in DC. Eventually tailor-made conditioning regimens, based on specific mutation analysis and designed to reduce specific organ toxicities, may become available. Currently all patients with DC should undergo a thorough pretransplant evaluation. In addition to the standard pretransplant work-up, this must include cancer screening as well as pulmonary and hepatic function tests, with tissue biopsies when indicated. The presence of certain cancers and significant pulmonary or hepatic dysfunction may preclude transplantation using even nonmyeloablative conditioning regimens. Matched-Related Donor Evaluation. We are not aware of any presymptomatic family members with DC being used as HSCT donors. However, in five of the families in the DCR, the identification of a family member with SAA and the stigmata of DC led to the posthumous diagnosis of DC in seven male family members who had died from SAA. Furthermore, patients with the presumed diagnosis of SAA have been transplanted, with the diagnosis of DC made only after the transplant (51,54,56). This suggests that there is a significant potential risk of using an asymptomatic, but genetically affected, sibling donor when performing an HSCT for DC. For this reason, the pretransplant donor evaluation should include a very careful physical and laboratory exam with a mutation analysis for either the X-linked recessive or autosomal dominant subtypes, particularly when the use of stem cells from a potentially presymptomatic newborn or young donor is contemplated.

Inherited Bone Marrow Failure Syndromes

347

Single Cytopenias Diamond Blackfan Anemia Pathophysiology and Clinical Features. DBA, first described in 1936 (61) and then more carefully categorized by Diamond and Blackfan (62), is a rare pure red cell aplasia predominantly of infancy and childhood (63) resulting from an intrinsic hematopoietic cell defect (64,65) in which erythroid progenitors and precursors are highly sensitive to death by apoptosis (2). In addition to anemia, other significant cytopenias have been reported (66). Elevated erythrocyte adenosine deaminase (eADA) activity, found in 85% of patients, macrocytosis, and elevated fetal hemoglobin are supportive but not diagnostic of DBA. The Diamond Blackfan Anemia Registry (DBAR) of North America, established in 1993 (67), has provided demographic, laboratory, and clinical data on DBA patients in the United States and Canada (68). For the 420 patients reported to the DBAR (68), the median age at presentation of anemia is two months, and the median age at diagnosis of DBA is three months. More than ninety percent of the patients present during the first year of life. Physical anomalies, excluding short stature, were found in 47% of the patients. Of these, 50% were of the face and head, 39% genitourinary, 38% upper limb and hand (including flat thenar eminences and thumb anomalies), and 30% of the heart. More than one anomaly was found in 21% of the patients. DBA has recently been recognized as a cancer predisposition syndrome. In the updated report, (69) 8 of 420 evaluable patients in the DBAR had a malignancy. Three patients had osteogenic sarcoma (ages 4, 13, and 22, years), two had MDS (ages 17 and 45 years), one had AML (age 44 years), one had colon carcinoma (age 34 years), and one had a soft tissue sarcoma (age 30 years). The cases in the DBAR occurred at younger than expected ages, and the all the patients died from toxicity of the chemotherapy or recurrent malignancy. A review of the literature revealed 23 additional cases of cancer. Among these were ten cases of AML/MDS, four lymphoid malignancies, two cases of osteosarcoma, two breast cancers, and five other cancers (70). Genetics. The first DBA gene, DBA1, has been cloned and is identified as RPS 19, a gene that codes for a ribosomal protein, located at chromosome 19q13.2 (71,72). Studies show that RPS19 mutations account for only 20–25% of both sporadic and familial cases. The function of this protein is not fully understood. A second gene, DBA2, has been inferred by linkage analysis to chromosome 8p22–23 (73). This second locus may account for 40–45% of patients. Almost 20% of families were inconsistent for linkage to 19q or 8p, strongly suggesting further genetic heterogeneity. Approximately 10% of families in the DBAR have more than one family member diagnosed with DBA. The majority of these cases appear to follow an autosomal dominant pattern of inheritance with no substantial evidence of an autosomal recessive pattern. An evaluation of family members mutated in RPS19 revealed autosomal dominant inheritance in approximately 45% of families (74). Within these pedigrees there exists considerable heterogeneity in the expression of the DBA phenotype, with some family members having no hematologic or nonhematologic manifestations of DBA (68). Frontline Therapy. Historically there have been two treatment options for patients with DBA. Since 1951 corticosteroids have been the mainstay of treatment (75), with red cell transfusions reserved for those patients who fail this modality as a consequence of either nonresponsiveness or toxicity. Approximately 80% of DBA patients will respond initially to corticosteroid therapy. The remaining 20% require transfusion therapy. Patients are usually started on prednisone at doses of 2 mg/kg and weaned to the lowest dose at which the hemoglobin concentration is both clinically acceptable and stable. Steroid-related side effects were observed in most patients, with 48%, 22%, and 12% manifesting Cushingoid features, pathologic fractures, and cataracts, respectively. The major complication of transfusion therapy is iron overload, the consequences of which include diabetes mellitus, cardiac and hepatic dysfunction, growth failure, as well as other endocrine problems. Iron chelation with desferioxamine is therefore an essential component of a transfusion program. Many patients, however, find nearly daily subcutaneous chelation therapy onerous and compliance is often poor. The oral chelator

348

Vlachos et al.

deferiprone (L1) has been associated with significant neutropenia in patients with DBA (76); however, clinical trials with a new oral chelator, deferasirox (Exjadew), have demonstrated efficacy without evidence of hematopoietic toxicity (77). At the time of the updated DBAR analysis, 37% of patients were receiving corticosteroids, and 31% were receiving red cell transfusions. Significantly, the DBAR has documented sustained hematologic remissions, defined as stable hemoglobin and no transfusion or steroid requirement for six months, in 20% of the patients. The overall actuarial survival at 40 years of age is approximately 75%: 87% for corticosteroid-maintainable patients, and 57% for transfusion dependent patients (69). The fact that both chronic corticosteroid therapy and chronic transfusion therapy may lead to a number of significant immediate and long-term complications supports a role for HSCT. Alternative Therapy. A number of treatments, including erythropoietin (78), immunoglobulin (79), megadose corticosteroids (80), and androgens (10), have been utilized in DBA patients with little success. Some responses have been confirmed with other agents. These include cyclosporine (81), interleukin-3 (IL-3) (82), and most recently metoclopramide (83). With the exception of metoclopramide, the toxicity seems unwarranted for most patients. More extensive trials with metoclopramide are underway. These agents should be explored on a case-by-case basis after evaluating the risk benefit ratio in individual patients.

Hematopoietic Stem-Cell Transplantation Indications, Outcomes, and the Approach to the Patient. The first “successful” bone marrow transplant for DBA was performed by August and colleagues in 1976 (84). The patient died of interstitial pneumonitis on day C55, but hematopoietic engraftment from donor bone marrow confirmed DBA as a transplantable disease. Since the initial case, 50 additional transplants, virtually all from HLA-matched sibling donors, have appeared in the literature (85– 102). Bone marrow was the most common stem cell source; however, UCB has also been used recently (99–101). A series of ten transplants (eight from HLA-matched siblings) was reported to the International Bone Marrow Transplant Registry (IBMTR), with a two-year survival of 58% for all patients, 72% for sibling HSCT patients (97). The French registry compiled another 13 transplants (11 from HLA-matched siblings) (102). In 1998, a review of HSCT in DBA by Alter (103) included 35 of 37 cases from the literature. The projected actuarial survival for HSCT, utilizing predominantly allogeneic HLA-matched related donors (33 siblings, one mother and one unrelated donor transplant), was 66%. The DBAR reported the results of 36 patients undergoing HSCT for DBA (66,69). The median age at transplant for all patients was 7.8 years; 7.3 years for the 21 HLA-matched sibling-matched related donor transplants versus 9.7 years for the 15 alternative donor HSCT. There was a trend towards alternative donor transplants being performed at an older age than the matched related transplants, although this difference was not statistically significant. Thirteen of the 15 alternative donor transplants were from unrelated donors, whereas two were from mismatched relatives. The major indication for HSCT was transfusion dependence. However, two patients had developed SAA, and one had significant thrombocytopenia. Twenty-eight of the 36 transplants were done using bone marrow; one used peripheral blood stem cells, six UCB, and one combined marrow and UCB as the stem cell source. All of the HLA-matched sibling HSCT were done using a nonirradiation-containing conditioning regimen, whereas the majority of alternative donor HSCT were performed using myeloablative chemotherapy and TBI. There were three transplants done using nonmyeloablative conditioning regimens, one with an unrelated marrow donor. The vast majority of patients received a cyclosporine-containing GVHD prophylaxis regimen. The actuarial survival for allogeneic HLA-matched sibling and alternative donor HSCT were 73G11% and 19G12%, respectively. Patients under ten years of age receiving sibling transplants had an improved survival of 92G8%. Toxicity of stem cell transplantation in DBA is identical to that seen in other nonmalignant diseases with only one

Inherited Bone Marrow Failure Syndromes

349

death occurring as a consequence of graft failure. Similar results have been reported to the IBMTR on an additional 61 patients undergoing HSCT for DBA (104). In general, favorable transplant outcomes are most likely if the patient is in good health at the time of HSCT, without complications of iron overload and allosensitization. Dianzani et al. (105) point out the similarity between DBA and thalassemia major and suggest that similar criteria for HSCT should be applied in both. Indeed, the ideal thalassemia major transplant candidate is a young patient who is minimally transfused or at least in good iron balance, with no evidence of significant hepatic damage, transplanted from an HLA-identical allogeneic sibling donor. Improvements in supportive care, GVHD prophylaxis and infection control have resulted in a marked decrease in HSCT transplant-related morbidity and mortality. Thus, sibling HSCT is recommended for young DBA patients, prior to the development of significant allosensitization or iron overload, when there is an available HLA-matched related donor. The outcomes for alternative donor HSCT are significantly inferior to those performed using HLA-matched sibling donors, arguing for a more conservative approach in patients without a matched related donor. A variable that complicates the decision to perform HSCT is the 20% likelihood that a DBA patient will enter a spontaneous remission by the age of 25; 72% of this total doing so by their tenth birthday. The true incidence of aplastic anemia, MDS, and hematopoietic malignancy—and the predisposition to nonhematopoietic malignancy in DBA—is being defined. Thus, the decision to undergo a HSCT in a patient with DBA must be based upon the availability of a suitable donor and an accurate assessment of the consequences of prolonged corticosteroid and transfusion therapy, as well as the natural history of DBA. Patients who develop other cytopenias, MDS or leukemia should be evaluated for alternative donor transplants on a case-by-case basis. Matched-Related Donor Evaluation. There must be a note of caution regarding allogeneic donor selection. Recently a significant number of apparently hematologically normal family members of DBA patients have been found to have a silent DBA phenotype by virtue of macrocytosis, elevated fetal hemoglobin, and/or eADA (106). An allogeneic HSCT inadvertently using one such donor, predictably, resulted in engraftment with persistent red cell aplasia (107). Allogeneic donor evaluations should therefore include assessment of eADA activity, mean corpuscular volume, fetal hemoglobin, and a thorough genetic analysis to determine the presence of a known mutation. In contrast to HLA-matched sibling HSCT, alternative donor transplantation, especially in light of a reasonable likelihood of spontaneous remission, should be considered on a case-bycase basis when individual circumstances justify the risk. Improvements in the control of GVHD as well as more precise high resolution HLA typing, and the availability of larger donor pools should lead to improved outcomes for transplants utilizing alternative donor sources. The risks of nonhematologic malignancy in patients with DBA (70) and the role of HSCT, particularly with radiation-based conditioning regimens (108), need to be investigated.

Kostmann Syndrome Pathophysiology and Clinical Features. The Severe Chronic Neutropenia International Registry (SCNIR) defines severe chronic neutropenia as three blood neutrophil counts of less than 500/mL obtained at least three months after birth within a six-month observation period; a typical pattern of recurrent fevers, chronic gingivitis, and infections at regular intervals; a bone marrow aspirate showing a “maturation arrest” at the promyelocyte or myelocyte stage; and a normal cytogenetic analysis (109). The manifestations of Kostmann Syndrome (KS) are virtually restricted to the granulocyte lineage. Classically KS was inferred when there was more than one affected child in a family with unaffected parents (suggesting an autosomal recessive mode of inheritance), whereas congenital neutropenia was reserved for a single “sporadic” case in a family. The ANC in KS is continuously less than 200/mL. Patients with SDS, described later in this chapter, are relatively easily distinguished on the basis of the clinical features of exocrine pancreatic insufficiency, malabsorption, and growth retardation. Autosomal dominant cyclic neutropenia is

350

Vlachos et al.

diagnosed when serial neutrophil counts reveal oscillations with a periodicity of approximately 21 days. There is considerable clinical overlap between what have previously been thought to be sporadic cases of congenital neutropenia and the autosomal recessive forms of KS. In this text, KS will continue to be used to refer to severe congenital neutropenia, the term preferred by some authors (110), that is neither cyclic nor SDS, regardless of its mode of inheritance. Recent advances in molecular genetics have resulted in clarification of the classification of these related disorders. Mutations of ELA2, the neutrophil elastase gene, predominate in both the autosomal dominant forms of KS (55–85%) and cyclic neutropenia (100%) (111). The defect in the ELA2 gene results in accelerated apoptosis at the promyelocyte/myelocyte stage of neutrophil differentiation. Although the molecular defect in KS is defined for the majority of cases, the precise pathophysiology is still unclear. Neutrophil elastase is synthesized and packaged in promyelocyte primary granules for release by neutrophils at sites of inflammation (112). The rather restricted tissue expression of ELA2 as compared to FANC (A-G), DKC1, hTR and DBA1 appears to correlate with the limited hematological and nonhematological manifestations of KS and cyclic neutropenia, in contrast to the other inherited bone marrow failure syndromes. Although KS has been associated with short stature, developmental delay, and microcephaly, it is unclear if these findings are a result of a genetic mutation, or secondary to the frequent infections associated with KS in the pre-G-CSF era (10). The severity and periodicity of neutropenia is a consequence of the location of the neutrophil elastase mutation and, perhaps to some degree, other genetic factors. One theory suggests that cyclic neutropenia mutations, for the most part, present at the active site of neutrophil elastase. In contrast, severe congenital neutropenia mutations largely cluster away from the active site, leading to conformational changes in the molecule, apparently resulting in more significant alterations in elastase function (111). There may however be other explanations (113) and the French Neutropenia Register fails to confirm the “active site” hypothesis (114). Prior to the availability of cytokine therapy, the prognosis for patients with KS was extremely poor. Only 30% of patients reported in Alter’s review of the literature were predicted to survive beyond their twentieth birthday, with a median and mean age at death of seven months and two years, respectively. No patients developed aplastic anemia (10). The clinical hallmark of KS is the early onset of severe bacterial infections. Omphalitis may present immediately after birth, followed by subsequent upper respiratory infections, otitis media, pneumonia, and abscesses of the skin and liver. In addition patients suffered from aphthous stomatitis, gingival hyperplasia, and consequent tooth loss (115). The vast majority died from infection, in particular pneumonia or sepsis. Occasional cases of leukemia were reported, but the high early death rate precluded a true assessment of the risk of leukemia in KS. Genetics. KS was originally described in 1956 in an extended Swedish family as an autosomal recessively inherited disorder (116). Subsequently, however, it has been shown that the majority of cases of KS and all cases cyclic neutropenia are the result of dominant-acting mutations of the neutrophil elastase (ELA2) gene mapped to chromosome 19p13.3 (117). Mutations in a zinc finger transcription factor GFI-1, which controls the expression of granulocyte specific genes and is an ELA-2 repressor, have been associated with KS (118). Frontline Therapy. Advances in our understanding of the regulation of granulopoiesis, as well as the molecular etiology and pathophysiology of KS, have been accompanied by improvements in clinical management. In particular the antiapoptotic effect of G-CSF has been exploited as a treatment strategy with dramatic results. A randomized multicenter phase III trial of recombinant human granulocyte-colony stimulating factor published in 1993 showed a 50% reduction in the incidence and duration of infection and a 70% reduction in antibiotic use (119). Data from the SCNIR on more than 300 patients indicate that more than 90% respond to G-CSF with an ANC of R1000/mL (115). A review by Zeidler and colleagues (120) describes a consensus dosing scheme for G-CSF, the goal being to find an acceptable dose that maintains

Inherited Bone Marrow Failure Syndromes

351

an ANC of R1000/mL. Nonresponders are those patients who do not have an increase in their neutrophil count sufficient to reduce infections even at a dose of up to 120 mg/kg/day. Partial responders are defined as patients whose ANC reaches 500/mL but continue to have infections. Patients whose ANC exceeds 5000/mL may have their dose reduced to the lowest effective level or change to an every other day dosing schedule. Those 5–10% of patients who fail G-CSF treatment are considered candidates for HSCT when a matched related or acceptable alternative donor exists. The use of HSCT in G-CSF-responders who have an HLA-matched sibling donor must be considered in the context of the natural history of G-CSF-treated KS and the side effects of G-CSF. The major consequences of long-term G-CSF therapy are osteoporosis, vasculitis, splenomegaly, and the appearance of leukemia/myelodysplasia (115,120,121). Rare cases of thrombocytopenia, responding to G-CSF dose adjustment, and an apparent propensity to immune complex disease have been identified. The major recognized consequence of therapy has been the emergence of a cohort of patients who develop AML/MDS (122). The predisposition to clonal disease may be inherent in KS; however, a role for G-CSF in the pathogenesis of AML/MDS cannot be ruled out. The most recent evaluation from the SCNIR reveals a cumulative incidence of AML/MDS of 21% after 10 years on G-CSF treatment, and 36% after 12 years (121). There are no survivors of AML treated with chemotherapy reported to the SCNIR (122).

Hematopoietic Stem-Cell Transplantation Indications, Outcomes, and the Approach to the Patient. Until 1993, when G-CSF was found to significantly increase the neutrophil count and ameliorate symptoms, HSCT was the only therapeutic modality for patients with KS (123). Recombinant human G-CSF has now become front-line therapy. Indeed an analysis of data from the SCNIR has resulted in the recommendation that the decision to undergo HSCT be individualized (115,120,121). In the transplant experience reported from the SCNIR (124), 11 patients were transplanted for indications other than malignancy (8 G-CSF partial or nonresponders, 1 with neutropenia prior to G-CSF availability, 1 with pancytopenia, and 1 with a G-CSF receptor mutation) between 1976 and 1998. Eight patients received an HLA-matched sibling donor HSCT. One of the eight received cyclophosphamide alone as the conditioning regimen and rejected the graft. Five of the remaining seven are alive and well. The remaining two are “cured” but have significant sequelae of HSCT: severe chronic GVHD and severe hemorrhagic cystitis requiring cystectomy. Two of the three patients transplanted from alternative donors died from transplant related complications. The final patient suffers from extensive chronic GVHD. In analyzing the decision to withhold HSCT from KS patients who have an HLAmatched related donor, three factors must be considered: first, the excellent outcome for HLA-matched sibling HSCT in the modern era; second, the actuarial risk of AML in G-CSF treated patients with KS; and third, to what extent the evolution to leukemia can be predicted. The actuarial risk of AML for G-CSF treated KS patients is 36% at 12 years. The treatment of AML in KS using conventional chemotherapy has been uniformly unsuccessful. The results for HSCT in patients with KS and MDS/AML are also poor (124). The SCINR has recorded 18 patients transplanted for leukemia, with only 3 surviving (122,124). Only one of these patients had an HLA-matched sibling donor. In view of these findings, the ability to reliably predict the emergence of AML would provide a valuable means of selecting who should undergo preemptive HSCT. Mutations in the G-CSF receptor are found in the leukemic cells of the majority of patients who evolve to AML (115,122,125). Most, but not all, patients acquire these mutations years prior to the development of overt leukemia (126). It is unknown, however, whether patients with G-CSF receptor mutations who undergo an HLA-matched

352

Vlachos et al.

sibling donor HSCT will have improved survival compared to those transplanted prior to the development of these mutations. These data suggest the need for a careful prospective evaluation to determine the reliability of G-CSF mutation analysis as a predictive assay for the development of AML and the outcome of early transplant intervention. It is reasonable to consider patients with KS who have an HLA-matched sibling donor as candidates for HSCT. Alternative donor transplants should be reserved for patients who develop G-CSF receptor mutations. Those patients refractory to G-CSF should be transplanted using the best available donor. Matched-Related Donor Evaluation. Potential HLA-matched sibling donors should be evaluated for neutropenia and, when feasible, ELA2 and GFI-1 gene mutations.

Shwachman Diamond Syndrome Pathophysiology and Clinical Features. SDS, first described in 1964, is a rare autosomal recessive disorder characterized by neutropenia, exocrine pancreatic insufficiency, and metaphyseal dysostosis (127,128). Most of the patients present in infancy or early childhood with failure to thrive and diarrhea. Classic diagnostic criteria require the documentation of pancreatic insufficiency, determined by either decreased levels of pancreatic enzymes or steatorrhea evidenced by an abnormal 72-hour fecal fat study in a patient with persistent, intermittent, or cyclic neutropenia (10). More recently low serum levels of trypsinogen and isoamylase (when these tests are available) in a neutropenic patient have been used to confirm the diagnosis. The serum isoamylase remains low throughout life, unlike serum trypsinogen, which tends to increase with age (129). An imaging study, CT, ultrasound, or MRI scan demonstrating pancreatic lipomatosis is highly suggestive of the diagnosis (130). A comprehensive review describes the variety of laboratory and clinical findings in patients with SDS (130). Neutropenia, found in 88–100% of patients, is the hematologic hallmark of SDS. In some patients there is also a defect in neutrophil chemotaxis (131). Anemia and reticulocytopenia are present in approximately 80% of patients. The anemia is usually mild; however, some patients do develop a significant macrocytic anemia. A platelet count !150,000/mm3 is common. Pancytopenia occurs in 10–65% of cases and may precede progressive bone marrow dysfunction characterized by SAA, MDS, or acute leukemia, usually of myeloid origin (10,129). Metaphyseal dysostosis and rib and/or thoracic cage abnormalities are found in 44–77% and 32–52% of patients, respectively. Poor weight gain, short stature, delayed bone maturation, delayed puberty, developmental delay and learning disabilities, hepatomegaly, elevated liver transaminases, icthyosis, frequent infections, dental caries, and dysplastic teeth are consistent features in patients with SDS (130,132). The differential diagnoses of cystic fibrosis, Pearson syndrome, cartilage hair hypoplasia, and celiac disease are relatively easily excluded. The pathophysiology of SDS is still unclear. However, diminished stem and progenitor cell numbers, manifest by reduced in vitro bone marrow progenitor cell proliferation (133–135), and a decreased CD34C count accompanied by defective marrow stroma (136) are features of SDS. As previously mentioned, the underlying pathophysiology of the hematopoietic defect in SDS, in common with the other inherited bone marrow failure syndromes, seems to be a progenitor cell predisposition to undergo apoptosis (4). Interestingly, the mean telomere length in leukocytes of SDS patients is significantly shorter than that found in normal leukocytes. The degree of telomere shortening does not, however, appear to affect the clinical severity of the disease and the significance of this observation is being investigated (137). The pancreatic insufficiency seen in SDS patients has been noted to improve with age. In many cases SDS patients are able to discontinue supplemental pancreatic enzymes as they get older. However hematologic dysfunction may progress with age (129). Patients with SDS should have regular hematologic monitoring. Dror and Freedman recommend a complete blood count every four months (130). For patients who are hematologically stable, a yearly bone marrow aspirate and biopsy for morphology and cytogenetics are suggested. Some patients may

Inherited Bone Marrow Failure Syndromes

353

require packed red cell and platelet transfusions, and the anemia may respond to erythropoietin (138). G-CSF has, as in KS, been used with a decrement in the number and severity of infections and improved quality of life for patients with critical neutropenia (109). G-CSF appears also to be effective when used during documented infections in SDS patients with less severe neutropenia (118). Acceptable acute side effects are observed with the use of G-CSF (121). However the incremental risk, if any, of MDS/AML in patients with SDS receiving the cytokine is not known. In patients who evolve to SAA or myelodysplasia, conventional medical management other than supportive care is ineffective. The time from peripheral blood count changes to myelodysplasia is unknown, as is the time from myelodysplasia to overt leukemia. The predictive value of bone marrow cytogenetic abnormalities is also unknown and controversial. Even the ability to define MDS in SDS is questionable, although Dror and Freedman make a strong case that SDS “. . . is a myelodysplastic disorder from its inception” (130). The true incidence of hematologic malignancy and myelodysplasia is unknown, although MDS and/or clonal cytogenetics and leukemia have been described in 10–44% and 5–24%, respectively, in retrospective reviews (139–142). The most common cytogenetic abnormalities are isochromosome 7 (i7q), monosomy 7 (-7), deletions involving the long arm of 7 (-7q), -5q, and other complex abnormalities involving chromosomes 7 or 5. Sometimes chromosomal abnormalities are seen only intermittently. The significance of these abnormalities is unclear as not all of the patients undergo leukemic transformation (143). In a longitudinal prospective study, 29% of patients were initially observed to have clonal marrow cytogenetic abnormalities (144). These abnormalities include i7q, -20q, and a combination of the two. At the time of the report, no patient had progressed into MDS or AML, and one of the patients had no detectable clone at two years of follow-up. Unfortunately once AML develops, the response to conventional chemotherapy is poor. In addition intolerance to chemotherapy with untoward side effects has been observed in SDS patients. For unknown reasons, the vast majority of leukemia occurs in males with SDS. Genetics. SDS is inherited as an autosomal recessive disorder. Goobie and colleagues defined the SDS locus as a 2.7centimorgan (cM) interval spanning the centromere of chromosome 7. Analysis of multiplex SDS families revealed linkage to this single locus (145). The same group refined the SDS locus to a 1.9 cM interval at 7q11 and ultimately identified the previously uncharacterized gene, SBDS (146) that may be involved in ribosome biogenesis (147).

Hematopoietic Stem-Cell Transplantation Indications, Outcomes, and the Approach to the Patient. HSCT is considered the only curative option for patients with critical neutropenia unresponsive to G-CSF, SAA, “true” MDS and leukemia. The first transplant for SDS was reported in 1989 in a 10-year-old boy with pancytopenia (7). He underwent HSCT from an HLA-identical sibling after receiving myeloablative conditioning with busulfan, cyclophosphamide, and ATG. He developed skin and gut GVHD. On day C12 he was found to have congestive heart failure. The patient engrafted on day C21 but died on day C23 of cardiac failure secondary to cardiomyopathy. Table 3 describes 21 HSCT for SDS reported in the literature to date; 10 from HLAidentical siblings, 1 from a mismatched mother, and 10 from unrelated donors. The indications for transplantation in these cases were pancytopenia/aplastic anemia and progression to myelofibrosis, MDS, or AML (7,39,140,148,149–161). Chromosome 7 abnormalities were found prior to transplant in more than 50% of the cases of MDS/AML. Eighteen patients engrafted; two patients had primary graft failure. GVHD complicated more than half of the transplants, both HLA-matched sibling and unrelated HSCT. Eleven patients are alive: 5/10 sibling HSCT, 0/1 maternal HSCT, and 6/10 unrelated HSCT. Three patients developed significant liver dysfunction, one requiring a liver transplant for veno-occlusive disease (152). One SDS patient received an HSCT using an unrelated cord blood donor (161). Two of the

HLA match

Sibling Sibling URD URD Sibling Sibling URD URD URD URD Sibling URD

Mother

URD Sibling URD Sibling Sibling

10/M 17/F 38/M 5/M 14/F 24/M ?/F 13/M 7/M 8/F 25/M 8/M

9/?

?/? 5/M 5/M 35/F 30/M

Bu/Cy/thiotepa Bu/Cy Bu/Cy/thiotepa/rabbit ALG TBI/Cy Bu/Cy/T-cell depletion

TBI/Cy/thiotepa/T-cell depletion

Bu/Cy/ATG TLI/Cy Bu/Cy TBI/Cy Bu/Cy/Campath Bu/Cy TBI/Cy/Ara-C/T-cell depletion TBI/Cy TBI/Cy TBI/Cy TBI/Cy/Campath TBI/Cy/thiotepa/T-cell depletion

Conditioning regimen

Stem-Cell Transplants for Shwachman Diamond Syndrome

Age/Sex

Table 3

Died (23 days) Cy-induced pancarditis Alive Alive hepatic dysfunction Died (1 year) BM failure Alive mild VOD Died (9 months) leukemic relapse Alive liver transplantation for severe VOD Alive Nonengraftment died (58 days) infection Died (2 months) ARDS Gut GVHD died (6 months) fungal pneumonia Died (93 days) Grade IV GVHD pulmonary hemorrhage Died (32 days) grade IV GVHD pulmonary hemorrhage Alive Alive Alive Died (135 days) liver failure Died (6 months) graft failure recurrent MDS

Outcome

Miano (1998) (39) Faber (1999) (155) Cesaro (2001) (156) Ritchie (2002) (157) Hsu (2002) (158)

Okcu (1998) (154)

Tsai (1990) (7) Barrios (1991) (148) Seymour (1993) (138) Smith (1995) (149) Smith (1996) (140) Arseniev (1996) (150) Bunin (1996) (151) Davies (1997) (152) Davies (1997) (152) Davies (1997) (152) Dokal (1997) (153) Okcu (1998) (154)

Reference

354 Vlachos et al.

Sibling Sibling Unrelated UCB Sibling URD URD ? Father Sibling Sibling URD URD Unrelated UCB ? URD Yes

TBI/Cy Melphalan/Etoposide/ATG Melphalan/TLI/Etoposide/ATG BMT #1 Bu/Cy BMT #2 TBI/Cy TBI/Cy TBI/Cy ? ? ? ? Cy/Fludarabine/ATG Fludarabine/low dose TBI/CsA/MMF ? TBI/Cy/ATG Fludarabine/low dose TBI/CSA/MMF Fludarabine/low dose TBI/CSA/MMF

Alive Alive Alive Graft failure (9 months) died (3 months) AML Died (3 months) VOD/GVHD liver Died (3 months) pulmonary infection/ARDS Died Alive Died hemorrhage Alive Died (330 days) infection Died graft failure Alive Died (10 days) fungal infection Died (7 months) graft failure sepsis Alive grade IV GVHD extensive chronic GVHD

Park (2002) (159) Fleitz (2002) (160) Fleitz (2002) (161) Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished

Abbreviations: Cy, cyclophosphamide; Bu, busulfan; GVHD, graft-versus-host disease; TBI, total body irradiation; TLI, total lymphoid irradiation; VOD, veno-occlusive disease; ATG, antithymocye globulin; CSA, cyclosporine; MMF, mycophenolate mofetil; URD, unrelated donor transplant; ARDS, adult respiratory distress syndrome; AML, acute myeloid leukemia; UCB, umbilical cord blood; MDS, myelodysplastic syndrome.

21/F 3/F 22m/F 7/F 14/M 22m/F 19/F ?/M ?/F ?/M 6/F 3/M ?/F 23/M 34/M 10/M

Inherited Bone Marrow Failure Syndromes 355

356

Vlachos et al.

patients noted above received a PBSCT, one from a matched sibling and one from a mother (154,155). Both patients died of severe GVHD. A mostly unpublished experience of five HSCT performed in four patients with SDS from a single institution utilized three sibling and two unrelated donors (7,162). All four patients died. The causes of death were cyclophosphamide-associated cardiomyopathy leading to cardiac failure, late graft failure at ten months, secondary leukemia after a second HSCT, and VOD/GVHD of the liver with acute respiratory distress syndrome. Unpublished data from the SDS International Registry report nine patients who have undergone HSCT (163). One matched-sibling HSCT is alive. Two related nonsibling HSCT have been performed, one is alive with GVHD, and one died of liver failure. Six patients received unrelated HSCT, with four receiving a myeloablative regimen. One is alive with gastrointestinal GVHD, two died of acute GVHD of the liver, and for one, the status is unknown. The remaining two unrelated HSCT were conditioned with a nonmyeloablative regimen consisting of TBI 200 cGy with mycophenolate mofetil/CSA post transplant. One adult died eight months post transplant of an infection, and one child with nonengraftment died after a second HSCT. Overall crude survival for the reported and unpublished HSCT for SDS is 43%: 7/14 for HLA-matched sibling HSCT, 1/3 for related nonsibling HSCT, and 6/16 for unrelated HSCT, with one unknown. HSCT for patients with SDS patients is fraught with many difficulties. As with all autosomal recessive genetic disorders, the probability of finding an unaffected HLA-matched sibling donor is only 3 in 16. Hepatic and cardiac toxicity, nutritional deficiencies, and preexisting infection, as well as refractory clonal hematologic disease, in particular, severely limit the efficacy of HSCT. Dror and Freedman (130) propose that the poor outcome is a consequence of a marrow stromal defect not corrected, and perhaps exacerbated by conditioning regimens, as well as critical organ apoptosis induced by radiation and/or chemotherapy. Furthermore, many transplants reported were done as a last resort for patients with advanced disease. Medical management with G-CSF and appropriate supportive care combined with surveillance for clonal hematologic disease and SAA is the preferred approach for the majority of less severely affected patients. HSCT should only be considered in SDS for patients with clinically significant aplastic anemia or myelodysplasia who fail medical management and for patients with leukemia. The best chance of a successful transplant is with an HLA-matched sibling donor or an unrelated HSCT from a molecularly matched donor. The patient must undergo a complete pretransplant evaluation specific for SDS. The assessment should include a cardiac evaluation with electrocardiogram and echocardiogram. A pulmonary evaluation is also essential with pulmonary function testing, diffusing lung capacity, and chest radiograph. A CT of the chest should also be considered for children who cannot cooperate for pulmonary function testing or for those whose work-up reveals pulmonary pathology. An evaluation of the liver is also vital and should include chemistries and possible liver biopsy (132). Documentation of underlying organ pathology necessitates individualization of the conditioning and GVHD prophylactic regimens. Clinical trials with less toxic, nonmyeloablative, preparatory regimens and tailor-made GVVD prophylaxis are indicated with specific attention given to the cardiac, hepatic, and pulmonary abnormalities found in these patients. Matched-Related Donor Evaluation. Potential HLA-matched sibling donors should be evaluated for neutropenia and perhaps pancreatic insufficiency and, when indicated, SBDS gene mutation analysis.

Congenital Amegakaryocytic Thrombocytopenia Pathophysiology and Clinical Features. CAT is the rarest of the inherited bone marrow failure syndromes discussed in this chapter. The disorder is characterized by severe hypo- or amegakaryocytic thrombocytopenia presenting in early childhood. Alter describes only 37 cases reported in the literature. In addition Alter reports physical anomalies in 16, some

Inherited Bone Marrow Failure Syndromes

357

consistent with FA or DC. She notes that those disorders could not be ruled out on the basis of a retrospective literature review. In addition, a number of cases can be reassigned as HoyeraalHreidarsson syndrome, a variant of DC (8). Thus, the number of reported cases is likely overestimated. In comparison there were, at the time of the compilation, approximately 170 reported cases of TAR, 850 FA, 200 DC, 450 DBA, 200 SDS, and 150 KS. A more recent update reveals 44 patients with presumed CAT (164); 18 of these had physical anomalies. Although many of the patients had abnormalities inconsistent with other known syndromes, it is most safe to restrict the discussion to those lacking anomalies. Furthermore, although a patient with cerebellar vermis hypoplasia has been described (165), the majority of cases of CAT confirmed by mutation analysis lack congenital anomalies. Thus, patients with amegakaryocytic thrombocytopenia and congenital anomalies must be carefully evaluated to rule out other inherited bone marrow failure syndromes. In particular it is critical that the HoyeraalHreidarsson variant of DC (characterized by cerebellar agenesis) as well as FA be ruled out in order to avoid life-threatening transplant conditioning regimen–related toxicity. TAR syndrome can be excluded on the basis of the defining congenital anomalies. Patients with TAR will improve, usually by one year of age, and can be managed with platelet transfusions (166) except under extreme circumstances (167). The 26 patients without anomalies presented with the signs and symptoms of thrombocytopenia at a median age of seven days of life. Eleven developed aplastic anemia at a median of three years. One developed AML and died, and one developed MDS (164). The median survival was only six years. Fifteen other patients died of bleeding and/or sepsis. The hematologic phenotype of CAT is variable. There appear to be two forms: one characterized by both early onset thrombocytopenia and rapid evolution to pancytopenia, the other with a later presentation of both thrombocytopenia and pancytopenia (168). Twenty one of the 26 patients reported by Alter developed thrombocytopenia in the first year of life, with 15 developing it during the first week of life. The other five cases were characterized by a later presentation, with thrombocytopenia developing between two and nine years of age. Particular mutations, described below, appear to correlate with severity. Genetics. Recently CAT was found to be associated with compound heterozygous mutations in exon 10 of the c-mpl gene, each contributed by one parent. Further evaluation showed that these mutations disrupt the function of the thrombopoietin receptor (165). Additional studies show CAT to be an autosomal recessive disorder in which virtually all cases are due to either homozygous or compound heterozygous mutations of c-mpl (168–170). Mutation analysis appears to indicate that those patients with complete loss of c-mpl function have more severe thrombocytopenia and more rapid progression to pancytopenia, in contrast to less severely affected patients who maintain some c-mpl activity (168).

Hematopoietic Stem-Cell Transplantation Indications, Outcomes, and the Approach to the Patient. The prognosis for patients with CAT is very poor. Alter’s review of the literature reports approximately 80% of bona fide patients succumbing to complications of thrombocytopenia or pancytopenia by ten years of age. Corticosteroids, androgens, cytokines, and cyclosporine have been tried with, at best, temporary responses (164,171–174). The only curative treatment has been HSCT, which is the treatment of choice. Two patients with CAT developed AML/MDS at 16 and 19 years, respectively. Transplants should be done early enough to avoid blood product sensitization as well as the development of pancytopenia or clonal disease. In FA, DC, SDS, and perhaps DBA, there is an increased risk to HSCT secondary to nonhematopoietic manifestations of the disorder. This appears not to be the case with CAT, which resembles KS in that c-mpl (like ELA2) expression appears limited (megakaryocyte progenitors, brain, and fetal liver) (175), resulting in no incremental organ toxicity that would

358

Vlachos et al.

complicate HSCT. This has been borne out by the clinical experience. We were able to find 17 reasonably well documented HSCT reported in the literature. Six unrelated and three HLA matched-related HSCT are reported as case reports (173,176–179). A variety of conditioning regimens and stem cell sources were utilized. Four of six unrelated and three of three matched related donor transplants were alive and well at the time of the reports. Five matched related, one haploidentical parent, and two unrelated HSCT (Table 4) are reported from a single center (180). All patients were severely thrombocytopenic, and two had aplastic anemia at ages 22 months and 6 years 2 months. The median age at HSCT was 2.7 years (range, 12 months to 6.5 years). The conditioning regimens included busulfan and cyclophosphamide in all cases. All but two also received ATG, with one receiving thiotepa. GVHD prophylaxis was shortcourse methotrexate and cyclosporine in all but the two peripheral blood stem cell transplants that were T-cell depleted. The outcome has been excellent. Five of five patients from the single institution and all eight who received an HSCT from an HLA-matched relative are alive and well with full engraftment, minimal acute GVHD and no chronic GVHD at the time of their report. The median follow-up for the single institution patients (including one patient transplanted with a haploidentical mother) was 17 months (range, 3–27 months). Of the nine alternative donor HSCT, five are alive at 7, 16, and 31 months for the three patients for whom such data were available. In conclusion patients with CAT should be transplanted as soon as an acceptable donor is found, prior to the development of platelet allosensitization, bleeding or neutropeniaassociated infection. Matched-Related Donor Evaluation. HLA-matched allogeneic donor selection should not present a problem, as it is unlikely those mild phenotypes exist within a family or that heterozygotes are in any way impaired. However, when related UCB or a young bone marrow donor are contemplated, c-mpl gene mutation analysis is warranted.

Table 4

Stem-Cell Transplants for Congenital Amegakaryocytic Thrombocytopenia

Age

HLA match

22 months

URD

24 months

Mother

4 years, 1 months 2 years 4 months

Sibling

12 months 3 years 6 months 6 years 6 months 4 years 4 months

Mother

URD

Conditioning regimen Bu 20 mg/kg Cy 100 mg/kg ATG Bu 20 mg/kg Cy 200 mg/kg ATG Bu 16 mg/kg Cy 200 mg/kg Bu 16 mg/kg Cy 200 mg/kg ATG

Bu 20 mg/kg Cy 120 mg/kg ATG Sibling Bu 16 mg/kg Cy 200 mg/kg ATG Sibling Bu 16 mg/kg Cy 200 mg/kg ATG Haploidentical Bu 16 mg/kg mother Cy 200 mg/kg thiotepa

Stem cell source BM

Outcome

BM

Died Bronchiolitis obliterans Alive fully engrafted

UCB

Alive fully engrafted

T-cell depleted PBSC BM

Alive fully engrafted

Died infection

BM

Alive fully engrafted

BM

Alive fully engrafted

T-cell depleted PBSC

Alive fully engrafted

Abbreviations: Cy, cyclophosphamide; Bu, busulfan; ATG, antithymocye globulin; BM, bone marrow; UCB, umbilical cord blood; PBSC, peripheral blood stem cells; URD, unrelated donor transplant. Source: From Ref. 180.

Inherited Bone Marrow Failure Syndromes

359

PERSPECTIVES ON THE FUTURE Over the last decade the “cure” of inherited bone marrow failure syndromes utilizing HSCT has been accomplished, in large part by the considerable reduction in morbidity and mortality associated with matched-related transplants. In addition, the availability of a variety of wellmatched nonrelated stem cell sources has made alternative donor transplantation a viable option under certain circumstances. Furthermore, a comprehensive understanding of the biology and clinical manifestations of the inherited bone marrow failure syndromes has led to necessary improvements in donor selection and conditioning regimens. The outcome for HSCT will undoubtedly continue to improve. Modern molecular genetics and advances in reproductive biology present another opportunity. We anticipate the cloning of virtually all the genes responsible for the inherited bone marrow failure syndromes. Mutation analysis should allow for genotype-phenotype correlations that will better define prognosis and perhaps lead to effective non-HSCT treatment strategies. When mutation analysis is available for a particular syndrome, it should be considered as part of the routine patient and donor evaluation. The young age at diagnosis of the majority of cases of inherited bone marrow failure also permits reproductive strategies. Random genetic screening of couples for the majority of these disorders is currently impractical. This will no doubt change in the future. However, for young families who already have a child diagnosed, the opportunity to have a subsequent unaffected sibling is increasing as specific mutations for many of the inherited bone marrow failure syndromes are described. Preimplantation genetic diagnosis combined with in vitro fertilization provides the chance to have not only an unaffected child but also one who is genetically selected to be an HLA-matched UCB donor. One case of a successful HSCT for inherited bone marrow failure using UCB stem cells from an HLA-matched, non-FA sibling conceived in this manner has been reported (181,182). The ethical arguments surrounding preimplantation genetics (183) and a risk-to-benefit analysis of in vitro fertilization are complex and beyond the scope of this discussion. However, more and more we, as transplanters, will be asked to weigh in as parents inquire about this approach. Knowledge of pathophysiology, natural history, nontransplant treatment options and outcomes, as well as the epidemiology and genetics of this group of complex disorders, is essential in order to guide affected families through the maze of options available to them (184). Statements regarding nonmyeloablative HSCT must be issued with a caveat. That is, this approach is still considered experimental and rapidly evolving when applied in the context of the inherited bone marrow failure syndromes. The technique has been useful in the elderly and impaired who would not otherwise be transplant candidates. As mentioned FA, DC, and SDS demand modified conditioning regimens due to disorder-associated intolerance to traditional myeloablative approaches. It remains to be seen whether such a strategy will be advantageous in other bone marrow failure syndromes for which there is no incremental intolerance to myeloablative therapy. This is of special concern in DBA, KS, and CAT, where the establishment of mixed hematopoietic chimerism may not prevent, or may even exacerbate the evolution to clonal hematopoietic disease as the remaining recipient cells may be predisposed to develop leukemia. Finally, for each of the inherited bone marrow failure syndromes discussed in this chapter, there is a spectrum of hematological as well as nonhematological manifestations. This may be the consequence of nonallelic genes within a “family” being mutated, as well as different allelic mutations. In addition the clear demonstration of variable expressivity of these disorders within a particular genotype supports a role for interacting genes and environmental factors. It is likely that there are a number of patients diagnosed with acquired cytopenias and transplanted for whom a genetic diagnosis would have been made if appropriately investigated. In some instances these patients may have unexpected toxicity (recipient effect) or graft failure (donor effect, if a genetically affected asymptomatic donor is used). These possibilities must be investigated, as technology permits, in presumed cases of acquired bone marrow failure.

360

Vlachos et al.

The choice of nontransplant treatment options, conditioning regimens, and donor selection may be influenced considerably. Allelic differences or even nonallelic polymorphisms may predict a predisposition to certain HSCT-related toxicities and, in the future, permit patient (genotype)specific conditioning regimens.

REFERENCES 1. Williams DA. Ex vivo expansion of hematopoietic stem and progenitor cells: robbing Peter to pay Paul. Blood 1993; 81:3169–3172. 2. Perdahl EB, Naprstek BL, Wallace WC, Lipton JM. Erythroid failure in Diamond-Blackfan anemia is characterized by apoptosis. Blood 1994; 83:645–650. 3. Aprikyan AG, Liles WC, Person RE, Rogers E, Dale DC. Accelerated apoptosis of bone marrow progenitor cells in severe congenital neutropenia. Blood 1999; 94:482a. 4. Dror Y, Freedman MH. Shwachman-Diamond syndrome marrow cells show abnormally increased apoptosis mediated through the fas pathway. Blood 2001; 97:3011–3016. 5. Pang Q, Christianson TA, Keeble W, Koretsky T, Bagby GC. The anti-apoptotic function of Hsp70 in the interferon-inducible double-stranded RNA-dependent protein kinase-mediated death signaling pathway requires the Fanconi anemia protein, FANCC. J Biol Chem 2002; 277:49638–49643. 6. Gluckman E, Devergie A, Dutreix J. Radiosensitivity in Fanconi anemia: application to the conditioning regimen for bone marrow transplantation. Br J Haematol 1983; 54:431–440. 7. Tsai PH, Sahdev I, Herry A, Lipton JM. Fatal cyclophosphamide induced congestive heart failure in a ten year-old boy with Shwachman Diamond syndrome and severe bone marrow failure treated with allogeneic bone marrow transplantation. Am J Pediatr Hematol/Oncol 1990; 12:472–476. 8. Dokal I. Dyskeratosis congenita in all its forms (Review). Br J Haematol 2000; 110:768–779. 9. Botnick LE, Hannon EC, Hellman S. A long lasting proliferative defect in the hematopoietic stem cell compartment following cytotoxic agents. Int J Radiat Oncol Biol Phys 1979; 5:1621–1625. 10. Young NS, Alter BP. Aplastic anemia acquired and inherited. Philadelphia: WB Saunders Co, 1994. 11. Fanconi G. Familiare infantile perniziosaartige ana¨mie (pernizioses blutbild und konstitution). Jahrbuch Kinderheil 1927; 117:257–280. 12. Giampietro PF, Adler-Brecher B, Verlander PC, Pavlakis SG, Davis JG, Auerbach AD. The need for more accurate and timely diagnosis in Fanconi anemia: a report from the International Fanconi Anemia Registry. Pediatrics 1993; 91:1116–1120. 13. Butturini A, Gale RP, Verlander PC, Adler-Brecher B, Gillio AP, Auerbach AD. Hematologic abnormalities in Fanconi anemia: an International Fanconi Anemia Registry study. Blood 1994; 84:1650–1655. 14. Kutler DI, Singh B, Satagopan J, et al. A 20 year prospective of the International Fanconi Anemia Registry (IFAR). Blood 2003; 101:1249–1256. 15. Rosenberg PS, Greene MH, Alter BP. Cancer incidence in persons with Fanconi anemia. Blood 2003; 101:822–826. 16. Auerbach AD, Rogatko A, Schroeder-Kurth TM. International Fanconi Anemia Registry: relation of clinical symptoms to diepoxybutane sensitivity. Blood 1989; 73:391–396. 17. Arkin S, Brodtman D, Alter BP, Lipton JM. A screening test for Fanconi anemia using flow cytometry. Blood 1993; 82:688. 18. Meetei AR, Levitus M, Xue Y, et al. X-linked inheritance of Fanconi anemia complementation group B. Nat Genet 2004; 36:1219–1224. 19. Joenje H, Patel KJ. The emerging genetic and molecular basis of Fanconi anaemia. Nat Rev Genet 2001; 2:446–457. 20. Grompe M, D’Andrea A. Fanconi anemia and DNA repair. Hum Mol Genet 2001; 10:2253–2259. 21. Collins N, Kupfer GM. Molecular pathogenesis of Fanconi anemia. Int J Hematol 2005; 82:176–183. 22. Levitus M, Rooimans MA, Steltenpool J, et al. Heterogeneity in Fanconi anemia: evidence for 2 new genetic subtypes. Blood 2004; 103:2498–2503. 23. Meetei AR, Medhurst AL, Ling C, et al. Human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M. Nat Genet 2005; 37:958–963. 24. Taniguchi T, D’Andrea AD. The Fanconi anemia protein, FANCE, promotes the nuclear accumulation of FANCC. Blood 2002; 100:2457–2462.

Inherited Bone Marrow Failure Syndromes

361

25. Howlett NG, Taniguchi T, Olson S, et al. Bi-allelic inactivation of BRCA2 in Fanconi anemia. Science 2002; 297:606–609. 26. D’Andrea AD, Grompe M. The Fanconi anaemia/BRCA pathway. Nat Rev Cancer 2003; 3:23–34. 27. Futaki M, Yamashita T, Yagasaki H, et al. The IVS4C4 A to T mutation of the Fanconi anemia gene FANCC is not associated with a severe phenotype in Japanese patients. Blood 2000; 95:1493–1498. 28. Faivre L, Guardiola P, Lewis C, et al. Association of complementation group and mutation type with clinical outcome in Fanconi anemia. European Fanconi anemia research group. Blood 2000; 96:4064–4070. 29. www.fanconi.org. 30. Alter B, Lipton J. Anemia, Fanconi. eMedicine Journal [serial online] 2002. (Available at: http:// www.emedicine.com/ped/topic3022.htm). 31. Harris R. Matched sibling donor hematopoietic cell transplantation. In: Owen J, Frohnmayer L, Eiler ME, eds. Fanconi Anemia, standards for clinical care. 2nd ed.. Eugene, O.R.: Fanconi Research Fund, Inc, 2003:95–108. 32. Gluckman E, Berger R, Dutreix J. Bone marrow transplantation for Fanconi anemia. Semin Hematol 1984; 21:20–26. 33. Guardiola P, Socie G, Pasquini R, et al. Allogeneic stem cell transplantation for Fanconi anaemia. Severe aplastic anaemia working party of the EBMT and EUFAR. European group for blood and marrow transplantation. Bone Marrow Transplant 1998; 21:S24–S27. 34. Kohli-Kumar M, Morris C, DeLaat C, et al. Bone marrow transplantation in Fanconi anemia using matched sibling donors. Blood 1994; 84:2050–2054. 35. Gluckman E, Auerbach AD, Horowitz MM, et al. Bone marrow transplantation for Fanconi anemia. Blood 1995; 86:2856–2862. 36. Davies SM, Khan S, Wagner JE, et al. Unrelated donor bone marrow transplantation for Fanconi anemia. Bone Marrow Transplant 1996; 17:43–47. 37. Guardiola P, Pasquini R, Dokal I, et al. Outcome of 69 allogeneic stem cell transplantations for Fanconi anemia using HLA-matched unrelated donors: a study on behalf of the European group for blood and marrow transplantation. Blood 2000; 95:422–429. 38. Kapelushnik J, Or R, Slavin S, Nagler A. A fludarabine-based protocol for bone marrow transplantation in Fanconi’s anemia. Bone Marrow Transplant 1997; 20:1109–1110. 39. Miano M, Porta F, Locatelli F, et al. Unrelated donor marrow transplantation for inborn errors. Bone Marrow Transplant 1998; 21:S37–S41. 40. MacMillan ML, Auerbach AD, Champagne MA, et al. High probability of survival after related and alternative donor hematopoietic cell transplantation for Fanconi anemia using fludarabine based preparative therapy. Blood 2003; 102:465a. 41. Marciniak R, Guarente L. Testing telomerase. Nature 2001; 413:370–373. 42. Connor JM, Gatherer D, Gray FC, Pirrit LA, Affara NA. Assignment of the gene for dyskeratosis congenita to Xq28. Human Genet 1986; 72:348–351. 43. Heiss NS, Knight SW, Vulliamy TJ, et al. X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar function. Nature Genet 1998; 19:32–38. 44. Vulliamy T, Marrone A, Goldman F, et al. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature 2001; 413:432–435. 45. Knight SW, Heiss NS, Vulliamy TJ, et al. Unexplained aplastic anaemia, immunodeficiency, and cerebellar hypoplasia (Hoyeraal-Hreidarsson syndrome) due to mutations in the dyskeratosis congenita gene, DKC1. Br J Haematol 1999; 107:335–339. 46. Yamaguchi H, Calado RT, Ly H, et al. Mutations in TERT, the gene for telomerase reverse transcriptase, in aplastic anemia. N Engl J Med 2005; 352:1413–1424. 47. Drachtman RA, Alter BP. Dyskeratosis congenita: clinical and genetic heterogeneity. Report of a new case and review of the literature. Am J Pediatr Hematol/Oncol 1992; 14:297–304. 48. Mahmoud HK, Schaefer UW, Schmidt CG, Becher R, Gotz GF, Richter HJ. Marrow transplantation for pancytopenia in dyskeratosis congenita. Blut 1985; 51:57–60. 49. Conter V, Johnson FL, Paolucci P, Ruggiero M, Janco RL. Bone marrow transplantation for aplastic anemia associated with dyskeratosis congenita. Am J Pediatr Hematol-Oncol 1988; 10:99–102. 50. Berthou C, Devergie A, D’Agay MF, et al. Late vascular complications after bone marrow transplantation for dyskeratosis congenita. Br J Haematol 1991; 79:335–336.

362

Vlachos et al.

51. Rocha V, Devergie A, Socie G, et al. Unusual complications after bone marrow transplantation for dyskeratosis congenita. Br J Haematol 1998; 103:243–248. 52. Dokal I, Bungey J, Williamson P, Oscier D, Hows J, Luzzatto L. Dyskeratosis congenita fibroblasts are abnormal and have unbalanced chromosomal rearrangements. Blood 1992; 80:3090–3096. 53. Phillips B, Judge M, Webb D, Harper JI. Dyskeratosis congenita: delay in diagnosis and successful treatment of pancytopenia by bone marrow transplantation. Br J Dermatol 1992; 127:278–280. 54. Chessells JM, Harper J. Bone marrow transplantation for dyskeratosis congenita. Br J Haematol 1992; 81:314. 55. Langston AA, Sanders JE, Deeg HJ, et al. Allogeneic marrow transplantation for aplastic anaemia associated with dyskeratosis congenita. Br J Haematol 1996; 92:758–765. 56. Ling NS, Fenske NA, Julius RL, Espinoza CG, Drake LA. Dyskeratosis congenita in a girl simulating chronic graft-versus-host disease. Arch Dermatol 1985; 121:1424–1428. 57. Storb R, Sanders JE, Pepe M, et al. Graft-versus-host disease prophylaxis with methotrexate/cyclosporine in children with severe aplastic anemia treated with cyclophosphamide and HLAidentical marrow grafts. Blood 1991; 78:1144–1145. 58. Yabe M, Yabe H, Hattori K, et al. Fatal interstitial pulmonary disease in a patient with dyskeratosis congenita after allogeneic bone marrow transplantation. Bone Marrow Transplant 1997; 19:389–392. 59. Ghavamzadeh A, Alimoghadam K, Nasseri P, Jahani M, Khodabandeh A, Ghahremani G. Correction of bone marrow failure in dyskeratosis congenita by bone marrow transplantation. Bone Marrow Transplant 1999; 23:299–301. 60. Bonfim CMS, Dokal I, de Medeiros CR, et al. Allogeneic bone marrow transplantation (allo-BMT) for patients (pts) with dyskeratosis congenita (DC). Blood 2001; 98:411a. 61. Josephs HW. Anaemia of infancy and early childhood. Medicine 1936; 15:307–402. 62. Diamond LK, Blackfan KD. Hypoplastic anemia. Am J Dis Child 1938; 56:464. 63. Lipton JM, Alter BP. Diamond blackfan anemia. In: Feig SA, Freedman MH, eds. Clinical Disorders and Experimental Models of Erythropoietic Failure. Boca Raton: CRC Press Inc, 1993:39–67. 64. Lipton JM, Kudisch M, Gross R, Nathan DG. Defective erythroid progenitor differentiation system in congenital hypoplastic (Diamond-Blackfan) anemia. Blood 1986; 67:963–968. 65. Tsai P, Arkin S, Lipton JM. An intrinsic progenitor defect in Diamond-Blackfan anemia. Br J Haematol 1989; 73:112–120. 66. Vlachos A, Federman N, Reyes-Haley C, Abramson J, Lipton JM. Hematopoietic stem cell transplantation for Diamond Blackfan anemia: a report from the Diamond Blackfan Anemia Registry. Bone Marrow Transplant 2001; 27:381–386. 67. Vlachos A, Alter B, Buchanan G, Freedman M, Glader B, Lipton JM. The Diamond Blackfan Anemia Registry (DBAR): preliminary data. Blood 1993; 82:88a. 68. Vlachos A, Klein GW, Lipton JM. The Diamond Blackfan Anemia Registry: tool for investigating the epidemiology and biology of Diamond Blackfan anemia. J Pediatr Hematol/Oncol 2001; 23:377–382. 69. Lipton JM, Atsidaftos E, Zyskind I, Vlachos A. Improving clinical care and elucidating the pathophysiology of Diamond Blackfan Anemia: an update from the Diamond Blackfan Anemia Registry. Pediatr Blood Cancer 2006; 46:558–564. 70. Lipton JM, Federman N, Khabbaze Y, et al. Osteogenic sarcoma associated with Diamond Blackfan Anemia: a report from the Diamond Blackfan Anemia Registry. J Pediatr Hematol/Oncol 2001; 23:39–44. 71. Gustavsson P, Willig T-N, van Haeringen A, et al. Diamond-Blackfan anemia: genetic homogeneity for a gene on chromosome 19q13 restricted to 1.8 Mb. Nat Genet 1997; 16:368–371. 72. Draptchinskaia N, Gustavsson P, Andersson B, et al. The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anemia. Nat Genet 1999; 21:169–175. 73. Gazda H, Lipton JM, Willig T-N, et al. Evidence for linkage of familial Diamond-Blackfan anemia to chromosome 8p23.2-23.1 and non-19q non-8p familial disease. Blood 2001; 97:2145–2150. 74. Orfali KA, Ohene-Abuakwa Y, Ball SE. Diamond Blackfan anaemia in the U.K.: clinical and genetic heterogeneity. Br J Haematol 2004; 125:243–252. 75. Gasser C. Aplastische ana¨mie (chronische erythroblastophthise) und cortison. Schweiz Med Wochenschr 1951; 81:1241–1242. 76. Hoffbrand AV, Al-Refaie F, Davis B, et al. Long-term trial of deferiprone in 51 transfusiondependent iron overloaded patients. Blood 1998; 91:295–300.

Inherited Bone Marrow Failure Syndromes

363

77. Porter J, Vichinsky E, Rose C, et al. A Phase II study with ICL670 (Exjadew), a once-daily oral iron chelator, in patients with various transfusion-dependent anemias and iron overload. Blood 2004; 104:872a. 78. Niemeyer CM, Baumgarten E, Holldack J, et al. Treatment trial with recombinant human erythropoietin in children with congenital hypoplastic anemia. In: Gurland HJ, Moran J, Samtleben W, Scigalla P, Wieczorek L, eds. In: Erythropoietin in Renal and Non-Renal Anemias, Vol. 88. Basel: Karger, 1991:276–280. 79. Sumimoto S-I, Kawai M, Kasajima Y, Hamamoto T. Intravenous g-globulin therapy in DiamondBlackfan anemia. Acta Paediatr Jpn 1992; 34:179–180. 80. Bernini JC, Carillo JM, Buchanan GR. High-dose methylprednisone therapy for patients with Diamond-Blackfan anemia refractory to conventional doses of prednisone. J Pediatr 1995; 127:654–659. 81. Alessandri AJ, Rogers PC, Wadsworth LD, Davis JH. Diamond-Blackfan anemia and cyclosporine therapy revisited. J Pediatr Hematol/Oncol 2000; 22:176–179. 82. Gillio AP, Faulkner LB, Alter BP, et al. Treatment of Diamond-Blackfan anemia with recombinant human interleukin-3. Blood 1993; 82:744–751. 83. Abkowitz JL, Schaison G, Boulad F, et al. Response of Diamond-Blackfan anemia to metoclopramide: evidence for a role for prolactin in erythropoiesis. Blood 2002; 100:2687–2691. 84. August CS, King E, Githens JH, McIntosh K, Humbert JR, Greensheer A, Johnson FB. Establishment of erythropoiesis following bone marrow transplantation in a patient with congenital hypoplastic anemia (Diamond-Blackfan syndrome). Blood 1976; 48:491–498. 85. Iriondo A, Garijo J, Baro J, et al. Complete recovery of hemopoiesis following bone marrow transplant in a patient with unresponsive congenital hypoplastic anemia (Blackfan-Diamond syndrome). Blood 1984; 64:348–351. 86. Ash RC, Montgomery T, Moreno H, Geil J, Greenwood M. Cure of steroid-refractory congenital hypoplastic anemia. Clin Res 1985; 33:334a. 87. Wiktor-Jedrzejczak W, Szczylik C, Pojda Z, et al. Success of bone marrow transplantation in congenital Diamond-Blackfan anaemia: a case report. Eur J Haematol 1987; 38:204–206. 88. Lenarsky C, Weinberg K, Guinan E, et al. Bone marrow transplantation for constitutional pure red cell aplasia. Blood 1988; 71:226–229. 89. Gluckman E, Esperou H, Devergie A, Traineau R, Leverger G, Schaison G. Pediatric bone marrow transplantation for leukemia and aplastic anemia. Nouv Rev Fr Hematol 1989; 31:111–114. 90. Skimada M, Mushigama H, Hara M. Complete recovery of hemopoiesis following bone marrow transplantation in a patient with unresponsive congenital pure red cell anemia. Jpn J Pediatr Hematol 1989; 3:276–382. 91. Zintl F, Hermann J, Fuchs D, et al. Korrektur lethal verlaufender genetischer erkrankungen mit hilfe der knochenmarktransplantation. Kinderarztl Prax 1991; 59:10–15. 92. Mori PG, Haupt R, Fugazza G, et al. Pentasomy 21 in leukemia complicating Diamond-Blackfan anemia. Cancer Genet Cytogenet 1992; 63:70–72. 93. Saunders EF, Olivieri N, Freedman MH. Unexpected complications after bone marrow transplantation in transfusion-dependent children. Bone Marrow Transplant 1993; 12:88–90. 94. Greinix HT, Storb R, Sanders JE, et al. Long-term survival and cure after marrow transplantation for congenital hypoplastic anaemia (Diamond-Blackfan syndrome). Br J Haematol 1993; 84:515–520. 95. Seip M. Malignant tumors in two patients with Diamond-Blackfan anemia treated with corticosteroids and androgens. Pediatr Hematol Oncol 1994; 11:423–426. 96. Lee AC, Ha SY, Yuen KY, Lau YL. Listeria septicemia complicating bone marrow transplantation for Diamond-Blackfan syndrome. Pediatr Hematol Oncol 1995; 12:295–299. 97. Mugishima H, Gale RP, Rowlings PA, et al. Bone marrow transplantation for Diamond-Blackfan anemia. Bone Marrow Transplant 1995; 15:55–58. 98. Van Dijken PJ, Verwijs W. Diamond Blackfan anemia and malignancy. Cancer 1995; 76:517–520. 99. Wagner JE, Rosenthal J, Sweetman R, et al. Successful transplantation of HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft-versus-hostdisease. Blood 1996; 88:795–802. 100. Bonno M, Azuma E, Nakano T, et al. Successful hematopoietic reconstitution by transplantation of umbilical cord blood cells in a transfusion-dependent child with Diamond-Blackfan anemia. Bone Marrow Transplant 1997; 19:83–85. 101. Vettenranta K, Saarinen UM. Cord blood stem cell transplantation for Diamond Blackfan anemia. Bone Marrow Transplant 1997; 19:507–508.

364

Vlachos et al.

102. Willig T-N, Niemeyer C, Leblanc T, et al. Identification of new prognosis factors from the clinical and epidemiologic analysis of a registry of 229 Diamond-Blackfan anemia patients. Pediatr Res 1999; 46:553–561. 103. Alter BP. Bone marrow transplant in Diamond-Blackfan anemia. Bone Marrow Transplant 1998; 21:965 letter. 104. Roy V, Perez WS, Eapen M, et al. Bone marrow transplantation for Diamond-Blackfan anemia. Biol Blood Marrow Transplant 2005; 11:600–608. 105. Dianzani I, Garelli E, Ramenghi U. Diamond Blackfan anemia: a congenital defect in erythropoiesis. Haematologica 1996; 81:560–572. 106. Willig TN, Pe´rignon JL, Gustavsson P, et al. High adenosine deaminase level among healthy probands of Diamond-Blackfan anemia (DBA) co-segregates with the DBA gene region on chromosome 19q13. Blood 1998; 92:4422–4427. 107. Orfali KA, Wynn RF, Stevens RF, Chopra R, Ball SE. Failure of red cell production following allogeneic BMT for Diamond Blackfan anemia (DBA) illustrates functional significance of high erythrocyte adenosine deaminase (eADA) activity in the donor. Blood 1999; 94:414a. 108. Deeg HJ, Socie G, Schoch R, et al. Malignancies after marrow transplantation for aplastic anemia and Fanconi anemia: a joint Seattle and Paris analysis of results in 700 patients. Blood 1996; 87:386–392. 109. Freedman MH, Bonilla MA, Fier C, et al. Myelodysplastic syndrome and acute myeloid leukemia in patients with congenital neutropenia receiving G-CSF therapy. Blood 2000; 96:429–436. 110. Dale DC. Introduction: severe chronic neutropenia. Semin Hematol 2002; 39:73–74. 111. Dale DC, Person RE, Bolyard AA, et al. Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia. Blood 2000; 96:2317–2322. 112. Berliner N. Molecular biology of neutrophil differentiation. Curr Opin Hematol 1998; 5:49–53. 113. Horwitz M, Benson KF, Duan Z, Li FQ, Person RE. Hereditary neutropenia: dogs explain human neutrophil elastase mutations. Trends Mol Med 2004; 10:163–170. 114. Bellanne-Chantelot C, Clauin S, Leblanc T, et al. Mutations in the ELA2 gene correlate with more severe expression of neutropenia: a study of 81 patients from the French Neutropenia Register. Blood 2004; 103:4119–4125. 115. Zeidler C, Welte K. Kostmann syndrome and severe congenital neutropenia. Semin Hematol 2002; 39:82–88. 116. Kostmann R. Infantile genetic agranulocytosis. Acta Pediatr Scand 1956; 45:1–78. 117. Horwitz M, Benson K, Person R, Aprikyan A, Dale D. Mutations in ELA2 encoding neutrophil elastase, define a 21 day biological clock in cyclic haematopoiesis. Nat Genet 1999; 23:433–436. 118. Person RE, Li FQ, Duan Z, et al. Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2. Nat Genet 2003; 34:308–312. 119. Dale DC, Bonilla MA, Davis MW, et al. A randomized controlled phase III trial of recombinant human granulocyte colony-stimulating factor (filgrastim) for treatment of severe chronic neutropenia. Blood 1993; 81:2496–2502. 120. Zeidler C, Boxer L, Dale DC, Freedman MH, Kinsey S, Welte K. Management of Kostmann syndrome in the G-CSF era. Br J Haematol 2000; 109:490–495. 121. Rosenberg PS, Alter BP, Bolyard AA, et al. The incidence of leukemia and mortality from sepsis in patients with severe congenital neutropenia receiving long-term G-CSF therapy. Blood 2006: Feb 23; [Epub ahead of Print]. 122. Freedman MH, Alter BP. Risk of myelodysplastic syndrome and acute myeloid leukemia in congenital neutropenia. Semin Hematol 2002; 39:128–133. 123. Rappeport JM, Parkman R, Newburger P, Camitta BM, Chusid MJ. Correction of infantile agranulocytosis (Kostmann’s syndrome) by allogeneic bone marrow transplantation. Am J Med 1980; 68:605–609. 124. Zeidler C, Welte K, Barak Y, et al. Stem cell transplantation in patients with severe congenital neutropenia without evidence of leukemic transformation. Blood 2000; 95:1195–1198. 125. Dong F, Brynes RK, Tidow N, Welte K, Lo¨wenberg B, Touw IP. Mutations in the gene for granulocyte colony-stimulating-factor receptor in patients with acute myeloid leukemia preceded by severe congenital neutropenia. N Eng J Med 1995; 333:487–493. 126. Tschan CA, Pilz C, Zeidler C, Welte K, Germeshausen M. Time course of increasing numbers of mutations in the granulocyte colony-stimulating factor receptor gene in a patient with congenital neutropenia who developed leukemia. Blood 2001; 97:1882–1884. 127. Shwachman H, Diamond LK, Oski FA, Khaw K-T. The syndrome of pancreatic insufficiency and bone marrow dysfunction. J Pediatr 1964; 65:645–663.

Inherited Bone Marrow Failure Syndromes

365

128. Bodian M, Sheldon W, Lightwood R. Congenital hypoplasia of the exocrine pancreas. Acta Paediatr 1964; 53:282–293. 129. Ip WF, Dupuis A, Ellis L, et al. Serum pancreatic enzymes define the pancreatic phenotype in patients with Shwachman-Diamond syndrome. J Pediatr 2002; 141:259–265. 130. Dror Y, Freedman MH. Shwachman-Diamond syndrome. Br J Haematol 2002; 118:701–713. 131. Dror Y, Ginzberg H, Dalal I, et al. Immune function in patients with Shwachman-Diamond syndrome. Br J Haematol 2001; 114:712–717. 132. Rothbaum R, Perrault J, Vlachos A, et al. Shwachman-Diamond syndrome: report from an international conference. J Pediatr 2002; 141:266–270. 133. Saunders EF, Gall G, Freedman H. Granulopoiesis in Shwachman’s syndrome (pancreatic insufficiency and bone marrow dysfunction). Pediatrics 1979; 64:515–519. 134. Woods WG, Krivit W, Lubin BH, Ramsay NKC. Aplastic anemia associated with Shwachman syndrome. In vivo and in vitro observations. Am J Pediatric Hematol Oncol 1981; 3:347–351. 135. Suda T, Mizoguchi H, Miura Y, et al. Hemopoietic colony-forming cells in Shwachman’s syndrome. Am J Pediatric Hematol Oncol 1982; 4:129–133. 136. Dror Y, Freedman MH. Shwachman-Diamond syndrome: an inherited preleukemic bone marrow failure disorder with aberrant hematopoietic progenitors and faulty microenvironment. Blood 1999; 94:3048–3054. 137. Thornley I, Dror Y, Sung L, Wynn RF, Freedman MH. Telomere shortening in leukocytes of children with Shwachman-Diamond syndrome. Br J Haematol 2002; 117:189–192. 138. Seymour JF, Escudier SM. Acute leukemia complicating bone marrow hypoplasia in an adult with Shwachman’s syndrome. Leuk Lymphoma 1993; 12:131–135. 139. Aggett PJ, Cavanagh NPC, Matthew DJ, Pincott JR, Sutcliffe J, Harries JT. Shwachman’s syndrome. A review of 21 cases. Arch Dis Child 1980; 55:331–347. 140. Smith OP, Hann IM, Chesells JM, Reeves BR, Milla P. Haematological abnormalities in Shwachman-Diamond syndrome. Br J Haematol 1996; 94:279–284. 141. Mack DR, Forstner GG, Wilschanski M, Freedman MH, Durie PR. Shwachman syndrome: exocrine pancreatic dysfunction and variable phenotypic expression. Gastroenterol 1996; 111:1593–1602. 142. Ginzberg H, Shin J, Ellis L, et al. Shwachman syndrome: phenotypic manifestations of sibling sets and isolated cases in a large patient cohort are similar. J Pediatr 1999; 135:81–88. 143. Smith A, Shaw PJ, Webster B, et al. Intermittent 20q- and consistent i(7q) in a patient with Shwachman-Diamond syndrome. Pediatr Hematol Oncol 2002; 19:525–528. 144. Dror Y, Durie P, Ginzberg H, et al. Clonal evolution in marrows of patients with ShwachmanDiamond syndrome: a prospective 5-year follow-up study. Exp Hematol 2002; 30:659–669. 145. Goobie S, Popovic M, Morrison J, et al. Shwachman-Diamond syndrome with exocrine pancreatic dysfunction and bone marrow failure maps to the centromeric region of chromosome 7. Am J Hum Genet 2001; 68:1048–1054. 146. Boocock GR, Morrison JA, Popovic M, et al. Mutations in SBDS are associated with ShwachmanDiamond syndrome. Nat Genet 2003; 33:97–101. 147. Savchenko A, Krogan N, Cort JR, et al. The Shwachman-Bodian-Diamond syndrome protein family is involved in RNA metabolism. J Biol Chem 2005; 280:19213–19220. 148. Barrios N, Kirkpatrick D, Regueira O, Wuttke B, McNeil J, Humbert J. Bone marrow transplant in Shwachman Diamond syndrome. Br J Haematol 1991; 79:337–338. 149. Smith OP, Chan MY, Evans J, Veys P. Shwachman-Diamond syndrome and matched unrelated donor BMT. Bone Marrow Transplant 1995; 16:717–718. 150. Arseniev L, Diedrich H, Link H. Allogeneic bone marrow transplantation in a patient with Shwachman-Diamond syndrome. Ann Hematol 1996; 72:83–84. 151. Bunin N, Leahey A, Dunn S. Related donor liver transplant for veno-occlusive disease following T-depleted unrelated donor bone marrow transplantation. Transplantation 1996; 61:664–666. 152. Davies SM, Wagner JE, Defor T, et al. Unrelated donor bone marrow transplantation for children and adolescents with aplastic anaemia or myelodysplasia. Br J Haematol 1997; 96:749–756. 153. Dokal I, Rule S, Chen F, Potter M, Goldman J. Adult onset of acute myeloid leukaemia (M6) in patients with Shwachman-Diamond syndrome. Br J Haematol 1997; 99:171–173. 154. Okcu F, Roberts WM, Chan KW. Bone marrow transplantation in Shwachman-Diamond syndrome: report of two cases and review of the literature. Bone Marrow Transplant 1998; 21:849–851. 155. Faber J, Lauener R, Wick F, et al. Shwachman-Diamond syndrome: early bone marrow transplantation in a high risk patient and new clues to pathogenesis. Eur J Pediatr 1999; 158:995–1000.

366

Vlachos et al.

156. Cesaro S, Guariso G, Calore E, et al. Successful unrelated bone marrow transplantation for Shwachman-Diamond syndrome. Bone Marrow Transplant 2001; 27:97–99. 157. Ritchie DS, Angus PW, Bhathal PS, Grigg AP. Liver failure complicating non-alcoholic steatohepatitis following allogeneic bone marrow transplantation for Shwachman-Diamond syndrome. Bone Marrow Transplant 2002; 29:931–933. 158. Hsu JW, Vogelsang G, Jones RJ, et al. Bone marrow transplantation in Shwachman-Diamond syndrome. Bone Marrow Transplant 2002; 30:255–258. 159. Park SY, Chae MB, Kwack YG, et al. Allogeneic bone marrow transplantation in ShwachmanDiamond syndrome with malignant myeloid transformation. A case report. Korean J Intern Med 2002; 17:204–206. 160. Fleitz J, Rumelhart S, Goldman F, et al. Successful allogeneic hematopoietic stem cell transplantation (HSCT) for Shwachman-Diamond syndrome. Bone Marrow Transplant 2002; 29:75–79. 161. Seymour JF, Escudier SM. Acute leukemia complicating bone marrow hypoplasia in an adult with Shwachman’s syndrome. Leuk Lymphoma 1993; 12:131–135. 162. Vlachos A, Lipton JM. Personal communication. 163. Shwachman Diamond Syndrome International. Personal communication. 164. Alter BP, Young NS. The bone marrow failure syndromes. In: Nathan DG, Orkin SH, eds. Nathan and Oski’s Hematology of Infancy and Childhood. Philadelphia, P.A.: WB Saunders, 1998:281– 282. 165. Ihara K, Ishii E, Eguchi M, et al. Identification of mutations in the c-mpl gene in congenital amegakaryocytic thrombocytopenia. PNAS 1999; 96:3132–3136. 166. Hedberg VA, Lipton JM. Thrombocytopenia with absent radii: a review of 100 cases. Am J Pediatr Hematol Oncol 1988; 10:51–64. 167. Brochstein JA, Shank B, Kernan NA, Terwilliger JW, O’Reilly RJ. Marrow transplantation for thrombocytopenia-absent radii syndrome. J Pediatr 1992; 121:587–589. 168. Ballmaier M, Germeshausen M, Schulze H, et al. c-mpl mutations as the cause of congenital amegakaryocytic thrombocytopenia. Blood 2001; 97:139–146. 169. Van den Oudenrijn S, Bruin M, Folman CC, et al. Mutations in the thrombopoietin receptor gene (c-mpl) in patients with congenital amegakaryocytic thrombocytopenia. Br J Haematol 2000; 110:441–448. 170. Tonelli R, Scardovi AL, Pession A, et al. Compound heterozygosity for two different amino-acid substitution mutations in the thrombopoietin receptor (c-mpl gene) in congenital amegakaryocytic thrombocytopenia (CAMT). Hum Genet 2000; 107:225–233. 171. Guinan EC, Lee YS, Lopez KD, et al. Effects of interleukin-3 and granulocyte-macrophage colonystimulating factor on thrombopoiesis in congenital amegakaryocytic thrombocytopenia. Blood 1993; 81:1691–1698. 172. Gillio AP, Gabrilove JL. Cytokine treatment of inherited bone marrow failure syndromes. Blood 1993; 81:1669–1674. 173. Henter JI, Winiarski J, Ljungman P, Ringden O, Ost A. Bone marrow transplantation in two children with congenital amegakaryocytic thrombocytopenia. Bone Marrow Transplant 1995; 15:799–801. 174. Hill W. Successful treatment of amegakaryocytic thrombocytopenic purpura with cyclosporine. N Eng J Med 1985; 312:1060–1061. 175. Columbyova L, Loda M, Scadden DT. Thrombopoietin receptor expression in human cancer cell lines and primary tissues. Cancer Res 1995; 55:3509–3512. 176. MacMillan ML, Davies SM, Wagner JE, Ramsay NK. Engraftment of unrelated donor stem cells in children with familial amegakaryocytic thrombocytopenia. Bone Marrow Transplant 1998; 21:735–737. 177. deVries DS, Bruin MC, Bierings M, Revesz T. Congenital amegakaryocytic thrombocytopenia: indication for allogenic stem cell transplantation. Ned Tijdschr Geneeskd 2000; 144:1596–1598. 178. Yesilipek MA, Hazar V, Kupesiz A, Yegin O. Peripheral stem cell transplantation in a child with amegakaryocytic thrombocytopenia. Bone Marrow Transplant 2000; 26:571–572. 179. Kudo K, Kato K, Matsuyama T, Kojina S. Successful engraftment of unrelated donor stem cells in two children with congenital amegakaryocytic thrombocytopenia. J Pediatr Hematol Oncol 2002; 24:79–80. 180. Lackner A, Basu O, Bierings M, Lassay L, Schaefer UW, Revesz T. Haematopoietic stem cell transplantation for amegakaryocytic thrombocytopenia. Br J Haematol 2000; 109:773–775.

Inherited Bone Marrow Failure Syndromes

367

181. Verlinsky Y, Rechitsky S, Schoolcraft W, Strom C, Kuliev A. Preimplantation diagnosis for Fanconi anemia combined with HLA matching. JAMA 2001; 285:3130–3133. 182. Verlinsky Y, Rechitsky S, Sharpova T, Morris R, Taranissi M, Kuliev A. Preimplantation HLA testing. JAMA 2004; 291:2079–2085. 183. Joffe S. The expanding indications for preimplantation genetic diagnosis. Oncol Spectr 2001; 2:687–696. 184. Wagner JE, Kahn JP, Wolf SM, Lipton JM. Preimplantation testing to produce an HLA-matched donor infant (letter). JAMA 2004; 292:803–804.

19 Hematopoietic Stem-Cell Transplantation for Acquired Aplastic Anemia Carole Paley Novartis Pharmaceuticals, East Hanover, New Jersey, U.S.A.

Adrianna Vlachos Division of Pediatric Hematology, Oncology and Stem Cell Transplantation, Schneider Children’s Hospital, New Hyde Park, New York, U.S.A.

Jeffrey Michael Lipton Division of Pediatric Hematology, Oncology and Stem Cell Transplantation, Schneider Children’s Hospital, New Hyde Park, New York, U.S.A.

INTRODUCTION The advent of clonal assays for pluripotent stem cells and hematopoietic progenitors, as well as the characterization of lymphohematopoietic cell surface antigens by flow cytometry, has resulted in a well-described developmental model of hematopoiesis. This schema provides a background in which to discuss marrow failure, both acquired and inherited. The proliferation and differentiation of pluripotent stem cells give rise to progeny that can populate the entire immunologic and hematopoietic systems (1). The immediate offspring of the stem cell are the committed progenitors of both the lymphoid and myeloid lineages. The multipotent myeloid progenitor, the CFU-GEMM, gives rise to the committed progenitors. In turn, each of these progenitors differentiates into the recognizable precursors of the granulocyte, erythrocyte, monocyte/macrophage, megakaryocyte, eosinophil, and basophil lineages. These progenitors appear as immature, undifferentiated mononuclear cells and are present in small numbers in the bone marrow. Cell culture and recombinant DNA technology have led to the identification and, in many cases, the cloning of the genes for a number of hematopoietic growth factors. These stimulatory and inhibitory factors influence the survival, differentiation, proliferation, and function of hematopoietic cells. Thus, in addition to hematopoietic stem cells and progenitors, the bone marrow contains a complex array of support elements or stroma populated with accessory lymphocytes, macrophages, fibroblasts, endothelial cells, and adipocytes, as well as growth agonists and antagonists. The marrow microenvironment can be compared to a garden in which stem cell “seeds” are anchored by receptor-ligand “roots” and nurtured in stromal “soil” by specific and nonspecific growth factor “fertilizer” and “nutrients”(2). The array of potential mechanisms for bone marrow failure includes faulty stem/progenitor cells, defective stroma and accessory cells, abnormal growth factors or deficient nonspecific nutrients, and immune mediated abnormalities. However, the precise pathophysiology of acquired aplastic anemia (AA) has not yet been elucidated and may, in fact, be the result of multiple different factors, or combinations of factors. 369

370

Paley et al.

Young and Alter’s comprehensive publication, in 1994, Aplastic Anemia Acquired and Inherited (3) is recommended to those readers desiring a more complete discussion of bone marrow failure. We will endeavor to provide a discussion of developments since 1994 relevant to the topic.

PATHOPHYSIOLOGY AA is a rare disorder characterized by peripheral pancytopenia and reduced or absent hematopoietic elements in the bone marrow. Bone marrow aspiration and biopsy reveal the marrow cavities to be filled with fat; the cellular components consist mainly of lymphocytes, plasma cells, and fibroblasts. The differential diagnosis for this marrow picture includes paroxysmal nocturnal hemoglobinuria (PNH), hypocellular myelodysplasia, and inherited forms of AA, and in rare instances acute lymphoblastic leukemia may present as spontaneously remitting aplastic anemia. Accelerated apoptosis (4) may play a role in each of these conditions; however, their clinical courses are distinct. It is essential to differentiate between them because their management and their pretransplant conditioning regimens differ significantly. AA must also be distinguished from the inherited marrow failure syndromes, including Fanconi anemia, dyskeratosis congenita, Shwachman Diamond Syndrome, Diamond Blackfan anemia, reticular dysgenesis, and amegakaryocytic thrombocytopenia. A more comprehensive review is provided in an excellent article by Young and Maciejewski (5). A number of agents have been implicated in the pathogenesis of AA. Radiation, drugs (chloramphenicol, antiepileptics, antineoplastics, gold), and chemical exposure (benzene, insecticides), viral infections (EBV, hepatitis, parvovirus, HIV), immunologic disorders, and clonal disorders, including myelodysplasia, PNH, and preleukemia, have all been implicated in marrow aplasia. When no etiologic agent is identified, the term “idiopathic” is applied. Longterm sequelae of AA include the evolution of clonal hematopoietic disorders, such as PNH, myelodysplasia, and leukemia. The variability in the ultimate outcomes and responses to therapy suggests that AA is actually an array of disorders having a common phenotypic expression. A variety of pathophysiologic mechanisms could, in theory, result in the marrow hypoplasia and pancytopenia characteristic of AA. These include deficient or damaged hematopoietic stem and progenitor cells, an abnormal marrow microenvironment, and immune mediated inhibition of marrow function. The observation, in the early 1970s, that infusion of syngeneic marrow without prior immunosuppression resulted in hematopoietic recovery in approximately 50% of patients provided support for a stem cell defect as the etiology of aplastic anemia (6). However, the remaining 50% of patients receiving syngeneic marrow who failed to engraft without immunosuppression gave support to the theory that other factors were also involved. In addition, some patients who received cyclophosphamide or antilymphocyte globulin as part of the transplant-conditioning regimen and failed to engraft with donor marrow subsequently recovered autologous hematopoiesis. These observations suggested an immune etiology and formed the basis for the immunosuppressive approach to the treatment of AA. Recent laboratory evidence also supports an immune mechanism. The supernatant of T-cell cultures from AA patients has increased amounts of g-interferon and tumor necrosis factor. Both of these cytokines inhibit hematopoietic colony formation and induce apoptosis in vitro (4). Theoretically AA could also result from a marrow microenvironment that fails to support hematopoiesis. However, following successful bone marrow transplantation, most of the stromal cells continue to be of host origin and are able to support hematopoiesis. Thus, current evidence does not suggest a stromal defect as an etiology for AA. An immune mechanism is unlikely in those instances where syngeneic stem cells can reconstitute hematopoiesis without immunosuppression. A genetic basis for some cases of apparent AA is suggested by the finding of germline telomerase reverse transcriptase (TERT) mutations in some patients (7). These mutations impair telomerase activity and may be a risk factor for bone marrow failure. It is notable that none of the patients in whom TERT mutations were found responded to immunosuppressive therapy (IST).

HSCT for Acquired Aplastic Anemia

371

EPIDEMIOLOGY The incidence of AA in the United States and Europe is 2–3 per million per year. Aplastic anemia is more common in East Asia, with an incidence of 6–15 per million per year. Geographic variations in incidence appear to be more a function of environmental exposure (e.g., solvent exposure in Thailand, increased incidence of hepatitis in Asia) than of genetic predisposition. Aplastic anemia is traditionally considered to be a disease of the young. The age distribution of AA, however, shows two peaks: one at 15 to 25 years of age, and one at greater than 60 years of age. The peak in those over 60 years of age likely includes cases of hypoplastic myelodysplastic syndrome (MDS).

CLINICAL FEATURES Camitta et al. in 1975, defined criteria for distinguishing severe AA (SAA) in order to more precisely analyze responses to treatment (8). SAA was defined as a marrow cellularity of less than 25% and at least two of the following criteria: absolute neutrophil count (ANC) !500/ul; platelets !20,000/ul; absolute reticulocyte count !40,000/ul. Subsequently, very SAA was defined in those patients with an ANC !200/ul. It was observed that these patients had the poorest outcome. The clinical manifestations of AA result from pancytopenia and consist of bleeding, infection, and the consequences of anemia. Historically, without definitive therapy, more than 70% of patients died within two years of diagnosis; for those with SAA more than 50% succumbed within six months. Advances in supportive therapy including transfusions and antibiotics significantly prolonged survival. With the advent of sibling-matched transplants in the mid 1970s, followed by the introduction of immunosuppressive therapies, survival rates continued to improve. Camitta’s early review (8) unequivocally demonstrated that transplantation from an human leukocyte antigen (HLA)-matched sibling was the treatment of choice for pediatric patients with SAA. With continued improvements in both transplantation and IST, the superiority of sibling-matched transplant continues to be demonstrated. Currently, front-line therapy consisting of either immunosuppressive treatment or matched sibling transplant has an overall response rate of 70–90% in children (9).

IMMUNOSUPPRESSIVE (IMMUNOMODULATORY) THERAPY FOR SEVERE APLASTIC ANEMIA Antithymocyte Globulin and Cyclosporine Only 20–30% of patients will have an available sibling matched donor, thus IST has been frontline treatment for the majority of patients diagnosed with SAA. Several excellent reviews detail the evolution of IST for SAA (9–12). The combination of Antithymocyte globulin (ATG) and Cyclosporine (CSP) has become standard therapy. Initial hematopoietic response rates are similar to those seen with allogeneic transplant; however, long-term sequelae differ. Hematologic responses have been categorized as either partial or complete. Partial response is generally defined as transfusion independence, and complete response is defined as normalization of blood counts. Response rates in several trials, including both pediatric and adult patients, range from 70–82% (13). In pediatric patients with very SAA response rates of 90% at 18 months (65% complete and 25% partial) were reported by the German/Austrian Pediatric Aplastic Anemia Working Group (13). Several trials evaluating the addition of various hematopoietic growth factors to IST have been undertaken. Most have been terminated secondary to adverse effects or lack of efficacy. G-CSF, however, has repeatedly been shown to hasten neutrophil recovery, and is commonly added to ATG/CSP regimens.

372

Paley et al.

Several clinical points become apparent when evaluating responses to IST. First, response is slow and proceeds incrementally. Although 50% of responders will demonstrate some response by three months and 75% by six months, responses may be seen as late as one year from the initiation of therapy. Results vary across the different trials, but a significant percentage of responses are partial with patients requiring ongoing maintenance therapy. Thirty percent of those who respond will eventually relapse (10). Relapses may respond to reinstitution of CSP or retreatment with ATG and CSP. Fuhrer et al. reported on eleven children who relapsed within twelve months of IST. Seven responded to CSP monotherapy (14). Young and colleagues report that for some patients, CSP therapy must be maintained chronically (13). The evolution of clonal disease following IST is not uncommon. In one series, laboratory evidence of PNH developed in 13% of patients, myelodysplasia in 10%, and AML in 7% (15). Results of the Pediatric Aplastic Anemia Cooperative Trials (PAACT) indicate that the rate of evolution of clonal disease following IST is lower in pediatric patients than in adults, thereby favoring frontline IST over alternative donor transplantation. For patients who relapse following IST, a second course of IST is commonly given; however, alternative donor transplant is also undertaken particularly if a well-matched unrelated donor (URD) is available. With ongoing improvements in outcome for alternative donor transplant this modality may become a frontline option in the future.

OTHER IMMUNOLOGIC THERAPIES Research is ongoing to identify new agents or combinations that may increase initial response rates and/or achieve hematologic response following relapse. Encouraged by reports of autologous marrow recovery in transplant patients following graft failure, Brodsky treated ten patients at diagnosis with high dose (HD) cyclophosphamide (45 mg/kg/day over 4 days) with or without CSP. Complete responses occurred in seven of these patients (16). Initial reports described a lower rate of evolution to clonal disorders with the use of cytoxan (CTX). Subsequent reports, however, have documented significant toxicity, particularly a high incidence of fungal sepsis, as well as late complications, including marrow cytogenetic abnormalities, potentially indicative of evolution towards MDS (17,18). Tisdale reported on a Phase III randomized trial comparing ATG/CSP with HD CTX (17,18). Secondary endpoints included relapse rate and clonal evolution. Thirty-one patients enrolled; 15 were assigned to CTX and 16 to ATG. All 31 patients received the full course of treatment; however, the trial was terminated prematurely after four cases of invasive fungal infection and three deaths in the CTX arm. Follow-up at a median of 38 months showed that 13 of 16 patients assigned to ATG (81%) and 8 of 15 patients assigned to CTX (53%) had hematologic responses. Relapses occurred in six (46%) of 13 responders in the ATG arm and two (25%) of eight responders in the CSP arm. Bone marrow cytogenetic abnormalities were observed in 2/14 treated with ATG and 1/12 treated with CTX. Based on these findings, Tisdale concluded that CTX confers no advantage in terms of hematologic response, relapse, or MDS risk, but it does confer a higher risk of morbidity and mortality. The National Heart, Lung, and Blood Institute is currently assessing the efficacy of a combined regimen of ATG, CSP, and mycophenolate mofetil in treating AA. Ongoing National Institute of Health (NIH) trials include: (1) a comparison of ATG/CSP with ATG/CSP and rapamycin (an immunosuppressive agent that arrests T-cell growth), and (2) in patients refractory to a single course of ATG, a comparison of rabbit ATG and Campath-1H, a monoclonal antibody directed at T cells.

HEMATOPOIETIC STEM-CELL TRANSPLANTATION FOR SEVERE APLASTIC ANEMIA It is important to begin the discussion of hematopoietic stem cell transplantation (HSCT) for SAA by underscoring the importance of a complete evaluation to rule out inherited bone

HSCT for Acquired Aplastic Anemia

373

Table 1 Five-Year Survival According to Stem Cell Source in 2002 Patients Transplanted from 1976–1998 (European Bone Marrow Transplant Group Data)

Stem cell source

N

Median age (years)

Identical twin HLA-identical sib Alternative donor

34 1699 269

22 19 15

Time from Dx. to SCT (days)

Graft failure (%)

5 year survival (%)

84 97 303

10 12 26

91 66 37

Abbreviations: Dx, diagnosis; HLA, human leukocyte antigen; SCT, stem cell transplant. Source: From Ref. 25.

marrow failure syndromes, such as Fanconi anemia, dyskeratosis congenita, and Shwachman Diamond syndrome prior to initiating transplant therapy. The failure to identify these disorders prior to transplant may result in significant toxicity as a consequence of inappropriate conditioning regimens. Stem cell transplantation provides curative therapy for SAA. Outcomes for siblingmatched transplants have improved progressively since their introduction in the mid-1970s. More recently outcomes are also improving for alternative donor transplants. Survival for HLA-matched sibling transplants has increased from 48% in the 1970s to 66% in the late 1980s and to 70–90% in current series (19,20–24). Bacigalupo’s analysis of 2002 patients transplanted in Europe between 1976 and 1998 (25) and the results from other groups provide a comprehensive overview of this subject (26–29). Table 1, based on Bacigalupo’s review, summarizes the extensive European Bone Marrow Transplant group (EBMT) data on transplantation for SAA from 1976 to 1998.

Syngeneic Transplant E. Donnell Thomas’s group in Seattle pioneered the use of bone marrow transplant for SAA. These early transplants used identical twin donors. In 1966 the Seattle group reported on the successful treatment of AA by infusion of syngeneic marrow (30). The reviews by Champlin, Lu, and Storb summarize the results of 70 syngeneic transplants for SAA (6,31,32). In these cumulative reviews, infusion of donor stem cells was adequate to restore hematopoiesis in 50% of patients. The remainder of patients failed to engraft unless HD cyclophosphamide or other immunosuppressive agents were employed. These results provided insight into the pathophysiology of AA and suggested that some cases result from primary hematopoietic stem cell defects, whereas others are secondary to immune mediated mechanisms. The fact that sustained hematopoiesis is established following successful engraftment indicates that a defect in the marrow microenvironment is unlikely.

Allogeneic Transplant The majority of transplants for AA over the past 30 years have used HLA-identical sibling donors. The first successful allogeneic transplant for SAA was reported in 1972; this transplant utilized a HD cyclophosphamide preparative regimen. Throughout the seventies and eighties, conditioning regimens for allogeneic transplants consisted of cyclophosphamide 40 or 50 mg/kg/day for four days or 60 mg/kg/day for two days, given alone or in combination with radiation. Various raditation regimens were employed, including total lymphoid, thoracoabdominal, or total body irradiation. In the late 1970s the combination of cyclophosphamide and ATG was shown to be successful in conditioning patients for second transplants following failure of first transplants where cyclophosphamide alone had been used. Based on the success

374

Paley et al.

of these second transplants, the Seattle team, in 1988, pioneered the use of cyclophosphamide and ATG for first transplants. Long-term follow-up of sibling allogeneic transplants for SAA has demonstrated sustained donor hematopoiesis 20 years following engraftment. Wagner and Storb summarized allogeneic transplant outcomes for SAA in the 1980s and 1990s (28). The mean age of patients was 25 years, with a range of 1 to 57 years. Nearly all patients had received transfusions prior to transplant, a factor that negatively impacts on outcome by increasing the chance of graft rejection. The major complications were graft failure, acute, and chronic graft-versus-host disease (GVHD) and late toxicities of the conditioning regimen, including endocrine and gonadal dysfunction, cataracts, and an increased rate of malignancies (particularly in patients who received radiation).

Graft Failure Graft rejection was observed in 30–60% of patients prior to 1975. Animal models and retrospective analyses suggested that these high rejection rates resulted in large part from sensitization of patients to minor histocompatibility antigens through blood transfusions (27). In the early 1980s, more intensive immunosuppressive regimens, including cyclophosphamide and TBI or total lymphoid irradiation for multiply transfused patients, were employed. In 1981 the International Bone Marrow Transplant Registry (IBMTR) reported on outcomes in 595 patients (33). Rejection rates were significantly lower with the more immunosuppressive regimens; however, there was no increase in overall survival because higher rates of interstitial pneumonitis and GVHD were seen. In addition, follow-up studies revealed increased rates of secondary malignancies, cataracts, and aseptic necrosis, as well as impaired growth, development, and fertility in patients receiving radiation-containing regiments (34). Following the introduction of CTX/ATG conditioning regimens for first transplants in the late 1980s, most centers adopted this regimen. A review of transplants using CTX/ ATG in 84 patients, including the Seattle experience, revealed a graft rejection rate of only 4% (35). A more recent approach to improving engraftment has been to maximize the number of stem cells (ideally O3!108 mononuclear cells/kg) transplanted. The use of G-CSF-mobilized peripheral blood stem cells results in more rapid engraftment (compared to marrow) that is, unfortunately, counterbalanced by higher rates of acute and chronic GVHD (36). In transplants for SAA therefore, the increased risk of GVHD must be weighed against the benefits of more rapid engraftment when considering the stem cell source.

GRAFT-VERSUS-HOST DISEASE Acute Graft-Versus-Host Disease GVHD is a major cause of morbidity and mortality following transplant, and many studies have focused on decreasing its incidence and severity. In 1992 the IBMTR reported on the impact of acute GVHD on the five-year actuarial probability of survival (33). Patients with Grades II, III, and IV acute GVHD had a five-year actuarial probability of survival of 31% compared to 80% for those with no or mild GVHD. The earliest attempts at decreasing acute GVHD utilized posttransplant methotrexate (MTX). In the 1980s some centers began using T-cell depletion in order to decrease GVHD. This modality successfully reduced the incidence of severe GVHD but resulted in increased graft failure (37). In a 1994 review, Storb et al. demonstrated that the combination of CSP and MTX was superior to the use of MTX alone (38). The randomized prospective Italian Bone Marrow Transplant Group/European Bone Marrow Transplant group (GITMO/EBMT) trial confirmed that patients with AA receiving MTX/CSP GVHD prophylaxis had significantly improved survival compared to those receiving CSP alone (94% compared to 78%) (39). In all reviews of the incidence of acute and chronic GVHD, children fare better than adults. In the IBMTR review of the incidence of acute and

HSCT for Acquired Aplastic Anemia

375

chronic GVHD following transplants for AA from 1991 to 1997 the incidence of Grade II-IV acute GVHD in children was 15–20% while in adults the incidence was 40–45%. Improvements in GVHD prophylaxis over the past thirty years have paralleled progress in the field in general. Although the incidence and severity of acute GVHD continue to decline, there is still room for further improvement, particularly in the setting of alternative donor transplants.

Chronic Graft-Versus-Host Disease In spite of the significant progress made in reducing the incidence of acute GVHD, chronic GVHD persists as a complication of HSCT for SAA, particularly in adults. The major risk factors for chronic GVHD are antecedent acute GVHD, older patient age, and the use of supplemental buffy coat infusions. In Storb’s 1977 review, the incidence of chronic GVHD ranged from 40–60% (40). Several more recent reviews of pediatric patients report the incidence of chronic GVHD following sibling donor transplants as ranging 0–25% (41,42). Since the omission of buffy coat infusions, the use of CTX/ATG conditioning regimens, and the use of CSP/MTX for GVHD prophylaxis, most current series report a combined pediatric and adult chronic GVHD incidence of 30% to 35% (35). Treatment of chronic GVHD requires prolonged IST, with the attendant complications of infection, avascular necrosis, and osteoporosis. Quality of life is profoundly impacted both by chronic GVHD as well as by long-term immunosuppressive regimens. Agents that have been used to treat chronic GVHD following transplant for SAA include prednisone, azathioprine, cyclophosphamide, procarbazine, and thalidomide. Further improvements in chronic GVHD prevention are needed in order to improve outcome for transplant in SAA.

SURVIVAL In the 1970s long-term survival rates following HLA-matched sibling transplants for SAA ranged from 40% to 45% (40). Survival rates have doubled over the past three decades. This improvement has resulted mainly from reductions in the rates of graft failure and of acute and chronic GVHD. The GITMO/EBMT reported 94% survival at a median follow-up of two years (39). Several smaller series have reported survival rates ranging from 87–100% (41,43,44).

HEMATOPOIETIC STEM-CELL TRANSPLANTATION COMPARED WITH IMMUNOSUPPRESSIVE TREATMENT Outcomes for both IST and HSCT have improved considerably. Currently both modalities have survival rates greater than 80%. The comparison of hematologic response rates, as well as longterm outcomes, firmly supports HSCT as the treatment of choice for children with SAA when an HLA-matched related donor is available. HSCT is curative for most patients but still poses a risk of early mortality and long-term sequelae. IST improves hematopoiesis to transfusion independence in the majority of patients, but time to response is long, hematopoietic response may be partial, and relapses are relatively common. In addition, clonal hematologic disorders, including PNH, myelodysplasia, and leukemia, develop in 10% of patients treated with IST (45). Several retrospective reports have compared the efficacy and long-term outcomes of IST and HSCT for AA (19,25,46,). Bacigalupo presented an analysis of 1765 patients with AA treated with either sibling transplant (nZ583) or IST (nZ1182) between 1974 and 1996 (25). The outcomes were analyzed in subsets according to age and neutrophil count. The data indicate that matched sibling donor HSCT is always superior in young patients (!20 years of age) at any neutrophil count. Immunosuppression was superior in older patients (41 to 50 years) with a neutrophil count greater than 0.5!109/L. However, in patients 41 to 50 years old with an ANC

376

Paley et al.

of 0.1!109/L the advantage for IST is significant only for the first three years; after this, the advantage disappears due to relapse following IST. For the 21 to 40 years age group, the differences were less clear. Thus patients in this age group must be carefully evaluated on a caseby-case basis. For all age groups there was a higher percentage of late failures for the IST patients. The difference in survival between patients treated with HSCT and IST is not constant but increases with time. For the younger group of patients, a 10% advantage in favor of BMT at one year post transplant became a 19% advantage at 5 years. These data confirm the higher risk of late deaths in patients treated with IST due to complications including relapse and evolution to clonal disorders. Kojima presented data on 100 children less than 17 years of age treated with IST (nZ63) or matched sibling HSCT (nZ37) (19). Because of the high relapse rate for IST, the data was analyzed in terms of failure free survival (FFS). The data showed a clear advantage for HSCT. The probability of FFS at 10 years was 97C/K 3% for the HSCT group and 40C/K 8% for the IST group (PZ0.0001). Seven patients, all treated with IST, evolved to MDS with monosomy 7, and the estimated cumulative incidence of MDS at 10 years from diagnosis was 20C/K 7% (Table 2 compares the outcomes of SCT and IST in several published series). Socie et al. (55) compared the rate of secondary malignancies following HSCT and IST. Forty-two malignancies developed in 860 patients receiving IST, compared to 9 in 748 patients who underwent HSCT. In this study, acute leukemia and myelodysplasia were seen exclusively in IST treated patients, whereas the incidence of solid tumors was similar in the two groups of patients. The data presented in the above studies is retrospective, and covers a period of nearly 30 years, during which time IST and transplant regimens have changed significantly. The Pediatric Blood and Marrow Transplant Consortium and the PAACT are currently conducting a prospective study comparing outcomes of IST and HSCT in children with SAA using immunosuppressive and transplant regimens. For patients lacking a sibling donor, a future prospective trial comparing alternative donor transplantation with IST might provide further insight into the appropriate choice for the front line therapy of AA.

Table 2 Published Trials Comparing Survival Following Stem-Cell Transplant or Immunosuppressive Therapy SCT

IST

Author, Year

Ped/Adult

N

2–6 year survival (%)

N

2–6 year survival (%)

Speck, 1981 (47) Bayever, 1984 (48) Bacigalupo, 1988 (49) Halperin, 1989 (50) Speck, 1990 (51) Doney, 1990 (52) Locasciulli, 1990 (53) Paquette, 1995 (54) Lawlor, 1997 (20) Gillio, 1997 (21) Fuhrer, 1998 (22) Pitcher, 1999 (23) Kojima, 2000 (19) Ahn, 2003 (24)

Ped/Adult Ped Ped/Adult Ped Ped/Adult Ped/Adult Ped Adult Ped Ped Ped Ped Ped Adult

18 35 218 14 34 131 171 55 9 25 26 10 37 64

44 72 63 79 50 68 63 72 75 76 84 93 97 79

32 22 291 12 111 139 133 155 18 23 86 25 63 156

69 45 61 25 71 52 48 45 92 74 87 86 54 65

Abbreviations: SCT, stem cell transplant; IST, immunosuppressive therapy; Ped, !25 years. Source: From Refs. 19, 23, 26.

HSCT for Acquired Aplastic Anemia

377

ALTERNATIVE DONOR TRANSPLANT The treatment of choice for SAA in children is HLA-matched sibling transplant if a donor is available. For the remainder of patients IST is instituted. Alternative donor transplant, using either matched URDs or partially mismatched family members, is currently recommended in SAA only after the failure of IST. Because it can take anywhere from several months to a year to see the full impact of IST, alternative donor transplant is undertaken relatively late in the course of the illness. Outcomes of alternative donor transplants are significantly inferior to those of HLA-matched sibling donor transplants. Two major factors contribute to the poor outcomes. First, there is a greater minor antigen disparity between donor and host, resulting in a higher incidence of acute and chronic GVHD. Second, emerging data suggest that the duration of time from IST failure to transplant is an important factor in determining a successful transplant outcome. Patients undergoing alternative donor transplant are more likely to have developed platelet refractoriness, infections, and organ toxicity, such as renal impairment, from prolonged CSP use. For this reason the failure of IST must be conceded early in the course of treatment so as not to preclude successful alternative donor transplants, particularly if a wellmatched URD is available.

UNRELATED DONOR TRANSPLANT In the 1990s, URD transplants for SAA had poor outcomes. In 1999 Deeg et al. published a retrospective review of 141 patients who had received matched URD transplants for SAA between 1988–1995. The survival rate for this cohort of patients was only 37% (56). This review and other series demonstrated that CTX/ATG conditioning was inadequate to support the sustained engraftment of URD marrow (57). In 2001, the Seattle group reported on a multicenter prospective trial to determine the minimal dose of TBI necessary to achieve sustained engraftment when combined with CTX/ATG (58). Forty patients ranging in age from 1.3 years to 46.5 years were transplanted. TBI was given in escalating doses from 1!200 cGy to 3!200 cGy. Pulmonary toxicity was highest in patients receiving 3!200 cGy concurrently with CTX/ATG. All patients receiving 3!200 cGy and 2!200 cGy achieved sustained engraftment; however, toxicity was higher in the former group. The study concluded that 2!200 cGy combined with CTX/ATG was adequate for sustained engraftment. The highest probability of survival (73%) was seen in patients who underwent transplant within one year of diagnosis. In addition, younger patients (!20 years of age) were more likely to survive than older patients. Kojima et al. reported on 15 patients less than 20 years of age who underwent URD transplant for SAA unresponsive to IST (59). Pretransplant conditioning consisted of CTX/ATG and 250 cGy TBI, with CSP/MTX used for GVHD prophylaxis. All patients engrafted and were alive at a median follow-up of 51 months. Moderate to severe acute GVHD occurred in 5 of 15 patients, and one patient developed extensive chronic GVHD. In 2001 Vassiliou described the successful transplant of eight children with SAA using alternative donors (seven matched URDs and one HLA-DP mismatched sibling) (60). Seven of the eight patients demonstrated 100% donor hematopoiesis, and one showed low-level recipient chimerism. None of the patients developed severe acute GVHD or chronic GVHD. The conditioning regimen employed Campath-1G (0.2 mg/kg/d for 5 days), cyclophosphamide (50 mg/kg/d for 4 days), and TBI (300 cGy as a single fraction) in seven of the patients. The youngest patient, a seven-month-old infant, received fludarabine (25 mg/m2/day for 5 days) instead of TBI. The positive outcomes in this small series indicate that intensive immunosuppression and effective GVHD prohylaxis may decrease the role of radiation in alternative donor transplants. This novel conditioning regimen will need to be studied in a larger series in order to confirm its efficacy. These results taken in conjunction with the increasingly refined molecular HLA testing currently available suggest that, in young patients with an excellent molecularly matched URD,

378

Paley et al.

URD transplantation may provide a reasonable alternative to IST. This approach requires further validation. This decision has to take into account the higher rates of clonal disease evolution following IST and the long-term toxicity of TBI-containing regimens. For those patients who fail IST, it is clear that results for URD transplant are superior when the transplant is undertaken early in the course of the disease.

HLA-NONIDENTICAL RELATED DONORS Data from the IBMTR reveal that only 5% of transplants performed for SAA over the past decade were from HLA-nonidentical related donors. In 1996 Wagner reviewed the Seattle experience and concluded that the addition of 1200 cGy TBI to the conditioning regimen improved survival in mismatched transplants by reducing graft rejection (61). In 2001, Margolis and Casper reviewed the status of alternative donor transplants for SAA (62). The probability of survival at three years was 45% for haploidentical family member transplants and 33% for unrelated matched donor transplants. The interval from diagnosis to transplant repeatedly was demonstrated to be an independent prognostic factor. Poor survival was secondary to a multitude of factors, including nonengraftment, GVHD, infection, and organ failure resulting from drug or radiation toxicity. The most important variables in the success of alternative donor transplant were patient age, time interval from diagnosis, and the degree of histocompatibility between donor and host. Currently, results for haploidentical transplants are clearly inferior to those for high resolution, molecularly matched URD transplants. Although a haploidentical donor is available for nearly all patients, the high rates of rejection, GVHD, and toxicity must be overcome before this type of transplant can be widely used in the treatment of SAA.

CONCLUSIONS AND FUTURE PERSPECTIVES Over the past three decades improvements in survival following matched sibling transplant for SAA have largely been due to improved conditioning regimens and advances in acute and chronic graft versus host disease prophylaxis. Conditioning with cyclophosphamide and ATG supports sustained donor hematopoiesis, and radiation therapy is not necessary for matched sibling transplants. The combination of CSP and MTX has significantly decreased acute and chronic GVHD; although chronic GVHD continues to be a problem, particularly in adults. Better methods for preventing and treating chronic GVHD in high-risk groups are still needed. Survival for alternative donor transplants has improved since the 1990s. Intensification of conditioning regimens by adding TBI has improved engraftment in both haploidentical and matched URD transplants. However these improvements come at the cost of the long-term sequelae of TBI, including impaired growth and development, endocrine dysfunction, organ toxicity, and the development of secondary malignancies. There is increasing evidence that fludarabine containing regimens may be able to support sustained engraftment in alternative donor transplants without the use of TBI. Lee et al. reported in 2005 on 13 patients with SAA who received alternate donor transplants utilizing non-TBI containing preparative regimens. Nine received CTX/ATG, two CTX/fludarabine, and two CTX/fludarabine/ATG (63). All patients engrafted at a median of 21 days. Grade III–IV acute GVHD developed in three (23%) and extensive chronic GVHD in four patients (31%). At a median follow-up of 3.1 years (range: 0.3–4.3 year) ten patients (74.6%) are alive. Kang et al. (64) reported on five patients with SAA who underwent alternative donor transplant with preparative regimens consisting of fludarabine, cyclophosphamide, and ATG. All patients attained hematologic recovery. Four of five are alive at a median of 1.6 years. The median time from diagnosis to transplant was six months, again demonstrating the advantage of proceeding to alternative donor transplant as soon as the necessity is realized. Trials are ongoing to further evaluate the role of fludarabine in alternative donor transplant.

HSCT for Acquired Aplastic Anemia

379

Refinements in molecular HLA typing, as well as improvement in preparative and antiGVHD regimens have contributed to improved outcomes for URD transplants for SAA over the past decade. In addition, it has become clear that URD transplants have the best outcome when done within one year of diagnosis. As more patients survive, the long-term sequelae of the conditioning and posttransplant immunosuppressive regimens persist as significant problems. In particular secondary malignancies (following radiation-containing regimens), osteoporosis, infertility, and endocrine failure profoundly impact on the quality of life of long-term survivors. Further refinements are needed in the conditioning regimens and posttransplant therapies in order to decrease the incidence and severity of these sequelae.

REFERENCES 1. Akashi K, Traver D, Miyamoto T, Weissman IL. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 2000; 404:193–197. 2. Lipton JM. The hematopoietic garden: how does it grow? (editorial) J Pediatr 1998; 132:565–567. 3. Young NS, Alter BP. Aplastic Anemia Acquired and Inherited. Philadelphia: WB Saunders Co, 1994. 4. Selleri C, Sato T, Anderson S, et al. Interferon-gamma and tumor necrosis factor-alpha suppress both early and late stages of hematopoiesis and induce programmed cell death. J Cell Physiol 1995; 165:538–546. 5. Young NS, Maciejewski J. The pathophysiology of acquired AA. N Engl J Med 1997; 336:1365–1372. 6. Champlin RE, Feig SA, Sparkes RS, Gale RP. Bone marrow transplantation from identical twins in the treatment of aplastic anemia: implications for the pathogenesis of the disease. Br J Haematol 1984; 56:455–463. 7. Yamaguchi H, Calado RT, Hinh L, et al. Mutations in tert, the gene for telomerase reverse transcriptase, in AA. N Engl J Med 2005; 352:14113–14124. 8. Camitta BM, Thomas ED, Nathan DG, et al. A prospective study of androgens and bone marrow transplantation for treatment of severe AA. Blood 1979; 53:504–514. 9. Ball SE. The modern management of severe aplastic anaemia. Br J Haematol 2001; 110:41–53. 10. Rosenfeld SJ, Kimball J, Vining D, Young NS. Intensive immunosuppression with antithymocyte globulin and cyclosporine as treatment for severe acquired AA. Blood 1995; 85:3058–3065. 11. Frickhofen N, Heimpel H, Kaltwasser JP, Schrezenmeier H. Antithymocyte globulin with or without cyclosporine A: 11-year follow-up of a randomized trial comparfing treatments of AA. Blood 2003; 101:1236–1242. 12. Frickhofen N, Rosenfeld SJ. Immunosuppressive treatment of AA with antithymocyte globulin and cyclosporine. Semin Hematol 2000; 37:56–68. 13. Young NS, Barrett AJ. The treatment of severe acquired AA. Blood 1995; 85:3367–3377. 14. Fuhrer M, Burdach S, Ebell W, et al. Relapse and clonal disease in children with aplastic anemia (AA) after immunosuppressive therapy (IST): the SAA 94 experience. German/Austrian pediatric aplastic anemia working group. Klin Padiatr 1998; 210:173–179. 15. Tichelli A, Gratwohl A, Nissen C, et al. Morphology in patients with severe AA treated with antilymphocyte globulin. Blood 1992; 80:337–345. 16. Brodsky RA, Sensenbrenner LL, Jones RJ. Complete remission in severe AA after high dose cyclophosphamide without bone marrow transplantation. Blood 1996; 87:491–494. 17. Tisdale JF, Dunn DE, Geller N, et al. High-dose cyclophosphamide in severe aplastic anemia: a randomized trial. Lancet 2000; 356:1554–1559. 18. Tisdale JF, Maciejewski JP, Nunez O, et al. Late complications for severe AA (SAA) with high-dose cyclophosphamide (Cy): follow-up of a randomized trial. Blood 2002; 100:4668–4670. 19. Kojima S, Horibe K, Inaba J, et al. Long term outcome of acquired AA in children: comparison between immunosuppressive therapy and bone marrow transplantation. Br J Haematol 2000; 111:321–328. 20. Lawlor ER, Anderson RA, Davis JH, et al. Immunosuppressive therapy: a potential alternative to bone marrow transplantation as initial therapy for acquired severe aplastic anemia in childhood? J Pediatr Hematol Oncol 1997; 19:115–123.

380

Paley et al.

21. Gillio AP, Boulad F, Small TN, et al. Comparison of long-term outcome of children with severe AA treated with immunosuppression versus bone marrow transplantation. Biol Blood Marrow Transplant 1997; 3:18–24. 22. Fuhrer M, Rampf U, Burdach S, et al. Immunosuppressive therapy (IST) and bone marrow transplantation (BMT) for AA in children. Blood 1998; 92:156a abstract 631. 23. Pitcher LA, Hann LM, Evans JP, et al. Improved prognosis for acquired AA. Arch Dis Child 1999; 80:158–162. 24. Ahn MJ, Choi JH, Lee YY, et al. Outcome of adult severe or very severe AA treated with immunosuppressive therapy compared with bone marrow transplantation: multicenter trial. Int J Hematol 2003; 78:133–138. 25. Bacigalupo A, Oneto R, Bruno B, et al. Current results of bone marrow transplantation in patients with acquired severe AA. Report of the European group for blood and marrow transplantation. Acta Haematol 2000; 103:19–25. 26. Bacigalupo A, Brand R, Oneto R, et al. Treatment of acquired severe AA: bone marrow transplantation compared with immunosuppressive therapy—the European group for blood and marrow transplantation experience. Semin Hematol 2000; 37:69–80. 27. Georges GE, Storb R. Stem cell transplantation for AA. Int J Hematol 2002; 75:141–146. 28. Wagner J, Storb R. Allogeneic transplantation for aplastic anemia. In: Thomas ED, Blume K, Forman S, eds. Hematopoietic Cell Transplantation. 2nd ed. Malden, MA: Blackwell Science, Inc, 1999:791–806. 29. Horowitz MM. Current status of allogeneic bone marrow transplantation in acquired aplastic anemia. Semin Hematol 2000; 37:30–42. 30. Pillow RP, Epstein RB, Buckner CD, et al. Treatment of bone marrow failure by isogeneic marrow infusion. N Engl J Med 1966; 275:94–97. 31. Lu DP. Syngeneic bone marrow transplantation for treatment of aplastic anemia: report of a case and review of the literature. Exp Hematol 1981; 9:257–263. 32. Storb R. Bone marrow transplantation for AA. Cell Transplant 1993; 2:365–379. 33. Gluckman E, Horowitz MM, Champlin RE, et al. Bone marrow transplantation for severe AA: influence of conditioning and graft-versus-host disease prophylaxis regimens on outcome. Blood 1992; 79:269–275. 34. Sullivan KM. Long-term follow-up and quality of life after hematopoietic stem cell transplantation. J Rheumatol Suppl 1997; 48:46–52. 35. Storb R, Blume KG, O’Donnell MR, et al. Cyclophosphamide and antithymocyte globulin to condition patients with AA for allogeneic marrow transplantations: the experience in four centers. Biol Blood Marrow Transplant 2001; 7:39–44. 36. Lipton JM. Peripheral blood as a stem cell source for hematopoietic cell transplantation in children; is the effort in vein? Pediatr Transplant 2003; 7:65–70. 37. Marmont AM, Horowitz MM, Gale RP, et al. T-cell depletion of HLA-identical transplants in leukemia. Blood 1991; 78:2120–2130. 38. Storb R, Leisenring W, Deeg HJ, et al. Long-term follow-up of a randomized trial of graft-versushost disease prevention by methotrexate/cyclosporine versus methotrexate alone in patients given marrow grafts for severe AA. Blood 1994; 83:2749–2750. 39. Locatelli F, Bruno B, Zecca M, et al. Cyclosporin A and short-term methotrexate versus cyclosporine A as graft versus host disease prophylaxis in patients with severe aplastic anemia given allogeneic bone marrow transplantation from an HLA-identical sibling: results of a GITMO/EBMT randomized trial. Blood 2000; 96:1690–1697. 40. Storb R, Prentice RL, Thomas ED. Marrow transplantation for severe AA. An analysis of factors associated with graft rejection. N Engl J Med 1977; 296:61–66. 41. Azuma E, Kojima S, Kato K, et al. Conditioning with cyclophosphamide/antithymocyte globulin for allogeneic bone marrow transplantation from HLA-matched siblings in children with severe AA. Bone Marrow Transplant 1997; 19:1085–1087. 42. Makipernaa A, Saarinen UM, Siimes MA. Allogeneic bone marrow transplantation in children: single institution experience from 1974 to 1992. Acta Paediatr 1995; 84:683–688. 43. Bunin N, Leahey A, Kamani N, August C. Bone marrow transplantation in pediatric patients with severe AA: cyclophosphamide and anti-thymocyte globulin conditioning followed by recombinant human granulocyte-macrophage colony stimulating factor. J Pediatr Hematol Oncol 1996; 18:68–71. 44. Horstmann M, Stockschlader M, Kruger W, et al. Cyclophosphamide/anti-thymocyte globulin conditioning of patients with severe AA for marrow transplantation from HLA-matched siblings: preliminary results. Ann Hematol 1995; 71:77–81.

HSCT for Acquired Aplastic Anemia

381

45. Young N. Aplastic anaemia. Lancet 1995; 346:228–232. 46. Doney K, Leisenring W, Storb R, Appelbaum FR. Primary treatment of acquired AA: outcomes with bone marrow transplantation and immunosuppressive therapy. Seattle bone marrow transplant team. Ann Intern Med 1997; 126:107–115. 47. Speck B, Grathwohl A, Nissen C, et al. Treatment of severe AA with antilymphocyte globulin or bone marrow transplantation. BMJ 1981; 282:860–863. 48. Bayever E, Champlin R, Ho E, et al. Comparison between bone marrow transplantation and antithymocyte globulin in treatment of young patients with severe AA. J Pediatr 1984; 105:920–925. 49. Bacigalupo A, Hows J, Gluckman E, et al. Bone marrow transplantation (BMT) versus immunosuppression for the treatment of severe AA (SAA): a report of the EBMT SAA working party. Br J Haematol 1988; 177:70–74. 50. Halperin DS, Grisaru D, Freedman MH, et al. Severe acquired AA in children: 11-year experience with bone marrow transplantation and immunosuppressive therapy. Am J Pediatr Hematol Oncol 1989; 11:304–309. 51. Speck B, Tichelli A, Grathwohl A, et al. Treatment of severe AA: a 12 year follow-up of patients after bone marrow transplantation or after therapy with antilymphocyte globulin. In: Shahidi T et al, ed. Aplastic Anemia and Other Bone Marrow Failure Syndromes. London: Springer, 1990:96. 52. Doney K, Kopecky K, Storb R, et al. Long-term comparison of immunosuppressive therapy with antithymocyte globulin to bone marrow transplantation in AA. In: Shahidi NT et al, ed. Aplastic Anemia and Other Bone Marrow Failure Syndromes. London: Springer, 1990:104–114. 53. Locasciulli A, Bacigalupo A, Van Lint MT, et al. Hepatitis B Virus (HBV) infection and liver disease after allogeneic bone marrow transplantation: a report of 30 cases. Bone Marrow Transplant 1990; 6:25–29. 54. Paquette RL, Tebyani N, Frane M, et al. Long-term outcome of AA in adults treated with antithymocyte globulin: comparison with bone marrow transplantation. Blood 1995; 85:283–290. 55. Socie G, Henry-Amar M, Bacigalupo A, et al. Malignant tumors occurring after treatment of AA. European bone marrow transplantation—severe aplastic anemia working party. N Engl J Med 1993; 329:1152–1157. 56. Deeg HJ, Seidel K, Casper J, et al. Marrow transplantation from unrelated donors for patients with severe AA who have failed immunosuppressive therapy. Biol Blood Marrow Transplant 1999; 5:243–252. 57. Deeg HJ, Anasetti C, Petersdorf E, et al. Cyclophosphamide plus ATG conditioning is insufficient for sustained hematopoietic reconstitution in patients with severe aplastic anemia transplanted with marrow from HLA-A,B,DRB matched unrelated donors. Blood 1994; 83:3417–3418. 58. Deeg HJ, Amylon ID, Harris RE, et al. Marrow transplants from unrelated donors for patients with AA: minimum effective dose of total body irradiation. Biol Blood Marrow Transplant 2001; 7:208–215. 59. Kojima S, Inaba J, Yoshimi A, et al. Unrelated donor marrow transplantation in children with severe aplastic anemia using cyclophosphamide, anti-thymocyte globulin and total body irradiation. Br J Haematol 2001; 114:706–711. 60. Vassiliou GJ, Webb DK, Pamphilon D, et al. Improved outcome of alternative donor bone marrow transplantation in children with severe AA using a conditioning regimen containing low-dose total body irradiation, cyclophosphamide, and Campath. Br J Haematol 2001; 114:701–705. 61. Wagner JL, Deeg HJ, Seidel K, et al. Bone marrow transplantation for severe AA from genotypically HLA-nonidentical relatives. An update of the Seattle experience. Transplantation 1996; 61:54–61. 62. Margolis DA, Casper JT. Alternative-donor hematopoietic stem-cell transplantation for severe AA. Semin Hematol 2000; 37:43–55. 63. Lee JH, Choi SJ, Lee JH, et al. Non-total body irradiation containing preparative regimen in alternative donor bone marrow transplantation for severe AA. Bone Marrow Transplant 2005; 35:755–761. 64. Kang HJ, Shin HY, Choi HS, et al. Fludarabine, cyclophosphamide plus thymoglobulin conditioning regimen for unrelated bone marrow transplantation in severe AA. Bone Marrow Transplant 2004; 34:939–943.

20 Hematopoietic Stem-Cell Transplantation for the Treatment of Beta Thalassemia Farid Boulad Department of Pediatrics, Bone Marrow Transplant Service, Memorial Sloan-Kettering Cancer Center, New York, New York, U.S.A.

INTRODUCTION Beta thalassemia is caused by a large number of various genetic mutations or deletions of the globin gene which cause a reduction in the synthesis and accumulation of the beta-globin polypeptide (1–3). This gives rise to an imbalance of the beta and alpha globin chains, which in turn, leads to the formation of significant amounts of unstable alpha globin tetramers (4–6). These unstable tetramers give rise to oxidative damage of the red cell membrane and a decrease in its lifespan. Thalassemias affect the populations of Mediterranean and South Asian Ancestry. Homozygous beta thalassemia represents the most frequent form of thalassemia in the US with an estimatd total number of 1,000 patients (7,8). The clinical course of patients with beta thalassemia is homogenous. All patients develop a major anemia requiring periodic transfusions (hypertransfusion) which in turn will give rise to iron overload requiring chelation, but eventually becoming a cause of significant morbidity and mortality in the third decade of life. The median survival of patients with beta thalassemia using this “palliative” therapeutic approach is 35 years (7–9). It is estimated that by their fourth decade, the vast majority of patients with thalassmia will have succumbed to the complications of their disease. The therapeutic approaches of beta thalassemia can be palliative or curative. The “standard of care” for the treatment of thalassemia is palliative with the use of hypertransfusion (7–9). This, however, will result in iron overload with secondary hemochromatosis, and eventually end-organ damage, involving mainly the heart, the liver and the pancreas. The use of chelation can only slow down this process. Other complications of this therapeutic approach include viral infections transmitted through the transfusion blood products, which may contribute to fasten the end organ damage of the patients. Recently in the mid 90’s, sodium phenylbutyrate and hydroxyurea are two agents that were found to increase the transcription of the gamma-globin chain and the consequent production of hemoglobin F (10). These “hemoglobin switching” agents were used alone or with erythropoietin in thalassemia with however overall limited success. Of note, in the last few years, gene therapy has been advancing significantly. It has been shown to be effective in the mouse model (11). Hopefully, in the next few years, trials will start on the use of that alternative curative approach in humans. 383

384

Boulad

Allogeneic hematopoietic stem cell transplantation for the treatment of thalassemia was performed for the first time 20 years ago, by E. Donall Thomas for thalassemia in 1982 (12). Approximately 1,500 patients with thalassemia have been transplanted worldwide since, the vast majority of whom were reported by a Transplant group in Pesaro Italy, led by Professor Guido Lucarelli. The curative approach to beta thalassemia is the replacement of the hematopoietic system with new hematopoietic stem cells that can be normal or heterozygous for the beta thalassemia gene mutation. The first report of marrow transplantation for the treatment of thalassemia came from one of the pioneers of hematopoietic stem cell transplantation (HSCT), E. Donall Thomas (12). His group in Seattle transplanted the first patient with thalassemia successfully in 1982. That patient was transplanted at the age of 16 months and received cytoreduction with dimethyl busulfan (5 mg/kg) and cyclophosphamide (50 mg/kg !4), followed by an unmodified human leukocyte antigen (HLA)-matched sibling graft and long course methotrexate posttransplant for graft-versus-host disease (GVHD) prophylaxis. This patient is now 22 years posttransplant; his last follow-up with the Seattle transplant team was in 2001, 20 years posttransplant. In the U.S., the second report on transplantation for thalassemia came from Memorial Sloan Kettering Cancer Center, which reported on two patients successfully transplanted after cytoreduction with total body irradiation (720 cGy) and cyclophosphamide (60 mg/kg!2). Both these patients are still alive, thalassemia-free (13). After these initial three transplants, the field of allogeneic stem cell transplantation for thalassemia has been subsequently developed by Professor Guido Lucarelli and his Pesaro transplant team in Italy. His team has performed more than 1000 transplants to date for patients with thalassemia and have published more than 100 reports on their experience (14–29). Their work in this field represents the majority of the information found in this chapter.

HEMATOPOIETIC STEM-CELL TRANSPLANTATION FROM HLA-MATCHED SIBLINGS The Pesaro Experience In 1984, the Pesaro transplant group reported on their first 13 transplanted thalassemia patients, of whom only two were alive, thalassemia-free (14). In 1990, 6 years and 20 reports later, Lucarelli et al. reported on 222 patients transplanted at their center (17). This report has become the cornerstone of transplantation of thalassemia patients. First, the investigators reported results on a variation on the established busulfan-cyclophosphamide (BU-CY) regimen, decreasing the dose of BU from 16 mg/kg to 14 mg/kg with the CY dose maintained at 200 mg/kg. Since this report, this regimen has become the most widely used cytoreductive regimen for thalassemia patients. Second, the investigators performed a multivariate analysis of pretransplant prognostic factors on the 116 consecutive patients treated on this regimen and established a new risk classification for the transplantation of thalassemia patients. In their initial analysis, portal fibrosis and either the presence of hepatomegaly or a history of inadequate chelation therapy was significantly associated with reduced probabilities of overall survival and event-free survival. The patients were divided into three risk classes on the basis of the presence or absence of either hepatomegaly or portal fibrosis. (Class 1 had neither factor, class 2 had one, and class 3 had both.) This classification was subsequently changed to the currently used risk factors (Table 1), which include three factors: (1) hepatomegaly, (2) portal fibrosis, and (3) inadequate chelation therapy. Class 1 represents patients who have no risk factors, Class 2, those patients who have one or two risk factors, and Class 3, those patients with all three risk factors. This classification also underscored the importance of a pretransplant staging liver biopsy for the determination of the absence or presence of fibrosis and the degree of iron overload as measured directly in liver tissue. Liver biopsy is now a major component of the standard of care for patients with thalassemia in preparation for transplantation.

HSCT for the Treatment of Beta Thalassemia Table 1

385

The Pesaro Thalassemia Risk Classification

Risk factors

Determination

Hepatomegaly Hepatic fibrosis Inadequate chelation

Physical examination Liver biopsy—pathology History, ferritin value, and iron quantitation by liver biopsy

RISK CLASSIFICATION Class 1: No risk factors Class 2: 1 or 2 risk factors Class 3: All 3 risk factors

More recently, a new method of measuring iron concentration by a noninvasive method was developed. Superconducting Quantum Interference Device (SQUID) biomagnetic liver susceptometry is now available in only a few centers in the world. It is not yet the standard of care for quantifying hepatic iron stores but may, in the future, replace the need for liver biopsies (30). Lucarelli and his team have published several updates on the results of transplants for the different risk classes of patients with thalassemia. The most recent detailed results were published in 2002, (Table 2) (28) but were updated in a less detailed fashion at the 2003 meeting of the American Society of Hematology meeting. The total number of patients transplanted is 1003, with ages ranging from 1–35 years. Results are as follows: the overall thalassemia-free survival for the entire cohort of was 68%, with an overall thalassemia-free survival of 87% and 84% for the 146 class I and 334 class 2 patients respectively. Class 3 patients had a thalassemia free survival of 58% for patients 17 years of age or less and 62% for adult patients. In 1997 a new preparative regimen was developed for class 3 pediatric patients that included myelosuppression and immunosuppression with azathioprine, hydroxyurea, and fludarabine, prior to a conditioning regimen of BU 14 mg/kg and CY 160 mg/kg (instead of 200 mg/kg) (31). Unpublished data, calculated on the basis of results in 38 patients as of March 2003, showed a substantial improvement of results, with an overall survival rate of 94%, a thalassemia-free survival rate of 80%, and a rejection incidence of 14%. The risk of GVHD in thalassemia patients who received unmodified marrow grafts from HLA-matched siblings was described in detail by Gaziev et al. (29). The overall incidence of grade II–IV and III–IV acute GVHD (aGVHD) was 26.9% and 13.5%, respectively. The cumulative incidence of grade II–IV aGVHD in patients treated with cyclosporine and methylprednisolone as compared to cyclosporine, methotrexate, and methylprednisolone was 32% and 17%, respectively. The incidence of chronic GVHD (cGVHD) was 27%, with the probability of cGVHD 2 years after bone marrow transplantation (BMT) in patients with grade 0, I, II, and III–IV aGVHD was 15, 32, 53, and 54%, respectively.

Table 2 Unmodified Marrow Transplants from HLA-Matched Siblings for the Treatment of Thalassemia—the Pesaro Experience

Overall Class 1 Class 2 Class 3 Adults

N

Rejection (%)

TRM (%)

OS (%)

EFS (%)

826 (now 1003) 121 (now 146) 272 (now 334) 122 109

5 4 30 4

5 15 18 36

78 95 85 79 66

72 90 81 58 62

Abbreviations: TRM, transplant related mortality; OS, overall survival; EFS, event-free survival.

386

Boulad

Table 3 Marrow Transplants from HLA-Matched Siblings for the Treatment of Thalassemia—the Experience of Centers and Countries Other Than Pesaro

IBMTR Italy (Cagliari) Italy (Pescara) Hong Kong U.K. (Birmingham) Malaysia U.S. (Stanford) U.S. (MSKCC) Thailand India Holland U.S. (Multicenter) France U.K. (Westminster) U.S. (FHCRC) Taiwan Greece Iran Israel (T-cell depleted)

N

Rejection (%)

TRM (%)

OS (%)

EFS (%)

139 37 102 25 50 28 11 18 26 16 19 27 17 16 11 12 8 67 12

26 0 1 8 13 33 27 6 19 14 55 24 18 8 27 17 50 11 17

26 11 8 8 5 NA 0 6 11 18 5 19 17 37 18 42 0 NA 3

73 88 91 88 94 81 100 94 89 76 90 80 82 63 81 64 100 82 75

58 88 87 84 82 75 73 72 70 68 64 59 58 56 54 50 50 NA 75

Abbreviations: N, number; IBMTR, International Bone Marrow Transplant Registry; FHCRC, Fred Hutchinson Cancer Research Center; TRM, transplant related mortality; OS, overall survival; EFS, event-free survival; MSKC, Memorial Sloan-Kettering Cancer Center.

Experience from Other Centers and Countries Many series from various countries and centers have been published over the years on stem cell transplantation for thalassemia (Table 3), with patient numbers varying from 8 patients to 102 patients (32–49). The risk of graft rejection ranged from a low of 0% in the Cagliari series to as high as 55% in the Dutch series. Similarly, transplant related mortality ranged from a low of 0% in the Stanford (U.S.) series to a high of 37% in the Westminster (U.K.) series. Thalassemiafree survival was the highest in the two Italian centers at Cagliari and Pescara, followed by Hong Kong and Birmingham (U.K.), with survival rates of 88, 87, 84, and 82% respectively. These somewhat heterogeneous results can be attributed to the smaller numbers of patients and the heterogeneity of patient risk classes in these different series. Most of these series used the standard BU/CY cytoreduction regimen with minor variations. Two centers, however, have attempted different transplant approaches. The Stanford Group used total body irradiation (1200 cGy) and cyclophosphamide (120 mg/kg) in a subgroup of patients with no mortality (as noted above) but a high rate of graft rejection (49). The transplant team from Hadassah Hospital in Israel used grafts that were T-cell depleted with Campath and a conditioning regimen using BU/CY/ATG or TLI/BU/CY. Their results are described in detail later in this chapter.

HEMATOPOIETIC STEM-CELL TRANSPLANTATION USING ALTERNATIVE DONORS HSCT for patients with thalassemia has been widely accepted as the treatment of choice when HLA-matched siblings are identified. Only a minority of such patients will have suitable donors however, making the use of alternative donors an important clinical question. Until recent

HSCT for the Treatment of Beta Thalassemia

387

publications, however, very little experience has been available on the use of alternative donors in thalassemia.

HLA-Closely-Matched Unrelated Donors The first report on the use of unrelated donors for transplantation of patients with thalassemia was published by La Nasa in 2002 (50). This report included 32 patients aged 2 to 28 years with four class 1 patients, 11 class 2 patients, and 17 class 3 patients. Extended haplotype analysis and family segregation studies were employed for identification of suitable donors. Of the 32 donor/recipient pairs, 24 were identical for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5, DQA1, and DQB1 loci. Seven pairs were identical for two extended haplotypes, and 15 pairs shared one extended haplotype. Patients were conditioned using busulfan, thiotepa, and cyclophosphamide followed by cyclosporine and methotrexate as GVHD prophylaxis. Grade II–IV aGVHD developed in 11 cases (41%) and cGVHD in 6 (25%) of 24 evaluable patients. With a median follow-up of 30 months, 22 patients (69%) were alive and thalassemia free. Six patients (19%) died of transplant related complications. In 4 cases (12.5%) graft rejection was observed, followed by autologous reconstitution. Nineteen of 22 patients with a donor identical for at least one extended haplotype survived, with 17 of these thalassemia-free. Conversely, among the 10 recipients who did not share any extended haplotype with the donor, only 5 were alive thalassemia-free. Hongeng et al. from Thailand recently published their experience with the transplantation of 11 thalassemia patients using unmodified unrelated donor marrow grafts (51). The conditioning regimen consisted of BU, CY, and antithymocyte globulin. Six patients (54%) developed grade II–IV aGVHD, and one patient (9%) developed grade III–IV aGVHD. Three of 11 evaluable patients (27%) had cGVHD (limited stage). With a median follow-up of 13 months, 11 patients were alive thalassemia-free. These studies support the consideration of unrelated donor transplantation as an acceptable alternative therapy for thalassemia patients. The results are expected to be comparable to those results obtained when using HLA-matched related donors, especially in patients who are young with minimal iron overload, and have closely matched unrelated donors.

HLA-Mismatched Related Donors Experience using HLA-mismatched related donors also has come primarily from the Pesaro transplant group. Gaziev et al. reported on the experience of 29 thalassemia patients who received transplants from related genotypically nonidentical donors (52). Six of the 29 donors were phenotypically identical, 13 were mismatched siblings, 8 were mismatched parents, and 2 mismatched relatives. Six patients were not antigenically mismatched, 15 patients were mismatched for one antigen, 5 for two antigens, and 3 for three antigens. Thirteen patients were given BU/CY, nine patients BU/CY plus ALG, six patients BU/CY plus TBI or TLI, and one patient BU/CY with prior cytoreductive-immunosuppressive treatment as conditioning. GVHD prophylaxis consisted of MTX alone for four patients, CsA/MTX/Methylprednisolone (MP) for 22 patients, and CsA/MP for three patients. Thirteen of 29 patients (45%) had sustained engraftment, although the probability of graft failure or rejection was 55%. There was no significant correlation between the number of antigen disparities and graft failure. The incidence of grade II–IV aGVHD was 47% and cGVHD was 38%. The probability of overall and event-free survival was 65% and 21%, respectively, with median follow-up of 7.5 years (range 0.6–17 years) for surviving patients. Transplant-related mortality was 34%. GVHD (acute and chronic) was the major contributing cause of death (50%), followed by infections (30%). It is clear from this study that transplantation of thalassemia patients with unmodified transplants from HLA-mismatched related donors are associated with poor outcome and are not a recommended therapeutic option for patients without HLA-matched donors. Future studies

388

Boulad

are warranted using mismatched related donors but with the addition of T-cell depleted, peripheral blood stem cell grafts, as has been shown to be successful in patients with leukemia.

HEMATOPOIETIC STEM-CELL TRANSPLANTATION USING ALTERNATIVE SOURCES OF STEM CELLS The majority of experience with allogeneic stem cell transplantation has been with the use of bone marrow derived stem cells. The use of umbilical cord blood was initially disappointing with a first report by Miniero et al. on 10 patients with hemoglobinopathies, including 7 patients with thalassemia. In this report, five patients suffered early graft rejection, and one patient suffered late rejection, with only five of the ten patients alive and disease-free (53). A more recent report by Locatelli et al. however, included 44 patients transplanted with allogeneic related cord blood for either thalassemia (nZ33) or sickle cell disease (nZ11). The median number of nucleated cells infused was 4.0!107/kg (range, 1.2–10!107/kg). Sixteen of the 33 patients with thalassemia received cytoreduction with BU/CY, with only 11 patients engrafting, confirming the prior disappointing results of Miniero. Conversely, however, the remaining 17 patients received cytoreduction with BU/CY with the addition of thiotepa or fludarabine. In this patient cohort, there was only one episode of graft rejection, with 79% disease-free survival (54). This report showed that cord blood transplant is an acceptable alternative to marrow grafts in thalassemia patients with HLA-matched siblings, as long as an acceptable number of stem cells are available and an “augmented” cytoreductive regimen is used. The use of cord blood units from unrelated donors for transplantation of patients with hemoglobinopathies has been described in two reports: one for three patients with sickle cell disease (55) and one for one patient with thalassemia (56). All four received cytoreduction with BU/CY and ATG, and all received 4/6 HLA-matched cord blood units. Two of the three patients with sickle cell disease and the one patient with thalassemia engrafted. Although this approach appears to be promising, it should be used with caution until further reports confirming its efficacy are available, as the potential for a higher risk of graft rejection remains significant.

MIXED CHIMERISM POSTTRANSPLANT FOR THALASSEMIA Ablation of all of the host hematopoietic cells appears to be necessary to establish conditions for complete marrow engraftment of donor stem cells or complete chimerism in thalassemia patients. However, mixed chimerism has not been an unusual finding in this patient population post transplant (57–59). Basically four groups of patients have emerged: (1) patients with complete donor chimerism acquired early posttransplant, (2) patients with early transient mixed chimerism posttransplant that evolves to complete engraftment, (3) patients with early mixed chimerism early posttransplant, that evolves into graft rejection, and (4) patients with persistent mixed chimerism. The latter is considered to be present when the coexistence of donor and recipient cells lasts longer than two years, producing a “functional graft” with hemoglobin levels sufficient to correct the genetic defect and abolish the need for red blood cell transfusions. The Pesaro group evaluated a group of 335 patients with two or more years of follow-up posttransplant (57). In this patient group, 108 individuals (33%) had mixed chimerism two months after transplantation and 227 patients had complete donor chimerism. Thirty-five of the 108 patients (33%) with mixed chimerism rejected their grafts, as compared to none of the 227 patients with complete chimerism. Interestingly, among the 227 patients with complete chimerism two months after transplant, 4% showed persistent mixed chimerism two years after transplant. Thirty-four patients cured of thalassemia after transplant were found to have periods of mixed chimerism lasting from 2–13 years but with continued transfusion independence with hemoglobin levels ranging from 8.3–14.7 g/dL.

HSCT for the Treatment of Beta Thalassemia

389

The Pesaro group has stated that patients with stable mixed chimerism posttransplant were no longer at risk of graft rejection (G. Lucarelli, personal communication). In our own experience, however, two of our first group of four patients transplanted prior to 1986 with a cytoreductive regimen of lower dose total body irradiation (720 cGy) and cyclophosphamide, developed long term posttransplant mixed chimerism. Both patients had hemoglobin levels of 8–10 g/dL for several years posttransplant. Recently, however, both patients went from a stable 10–15% donor cells status to complete graft rejection with the complete absence of donor cells and a return to transfusion dependence 18 years posttransplant. One of these patients also had the misfortune of having his residual host cells transform into a myelodysplastic syndrome—likely secondary to the exposure of host cells to radiation and chemotherapy. This patient eventually underwent a second transplant 19 years after his first transplant and is alive and disease-free. Nevertheless, the potential for malignant transformation in residual host cells exposed to high doses of chemoradiotherapy is a potentially very important issue that must be considered among the late effects of patients in whom a long-term mixed chimeric state exists. The reasons for this variability in donor chimerism in a relatively homogeneous patient population are unclear. Potential factors that influence donor chimerism are: (1) the cytoreductive regimen and possible variations in busulfan pharmacokinetics giving a category of patients who receive a less effective myeloablative regimen than others, thus allowing residual host cells to survive conditioning therapy, and (2) the possible existence regulatory T-cell clones that maintain donor/host tolerance in the category of patients who have stable mixed chimerism (58).

ALTERNATIVE APPROACHES FOR ALLOGENEIC STEM-CELL TRANSPLANTATION FOR THALASSEMIA T-Cell Depletion Because of the relatively high risk of graft rejection associated with the transplantation of nonmalignant hematological disorders, such as thalassemia, T-cell depletion (TCD) has not been widely used as GVHD prophylaxis. Only the Hadassah hospital group in Israel has used such an approach. This group has published several reports on the use of TCD stem cell transplantation for thalassemia (60–64). The cytoreductive regimens used were BU/CY with the addition of either TLI or Thiotepa. TCD was performed using Campath either ex vivo or in vivo. The most recent report includes 22 patients with thalassemia. Summary results from this group’s experience include an overall survival and disease-free survival of 70–75% with a risk of graft rejection of 17%. Mixed chimerism was a more frequent occurrence with 50% of patients reported to have mixed chimerism. The use of a “mega dose” of stem cells obtained by mobilization of these cells from the peripheral blood, together with TCD, could potentially decrease the risk of graft rejection and bring T-cell depletion into stem cell transplantation for patients with thalassemia, especially in the context of mismatched related transplants. This has been successfully done for patients with leukemia (65), but at this time, there are no such reports for patients with hemoglobinopathies.

Nonmyeloablative Cytoreductive Regimens The clinical resolution of disease in patients with mixed chimerism has encouraged the use of reduced intensity stem cell transplants (RIST) in which investigators have based the cytoreductive approach on a nonmyeloablative regimen which uses a less aggressive conditioning regimen that is primarily immunosuppressive, allowing a stable mixed chimeric state that would theoretically render patients transfusion independent with “some” donor cells. The cytoreductive regimens used in these transplants have included low dose TBI, low dose busulfan, fludarabine, and/or ATG. Most of the hemoglobinopathy patients treated with this approach were patients with sickle cell disease. There are several important issues that may

390

Boulad

confound the use of RIST for thalassemias, however. Mixed chimerism is an “unpredictable” phenomenon posttransplant. Because the factors that allow stable mixed chimerism to occur are unknown, the risk of such an approach is an increase in the risk of graft rejection. If mixed chimerism leads to graft rejection then, the chances of success with a second transplant are greatly reduced. This may be secondary to recipient sensitization. In other words, by attempting to decrease transplant related mortality, one could end up with irreversible life-threatening graft rejection. Although some investigators have tried to use donor leukocyte infusions to increase the percentage of donor cells (66), the likelihood of this approach to be successful is also unpredictable. Finally, as mentioned earlier, there is a potential risk of late posttransplant myelodysplastic syndromes arising in host cells exposed to chemoradiotherapy. As expected, the final conclusion of two recent reports on RIST for hemoglobinopathies is that stable donor engraftment is difficult to achieve among immunocompetent (and often multiply transfused) patients with hemoglobinopathies (67,68).

LATE EFFECTS POSTTRANSPLANTATION—THE “EX-THALASSEMIC” PATIENT Patients with thalassemia who are alive and thalassemia-free posttransplant have been labeled by the Pesaro transplant team as the “ex-thalassemic after BMT,” a term that is clinically appropriate because it refers to patients who are cured of their genetic defect but continue to have residual organ damage secondary to both iron overload, as well as the effects of the transplant itself. The major areas of concern in these ex-thalassemic patients includes the status of the liver with issues of iron overload and possible transfusion related hepatitis, other organs affected by iron overload, such as the heart, and posttransplant endocrine dysfunction secondary to both iron overload and the effects of the transplant. Specifically, common areas of endocrine dysfunction include linear growth, gonadal function, and thyroid function (69).

The Liver: Iron and Hepatitis Iron The Pesaro group initially treated the iron overload of “ex-thalassemic” patients with subcutaneous desferioxamine (70–72). Although this approach showed efficacy, it did not appear to be sufficient. For this reason, the group established a posttransplant approach to decrease iron overload based on phlebotomy (73,74). Patients underwent a moderate intensity phlebotomy program, with the withdrawal of 6 ml/kg of blood at 14-day intervals. The efficacy of this approach has been published in several reports, with the most recent data showing an average ferritin decrease from 2600 (range: 2130–4820) to 280 (range: 130–920) micrograms/l. Total transferrin increased from 2.3 to 2.9 g/l, while saturation decreased from 90% to 40%. Finally, liver iron concentration as measured by biopsy decreased from 21 (range: 15–28) to 3 (range: 1–15) mg/g dry weight. Other results from this work included the finding that hepatic iron concentration is a reliable indicator of total body iron stores in patients with thalassemia major. Therefore, in patients with transfusion-related iron overload, repeated determinations of the hepatic iron concentration can provide a quantitative means of measuring long-term iron balance (75,76). There was some degree of reversible liver cirrhosis noted in “ex-thalassemic” patients who underwent aggressive phlebotomy with subsequently decreased hepatic iron content (77). It was also noted that the presence of hepatitis C infection is an independent prognostic factor associated with progression of hepatic fibrosis in the “ex-thalassemic” patients despite aggressive phlebotomy (78).

HSCT for the Treatment of Beta Thalassemia

391

Hepatitis In one study from the Pesaro group, antibodies to hepatitis C were detected in 51% of 98 patients with beta thalassemia transplanted between May 1990 and March 1992. The high prevalence of anti-HCV positivity in thalassemic patients is related to the continuous requirement for blood transfusions. In the Pesaro experience, the presence of HCV hepatitis did not influence the outcome of BMT. However, as expected, a strong correlation between biochemical and histological evidence of liver damage and anti-HCV positivity was found in these multitransfused patients (79). Based on the facts that the inflammatory activity related to HCV infection results in a chronic fibrogenous mechanism potentially leading to liver cirrhosis and hepatocellular carcinoma and that HCV infection contributes to higher level of iron deposition and the inability to remove this iron successfully, the treatment of such “ex-thalassemic” patients with interferon was attempted (80). Eleven patients with definitive evidence of HCV infection were treated for 6–12 months with recombinant alpha-interferon, 2–5 years post BMT. Ten patients completed the treatment and five patients were responders. In these patients, liver histology showed a reduction of inflammation and necrosis and a decline in the inflammatory activity from a state of chronic active hepatitis to a chronic persistent hepatitis or minimal residual inflammatory activity. All responders had a negative serum HCV-RNA at the end of treatment. The treatment with interferon was well tolerated with no major complications and no influence on marrow engraftment parameters. Better results have been obtained in the treatment of HCV infections in nontransplanted thalassemia patients using a combination of alpha-interferon and ribavirin (81). This combination has become the standard of care for patients with HCV and is now the recommended treatment also for patients with thalassemia posttransplant.

Organs Other Than the Liver Very little data has been published on the recovery of organ function (other than hepatic) in these “ex-thalassemic” patients. One study showed a reversal of cardiac dysfunction in 32 patients who underwent phlebotomy posttransplant (82–84). A similar finding was also noted in the Memorial Sloan-Kettering Cancer Center (MSKCC) patient series, where serial echocardiograms revealed normal cardiac function in 8 patients who had undergone phlebotomy posttransplant with subsequent normalization of iron status. There have also been reports of diabetes mellitus (85) in a patient from China and one in the MSKCC series (unpublished data). These findings may suggest the importance of measuring pancreatic function and glucose/insulin status in the posttransplant “ex-thalassemic” patient.

Endocrine Dysfunction Gonadal Function Very little data is available on gonadal function in the “ex-thalassemic” patient (86–89). One report was published on 50 thalassemia patients who were prepubertal at the time of transplant; however, very few conclusions could be drawn from this series other than the fact that only 40% of patients subsequently entered puberty. In one study from China (85), all ten female patients transplanted showed evidence of ovarian failure posttransplant, whereas eight of ten boys entered puberty spontaneously. In the smaller MSKCC series, all three females transplanted at ages 10–11 years, who were evaluable at a postpubertal age had evidence of gonadal failure (with high FSH and LH and low estradiol levels) requiring hormonal replacement. Five of seven male patients who were postpubertal had evidence of gonadal failure with high FSH and LH but normal testosterone levels, and two patients also had evidence of Leydig cell failure with a low testosterone level. Both these patients required

392

Boulad

androgen replacement therapy. One patient eventually recovered normal Leydig cell function spontaneously, and the second patient continues to require androgen replacement therapy.

Growth At the time of transplant most thalassemia patients already show evidence of growth retardation. In the Pesaro patient series, patients older than seven years at the time of BMT showed a significant worsening of their growth delay at four years post BMT. In comparison, patients younger than seven years at the time of BMT had more constant growth retardation over time after transplantation. Of 22 patients followed for growth status, 6 of 11 younger patients showed an improvement of their growth delay compared with only 1 of 11 older patients (90–92). This finding of the better prognosis of growth in the younger patient group was confirmed in one study from China (85). Similarly, in our own series of “ex-thalassemic” patients, three of four patients transplanted at five years of age were found to be growing at the 25–50th percentiles for height, three of four patients transplanted at the ages of 5–10 years were growing at the 5–10th percentiles for height, and five of five patients transplanted at age O10 years were growing at !5th percentile for height. In the Pesaro study, not surprisingly, the outcome of height standard deviation score at two and four years posttransplant BMT was also correlated with the patients’ hepatic status. A cohort of thalassemia patients from the Pesaro Group was treated with recombinant human growth hormone (rhGH) posttransplant (93). After 2 years of treatment, two groups were identified: the responders had (1) a significant high rise in the plasma levels of IGF-1, and (2) a growth rate after two years of treatment significantly higher than at the start of treatment. In contrast, the nonresponders had no rise in IGF-1 levels and a worsening growth rate over time during rhGH treatment. The outcome of treatment with growth hormone was also correlated with the patients’ pre- and posttransplant hepatic status.

SUMMARY In summary, over the last 20 years, HSCT has been well established as the treatment of choice for patients with thalassemia who have HLA-matched siblings. The standard of care for these patients include: 1. When there is an HLA-matched sibling, proceeding to transplant at an early age—at a class 1 status—is important. The optimal age at which to proceed to transplant is not well established. The options are transplantation at a very early age (before one year) or waiting for an age of 3–5 years 2. Adequate chelation should be performed in older patients prior to transplantation 3. Pretransplant risk classification should be performed prior to transplantation and should include liver biopsy 4. Unmodified marrow transplant using a busulfan and cyclophosphamide–based cytoreductive regimen. Alternatively, cord blood transplant from an HLA-matched related cord blood using a busulfan cyclophosphamide C thiotepa or fludarabine could be performed only if the cord blood unit contains adequate cell doses 5. It is extremely important to follow chimerism status posttransplant carefully. There is no recommended therapeutic approach if progressive host chimerism occurs. Alternative options are: donor leukocyte infusions, immunosuppression followed by a stem cell boost, or secondary transplant 6. Use of a phlebotomy program for patients with iron overload 7. Close monitoring of organ function including endocrine evaluation posttransplant. Consideration of the use of growth hormone for patients with growth hormone deficiency.

HSCT for the Treatment of Beta Thalassemia

393

Future directions in the management and treatment of this disease include: Management: 1. The standardization of SQUID testing for the quantitation of iron overload and follow-up in lieu of liver biopsies 2. The possible use of the new oral iron chelators posttransplant in lieu of phlebotomies. Treatment: 3. Improvement of stem cell transplantation from HLA-mismatched related donors with mega-dose stem cells obtained from peripheral blood and T-cell depleted grafts 4. Improvement of stem cell transplantation from unrelated donors either from cord blood or from volunteer donors with the use of an approach similar to that for mismatched related donor transplant 5. Gene therapy.

REFERENCES 1. Collins FS, Weissman SM. The molecular genetics of human hemoglobin. Prog Nucl Acid Res Mol Biol 1984; 31:3. 2. Arcasoy MO, Gallagher PG. Molecular diagnosis of hemoglobinopathies and other red blood cell disorders. Semin Hematol 1999; 36:328–339. 3. Schwartz E, Cohen A, Surrey S. Overview of the beta thalassemias; genetic and clinical aspects. Hemoglobin 1988; 12:551–564. 4. Nathan DG, Gunn RB. Thalassemia: the consequences of unbalanced hemoglobin synthesis. Am J Med 1966; 41:8. 5. Nathan DG, Stossel TB. Influence of hemoglobin precipitation on erythrocyte metabolism in a and b-thalassemia. J Clin Invest 1969; 48:33. 6. Fessas P. Inclusions of hemoglobin in erythroblasts and erythrocytes of thalassemia. Blood 1963; 21:2. 7. Giardina PJ, Hilgartner HW. Update on thalassemia. Pediatr Rev 1992; 13:55. 8. Lo L, Singer ST. Thalassemia: current approach for an old disease. Ped Clin North America 2002; 49:1165–1191. 9. Calleja EM, Shen JY, Lesser M, Grady RW, New MI, Giardina PJ. Survival and morbidity in transfusion-dependent thalassemia patients on subcutaneous desferioxamine chelation. Nearly two decades of experience. Ann N Y Acad Sci 1998; 850:469–470. 10. Dover GJ. Hemoglobin switching protocols in thalassemia. Experience with sodium phenyl butyrate and hydroxyurea. Ann N Y Acad Sci Jun 30 1998; 850:80–86. 11. Sadelain M, Rivella S, Lisowski L, Samakoglu S, Riviere I. Globin gene transfer for treatment of the beta-thalassemias and sickle cell disease. Best Pract Res Clin Haematol 2004. 12. Thomas ED, Buckner CD, Sanders JE, et al. Marrow transplantation for thalassaemia. Lancet 1982; 2:227–229. 13. Brochstein JA, Kirkpatrick D, Giardina PJ, et al. Bone marrow transplantation in two multiply transfused patients with thalassaemia major. Br J Haematol 1986; 63:445–456. 14. Lucarelli G, Polchi P, Izzi T, et al. Allogeneic marrow transplantation for thalassemia. Exp Hematol 1984; 12:676–681. 15. Lucarelli G, Polchi P, Galimberti M, et al. Marrow transplantation for thalassaemia following busulphan and cyclophosphamide. Lancet 1985; 1:1355–1357. 16. Lucarelli G, Galimberti M, Polchi P, et al. Marrow transplantation in patients with advanced thalassemia. N Engl J Med 1987; 316:1050–1055. 17. Lucarelli G, Galimberti M, Polchi P, et al. Bone marrow transplantation in patients with thalassemia. N Engl J Med 1990; 322:417–421. 18. Lucarelli G, Galimberti M, Polchi P, et al. Bone marrow transplantation in thalassemia. Hematol Oncol Clin North Am 1991; 5:549–556. 19. Lucarelli G, Galimberti M, Polchi P, et al. Bone marrow transplantation in adult thalassemia. Blood 1992; 80:1603–1607.

394

Boulad

20. Baronciani D, Galimberti M, Lucarelli G, et al. Bone marrow transplantation in class 1 thalassemia patients. Bone Marrow Transplant 1993; 12:56–58. 21. Giardini C, Galimberti M, Lucarelli G, Polchi P, Baronciani D, Angelucci E. Bone marrow transplantation in class 2 thalassemia patients. Bone Marrow Transplant 1993; 12:59–62. 22. Angelucci E, Baronciani D, Lucarelli G, et al. Bone marrow transplantation in class 3 thalassaemia patients. Bone Marrow Transplant 1993; 12:63–64. 23. Erer B, Galimberti M, Lucarelli G, et al. Bone marrow transplantation in adult thalassemia. Bone Marrow Transplant 1993; 12:65–66. 24. Lucarelli G, Clift RA, Galimberti M, et al. Marrow transplantation for patients with thalassemia: results in class 3 patients. Blood 1996; 87:2082–2088. 25. Andreani M, Manna M, Nesci S, Fattorini P, Graziosi G, Lucarelli G. Persistence of mixed chimerism in class 3 thalassemic patients following BMT. Bone Marrow Transplant 1991; 7:75. 26. Lucarelli G, Galimberti M, Giardini C, et al. Bone marrow transplantation in thalassemia. The experience of Pesaro. Ann NY Acad Sci 1998; 850:270–275. 27. Lucarelli G, Andreani M, Angelucci E. The cure of thalassemia with bone marrow transplantation. Bone Marrow Transplant 2001; 28:S11–S13. 28. Lucarelli G, Andreani M, Angelucci E. The cure of thalassemia by bone marrow transplantation. Blood Rev 2002; 16:81–85. 29. Gaziev D, Polchi P, Galimberti M, et al. Graft-versus-host disease after bone marrow transplantation for thalassemia: an analysis of incidence and risk factors. Transplantation 1997; 63:854–860. 30. Fischer R, Longo F, Nielsen P, Engelhardt R, Hider RC, Piga A. Monitoring long-term efficacy of iron chelation therapy by deferiprone and desferrioxamine in patients with beta-thalassaemia major: application of SQUID biomagnetic liver susceptometry. Br J Haematol 2003; 121:938–948. 31. Sodani P, Gaziev D, Polchi P, et al. New approach for bone marrow transplantation in patients with class 3 thalassemia aged younger than 17 years. Blood 2004; 104:1201–1203. 32. Barrett AJ, Lucarelli G, Gale RP, Sobocinski A, Horowitz M, Bortin MM. Bone marrow transplantation for thalassaemia—a preliminary report from the international bone marrow transplant registry. Prog Clin Biol Res 1989; 309:173–185. 33. Hugh-Jones K, Vellodi A, Jones ST, Hobbs JR, Rogers JR, Abdul had A. Bone marrow transplantation for thalassaemia: Westminster children’s hospital and United Kingdom experience. Prog clin Biol Res 1989; 309:201–205. 34. Frappaz D, Gluckman E, Souillet G, et al. Bone marrow transplantation for thalassemia major. The French experience. Prog clin Biol Res 1989; 309:207–216. 35. Lin KH, Lin KS. Allogeneic one marrow transplantation for thalassemia in Taiwan: factors associated with graft failure. Am J Pediatr Hematol Oncol 1989; 11:417–423. 36. Clift RA. Marrow transplantation for thalassemia: the Seattle experience. Seattle marrow transplant team. Bone Marrow Transplant 1993; 12:47–48. 37. Issaragrisil S, Visudhisakchai S, Suvatte V, et al. Bone marrow transplantation for thalassemia in Thailand. Transplant Proc 1994; 26:2348–2349. 38. Vellodi A, Picton S, Downie CJ, Eltumi M, Stevens R, Evans DI. Bone marrow transplantation for thalassaemia: experience of two British centres. Bone Marrow Transplant 1994; 13:559–562. 39. Walters MC, Sullivan KM, O’Reilly RJ, et al. Bone marrow transplantation for thalassemia. The U.S.A. experience. Am J Pediatr Hematol Oncol 1994; 16:11–17. 40. Di Bartolomeo P, Di Girolamo G, Olioso P, et al. The Pescara experience of allogeneic bone marrow transplantation in thalassemia. Bone Marrow Transplant 1997; 19:48–53. 41. Li CK, Yuen PMP, Shing MK, et al. Stem cell transplant for thalassaemia patients in Hong Kong. Bone Marrow Transplant 1997; 19:62–67. 42. Graphacos S, Kitra V, Peristeri J, Goussetis E, Papadakis V. Haemopoietic transplantation for thalassaemic children: the Greek experience. Bone Marrow Transplant 1997; 19:68–69. 43. Dennison D, Srivastava A, Chandy M. Bone marrow transplantation for thalassaemia in India. Bone Marrow Transplant 1997; 19:70. 44. Lin HP, Chan LL, Lam SK, Ariffin W, Menaka N, Looi LM. Bone marrow transplantation for thalassaemia. The experience from Malaysia. Bone Marrow Transplant 1997; 19:74–76. 45. Boulad F, Giardina P, Gillio A, et al. Bone marrow transplantation for homozygous beta-thalassemia. The memorial Sloan-Kettering cancer center experience. Ann NY Acad Sci 1998; 850:498–502. 46. Ghavamzadeh A, Nasseri P, Eshraghian MR, et al. Prognostic factors in bone marrow transplantation for beta thalassemia major: experiences from Iran. Bone Marrow Transplant 1998; 22:1167–1169.

HSCT for the Treatment of Beta Thalassemia

395

47. Lawson SE, Roberts IA, Amrolia P, Dokal I, Szydlo R, Darbyshire PJ. Bone marrow transplantation for beta-thalassaemia major: the U.K. experience in two paediatric centres. Br J Haematol 2003; 120:289–295. 48. Ball LM, Lankester AC, Giordano PC, et al. Paediatric allogeneic bone marrow transplantation for homozygous beta-thalassaemia, the Dutch experience. Bone Marrow Transplant 2003; 31:1081–1087. 49. Lee YS, Kristovich KM, Ducore JM, et al. Bone marrow transplant in thalassemia. A role for radiation. Ann NY Acad Sci 1998; 850:503–505. 50. La Nasa G, Giardini C, Argiolu F, et al. Unrelated donor bone marrow transplantation for thalassemia: the effect of extended haplotypes. Blood 2002; 99:4350–4356. 51. Hongeng S, Pakakasama S, Chaisiripoomkere W, et al. Outcome of transplantation with unrelated donor bone marrow in children with severe thalassaemia. Bone Marrow Transplant 2004; 33:377–379. 52. Gaziev D, Galimberti M, Lucarelli G, et al. Bone marrow transplantation from alternative donors for thalassemia: HLA-phenotypically identical relative and HLA non-identical sibling or parent transplants. Bone Marrow Transplant 2000; 25:815–821. 53. Miniero R, Rocha V, Saracco P, et al. Cord blood transplantation (CBT) in hemoglobinopathies. Eurocord. Bone Marrow Transplant 1998; 22:S78–S79. 54. Locatelli F, Rocha V, Reed W, et al. Related umbilical cord blood transplantation in patients with thalassemia and sickle cell disease. Blood 2003; 101:2137–2143. Epub 2002 Nov 07. 55. Adamkiewicz TV, Mehta PS, Boyer MW, et al. Transplantation of unrelated placental blood cells in children with high-risk sickle cell disease. Bone Marrow Transplant 2004; 34:405–411. 56. Hall JG, Martin PL, Wood S, Kurtzberg J. Unrelated umbilical cord blood transplantation for an infant with beta-thalassemia major. J Pediatr Hematol Oncol 2004; 26:382–385. 57. Andreani M, Nesci S, Lucarelli G, et al. Long-term survival of ex-thalassemic patients with persistent mixed chimerism after bone marrow transplantation. Bone Marrow Transplant 2000; 25:401–404. 58. Battaglia M, Andreani M, Manna M, et al. Coexistence of two functioning T-cell repertoires in healthy ex-thalassemics bearing a persistent mixed chimerism years after bone marrow transplantation. Blood 1999; 94:3432–3438. 59. Nesci S, Manna M, Andreani M, Fattorini P, Graziosi G, Lucarelli G. Mixed chimerism in thalassemic patients after bone marrow transplantation. Bone Marrow Transplant 1992; 10:143–146. 60. Or R, Naparstek E, Cividalli G, et al. Bone marrow transplantation in beta-thalassemia major. The Israeli experience. Hemoglobin 1988; 12:609–614. 61. Or R, Naparstek E, Aker M, et al. Bone marrow transplantation with T-cell depleted allografts for the treatment of severe beta thalassemia major. Prog Clin Biol Res 1989; 309:217–222. 62. Kapelushnik J, Or R, Filon D, et al. Analysis of beta-globin mutations shows stable mixed chimerism in patients with thalassemia after bone marrow transplantation. Blood 1995; 86:3241–3246. 63. Naparstek E, Delukina M, Or R, et al. Engraftment of marrow allografts treated with campath-1 monoclonal antibodies. Exp Hematol 1999; 27:1210–1218. 64. Rosales F, Peylan-Ramu N, Cividalli G, et al. The role of thiotepa in allogeneic bone marrow transplantation for genetic diseases. Bone Marrow Transplant 1999; 23:861–865. 65. Yeisner Y, Martelli MF. Transplantation tolerance induced by “mega-dose” CD34C stem cell transplants. Exp Hematol 2000; 28:119–127. 66. Aker M, Kapelushnik J, Pugatsch T, et al. Donor lymphocyte infusions to displace residual host hematopoietic cells after allogeneic bone marrow transplantation for beta-thalassemia major. J Pediatr Hematol Oncol 1998; 20:145–148. 67. Horan JT, Liesveld JL, Fenton P, Blumberg N, Walters MC. Hematopoietic stem cell transplantation for multiply transfused patients with sickle cell disease and thalassemia after low-dose total body irradiation, fludarabine, and rabbit anti-thymocyte globulin. Bone Marrow Transplant 2004:1–7. 68. Iannone R, Casella JF, Fuchs EJ, et al. Results of minimally toxic nonmyeloablative transplantation in patients with sickle cell anemia and beta-thalassemia. Biol Blood Marrow Transplant 2003; 9:519–528. 69. Piga A, Longo F, Voi V, Facello S, Miniero R, Dresow B. Late effects of bone marrow transplantation for thalassemia. Ann NY Acad Sci 1998; 850:294–299. 70. Lucarelli G, Angelucci E, Giardini C, et al. Fate of iron stores in thalassaemia after bone-marrow transplantation. Lancet 1993; 342:1388–1391. 71. Giardini C, Galimberti M, Lucarelli G, et al. Desferrioxamine therapy accelerates clearance of iron deposits after bone marrow transplantation for thalassaemia. Br J Haematol 1995; 89:868–873.

396

Boulad

72. Giardini C, Galimberti M, Lucarelli G, et al. Desferioxamine therapy of secondary hemochromatosis after BMT for thalassemia. Bone Marrow Transplant 1997; 19:119–122. 73. Angelucci E, Muretto P, Lucarelli G, et al. Treatment of iron overload in the “ex-thalassemic.” Report from the phlebotomy program. Ann NY Acad Sci 1998; 850:288–293. 74. Angelucci E, Muretto P, Lucarelli G, et al. Phlebotomy to reduce iron overload in patients cured of thalassemia by bone marrow transplantation. Italian cooperative group for phlebotomy treatment of transplanted thalassemia patients. Blood 1997; 90:994–998. 75. Angelucci E, Brittenham GM, McLaren CE, et al. Hepatic iron concentration and total body iron stores in thalassemia major. N Engl J Med 2000; 343:327–331. 76. Angelucci E, Baronciani D, Lucarelli G, et al. Needle liver biopsy in thalassaemia: analyses of diagnostic accuracy and safety in 1184 consecutive biopsies. Br J Haematol 1995; 89:757–761. 77. Muretto P, Angelucci E, Lucarelli G. Reversibility of cirrhosis in patients cured of thalassemia by bone marrow transplantation. Ann Intern Med 2002; 136:667–672. 78. Angelucci E, Muretto P, Nicolucci A, et al. Effects of iron overload and hepatitis C virus positivity in determining progression of liver fibrosis in thalassemia following bone marrow transplantation. Blood 2002; 100:17–21. 79. Erer B, Angelucci E, Lucarelli G, et al. Hepatitis C virus infection in thalassemia patients undergoing allogeneic bone marrow transplantation. Bone Marrow Transplant 1994; 14:369–372. 80. Giardini C, Galimberti M, Lucarelli G, et al. Alpha-interferon treatment of chronic hepatitis C after bone marrow transplantation for homozygous beta-thalassemia. Bone Marrow Transplant 1997; 20:767–772. 81. Wonke B, Telfer P, Garson JA, et al. Alpha-interferon alone and in combination with ribavirin for hepatitis C infection in multiply transfused patients with thalassemia major—the U.K. experience. Bone Marrow Transplant 1997; 19:163–165. 82. Mariotti E, Agostini A, Angelucci E, Lucarelli G, Sgarbi E. Reversal of the initial cardiac damage in thalassemic patients treated with bone marrow transplantation and phlebotomy. Bone Marrow Transplant 1997; 19:139–141. 83. Manenti F, Galimberti M, Lucarelli G, et al. Growth and endocrine function after bone marrow transplantation for thalassemia major. Prog Clin Biol Res 1989; 309:273–280. 84. Mariotti E, Agostini A, Angelucci E, Lucarelli G, Sgarbi E. Echocardiographic study in ex-thalassemic patients with iron overload, preliminary observations during phlebotomy therapy. Bone Marrow Transplant 1993; 12:106–107. 85. Li CK, Chik KW, Wong GW, Cheng PS, Lee V, Shing MM. Growth and endocrine function following bone marrow transplantation for thalassemia major. Pediatr Hematol Oncol 2004; 21:411–419. 86. Galimberti M, De Sanctis V, Lucarelli G, et al. Endocrine function after bone marrow transplantation for thalassemia. Bone Marrow Transplant 1991; 7:74. 87. De Sanctis V, Galimberti M, Lucarelli G, et al. Pubertal development in thalassaemic patients after allogenic bone marrow transplantation. Eur J Pediatr 1993; 152:993–997. 88. De Sanctis V, Galimberti M, Lucarelli G, Polchi P, Ruggiero L, Vullo C. Gonadal function after allogenic bone marrow transplantation for thalassaemia. Arch Dis Child 1991; 66:517–520. 89. Ogilvy-Stuart AL, Shalet SM. Gonadal function after allogeneic bone marrow transplantation for thalassaemia. Arch Dis Child 1991; 66:1365–1366. 90. Ponte G, Ferrara M, Esposito L. Growth in homozygous beta-thalassemia after bone marrow transplantation. Bone Marrow Transplant 1991; 8:68–69. 91. Cohen A, van Lint MT, Uderzo C, et al. Growth in patients after allogeneic bone marrow transplant for hematological diseases in childhood. Bone Marrow Transplant 1995; 15:343–348. 92. De Simone M, Olioso P, Di Bartolomeo P, et al. Growth and endocrine function following bone marrow transplantation for thalassemia. Bone Marrow Transplant 1995; 15:227–233. 93. De Simone M, Di Bartolomeo P, Olioso P, et al. Growth after recombinant human growth hormone (rhGH) treatment in transplanted thalassemic patients. Bone Marrow Transplant 1997; 20:567–573.

21 Hematopoietic Stem-Cell Transplantation for Sickle Cell Disease Paul Woodard Hematology/Oncology, Division of Stem Cell Transplantation, St. Jude Children’s Research Hospital, Memphis, Tennessee, U.S.A.

INTRODUCTION Studies of the natural history of sickle cell disease (SCD) have shown a median life expectancy for adults with SCD of 42–53 years for men and 48–59 years for women (1,2). Patients with hemoglobin (Hgb) SS disease have a higher incidence of death than other sickle disorders, such as Hgb SC or Hgb Sb0 thalassemia (3). The proportion of patients with Hgb SS surviving to age 20 years was 85%, when reported by Leikin in 1989 (3). More contemporary survival and natural history studies are lacking. Lower steady-state Hgb, higher reticulocyte counts, white blood cell counts O15,000/mm3, and Hgb F !15% are predictive of a higher rate of death (3). Similar analyses for patients at two years of age have shown that leukocytosis, early dactylitis, and severe anemia (Hgb !7 gm/dl) were predictive for severe SCD (4). Up to 11% of patients suffer clinical strokes, and an additional 17–22% will suffer a subclinical “silent” infarction evident on brain magnetic resonance imaging (MRI) (5). Treatment with chronic red blood cell transfusions to prevent additional strokes or acute chest syndrome can lead to red blood cell alloantibodies and autoantibodies (6) and iron overload (7).

BONE MARROW TRANSPLANTATION FOR SICKLE CELL DISEASE The first bone marrow transplant for SCD was reported in 1984 by Johnson (8). The patient was an 8-year-old girl with both SCD and acute myelogenous leukemia (AML). She received a matched sibling donor (MSD) graft from her 4-year-old brother who had sickle cell trait. Conditioning consisted of cyclophosphamide 120 mg/kg and total body irradiation (TBI) 1150 cGy. Her course was complicated by acute and chronic graft-versus-host disease (GVHD). This patient was converted to sickle cell trait, had no further complications of SCD, and remains in remission for AML at present. Investigators in Belgium then reported successful allogeneic hematopoietic stem cell transplantation (HSCT) for five African children with SCD who had experienced up to four vaso-occlusive crises (VOC) per year and up to four transfusions per year (9). These patients were transplanted prior to the development of chronic organ damage but were selected due to a combination of factors: disease severity (VOC and transfusion frequency) and inability to receive advanced medical care when they returned to their home in a developing country. 397

398

Woodard

Patients were conditioned with busulfan (4 mg/kg/day for four days) and cyclophosphamide (50 mg/kg/day for four days). Cyclosporine and short-course methotrexate (10 mg/m2 on days 1, 3, 6, and 11) were administered for GVHD prophylaxis. All five patients engrafted initially; however, one suffered secondary graft rejection. This patient engrafted after a second transplant two months later using cyclophosphamide (50 mg/kg/day for four days), thoraco-abdominal irradiation (750 cGy in one fraction over 6 hours, with lung and ovarian shielding) and horse antithymocyte globulin [(ATG), 30 mg/kg/day for three days]. All donors were human leukocyte antigen (HLA) identical. Two donors had a normal Hgb electrophoresis (Hgb AA), while three had sickle cell trait (Hgb AS). Two patients had mild grade I acute GVHD. One patient had transient autoimmune thrombocytopenia one year after transplant, which was responsive to immunosuppressive therapy. The Hgb electrophoreses after transplantation demonstrated a similar pattern as the donors. Prior to the development of clinical trials of HSCT for SCD in North America, a symposium was held at Fred Hutchinson Cancer Research Center in Seattle in August 1990. Experts in the care of patients with SCD and transplantation reviewed the current state of their respective fields (10). Complications agreed upon as considerations for HSCT included stroke, alloimmunization, recurrent pain crises, renal involvement, and pulmonary involvement. Patients with irreversible organ damage were not deemed candidates for HSCT (11). The application of HSCT for hematological disease has used myeloablative conditioning prior to HLA-MSD bone marrow transplantation to provide normal erythropoiesis for patients with SCD (12,13) and b-thalassemia (14). In European and American studies, the most frequently used eligibility criteria for transplantation for SCD have included the following: 1. 2. 3. 4.

Hgb SS, SC, or Sb0 thalassemia Age %16 years HLA-identical sibling donor At least one of the following: i. Stroke ii. Recurrent acute chest syndrome iii. Recurrent severe pain episodes.

Worldwide, over 200 patients with SCD have been transplanted with MSDs using a myeloablative approach. In the multicenter investigation of HSCT for SCD, patients received a myeloablative regimen of busulfan 14–16 mg/kg or 500 mg/m2, cyclophosphamide 200 mg/kg, and either horse ATG 90 mg/kg, rabbit ATG 20 mg/kg, or CAMPATH 10 mg for five days (12). Many of the patients had targeted dosing of busulfan to achieve steady state concentrations of 400–600 ng/ml. Patients received methotrexate and cyclosporine as GVHD prophylaxis. Patients not receiving chronic transfusion therapy received partial exchange transfusions to decrease the percentage of HbS to !30% prior to conditioning. Patients received penicillin prophylaxis for at least two years after HSCT or until splenic recovery was documented by scintigraphy. Due to an increased incidence of neurologic complications, such as seizures and hemorrhage, in the early experience, patients received anticonvulsants from the beginning of busulfan administration until six months after HSCT, platelets counts were maintained O50,000/mL, and Hgb concentrations were maintained between 9 and 11 gm/dl. Results have been promising with 91–93% overall survival and 82% disease-free survival (Figs. 1 and 2) (12,13). Many of these patients received chronic transfusion programs prior to transplantation, leading to a rejection rate of approximately 8%. Although causes of rejection were not specifically assessed, one can speculate that alloimmunization to red blood cells, potential HLA alloimmunization through exposure to transfusion antigens, or insufficient ablation may have contributed to rejection. Chronic GVHD occurred in 12%. The transplantrelated mortality was 9%, largely related to complications of GVHD. Better results were obtained in a group of young asymptomatic patients in Belgium. These patients were transplanted early due to their family’s return to Africa, where adequate supportive

HSCT for Sickle Cell Disease

399

Figure 1 Kaplan-Meier estimates of survival and event-free survival after bone marrow transplantation in 22 patients with sickle cell disease. Source: Adapted from Ref. 12.

care would be difficult to obtain (13). This group of patients with less severe disease had improved overall survival (OS), and disease-free survival (DFS) rates of 100% and 93%, respectively.

UMBILICAL CORD BLOOD TRANSPLANTATION FOR SICKLE CELL DISEASE The Eurocord Transplant Group reported the results of related umbilical cord blood (UCB) transplantation in 44 patients with thalassemia (nZ33) or SCD (nZ11) (15). Patients received varying myeloablative conditioning regimens, consisting of busulfan and cyclophosphamide (Bu/Cy)CATG or antilymphocyte globulin (nZ26), Bu/Cy or fludarabine/BuCthiotepa (nZ17), or Bu/Cy/fludarabine (nZ1). Ten of 11 with SCD obtained durable engraftment. One patient suffered acute rejection at day C28 and underwent a successful second transplant from the same donor. The rate of grade II acute GVHD was 11%. Six percent developed limited chronic GVHD. Overall survival was 100%, and event-free survival (EFS) was 90% for patients with SCD (Fig. 3). The 2-year Kaplan-Meier estimate for EFS was 90% for SCD. Analysis of factors associated with outcome showed that the use of methotrexate adversely affected outcome, while the use of a regimen of Bu/Cy/thiotepa or fludarabine/Bu/thiotepa rather than Bu/Cy was associated with a better rate of engraftment (PZ0.0003) and better EFS (PZ0.03). Results such as these led to the development of national cord blood banking programs for families with children who might benefit from UCB transplantation. One such program was developed at the Children’s Hospital Oakland Research Institute (16). More than 500 UCB donations have been collected from remote sites (primarily at community hospitals) in 42 states. These stored UCB units have a low bacterial contamination rate (!3%) and adequate cellular content for transplantation (O1.5!107 cells/kg recipient weight) in 90% of the cases. To date, approximately 3.4% of the UCB units have been used for transplantation.

ACUTE TOXICITIES OF HEMATOPOIETIC STEM-CELL TRANSPLANTATION Failure to Engraft/Rejection Approximately 90% of patients treated by MSD bone marrow or related UCB transplantation had durable donor cell engraftment. In the Belgian study, 47 of 50 patients had donor engraftment (94%). Failure to engraft led to recovery of autologous hematopoiesis; however, these patients had

Survival (%)

400

Woodard

100 90 80 70 60 50 40 30 20 10 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 Time (months)

Figure 2 Overall survival (—), EFS (—) and DFS (.) in 50 patients transplanted in Belgium for sickle cell anemia. The results were respectively 93%, 82%, and 85%. Events were defined as death, absence of engraftment, rejection, and recurrence of disease. Disease-free survival was defined as the absence of clinical and biological signs of sickle cell anemia. Source: Adapted from Ref. 13.

prolonged asymptomatic periods due to a persistent high level of fetal (Hgb F). Two patients (4%) had secondary graft failure 1 and 18 months after HSCT. The increased incidence of rejection in patients with SCD is likely due, in part, to chronic red blood cell transfusions. Patients are presented with different minor histocompatibility antigens expressed on leukocytes that accompany these transfusions. The incidence of alloantibody formation in children with SCD is approximately 5–36%, leading to hemolytic transfusion reactions in nontransplanted patients (6,17). Persistent alloantibodies or host B-cells producing alloantibodies may contribute to rejection. Techniques, such as anti-CD20 antibody, plasmapheresis, and anti-T-cell antibodies, may reduce rejection in alloimmunized hosts, but these have not been extensively studied in HSCT (18–20).

Graft-Versus-Host Disease Acute GVHD grade II–III developed in 2 (9%) of the multicenter cohort (12). In the Belgian experience 10 patients (20%) had grade I, 9 (18%) grade II, and 1 (2%) grade III acute GVHD (13). Eleven and one-half percent developed chronic GVHD in the multicenter cohort, and 20% developed chronic GVHD in the Belgian cohort (7 limited, 3 extensive).

Neurologic Toxicities Two of 50 patients (4%) transplanted by the multicenter group had episodes of intracranial hemorrhage (ICH). This was likely due, in part, to preexisting severe cerebral vasculopathy in conjunction with low platelet counts and/or hypertension after HSCT. These episodes occurred in the early years of the protocol, leading to treatment modifications including the maintenance of platelet counts at greater than 50,000/uL and strict control of hypertension. These modifications have led to a marked reduction in ICH: approximately 25% of patients had seizures after HSCT. These were often seen in association with hypertension, hypomagnesemia, and/or subtherapeutic anticonvulsant levels. Correction of these abnormalities, as well as administration of anticonvulsants for six months after HSCT, have decreased the incidence of seizures.

EFFECTS ON ORGANS Hematologic Effects Successful donor cell engraftment led to resolution of hemolytic anemia and freedom from acute sickle-related events. Engrafted patients have not experienced recurrent strokes, acute

HSCT for Sickle Cell Disease

401

1.0 Sickle cell disease 0.8 Thalassemia EFS

0.6 n

events

Thalassemia

33

7

Sickle cell disease

11

1

0.4 0.2

P = .05 0.0 0

500

1000

1500 Days

2000

2500

Figure 3 Kaplan-Meier estimate of the probability of event-free survival according to the original disease. Source: Adapted from Ref. 15.

chest syndrome, painful crises, sequestration, or other events. Levels of Hgb S paralleled those seen in the donors (i.e., trait donors led to trait levels of Hgb S while normal donors led to normal Hgb A levels).

Effects on Osteonecrosis Hernigou reported improvement of osteonecrosis after successful HSCT (21). Prior to HSCT, the patient had osteonecrosis of the humerus leading to pain and impaired function. Three months after HSCT, MRI showed improvement in the epiphysis. Four years later there was continued normalization of the marrow signal in the proximal humerus. Clinically, movement of the joint had improved and there was only slight residual pain.

Recovery of Splenic Function Although not prospectively studied, there is anecdotal retrospective data reporting restoration of splenic function after successful HSCT. Ferster reported three patients 10–14 years of age with absent splenic function, as noted by the presence of Howell-Jolly bodies and absence of splenic uptake on radionuclide scanning (22). Howell-Jolly bodies disappeared within three months, and splenic uptake was seen at three months in one patient. When measured at 1 and 1.5 years later in two other patients, splenic uptake was seen. Vermylen reported recovery of splenic uptake in seven patients (13). Three patients did not show splenic recovery, including two patients with chronic GVHD who had no function or decreased function after HSCT.

Growth and Endocrine Effects Late effects on growth and endocrine parameters in children transplanted for SCD have been reported. Twenty-six patients with a median follow-up of 56.6 months after transplantation were assessed in detailed fashion (23). All patients had normal thyroid function after HSCT. Growth improved from a median of K0.7 standard deviations prior to HSCT to K0.2 after HSCT. One concern voiced among families and providers is the potential for gonadal dysfunction after HSCT. Five of seven females had primary amenorrhea after HSCT. Five had elevated LH and FHS levels, four of whom had low estradiol as well. Similar findings

402

Woodard

were seen among prepubertal girls in Belgium (13). Two postpubertal girls had secondary amenorrhea. Among males in the multicenter study, 0/4 had elevated gonadotropins, although one had low testosterone levels. Six adolescent boys in the Belgian cohort had normal sexual maturation, although four had decreased testosterone levels and increased FSH. One had elevated LH.

Neurologic Effects SCD may lead to central nervous system injury, manifested as stroke, silent infarction, cerebrovascular disease, or abnormal neuropsychometric testing. One of the most important questions is the effect of HSCT on the central nervous system (CNS). Stabilization of parenchymal lesions measured by brain MRI has been reported (23). Thirty-one of 59 patients treated on the multicenter study were transplanted for stroke or other significant CNS disease. Of these 31 patients, there were 3 who rejected their graft and 1 death due to ICH. The remaining 27 patients all experienced donor engraftment and had no further CNS events. At a median follow-up of 32 months, follow-up cerebral MRIs were stable in all but one patient, who showed improvement. Nine additional patients had evidence of silent infarction prior to HSCT. Seven of the nine were reported as stable, and two showed improvement on postHSCT MRI. Improvement in the diameter of cerebral arteries and improvement in cerebral blood flow velocity has been reported in some patients by Steen et al. (24) A retrospective, blinded analysis of serial magnetic resonance angiography (MRA) scans was performed for 24 patients with SCD, including four who received allogeneic HSCT. Patients who underwent HSCT had improved (increased) arterial diameter after HSCT, compared to those who received chronic transfusions, hydroxyurea, or no therapy. Importantly, two stenosis had normalization of the stenotic artery after HSCT. A subsequent report prospective study of nine patients from the same institution, followed for a minimum of three years after HSCT, complicates the picture (25). Patients were followed prospectively, had uniform, serial brain MRI and MRAs and serial neuropsychometric testing. A scale was developed to classify the severity of pre- and postHSCT parenchymal or vascular abnormalities. While patients had stable IQ and reading and arithmetic scores over three years of follow-up, those with ischemic brain injury prior to HSCT developed additional parenchymal changes. The severity of pretransplant brain parenchymal abnormalities were predictive of subsequent brain parenchymal changes (PZ0.03), but pretransplant MRAs were not predictive of subsequent vascular changes (PZ0.21). Interestingly, two previously reported patients who had demonstrated improvement (24), subsequently had worsening of cerebral arterial stenosis. The explanation for these changes is currently under investigation. It is important to note that no patient had a recurrent clinical stroke after donor engraftment. Other investigators have reported worsening CNS status after HSCT in one patients with severe CNS vasculopathy prior to HSCT (26). This patient, a nine-year-old with a prior left frontal lobe infarction, multiple cerebral vascular stenoses, and moyamoya, developed seizures with extension of the previous infarction at day C38 after HSCT. At day C58, the patient had recurrent seizures and a new right parietal subarachnoid hemorrhage. At 16 months, this patient developed worsening cognitive and memory deficits. An MRI showed deterioration of the vessels with progression of occlusion of the M1 segments of the middle cerebral arteries. Interestingly, the patient had markedly increased levels of Factor VIII (332.8%) and vWF antigen (213%); a significant decrease of high molecular weight multimers was seen. The authors speculated that the combination of higher Hgb with increased viscosity, preexisting moyamoya, and decreased levels of high molecular weight VWF led to ongoing vasculopathy. More detailed analyses of pre- and post-HSCT MRI, MRA, neuropsychometric testing results, and neurologic exams are underway.

HSCT for Sickle Cell Disease

403

Pulmonary Effects Pulmonary function testing (PFT) was performed after HSCT in 25 patients in the multicenter study (23). Twenty-one patients had stable or normal PFTs after HSCT. One patient who subsequently died of bronchiolitis obliterans had obstructive disease, one developed restrictive disease, and two with prior restrictive disease worsened after HSCT. Seven of eight patients with recurrent acute chest syndrome (ACS) had stable PFTs, and none experienced recurrent ACS.

Quality of Life Quality of life was also assessed in the study of Walters (23). All patients with donor engraftment had post HSCT Karnofsky or Lansky Performance Scores of 100. Vermylen reported a Lansky score of 100 in all patients except two with bronchiolitis obliterans (scoreZ90) and one with chronic GVHD and AML (scoreZ60).

CHALLENGES TO TRANSPLANTATION Safety Some providers remain concerned that the approximately 6% mortality associated with HSCT for SCD is too high. SCD, unlike malignancies, is not immediately life threatening; therefore, concerns about safety are paramount. Future efforts will likely be focused on providing durable engraftment with reduced toxicity. One approach to address these issues is less toxic conditioning.

Lack of Human Leukocyte Antigen Matched Sibling Donors The major barrier to HSCT for SCD has been the lack of an HLA MSD. Even among those who meet eligibility criteria, a minority of patients have been HLA typed. In one review of 4848 patients with SCD treated at 22 centers, 6.5% met transplant eligibility criteria (27). Only 41% of these had HLA typing performed, and only 14% of those meeting eligibility criteria had an HLA identical sibling. Lack of a sibling donor (24%), lack of financial or psychosocial support (10.5%), parental refusal (9.5%), physician refusal (4%), medical noncompliance (!1%), and other reasons (10.5%) were given as reasons for not receiving bone marrow transplantation (BMT) among those eligible. Mentzer reviewed records of 143 patients with sickle cell anemia under age 16 at the Northern California Comprehensive Sickle Cell Center (28). Approximately 16% of patients had stroke or recurrent painful crises, and 38% met the criteria of the multicenter study. Only 18% had an HLA MSD.

Decision-Making in Children with Sickle Cell Disease Kodish studied parental decision-making regarding HSCT for SCD in 67 parents of children with SCD (29). Parental preferences were widely divergent. Twenty-four percent would not tolerate any risk and would not pursue BMT, even with a 100% probability of cure and 0% mortality. Twenty-two percent would accept BMT if the risk of mortality was less than 5%. On the other hand, 37% would accept a mortality rate of 15% or greater, and 12% would accept up to 50% mortality. Parents who were employed, had a high school education, or had more than one affected child were more willing to accept risk associated with BMT. A clinical trial of parental choices among BMT, hydroxyurea, and chronic red blood cell transfusion therapy is in progress at St. Jude Children’s Research Hospital. It is clear that a major element of providing HSCT to children with severe SCD is appropriate education of parents and patients so that truly informed consent is given. Similarly, education of primary care providers and hematologists regarding this therapy are important.

404

Woodard

In similar study of 100 adult patients with SCD, 28 were unwilling to accept any risk of death within 30 days (30). Nine agreed to a 5% risk of mortality, 46 to O15% risk, 20 to O30% risk, and 12 to O40% risk. In contrast to the pediatric study, there was no association between patient or disease characteristics and the degree of risk accepted. In a subsequent interview of health care providers, there was no agreement between provider recommendations and patient decisions. The STOP stroke prevention trial demonstrated that chronic transfusion therapy was effective in reducing the risk of stroke in patients with elevated cerebral blood velocity (O200 cm/sec as measured by transcranial Doppler ultrasonography) when compared to observation only (31). Nietert performed a decision analysis to compare HSCT with chronic blood transfusion therapy for patients with elevated cerebral blood velocity (32). This was a computer simulation of the projected impact of HSCT and chronic transfusion therapy on subsequent quality of life, using estimated probabilities of various risks of the two treatments in hypothetical cohorts of 100,000 patients. An analysis of actual treatment received (HSCT versus chronic blood transfusion) gave a slight advantage to HSCT. Similar analyses may yield intriguing results if performed for patients with traditional transplant indications, such as stroke, silent infarction, recurrent acute chest syndrome, or other disease states.

MIXED CHIMERISM AFTER TRANSPLANTATION Although replacement of defective hematopoiesis with full donor chimerism of normal stem cells is the goal of myeloablative HSCT, a small proportion of patients develop mixed donor-host hematopoietic chimerism. This occurs due to survival of host cells after insufficiently ablative conditioning. When a state of tolerance develops to allow persistent donor and host hematopoiesis after HSCT, this is called stable mixed chimerism. Tolerance generally develops after destruction of host T cells with conditioning and suppression of donor T cells by immunosuppressive medications, such as cyclosporine or tacrolimus. Failure to achieve tolerance can lead to rejection by active host T cells or GVHD by donor T cells. Several investigators have analyzed mixed chimerism after myeloablative HSCT for malignant disease. Bader prospectively analyzed 55 patients with acute leukemias by variable nucleotide tandem repeat analysis after HSCT (33). Mixed chimerism was seen in 18 patients. The probability of relapse-free survival for patients with decreasing host MC was 100% (nZ8) compared to 10% for children with increasing host mixed chimerism (MC) (nZ10). GVHD grades II–IV was seen in 22% of those with MC compared to 44% of those with full donor chimerism. Lineage differences in chimerism can also be seen. Winiarski reported mixed chimerism in 6/33 (18%) patients greater than one year after HSCT (34). Three of four patients with aplastic anemia had mixed chimerism of the T-cell lineage only, which caused no clinical problems. These patients had received conditioning with cyclophosphamide 200 mg/kg and ATG, followed by matched sibling grafts in two or a matched unrelated graft in one. Two patients with genetic diseases who had mixed chimerism, which included B cells, developed immune hemolytic anemia. Other investigators have reported decreased rates of moderate to severe acute GVHD when mixed chimerism or mixed T-cell chimerism is seen. Huss reported that the relative risk of developing grade II–IV acute GVHD in patients with severe aplastic anemia receiving MSD transplantation was 0.48 in mixed chimeras compared to full donor chimeras (35). Mattsson analyzed the effect of mixed T-cell chimerism on GVHD in 102 patients (36). Fifty-seven percent had mixed T-cell chimerism before day C21. Eighteen had mixed T-cell chimerism persisting beyond three months after HSCT. The probability of grade II–IV acute GVHD was markedly lower in patients with mixed T-cell chimerism (5% vs. 52%, P!0.001). Although mixed chimerism may be undesirable in malignant disease due to the increased risk of relapse, it may allow for correction of nonmalignant diseases, including red blood cell

HSCT for Sickle Cell Disease

405

disorders or metabolic enzyme deficiencies, with decreased GVHD. Mixed chimerism has been reported in approximately 10% of patients with b-thalassemia undergoing MSD transplantation (37). This chimerism appears to be durable, persisting for 2–11 years at the time of the report (38). The level of donor chimerism ranged from 25–90% donor, leading to transfusion-independence with Hgb levels of 8.3–14.7 gm/dl. Mixed T-cell chimerism has also been reported in b-thalassemia (39). Although this may lead to a skewed T-cell repertoire due to expansion of T-cell clones responsible for maintaining tolerance, upon T-cell activation, a complex T-cell receptor repertoire can be seen. Similar to b-thalassemia, approximately 10% of those with SCD transplanted by the multicenter group developed stable mixed chimerism (40). Among the 13 patients with stable mixed chimerism, none had acute GVHD, and none developed graft rejection. Eight patients had levels of donor chimerism between 90% and 99%, whereas five patients had levels of donor chimerism of 11–74%. Levels of donor chimerism in three patients with Hgb AA donors were 67%, 74%, and 11%, which translated into Hgb S percentages of 0%, 0%, and 7%, respectively. Two patients with Hgb AS (sickle trait) donors had donor chimerism levels of 25% and 60%, which corresponded to levels of HgbS of 36% and 37%, respectively. Hgb levels in these patients ranged from 11.2 to 14.7 gm/dl. More importantly, there were no clinical sickle events in these patients. Although prior iron chelation therapy was associated with increased incidence of rejection, there was no association with stable mixed chimerism. In this study, iron chelation can be considered a surrogate marker for quantity of transfusions received. One would expect a greater risk of alloimmunization in patients with significant iron overload, such as those receiving iron chelation. It is likely not desferal that is associated with increased rejection but exposure to antigens through transfusions. Patients younger than 10 years of age were more likely to develop stable mixed chimerism than those older than 10 (PZ0.001). These observations suggest that partial or mixed chimerism, even with a minority of donor cells might have a curative effect if it is stably maintained. Full engraftment of donor cells does not appear to be necessary to derive benefit from transplantation. For patients with declining or unstable mixed chimerism, low-dose DLI may prevent graft loss. Baron reported a 4 year-old with SCD who received MSD HSCT due to recurrent vaso-occlusive crises and two splenic crises (41). The patient engrafted after myeloablative conditioning with busulfan 16 mg/kg, cyclophosphamide 200 mg/kg, and horse ATG 90 mg/kg. Between 73 and 200 days after transplantation, the donor chimerism declined, leading to a DLI (1!107 CD7C cells/kg) on day C234. On day C243, the patient experienced a further decrease in donor cells (60% donor) and required transfusions. A second DLI (2!107 CD7C cells/kg) was given on day C267. This led to overall grade II acute GVHD (stage 2 skin and stage 1 liver), responsive to steroids and cyclosporine. By day C297, full donor chimerism was achieved. Immunosuppressive medications were discontinued by day C451. This anecdotal report raises the possibility of using low-dose DLI to reverse unstable chimerism. Another useful tool to assess chimerism will be the use of lineage-specific measures of chimerism. A recent report by Wu demonstrated the potential for measuring erythroid lineage chimerism by quantifying the relative levels of normal and sickle b-globin messenger RNA (42). These investigators measured erythroid chimerism in two adults after nonmyeloablative HSCT for SCD. In these patients levels of donor myeloid chimerism in the peripheral blood were 25–50%, which correlated to normal b-globin mRNA levels of 55–100%. Studies such as these demonstrate an advantage for red blood cells with normal b-globin.

Animal Studies of Stable Mixed Chimerism in Sickle Cell Disease Although it is possible that stable mixed chimerism may correct the hematologic manifestations of SCD, critical questions remain regarding the effects of mixed chimerism on chronic organ damage. Iannone used a murine model of SCD to examine the effects of mixed chimerism on hematologic and organ manifestations of the disease (43). Lethally irradiated normal mice were

406

Woodard

reconstituted with varying ratios of T-cell depleted marrow from normal and transgenic sickle cell mice. The transgenic sickle cell mice demonstrated anemia and marked abnormalities of the liver and spleen. Normal Hgb values were obtained when at least 25% of myeloid chimerism was derived from normal mice, but a higher percentage of normal myeloid chimerism was necessary to reverse other organ damage. For example, liver infarctions were still seen in mice with 33% Hgb S and in one mouse with 16.8% Hgb S. These intriguing studies suggest there may be different thresholds for correction of anemia and correction/prevention of organ damage. One must consider these animals studies in the context of chronic transfusion therapy in humans, which seeks to reduce the percentage of Hgb S to less than 30%, because these levels are protective against subsequent strokes.

Techniques to Induce Stable Mixed Chimerism Myeloablative transplantation has predictable short and long-term toxicities. Short-term toxicities include mucositis, veno-occlusive disease, and acute GVHD, whereas long-term toxicities may include infertility, endocrine dysfunction, and chronic GVHD. Although results to date with myeloablative HSCT for SCD have been good, many providers remain concerned about the risks of mortality, GVHD, and infertility. If it were possible to reduce the toxicity of HSCT, this therapy might be expanded to larger numbers of patients or to patients prior to the onset of organ damage. To circumvent these toxicities, several groups have attempted to use nonmyeloablative conditioning for transplantation of patients with SCD. If successful, such approaches may make HSCT a more widely accepted therapeutic option for SCD. There are limited reports of successful reduced-intensity transplantation with MSDs for SCD (44,45). In one report, an eight-year old with Hgb SS with a history of stroke, recurrent acute chest syndrome, and recurrent pain crises was transplanted with an HLA-matched unrelated donor. The patient was receiving chronic transfusion therapy and iron chelation therapy at the time of HSCT. Conditioning consisted of fludarabine 175 mg/m2, horse ATG 150 mg/kg, and total lymphoid irradiation 500 cGy in a single fraction. GVHD prophylaxis consisted of cyclosporine and mycophenolate mofetil (MMF). The ANC recovered to O500/mm3 by day C9. The patient initially developed mixed chimerism, with 67% donor cells at day C12. Over 60 days, the proportion of donor cells increased to 96% and was 100% by one year. The Hgb was sufficient to support a phlebotomy program to reduce iron overload by one year after HSCT. In the only successful report of an adult with SCD, Schleuning reported successful transplantation in a 22-year-old using reduced intensity conditioning and G-CSF mobilized peripheral blood stem cells from a MSD (44). Conditioning consisted of fludarabine 120 mg/m2 and cyclophosphamide 120 mg/kg. The CD34C cell dose was 25.9!106 cells/kg. GVHD prophylaxis consisted of cyclosporine (CSA) and MMF. The patient initially demonstrated 73% donor chimerism of peripheral blood 30 days after HSCT. MMF was discontinued on day C35 and CSA on day C120. The patient demonstrated full donor chimerism at 120 days. After discontinuation of CSA, the patient developed mild chronic GVHD, which was responsive to CSA, low-dose corticosteroids, and isotretinoin. At the time of the report, 315 days after HSCT, the patient had been tapered off immunosuppressive medication and was doing well. Although these anecdotal reports are illustrative of the potential for cure with minimal toxicity using reduced intensity conditioning, additional study is required to assess engraftment and mechanisms of immune tolerance. Other investigators have reported significant graft failure after less intensive conditioning. Iannone reported the use of nonmyeloablative HSCT from MSD in six patients with SCD and one patient with thalassemia (46). Patients received a minimally toxic regimen of fludarabine 90–150 mg/m2 and 200 cGy TBIChorse ATG (40 mg/kg) in two patients. GVHD prophylaxis consisted of CSA or tacrolimus with MMF. The regimen was minimally myelosuppressive, with the median duration of an absolute neutrophil count !0.5!109/L being five days. Six of seven had donor engraftment ranging

HSCT for Sickle Cell Disease

407

from 25–85%, which was associated with increased total Hgb and Hgb A as well as reduced transfusion requirements and reticulocytosis. One patient had grade II acute GVHD responsive to treatment. Unfortunately, all patients lost donor engraftment after immune suppression was discontinued. Another trial by Horan evaluated minimal conditioning in three patients with SCD and one with thalassemia (47). Conditioning consisted of fludarabine 125 mg/m2, rabbit ATG (Thymoglobulin) (variable dosing), and a single fraction (200 cGy), followed by matched sibling PBSC in one and marrow in three. Postgrafting immunosuppression consisted of cyclosporine and MMF. The patient with thalassemia engrafted but died of RSV. One patient with SCD developed mixed chimerism complicated by red cell aplasia, which resolved with immunosuppression and DLI. Two experienced graft rejection. It is possible that modest increases in conditioning therapy, augmentation of the stem cell dose, manipulation of posttransplant immunosuppression, or low-dose DLI may lead to improved long-term engraftment and/or stable mixed chimerism. Clinical trials to assess the utility of nonmyeloablative HSCT in SCD are currently underway.

ALTERNATE SOURCES OF ALLOGENEIC HEMATOPOIETIC STEM CELLS There are no reports of successful unrelated donor HSCT bone marrow or peripheral blood stem cell transplantation for SCD; however, Adamkiewicz reported the use of unrelated cord blood transplantation in three children with high-risk SCD and prior strokes (48). All three had prior strokes. All were conditioned with myeloablative doses of busulfan, cyclophosphamide, and equine ATG. Cord blood units were matched at four of six HLA-A, -B, and -DRB1 antigens. All three engrafted at 23–42 days. All and developed grade II–III acute GVHD, whereas one developed extensive chronic GVHD. One patient developed graft failure and autologous recovery. Two patients survive free of disease at 40 and 61 months after transplantation, while one patient developed graft failure and autologous recovery. Although there is only one report of successful unrelated donor transplantation for SCD using UCB, results obtained for another hemoglobinopathy, b-thalassemia, are relevant. Recent results using unrelated donors with extended HLA haplotype matching (HLA C, DRB3, DRB4, DRB5, DQA1, or DQB1) have shown that improved matching was associated with improved durable engraftment and survival (49). Patients with a donor identical for at least one extended haplotype had a 77% event-free survival. Failure to engraft and death occurred more frequently in patients who did not share extended haplotypes with their donors and patients with more advanced iron overload. Although extended haplotype matching may lead to improved results, more refined matching will lead to fewer matches, particularly for minorities who are underrepresented on unrelated donor registries. To overcome this problem, unrelated donor registries are targeting minority donors to improve the likelihood of finding matched unrelated donors for minorities. It has been estimated that an increase in the African-American donor pool by 100,000 donors would lead to an 18% increase in the probability of finding a match for AfricanAmerican patients (50). Recent discoveries regarding natural killer (NK) cell alloreactivity and killerimmunoglobulin-like receptors may lead to improved engraftment with decreased GVHD in the context of both unrelated and mismatched related donor transplantation. NK alloreactivity appears to lead to improved engraftment, decreased GVHD, decreased leukemic relapse for AML patients, and decreased transplant-related mortality (51–54). Haploidentical HSCT using stem cell selection processing techniques that enrich for hematopoietic stem cells and deplete alloreactive T cells can are currently under study in children with SCD. If mismatched related donor transplantation could be offered safely, potential benefits would include: (1) readily available donors, (2) large stem cell doses or megadoses of stem cells collected after mobilization of PBSC, and (3) rapid availability of

408

Woodard

donor lymphocytes or stem cell boosts. Historical disadvantages of mismatched related donor transplantation have included high rates of GVHD and infection, coupled with delayed immune reconstitution and increased rates of graft rejection (55,56). Current technology allows purification of very large doses of allogeneic peripheral blood stem cell grafts, coupled with depletion of alloreactive T cells. These advances offer the option of haploidentical SCT without significant GVHD for patients who otherwise lack suitable donors. Using this approach in patients with malignant disease, Aversa reported a dramatic reduction in acute GVHD with haploidentical donors due to the profound T-cell depletion that resulted from positive selection of hematopoietic stem cells (CD34C) cells (57). Another strategy to improve engraftment of mismatched related stem cells is to infuse very large doses of purified hematopoietic stem cells. Very large doses, or megadoses, of purified CD34C stem cells were able to overcome engraftment resistance in animal models (58–61). This may in part result from a “veto” effect in which purified stem cells down regulate anti-donor alloreactivity. In children with nonmalignant disease, megadoses of purified CD34C cells from mismatched related donors have been applied clinically. In twenty-five children with a variety of nonmalignant diseases transplanted from related and unrelated donors, initial engraftment occurred in 84% (62). Three of four patients who did not engraft had a successful second transplant. In total 24/25 (96%) achieved donor engraftment. The CD34C cell purity was 97.6%. The median CD34C stem cell dose was 12.9!106/kg, with a median CD3C dose of 6.1!103/kg. No posttransplant immunosuppression was given, except to the first two patients. Three patients (13%) had grade I acute GVHD and two (8%) had grade II acute GVHD. Two patients (8%) had transient, limited chronic GVHD. Seventy-six percent survive free of disease at a median of 3.7 years. Three patients died, one each from non-engraftment relapse and progressive chronic pulmonary disease (nZ1). A similar approach in children with severe aplastic anemia refractory to immunosuppressive therapy has led to long-term survival free of disease and GVHD in 8/9 patients. Delayed immune reconstitution is a concern after T-cell depleted HCT from haploidentical donors (63,64). In the context of highly purified CD34C stem cell transplants, patients who received greater than 20!106 CD34C cells/kg had more rapid T-cell recovery than those who received a lower dose (65). Natural killer cells recovered rapidly, whereas T-lymphocyte recovery of O0.1!109 CD3C cell/l required a median of 2.5 months. Vigilant monitoring for opportunistic infections is required, using highly sensitive techniques such as polymerase chain reaction to detect viral sequences. A phase 1 trial of haploidentical transplantation underway at St. Jude Children’s Research Hospital. Preliminary results in five children who received haploidentical HSCT have shown that engraftment without servere GVHD is possible for children with severe SCD.

USE OF PERIPHERAL BLOOD STEM CELLS Patients who have experienced graft rejection after HSCT have had autologous reconstitution with recurrent SCD. Autologous “back-up” stem cells are collected and cryopreserved in case patients have aplasia after graft loss. Although the collection of autologous bone marrow has been performed without incident, the collection of peripheral blood stem cells after G-CSF mobilization in patients with SCD has been associated with severe complications and should be avoided. Wei reported severe hypoxia with myocardial ischemia, elevated creatinine, abnormal liver function tests, and encephalopathy in a 58-year-old with SCD and breast cancer who received G-CSF after chemotherapy to mobilized PBSC (66). Abboud reported onset of acute chest syndrome in association with marked leukocytosis after G-CSF administration to a 34-year-old with SCD (67). Symptoms responded to cessation of G-CSF and treatment with hydroxyurea. Adler reported a fatal sickle cell crisis after G-CSF and dexamethasone mobilization to donate PBSC for a sibling with chronic myelogenous leukemia (68). Although G-CSF is not safe for patients with SCD, it appears to be safe to administer to patients with sickle cell trait for PBSC mobilization. Kang administered 10 mcg/kg/day for five

HSCT for Sickle Cell Disease

409

days G-CSF to eight donors with sickle cell trait Donors were pheresed on day 5 (69). The CD34C stem cell content was comparable to control donors. There was no gelling of cellular products from trait donors. There was no difference in symptoms between controls and sickle cell trait donors. The use of PBSC has several potential differences compared to bone marrow, including shorter time to neutrophil and platelet engraftment, shorter hospitalizations, reduced blood product utilization and fewer infections (70). Unfortunately, other reports have found higher rates of chronic GVHD due to increased numbers of T cells in unmanipulated products. One report of PBSC transplantation for beta-thalassemia reported overall and event-free survival of 87% in 15 patients (71). Three patients (20%) developed chronic GVHD. The role of PBSC transplantation from MSDs for nonmalignant diseases requires further study.

FUTURE DIRECTIONS There are many questions remaining to be answered regarding HSCT for SCD. Among the important questions are the following: 1. Will the use of reduced intensity conditioning reliably induce stable mixed chimerism and reduce toxicity? Will modifications in conditioning and/or posttransplant immunosuppression overcome rejection? 2. Will matched unrelated donors, mismatched related donors, and/or unrelated cord blood transplantation be performed safely? 3. Will peripheral blood stem cells be used in children with SCD? 4. Will new techniques or medications be developed to prevent GVHD and other transplant-related complications, rendering HSCT a safer procedure? 5. What will more detailed analyses of chronic organ damage after transplantation yield? 6. Should HSCT be applied to patients with silent ischemia, abnormal transcranial Doppler ultrasound, alloimmunization, or other disease complications?

SUMMARY Myeloablative conditioning and MSD HSCT is curative for approximately 85% of patients with SCD. The largest risks, rejection and mortality, are approximately 10% and 6%, respectively. In addition to correction of hematologic abnormalities, donor engraftment may lead to improved growth and stabilization of chronic organ injury in the CNS and lungs. Although these data are encouraging, only a small minority (!20%) have appropriate MSD. Current efforts are centering on reducing toxicity through reduced intensity conditioning and use of related cord blood transplantation. Preliminary studies are seeking to expand HSCT to patients without MSD by using new stem cell selection technologies or unrelated cord blood for alternate donor transplantation. Hopefully, advances in transplantation and carefully designed trials will lead to improved safety and answers to many intriguing questions in the decade to come.

REFERENCES 1. Platt OS, Brambilla DJ, Rosse WF, et al. Mortality in sickle cell disease. Life expectancy and risk factors for early death. N Engl J Med 1994; 330:1639–1644. 2. Wierenga KJ, Hambleton IR, Lewis NA. Survival estimates for patients with homozygous sickle-cell disease in Jamaica: a clinic-based population study. Lancet 2001; 357:680–683.

410

Woodard

3. Leikin SL, Gallagher D, Kinney TR, et al. Mortality in children and adolescents with sickle cell disease. Pediatrics 1989; 84:500–508. 4. Miller ST, Sleeper LA, Pegelow CH, et al. Prediction of adverse outcomes in children with sickle cell disease. N Engl J Med 2000; 342:83–89. 5. Ohene-Frempong K, Weiner SJ, Sleeper LA, et al. Cerebrovascular accidents in sickle cell disease: rates and risk factors. Blood 1998; 91:288–294. 6. Aygun B, Padmanabhan S, Paley C, Chandrasekaran V. Clinical significance of RBC alloantibodies and autoantibodies in sickle cell patients who received transfusions. Transfusion 2002; 42:37–43. 7. Ballas SK. Iron overload is a determinant of morbidity and mortality in adult patients with sickle cell disease. Semin Hematol 2001; 38:30–36. 8. Johnson FL, Look AT, Gockerman J, et al. Bone-marrow transplantation in a patient with sickle-cell anemia. N Engl J Med 1984; 311:780–783. 9. Vermylen C, Fernandez Robles E, Ninane J, Corno G. Bone marrow transplantation in five children with sickle cell anemia. Lancet 1988; 1:1427–1428. 10. Sullivan KM, Reid CD. Introduction to a symposium on sickle cell anemia: current results of comprehensive care and the evolving role of bone marrow transplantation. Semin Hematol 1991; 28:177–179. 11. Thomas ED. The pros and cons of marrow transplantation for sickle cell anemia. Semin Hematol 1991; 28:260–262. 12. Walters MC, Patience M, Leisenring W, et al. Bone marrow transplantation for sickle cell disease. N Engl J Med 1996; 335:369–376. 13. Vermylen C, Cornu G, Ferster A, et al. Haematopoietic stem cell transplantation for sickle cell anemia: the first 50 patients transplanted in Belgium. Bone Marrow Transplant 1998; 22:1–6. 14. Lucarelli G, Galimberti M, Giardini C, et al. Bone marrow transplantation in thalassemia. The experience of Pesaro. Ann NY Acad Sci 1998; 850:270–275. 15. Locatelli F, Rocha V, Reed W, et al. Related umbilical cord blood transplantation in patients with thalassemia and sickle cell disease. Blood 2003; 101:2137–2143. 16. Reed W, Smith R, Dekovic F, et al. Comprehensive banking of sibling donor cord blood for children with malignant and nonmalignant disease. Blood 2003; 101:351–357. 17. Rosse WF, Gallagher D, Kinney TR, et al. Transfusion and alloimmunization in sickle cell disease. The cooperative study of sickle cell disease [abstract]. Blood 1990; 76:1431–1437. 18. Li CK, Shing MM, Chik KW, et al. Second transplant for a thalassemia patient after graft rejection: with immunosuppression and allogeneic peripheral blood stem cell. Pediatr Hematol Oncol 2002; 19:267–271. 19. Myers SN, Zeevi A, Zorich GP, et al. Successful engraftment following unrelated donor transplant in an alloimmunized patient with Kostmann syndrome. Pediatr Blood Cancer 2005; 44:508–510. 20. Usuda M, Fujimori K, Koyamada N, et al. Successful use of anti-CD20 monoclonal antibody (rituximab) for ABO-incompatible living-related liver transplantation. Transplantation 2005; 79:12–16. 21. Hernigou P, Bernaudin F, Reinhardt D, Kuentz M, Vernant JP. Bone-marrow transplantation in sickle-cell disease. J Bone Joint Surgery 1997; 79:1726–1730. 22. Ferster A, Bujan W, Corazza F, et al. Bone marrow transplantation corrects the splenic reticuloendothelial dysfunction in sickle cell anemia. Blood 1993; 81:1102–1105. 23. Walters MC, Storb R, Patience M, et al. Impact of bone marrow transplantation for symptomatic sickle cell disease: an interim report. Blood 2000; 95:1918–1924. 24. Steen RG, Helton KJ, Horwitz EM, et al. Improved cerebrovascular patency following therapy in patients with sickle cell disease: initial results in 4 patients who received HLA-identical hematopoietic stem cell allografts. Ann Neurol 2001; 49:222–229. 25. Woodard P, Helton KJ, Khan RB, et al. Brain parenchymal damage after haematopoietic stem cell transplantation for severe sickle cell disease. Br J Haematol 2005; 129:550–552. 26. Abboud MR, Jackson SM, Barredo J, et al. Neurologic complications following bone marrow transplantation for sickle cell disease. Bone Marrow Transplant 1996; 17:405–407. 27. Walters MC, Patience M, Leisenring W, et al. Barriers to bone marrow transplantation for sickle cell anemia. Biol Blood Marrow Transplant 1996; 2:100–104. 28. Mentzer WC, Heller S, Pearle PR, Hackney E, Vichinsky E. Availability of related donors for bone marrow transplantation in sickle cell anemia. Am J Pediatr Hematol Oncol 1994; 16:27–29. 29. Kodish E, Lantos J, Stocking C, Singer PA, Siegler M, Johnson FL. Bone marrow transplantation for sickle cell disease: a study of parents decisions. N Engl J Med 1991; 325:1349–1353.

HSCT for Sickle Cell Disease

411

30. van Besien K, Koshy M, Anderson-Shaw L, et al. Allogeneic stem cell transplantation for sickle cell disease. A study of patients decisions. Bone Marrow Transplant 2001; 28:545–549. 31. Adams RJ, McKie VC, Hsu L, et al. Prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial doppler ultrasonography. N Engl J Med 1998; 339:5–11. 32. Nietert PJ, Abboud MR, Silverstein MD, Jackson SM. Bone marrow transplantation versus periodic prophylactic blood transfusion in sickle cell patients at high risk of ischemic stroke: a decision analysis. Blood 2000; 95:3057–3064. 33. Bader P, Beck J, Frey A, et al. Serial and quantitative analysis of mixed hematopoietic chimerism by PCR in patients with acute leukemias allows the prediction of relapse after allogeneic BMT. Bone Marrow Transplant 1998; 21:487–495. 34. Winiarski J, Gustafsson A, Wester D, Dalianis T. Follow-up of chimerism, including T- and B-lymphocytes and granulocytes in children more than one year after allogeneic bone marrow transplantation. Pediatr Transplant 2000; 4:132–139. 35. Huss R, Deeg HJ, Gooley T, et al. Effect of mixed chimerism on graft-versus-host disease, disease recurrence and survival after HLA-identical marrow transplantation for aplastic anemia or chronic myelogenous leukemia. Bone Marrow Transplant 1996; 18:767–776. 36. Mattsson J, Uzunel M, Remberger M, Ringden O. T cell mixed chimerism is significantly correlated to a decreased risk of acute graft-versus-host disease after allogeneic stem cell transplantation. Transplantation 2001; 71:433–439. 37. Andreani M, Manna M, Lucarelli G, et al. Persistence of mixed chimerism in patients transplanted for the treatment of thalassemia. Blood 1996; 87:3494–3499. 38. Andreani M, Nesci S, Lucarelli G, et al. Long-term survival of ex-thalassemic patients with persistent mixed chimerism after bone marrow transplantation. Bone Marrow Transplant 2000; 25:401–404. 39. Battaglia M, Andreani M, Manna M, et al. Coexistence of two functioning T-cell repertoires in healthy ex-thalassemics bearing a persistent mixed chimerism years after bone marrow transplantation. Blood 1999; 94:3432–3438. 40. Walters MC, Patience M, Leisenring W, et al. Stable mixed hematopoietic chimerism after bone marrow transplantation for sickle cell anemia. Biol Blood Marrow Transplant 2001; 7:1–9. 41. Baron F, Dresse MF, Beguin Y. Donor lymphocyte infusion to eradicate recurrent host hematopoiesis after allogeneic BMT for sickle cell disease. Transfusion 2000; 40:1071–1073. 42. Wu CJ, Hochberg E, Rogers SA, et al. Molecular assessment of erythroid lineage chimerism following nonmyeloablative allogeneic stem cell transplantation. Exp Hematol 2003; 31:924–933. 43. Iannone R, Luznik L, Engstrom LW, et al. Effects of mixed hematopoietic chimerism in a mouse model of bone marrow transplantation for sickle cell anemia. Blood 2001; 97:3960–3965. 44. Krishnamurti L, Blazar BR, Wagner JE. Bone marrow transplantation without myeloablation for sickle cell disease. N Engl J Med 2001; 344:68. 45. Schleuning M, Stoetzer O, Waterhouse C, Schlemmer M, Ledderose G, Kolb H-J. Hematopoietic stem cell transplantation after reduced-intensity conditioning as treatment of sickle cell disease. Exp Hematol 2002; 30:7–10. 46. Iannone R, Casella JF, Fuchs EJ, et al. Results of minimally toxic nonmyeloablative transplantation in patients with sickle cell anemia and Beta-thalassemia. Biol Blood Marrow Transplant 2003; 9:519–528. 47. Horan JT, Liesveld JL, Fenton P, Blumberg N, Walters MC. Hematopoietic stem cell transplantation for multiply transfused patients with sickle cell disease and thalassemia after low-dose total body irradiation, fludarabine, and rabbit anti-thymocyte globulin [abstract]. Bone Marrow Transplant 2005; 35:171–177. 48. Adamkiewicz TV, Mehta PS, Boyer MW, et al. Transplantation of unrelated placental blood cells in children with high-risk sickle cell disease. Bone Marrow Transplant 2004; 34:405–411. 49. La Nasa G, Giardini C, Argiolu F, et al. Unrelated donor bone marrow transplantation for thalassemia: the effect of extended haplotypes. Blood 2002; 99:4350–4356. 50. Beatty PG, Mori M, Milford E. Impact of racial genetic polymorphism on the probability of finding an HLA-matched donor. Transplantation 1995; 60:778–783. 51. Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295:2097–2100. 52. Ruggeri L, Capanni M, Casucci M, et al. Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation. Blood 1999; 94:333–339.

412

Woodard

53. Giebel S, Locatelli F, Lamparelli T, et al. Survival advantage with KIR ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors. Blood 2003; 102:814–819. 54. Farag SS, Fehniger TA, Ruggeri L, Velardi A, Caligiuri MA. Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect. Blood 2002; 100:1935–1947. 55. Kernan NA, Flomenberg N, Dupont B, O’Reilly RJ. Graft rejection in recipients of T-cell-depleted HLA-nonidentical marrow transplants for leukemia. Identification of host-derived antidonor allocytotoxic T lymphocytes. Transplantation 1987; 43:842–847. 56. Mitsuyasu RT, Champlin RE, Gale RP, et al. Treatment of donor bone marrow with monoclonal antiT-cell antibody and complement for the prevention of graft-versus-host disease. A prospective, randomized, double-blind trial. Ann Intern Med 1986; 105:20–26. 57. Aversa F, Tabilio A, Terenzi A, et al. Successful engraftment of T-cell-depleted haploidentical “three-loci” incompatible transplants in leukemia patients by addition of recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells to bone marrow innoculum. Blood 2002; 84:3948–3955. 58. Reisner Y, Bachar-Lustig E, Li H-W, et al. The role of megadose CD34C progenitor cells in the treatment of leukemia patients without a matched donor and in tolerance induction for organ transplantation. Ann NY Acad Sci 1999; 872:336–348. 59. Reisner Y, Gur H, Reich-Zeliger S, Martelli MF, Bachar-Lustig E. Hematopoietic stem cell transplantation across major genetic barriers: tolerance induction by megadose CD34 cells and other veto cells. Ann NY Acad Sci 2003; 996:72–79. 60. Reisner Y, Martelli MF. Tolerance induction by ‘megadose’ transplants of CD34C stem cells: a new option for leukemia patients without an HLA-matched donor. Curr Opin Immunol 2000; 12:536–541. 61. Reisner Y, Martelli MF. Transplantation tolerance induced by “mega dose” CD34C cell transplants. Exp Hematol 2000; 28:119–127. 62. Lang P, Klingebiel T, Bader P, et al. Transplantation of highly purified peripheral-blood CD34(C) progenitor cells from related and unrelated donors in children with nonmalignant diseases. Bone Marrow Transplant 2004; 33:25–32. 63. Small TN, Papadopoulos EB, Boulad F, et al. Comparison of immune reconstitution after unrelated and related T-cell-depleted bone marrow transplantation: effect of patient age and donor leukocyte infusions. Blood 1999; 93:467. 64. Vossen J, Handgretinger R. Immune recovery and immunotherapy after stem cell transplantation in children. Bone Marrow Transplant 2001; 28:S14–S15. 65. Handgretinger R, Klingebiel T, Lang P, et al. Megadose transplantation of purified peripheral blood CD34C progenitor cells from HLA-mismatched parental donors in children. Bone Marrow Transplant 2001; 27:777–783. 66. Wei A, Grigg A. Granulocyte colony-stimulating factor-induced sickle cell crisis and multiorgan dysfunction in a patient with compound heterozygous sickle cell/betaC thalassemia. Blood 2001; 97:3998–3999. 67. Abboud M, Laver J, Blau CA. Granulocytosis causing sickle-cell crisis. Lancet 1998; 351:959. 68. Adler BK, Salzman DE, Carabasi MH, et al. Fatal sickle cell crisis after granulocyte colonystimulating factor administration. Blood 2001; 97:3313–3314. 69. Kang EM, Areman EM, David-Ocampo V, et al. Mobilization, collection, and processing of peripheral blood stem cells in individuals with sickle cell trait. Blood 2002; 99:850–855. 70. Mavroudis D, Read E, Cottler-Fox M, et al. CD34C cell dose predicts survival, post transplant morbidity, and rate of hematologic recovery after allogeneic marrow transplants for hematologic malignancies. Blood 1996; 88:3223–3229. 71. Yesilipek MA, Hazar V, Kupesiz A, et al. Peripheral blood stem cell transplantation in children with beta-thalassemia. Bone Marrow Transplant 2001; 28:1037–1040.

22 Metabolic Diseases Charles Peters Hematopoietic Stem Cell Transplantation, Division of Hematology/Oncology, Children’s Mercy Hospital, Kansas City, Missouri, U.S.A.

INTRODUCTION Metabolic diseases are a diverse, complex group of disorders that includes mucopolysaccharidoses (MPS), leukodystrophies, glycoprotein metabolic disorders, and such miscellaneous diseases as neuronal ceroid lipofuscinosis (NCL), Niemann-Pick, mucolipidoses, gangliosidoses, Wolman syndrome, Farber lipogranulomatosis, and osteopetrosis. Individually, many of these disorders are considered rare, although high prevalence rates have been reported in some populations (1). Most of these genetic disorders arise from a deficiency of a specific hydrolytic enzyme found in or associated with the lysosome leading to the term lysosomal storage disorder. X-linked adrenoleukodystrophy (X-ALD) is likely a peroxisomal disorder. Osteopetrosis arises from defective osteoclast function. Following this introduction, a general overview of hematopoietic stem cell transplantation (HSCT) will be presented with attention to metabolic disease issues. Disease-specific discussions will follow. Finally, developing therapies and novel approaches will be described for this rapidly evolving field of medicine.

Lysosomes and Peroxisomes Lysosomes Lysosomes are found in all cells, including leukocytes, erythrocytes, and platelets. The formation of lysosomes begins in ribosomes attached to the limiting membrane of the rough endoplasmic reticulum (RER). Proteins synthesized in RER proceed to the smooth ER, where further processing of proteins destined to become enzymes occurs and mannose-rich oligosaccharide chains are added. Following modification in the Golgi apparatus and the addition of mannose-6-phosphate residues, the proenzyme is released and packaged into the lysosomal vesicle (2). Lysosomal hydrolytic enzymes function in a digestive manner while normal cell constituents are protected by the lysosomal membrane. At least 50 enzymes acting on carbohydrates, lipids, proteins, and nucleotides reside in lysosomes and function with specificity. Active two-way exchange of lysosomal enzymes occurs within and between cells and the extracellular space (3). Enzyme release from normal cells with uptake by enzymedeficient cells can occur. Metabolic correction of lysosomal storage disorders is based upon mannose 6-phosphate (M6P) receptor-mediated endocytosis of secreted enzyme or by direct transfer of enzyme from adjacent cells (4–7). Both mechanisms provide enzymatic correction following HSCT. Receptor-mediated endocytosis occurs when a lysosomal hydrolase bearing a M6P residue is secreted by the donor cell and binds to the host cell M6P receptor. Enzyme is 413

414

Peters

internalized into the cytoplasm and transferred to the lysosomal compartment (4–7). Variability in receptor-mediated enzyme endocytosis in cells and tissues may affect hydrolase uptake after HSCT (8). For example, monocytes and tissue macrophages have receptors for N-acetylglucosamine and mannose whereas glial cells have receptors for sialic acid (9). Direct enzyme transfer to the intracellular space occurs independently of receptor-mediated endocytosis and requires cell-cell contact (4,10,11) through various adhesion molecules (4,11).

Peroxisomes Peroxisomes are subcellular components responsible for fatty acid metabolism. X-ALD is a peroxisomal disorder of very long chain fatty acid (VLCFA) b-oxidation. Rather than a metabolic cross-correction, the likely mechanism by which HSCT effectively halts the cerebral demyelination of X-ALD is by replacement of metabolically abnormal cell populations rather than by transfer of biochemical mediators from normal donor cells; decreased perivascular inflammation also contributes. The benefit from normalization of plasma VLCFA is unclear. Pathogenesis Deficiency of a single lysosomal enzyme activity results in substrate accumulation within lysosomal membrane-bound vesicles. Continued lysosomal uptake of the nonmetabolized material leads to lysosomal hypertrophy and excessive “storage” material. Signs and symptoms in each of the metabolic storage diseases reflect the pattern of distribution of the nondegraded material. For example, in Gaucher and Niemann-Pick diseases, the turnover of erythrocyte and leukocyte membranes leads to the formation of lipids that become trapped within visceral organs. However, in the gangliosidoses, the primary pathologic changes occur in the central nervous system (CNS) because under normal circumstances ganglioside concentrations are much greater here. Glycoproteins are present in nearly all tissues, inside and outside the CNS. Consequently, diseases arising from storage of mucopolysaccharides and other complex carbohydrates derived from glycoproteins affect many organ systems.

Genetics Nearly all lysosomal storage diseases, are autosomal recessive disorders. Two exceptions, Fabry disease and Hunter syndrome, are X-linked disorders, as is the peroxisomal disease X-ALD. Genes responsible for these single gene disorders have been sequenced, and their enzyme products have been characterized. Furthermore, to a limited degree, genotype/phenotype correlations have been described. However, for a large proportion of patients and diseases, the mutation is a private one that does not allow precise prediction of the disorder’s course. Prenatal diagnosis is possible and allows families to make reproductive choices about an affected fetus. The chromosome assignment for genes coding for certain lysosomal and peroxisomal enzymes and proteins are presented (Table 1).

Clinical Evaluation Clinical evaluation of lysosomal and peroxisomal disorders should include a complete medical history, including a detailed family history and physical examination. Dysmorphic facial features and hepatosplenomegaly are common. Many disorders display connective tissue infiltration, such as joint contractures, skeletal dysplasia, and corneal clouding. Signs of CNS involvement with progressive neurocognitive developmental delay are often evident. Knowledge of the patient’s family history can be helpful. In the clinical evaluation of patients with possible metabolic storage disease, close attention should be given to the chronology of the disease, the family background and neurologic, neuropsychological, visceral, and skeletal manifestations. In general, the later in life that clinical signs appear, the slower the rate of progression and consequently the more attenuated the disease process. Despite clear clinical signs, laboratory and imaging modalities are important for assessment and diagnosis.

Metabolic Diseases

415

Table 1 Chromosomal Assignment of Genes Coding for Certain Lysosomal and Peroxisomal Enzymes and Proteins Disease and enzyme Mucopolysaccharidoses MPS I: alpha-L-iduronidase MPS II: iduronate sulfatase MPS III A: Heparan-N-sulfatase B: a-N-acetyl-glucosaminidase C: acetyl CoA: a-glucosaminide-acetyltransferase D: N-acetylglucosamine 6-sulfatase MPS IV A: Galactose-6-sulfatase B: b-galactosidase MPS VI: arylsulfatase B MPS VII:b-glucuronidase Multiple sulfatase deficiency: Leukodystrophies X-linked adrenoleukodystrophy: ALDP Globoid-cell: galactocerebrosidase Metachromatic: arylsulfatase A Glycoprotein metabolic disorders Fucosidosis: fucosidase Gaucher: glucocerebrosidase Mannosidosis Alpha: alpha-mannosidase Aspartyglucosaminuria: aspartylglucosaminidase Miscellaneous disorders Neuronal ceroid lipofuscinosis Infantile type (CLN-1): palmitoyl protein thioesterase Late infantile type (CLN-2): tripeptidyl peptidase I Early juvenile type (CLN-5): lysosomal membrane associated protein Juvenile type (CLN-3): lysosomal membrane associated protein Adult type (CLN-4 and 6): unknown Niemann-Pick A and B: acid sphingomyelinase C1 and C2: cholesterol trafficking Mucolipidosis II: phosphotransferase Gangliosidosis GM1 (type 1, infantile GM1; type 2, late infantile/juvenile; type 3, adult): beta-galactosidease GM2 Tay-Sachs: hexosaminidase A Sandhoff: hexosamindase B GM2 activator deficiency Sialidosis: neuraminidase Wolman: acid lipase Farber lipogranulomatosis: acid ceramidase Osteopetrosis (OP) Carbonic anhydrase II deficiency (CAD)

Chromosome 4p16.3 Xq27–28 17q25.1 17q21.1 Not known 12q14 16q24.3 3p21.33 5q13–q14 7q21.11 Multiple chromosomes Xq28 14q13 22q13 1p24 1q21 19p13.2–q12 4q34–35

1p32 11p15

16p12

11p15.1–15.4 C1: 18q11 12p and 16p 3p21.33

6p21 10q23.2–q23.3 8p21.3/22 8q22

416

Peters

Laboratory and Imaging Evaluation and Diagnosis Laboratory evaluation of metabolic storage disorders may involve evaluation of the urine for an increase in complex carbohydrates, such as glycosaminoglycans (GAGs), followed by definitive testing of specific hydrolytic enzyme activity levels in leukocytes. Molecular studies are commonplace and can identify the specific mutation in the gene of interest. Furthermore, molecular characterization is critical to determine carrier status due to the overlap between homozygous normal and heterozygous carrier enzyme activity levels. Nuances in diagnosis will be presented for individual disorders. Skeletal dysplasia may be evident on radiographic studies (e.g., dysostosis multiplex of MPS disorders). Imaging of the brain may show evidence of Virchow-Robin space abnormalities (e.g., MPS disorders), increased signal on T2-weighted images consistent with leukodystrophy, spectroscopy may demonstrate abnormal metabolite ratios (e.g., Canavan disease).

Treatment and Principal of Cross Correction This chapter focuses on hematopoietic cell transplantation for selected inherited metabolic storage disorders. The principal underlying its use is that of cross-correction. In 1968, Fratantoni and Neufeld established the basis of our understanding of transferable lysosomal enzymes by demonstrating metabolic cross-correction of defects in cocultures of fibroblasts from Hurler and Hunter syndrome patients (12). This observation and the later demonstration of correction by lymphocyte extracts or serum (13,14) led Hobbs to employ HSCT as a permanent source of enzyme in a Hurler patient (15). Dramatic improvement in disease phenotype paved the way for HSCT in many inherited metabolic diseases.

Prevention Prenatal diagnosis has been accomplished or is theoretically possible for each of the metabolic storage diseases. It is important that parents of children with sphingolipidoses, MPS, or oligosaccharidoses be counseled and informed of the mode of inheritance and the availability of prenatal diagnosis. Screening of large populations for heterozygotes has been performed for Tay-Sachs, Niemann-Pick, and Gaucher disease as well as Fanconi anemia. This has been feasible due to the relatively high gene frequency in a small subgroup of the population, namely, Ashkenazic Jews, and the availability of a simple, dependable, and economical test for heterozygotes. Testing for other metabolic storage disorders cannot meet these conditions either because the disease is very rare and distributed equally throughout the general population or because the enzyme assay for heterozygotes is complex and/or unreliable. At this time, prevention of metabolic storage disorders relies on early case identification, screening of close relatives for heterozygotes, and prenatal testing during pregnancies in which both parents are known to be carriers for the same recessive trait.

HEMATOPOIETIC-CELL TRANSPLANTATION Patient Evaluation Evaluation of a prospective patient with a metabolic storage disease prior to HSCT should include not only a comprehensive assessment that would be performed for any potential transplant recipient but also a disease-specific evaluation that evaluates the stage of the disease process and its rate of progression. Specific details for such an evaluation will be included in the discussion of the particular diseases (see below).

Hematopoietic Stem Cells, Timing of Transplantation, and Donor Selection Sources of hematopoietic stem cells include bone marrow, peripheral blood, and umbilical cord blood. Cell dose, preparative regimen, potential for graft manipulation, and timing of the

Metabolic Diseases

417

transplant are important factors. Consideration must be given to stage of disease in the potential recipient, rate of disease progression, and potential for stabilization or reversal of the disease process. Whenever possible, the enzyme activity level appropriate for the disease being treated should be evaluated in potential donors. For example, if the recipient has Hurler syndrome, the potential donor’s alpha-L-iduronidase enzyme activity level can be considered during the donor selection process.

Preparative Regimens, Graft Engineering, and Stem-Cell Selection Preparative regimens vary along a continuum with respect to their intensity from fully myeloablative to minimally myelosuppressive and maximally immunosuppressive. The risks and benefits should be considered for the particular disease and patient. To date, the clinical experience with reduced intensity preparative regimens is limited, though reports of successful transplant have appeared in the literature.

Graft-Versus-Host Disease Prophylaxis and Treatment Graft-versus-host disease (GVHD) prophylaxis can take the form of various peri- and posttransplant regimens, including antithymocyte globulin, cyclosporine, steroids, methotrexate, etc. Furthermore, graft manipulation especially with bone marrow and peripheral blood stem cells can lead to a significant depletion of lymphocytes from the graft in the hope of reducing the incidence and severity of GVHD. Treatment for clinically evident GVHD includes standard therapies, such as high dose steroids, ATG, and other potent immunosuppressive regimens. It should be noted that GVHD is of no clinical benefit to patients with inherited metabolic storage disorders as there is no comparable graft-versus-disease effect, as is seen in some malignant conditions.

Regimen- and Disease-Related Complications Failure after HSCT may be due to primary or secondary graft failure, with loss of donor cells and their accompanying enzyme activity or a regimen- or disease-related complication. Regimen-related toxicities are well recognized after HSCT. However, patients with inherited metabolic disorders have also demonstrated many disease-specific complications, which may involve the cardiopulmonary system and the CNS.

Multi-Disciplinary Follow-Up Due to the multiple organ systems that can be affected by the underlying disease, as well as the potential late effects of the HSCT, multidisciplinary, multispecialty, comprehensive, coordinated follow-up at an experienced medical center in these complex diseases is both highly desirable and essential.

MUCOPOLYSACCHARIDOSES The MPS are a group of lysosomal disorders caused by deficiency of degradative enzymes of GAGs (16). GAGs, such as heparan, dermatan, keratan, and chondroitin sulfates, individually or in combination accumulate or are “stored” intracellularly and lead to cellular, tissue, and organ dysfunction. These GAGs are typically excreted in urine and are often detected in the initial diagnostic screening tests. The genes and the cDNAs encoding most of these enzymes have been cloned leading to characterization of their primary structures, production of recombinant enzymes, and identification of disease-causing mutations. The MPS disorders and their respective enzyme deficiency include MPS I (Hurler—severe form; Hurler-Scheie and Scheie—attenuated forms; a -L-iduronidase), MPS II (severe and attenuated forms of Hunter; MPS IIA and B, respectively; iduronate sulfatase), MPS III (Sanfilippo IIIA, sulfamidase; IIIB,

418

Peters

a-N-acetylglucosaminidase; IIIC, acetyl-CoA: a-glucosaminide acetyltransferase; IIID, N-acetylglucosamine 6-sulfatase), MPS IV (Morquio A, N-acetylgalactosamine 6-sulfatase; Morquio B, b-galactosidase), MPS VI (Maroteaux-Lamy, arylsulfatase B), and MPS VII (Sly, b-glucuronidase). There are many shared clinical features among the MPS disorders, including chronic, progressive courses; multiorgan system involvement; and organomegaly, dysostosis multiplex and facial dysmorphia. Hearing, vision, pulmonary, and cardiovascular function are often affected. Profound psychomotor retardation is observed in Hurler syndrome (MPS IH), the severe form of Hunter syndrome (MPS IIA), and Sanfilippo syndrome (MPS III). All of the MPS disorders, with the exception of Hunter syndrome, which is X-linked, are inherited in an autosomal recessive manner. Although many mutations have been identified and found to be associated with a severe phenotype for the various MPS disorders, a precise correlation between all genotypes and phenotypes remains elusive. Enzyme assays are available for diagnosis, including prenatal evaluation, for all MPS disorders. Definitive identification of heterozygotes by enzyme determination is often not possible. Characterization of the mutation(s) within a family can be informative. In addition to naturally occurring disease models observed in dogs, cats, rats, mice, and goats, there are mouse models created by targeted gene disruption. The biochemical and pathologic features of these “knock-out” animal models can be similar to the human disease condition. To date, HSCT represents the most effective long-term therapy for selected MPS disorders. MPS disorders that benefit from HSCT include Hurler (MPS IH), Maroteaux-Lamy (MPS VI), and Sly (MPS VII) syndromes. Unequivocal benefit following HSCT for the CNS or the skeletal system has not been shown for patients with Hunter (MPS II), Sanfilippo (MPS III), and Morquio (MPS IV) syndromes. Clinical data are also available now for shorter-term enzyme replacement therapy (ERT) for selected MPS disorders including MPS I, II, and VI.

Mucopolysaccharidoses I Mucopolysaccharidoses IH (Hurler Syndrome, a-L-Iduronidase Deficiency) Preclinical Models. Naturally occurring preclinical models of MPS I include the dog, cat, the murine model generated by targeted disruption of the murine iduronidase gene (16). In the Plott hound, HSCT leads to normalization of tissue GAGs (17). Despite low iduronidase enzyme activity in the CNS, GAG is cleared from neurons and glial cells with decreased meningeal thickening (18,19). Improvements in corneal clouding (20) and cardiac valvular thickening (21) were also noted. Compared to untransplanted littermates, skeletal abnormalities were ameliorated to some degree (22). Detailed identification and characterization of the molecular lesion causing MPS I in cats has also been performed (23). More recently, the development of the MPS I mouse by targeted disruption of the murine iduronidase gene (24) has led to further insights into the pathogenesis of Hurler syndrome (25), particularly the skeletal and CNS manifestations. Analysis of the CNS revealed the novel finding of progressive neuronal loss in the cerebellum and increased levels of GM2 and GM3 gangliosides in brain tissue. Clinical Experience. Hurler syndrome represents the most severe phenotype of the three MPS I entities. Various approaches have been used to assess phenotype severity, including genotyping, iduronidase enzyme kinetics, immunoquantitation, in vitro turnover studies (26), mutational analysis (27), and quantitation of urinary GAGs (28). Although the W402X and Q70X mutations have been recognized as the most common mutations in MPS IH patients (29,30), numerous other mutations have been observed in patients with MPS IH, further underscoring the genetic and clinical heterogeneity of MPS I (31). MPS IH, is characterized by progressive mental retardation and hydrocephalus, frequent ear infections, rhinorrhea, auditory impairment, corneal clouding, sleep apnea (32,33), cardiopulmonary disease (including thickened valves, coronary artery narrowing, pulmonary hypertension,

Metabolic Diseases

419

and congestive heart failure) (34), hepatosplenomegaly, and severe skeletal abnormalities (termed dysostosis multiplex) (35). These result in substantial morbidity and early death in untreated MPS IH patients, yielding a median survival of 5 years with few children surviving beyond 12 years (36). During the more than two decades since the first allogeneic HSCT for MPS IH (37), more than 300 MPS IH patients have been transplanted. Large HSCT experiences with both unrelated (38) and related donors (39) have provided guidance for patient selection and timing of HSCT and a realistic assessment of the effects of HSCT on various organs and tissues in MPS IH. Due to the nature of the small clinical studies performed to date, it is difficult to provide a precise estimate of long-term engrafted survival for Hurler HSCT patients. However, the likelihood of long-term engrafted survival would be estimated to be as high as 80% using bone marrow or umbilical cord blood (UCB) (40,41). When performed early in the disease course or in conjunction with intensive neuropsychological and speech therapy, HSCT can preserve intellectual function and prevent the severe phenotypic manifestations associated with “classic” Hurler syndrome. The mechanism by which progressive mental retardation occurs in Hurler syndrome remains unclear; however, communicating hydrocephalus and increased intracranial pressure arising from GAG deposition in the meninges and periadventitial (i.e., Virchow-Robin) spaces is wellrecognized particularly late in the disease course (16). After successful HSCT, children with MPS IH have not required ventriculoperitoneal shunting to treat hydrocephalus (42,43). Age, cardiopulmonary, and neurodevelopmental status are important determinants of outcome in Hurler patients following HSCT (44). Children with Hurler syndrome who are less than 18 months of age at the time of HSCT and who have no prior history of pulmonary complications (i.e., upper or lower airway obstructive disease, pneumonias, oxygen requirement, or ventilator support), enjoy a long-term survival rate after HSCT of approximately 80%. The incidence of intubation in children with Hurler syndrome after HSCT is 31% by day C100 (44) with pulmonary hemorrhage observed in 14% of patients with Hurler syndrome by this time after HSCT. The risk factors that appear to predict requirement for ventilator support include lower airway obstruction, age greater than 18 months, and severe acute GVHD. Other contributing factors include quality and quantity of developmental services and therapies (e.g., speech therapy) as well as the post-HSCT course (38,39,45). HSCT is able to preserve neurocognitive development in Hurler children with relatively normal intellectual function (28,46–48). Although a goal of the first HSCT is to achieve full donor chimerism, in some cases this is not possible. Graft failure in Hurler patients requires further investigation. Nevertheless, a timely second HSCT can be well-tolerated and beneficial in stabilizing neuropsychological function (49). It has been shown that busulfan pharmacokinetics are not altered in children with inherited metabolic storage diseases, including Hurler syndrome, and that rates of donor-derived engraftment are comparable for patients with storage diseases, including Hurler syndrome (Tables 2 and 3) (50). There continues to be considerable discussion regarding the optimal stem cell source and iduronidase enzyme activity level for HSCT. Although levels of iduronidase can be measured in the WBC of marrow donors as well as in UCB (51), the latter remains problematic because a satisfactory WBC sample from the UCB unit is often not available to be analyzed in a timely manner. The nature of the preparative regimen, including less intensive therapy, is also being critically reviewed (52) as well as the use of haploidentical PBSC (53).Centers caring for large numbers of high-risk Hurler patients based upon their age and/or cardiopulmonary status (44,54) have been examining the capability of reduced intensity regimens, followed by UCB or PBSC transplantation to achieve significant, stable long-term donor chimerism while minimizing regimen- and disease-related toxicities. A recent report by Grigull and coworkers

420

Peters

in Hannover, Germany, described the successful use of a fludarabine-based conditioning regimen for five children with Hurler syndrome (54a). With successful donor-derived engraftment, Hurler patients can experience favorable long-term CNS outcomes. These outcomes include stabilization of the CNS with preservation of neurocognitive function and motor development within the range of normal and capacity for independence in activities of daily living. Careful neuropsychological and neuroradiologic assessments are required (55–57). Hepatosplenomegaly, joint mobility, and upper airway obstruction with sleep apnea (58) resolve within months of the HSCT; GAG disappears from hepatocytes and Kupffer cells (59). Table 2 Guidelines for the Evaluation of Patients with Hurler Syndrome Prior to and Long-Term Following Hematopoietic-Cell Transplantation Pretransplant evaluation Blood and marrow transplant team: physician, nurse practitioner, nurse, social worker Neurology including examination Neuropsychology: test of cognitive ability according to age and developmental level, i.e., Mullen scales of early learning to obtain early learning composite or WISC III Language assessment to obtain receptive and expressive language scores Vineland adaptive behavior scales Neuroradiology: MRI of brain and upper C-spine (attention to odontoid process) Cardiology, including echocardiogram; EKG Pulmonology, including chest radiograph and possibly polysomnogram Audiology: ENT (as needed), BAER if requested by audiology Ophthalmology and possible ERG Genetic counseling Electrophysiology studies (EMG) with surface electrodes Occupational, physical, and speech therapy: assessment and therapies as needed Donor studies: Donor’s enzyme activity level Molecular biology studies: on admission blood sample to obtain DNA (e.g., mutation analysis, pharmacogenomics) Lumbar puncture: opening and closing pressures, CSF glucose, and protein Bronchoscopy and collection of sample (bronchoalveolar lavage while patient is in or for central line placement or other or procedure) Follow-up orders Blood and marrow transplant team: physician, nurse practitioner, nurse, social worker Neurology, including examination Neuropsychology: test of cognitive ability according to age and developmental level, i.e., Mullen Scales of Early Learning (early learning composite), or Stanford Binet Intelligence Scale (composite score), or Wechsler Preschool and Primary Scale of Intelligence (full scale IQ); language assessment to obtain receptive and expressive language scores; Vineland Adaptive Behavior Scales Neuroradiology: MRI of brain and upper C-spine (attention to odontoid process) Cardiology, including echocardiogram; EKG Pulmonology, including chest radiograph, consider follow-up polysomnogram as indicated Audiology: ENT (as needed), BAER if requested by audiology Ophthalmology, consider ERG Orthopedic surgery: orthopedic evaluation of spine, hips, knees, and hand surgery assessment for carpal tunnel syndrome Endocrinology: growth hormone stimulation test, Free T4, TSH levels, DEXA scan, bone age radiograph Electrophysiology studies (EMG) with surface electrodes Occupational, physical, and speech therapy: assessment and therapies as needed Engraftment and chimerism studies: patient’s VNTR (variable nucleotide tandem repeat) and enzyme level (days C21, 60, 100, 6 months, 1 year, yearly post-HSCT thereafter)

Metabolic Diseases Table 3

421

Busulfan Clearance in Pediatric Patients with Respect to Age

Age (years)

n

Cl/F (ml/min/kg) Mean G s.d.

!3 R3 !5

26 13 4

6.2G2.2 4.3G1.3 7.3

5–16 O16 0.5–3 7–19

5 18 10 6

3.02 2.7 10.3G4

%3 4–14.2 0.2–3.6

13 7 14

6.6G1.7 12.2 4.0 8.4G4.3

0.2–2.75

33

6.8G3.0

Disease Metabolic storage diseases

Ref. Our study

Lymphohematopoietic malignancies, leukodystrophy

12

Lymphohematopoietic malignancies, bone marrow failure syndromes, thalassemia

11

Lymphohematopoietic malignancies, bone marrow failure syndromes Leukemia, bone marrow failure syndromes, Hurler syndrome Bone marrow failure syndromes, metabolic storage diseases, leukemia

29 28 27

Corneal clouding stabilizes or slowly resolves in many patients; ocular pressures may normalize; however, for many patients, electroretinogram abnormalities are evident long-term (60–62). HSCT does not reverse the progressive, profound conductive and sensorinerual hearing observed in many Hurler patients, though between 30% and 40% of children do show stabilized or improved auditory acuity after transplant (36). Heart failure and tachyarrhythmias are eliminated by 1 year after successful HSCT (63,64). Myocardial muscle function is stabilized or improved and coronary artery patency has been demonstrated up to 14 years after HSCT (65). The long-term outcome of cardiac valvular thickening and insuffiency require continued monitoring. Early cardiac death in children with Hurler syndrome often stems from progressive deposition of GAGs within the myointima of the coronary arteries and airways; however, cardiac ultrasound findings of patients successfully transplanted 9 to 15 years before shows cardiac function is preserved, hypertrophy regressed, and that chamber dimensions trended toward normal; in some patients, the left-sided cardiac valves continued to thicken and develop insufficiency (66). However, some disease features show much poorer response due to poor penetration of iduronidase into the relevant tissue. Principal among these are the skeletal abnormalities known as dysostosis multiplex (67). Successfully transplanted children often require major orthopedic surgical procedures for genu valgum, acetabular hip dysplasia, kyphoscoliosis, carpal tunnel syndrome, and trigger digits by 6 years of age (68–71). Interestingly, stabilization of the upper cervical spine has been observed in the long-term outcomes of many donor-engrafted Hurler patients (Fig. 1) (72). However, severe spinal cord injury has been observed 6 years after successful HSCT (73). Orthopedic problems merit careful monitoring and appropriate, timely intervention, although the optimal timing of the latter remains under debate (Fig. 2). The decision of whether to proceed with HSCT for patients with Hurler syndrome is a complex one and demands that a comprehensive pretransplant evaluation be performed and that detailed information be provided to families with particular attention to transplant- and diseaserelated complications, as well as Hurler characteristics that are typically refractory to HSCT, such as the skeletal abnormalities, some ocular and auditory features, and cardiac aspects (Table 2). Complications of HSCT in Hurler patients can include hemolytic anemia, pulmonary hemorrhage, gallbladder hydrops, ventriculoperitoneal-shunt infection, and graft failure (41). Detailed analysis of risk factors will enable transplant physicians, health care providers, and

422

Peters

families of these children to determine the safest and most effective HSCT strategy (44). A novel approach that is under study is the combination of ERT with laronidase in conjunction with HSCT. The rationale for this approach is as follows: reduction of the total body burden of GAGs after multiple weekly doses of enzyme will lead to reduction in regimen- and diseaserelated morbidity and mortality associated with HSCT. Since the fall of 2003, more than 40 children with Hurler syndrome have been treated in this manner. Preliminary results suggest that safe and effects transplants are possible; however, failures have been observed in cases of reduced intensity preparative regimens and umbilical cord blood (74). Long-term, coordinated, comprehensive monitoring and follow-up, together with close collaboration with educators and therapists (e.g., physical, occupational and speech), is paramount (Figs. 3 and 4).

Figure 1 (A) Patient with Hurler syndrome, age 2.3 years prior to BMT. Lateral view of the cervical spine in neutral position. This was the initial evaluation at the time of BMT. The odontoid morphology shows severe dysplasia. The odontoid has a triangular configuration with a broad base and decreased vertical height. (B) The same patient at age 12.5 years (10 years after BMT). Sagittal T2-weighted image through the craniocervical junction (TRZ2500, TEZ90). This study was obtained 10 years following successful BMT. The odontoid process of C2 has normal morphology. A dural low-signal intensity mass is identified producing anteroposterior indentation on the subarachnoid space at the level of the upper cervical canal (arrow). Source: From Ref. 72.

Metabolic Diseases

423

Figure 2 Patient with Hurler syndrome, anteroposterior radiographs of the knees. (A) At age 22 months, the left knee alignment was neutral, whereas the right knee showed varus deformity. (B) Valgus deformities of the untreated knees in this patient at age 10 years. Source: From Ref. 69.

Mucopolysaccharidoses IH/S and Mucopolysaccharidoses IS (Hurler-Scheie and Scheie Syndromes) Although there is no definitive animal model for MPS IH/S or MPS IS, it is felt that the iduronidase-deficient dog is more representative of MPS IH/S than MPS IH. HSCT for this model has been discussed (75). Hurler-Scheie syndrome demonstrates an intermediate phenotype between Hurler and Scheie syndromes with normal to mildly delayed neurocognitive development and survival beyond the first decade and often into the third decade (16). A 10-year-old child with MPS IH/S underwent HSCT and demonstrated the resolution of hepatosplenomegaly, amelioration of facial features, stabilized cardiac function, and improved joint range of motion, with persistent and progressive skeletal abnormalities and unchanged corneal clouding, though visual acuity improved. Neurocognitive function was preserved at the below average level (76). Although HSCT can successfully treat MPS IH/S or MPS IS as well, the typical approach is to consider such patients for laronidase ERT (77). Future Directions for Mucopolysaccharidoses I Although timely diagnosis of children with MPS I is desirable, it is uncommon. Through the development and implementation of newborn screening methods, timely diagnosis would be possible (78). For some patients with MPS I (e.g., Hurler/Scheie and Scheie syndromes), longterm ERT may be effective therapy (77,79–81). A Hurler fibroblast cell line heterozygous for the iduronidase stop mutations Q70X and W402X showed a significant increase in iduronidase activity when cultured in the presence of gentamicin, resulting in the restoration of 2.8% of normal iduronidase activity (81). The clinical significance remains unclear; in the meantime, efforts continue to develop gene therapy for Hurler syndrome (82–84).

424

Peters

Mental Age Equivalent (months)

(A) 144 slope ≥ .50, N=9 slope .25-.49, N=5

120 96 72 48 24 0

0

24

72 96 48 Chronological age (months)

120

144

120

144

Mental Age Equivalent (months)

(B) 144 slope ≥ .50, N=3 slope .25-.49, N=4 slope<.25, N=5

120 96 72 48 24 0

0

24

72 96 48 Chronological age (months)

Figure 3 (A) Mental age-equivalent scores of patients with Hurler syndrome receiving relateddonor BMT before 24 months of age (normal, K; slope R 0.50, O; slope 0.25 to 0.49, ,; solid symbol denotes most recent neuropsychologic evaluation). (B) Mental age-equivalent scores of patients with Hurler syndrome receiving related-donor BMT after 24 months of age. Abbreviations: normal, K; slopeR0.50, O; slope 0.25 to 0.49, ,; slope !0.25, >; solid symbol denotes most recent neuropsychologic evaluation. *Latest neuropsychologic evaluation at 159 months of age and a mental age-equivalent score of 108 months. Source: From Ref. 39.

Mucopolysaccharidoses II (Hunter Syndrome) There are two distinct clinical phenotypes of MPS II; the more common severe form (i.e., MPS IIA) presents before the age of 3 years with profound neurocognitive and developmental delay and shares clinical similarities with MPS IH, including facial dysmorphia, progressive conductive and sensorineural hearing loss, upper airway obstruction with sleep apnea, cardiopulmonary dysfunction, hepatosplenomegaly, joint stiffness, and short stature. Dysostosis multiplex is less severe, and corneal clouding is absent in Hunter syndrome. The attenuated form of MPS II (i.e., MPS IIB) is associated with less neurocognitive impairment and, in some cases, normal intelligence. Survival can extend into the fifth and sixth decades, in contrast to MPS IIA with its shortened life expectancy of one to two decades. Although HSCT seems to help patients with MPS IIA and B with respect to organomegaly, airway obstruction, and cardiac function, boys with MPS IIA have still demonstrated profound neurocognitive disabilities despite early, successful transplant (55,85–90). The failure of HSCT to favorably alter the long-term neurocognitive outcome in MPS IIA suggests that enzyme is not being effectively delivered to essential components of the CNS. In cases of HSCT-treated MPS IIB

425

72

72

60

60 Age Equivalent Score

Age Equivalent Score

Metabolic Diseases

48 36 24 12 0

48 36 24 12 0

0

12

24

36

48

60

72

Chronological age in months at testing

0

12

24

36

48

60

72

Chronological age in months at testing

Figure 4 (Left) Age-equivalent scores of patients with Hurler syndrome receiving unrelated donor BMT, showing baseline mental developmental index scores greater than 70 (normal:K, PI 1:K>K, PI 4:K-K, PI 15:KOK, PI 22:K%K, PI 30:KCK, PI 32:K,K). (Right) Age-equivalent scores of patients with Hurler syndrome receiving unrelated donor BMT, showing baseline mental developmental index scores less than 70 (normal:K, PI 5:KCK, PI 8:KOK, PI 19:K%K, PI 23:K-K, PI 27:K,K). Source: From Ref. 38.

patients there have been somatic benefits and intellectual function has remained intact as expected based upon the natural history of the disorder. Because HSCT offers limited somatic benefit to patients with Hunter syndrome and because boys or men with the attenuated form of Hunter syndrome (i.e., MPS IIB) have normal neurocognitive function, performing HSCT for such patients has raised concerns as to its indication. Some of these ethical issues pertaining to the use of HSCT in patients with MPS IIB have been addressed (91,92).

Mucopolysaccharidoses III (Sanfilippo Syndrome) Neurobehavioural manifestations are the hallmarks of all 4 types of MPS III. Characteristic findings include extreme hyperactivity, aggressive behaviours, attention deficits, and progressive neurocognitive delay often associated with expressive language disabilities. As in MPS II, HSCT is able to effectively treat the various somatic aspects of MPS III; however, the uniformly poor neurocognitive and developmental outcomes despite fully donor-engrafted marrow performed early in the disease (55,93–96) has been attributed to the reduced efficiency in uptake of the MPS III-specific hydrolases and, therefore, diminished substrate clearance (97). A recent report on the use of unrelated donor umbilical cord blood for HSCT in MPS III patients once again failed to demonstrate neuropsychologic benefits in the setting of significant morbidity and mortality. HSCT has not been evaluated in the Nubian goat model of MPS IIID which is characterized by the development of neurologic complications in the neonatal period (98).

Mucopolysaccharidoses IV (Morquio Syndrome) The two distinct biochemical forms of MPS IV have similar clinical features, including severe dysostosis multiplex, dwarfism, atlantoaxial instability, short trunk, and hyperextensible joints with ligamentous laxity. Corneal clouding is mild, hepatosplenomegaly moderate, cardiac abnormalities are unusual, and intelligence is preserved (16). HSCT is unable to ameliorate the severe skeletal deformities. Therefore, this shortcoming of HSCT and the MPS IV clinical features, make HSCT a suboptimal intervention for this disease (99–101). Recently, a mouse model of MPS IV developed through targeted mutagenesis of the N-acetylgalactosamine-6sulfate sulfatase gene was reported (102).

426

Peters

Mucopolysaccharidoses VI (Maroteaux-Lamy Syndrome) The principal clinical features of MPS VI children are dysostosis multiplex with severe short stature, corneal clouding, pulmonary problems related to decreased intrathoracic volume and limited expansion due to hepatosplenomegaly, cardiac valvular abnormalities (16). Although intelligence is felt to be preserved, there is experience from the University of Minnesota documenting neurocognitive delay in some children with MPS VIH (103). HSCT has been used successfully to treat MPS VI for two decades (104–106), with resolution of hepatosplenomegaly, airway obstruction, and sleep apnea, with the prevention of further cardiopulmonary deterioration and improved joint mobility (Fig. 5). Visual acuity improved in some cases (88,104,105). As in other MPS disorders, HSCT has not been able to treat effectively the skeletal abnormalities, and consequently these successfully transplanted children still required orthopedic surgical interventions, including medial femoral epiphyseal stapling for genu valgum and osteotomies of the femoral heads for acetabular hip dysplasia. ERT has been used for MPS VI, and the results reported in six patients (107). Recombinant human arylsulfatase B treatment was well tolerated and reduced urinary GAGs. Clinical responses were present in all patients with the largest gains occurring in patients with advanced disease who received high dose ERT. HSCT leads to resolution of corneal clouding and improved facies, gait, movement of the head and neck (108), and bone histopathology (109) in Siamese cats with MPS VI. The use of HSCT in neonatal rats with MPS VI was associated with high peri-transplant mortality and does not treat the dysostosis multiplex (110). Genetically manipulated mice with deficiency of arylsulfatase B have only a slight reduction in life span and maintain fertility; HSCT has not been studied in this model.

Mucopolysaccharidoses VII (Sly Syndrome) The use of HSCT for MPS VII is limited by the rarity of the disorder and the predilection toward hydrops fetalis though there are mild adult forms (16). The neonatal form of MPS VII is one of the few lysosomal storage diseases with clinical manifestations in utero or at birth (111). MPS VII, in certain circumstances, can be ameliorated by HSCT, provided that the neuropsychological and clinical status of the patient is good at the time of HSCT (112). In this case report, the 12-year-old MPS VII patient experienced major improvements in motor function and pulmonary function with diminished upper respiratory tract and middle ear infections, leading to an improved quality of life. The two preclinical models of MPS VII, the naturally occurring b-glucuronidasedeficient mouse and dog, have been studied extensively following HSCT as well as ERT. HSCT has led to an increased life span and correction of the metabolic defect (113), including reduction in hearing loss (114); combined early ERT and HSCT have been shown to convey greater safety and long-term benefits (115). Other therapeutic modalities tested included stem cell and neural progenitor cell transplantation as well as a variety of viralmediated gene therapies (116). Due to the high mortality of early neonatal HSCT and an ablative preparative regimen, nonablative regimens as well as multiple infusions of bone marrow have been tried with success in reducing bone pathology, GAG storage, and improving both visceral organs and hematopoietic tissues (117). In the canine MPS VII model, HSCT led to improved echocardiographic findings as well as histopathologic and ultrastructural changes (118).

Multiple Sulfatase Deficiency Multiple sulfatase deficiency is a rare autosomal recessive disorder characterized by deficiencies of all 12 known sulfatases, which leads to a clinical presentation that is similar to that of the late infantile form of metachromatic leukodystrophy (MLD) (119). The diagnosis

Metabolic Diseases

427

Figure 5 Lysosomes of hepatic Ito cells. (A) Mucopolysaccharide accumulation was observed 43 days after BMT. The material was variably dense and flocculent with parallel membranous arrays. Neutral lipid was also present in the Ito cells (!13,800). (B) No accumulated mucopolysaccharide was observed in Ito cells 148 days after BMT or in biopsy specimens obtained at 342 and 600 days after BMT. The Ito cells also contained small amounts of glycogen and variably sized lipid droplets (!7860). Abbreviations: I denotes Ito cell, D Disse’s space, H hepatocyte, and S sinus. BarZ1 mm. Source: From Ref. 104.

of multiple sulfatase deficiency can be made biochemically through observation of the typical pattern of sulfatase deficiencies. Affected patients demonstrate abnormal urinary oligosaccharide, mucopolysaccharide, and glycopeptide profiles. This disease should be considered in the differential diagnosis of young children with signs and symptoms that suggest a mucopolysaccharide disorder. Prenatal diagnosis is possible as is carrier detection. Although there is no definitive treatment for patients with multiple sulfatase deficiency, hematopoietic cell transplantation can be considered.

LEUKODYSTROPHIES Cerebral X-Adrenoleukodystrophy X-ALD is a peroxisomal disorder with impaired beta-oxidation of VLCFA and reduced function of very long chain fatty acyl-CoA synthase. The minimum frequency of X-ALD in the United States is estimated to be one in 16,800 in the total population and one in 21,000 males (Table 4) (120). The clinical spectrum of X-ALD ranges from the rapidly progressive childhood or juvenile onset cerebral form (CALD), which typically leads to severe disability and death within 2 to 5 years of the onset of symptoms, to the milder adrenomyeloneuropathy (AMN) that usually manifests between 20 and 30 years of age, affecting mainly the spinal cord and may be compatible with survival into the eighth decade, to pure Addison’s disease with its adrenal insufficiency (121,122). The principal biochemical abnormality is the accumulation of VLCFAs, particularly in brain and adrenal cortex. The ALD gene, identified in 1993 (122,123), is now referred to as ABCD1 and codes for a peroxisomal membrane protein, termed the ALD protein, a member of the ATP-binding cassette transporter superfamily. Cerebral X-ALD is characterized by an inflammatory demyelinating process that appears to be immunologically

428

Peters

mediated (124,125) with CD8 cytotoxic T cells operative in the early stages of demyelination during cytolysis of oligodendrocytes. Despite the X-linked inheritance pattern, the various phenotypes co-occur frequently within the same family or kindred. The illness does not manifest clinically before the age of 3 years. Biochemical or genetic studies can diagnose it at birth or prenatally; however, those tests do not permit phenotype prediction. Approximately 40% of boys with X-ALD will develop CALD during childhood or adolescence (126). The importance of testing for ALD in all males in the pediatric age range with Addison’s disease cannot be overstated (127). Clinical manifestations of CALD depend upon the pattern and extent of demyelination. More than 80% of boys exhibit a parietal-occipital distributation, approximately 15% will demonstrate predominantly frontal lobe lesions, and less than 5% will have abnormality evident primarily in the fronto-pontine-corticospinal tracts (Figs. 6 and 7) (130). Characteristic clinical features of these three patterns of demyelination are as follows: (1) parieto-occipital—graphomotor, spatial perception, and visual memory difficulties, visual agnosia ending in cortical blindness, (2) frontal—“acquired” ADHD-like presentation, behavioral disinhibition, verbal fluency problems, memory and new learning difficulties, (3) fronto-pontine-corticospinal—corticospinal signs in the extremites; Complete evaluation of a boy with cerebral X-ALD includes a thorough neurologic examination; comprehensive neuropsychological assessment, with particular attention to the performance IQ (PIQ), a sensitive indicator of deficits in visual perceptual and spatial processing, speed and efficiency of task completion, and novel problem solving; MRI of the brain; and assessment of the degree of disability (Table 5). Boys diagnosed with X-ALD because of a family history, merit careful, complete serial monitoring beginning at age 3 years and continuing into adolescence, with the objective being to detect cerebral demyelination at an early stage and to intervene with HSCT in a timely and presumably effective manner. It should be noted that brainstem evoked auditory response abnormalies are common in patients with X-ALD but do necessarily represent changes due to cerebral demyelination. Under no circumstances should HSCT be performed in the absence of cerebral disease on the basis of detailed neuroimaging. The rationale for this strongly worded guideline is as follows: only approximately 35–40% of boys with the biochemical diagnosis of X-ALD are destined to developed cerebral demyelination in childhood; the remaining 60–65% of boys with X-ALD are likely to develop the spinal form of the disease (i.e., AMN) during the second, third, and fourth decades of life). There is no evidence to date that HSCT is effective in treating or preventing the development of AMN. There is clear evidence that timely HSCT for early stage cerebral is both safe and effective (see below). Based upon the long-term outcomes of HSCT for cerebral X-ALD, the international HSCT experience for this disease, and knowledge of the natural history, the following guidelines can be promulagated: (1) evidence of demyelination on MRI of the brain and gadolinium enhancement are highly predictive of the likelihood of disease progression, e.g., approximately 80% to 90% likelihood of severe, progressive dysmyelination (131–134), (2) boys with advanced disease manifested by low PIQ, i.e., PIQ !80 characterized by impaired visual processing, neurologic impairments, significant disability (Table 6) and high MRI severity score (R9) are poor candidates for HSCT (128), (3) boys with early stage cerebral X-ALD defined by MRI score !9 typically without neurologic and neuropsychological abnormalities are the best candidates for HSCT (Fig. 6). The 5- to 10-year follow-up of 12 boys with cerebral X-ALD shows the long-term beneficial effect of HSCT when the transplant is Table 4

Minimum Frequency of X-Linked Adrenoleukodystrophy in the United States Male population

Hemizygotes (data based) Heterozygotes (calculated) Hemizygotes C heterozygotes (calculated)

Female population

Total population

1:14,000

1:42,000 1:28,000 1:16,800

1:21,000

Metabolic Diseases

429 P<.01

1.0

Probability

0.8 0.6 0.4 0.2 0.0 0

1

2

3

4

5

Time (y)

Figure 6 Kaplan-Meier estimate of survival for cerebral X-linked adrenoleukodystrophy following hematopoietic-cell transplantation by number of neurologic deficits and MRI severity score before transplantation. Solid line indicates patients with 0 to 1 neurologic deficits and MRI severity score less than nine (nZ25). Dashed line indicates patients with two or more neurologic deficits or MRI severity score nine or greater (nZ37). Ticks on probability lines indicate dates of censoring at last follow-up. Source: From Ref. 128.

done at an early stage of disease (128,133,135). The entire University of Minnesota childhoodand adolescent-onset cerebral X-ALD HSCT experience extended from 1991 to 2004. These 59 boys had a median age at HSCT of 9 years. There were 22 recipients of related marrow transplants, 23 of unrelated donor marrow, and 14 recipients of unrelated umbilical cord blood. The diagnosis was made on the basis of family history in 36% of cases. Survival was analyzed based upon age at HSCT (4–9 years vs. O9 years, PZNS), donor type (related donor marrow, matched unrelated donor marrow, mismatched unrelated donor marrow, and UCB, PZNS), reason for X-ALD diagnosis (family history vs. disease manifestation, PZ0.08). Analysis of survival according to the baseline MRI severity score was significant (P!0.01). Specifically, boy with an MRI severity score !6 (nZ12) enjoyed 100% survival at 5 years compared to boys with an MRI severity score O10 (nZ29), whose survival at 5 years was only 35% (95% CI, 9–61%) (136). Boys with an MRI severity score between six and 10 experience an intermediate probability of survival. Outcome measures included neuroradiologic assessment of demyelination, neurologic examination, neurocognitive testing, including verbal intelligence and performance (nonverbal) abilities. Unfortunately, the typical boy with cerebral X-ALD has parietal-occipital demyelination and is diagnosed due to clinical symptomatology (i.e., not at an early stage of disease) has relatively intact verbal intelligence, visual processing difficulties, neurologic impairments in one or more of the following areas—vision, hearing, speech, and gait, and an MRI severity scores that is usually O9 and often R13. The HSCT- and diseasespecific outcomes in these boys have been discouraging, with many dying of progressive X-ALD (130,131,137). For survivors, there are permanent, severe neurologic and neuropsychologic sequelae; quality of life is compromised. As currently practiced, HSCT has not been successful for these boys with advanced stage cerebral X-ALD. Alternative therapies are under investigation and may shed light on the mechanism by which the cerebral demyelination process can be prevented, halted, or perhaps reversed. These therapies include Lorenzo’s oil (138,139), immunosuppression, lovastatin (140), 4-phenylbutyrate (141), and co-enzyme Q-10.

Globoid-Cell Leukodystrophy Globoid-cell leukodystrophy is an autosomal recessive disorder caused by deficiency of lysosomal galactocerebrosidase enzyme activity. The severe, rapidly progressive infantile form of GLD (i.e., Krabbe disease) is the most common type with an incidence of one in 100,000 in the

430

Peters

Figure 7 Different patterns recognized in males with cerebral X-linked adrenoleukodystrophy. (A) White matter in the parieto-occipital lobe or splenium of the corpus callosum; (B) white matter in the frontal lobe or genu of the corpus callosum; (C) primary involvement of the frontopontine or corticospinal projection fibers with affecting periventricular white matter; (D) primary involvement of cerebellar white matter; (E) combined but separate initial involvement of frontal and parietooccipital white matter. Source: From Ref. 129.

general population, though there is also a late-onset form of GLD that is tenfold less common. Decreased ability to degrade galactolipids found almost exclusively in myelin leads to injury to the central and peripheral nervous systems, the presence of globoid cells, and decreased myelin stemming from the toxicity of psychosine and accumulation of galactosylceramide. MRI of the brain is able to distinguish early-onset from late-onset disease, with the former characterized by cerebellar white matter involvement, deep gray matter involvement, and cerebral atrophy (142). Murine, canine, and nonhuman primate models of GLD have been studied to better understand the pathophysiology of the disorder and its response to such treatment as HSCT (143). HSCT has been studied extensively in the naturally occurring mouse model of GLD, namely the twitcher

Metabolic Diseases Table 5

431

X-Linked Adrenoleukodystrophy-Disability Rating Scale (ALD-DRS)

Scale level

Description

0 I

No difficulties Mild learning or coordination difficulties from ALD; patient does not require support or intervention Moderate learning, sensory, and/or neurologic abnormality; patient requires support or intervention in a few areas Severe learning, sensory, and/or neurologic abnormality; patient requires support or intervention in many areas Loss of cognitive ability and disorientation; patient requires constant supervision

II III IV

mouse (144), which most closely resembles human Krabbe disease. HSCT in the twitcher mouse leads to prolonged survival, clinical improvements, attenuation of hind limb paralysis, remyelination of peripheral nerves and the CNS, and stabilization of motor nerve conduction velocities in mice prior to onset of symptoms but not in symptomatic animals (145–149). After HSCT, enzyme is present in the CNS and nonneural tissues and levels of psychosine are reduced in the CNS. BMT led to disappearance of globoid cells in the CNS; however, postnatal transplant was not curative for twitcher mice, primarily due to the fact that repopulation of the central and peripheral nervous systems is not fast enough to stabilize and halt the rapidly progressive dysmyelination (150–153). With appropriate timing and use of HSCT, GLD can be effectively treated in symptomatic late onset cases leading to normalization of CSF protein, stabilization of the neurologic examination, neuropsychologic function, and the extent of dysmyelination on MRI (Fig. 8) (154). However, to date, classic infantile onset GLD, which is characterized by profound psychomotor retardation, failure to thrive, spasticity, seizures, optic atrophy, and cortical blindness, if diagnosed antenatally, can only be ameliorated if HSCT is performed in the neonatal period (154,155). The later onset forms (i.e., after age 2 years and including juvenile and adult onset forms) are characterized by loss of vision, progressive spasticity of the lower extremities, neurocognitive decline albeit in some cases very slowly, gait disturbances,

Table 6 X-Adrenoleukodystrophy-Disability Rating Scale Levels Before and After Hematopoietic-Cell Transplantation (HCT) in 94 Patients with Cerebral X-Adrenoleukodystrophy Level after HCT, no. (%) Level before HCT

No.

0

I

II

0 I II III IV Missing data Totals Deaths

13 21 36 12 1 11 94 35

8(62)a 1(4.8) 1(2.8)

3(23) 6(28)b 1(2.8)

1(7.6) 4(19)b 10(28)c

a

Denotes 1 death. Denotes 2 deaths. c Denotes 3 deaths. d Denotes 9 deaths. e Denotes 4 deaths. b

10 1

10 2

1(9) 16 5

III

IV

3(14) 1(2.8) 1(8.3)

1(7.6) 6(28)b 19(53)d 10(83)e 1(100)a

5 0

37 16

No data, because of death 1(4.8) 4(11) 1(8.3) 5(45) 11 11

432

Peters

long-tract signs, extremity weakness that can be asymmetric, and difficulties with coordination and balance.

Metachromatic Leukodystrophy MLD is an autosomal recessive inherited disorder of myelin metabolism characterized by accumulation of cerebroside sulfate in white matter of the CNS and PNS. MLD is one of the more common lysosomal storage disorders, with an estimated incidence of one in 25,000 to one in 40,000 with a gene frequency of 0.5% (156–158). Documentation of both deficiency of leukocyte arylsulfatase A enzyme activity and elevation of urinary sulfatides is needed to ensure an accurate diagnosis of MLD and to exclude the possibility of the pseudodeficiency state (159). The arylsulfatase A pseudodeficiency allele is common in the general population, with nearly 30% being carriers. Furthermore, in MLD kindreds, up to 15% of family members are heterozygous for the arylsulfatase A pseudodeficiency allele. MLD may have its onset at any age. The late infantile form is first recognized in the first or second year of life with the recognition of loss of motor milestones, abnormal speech, and loss of neurocognitive skills, culminating in dementia and death within several years and usually by age 10. HSCT does not reverse or even stabilize demyelination in this rapidly progressive form of MLD when children are already symptomatic. Even in the few cases of presymptomatic HSCT for late infantile MLD, the results have been disappointing. Late onset forms of MLD with disease manifesting during school age, adolescence or adulthood present with progressive motor signs and symptoms, including gait disturbance, clumsiness, tremor, and dysarthria. Gradual decline in neurocognitive function is also observed. Neurocognitive and neurobehavioral symptoms predominate in patients whose disease first manifests itself in the late teenage years or early to middle adulthood. In fact, many such cases are misdiagnosed as schizophrenia or major psychosis. Attention deficits, impulsive behavior, disinhibition, impulsivity, loss of spatial skills, memory loss, and personality changes, together with features of frontal lobe dysfunction often progress over years to even decades. Peripheral nervous system disease is variably present. This indolent course in adult cases and the potential for intervening at an appropriate stage of disease for juvenile onset cases permits the effective use of HSCT for these forms of MLD. HSCT succeeds in stabilizing CNS disease and function while having little to no beneficial effect on the peripheral nervous system (36,160–173). 150 Patient 1 Patient 3 Patient 4 Patient 5

500 400

125 100

300

75

200

50

100

25

Cerebrospinal Fluid Total Protein in Patient 1,3, and 4 (mg/dl)

Cerebrospinal Fluid Total Protein in Patient 5 (mg/dl)

600

0

0 0

3

6

9

12

15

18

21

24

Months after Transplantation

Figure 8 Cerebrospinal fluid total protein levels in four patients with globoid-cell leukodystrophy before and after hematopoietic cell transplantation (patients one, three, and four: late onset disease; patient five: early onset disease). Source: From Ref. 153.

Metabolic Diseases

433

Other Leukodystrophies: Pelizeaus-Merzbacher, Zellweger Syndrome, Vanishing White Matter Disease, Canavan Disease To date, there has been neither experience with HSCT nor a rationale developed for its use for the following leukodystrophies: Pelizaeus-Merzbacher (174) Zellweger syndrome (175,176) vanishing white matter disease (177,178) and Canavan disease (178).

GLYCOPROTEIN METABOLIC DISORDERS Fucosidosis Fucosidosis, an autosomal recessive disorder resulting from deficiency of the lysosomal hydrolase alpha fucosidase, is characterized by, in severely affected patients, onset of psychomotor retardation in the first year of life, growth retardation, dysostosis multiplex, and increased sweat chloride (179). Detailed studies of a valid animal model for this disorder namely the springer spaniel include the use of HSCT after total lymphoid irradiation to correct the enzymatic deficiency (180). There is very limited experience with HSCT in children with fucosidosis (181–183). Due to disease variability, a definitive conclusion regarding the benefits of HSCT cannot be reached at this time.

Gaucher Disease: Type 1, Non-Neuronopathic; Type 2, Acute Neuronopathic; Type 3, Sub-Acute Neuronopathic Gaucher disease is an autosomal recessive lysosomal glycolipid storage disorder characterized by the accumulation of glucosylceramide due to deficiency of glucocerebrosidase enzyme activity (184). Three clinical phenotypes of Gaucher disease have been identified based upon the presence and severity of neurological disease. Type 1, the most common with a prevalence of one in 40,000, is distinguished from types 2 and 3 disease by the absence of primary CNS involvement. Type 2, the acute neuronopathic form of Gaucher disease, has an early onset with severe CNS involvement and death usually in the first 2 years of life. Type 3, subacute neuronopathic Gaucher disease, demonstrates neurologic manifestations that are later in onset and more chronic than those seen in type 2 Gaucher disease. Classically described as the Norrbottnian form of Gaucher disease from a northern region of Sweden, type 3 disease is associated with a life expectancy that extends into the third to fifth decades. Hepatosplenomegaly, bone lesions, and occasionally involvement of lungs and other organs occur in all forms of Gaucher disease. The characteristic substrate-filled macrophages (i.e., Gaucher cells) are abundant in the marrow. The quality of life of Gaucher patients can be improved by a variety of medical and surgical interventions, including exogenous ERT, joint replacement, and splenectomy. The accumulation of glucosylceramide and many of the associated clinical manifestations can be reversed by ongoing infusions of modified acid beta-glucosidase (alglucerase). ERT has been used extensively and effectively in type 1 disease and to a lesser extent and with much less efficacy for type 2 and 3 patients. Issues of intravenous access and associated complications, patient comfort and lifestyle, as well as resources have led to a critical review of the risks and benefits of ERT. Infusions of alglucerase are effective in type 1 Gaucher disease and lead to regression of hepatosplenomegaly and normalization of hematologic parameters (185,186), though at a significant cost, depending upon patient weight and dosing schedule, that could prove prohibitive (e.g., $380,000 to more than $750,000 per year). Furthermore, evidence is accumulating that some organs or tissues, such as the lungs, lymphoid tissue, and the nervous system, may derive little benefit from ERT. In fact, progressive dementia and myoclonic encephalopathy have been observed in type 3 Gaucher patients treated long-term on ERT (187).

Alpha-Mannosidosis Alpha-mannosidosis is an autosomal recessive lysosomal storage disease caused by deficiency of alpha-mannosidase enzyme activity (179). Defective glycoprotein degradation leads to

434

Peters

excretion of mannose-rich oligosaccharides in urine and accumulation of oligosaccharides in various tissues, including CNS, liver, and bone marrow. The infantile form of alphamannosidosis closely resembles Hurler syndrome and includes onset of symptoms before age 12 months, macrocephaly, facial dysmorphia, hepatosplenomegaly, dysostosis multiplex, loss of previously acquired developmental skills, and recurrent infections. There is progressive deterioration leading to early demise in the first or second decade of life. Walkley et al. demonstrated that HSCT is effective in the feline model of mannosidosis (188). The first reported case of alpha-mannosidosis undergoing HSCT was reported by Will and colleagues (189). A subsequent successful case of HSCT with long-term survival was reported by Wall et al., with description of the resolution of sinopulmonary infections and organomegaly, improved dysostosis multiplex, and stabilization of neurocognitive function (190). More recently, four surviving, fully engrafted patients with a-mannosidosis transplanted at the University of Minnesota are demonstrating a very good to excellent quality of life with preservation of normal neurocognitive and cardiopulmonary function (191) and improved sensorineural hearing (Fig. 9). Patients in this and other reports have experienced pulmonary complications from 10 to 20 weeks after HSCT. No infectious etiologies were identified; it appears that selected storage disease patients are at increased risk for pulmonary complications, including hemorrhage and/or bronchiolitis obliterans. Generally, patients transplanted early in their disease course prior to the onset of significant disease-related complications are the best candidates for HSCT.

Aspartylglucosaminuria Aspartylglucosaminuria (AGU) is caused by deficiency of aspartylglucosaminuridase leading to interruption of the orderly breakdown of lysosomal glycoproteins (179) As a consequence of abnormal glycoprotein catabolism, patients with AGU exhibit severe cell injury, especially in the CNS. The relatively uniform phenotype observed in AGU patients should make effective evaluation of treatment trials feasible. The medical center in Helsinki, Finland, has experience with HSCT in three patients with follow-up ranging from one to more than 5 years, including serial MRI, biochemical, and clinical examinations (192). Transplanted patients’ MRIs of the brain with at least 2 years of follow-up showed nearly normal gray-white matter relationships and improved neuropsychological function also occurred (193) More recently, longer-term follow-up raised questions about the efficacy of HSCT in these patients (194).

MISCELLANEOUS DISORDERS Neuronal Ceroid Lipofuscinoses (NCL): Infantile, Late Infantile, Early Juvenile, Juvenile, and Adult Types Two forms of NCL, i.e., NCL one and two, are recognized as true lysosomal enzyme storage disorders implying that HSCT might be effective therapy (195). However, this has not been borne out by the only presymptomatic patient with NCL two who has been transplanted or by the existing animal model studies (196–199).

Fabry Disease Fabry disease is an X-linked disorder of glycosphingolipid catabolism resulting from deficiency of alpha-galactosidase activity. Hemizygous males have extensive deposition of these glycosphingolipid substrates in body fluids and in lysosomes of endothelial, perithelial and smooth muscle cells of blood vessels. Deposition also occurs in ganglion cells and in various cell types including heart, kidney, and eyes (200). Clinical manifestations include childhood or adolescence onset of pain and paresthesias in the extremities, angiokeratoma of the skin and

Metabolic Diseases

435

Frequencies tested (x1000 Hz)

(A)

dBHL .125 KHZ .250

.500 .750 1

1.5 2

3

4

6 8

-10 Normal hearing

0 10

Speech hearing

Decibel hearing loss

20 Mild loss

30 40

Moderate loss 50 Severe loss

60 70 80 90 100 110

(B)

Frequencies tested (x1000 Hz) dBHL .125 KHZ .250

-10 0

Decibel hearing loss

10

.500 .750 1

1.5 2

3

Speech hearing

4

6

8

Normal hearing

20 30

Mild loss

40 50 60

Moderate loss Severe loss

70 80 90 100 110

Figure 9 Pure tone audiometry in a patient with alpha-mannosidosis. (A) At pretransplant evaluation, and (B) 2 years after HSCT. Abbreviations: X, left ear; O, right ear; !, bone conduction (left); O, bone conduction (right). Bone conduction studies unmasked a sensorineural hearing loss at both evaluations without a significant conductive component. Hearing impairment was classified as mild (20–40 dB), moderate (40–55 dB), moderately severe (55–70 dB), severe (70–90 dB), and profound (O90 dB). The most important frequencies for speech comprehension are in the 500 to 3000 Hz (0.5–3.0 kHz) range. Source: From Ref. 190.

mucous membranes, and hypohidrosis. Corneal and lenticular opacities are early signs. With aging, proteinuria, hyposthenuria, and lymphedema develop. Severe renal dysfunction leads to hypertension and uremia. The cause of death is often renal failure or cardiac or cerebrovascular disease. Atypical hemizygous males (i.e., those with residual enzyme activity) may be asymptomatic or have late-onset manifestations that are milder and limited to the heart.

436

Peters

Heterozygous females may have an attenuated form of the disease and are usually asymptomatic. The most common clinical finding in females is the characteristic whorl-like corneal epithelial dystrophy noted on slit-lamp examination. Confirmation of the diagnosis in affected males rests on the demonstration of deficiency of alpha-galactosidase activity in plasma, leukocytes, or tears. Accurate diagnosis of heterozygous females rests upon molecular characterization. The gene is localized to Xq22.1; there is significant molecular heterogeneity in this disorder. Prenatal diagnosis is possible. Clinical trials and now widespread clinical administration of direct enzyme replacement has been therapeutic. The absence of neurocognitive deficits and the pathophysiology of Fabry disease are the primary factors that have led to the use of ERT rather than HSCT as the treatment of choice.

Niemann-Pick Disease: Types A, B, and C Niemann-Pick disease types A and B are lysosomal storage disorders resulting from deficient acid sphingomyelinase enzyme activity (201). Niemann-Pick type A is a rapidly progressive disorder of infancy characterized by neurodegeneration, failure to thrive, hepatosplenomegaly, and death often by 2 to 3 years of age. Generally, HSCT has not been effective in preventing the inexorable neurodevelopmental decline (202), though if HSCT were performed early in the first year of life it might be effective (C. Peters, personal communication, September 2004). NiemannPick type B is a phenotypically variable disorder that is usually diagnosed in childhood due to marked hepatosplenomegaly. HSCT appears to effectively treat the somatic manifestations of Niemann-Pick type B disease (203). Pulmonary as well as hepatic complications following HSCT can be severe (W. Krivit and C. Peters, personal communication, September 2004). Niemann-Pick disease, type C, is an autosomal recessive lipidosis resulting from a unique error in cellular trafficking of exogenous cholesterol and is associated with lysosomal accumulation of unesterified cholesterol (204). A case report of a 3-year-old girl with NiemannPick type C disease who underwent allogeneic HLA-identical sibling donor BMT documented engrafted survival with improvement in somatic disease features including hepatosplenomegaly, marrow and lung infiltration; however, neurological status continued to deteriorate (205). It is therefore unclear at this time whether HSCT can play an effective role in the treatment of the CNS in Niemann-Pick disease type C.

Mucolipidosis: Type II Mucolipidosis II or I-cell disease is an autosomal recessive disorder characterized by severe, progressive psychomotor retardation and by many of the clinical features and radiologic features observed in Hurler syndrome, such as facial dysmorphia, severe skeletal abnormalities, hernias, gingival hyperplasia, corneal haze, cardiac abnormalities, and frequent respiratory tract infections. Important differences exist however. I-cell disease patients do not demonstrate mucopolysacchariduria, and the deficient enzyme is a phosphotransferase that is responsible for the targeting of lysosomal enzymes to lysosomes (206) Historical experience with HSCT for this disorder was initially only in patients with end-stage cardiopulmonary disease (88,207,208). Neurodevelopmental gains and prevention of cardiopulmonary complications were noted five years following HLA-matched carrier donor sibling HSCT in a child with I-cell disease (209).

Gangliosidoses: GM2 (Tay-Sachs, Sandhoff, GM2 Activator Deficiency) and GM1 The GM2 gangliosidoses are a group of inherited disorders caused by excessive accumulation of ganglioside GM2 and related glycolipids in lysosomes, especially in neurons (210). Infantile onset, rapidly progressive neurodegenerative disease leads to death by 4 years (classic

Metabolic Diseases

437

Tay-Sachs, Sandhoff, and GM2 activator deficiency), whereas later onset subacute or chronic forms show more slowly progressive neurologic conditions compatible with survival into late childhood, adolescence, or even adulthood. HSCT does not appear to successfully treat these disorders; however, future therapy that combines a direct CNS intervention with systemic therapy, such as HSCT, may prove beneficial for these disorders (43,210).

Wolman Disease Wolman disease is an autosomal recessive disorder due to deficient enzyme activity of acid lipase resulting in massive accumulation of cholesteryl esters and triglycerides in most body tissues (211). In 1956, Abramov, Schorr, and Wolman described an infant with abdominal distention, hepatosplenomegaly, and massive calcification of the adrenal glands (212). The disease occurs in infancy and is typically fatal in the first year of life. HSCT has been performed in a small number of Wolman patients. To date, a patient who underwent HSCT in 1996 at the University of Minnesota is one of the few long-term survivors (213).

Farber Disease Infantile ceramidase deficiency (Farber disease) is a rare, progressive lysosomal storage disorder characterized by lipogranulomata in subcutaneous tissues, painful periarticular swelling, psychomotor retardation, and varying degrees of organ involvement of eyes, lungs, and liver (214). HSCT in selected cases particularly those transplanted early may be effective.

Osteopetrosis Osteopetrosis represents a heterogeneous group of inherited skeletal disorders characterized by defective bone resorption by osteoclasts. Since the original description by Dr. AlbersSchonberg in 1904, the spectrum of clinical severity has been well documented. Autosomal recessive osteopetrosis is associated with the most severe phenotype. There is reduced medullary space with compensatory extramedullary hematopoiesis, which often leads to massive hepatosplenomegaly beginning in infancy. There can be cranial nerve dysfunction in severely affected children when the skull’s bony foramina fail to grow. Visual deficits are common. Further disease-related complications include thrombocytopenia, anemia, and infection, which can prove fatal. This form of osteopetrosis, termed “malignant,” contrasts with attenuated forms of this autosomal recessive disorder. Autosomal dominant osteopetrosis can be classified into two forms: (1) type 1, which demonstrates profound sclerosis of the cranial vault with uniformly increased density of the spine, and (2) type 2, which is characterized by sclerosis of the base of the skull and end-plate thickening in the vertebrae and iliac wings of the pelvis. A normal life expectancy can be anticipated in these forms of osteopetrosis. With the increasing awareness of the molecular basis of increased bone density, there is interest in revising this classification system. The majority of osteopetrosis patients have defects in one of three genes involved in the osteoclast acidification pathway. Defects of human osteoclasts associated with osteopetrosis include: (1) carbonic anhydrase II deficiency, (2) osteoclast “proton pump” deficiency, (3) chloride channel defects, and (4) other genotypes associated with clinical increases in bone density (215). Therapy for osteopetrosis has included HSCT (216–227) as well as interferon-gamma (228–230).

DEVELOPING THERAPIES AND FUTURE DIRECTIONS Efforts are ongoing to reduce morbidity and mortality associated with HSCT, including the use of reduced intensity preparative regimens. In addition, alternative and complementary therapies

438

Peters

to HSCT for these inherited metabolic storage disorders are either in place or being developed. ERT has been available for patients with Gaucher disease type 1 for more than a decade (231). More recently, enzyme replacement has been making the transition from clinical trial to proven therapy for Fabry, Pompe, Niemann-Pick, type B, and selected MPS including MPS I, II, and VI. In the case of Fabry disease, ERT produced a reduction of severe neuropathic pain, stabilization of renal disease, and improved vascular function and structure (232). In Pompe disease, a fatal cardiac and skeletal muscle disorder due to acid maltase deficiency, ERT has improved cardiac function and structure and increased overall muscle strength (231–233). Important lessons from these experiences include the variability of the clinical response to ERT depending upon the disease, the organ or tissue, the dose of enzyme, and the schedule of administration. New approaches to deliver enzyme to the CNS are also being investigated. This is of particular importance because there is limited to no penetration of exogenous, intravenous enzyme into the nervous system. In the area of substrate depletion or deprivation, a number of disorders have been investigated, including Gaucher, GM2, Fabry, and cystinosis. Drugs that slow the rate of formation of accumulating glycolipids are being developed. One such agent is N-butyldeoxynojirimycin (OGT-918) and is showing promise in Gaucher disease patients. Although benefits have been observed in these various disorders, the extent of amelioration has been limited in both degree and scope. Alternative stem cells, including both embryonic and adult progenitor and stem cells, are under investigation for both transplantation and gene transfer/therapy protocols. This is important in light of the observation that bone marrow-derived mesenchymal stem cells (MSC) remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases (234). Investigators have hypothesized that correction of such a ubiquitous cell type as mesenchyme would have widespread impact and greatly extend the therapeutic benefit for these disorders. Verfaillie and colleagues have identified a class of cells which they have termed multipotent adult progenitor cells (MAPC) in postnatal human and rodent bone marrow that copurify with MSC (235). These cells are recovered from bone marrow mononuclear cells depleted of CD45C and glycophorin AC cells. Differentiation potential has included osteoblasts, chondrocytes, adipocytes, stroma cells, skeletal myoblasts, and endothelial cells (236). Further studies have demonstrated that MAPC is a progenitor of angioblasts, with subsequent differention into cells that express endothelial cell markers (237). There is evidence that human postnatal bone marrow stem cells exhibit neural phenotypes including expression of astrocyte, oligodendrocyte, and neuronal markers (238). Differentiation into functional hepatocyte-like cells has been observed as well (239). Finally, Verfaillie and coworkers have reported that cells co-purifying with MSCs, which they have termed MAPCs, differentiate at the single cell level, not only into mesenchymal cells but also into cells with visceral mesoderm, neuroectoderm, and endoderm characteristics in vitro (240,241). The scope of gene transfer applications in therapy for human diseases has expanded greatly over the past 15 years and includes the genetic storage diseases. Hematopoietic stem cells have been considered to be excellent targets for therapeutic gene transfer due to their ability to self-renew as well as differentiate into multiple cell lineages. The storage diseases, such as Gaucher disease, Hurler syndrome, MLD and other enzyme deficiency states, may be amenable to treatment by gene transfer into hematopoietic cells due to the previously discussed process of enzyme production, release, and update leading to metabolic cross-correction (242).

REFERENCES 1. Meikle P, Hopwood J, Clague A, Carey W. Prevalence of lysosomal storage disorders. JAMA 1999; 281:249–254. 2. Goldberg D, Kornfeld S. Evidence for extensive subcellular organization of asparagine-linked oligosaccharide processing and lysosomal enzyme phosphorylation. J Biol Chem 1983; 258:3159–3165. 3. Kornfeld S. Trafficking of lysosomal enzymes in normal and disease states. J Clin Invest 1986; 77:1–6.

Metabolic Diseases

439

4. Bou-Gharios G, Abraham D, Olsen I. Lysosomal storage diseases: mechanisms of enzyme replacement therapy. Histochem J 1993; 25:593–605. 5. Neufeld E. Lysosomal storage diseases. Annu Rev Biochem 1991; 60:257–280. 6. Chao H-J, Waheed A, Polhmann R, Hille A, von Figuera K. Mannose-6-phosphate receptor dependent secretion of lysosomal enzymes. EMBO J 1990; 9:3507–3513. 7. Pfeffer S. Targeting of proteins to the lysosome. Curr Top Microbiol Immunol 1991; 170:43–65. 8. Rodman J, Mercer R, Stahl P. Endocytosis and trancytosis. Curr Opin Cell Biol 1990; 2:664–672. 9. Jenkins H, Martin J, Dean M. Receptor-mediated uptake of beta-glucuronidase into primary astrocytes and C6 glioma cells from rat brain. Brain Res 1988; 462:265–274. 10. Olsen I, Dean M, Harris G, Muir H. Direct transfer of a lysosomal enzyme from lymphoid cells to deficient fibroblasts. Nature 1981; 291:244–247. 11. Olsen I, Oliver T, Muir H, Smith R, Partridge T. Role of cell adhesion in contact-dependent transfer of a lysosomal enzyme from lymphocytes to fibroblasts. J Cell Sci 1986; 85:231–244. 12. Fratantoni J, Hall C, Neufeld E. The defect in Hurler and Hunter syndromes. II. Deficiency of specific factors involved in mucopolysaccharide degradation. Proc Natl Acad Sci USA 1969; 64:360–366. 13. Di Ferrante N, Nichols B, Donnelly P, Neri G, Hrgovcic R, Berglund R. Induced degradation of glycosaminoglycans in Hurler’s and Hunter’s syndromes by plasma infusion. Proc Natl Acad Sci USA 1971; 68:303–307. 14. Knudson A, Di Ferrante N, Curtis J. Effect of leukocyte transfusion in a child with type II mucopolysaccharidosis. Proc Natl Acad Sci USA 1971; 68:1738–1741. 15. Hobbs J, Hugh-Jones K, Barrett A, et al. Reversal of clinical features of Hurler’s disease and biochemical improvement after treatment by bone-marrow transplantation. Lancet 1981; 2:709–712. 16. Neufeld E, Muenzer J. The mucopolysaccharidoses. In: Scriver C, Beaudet A, Sly W, Valle D, eds. The Metabolic and Molecular Basis of Inherited Disease. New York: McGraw-Hill, 2001:3421–3452. 17. Brieder M, Shull R. Long-term effects of bone marrow transplantation in dogs with mucopolysaccharidosis I. Am J Pathol 1989; 134:677–692. 18. Shull R, Breider M, Constantopoulos G. Long-term neurological effects of bone marrow transplantation in a canine lysosomal storage disease model. Pediatr Res 1988; 24:347–352. 19. Shull R, Hastings N, Selcer R, et al. Bone marrow transplantation in canine mucopolysaccharidosis I. J Clin Invest 1987; 79:435–443. 20. Constantopoulos G, Scott J, Shull R. Corneal opacity in canine MPS I. Changes after bone marrow transplantation. Invest Ophthalmol Visual Sci 1989; 30:1802–1807. 21. Gompf R, Shull R, Breider M, Scott J, Constantopoulos G. Cardiovascular changes after bone marrow transplantation in dogs with mucopolysaccharidosis I. Am J Vet Res 1990; 51:2054–2060. 22. Shull R, Walker M. Radiographic findings in a canine model of mucopolysaccharidosis I. Changes associated with bone marrow transplantation. Invest Radiol 1988; 23:124–130. 23. He X, Li C, Simonaro C, et al. Identification and characterization of the molecular lesion causing mucopolysaccharidosis type I in cats. Mol Genet Metab 1999; 67:106–112. 24. Clarke L, Russell C, Pownall S, et al. Murine mucopolysaccharidosis type I: targeted disruption of the murine alpha-L-iduronidase gene. Hum Mol Genet 1997; 6:503–511. 25. Russell C, Hendson G, Jevon G, et al. Murine MPS I: insights into the pathogenesis of Hurler syndrome. Clin Genet 1998; 53:349–361. 26. Bunge S, Clements PR, Byers S, Kleijer WJ, Brooks DA, Hopwood JJ. Genotype-phenotype correlations in mucopolysaccharidosis type I using enzyme kinetics, immunoquantification and in vitro turnover studies. Biochim Biophys Acta 1998; 1407:249–256. 27. Beesley CE, Meaney CA, Greenland G, et al. Mutational analysis of 85 mucopolysaccharidosis type I families: frequency of known mutations, identification of 17 novel mutations and in vitro expression of missense mutations. Hum Genet 2001; 109:503–511. 28. Guffon N, Souillet G, Maire I, Straczek J, Guibaud P. Follow-up of nine patients with Hurler syndrome after bone marrow transplantation. J Pediatr 1998; 133:119–125. 29. Scott HS, Bunge S, Gal A, Clarke LA, Morris CP, Hopwood JJ. Molecular genetics of mucopolysaccharidosis type I: diagnostic, clinical, and biological implications. Hum Mutat 1995; 6:288–302. 30. Whitley CB, Krivit W, Ramsay NKC, et al. Mutation analysis and clinical outcome of patients with Hurler syndrome (mucopolysaccharidosis type I-H) undergoing bone marrow transplantation. Am J Hum Genet 1993; 53:101. 31. Beck M. Variable clinical presentation in lysosomal storage disorders. J Inherit Metab Dis 2001; 24:47–51 discussion 45–46.

440

Peters

32. Leighton SE, Papsin B, Vellodi A, Dinwiddie R, Lane R. Disordered breathing during sleep in patients with mucopolysaccharidoses. Int J Pediatr Otorhinolaryngol 2001; 58:127–138. 33. Shih S, Lee Y, Lin S, Sheu C, Blickman J. Airway changes in children with mucopolysaccharidoses. Acta Radiol 2002; 43:40–43. 34. Dangel J. Cardiovascular changes in children with mucopolysaccharide storage diseases and related disorders—clinical and echocardiographic findings in 64 patients. Eur J Pediatr 1998; 157:534–538. 35. Peters C, Steward C. Hematopoietic cell transplantation for inherited metabolic diseases: an overview of outcomes and practice guidelines. Bone Marrow Transplant 2003; 31:229–239. 36. Krivit W, Lockman L, Watkins P, Hirsch J, Shapiro E. The future for treatment by bone marrow transplantation for adrenoleukodystrophy, metachromatic leukodystrophy, globoid cell leukodystrophy and Hurler syndrome. J Inherit Metab Dis 1995; 18:398–412. 37. Hobbs J, Hugh-Jones K, Barrett A, et al. Reversal of clinical features of Hurler’s disease and biochemical improvement after treatment by bone marrow transplantation. Lancet 1981; 2:709–712. 38. Peters C, Balthazor M, Shapiro E, et al. Outcome of unrelated donor bone marrow transplantation in 40 children with Hurler syndrome. Blood 1996; 87:4894–4902. 39. Peters C, Shapiro E, Anderson J, et al. Hurler syndrome: II. Outcome of HLA-genotypically identical sibling and HLA-haploidentical related donor bone marrow transplantation in fifty- four children. The storage disease collaborative study group. Blood 1998; 91:2601–2608. 40. Peters C, Orchard P, Defor T, et al. Hematopoietic cell transplantation (HSCT) for Hurler syndrome: the university of minnesota experience from 1983 to 2001 (abstr). Blood 2001; 98:667a. 41. Staba SL, Escolar ML, Poe M, et al. Cord-blood transplants from unrelated donors in patients with Hurler’s syndrome. N Engl J Med 2004; 350:1960–1969. 42. Whitley C, Belani K, Chang P, et al. Long-term outcome of Hurler syndrome following bone marrow transplantation. Am J Med Genet 1993; 46:209–218. 43. Krivit W, Sung J, Lockman L, Shapiro E. Bone marrow transplantation for treatment of lysosomal and peroxisomal storage diseases: focus on central nervous system reconstitution. In: Rich R, Fleisher T, Schwartz B, Shearer W, Strober W, eds. Principles of Clinical Immunology. St. Louis: Mosby, 1995:1852–1864. 44. Orchard P, Grewal S, Milla C, et al. Pulmonary risk factors in allogeneic transplantation for Hurler syndrome (abstr). Blood 2004; 104:592a. 45. Phipps S, Mulhern R. Developmental outcome of unrelated donor bone marrow transplantation in children with Hurler syndrome [letter]. Blood 1997; 89:732–734. 46. Peters C, Shapiro E, Krivit W. Hurler syndrome: past, present, and future. J Pediatr 1998; 133:7–9. 47. Fleming D, Henslee-Downey P, Ciocci G, et al. The use of partially HLA-mismatched donors for allogeneic transplantation in patients with mucopolysaccharidosis-I. Pediatr Transplant 1998. 48. Peters C, Shapiro EG, Krivit W. Neuropsychological development in children with Hurler syndrome following hematopoietic stem cell transplantation. Pediatr Transplant 1998; 2:250–253. 49. Grewal S, Krivit W, Defor T, et al. Outcome of second hematopoietic cell transplantation in Hurler syndrome. Bone Marrow Transplant 2002; 29:491–496. 50. Jacobson P, Park J, DeFor T, et al. Oral busulfan pharmacokinetics and engraftment in children with Hurler syndrome and other inherited metabolic storage diseases undergoing hematopoietic cell transplantation. Bone Marrow Transplant 2001; 27:855–861. 51. deGasperi R, Raghavan S, Sosa M, et al. Measurements from normal umbilical cord blood of four lysosomal enzymatic activities: alpha-L-iduronidase (Hurler), galactocerebrosidase (globoid cell leukodystrophy), arylsulfatase A (metachromatic leukodystrophy), arylsulfatase B (MaroteauxLamy). Bone Marrow Transplant 2000; 25:541–544. 52. Rosales F, Peylan-Ramu N, Cividalli G, et al. The role of thiotepa in allogeneic bone marrow transplantation for genetic diseases. Bone Marrow Transplant 1999; 23:861–865. 53. Kapelushnik J, Mandel H, Varadi G, Nagler A. Fludarabine-based protocol for haploidentical peripheral blood stem cell transplantation in Hurler syndrome. J Pediatr Hematol Oncol 2000; 22:433–436. 54. Drew B, Peters C, Rimell F. Upper airway complications in children after bone marrow transplantation. Laryngoscope 2000; 110:1446–1451. 54a. Grigull L, Beilken A, Schrappe M, et al. Transplantation of allogeneic CD34-selected stem cells after fludarabine-based conditioning regimen for children with mucopolysaccharidosis 1H (M. Hurler). Bone Marrow Transplant 2005; 35:265–269. 55. Shapiro EG, Lockman L, Balthazor M, Krivit W. Neuropsychological outcomes of several storage diseases with and without bone marrow transplantation. J Inherit Metab Dis 1995; 18:413–429.

Metabolic Diseases

441

56. Blaser S, Clarke J, Becker L. Neuroradiology of lysosomal disorders. Neuroimaging Clin N Am 1994; 4:283–298. 57. Takahashi Y, Sukegawa K, Aoki M, et al. Evaluation of accumulated mucopolysaccharides in the brain of patients with mucopolysaccharidoses by (1)H-magnetic resonance spectroscopy before and after bone marrow transplantation. Pediatr Res 2001; 49:349–355. 58. Malone B, Whitley C, Duvall A, et al. Resolution of obstructive sleep apnea in Hurler syndrome after bone marrow transplantation. Int J Pediatr Otorhinolaryngol 1988; 15:23–31. 59. Resnick J, Krivit W, Snover D, et al. Pathology of the liver in mucopolysaccharidosis: light and electron microscopic assessment before and after bone marrow transplantation. Bone Marrow Transplant 1992; 10:273–280. 60. Summers C, Purple R, Krivit W, et al. Ocular changes in the mucopolysaccharidoses after bone marrow transplantation. A preliminary report. Ophthalmology 1989; 96:977–984. 61. Christiansen SP, Smith TJ, Henslee-Downey PJ. Normal intraocular pressure after a bone marrow transplant in glaucoma associated with mucopolysaccharidosis Type I-H. Am J Ophthalmol 1990; 109:230–231. 62. Gullingsrud EO, Krivit W, Summers CG. Ocular abnormalities in the mucopolysaccharidoses after bone marrow transplantation. Longer follow-up. Ophthalmology 1998; 105:1099–1105. 63. Braunlin EA, Hunter DW, Krivit W, et al. Evaluation of coronary artery disease in the Hurler syndrome by angiography. Am J Cardiol 1992; 69:1487–1489. 64. Du Cret R, Weinberg E, Jackson C, et al. Resting TI-201 scintigraphy in the evaluation of coronary artery disease in children with Hurler Syndrome. Clin Nucl Med 1994; 19:975–978. 65. Braunlin E, Rose A, Hopwood J, Candel R, Krivit W. Coronary artery patency following long-term successful engraftment 14 years after bone marrow transplantation in the Hurler syndrome. Am J Cardiol 2001; 88:1075–1077. 66. Braunlin E, Stauffer N, Peters C, et al. Usefulness of bone marrow transplantation in the Hurler syndrome. Am J Cardiol 2003; 92:882–886. 67. Field R, Buchanan J, Copplemans M, Aichroth P. Bone-marrow transplantation in Hurler’s syndrome. Effect on skeletal development. J Bone Joint Surg Br 1994; 76:975–981. 68. Masterson EL, Murphy PG, O’Meara A, Moore DP, Dowling FE, Fogarty EE. Hip dysplasia in Hurler’s syndrome: orthopaedic management after bone marrow transplantation. J Pediatr Orthop 1996; 16:731–733. 69. Odunusi E, Peters C, Krivit W, Ogilvie J. Genu valgum deformity in Hurler syndrome after hematopoietic stem cell transplantation: correction by surgical intervention. J Pediatr Orthop B 1999; 19:270–274. 70. Van Heest A, House J, Krivit W, Walker K. Surgical treatment of carpal tunnel syndrome and trigger digits in children with mucopolysaccharide storage disorders. J Hand Surg 1998; 23A:236–243. 71. Krivit W, Shapiro EG, Balthazor M, et al. Hurler syndrome: outcomes and planning following bone marrow transplantation. In: Steward CG, Hobbs JR, eds. Correction of Genetic Diseases by Transplantation III. London: COGENT, 1995:25–40. 72. Hite SH, Peters C, Krivit W. Correction of odontoid dysplasia following bone-marrow transplantation and engraftment (in Hurler syndrome MPS 1H). Pediatr Radiol 2000; 30:464–470. 73. Kachur E, Del Maestro R. Mucopolysaccharidoses and spinal cord compression: case report and review of the literature with implications of bone marrow transplantation. Neurosurgery 2000; 47:223–228 discussion 228–229. 74. Grewal SS, Wynn R, Abdenur JE, et al. Safety and efficacy of enzyme replacement therapy in combination with hematopoietic stem cell transplantation in Hurler syndrome. Genet Med 2005; 7:143–146. 75. Breider MA, Shull RM, Constantopoulos G. Long-term effects of bone marrow transplantation in dogs with mucopolysaccharidosis I. Am J Pathol 1989; 134:677–692. 76. Navarro C, Dominguez C, Costa M, Ortega J. Bone marrow transplant in a case of mucopolysaccharidosis I scheie phenotype: skin ultrastructure before and after transplantation. Acta Neuropathol 1991; 82:33–38. 77. Kakkis ED, Schuchman E, He X, et al. Enzyme replacement therapy in feline mucopolysaccharidosis I. Mol Genet Metab 2001; 72:199–208. 78. Chamoles NA, Blanco MB, Gaggioli D, Casentini C. Hurler-like phenotype: enzymatic diagnosis in dried blood spots on filter paper. Clin Chem 2001; 47:2098–2102. 79. Zhaol K, Faull K, Kakkis E, Neufeld E. Carbohydrate structures of recombinant human alpha-Liduronidase secreted by Chinse hamster ovary egg. J Biol Chem 1997; 272:22758–22765.

442

Peters

80. Wraith JE. Enzyme replacement therapy in mucopolysaccharidosis type I: progress and emerging difficulties. J Inherit Metab Dis 2001; 24:245–250. 81. Keeling KM, Brooks DA, Hopwood JJ, Li P, Thompson JN, Bedwell DM. Gentamicin-mediated suppression of Hurler syndrome stop mutations restores a low level of alpha-L-iduronidase activity and reduces lysosomal glycosaminoglycan accumulation. Hum Mol Genet 2001; 10:291–299. 82. Fairbairn LJ, Lashford LS, Spooncer E, et al. Towards gene therapy of Hurler syndrome. Cas Lek Cesk 1997; 136:27–31. 83. Huang MM, Wong A, Yu X, Kakkis E, Kohn DB. Retrovirus-mediated transfer of the human alphaL-iduronidase cDNA into human hematopoietic progenitor cells leads to correction in trans of Hurler fibroblasts. Gene Ther 1997; 4:1150–1159. 84. Baxter MA, Wynn RF, Deakin JA, et al. Retrovirally mediated correction of bone marrow-derived mesenchymal stem cells from patients with mucopolysaccharidosis type I. Blood 2002; 99:1857–1859. 85. Warkentin PI, Dixon MJ, Schafer I, Strandjord SE, Coccia PF. Bone marrow transplantation in Hunter syndrome: a preliminary report. Birth Defects 1986; 22:31–39. 86. Bergstrom SK, Quinn JJ, Greenstein R, Ascensao J. Long-term follow-up of a patient transplanted for Hunter’s disease type IIB: a case report and literature review. Bone Marrow Transplant 1994; 14:653–658. 87. Coppa GV, Gabrielli O, Zampini L, et al. Bone marrow transplantation in Hunter syndrome (mucopolysaccharidosis type II): two-year follow-up of the first Italian patient and review of the literature. Pediatr Med Chir 1995; 17:227–235. 88. Imaizumi M, Gushi K, Kurobane I, et al. Long-term effects of bone marrow transplantation for inborn errors of metabolism: a study of four patients with lysosomal storage diseases. Acta Paediatr Jpn 1994; 36:30–36. 89. McKinnis EJ, Sulzbacher S, Rutledge JC, Sanders J, Scott CR. Bone marrow transplantation in Hunter syndrome. J Pediatr 1996; 129:145–148. 90. Vellodi A, Young E, Cooper A, Lidchi V, Winchester B, Wraith JE. Long-term follow-up following bone marrow transplantation for Hunter disease. J Inherit Metab Dis 1999; 22:638–648. 91. Peters C, Krivit W. Hematopoietic cell transplantation for mucopolysaccharidosis IIB (Hunter syndrome): an ethical commentary. Bone Marrow Transplant 2000; 25:1097–1099. 92. Mullen C, Thompson J, Richard L, Chan K. Unrelated umbilical cord blood transplantation in infancy for mucopolysaccharidosis type IIB (Hunter syndrome) complicated by autoimmune hemolytic anemia. Bone Marrow Transplant 2000; 25:1093–1097. 93. Bordigoni P, Vidalilbet M, Lena M, Maire I, Gelot S. Bone marrow transplantation for Sanfilippo Syndrome. In: Hobbs J, ed. Correction of Certain Genetic Diseases by Transplantation. COGENT Press: London, 1989:114–119. 94. Klein K, Krivit W, Whitley C, et al. Poor cognitive outcome of nine children with Sanfilippo syndrome following bone marrow transplantation and successful engraftment. Bone Marrow Transplant 1995; 15:S176–S181. 95. Vellodi A, Young E, New M, Pot-Mees C, Hugh-Jones K. Bone marrow transplantation for Sanfilippo disease type B. J Inherit Metab Dis 1992; 15:911–918. 96. Gungor N, Tuncbilek E. Sanfilippo disease type B. A case report and review of the literature on recent advances in bone marrow transplantation. Turk J Pediatr 1995; 37:157–163. 97. O’Brien JS, Miller AL, Loverde AW, Veath ML. Sanfilippo disease type B: enzyme replacement and metabolic correction in cultured fibroblasts. Science 1973; 181:753–755. 98. Thompson JN, Jones MZ, Dawson G, Huffman PS. N-acetylglucosamine 6-sulphatase deficiency in a Nubian goat: a model of Sanfilippo syndrome type D (mucopolysaccharidosis IIID). J Inherit Metab Dis 1992; 15:760–768. 99. Kato S, Kubota C, Yabe H, et al. Bone marrow transplantation in Morquio’s disease. In: Hobbs J, ed. Correction of Certain Genetic Diseases by Transplantation. London: The COGENT Trust, 1989:120–126. 100. Desai S, Hobbs J, Hugh-Jones K. Morquio’s disease (mucopolysaccharidosis Type IV) treated by bone marrow transplant. Exp Hematol 1983;2. 101. Kato S, Yabe H, Yabe M, et al. Bone marrow transplantation in children. Tokai J Exp Clin Med 1986; 11:43–47. 102. Tomatsu S, Orii K, Vogler C, et al. Mouse model of morquio a syndrome produced by targeted mutagenesis. 7th international symposium on MPS and related disorders, Paris, France, June 20–23, 2002. 103. Delaney K, Gray R, Charnas L, Peters C, Abel S, Shapiro E. Neuropsychological characteristics of MPS VI with and without treatment. 30th annual meeting of the international neuropschological society, Toronto, Canada, Febuary 13–16, 2002.

Metabolic Diseases

443

104. Krivit W, Pierpont M, Ayaz K, et al. Bone-marrow transplantation in the Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI). Biochemical and clinical status 24 months after transplantation. N Engl J Med 1984; 311:1606–1611. 105. Krivit W. Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI): treatment by allogeneic bone marrow transplantation in 6 patients and potential for autotransplantation bone marrow gene insertion. Int Pediatr 1992; 7:47–52. 106. Herskhovitz E, Young E, Rainer J, et al. Bone marrow transplantation for Maroteaux-Lamy syndrome (MPS VI): long-term follow-up. J Inherit Metab Dis 1999; 22:50–62. 107. Harmatz P, Whitley CB, Waber L, et al. Enzyme replacement therapy in mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). J Pediatr 2004; 144:574–580. 108. Gasper PW, Thrall MA, Wenger DA, et al. Correction of feline arylsulphatase B deficiency (mucopolysaccharidosis VI) by bone marrow transplantation. Nature 1984; 312:467–469. 109. Norrdin RW, Moffat KS, Thrall MA, Gasper PW. Characterization of osteopenia in feline mucopolysaccharidosis VI and evaluation of bone marrow transplantation therapy. Bone 1993; 14:361–367. 110. Simonaro CM, Haskins ME, Kunieda T, Evans SM, Visser JW, Schuchman EH. Bone marrow transplantation in newborn rats with mucopolysaccharidosis type VI: biochemical, pathological, and clinical findings. Transplantation 1997; 63:1386–1393. 111. Machin G. Hydrops revised: literature review of 1414 cases published in the 1980s. Am J Med Genet 1992; 34:366–390. 112. Yamada Y, Kato K, Sukegawa K, et al. Treatment of MPS VII (Sly disease) by allogeneic BMT in a female with homozygous A619V mutation. Bone Marrow Transplant 1998; 21:629–634. 113. Birkenmeier EH, Barker JE, Vogler CA, et al. Increased life span and correction of metabolic defects in murine mucopolysaccharidosis type VII after syngeneic bone marrow transplantation. Blood 1991; 78:3081–3092. 114. Sands MS, Erway LC, Vogler C, Sly WS, Birkenmeier EH. Syngeneic bone marrow transplantation reduces the hearing loss associated with murine mucopolysaccharidosis type VII. Blood 1995; 86:2033–2040. 115. Sands M, Vogler C, Torrey A, et al. Murine mucopolysaccaridosis type VII: long term therapeutic effects of enzyme replacement and enzyme replacement followed by bone marrow transplant. J Clin Invest 1997; 99:1596–1605. 116. Vogler C, Barker J, Sands MS, Levy B, Galvin N, Sly WS. Murine mucopolysaccharidosis VIL: impact of therapies on the phenotype, clinical course, and pathology in a model of a lysosomal storage disease. Pediatr Dev Pathol 2001; 4:421–433. 117. Soper BW, Lessard MD, Vogler CA, et al. Nonablative neonatal marrow transplantation attenuates functional and physical defects of beta-glucuronidase deficiency. Blood 2001; 97:1498–1504. 118. Sammarco C, Weil M, Just C, et al. Effects of bone marrow transplantation on the cardiovascular abnormalities in canine mucopolysaccharidosis VII. Bone Marrow Transplant 2000; 25:1289–1297. 119. Hopwood J, Ballabio A. Multiple sulfatase deficiency and the nature of the sulfatase family. In: Scriver S, Beaudet A, Sly W, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. McGrath-Hill: New York, 2001:3733–3774. 120. Bezman L, Moser AB, Raymond GV, et al. Adrenoleukodystrophy: incidence, new mutation rate, and results of extended family screening. Ann Neurol 2001; 49:512–517. 121. Moser HW. Adrenoleukodystrophy: phenotype, genetics, pathogenesis and therapy. Brain 1997; 120:1485–1508. 122. Mosser J, Douar AM, Sarde CO, et al. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature 1993; 361:726–730. 123. Mosser J, Lutz Y, Stoeckel ME, et al. The gene responsible for adrenoleukodystrophy encodes a peroxisomal membrane protein. Hum Mol Genet 1994; 3:265–271. 124. Powers JM, Liu Y, Moser AB, Moser HW. The inflammatory myelinopathy of adrenoleukodystrophy: cells, effector molecules, and pathogenetic implications. J Neuropathol Exp Neurol 1992; 51:630–643. 125. Ito M, Blumberg BM, Mock DJ, et al. Potential environmental and host participants in the early white matter lesion of adreno-leukodystrophy: morphologic evidence for CD8 cytotoxic T cells, cytolysis of oligodendrocytes, and CD1-mediated lipid antigen presentation. J Neuropathol Exp Neurol 2001; 60:1004–1019. 126. Moser H, Smith K, Watkins P. X-linked adrenoleukodystrophy. In: Scriver S, Beaudet A, Sly W, Valle D, eds. The Metabolic and Molecular Basis of Inherited Disease. New York: McGraw-Hill, 2001:3257–3301.

444

Peters

127. Ronghe MD, Barton J, Jardine PE, et al. The importance of testing for adrenoleucodystrophy in males with idiopathic Addison’s disease. Arch Dis Child 2002; 86:185–189. 128. Peters C, Charnas LR, Tan Y, et al. Cerebral X-linked adrenoleukodystrophy: the international hematopoietic cell transplantation experience from 1982 to 1999. Blood 2004; 104:881–888. 129. Loes DJ, Fatemi A, Melhem, et al. Analysis of MRI patterns aids prediction of progression in X-linked adrenoleukodystrophy. Neurology 2003; 61:369–374. 130. Loes DJ, Hite S, Moser H, et al. Adrenoleukodystrophy: a scoring method for brain MR observations. AJNR Am J Neuroradiol 1994; 15:1761–1766. 131. Loes DJ, Stillman AE, Hite S, et al. Childhood cerebral form of adrenoleukodystrophy: short-term effect of bone marrow transplantation on brain MR observations. AJNR Am J Neuroradiol 1994; 15:1767–1771. 132. Melhem ER, Loes DJ, Georgiades CS, Raymond GV, Moser HW. X-linked adrenoleukodystrophy: the role of contrast-enhanced MR imaging in predicting disease progression. AJNR Am J Neuroradiol 2000; 21:839–844. 133. Shapiro E, Krivit W, Lockman L, et al. Long-term effect of bone-marrow transplantation for childhood-onset cerebral X-linked adrenoleukodystrophy. Lancet 2000; 356:713–718. 134. Moser HW, Loes DJ, Melhem ER, et al. X-linked adrenoleukodystrophy: overview and prognosis as a function of age and brain magnetic resonance imaging abnormality. A study involving 372 patients. Neuropediatrics 2000; 31:227–239. 135. Aubourg P, Blanche S, Jamabaque I, et al. Reversal of early neurologic and neuroradiologic manifestations of X-linked adrenoleukodystrophy by bone marrow transplantation. N Engl J Med 1990; 322:1860–1866. 136. Peters C, Charnas L, Defor T, et al. Cerebral X-linked adrenoleukodystrophy: the university of Minnesota hematopoietic cell transplantation experience from 1991 to 2004 (abstr). Blood 2004;104. 137. Shapiro E, Lockman L, Balthazor M, et al. Neuropsychological and neurological function and quality-of-life before and after bone marrow transplantation for adrenoleukodystrophy. In: Ringden O, Hobbs J, Steward C, eds. Correction of Genetic Diseases by Transplantation 1997. London, U.K.: The COGENT Press, 1997. 138. Moser H, Raymond G, Koehler W, Sokolowski P. Evaluation of the preventive effect of glyceryl trioleate-trierucate (Lorenzo’s oil) therapy in X-linked adrenoleukodystrophy: results of two concurrent trials. In: Roels F, Baes M, de Bie S, eds. Peroxisomal Disorders and Regulation of Genes. New York: Kluwer Plenum, 2003. 139. van Geel BM, Assies J, Haverkort EB, et al. Progression of abnormalities in adrenomyeloneuropathy and neurologically asymptomatic X-linked adrenoleukodystrophy despite treatment with “Lorenzo’s oil”. J Neurol Neurosurg Psychiatry 1999; 67:290–299. 140. Singh I, Khan M, Key L, Pai S. Lovastatin for X-linked adrenoleukodystrophy. N Engl J Med 1998; 339:702–703. 141. Kemp S, Wei H-M, Lu J-F, et al. Gene redundancy and pharmacological gene therapy: implications for X-linked adrenoleukodystrophy. Nat Med 1998; 4:1261–1268. 142. Loes DJ, Peters C, Krivit W. Globoid cell leukodystrophy: distinguishing early-onset from late-onset disease using a brain MR imaging scoring method. AJNR Am J Neuroradiol 1999; 20:316–323. 143. Wenger DA. Murine, canine and non-human primate models of Krabbe disease. Mol Med Today 2000; 6:449–451. 144. Suzuki K, Suzuki K. The twitcher mouse: a model for Krabbe disease and for experimental therapies. Brain Pathol 1995; 5:249–258. 145. Yeager AM, Brennan S, Tiffany C, Moser HW, Santos GW. Prolonged survival and remyelination after hematopoietic cell transplantation in the twitcher mouse. Science 1984; 225:1052–1054. 146. Yeager AM, Shinohara M, Shinn C. Hematopoietic cell transplantation after administration of highdose busulfan in murine globoid cell leukodystrophy (the twitcher mouse). Pediatr Res 1991; 29:302–305. 147. Kondo A, Hoogerbrugge PM, Suzuki K, Poorthuis BJ, Van Bekkum DW. Pathology of the peripheral nerve in the twitcher mouse following bone marrow transplantation. Brain Res 1988; 460:178–183. 148. Suzuki K, Hoogerbrugge PM, Poorthuis BJ, Bekkum DW. The twitcher mouse. Central nervous system pathology after bone marrow transplantation. Lab Invest 1988; 58:302–309. 149. Toyoshima E, Yeager AM, Brennan S, Santos GW, Moser HW, Mayer RF. Nerve conduction studies in the twitcher mouse (murine globoid cell leukodystrophy). J Neurol Sci 1986; 74:307–318.

Metabolic Diseases

445

150. Hoogerbrugge P, Suzuki K, Suzuki K, et al. Donor-derived cells in the central nervous system of twitcher mice after bone marrow transplantation. Science 1988; 239:1035–1038. 151. Ichioka T, Kishimoto Y, Brennan S, Santos GW, Yeager AM. Hematopoietic cell transplantation in murine globoid cell leukodystrophy (the twitcher mouse): effects on levels of galactosylceramidase, psychosine, and galactocerebrosides. Proc Natl Acad Sci USA 1987; 84:4259–4263. 152. Yeager AM, Shinn C, Shinohara M, Pardoll DM. Hematopoietic cell transplantation in the twitcher mouse. The effects of pretransplant conditioning with graded doses of busulfan. Transplantation 1993; 56:185–190. 153. Igisu H, Suzuki K. Progressive accumulation of toxic metabolite in a genetic leukodystrophy. Science 1984; 224:753–755. 154. Krivit W, Shapiro EG, Peters C, et al. Hematopoietic stem-cell transplantation in globoid-cell leukodystrophy. N Engl J Med 1998; 338:1119–1126. 155. Kurtzberg J, Richards K, Wenger D. Correction of Krabbe disease with neonatal hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2002; 8:97a. 156. Von Figura K, Gieselman V, Jaeken J. Metachromatic leukodystrophy. In: Scriver C, Beaudet A, Sly W, Valle D, eds. The Metabolic and Molecular Basis of Inherited Disease. New York: McGrawHill, 2001:3695–3724. 157. Kolodny E. Metachromatic leukodystrophy and multiple sulfatase deficiency: sulfatide lipidosis. In: Barchi R, ed. The Molecular and Genetic Basis of Neurological Diseases. Boston: ButterworthHeinemann, 1997:433–442. 158. Kolodny E. Metachromatic leukodystrophy and multiple sulfatase deficiency: sulfatide lipidosis. In: Rosenberg R, Prusiner S, DiMauro S, Barchi R, eds. The Molecular and Genetic Basis of Neurological Diseases. Boston: Butterworth-Heinemann, 1997:433–442. 159. Tylki-Szymanska AT, Czartoryska B, Lugowska A. Practical suggestions in diagnosing metachromatic leukodystrophy in probands and in testing family members. Eur Neurol 1998; 40:67–70. 160. Krivit W, Shapiro E, Lockman L, et al. Bone marrow transplantation treatment for globoid cell leukodystrophy, metachromatic leukodystrophy, adrenoleukodystrophy, and Hurler syndrome. In: Moser HW, ed. Handbook of Clincial Neurology, 1996:87–106. 161. Hoogerbrugge PM, Brouwer OF, Bordigoni P, et al. Allogeneic bone marrow transplantation for lysosomal storage diseases. The European group for bone marrow transplantation. Lancet 1995; 345:1398–1402. 162. Krivit W, Shapiro E, Kennedy W, et al. Treatment of late infantile metachromatic leukodystrophy by bone marrow transplantation. N Engl J Med 1990; 322:28–32. 163. Krivit W, Lipton ME, Lockman LA, et al. Prevention of deterioration in metachromatic leukodystrophy by bone marrow transplantation. Am J Med Sci 1987; 294:80–85. 164. Dhuna A, Toro C, Torres F, Kennedy W, Krivit W. Longitudinal neurophysiologic studies in a patient with metachromatic leukodystrophy following bone marrow transplantation. Arch Neurol 1994; 49:1088–1092. 165. Stillman AE, Krivit W, Shapiro E, Lockman L, Latchaw RE. Serial MR after bone marrow transplantation in two patients with metachromatic leukodystrophy. AJNR Am J Neuroradiol 1994; 15:1929–1932. 166. Shapiro EG, Lipton ME, Krivit W. White matter dysfunction and its neuropsychological correlates: a longitudinal study of a case of metachromatic leukodystrophy treated with bone marrow transplant. J Clin Exp Neuropsychol 1992; 14:610–624. 167. Pridjian G, Humbert J, Willis J, Shapira E. Presymptomatic late-infantile metachromatic leukodystrophy treated with bone marrow transplantation. J Pediatr 1994; 125:755–758. 168. Guffon N, Souillet G, Maire I, Dorche C, Mathieu M, Guibaud P. Juvenile metachromatic leukodystrophy: neurological outcome two years after bone marrow transplantation. J Inherit Metab Dis 1995; 18:159–161. 169. Shapiro EG, Lockman LA, Knopman D, Krivit W. Characteristics of the dementia in late-onset metachromatic leukodystrophy. Neurology 1994; 44:662–665. 170. Navarro C, Fernandez JM, Dominguez C, Fachal C, Alvarez M. Late juvenile metachromatic leukodystrophy treated with bone marrow transplantation; a 4-year follow-up study. Neurology 1996; 46:254–256. 171. Kapaun P, Dittmann RW, Granitzny B, et al. Slow progression of juvenile metachromatic leukodystrophy 6 years after bone marrow transplantation. J Child Neurol 1999; 14:222–228.

446

Peters

172. Yeager A, Moser H, Forte K. Allogeneic bone marrow transplantation provides biochemical improvement and stabilizes the neurocognitive status in the adult form of metachromatic leukodystrophy. Blood 1994; 86:974a. 173. Solders G, Celsing G, Hagenfeldt L, Ljungman P, Isberg B, Ringden O. Improved peripheral nerve conduction, EEG and verbal IQ after bone marrow transplantation for adult metachromatic leukodystrophy. Bone Marrow Transplant 1998; 22:1119–1122. 174. Seitelberger F, Urbanits S, Nave. Pelizaeus-Merzbaucher disease. In: Vinken P, Bruyn G, eds. Handbook of Clinical Neurology: Neurodystrophies and Neurolipidoses. Amsterdam: Elsevier, 1996:559–579. 175. Wilson GN, Holmes RG, Custer J, et al. Zellweger syndrome: diagnostic assays, syndrome delineation, and potential therapy. Am J Med Genet 1986; 24:69–82. 176. Goldfischer S, Moore CL, Johnson AB, et al. Peroxisomal and mitochondrial defects in the cerebrohepato-renal syndrome. Science 1973; 182:62–64. 177. Leegwater PA, Vermeulen G, Konst AA, et al. Subunits of the translation initiation factor eIF2B are mutant in leukoencephalopathy with vanishing white matter. Nat Genet 2001; 29:383–388. 178. van der Knaap MS, Barth PG, Gabreels FJ, et al. A new leukoencephalopathy with vanishing white matter. Neurology 1997; 48:845–855. 179. Thomas GH, Beaudet AL. Disorders of glycoprotein degradation and structure: alphamannosidosis, beta-mannosidosis, fucosidosis, sialidosis, aspartylglucosaminuria, and carbohydrate-deficient glycoprotein syndrome. In: Valle D, ed. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill, 1995:2529–2561. 180. Taylor RM, Farrow BRH, Stewart GJ, Healy PJ. Enzyme replacement in nervous tissue after allogenic bone-marrow transplantation for fucosidosis in dogs. Lancet 1986; 2:772–774. 181. Miano M, Lanino E, Gatti R, et al. Four year follow-up of a case of fucosidosis treated with unrelated donor bone marrow transplantation. Bone Marrow Transplant 2001; 27:747–751. 182. Krivit W, Peters C, Shapiro EG. Bone marrow transplantation as effective treatment of central nervous system disease in globoid cell leukodystrophy, metachromatic leukodystrophy, adrenoleukodystrophy, mannosidosis, fucosidosis, aspartylglucosaminuria, hurler, maroteauxlamy, and sly syndromes, and Gaucher disease type III. Curr Opin Neurol 1999; 12:167–176. 183. Vellodi A, Cragg H, Winchester B, et al. Allogeneic bone marrow transplantation for fucosidosis. Bone Marrow Transplant 1995; 15:153–158. 184. Beutler E, Grabowski GA. Gaucher disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill, 1995:3635–3668. 185. Barton NW, Brady RO, Dambrosia JM, et al. Replacement therapy for inherited enzyme deficiency—macrophage-targetedglucocerebrosidase for Gaucher’s disease. N Engl J Med 1991; 324:1464–1470. 186. Figueroa ML, Rosenbloom BE, Kay A, et al. A less costly regimen of alglucerase to treat Gaucher’s disease. N Engl J Med 1992; 327:1632–1636. 187. Schiffmann R, Heyes MP, Aerts JM, et al. Prospective study of neurological responses to treatment with macrophage-targeted glucocerebrosidase in patients with type 3 Gaucher’s disease. Ann Neurol 1997; 42:613–621. 188. Walkley SU, Thrall MA, Dobrenis K, et al. Bone marrow transplantation corrects the enzyme defect in neurons of the central nervous system in a lysosomal storage disease. Proc Natl Acad Sci USA 1994; 91:2970–2974. 189. Will A, Cooper A, Hatton C, Sardharwalla IB, Evans DI, Stevens RF. Bone marrow transplantation in the treatment of alpha-mannosidosis. Arch Dis Child 1987; 62:1044–1049. 190. Wall DA, Grange DK, Goulding P, Daines M, Luisiri A, Kotagal S. Bone marrow transplantation for the treatment of alpha-mannosidosis. J Pediatr 1998; 133:282–285. 191. Grewal SS, Shapiro EG, Krivit W, et al. Effective treatment of alpha-mannosidosis by allogeneic hematopoietic stem cell transplantation. J Pediatr 2004; 144:569–573. 192. Autti T, Santavuori P, Raininko R, et al. Bone marrow transplantation in aspartylglucosaminuria: MRI of the brain suggests normalizing myelination. In: Ringden O, Hobbs JR, Steward CG, eds. Correction of Genetic Diseases by Transplantation. London: COGENT, 1997:92. 193. Autti T, Rapola J, Santavuori P, et al. Bone marrow transplantation in aspartylglucosaminuria— histopathological and MRI study. Neuropediatrics 1999; 30:283–288. 194. Arvio M, Sauna-Aho O, Peippo M. Bone marrow transplantation for aspartylglucosaminuria: follow-up study of transplanted and non-transplanted patients. J Pediatr 2001; 138:288–290. 195. Santavuori P, Lauronen L, Kirveskari E, Aberg L, Sainio K, Autti T. Neuronal ceroid lipofuscinoses in childhood. Neurol Sci 2000; 21:S35–S41.

Metabolic Diseases

447

196. Lake BD, Steward CG, Oakhill A, Wilson J, Perham TG. Bone marrow transplantation in late infantile Batten disease and juvenile Batten disease. Neuropediatrics 1997; 28:80–81. 197. Lipman RD, Donohue LR, Hoppe P, Bronson RT. Evidence that lysosomal storage of proteolipids is a cell autonomous process in the motor neuron degeneration (mnd) mouse, a model of neuronal ceroid lipofuscinosis. Neurosci Lett 1996; 219:111–114. 198. Westlake VJ, Jolly RD, Jones BR, et al. Hematopoietic cell transplantation in fetal lambs with ceroid-lipofuscinosis. Am J Med Genet 1995; 57:365–368. 199. Deeg HJ, Shulman HM, Albrechtsen D, Graham TC, Storb R, Koppang N. Batten’s disease: failure of allogeneic bone marrow transplantation to arrest disease progression in a canine model. Clin Genet 1990; 37:264–270. 200. Desnick R, Ioannou Y,, Eng C. Alpha-Galactosidase a deficiency: fabry disease. In: Scriver S, Beaudet A, Sly W, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill, 2001:3733–3774. 201. Schuchman EH, Desnick RJ. Niemann-Pick disease types A and B: acid sphingomyelinase deficiencies. In: Valle D, ed. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill, 1995:2601–2624. 202. Bayever E, Kamani N, Ferreira P, et al. Bone marrow transplantation for Niemann-Pick type IA disease. J Inherit Metab Dis 1992; 15:919–928. 203. Vellodi A, Hobbs JR, O’Donnell NM, Coulter BS, Hugh JK. Treatment of Niemann-Pick disease type B by allogeneic bone marrow transplantation. Br Med J (Clin Res Ed) 1987; 295:1375–1376. 204. Pentchev PG, Vanier MT, Suzuki K, Patterson MC. Niemann-Pick disease type C: a cellular cholesterol lipidosis. In: Valle D, ed. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill, 1995:2625–2639. 205. Hsu YS, Hwu WL, Huang SF, et al. Niemann-Pick disease type C (a cellular cholesterol lipidosis) treated by bone marrow transplantation. Bone Marrow Transplant 1999; 24:103–107. 206. Kornfeld S, Sly W. I-cell disease and pseudo-Hurler polydystrophy: disorders of lysosomal enzyme phosphorylation and localization. In: Scriver C, Beaudet A, Sly W, Valle D, eds. The Metabolic and Molecular Basis of Inherited Disease. New York: McGraw-Hill, 2001:3469–3482. 207. Kurobane I, Inoue S, Gotoh Y, et al. Biochemical improvement after treatment by bone marrow transplantation in I-cell disease. Tohoku J Exp Med 1986; 150:63–68. 208. Yamaguchi K, Hayasaka S, Hara S, Kurobane I, Tada K. Improvement of tear lysosomal enzyme levels after treatment with bone marrow transplantation in a patient with I-cell disease. Ophthalmic Res 1989; 21:226–229. 209. Grewal S, Shapiro E, Braunlin E, et al. Continued neurocognitive development and prevention of cardiopulmonary complications after successful BMT for I-cell disease: a long-term follow-up report. Bone Marrow Transplant 2003; 32:957–960. 210. Gravel RA, Clarke JTR, Kaback MM, Mahuran D, Sandhoff K, Suzuki K. The GM2 gangliosidoses. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill, 1995:2839–2879. 211. Assmann G, Seedorf U. Acid lipase deficiency: Wolman disease and cholesteryl ester storage disease. In: Valle D, ed. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill, 1995:2563–2587. 212. Abramov A, Schorr S, Wolman M. Generalized xanthomatosis with calcified adrenals. J Dis Child 1956; 91:282. 213. Krivit W, Peters C, Dusenbery K, et al. Wolman disease successfully treated by bone marrow transplantation. Bone Marrow Transplant 2000; 26:567–570. 214. Yeager AM, Uhas KA, Coles CD, Davis PC, Krause WL, Moser HW. Bone marrow transplantation for infantile ceramidase deficiency (Farber disease). Bone Marrow Transplant 2000; 26:357–363. 215. Tolar J, Teitelbaum S, Orchard P. Molecular etiology of osteopetrosis. N Engl J Med 2004; 351:2839–2849. 216. Coccia PF, Krivit W, Cervenka J, et al. Successful bone-marrow transplantation for infantile malignant osteopetrosis. N Engl J Med 1980; 302:701–708. 217. Gerritsen EJ, Vossen JM, Fasth A, et al. Bone marrow transplantation for autosomal recessive osteopetrosis. A report from the working party on inborn errors of the european bone marrow transplantation group. J Pediatr 1994; 125:896–902. 218. Locatelli F, Beluffi G, Giorgiani G, et al. Transplantation of cord blood progenitor cells can promote bone resorption in autosomal recessive osteopetrosis. Bone Marrow Transplant 1997; 20:701–705. 219. Eapen M, Davies SM, Ramsay NK, Orchard PJ. Hematopoietic stem cell transplantation for infantile osteopetrosis. Bone Marrow Transplant 1998; 22:941–946.

448

Peters

220. Fasth A, Porras O. Human malignant osteopetrosis: pathophysiology, management and the role of bone marrow transplantation. Pediatr Transplant 1999; 3:102–107. 221. McMahon C, Will A, Hu P, Shah GN, Sly WS, Smith OP. Bone marrow transplantation corrects osteopetrosis in the carbonic anhydrase II deficiency syndrome. Blood 2001; 97:1947–1950. 222. Kapelushnik J, Shalev C, Yaniv I, et al. Osteopetrosis: a single centre experience of stem cell transplantation and prenatal diagnosis. Bone Marrow Transplant 2001; 27:129–132. 223. Schulz AS, Classen CF, Mihatsch WA, et al. HLA-haploidentical blood progenitor cell transplantation in osteopetrosis. Blood 2002; 99:3458–3460. 224. Driessen GJ, Gerritsen EJ, Fischer A, et al. Long-term outcome of haematopoietic stem cell transplantation in autosomal recessive osteopetrosis: an EBMT report. Bone Marrow Transplant 2003; 32:657–663. 225. Steward CG, Pellier I, Mahajan A, et al. The working party on inborn errors of the european blood and marrow transplantation group. Br J Haematol 2004; 124:63–71. 226. Steward CG, Blair A, Moppett J, et al. High peripheral blood progenitor cell counts enable autologous backup before stem cell transplantation for malignant infantile osteopetrosis. Biol Blood Marrow Transplant 2005; 11:115–121. 227. Tsuji Y, Ito S, Isoda T, et al. Successful nonmyeloablatve cord blood transplantation for an infant with malignant infantile osteopetrosis. J Pediatr Hematol Oncol 2005; 27:495–498. 228. Key LL, Jr., Ries WL, Rodriquiz RM, Hatcher HC. Recombinant human interferon gamma therapy for osteopetrosis. J Pediatr 1992; 121:119–124. 229. Key LL, Jr., Rodriguiz RM, Willi SM, et al. Long-term treatment of osteopetrosis with recombinant human interferon gamma. N Engl J Med 1995; 332:1594–1599. 230. Kubo T, Tanaka H, Ono H, Moriwake T, Kanzaki S, Seino Y. Malignant osteopetrosis treated with high doses of 1 alpha-hydroxyvitamin D3 and interferon gamma. J Pediatr 1993; 123:264–268. 231. Weinreb N, Charrow J, Andersson H, et al. Effectiveness of enzyme replacement therapy in 1028 patients with Type 1 Gaucher disease after 2 to 5 years of treatment: a report from the Gaucher registry. Am J Med 2002; 113:112–119. 232. Schiffmann R, Brady RO. New prospects for the treatment of lysosomal storage diseases. Drugs 2002; 62:733–742. 233. Kaye EM. Therapeutic approaches to lysosomal storage diseases. Curr Opin Pediatr 1995; 7:650–654. 234. Kaye E. Lysosomal storage diseases. Curr Treat Options Neurol 2001; 3:249–256. 235. Koc ON, Peters C, Aubourg P, et al. Bone marrow-derived mesenchymal stem cells remain hostderived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases. Exp Hematol 1999; 27:1675–1681. 236. Jiang Y, Vaessen B, Lenvik T, Blackstad M, Reyes M, Verfaillie CM. Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp Hematol 2002; 30:896–904. 237. Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001; 98:2615–2625. 238. Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 2002; 109:337–346. 239. Zhao LR, Duan WM, Reyes M, Keene CD, Verfaillie CM, Low WC. Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats. Exp Neurol 2002; 174:11–20. 240. Schwartz RE, Reyes M, Koodie L, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 2002; 109:1291–1302. 241. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002; 418:41–49. 242. McIvor RS. Gene therapy of genetic diseases and cancer. Pediatr Transplant 1999; 3:116–121.

23 Hematopoietic Stem-Cell Transplantation for Autoimmune Diseases in Children Richard K. Burt and Larissa Verda Division of Immunotherapy, Feinberg School of Medicine, Northwestern University Medical Center, Chicago, Illinois, U.S.A.

I. M. de Kleer and Nico Wulffraat Pediatric BMT Unit, University Medical Center Utrecht, Utrecht, The Netherlands

INTRODUCTION Hematopoietic stem cell transplantation (HSCT) was first proposed as a therapy for autoimmune diseases in 1995 (1,2). Since then approximately 800 HSCT for autoimmune disease have been reported to the European Bone Marrow Transplant/European League Against Rheumatism (verbal communication—Passweg J., Basel) and International Bone Marrow Transplantation registries (verbal communication—Bredeson C., Milwaukee). While most HSCT for autoimmune disease have been performed in adults, indications for HSCT of childhood autoimmune diseases include juvenile idiopathic arthritis (JIA), systemic lupus erythematosus (SLE), Crohn’s disease (CD), and juvenile dermatomyositis (JDM).

RATIONALE The standard of treatment for autoimmune diseases is immune suppressive or immune modulating medications. Autologous HSCT is an extension of immune suppression to the point of immune ablation. In theory, the transplant conditioning regimen ablates the aberrant disease causing immune cells, whereas hematopoietic stem cells (HSC) regenerate a new and antigen naı¨ve immune system. The de novo development of the T- and B-cell repertoire from uncommitted progenitor cells in the presence of autoantigens is thought to reintroduce selftolerance similar to the normal ontogeny of the immune system during fetal development. If autologous HSCT results in an “immune reset” with reconstitution of a normal nondisease causing immune repertoire, then a sustained remission of active inflammation would be anticipated (3,4). Similar to autologous HSCT, after an allogeneic HSCT, reconstitution of the immune system will occur from the stem cell compartment. However, allogeneic HSCT by providing HSC from a disease resistant donor will also alter the patient’s genetic predisposition to disease susceptibility. Because allogeneic HSCT of malignancies has been traditionally complicated by 449

450

Burt et al.

relatively high mortality from graft versus host disease (GVHD), to date most HSCT for autoimmune diseases have been autologous HSCT (4,5).

ANIMAL MODELS Animal autoimmune diseases may arise spontaneously or be environmentally induced. Spontaneous onset diseases are genetically predetermined and occur without known environmental manipulation. Environmentally induced autoimmune diseases may arise from infections, immunization with autologous antigen(s), or adoptive transfer of lymphocytes from a diseased animal. Bone marrow HSC are acquired from a euthanized animal of a different animal strain (allogeneic HSCT), of the same highly inbred strain (syngeneic HSCT), or from a syngeneic animal with the same stage of disease (pseudo-autologous HSCT). Because bone marrow is collected from a surgically removed femur of a euthanized murine donor, syngeneic or pseudoautologous HSCT are performed in place of autologous marrow transplants (4,6). Spontaneous autoimmune diseases include diabetes in nonobese diabetic (NOD) mice (7), arthritis-colitis in HLA-B27 mice (8), and a lupus-like illness in numerous murine strains (9–13). HSCT from a nondisease susceptible donor will prevent and/or cure spontaneous onset autoimmune disease by changing genetic predisposition to disease from recipient to donor genotype (9,14–16). NOD mice develop subclinical insulitis by 90–100 days of age, clinical diabetes with glycosuria by 150–200 days old, and die within one month of glycosuria onset (14). NOD mice transplanted with T-cell depleted BALB/c bone marrow cells do not develop insulitis or become diabetic (14). NZW mice cross-bred with BXSB mice (abbreviated W/BF1) develop lupus nephritis, anti-cardiolipin antibodies, thrombocytopenia and myocardial infarction. Bone marrow transplantation from a normal mouse to a W/BF1 mouse cures these complications (16). MRL/lpr mice have a mutant Fas receptor (CD95). CD95/CD95 ligand interaction is involved in apoptosis of activated peripheral (postthymic) lymphocytes and in negative selection of thymic (central) lymphocytes. MRL/lpr mice develop lymphoid tissue hyperplasia and a lupus-like autoimmune disease (10). Allogeneic HSCT from a normal mouse cures the MRL/lpr phenotype (9). Although MRL/lpr lupus-like disease is due to a single gene defect, most animal autoimmune diseases are polygeneic. SLE susceptible loci have been identified and named according to the strain of mice in which they were discovered, such as the Bxs loci in BXSB mice, Nba loci in NZB mice, and Nwa loci in NZW mice (17,18). Allogeneic transplant of bone marrow cells from a normal strain of mice into NZW/NZB mice cures the autoimmune phenotype (19). Although allogeneic HSCT from a nondisease prone strain prevents or cures spontaneous onset autoimmune diseases, syngeneic HSCT, even if from predisease mice, has no effect or results in only transient amelioration of symptoms (20,21). Syngeneic HSCT of either NZB/DBA mice or HLA-B27 rats has no effect on lupus-like disease or arthritis-colitis, respectively (8,19). Syngeneic HSCT of MRL/lpr mice resulted in only transient remission of lupus-like manifestations (10). These data imply that genetically preordained autoimmune disorders require an allogeneic stem cell source for cure. In contrast to spontaneous onset autoimmune disorders, environmental induced autoimmune disease may be prevented or cured by syngeneic HSCT (20). Experimental autoimmune encephalomyelitis (EAE) is an autoimmune central nervous system (CNS) demyelinating disease that is an animal model for multiple sclerosis (MS). EAE may be induced in Lewis rats, several strains of mice, and in primates. One common murine strain in which to study EAE is Swiss Jackson Lewis/Jackson (SJL/J) mice. SJL/J mice develop a relapsing remitting disease clinically similar to relapsing remitting MS, following either immunization with myelin peptides, such as proteolipid protein amino acid sequence 139–151, or following adoptive transfer of lymphocytes from immunized mice. Syngeneic HSCT is capable of inducing remission and preventing relapse when performed either before disease onset but after environmental exposure (immunization or adoptive transfer) or early after disease onset during

HSCT for Autoimmune Diseases in Children

451

peak of disease or 1st remission (22–25). In contrast, syngeneic HSCT is not effective therapy for late stage or chronic progressive EAE (22). HSCT is, therefore, effective for inflammatory relapsing experimental autoimmune encephalomyelitis (R-EAE) but not progressive or chronic degenerative EAE. This has proven predictive for results of autologous HSCT for MS in which improvement occurs for patients with relapsing disease and inflammatory activity demonstrated by gadolinium enhancement on magnetic imaging resonance (MRI) scans but does not improve symptoms in patients with late progressive MS (26). Another animal model of MS is Theiler’s murine encephalomyelitis virus (TMEV)–induced CNS demyelinating disease manifest at onset as progressive neurologic deterioration (27,28). TMEV is a small RNA virus (picornavirus) acquired in the wild by oral inoculation, whereas in the laboratory infection, it is via direct intracerebral inoculation resulting in a higher proportion of diseased animals. Disease resistant strains of mice clear the infection within two weeks of infection, where as disease susceptible strains have a persistent CNS infection. Both virus—and myelinspecific T-cell responses—occur in TMEV-induced demyelinating disease (27,28). Unlike the beneficial effect of HSCT on R-EAE, syngeneic HSCT of TMEV-infected mice results in exacerbation of neurologic disability and high mortality due to CNS viral hyperinfection following immune ablation (29). HSCT using marrow from disease-resistant but previously infected donors ameliorated HSCT-related neurologic mortality, presumably by transfer of virus specific cytotoxic T cells along with the marrow graft (29). Therefore, a functional immune system appears important to prevent lethal neuropathic effects from a persistent viral-induced CNS demyelinating disease. Because several hundred patients with autoimmune diseases, including MS, have undergone HSCT worldwide without experiencing infection-related disease reactivation, it is unlikely that patients with autoimmune diseases harbor a persistent disease associated subclinical infection. Other animal autoimmune diseases cured by syngeneic HSCT include experimental autoimmune myasthenia gravis (30), a model of myasthenia gravis, and adjuvant arthritis (AA), a Freund’s adjuvant induced arthritis and model for JIA or rheumatoid arthritis (RA) (20,31). In AA, to prevent permanent deformities, HSCT needs to be performed before onset of irreversible chondro-cartilage destruction and osteonecrosis (20,31). Besides autologous transplants, allogeneic HSCT from a normal animal may also cure environmental-induced autoimmune diseases, such as EAE or AA (21,31,32). Environmental induced relapse may be cured after either autologous or allogeneic HSCT but can be reinduced by the initial environmental trigger in a higher percentage of animals after autologous compared to allogeneic HSCT (20,21). In summary, animal models suggest that: (1) genetically preordained autoimmune diseases may be cured by an allogeneic HSCT, (2) environmental induced autoimmune diseases may be cured with either an autologous or allogeneic HSCT, (3) to be effective HSCT should be performed in the inflammatory phase of autoimmune disease and before onset of irreversible tissue degeneration, while the disease is still an immune mediated inflammatory process rather than a chronic degenerative disease, and (4) relapses may occur upon reexposure to diseaseinitiating stimuli, and relapse is more common after autologous compared to allogeneic HSCT. Whether human autoimmune diseases are similar to spontaneous onset or induced animal autoimmune disease is unclear because the role of genetics versus environment in human autoimmune disorders is unknown. Murine models are selected for genetic predisposition by successive generations of inbreeding for autoimmune phenotypes unlike human populations that are generally highly outbred. Therefore, most human autoimmune diseases are probably polygenetic. Exceptions include single gene mutations in the autoimmune regulator gene that causes autoimmune polyendocrinopathy candidiasis, ectodermal dystrophy (APECED) (33) and the single Fas gene mutation that causes autoimmune lymphoproliferative syndrome (ALPS) similar to the single gene Fas mutation in MRL/lpr mice (34). The concordance of identical (syngeneic) twins for autoimmune disease is approximately 33% regardless of whether the disease is type I diabetes, RA, SLE, MS, or CD (35). Therefore, for most autoimmune diseases, environmental influences, even in identical twins, are important for clinical manifestation.

452

Burt et al.

STEM-CELL MOBILIZATION IN PATIENTS WITH AUTOIMMUNE DISEASES The most common method of collecting HSC is by mobilization from the peripheral blood. In small or underweight children weighing less than 30 kg, unprimed bone marrow aspiration under general anesthesia is also used. Because negligible HSC are detectable in the peripheral blood during steady state, either a hematopoietic growth factor, such as granulocyte colony stimulating factor (G-CSF), or chemotherapy (usually cyclophosphamide) with or without G-CSF are necessary to mobilize HSC into and subsequently collect HSC from the peripheral blood (36). Hematopoietic growth factors used to mobilize HSC also have immune modulating effects and depending upon growth factor may exacerbate or ameliorate disease (37,38). The effects of various growth factors on disease activity have been evaluated in EAE. G-CSF as well as Flt-3 ligand and stem cell factor (SCF) exacerbates EAE, whereas thrombopoietin mobilizes HSC without affecting disease severity (39). In clinical practice, G-CSF is the predominant growth factor used to mobilize HSC. G-CSF is used to treat Crohn’s fistulae and may contribute to amelioration of CD during mobilization (40). On the other hand, G-CSF may precipitate clinical flares of MS sometimes with significant and irreversible neurologic deterioration (41). G-CSF may also cause a transient increase in the number of swollen and/or tender joints in patients with RA that improves with corticosteroids (36). In autoimmune diseases, a G-CSF induced flare may be prevented by either administration of corticosteroids or mobilization with combined cyclophosphamide and G-CSF (36,41). HSC may be mobilized from the peripheral blood using between 5 to 10 mcg/kg/day of subcutaneous G-CSF along with 0.5 to 1.0 mg/kg/day of oral prednisone. Apheresis to collect progenitor cells begins on either day 4 or 5 of G-CSF administration. Ten to 15 liters peripheral blood apheresis performed on one day is usually adequate for collection of sufficient numbers of HSC. Occasionally a consecutive second or third day of apheresis may be necessary. HSC may also be collected by administration of cyclophosphamide (2.0 to 4.0 g/m2) and daily G-CSF beginning 72 hours after cyclophosphamide. Apheresis is performed when the white blood cell count rebounds, usually 10 days after cyclophosphamide infusion (36,42). G-CSF mobilization is not associated with neutropenia or risk of neutropenic infections. Mobilization with cyclophosphamide and G-CSF may cause one to two days of neutropenia. Infection risk during this interval may be minimized with prophylactic antibiotics. Advantages for cyclophosphamide / G-CSF mobilization are higher stem cell yields, an in vivo purge effect by selectively killing lymphocytes in cell cycle, and a cyclophosphamide-mediated diseaseameliorating effect (36,42).

EX VIVO STEM-CELL SELECTION The majority of mononuclear cells collected by peripheral blood apheresis (or bone marrow harvest) are immune cells, such as lymphocytes and monocytes, not HSCs. Although the true identity of human HSCs remains elusive, either purified CD34C or AC133C hematopoietic progenitor cells are sufficient for hematopoietic and immune reconstitution. In general, a minimum of 2!106 CD34C cells/kg recipient weight will ensure engraftment. HSC may be positively selected or enriched by three to four logarithms using antibodies to CD34 or AC133 or purified by negative selection to remove lymphocytes. In practice the most common method of purging lymphocytes is via CD34C enrichment using either the Miltenyi CliniMACS or Baxter Isolex cell separator device (36,42). Whether enrichment of the graft for CD34C HSC is necessary or even superior to an unmanipulated graft remains unclear (43). Reinfusion of lymphocytes has the theoretical risk of reinfusing autoreactive cells and might thus induce autoimmune disease. CD34C selection by removing lymphocytes is

HSCT for Autoimmune Diseases in Children

453

perhaps best viewed as another method of immune suppression. For an intense conditioning regimen, CD34C selection may not be necessary or even detrimental by increasing the risk of post HSCT infection.

RATIONALE FOR DESIGN OF AUTOLOGOUS AUTOIMMUNE HEMATOPOIETIC STEM-CELL TRANSPLANTATION (HSCT) REGIMENS Unlike malignancies where visceral organ impairment is a contraindication to HSCT, diseaserelated organ dysfunction is often the indication for HSCT of autoimmune diseases. Implicit in using HSCT in treating end organ damage is that the conditioning regimen should avoid agents that can potentially damage the organs that the treatment is designed to salvage. For example, such agents as bleomycin, BCNU (carmustine), and radiation, whose significant adverse effects are pulmonary fibrosis, would not be logical conditioning agents for immune-mediated diseases, such as scleroderma, mixed connective tissue disease, or dermatomyositis, in which a major cause of death is related to pulmonary fibrosis (44). Conditioning regimen design should also avoid agents that could damage organ specific stem cell compartments that may contribute to organ repair after the disease remits. The effect of conditioning agents on tissue specific stem cell viability and proliferative potential is unknown. However, even low dose total body irradiation (TBI) is myeloablative, (i.e., lethal for HSC), and in animal models, cranial radiation (10 cGy) impairs mechanism of CNS repair. Radiation mediated neural stem cell injury may occur by such mechanisms as apoptosis, alteration in cell cycle progression, and destruction of the neural stem cell niche or milieu through invasion of macrophages and microglia (45). It is, therefore, reasonable to assume that myeloablative agents, which by definition are cidal for marrow HSC, may damage or be lethal for HSC in other organ systems. The rationale for autologous HSCT of autoimmune diseases is to regenerate a new (i.e., antigen naı¨ve immune system), from the patient’s own HSC. Immune reconstitution will require the reemergence of virgin (antigen naı¨ve) recent thymic emigrant T cells (4,46,47). We recently showed renewed thymopoiesis of CD4CCD25C regulatory T cells in the first months after autologous stem cell transplantation (ASCT), resulting in a complete recovery of the CD4CCD25C regulatory T cell network. Also, we demonstrated that, independent of the CD4CCD25C regulatory T cells, HSCT induces arthritis related autoimmune T cells to deviate from a proinflammatory phenotype to a tolerant phenotype (48). The goal of autologous stem cell conditioning regimens is to reduce or eliminate the immune cells that contribute to the pathologic process. In contrast to conditioning regimens for hematologic malignancies, there is no need to sterilize the marrow stem cell population. Hematopoietic stem cell destruction from myeloablative agents, such as TBI, would be an unwanted side effect of autologous regimens in which the goal is immune ablation not myeloablation. Intense immune ablation without myeloablative side effects could be accomplished with such agents as cyclophosphamide, fludarabine, and antibodies to T cells [antithymocyte globulin (ATG)] and/or B cells (rituximab) or both T and B cells (CAMPATH or alemtuzumabw) (42,44). In summary, for autologous HSCT of autoimmune disease the rationale behind the conditioning regimen is to: (1) dose escalate agents that work as conventional therapy, (2) maximize immune suppression without myeloablation, (3) avoid conditioning regimen agents that may cause injury to already disease affected and damaged tissue, (4) avoid injury to tissue specific stem cell compartments that may be important for organ repair, (5) design regimens that are justified for the risk of the disease being treated, and (6) finally for children avoid agents, such as TBI, with a known tendency towards growth retardation or secondary malignancies.

454

Burt et al.

JUVENILE IDIOPATHIC ARTHRITIS The first reported use of autologous HSCT in JIA included children with the most severe and longstanding systemic disease, with significant erosive and irreversible joint destruction already presents (49). Since this report, more than 50 cases have been reported by nine pediatric bone marrow transplant units registered in the database of the working party for autoimmune diseases of the European Blood and Marrow Transplantation (EBMT) group (47,49–51).

Introduction Childhood chronic arthritis may be referred to as either JIA, juvenile rheumatoid arthritis (JRA), or juvenile chronic arthritis (JCA) due to different but overlapping terminologies developed by three different committees. The JRA classification system was developed by the American College of Rheumatology, JCA by the European League Against Rheumatism, and JIA by the International League of Associations for Rheumatology. For this review, we will use the terminology JIA (50). By definition JIA must begin before age 16 and duration of arthritis must be O6 weeks. Although a childhood disease, JIA may persist into adult life. Subtypes of JIA include systemic onset, polyarticular RF negative, polyarticular RF positive, oligoarticular, psoriatic-related arthritis, and enthesitis-related arthritis. Signs and symptoms of JIA are morning stiffness, joint pains or aches, anorexia, weight loss, growth failure, and deformity. Any joint may be affected, although large joints, such as knee joints, are most commonly involved. Systemic onset disease is more frequently associated with pericarditis, pericardial effusions, myocarditis, interstitial pulmonary fibrosis, hepatomegaly, splenomegaly, lymphadenopathy, and amyloidosis. Chronic uveitis is more common with oligoarticular arthritis and may manifest as ocular pain, redness, photophobia, headache, and/or change or loss of vision (52). There are 32,000 children in the United States with JIA. The peak age of onset is 1–3 years old (53). Overall mortality is approximately 1% but is up to 10–15% in systemic onset with severe functional status limitations. Death is due to infections, amyloid-related renal or heart failure, or myocardial infarction from corticosteroid related atherosclerosis (54).

Outcome Measures For evaluation after HSCT, a set of outcome variables for clinical trials in childhood arthritis [proposed by Giannini and the Pediatric Rheumatology International Trials Organization (PRINTO)] is used. PRINTO outcome measures for JIA include: CHAQ, a physicians’ global assessment of disease activity, a parent/patient assessment of overall well being, Fuchs swelling index (FSI), i.e., number of joints with active arthritis, Escola Paulista de Medicina range of motion, i.e., number of joints with limited range of motion, and ESR (erythrocyte sedimentation rate) (55–59).

Hematopoietic Stem-Cell Transplantation for Juvenile Idiopathic Arthritis Eligibility for autologous HSCT are polyarticular or systemic JIA that fails to respond to or has unacceptable toxicity to antitumor necrosis alpha therapy and high dose methotrexate (MTX) given by the intramuscular or subcutaneous routes (1.0 mg/kg/wk), and corticosteroid dependency. Exclusion criteria should include cardiorespiratory insufficiency, chronic active infection from Epstein-Barr virus (EBV), cytomegalovirus (CMV), or toxoplasmosis, or ongoing spiking fever and other signs of systemic disease activity despite steroids, and end-stage disease (Steinbrocker IV) or poor compliance (55).

HSCT for Autoimmune Diseases in Children

455

HSC in patients with JIA have been harvested from either bone marrow or by peripheral stem cell mobilization using a single infusion of cyclophosphamide between 1.5–3.0 g/m2 and G-CSF at 10 mg/kg/d. In general, the harvested cells were either purged by T-cell depletion with CD2 and CD3 antibodies or by positive stem cell selection using CD34C cell separation device. Immunorosette T-cell depletion, with CD2/CD3 tetrameric complexes, resulted in a mean 2.5 log depletion of T cells with a yield of 50% of the CD34C progenitor cell population. Stem cell selection using one of the CD34 selection procedures (either Isolex 300SA or CliniMax) resulted in a 4.5 log depletion of T cells for both systems but with different results for the recovery of CD34C cells. An increased yield of CD34C cells was obtained with the CliniMax procedure (57.9C/K9.0%) in comparison to the Isolex procedure (40.1C/K12.5%). In the cases of peripheral bone marrow collection these techniques yielded a final suspension with a CD34C cell count of 2.9–10.9!106 cells per kg (mean 5.2!106 cells per kg) and with a CD3C cell count of 0–1.4!105 cells per kg (mean 0.3!105 cells per kg). In the 25 cases of bone marrow collection, these techniques yielded a final suspension with a CD34C cell count of 0.44–6.0!106 cells per kg (mean 2.2!106 cells per kg) and with a CD3C cell count of 0–3.5!105 cells per kg (mean 0.7!105 cells per kg) (50,51,60,61). Four different conditioning regimens have been used for HSCT of children with JIA (Table 1). The first conditioning regimen (23 patients) included four days of rabbit antithymocyte globulin (ATG, Sangstat), 5 mg/kg from day -9 to -6, cyclophosphamide, 50 mg/kg/day from day -5 to -2 and low dose TBI (four gray, single fraction) on day -1. The second conditioning regimen (eight patients) included the above scheme without TBI. The third conditioning regimen (three patients) included fludarabine (30 mg/m2) on day -7 and -6, cyclophosphamide (50 mg/kg/day) from day -5 to -2, ATG (5 mg/kg) from day -6 to -3, methylprednisolone (1 g/m2) from day -4 to -2 and no TBI. MTX and cyclosporine A (CsA) were stopped before HSCT and prednisone was tapered, when possible, starting not earlier than two months after HSCT. Disease flares or relapses were treated as before HSCT. A total of 34 children were treated with these regimes. The results of autologous HSCT were striking, with a prolonged drug free follow-up of 6 to 60 months (Figs. 1 and 2), 50% are still in drug-free remission (47,51,60,62). One quarter of the patients showed a transient or mild relapse of active arthritis, and 13% developed a persistent relapse of arthritis. Seventeen of 32 children (53%) showed a drug-free improvement of more than 50% after a follow-up of 4 to 60 months, with a marked decrease in the scores of the Childhood Health Assessment questionnaire, the Physicians Global Assessment and in swollen joint count (47,51,60,62). The measurement of limitation of movement largely reflects permanent joint destruction and did not, as expected, change considerably. The ESR, CRP, and hemoglobin levels returned to near normal values with six weeks. Because the response rate was not improved with a TBI containing regimen compared to non-TBI regimen and due to concerns over the TBI-related growth retardation and late malignancies, future conditioning regimens will probably not contain TBI (51). Post HSCT the neutrophils recovered (O0.5!109/l) between day 12–30, and the platelet count reached 20!109/l between days 16 to 35. Five to nine months after HSCT the numbers of circulating T cells were normal, with normal in vitro mitogenic responses at 6 to 18 months after HSCT (47,51). Patients with JIA who were transplanted after conditioning with high-dose cyclophosphamide, ATG- and low-dose TBI, however, showed a prolonged lymphopenia, especially of CD4C lymphocytes, persisting for 6 to 12 months after HSCT (Fig. 3) (51,60,62). Relapse was noted in seven children 18 months after autologous HSCT. Some of these relapses have been mild with oligoarthritis and sporadic fever, which could be controlled easily with a 3-month course of low dose prednisone and nonsteroidal antiinflammatory agents. Even these seven patients showed a 30% improvement in their disease. Four children were refractory to autologous HSCT and showed a persistent recurrence of disease that was as severe as before the HSCT (51). Recently, durable remission was also reported in two patients with JIA after conditioning with etoposide (VP16) (2 g/m2), thiotepa (300 mg/m2), and ATG (40 mg/kg), followed by reinfusion of purified CD34C HSC selected from peripheral blood mononuclear cells (47,49–51,60,62).

Cy, ATG Cy, Flu, ATG Cy, VP16,TT, ATG

2 3 4

Same as above

Exclusion criteria

Chronic infections, active systemic disease at time of ASCT

Exclusion criteria

Isolation during aplastic period IVIG (0.4 mg/kg!3 weeks) until CD3 countO500/mL Cotrimoxazole prophylaxis. Give high-dose steroids (2 mg/kg) during conditioning to prevent MAS

Supportive care

No specific rules for IVIG or antimicrobial prophylaxis

Supportive care

Abbreviations: ASCT, autologous stem cell transplantation; ATG, antithymocyte globulin; CD2/3, negative selection by monoclonal antibodies to CD2C or CD3C lymphocytes; CD34, positive selection of CD34C stem cells; Cy, cyclophosphamide; FLU, fludarabine; IVIG, intravenous immunoglobulin suppletion; JIA, juvenile idiopathic arthritis; MAS, macrophage activation syndrome; TBI, total body irradiation; TCD, T-cell depletion; TNF, tumor necrosis factor; TT, thiotepa; VCR, ex vivo T-cell depletion using vincristine; VP16, etoposide.

Same as above

CD34 selection. CD34O 1!106/kg and add back CD3 1!105/kg

Proposed new protocol

Drug resistant, including antiTNF therapy

Inclusion criteria

Inclusion criteria

24 24–36 36

4–60

Follow-up (month)

Currently Used Protocols of ASCT for JIA

TCD

CD34 VCR or CD34 CD34

CD2/3

TCD

Cy (50 mg/kg/day at day -7 and -6 ATG 5 mg/kg /day at day -9 to -6 Flu 30 mg/m2/day at day -5 to -1 Reinfusion of stem cells at day 0

Conditioning

Cy, ATG, TBI (low dose, 4Gy)

Conditioning

1

No

Table 1 Protocols of ASCT for JIA

456 Burt et al.

HSCT for Autoimmune Diseases in Children

457

Swollen Joint Count 50

40

30

20

10

0

pre

+3m

+6m

+9m

+12m +24m +36m +48m

Figure 1 Active joint count before and after autologous stem cell transplantation. Patient data were obtained from eight European pediatric transplant centers. Solid black triangles represent children transplanted without use of total body irradiation as part of the conditioning regimen. Solid triangles represent no TBI.

Systemic JIA may be complicated by macrophage activation syndrome (MAS). Recurrence of fever, hepatomegaly, pancytopenia, clotting disorders, and hemophagocytosis in the marrow are symptoms of MAS, which must be promptly treated with high-dose corticosteroids (intravenous methylprednisolone 15 mg per kg per day) and cyclosporine, and in more severe cases with etoposide. Because MAS has occurred after autologous HSCT, etoposide-containing conditioning regimens, or other antimacrophage agents, such as CAMPATH-1H, may help prophylaxis against MAS (63). Prior to the onset of their arthritis the Dutch children in this study had heights between K0.2 and C2 standard deviations (SD) of the mean length for their age. During the course of their disease they lost three to five SDs. After autologous HSCT some children, (mostly the younger ones) showed a catch up growth of one to two SDs (51). De Kleer et al. summarized the European results with regard to infectious complications after HSCT (47,51). Most of the 34 children (71%) developed at least one infection, mainly during the aplastic period (mostly coagulase negative Staphylococcus and Streptococcus mitis).

Proportion of patients

1.00

0.75

Survival

0.50

Event−free survival

0.25

0.00 0

Figure 2

10

20 30 40 Months after ASCT

Kaplan-Meier curves.

50

60

458

Burt et al.

3000

Pre ASCT +3m

Number of cells per μl

+6m +9m 2000

+12m +24m

1000

0 CD3

CD4

CD8

CD19

CD16

CD4CD45RA CD4CD45RO CD8CD45RA CD8CD45RO

Figure 3 Reconstitution of different lymphocyte subsets in 29 patients with juvenile idopathic arthritis (JIA) after autologous stem cell transplantation for 24 months (mean G SE).

Viral infections, mostly varicella zoster virus reactivation 3 to 18 months after autologous HSCT but also CMV (both reactivation and a primary infection), occurred frequently. More rarely seen were opportunistic infections from atypical Mycobacterial infection (nZ1), Legionella pneumoniae (nZ1), hepatitis A infection (nZ1), and herpes simplex virus hepatitis (nZ1); all resolved completely (51). Four patients died. Causes of death were mostly due to infection associated with bone marrow suppression (51). In three of these children a MAS, a well-known complication of systemic JIA, reflecting marked macrophage activation due to loss of T-cell control and perhaps an underlying abnormality of macrophage function, was also present (51,63,64). This complication was preceded by infections, including EBV reactivation and disseminated toxoplasmosis (51,64). The unexpected occurrence of MAS, also known as hemophagocytic lymphohistiocytosis, after autologous HSCT remains unexplained. This complication resembles the active phases of familial hemophagocytic lymphohistiocytosis (FHL), in which an immunological imbalance between regulatory T cells and macrophages has been postulated (65,66). FHL is an autosomal recessive disorder characterized by uncontrolled activation of T cells and macrophages and excessive production of inflammatory cytokines (66). Recently a defect in the perforin gene, whose product is involved in the immunoregulatory process of target cell killing, was described as the underlying cause of FHL (67). Perforin expression appears to often be severely reduced in systemic JIA (62). Prolonged immunosuppression and dysregulation of perforin expression on cytotoxic effector and natural killer (NK) cells and a defective NK cell function during early immune reconstitution after HSCT may explain development of MAS after stem cell therapy (62).

CROHN’S DISEASE Introduction CD usually presents with diarrhea, abdominal pain and weight loss, although symptoms reflect the location and extent of gastrointestinal (GI) tract involved. For example, small volume diarrhea with frequent stools occurs in colonic involvement, whereas large volume stools with less frequent bowel movements occur in small intestinal disease. Malabsorption arises with small bowel disease, nausea, and vomiting with upper GI involvement, whereas pancreatitis may occur secondary to inflammation and obstruction involving the sphincter of Odi (68–70). Other symptoms include perianal skin tags, fissures, fistulae, rectal bleeding, abdominal masses, percutaneous or intra-abdominal fistulae, peritoneal sterile or infected abscesses, arthralgias, and disease-related fever without infection. CD may also involve the mouth,

HSCT for Autoimmune Diseases in Children

459

stomach, esophagus, and pancreatobiliary system. Recurrent surgeries to palliate symptoms may result in short bowel syndrome and require chronic total parenteral nutrition (TPN) (69,70). In children, CD may result in growth retardation (71). The clinical course is variable and be persistent, progressive, or may wax and wane (68–70). The fluctuating nature of CD makes clinical trials suspect unless a control arm is included or a dramatic salutary effect occurs as has been seen following autologous HSCT. General mortality from CD does not seem to be excessively high in the available literature, but subgroup analysis in severe refractory cases has not been performed. The mortality directly related to CD in one large series was 6%, and 3% in another study confined to Crohn’s colitis (68–70,72,73). A study from Minnesota showed a statistically increased mortality above expected in patients with CD (pZ0.007), although the numerical increase was small (74). Risk factors for Crohn’s-related mortality have not yet been established. Therefore, the mortality in severe and treatment refractory cases is unknown but is believed to be higher than available data would suggest from such figures. In addition, patients with severe disease suffer from being unable to eat, frequent nausea, vomiting, diarrhea, malnutrition, growth retardation, fistula formation, abdominal pain, multiple surgeries, extraintestinal symptoms, a grim quality of life, and significant and sometimes life-threatening side effects from currently approved therapy, including anaphylaxis to infliximab, and pancreatitis and prolonged bone marrow suppression with cytopenias from 6-mercatopurine or MTX. Children represent a subgroup of CD patients in which disease morbidity can be devastating to the quality of life (71,75–78). Growth failure is a common presentation of CD in children (79–83). It is probably the result of chronic inflammation and the effect of inflammatory cytokines on the growth hormone axis. In particular, there is a defect in the synthesis of insulin-like growth factor 1 in response to growth hormone as long as active CD inflammation persists (84). There are also direct inhibitory effects of inflammatory cytokines, particularly tumour necrosis factor alpha (TNFa), on long-bone growth plates (85,86). The cumulative effect of these abnormalities is to inhibit long-bone growth and maturation. If the disease is not held in remission, adult height will be stunted (81). The therapy CD in children depends, in large part, on the use of corticosteroids. In most treatment paradigms, corticosteroids are used to induce remission, with the expectation that they can be discontinued or reduced in dosage once the disease is in remission (87). Other drugs are used to maintain remission, allowing decreased used of steroids; however, a significant proportion of children with CD are dependent on steroids to keep their disease in control. The use of daily steroids is associated with poor growth and osteopenia. As a result, some children with hard to manage CD have complications of bone disease related to both the disease itself and corticosteroid therapy. These complications include pathological fractures of long bones and compression fractures of vertebrae, as well as marked impairment of bone calcium accrual that persists into adulthood. Dependence on daily steroids to maintain reasonable disease control is as disastrous to the quality of life as is uncontrolled disease. In either case, children fail to grow, have delayed puberty, and cannot enjoy anything like a normal adolescence. The etiology of CD is unknown. No intestinal self-antigen (initiating or spread epitope) that is pathogenic has been identified. On the other hand, several animal gene knockout models suggest that inflammatory bowel disease may be a result of immune dysregulation between Th1 and Th2 cytokines. Deficiency of multiple Th2 cytokines may cause colitis in animal models. Interleukin-10 deficient mice develop acute and chronic colitis (88). IL-2 deficient, double mutant IL-2, and IL-4 deficient and transforming growth factor beta deficient mice also develop colitis (89,90). When raised in a germ free environment, these gene knockout mice remain disease free. Animal models, therefore, demonstrate the need for both cytokine imbalance and intestinal flora as disease triggers. CD responds to anti-inflammatory agents, antibiotics, and immunosuppressive drugs, such as 5-ASA products (91), metronidazole (92), quinolones, corticosteroids, 6-mercaptoprine (93,94), azathioprine (95) and MTX (96), cyclosporine (97) and intravenous pulse cyclophosphamide (98). A TNF inhibitor, infliximab, has also shown consistent benefit (99). Unfortunately, none of these treatments is curative. In addition, many patients with severe

460

Burt et al.

disease will have recurrence after some response or will not respond. This leads to significant morbidity from disease and treatment complications. The treatment of CD in children differs significantly from treatment of adults because of the imperative to quickly achieve remission to permit growth. Corticosteroids are most effective in inducing remission, and as a result, they are used as first line therapy in essentially all children with CD. Once the disease is brought into remission, steroid sparing agents are added to the therapeutic regimen. Most frequently, a combination of 5-ASA compound and azathioprine or 6-mercaptopurine (6-MP) is employed. Steroids are then carefully weaned in hopes of maintaining remission. Failure to achieve remission and inability to wean from steroids constitute the main indications for the use of Infliximab. Failure of Infliximab to induce remission constitutes the main indication to introduce long term TPN and surgical resection.

Outcome Measures Various scoring systems have been developed to qualify response to therapy and follow natural course of CD (100). Two commonly used indices are the Crohn’s disease activity index (CDAI) and inflammatory bowel disease questionnaire that address quality of life issues (99,101). Laboratory values to monitor include albumin, anemia, sedimentation rate, C-reactive protein, weight, and in children height and pubertal development.

Hematopoietic Stem-Cell Transplantation for Crohn’s Disease in Adults Anecdotal case reports from hematopoietic stem cell transplant recipients who had coexistent CD in addition to their malignancies suggest that HSCT is highly effective and could provide durable long-term remission. Six patients with CD have been reported who underwent allogeneic bone marrow transplantation for other reasons (102). One was in remission at the time of allogeneic HSCT and remained in remission for 15 years despite discontinuation of immunosuppression. Three of five with active CD went into remission and maintained their remission for 6–10 years at the time of most recent post-HSCT follow-up. The fourth had continued fistulous disease and required post-HSCT ileac resection. The fifth patient died of sepsis three months post HSCT, and CD activity was not reported (103). Another report of allogeneic HSCT in a patient with coincidental CD described post-HSCT improvement (103). Two patients with CD have been reported who underwent autologous HSCT for other reasons (104,105). One patient with non-Hodgkin’s lymphoma entered a post autologous transplant remission for both lymphoma and CD for seven years at the time of report (104). The second patient entered a prolonged clinical remission after autologous HSCT although subclinical colonic inflammation remained on colonoscopy (105). At Northwestern University (Chicago, IL), twelve patients have undergone autologous HSCT for the sole indication of CD (68,106,107). HSC were collected from the peripheral blood with 2.0 g/m2 of cyclophosphamide and10 mcg/kg/day G-CSF. Because CD results in mucosal inflammation and ulceration, the conditioning regimen sought to avoid mucositis causing agents. Conditioning was therefore with a nonmyeloablative regimen of cyclophosphamide 200 mg/kg/day divided over four consecutive days and ATG 90 mg/kg divided over three days, with solumedrol (one gram) infused 30 minutes prior to each dose of ATG. The regimen also tried to maintain fertility. The risk of infertility from 200 mg/kg of cyclophosphamide is age dependent. Although only 1/3 of women over age 26 regain fertility, virtually all women under age 26 remain fertile. Because CD may cause fever and sterile granulomatous abscesses, it is also important to rule out infection before starting the conditioning regimen. Intestinal perforation, toxic megacolon, or a suppurative problem that may require urgent surgery should be considered contraindications to HSCT. Abdominal pain and diarrhea markedly improve or resolve prior to HSCT hospital discharge. However, most patients still show minor signs of residual inflammation on colonoscopy despite clinical remission. The significance of this finding is unclear. Only one

HSCT for Autoimmune Diseases in Children

461

patient developed clinical recurrence, whereas all other patients have maintained a clinical and drug-free remission. Large bowel abdominal masses and narrowing resolved after HSCT. Small bowel stricture in one patient required surgical resection two months after HSCT for obstruction. Histopathology of the surgically resected specimen revealed no evidence of active Crohn’s. The Northwestern protocol has recently been extended to pediatric patients. To date the youngest patient treated at Northwestern was 14 years old.

Hematopoietic Stem-Cell Transplantation Eligibility for Pediatric Patients Candidates should have an established diagnosis of CD and biopsy proven inflammatory bowel disease. Eligibility should include a CDAI of 250–400 or pediatric CDAI of 25–40, despite treatment with appropriate corticosteroids, azathioprine or 6-MP, 5ASA products, and metronidazole for a period of at least one year without achieving and/or maintaining remission. The patient should have received at least one course of infliximab (one course usually involves three infusions), without sustained benefit or intolerable side effects. The eligibility age is between 10 and 18, which includes most CD patients who are at a critical age regarding therapy and can benefit from definitive therapy that would induce remission and permit pubertal growth and an increase in bone density. Other eligibility may include any of the following: (1) corticosteroid-dependence having required daily dosing at O0.5 mg/kg for at least six months to maintain disease remission (CDAI !150) and having failed corticosteroid withdrawal (flare of active disease), (2) TPN dependent, (3) severe growth restriction—current and predicted adult height are !5 percentile and/or growth Z-score for the year prior to pretransplant evaluation is !K2, (4) severe restriction of bone accrual—lumbar bone density is O2SD below normal—and/or has suffered one or more pathological fractures, (5) debilitating or deforming perianal disease or other disabling fistulizing disease (e.g., to the urinary bladder), or (6) the patient is being considered for surgical resection because of failure of medical therapy.

SYSTEMIC LUPUS ERYTHEMATOSUS Introduction SLE is a multisystem, inflammatory disorder characterized by the production of antibodies that react with many different self-antigens. Despite differences in presentation, manifestations, and clinical course, the common factor for patients with SLE is hyperreactivity of T and B cells to endogenous stimuli (108–110). Although the usual SLE patient is a woman of child-bearing age [approximate ratio of women to men is 10:1 (111)], the disease can affect patients of all ages, ethnic backgrounds, and both sexes. About 20% of all lupus cases are diagnosed during first two decades of life (112). SLE is a clinically heterogeneous disease with manifestations that may affect predominately one or combinations of organ systems and varies between patients. Some patients have predominately a single system affected [e.g., nephritis, serositis, pneumonitis, cerebritis, vasculitis, antiphospholipid antibody syndrome with venous and vascular thrombi, arthralgias, myalgias, cutaneous symptoms (rash, livedo reticularis, ulcerations), or immune-mediated cytopenias]. A number of factors are associated with poor prognosis; these include the presence of renal disease causing an elevation in serum creatinine or urine protein excretion above normal, hypertension, diffuse proliferative glomerulonephritis on renal biopsy, chronic changes on biopsy, lung involvement, a high systemic lupus erythematosus disease activity index (SLEDAI) score, anemia, thrombocytopenia, and antibodies to phospholipids (113–116). General clinical manifestations and organ involvements in childhood are similar to adult SLE; however, the disease in children appears to have more severe course (112,117). It has been shown that renal involvement is more common and severe in pediatric SLE patients

462

Burt et al.

compared to adults, with estimates of prevalence ranging from 50% to 80% (118,119). Clinical manifestations of SLE in childhood include broad variations between rash, arthritis, renal disease (most common are grades III and IV according to the World Health Organization), pulmonary involvement (most frequent type is restrictive lung disease), cardiovascular manifestations, and neuropsychiatric involvement (118–120). SLE is polygenetic disease with multiple potential combinations predisposing to disease development. Genetic analysis predicts that many genes are involved in its expression in humans (121,122). Class II molecules, DRB2 and DRB3 have been associated with SLE in North American Caucasians (123). Various combinations of SLE-prone genes among different patients may explain why patients with SLE can have highly variable organ involvement and clinical symptoms (124). Additionally, it is believed that genetic predisposition alone is not sufficient to induce SLE in humans. In monozygotic twins, 30–50% are concordant for disease (125). A multifactorial etiology, including genetic predisposition, permissive sex hormone status, and environmental interaction, are probably necessary for development of clinical disease. Mortality from SLE has markedly improved due to more aggressive anti-inflammatory and immunosuppressive treatments as well as improved supportive care from dialysis, apheresis, and newer antihypertensive and antibiotic medications (126,127). For patients with diffuse proliferative glomerulonephritis, 5-year survival in the 1950s was almost zero (128). With the introduction of high-dose corticosteroids in the 1960s, the 5-year survival for this subset improved to 25% (128). Following the addition of oral cyclophosphamide and azathioprine, the 5-year survival in severe lupus nephritis improved to 40–70% (129). In the 1980s, introduction of intravenous pulse cyclophosphamide (500–1000 mg/m2 monthly for six months and then every three months for six months) improved 5-year survival to 80% (109,129). More recent therapies for SLE include cyclosporine, mycophenolate mofetil, and rituximab (130,131). Twelve-month survival rates and response rates were similar in patients with lupus nephritis (approximately 80%) treated with either daily oral cyclophosphamide for six months (followed by daily oral azathioprine for six months) compared to mycophenolate mofetil daily for 12 months. Despite significant improvement in the treatment of high risk SLE, the morbidity still remains high. The current treatment of children with SLE does not significantly differ from that in adults and depends on the clinical expression of the disease. Major manifestations can endanger the patient’s life and require early, aggressive treatment: usually combinations of high doses of immunosuppressants, such as corticosteroids, azathioprine, MTX, cyclosporine, and cyclophosphamide. The immunosuppression achieved with these treatments is not always effective and is associated with significant toxicities, including growth retardation, accelerated atherosclerosis, and severe infectious complications (132).

Outcome Measures Multiple indices exist to measure or characterize disease activity (133). Activity instruments include the British Isles Lupus Assessment Group (BILAG) scale (134), SLEDAI (135), systemic lupus activity measure (136), and the lupus activity index (133). The instrument employed depends on institutional and investigator familiarity. The BILAG has developed a scoring system to evaluate the current disease activity and the changes in disease activity from the last assessment. The evaluation is based on a five-category classification characterizing the degree of symptoms attributed to active lupus, with 86 questions based on the patient’s history, examination, and laboratory results. The 86 questions are grouped into eight systems: general, mucocutaneous, neurological, musculoskeletal, cardiovascular and respiratory, vasculitis, renal, and hematological. For each of the eight systems, a severity grade (A–E) is calculated based on the scores. The following list indicates interpretation of each of the grades for each system:

HSCT for Autoimmune Diseases in Children

1. 2. 3. 4. 5.

463

Disease is active enough to need treatment Disease has the potential to need treatment soon Disease currently does not meet grade A or B criteria Disease has satisfactorily resolved Disease has never occurred in this system.

The BILAG is one of the more useful instruments for characterizing disease stage because the BILAG score correlates with intention to treat. The BILAG has been validated as an instrument to measure disease activity (133).

Hematopoietic Stem-Cell Transplantation for Systemic Lupus Erythematosus in Adults A study of lymphocyte depleted autologous HSCT for patients with poor prognosis SLE was initiated in 1996 at Northwestern University (137). Forty-three patients have undergone transplant (unpublished data). The first patient was reported as a letter to the New England Journal of Medicine (137) and subsequently further results as articles in The Lancet (138) and Arthritis & Rheumatism (139). Cyclophosphamide was chosen as the immunosuppressive regimen because of its established effectiveness at conventional dosage in the treatment of lupus. All transplant candidates had to have failed monthly pulse cyclophosphamide at 500–1000 mg/m2. No patient who received a HSCT died of treatment and virtually all patients seem to markedly improve initially, although relapses have occurred, and four patients have died of relapsed disease. Relapse increases with time after autologous HSCT. In the most recent 22 patients who have undergone autologous HSCT within the last two years, 3 (15%) have relapsed. In the first 20 patients who underwent autologous HSCT from two to six years earlier, 9 (45%) have relapsed. Therefore, the estimated relapse within three to five years after autologous HSCT is 40–50%. Thus, autologous HSCT infusion after conditioning with a cyclophosphamide/ATG regimen appears capable of long-term disease control or may possibly be considered curative; the long sought goal of treatment for SLE is 50% of patients. Although ours is the largest single-center experience reporting on 43 patients with no treatment-related mortality and a 50–60% durable remission, in a report from the EBMT registry on 53 patients from 23 European centers using a variety of different conditioning regimens and different eligibility criteria with a mean follow-up of 26 months (range 0–78 months) 12 patients died, 7 from the treatment and 5 from disease (140). Disease remission (SLEDAI !3) occurred in 66%; however, 32% relapsed after only six months (range 0–40 months). Because therapeutic efficacy is due to conditioning regimen and the autologous HSC are only a supportive blood transfusion, the difference in efficacy between the European registry data and the single center Northwestern experience may be attributed to differences in conditioning regimens. Differences in toxicity may be due not only to conditioning regimen toxicity but also disease heterogeneity and differences in patient selection as well as clinical experience and supportive care (44,139). Infection may mimic active SLE. For example, infectious peritonitis is clinically similar to SLE-related serositis. CNS viral or fungal infections may be difficult to distinguish from lupus cerebritis. CMV or pneumocystis carinii pneumonia (PCP) may on radiologic and clinical examination be confused with lupus pneumonitis. SLE is prone to infectious complications due to chronic and often high-dose immune suppressive therapies as well as disease-related immune abnormalities. Low C3 and C4 complement levels predispose to bacterial infections, diseaserelated Th2 skewing increases the risk of viral and fungal infections, and a narrowed and skewed T-cell receptor (TCR) repertoire increases infectious risk. This susceptibility to infections is important in evaluation of transplant candidates because in our experience: (1) routine preadmission blood cultures have documented unsuspected bacteremia, (2) a patient with pretransplant ambulation associated oxygen desaturation and a normal chest radiograph

464

Burt et al.

had bronchoscopy documented PCP, and (3) a HSCT candidate whose seizures were attributed to lupus cerebritis was found on autopsy to have cerebral mucormycosis (44,139,141). Because fever may be moderated by high-dose steroids or attributed to lupus, SLE patients need careful pretransplant infectious screening. Documentation of active infection would be a contraindication to HSCT. Prophylactic fungal, bacterial, PCP, and viral antibiotics should be used during mobilization or HSCT induced neutropenia. Some SLE patients are unusually prone to rash or even disease flare when treated with sulfa drugs. In these patients, aerosolized pentamidine should be substituted for Bactrim as PCP prophylaxis. Fungal prophylaxis should include coverage for aspergillus and mucor with either voriconazole or liposomal amphotericin. Infections are the most common cause of death for patients with active SLE. Therefore, unless careful screening and aggressive prophylactic antibiotics are part of lupus HSCT supportive care guidelines, a high rate of transplant-related infectious deaths will occur (44,141). High-dose cyclophosphamide infusion is accompanied by aggressive intravenous hydration and forced diuresis to prevent hemorrhagic cystitis. In patients with lupus nephritis, volume overload refractory to diuresis can quickly result in pulmonary edema and mechanical ventilation. Early intervention with dialysis or ultrafiltration to maintain dry weight will prevent this complication. Alternatively, using Mesna and bladder irrigation as uroprotectants may allow less aggressive hydration. Because the metabolism of cyclophosphamide in renal insufficiency is unknown, patients with renal insufficiency should undergo dialysis every morning following the prior day’s infusion of cyclophosphamide to remove toxic cyclophosphamide metabolites. Lupus nephritis may also be accompanied during the peri-transplant period by renal tubular acidosis and, therefore, serum bicarbonate (HCO3) levels should be monitored (44). Lupus nephritis may result in refractory hypertension. It has been our experience that patients on multiple hypertensive medications who remain poorly controlled often respond to the addition of minoxidil. Because hypertension during chemotherapy-induced thrombocytopenia may increase the risk of an intracranial bleeding, platelet counts are maintained O30,000/ul by transfusion. Another manifestation of SLE is antiphospholipid antibody syndrome with a history of lower extremity thrombosis and/or pulmonary emboli. During transplant we have maintained these patients on prophylactic subcutaneous daily injection of fragmin or twice-daily lovenox without bleeding or thrombotic events, although platelets are transfused for levels below 30,000/ul (44). High-dose cyclophosphamide with or without stem cell infusion appears to be a potent salvage regimen for treatment of refractory SLE. Brodsky et al. have demonstrated clinical remission in SLE after high-dose cyclophosphamide (200 mg/kg) without HSC (142,143). This approach avoids the expense of collecting and infusing HSC that may be contaminated with disease causing lymphocytes. Conversely, advantages of HSCT include a total (combined mobilization and conditioning) cyclophosphamide dose of 255 mg/kg compared to 200 mg/kg in the Hopkins protocol. Besides a higher dose of cyclophosphamide, the Northwestern HSCT protocol includes additional immune suppression with 90 mg/kg ATG, whereas the mobilized HSC shorten the duration of neutropenia by four to five days compared to high-dose cyclophosphamide alone (139). For patients with active lupus on high dose corticosteroids, a short neutropenic interval is essential to minimize life-threatening infectious complications. Although it is unclear whether graft purging minimizes the risk of relapse, CD34C selection using either the Miltenyi Clinimacs or Baxter Isolex results in a 4-log depletion of lymphocytes in the graft.

Hematopoietic Stem-Cell Transplantation for Systemic Lupus Erythematosus in Children Experience with autologous HSCT for childhood SLE is very limited. The first two autologous HSCT for refractory childhood SLE were reported to EBMT (144). Both children were

HSCT for Autoimmune Diseases in Children

465

disease-free until one developed a coagulation disorder and thrombocytopenia, which responded to steroids (140,144). Chen et al. reported successful treatment of two children with refractory grade III and IV lupus nephropathy with autologous peripheral progenitor stem cell transplantation (145). Mobilized with G-CSF HSC were positively selected for CD34, the conditioning regimen included cyclophosphamide and ATG. At 13 and 6 months after HSCT respectively, these two children were disease free: neither original symptoms, such as thrombocytopenia, proteinuria, and pleural effusion, nor serum autoantibodies were observed. Long-term effects remain to be confirmed by further studies on more cases (145). Finally, it is important to recognize that, unlike adults, children under the age of 10 may have a lupus-like disease due to hereditary deficiency of complement components, (i.e., C3 or C4). In patients with a genetic deficiency of complement, an autologous HSCT is unlikely to be beneficial because of the unresolved continuing complement deficiency. An allogeneic HSCT from a normal HLA-matched sibling may be considered because of the potential for HSC to transdifferentiate into hepatocytes (146) and thereby synthesize the deficient complement component. This concept is still controversial, and we are unaware of any allogeneic transplant being performed for SLE in the clinical setting of an inherited complement deficiency.

TYPE I DIABETES Introduction Type I diabetes is an autoimmune disease with B-cell immunoglobulins and T-cell responses against islet cell peptides. Insulin therapy (IT) changed diabetes from an acute disease, lethal within months of onset, to a chronic disease with significant morbidity and increased mortality occurring decades after onset compared to nondiabetics (147–149). Diabetic mortality is related to hyperglycemia. For every 1% increase in hemoglobin A1C (HgbA1C), above normal, mortality increases 11%. Near normalization of blood glucose with intensive IT delays diabetic secondary complications. IIT requires close monitoring of blood glucose, frequent injections (five times a day) or continuous insulin infusion pump, reliable access to the medical health system, and is complicated by an increased incidence of hypoglycemic reactions, and even in developed countries only practiced by 20% of type I diabetics. Consequently, in America, diabetes remains the leading cause of blindness and renal failure and the sixth most common cause of death (147–151). The prevalence of type I diabetes is 0.2% (152). Forty percent of type I diabetes occurs before 14 years of age (153). Acute complications of type I diabetes include diabetic ketoacidosis (DKA) and hyperosmolar coma. Long-term morbidity and mortality results from vascular complications, which manifest as retinopathy, nephropathy, cerebrovascular disease (hemorrhage, infarcts, aneurysms), myocardial infarctions, peripheral vascular disease (claudication, ulcers, gangrene, extremity amputation), and autonomic and peripheral neuropathy. Diabetics also suffer from a higher incidence of infections, urinary tract infections, pneumonia, wound infections, osteomyelitis, and mucocutaneous and disseminated fungal infections. Immune-mediated b-islet cell destruction often becomes clinically manifest with onset of DKA, but islet cell loss is not complete until weeks to months after onset of DKA. Measurement of C-peptide, a marker for endogenous insulin production, indicates persistence of islet cells and low normal C-peptide levels for up to one year after onset of DKA. Immune suppression with cyclosporine and/or azathioprine if started within eight weeks of DKA delays onset of insulin dependence. A study of anti-CD3 antibody infused for 14 days within six weeks of DKA onset demonstrated improved endogenous insulin production, reduced HgbA1C, and reduced endogenous insulin requirements for up to one year after treatment (154–159).

466

Burt et al.

Hematopoietic Stem-Cell Transplantation Protocols for Type I Diabetes Mellitus Immune suppressive trials and the higher mortality of diabetes in third world countries compared to the United States have provided the rationale to attempt immune ablation and autologous HSCT within six weeks of DKA onset in an attempt to preserve remaining b-islet cells (160). A protocol using autologous HSCT for new onset type I diabetes is currently ongoing in Brazil (verbal communication—Voltarelli J., Brazil). HSC are mobilized with cyclophosphamide 2.0 g/m2 and G-CSF. The transplant conditioning regimen is 200 mg/kg cyclophosphamide and 6.5 mg/kg rabbit ATG (rATG) without HSC enrichment or manipulation. This regimen does not cause infertility in pediatric patients (161), is not associated with an increase in malignancies, and has a track record of safety in other autoimmune diseases. Corticosteroids induce apoptosis of b-islet cells. Because HSCT is designed to salvage islet cells, the use of steroids during transplant should be minimized. However, it is common to premedicate patients with high-dose steroids prior to rATG to prevent ATG-related febrile reactions. In patients with new onset diabetes this practice should be avoided. Using a lower first dose of rATG (0.25 mg/kg) helps minimize ATG-related cytokine release independent of corticosteroid pretreatment (160,162). Suggested eligibility criteria for HSCT include age less than 26, treatment within six weeks of diagnosis, and financial, social, or economic inability to comply with IT or maintain long-term close and frequent access to medical facilities required for IT. Study endpoints should include insulin requirements (units/kg/day), glycosylated hemoglobin (HgbA1C), fasting C-peptide levels, antiglutamic acid decarboxylase (GAD) antibodies, and T-cell GAD peptide responses (160).

JUVENILE DERMATOMYOSITIS Introduction JDM is the most common form of idiopathic myositis that by definition has its onset prior to 18 years of age. The mean age of onset is 6.7 years old, with 25% of cases occurring before four years of age. JDM has an incidence of 3.0 new cases/million children/year (163–166). The typical JDM rash involves lilac discoloration of the eyelids (heliotrope rash), metacarpal pharyngeal joints (Gottron’s papules), the malar surface, knees, and/or elbows. Classical diagnostic criteria by Bohan and Peter (1975) include the typical rash and any three of the following: (1) symmetrical proximal weakness of limb girdle and anterior neck flexors, (2) histopathologic evidence of muscle inflammation, (3) elevated muscle enzymes (aldolase, creatinine phosphokinase, alanine or aspartate aminotransferase, lactate dehydrogenase), and (4) electromyographic changes of myositis (small polyphasic motor units, spontaneous fibrillation, insertional irritability) (163,164,167,168). Recently, MRI has replaced muscle biopsies (169). JDM may have distinct clinical courses, monocyclic, polycyclic, chronic unremitting, and ulcerative. JDM is an inflammatory vasculitis with T and B cells in a perivascular distribution with small vessel thrombosis (163,164,167). Myositis-specific autoantibodies (MSA) have been documented only in patients with myositis and not with other disorders. Myositis-associated antibodies (MAA) are present in myositis as well as other connective tissue diseases. MSA and/or MAA are found in only 20% of JDM. Myositis-specific antibodies include anti–aminoacyl-tRNA synthetases (Jo1, PL-12, Pl-7), anti–signal recognition particle (SNP), anti-Mi-2. Jo1 (histidyl-tRNA synthetase), PL-7 (threonyl-tRNA synthetase, PL-12 (alanyl-tRNA synthetase) are associated with more severe clinical manifestations, including unremitting myositis, interstitial lung disease, fever, and Raynaud’s phenomena. SRP autoantibodies are associated with severe polymyositis. In contrast, anti-Mi-2 antibodies are generally associated with a good prognosis (170–172). Prior to the introduction of corticosteroid therapy one-third of children died, one-third developed crippling calcifications, and one-third survived without severe long-term

HSCT for Autoimmune Diseases in Children

467

complications (173). Current JDM mortality is 3%. However, significant long-term morbidity from weakness, contractures, functional disability, growth retardation, myocarditis, myocardial infarctions, dysphagia, or dysphonia remains a problem in 30% of patients. Recurrent or unremitting courses are predictive for calcinosis. There is little data on long-term outcome in adults who suffered JDM as children. Most follow-up studies cover durations of only 5–7 years. Because JDM is a vasculopathy with calcinosis and is treated with corticosteroids, it is likely that adult survivors of JDM have a higher risk for mortality from complications of atherosclerosis (163,164,167,173,174).

Hematopoietic Stem-Cell Transplantation for Idiopathic Inflammatory Myositis Three cases of autologous transplant for polymyositis have been reported in Europe (175–178). A 28-year-old female with Jo-1 positive polymyositis and severe lung involvement (after failure of high dose steroids, azathioprine, MTX, and cyclophosphamide) underwent high-dose chemotherapy (busulfan, cyclophosphamide and ATG) and peripheral blood selected stem cell transplantation. At 15 months after HSCT the patient had significantly improved, including her pulmonary function, findings on CT scan, and muscle strength, in spite of discontinuation of all immunosuppressive medications (175). Another case report of rapidly progressive Jo-1-positive polymyositis described early improvement after nonmyeloablative HSCT and recurrence on day 21 post transplant (176). Chakraverty et al. reported the case of a 46-year old male who developed chemotherapy resistant dermatomyositis and sarcoid-like reaction in association with testicular relapse of multiple myeloma. Dramatic and sustained response (follow-up 18 months) was observed after high dose chemotherapy (melphalan 200 mg/m2) and PBSCT (177).

Hematopoietic Stem-Cell Transplantation Approach for Juvenile Dermatomyositis To date, there is one unpublished case of JDM treated with autologous HSCT (verbal communication—Wedderburn LR, Great Ormond Street Hospital, London). The disease stabilized in this girl, but she still suffers from severe preexisting muscle damage. This case illustrates that such transplants should be performed as early in the disease course as possible. Eligible candidates for autologous HSCT should have either recurrent relapses or unremitting disease with high disease activity scores despite corticosteroids and either MTX, azathioprine, or cyclosporine. Because JDM may involve the heart with arrhythmias, myocarditis, pericardial effusions, or congestive heart failure, the regimen should minimize further cardiac stress. A possible regimen is fludarabine (150 mg/m2), cyclophosphamide (dose decreased to 120 mg/kg) and CAMPATH-1H. To further minimize cardiovascular stress, mesna and bladder irrigation should be substituted for hyperhydration.

IMMUNOLOGIC MECHANISMS OF HEMATOPOIETIC STEM-CELL TRANSPLANTATION If HSCT is only transient immune suppression, the same immune phenotype and repertoire might be preserved, although immune cell numbers will be diminished. On the other hand, if HSCT results in a new immune system (i.e., “immune reset”), the posttransplant immune system should be characterized by an increase in phenotypically naı¨ve lymphocytes, an increase in recent thymic emigrants, and differences in T- and B-cell repertoire distribution compared to pre-HSCT. Recent production of naı¨ve lymphocytes from the thymus may be determined by TCR excision circles and/or by coexpression of phenotypic markers, such as CD27 and CD45RA (or lack of CD45RO). Indeed this was shown recently by the occurrence

468

Burt et al.

after HSCT of CD4/CD25 bright regulatory T cells with a suppressor phenotype (48). The composition of TCR repertoires of CD4C and CD8CT-cell subsets can be determined before and after HSCT by flow cytometry using a panel of TCRVb-specific monoclonal antibodies and/or TCR CDR3 spectratyping analysis (178). Autologous HSCT for one autoimmune disease (MS) is consistent with a post-HSCT “immune reset” (verbal communication—Muraro P, NIH, DC). These data indicate that the mechanism of action of HSCT includes immune rejuvenation or regeneration and is not a result of just transient immune suppression.

ALLOGENEIC HEMATOPOIETIC STEM-CELL TRANSPLANTATION The objective of allogeneic HSCT in autoimmune diseases is to change the genetic susceptibility towards disease by inducing donor-recipient mixed chimerism (coexisting recipient and donor hemato-immunopoiesis). In animal models of type I diabetes, mixed chimerism results in prolonged disease remission by replacing autoimmune susceptible hematopoietic-derived immune cells with autoimmune resistant donor cells (14,21). Mixed chimerism in patients with malignancies is associated with disease relapse. In contrast, in the context of autoimmune diseases, allogeneic nonmyeloablative (NST) trials that are designed to achieve mixed chimerism without GVHD may be capable of durable disease remission (179). In a patient with RA, allogeneic NST from an HLA-matched sibling has resulted in mixed chimerism without GVHD (180). At 14 months post NST, the patient is a stable mixed chimera [both donor and host myeloid (CD33) and T (CD3) cells], in complete remission, and rheumatoid factor negative. The patient is off all immune suppressive or modulating therapy and has had no GVHD or infections (180). Although this result is encouraging, more experience with mixed chimerism in autoimmune diseases is needed to confirm that mixed chimerism may confer “graft versus autoimmunity” without GVHD. Allogeneic NST regimens for autoimmune diseases need to be designed to prevent GVHD either by ex vivo CD34C enrichment (i.e., lymphocyte depletion) of the donor graft or in vivo depletion of antigen presenting cells (T- and B-lymphocytes and dendritic cells) with CAMPATH-1H. Current allogeneic NST trials for autoimmune disease involve HLA-matched sibling donors because complications are more severe with HLA-mismatched or unrelated donor transplants. Despite numerous genetic differences, an HLA-identical donor will confer at least one similar recipient genetic risk factor, that is MHC phenotype. In addition, limiting NST to only those patients with the availability of a healthy HLA-matched sibling would limit the number of potential donors. In such instances, an alternative stem cell source, such as embryonic stem cells (ESC), could be a promising advance. ESC lines are derived from the inner cell mass of the blastocyst and are totipotent and immortal. ESC can be expanded ex vivo as undifferentiated cells that retain a normal karyotype or, alternatively, can be differentiated ex vivo into cell types of all three germ layers. We have demonstrated that murine ESC induced to differentiate towards HSC by removal of leukemia inhibitory factor and addition of SCF, interleukin-3, and interleukin-6 reconstitute hematopoiesis in lethally irradiated MHC mismatched mice. This occurs without clinical or histological evidence of GVHD with bidirectional host/donor tolerance and intact 3rd party immune responses (181). If human ESCs have the same potential, then a renewable wellidentified source of allogeneic HSCs capable of reconstituting a normal immune system without GVHD and without genetic predisposition towards autoimmune disease would be available to treat autoimmune disorders.

REFERENCES 1. Burt RK, Burns W, Hess A. Bone marrow transplantation for multiple sclerosis. Bone Marrow Transplant 1995; 16:1–6.

HSCT for Autoimmune Diseases in Children

469

2. Marmont AM. Immune ablation with stem-cell rescue: a possible cure for systemic lupus erythematosus? Lupus 1993; 2:151–156. 3. Kashyap A, Passweg J, Tyndall A. Autologous stem cell transplant regimens for the treatment of severe autoimmune diseases. Autologous Blood and Marrow Transplantation: Proceedings of the Tenth International Symposium Dallas, Texas. Charlottesville, VA: Garden Jennings Publishing, 2001 pp. 219–225. 4. Burt RK, Slavin S, Burns W, Marmont A. Induction of tolerance in autoimmune diseases by hematopoietic stem cell transplantation: getting closer to cure? Blood 2002; 99:768–784. 5. Marmont AM. Historical perspectives and rationale of HSCT for autoimmune diseases. In: Burt RK, Marmont AM, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:223–231. 6. Ikehara S, Good RA, Nakamura T, et al. Rationale for bone marrow transplantation in the treatment of autoimmune diseases. Proc Natl Acad Sci USA 1985; 82:2483–2487. 7. Makino S, Kunimoto K, Muraoka Y, et al. Breeding of a non-obese, diabetic strain of mice. Exp Anim 1980; 29:1–13. 8. Breban M. Transfer of inflammatory disease of HLA-B27 transgenic rats by bone marrow engraftment. J Exp Med 1993; 178:1607–1616. 9. Kushida T, Inaba M, Takeuchi K, et al. Treatment of intractable autoimmune diseases in MRL/lpr mice using a new strategy for allogeneic bone marrow transplantation. Blood 2000; 95:1862–1868. 10. Karussis DM. Immunomodulation of autoimmunity in MRL/lpr mice with syngeneic bone marrow transplantation (sBMT). Clin Exp Immunol 1995; 100:111–117. 11. Akizuki M, Reeves JP, Steinberg AD. Expression of autoimmunity by NZB/NZW marrow. Clin Immunol Immunopathol 1978; 10:247–250. 12. Jyonouchi H, Kincade PW, Good RA, et al. Reciprocal transfer of abnormalities in clonable B-lymphocytes and myeloid progenitors between NZB and DBA/2 mice. J Immunol 1981; 127:1232–1238. 13. Eisenberg RA, Izui S, McConahey PJ, et al. Male determined accelerated autoimmune disease in BXSB mice: transfer by bone marrow and spleen cells. J Immunol 1980; 125:1032–1036. 14. Ikehara S, Ohtsuki H, Good RA, et al. Prevention of type I diabetes in nonobese diabetic mice by allogeneic bone marrow transplantation. Proc Natl Acad Sci USA 1985; 82:7743–7747. 15. van Gelder M, Mulder AH, van Bekkum DW. Treatment of relapsing experimental autoimmune encephalomyelitis with largely MHC-mismatched allogeneic bone marrow transplantation. Transplant 1996; 62:810–818. 16. Adachi Y, Inaba M, Amoh Y, et al. Effect of bone marrow transplantation on anti-phospholipid antibody syndrome in murine lupus mice. Immunobiology 1995; 192:218–230. 17. Kono DH, Theophilopoulos AN. The genetics of murine systemic lupus erythematosus. In: Wallace DJ, Hahn BH, eds. Dubois’ Lupus Erythematosus. Philadelphia: Lippincott Williams & Wilkins, 2002:97–120. 18. Encinas JA, Kuchroo VK. Mapping and autoimmunity genes. Autoimmunity 2001; 12:91–97. 19. Morton JI, Siegel BV. Transplantation of autoimmune potential. Reversal of the NZB autoimmune syndrome by bone marrow transplantation. Transplant 1979; 27:133–134. 20. van Bekkum DW. Autologous stem cell transplantation in animal models of autoimmune diseases. In: Burt RK, Marmont AM, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, Texas: Landes Bioscience, 2004:237–244. 21. Ikehara S. Allogeneic hematopoietic stem cell transplantation in animal models of autoimmune disease. In: Burt RK, Marmont AM, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:245–252. 22. Burt RK, Padilla J, Begolka WS, Dal Canto MC, Miller SD. Effect of disease stage on clinical outcome after syngeneic bone marrow transplantation for relapsing experimental autoimmune encephalomyelitis. Blood 1998; 91:2609–2616. 23. Karussis DM, Slavin S, Lehmann D, Mizrachi-Koll R, Adamsky O, Ben-Nun A. Prevention of experimental autoimmune encephalomyelitis and induction of tolerance with acute immunosuppression followed by syngeneic bone marrow transplantation. J Immunol 1992; 148:1693–1698. 24. van Gelder M, van Bekkum DW. Effective treatment of relapsing experimental autoimmune encephalomyelitis with pseudoautologous bone marrow transplantation. Bone Marrow Transplant 1996; 18:1029–1034. 25. Karussis DM, Vourka-Karussis U, Lehmann D, et al. Prevention and reversal of adoptively transferred, chronic relapsing experimental autoimmune encephalomyelitis with a single high dose

470

26.

27.

28. 29.

30. 31. 32.

33.

34. 35. 36. 37. 38. 39.

40. 41. 42.

43.

44. 45. 46.

47.

Burt et al. cytoreductive treatment followed by syngeneic bone marrow transplantation. J Clin Invest 1993; 92:765–772. Mancardi GL, Saccardi R, Murialdo A, et al. Intense immunosuppression followed by autologous stem cell transplantation in severe multiple sclerosis cases: MRI and clinical data. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:302–307. Croxford JL, Miller SD. Evidence for a role of infections in the activation of autoreactive T cells and the pathogenesis of autoimmunity. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:182–193. Miller SD, Vanderlugt CL, Begolka WS, et al. Persistent infection with Theiler’s virus leads to CNS autoimmunity via epitope spreading. Nat Med 1997; 3:1133–1136. Burt RK, Padilla J, Dal Canto MC, Miller SD. Viral hyperinfection of the central nervous system and high mortality after hematopoietic stem cell transplantation for treatment of Theiler’s murine encephalomyelitis virus-induced demyelinating disease. Blood 1999; 94:2915–2922. Pestronk A. Combined short-term immunotherapy for experimental autoimmune myasthenia gravis. Ann Neurol 1983; 14:235–241. van Bekkum DW. Regression of adjuvant-induced arthritis in rats following bone marrow transplantation. Proc Natl Acad Sci USA 1989; 86:10090–10094. van Gelder M, Mulder AH, van Bekkum DW. Treatment of relapsing experimental autoimmune encephalomyelitis with largely MHC-mismatched allogeneic bone marrow transplantation. Transplant 1996; 62:810–818. The Finnish-German APECED Consortium. An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc-finger domains. Nat Genet 1997; 17:393–398. Straus SE, Sneller M, Lenardo MJ, et al. An inherited disorder of lymphocyte apoptosis: the autoimmune lymphoproliferative syndrome. Ann Intern Med 1999; 130:591–601. Theofilopoulos AN, ed. Genes and Genetics of Autoimmunity. In: Curr Dir Autoimmunity. Basel: Karger, 1999:1–296. Burt RK, Fassas A, Snowden J, et al. Collection of hematopoietic stem cells from patients with autoimmune diseases. Bone Marrow Transplant 2001; 28:1–12. Zavala F, Abad S, Ezine S, et al. G-CSF therapy of ongoing experimental allergic encephalomyelitis via chemokine- and cytokine-based immune deviation. J Immunol 2002; 168:2011–2019. Lock C, Hermans G, Pedotti R, et al. Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat Med 2002; 8:500–508. Verda L, Luo K, Kim DA, et al. Effect of colony stimulating factors on relapse and severity of experimental autoimmune encephalomyelitis: a model for human autoimmune diseases. Bone Marrow Transplant 2006; Submitted. Vaughan D, Drumm B. Treatment of fistulas with granulocyte colony-stimulating factor in a patient with Crohn’s disease. N Engl J Med 1999; 340:239–240. Openshaw H, Stuve O, Antel JP, et al. Multiple sclerosis flares associated with recombinant granulocyte colony-stimulating factor. Neurology 2000; 54:2147–2150. Carrier E, Burt RK. Mobilization and conditioning regimens in stem cell transplant for autoimmune diseases. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:253–261. Moore JJ, Brooks P, Milliken ST, et al. A pilot randomised trial comparing CD34 selected versus unmanipulated haemopoietic stem cell transplantation for severe resistant rheumatoid arthritis. Arthritis Rheum 2002; 46:2301–2309. Burt RK, Verda L, Statkute L, Oyama Y. Unique aspects of hematopoietic stem cell transplantation for autoimmune diseases. Curr Opin Organ Transplant 2004; 9:49–53. Monje ML, Mizumatsu S, Fike JR, et al. Irradiation induces neural precursor-cell dysfunction. Nat Med 2002; 8:955–962. Muraro PA, McFarland HF, Martin R. Immunological aspects of multiple sclerosis with emphasis on the potential use of autologous hemopoietic stem cell transplantation. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:277–283. De Kleer IM, Brinkman D, Fester A, et al. Autologous stem cell transplantation for refractory juvenile idiopathic arthritis: analysis of mortality and transplant related morbidity. Ann Rheum Dis 2004; 63:1318–1326.

HSCT for Autoimmune Diseases in Children

471

48. de Kleer IM, Vastert B, Klein M, et al. Autologous stem cell transplantation for autoimmunity induces immunologic self-tolerance by reprogramming autoreactive T-cells and restoring the CD4CCD25C immune regulatory network. Blood 2006; 107:1696–1702. 49. Wulffraat NM, van Royen A, Bierings M. Autologous haemopoietic stem cell transplantation in four patients with refractory juvenile chronic arthritis. Lancet 1999; 353:550–553. 50. Wulffraat N. Autologous stem cell transplantation for refractory juvenile idiopathic arthritis (JIA). In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:378–387. 51. Wulffraat NM, Brinkman D, Ferster A, et al. Long-term follow-up of autologous stem cell transplantation for refractory juvenile idiopathic arthritis. Bone Marrow Transplant 2003; 32:S61–S64. 52. Ansell BM, Wood PHN. Prognosis in juvenile chronic arthritis. Clin Rheum Dis 1976; 2:397. 53. Gewanter HL, Roghmann KJ, Baum J. The prevalence of juvenile arthritis. Arthritis Rheum 1983; 26:599–603. 54. Baum J, Gutowska G. Death in juvenile rheumatoid arthritis. Arthritis Rheum 1977; 20:253. 55. Wulffraat NM, Kuis W, Petty R. Addendum: proposed guidelines for autologous stem cell transplantation in juvenile chronic arthritis. Paediatric rheumatology workshop. Rheumatology 1999; 38:777–778. 56. Giannini EH, Ruperto N, Ravelli A, et al. Preliminary definition of improvement in juvenile arthritis. Arthritis Rheum 1997; 40:1202–1209. 57. Ruperto N, Ravelli A, Falcini F, et al. Performance of the preliminary definition of improvement in juvenile chronic arthritis patients treated with methotrexate. Ann Rheum Dis 1998; 57:38–41. 58. Fuchs HA, Pincus T. Reduced joint counts in controlled clinical trials in rheumatoid arthritis. Arthritis Rheum 1994; 37:470–475. 59. Len C, Ferraz MB, Goldenberg J, et al. Paediatric escola paulista de medicina rabge of motion scale: a reduced joint count scale for general use in juvenile rheumatoid arthritis. J Rheumatol 1999; 26:909–913. 60. Wulffraat M, de Kleer I, Brinkman D, et al. Autologous stem cell transplantation for refractory juvenile idiopathic arthritis: current results and perspectives. Transplant Proc 2002; 34:2925–2926. 61. Brinkman DM, ten Cate R, Vossen JM, et al. Decrease in synovial cellularity and cytokine expression after autologous stem cell transplantation in patients with juvenile idiopathic arthritis. Arthritis Rheum 2002; 46:1121–1123. 62. Wulffraat NM, Rijkers GT, Elst E, et al. Reduced perforin expression in systemic juvenile idiopathic arthritis is restored by autologous stem-cell transplantation. Rheumatology 2003; 42:375–379. 63. ten Cate R, Brinkman DM, van Rossum MA, et al. Macrophage activation syndrome after autologous stem cell transplantation for systemic juvenile idiopathic arthritis. Eur J Ped 2002; 161:685–686. 64. Quartier P, Prieur AM, Fischer A. Disseminated toxoplasmosis following autologous bone marrow transplantation for systemic onset juvenile chronic arthritis. Lancet 1999; 353:1885–1886. 65. Ramanan AV, Baildam EM. Macrophage activation syndrome is hemophagocytic lymphohistiocytosis—need for the right terminology. J Rheumatol 2002; 29:1105. 66. Arico M, Danesino C, Pende D, et al. Pathogenesis of haemophagocytic lymphohistiocytosis. Br J Haematol 2001; 111:761–769. 67. Goransdotter EK, Fadeel B, Nilsson-Ardnor S, et al. Spectrum of perforin gene mutations in familial hemophagocytic lymphohistiocytosis. Am J Hum Genet 2001; 68:590–597. 68. Craig RM, Burt RK. Autologous hematopoietic stem cell transplantation for Crohn’s disease. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Landes Bioscience: Georgetown, TX, 2004:448–456. 69. Farmer FG, Hawk WA, Turnbull RB. Clinical patterns in Crohn’s disease: a statistical study of 615 cases. Gastroenterology 1975; 68:627–639. 70. Ogorek CP, Caraoline DF, Fisher RS. Presentation, evaluation and natural history of inflammatory bowel disease. In: MacDermott RP, Stenson WF, eds. Inflammatory Bowel Disease. New York: Elsevier, 1992:355–386. 71. Hyams JS. Crohn’s disease in children. Pediatr Clin North Am 1996; 43:255–277. 72. Persson PG, Bernell O, Leijonmarck CE, Farahmand BY, Hellers G, Ahlbom A. Survival and cause-specific mortality in inflammatory bowel disease: a population-based cohort study. Gastroenterology 1996; 110:1339–1345.

472

Burt et al.

73. Jess T, Wnither KV, Munkholm P, Langholz E, Binder V. Mortality and causes of death in Crohn’s disease: follow-up of a population-based cohort in Copenhagen county, Denmark. Gastroenterology 2002; 122:1808–1814. 74. Loftus EV, Silverstein MD, Sandborn WJ, et al. Crohn’s disease in Olmsted county, Minnesota, 1940–1993: incidence, prevalence, and survival. Gastroenterology 1998; 114:1161–1168. 75. Otley A, Smith C, Nicholas D, et al. The IMPACT questionnaire: a valid measure of health-related quality of life in pediatric inflammatory bowel disease. J Pediatr Gastroenterol Nutr 2002; 35:557–563. 76. Richardson G, Griffiths AM, Miller V, Thomas AG. Quality of life in inflammatory bowel disease: a cross-cultural comparison of English and Canadian children. J Pediatr Gastroenterol Nutr 2001; 32:573–578. 77. Griffiths AM, Nicholas D, Smith C, et al. Development of a quality-of-life index for pediatric inflammatory bowel disease: dealing with differences related to age and IBD type. J Pediatr Gastroenterol Nutr 1999; 28:S46–S52. 78. MacPhee M, Hoffenberg EJ, Feranchak A. Quality-of-life factors in adolescent inflammatory bowel disease. Inflamm Bowel Dis 1998; 4:6–11. 79. Hildebrand H, Karlberg J, Kristiansson B. Longitudinal growth in children and adolescents with inflammatory bowel disease. J Pediatr Gastroenterol Nutr 1994; 18:165–173. 80. Hyams JS. Extraintestinal manifestations of inflammatory bowel disease in children. J Pediatr Gastroenterol Nutr 1994; 19:7–21. 81. Kirschner BS. Permanent growth failure in pediatric inflammatory bowel disease. J Pediatr Gastroenterol Nutr 1993; 16:368–369. 82. Markowitz J, Grancher K, Rosa J, Aiges H, Daum F. Growth failure in pediatric inflammatory bowel disease. J Pediatr Gastroenterol Nutr 1993; 16:373–380. 83. Homer DR, Grand RJ, Colodny AH. Growth, course, and prognosis after surgery for Crohn’s disease in children and adolescents. Pediatrics 1977; 59:717–725. 84. Kirschner BS, Sutton MM. Somatomedin-C levels in growth-impaired children and adolescents with chronic inflammatory bowel disease. Gastroenterology 1986; 91:830–836. 85. Murch SH, Lamkin VA, Savage MO, Walker-Smith JA, MacDonald TT. Serum concentrations of tumour necrosis factor alpha in childhood chronic inflammatory bowel disease. Gut 1991; 32:913–917. 86. Breese EJ, Michie CA, Nicholls SW, et al. Tumor necrosis factor alpha-producing cells in the intestinal mucosa of children with inflammatory bowel disease. Gastroenterology 1994; 106:1455–1466. 87. Whittington PF, Barnes HV, Bayless TM. Medical management of Crohn’s disease in adolescence. Gastroenterology 1977; 72:1338–1344. 88. Kuhn R, Lohler J, Rennick D, et al. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 1993; 75:263–274. 89. Ehrhardt RO, Ludviksson B. Induction of colitis in IL2-deficient-mice: the role of thymic and peripheral dysregulation in the generation of autoreactive T-cells. Res Immunol 1997; 148:582–588. 90. Kulkarni AB, Ward JM, Yaswen L, et al. Transforming growth factor-beta 1 null mice. An animal model for inflammatory disorders. Am J Pathol 1995; 146:264–275. 91. Rao SS, Cann PA, Holdsworth CD. Clinical experience of the tolerance of mesalazine and olsalazine in patients intolerant of sulfasalazine. Scand J Gastroenterol 1987; 22:332–336. 92. Bernstein LH, Frank MS, Brant LJ, et al. Healing of perianal Crohn’s disease with metronidazole. Gastroenterology 1980; 79:357–365. 93. Present DH, Korelitz BI, Wisch N, et al. Treatment of Crohn’s disease with 6-mercaptopurine. A long-term, randomized, double-blinded study. N Engl J Med 1980; 302:981–987. 94. Markowitz J, Grancher K, Kohn N, et al. A multicenter trial of 6-mercaptopurine and prednisone in children with newly diagnosed Crohn’s disease. Gastroenterology 2000; 119:895–902. 95. Willoughby JM, Becket J, Kumar PJ, et al. Controlled trial of azathioprine in Crohn’s disease. Lancet 1971; 2:944–947. 96. Kozarek RA, Patterson DJ, Gelfand MD, et al. Methotrexate induces clinical and histologic remission in patients with refractory inflammatory bowel disease. Ann Intern Med 1989; 110:353–356. 97. Brynshov J, Freund L, Rasmussen SN, et al. A placebo-controlled, double-blinded randomized trial of cyclosporine therapy in active chronic Crohn’s disease. N Engl J Med 1989; 321:845–850.

HSCT for Autoimmune Diseases in Children

473

98. Stallmach A, Wittig BM, Moser C, Fischinger J, Duchmann R, Zeitz M. Safety and efficacy of intravenous pulse cyclophosphamide in acute steroid refractory inflammatory bowel disease. Gut 2003; 52:377–382. 99. Present DP, Rutgeerts P, Targan S, et al. Infliximab for the treatment of fistulas in patients with Crohn’s disease. N Engl J Med 1999; 340:1398–1405. 100. Irvine EJ, Feagen B, Rochon J, et al. Quality of life: a valid and reliable measure of therapeutic efficacy in the treatment of inflammatory bowel disease. Gastroenterology 1994; 106:287–296. 101. Best WR, Becktel JM, Singleton JW, et al. Development of a Crohn’s disease activity index. National Cooperative Crohn’s Disease Study. Gastroenterology 1976; 70:843–850. 102. Lopez-Cubero SO, Sullivan KM, McDonald GB. Course of Crohn’s disease after allogeneic marrow transplantation. Gastroenterology 1998; 114:433–440. 103. Talbot DC, Montes A, Teh WL, Nandi A, Powles RL. Remission of Crohn’s disease following allogeneic bone marrow transplant for acute leukaemia. Hosp Med 1998; 59:580–581. 104. Kashyap A, Foreman SJ. Autologous bone marrow transplantation for non-Hodgkin’s lymphoma resulting in long term remission of coincidental Crohn’s disease. Br J Haematol 1998; 103:651–652. 105. Castro J, Benich HI, Smith HL. Prolonged clinical remission in patients with inflammatory bowel disease after high dose chemotherapy and autologous bone marrow stem cell transplantation. Blood 1996; 88:133A. 106. Burt RK, Traynor A, Oyama Y, Craig R. High dose immune suppression and autologous hematopoietic stem cell transplantation in refractory Crohn’s disease. Blood 2003; 101:2064–2066. 107. Oyama Y, Craig RM, Traynor AE, et al. Autologous hematopoietic stem cell transplantation in patients with refractory Crohn’s disease. Gastroenterology 2005; 128:552–563. 108. Rothfield N. Clinical features of systemic lupus eythematosus. In: Kelley WN, Harris ED, Ruddy S et al. eds. Textbook of Rheumatology. Philadelphia: W.B. Saunders, 1981. 109. Suh AY, Rosa RM. Lupus nephritis. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Landes Bioscience: Georgetown, TX, 2004:339–346. 110. Traynor AE, Burt RK, Marmont A. Hematopoietic stem cell transplantation for systemic lupus erythematosus. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:347–357. 111. Lahita RG. The role of sex hormones in systemic lupus erythematosus. Curr Opin Rheumatol 1999; 11:352–356. 112. Klein-Gitelman M, Reiff A, Silverman ED. Systemic lupus erythematosus in childhood. Rheum Dis Clin North Am 2002; 28:561–577. 113. Esdaile JM. Prognosis in systemic lupus erythematosus. Springer Semin Immunopathol 1994; 16:337–355. 114. Ginzler EM, Schorn K. Outcome and prognosis in SLE. Rheum Dis Clin North Am 1988; 14:67–68. 115. Ward MM, Pyun E, Studenski S. Long-term survival in SLE: patient characteristics associated with poorer outcomes. Arthritis Rheum 1995; 38:274–283. 116. Jacobsen S, Petersen J, Ullman S, et al. A multicentre study of 513 Danish patients with SLE II. Disease mortality and clinical factors of prognostic values. Clin Rheumatol 1998; 17:478–484. 117. Tucker LB, Menon S, Schaller JG, et al. Adult- and childhood-onset systemic lupus erythematosus: a comparison of onset, clinical features, serology, and outcome. Br J Rheumatol 1995; 34:866–872. 118. Marini R, Constallat LT. Young age at onset, renal involvement and arterial hypertension are of adverse prognostic significance in juvenile systemic lupus erythematosus. Rev Rheum Engl Ed 1999; 66:303–309. 119. Emre S, Bilge I, Sirin A, et al. Lupus nephritis in children: prognostic significance of clinicopathological findings. Nephron 2001; 87:118–126. 120. Lee T, von Scheven E, Sandborg C. Systemic lupus erythematosus and antiphospholipid syndrome in children and adolescents. Curr Opin Rheumatol 2001; 13:415–421. 121. Tsokos GC, Juang YT, Tsokos CG, et al. Molecular and cellular pathogenesis of systemic lupus erythematosus. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:320–327. 122. Tsao BP. The genetics of human lupus. In: Wallace DJ, Hahn BH, eds. Dubois Lupus Erythematosus. Philadelphia: Lippincott Williams & Wilkins, 2002:97–120. 123. Nepom GT, Erlich H. MHC class-II molecules and autoimmunity. Ann Rev Immunol 1991; 9:493–525. 124. Herrman M, Winkler T, Gaipl U, et al. Etiopathogenesis of systemic lupus erythematosus. Int Arch Allergy Immunol 2000; 123:28–35.

474

Burt et al.

125. Deapen D, Escalante A, Weinrib L, et al. A revised estimate of twin concordance in systemic lupus erythematosus. Arthritis Rheum 1992; 35:311–318. 126. Strand V. New therapies for systemic lupus erythematosus. Rheum Dis Clin North Am 2000; 26:389–405. 127. Schur PH, Kammer GA. Treatment of systemic lupus erythematosus. In: Tsokos GC, ed. Modern Therapeutics in Rheumatic Diseases. Totowa, NJ: Humana Press, 2002:259–295. 128. Badsha H, Edwards CJ. Intravenous pulses of methylprednisolone for systemic lupus erythematosus. Semin Arthritis Rheum 2003; 32:370–377. 129. Steinberg AD. The treatment of lupus nephritis. Kidney Int 1986; 30:769–787. 130. Feutren G, Querin S, Noel LH, et al. Effects of cyclosporine in severe systemic lupus erythematosus. J Pediatr 1987; 111:1063–1068. 131. Chan TM, Li FK, Tang CS, et al. Efficacy of mycophenolate mofetil in patients with diffuse proliferative lupus nephritis. N Engl J Med 2000; 343:1156–1162. 132. Carreno L, Lopez-Longo FJ, Gonzalez CM, Monteagudo I. Treatment options for juvenile-onset systemic lupus erythematosus. Paediatr Drugs 2002; 4:241–256. 133. Grossman JM, Kalunian KC. Definition, classification, activity and damage indices in systemic lupus erythematosus. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:328–338. 134. Symmons DPM, Coopock JS, Bacon PA, et al. Development and assessment of a computerized index of clinical disease activity in systemic lupus erythematosus. Q J Med 1988; 68:927–937. 135. Bombardier C, Gladman DD, Urowitz MB, et al. Derivation of the SLEDAI. A disease activity index for lupus patients. The Committee of Prognosis Studies in SLE. Arthritis Rheum 1992; 35:630–640. 136. Liang MH, Socher SA, Roberts WN, et al. Measurement of systemic lupus erythematosus activity in clinical research. Arthritis Rheum 1988; 31:817–825. 137. Burt RK, Traynor AE, Ramsey-Goldman R. Hematopoietic stem-cell transplantation for systemic lupus erythematosus. N Engl J Med 1997; 337:1777–1778. 138. Traynor AE, Schroeder J, Rosa RM, et al. Treatment of severe systemic lupus erythematosus with high-dose chemotherapy and haemopoietic stem-cell transplantation: a phase I study. Lancet 2000; 356:701–707. 139. Traynor AE, Barr WG, Rosa RM, et al. Hematopoietic stem cell transplantation for severe and refractory lupus. Analysis after five years and fifteen patients. Arthritis Rheum 2002; 46:2917–2923. 140. Jayne D, Passweg J, Marmont A, for European Group for Blood and Marrow Transplantation and European League against Rheumatism, et al. Autologous stem cell transplantation for systemic lupus erythematosus. Lupus 2004; 13:168–176. 141. Stosor V, Zembower TR. Infection in the hematopoietic stem cell transplant recipient with autoimmune disease. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:263–276. 142. Petri M, Jones RJ, Brodsky RA. High dose cyclophosphamide without stem cell transplantation in systemic lupus erythematosus. Arthritis Rheum 2003; 48:166–173. 143. Brodsky RA. High-dose immune suppression without hematopoietic stem cells for autoimmune diseases. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:232–236. 144. Wulffraat NM, Sanders EAM, Lilien M, et al. Prolonged treatment free remission after autologous stem cell transplantation (ASCT) for refractory childhood systemic lupus erythematosus (SLE). Arthritis Rheum 2001; 44:728–731. 145. Chen J, Gu LJ, Zhao HJ, et al. Application of CD34C autologous peripheral progenitor cell transplant in the treatment of children with refractory SLE. Zhonghua Er Ke Za Zhi 2003; 41:426–429. 146. Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic oval cells. Science 1999; 284:1168–1170. 147. Miller WA. Diabetes mellitus—long-term survival. J Insur Med 1998; 30:17–27. 148. Wibell L, Nystrom L, Ostman J, et al. Increased mortality in diabetes during the first 10 years of the disease. A population-based study (DISS) in Swedish adults 15–34 years old at diagnosis. J Intern Med 2001; 249:263–270. 149. Edge JA, Ford-Adams ME, Dunger DB. Causes of death in children with insulin dependent diabetes 1990–96. Arch Dis Child 1999; 81:318–323.

HSCT for Autoimmune Diseases in Children

475

150. Laing SP, Swerdlow AJ, Slater SD, et al. The British Diabetic Association Cohort Study, II: cause-related mortality in patients with insulin-treated diabetes mellitus. Diabet Med 1999; 16:466–471. 151. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications of insulin-dependent diabetes mellitus. N Engl J Med 1993; 329:977–986. 152. Green A, Gale EA, Patterson CC. Incidence of childhood-onset insulin-dependent diabetes mellitus: The EURODIABACE Study. Lancet 1992; 339:905–909. 153. DPT-1 Study Group. Diabetes in America. 2nd ed. Bethesda: National Institute of health, 1995. 154. Bougneres PF, Carel JC, Castano L, et al. Factors associated with early remission of type I diabetes in children treated with cyclosporine. N Engl J Med 1988; 318:663–670. 155. Assan R, Feutren G, Sirmai J, et al. Plasma C-peptide levels and clinical remissions in recent-onset type I diabetic patients treated with cyclosporine A and insulin. Diabetes 1990; 39:768–774. 156. The Canadian European Randomized Trial Group. Cyclosporin-induced remission of IDDM after early intervention. Diabetes 1988; 37:1574–1582. 157. Cook JJ, Hudson I, Harrison LC, et al. Double-blinded controlled trial of azathioprine in children with newly diagnosed type I diabetes. Diabetes 1989; 38:779–783. 158. Silverstein J, Maclaren N, Riley W, et al. Immunosuppression with azathioprine and prednisone in recent-onset insulin-dependent diabetes mellitus. N Engl J Med 1988; 319:599–604. 159. Herold KC, Hagopian W, Auger JA, et al. Anti-CD3 monoclonal antibody in new-onset type I diabetes mellitus. N Engl J Med 2002; 346:1692–1698. 160. Voltarelli JC, Burt RK, Kenyon N, et al. Hematopoietic stem cell transplantation as treatment for type 1 diabetes. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:442–447. 161. Sanders JE, Hawley J, Lewy W, et al. Pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total body irradiation and bone marrow transplantation. Blood 1996; 87:3045–3052. 162. Burt RK, Oyama Y, Traynor A, Kenyon NS. Hematopoietic stem cell therapy for type 1 diabetes: induction of tolerance and islet cell neogenesis. Autoimmunity Rev 2002; 1:133–138. 163. Peloro TM, Miller F, Hahn TF, et al. Juvenile dermatomyositis: a retrospective review of 30-year experience. J Am Acad Dermatol 2001; 45:28–34. 164. Spencer CH, Hanson V, Singsen BH, et al. Course of treated juvenile dermatomyositis. J Pediatr 1984; 105:399–408. 165. Mendez EP, Lipton R, Ramsey-Goldman R, et al. NIAMS Juvenile DM Registry Physician Referral Group. U.S. incidence of juvenile dermatomyositis, 1995–1998: results from the National Institute of Arthritis and Musculoskeletal and Skin Diseases Registry. Arthritis Rheum 2003; 49:300–305. 166. Symmons DP, Sills JA, Davis SM. The incidence of juvenile dermatomyositis: results from a nation-wide study. Br J Dermatol 1995; 34:732–736. 167. Ramanan AV, Feldman BM. Clinical features and outcomes of juvenile dermatomyositis and other childhood onset myositis syndromes. Rheum Dis Clin North Am 2002; 28:833–857. 168. Ruperto N, Ravelli A, Murray KJ, et al. Paediatric Rheumatology International Trials Organization (PRINTO). Pediatric Rheumatology Collaborative Study Group (PRCSG). Preliminary core sets of measures for disease activity and damage assessment in juvenile systemic lupus erythematosus and juvenile dermatomyositis. Rheumatology 2003; 42:1452–1459. 169. Maillard SM, Jones R, Owens S, et al. Quantitative assessment of MRI T2 relaxation time of thigh muscles in juvenile dermatomyositis. Rheumatology (Oxford) 2004; 43:603–608. 170. Love LA, Leff RL, Fraser DD, et al. A new approach to the classification of idiopathic inflammatory myopathy: myositis-specific autoantibodies define useful homogenous patient groups. Medicine (Baltimore) 1991; 70:360–374. 171. Wargula JC. Update on juvenile dermatomyositis: new advances in understanding its etiopathogenesis. Curr Opin Rheum 2003; 15:595–601. 172. Feldman BM, Reichlin M, Laxer RM, et al. Clinical significance of specific autoantibodies in juvenile dermatomyositis. J Rheumatol 1996; 23:1794–1797. 173. Carpenter JR, Bunch TW, Engel AG, et al. Survival in polymyositis: corticosteroid and risk factors. J Rheumatol 1977; 4:207–214. 174. Oyama Y, Barr WG, Burt RK. Autologous hematopoietic stem cell transplantation for idiopathic inflammatory myositis. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:437–441.

476

Burt et al.

175. Baron F, Ribbens C, Kaye O, et al. Effective treatment of Jo-1-associated polymyositis with T-cell depleted autologous peripheral blood stem cell transplantation. Br J Haematol 2000; 110:339–342. 176. Bingham S, Griffith B, McGonalde D, et al. Autologous stem cell transplantation for rapidly progressive Jo-1-positive polymyositis with long-term follow-up. Br J Haematol 2001; 113:839–842. 177. Chakraverty R, Rabin N, Peggs K, et al. Dermatomyositis and sarcoid-like reaction associated with multiple myeloma treated effectively by high-dose chemotherapy and autologous peripheral blood stem cell transplantation. Bone Marrow Transplant 2001; 27:1215–1217. 178. Thiel A, Alexander T, Schmidt CA, et al. Immune reconstitution after hematopoietic stem cell transplantation. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:206–222. 179. Slavin S, Marmont A, Burt RK. Allogeneic hematopoietic stem cell transplantation for autoimmune diseases. In: Burt RK, Marmont A, eds. Stem Cell Therapy for Autoimmune Diseases. Georgetown, TX: Landes Bioscience, 2004:468–473. 180. Burt RK, Oyama Y, Verda L, et al, Remission of severe and refractory rheumatoid arthritis by allogeneic mixed chimerism. Arthritis Rheum 2004; 50:2466–2470. 181. Burt RK, Verda L, Kim DA, et al. Embryonic stem cells as an alternate marrow donor Source: engraftment without graft-versus-host disease. J Exp Med 2004; 199:895–904.

SECTION III: MALIGNANT DISEASES

24 Transplantation for Childhood Acute Lymphoblastic Leukemia Donna A. Wall Pediatric Blood and Marrow Transplant Program, Methodist Children’s Hospital/Texas Transplant Institute, San Antonio, Texas, U.S.A.

Kirk R. Schultz Division of Hematology/Oncology, Blood and Marrow Transplantation Program, British Columbia Children’s Hospital, University of British Columbia, Vancouver, British Columbia, Canada

Gregor S. D. Reid Department of Oncology, The Children’s Hospital of Philadelphia, Joseph Stokes, Jr. Research Institute, Abramson Research Center, Philadelphia, Pennsylvania, U.S.A.

INTRODUCTION Despite advances in the treatment of childhood acute lymphoblastic leukemia (ALL), relapsed ALL remains the third most common cancer in children. Because transplantation is frequently used as therapy for recurrent disease, ALL is one of the most common indications for transplantation in pediatrics. With improved frontline therapies for childhood ALL, there are fewer children presenting to transplant; but those who do are frequently more heavily pretreated and have more drug-resistant disease than those transplanted in earlier years. This makes the child with recurrent ALL one of the more challenging of transplant patients today. The theory behind transplantation is to destroy resistant leukemia cells with high-dose radiation therapy and chemotherapy and to generate a cellular graft-versus-leukemia (GVL) effect.

BIOLOGY OF TRANSPLANTATION FOR ACUTE LYMPHOBLASTIC LEUKEMIA Hematopoietic stem cell transplantation (HSCT) is the only successful form of immune therapy for ALL. Although intensive chemotherapy or irradiation has an impact on ALL, the GVL effect is very important, as evidenced by the high relapse rate in autologous HSCT (1) and identical twin HSCT (2). The GVL effect appears to have the strongest correlation with the development of chronic graft-versus-host disease (GVHD) post HSCT, although acute GVHD, donor mismatch, and possibly donor source appear to play a role in development of GVL effects as well (3–5). The GVL effect in ALL appears to be of less consequence than in AML, CML, and JMML (3). The therapeutic impact of the GVL effect appears to be equivalent for both B- and T-cell phenotype ALL (1,5). Because of the significant morbidity of chronic GVHD and 477

478

Wall et al.

its strong association with the GVL effect, one of the major therapeutic goals of allogeneic HSCT in the treatment of ALL is to perform an HSCT that has no chronic GVHD and yet maintains or increases the GVL effect. Improved understanding of the donor immune response to ALL is imperative to better understand how HSCT can successfully treat ALL.

IMPORTANCE OF DISEASE CONTROL PRETRANSPLANT It is becoming increasingly apparent that disease burden prior to transplant for ALL is directly correlated with disease-free survival (DFS). Published series that report long term DFS in patients with leukemia in relapse or with patients with leukemia in relapse or with refractory disease are generally reporting successful transplant in myeloid disorders. Outcome for transplant of ALL in relapse is uniformly poor with a relapse rate of more than 70% (6–8). For patients in remission at time of transplant, reports of pretransplant minimal residual disease (MRD) augur a poor prognosis (6). Knechtli and colleagues analyzed PCR-based MRD analysis, in 64 patients receiving allogeneic HSCT for childhood ALL. MRD was analyzed by polymerase chain reaction (PCR) of Ig or T-cell receptor delta or gamma rearrangements, electrophoresis, and allele-specific oligoprobing (9). For patients in microscopic remission at time of transplant, 12 patients had MRD detectable to a sensitivity of 10K2 to 10K3, and 11 had MRD detectable at lower levels (sensitivity 10K3 to 10K5). MRD was undetectable in 33. Two-year event-free survival (EFS) for these groups was 0%, 36%, and 73%, respectively (P !0.001). In a small series reported by Uzunel and colleagues, the only survivors in patients with pretransplant MRD positivity were those who developed GVHD (10). Among MRD negative patients, the probability of relapse was zero in five, compared to MRD positive patients (13 of 25) (PZ0.05). No major difference was seen between the high- and low-level MRD groups. Among the MRD positive patients, only 2 of 11 with acute and chronic GVHD had a relapse compared to 11 of 14 without any GVHD (PZ0.005). There remain many unanswered questions in the application of pretransplant MRD monitoring: is there a benefit to continuing pretransplant chemotherapy with the goal of lowering pretransplant disease load; will patients who are MRD negative at end of induction benefit from pretransplant consolidation therapy; and should maneuvers to increase allogenicity/GVHD be attempted in patients who are MRD positive pretransplant?

WHEN TO TRANSPLANT IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA First Remission With improvements in outcome of frontline chemotherapy there are few children for whom transplantation is indicated in CR1. The indications for transplant in CR1 include failure to enter remission after the first course of induction therapy, chromosomal abnormalities, including t(1:14), t(1;19), and hypodiploidy. Other cytogenetic abnormalities, slow early response to steroids, and very high white count have variably been associated with poor outcome (11). Currently the definition of very high risk (VHR) disease is 35–40% DFS with conventional chemotherapy. Improvements in frontline therapy will change the makeup of this group of patients, which currently include 4–8% of children with newly diagnosed ALL. In the future it is likely that minimal disease monitoring will help to identify children at high risk for relapse early on in therapy—perhaps this will be an alternative approach to identifying children who will benefit from earlier allogeneic transplant (9–12). Numerous reports have been published exploring HLA-matched sibling donor (MSD) HSCT for adult patients with high-risk ALL in first continuous remission. The strongest pediatric data is in children with Philadelphia chromosome positive (PhC) ALL. An international retrospective study of the outcome for 267 patients with PhC ALL showed improved outcome at two years for those who underwent matched related donor (MRD) HSCT (EFSZ71%) compared

Transplantation for Childhood Acute Lymphoblastic Leukemia

479

with to those patients treated with mismatched related donor HSCT (EFSZ56%), unrelated donor HSCT (EFSZ37%), or chemotherapy (EFSZ44%) (13). Although low white blood cell (WBC) at diagnosis may identify a lower risk population of children with PhC ALL who may not require HSCT, this factor does not appear to result in a long-term EFS that is O45% in the larger international analysis (13). In a recent single institution review, Sharathkumar and colleagues reported their experience with HSCT using MRD or matched unrelated donor (MUD) versus chemotherapy in children with Ph (C) ALL in CR1 (14). Of 21 children treated from 1985 to 2001, 10 received chemotherapy, and 11 received allogeneic BMT, four MFD, and seven MUD. The 4-year EFS for the HSCT group was 53G15%. The 4-year EFS for the chemotherapy and MUD groups was 33G17 and 35.7G20%, respectively. Four chemotherapy patients received CR2 transplants; all died. This difference was not statistically significant. The challenge here is that the children with PhC ALL who relapse following chemotherapy are often difficult to get into durable second remission. Previously other biological markers have identified children that may benefit from HSCT in first CR1. These include MLL gene rearrangements (15) including t(4;11) and monosomy 7 (16). With current frontline chemotherapy regimens, none of these biological markers meet the criteria of identifying children with an expected outcome of !45% expected EFS and thus should not be considered for HSCT. Among other VHR patients defined herein, information regarding the impact of HSCT is sparse and incomplete. No CCG or POG data were available for patients with hypodiploidy (%44 chromosomes) who underwent HSCT. Patients with an M3 bone marrow (BM) at the end of induction therapy had a 4-year EFS of 39% (11). Evaluation of therapeutic approaches for children with these poor prognostic features will require the comparison of consistent HSCT and chemotherapeutic regimens. At this time, the best outcome for children with PhC appears to be with HLA-matched or one antigen mismatched related donor HSCT in first complete remission. The benefits of using HSCT for other very high-risk features are less clear. The role of unrelated HSCT, as a therapeutic approach in ALL-CR1 is still controversial. Although the expected outcome for a sibling donor transplant performed in CR1 can be expected to be 60–70% at 5 years (13,17–19), the outcome for most unrelated HSCT appears to be approximately 40%, (20) but range up to 70% in single institution trials (19). Utilizing the criteria that an HSCT should be performed if it offers a potential outcome that is 10–15% greater than that expected for chemotherapy, the only two groups that may be considered are those patients that do not achieve a remission after induction and PhC ALL (22). There is very little published data supporting unrelated HSCT for induction failure. One of the main reasons for this is that the induction failure in children with ALL is 0.1% for NCI standard risk and 1% for NCI high risk (unpublished COG data), meaning that there are only about 10–15 induction failures in North America each year. The role of alternative donor HSCT for ALL patients that cannot achieve a remission before transplantation is controversial. A high-risk subset of children with PhC ALL appears to be identified by older age at presentation, high presenting WBC count, or a slow early response (13,21). In this population, unrelated donor transplant is recommended despite the potential of increased transplant related morbidity and mortality. A comparison of chemotherapy versus MSD transplant in pediatric patients with VHR ALL was performed prospectively by Wheeler and colleagues in the Medical Research Council (MRC) trials UKALL X and XI from 1985 to 1997 (17). Of 3676 patients aged 1 to 15 years, 473 patients (13%) were classified as VHR and were eligible for a HSCT from a MSD. The study was challenged by the use of unrelated donor HSCT outside the study design. The 10-year EFS (adjusted for the time to HSCT, diagnostic WBC count, Ph chromosome status, and ploidy) was 6.0% higher [95% confidence interval (CI) -10.5% to 22.5%] for 101 patients who received a first-remission HSCT (MSD and MUD) than for the 351 patients treated with chemotherapy (HSCT, 45.3%, vs. chemotherapy, 39.3%). The HSCT group had fewer relapses (31%) than the chemotherapy group (55%), but it also had more remission deaths (18%) than the chemotherapy group (3%). The adjusted 10-year EFS was 10.7% higher (95% CI, -2.6% to 24.0%) for patients without an HLA-matched donor than for those patients with a donor (no donor, 50.4%, vs. donor, 39.7%). In this series, for the majority of children with VHR ALL,

480

Wall et al.

first-remission HSCT did not improve EFS. Uderzo and colleagues, with the Associazione Italiana de Ematologia de Oncologia Pediatrica (AIEOP) trials at 13 Italian centers from 1986 to 1994 compared related HLA-matched HSCT with chemotherapy for children (!16 years) with ALL in CR1 (18). High-risk patients were defined by: (1) high-risk cytogenetic abnormalities (e.g., t(9;22) or t(4;11)), (2) BFM risk index (18) O1.7, (3) T-cell immunophenotype and WBC O100,000/mm3 or day 7 steroid resistance, or (4) failure to achieve remission by day 42 of induction. Thirty HSCT patients were matched to 130 controls. At a median follow-up of 4 years, the 4-year unadjusted DFS was not significantly different; 58.5% in the HSCT group and 47.7% in the chemotherapy group. Supporting improved outcome with HSCT are the results of Saarinen et al. who performed a matched case-control study of VHR pediatric patients (!16 years) with ALL selected from a population-based registry of all children with ALL diagnosed between 1981 and 1991 from five Nordic countries (23). Twenty-two children with VHR-ALL who underwent allo-HSCT in CR1 between 1981–1991 were compared to 44 matched control patients who received conventional chemotherapy on high risk ALL protocols, as well as with a group of 405 children representing the remaining high risk ALL patients in the Nordic ALL database. The DFS at 10 years was 73% in children receiving allo-HSCT in CR1, 50% in the matched controls (PZ0.02), and 59% in the remaining high risk ALL patients. The HSCT group had a lower relapse rate of 9% compared to 41% in the group of matched controls. The group of patients who had the greatest benefit were those with a WBC O100!109/L at diagnosis.

Second Remission Acute Lymphoblastic Leukemia The outcome of HSCT in children with ALL in CR2 is affected by the length of the first remission and the donor source. With more intensive frontline therapies, better characterization of residual disease, and improvements in unrelated donor transplantation, it is likely that the decision to transplant will continue to evolve. Salvage treatment utilizing secondary chemotherapy regimens for treatment of children with isolated testicular or late central nervous system (CNS) relapse have had excellent outcomes and in general the risk of early HSCT-related morbidity and mortality are not justified (24). Gaynon and colleagues reviewed 1144 relapses among the 3712 eligible patients enrolled on Children’s Cancer Group trials between 1983 and 1989 (24). The details of treatment after relapse were not accessible. Rates of 6-year survival (G standard error) after isolated BM, isolated CNS, and isolated testis relapse were 20%G2%, 48%G4%, and 70%G5%, respectively. Rates of survival after isolated BM relapse at 0–17 months, 18–35 months, and 36 months after diagnosis were 6%G2%, 11%G 2%, and 43%G4%, respectively. Rates of survival after isolated CNS relapse at 0–17 months, 18–35 months, and greater than 36 months were 33%G4%, 59%G5%, and 72%G8%, respectively. Rates of survival after isolated testis relapse at 0–17 months, 18–35 months, and greater than 36 months were 52%G11%, 57%G10%, and 81%G5%, respectively. Rates of survival after combined BM and CNS or testis relapse at 0–17 months, 18–35 months, and greater than 36 months were 9%G5%, 11%G6%, and 49%G7%, respectively. Poor prognosis relapses were those with very early relapse (!18 months after diagnosis) or recurrent T-cell disease. With the new generation of intensive front line chemotherapy regimens, there is concern that this level of survival will not be seen in these more intensively treated children. There is consensus that children who relapse early (!36 months) following the start of conventional chemotherapy have poor salvage with second line chemotherapy. However, this group of children also has poor transplant outcomes—attributable to a combination of transplant related morbidity/mortality and relapse (Table 1). Feig and colleagues reported the experience on CCG 1884, which compared the outcome of chemotherapy versus HSCT in 62 children with ALL with marrow relapses while on, or within 1 year, of completing initial therapy and who were able to achieve a second remission (27). Nineteen patients underwent HSCT in second remission (11 MSD, 7 autologous, 1 MUD). The actuarial 2-year EFS of HSCT patients was 37G22% (95% CI) compared to 18G13% for chemotherapy-treated patients (PZ0.017). In this small transplant series, there was similar

179/179 115/51 142/29 33/19 26/13 30/14 21/11

N (Chemo/ HSCT) 10G3% (5 year LFS) 22G4% (7 year EFS) 33.4G8.6% (3 year DFS) 18G13% (2 year EFS) 10% (8 year EFS) 6.9G4.7% (O10 year DFS) 9G9% (5 year DFS)

Chemotherapy outcome

35G4% (5 year LFS) 52G8% (7 year EFS) 16.1G4.5% (3 year DFS) 37G22% (2 year EFS) 54% (8 year EFS) 36G12.8 (O10 year DFS) 48G11% (5 year DFS)

HSCT outcome

!0.001 !0.01 0.002 0.017 !0.02 0.01 !0.01

p value

Abbreviations: HSCT, hematopoietic stem cell transplantation; MSD, matched sibling donor; LFS, leukemia-free survival; EFS, event-free survival; DFS, disease-free survival; POG, Pediatric Oncology Group; BFM, Berlin Frankfurt Munster; GITMO, Gruppo Italiano Trapianto di Midollo Osseo; AIEOP, Associazione Italiana de Ematologia de Oncologia Pediatrica; CCG, Children’s Cancer Group; MSKCC, Memorial Sloan-Ketting Cancer Center; IBMTR, International Bone Marrow Transplant Registry.

Remission !36 m, MSD Relapse!6 m off therapy Remission!30 m, MSD Early Rel., mostly MSD Remission !36 m, MSD Remission !30 m, MSD Relapse !24 m, MSD

Population

Matched Sibling Hematopoietic Stem-Cell Transplantation vs. Chemotherapy for Early Bone Marrow Relapse

IBMTR/POG (25) BFM (26) GITMO/AIEOP (18) CCG 1884 (27) Australian (28) Spanish (29) MSKCC (30)

Study

Table 1

Transplantation for Childhood Acute Lymphoblastic Leukemia 481

482

Wall et al.

outcome in the allogeneic and autologous recipients. HSCT had an improved EFS over continued chemotherapy after adjustment for intensity of prior chemotherapy regimens and length of prior remission (RRZ2.59, pZ0.01). With later marrow relapse, with improved outcomes from salvage chemotherapy, the benefit of allogeneic transplant, especially alternative donor transplant, is less clear. Wheeler and colleagues retrospectively reviewed the outcomes of 432 children diagnosed between 1985 and 1990 with ALL who received induction therapy in the MRC UKALL X trial, who had a relapse and were able to achieve second remission and received either maintenance chemotherapy or HSCT (31). There were 110 allogeneic HSCT (83 related, 27 MUD), 61 autologous HSCT, and 261 children who received maintenance chemotherapy. DFS at 4 years, adjusted for duration of first remission and site or relapse, showed a modest improvement in survival after allogeneic HSCT over chemotherapy (42.6% vs. 28.2%, pZ0.05). There was no benefit to autologous transplant over continued chemotherapy. In the retrospective comparison of IBMTR, and POG transplants, Barrett and colleagues demonstrated a benefit of MSD transplant at all durations of CR1 evaluated (25). However in a matched-pair analysis performed by Borgmann and colleagues of children treated according to ALL Relapse BerlinFrankfurt-Munster (ALL-REZ BFM) Study Group protocols after first relapse with chemotherapy or MUD HSCT, there was no significant difference in the probability of EFS between MUD HSCT or chemotherapy for the 28 pairs of children with intermediate risk prognosis (those with late relapse) (0.39G0.10 vs. 0.49G0.11, pZ0.105) (32).

Beyond Second Remission or Relapse Transplants in greater than second remission have both high transplant-related morbidity/mortality and high relapse rates. Woolfrey and colleagues evaluated outcomes after 88 unrelated donor HSCT (56 HLA-matched, 32 HLA–partially matched) in children with ALL (19). All received cyclophosphamide (Cy) and fractionated total body irradiation (TBI) as conditioning treatment, and all received GVHD prophylaxis with cyclosporine and methotrexate. Three year leukemia-free survival (LFS) according to disease state at time of transplant were 70% for CR1, 46% for CR2, 20% for CR3 and 9% for HSCT in relapse (p!0.0001). The 3-year cumulative relapse rates were 10%, 33%, 20%, and 50%, respectively, and the 3-year cumulative rates of death not attributed to relapse were 20%, 22%, 60%, and 41%, respectively.

PREPARATIVE REGIMENS Total Body Irradiation—Importance of Fractionation/Total Dose Transplant preparative regimens for adults with ALL have tended to be based on TBI. There has been an attempt in childhood ALL to limit exposure to TBI in young children; however, several retrospective and prospective trials support the use of a TBI-based regimen over busulfan (Bu)/Cy (30,33–36). A retrospective review of the IBMTR was conducted by Davies and colleagues of patients less than 20 years of age, transplanted between 1988 and 1995, who had received TBI based regimens (total nZ451, fractionated !1200 cGy nZ253, fractionated O1200 cGy nZ117, unfractionated !1000 cGy nZ72, unfractionated O1000 nZ9) or BuCCy (nZ176) as conditioning regimens (36). The 3-year probabilities of survival were 55% with TBI/Cy and 40% with Bu/Cy, with a similar difference between the groups for LFS. Transplant-related mortality (TRM) was higher in the Bu/Cy group. Overall, the study showed a superior survival with Cy/TBI conditioning, compared with Bu/Cy conditioning, for HLA-identical sibling HSCT in children with ALL. Early TBI experience was with single fraction dosing. This has been replaced with fractionated radiation therapy in pediatrics because of a lower rate of long-term sequelae. Dose escalation trials with TBI have demonstrated improved antileukemic efficacy with higher doses, but this has been tempered by increased transplant related morbidity and mortality, mostly pulmonary toxicity (37,38).

Transplantation for Childhood Acute Lymphoblastic Leukemia

483

Radiation therapy approaches utilize a variety of fractionations, blocks, and boosts, but most programs are delivering between 1200 and 1400 cGy total dose. Several groups have attempted to modify the basic TBI/Cy regimen with the goal of introducing therapies with nonoverlapping toxicities with the potential of improving disease control. Pilot experience from POG 9710 (39) and a PBMTC trial (40) have demonstrated that fractionated TBI (1200 cGy in six fractions) followed by VP16 (1500 mg/m2) and Cy (60 mg/ kg!2 doses) is well tolerated and supports engraftment with a variety of stem cell sources. Zecca and colleagues reported excellent outcomes with the addition of Thiotepa into the TBI/Cy regimen (41). Thiotepa is attractive because of its nonoverlapping nonhematopoietic toxicities and CNS penetration. They treated 40 children undergoing MSD HSCT for ALL in CR 1 or 2 with a combination of fractionated TBI, thiotepa (10 mg/kg) and Cy (120 mg/kg over 2 days). GVHD prophylaxis used cyclosporine as a single agent. There was only one toxic death, and the relapse rate was 23% with 26/40 (65%) of children alive and in remission with a median follow-up of 36 months (range 14–57 months). Similarly good results have been reported with the pairing of Ara C with TBI (42,43) or with the addition/substitution of Bu, melphalan, and/or fludarabine (44). Submyeloablative approaches for the transplant preparative regimen are being studied. Because relapse occurs rapidly with ALL, the challenge is to achieve rapid immunologic recovery post transplant. Initial reports of submyeloablative transplants for all have had poor outcomes, but the trials are still in the early phases (45).

HEMATOPOIETIC STEM CELL SOURCE Hematopoietic cell sources utilized for the transplantation of ALL in children have included autologous cells and related and unrelated donors. Autologous transplantation, usually combined with a purging strategy, has been explored as an alternative for children without MSD. Given the high relapse rate following autologous transplant, current practice trends to use of allogeneic stem cell sources, including BM, G-CSF mobilized peripheral blood, and umbilical cord blood (UCB).

Autologous Donor Sources The use of autologous cells, either BM or G-CSF mobilized peripheral blood, is challenging in ALL because of the concern for infusing autologous leukemic cells. Many purging approaches have been studied, including chemotherapy (e.g., mafosfamide, vincristine, and prednisolone) (46) and monoclonal antibodies (anti-CD19 and/or 10 linked with B4-ricin) (39,47) as well as strategies of in vivo purging prior to stem cell collection (48). Rescue overall has been relatively unsuccessful compared to allogeneic sources. On the other hand, because of the lower toxicity associated with autologous transplantation, some groups have reported similar results between autologous and allogeneic HSCT (49). Although results with autologous transplantation have been associated with high relapse rates, there are possibly subgroups of patient who may benefit from dose intensification and autologous stem cell rescue, utilizing either in vivo or ex vivo approaches to decreasing the risk of transferring disease. These include patients with late relapse as reported by the Billet and colleagues from the Dana Farber (47,50). Fifty-one children with ALL in CR2 or subsequent remission after a first remission of at least 24 months underwent purged, autologous HSCT. BM was harvested in remission and purged in vitro with monoclonal antibodies specific for leukemia-associated antigens. Ablative chemotherapy included cytarabine, teniposide, and Cy, followed by TBI. Of the 51 patients treated between November 1980 and June 1991, 5 died of treatment-related complications, 18 relapsed, 1 died of a second tumor at 6.7 years, and 27 remained in continuous complete remission for a median of 39 months (range, 9C to 124C). EFS (GSE) at 3 years after autologous HSCT was 53%G7%. In multivariate analysis, the most significant predictors of

484

Wall et al.

EFS were duration of longest pre-ABMT remission and remission duration immediately before ABMT. Their series of patients was compared to 17 patients who underwent allogeneic BMT for B-cell lineage ALL after at least one marrow relapse. The allogeneic BMT cohort included only those who would also have been eligible for autologous HSCT had they not had a MSD. Specifically, patients who were not in complete remission, those with T-cell positive leukemia, t(9;22) or those with only an extramedullary relapse were excluded from both groups. Conditioning regimens, age, WBC at diagnosis, and duration of first and longest complete remissions were comparable for the two groups. The relapse rate was higher in the autologous HSCT group and the incidence of nonleukemic deaths higher in the allogeneic HSCT group. EFS at 3 years was comparable for the two groups (47%G7 vs. 53%G12, autologous vs. allogeneic, respectively; PZ0.77). Similar equivalency in outcome for autologous transplant compared to allogeneic sources has been reported by others (1,51,52). Messina and colleagues assessed the role of autologous HSCT in children with ALL who were in 2nd CR after an early isolated CNS relapse (46). All children experiencing an isolated CNS relapse at 10 AIEOP centers from 1986 to 1992 were eligible for this study. The series included 69 patients who relapsed within 3 years from diagnosis: 19 underwent autologous HSCT, 9 patients underwent allogeneic HSCT from an HLA-identical sibling, and 41 received conventional chemotherapy. The 5-year DFS was 56.3% for patients in the autologous HSCT group compared to 12.6% in the chemotherapy group. The risk of relapse was reduced by onethird in the autologous HSCT group as compared to the chemotherapy group in the multivariate analysis (P !0.01). In the allogeneic group four out of nine patients were in CR 4–5 years post transplant.

Peripheral Blood Progenitor Cells A recent retrospective review of the IBMTR early experience with G-CSF mobilized peripheral blood or peripheral blood stem cells (PBSC) compared to BM transplant in children with acute leukemia demonstrated a disturbing finding of a worse outcome in children receiving PBSC as the donor source (53). In this retrospective review of 143 PBSC and 630 BM HLA-identical sibling donor transplants in children with leukemia from 8–20 years, Eapen and colleagues found a higher rate of chronic GVHD and TRM with a similar relapse risk in children receiving PBSC compared to BM. Overall, there was poorer survival when PBSC was used as the donor source, even after adjusting for the relative risks of the biologic differences between the two stem cell products, including differences in both intermediate hematopoietic progenitors and T cells. In multiple animal and human trials there has consistently been a strong correlation between the number of T cells in the transplanted graft and the development of GVHD. The tenfold increase in T cells in a PBSC graft initially made allogeneic transplant physicians hesitant to use PBSC as a stem cell product, but experience has shown that PBSC can be used as an allogeneic stem cell source safely (54). In the process of mobilization, the T cells have been exposed to G-CSF, which is known to modify T-cell responses in the direction of the TH2 response—potentially explaining the tolerable GVHD observed. It is important to appreciate that this report (53) captures the early experience in pediatrics with the use of PBSC in the allogeneic setting. This series has a high representation of patients for whom transplant physicians felt the benefits of more rapid engraftment would outweigh the possible risk of GVHD. Some of these factors are captured and controlled for in this analysis (e.g., disease state at time of transplant); however, other risk features are more difficult to define in retrospective surveys [e.g., prior serious infections, donor recipient size discrepancy, or more aggressive leukemia (i.e., all second remission patients with ALL are not alike)]. The concept that infusing a greater number of T cells would enhance the GVL effect of the graft is frequently the reason for using PBSC in children. There is currently a BMT Clinical Trials Network study comparing PBSC versus marrow in the unrelated donor setting. It is

Transplantation for Childhood Acute Lymphoblastic Leukemia

485

critical that a similar trial comparing children with HLA-matched siblings donors, controlling for disease (leukemia, nonmalignant disorders) and disease state at time of transplant, also be studied before PBSC is considered the standard of care in pediatric MSD allogeneic transplantation. To date there is only a limited set of pediatric data, and based on this retrospective review the benefit of PBSC is in question.

Bone Marrow (Unstimulated and G-CSF Stimulated) When BM is used as the donor source, the donor, who is likely close in age, will also be young and tend to have a rich harvest. This translates into children routinely receiving 3–5!108 cells/kg in the marrow inoculum—the equivalent of a “rich BM,” which was reported by Gorin and colleagues as being the ideal stem cell source (55). Thus the pediatric population is starting with a better BM product compared to those used in adult transplants series. One alternative being explored is the use of G-CSF mobilized BM. With this strategy, the goal is to engineer a graft that has the increased number of intermediately committed hematopoietic progenitors found in PBSC grafts but fewer mature T cells.

Umbilical Cord Blood Increasingly UCB is being used as an alternative donor source—especially in children, where the cell dose in the UCB unit approaches conventional minimal cell doses achieved by BM (56–58). The major benefits of UCB are the ready availability and ability to transplant at time of optimal disease control and the lower risk of transferring blood-borne infection. The lower risk of GVHD, especially chronic GVHD, has raised the concern that UCB will have inferior GVL activity. Fewer T lymphocytes are infused in a UCB graft and the phenotype of natural killer (NK) cells and T cells are more immature in cord blood (59–62). This raises the question of whether the early recovery of immune cells produced by UCB will be functional. Joshi and colleagues demonstrated strong tumor cell killing by UCB NK cells, that was enhanced by the addition of IL-2 (63,64). Harris and colleagues demonstrated an antileukemic effect of CB in both in vitro and animal models (65). Clinically, several anecdotal reports have described leukemia regression following withdrawal of immunosuppression following CB transplantation (66). Because there are as yet no prospective comparisons of UCB to other HSCT sources, it is difficult to address the GVL effect. Because UCB transplants can be arranged within days, there has been a greater representation of patients with resistant disease in the UCB series. However, disease control appears to be similar to other unrelated donor sources—perhaps related to the fact that major HLA mismatches are routinely accepted for UCB transplantation. In a retrospective review comparing matched sibling UCB transplant to BM transplant in children, Rocha and colleagues reported similar survivals with persistent or recurrent disease, accounting for 48% of deaths in the UCB group and 49% in the BM group (58). The fact that there tends to be a greater HLA-mismatch accepted between donor and recipient in UCB transplant increases the antigenic single differences that could potentially act as targets for a GVL effect. Recent single institution experiences support the efficacy of UCB as an alternative donor source in the treatment of childhood ALL. Sawczyn and colleagues reported a series of 26 consecutive UCB HSCT for ALL performed from 1996 to 2002 (67). Median patient age was 8.5 years, and median UCB nucleated cell dose was 3.26!107/kg (range, 0.8–12.9). With a median follow-up of 548 days, 16/26 patients (62%) are surviving without evidence of disease. Acute GVHD developed in 14/24 evaluable patients (7—grade III–IV). Chronic GVHD occurred in 10/22 evaluable patients. Multivariate analysis showed higher total nucleated cell dose per kilogram to be the strongest predictor of EFS. Jacobsohn and colleagues retrospectively looked at their institutional experience in transplantation for ALL from 1992–2003 (56). All patients were either in CR1 with high-risk features (nZ21) or in CR2 (nZ28) with an initial remission lasting less than 36 months. Patients received myeloablation

486

Wall et al.

with fractionated TBI, Cy, and Etoposide, and GVHD prophylaxis with cyclosporine and methotrexate. ATG was added for UCB recipients. In all, 23 patients underwent allogeneicrelated HSCT, and 26 underwent UCB HSCT. The 3-year overall survival is 64%, and 3-year EFS is 60% for both groups. Rates of GVHD and TRM were similar. These results are being supported by the larger prospective experience of the NHLBI-sponsored COBLT trial.

Haploidentical Donor Source The use of a partially matched family member donor has been a focus of research by several groups (68–71,73). Klingebiel and colleagues have the largest series of children transplanted for ALL (68,69,72). Their approach was to utilize very high doses of purified positively selected mobilized peripheral blood CD34C hematopoietic stem cells from adult donors. Of the 27 children with ALL reported, 7 were in CR1, 10 in CR2, 4 in CR3, and 6 had refractory leukemia. The patients received a mean number of 19.1G11.3!106/kg purified CD34C and a mean number of 15.5G24.2!103/kg CD3C cells. No GVHD prophylaxis was used post transplant after the first three patients. The conditioning therapy was either Bu-based in 12 or TBI-based in 14 patients. Engraftment was rapid in the 26 patients with only two graft rejections. Overall survival was 34%, with the 12 patients transplanted in remission having a 44% survival. None of the patients transplanted with active disease survived. There was no statistical difference in survival for patients with a one, two, or three antigen-mismatched donor (out of 6 HLA antigens) or for patients in 1st, 2nd or 3rd remission. Relapse was the major cause of death.

Comparison of Stem Cell Sources The choice of stem cell sources for a child requiring HSCT for ALL is a frequent clinical question. With the maturation of the adult unrelated donor and UCB registries, an unrelated donor option is available in a timely fashion for a majority of children. In one direct comparison, Eapen and colleagues utilized the CIBMTR database to study the outcomes of unrelated donor HSCT (85 BM, 81 CB) in comparison to 101 MSD HSCT in children less than 18 months of age at diagnosis) with acute leukemia (74). The focus on infants is important since for the most part they received relatively high cell doses of cord blood. In their series, unrelated donor HSCT recipients were younger, more likely to have MLL gene rearrangements, to have advanced leukemia, and to receive irradiation before HSCT. Treatment-related mortality rates were 6%, 15%, and 31% after MSD, MUD marrow, and UCB, respectively. Risks of relapse, overall, and LFS were significantly associated with disease status at transplantation. Overall survival and LFS rates were similar after MSD and unrelated donor HSCT, after adjustment for disease status. Relapse, overall, and LFS did not differ by graft type (BM vs. CB) or type of leukemia. Three-year probabilities of LFS were 49% and 54% after HLA-matched sibling and unrelated donor transplantation in first CR, respectively. Corresponding rates for those with advanced leukemia were 20% and 30%. They concluded that unrelated donor HSCT should be considered for infants with acute leukemia in first CR using the same eligibility criteria as are currently used for those with HLA MSDs.

GRAFT-VERSUS-LEUKEMIA IN ACUTE LYMPHOBLASTIC LEUKEMIA: FACT OR FICTION Graft-Versus-Leukemia Despite reports of ALL resolution following withdrawal of immunosuppression or development of GVHD, the immune-mediated destruction of recipient leukemic blasts by donor lymphocytes, the GVL effect, was long thought to be irrelevant for ALL. Direct evidence for GVL activity was eventually attained through the many studies comparing HSCT donor

Transplantation for Childhood Acute Lymphoblastic Leukemia

487

sources and outcome. The consistently higher relapse rates associated with T-cell depleted versus nondepleted grafts, with autologous or identical twin transplants compared to allogeneic transplants and with syngeneic compared to allogeneic transplants clearly demonstrate significant therapeutic immune activity (1–3,20,58,75,76). The influence of donor lymphocytes is further supported by the strong correlation between the development of GVHD and lower relapse rates (4,5,10,77,78). In pediatric transplants, the development of chronic GVHD has the strongest correlation with decreased relapse, although GVL in the absence of overt GVHD may also occur. Consistent with a therapeutic role for GVL activity, posttransplant immune suppression has been shown to influence the risk of relapse (78–80).

Mechanisms of Graft-Versus-Leukemia Studies using model systems and clinical observations from a variety of hematological malignancies indicate that both T cells and NK cells are involved in mediating GVL activity. Although GVL effects specifically against ALL have not been as extensively studied, the data obtained have been consistent with this model. The strong correlation between chronic GVHD and a GVL effect implicates donor T cells in the anti-ALL activity. The development of donorderived cytotoxic T-lymphocyte activity specifically directed against pediatric acute leukemic blasts has been reported to contribute to the maintenance of remission (81). Although T cells capable of recognizing leukemia-specific antigens can be isolated from pediatric ALL patients (82,83), there have been no reports of successful activation and expansion of such T cells after transplant. However, consistent with the greater GVL activity after allogeneic transplant, T cells specific for minor histocompatibility antigens have been isolated from ALL patients after transplant (84,85). A role for NK cells in control of ALL was suggested by reports showing that a dramatic loss of autologous NK activity preceded relapse in ALL patients (86,87). The role of NK cells in posttransplant control of ALL has been most directly demonstrated by the development of a haplo-identical transplantation strategy designed to optimize NK cell activity, on the basis of killer cell Ig-like receptor (KIR) expression. Although initially NK activity in this setting was reported to be minimal for ALL (88), recent findings that use a novel method of defining mismatches in the graft-versus-host direction significantly correlated with remission in pediatric ALL patients undergoing haplo-identical HSCT (89). The importance of sustained NK cell activity and blast cell sensitivity in pediatric ALL patient post transplant has not yet been defined.

Donor Lymphocyte Infusions A second source of posttransplant antileukemia immune activity is the use of donor lymphocyte infusion (DLI). As is the case for the GVL effect, DLI is more successful in the treatment of myelogenous leukemia than lymphoblastic leukemia. Despite several reports of successful treatment of small numbers of ALL patients with DLI, such positive effects have not been observed in larger studies, with response rates of less than 15% being reported, compared to 70% for chronic phase CML and 25% for AML (90,91). Two recent reports, however, suggest that improved DLI responses may be obtainable in pediatric ALL patients (92,93). These studies used the early detection of increasing mixed chimerism as the indicator to initiate immune therapy, DLI or withdrawal of immune suppression, and in both reports significantly improved EFS was obtained. These findings suggest that timing may be one of the most important variables in the success of immune therapy for ALL.

Immune Subversion by Acute Lymphoblastic Leukemia Although the general mechanisms mediating GVL effects appear to be common to different leukemias, the activity is weaker against ALL than against other leukemias. Because 20–50% of children will experience a posttransplant relapse frequently, there is a failure to establish or maintain immune-mediated control of the leukemia. The development of strategies to augment

488

Wall et al.

anti-ALL immune activity will require an understanding of mechanisms underlying the frequent failure of GVL activity. Although extensively investigated in other tumor systems, reports of mechanisms by which ALL cells evade the immune system are rare (94–97). These studies, together with observations from other leukemia models, (98–100) implicate a failure of T-cell stimulation by leukemic blasts as a factor in disease progression. T-cell activation requires presentation of suitable peptide antigens by MHC complexes. Similar levels of GVL activity have been reported against T- and B-cell ALL (5). Given the lack of MHC Class II expression by T-ALL, this suggests that the helper T-cell ligand is not essential for GVL generation. In contrast to observations made in solid tumor settings, MHC class I down-regulation is not often observed in ALL (97,101), perhaps as a result of selective pressure exerted by NK cell activity. T-cell activation also requires the expression of costimulatory molecules by the antigen presenting cells. Studies of pediatric pre-B-ALL have shown an absence of CD80 expression, but CD86 has been detected at variable levels (102,103). CD80 has been shown in model systems to be superior for the generation of antileukemia T-cell activity (104), and its absence from the surface of ALL cells may contribute to the inefficient stimulation of T cells, or induction of T-cell anergy (102,105). Adult ALL cells are generally poor targets of NK cell-mediated killing, and this is thought to be the result of deficient expression of necessary adhesion molecules (88,106). Pediatric ALL blasts are not deficient in adhesion molecule expression, and no correlation between resistance to NK killing and adhesion molecule expression has been described (89,107–109). The accuracy of the recently described KIR mismatch prediction method as applied to pediatric ALL transplants suggest that NK activation deficiencies should be addressed in future trials.

STRATEGIES TO AUGMENT POSTTRANSPLANT IMMUNE ACTIVITY The transplant outcomes associated with different donor sources demonstrate that immunemediated control, and in many cases cure, of relapsed pediatric ALL is achievable. However, the aggressive nature of the disease, combined with inherent properties of the leukemic blasts that remain poorly understood, undermine the efficiency of immune responses against ALL. Our improved knowledge of the immune system has led to the design of several approaches to enhance immune responses against ALL.

Cytokines One of the earliest post-transplant interventions designed to improve antileukemia immune responses was the use of IL-2 (110). In addition to stimulating T-cell proliferation, IL-2 is a potent activator of NK cells, producing highly cytotoxic lymphokine activated killer (LAK) cells. LAK cells be expanded cells from peripheral blood mononuclear cells of ALL patients and demonstrate enhanced killing of autologous and allogeneic ALL blasts (111). To date, the clinical use of IL-2 has achieved the goal of increasing numbers of activated T, NK, and LAK cells but is associated with significant toxicity and achieves limited remission rates in children (112,113). Recently, the use of additional cytokines to generate anti-ALL activity has met with early success (114,115), suggesting that further advances with this approach may be forthcoming.

Retargeting Effector Cells Inefficient targeting of ALL blasts by effector cells is a limiting step in the development of immune therapy. One approach to address this issue is the use of chimeric antibodies that form a bridge between target and effector cells. Although still in preclinical testing, promising results have been obtained using antibodies against the B-cell markers, CD19, and CD20, to enhance antibody-dependent cellular cytotoxicity against pediatric ALL (116,117). An alternative

Transplantation for Childhood Acute Lymphoblastic Leukemia

489

strategy to address this issue has been to transduce effector cells with receptors specific for ALL cells. The feasibility of this approach has been demonstrated for both T cells and NK cells, and significantly improved levels of pediatric ALL killing have been reported (118,119). The application of these reagents in the post-HSCT environment may provide enhanced anti-ALL activity with minimal complications from GVHD.

Ex Vivo Expansion of Acute Lymphoblastic Leukemia-Specific T Cells Several strategies to generate anti-ALL specific T cells for adoptive transfer have been developed. One approach involves the presentation of leukemia-specific antigens to T cells by professional APC, such as dendritic cells (DC), an approach that circumvents any APC deficiencies present in the leukemia cells. The generation of donor-derived specific cytotoxic T cells (CTL) by in vitro priming with patient-derived DC in the presence of apoptotic ALL cells has been achieved (120). Alternatively, the ALL blast can be modified to enhance its ability to stimulate T cells directly. The necessary increase in costimulation by ALL cells has been achieved by inducing the leukemia cells to differentiate into DC (121,122), by ligation of CD40 expressed on ALL cells (82,83,123–125), and by stimulation of ALL blasts with oligodeoxynucleotides containing immunostimulatory CpG motifs (126). These modifications result in the production of strong inflammatory (Th1) T-cell responses directed against ALL cells. It remains to be determined whether such approaches remove all the inherent APC deficiencies of ALL blasts (127). The recent success of adoptive T-cell therapy against melanoma raises hope that antileukemia T cells generated in this fashion will be clinically useful (128).

Vaccination The development of vaccines is limited by the need to identify effective peptide epitopes. Using a variety of approaches, several groups have identified candidate antigens that generate ALL specific T-cell responses (129–131). The in vivo efficacy of such peptide-specific immune therapy for ALL remains to be tested. A whole cell vaccine approach eliminates the need for peptide antigen identification. Promising early clinical trial results using ALL blasts mixed with fibroblasts expressing CD40 ligand and IL-2 have recently been reported for high-risk pediatric ALL (132). Early success has also been achieved in murine models, where infusion of ALL cells transfected with CD80 and GM-CSF into mice led to efficient clearance of nontransfected ALL cells (133). This approach has been shown to be feasible in human ALL (134). Alternatively, DC primed in the presence of apoptotic leukemia cells could be administered to the patient to stimulate immune responses in vivo (135). As with the use of DLI post transplant, it seems likely that the timing of the vaccine interventions will be critical to the success of these approaches (136).

REFERENCES 1. Weisdorf D, Bishop M, Dharan B, et al. Autologous versus allogeneic unrelated donor transplantation for acute lymphoblastic leukemia: comparative toxicity and outcomes. Biol Blood Marrow Transplant 2002; 8:213–220. 2. Ringden O, Labopin M, Gorin NC, et al. Is there a graft-versus-leukaemia effect in the absence of graft-versus-host disease in patients undergoing bone marrow transplantation for acute leukaemia? Br J Haematol 2000; 111:1130–1137. 3. Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990; 75:555–562. 4. Gustafsson Jernberg A, Remberger M, Ringden O, et al. Graft-versus-leukaemia effect in children: chronic GVHD has a significant impact on relapse and survival. Bone Marrow Transplant 2003; 31:175–181. 5. Passweg JR, Tiberghien P, Cahn JY, et al. Graft-versus-leukemia effects in T lineage and B lineage acute lymphoblastic leukemia. Bone Marrow Transplant 1998; 21:153–158.

490

Wall et al.

6. Dworzak MN, Froschl G, Printz D, et al. Prognostic significance and modalities of flow cytometric minimal residual disease detection in childhood acute lymphoblastic leukemia. Blood 2002; 99:1952–1958. 7. Brochstein JA, Kernan NA, Groshen S, et al. Allogeneic bone marrow transplantation after hyperfractionated total-body irradiation and cyclophosphamide in children with acute leukemia. N Engl J Med 1987; 317:1618–1624. 8. Harrison G, Richards S, Lawson S, et al. Comparison of allogeneic transplant versus chemotherapy for relapsed childhood acute lymphoblastic leukaemia in the MRC UKALL R1 trial. MRC childhood leukaemia working party. Ann Oncol 2000; 11:999–1006. 9. Knechtli CJ, Goulden NJ, Hancock JP, et al. Minimal residual disease status before allogeneic bone marrow transplantation is an important determinant of successful outcome for children and adolescents with acute lymphoblastic leukemia. Blood 1998; 92:4072–4079. 10. Uzunel M, Mattsson J, Jaksch M, et al. The significance of graft-versus-host disease and pretransplantation minimal residual disease status to outcome after allogeneic stem cell transplantation in patients with acute lymphoblastic leukemia. Blood 2001; 98:1982–1984. 11. Schultz KR, Pullen J, Sather H, et al. Camitta biology-driven classification of childhood acute lymphoblastic leukemia: a combined analysis of prognostic markers from the pediatric oncology group (POG) and children’s cancer group (CCG). Blood 2004; 104:519 (ASH Abstracts). 12. Knechtli CJ, Goulden NJ, Hancock JP, et al. Minimal residual disease status as a predictor of relapse after allogeneic bone marrow transplantation for children with acute lymphoblastic leukaemia. Br J Haematol 1998; 102:860–871. 13. Arico M, Valsecchi MG, Camitta B, et al. Outcome of treatment in children with Philadelphia chromosome-positive acute lymphoblastic leukemia. N Engl J Med 2000; 342:998–1006. 14. Sharathkumar A, Saunders EF, Dror Y, et al. Allogeneic bone marrow transplantation versus chemotherapy for children with Philadelphia chromosome-positive acute lymphoblastic leukemia. Bone Marrow Transplant 2004; 33:39–45. 15. Pui CH, Chessells JM, Camitta B, et al. Clinical heterogeneity in childhood acute lymphoblastic leukemia with 11q23 rearrangements. Leukemia 2003; 17:700–706. 16. Heerema NA, Nachman JB, Sather HN, et al. Deletion of 7p or monosomy 7 in pediatric acute lymphoblastic leukemia is an adverse prognostic factor: a report from the children’s cancer group. Leukemia 2004; 18:939–947. 17. Wheeler KA, Richards SM, Bailey CC, et al. Bone marrow transplantation versus chemotherapy in the treatment of very high-risk childhood acute lymphoblastic leukemia in first remission: results from medical research council UKALL X and XI. Blood 2000; 96:2412–2418. 18. Uderzo C, Valsecchi MG, Balduzzi A, et al. Allogeneic bone marrow transplantation versus chemotherapy in high-risk childhood acute lymphoblastic leukaemia in first remission. Associazione Italiana di ematologia ed oncologia pediatrica (AIEOP) and the gruppo Italiano trapianto di midollo osseo (GITMO). Br J Haematol 1997; 96:387–394. 19. Woolfrey AE, Anasetti C, Storer B, et al. Factors associated with outcome after unrelated marrow transplantation for treatment of acute lymphoblastic leukemia in children. Blood 2002; 99:2002–2008. 20. Bunin N, Carston M, Wall D, et al. Unrelated marrow transplantation for children with acute lymphoblastic leukemia in second remission. Blood 2002; 99:3151–3157. 21. Balduzzi A, Valsecchi MG, Uderzo C, et al. Chemotherapy versus allogeneic transplantation for very-high-risk childhood acute lymphoblastic leukaemia in first complete remission: comparison by genetic randomisation in an international prospective study. Lancet 2005; 366:635–642. 22. Talano JM, Casper JT, Camitta BM, et al. Alternative donor bone marrow transplant for children with Philadelphia chromosome ALL. Bone Marrow Transplant 2006; 37:135–141. 23. Saarinen UM, Mellander L, Nysom K, et al. Allogeneic bone marrow transplantation in first remission for children with very high-risk acute lymphoblastic leukemia: a retrospective casecontrol study in the Nordic countries. Nordic society for pediatric hematology and oncology (NOPHO). Bone Marrow Transplant 1996; 17:357–363. 24. Gaynon PS, Qu RP, Chappell RJ, et al. Survival after relapse in childhood acute lymphoblastic leukemia: impact of site and time to first relapse—the children’s cancer group experience. Cancer 1998; 82:1387–1395. 25. Barrett AJ, Horowitz MM, Pollock BH, et al. Bone marrow transplants from HLA-identical siblings as compared with chemotherapy for children with acute lymphoblastic leukemia in a second remission. N Engl J Med 1994; 331:1253–1258.

Transplantation for Childhood Acute Lymphoblastic Leukemia

491

26. Henze G, Fengler R, Hartmann R, et al. Six-year experience with a comprehensive approach to the treatment of recurrent childhood acute lymphoblastic leukemia (ALL-REZ BFM 85). A relapse study of the BFM group. Blood 1991; 78:1166–1172. 27. Feig SA, Harris RE, Sather HN. Bone marrow transplantation versus chemotherapy for maintenance of second remission of childhood acute lymphoblastic leukemia: a study of the children’s cancer group (CCG-1884). Med Pediatr Oncol 1997; 29:534–540. 28. Bleakley M, Shaw PJ, Nielsen JM. Allogeneic bone marrow transplantation for childhood relapsed acute lymphoblastic leukemia: comparison of outcome in patients with and without a matched family donor. Bone Marrow Transplant 2002; 30:1–7. 29. Torres A, Alvarez MA, Sanchez J, et al. Allogeneic bone marrow transplantation versus chemotherapy for the treatment of childhood acute lymphoblastic leukaemia in second complete remission (revisited 10 years on). Bone Marrow Transplant 1999; 23:1257–1260. 30. Boulad F, Steinherz P, Reyes B, et al. Allogeneic bone marrow transplantation versus chemotherapy for the treatment of childhood acute lymphoblastic leukemia in second remission: a singleinstitution study. J Clin Oncol 1999; 17:197–207. 31. Wheeler K, Richards S, Bailey C, et al. Comparison of bone marrow transplant and chemotherapy for relapsed childhood acute lymphoblastic leukaemia: the MRC UKALL X experience. Medical research council working party on childhood leukaemia. Br J Haematol 1998; 101:94–103. 32. Borgmann A, von Stackelberg A, Hartmann R, et al. Unrelated donor stem cell transplantation compared with chemotherapy for children with acute lymphoblastic leukemia in a second remission: a matched-pair analysis. Blood 2003; 101:3835–3839. 33. IBMTR/ABMTR Newsletter. 2002:6. 34. Ringden O, Ruutu T, Remberger M, et al. A randomized trial comparing busulfan versus total body irradiation in allogeneic marrow transplant recipients with hematological malignancies. Transplant Proc 1994; 26:1831–1832. 35. Ringden O, Remberger M, Ruutu T, et al. Increased risk of chronic graft-versus-host disease, obstructive bronchiolitis, and alopecia with busulfan versus total body irradiation: long-term results of a randomized trial in allogeneic marrow recipients with leukemia. Nordic bone marrow transplantation group. Blood 1999; 93:2196–2201. 36. Davies SM, Ramsay NK, Klein JP, et al. Comparison of preparative regimens in transplants for children with acute lymphoblastic leukemia. J Clin Oncol 2000; 18:340–347. 37. Petersen FB, Deeg HJ, Buckner CD, et al. Marrow transplantation following escalating doses of fractionated total body irradiation and cyclophosphamide—a phase I trial. Int J Radiat Oncol Biol Phys 1992; 23:1027–1032. 38. Demirer T, Petersen FB, Appelbaum FR, et al. Allogeneic marrow transplantation following cyclophosphamide and escalating doses of hyperfractionated total body irradiation in patients with advanced lymphoid malignancies: a phase I/II trial. Int J Radiat Oncol Biol Phys 1995; 32:1103–1109. 39. Sandler E. POG 9410, Personal communication, 2003. 40. Bunin N, Aplenc R, Kamani N, et al. Randomized trial of busulfan versus total body irradiation containing conditioning regimens for children with acute lymphoblastic leukemia: a pediatric blood and marrow transplant consortium study. Bone Marrow Transplant 2003; 32:543–548. 41. Zecca M, Pession A, Messina C, et al. Total body irradiation, thiotepa, and cyclophosphamide as a conditioning regimen for children with acute lymphoblastic leukemia in first or second remission undergoing bone marrow transplantation with HLA-identical siblings. J Clin Oncol 1999; 17:1838–1846. 42. Gordon BG, Warkentin PI, Strandjord SE, et al. Allogeneic bone marrow transplantation for children with acute leukemia: long-term follow-up of patients prepared with high-dose cytosine arabinoside and fractionated total body irradiation. Bone Marrow Transplant 1997; 20:5–10. 43. Coccia PF, Strandjord SE, Warkentin PI, et al. High-dose cytosine arabinoside and fractionated total-body irradiation: an improved preparative regimen for bone marrow transplantation of children with acute lymphoblastic leukemia in remission. Blood 1988; 71:888–893. 44. Petropoulos D, Worth LL, Mullen CA, et al. Total body irradiation, fludarabine, melphalan, and allogeneic hematopoietic stem cell transplantation for advanced pediatric hematologic malignancies. Bone Marrow Transplant 2006. 45. Gomez-Almaguer D, Ruiz-Arguelles GJ, Tarin-Arzaga Ldel C, et al. Reduced-intensity stem cell transplantation in children and adolescents: the Mexican experience. Biol Blood Marrow Transplant 2003; 9:157–161.

492

Wall et al.

46. Messina C, Valsecchi MG, Arico M, et al. Autologous bone marrow transplantation for treatment of isolated central nervous system relapse of childhood acute lymphoblastic leukemia. AIEOP/FONOP-TMO group. Associzione Italiana emato-oncologia pediatrica. Bone Marrow Transplant 1998; 21:9–14. 47. Billett AL, Kornmehl E, Tarbell NJ, et al. Autologous bone marrow transplantation after a long first remission for children with recurrent acute lymphoblastic leukemia. Blood 1993; 81:1651–1657. 48. Corbacioglu S, Eber S, Gungor T, et al. Induction of long-term remission of a relapsed childhood B-acute lymphoblastic leukemia with rituximab chimeric anti-CD20 monoclonal antibody and autologous stem cell transplantation. J Pediatr Hematol Oncol 2003; 25:327–329. 49. Badell I, Munoz A, Ortega JJ, et al. Long-term outcome of allogeneic or autologous haemopoietic cell transplantation for acute lymphoblastic leukaemia in second remission in children. GETMON experience 1983–1998. Bone Marrow Transplant 2005; 35:895–901. 50. Parsons SK, Castellino SM, Lehmann LE, et al. Relapsed acute lymphoblastic leukemia: similar outcomes for autologous and allogeneic marrow transplantation in selected children. Bone Marrow Transplant 1996; 17:763–768. 51. Weisdorf DJ, Billett AL, Hannan P, et al. Autologous versus unrelated donor allogeneic marrow transplantation for acute lymphoblastic leukemia. Blood 1997; 90:2962–2968. 52. Ramsay N, Le Bien T, Nesbit M, et al. Autologous bone marrow transplantation for patients with acute lymphoblastic leukemia in second or subsequent remission: results of bone marrow treated with monoclonal antibodies BA-1, BA-2, and BA-3 plus complement. Blood 1985; 66:508–513. 53. Eapen M, Horowitz MM, Klein JP, et al. Higher mortality after allogeneic peripheral-blood transplantation compared with bone marrow in children and adolescents: the histocompatibility and alternate stem cell source working committee of the international bone marrow transplant registry. J Clin Oncol 2004; 22:4872–4880. 54. Favre G, Beksac M, Bacigalupo A, et al. Differences between graft product and donor side effects following bone marrow or stem cell donation. Bone Marrow Transplant 2003; 32:873–880. 55. Gorin NC, Labopin M, Rocha V, et al. Marrow versus peripheral blood for geno-identical allogeneic stem cell transplantation in acute myelocytic leukemia: influence of dose and stem cell source shows better outcome with rich marrow. Blood 2003; 102:3043–3051. 56. Jacobsohn DA, Hewlett B, Ranalli M, et al. Outcomes of unrelated cord blood transplants and allogeneic-related hematopoietic stem cell transplants in children with high-risk acute lymphocytic leukemia. Bone Marrow Transplant 2004; 34:901–907. 57. Locatelli F, Rocha V, Chastang C, et al. Factors associated with outcome after cord blood transplantation in children with acute leukemia. Eurocord-cord blood transplant group. Blood 1999; 93:3662–3671. 58. Rocha V, Cornish J, Slevers EL, et al. Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood 2001; 97:2962–2971. 59. Theilgaard-Monch K, Raaschou-Jensen K, Palm H, et al. Flow cytometric assessment of lymphocyte subsets, lymphoid progenitors, and hematopoietic stem cells in allogeneic stem cell grafts. Bone Marrow Transplant 2001; 28:1073–1082. 60. Chaisiripoomkere W, Jootar S, Ungkanont A, et al. Study of hematopoietic progenitor cells, hematological values and lymphocyte subsets in cord blood: application for cord blood transplantation. Southeast Asian J Trop Med Public Health 1999; 30:781–785. 61. Krampera M, Tavecchia L, Benedetti F, et al. Intracellular cytokine profile of cord blood T-, and NK- cells and monocytes. Haematologica 2000; 85:675–679. 62. D’Arena G, Musto P, Cascavilla N, et al. Flow cytometric characterization of human umbilical cord blood lymphocytes: immunophenotypic features. Haematologica 1998; 83:197–203. 63. Joshi SS, Babushkina-Patz NN, Verbik DJ, et al. Antitumor activity of human umbilical cord blood cells: a comparative analysis with peripheral blood and bone marrow cells. Int J Oncol 1998; 13:791–799. 64. Joshi SS, Tarantolo SR, Kuszynski CA, et al. Antitumor therapeutic potential of activated human umbilical cord blood cells against leukemia and breast cancer. Clin Cancer Res 2000; 6:4351–4358. 65. Harris DT. In vitro and in vivo assessment of the graft-versus-leukemia activity of cord blood. Bone Marrow Transplant 1995; 15:17–23. 66. Howrey RP, Martin PL, Driscoll T, et al. Graft-versus-leukemia-induced complete remission following unrelated umbilical cord blood transplantation for acute leukemia. Bone Marrow Transplant 2000; 26:1251–1254.

Transplantation for Childhood Acute Lymphoblastic Leukemia

493

67. Sawczyn KK, Quinones R, Malcolm J, et al. Cord blood transplant in childhood ALL. Pediatr Blood Cancer 2005; 45:964–970. 68. Klingebiel T, Handgretinger R, Lang P, et al. Haploidentical transplantation for acute lymphoblastic leukemia in childhood. Blood Rev 2004; 18:181–192. 69. Lang P, Greil J, Bader P, et al. Long-term outcome after haploidentical stem cell transplantation in children. Blood Cells Mol Dis 2004; 33:281–287. 70. Singhal S, Henslee-Downey PJ, Powles R, et al. Haploidentical versus autologous hematopoietic stem cell transplantation in patients with acute leukemia beyond first remission. Bone Marrow Transplant 2003; 31:889–895. 71. Umeda K, Adachi S, Ishihara H, et al. Successful T-cell-replete peripheral blood stem cell transplantation from HLA-haploidentical microchimeric mother to daughter with refractory acute lymphoblastic leukemia using reduced-intensity conditioning. Bone Marrow Transplant 2003; 31:1061–1063. 72. Klingebiel T, Lang P, Schumm M, et al. Experiences with haploidentical stem cell transplantation in children with acute lymphoblastic leukemia. Pathol Biol (Paris) 2005; 53:159–161. 73. Henslee-Downey PJ. Allogeneic transplantation across major HLA barriers. Best Pract Res Clin Haematol 2001; 14:741–754. 74. Eapen M, Rubinstein P, Zhang MJ, et al. Comparable long-term survival after unrelated and HLAmatched sibling donor hematopoietic stem cell transplantations for acute leukemia in children younger than 18 months. J Clin Oncol 2006; 24:145–151. 75. Marmont AM, Horowitz MM, Gale RP, et al. T-cell depletion of HLA-identical transplants in leukemia. Blood 1991; 78:2120–2130. 76. Barrett AJ, Ringden O, Zhang MJ, et al. Effect of nucleated marrow cell dose on relapse and survival in identical twin bone marrow transplants for leukemia. Blood 2000; 95:3323–3327. 77. Ringden O, Labopin M, Gluckman E, et al. Graft-versus-leukemia effect in allogeneic marrow transplant recipients with acute leukemia is maintained using cyclosporin a combined with methotrexate as prophylaxis. Acute leukemia working party of the European group for blood and marrow transplantation. Bone Marrow Transplant 1996; 18:921–929. 78. Nordlander A, Mattsson J, Ringden O, et al. Graft-versus-host disease is associated with a lower relapse incidence after hematopoietic stem cell transplantation in patients with acute lymphoblastic leukemia. Biol Blood Marrow Transplant 2004; 10:195–203. 79. Locatelli F, Zecca M, Rondelli R, et al. Graft versus host disease prophylaxis with low-dose cyclosporine-A reduces the risk of relapse in children with acute leukemia given HLA-identical sibling bone marrow transplantation: results of a randomized trial. Blood 2000; 95:1572–1579. 80. Teuffel O, Schrauder A, Sykora KW, et al. The impact of cyclosporin A on acute graft-versus-host disease after allogeneic bone marrow transplantation in children and adolescents with acute lymphoblastic leukemia. Bone Marrow Transplant 2005; 36:145–150. 81. Montagna D, Locatelli F, Calcaterra V, et al. Does the emergence and persistence of donor-derived leukaemia-reactive cytotoxic T lymphocytes protect patients given an allogeneic BMT from recurrence? Results of a preliminary study. Bone Marrow Transplant 1998; 22:743–750. 82. Yotnda P, Garcia F, Peuchmaur M, et al. Cytotoxic T cell response against the chimeric ETV6AML1 protein in childhood acute lymphoblastic leukemia. J Clin Invest 1998; 102:455–462. 83. Cardoso AA, Seamon MJ, Afonso HM, et al. Ex vivo generation of human anti-pre-B leukemiaspecific autologous cytolytic T cells. Blood 1997; 90:549–561. 84. Warren EH, Greenberg PD, Riddell SR. Cytotoxic T-lymphocyte-defined human minor histocompatibility antigens with a restricted tissue distribution. Blood 1998; 91:2197–2207. 85. Dolstra H, Fredrix H, Preijers F, et al. Recognition of a B cell leukemia-associated minor histocompatibility antigen by CTL. J Immunol 1997; 158:560–565. 86. Tratkiewicz JA, Szer J. Loss of natural killer activity as an indicator of relapse in acute leukaemia. Clin Exp Immunol 1990; 80:241–246. 87. Lowdell MW, Craston R, Samuel D, et al. Evidence that continued remission in patients treated for acute leukaemia is dependent upon autologous natural killer cells. Br J Haematol 2002; 117:821–827. 88. Ruggeri L, Capanni M, Casucci M, et al. Role of natural killer cell alloreactivity in HLAmismatched hematopoietic stem cell transplantation. Blood 1999; 94:333–339. 89. Leung W, Iyengar R, Turner V, et al. Determinants of anti-leukemia effects of allogeneic NK cells. J Immunol 2004; 172:644–650. 90. Collins RH, Jr., Goldstein S, Giralt S, et al. Donor leukocyte infusions in acute lymphocytic leukemia. Bone Marrow Transplant 2000; 26:511–516.

494

Wall et al.

91. Kolb HJ, Schattenberg A, Goldman JM, et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood 1995; 86:2041–2050. 92. Gorczynska E, Turkiewicz D, Toporski J, et al. Prompt initiation of immunotherapy in children with an increasing number of autologous cells after allogeneic HCT can induce complete donor-type chimerism: a report of 14 children. Bone Marrow Transplant 2004; 33:211–217. 93. Bader P, Kreyenberg H, Hoelle W, et al. Increasing mixed chimerism is an important prognostic factor for unfavorable outcome in children with acute lymphoblastic leukemia after allogeneic stem-cell transplantation: possible role for pre-emptive immunotherapy? J Clin Oncol 2004; 22:1696–1705. 94. Sondel PM, Hank JA, Molenda J, et al. Relapse of host leukemic lymphoblasts following engraftment by an HLA-mismatched marrow transplant: mechanisms of escape from the “graft versus leukemia” effect. Exp Hematol 1985; 13:782–790. 95. Wolff D, Knopp A, Weirich V, et al. Loss of the GVL effect by loss of the Y-chromosome as putative mechanism of immune escape in ALL. Bone Marrow Transplant 2005; 35:101–102. 96. Barbaric D, Wynne K, Aslanian S, et al. Immune evasion strategies of pediatric precursor-B acute lymphoblastic leukemia after allogeneic bone marrow transplantation-a case study. Leuk Res 2005; 29:711–714. 97. Brouwer RE, van der Heiden P, Schreuder GM, et al. Loss or downregulation of HLA class I expression at the allelic level in acute leukemia is infrequent but functionally relevant, and can be restored by interferon. Hum Immunol 2002; 63:200–210. 98. Dermime S, Mavroudis D, Jiang YZ, et al. Immune escape from a graft-versus-leukemia effect may play a role in the relapse of myeloid leukemias following allogeneic bone marrow transplantation. Bone Marrow Transplant 1997; 19:989–999. 99. Molldrem JJ, Lee PP, Kant S, et al. Chronic myelogenous leukemia shapes host immunity by selective deletion of high-avidity leukemia-specific T cells. J Clin Invest 2003; 111:639–647. 100. Wang J, Shaw JL, Mullen CA. Down-regulation of anti-host alloreactivity after bone marrow transplant permits relapse of hematological malignancy. Cancer Res 2002; 62:208–212. 101. Reid GS, Terrett L, Alessandri AJ, et al. Altered patterns of T cell cytokine production induced by relapsed pre-B ALL cells. Leuk Res 2003; 27:1135–1142. 102. Cardoso AA, Schultze JL, Boussiotis VA, et al. Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen. Blood 1996; 88:41–48. 103. Alessandri AJ, Reid GS, Bader SA, et al. ETV6 (TEL)-AML1 pre-B acute lymphoblastic leukaemia cells are associated with a distinct antigen-presenting phenotype. Br J Haematol 2002; 116:266–272. 104. Matulonis U, Dosiou C, Freeman G, et al. B7-1 is superior to B7-2 costimulation in the induction and maintenance of T cell-mediated anti-leukemia immunity. Further evidence that B7-1 and B7-2 are functionally distinct. J Immunol 1996; 156:1126–1131. 105. Nijmeijer BA, van Schie ML, Verzaal P, et al. Responses to donor lymphocyte infusion for acute lymphoblastic leukemia may be determined by both qualitative and quantitative limitations of antileukemic T-cell responses as observed in an animal model for human leukemia. Exp Hematol 2005; 33:1172–1181. 106. Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295:2097–2100. 107. Mengarelli A, Zarcone D, Caruso R, et al. Adhesion molecule expression, clinical features and therapy outcome in childhood acute lymphoblastic leukemia. Leuk Lymphoma 2001; 40:625–630. 108. Reid GS, Bharya S, Klingemann HG, et al. Differential killing of pre-B acute lymphoblastic leukaemia cells by activated NK cells and the NK-92 ci cell line. Clin Exp Immunol 2002; 129:265–271. 109. Romanski A, Bug G, Becker S, et al. Mechanisms of resistance to natural killer cell-mediated cytotoxicity in acute lymphoblastic leukemia. Exp Hematol 2005; 33:344–352. 110. Foa R. Does interleukin-2 have a role in the management of acute leukemia? J Clin Oncol 1993; 11:1817–1825. 111. Parrado A, Rodriguez-Fernandez JM, Casares S, et al. Generation of LAK cells in vitro in patients with acute leukemia. Leukemia 1993; 7:1344–1348. 112. Messina C, Zambello R, Rossetti F, et al. Interleukin-2 before and/or after autologous bone marrow transplantation for pediatric acute leukemia patients. Bone Marrow Transplant 1996; 17:729–735. 113. Maraninchi D, Vey N, Viens P, et al. A phase II study of interleukin-2 in 49 patients with relapsed or refractory acute leukemia. Leuk Lymphoma 1998; 31:343–349. 114. Torelli GF, Guarini A, Maggio R, et al. Expansion of natural killer cells with lytic activity against autologous blasts from adult and pediatric acute lymphoid leukemia patients in complete hematologic remission. Haematologica 2005; 90:785–792.

Transplantation for Childhood Acute Lymphoblastic Leukemia

495

115. Gruber TA, Skelton DC, Kohn DB. Recombinant murine interleukin-12 elicits potent antileukemic immune responses in a murine model of Philadelphia chromosome-positive acute lymphoblastic leukemia. Cancer Gene Ther 2005; 12:818–824. 116. Lang P, Barbin K, Feuchtinger T, et al. Chimeric CD19 antibody mediates cytotoxic activity against leukemic blasts with effector cells from pediatric patients who received T-cell-depleted allografts. Blood 2004; 103:3982–3985. 117. Pfeiffer M, Stanojevic S, Feuchtinger T, et al. Rituximab mediates in vitro antileukemic activity in pediatric patients after allogeneic transplantation. Bone Marrow Transplant 2005; 36:91–97. 118. Imai C, Iwamoto S, Campana D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood 2005; 106:376–383. 119. Cooper LJ, Topp MS, Serrano LM, et al. T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-B-lineage leukemia effect. Blood 2003; 101:1637–1644. 120. Montagna D, Maccario R, Locatelli F, et al. Ex vivo priming for long-term maintenance of antileukemia human cytotoxic T cells suggests a general procedure for adoptive immunotherapy. Blood 2001; 98:3359–3366. 121. Mohty M, Isnardon D, Charbonnier A, et al. Generation of potent T(h)1 responses from patients with lymphoid malignancies after differentiation of B lymphocytes into dendritic-like cells. Int Immunol 2002; 14:741–750. 122. Kohler T, Plettig R, Wetzstein W, et al. Cytokine-driven differentiation of blasts from patients with acute myelogenous and lymphoblastic leukemia into dendritic cells. Stem Cells 2000; 18:139–147. 123. Cardoso AA, Veiga JP, Ghia P, et al. Adoptive T-cell therapy for B-cell acute lymphoblastic leukemia: preclinical studies. Blood 1999; 94:3531–3540. 124. Lee AJ, Haworth C, Hutchinson RM, et al. Enhancement of cALL immunogenicity by co-culture with a CD154 expressing 293 cell line. Clin Exp Immunol 2001; 124:359–368. 125. Todisco E, Gaipa G, Biagi E, et al. CD40 ligand-stimulated B cell precursor leukemic cells elicit interferon-gamma production by autologous bone marrow T cells in childhood acute lymphoblastic leukemia. Leukemia 2002; 16:2046–2054. 126. Reid GS, She K, Terrett L, et al. CpG stimulation of precursor B-lineage acute lymphoblastic leukemia induces a distinct change in costimulatory molecule expression and shifts allogeneic T cells toward a Th1 response. Blood 2005; 105:3641–3647. 127. D’Amico G, Vulcano M, Bugarin C, et al. CD40 activation of BCP-ALL cells generates IL-10producing, IL-12-defective APC’s that induce allogeneic T-cell anergy. Blood 2004; 104:744–751. 128. Dudley ME, Wunderlich JR, Robbins PF, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 2002; 298:850–854. 129. Xiaoling G, Ying L, Jing L, et al. Induction of anti B-cell malignance CTL response by subfamilyshared peptides derived from variable domain of immunoglobulin heavy chain. Cancer Immunol Immunother 2005; 54:1106–1114. 130. Kircher B, Wolf M, Stevanovic S, et al. Hematopoietic lineage-restricted minor histocompatibility antigen HA-1 in graft-versus-leukemia activity after donor lymphocyte infusion. J Immunother 2004; 27:156–160. 131. de Rijke B, Fredrix H, Zoetbrood A, et al. Generation of autologous cytotoxic and helper T-cell responses against the B-cell leukemia-associated antigen HB-1: relevance for precursor B-ALLspecific immunotherapy. Blood 2003; 102:2885–2891. 132. Rousseau RF, Biagi E, Dutour A, et al. Immunotherapy of high-risk acute leukemia with a recipient (autologous) vaccine expressing transgenic human CD40L and IL-2 after chemotherapy and allogeneic stem cell transplantation. Blood 2006; 107:1332–1341. 133. Vereecque R, Buffenoir G, Preudhomme C, et al. Gene transfer of GM-CSF, CD80 and CD154 cDNA enhances survival in a murine model of acute leukemia with persistence of a minimal residual disease. Gene Ther 2000; 7:1312–1316. 134. Stripecke R, Cardoso AA, Pepper KA, et al. Lentiviral vectors for efficient delivery of CD80 and granulocyte-macrophage-colony-stimulating factor in human acute lymphoblastic leukemia and acute myeloid leukemia cells to induce antileukemic immune responses. Blood 2000; 96:1317–1326. 135. Montagna D, Maccario R, Montini E, et al. Generation and ex vivo expansion of cytotoxic T lymphocytes directed toward different types of leukemia or myelodysplastic cells using both HLAmatched and partially matched donors. Exp Hematol 2003; 31:1031–1038. 136. Haining WN, Cardoso AA, Keczkemethy HL, et al. Failure to define window of time for autologous tumor vaccination in patients with newly diagnosed or relapsed acute lymphoblastic leukemia. Exp Hematol 2005; 33:286–294.

25 Bone Marrow Transplantation for Acute Myeloid Leukemia in Children Allen R. Chen and Robert J. Arceci Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins University, Baltimore, Maryland, U.S.A.

HISTORICAL BACKGROUND Acute myeloid leukemia (AML) was one of the first indications for bone marrow transplantation (BMT). The Seattle experience with HLA-identical sibling BMT for refractory acute leukemia from 1971 to 1975 included 54 patients with AML (1). Of this initial cohort, seven patients survived more than 18 months after BMT, including five very long-term survivors who remained in continuous complete remission 20 years after BMT. These results demonstrated the curative potential of high-dose cyclophosphamide (Cy) and total body irradiation (TBI) with marrow transplantation for chemotherapy-refractory AML. Applying BMT earlier in the course of disease was expected to improve results because the leukemia would be less resistant and patients in better clinical condition would likely experience reduced transplant-related mortality. For patients with AML in first remission, matched sibling BMT has produced long-term overall survival (OS) of 50–60%. Another milestone was the development of the busulfan (Bu)/Cy preparative regimen in an effort to make BMT more accessible and in the hope of avoiding some of the irreversible endorgan toxicities associated with TBI (2,3). Most patients lack an HLA-identical sibling who can serve as a marrow donor. For these patients, autologous transplantation using marrow cryopreserved in first remission was developed as an method to allow the use of marrow ablative therapy as consolidation (4). The National Marrow Donor Program was founded in 1987 to establish a registry of volunteer donors willing to provide marrow for unrelated recipients who were phenotypically matched for the MHC antigens. As the registry has grown to more than five million donors, the probability of finding at least one suitably matched unrelated donor has increased to approximately 30%. However, for many patients, particularly those of mixed ethnicity, a suitably HLA-matched unrelated donor cannot be identified. Placental blood, although of limited volume, is rich in cells of particularly high repopulating activity, and the reduced immunologic activity of these products allows their use with a greater degree of HLA disparity (5). Nonablative BMT was developed as a way to provide the benefits of adoptive allogeneic immunotherapy for patients too old or too sick to undergo standard BMT (6,7). This strategy may prove fruitful when applied to patients with more favorable prognosis as a way to reduce the mortality and long-term adverse effects of BMT. 497

498

Chen and Arceci

RISK GROUPS AND PROGNOSTIC FACTORS To date, the various cooperative groups have not produced consensus stratification of risk groups among patients with AML. Some differences in the factors identified as prognostically significant among trials may reflect inadequate sample size for sensitive subgroup analyses. On the other hand, there may be interactions between therapy and prognostic factors, so differences in prognosis from one trial to another may generate hypotheses regarding preferred therapeutic approaches for subgroups of patients with specific prognostic factors. For example, presentation in infancy does not carry the same prognostic importance in AML as in ALL. The Pediatric Oncology Group (POG) showed that age under two was associated with significantly poorer prognosis (8). The Berlin-Frankfurt-Mu¨nster (BFM) study group also found that children under two had a poorer prognosis, but this difference did not persist after stratifying by morphologically defined risk group (9). However, given more recent, intensified therapeutic approaches such as the Medical Research Council (MRC) 10 trial and the Children’s Cancer Group (CCG) intensively timed DCTER (CCG-2891 Trial), infants have not fared worse. Infants with AML treated intensively in the Japan Infant Leukemia Study Group had 72% 3-year event-free survival (EFS), censoring at the time of allogeneic BMT (10), and all six patients who received allogeneic BMT remain in remission; the small number of patients in this study precludes a definitive conclusion that the results are superior to other trials using intensive treatment approaches (11). In the MRC 10 trial, on which children and adults up to age 55 were treated, cytogenetics were identified as the most important prognostic factor, and patients with t(8;21), t(15;17), and inv(16) had a favorable prognosis with respect to response to induction, freedom from relapse, and survival (12). An analysis of the POG 8821 study, excluding patients with t(15;17) because they were known to benefit from treatment with all-trans retinoic acid (ATRA), showed that patients with the t(8;21) and inv(16) had significantly better EFS than others (8). An earlier BFM analysis identified a low-risk group on the basis of morphology, including the FAB types with granulocytic differentiation and specific additional features: FAB M1 with Auer rods, FAB M2 with white blood cell count of less than 20!109/L, all patients with FAB M3, and FAB M4 with eosinophilia (13); M3 promyelocytic morphology is associated with the t(15;17) translocation, whereas the other phenotypes are associated with core binding factor leukemias, characterized by the t(8;21) and inv(16) cytogenetics. However, these cytogenetic abnormalities did not confer a significant survival advantage in the CCG studies using intensively timed DCTER induction chemotherapy (14) that was based on a continuous infusion of low-dose cytarabine, although there was a trend toward improvement noted. Moreover, the St. Jude experience suggests that the outcome of patients with inv(16) improved when high-dose cytarabine was added to their treatment regimen (15). Taken together, these findings suggest that the chemotherapy regimen interacts with the biologic features of the disease to affect prognosis. The first molecular mechanism identified in adults as a poor prognostic feature in AML was the expression level of the multidrug resistance energy-dependent transport protein, MDR1; however, while it is correlated with functional drug efflux, there are many discordant cases (16) that can be explained by the presence of other transport mechanisms. In addition, the CCG could not confirm the prognostic value of MDR1 expression (17), suggesting that this mechanism may be overcome by intensively timed infusional chemotherapy. The search for molecular abnormalities that identify patients with poorer prognosis has uncovered new potential targets for therapy. An internal tandem duplication of FLT3, the receptor for FLT3 ligand, is associated with poorer survival (18). This mechanism produces chemotherapy resistance in vitro that can be overcome by specific small molecule inhibitors (19). However, these specific mutations affect a relatively small proportion of patients (18). Activating mutations in receptor tyrosine kinases and RAS signal transduction pathways are common in aggregate (46%) and are associated with poor prognosis in pediatric AML, with the exception of activating loop mutations of FLT3 (20).

BMT for Acute Myeloid Leukemia

499

Prognosis Among Transplanted Patients Disease status is the paramount prognostic factor for patients undergoing BMT for AML. It is consistently significant for all sources of marrow, including unrelated donors (21,22) and cord blood (23).

Impact of Prior Chemotherapy The CCG has demonstrated that the intensity of the induction regimen affects the prognosis after subsequent allogeneic or autologous BMT (24). All patients with HLA-identical siblings or 5/6 antigen matched first degree relatives were assigned to consolidation with BMT in CR1, after four cycles (two courses) of DCTER (dexamethasone, cytarabine, thioguanine, etoposide, and daunorubicin) induction chemotherapy. Patients randomized to intensively timed DCTER induction had significantly better EFS after allogeneic BMT than those randomized to receive the same induction with standard timing. This result may be explained by several models: any intensification of induction therapy may reduce the total burden of leukemic cells; the less intensive, standard timed induction may select for resistant cells that are cross-resistant to transplant therapy; or there may be a critical interval after the first cycle of chemotherapy during which leukemic stem cells are induced to cycle and therefore are sensitized to the second cycle of chemotherapy in a timed-sequential regimen. Additional support for the first model comes from a small institutional study that suggested that the detection of minimal residual disease (MRD) by multiparameter flow cytometry immediately before BMT was associated with a higher relapse rate (46% vs. 25%) among 21 patients undergoing BMT for acute leukemia & MDS (25). When the analysis was restricted to patients with AML, OS was 33% for those with negative multiparameter flow cytometry versus 0% for those with detectable MRD. The conclusions of this study must be regarded as tentative given the small number of patients and the numerous confounding factors, including different remission status, preparative regimens, graft-versus-host disease (GVHD) prophylactic regimens, and the inclusion of both autologous and allogeneic BMTs. The Hoˆpital St.-Louis in Paris retrospectively analyzed their results in patients with AML receiving HLA-identical sibling BMTs in 1st or 2nd remission to identify risk factors for nonrelapse mortality (NRM). Gram negative bacteremia before transplantation was identified as a significant risk factor for poorer EFS. In addition, among patients transplanted in first remission, a greater number of courses of chemotherapy was associated with significantly poorer EFS (RR 4.1) (26). Although this observation was partly explained by greater NRM in patients who were more heavily pretreated, it also reflects greater risk for relapse in patients selected for more resistant disease, either because they required more cycles of chemotherapy to achieve a remission or because the chemotherapy itself selected for resistant disease, consistent with the second model. For patients being treated for recurrent AML after primary therapy on the BFM 87 or 93 protocols, the duration of first remission predicted both the likelihood of achieving a second remission and long-term survival after BMT. In a dichotomized analysis, patients whose first remission exceeded 1.5 year enjoyed 40% 5-year survival, as compared to 10% 5-year survival for those with shorter first remissions (27). Prognostic Factors Present at Diagnosis and Bone Marrow Transplantation Outcomes Few studies have examined the prognostic value of factors present at initial diagnosis on ultimate outcome after transplantation. Such studies are of interest for several reasons. First, they provide the basis for stratified analysis so that transplant outcomes can be fairly compared across different cohorts of patients. Second, they allow assessment of the impact of patient selection in studies in which not all patients are treated as intended. Importantly, the comparison of factors that are prognostically important for patients who are treated with

500

Chen and Arceci

conventional chemotherapy to those that are prognostically important for patients who treated with BMT can provide insight into which subsets of patients who benefit most and least from BMT. That is, if all patients benefit equally from BMT, the same factors would predict outcomes after BMT and after conventional chemotherapy. However, if BMT is effective for a subset of patients with high-risk disease, then the factor that identified that subgroup as “highrisk” for conventional chemotherapy will lose its significance among BMT patients, and there will be a statistical interaction between type of treatment and prognostic factors. The best available data of this type come from the European BMT group, which reviewed the results of BMT for AML in CR1 from 1980–1995. This study has the virtue of large numbers, encompassing 846 autologous and 826 matched sibling BMTs in patients of all ages. Cytogenetic results were categorized as “good” if t(15;17), t(8;21), or inv(16) karyotypic abnormalities were present; “poor” if an abnormality of chromosome 5 and/or 7 was identified, or if there was an abnormality of 11q, or hypodiploidy; all others were categorized as “standard” (28). EFS (46%) was identical for patients with “good” or “standard” cytogenetics receiving autografts. Among patients receiving allografts, those with “good” cytogenetics fared slightly better than those with “standard” cytogenetics, with 62% vs. 58% EFS and 12% vs. 24% incidence of relapse, respectively. “Poor” cytogenetics was associated with significantly worse EFS regardless of transplant type, with 26% EFS after autologous and 27% EFS after allogeneic BMT. The incidence of relapse was 73% vs. 49% for patients receiving autografts and 62% vs. 24% for patients receiving allografts with “poor” cytogenetics as compared to standard cytogenetics. In multivariate analysis, longer interval from diagnosis to CR1 than 39 days was significantly associated with poorer EFS after both autologous (RR 1.25, p!0.05) and allogeneic BMT (RR 1.5, p!0.05). The only analysis specific for children comes from the Italian Association of Pediatric Hematology Oncology (l’Associazione Italiana Ematologia Oncologia Pediatrica or AIEOP) and the Italian Bone Marrow Transplant Group (Gruppo Italiano Trapianto di Midollo Osseo). This study reported results for all children ages 1–15 year who received matched sibling or identical twin BMT for AML in CR1 in Italy in the 1980s. Among 59 patients, relapse-free survival was 58% with 5–11 year follow-up, and the principal factor associated with lower RFS was high white blood cell (WBC) at initial diagnosis in an analysis dichotomized as above or below 14,000 (29). FAB type, age, time to achieve remission, and year of BMT were not significant, but the number of patients in the study limited its power to detect small to moderatesized effects. Thus, it would be desirable to analyze the North American results with respect to prognostic factors present at initial diagnosis among patients receiving BMT versus those receiving conventional chemotherapy. If the results of the European BMT group are confirmed, one conclusion would be that patients with “good” cytogenetics benefit least from BMT in first remission.

Prognosis in Secondary and Therapy-Related Acute Myeloid Leukemia Therapy-related AML carries a poor prognosis, even if treated with BMT (30,31). Of 21 patients who received allogeneic BMT for therapy-related AML after treatment for a lymphoid malignancy at St. Jude between 1990 and 1997, only four survive long-term (3-year DFS, 19%). This study provided no evidence that induction therapy benefits such patients: 2/13 patients who received induction chemotherapy and 2/8 patients who proceeded directly to BMT were among the long-term survivors (30). Similarly, the Seattle experience in therapy-related and secondary AML revealed no difference in outcome comparing 46 previously untreated patients with 20 patients who had responded to induction chemotherapy (although 11 of these patients were transplanted in first untreated relapse) (31). In contrast, a French Society of Pediatric Oncology (SFOP) analysis of 70 adolescent and younger adult patients with therapy-related MDS or AML found that chemotherapy prior to BMT was favorable (32). In this series, 33 patients who had received chemotherapy (24 of whom achieved CR) had an EFS of about 40% (45% if they achieved CR), versus under 20% among 37 patients who had not

BMT for Acute Myeloid Leukemia

501

received induction chemotherapy. In addition, younger age (HR 4.5 for age O37), CR, and cytogenetics without chromosome 7 or complex abnormalities were all favorable and each separated a population with 40–50% 2-year EFS from a remaining population with 15–20% EFS. Intensive preparative regimens cannot be recommended, at least in this older patient population, because they were associated with increased treatment related mortality (TRM) (HR 2.45, p!0.05) without any improvement in the relapse rate (32). The EBMT has surveyed the results of matched sibling (33) and unrelated donor (34) BMT for MDS and secondary AML in patients of all ages from infancy to age 55. The matched sibling BMT analysis was restricted to patients who had not received prior chemotherapy. Regardless of donor type, younger age was favorable due to lower TRM. For patients receiving matched sibling BMTs, DFS was 45% among patients through age 20 as compared to 32% for patients ages 21–40. For patients with unrelated donors, DFS was 41% for patients under 18, with 40% TRM, as compared to 28% DFS among patients of all ages, with 70% TRM. A shorter duration from diagnosis to BMT was favorable, confirming the results of an earlier single-institutional analysis (35). For patients receiving unrelated donor BMT, there was a monotonic decrease in DFS with time from diagnosis to BMT, from 55% with an interval !6 months, to 34% with an interval of 6–12 months, to 16% when the interval was O12 months, p!0.05. For patients receiving matched sibling BMT, there was a biphasic relationship between interval from diagnosis to BMT and outcome. DFS was 54% when the interval was less than 4 months, 27% with an interval between 4 and 12 months, and 43% DFS with an interval greater than 12 months. A shorter interval from diagnosis to BMT was associated with much less TRM (29% vs. 51–58% for the various FAB morphologic types of MDS), but a longer interval from diagnosis to BMT in the matched sibling setting was associated with a lower relapse rate (14% vs. 46–51%), perhaps due to temporal selection for less aggressive disease (33). Both acute and chronic GVHD were associated with a lower relapse rate (26% with GVHD vs. 41–42% without) in the unrelated donor setting (34). The greater likelihood of GVHD after unrelated donor BMT may be associated with a more potent graft-versus-leukemia (GVL) effect and account for the lack of reduction in risk of relapse with longer interval from diagnosis to BMT in this setting. On the basis of these results, BMT can be recommended as soon as feasible for pediatric patients with MDS or secondary AML. Induction chemotherapy is probably required for those patients with secondary AML to facilitate a donor search, but otherwise may not be indicated.

PREPARATIVE REGIMENS Total Body Irradiation-Based Regimens The first regimen used to prepare patients with AML for BMT consisted of Cy with TBI (1,36). To find the optimal dosage of TBI for AML, the Seattle group conducted a trial that randomized patients to 12 Cy vs. 15.75 Cy TBI in the context of HLA-identical sibling BMT with cyclosporine and methotrexate as GVHD prophylaxis (37). Although the higher dose of TBI was associated with reduced risk of relapse, the benefit was offset by an increased risk of transplant-related mortality, resulting in equal long-term survival. A retrospective analysis of TBI dosage in a single-institutional experience that was heterogeneous with respect to diagnosis, disease status, additional agents used in combination with TBI, and the use of T-cell depletion (TCD) produced similar results, but there was a trend toward worse survival with higher doses of TBI, that ranged from 10 Cy to 13.5 Cy (38). A randomized prospective trial was conducted comparing a regimen that substituted melphalan for Cy in 63 patients with AML in first complete remission receiving HLA-identical sibling BMT (39). The overall relapse-free survival on the two arms was essentially identical, 55% for patients receiving melphalan/TBI and 53% for patients receiving Cy/TBI. However, there was a substantial difference in the cause of treatment failure between the two arms that did not achieve statistical significance: the actuarial probability of remaining in remission was 94%

502

Chen and Arceci

in the melphalan/TBI arm vs. 66% in the Cy/TBI arm. This benefit was offset by greater regimen-related mortality, with a high incidence of nephrotoxicity associated with the combination of melphalan and cyclosporine. If this nephrotoxicity is preventable, and the superior antileukemic activity of melphalan is confirmed, the use of melphalan may be an approach to improve the results of TBI-based preparative regimens for AML. Another alternative TBI-based preparative regimen used etoposide after TBI and was also active in a variety of hematologic malignancies (40). Children have fared particularly well with this regimen, with 84% 7-year EFS in an institutional cohort of 33 patients with early highrisk hematologic malignancies, including 18 patients with AML in CR1 (41). Although a randomized controlled study was conducted comparing TBI/etoposide to TBI/Cy (42), the results have been published only in abstract form. Attempts to improve the delivery of TBI to increase the homogeneity of dosing have included more complex field planning (43) and higher energy sources (44). In addition, the use of monoclonal antibodies to target radioisotopes to the marrow space has been tested in pilot fashion in AML, with encouraging preliminary results (45,46).

Busulfan-Based Regimens The Bu/Cy regimen was first reported in the early 1980s as an alternative to radiation-based preparative regimens and offered easier administration, as well as the potential to avoid radiation effects, such as cataracts, secondary malignancies, interstitial pneumonitis, growth failure, and other endocrinologic disturbances (47). A common variant of this regimen reduced the Cy from 200 mg/kg (50 mg/kg/day!4 days) to 120 mg/kg (60 mg/kg/day!2 days) in an effort to reduce toxicity without affecting efficacy (48). Substitution of melphalan (60–70 mg/ m2/day!3 days) for Cy in combination with standard oral Bu has been piloted in a singleinstitution study for children with acute leukemia undergoing HLA-identical sibling BMT primarily in CR1 (49). The results were encouraging, with 90% 5-year EFS and relatively mild late effects, including height Z scores that decreased from -0.47 to -0.98 at a median of 4.2 (2.6–8.4) years after BMT. As in the case of TBI-based preparative regimens, this pilot experience suggests that melphalan may have superior antileukemic efficacy as compared with Cy.

Pharmacokinetic Targeting The rationale for pharmacokinetic adjustment of Bu dosing is that (1) there is substantial interindividual variation in exposure; (2) the therapeutic index is narrow; (3) pharmacokinetic parameters correlate with toxicity; and (4) pharmacokinetic parameters correlate with efficacy. There is substantial interindividual variation in the exposure of patients to Bu when a standard dose is calculated on the basis of weight. In children, bioavailability varies fivefold between individuals (50), and clearance varies six- to twenty-fold (51–55). Moreover, dosing designed for adults is not optimal for children. Young children were noted to have significantly less toxicity and less therapeutic efficacy than adults treated with a standard Bu/Cy regimen, and this difference was associated with significantly lower Bu AUC in young children as compared to older children and adults (3). This study demonstrated that for children under age 6, a dose of 160 mg/m2/day PO divided every 6 hours produced an AUC equivalent to the standard dosage for adults, 4 mg/kg/day orally divided every 6 hours. This finding was confirmed using a regimen of single daily oral dosing, in which a dose of 150 mg/m2/day PO avoided lower systemic exposure in younger children (52). These dosing guidelines do not substitute for therapeutic drug monitoring of Bu. Bu clearance was higher in children under age 10, regardless of normalization to actual weight, ideal body weight, or body surface area (56). In addition, although clearance and volume of distribution are significantly associated with patient age, the coefficient of determination is modest, 0.04–0.12, indicating that only 4–12% of the variance in the major

BMT for Acute Myeloid Leukemia

503

pharmacokinetic parameters is explained by age, when patients between 2 months and 59 years were studied (57). Higher exposure to Bu, as measured by average steady state concentration, is significantly correlated with regimen-related toxicity (RRT) (rZ0.72) (56) and is associated with 100-day mortality (58). Of note, threshold exposure levels of Bu have been identified, above which adults experience an increased risk for veno-occlusive disease (VOD) of the liver (59,60). In contrast, in a study of 61 children, of whom 24 developed reversible VOD, the patients who developed VOD tended to have higher Bu exposure (AUC 6811C/K 2943 ng*hr/ml) than patients who did not develop VOD (5760C/K 1891 ng*hr/ml, pZ0.10), but no toxic level could be identified (53). This study was limited by lack of standardization of the preparative regimen. Pharmacokinetic parameters of Bu exposure are also correlated with efficacy. A large retrospective analysis showed a highly significant correlation between Bu 9 exposure and engraftment. A threshold average steady-state concentration (C ss ) of 200 ng/ml was sufficient for engraftment of HLA-identical sibling marrow, and a threshold C ss of 600 ng/ml was required for engraftment of marrow from unrelated donors or partially matched related donors (56). In a prospective trial in children undergoing BMT, pharmacokinetics were performed on a test dose of Bu prior to admission, and the results were used to adjust Bu doses for a target Bu C ss of 600–900 ng/ml. Noteworthy were the success rate of 94% of patients achieving target concentrations and the 2.7-fold range of doses required, from 10.9 to 29 mg/kg/course. The rate of engraftment improved from 74% in historical controls to 94% on the study, with 21% grade 3 or 4 RRT (61). Given the goal of achieving a target Bu exposure, what is the most efficient approach? Limited sampling methods have advantages of lower cost and feasibility in young children in whom large volume blood drawing is contraindicated. A method to determine the AUC by combining three Bu concentration measurements with Bayesian prior assumptions has been validated and very strongly correlates with standard, 9-sample pharmacokinetics (Pearson rZ0.98) (62). Another trial of a test dose strategy indicated that only 6/29 (21%) patients receiving a standard dose of 1 mg/kg would have achieved a steady state AUC within the therapeutic window of 3600–5400 ng*hr/ml (C ss 600–900 ng/ml), as compared to 28% using pharmacokinetic monitoring after a test dose and 45% using pharmacokinetic measurements after their first dose (63). The IV administration of Bu avoids the uncertainties engendered by delayed absorption or emesis after oral Bu. The contribution of oral bioavailability to interpatient variability is eliminated, and 86% of adults achieved an AUC between 800 and 1500 mMol*min after Bu 0.8 mg/kg/dose IV (64). However, even with IV Bu, the risk of toxicity is related to systemic exposure. In patients with CML undergoing matched sibling BMT, the probability of toxicities including gastrointestinal (GI) toxicity, hepatotoxicity, mucositis, and aGVHD all increased with increasing Bu AUC (65). In children receiving IV Bu with Cy as the preparative regimen for allogeneic BMT, a retrospective population pharmacokinetic analysis showed very little intraindividual variation of Bu exposure (CV 9%) (66). Nevertheless, the interindividual variability was substantial, with a CV 56% because of a significant log-linear response to body weight. The authors therefore propose that instead of dosing Bu as a constant multiple of the patient’s weight, the dosage of Bu should differ for various weight ranges. Although their simulation suggests that this approach would reduce the CV to 19%, this model awaits validation by an independent dataset.

Comparisons of Total Body Irradiation and Busulfan There has been a strong desire to avoid the use of TBI in preparative regimens for young children, primarily because of concerns about long-term effects on the developing brain. Of particular relevance is the retrospective comparison of single-fraction TBI vs. Bu/Cy in 44 children under 7 years old with hematologic malignancies conducted at Bristol Hospital, U.K. (67). Overall survival was 43.3% for patients who received TBI as compared with 33.3%

504

Chen and Arceci

among patients treated with Bu/Cy (pZ0.6). It appeared that TBI produced better survival for patients with high-risk disease. There were different patterns of long-term effects, including more hypothyroidism, GH deficiency, learning problems, and cataract formation with TBI. However, patients who received Bu were at greater risk of early cardiac toxicity, behavioral problems, and seizures. Because young children have higher Bu clearance per kg than adults, it is possible that the efficacy of Bu would have been improved by pharmacokinetic targeting but equally possible that the endocrinologic and neurodevelopmental effects would have increased to match the results of TBI. Because patients were not randomly assigned to these regimens, patient selection bias may also contribute to apparent differences. Small institutional studies have produced conflicting results regarding the efficacy of TBI as compared to Bu. In the Huddinge Hospital cohort, a fractionated TBI regimen was associated with a higher risk of relapse after allogeneic BMT than Bu in 180 patients with hematological malignancies (44% vs. 16% respectively, pZ0.01) (68). In contrast, the University of Minnesota conducted a randomized trial of Cy/TBI versus Bu/Cy that enrolled 35 patients with AML in CR1 or after relapse. This study revealed clinically important but statistically insignificant differences in favor of TBI, with 2-year DFS of 50% versus 24% (pZ0.12) and a relapse rate of 43% versus 70% (pZ0.17). A particularly significant advantage for TBI was observed for patients with more advanced disease beyond CR1, with a DFS of 42% versus 9% compared to Bu (pZ0.07) (69). By combining these patients with additional patients who were nonrandomly assigned to TBI or Bu containing preparative regimens for 4HC-purged autologous BMT, they were able to demonstrate a statistically significant survival advantage and with less VOD among patients prepared with TBI-based preparative regimens (70). To resolve these conflicting results from single institutions, the EBMTR has addressed the effectiveness of TBI in a very large retrospective analysis of 1672 patients transplanted for AML in CR1 1980–1995, including patients of all ages, from 1–69 years old. The population was equally divided, with 846 autologous transplants and 826 matched sibling BMT. Among allogeneic BMT patients, their multivariate analysis showed that receiving a non-TBI preparative regimen was associated with significantly greater risk of relapse, a RR of 2.56, p!0.01 (28). The two major limitations of this study were the nonrandom assignment of patients and the variations in technique among institutions. Selection bias was in part mitigated by the fact that the most important risk factor for relapse is status of disease, and the analysis was restricted to patients in first CR. In addition, there were sufficient patients to adjust for major confounding risk factors in the multivariate analysis. However, young children would have been disproportionately assigned to Bu due to investigator bias, without a comparable population treated with TBI. With respect to variations in technique, this study accurately reflects the effectiveness of the preparative regimens as practiced, and there is little risk of misclassification of the endpoint. An important meta-analysis (71) has combined the primary data of four prospective randomized controlled trials that compared Cy/TBI with Bu/Cy for patients with myeloid leukemia and extended the follow-up to a median of 7 years. Unfortunately, only two of the studies enrolled patients with AML, younger patients were nonrandomly assigned to receive Bu, and there was no pharmacokinetic targeting of Bu (72,73). All patients received Cy 120 mg/kg/course IV over 2 days as part of the preparative regimen. This study showed that survivors generally enjoyed good performance status regardless of preparative regimen: 88% and 85% of survivors were working after Bu and TBI, respectively. However, this study does not address whether differences in developmental outcome would appear in patients treated early in childhood. Late effects were comparable except that cataracts were associated with TBI, and irreversible alopecia was associated with Bu. The duration of follow-up was not sufficient to comment on the relative risk of second malignant neoplasms. There was a trend toward better 10-year survival in patients treated with TBI (63% vs. 51%, not statistically significant) compared to patients treated with Bu. This trend was attributable to more TRM in Bu-treated patients in one cohort (73) and more relapse in the other study (72). Thus, one may speculate that pharmacokinetic targeting of Bu may eliminate any difference in survival outcomes as compared to TBI.

BMT for Acute Myeloid Leukemia

505

Intensification of Regimens Attempts to combine Bu with TBI have shown the combination to be active but toxic. Bu (7 mg/kg/course) was combined with TBI (12 Gy) in a single-institution study for MDS and secondary AML that included 60 adult and pediatric patients with a median age of 40 years (range, 12–62). Twenty patients had related donors and 40 had unrelated donors. The 100-day NRM of 38% far exceeded the 25% cumulative incidence of relapse, with the result that 3-year overall survival was 26% (74), no better than the results of a standard Cy/TBI preparative regimen. A 20-patient pilot trial of TBI/Bu/Mel in children with high-risk hematologic malignancies was successful for matched sibling BMT, with 9/12 patients alive, but NRM was high among the eight patients who received grafts from unrelated or mismatched donors (75). The most successful trial of this type was a single institutional study in Japan that combined Bu (8 mg/kg) and TBI (10 Gy) with Cy (120 mg/kg) for patients with myeloid leukemia undergoing allogeneic BMT (76). A total of 50 patients, ages 3–52 years old, were enrolled, including 28 patients with AML, only 9 of whom were in CR1. The cumulative incidence of relapse at 5 years was 0% among patients in CR1 and 28% in those with more advanced disease, so although there was significant RRT with 14% NRM, the results were encouraging with 85% 5-year survival in CR1 and 50% for patients in CR2 or greater. Because of the relatively small numbers of patients, the confidence intervals around these survival estimates are wide, so there is not strong evidence that the true effectiveness of this regimen is very different from other similar intensified Bu/TBI regimens. Addition of a third alkylating agent is justified on the basis that alkylating agents are generally not cross-resistant. However, it is difficult because of overlapping nonhematopoietic toxicity. Addition of thiotepa, 5 mg/kg/day!2 days, to a standard Bu 16/Cy 120, regimen was tested in patients with acute leukemia undergoing autologous BMT. Most of the patients were in remission, including 26 with AML and 2 with ALL, whereas 5 patients were transplanted in early relapse (1 AML, 4 ALL). The addition of thiotepa did not reduce the relapse rate (61% vs. 52% in historical controls) or improve survival (33% vs. 38% in historical controls) (77). A similar single institution study altered the Bu/Cy 120 regimen by reducing the Bu to 12 doses and incorporating a significantly higher dose of thiotepa, 250 mg/m2/day!3 days (78). Children with high-risk myeloid disease were eligible, including those with active disease, secondary MDS/AML, or second or later remission. Seven of 17 patients had alternative donors, and all engrafted. Nine patients had active disease, and all achieved remission. EFS was 51% at 3 years, with only one relapse, and 41% TRM, primarily due to infection. Given the high-risk patient population studied, the EFS is encouraging and suggests that the regimen warrants additional consideration, if the TRM can be reduced. An alternative strategy is to add an agent that is synergistic with the alkylating agents that form the backbone of the regimen. Topoisomerase inhibitors interfere with DNA repair after damage by alkylating agents, so although they are schedule dependent and cannot be escalated to marrow ablative doses, the Bu/Cy regimen has been intensified by the addition of etoposide to full doses of Bu (640 mg/m2/course over 16 doses) and Cy (120–150 mg/kg/course in two doses). In a Phase I study conducted by the Pediatric Blood and Marrow Transplant Consortium, the dose limiting toxicity was hepatic toxicity, including both VOD and other patterns of hepatic toxicity, and the maximum tolerated dose (MTD) of etoposide was 60 mg/kg/day, given as a single IV dose. The regimen produced good survival in allogeneic (8/14) but not autologous (1/6) BMT, a disappointing result because the purpose of intensifying preparative regimen is to reduce the relapse rate in the absence of an immunologic graft-versusleukemia effect (79). Similarly, both Bu/Cy and Cy/TBI have been intensified by the addition of idarubicin, 21 mg/m2/day!2 days, for unpurged autologous BMT for AML in CR1. Compared to historical controls, the relapse rate was significantly lower than expected (7% vs. 45%, p!0.05), and survival was markedly improved (82% vs. 46%, p!0.05) (80). These results warrant larger scale testing of this regimen. Unfortunately, many patients have had substantial cumulative anthracycline exposure prior to BMT.

506

Chen and Arceci

Nonmyeloablative Regimens The concept of nonmyeloablative BMT was pioneered by Storb and colleagues in the canine model, based on the premise that because host-versus-graft and graft-versus-host reactions are mediated by T cells, improving posttransplant immune suppression would control both reactions and allow reduction in the intensity of the preparative regimen (81). Reduction in preparative regimen intensity was expected to be advantageous for patients too ill or too old to undergo standard BMT, provided that their leukemia is susceptible to immunologic GVL effector cells. This approach has been validated extensively in adults (7), but few data have been published in pediatric AML to date. The morbidity and NRM associated with HLAmatched related BMT were significantly less in patients treated with nonmyeloablative preparative regimens, as compared to those treated with standard ablative preparative regimens. This is a remarkable success, considering that most patients treated with nonmyeloablative preparative regimens were ineligible for standard ablative preparative regimens because of advanced age or poor clinical condition (82,83). In addition, less grade II–IV acute GVHD (aGVHD) (12% vs. 36%) and less chronic (cGVHD) (14% vs. 40%) was observed after nonmyeloablative preparative regimens (83), perhaps because the reduction in tissue injury leads to a reduction in inflammatory mediators that activate antigen-presenting cells. A retrospective analysis by the Cooperative German Transplant Study Group indicated that among patients ineligible for ablative BMT the strongest prognostic factor for survival is the percentage of blasts; other significant prognostic factors were performance status and matched sibling versus alternative donor (84). This observation suggests the hypothesis that nonmyeloablative transplantation may be particularly useful for patients with myelodysplastic syndromes.

ALLOGENEIC TRANSPLANTATION Allogeneic Effect The seminal observation demonstrating the existence of an immunologic graft-versus-leukemia effect was the recognition of an inverse relationship between the development of either acute or chronic GVHD and subsequent risk of relapse (85,86). This observation has been extended specifically to children with AML. In the CCG protocol 2891, which assigned patients with a matched related donor to BMT in first complete remission, grade one or two aGVHD was associated with superior relapse-free survival, as compared to patients without GVHD (87). Moreover, the relapse rate is significantly less in recipients of allogeneic BMT, as compared with syngeneic BMT, 62% vs. 75%, p!0.0001 (88). This allogeneic effect has been validated in a large analysis of the IBMTR, and overt GVHD is not required for a reduction in the relapse rate in recipients of BMT from allogeneic as compared to syngeneic donors, 16% vs. 52%, p!0.001 (89,90). The most direct demonstration of potency of alloreactive immune cells is that patients who relapse after allogeneic BMT can sometimes be induced into remission by simple infusions of lymphocytes from their marrow donors (91). Considerable attention has therefore focused on the potential to dissociate GVL from GVHD and to augment GVL effects. Early studies focused on the effector cells, such as CTL precursors in the circulation of children following allogeneic BMT for acute leukemia. These CTL precursors were assayed by stimulating them in culture with autologous leukemic blasts and measuring specific cytotoxicity against leukemic blasts, in comparison to such control cells as lymphoblastoid cell lines, PHA-stimulated blasts, or bone marrow cells collected in remission. The leukemia-specific CTL precursor cell frequency was expanded more than an order of magnitude compared with the donor circulation, and there was no correlation between CTL precursor frequency against leukemic blasts and aGVHD. Of note, the CTL precursor frequency against autologous leukemia declined a few weeks before relapse in the one patient who relapsed in this study of nine patients (92).

BMT for Acute Myeloid Leukemia

507

More recent studies have begun to assess the afferent arm of the immune system, antigen presentation. M5-AML cells have been shown in the majority of cases (8/10) to differentiate in vitro, in the presence of GM-CSF and IL-4, or with stimulation by CD40L, into cells that express the phenotype of mature dendritic cells (DC). These leukemia-derived DC produce IL-12 and induce the differentiation of CTL directed against autologous leukemic blasts (93). This observation is important because it is hypothesized that graft-versus-leukemia effects are likely to be most effective early after BMT, in the setting of MRD. Unfortunately, the pace of donor-derived dendritic cell recovery is slow and is further delayed in patients who develop aGVHD (94). When PBSC are the source of the graft, the number of plasmacytoid DC2 is increased. This increase may be an advantage in eliciting more robust GVL effects.

Natural Killer as Effector Cells Natural killer (NK) cells were described in 1975 as cells that mediated contact-dependent, MHC-independent cytotoxicity without the need for priming (95). The standard assay in humans uses the K562 erythroleukemia cell line as the target cells. NK cells were hypothesized to play a role in tumor surveillance (96) and in the regulation of hematopoiesis (97,98). There has been a dramatic resurgence of interest in the effector function of these cells after the elucidation of the basis for target recognition by NK cells: loss of self-antigen expression (99). NK cells express both activating and inhibitory receptors, the best understood of which are the killer-cell immunoglobulin-like receptors (KIR). The ligand specificities of some of the inhibitory KIR have been identified as public epitopes of class I HLA antigens. The genes encoding the KIR are organized in tandem on chromosome 19q13.4, and KIR haplotypes differ both in the number of genes expressed as well as in allelic differences (100). In the setting of allogeneic BMT, it is possible for the recipient to lack a class I HLA antigen that is the ligand for inhibitory KIR receptors on donor NK cells, in which case allogeneic NK may be an important effector mechanism. Ruggeri et al. were the first to demonstrate a dramatic relationship between potential graft-versus-host NK alloreactivity and a reduction in the risk of relapse in patients with AML undergoing stringently T-cell depleted haploidentical BMT (101). A subsequent analysis, restricted to children, showed that in addition to the absence of the HLA class I antigens that are the ligands for inhibitory KIR, the presence of the corresponding inhibitory KIR on donor cells was important in predicting a low risk of relapse after T-cell depleted haploidentical BMT (102). Patients who lacked a ligand for donor inhibitory KIR had a 13% risk of relapse, as compared to 54% among patients whose cells express ligands for all donor inhibitory KIR (p!0.01). However, no such benefit from KIR ligand mismatch was observed in other series of patients who received haploidentical transplants with less rigorous T-cell depletion (103, 104). In these patients, the presence of HLA mismatches that would allow donor antihost NK alloreactivity was associated with an increased risk of severe GVHD and poorer survival, thus masking any potential benefit in terms of prevention of relapse and of graft failure. Similarly, an analysis of partially mismatched unrelated donor BMT showed no benefit of KIR ligand incompatibility for reducing relapse, but these patients generally received cyclosporine and methotrexate as GVHD prophylaxis instead of T-cell depletion (105). Thus, it appears that T-cell alloreactivity dominates NK alloreactivity unless the graft is rigorously T-cell depleted (104). It remains to be seen whether prospective selection of donors for NK alloreactivity can improve BMT outcomes.

Donor Sources and Relevance Importance of Marrow Cell Dose The effect of BM cell dose among BMT recipients has been retrospectively analyzed. Higher cell doses are associated with better survival for the full range of donor types, including autologous marrow with or without purging, syngeneic twins, HLA-matched siblings, unrelated donor marrow, and unrelated donor cord blood (23, 106–110). In various systems, both a

508

Chen and Arceci

reduced relapse rate (109) and reduced transplant-related mortality (106–111) contribute to improved outcomes. In the haploidentical setting, doses of progenitor cells tenfold higher than usual have been used to overcome the immunologic barrier to engraftment and the high risk of graft failure associated with TCD of the graft (112,113).

Peripheral Blood Stem Cell Versus Bone Marrow If higher cell doses within a type of product are associated with better transplant outcomes, perhaps the use of a different source of stem cells that allows higher cell doses may also improve outcomes. In the autologous setting, peripheral blood stem cell (PBSC) grafts typically contain an order of magnitude more CD34C progenitor cells than marrow grafts. Single institutional studies demonstrated the feasibility of HLA-identical sibling PBSC transplantation without excessive rates of aGVHD despite the large dose of T cells (114–116), with faster hematopoietic recovery (117,118). Because these small studies showed a trend toward reduced relapse (116) and improved survival (115), several larger prospective randomized trials were launched that compared PBSC to bone marrow (BM) for allogeneic transplantation for adult patients with hematologic malignancies (119–124). The use of mobilized peripheral blood stem cells as an alternative to BM is clearly associated with more rapid hematopoietic recovery without excessive rates of aGVHD. Some of the studies have suggested a lower relapse rate in recipients of PBSC (119,123). A unifying hypothesis is that patients with myeloid malignancies and higher risk of relapse benefit from the use of PBSC over BM (118). However, the use of PBSC is associated with a significantly higher rate of chronic GVHD (121,124,125), and there is preliminary evidence for poorer quality of life in survivors of allogeneic PBSCT compared with BMT (126). Therefore, it will be important to confirm that early survival benefits associated with the use of PBSC persist after sufficient follow-up to observe events due to chronic GVHD. In children, the feasibility of collecting mobilized PBSC has been demonstrated in the setting of autologous transplantation for patients with high-risk malignancies (127), but the issues of treating healthy, minor donors with G-CSF and of exposing these donors to allogeneic blood used to prime the leukapheresis equipment have resulted in sparse literature regarding allogeneic PBSCT in children. Small case series suggest the feasibility of allogeneic PBSCT in children, and reports of long-term survival in patients who relapse after BMT and are salvaged with PBSCT from the same donor suggest that the allogeneic effect may be enhanced (128). If the use of PBSC produces a survival advantage compared with BM for patients with a high risk of relapse, it will be important to consider whether an enhanced BM progenitor cell dose might offer similar benefits without the same morbidity from chronic GVHD. The EBMTR has analyzed the impact of the source and dose of stem cells on the outcome of patients with AML undergoing matched sibling BMT in first remission. The study included 881 adults who underwent transplantation without T-cell depletion, either with BM or PBSC, and showed that leukemia-free survival was better in patients who received BM with a good cell dose (72%) as compared with those who received PBSC (61%) (129). Priming of the marrow donor with a colony stimulating factor prior to harvest may be a method to evaluate the benefit of increased BM cell doses prospectively (130).

Use of Cytokines After Bone Marrow Transplantation Although marrow cell dose is important, there is little reason to recommend administration of hematopoietic cytokines after transplantation to accelerate hematopoietic recovery. One randomized prospective trial of G-CSF in 221 children undergoing allogeneic or autologous BMT or PBSCT showed faster myeloid engraftment in the arm receiving G-CSF (14 vs. 20 days), both for autologous and allogeneic BMT, and shorter time to discharge after BMT (131). However, a large retrospective analysis of EBMT data raised significant concerns about the use of G-CSF after allogeneic BMT. This analysis showed that although patients who

BMT for Acute Myeloid Leukemia

509

received G-CSF had faster neutrophil recovery, this benefit was offset by slower platelet recovery, a higher incidence of GVHD, and greater transplant-related mortality than patients who did not receive G-CSF after BMT (132). However, the study does not demonstrate a causal relationship between G-CSF administration and increased transplant-related mortality, because the groups differed significantly in important respects other than the use of growth factors. For example, the patients treated with G-CSF were younger, had higher risk disease, and more often received single-agent GVHD prophylaxis. Moreover, this finding was confined to BMT and did not extend to recipients of PBSC grafts; furthermore, the observations were not consistent with the results observed in other large datasets. A meta-analysis of studies of G-CSF or GM-CSF after allogeneic BMT was designed to test the hypothesis that G-CSF, which produces Th2 T cell polarization, would reduce aGVHD, whereas GM-CSF, which produces Th1 T cell polarization, would increase aGVHD. The study included more than 1000 patients on nine randomized prospective trials, eight retrospective cohort studies, and one case-control study. In contrast to the EBMTR analysis, this study found no association between the use of G-CSF or GM-CSF with acute or chronic GVHD (133).

T-Cell Depletion Versus Immune Suppression TCD is the most effective method to prevent GVHD (113,134,135). However, this benefit is offset by higher rates of graft failure and delayed immune reconstitution, with consequent risk of opportunistic infection, lymphoproliferative disease, and relapse. There has not been a randomized controlled trial of TCD versus immune suppression as prophylaxis of GVHD. Therefore, the best available comparison data come from registry studies. An analysis performed by the EBMTR of 826 patients with AML who received matched sibling transplants in first complete remission from 1980–1995 showed that TCD was associated with significantly poorer leukemia-free survival, with a relative risk of 1.63 (pZ0.01) (28). The stringency of TCD depends on the specific technology used. For example, the use of elutriation to produce a graft with 5!105 CD3CT cells/kg for unrelated donor BMT was associated with an unacceptably high (4/6) graft failure rate after a standard Bu/Cy preparative regimen. When the immune suppressiveness of the preparative regimen was increased by the use of TBI and ATG in addition to CSA/prednisone GVHD prophylaxis, the engraftment rate improved (10/12), but the risk of AGVHD remained higher than is expected after matched sibling BMT (136). Other techniques can reduce the T-cell content of the graft below 5!104 CD3C T cells/kg (113,134). Although more stringent TCD may reliably reduce the risk of GVHD, it would be preferable to selectively eliminate only the subset of cells responsible for GVHD, leaving intact other cells that can contribute to rapid immunologic reconstitution and robust GVL. Similarly, optimization of immune suppression may also improve BMT outcomes significantly. The tailoring of immune suppressive GVHD prophylaxis according to the risk of GVHD, and the early withdrawal of CSA for patients who do not develop aGVHD, was associated with more cGVHD (53% vs. 25%) and less relapse (20% vs. 52%) and therefore better 5-year DFS (66% vs. 41%, pZ0.07) in patients with leukemia in first complete remission (137).

Alternative Donor Transplantation The feasibility of BMT from donors other than HLA-identical siblings has been explored since the early days of BMT. Relatives other than matched siblings who are phenotypically matched except for at most one HLA antigen have also produced acceptable results given standard immune suppression as GVHD prophylaxis. Experimental approaches, including in vitro and in vivo TCD (113,138,139), have produced encouraging results with related donors with greater degrees of mismatch. A more elegant approach is specific tolerization of the graft by ex vivo coculture with recipient cells in the presence of CTLA4-Ig to block costimulatory signals (140).

510

Chen and Arceci

Unrelated donor BMT is feasible for about 30% of patients (141). Limitations include the time and expense involved in identifying a well-matched donor, burdens that few patients can afford. In addition, the great diversity of the HLA genetic variation, combined with the demonstrable clinical impact of even single allele-level mismatches (21,142–144), makes it difficult to provide a well-matched unrelated donor for every patient who would benefit from allogeneic BMT. Unrelated cord blood banks represent an alternative that is appealing because of greater latitude in the degree of HLA matching and the ability to collect products at no risk to the donor. However, the limited progenitor cell dose in cord blood units tends to result in a longer period of pancytopenia and consequent increases in NRM for larger patients who receive a lower cord blood cell dose relative to their ideal body weight. In addition, the potential for robust graft-versus-leukemia immunotherapy may be inherently limited compared to the potential with marrow donors. After cord blood transplantation, there is a unique pattern of immune reconstitution (145) with a prolonged, low CD4-CD8 ratio (146), and there is no opportunity for the infusion of supplemental doses of donor lymphocytes. The first prospective studies of unrelated donor BMT, matched serologically for HLA-A and B antigens and matched using early DNA techniques for HLA-DRB1, demonstrated inferior outcomes as compared to HLA-identical sibling BMT (147). These differences were largely attributable to undetected HLA mismatches (21,148). One strategy for avoiding such mismatches is to match for blocks within the major histocompatibility complex that do not encode HLA, taking advantage of linkage dysequilibrium. A prospective analysis of 40 consecutive serologic A, B, and DNA DRB1 matched BMTs showed patients whose donors matched for non-HLA blocks within the MHC have significantly less severe aGVHD and significantly better survival (67% vs. 29%) than those with mismatches, and that matching these blocks correlates with allele level matching of HLA B, C, DRB1, and DQB1 (149). Matching at the allele level by directly sequencing the HLA alleles produces comparable improvement in outcomes (150). Conversely, others have shown that in children with acute leukemia, increased transplant-related mortality associated with the use of matched unrelated donors as compared with matched sibling donors is offset by lower relapse, so that leukemia-free survival is not different (151,152). Similarly, for young recipients, an antigen-level mismatch in an unrelated donor may be acceptable because improved GVL offsets the increased risks of GVHD and graft failure, leading to similar leukemia-free survival (21). However, even though the risk-benefit ratio may appear to be the same for a mismatched unrelated donor BMT that is both more toxic and more effective, optimal patient selection does differ. The threshold risk of relapse that justifies BMT must increase as the probability of salvage after relapse increases. Another strategy to reduce the morbidity of unrelated donor BMT is to deplete T cells in vivo with the administration of antibodies to T cells during the preparative regimen. In a single institution trial, ATG was given before unrelated BMT to 98 recipients of all ages (range, 1–56), including 84 patients matched for A, B, DR, and 14 with a mismatch. The results closely approximated those of matched sibling BMT, with only one primary graft failure, a 37% incidence of grade II–IV aGVHD, and a 17% incidence of extensive cGVHD (153). A matched cohort study demonstrated an advantage for the administration of ATG with respect to GVHD and NRM in patients with leukemia undergoing unrelated donor BMT (154).

AUTOLOGOUS TRANSPLANTATION Use of Autologous Bone Marrow Transplantation as Consolidation of First Remission Randomized comparisons of autologous BMT versus chemotherapy consolidation have generally demonstrated equivalent survival when analyzed by intent-to-treat (155–158). A meta-analysis of these randomized trials concluded that the pooled data are insufficient to demonstrate an advantage for autologous BMT as compared to conventional chemotherapy as

BMT for Acute Myeloid Leukemia

511

consolidation for pediatric AML in first remission (159). However, many of the patients assigned to BMT relapsed before transplantation, and cross-over from the assigned arm to an alternative arm was common on these trials; thus, when analyzed as treated, patients who actually underwent autologous BMT fared slightly better than patients who actually received conventional chemotherapy as consolidation. Therefore, one of the pitfalls in autologous BMT as studied is the delay involved in harvesting autologous marrow after full hematopoietic recovery during a course of intensive chemotherapy. In addition, the preparative regimen was untargeted Bu/Cy. This is a standard regimen used for allogeneic BMT; as such, it is unnecessarily immune suppressive for autologous BMT. Because the MTD of the preparative regimen was determined in the presence of posttransplant immune suppression and a significant incidence of GVHD, it is plausible that in the autologous setting, the preparative regimen intensity could be safely escalated to improve its antileukemic efficacy. Moreover, no large multicenter randomized study comparing autologous BMT to chemotherapy consolidation incorporated pharmacokinetic targeting of Bu, purging of the graft, or the use of mobilized PBSC as the graft. Several studies have incorporated one or more of these strategies and have produced superior outcomes. A single-institutional study from Barcelona produced exceptional results with the use of mafosfamide purging. In this study, 79 consecutive children diagnosed with AML from 1988 through 2001 were enrolled in the AML-88 trial, in which all high-risk patients were assigned to receive consolidation with BMT after induction chemotherapy and two consolidation courses. The 31 patients who did not have an HLA-matched sibling donor received mafosfamide-purged autologous BM grafts and achieved the same high EFS of 74% as the 17 patients who underwent allogeneic BMT, with a median follow-up exceeding 7 years (160). This was a nearly population-based study and thus avoided selection bias, in that all eligible patients consented to treatment on protocol, and 69 of 79 patients were from the local region, Catalonia, whereas the other 10 patients were referred from two hospitals of other regions. Another multicenter study of purging with the use of monoclonal antibodies to CD15 and CD14 plus complement included 138 patients with AML of all ages, in first through third remission or first relapse. For the 23 patients transplanted in first complete remission, the 3-year DFS was 50% (161,162). These results are comparable to the Minnesota experience, in which autologous marrow was purged with 4-hydroperoxycyclophosphamide (4HC) in 69 of 75 cases. The 44 patients transplanted in CR1 with 4HC purged autologous grafts achieved 59% 2-year DFS (70). Several groups have explored the use of G-CSF mobilized autologous PBSCT in children with AML in CR1 (163–165). This approach is feasible even in infants (163,164). The use of PBSC leads to rapid neutrophil recovery above 0.5!109/L in 9 days (range, 7–13 days) (163). Analysis of the EBMT data, including 387 children undergoing autologous BMT for AML, confirms the significant association of PBSC with more rapid neutrophil recovery (166). These studies indicate that the timing of PBSC collection is critical for outcome. A retrospective analysis of 32 patients who received autologous PBSCT for AML in CR1 showed that the 22 patients enrolled on a pilot trial that added a course of intensification chemotherapy before PBSC collection had significantly better DFS, 69% with intensification compared to 36% without; this was due to a reduced rate of relapse of 31% versus 65% respectively (165). Similarly, the Japanese Cooperative Study Group for PBSCT & Children’s Cancer and Leukemia Study Group treated a series of 28 children with AML in CR (24 CR1, 4 CR2) with G-CSF mobilized PBSCT (164). On these two prospective controlled trials, high-risk patients without an HLA-matched sibling marrow donor were assigned to autologous PBSCT if they were being managed at a transplant institution. Those patients whose PBSC were collected less than 2.5 months after achieving CR had a significantly poorer outcome (12% vs. 73% RFS, p!0.05). The high relapse rate in patients whose grafts were collected earlier was independent of the timing of BMT (164). Taken together, these results suggest that in vivo purging of the PBSC graft is important. The use of regimens designed exclusively for high-dose consolidation with autologous stem cell rescue has also generated encouraging results. Patients treated for AML in CR1 with

512

Chen and Arceci

high-dose melphalan and autologous HSCT have been analyzed retrospectively by the Italian Association of Pediatric Hematology Oncology-BMT group (AIEOP-TMO). This was a small, heterogeneous group of 20 patients treated at six centers from 1994–1999, with some grafts purged in vitro with mafosfamide and others in vivo with IL-2. Melphalan was administered as a single agent at doses that ranged from 150–220 mg/m2 IV. Nevertheless, the results were encouraging, with no toxic deaths, a 62% OS, and 56% EFS at 3 years (167). The advantage of this regimen is that it appears to be less toxic than standard Bu or TBI-based BMT preparative regimens. The combination of melphalan with TBI appears to be even more interesting for autologous BMT because of greater antileukemic effectiveness. In a phase II trial in pediatric AML that included 10 patients in CR1 and 9 patients in CR2, the patients in CR1 had a 100% projected 6-year EFS (168). A retrospective analysis of the AIEOP-BMT registry data from 1984 to 1996 for pediatric patients with AML treated with autologous BMT identified a subgroup of 21 patients who received melphalan-TBI for AML in CR1 and achieved 85% 7-year EFS (169). These studies suggest that highly active regimens tailored for autologous BMT warrant further attention in larger prospective trials.

Use After Relapse An alternative to the use of autologous BMT as consolidation in first remission is to reserve autologous BMT for use as salvage therapy in the event of relapse, using a graft cryopreserved in first remission or harvested after inducing a second remission. When the Seattle results were reviewed retrospectively, 47 cases were identified in which patients received autologous BMT for AML beyond first remission, with a trend toward better RFS in first untreated relapse (45%) compared to second remission (32%). This observation led to the suggestion that chemotherapy to induce a second remission is not indicated (170). Similarly, in the multicenter trial of monoclonal-antibody purged autologous BMT, the patients transplanted in first relapse achieved better 5-year RFS (37%) than those transplanted in second or third remission (23%) (162). However, only 4 of 26 patients in the Seattle series transplanted in second remission had autologous marrow stored in first remission, so the patient populations were inherently different other than in treatment with salvage induction chemotherapy. In addition, it is likely that there was selection among patients in relapse for patients in better clinical condition, such as those in early relapse, to proceed directly to BMT. When marrow was prospectively stored in first remission for use in the event of relapse, 65 of 98 (66%) patients relapsed, and their 4-year survival was only 8%. Again, there was a trend toward better survival in those who proceeded directly to BMT in untreated relapse, with 8 of 38 patients surviving, as compared to 2 of 24 patients surviving after receiving chemotherapy as initial therapy for relapse (4). Although this study was prospectively designed, and all patients had autologous marrow storage in first remission, a limitation is that clinical patient selection for chemotherapy may account for poorer outcomes in the patients treated with induction chemotherapy. This potential selection bias prevents a firm conclusion that induction chemotherapy is not beneficial before autologous BMT in the event of recurrent AML.

Role of Purging The role of purging in autologous BMT for AML is controversial, and has yet to be tested in a randomized controlled trial. Certainly, many cohorts of patients have been treated with unpurged autologous BMT and have achieved excellent survival (80). However, several lines of evidence suggest outcomes can be improved with the use of purging. First, the data of the Autologous Blood and Marrow Transplant Registry were analyzed, including 294 patients transplanted for AML in first (209) or second (85) remission, within 6 months of achieving remission (171). A Cox proportional hazards analysis was performed to adjust for important prognostic factors and showed that patients who received purged grafts had better 3-year leukemia-free survival than those who received unpurged grafts: 56% versus 31% in first

BMT for Acute Myeloid Leukemia

513

remission and 39% vs. 10% in second remission respectively. Another registry study, conducted by the EBMT in 387 children who underwent autologous BMT for AML, showed that the use in vivo purging reduced the risk of relapse from 43% to 30% (p!0.05). Purging was associated with significant protection in multivariate analyses for relapse (RR 0.65, p!0.05) and for overall survival (RR 0.64, p!0.05) (166). The results of 229 consecutive patients at Hoˆpital St. Antoine receiving mafosfamidepurged autologous marrow transplants for acute leukemia provides additional support for the use of purging. This series included 165 patients with AML, 123 in CR1, 32 in CR2, and 10 with more advanced disease. In the analysis of prognostic factors, a higher prepurging cell dose (Omedian 5.46!104 CFU-GM/kg) was significantly associated with less TRM (6% vs. 30%), better LFS (59% vs. 42%), and better OS (65% vs. 43%) (p!0.001), but a lower post-purging progenitor cell dose was associated with less relapse, RRZ0.5, p!0.01 (109). These data imply that higher intensity purging significantly reduced the relapse rate. Finally, the studies discussed above that show better results when PBSC grafts are collected after intensification therapy (164,165) are consistent with the hypothesis that in vivo purging improves outcome and that viable leukemia cells contaminating the graft may contribute to relapse. However, it must be noted that an alternative interpretation is that these patients are temporally selected for freedom from relapse during longer conventional therapy.

QOL/LATE EFFECTS As various treatment strategies produce comparable long-term survival, quality of life (QOL) issues both during and after therapy become increasingly important. In general, although BMT produces major impact on short-term QOL, long-term QOL is nearly intact except for sexual function (172). However, relatively few quality of life studies have focused on patients transplanted during childhood. The SFOP has studied late effects of allogeneic BMT for AML in CR1 in children, comparing the results of Bu and TBI based preparative regimens. Of 45 children, 26 received Bu/Cy and 19 received TBI (9 fractionated, 10 single dose) and were studied at a median follow-up of 5.9 year after transplantation. Even though none of the patients in the TBI group had received prior cranial irradiation, although four patients in the Bu group had received cranial irradiation, TBI was associated with greater declines in height Z score (-0.86 and -1.56 at 3 and 5 year) than Bu (-0.05, -0.17), a higher incidence of hypothyroidism (43% vs. 9%) and of cataracts (70% vs. 0% at 6 year) (173). Although mean height Z scores may be relatively stable in infants and children treated with Bu, they are at risk for myriad endocrine disturbances. About 35% of patients have an unexplained growth disturbance, growth hormone levels are low in approximately 40%, 19% have compensated hypothyroidism, 33% have subclinical hyperparathyroidism, and half have gonadal failure after high-dose Bu (174). The Children’s Hospital of Philadelphia in 1999 reported a comparison of late effects of chemotherapy or BMT among patients seen in their long-term follow-up facility after either the initial diagnosis of AML or referral for marrow transplantation of MDS or AML. In their cohort, although more patients had gonadal failure requiring estrogen replacement after BMT, they observed similar growth, renal function, and cardiac function among longterm survivors treated with BMT as compared to those treated with conventional chemotherapy (175). Therefore, they concluded that probability of cure should be the primary basis for recommendations regarding therapeutic choices for AML. The cohort of 10-year survivors of childhood AML treated at St. Jude Children’s Research Hospital from 1976 to 1989 included 77 patients, of whom 15 had undergone allogeneic BMT with TBI. As in the Philadelphia cohort, these BMT survivors had a higher rate of gonadal endocrine failure than did chemotherapy recipients and similar cardiac function. However, they observed significantly poorer growth (height Z scores -1.33 vs. -0.21), increased incidence

514

Chen and Arceci

of cataracts (9/15 vs. 0/44), and increased academic difficulties (5/15 vs. 2/44), all significantly different from the patients treated with intensive chemotherapy, but similar to the patients treated with chemotherapy and cranial radiation (176). These findings are quite consistent with the differences in late effects associated with Bu versus TBI described by the SFOP (173). A quality of life analysis of patients treated on the POG 8821 trial was dominated by survival outcomes, similar to the Philadelphia study. In this study, the endpoint was time without symptoms or toxicity of treatment (TWiST). Measuring quality of life was not a prospective objective of the study, but the analysis used prospectively recorded toxicity data to score the time with symptoms grade three and above and the time of relapse, making the assumption that all time after relapse was compromised by symptoms of disease. Using this approach, the best TWiST was achieved in patients with HLA-matched related donors, who were assigned to allogeneic BMT in CR1 and attained the best OS and RFS. The patients randomized to autologous BMT had a trend toward poorer Q-TWiST than those randomized to conventional chemotherapy (177), although OS and RFS were equivalent in these two groups. A cross-sectional analysis of 479 survivors over age 15 treated on MRC AML 10 trial showed survivors of either allogeneic or autologous BMT were significantly more likely to report diminished sexual functioning and infertility compared with survivors of consolidation with chemotherapy. Women were more profoundly affected than men (178). As with any study using self-reporting by questionnaire, this study has limitations, including recall bias and potential misclassification bias. In addition, this study did not correct for baseline patient characteristics and possible survival bias, because higher risk patients were more likely to survive after BMT. Nevertheless, the conclusions are plausible, and implications include potential intervention with counseling and banking of gametes before BMT and hormone replacement and psychological treatment after BMT.

FUTURE DIRECTIONS AND CONTROVERSIES Who Should Undergo Bone Marrow Transplantation in CR1? The typical approach to AML has been derived from a study design in which patients who had HLA-identical sibling donors were assigned to BMT in first complete remission, and those without HLA-identical sibling donors received either chemotherapy consolidation or autologous BMT. Whether this is an appropriate or optimal strategy has recently become the subject of debate (179–182). We propose that risk stratification and molecular diagnosis be used to assign therapy. For example, activating mutations in receptor tyrosine kinase and Ras pathways are common (46%) and are associated with poor prognosis in pediatric AML, in patients treated on the CCG 2891 protocol. Moreover, there is a striking interaction between the presence of mutations in receptor tyrosine kinases and the benefit of allogeneic BMT. In mutation-positive patients, allogeneic BMT produced 72% DFS, whereas these patients achieved only 23% DFS when consolidated with chemotherapy or autologous BMT. In contrast, in mutation-negative patients, allogeneic BMT produced only 55% DFS, as compared to 40% DFS with chemotherapy or autologous BMT (20). Furthermore, complex cytogenetic abnormalities are associated with a poor prognosis, both when treated with conventional chemotherapy and with allogeneic BMT (183). On this basis, the presence of receptor tyrosine kinase mutations, but not the presence of complex cytogenetic abnormalities, would be useful for assignment of patients to allogeneic BMT in first remission regardless of the availability of a matched related donor. However, prospective clinical trials have yet to be done to demonstrate that such strategies improve on traditional treatment assignment based on donor availability.

BMT for Acute Myeloid Leukemia

515

Bone Marrow Transplantation in Untreated Relapse Versus Attempted Induction? Several studies have demonstrated that BMT is potentially curative therapy for AML in first untreated relapse, both with allogeneic donors (184) and with autologous marrow grafts (4,170,184). These studies have shown that the survival of patients treated in first untreated relapse is no worse than the survival of those treated in second remission. These studies have led to the suggestion that one need not attempt induction therapy for a patient with recurrent AML but should proceed directly to BMT. Induction therapy may be of no value, if it is cross-resistant with the patient’s prior therapy or with the transplant therapy or if it has significant cumulative toxicity with the transplant regimen. On the other hand, it is possible that the patients selected to proceed directly to BMT in these retrospective analyses had a much more favorable prognosis than those who were treated with induction chemotherapy first. For example, some patients referred for BMT as consolidation in first remission who were found to be in early asymptomatic relapse during their pre-BMT workup may have comprised a favorable prognosis subset of the patients transplanted in first relapse. Similarly, among patients undergoing autologous BMT in first untreated relapse, those with absolute blast counts above 4!109 blasts/L did not achieve long-term survival (170), indicating the need for patient selection to achieve favorable results in untreated relapse. For patients without a suitable related donor or stored autologous marrow, induction chemotherapy is required to permit identification of an unrelated donor. In addition, successful induction of second remission may permit the use of a less morbid transplant strategy. A high relapse rate of 70% was observed when more effective GVHD prophylaxis with cyclosporine and methotrexate was used, as compared with 25% when methotrexate was given alone as GVHD prophylaxis (184). Optimal EFS (38% vs. 18%, p!0.05) required accepting 40% transplant-related mortality by using methotrexate as the sole GVHD prophylaxis for patients transplanted in first untreated relapse (184). If a non-cross-resistant induction regimen is available, mild or even subclinical GVHD may be sufficient to produce an adequate GVL effect. The BFM has taken a very different approach, and the outcomes of 134 patients who relapsed after treatment on the BFM 87 and 93 protocols have been analyzed to identify prognostic factors for second remission and for long-term survival in an unselected patient population (27). In this series, only four patients were transplanted in first untreated relapse, and none survived long term. Of 102 patients treated with salvage induction chemotherapy, typically with mitoxantrone and etoposide, 50% achieved a second complete remission. Patients went on to consolidation with allogeneic (nZ27) or autologous (nZ23) BMT in CR (nZ43) or PR (nZ7). Projected 7-year survival is 21%. Interestingly, the long-term survival is similar after allogeneic and autologous BMT, and the major prognostic factor both for achievement of a second remission and for long-term survival is the duration of first remission. The success of mitoxantrone/high-dose cytarabine, with a remission induction rate of 76% for 101 pediatric patients with relapsed or refractory AML, and only 3% toxic mortality (185) and of fludarabine/cytarabine/G-CSF/idarubicin (FLAG-Ida), with remission induction rates of 44% to 52% (186–188) with 6% toxic mortality offers reasonable alternatives for most patients with recurrent AML who might benefit from salvage induction. New approaches employing nonanthracycline-based regimens or combinations of molecularly targeted therapies and chemotherapy are currently being tested. Acute promyelocytic leukemia represents a distinct clinical situation because of the success of molecularly targeted therapy with ATRA and the distinctive sensitivity to anthracyclines but not cytarabine. Therefore, patients with APL are no longer considered as candidates for BMT in first remission. When patients relapse after treatment with ATRA and chemotherapy, appropriate salvage therapy may include BMT versus additional chemotherapy versus arsenic trioxide alone or in combination with idarubicin. A study comparing these approaches showed that the toxic mortality was highest with chemotherapy, and although there was no toxic mortality in patients treated with arsenic trioxide, 38% of patients suffered a

516

Chen and Arceci

second relapse if they received no further definitive therapy. Thus, the 2-year OS was 83% for patients treated with arsenic trioxide-based chemotherapy, 43% for patients undergoing BMT, and 23% for patients treated with conventional chemotherapy (189). These results reflect in part the high mortality associated with BMT in adult patients. Thus, patients with APL in first CR should be followed closely for the presence of persistent or increasing evidence for MRD. Such patients will relapse and early therapeutic intervention is indicated (190–192). Once a molecular remission is obtained, usually with arsenic trioxide treatment alone, a matched sibling donor BMT is recommended (193). If such a donor is not available, then autologous BMT should be considered, whereas MUD donors should be considered for higher-risk patients, such as those who are not able to achieve a MRD status (194). For patients who are not able to undergo BMT, some studies are ongoing in adults that employ combinations of arsenic, ATRA, and immunologically targeted therapy with agents such as gemtuzumab ozogamicin (GO) (195,196).

Therapy for Relapse After Bone Marrow Transplantation For patients who relapse after autologous BMT, alternatives include palliative chemotherapy, with 10% 2-year survival (197), treatment with novel agents, or second marrow transplantation. Therapy with the mAb-targeted toxin, GO, is illustrative of the risks and benefits of treatment with novel agents. It is associated with an increased risk of VOD, both when used before or after BMT (198,199). However, as a non-cross-resistant agent, it produced responses in approximately 38% of relapsed patients. Similar results have been observed in children with relapsed and/or refractory AML. Of note, similar response rates were observed in patients who had relapsed following a remission and those with primary refractory AML, suggesting that GO was able to circumvent different chemotherapy resistance mechanisms (200). GO is now being tested in randomized Phase III trials in children and adults. The EBMTR has retrospectively reviewed the results of second transplants done for patients who relapsed after autologous BMT and who are in CR. Only 168/2752 patients with acute leukemia who relapsed after autologous BMT underwent a second transplant procedure, 94 with an allogeneic transplant and 74 with a second autologous transplant, so this population was heavily selected, both for ability to achieve a complete remission and for survival long enough to identify a suitably matched unrelated donor. A longer interval from first autologous BMT to relapse, a longer interval from relapse to second transplant, and younger age were all associated with better survival. Long-term survival was superior with a matched allogeneic transplant than with a second autologous BMT, because the much lower relapse rate (34% vs. 57%) offset higher transplant-related mortality (44% vs. 31%). Surprisingly, however, there was an interaction with the preparative regimen for the first autologous BMT: patients treated with TBI during the first autologous BMT attained better survival with a second autologous BMT, whereas those who did not receive TBI during their first autologous BMT achieved better survival with a matched allogeneic BMT. Outcomes of second autologous BMT were significantly better when the graft was harvested before the second autologous BMT, rather than using material stored before the first autologous BMT (197). The results of second marrow transplants in children are improving with appropriate patient selection, in accordance with these findings. The St. Jude cohort of 23 children treated with allogeneic BMT for failure of autologous BMT included 21 patients with AML. The majority of patients received unrelated donor BMT. DFS was 39% at 4 years equal numbers of deaths occurred due to RRT and due to relapse (201). The Seattle experience with second transplants in pediatric AML using Cy/TBI, following relapse after a Bu/Cy preparative regimen, included 25 patients whose first BMT was allogeneic in 14 cases and autologous in the remaining 11. All second BMT were allogeneic, 12 from HLA-matched relatives, 9 from mismatched relatives, and 4 from unrelated donors. All patients engrafted, and 76% developed grade II-IV GVHD. The NRM was 12% at day 100, and nine patients relapsed at a median of 5.4 months (range, 1.8–34 months) after BMT. Twelve patients were alive a median 9.1 years

BMT for Acute Myeloid Leukemia

517

(range, 7–14 years) after 2nd BMT (44% 10-year DFS) (202). There was a higher risk of relapse for patients not in remission at the time of 2nd BMT (HR 7.8) or if the interval from first BMT was less than 6 months. Donor lymphocyte infusions (DLI) remain useful, particularly for patients who relapse early after allogeneic BMT. DLI response has been similar in Japan as in western countries; in AML, 8/21 (38%) achieved remission after cell doses of at least 1!108 mononuclear cells/kg (203); however, 3-year survival is only 7%. Chemotherapy prior to DLI can improve response for many reasons, including direct antileukemic activity, improvement in the effector: target ratio, elimination of regulatory T cells, and creation of a cytokine milieu that favors GVL. One such series includes three pediatric patients with AML in relapse after matched-sib BMT who were reinduced with chemotherapy and consolidated with DLI. All developed grade III/IV aGVHD or extensive cGVHD, and all remained in remission with good QOL (204).

Preemptive Immunotherapy In chronic myeloid leukemia, DLI is more effective when applied during molecular or cytogenetic relapse than after overt hematologic relapse (205). Because the induction rate of DLI is considerably less in AML than in CML (91), there has been considerable interest in the prophylactic use of DLI before hematologic relapse (206,207). In the setting of cord blood transplantation, alternatives to DLI include administration of cytokines (208) and immunostimulatory medications (171). The measurement of MRD is highly relevant to such preemptive immunotherapy strategies, both for the selection of patients who need treatment and for monitoring the success of immunotherapy by the disappearance of MRD. In CML, the Philadelphia chromosome provides a consistent marker that can be detected and quantified with great sensitivity and specificity by various molecular assays. In an analogous fashion, MRD can be detected by quantitative RT-PCR for the subset of patients with AML who have fusion oncogenes, and MRD detected by this method has prognostic importance for patients with APL with the characteristic t(15;17) (210) and for some patients with core binding factor leukemias (211–213). A small study explored RT-PCR to measure CBFb/MYH11 as an index of MRD in patients with AML with inv(16), during consolidation with high-dose cytarabine or unrelated donor BMT (214). In five of six patients, levels of the fusion oncogene declined by more than four orders of magnitude, and the patients remained in continuous complete remission. In one patient, the level declined by only two orders of magnitude, and the patient relapsed. Moreover, detectable MRD temporarily reappeared during the treatment of GVHD and subsequently declined after reduction of immune suppression (214), demonstrating the potential value both of this assay and of GVL for the prevention of relapse. An alternative approach, applicable to most patients with AML without a requirement for fusion genes, is multiparameter flow cytometry (215,216). In patients whose leukemic blasts exhibit an abnormal phenotype, MRD can be detected and can provide lead time before clinical and hematologic relapse (25,217,218). Moreover, the presence of MRD detectable by multiparameter flow cytometry before BMT identifies a poor prognosis cohort of patients who may be candidates for more aggressive posttransplant immunotherapy (25). Another approach, applicable to all patients undergoing allogeneic BMT, is the analysis of hematopoietic chimerism; increasing host chimerism is associated with an increased risk of relapse (219,220). This approach has been used in a limited institution study in Tubingen and Freiburg, involving 22 pediatric patients with acute leukemia or MDS. Peripheral blood chimerism was monitored weekly until day 100 and monthly thereafter, by semiquantitative PCR based on VNTR & STR. Whenever increasing host chimerism was detected, subpopulation analyses were performed to determine whether this was due to detection of the malignant clone or because of detection of normal cells. In most cases, host chimerism included normal hematopoietic lineages, so the presence of host hematopoiesis may predict relapse because it reflects less GVL or the host cells may directly inhibit GVL. The first nine

518

Chen and Arceci

patients who demonstrated increasing host chimerism were not given further therapy, and all relapsed, with lead time that ranged from 32 to 296 days. Subsequent patients with increasing host chimerism had their immune suppression withdrawn. Of five patients treated this way, three responded with a reduction in host chimerism and two had a further increase of host chimerism and, therefore, were given DLI. Only one of these patients relapsed, after receiving immune suppression for GVHD (221). In summary, these studies demonstrate that measurement of MRD is feasible, that it has prognostic value, and that immunotherapy can be effective when MRD is detected.

REFERENCES 1. Thomas ED, Buckner CD, Banaji M, et al. One hundred patients with acute leukemia treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation. Blood 1977; 49:511–533. 2. Geller RB, Saral R, Piantadosi S, et al. Allogeneic bone marrow transplantation after high-dose busulfan and cyclophosphamide in patients with acute nonlymphocytic leukemia. Blood 1989; 73:2209–2218. 3. Yeager AM, Wagner JE, Jr., Graham ML, Jones RJ, Santos GW, Grochow LB. Optimization of busulfan dosage in children undergoing bone marrow transplantation: a pharmacokinetic study of dose escalation. Blood 1992; 80:2425–2428. 4. Schiffman K, Clift R, Appelbaum FR, et al. Consequences of cryopreserving first remission autologous marrow for use after relapse in patients with acute myeloid leukemia. Bone Marrow Transplant 1993; 11:227–232. 5. Wagner JE, Broxmeyer HE, Byrd RL, et al. Transplantation of umbilical cord blood after myeloablative therapy: analysis of engraftment. Blood 1992; 79:1874. 6. McSweeney PA, Wagner JL, Maloney DG, et al. Outpatient PBSC allografts using immunosuppression with low-dose TBI before, and cyclosporine (CSP) and mycophenolate mofetil (MMF) after transplant. Blood 1998; 92:519a. 7. McSweeney PA, Niederwieser D, Shizuru JA, et al. Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graft-versustumor effects. Blood 2001; 97:3390–3400. 8. Chang M, Raimondi SC, Ravindranath Y, et al. Prognostic factors in children and adolescents with acute myeloid leukemia (excluding children with Down syndrome and acute promyelocytic leukemia): univariate and recursive partitioning analysis of patients treated on pediatric oncology group (POG) study 8821. Leukemia 2000; 14:1201–1207. 9. Vormoor J, Ritter J, Creutzig U, et al. Acute myelogenous leukaemia in children under 2 years— experiences of the West German AML studies BFM-78, -83 and -87. Br J Cancer Suppl 1992; 18:S63–S67. 10. Kawasaki H, Isoyama K, Eguchi M, et al. Superior outcome of infant acute myeloid leukemia with intensive chemotherapy: results of the Japan infant leukemia study group. Blood 2001; 98:3589–3594. 11. Loeb DM, Arceci RJ. Treatment and outcome of infants with acute myeloid leukemia. Blood 2002; 99:2626–2627. 12. Grimwade D, Walker H, Oliver F, et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1612 patients entered into the MRC AML 10 trial. The medical research council adult and children’s leukaemia working parties. Blood 1998; 92:2322–2333. 13. Creutzig U, Ritter J, Schellong G. Identification of two risk groups in childhood acute myelogenous leukemia after therapy intensification in study AML-BFM-83 as compared with study AML-BFM78. Blood 1990; 75:1932–1940. 14. Woods WG, Kobrinsky N, Buckley J, et al. Intensively timed induction therapy followed by autologous or allogeneic bone marrow transplantation for children with acute myeloid leukemia or myelodysplastic syndrome: a childrens cancer group pilot study. J Clin Oncol 1993; 11:1448–1457. 15. Razzouk BI, Raimondi SC, Srivastava DK, et al. Impact of treatment on the outcome of acute myeloid leukemia with inversion 16: a single institution’s experience. Leukemia 2001; 15:1326–1330.

BMT for Acute Myeloid Leukemia

519

16. Leith CP, Chen IM, Kopecky KJ, et al. Correlation of multidrug resistance (MDR1) protein expression with functional dye/drug efflux in acute myeloid leukemia by multiparameter flow cytometry: identification of discordant MDR-/effluxC and MDR1C/efflux- cases. Blood 1995; 86:2329–2342. 17. Sievers EL, Smith FO, Woods WG, et al. Cell surface expression of the multidrug resistance P-glycoprotein (P-170) as detected by monoclonal antibody MRK-16 in pediatric acute myeloid leukemia fails to define a poor prognostic group: a report from the children’s cancer group. Leukemia 1995; 9:2042–2048. 18. Meshinchi S, Woods WG, Stirewalt DL, et al. Prevalence and prognostic significance of Flt3 internal tandem duplication in pediatric acute myeloid leukemia. Blood 2001; 97:89–94. 19. Levis M, Pham R, Smith BD, Small D. In vitro studies of a FLT3 inhibitor combined with chemotherapy: sequence of administration is important in order to achieve synergistic cytotoxic effects. Blood 2004. 20. Meshinchi S, Stirewalt DL, Alonzo TA, et al. Activating mutations of RTK/ras signal transduction pathway in pediatric acute myeloid leukemia. Blood 2003; 102:1474–1479. 21. Balduzzi A, Gooley T, Anasetti C, et al. Unrelated donor marrow transplantation in children. Blood 1995; 86:3247–3256. 22. Casper J, Camitta B, Truitt R, et al. Unrelated bone marrow donor transplants for children with leukemia or myelodysplasia. Blood 1995; 85:2354–2363. 23. Locatelli F, Rocha V, Chastang C, et al. Factors associated with outcome after cord blood transplantation in children with acute leukemia. Eurocord-cord blood transplant group. Blood 1999; 93:3662–3671. 24. Woods WG, Kobrinsky N, Buckley JD, et al. Timed-sequential induction therapy improves postremission outcome in acute myeloid leukemia: a report from the children’s cancer group. Blood 1996; 87:4979–4989. 25. Nagler A, Condiotti R, Rabinowitz R, Schlesinger M, Nguyen M, Terstappen LW. Detection of minimal residual disease (MRD) after bone marrow transplantation (BMT) by multi-parameter flow cytometry (MPFC). Med Oncol 1999; 16:177–187. 26. Robin M, Guardiola P, Dombret H, et al. Allogeneic bone marrow transplantation for acute myeloblastic leukaemia in remission: risk factors for long-term morbidity and mortality. Bone Marrow Transplant 2003; 31:877–887. 27. Stahnke K, Boos J, Bender-Gotze C, Ritter J, Zimmermann M, Creutzig U. Duration of first remission predicts remission rates and long-term survival in children with relapsed acute myelogenous leukemia. Leukemia 1998; 12:1534–1538. 28. Cahn JY, Labopin M, Sierra J, et al. No impact of high-dose cytarabine on the outcome of patients transplanted for acute myeloblastic leukaemia in first remission. Acute leukaemia working party of the European group for blood and marrow transplantation (EBMT). Br J Haematol 2000; 110:308–314. 29. Dini G, Boni L, Abla O, et al. Allogeneic bone marrow transplantation in children with acute myelogenous leukemia in first remission. Associazione Italiana di ematologia e oncologia pediatrica (AIEOP) and the gruppo Italiano per il trapianto di midollo osseo (GITMO). Bone Marrow Transplant 1994; 13:771–776. 30. Hale GA, Heslop HE, Bowman LC, et al. Bone marrow transplantation for therapy-induced acute myeloid leukemia in children with previous lymphoid malignancies. Bone Marrow Transplant 1999; 24:735–739. 31. Anderson JE, Gooley TA, Schoch G, et al. Stem cell transplantation for secondary acute myeloid leukemia: evaluation of transplantation as initial therapy or following induction chemotherapy. Blood 1997; 89:2578–2585. 32. Yakoub-Agha I, de La Salmoniere P, Ribaud P, et al. Allogeneic bone marrow transplantation for therapy-related myelodysplastic syndrome and acute myeloid leukemia: a long-term study of 70 patients-report of the French society of bone marrow transplantation. J Clin Oncol 2000; 18:963–971. 33. Runde V, de Witte T, Arnold R, et al. Bone marrow transplantation from HLA-identical siblings as first-line treatment in patients with myelodysplastic syndromes: early transplantation is associated with improved outcome. Chronic leukemia working party of the European group for blood and marrow transplantation. Bone Marrow Transplant 1998; 21:255–261. 34. Arnold R, de Witte T, van Biezen A, et al. Unrelated bone marrow transplantation in patients with myelodysplastic syndromes and secondary acute myeloid leukemia: an EBMT survey. European blood and marrow transplantation group. Bone Marrow Transplant 1998; 21:1213–1216.

520

Chen and Arceci

35. Anderson JE, Appelbaum FR, Fisher LD, et al. Allogeneic bone marrow transplantation for 93 patients with myelodysplastic syndrome. Blood 1993; 82:677–681. 36. Thomas ED, Buckner CD, Clift RA, et al. Marrow transplantation for acute non-lymphoblastic leukemia in first remission. N Engl J Med 1979; 301:597–599. 37. Clift RA, Buckner CD, Appelbaum FR, et al. Allogeneic marrow transplantation in patients with acute myeloid leukemia in first remission: a randomized trial of two irradiation regimens [see comments]. Blood 1990; 76:1867–1871. 38. Bieri S, Helg C, Chapuis B, Miralbell R. Total body irradiation before allogeneic bone marrow transplantation: is more dose better? Int J Radiat Oncol Biol Phys 2001; 49:1071–1077. 39. Helenglass G, Powles RL, McElwain TJ, et al. Melphalan and total body irradiation (TBI) versus cyclophosphamide and TBI as conditioning for allogeneic matched sibling bone marrow transplants for acute myeloblastic leukaemia in first remission. Bone Marrow Transplant 1988; 3:21–29. 40. Blume KG, Forman SJ, O’Donnell MR, et al. Total body irradiation and high-dose etoposide: a new preparatory regimen for bone marrow transplantation in patients with advanced hematologic malignancies. Blood 1987; 69:1015–1020. 41. Amylon MD, Co JP, Snyder DS, Donaldson SS, Blume KG, Forman SJ. Allogeneic bone marrow transplant in pediatric patients with high-risk hematopoietic malignancies early in the course of their disease. J Pediatr Hematol Oncol 1997; 19:54–61. 42. Blume KG, Long GD, Negrin RS, Chao NJ, Kusnierz-Glaz C, Amylon MD. Role of etoposide (VP-16) in preparatory regimens for patients with leukemia or lymphoma undergoing allogeneic bone marrow transplantation. Bone Marrow Transplant 1994; 14:S9–S10. 43. Malicki J. Doses in critical organs during total body irradiation before bone marrow transplantation. Ann Transplant 1998; 3:14–19. 44. Bradley J, Reft C, Goldman S, et al. High-energy total body irradiation as preparation for bone marrow transplantation in leukemia patients: treatment technique and related complications. Int J Radiat Oncol Biol Phys 1998; 40:391–396. 45. Matthews DC, Appelbaum FR, Eary JF, et al. Development of a marrow transplant regimen for acute leukemia using targeted hematopoietic irradiation delivered by 131I-labeled anti-CD45 antibody, combined with cyclophosphamide and total body irradiation. Blood 1995; 85:1122–1131. 46. Matthews DC, Appelbaum FR, Eary JF, et al. Phase I study of (131)I-anti-CD45 antibody plus cyclophosphamide and total body irradiation for advanced acute leukemia and myelodysplastic syndrome. Blood 1999; 94:1237–1247. 47. Santos GW, Tutschka PJ, Brookmeyer R, et al. Marrow transplantation for acute non-lymphocytic leukemia after treatment with busulfan and cyclophosphamide. N Engl J Med 1983; 309:1347–1353. 48. Tutschka PJ, Copelan EA, Klein JP. Bone marrow transplantation for leukemia following a new busulfan and cyclophosphamide regimen. Blood 1987; 70:1382–1388. 49. Matsuyama T, Kojima S, Kato K. Allogeneic bone marrow transplantation for childhood leukemia following a busulfan and melphalan preparative regimen. Bone Marrow Transplant 1998; 22:21–26. 50. Hassan M, Ljungman P, Bolme P, et al. Busulfan bioavailability. Blood 1994; 84:2144–2150. 51. Grochow LB, Krivit W, Whitley CB, Blazar B. Busulfan disposition in children. Blood 1990; 75:1723–1727. 52. Shaw PJ, Scharping CE, Brian RJ, Earl JW. Busulfan pharmacokinetics using a single daily highdose regimen in children with acute leukemia. Blood 1994; 84:2357–2362. 53. Vassal G, Koscielny S, Challine D, et al. Busulfan disposition and hepatic veno-occlusive disease in children undergoing bone marrow transplantation. Cancer Chemother Pharmacol 1996; 37:247–253. 54. Hassan M, Fasth A, Gerritsen B, et al. Busulphan kinetics and limited sampling model in children with leukemia and inherited disorders. Bone Marrow Transplant 1996; 18:843–850. 55. Pawlowska AB, Blazar BR, Angelucci E, Baronciani D, Shu XO, Bostrom B. Relationship of plasma pharmacokinetics of high-dose oral busulfan to the outcome of allogeneic bone marrow transplantation in children with thalassemia. Bone Marrow Transplant 1997; 20:915–920. 56. Slattery JT, Sanders JE, Buckner CD, et al. Graft-rejection and toxicity following bone marrow transplantation in relation to busulfan pharmacokinetics. Bone Marrow Transplant 1995; 16:31–42. 57. Bostrom B, Enockson K, Johnson A, Bruns A, Blazar B. Plasma pharmacokinetics of high-dose oral busulfan in children and adults undergoing bone marrow transplantation. Pediatr Transplant 2003; 7:12–18.

BMT for Acute Myeloid Leukemia

521

58. Ljungman P, Hassan M, Bekassy AN, Ringden O, Oberg G. High busulfan concentrations are associated with increased transplant-related mortality in allogeneic bone marrow transplant patients. Bone Marrow Transplant 1997; 20:909–913. 59. Dix SP, Wingard JR, Mullins RE, et al. Association of busulfan area under the curve with venoocclusive disease following BMT. Bone Marrow Transplant 1996; 17:225–230. 60. Grochow LB, Jones RJ, Brundrett RB, et al. Pharmacokinetics of busulfan: correlation with venoocclusive disease in patients undergoing bone marrow transplantation. Cancer Chemother Pharmacol 1989; 25:55–61. 61. Bolinger AM, Zangwill AB, Slattery JT, et al. Target dose adjustment of busulfan in pediatric patients undergoing bone marrow transplantation. Bone Marrow Transplant 2001; 28:1013–1018. 62. Chattergoon DS, Saunders EF, Klein J, et al. An improved limited sampling method for individualised busulphan dosing in bone marrow transplantation in children. Bone Marrow Transplant 1997; 20:347–354. 63. Lindley C, Shea T, McCune J, et al. Intraindividual variability in busulfan pharmacokinetics in patients undergoing a bone marrow transplant: assessment of a test dose and first dose strategy. Anticancer Drugs 2004; 15:453–459. 64. Andersson BS, Kashyap A, Gian V, et al. Conditioning therapy with intravenous busulfan and cyclophosphamide (IV BuCy2) for hematologic malignancies prior to allogeneic stem cell transplantation: a phase II study. Biol Blood Marrow Transplant 2002; 8:145–154. 65. Andersson BS, Thall PF, Madden T, et al. Busulfan systemic exposure relative to regimen-related toxicity and acute graft-versus-host disease: defining a therapeutic window for i.v. BuCy2 in chronic myelogenous leukemia. Biol Blood Marrow Transplant 2002; 8:477–485. 66. Nguyen L, Fuller D, Lennon S, Leger F, Puozzo C. I.V. busulfan in pediatrics: a novel dosing to improve safety/efficacy for hematopoietic progenitor cell transplantation recipients. Bone Marrow Transplant 2004; 33:979–987. 67. Lahteenmaki PM, Chakrabarti S, Cornish JM, Oakhill AH. Outcome of single fraction total body irradiation-conditioned stem cell transplantation in younger children with malignant disease— comparison with a busulphan-cyclophosphamide regimen. Acta Oncol 2004; 43:196–203. 68. Mattsson J, Uzunel M, Remberger M, Hassan M. Fractionated TBI correlates with less T cell mixed chimerism but increased risk of relapse compared to busulphan in patients with haematological malignancies after allogeneic stem cell transplantation. Bone Marrow Transplant 2003; 32:477–483. 69. Dusenbery KE, Daniels KA, McClure JS, et al. Randomized comparison of cyclophosphamide-total body irradiation versus busulfan-cyclophosphamide conditioning in autologous bone marrow transplantation for acute myeloid leukemia. Int J Radiat Oncol Biol Phys 1995; 31:119–128. 70. Dusenbery KE, Steinbuch M, McGlave PB, et al. Autologous bone marrow transplantation in acute myeloid leukemia: the university of Minnesota experience. Int J Radiat Oncol Biol Phys 1996; 36:335–343. 71. Socie G, Clift RA, Blaise D, et al. Busulfan plus cyclophosphamide compared with total-body irradiation plus cyclophosphamide before marrow transplantation for myeloid leukemia: long-term follow-up of 4 randomized studies. Blood 2001; 98:3569–3574. 72. Blaise D, Maraninchi D, Archimbaud E, et al. Allogeneic bone marrow transplantation for acute myeloid leukemia in first remission: a randomized trial of a busulfan-cytoxan versus cytoxan-total body irradiation as preparative regimen: a report from the group d’Etudes de la greffe de moelle osseuse. Blood 1992; 79:2578–2582. 73. Ringden O, Ruutu T, Remberger M, et al. A randomized trial comparing busulfan with total body irradiation as conditioning in allogeneic marrow transplant recipients with leukemia: a report from the nordic bone marrow transplantation group. Blood 1994; 83:2723–2730. 74. Jurado M, Deeg HJ, Storer B, et al. Hematopoietic stem cell transplantation for advanced myelodysplastic syndrome after conditioning with busulfan and fractionated total body irradiation is associated with low relapse rate but considerable nonrelapse mortality. Biol Blood Marrow Transplant 2002; 8:161–169. 75. Yoshihara T, Naya M, Tsunamoto K, et al. A preparatory regimen of total body irradiation, busulphan and melphalan for allogeneic bone marrow transplantation in childhood high-risk leukemia and lymphoma. Anticancer Res 2003; 23:1739–1742. 76. Hirabayashi N, Goto S, Ishii M, Yuge M, Mitsuma A, Noda N. Busulfan, cyclophosphamide and total body irradiation as conditioning for allogeneic bone marrow transplantation for acute and chronic myeloid leukemia. Bone Marrow Transplant 1998; 21:1079–1083.

522

Chen and Arceci

77. Nagler A, Finlander R, Or R, Naparstek E, Varadi G, Slavin S. The role of thiotepa in autologous bone marrow transplantation for acute leukemia. Leuk Res 1998; 22:991–995. 78. Worth L, Tran H, Petropoulos D, et al. Hematopoietic stem cell transplantation for childhood myeloid malignancies after high-dose thiotepa, busulfan and cyclophosphamide. Bone Marrow Transplant 1999; 24:947–952. 79. Sandler ES, Hagg R, Coppes MJ, et al. Hematopoietic stem cell transplantation (HSCT) with a conditioning regimen of busulfan, cyclophosphamide, and etoposide for children with acute myelogenous leukemia (AML): a phase I study of the pediatric blood and marrow transplant consortium. Med Pediatr Oncol 2000; 35:403–409. 80. Jerjis S, Roovers E, Muus P, Schaap N, de Witte T. Idarubicin to intensify the conditioning regimens of autologous bone marrow transplantation for patients with acute myeloid leukemia in first complete remission. Bone Marrow Transplant 1998; 22:13–19. 81. Storb R, Yu C, Wagner JL, et al. Stable mixed hematopoietic chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation. Blood 1997; 89:3048–3054. 82. Diaconescu R, Flowers CR, Storer B, et al. Morbidity and mortality with nonmyeloablative compared to myeloablative conditioning before hematopoietic cell transplantation from HLA matched related donors. Blood 2004. 83. Couriel DR, Saliba RM, Giralt S, et al. Acute and chronic graft-versus-host disease after ablative and nonmyeloablative conditioning for allogeneic hematopoietic transplantation. Biol Blood Marrow Transplant 2004; 10:178–185. 84. Sayer HG, Kroger M, Beyer J, et al. Reduced intensity conditioning for allogeneic hematopoietic stem cell transplantation in patients with acute myeloid leukemia: disease status by marrow blasts is the strongest prognostic factor. Bone Marrow Transplant 2003; 31:1089–1095. 85. Weiden PL, Flournoy N, Thomas ED, et al. Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 1979; 300:1068–1073. 86. Sullivan KM, Fefer A, Witherspoon R, et al. Graft-versus-leukemia in man: relationship of acute and chronic graft-versus-host disease to relapse of acute leukemia following allogeneic bone marrow transplantation. Prog Clin Biol Res 1987; 244:391–399. 87. Neudorf S, Sanders J, Kobrinsky N, et al. Allogeneic bone marrow transplantation for children with acute myelocytic leukemia in first remission demonstrates a role for graft versus leukemia in the maintenance of disease-free survival. Blood 2004. 88. Fefer A, Sullivan KM, Weiden P, et al. Graft versus leukemia effect in man: the relapse rate of acute leukemia is lower after allogeneic than after syngeneic marrow transplantation. Prog Clin Biol Res 1987; 244:401–408. 89. Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990; 75:555–562. 90. Gale RP, Horowitz MM, Ash RC, et al. Identical-twin bone marrow transplants for leukemia. Ann Intern Med 1994; 120:646–652. 91. Kolb HJ, Schattenberg A, Goldman JM, et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. European group for blood and marrow transplantation working party chronic leukemia. Blood 1995; 86:2041–2050. 92. Montagna D, Locatelli F, Calcaterra V, et al. Does the emergence and persistence of donor-derived leukaemia-reactive cytotoxic T lymphocytes protect patients given an allogeneic BMT from recurrence? Results of a preliminary study. Bone Marrow Transplant 1998; 22:743–750. 93. Charbonnier A, Gaugler B, Sainty D, Lafage-Pochitaloff M, Olive D. Human acute myeloblastic leukemia cells differentiate in vitro into mature dendritic cells and induce the differentiation of cytotoxic T cells against autologous leukemias. Eur J Immunol 1999; 29:2567–2578. 94. Porta MD, Rigolin GM, Alessandrino EP, et al. Dendritic cell recovery after allogeneic stem-cell transplantation in acute leukemia: correlations with clinical and transplant characteristics. Eur J Haematol 2004; 72:18–25. 95. Kiessling R, Klein E, Wigzell H. “Natural” killer cells in the mouse. I Cytotoxic cells with specificity for mouse moloney leukemia cells. Specificity and distribution according to genotype. Eur J Immunol 1975; 5:112–117. 96. Herberman RB, Holden HT. Natural killer cells as anti-tumor effector cells. J Natl Cancer Inst 1979; 62:441–445. 97. Lotzova E, Savary CA. Possible involvement of natural killer cells in bone marrow graft rejection. Biomedicine 1977; 27:341–344.

BMT for Acute Myeloid Leukemia

523

98. O’Brien TK, Kendra JA, Stephens HA, Knight RA, Barrett AJ. Recognition of marrow elements by natural killer cells: are NK cells involved in haemopoietic regulation? Br J Haematol 1983; 53:161–164. 99. Karre K, Ljunggren HG, Piontek G, Kiessling R. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 1986; 319:675–678. 100. Carrington M, Norman PJ. The KIR gene cluster. National Center for Biotechnology Information. Bethesda, MD: National Library of Medicine (U.S.), 2003. 101. Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295:2097–2100. 102. Leung W, Iyengar R, Turner V, et al. Determinants of antileukemia effects of allogeneic NK cells. J Immunol 2004; 172:644–650. 103. Bishara A, De Santis D, Witt CC, et al. The beneficial role of inhibitory KIR genes of HLA class I NK epitopes in haploidentically mismatched stem cell allografts may be masked by residual donoralloreactive T cells causing GVHD. Tissue Antigens 2004; 63:204–211. 104. Lowe EJ, Turner V, Handgretinger R, et al. T-cell alloreactivity dominates natural killer cell alloreactivity in minimally T-cell-depleted HLA-non-identical paediatric bone marrow transplantation. Br J Haematol 2003; 123:323–326. 105. Davies SM, Ruggieri L, DeFor T, et al. Evaluation of KIR ligand incompatibility in mismatched unrelated donor hematopoietic transplants. Killer immunoglobulin-like receptor. Blood 2002; 100:3825–3827. 106. Mehta J, Powles R, Singhal S, et al. Autologous bone marrow transplantation for acute myeloid leukemia in first remission: identification of modifiable prognostic factors. Bone Marrow Transplant 1995; 16:499–506. 107. Mehta J, Powles R, Treleaven J, et al. Long-term follow-up of patients undergoing allogeneic bone marrow transplantation for acute myeloid leukemia in first complete remission after cyclophosphamide-total body irradiation and cyclosporine. Bone Marrow Transplant 1996; 18:741–746. 108. Sierra J, Storer B, Hansen JA, et al. Transplantation of marrow cells from unrelated donors for treatment of high-risk acute leukemia: the effect of leukemic burden, donor HLA- matching, and marrow cell dose. Blood 1997; 89:4226–4235. 109. Gorin NC, Labopin M, Laporte JP, et al. Importance of marrow dose on post-transplant outcome in acute leukemia: models derived from patients autografted with mafosfamide-purged marrow at a single institution. Exp Hematol 1999; 27:1822–1830. 110. Anasetti C. Transplantation of hematopoietic stem cells from alternate donors in acute myelogenous leukemia. Leukemia 2000; 14:502–504. 111. Barrett AJ, Ringden O, Zhang MJ, et al. Effect of nucleated marrow cell dose on relapse and survival in identical twin bone marrow transplants for leukemia. Blood 2000; 95:3323–3327. 112. Aversa F, Tabilio A, Terenzi A, et al. Successful engraftment of T-cell-depleted haploidentical “three-loci” incompatible transplants in leukemia patients by addition of recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells to bone marrow inoculum. Blood 1994; 84:3948–3955. 113. Handgretinger R, Klingebiel T, Lang P, et al. Megadose transplantation of purified peripheral blood CD34(C) progenitor cells from HLA-mismatched parental donors in children. Bone Marrow Transplant 2001; 27:777–783. 114. Bensinger WI, Weaver CH, Appelbaum FR, et al. Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor. Blood 1995; 85:1655. 115. Bensinger WI, Clift R, Martin P, et al. Allogeneic peripheral blood stem cell transplantation in patients with advanced hematologic malignancies: a retrospective comparison with marrow transplantation. Blood 1996; 88:2794–2800. 116. Ustun C, Arslan O, Beksac M, et al. A retrospective comparison of allogeneic peripheral blood stem cell and bone marrow transplantation results from a single center: a focus on the incidence of graftversus-host disease and relapse. Biol Blood Marrow Transplant 1999; 5:28–35. 117. Pavletic ZS, Bishop MR, Tarantolo SR, et al. Hematopoietic recovery after allogeneic blood stemcell transplantation compared with bone marrow transplantation in patients with hematologic malignancies. J Clin Oncol 1997; 15:1608–1616. 118. Russell JA, Larratt L, Brown C, et al. Allogeneic blood stem cell and bone marrow transplantation for acute myelogenous leukemia and myelodysplasia: influence of stem cell source on outcome. Bone Marrow Transplant 1999; 24:1177–1183.

524

Chen and Arceci

119. Bensinger WI, Martin PJ, Storer B, et al. Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives in patients with hematologic cancers. N Engl J Med 2001; 344:175–181. 120. Schmitz N, Bacigalupo A, Hasenclever D, et al. Allogeneic bone marrow transplantation versus filgrastim-mobilised peripheral blood progenitor cell transplantation in patients with early leukaemia: first results of a randomised multicenter trial of the European group for blood and marrow transplantation. Bone Marrow Transplant 1998; 21:995–1003. 121. Schmitz N, Beksac M, Hasenclever D, et al. Transplantation of mobilized peripheral blood cells to HLA-identical siblings with standard-risk leukemia. Blood 2002; 100:761–767. 122. Remberger M, Ringden O, Blau IW, et al. No difference in graft-versus-host disease, relapse, and survival comparing peripheral stem cells to bone marrow using unrelated donors. Blood 2001; 98:1739–1745. 123. Couban S, Simpson DR, Barnett MJ, et al. A randomized multicenter comparison of bone marrow and peripheral blood in recipients of matched sibling allogeneic transplants for myeloid malignancies. Blood 2002; 100:1525–1531. 124. Mohty M, Kuentz M, Michallet M, et al. Chronic graft-versus-host disease after allogeneic blood stem cell transplantation: long-term results of a randomized study. Blood 2002; 100:3128–3134. 125. Flowers ME, Parker PM, Johnston LJ, et al. Comparison of chronic graft-versus-host disease after transplantation of peripheral blood stem cells versus bone marrow in allogeneic recipients: longterm follow-up of a randomized trial. Blood 2002; 100:415–419. 126. De Souza CA, Duraes MI, Vigorito AC, et al. Quality of life in patients randomized to receive a bone marrow or a peripheral blood allograft. Haematologica 2002; 87:1281–1285. 127. Benito AI, Gonzalez-Vicent M, Garcia F, et al. Allogeneic peripheral blood stem cell transplantation (PBSCT) from HLA-identical sibling donors in children with hematological diseases: a single center pilot study. Bone Marrow Transplant 2001; 28:537–543. 128. Matsubara H, Makimoto A, Takayama J, et al. Possible clinical benefits of the use of peripheral blood stem cells over bone marrow in the allogeneic transplantation setting for the treatment of childhood leukemia. Jpn J Clin Oncol 2001; 31:30–34. 129. Gorin NC, Labopin M, Rocha V, et al. Marrow versus peripheral blood for geno-identical allogeneic stem cell transplantation in acute myelocytic leukemia: influence of dose and stem cell source shows better outcome with rich marrow. Blood 2003; 102:3043–3051. 130. Mavroudis DA, Read EJ, Molldrem J, et al. T cell-depleted granulocyte colony-stimulating factor (G-CSF) modified allogeneic bone marrow transplantation for hematological malignancy improves graft CD34C cell content but is associated with delayed pancytopenia. Bone Marrow Transplant 1998; 21:431–440. 131. Dallorso S, Rondelli R, Messina C, et al. Clinical benefits of granulocyte colony-stimulating factor therapy after hematopoietic stem cell transplant in children: results of a prospective randomized trial. Haematologica 2002; 87:1274–1280. 132. Ringden O, Labopin M, Gorin NC, et al. Treatment with granulocyte colony-stimulating factor after allogeneic bone marrow transplantation for acute leukemia increases the risk of graft-versus-host disease and death: a study from the acute leukemia working party of the European group for blood and marrow transplantation. J Clin Oncol 2004; 22:416–423. 133. Ho VT, Mirza NQ, Junco Dd D, Okamura T, Przepiorka D. The effect of hematopoietic growth factors on the risk of graft-versus-host disease after allogeneic hematopoietic stem cell transplantation: a meta-analysis. Bone Marrow Transplant 2003; 32:771–775. 134. Papadopoulos EB, Carabasi MH, Castro-Malaspina H, et al. T-cell-depleted allogeneic bone marrow transplantation as post-remission therapy for acute myelogenous leukemia: freedom from relapse in the absence of graft-versus-host disease. Blood 1998; 91:1083–1090. 135. Mehta J, Singhal S, Gee AP, et al. Bone marrow transplantation from partially HLA-mismatched family donors for acute leukemia: single-center experience of 201 patients. Bone Marrow Transplant 2004. 136. Neudorf SM, Rybka W, Ball E, et al. The use of counterflow centrifugal elutriation for the depletion of T cells from unrelated donor bone marrow. J Hematother 1997; 6:351–359. 137. Aschan J, Carlens S, Hagglund H, Klaesson S, Mattsson J, Remberger M. Improved survival after bone marrow transplantation for early leukemia using busulfan-cyclophosphamide and individualized prophylaxis against graft-versus-host disease: a long-term follow-up. Clin Transplant 1999; 13:512–519. 138. Aversa F, Terenzi A, Carotti A, et al. Improved outcome with T-cell-depleted bone marrow transplantation for acute leukemia. J Clin Oncol 1999; 17:1545–1550.

BMT for Acute Myeloid Leukemia

525

139. Godder KT, Hazlett LJ, Abhyankar SH, et al. Partially mismatched related-donor bone marrow transplantation for pediatric patients with acute leukemia: younger donors and absence of peripheral blasts improve outcome. J Clin Oncol 2000; 18:1856–1866. 140. Guinan EC, Boussiotis VA, Neuberg D, et al. Transplantation of anergic histoincompatible bone marrow allografts [see comments]. N Engl J Med 1999; 340:1704–1714. 141. Kernan NA, Bartsch G, Ash RC, et al. Analysis of 462 transplantations from unrelated donors facilitated by the national marrow donor program. N Engl J Med 1993; 328:593–602. 142. Beatty PG, Anasetti C, Hansen JA, et al. Marrow transplantation from unrelated donors for treatment of hematologic malignancies: effect of mismatching for one HLA locus. Blood 1993; 81:249–253. 143. Petersdorf EW, Longton GM, Anasetti C, et al. Association of HLA-C disparity with graft failure after marrow transplantation from unrelated donors. Blood 1997; 89:1818–1823. 144. Petersdorf EW, Hansen JA, Martin PJ, et al. Major-histocompatibility-complex class I alleles and antigens in hematopoietic-cell transplantation. N Engl J Med 2001; 345:1794–1800. 145. Locatelli F, Maccario R, Comoli P, et al. Hematopoietic and immune recovery after transplantation of cord blood progenitor cells in children. Bone Marrow Transplant 1996; 18:1095–1101. 146. Elhasid R, Ben Arush MW, Pollack S, et al. Immune and hematopoietic reconstitution after transplantation of cord blood progenitor cells: case report and review of the literature. Leukemia 2000; 14:931–934. 147. Hows J, Bradley BA, Gore S, Downie T, Howard M, Gluckman E. Prospective evaluation of unrelated donor bone marrow transplantation. The international marrow unrelated search and transplant (IMUST) study. Bone Marrow Transplant 1993; 12:371–380. 148. Sasazuki T, Juji T, Morishima Y, et al. Effect of matching of class I HLA alleles on clinical outcome after transplantation of hematopoietic stem cells from an unrelated donor. Japan marrow donor program [see comments]. N Engl J Med 1998; 339:1177–1185. 149. Witt C, Sayer D, Trimboli F, et al. Unrelated donors selected prospectively by block-matching have superior bone marrow transplant outcome. Hum Immunol 2000; 61:85–91. 150. Speiser DE, Tiercy JM, Rufer N, et al. High resolution HLA matching associated with decreased mortality after unrelated bone marrow transplantation. Blood 1996; 87:4455–4462. 151. Bearman SI, Mori M, Beatty PG, et al. Comparison of morbidity and mortality after marrow transplantation from HLA-genotypically identical siblings and HLA-phenotypically identical unrelated donors. Bone Marrow Transplant 1994; 13:31–35. 152. Saarinen-Pihkala UM, Gustafsson G, Ringden O, et al. No disadvantage in outcome of using matched unrelated donors as compared with matched sibling donors for bone marrow transplantation in children with acute lymphoblastic leukemia in second remission. J Clin Oncol 2001; 19:3406–3414. 153. Kroger N, Zabelina T, Kruger W, et al. Anti-thymocyte-globulin as part of the preparative regimen prevents graft failure and severe graft versus host disease (GVHD) in allogeneic stem cell transplantation from unrelated donors. Ann Hematol 2001; 80:209–215. 154. Remberger M, Storer B, Ringden O, Anasetti C. Association between pre-transplant thymoglobulin and reduced non-relapse mortality rate after marrow transplantation from unrelated donors. Bone Marrow Transplant 2002; 29:391–397. 155. Amadori S, Testi AM, Arico M, et al. Prospective comparative study of bone marrow transplantation and postremission chemotherapy for childhood acute myelogenous leukemia. The Associazione Italiana Ematologia ed Oncologia Pediatrica Cooperative Group. J Clin Oncol 1993; 11:1046–1054. 156. Ravindranath Y, Yeager AM, Chang MN, et al. Autologous bone marrow transplantation versus intensive consolidation chemotherapy for acute myeloid leukemia in childhood. Pediatric Oncology Group. N Engl J Med 1996; 334:1428–1434. 157. Stevens RF, Hann IM, Wheatley K, Gray RG. Marked improvements in outcome with chemotherapy alone in paediatric acute myeloid leukemia: results of the United Kingdom Medical Research Council’s 10th AML trial. MRC childhood leukaemia working party. Br J Haematol 1998; 101:130–140. 158. Woods WG, Neudorf S, Gold S, et al. A comparison of allogeneic bone marrow transplantation, autologous bone marrow transplantation, and aggressive chemotherapy in children with acute myeloid leukemia in remission: a report from the children’s cancer group. Blood 2001; 97:56–62. 159. Bleakley M, Lau L, Shaw PJ, Kaufman A. Bone marrow transplantation for paediatric AML in first remission: a systematic review and meta-analysis. Bone Marrow Transplant 2002; 29:843–852.

526

Chen and Arceci

160. Ortega JJ, Diaz de Heredia C, Olive T, et al. Allogeneic and autologous bone marrow transplantation after consolidation therapy in high-risk acute myeloid leukemia in children. Towards a risk-oriented therapy. Haematologica 2003; 88:290–299. 161. Selvaggi KJ, Wilson JW, Mills LE, et al. Improved outcome for high-risk acute myeloid leukemia patients using autologous bone marrow transplantation and monoclonal antibody-purged bone marrow. Blood 1994; 83:1698–1705. 162. Ball ED, Wilson J, Phelps V, Neudorf S. Autologous bone marrow transplantation for acute myeloid leukemia in remission or first relapse using monoclonal antibody-purged marrow: results of phase II studies with long-term follow-up. Bone Marrow Transplant 2000; 25:823–829. 163. Madero L, Gonzalez-Vicent M, Ramirez M, Diaz MA. G-CSF-mobilized PBSCT in children with AML in first complete remission. Bone Marrow Transplant 1999; 23:975–976. 164. Horikoshi Y, Mimaya J, Amano K, et al. Feasibility study of autologous peripheral blood stem cell transplantation for the treatment of childhood acute myelogenous leukemia. Jpn J Clin Oncol 2000; 30:137–145. 165. Martin C, Torres A, Leon A, et al. Autologous peripheral blood stem cell transplantation (PBSCT) mobilized with G-CSF in AML in first complete remission. Role of intensification therapy in outcome. Bone Marrow Transplant 1998; 21:375–382. 166. Locatelli F, Labopin M, Ortega J, et al. Factors influencing outcome and incidence of long-term complications in children who underwent autologous stem cell transplantation for acute myeloid leukemia in first complete remission. Blood 2003; 101:1611–1619. 167. Cesaro S, Meloni G, Messina C, et al. High-dose melphalan with autologous hematopoietic stem cell transplantation for acute myeloid leukemia: results of a retrospective analysis of the Italian pediatric group for bone marrow transplantation. Bone Marrow Transplant 2001; 28:131–136. 168. Bonetti F, Montagna D, Porta F, et al. Autologous bone marrow transplantation for acute myeloid leukaemia in children using total body irradiation and melphalan as conditioning regimen. Leukemia 1995; 9:570–575. 169. Vignetti M, Rondelli R, Locatelli F, et al. Autologous bone marrow transplantation in children with acute myeloblastic leukemia: report from the Italian national pediatric registry (AIEOP-BMT). Bone Marrow Transplant 1996; 18:59–62. 170. Petersen FB, Lynch MH, Clift RA, et al. Autologous marrow transplantation for patients with acute myeloid leukemia in untreated first relapse or in second complete remission. J Clin Oncol 1993; 11:1353–1360. 171. Miller CB, Rowlings PA, Zhang MJ, et al. The effect of graft purging with 4-hydroperoxycyclophosphamide in autologous bone marrow transplantation for acute myelogenous leukemia. Exp Hematol 2001; 29:1336–1346. 172. Redaelli A, Stephens JM, Brandt S, Botteman MF, Pashos CL. Short- and long-term effects of acute myeloid leukemia on patient health-related quality of life. Cancer Treat Rev 2004; 30:103–117. 173. Michel G, Socie G, Gebhard F, et al. Late effects of allogeneic bone marrow transplantation for children with acute myeloblastic leukemia in first complete remission: the impact of conditioning regimen without total-body irradiation—a report from the societe francaise de greffe de moelle. J Clin Oncol 1997; 15:2238–2246. 174. Bakker B, Oostdijk W, Bresters D, Walenkamp MJ, Vossen JM, Wit JM. Disturbances of growth and endocrine function after busulphan-based conditioning for haematopoietic stem cell transplantation during infancy and childhood. Bone Marrow Transplant 2004; 33:1049–1056. 175. Leahey AM, Teunissen H, Friedman DL, Moshang T, Lange BJ, Meadows AT. Late effects of chemotherapy compared to bone marrow transplantation in the treatment of pediatric acute myeloid leukemia and myelodysplasia. Med Pediatr Oncol 1999; 32:163–169. 176. Leung W, Hudson MM, Strickland DK, et al. Late effects of treatment in survivors of childhood acute myeloid leukemia. J Clin Oncol 2000; 18:3273–3279. 177. Parsons SK, Gelber S, Cole BF, et al. Quality-adjusted survival after treatment for acute myeloid leukemia in childhood: a Q-TWiST analysis of the pediatric oncology group study 8821. J Clin Oncol 1999; 17:2144–2152. 178. Watson M, Wheatley K, Harrison GA, et al. Severe adverse impact on sexual functioning and fertility of bone marrow transplantation, either allogeneic or autologous, compared with consolidation chemotherapy alone: analysis of the MRC AML 10 trial. Cancer 1999; 86:1231–1239. 179. Burnett AK. Current controversies: which patients with acute myeloid leukaemia should receive a bone marrow transplantation?—an adult treater’s view Br J Haematol 2002; 118:357–364.

BMT for Acute Myeloid Leukemia

527

180. Chen AR, Alonzo TA, Woods WG, Arceci RJ. Current controversies: which patients with acute myeloid leukaemia should receive a bone marrow transplantation?—an American view. Br J Haematol 2002; 118:378–384. 181. Creutzig U, Reinhardt D. Current controversies: which patients with acute myeloid leukaemia should receive a bone marrow transplantation?—a European view. Br J Haematol 2002; 118:365–377. 182. Wheatley K. Current controversies: which patients with acute myeloid leukaemia should receive a bone marrow transplantation?—a statistician’s view. Br J Haematol 2002; 118:351–356. 183. Erben U, Thiel E, Bittroff-Leben A, et al. CS-1, a novel c-kithiC acute myeloid leukemia cell line with dendritic cell differentiation capacity and absent immunogenicity. Int J Cancer 2003; 105:232–240. 184. Clift RA, Buckner CD, Appelbaum FR, et al. Allogeneic marrow transplantation during untreated first relapse of acute myeloid leukemia. J Clin Oncol 1992; 10:1723–1729. 185. Wells RJ, Adams MT, Alonzo TA, et al. Mitoxantrone and cytarabine induction, high-dose cytarabine, and etoposide intensification for pediatric patients with relapsed or refractory acute myeloid leukemia: Children’s Cancer Group Study 2951. J Clin Oncol 2003; 21:2940–2947. 186. Yalman N, Sarper N, Devecioglu O, et al. Fludarabine, cytarabine, G-CSF and idarubicin (FLAGIDA) for the treatment of relapsed or poor risk childhood acute leukemia. Turk J Pediatr 2000; 42:198–204. 187. Luczynski W, Muszynska-Roslan K, Krawczuk-Rybak M, Kuzmicz M, IwaszkiewiczPawlowska A, Kaliszewski J. Results of IDA-FLAG programme in the treatment of recurrent acute myeloblastic leukaemia—preliminary report. Med Sci Monit 2001; 7:125–129. 188. Pastore D, Specchia G, Carluccio P, et al. FLAG-IDA in the treatment of refractory/relapsed acute myeloid leukemia: single-center experience. Ann Hematol 2003; 82:231–235. 189. Au WY, Lie AK, Chim CS, et al. Arsenic trioxide in comparison with chemotherapy and bone marrow transplantation for the treatment of relapsed acute promyelocytic leukaemia. Ann Oncol 2003; 14:752–757. 190. Dombret H, Fenaux P, Soignet SL, Tallman MS. Established practice in the treatment of patients with acute promyleocytic leukemia and the introduction of arsenic trioxide as a novel therapy. Semin Hematol 2002; 39:8–13. 191. Soignet S, Maslak P. Therapy of acute promyelocytic leukemia. Adv Pharmacol 2004; 51:35–58. 192. Soignet SL, Frankel SR, Douer D, et al. United States multicenter study of arsenic trioxide in relapsed acute promyelocytic leukemia. J Clin Oncol 2001; 19:3852–3860. 193. Bourquin JP, Thornley I, Neuberg D, et al. Favorable outcome of allogeneic hematopoietic stem cell transplantation for relapsed or refractory acute promyelocytic leukemia in childhood. Bone Marrow Transplant 2004; 34:795–798. 194. Santana VM, Mirro J, Jr., Harwood FC, et al. A phase I clinical trial of 2-chlorodeoxyadenosine in pediatric patients with acute leukemia. J Clin Oncol 1991; 9:416–422. 195. Estey EH. Treatment options for relapsed acute promyelocytic leukaemia. Best Pract Res Clin Haematol 2003; 16:521–534. 196. Estey EH, Giles FJ, Beran M, et al. Experience with gemtuzumab ozogamicin (“mylotarg”) and alltrans retinoic acid in untreated acute promyelocytic leukemia. Blood 2002; 99:4222–4224. 197. Ringden O, Labopin M, Frassoni F, et al. Allogeneic bone marrow transplant or second autograft in patients with acute leukemia who relapse after an autograft. Acute leukaemia working party of the European group for blood and marrow transplantation (EBMT). Bone Marrow Transplant 1999; 24:389–396. 198. Bross PF, Beitz J, Chen G, et al. Approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia. Clin Cancer Res 2001; 7:1490–1496. 199. Giles FJ, Kantarjian HM, Kornblau SM, et al. Mylotarg (gemtuzumab ozogamicin) therapy is associated with hepatic venoocclusive disease in patients who have not received stem cell transplantation. Cancer 2001; 92:406–413. 200. Arceci RJ, Sande J, Lange B, et al. Safety and efficacy of gemtuzumab ozogamicin in pediatric patients with advanced CD33C acute myeloid leukemia. Blood 2005; 106:1183–1188. 201. Hale GA, Tong X, Benaim E, et al. Allogeneic bone marrow transplantation in children failing prior autologous bone marrow transplantation. Bone Marrow Transplant 2001; 27:155–162. 202. Meshinchi S, Leisenring WM, Carpenter PA, et al. Survival after second hematopoietic stem cell transplantation for recurrent pediatric acute myeloid leukemia. Biol Blood Marrow Transplant 2003; 9:706–713.

528

Chen and Arceci

203. Shiobara S, Nakao S, Ueda M, et al. Donor leukocyte infusion for Japanese patients with relapsed leukemia after allogeneic bone marrow transplantation: indications and dose escalation. Ther Apher 2001; 5:40–45. 204. Vettenranta K, Hovi L, Saarinen-Pihkala UM. Adoptive immunotherapy as consolidation of remission in pediatric AML relapsing post-transplant. Pediatr Transplant 2003; 7:446–449. 205. Raiola AM, Van Lint MT, Valbonesi M, et al. Factors predicting response and graft-versus-host disease after donor lymphocyte infusions: a study on 593 infusions. Bone Marrow Transplant 2003; 31:687–693. 206. Beck JF, Klingebiel T, Kreyenberg H, et al. Relapse of childhood ALL, AML and MDS after allogeneic stem cell transplantation can be prevented by donor lymphocyte infusion in a critical stage of increasing mixed chimerism. Klin Padiatr 2002; 214:201–205. 207. de Lima M, Bonamino M, Vasconcelos Z, et al. Prophylactic donor lymphocyte infusions after moderately ablative chemotherapy and stem cell transplantation for hematological malignancies: high remission rate among poor prognosis patients at the expense of graft-versus-host disease. Bone Marrow Transplant 2001; 27:73–78. 208. Worth LL, Mullen CA, Choroszy M, Koontz S, Chan K. Treatment of leukemia relapse with recombinant granulocyte-macrophage colony stimulating factor (rhGM-CSF) following unrelated umbilical cord blood transplant: induction of graft-versus-leukemia. Pediatr Transplant 2002; 6:439–442. 209. Okada H, Nomi K, Hamatani S, et al. Induction of graft-versus-host disease and a graft-versusleukemia effect using ubenimex in a patient with infantile leukemia relapsing after an unrelated cord blood transplant. Bone Marrow Transplant 2002; 30:463–465. 210. Martinelli G, Ottaviani E, Visani G, Testoni N, Montefusco V, Tura S. Long-term disease-free acute promyelocytic leukemia patients really can be cured at molecular level. Haematologica 1998; 83:860–863. 211. van der Reijden BA, Simons A, Luiten E, et al. Minimal residual disease quantification in patients with acute myeloid leukaemia and inv(16)/CBFB-MYH11 gene fusion. Br J Haematol 2002; 118:411–418. 212. Marcucci G, Caligiuri MA, Dohner H, et al. Quantification of CBFbeta/MYH11 fusion transcript by real time RT-PCR in patients with INV(16) acute myeloid leukemia. Leukemia 2001; 15:1072–1080. 213. Mitterbauer M, Mitterbauer-Hohendanner G, Sperr WR, et al. Molecular disease eradication is a prerequisite for long-term remission in patients with t(8;21) positive acute myeloid leukemia: a single center study. Leuk Lymphoma 2004; 45:971–977. 214. Laczika K, Novak M, Hilgarth B, et al. Competitive CBFbeta/MYH11 reverse-transcriptase polymerase chain reaction for quantitative assessment of minimal residual disease during postremission therapy in acute myeloid leukemia with inversion(16): a pilot study. J Clin Oncol 1998; 16:1519–1525. 215. Terstappen LW, Safford M, Konemann S, et al. Flow cytometric characterization of acute myeloid leukemia. Part II. Phenotypic heterogeneity at diagnosis. Leukemia 1992; 6:70–80. 216. Weir EG, Borowitz MJ. Flow cytometry in the diagnosis of acute leukemia. Semin Hematol 2001; 38:124–138. 217. Sievers EL, Lange BJ, Buckley JD, et al. Prediction of relapse of pediatric acute myeloid leukemia by use of multidimensional flow cytometry. J Natl Cancer Inst 1996; 88:1483–1488. 218. Sievers EL, Lange BJ, Alonzo TA, et al. Immunophenotypic evidence of leukemia after induction therapy predicts relapse: results from a prospective children’s cancer group study of 252 patients with acute myeloid leukemia. Blood 2003; 101:3398–3406. 219. Bader P, Holle W, Klingebiel T, et al. Mixed hematopoietic chimerism after allogeneic bone marrow transplantation: the impact of quantitative PCR analysis for prediction of relapse and graft rejection in children. Bone Marrow Transplant 1997; 19:697–702. 220. Bader P, Beck J, Frey A, et al. Serial and quantitative analysis of mixed hematopoietic chimerism by PCR in patients with acute leukemias allows the prediction of relapse after allogeneic BMT. Bone Marrow Transplant 1998; 21:487–495. 221. Bader P, Stoll K, Huber S, et al. Characterization of lineage-specific chimaerism in patients with acute leukaemia and myelodysplastic syndrome after allogeneic stem cell transplantation before and after relapse. Br J Haematol 2000; 108:761–768.

26 Hematopoietic Stem-Cell Transplantation for Children with Hodgkin’s and Non-Hodgkin’s Lymphoma Bruce Gordon Pediatric Hematology/Oncology and Stem Cell Transplantation, University of Nebraska Medical Center, Omaha, Nebraska, U.S.A.

K. Scott Baker Pediatric Blood and Marrow Transplant Program, University of Minnesota, Minneapolis, Minnesota, U.S.A.

NON-HODGKIN’S LYMPHOMA Introduction Non-Hodgkin’s lymphomas (NHL) account for 7% of all cancers in children younger than 20 years of age (1), with about 800 new cases diagnosed each year in the United States, an incidence of approximately 1 per 100,000. The incidence of NHL rises steadily throughout life, though the agespecific incidence of various subcategories varies markedly. Overall, more than 70% of children and adolescents with NHL will survive at least 5 years. Outcome is variable depending on a number of factors, including disease subtype and extent of disease at diagnosis (2). The distribution of histologic subtypes of NHL in children is clearly different from that seen in adults. Lymphomas in adults are more commonly of low or intermediate grade, and those that occur in children are usually high grade. The large majority of childhood lymphomas can be classified into one of four categories in the Revised European-American Lymphoma Classification classification: (1) Burkitt’s and Burkitt’s-like lymphoma (small noncleaved B-cell lymphoma); (2) lymphoblastic lymphomas; (3) diffuse large cell lymphomas (LCL); and (4) anaplastic large cell lymphoma.

Lymphoblastic Lymphomas Lymphoblastic lymphomas make up approximately 30% of childhood NHL. Nearly 75% of patients with lymphoblastic lymphoma have an anterior mediastinal mass. There may also be involvement of bone, skin, lymph nodes, bone marrow, central nervous system (CNS), liver, and spleen. Lymphoblastic lymphomas are usually positive for the enzyme terminal deoxynucleotidyl transferase and have a T-cell immunophenotype. About 10% to 15% of lymphoblastic lymphomas have non-T immunologic characteristics (3). 529

530

Gordon and Baker

Overall survival (OS) for localized disease is near 90% with short duration (9 weeks) leukemia-like therapy, including CNS chemoprophylaxis (4,5). Results in patients with more advanced disease are equally good. The BFM group has reported event-free survival (EFS) as high as 90% for patients with stage III and IV lymphoblastic lymphoma (6).

Small Noncleaved Cell Lymphoma Small noncleaved cell lymphoma (SNCL) (Burkitt’s and non-Burkitt’s) accounts for 40% to 50% of childhood NHL. Up to 90% of these tumors are intra-abdominal, with other sites of involvement including testis, lymphoid tissue of Waldeyer’s ring, nasal sinuses, bone, peripheral lymph nodes, skin, bone marrow, and CNS. SNCLs are of B-cell origin; they usually express surface immunoglobulin (most commonly IgM of either kappa or lambda light chain subtype). These tumors also express a characteristic chromosomal translocation, usually t(8;14) and more rarely t(8;22) or t(2;8). Localized SNCL can be effectively treated with short duration chemotherapy, producing EFS of 90% or greater (4,7). With more intensive chemotherapy, equally good outcome can be achieved for children with more advanced (stage III or IV) disease (8,9).

Large Cell Lymphoma LCL (consisting of anaplastic large cell and diffuse large cell type) account for approximately 20% to 25% of childhood NHL. In the case of LCLs, more relevant than the histologic distinction is the tumor immunophenotype. B lineage LCL may present clinically like the small noncleaved lymphomas (to which it bears certain biological similarities), although it is most often localized, frequently involving the mediastinum (10). T-lineage LCL include CD30-positive anaplastic LCL (ALCL) (11) as well as other mature T-cell lymphomas that are not clearly anaplastic (though often express CD30) (12,13). The nonrandom translocation (2;5) (p23;q35) is a characteristic finding in CD30-positive lymphoma. Clinically, ALCL (and other peripheral T-cell lymphomas) have a broad range of presentations, including involvement of lymph nodes and a variety of extranodal sites, particularly skin and bone. These tumors are often associated with constitutional symptoms and a prolonged waxing and waning course. Null cell LCLs (non-T, non-B) are often, but not always, CD30-positive, are usually classified as ALCLs, and have a clinical outcome similar to the T-cell LCLs (10). The prognosis for children with localized large cell lymphoma is as good as that for other histologies, with EFS of 90% (14). Patients with more advanced disease have long-term survival of 60% to 80%. B-LCL appear to have superior outcome (10). EFS for children with advanced CD30-positive ALCL are around 70% to 75% (15,16).

High-Dose Therapy with Stem-Cell Transplantation for Non-Hodgkin’s Lymphoma Despite these overall excellent treatment results, a proportion of children with NHL will relapse from their disease. Salvage chemotherapy may be effective in producing remissions, but only a small number are cured (17–19). Even worse outcomes in adults with relapsed NHL encouraged investigators in the 1980s to consider dose escalation with hematopoietic stem cell transplantation (HSCT) as a therapeutic option in older patients with relapsed lymphoma. A brief review of the data supporting this modality in adults is in order, before considering its applicability to children.

HSCT for Hodgkin’s and Non-Hodgkin’s Lymphoma

531

Adult Studies Hematopoietic Stem-Cell Transplantation as Salvage Therapy Following the demonstration of the efficacy of autologous transplantation in Burkitt’s lymphoma (BL) (20), numerous groups began exploring the use of this modality in other histologies of NHL (21,22). Philip, Armitage, and coworkers showed that, in a large group of adults with relapsed diffuse NHL (excluding BL), sensitivity to salvage chemotherapy was an significant determinant of response and cure with high-dose therapy and autologous stem cell rescue (23). The superiority of high-dose therapy with stem cell rescue over conventional salvage chemotherapy for relapsed NHL was definitively shown by the international PARMA trial (24). This study involved 215 patients between 18 and 60 years old with intermediate or high-grade NHL in first (nZ188) or second (nZ27) relapse. Patients were treated with two courses of DHAP (dexamethasone, cytarabine, and cisplatinum) salvage chemotherapy (25). The 109 patients who responded to salvage therapy [45 complete response (CR), 64 partial response (PR)] were randomized to either continued chemotherapy or to high dose chemotherapy with HSCT. Patients randomized to chemotherapy received four additional courses of DHAP, followed by 35 Gy involved field radiotherapy to residual bulky disease. Patient randomized to high-dose therapy were treated with 26 Gy involved field radiotherapy to residual bulky disease followed by BEAC (BCNU, etoposide Ara-C, cyclophosphamide) chemotherapy and autologous bone marrow transplantation. With a median follow-up of 63 months, the response rate was 84% after HSCT compared with 44% after chemotherapy alone. The EFS was 46% in the HSCT arm versus 12% in the chemotherapy arm (pZ0.001). OS was 53% versus 32% for the HSCT and chemotherapy arms respectively (pZ0.04). Subsequent analyses have confirmed the continued survival advantage of HSCT over conventional chemotherapy (26) but noted that the advantage was limited to those with higher risk disease [age adjusted International Prognostic Index (IPI) score greater than zero] (27). Hematopoietic Stem-Cell Transplantation as Consolidation Therapy The role of high dose therapy with stem cell transplantation for adults in first remission of their high risk NHL is less clear. Haioun reported the updated results of a large randomized trial (LNH-84) that compared consolidative sequential treatment versus the high-dose chemotherapy [cyclophosphamide, BCNU, and VP16 (CBV)] followed by autologous stem cell transplantation in 541 patients with aggressive non-Hodgkin’s lymphoma in first complete remission (28). For the entire group, disease-free survival (DFS) and survival did not differ significantly between the two consolidative treatment arms. However, among patients with two or three IPI risk factors, (29) CBV was superior to sequential chemotherapy, with 5-year DFS rates of 59% and 39%, respectively (pZ0.01). The 5-year survival rate was also superior in the CBV group at 65% compared with 52% in the sequential chemotherapy group (pZ0.06). Similarly, the Italian Non-Hodgkin’s Study Group reported 124 patients with bulky stage II or stage III/IV NHL randomized to receive 12 weeks of standard induction therapy (etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin; VACOP-B) alone or followed by autologous HSCT. For patients with high-intermediate and high-risk disease by age-adjusted IPI, DFS was superior in the autologous HSCT arm (87% vs. 48%, pZ0.008) (30). Both of these studies used full course induction therapy. Other trials using abbreviated induction therapy have failed to show an advantage for high-dose therapy in first remission. The LNH-93 trial randomized 370 patients with high-intermediate and high-risk disease to standard induction chemotherapy versus three cycles of abbreviated induction chemotherapy and autologous HSCT. Patients randomized to standard induction therapy had a superior outcome (EFS 54% vs. 41%, pZ0.01) (31). Similarly, a German High Grade Lymphoma Study Group trial found no advantage of abbreviated induction therapy with autologous HSCT over standard

532

Gordon and Baker

induction chemotherapy (32). Therefore, no definitive conclusions can be drawn regarding the utility of HSCT in adults in first CR of their NHL.

Autologous Versus Allogeneic Hematopoietic Stem-Cell Transplantation Autologous bone marrow (or blood stem cells) has been the preferred source of stem cells for rescue after high dose therapy for NHL. Although the presence of a graft-versus-leukemia effect has been well documented, there has been considerable debate whether a similar effect occurs following transplantation for NHL. Chopra and colleagues have published the results of a case control study based on data from the European Bone Marrow Transplant Group registry. The study compared the outcome of 101 patients undergoing high-dose therapy with allogeneic bone marrow transplantation to a matched control group of patients undergoing autologous HSCT. Subjects were matched to controls based on four prognostic factors: disease status at transplant, stage at transplant, histology, and conditioning regimen. They found no difference in the progression free survival (PFS) or relapse rate when comparing these two groups as a whole. PFS for patients undergoing allogeneic transplantation was 49%, compared with a PFS of 46% for autologous HSCT. Relapse rate was 23% for allogeneic HSCT versus 38% for autologous HSCT (not statistically significant). Subgroup analysis, however, showed a significantly lower relapse rate for patients undergoing allogeneic HSCT for lymphoblastic lymphoma, compared with those undergoing autologous HSCT (24% vs. 48% respectively, pZ0.035), suggesting the presence of a graft-versus-lymphoma effect. This advantage was negated by increased treatment related mortality in the allogeneic group (24% allogeneic vs. 10% autologous, pZ0.06), and there was no difference in PFS between the two groups (44% autologous vs. 57% allogeneic, pZ0.1). When the lymphoblastic lymphoma group was further subdivided, a PFS advantage was shown for patients in CR2 or greater who were treated with allogeneic HSCT (19% autologous vs. 40% allogeneic, pZ0.03) (33). Ratanatharathorn and colleagues conducted a prospective comparative trial of allogeneic versus autologous bone marrow transplant for 66 adults with relapsed or refractory NHL. Patients were prepared with either CBV or cyclophosphamide/total body irradiation (TBI). The probability of disease progression was significantly higher in the autologous group (69%) than in the allogeneic group (20%, pZ0.001), but again there was no significant advantage in PFS (24% autologous vs. 47% allogeneic, pZ0.21) (34). Both these studies suggested the presence of a graft-versus-lymphoma effect, with significant reduction in the risk of relapse in patients receiving allografts (at least in certain subgroups). In older patients, this effect counteracted by increased transplant related mortality in the allogeneic group, producing no net survival advantage. Both authors suggest that an advantage of allografts might be more prominent in younger patients for whom transplant related mortality is lower, though no direct data is available to support this conjecture.

Hematopoietic Stem-Cell Transplantation for Childhood Non-Hodgkin’s Lymphomas Though young patients are included in most of the large adult series, the differences in distribution of histologies and in tumor biology between adults and children provide a rationale for studies focusing on NHL in children and adolescents. There are, however, few large series available, and interpretation of these results are difficult. In general, because of the low incidence of relapse after primary treatment of NHL in children, single center studies have required accrual of subjects over the course of many years, and the intensity and efficacy of primary therapies have varied considerably. As early as 1983, O’Leary and coworkers reported use of high-dose therapy and allogeneic transplantation in 10 children and young adults (median age 16 years, range 4 to 29) with disseminated [Murphy stage III-IV BL (six cases) or relapsed T-cell lymphoblastic

HSCT for Hodgkin’s and Non-Hodgkin’s Lymphoma

533

lymphoma (four cases)]. Patients were prepared with BCNU, Ara-C, cyclophosphamide, and TBI. At a median of 29 months, five of 10 patients were alive without disease (35). Hartmann and colleagues reported 16 children with relapsed non-Hodgkin’s lymphoma who were treated with high-dose BCNU, cyclophosphamide, cytarabine, and thioguanine (BACT) followed by autologous bone marrow transplantation. Only 2 of 16 were in complete remission at the time of high dose therapy. Eleven of 15 evaluable patients achieved (or continued in) complete remission following high dose therapy. Five patients remained in prolonged complete remission 77C to 152C weeks after treatment. Patients who were treated in CR or PR had superior outcome (36). Loiseau and coworkers reported an additional 24 children with relapsed or refractory non-Hodgkin’s lymphoma (including 16 B-cell and eight T-cell lymphomas). Patients were prepared with busulfan with cyclophosphamide and/or melphalan. Nineteen of 23 evaluable at day 30 post HSCT had obtained complete remission. Eight patients were still surviving disease free 62 to 296 weeks after HSCT. Among the seven children with resistant disease before HDC, only one remained free of disease (37). Bureo and colleagues reported a retrospective analysis of 46 pediatric patients with NHL who underwent high-dose therapy and stem cell transplantation in six Spanish centers (38). Cases were collected between 1982 and 1993. The median age of the patients was 9 years, with a range of 1 to 17 years. Twenty-one cases were of lymphoblastic lymphoma, 19 were BL and 6 were large cell lymphoma (not further defined). Reflecting the long accrual period, patients were initially treated with varied regimens (with or without radiotherapy). At time of high-dose therapy, 13 were in first complete remission. These patients were felt to be candidates for high dose therapy because of poor risk features: two had slow response to first-line therapy, seven had only partial response to first-line therapy, and four had Murphy stage IV disease at diagnosis. Ten of the 13 had high lactate dehydrogenase (LDH) at diagnosis, and all had bulky disease at diagnosis. An additional 21 patients were transplanted in second complete remission, 7 in third CR, 4 with chemotherapy-sensitive active disease, and 1 with refractory disease. Fourteen patients underwent allogeneic bone marrow transplantation and 32 underwent autologous HSCT. The conditioning regimen was cyclophosphamide and TBI (23 cases), cyclophosphamide, Ara-C, and TBI (10 cases) or chemotherapy alone [busulfan, VP16, Ara-C, and melphalan (BEAM) in 6 cases, CBV in 4 cases, busulfan and cyclophosphamide in 2 cases, and BACT in 1 case]. Four patients had some form of ex vivo marrow treatment, either chemotherapy or monoclonal antibody and complement. Three of 14 patients who underwent allogeneic HSCT, and three of 32 who underwent autologous HSCT died from procedure related toxicities [predominately infection or graftversus-host disease (GVHD)]. There were no early deaths in the 13 patients undergoing HSCT in first CR. Event-free survival for all 46 patients was 58%, with a median follow-up of 33 months. Twelve patients relapsed one to seven months after HSCT (including 2 of 13 transplanted in CR1, 2 of 21 transplanted in CR2, and 8 of 12 transplanted in CR3 or with more advanced disease). In a multivariate analysis, disease status at the time of transplant was the only predictive factor for EFS. Patients transplanted in CR1 had an EFS of 83%, those in CR2 had an EFS of 68% (not different than CR1), those in CR3 29% (p!0.001 compared to CR1), and those with more advanced disease 0% (p!0.001 compared to CR1) (Fig. 1). EFS was similar for allogeneic HSCT and autologous patients (Fig. 2). The Pediatric Blood and Marrow Transplant Consortium reported a retrospective multicenter review of children undergoing HSCT for non-Hodgkin’s lymphoma (39). Clinical and outcome data were available for 28 children undergoing 29 transplants at 11 institutions between 1994 and 1997. Patients included in this series were 22 boys and 6 girls with the median age at time of stem cell transplant 12 years, with a range of one to 20 years. Eight patients had large cell lymphoma, seven had anaplastic large cell lymphoma (including six who were CD30 positive), six had lymphoblastic lymphoma, four had SNCL, and four had other histologies.

534

Gordon and Baker 100 80

S(t)

60 40 20 0E+0 0E+0 12

24

36

48

60

72

84

96

108 120

132

Months

Figure 1 EFS and disease status at time of HSCT. CR1 (____); CR2 (__.__); more advanced disease (- - -). Abbreviations: EFS, event-free survival; HSCT, hematopoietic stem-cell transplantation. Source: From Ref. 38.

One transplant was performed in CR1, 16 in CR2 and one in CR3. Eight transplants were preformed in patients with partial response or stable disease. Three transplants were performed on patients with progressive disease after two to three salvage regimens. A variety of preparative regimens were utilized based on institutional protocols. Nineteen patients were prepared with TBI-containing regimens. VP16, cyclophosphamide, and TBI were used in nine transplants, and VP16, thioTEPA, and TBI was used in four transplants. Five other regimens were used for the remaining six patients. Ten patients were prepared with non-TBI-containing regimens: CBV in five transplants, and busulfan, cyclophosphamide, and thioTEPA were used in two transplants. Three other regimens were used in the remaining three patients. Eighteen patients received autologous stem cells, obtained from the peripheral blood in eleven cases, bone marrow in four cases, and both bone marrow and blood in three cases. None of the autologous products were purged. Eleven patients received allogeneic stem cells. Stem cells were obtained from bone marrow in ten cases and cord blood in one case. The allogeneic 100 80

S(t)

60 40 20 0E+0 0E+0 12

24

36

48

60

72

84

96

108 120

132

Months

Figure 2 EFS for 14 patients undergoing allogeneic (____) or 32 patients undergoing autologous (- - -) HSCT. Abbreviations: EFS, event-free survival; HSCT, hematopoietic stem-cell transplantation. Source: From Ref. 38.

HSCT for Hodgkin’s and Non-Hodgkin’s Lymphoma

535

stem donor was a matched sibling in two cases, a mismatched parent in three cases, a matched unrelated donor in five cases, and a mismatched unrelated donor in one case. Comparing patients receiving allogeneic stem cells versus autologous stem cells, patients who underwent allogeneic transplants were more likely to have had BM or CNS involvement with lymphoma at some time during the course of their disease or have had lymphoblastic histology. There was no statistically significant difference between patients receiving allogeneic stem cells and those receiving autologous stem cells, in terms of stage at diagnosis, time of relapse (that is, on-therapy or off-therapy) or remission status at time of transplant. Overall, at the time of analysis, 12 of 29 patients were alive without disease a median of 18 months after transplant. EFS for the entire group was 33% (Fig. 3). Twelve of the remaining patients have relapsed, a median of 1.5 months after transplant. Eight of twelve relapses occurred within 100 days of transplant, and 11 of 12 within six months of transplant. Nine of the twelve patients who relapsed have subsequently died of progressive disease, and one was alive with disease. Two were alive without evidence of lymphoma, one 27 months after a 2nd stem cell transplant, and the other 36 months after salvage chemotherapy. The remaining five patients died without evidence of lymphoma, one each of sepsis, veno-occlusive disease of the liver, hepatic failure, chronic GVHD, and pneumonitis. Eight of 18 transplanted in CR were alive without disease a median of 13 months after transplant, and four of seven patients transplanted in PR were alive without disease a median of 23 months after transplant. EFS for the 25 patients transplanted in CR or PR was 38% (Fig. 4). In contrast, all four patients transplanted with stable or progressive disease relapsed or died without disease. In contrast to the predominately positive findings of the noncomparative retrospective series provided, Kobrinsky and colleagues reported the results of the CCG 5912 study (18). Children with recurrent NHL or Hodgkin’s disease were treated on this trial with DECAL (dexamethasone, etoposide, cisplatinum, Ara-C, and L-asparaginase) as salvage induction therapy.

Figure 3 Event-free survival from time of stem cell transplant for all patients (nZ29). Abbreviation: HSCT, hematopoietic stem-cell transplantation.

536

Gordon and Baker

Figure 4 Event-free survival from time of stem cell transplant for patients in CR/PR (nZ25). Abbreviations: CR, complete response; PR, partial response; HSCT, hematopoietic stem-cell transplantation.

Patients achieving remission were further treated with four maintenance cycles of ifosphamide and VP16, alternating with DECAL. Though not a formal part of the study, patients who responded to induction or subsequent maintenance therapy were allowed to proceed to allogeneic or autologous stem cell transplantation after induction therapy at the discretion of the treating physician. Fifty patients underwent stem cell transplantation (though the distribution between NHL and Hodgkin disease was not provided). Forty-two of these were performed in patients responsive to therapy. Overall, 17 of 42 patients who underwent transplant with responsive disease were event-free survivors. Cox regression analysis of patients with responsive disease did not show a significant benefit for patients who underwent stem cell transplantation as compared with those who received only maintenance therapy. However, as the authors admit, comparison of outcome between these groups is difficult. Selection of therapy was not random, and stem cell transplant was not performed in a standardized fashion, leaving uncontrolled such variables as source of stem cells (peripheral blood or bone marrow; autologous or allogeneic; related or unrelated), the stem-cell dose, and the conditioning regimen.

Transplantation for Specific Histologies In addition to these broader studies, there have been several other series reported regarding transplantation for specific NHL histologies. Small Noncleaved Cell Lymphoma (Burkitt Lymphoma) The prognosis for the child with recurrent or progressive SNCL is extremely poor. Atra and colleagues reported 26 children who failed one of two front-line, short, multiagent intensive

HSCT for Hodgkin’s and Non-Hodgkin’s Lymphoma

537

chemotherapy regimens. Only eight (30%) achieved remission with second line therapy and only three remained alive without disease (19). High-dose therapy may play a role in a proportion of children with relapsed or primary refractory BL. Ladenstein reported 89 children from the European Lymphoma BMT registry who underwent high-dose chemotherapy with stem cell rescue between 1979 and 1991 (40). The major determinant of survival in this study was the remission status of patients before highdose therapy. Only patients with sensitive disease benefited from the procedure: the 5-year EFS was 56% for patients with partial (O50%) response to primary induction therapy, and 49% for those in complete or partial response to salvage chemotherapy. In contrast, no patient with primary refractory disease or with disease refractory to salvage therapy survived. This observation was in line with previous reports of adult patients showing that resistance to chemotherapy is associated with very poor outcome (41–43). Even the positive results in this retrospective trial need to be taken cautiously. About 50% of patients with poor risk BL escape dose escalation due to rapid progression of disease after relapse. Philip reported that of 27 patients relapsing from front-line intensive chemotherapy (French LMB84), only 24 received rescue chemotherapy and only 15 finally underwent high-dose therapy (43). Therefore, those patients who actually achieved prolonged EFS following high-dose therapy represent a considerably smaller subgroup of relapsed BL patients that is suggested by Ladenstein and coworkers. Perhaps more importantly, a sizable proportion of children reported by Ladenstein (60 of 89 patients) had received less intensive initial chemotherapy than is the current standard, such as COPAD chemotherapy (cyclophosphamide, vincristine, prednisone, doxorubicin) (44) LMB 81/84 (COPADCmethotrexate 3 gm/m2Cinfusion Ara-C) (45,46). As might be expected, many patients failing this front-line therapy were responsive to high-dose chemotherapy. In contrast, none of the patients who failed more intensive LMB 86/89 (including methotrexate 8 gm/m2Chigh dose Ara-CC VP16) were salvaged by HDC with stem cell transplant. Similar observations have been made by others (19). Thus, with modern dose intensive induction therapy, as primary refractory disease becomes numerically a more important reason for failure than relapse, the role of high dose therapy for BL becomes less apparent.

Lymphoblastic Lymphoma The prognosis for the child with recurrent or progressive lymphoblastic non-Hodgkin’s lymphoma is poor. Patients relapsing after treatment on the CCG 551 study had a 5-year OS rate of only 10% (17). In the early 1980s, several groups reported remissions and long-term survival of children with relapsed lymphoblastic lymphoma (35,36). More recently, the series from the Spanish Working Party (38) included 21 children with lymphoblastic lymphoma. Six were transplanted after either failing induction chemotherapy (partial or no response to LSA2L2 therapy) or relapsing after an initial response to therapy [11 in CR2 (after LSA2L2C surgery), and four in CR3 or with sensitive persistent disease]. Overall, 13 of 21 patients remained alive without lymphoma at the time of this report. As would be expected, patients transplanted earlier in their course had a more favorable outcome (including all three transplanted in CR1). As mentioned above regarding BL, these results need to be interpreted cautiously, considering the intensity of initial chemotherapy utilized. LSA2L2 type therapy produces between 40 and 67% EFS in children with newly diagnosed disseminated lymphoblastic lymphoma (17,47,48). It is unclear whether patients relapsing after more initial intensive therapy [such as BFM 90 with a 90% EFS (6)] would be as likely to be salvaged with high-dose therapy.

Large Cell Lymphoma As with the other high-grade NHLs in children, prognosis for the child with recurrent or progressive LCL is, in general, poor. However, some subgroups, particularly recurrent ALCL

538

Gordon and Baker

have a more optimistic outlook. Brugieres reported 41 children with ALCL who relapsed after front line therapy for LCL (COPAD or HM89 or HM91) (49). The second complete remission rate was 88%. Of the 36 who obtained CR2, 11 remained in continuous CR2 and 10 relapsed. Interestingly, of the 10 who suffered a second relapse, five remain in CR3 through CR6 after further salvage therapies. The role of high dose therapy in children with relapsed LCL remains unclear. Gordon and coworkers reported 15 children with CD30 (Ki-1) positive T-cell NHL who underwent transplantation after preparation with high dose thioTEPA, TBICVP16 (50). Patients ranged between 2 and 18 years of age, with a median age of 8 years. Stem cell source was autologous bone marrow (five patients), autologous peripheral blood stem cells (PBSC) (six patients) or allogeneic bone marrow (four patients). Six tumors had anaplastic large cell histology, three mixed large and small cell, and three large cell. Three were not further characterized. All 15 expressed a panTcell marker and all were CD30 antigen positive. Seven of ten tumors evaluated cytogenetically had a t(2;5) (p23;q35). Twelve of 15 patients had experienced a CR to initial multiagent chemotherapy and had subsequently suffered a relapse a median of five months after initial complete remission (four while on therapy and another seven within six months of completing therapy). Eight were transplanted in CR2, three in 2nd relapse, and one in 3rd PR. Three of 15 patients had only a partial response to initial chemotherapy, and all had disease progression by one to three months after diagnosis. All three had subsequent response to salvage therapy, and two were transplanted in CR2 and one in CR3. Overall, ten were transplanted in CR2, one in CR3, three in 2nd relapse, and one in 3rd PR. At the time of last follow-up, with a median follow-up of more than five years, the estimated five-year EFS was 70%, with 11 of 15 alive without disease 21 to 109 months after HSCT. Three patients have relapsed (1,1 and 2 months after HSCT), and one died without disease 52 months after HSCT. Eleven of the 15 had no evidence of disease at time of HSCT. Nine of these 11 remained in complete remission 21 to 102 (median 65) months after HSCT, with an EFS of 82% at four years. Four of 15 had measurable disease at time of HSCT. Two of four are in CR 37 and 109 months after HSCT, one relapsed at one month after HSCT, and one died in remission. EFS for this group was 38%. Fifteen of the patients reported by Brugieres underwent high-dose chemotherapy in CR2, using various regimensCTBI (49). Nine of the 15 remain in CR2, one died of transplant related toxicity, and five have relapsed. Four of these five remain in CR3 to CR4 after treatment with low-dose single agent chemotherapy. High-dose chemotherapy did not seem to be effective for patients relapsing after salvage therapy: of the six patients who were transplanted in PR2, PR3 or CR3, five have relapsed. These authors also make an interesting observation. As noted above five patients having a second relapse after conventional therapy, and four patients relapsing after HSCT, remain in CR3 through CR6 after further salvage therapies. Moreover, ten patients had CRs to single agent vinblastine (including six patients who relapsed after HSCT), and five patients have maintained their remission after completion of a median of 27 months of vinblastine therapy. Similar responses to intermittent vinblastine have been reported in patients with relapsed Hodgkin’s disease after HSCT (51). The authors justifiably raise the question whether prolonged conventional therapy might be as effective as short intensive therapy with HSCT in some patients.

Conclusions The large majority of children with NHL can be cured with conventional dose chemotherapy. For the small minority who suffer relapse, historical data have suggested a role for high-dose chemotherapy with HSCT. However, as front-line therapies have become more intense (with the intention of inducing and maintaining initial remission), the role of high-dose salvage

HSCT for Hodgkin’s and Non-Hodgkin’s Lymphoma

539

therapy has become less clear. As will be discussed below, consideration of newer approaches, including tandem high dose cycles (52) and nonmyeloablative allogeneic HSCT (53) seems warranted.

HODGKIN’S DISEASE Introduction Treatment advances in pediatric Hodgkin’s lymphoma have progressed to the point that most children and adolescents diagnosed with Hodgkin’s disease (HD) will enjoy long-term diseasefree survival. Contemporary treatment protocols produce disease-free survival in 70–90% of patients with advanced stage disease and in 85–100% of patients with localized disease. The success of these protocols is attributed to the improved accuracy in diagnostic imaging techniques, advances that have been made in the development of more effective combination chemotherapy regimens and the improved techniques in the delivery and administration of radiation therapy. As a consequence of the fact that primary therapy results in very few failures, aggressive treatment modalities such as HSCT are generally reserved only for patients with relapsed or refractory disease.

Chemotherapy Approaches The development of the combination chemotherapy regimen of mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) was one the first effective chemotherapy regimens utilized for HD and achieved greater than 50% long-term disease-free survival in most patients (54). However, in children, the late effects of this regimen quickly became apparent including secondary acute myeloid leukemia (AML) associated with the high doses of the alkylating agent mechlorethamine administered, as well as significant gonadal toxicity, particularly in males who received six to eight cycles of this combination (55,56). Subsequently, the development of the ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) regimen was found to provide very effective therapy and avoided the increased risk of secondary leukemia or infertility, although with an anthracycline and bleomycin, was more concerning for cardiopulmonary toxicity. With the goal of reducing the potential for late effects, as well as improving the effectiveness of systemic chemotherapy by utilizing alternating cycles of multiagent chemotherapy, investigators then began to explore regimens of alternating cycles of MOPP/ABVD as well as other alternating cycle chemotherapy combinations. In children, trials with mostly small numbers of patients demonstrated that this approach with 6–12 total cycles of chemotherapy was very effective producing EFS rates of 75% to 91%, (57–60), although children with advanced stage disease generally fared less well. In order to limit the dose-related toxicity of these multiagent chemotherapy regimens further, and to improve on the outcome of patients with more advanced disease, standard dose extended or involved field radiation therapy was added to alternating cycle chemotherapy regimens and multiple published trials on this approach report EFS rates for advanced stage disease of 60% to 86% (61–64). Recently, for children with early stage, favorable HD (stage I, II, nonbulky), therapy with four cycles of vinblastine, doxorubicin, methotrexate, prednisone (VAMP), with low-dose (15 Gy) involved field radiation resulted in 5-year EFS of 93% and OS of 99% without any serious or late toxicity reported to date (65). This regimen was specifically designed with agents that were less likely to cause leukemia, sterility, or cardiac or pulmonary dysfunction. Investigators from the Pediatric Oncology Group determined for advanced stage HD that eight cycles of alternating MOPP and ABVD plus low-dose (21 Gy) total nodal radiation or subtotal nodal radiation were effective and achieved 5-year EFS and OS rates of 77% and 90% respectively (66). A subsequent study compared the above approach to that of the same chemotherapy without the addition of any radiation therapy. This study found that the EFS at 5 years was 80% for patients who received combined modality therapy compared to 79% for

540

Gordon and Baker

those patients who received MOPP-ABVD only (67). It remains to be seen whether the late effects of treatment in this chemotherapy only regimen are acceptable or if the delivery of 8 cycles of chemotherapy will increase the late effects of treatment such as pulmonary toxicity secondary to bleomycin, cardiac toxicity secondary to anthracyclines, and gonadal dysfunction secondary to nitrogen mustard and procarbazine. Similar approaches have been utilized at Stanford University incorporating six cycles of alternating MOPP-ABVD plus low-dose involved field RT with a reported EFS at 10 years of 100% for patients with stage I to III disease but only 69% for those with stage IV disease (68). This same approach has been substantiated by investigators from the Children’s Cancer Group and others, and in both of these studies patients with more advanced stage disease did not fare as well with this lower intensity treatment (61,69). Currently, many studies are focusing on a risk-adapted approach to therapy for HD where the duration and intensity of therapy is directed based upon a “favorable” versus an “unfavorable” clinical presentation. Unfavorable disease is generally defined as bulky mediastinal or peripheral lymphadenopathy, extranodal extension of disease and advance stage (IIIB or IV). Prospective clinical trials will be required to determine if these “unfavorable” risk patients would benefit from HSCT as part of their initial therapy in first CR.

Indications for Hematopoietic Stem-Cell Transplantation The overall excellent outcome after primary therapy for children with HD has limited the number of children for which effective salvage treatments are necessary. These excellent rates of disease-free survival, even for advanced-stage disease, limit the consideration for HSCT to those patients who have relapsed after multi-modal therapy or for those who develop refractory disease during treatment. Although unlikely to be encountered with current treatment standards, relapsing patients who may have been treated with radiation therapy only can frequently be salvaged by treatment with combination chemotherapy, and similarly, patients who have been treated with such a regimen as ABVD alone, can be successfully be salvaged with MOPP but with only a 30% freedom from progression and less than 20% in the reverse sequence (70). Equally poor survival has been reported by Longo, et al. in a long-term followup study of patients with relapsed HD where the long term survival was !20%, and there were no long-term survivors after salvage therapy in patients who had not achieved a remission with initial treatment, and a 10% chance or survival in patients who had an initial duration of remission of less than 1 year (71). In relapsed patients, two independent factors were found to significantly affect survival and DFS: (1) interval from end of treatment to relapse (P!0.0001) and (2) stage III or IV disease at relapse (pZ0.0013). For patients with at least one adverse prognostic factor, high-dose therapy gave the best survival results (72). A comparison of conventional salvage therapy to high-dose chemotherapy and HSCT for relapsed or refractory patients with HD found that patients who underwent HSCT had superior EFS (53% vs. 47%, p!0.01) and superior freedom from progression (FFP, 62% vs. 32%, p!0.01) compared to conventional salvage treatment (73). The use of high-dose therapy at relapse, a longer duration of remission, and favorable response to cytoreductive or salvage therapy were most predictive of superior EFS and FFP. For patients with late relapses (O1 year remission duration), low stage disease at relapse, and who achieve a second complete remission, salvage chemotherapy is a reasonable consideration, however, for all others, it appears that HSCT offers a greater chance of disease free survival. Autologous Hematopoietic-Cell Transplant A summary of the published results on HSCT for patients with HD is provided in Table 1. What is clear from this data is that in general, about 30–60% of patients with relapsed or refractory HD can be successfully salvaged with autologous HSCT and that there is little data available that specifically provides information on the effectiveness of this treatment modality for children. The majority of all patients in these studies have received at least one or more attempts at salvage chemotherapy prior to undergoing autologous HSCT, and as one can see

Reece (80) (1996) Weaver (81) (1995) Reece (82) (1995)

Bierman (79) (1996)

O’Brien (78) (1996)

58 28 30

BVAC

CBVGP

10 11 61 85

CBV

M MB MBV CBV

26 74 19 27 4 4 3

TBI/VC CBV CCNU/VC CBV BVAM BVAC C/TBI

Sureda (77) (1997)

102

CBV

Wheeler (75) (1997) Horning (76) (1997)

6

47

CBV Other regimens

Na

Baker (74) (1999)

Regimen

42%

64%

61%

40%

62%

42

PFSb

40%

72%

48%

31%

DFSb or EFSb

Autologous Hematopoietic-Cell Transplant Literature

Author (year)

Table 1

69%

64%

51%

41%

93%

52%

65

43%

OSb

3.6 year

3 year

7 year

5 year

5 year

7 year

4 year

3 year

5 year

Follow-up

PB/PB

PB

PB/PB

BM

BM/PB

BM/PB

BM/PB

BM/PB

Hematopoietic cell source

na

na

na

na

na

0%

na

na

10%

Transplantrelated mortality

(Continued)

All pts induction failures

Relapsed or refractory pts

HSCT in first CR for advanced stage, bulky disease, BM involvement or extranodal disease Relapsed or refractory pts, PFS 75% if CR at time of HSCT FFS 90% for pts with no salvage chemotherapy prior to HSCT All after first relapse only

All patients %21 year, in comparison to adult cohort no statistical difference in FFS or OS 19 pts !20 year, relapsed or refractory pts Relapsed or refractory pts

Comments

HSCT for Hodgkin’s and Non-Hodgkin’s Lymphoma 541

Crump (88) (1993) Williams (89) (1993)

73 81p 81a

CBV BVAM (Cothers)

128

CBV

VM

24

FTBI/CV

62

CBV

Horning (86) (1994) Bierman (87) (1993)

4

CVR

Matsuzaki (84) (1995) Burns (85) (1995)

22 63

Na

FTBI/CV CBV

Regimen

Nademanee (83) (1995)

Author (year)

39% 48%

55%

PFSb

39%

25%

47%

58%

DFSb or EFSb

45%

38%

75%

OSb

Table 1 Autologous Hematopoietic-Cell Transplant Literature (Continued)

36 months 34 months

4 year

4 year

3 year

3 year

2 year

Follow-up

BM

BM/PB

PB/PB

PB/PB

Hematopoietic cell source

11%

9.6%

8.6%

na

na

11%

Transplantrelated mortality

Case-matched PFS comparison of pediatric (p) to adult (a) patients with HD, which was

Long-term follow-up include late relapses, secondary MDS/leukemia Relapsed or refractory pts

51% HSCT in first relapse or second CR, 39% after second or subsequent relapse. Regimens equivalent All !20 year, 3 in CR, 1 in relapse. 2/4 DFS Recurrent of refractory pts. If in CR at HSCT, 50% DFS Recurrent/refractory pts

Comments

542 Gordon and Baker

CBV

Hurd (91) (1990)

33

56

32%

37%

56%

28 months

39 months

PB/PB

PB

0%

5%

Regimen: C, cyclophosphamide; B(CNU), carmustine; V, etoposide; P, Cisplatinum; A, Cytosine arabinoside; M, Melphalan; R, Ranimustine Hematopoietic Cell; BM, bone marrow; PB, peripheral blood hematopoietic cells; TBI, total body irradiation. a a, adult patients; p, pediatric patients; na, not available; pts, patients. b For all regimens combined if more than one listed for each study. Abbreviations: FFS, failure free survival; OS, overall survival; HSCT, hematopoietic stem cell transplantation; PFS, progression-free survival; DFS, disease-free survival; HD, Hodgkin’s disease; PBSC, peripheral blood stem cells; EFS, event-free survival; CR, complete response.

CBV

Kessinger (90) (1991)

not statistically different. PFS for chemoresponsive pts 55% pediatric, 58% adult PBSC only, included pts with CPB disease or marrow hypocellularity that would have not been eligible for PBT DFSZ33% if PB involvement at time of HSCT versus 56% if BM free of disease

HSCT for Hodgkin’s and Non-Hodgkin’s Lymphoma 543

544

Gordon and Baker

from the survival presented in Table 1, patients with chemosensitive disease are more successfully treated with this approach. It is interesting to point out, however, that at least two of these studies have small numbers of patients who were taken directly to HSCT at the time of relapse without being first treated with salvage chemotherapy and that the outcome of these patients was superior to others (74,79). In large part, this may be secondary to the fact that most of these patients had low disease burden and had previously demonstrated responsiveness to chemotherapy.

Preparative Regimens The majority of studies published on HSCT for HD have utilized a chemotherapy only preparative regimen, the most of common of which is the “CBV” regimen that includes cyclophosphamide, carmustine (BCNU), and etoposide. Variations of this regimen have also been utilized, such as BEAM (carmustine, etoposide, cytarabine, melphalan) (93,94) or BEAC (carmustine, etoposide, cytarabine, cyclophosphamide) (92,94,95), with overall similar response rates. Other studies have included patients who have received preparative regimens that have included fractionated TBI in combination with cyclophosphamide and etoposide but these studies do not report response or survival rates that are superior (or inferior) to those of chemotherapy only regimens (76,83,86).

Stem Cell Source As indicated in Table 1, the majority of studies have included patients who have received either PBSC or bone marrow and none make any comparisons regarding a benefit of one over the other. The choice of stem cell source is usually driven by a combination of factors that includes the presence or absence of bone marrow involvement with HD and the ability (or lack thereof) to successfully mobilize PBSC, particularly in patients who have been heavily pretreated prior to the time of mobilization.

Prognostic Factors Affecting Survival After Hematopoietic Stem-Cell Transplantation The literature on HSCT for patients with HD is, for the most part, limited to patients who have either relapsed disease or who are refractory to more than one attempt at remission induction at the time of diagnosis. There are multiple different factors that have been shown to be associated with adverse outcome after HSCT, and these are detailed in Table 2. One of the most consistent predictors of a poor prognosis is disease responsiveness prior to HSCT; with patients who are not responsive to chemotherapy prior to SCT have a uniformly poor prognosis (74,75,78,79,83). Other factors predicting a greater extent of disease burden as predicted by O1 extranodal site of disease (75), disseminated pulmonary or bone marrow involvement (76,83), or an elevated level of LDH (74) have also been associated with a poor prognosis after HSCT. Additionally, factors that may predict a more aggressive form of disease, such as shorter time from diagnosis to HSCT (75,94) or the presence of “B” symptoms (75,76,85), have also been associated with an unfavorable prognosis. Despite this knowledge regarding adverse prognostic features at the time of HSCT, disease features prior to HSCT have not been able to consistently predict which patient are most likely to fail standard treatment approaches, and therefore HSCT has generally not been offered to “high risk” patients that are in a first CR. However, one published study that did perform autologous HSCT in first CR for patients with advanced stage disease, bulky disease, bone marrow involvement, or extranodal disease did result in an excellent DFS of 72% and OS of 93% at 7 years for this group of high-risk patients (77). Comparative randomized clinical trials will need to be performed before this approach can routinely be justified for all advanced HD patients.

HSCT for Hodgkin’s and Non-Hodgkin’s Lymphoma

545

Table 2 Unfavorable Prognostic Features for Survival After Hematopoietic Stem-Cell Transplantation Disease burden (74–76,83) O1extranodal site of disease, or presence of extranodal disease at transplant Disseminated pulmonary or bone marrow disease at relapse Disease burden Ominimal at time of HSCT Elevated LDH Disease responsiveness (74,75,78,83,87) Number of previous chemotherapy treatments Lack of previous chemosensitivity Lack of response to treatment prior to HSCT Progressive disease at HSCT Other (74–76,87,88) Nodular sclerosis histology Abnormal performance status Shorter time from diagnosis to HSCT B symptoms Relapse in previous radiation field Abbreviation: HSCT, hematopoietic stem-cell transplantation.

Comparisons of Pediatric Versus Adult Patients In 1993 Williams et al. published the results of a case-matched comparison of pediatric HD with adult patients from the European Bone Marrow Transplant Group Lymphoma Registry (89). This study compared 81 children to 81 adult patients where case matching was accomplished by matching of the main prognostic factors for progression-free survival (PFS) by multivariate analysis. The pediatric group consisted of patients who were less than 16 years of age at the time of diagnosis (median 13.8, range 3.1–16.0) and had a median age at time of HSCT of 16.9 years (range 5.2–32.6). Response rates and the number of procedure related deaths were similar between the two groups, although veno-occlusive disease was more common in the pediatric group and cardiac complications were more common in the adult group. The PFS rate at a median follow-up of 36 months was 39% for the pediatric group and 48% in the adult group (PZ0.64). The relapse and progression rates were also similar between the two groups. This study thus suggests that patients with pediatric HD have the same outcome with autologous HSCT as their adult counterparts. Another study of 102 patients undergoing autologous HSCT included 19 patients who were !20 years of age at initial diagnosis, but in the analysis only evaluated age !30 years at time of diagnosis or at time of HSCT and found no difference in PFS or OS between these age groups (75). The largest study of relapsed or refractory pediatric HD patients treated with autologous HSCT was published from the University of Nebraska in 1999 (74). In this study, 53 patients were grouped by age at time of HSCT into children !13 years (nZ6), early adolescents 13–18 years (nZ18), and late adolescents 19–21 years (nZ29). The actuarial failure-free survival (FFS) of all 53 patients at 5 years post transplant was 31%, and the OS was 43%. There was not any significant difference in FFS or OS between the three age categories. Prognostic factors significantly influencing survival included and elevated level of LDH, and disease sensitivity at the time of HSCT, with the FFS in untreated relapse, sensitive disease, and resistant disease of 44%, 35%, and 9% respectively. To further explore the potential impact of age on survival, this pediatric cohort was compared with a historical group of adult patients age 21 years or older (nZ282) who had received autologous HSCT over the same time period at the University of Nebraska and found no significant difference in FFS or OS between these groups. In this study six surviving patients who underwent HSCT prior to their 18th birthday were found to have no significant late complications, with Karnofsky performance scores of 100% and were all either in school full-time or employed. Normal growth was achieved without growth hormone supplementation, although two females did have delayed onset of

546

Gordon and Baker

puberty and/or menses but progressed normally without hormone supplementation. Three patients developed late relapses at 26, 30, and 61 months post transplant and two patients developed secondary AML or MDS.

Allogeneic Hematopoietic-Cell Transplant There are only a few reports of allogeneic hematopoietic cell transplant for patients with HD and these have, in most cases, been associated with a high degree of transplant-related morbidity and mortality and survival rates of 0% to 25%, which has limited the application of this approach for HD patients. One of the largest series reported to date was from the International Bone Marrow Transplant Registry where the outcome of 100 patients who underwent HLA-identical sibling bone marrow transplant has been reported (96). The majority of these patients (89%) were not in remission at the time of transplant, 50 had pretransplant Karnofsky scores !90%, and 27 had active infections in the week before transplant. For these patients the OS was 21% at 3 years, and the DFS was only 15%. There was a significant incidence of acute GVHD of 35% and a 45% incidence of chronic GVHD. The treatment related mortality rate in this study was 61%. This study failed to demonstrate a statistically significant decrease in relapse rate in patients who developed acute or chronic GVHD, but the treatment related mortality was very high and makes assessment of this potential advantage of allogeneic HSCT difficult to determine. This study also failed to demonstrate any survival advantage to allogeneic HSCT in comparison to autologous HSCT, although the patients in this study were all very high-risk patients who would likely have faired poorly with autologous transplants also. A similar study from the European Bone Marrow Transplant registry utilizing a matched cohort design evaluated 45 HD patients undergoing allogeneic HSCT to 45 that were treated with autologous HSCT (97). Matching criteria included: sex, age, disease stage, bone marrow involvement at diagnosis and at transplantation, year of transplantation, disease status at time of transplantation, time from diagnosis, and conditioning regimen with or without TBI. Response rates were similar between the two groups. The four-year actuarial probability of survival, PFS, and relapse were not significantly different between the two groups; however, transplant-related mortality was significantly higher in the allogeneic transplant group (65% vs. 12%, pZ0.005). Acute GVHD greater than or equal to grade II was associated with a significantly lower risk or relapse and with a significantly lower OS rate. The results of this study provided no support that there was any advantage to myeloablative allogeneic HSCT for patients with relapsed HD. Current approaches to allogeneic hematopoietic cell transplant utilizing lower intensity nonmyeloablative preparative regimens (discussed below) may allow further evaluation of the effectiveness of the allogeneic effect in HD with a lower toxicity profile.

New Approaches to Hematopoietic-Cell Transplant for Hodgkin’s Disease Tandem Autologous Transplants As a mean to further intensify chemotherapy and improve outcomes, some investigators have utilized tandem transplants for patients with high-risk, chemoresponsive tumors, including HD. A small pilot study from French investigators reported on 15 patients with non-Hodgkin’s lymphoma and nine patients with poor prognosis HD who received a preparative regimen of BCNU, cyclophosphamide, VP-16, and mitoxantrone, followed at a median of 56 days by a second preparative regimen of busulfan, aracytine, and melphalan, or TBI with aracytine and melphalan (52). These tandem procedures were associated with a high degree of mucositis and three cases of veno-occlusive disease (after the second transplant). Hematopoietic recovery was prompt after each procedure, and 14/24 (58%) of patients remained in a CR. Other investigators have also utilized this approach for HD patients with resistant or refractory disease. A recent

HSCT for Hodgkin’s and Non-Hodgkin’s Lymphoma

547

report of tandem autologous transplants included 29 HD patients and found that 32% of those with refractory HD survived long term (98). Another study that included HD patients with induction failure or unfavorable relapse reported response rates of 63% overall. After the second transplant, 83% of unfavorable relapse patients remained in remission. Two-year OS was 74% (99). A larger study that included 102 relapsed or refractory HD patients treated with high-dose sequential chemotherapy had freedom from second failure and OS of 59% and 78%, respectively. Even for those with progressive disease or multiple relapses, OS was 48% (100). Larger cohorts of patients will be required in order to fully assess this approach and to see if improved outcomes in these poor risk patients can be achieved with acceptable toxicity.

Reduced Intensity Conditioning Hematopoietic-Cell Transplants The use of reduced intensity conditioning (RIC) allogeneic HSCT for lymphoma (both nonHodgkin’s lymphoma and HD) is increasing. No prospective randomized clinical trials have been performed, although comparisons typically show a lower relapse rate when allogeneic transplant results are compared to results of autologous transplants. This data supports that a graft-versus-lymphoma effect does exist. However, in most comparisons that have been published, the lower relapse rates following allogeneic HSCT are offset by higher transplant related mortality. It is for this reason that investigators have included HD in most of the early studies of nonmyeloablative HSCT as a means to gain the potential benefit of a graft-versuslymphoma effect with less morbidity and mortality. One study recently published provides preliminary evidence that this approach may be beneficial. In this study 40 patients with relapsed or refractory HD underwent RIC allogeneic HSCT with either an HLA-identical sibling (nZ20) or unrelated donor (nZ20). Median age was 31 (range 18–58). Conditioning regimens were either fludarabine, cyclophosphamide (flu/cy) C/K antithymocyte globulin (nZ14) or fludarabine with melphalan (flu/mel, nZ26). Transplant related mortality was 5% at day C100 and 22% at 18 months post HSCT. OS at 18 months was 73% in the group that received flu/mel versus 39% of those receiving flu/cy (pZ0.03), although PFS was not significantly different between the two groups (37% vs. 21%, pZ0.2) (101). Another such study in 49 patients with multiply relapsed HD, 44 (90%) who had progression of disease after previous autologous HSCT (102). Thirty-one patients had related donors, and 18 had unrelated donors. All patients engrafted, and 16 (33%) received donor lymphocyte infusions for residual disease or progression (nine responded to this). Nonrelapse mortality was 16.3% at 730 days (7.2% for related vs. 34.1% for unrelated donors, pZ0.02). Four-year OS was 55.7%, and PFS was 39% (62% and 41.5% for related donors). This study demonstrated a significant salvage rate for very poor risk patients, even for those who had failed prior auto-HSCT. One small study has been published in children (nZ6) with primary refractory or multiply relapsed HD (2 after auto-HSCT) who received fludarabine based preparative regimens and allogeneic PBSC. Two patients died, one from infection and the other from disease relapse. Two additional patients relapsed but responded after withdrawal of immunosuppression, chemotherapy, or donor lymphocyte infusions and were alive at 22 and 36 months post-HSCT (103). Overall this data is encouraging although studies that report on larger number of patients with longer follow-up will be necessary to determine whether this treatment approach for patients with relapsed or refractory HD will be advantageous and whether it could be considered earlier in the course of the disease. An area of active investigation is the coupling of a nonmyeloablative allogeneic HSCT to directly follow an autologous HSCT. In this form of “tandem transplant” the autologous transplant is performed first in order to achieve a CR, and this is then followed by a nonmyeloablative allogeneic transplant with the intent of eradicating any residual tumor cells by means of an allogeneic antitumor response. These studies are in a very early stage of development and will need to be assessed in terms of both efficacy and toxicity. They have been designed with the intent of harnessing the benefit of an allogeneic immune response but

548

Gordon and Baker

avoiding the high mortality rates that have been associated with standard myeloablative allogeneic transplants. At this point, this approach has only been utilized in patients who are very high risk by virtue of multiple relapses or primary refractory disease having failed several attempts at remission induction. A small series reported recently that included 15 patients (10 with HD, 5 with non-Hodgkin’s lymphoma) who underwent high-dose chemotherapy with autologous hematopoietic cells and then subsequently at a median of 61 days later underwent a nonmyeloablative regimen of fludarabine and cyclophosphamide, followed by the infusion of unmanipulated allogeneic hematopoietic cells (53). Thirteen patients had 100% donor cell engraftment. Eleven patients achieved complete remission after the combined procedure. Nine patients, who were in partial remission after autografting, achieved CR after mini-allografting. Ten patients survived, five in continuous CR. Seven patients received donor lymphocyte infusions with five CR’s. Five patients died, one of progressive disease, two of progressive disease combined with acute GVHD or chronic GVHD, and one from infection. This study demonstrated fairly high response rates for this group of refractory or heavily pretreated patients and similar protocols are currently undergoing clinical trials in many transplant centers.

Late Effects Some of the most extensive knowledge that is available on long-term effects of cancer treatment comes from studies in survivors of pediatric HD. Multiple reports detail the significant late effects, which include soft tissue and bone growth abnormalities, infertility, infection, thyroid abnormalities, pulmonary, and cardiac disease, psychosocial abnormalities, and second malignancies (particularly secondary breast cancer and AML) (104). However, major modifications in treatment modalities included: (1) decrease in the use of surgical staging with splenectomy to reduce surgically related complications and infection rates, (2) decrease in radiation doses and volumes by improved radiation techniques, and (3) changes in chemotherapy protocols to multiagent combinations using less toxic drugs delivered over shorter time periods have all led to a reduction in the incidence and severity of late effects (105). Unfortunately, there is comparatively little specific information that has been published on the additional late effects that a HSCT might impose on HD patients who have already been heavily treated with chemotherapy and radiation therapy prior to undergoing additional high dose chemotherapy associated with a transplant. Recent data from the University of Minnesota indicates that the risk of AML or myelodysplastic syndrome is 300-fold higher than expected in the general population for patients with HD or NHL who are undergoing autologous HSCT (106). In this cohort, 76% of all cases of AML/MDS occurred in patients with a primary diagnosis of HD or NHL, as did 18% of the solid tumors that were seen. Data suggest that exposures, such as the duration of pretransplant therapy, treatments with alkylator therapy of MOPP therapy for HD, and TBI in the preparative regimen, are all risk factor for the development of posttransplant AML/MDS (107–109), although it is difficult to separate out the additional impact of the preparative regimen for HSCT from that of pretransplant therapy received. Regardless of the exact mechanism or etiology, it is clear that these patients require very close follow-up after HSCT for the development of late complications of therapy, particularly second malignant neoplasms.

Conclusions The majority of children with HD can enjoy long-term disease free survival without the need for either autologous or allogeneic HSCT. However, for the minority of patients with either primary refractory or relapse HD, autologous HSCT provides an excellent opportunity for achieving a long-term remission, and the role of allogeneic HSCT and nonmyeloablative HSCT in particular needs to be further defined.

HSCT for Hodgkin’s and Non-Hodgkin’s Lymphoma

549

REFERENCES 1. Percy CL, Smith MA, Linet M, et al. Lymphomas and reticuloendothelial neoplasms. In: Ries LA, Smith MA, Gurney JG et al, eds. Cancer Incidence and Survival Among Children and Adolescents: United States SEER Program 1975–1995. Bethesda: National Cancer Institute SEER Program, 1999:35–50. 2. Pinkerton CR. The continuing challenge of treatment for non-Hodgkin’s lymphoma in children. Br J Haematol 1999; 107:220–234. 3. Neth O, Seidemann K, Jansen P, et al. Precursor B-cell lymphoblastic lymphoma in childhood and adolescence: clinical features, treatment, and results in trials NHL-BFM 86 and 90. Med Pediatr Oncol 2000; 35:20–27. 4. Link MP, Shuster JJ, Donaldson SS, Berard CW, Murphy SB. Treatment of children and young adults with early-stage non-Hodgkin’s lymphoma. N Engl J Med 1997; 337:1259–1266. 5. Magrath IT, Janus C, Edwards BK, et al. An effective therapy for both undifferentiated (including Burkitt’s) lymphomas and lymphoblastic lymphomas in children and young adults. Blood 1984; 63:1102–1111. 6. Reiter A, Schrappe M, Ludwig WD, et al. Intensive all-type therapy without local radiotherapy provides a 90% event-free survival for children with T-cell lymphoblastic lymphoma: a BFM group report. Blood 2000; 95:416–421. 7. Meadows AT, Sposto R, Jenkin RD, et al. Similar efficacy of 6 and 18 months of therapy with four drugs (COMP) for localized non-Hodgkin’s lymphoma of children: a report from the children’s cancer study group. J Clin Oncol 1989; 7:92–99. 8. Atra A, Imeson JD, Hobson R, et al. Improved outcome in children with advanced stage B-cell nonHodgkin’s lymphoma (B-NHL): results of the United Kingdom children cancer study group (UKCCSG) 9002 protocol. Br J Cancer 2000; 82:1396–1402. 9. Patte C, Auperin A, Michon J, et al. The societe francaise d’Oncologie pediatrique LMB89 protocol: highly effective multiagent chemotherapy tailored to the tumor burden and initial response in 561 unselected children with B-cell lymphomas and L3 leukemia. Blood 2001; 97:3370–3379. 10. Hutchison RE, Berard CW, Shuster JJ, et al. B-cell lineage confers a favorable outcome among children and adolescents with large-cell lymphoma: a pediatric oncology group study. J Clin Oncol 1995; 13:2023–2032. 11. Sandlund JT, Pui CH, Santana VM, et al. Clinical features and treatment outcome for children with CD30C large-cell non-Hodgkin’s lymphoma. J Clin Oncol 1994; 12:895–898. 12. Agnarsson BA, Kadin ME. Peripheral T-cell lymphomas in children. Semin Diagn Pathol 1995; 12:314–324. 13. Gordon BG, Weisenburger DD, Warkentin PI, et al. Peripheral T-cell lymphoma in childhood and adolescence. A clinicopathologic study of 22 patients. Cancer 1993; 71:257–263. 14. Link MP, Donaldson SS, Berard CW, Shuster JJ, Murphy SB. Results of treatment of childhood localized non-Hodgkin’s lymphoma with combination chemotherapy with or without radiotherapy. N Engl J Med 1990; 322:1169–1174. 15. Seidemann K, Tiemann M, Schrappe M, et al. Short-pulse B-non-Hodgkin lymphoma-type chemotherapy is efficacious treatment for pediatric anaplastic large cell lymphoma: a report of the Berlin-Frankfurt-Munster group trial NHL-BFM 90. Blood 2001; 97:3699–3706. 16. Massimino M, Gasparini M, Giardini R. Ki-1 (CD30) anaplastic large-cell lymphoma in children. Ann Oncol 1995; 6:915–920. 17. Anderson JR, Jenkin RD, Wilson JF, et al. Long-term follow-up of patients treated with COMP or LSA2L2 therapy for childhood non-Hodgkin’s lymphoma: a report of CCG-551 from the children’s cancer group. J Clin Oncol 1993; 11:1024–1032. 18. Kobrinsky NL, Sposto R, Shah NR, et al. Outcomes of treatment of children and adolescents with recurrent non-Hodgkin’s lymphoma and Hodgkin’s disease with dexamethasone, etoposide, cisplatin, cytarabine, and l-asparaginase, maintenance chemotherapy, and transplantation: children’s cancer group study CCG-5912. J Clin Oncol 2001; 19:2390–2396. 19. Atra A, Gerrard M, Hobson R, et al. Outcome of relapsed or refractory childhood B-cell acute lymphoblastic leukaemia and B-cell non-Hodgkin’s lymphoma treated with the UKCCSG 9003/9002 protocols. Br J Haematol 2001; 112:965–968. 20. Appelbaum FR, Deisseroth AB, Graw RG, Jr., et al. Prolonged complete remission following high dose chemotherapy of Burkitt’s lymphoma in relapse. Cancer 1978; 41:1059–1063.

550

Gordon and Baker

21. Phillips GL, Herzig RH, Lazarus HM, et al. Treatment of resistant malignant lymphoma with cyclophosphamide, total body irradiation, and transplantation of cryopreserved autologous marrow. N Engl J Med 1984; 310:1557–1561. 22. Philip T, Biron P, Maraninchi D, et al. Massive chemotherapy with autologous bone marrow transplantation in 50 cases of bad prognosis non-Hodgkin’s lymphoma. Br J Haematol 1985; 60:599–609. 23. Philip T, Armitage JO, Spitzer G, et al. High-dose therapy and autologous bone marrow transplantation after failure of conventional chemotherapy in adults with intermediate-grade or high-grade non-Hodgkin’s lymphoma. N Engl J Med 1987; 316:1493–1498. 24. Philip T, Guglielmi C, Hagenbeek A, et al. Autologous bone marrow transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive non-Hodgkin’s lymphoma. N Engl J Med 1995; 333:1540–1545. 25. Velasquez WS, Cabanillas F, Salvador P, et al. Effective salvage therapy for lymphoma with cisplatin in combination with high-dose Ara-C and dexamethasone (DHAP). Blood 1988; 71:117–122. 26. Chakraverty RK, Goldstone AH, McMillan AK, Chopra R. High-dose therapy for the treatment of non-Hodgkin’s lymphoma. In: Armitage JO, Antman K, eds. High-Dose Cancer Therapy. In: Pharmacology, Hematopoietins, Stem Cells. Philadelphia: Lippincott Williams & Wilkins, 2000:775–795. 27. Blay J, Gomez F, Sebban C, et al. The international prognostic index correlates to survival in patients with aggressive lymphoma in relapse: analysis of the PARMA trial. Parma group. Blood 1998; 92:3562–3568. 28. Haioun C, Lepage E, Gisselbrecht C, et al. Benefit of autologous bone marrow transplantation over sequential chemotherapy in poor-risk aggressive non-Hodgkin’s lymphoma: updated results of the prospective study LNH87-2. Groupe d’Etude des lymphomes de l’Adulte. J Clin Oncol 1997; 15:1131–1137. 29. The International Non-Hodgkin’s Lymphoma Prognostic Factors Project. A predictive model for aggressive Non-Hodgkin’s lymphoma. N Engl J Med 1993; 329:987–994. 30. Santini G, Salvagno L, Leoni P, et al. VACOP-B versus VACOP-B plus autologous bone marrow transplantation for advanced diffuse non-Hodgkin’s lymphoma: results of a prospective randomized trial by the non-Hodgkin’s lymphoma cooperative study group. J Clin Oncol 1998; 16:2796–2802. 31. Reyes F, Lepage E, Morel P, et al. Failure of first-line inductive high-dose chemotherapy (HDC) in poor-risk patients with aggressive lymphoma: updated results of the randomized LNH93-3 study. Blood 1997; 90:594a. 32. Kaiser U, Uebelacker I, Havemann K, et al. High dose chemotherapy with autologous stem cell transplantation in high grade NHL: first analysis of a randomized multicenter study [abstract]. Bone Marrow Transpl 1998; 21:S177. 33. Chopra R, Goldstone AH, Pearce R, et al. Autologous versus allogeneic bone marrow transplantation for non-Hodgkin’s lymphoma: a case-controlled analysis of the European bone marrow transplant group registry data. J Clin Oncol 1992; 10:1690–1695. 34. Ratanatharathorn V, Uberti J, Karanes C, et al. Prospective comparative trial of autologous versus allogeneic bone marrow transplantation in patients with non-Hodgkin’s lymphoma. Blood 1994; 84:1050–1055. 35. O’Leary M, Ramsay NK, Nesbit ME, Jr., et al. Bone marrow transplantation for non-Hodgkin’s lymphoma in children and young adults: a pilot study. Am J Med 1983; 74:497–501. 36. Hartmann O, Pein F, Beaujean F, et al. High-dose polychemotherapy with autologous bone marrow transplantation in children with relapsed lymphomas. J Clin Oncol 1984; 2:979–985. 37. Loiseau HA, Hartmann O, Valteau D, et al. High-dose chemotherapy containing busulfan followed by bone marrow transplantation in 24 children with refractory or relapsed non-Hodgkin’s lymphoma. Bone Marrow Transplant 1991; 8:465–472. 38. Bureo E, Ortega JJ, Munoz A, et al. Bone marrow transplantation in 46 pediatric patients with nonHodgkin’s lymphoma. Spanish working party for bone marrow transplantation in children. Bone Marrow Transplant 1995; 15:353–359. 39. Gordon B. For the pediatric blood and marrow transplant consortium: HSCT for children with nonHodgkin’s lymphoma (NHL): a multi-institutional analysis from the pediatric blood and marrow transplant consortium (PBMTC). Presented at marrow transplantation in children: current results and controversies, June 1998. (unpublished data).

HSCT for Hodgkin’s and Non-Hodgkin’s Lymphoma

551

40. Ladenstein R, Pearce R, Hartmann O, et al. High-dose chemotherapy with autologous bone marrow rescue in children with poor-risk Burkitt’s lymphoma: a report from the European lymphoma bone marrow transplantation registry. Blood 1997; 90:2921–2930. 41. Philip T, Pinkerton R, Hartmann O, et al. The role of massive therapy with autologous bone marrow transplantation in Burkitt’s lymphoma. Clin Haematol 1986; 15:205–217. 42. Vose JM, Anderson JR, Kessinger A, et al. High-dose chemotherapy and autologous hematopoietic stem-cell transplantation for aggressive non-Hodgkin’s lymphoma. J Clin Oncol 1993; 11:1846–1851. 43. Philip T, Hartmann O, Pinkerton R, et al. Curability of relapsed childhood B-cell non-Hodgkin’s lymphoma after intensive first line therapy: a report from the societe francaise d’Oncologie pediatrique. Blood 1993; 81:2003–2006. 44. Lemerle J. Treatment of B-cell non-Hodgkin’s malignant lymphomas of childhood in Europe: recent and on-going studies. IARC Sci Publ 1985:383–398. 45. Patte C, Philip T, Rodary C, et al. Improved survival rate in children with stage III and IV B cell non-Hodgkin’s lymphoma and leukemia using multi-agent chemotherapy: results of a study of 114 children from the French pediatric oncology society. J Clin Oncol 1986; 4:1219–1226. 46. Patte C, Bernard A, Hartmann O, et al. High-dose methotrexate and continuous infusion Ara-C in children’s non-Hodgkin’s lymphoma: phase II studies and their use in further protocols. Pediatr Hematol Oncol 1986; 3:11–18. 47. Sullivan MP, Boyett J, Pullen J, et al. Pediatric oncology group experience with modified LSA2-L2 therapy in 107 children with non-Hodgkin’s lymphoma (Burkitt’s lymphoma excluded). Cancer 1985; 55:323–336. 48. Tubergen DG, Krailo MD, Meadows AT, et al. Comparison of treatment regimens for pediatric lymphoblastic non-Hodgkin’s lymphoma: a children’s cancer group study. J Clin Oncol 1995; 13:1368–1376. 49. Brugieres L, Quartier P, Le Deley MC, et al. Relapses of childhood anaplastic large-cell lymphoma: treatment results in a series of 41 children-a report from the French society of pediatric oncology. Ann Oncol 2000; 11:53–58. 50. Gordon B, Weisenburger D, Harper J, et al. Hematopoietic stem cell transplantation (HSCT) for relapsed Ki-1 positive T-cell non-Hodgkin lymphoma (NHL) in children: long term follow-up of patients prepared with a thioTEPA (TT) and total body irradiation (TBI) based regimen. Presented at the meeting of the American Society of Pediatric Hematology/Oncology, September 1998. J Pediatr Hematol Oncol 1998; 20:393. Abstract 656. 51. Little R, Wittes RE, Longo DL, Wilson WH. Vinblastine for recurrent Hodgkin’s disease following autologous bone marrow transplant. J Clin Oncol 1998; 16:584–588. 52. Fitoussi O, Simon D, Brice P, et al. Tandem transplant of peripheral blood stem cells for patients with poor-prognosis Hodgkin’s disease or non-Hodgkin’s lymphoma. Bone Marrow Transplant 1999; 24:747–755. 53. Carella AM, Cavaliere M, Lerma E, et al. Autografting followed by nonmyeloablative immunosuppressive chemotherapy and allogeneic peripheral-blood hematopoietic stem-cell transplantation as treatment of resistant Hodgkin’s disease and non-Hodgkin’s lymphoma. J Clin Oncol 2000; 18:3918–3924. 54. DeVita VT, Jr., Serpick AA, Carbone PP. Combination chemotherapy in the treatment of advanced Hodgkin’s disease. Ann Intern Med 1970; 73:881–895. 55. Donaldson SS, Link MP. Combined modality treatment with low-dose radiation and MOPP chemotherapy for children with Hodgkin’s disease. J Clin Oncol 1987; 5:742–749. 56. Jenkin D, Doyle J, Berry M, et al. Hodgkin’s disease in children: treatment with MOPP and lowdose, extended field irradiation without laparotomy. Late results and toxicity. Med Pediatr Oncol 1990; 18:265–272. 57. Hutchinson RJ, Fryer CJ, Davis PC, et al. MOPP or radiation in addition to ABVD in the treatment of pathologically staged advanced Hodgkin’s disease in children: results of the children’s cancer group phase III trial. J Clin Oncol 1998; 16:897–906. 58. van den Berg H, Zsiros J, Behrendt H. Treatment of childhood Hodgkin’s disease without radiotherapy. Ann Oncol 1997; 8:15–17. 59. Baez F, Ocampo E, Conter V, et al. Treatment of childhood Hodgkin’s disease with COPP or COPP-ABV (hybrid) without radiotherapy in nicaragua. Ann Oncol 1997; 8:247–250. 60. Sripada PV, Tenali SG, Vasudevan M, et al. Hybrid (COPP/ABV) therapy in childhood Hodgkin’s disease: a study of 53 cases during 1989–1993 at the cancer institute, Madras. Pediatr Hematol Oncol 1995; 12:333–341.

552

Gordon and Baker

61. Oberlin O, Rubie H, Bertrand Y, et al. Low-dose radiation therapy and reduced chemotherapy in childhood Hodgkin’s disease: the experience of the French society of pediatric oncology. J Clin Oncol 1992; 10:1602–1608. 62. Schellong G, Bramswig JH, Hornig-Franz I. Treatment of children with Hodgkin’s disease—results of the German pediatric oncology group. Ann Oncol 1992; 3:73–76. 63. Schellong G. The balance between cure and late effects in childhood Hodgkin’s lymphoma: the experience of the German-Austrian study-group since 1978. Ann Oncol 1996; 7:S67–S72. 64. Vecchi V, Di Tullio MT, Rosati D, et al. Treatment of pediatric Hodgkin’s disease tailored to stage, mediastinal mass, and age. An Italian (AIEOP) multicenter study on 215 patients. Cancer 1993; 72:2049–2057. 65. Donaldson SS, Hudson MM, Lamborn KR, et al. VAMP and low-dose, involved-field radiation for children and adolescents with favorable, early-stage Hodgkin’s disease: results of a prospective clinical trial. J Clin Oncol 2002; 20:3081–3087. 66. Weiner MA, Leventhal BG, Marcus R, et al. Intensive chemotherapy and low-dose radiotherapy for the treatment of advanced-stage Hodgkin’s disease in pediatric patients: a pediatric oncology group study. J Clin Oncol 1991; 9:1591–1598. 67. Weiner MA, Leventhal B, Brecher ML, et al. Randomized study of intensive MOPP-ABVD with or without low dose total-nodal radiation therapy in the treatment of stages IIB, IIIA2, IIIB, and IV Hodgkin’s disease in pediatric patients: a pediatric oncology group study. J Clin Oncol 1997; 15:2769–2779. 68. Hunger SP, Link MP, Donaldson SS. ABVD/MOPP and low-dose involved-field radiotherapy in pediatric Hodgkin’s disease: the stanford experience. J Clin Oncol 1994; 12:2160–2166. 69. Fryer CJ, Hutchinson RJ, Krailo M, et al. Efficacy and toxicity of 12 courses of ABVD chemotherapy followed by low-dose regional radiation in advanced Hodgkin’s disease in children: a report from the children’s cancer study group. J Clin Oncol 1990; 8:1971–1980. 70. Canellos GP, Anderson JR, Propert KJ, et al. Chemotherapy of advanced Hodgkin’s disease with MOPP, ABVD, or MOPP alternating with ABVD. N Engl J Med 1992; 327:1478–1484. 71. Longo DL, Duffey PL, Young RC, et al. Conventional-dose salvage combination chemotherapy in patients relapsing with Hodgkin’s disease after combination chemotherapy: the low probability for cure. J Clin Oncol 1992; 10:210–218. 72. Brice P, Bastion Y, Divine M, et al. Analysis of prognostic factors after the first relapse of Hodgkin’s disease in 187 patients. Cancer 1996; 78:1293–1299. 73. Yuen AR, Rosenberg SA, Hoppe RT, Halpern FD, Horning SJ. Comparison between conventional salvage therapy and high-dose therapy with autografting for recurrent or refractory Hodgkin’s disease. Blood 1997; 89:814–822. 74. Baker KS, Gordon BG, Gross TG, et al. Autologous hematopoietic stem-cell transplantation for relapsed or refractory Hodgkin’s disease in children and adolescents. J Clin Oncol 1999; 17:825–831. 75. Wheeler C, Eickhoff C, Elias A, et al. High-dose cyclophosphamide, carmustine, and etoposide with autologous transplantation in Hodgkin’s disease: a prognostic model for treatment outcomes. Biol Blood Marrow Transpl 1997; 3:98–106. 76. Horning SJ, Chao NJ, Negrin RS, et al. High-dose therapy and autologous hematopoietic progenitor cell transplantation for recurrent or refractory Hodgkin’s disease: analysis of the Stanford university results and prognostic indices. Blood 1997; 89:801–813. 77. Sureda A, Mataix R, Hernandez-Navarro F, et al. Autologous stem cell transplantation for poor prognosis Hodgkin’s disease in first complete remission: a retrospective study from the Spanish GEL-TAMO cooperative group. Bone Marrow Transplant 1997; 20:283–288. 78. O’Brien MER, Milan S, Cunningham D, et al. High-dose chemotherapy and autologous bone marrow transplant in relapsed Hodgkin’s disease-a pragmatic prognostic index. Br J Cancer 1996; 73:1272–1277. 79. Bierman PJ, Anderson JR, Freeman MB, et al. High-dose chemotherapy followed by autologous hematopoietic rescue for Hodgkin’s disease patients following first relapse after chemotherapy. Ann Oncol 1996; 7:151–156. 80. Reece DE, Phillips GL. Intensive therapy and autologous stem cell transplantation for Hodgkin’s disease in first relapse after combination chemotherapy. Leuk Lymphoma 1996; 21:245–253. 81. Weaver CH, Schwartzberg L, Li W, Hazelton B, West W. High-dose chemotherapy and autologous peripheral blood progenitor cell transplant for the treatment of Hodgkin’s disease. Bone Marrow Transplant 1996; 17:715–721.

HSCT for Hodgkin’s and Non-Hodgkin’s Lymphoma

553

82. Reece DE, Barnett MJ, Shepherd JD, et al. High-dose cyclophosphamide, carmustine (BCNU), and etoposide (VP16-213) with or without cisplatin (CBV CP) and autologous transplantation for patients with Hodgkin’s disease who fail to enter a complete remission after combination chemotherapy. Blood 1995; 86:451–456. 83. Nademanee A, O’Donnell MR, Snyder DS, et al. High-dose chemotherapy with or without total body irradiation followed by autologous bone marrow and/or peripheral blood stem cell transplantation for patients with relapsed and refractory Hodgkin’s disease: results in 85 patients with analysis of prognostic factors. Blood 1995; 85:1381–1390. 84. Matsuzaki A, Okamura J, Nagatoshi Y, et al. Treatment of young relapsed Hodgkin’s disease patients with high-dose chemotherapy followed by peripheral blood stem cell transplantation. Leuk Lymphoma 1995; 18:505–509. 85. Burns LJ, Daniels KA, McGlave PB, et al. Autologous stem cell transplantation for refractory and relapsed Hodgkin’s disease: factors predictive of prolonged survival. Bone Marrow Transplant 1995; 16:13–18. 86. Horning SJ, Negrin RS, Chao NJ, et al. Fractionated total-body irradiation, etoposide, and cyclophosphamide plus autografting in Hodgkin’s disease and non-Hodgkin’s lymphoma. J Clin Oncol 1994; 12:2552–2558. 87. Bierman PJ, Bagin RG, Jagannath S, et al. High dose chemotherapy followed by autologous hematopoietic rescue in Hodgkin’s disease: long term follow-up in 128 patients. Ann Oncol 1993; 4:767–773. 88. Crump M, Smith AM, Brandwein J, et al. High-dose etoposide and melphalan, and autologous bone marrow transplantation for patients with advanced Hodgkin’s disease: importance of disease status at transplant. J Clin Oncol 1993; 11:704–711. 89. Williams CD, Goldstone AH, Pearce R, et al. Autologous bone marrow transplantation for pediatric Hodgkin’s disease: a case matched comparison with adult patients by the European bone marrow transplant group registry. J Clin Oncol 1993; 11:2243–2249. 90. Kessinger A, Bierman PJ, Vose JM, Armitage JO. High dose cyclophosphamide, carmustine, and etoposide followed by autologous peripheral stem cell transplantation for patients with relapsed Hodgkin’s disease. Blood 1991; 77:2322–2325. 91. Hurd DD, Haake RJ, Lasky LC, et al. Treatment of refractory and relapsed Hodgkin’s disease: intensive chemotherapy and autologous bone marrow or peripheral blood stem cell support. Med Pediatr Oncol 1990; 18:447–453. 92. Nachbaur D, Greinix HT, Koller E, et al. Long-term results of autologous stem cell transplantation for Hodgkin’s disease (HD) and low-/intermediate-grade B non-Hodgkin’s lymphoma (NHL): a report from the Austrian stem cell transplantation registry (ASCTR). Ann Hematol 2005; 84:462–473. 93. Wang EH, Chen YA, Corringham S, et al. High-dose CEB versus BEAM with autologous stem cell transplant in lymphoma. Bone Marrow Transplant 2004; 34:581–587. 94. Lancet JE, Rapoport AP, Brasacchio R, et al. Autotransplantation for relapsed or refractory Hodgkin’s disease: long-term follow-up and analysis of prognostic factors. Bone Marrow Transplant 1998; 22:265–271. 95. Vose JM, Bierman PJ, Lynch JC, et al. Transplantation of highly purified CD34CThy-1C hematopoietic stem cells in patients with recurrent indolent non-Hodgkin’s lymphoma. Biol Blood Marrow Transplant 2001; 7:680–687. 96. Gajewski JL, Phillips GL, Sobocinski KA, et al. Bone marrow transplants from HLA-identical sibling in advanced Hodgkin’s disease. J Clin Oncol 1996; 14:572–578. 97. Millpied N, Fielding AK, Pearce RM, Ernst P, Goldstone AH. Allogeneic bone marrow transplant is not better than autologous transplant for patients with relapsed Hodgkin’s disease. J Clin Oncol 1996; 14:1291–1296. 98. Ahmed T, Rashid K, Waheed F, et al. Long-term survival of patients with resistant lymphoma treated with tandem stem cell transplant. Leuk Lymphoma 2005; 46:405–414. 99. Brice P, Divine M, Simon D, et al. Feasibility of tandem autologous stem-cell transplantation (ASCT) in induction failure or very unfavorable (UF) relapse from Hodgkin’s disease (HD). Ann Oncol 1999; 10:1485–1488. 100. Josting A, Rudolph C, Mapara M, et al. Cologne high-dose sequential chemotherapy in relapsed and refractory Hodgkin lymphoma: results of a large multicenter study of the German Hodgkin lymphoma study group (GHSG). Ann Oncol 2005; 16:116–123.

554

Gordon and Baker

101. Anderlini P, Saliba R, Acholonu S, et al. Reduced-intensity allogeneic stem cell transplantation in relapsed and refractory Hodgkin’s disease: low transplant-related mortality and impact of intensity of conditioning regimen. Bone Marrow Transplant 2005; 35:943–951. 102. Peggs KS, Hunter A, Chopra R, et al. Clinical evidence of a graft-versus-Hodgkin’s-lymphoma effect after reduced-intensity allogeneic transplantation. Lancet 2005; 365:1934–1941. 103. Claviez A, Klingebiel T, Beyer J, et al. Allogeneic peripheral blood stem cell transplantation following fludarabine-based conditioning in six children with advanced Hodgkin’s disease. Ann Hematol 2004; 83:237–241. 104. Bhatia S, Robison LL, Oberlin O, et al. Breast cancer and other second neoplasms after childhood Hodgkin’s disease. N Engl J Med 1996; 334:745–751. 105. Donaldson SS, Hancock SL, Hoppe RT. The Janeway lecture. Hodgkin’s disease—finding the balance between cure and late effects. Cancer J Sci Am 1999; 5:325–333. 106. Baker KS, DeFor TE, Burns LJ, et al. New malignancies following blood or marrow stem cell transplantation in children and adults: incidence and risk factors. J Clin Oncol. 2003; 21:1352–1358. 107. Milligan DW, Ruiz De Elvira MC, Kolb HJ, et al. Secondary leukaemia and myelodysplasia after autografting for lymphoma: results from the EBMT. EBMT lymphoma and late effects working parties. European group for blood and marrow transplantation. Br J Haematol 1999; 106:1020–1026. 108. Harrison CN, Gregory W, Hudson GV, et al. High-dose BEAM chemotherapy with autologous haemopoietic stem cell transplantation for Hodgkin’s disease is unlikely to be associated with a major increased risk of secondary MDS/AML. Br J Cancer 1999; 81:476–483. 109. Stone RM, Neuberg D, Soiffer R, et al. Myelodysplastic syndrome as a late complication following autologous bone marrow transplantation for non-Hodgkin’s lymphoma. J Clin Oncol 1994; 12:2535–2542.

27 Hematopoietic Stem-Cell Transplantation for Pediatric Malignant Brain Tumors Sharon L. Gardner The Steven D. Hassenfeld Center for Children with Cancer and Other Blood Disorders, NYU Medical Center, New York, New York, U.S.A.

Ira J. Dunkel The Steven D. Hassenfeld Center for Children with Cancer and Other Blood Disorders, NYU Medical Center and Memorial Sloan-Kettering Cancer Center, New York, New York, U.S.A.

INTRODUCTION Historically, brain tumors in children were treated with neurosurgical resection and radiation therapy. The addition of chemotherapy to the treatment regimen has more recently been proven to be beneficial for many tumor types. However, certain brain tumors are still associated with a poor prognosis, and most patients with recurrent malignant brain tumors will die of their disease despite conventional retrieval strategies. The recognition of chemosensitivity of many types of brain tumors has led to the exploration of the use of hematopoietic cell support as a means of increasing chemotherapy dose intensity for patients with high-risk or recurrent tumors. In this chapter we will review the current status of high-dose chemotherapy with stem cell support for pediatric brain tumors.

GLIOMAS High grade gliomas comprise nearly 40% of the central nervous system tumors in adults; however, their frequency is less than 12% in children (1,2). Unfortunately the prognosis is as poor in children as it is in adults. Historically, radiation therapy has been the standard treatment for patients with these tumors with three-year survival rates of approximately 35% for patients with anaplastic astrocytoma and 15% for those with glioblastoma multiforme (1). The first study that demonstrated efficacy of chemotherapy in children with malignant brain tumors was performed by the children’s cancer group between 1976 and 1981 and examined the use of adjuvant chemotherapy (lomustine, vincristine and prednisone) with irradiation versus irradiation alone in 58 children with high-grade astrocytoma (3). The five-year event-free survival rates for children treated with chemotherapy and irradiation versus irradiation alone were 46% and 18%, respectively. Unfortunately there were no long-term survivors amongst the small number of children treated with chemotherapy at the time of recurrence. The earliest studies using high-dose chemotherapy with autologous stem cell rescue in adults with malignant gliomas primarily involved single agents. Several investigators used 555

556

Gardner and Dunkel

carmustine at high doses with autologous bone marrow rescue in adults with malignant glioma (4–6). The dose of carmustine ranged from 600 to 1400 mg/m2. Although there were some responses seen, there also was significant pulmonary, hepatic, and neurologic toxicity, particularly at the highest doses of carmustine. Other drugs which have been given as single agents at high doses include aziridinylbenzoquinone (AZQ), etoposide, and thiotepa. In the report by Abrams et al. patients with a variety of refractory central nervous system tumors were treated with AZQ (7). There were no radiographic responses seen in any patients with recurrent anaplastic astrocytoma. Etoposide has been administered at doses ranging from 1800 to 2400 mg/m2 with autologous bone marrow rescue. Giannone and Wolff reported objective responses in three of 16 patients with progressive astrocytoma (8). One was a long-term survivor at 54 months. Leff et al. also used high etoposide in patients with progressive astrocytoma (nine with high-grade astrocytoma and four with low grade astrocytoma) (9,10). Two of 11 evaluable patients had clinical improvement, although no patients had significant radiographic responses. Furthermore, there was significant but transient neurotoxicity and three toxic deaths. Thiotepa is one of the most promising drugs given as a single agent to patients with anaplastic astrocytoma. Ahmed et al. reported four complete responses and five partial responses in 16 patients with newly diagnosed anaplastic astrocytoma treated with thiotepa 600 to 900 mg/m2 with autologous bone marrow rescue (11). The majority of high-dose chemotherapy approaches in children with glioma have involved multidrug regimens. Several different combinations have been used, including thiotepa/cyclophosphamide, melphalan/cyclophosphamide, and thiotepa/etoposide alone and with the addition of carmustine or carboplatin. Heideman et al. used high-dose thiotepa (300 mg/m2/day) and cyclophosphamide (2 gm/m2/day) for three days with autologous bone marrow rescue in the treatment of 13 children with high grade glioma (11 newly diagnosed; 2 recurrent) (12). Prior to chemotherapy, three patients had near total resections (O90% tumor removal); four patients had subtotal resections (O50% but !90% tumor removal); six patients had biopsy only. At day 60, the 11 patients who had at least stable disease received hyperfractionated (nZ9) or conventional external beam radiation (nZ2). Prior to irradiation two patients received local radioactive iodine 125 implantation, and one patient had radiosurgery. One complete response and three partial responses were seen. After combined modality therapy, the median progression-free survival was nine months (range, 0 to 30C months). Kedar et al. also used the combination of thiotepa 250 to 300 mg/m2/day for three days and cyclophosphamide 750 to 975 mg/m2/day for four days in three patients with newly diagnosed high-grade glioma (13). Hyperfractionated radiation therapy was given following recovery from chemotherapy. One patient survived disease-free 22 months from diagnosis. Mahoney et al. reported the POG experience using cyclophosphamide in a dose escalation fashion with melphalan in children with recurrent or progressive malignant brain tumors (14). Cyclophosphamide was administered at a dose of 750 to 1500 mg/m2/day for four days followed by melphalan 60 mg/m2/day for three days with autologous bone marrow rescue. There were four children with high-grade gliomas treated with this approach: two with anaplastic astrocytoma; one with glioblastoma multiforme; and one with a brainstem glioma. All four children died within seven months of high-dose therapy; one from treatment-related toxicity and three from disease. Investigators at Duke University used three different high-dose regimens in children with recurrent or high-risk brain tumors (15). The regimens included cyclophosphamide 1.5 gm/m2 daily for four days followed by melphalan 25 to 60 mg/m2/day for three days; busulfan 37.5 mg/m2/dose every six hours for 16 doses and melphalan 140 or 180 mg/m2; and carboplatin 700 mg/m2/day for three days, alternating with etoposide 500 mg/m2/day for three days. There were 12 children with glial tumors included in their report; 6 with glioblastoma multiforme, 2 with brainstem glioma, and 1 each with spinal cord glioblastoma, spinal cord astrocytoma, pilocytic astrocytoma, and gliomatosis cerebri. There were two patients who were

Pediatric Malignant Brain Tumors

557

disease-free survivors, one with glioblastoma multiforme and one with a brainstem glioma at 32C and 55C months following autologous bone marrow rescue. Finlay et al. initially piloted a two-drug regimen, including thiotepa 300 mg/m2/day for three days with etoposide 500 mg/m2/day on the same three days with autologous bone marrow rescue in children and young adults with recurrent malignant brain tumors (16). This regimen then became part of a cooperative group study within the children’s cancer group (CCG 9883). Objective responses were seen in 4 of 14 evaluable patients with high-grade glioma. Four patients were not evaluable for disease response; three patients had no evidence of disease at the time of high-dose chemotherapy; and one patient received concomitant radiation therapy. Five of 18 patients were disease free survivors 39C to 59C months following autologous bone marrow rescue. There were seven (16%) toxic deaths amongst the entire cohort. Because of these encouraging responses, carmustine was added to the thiotepa/etoposide combination. The dose of carmustine was 600 mg/m2 given over three or four days followed by thiotepa 300 mg/m2/day and etoposide 250 or 500 mg/m2/day on the same three days. Twentyone newly diagnosed patients also received local irradiation following autologous bone marrow rescue. Papadakis et al. reported results of 42 patients (29 newly diagnosed; 13 recurrent) treated with this regimen (17). Diagnoses included high grade glioma (nZ37), medulloblastoma (nZ2), and nonbiopsied tumors (nZ3). Twenty-one patients with newly diagnosed disease received irradiation post high-dose chemotherapy. Patients ranged in age from 0.7 to 46.8 years (median 12.2 years). Three newly diagnosed patients and one patient treated for recurrent disease were alive without disease progression 64, 67, 86, and 110 months, respectively, following autologous bone marrow rescue. Unfortunately toxicity was significant with nine early deaths. The deaths were due to multiorgan system failure. The preceding event included respiratory failure (three patients), pulmonary hemorrhage (one patient), renal failure (one patient), brainstem necrosis (one patient), infection (two patients), and tumor invasion (two patient). Patients older than 18 years of age had a significantly higher toxic mortality rate (50%) compared with those less than 18 years of age (15%). Grovas et al. reported similar results for the children’s cancer group (CCG) study using the same approach in children newly diagnosed with glioblastoma multiforme (18). Eleven children received carmustine 100 mg/m2 every twelve hours for six doses followed by thiotepa 300 mg/m2/day and etoposide 250 mg/m2/day with 5400 cGy radiotherapy beginning 42 days following autologous stem cell rescue. Two-year progression-free survival rate for 11 patients was 46%C/K 14%. Three children (27%) had complete radiographic response 2.9, 3.9 and 5.1 years post autologous bone marrow rescue. However, once again toxicity was significant with five patients (45%) developing grade III or IV pulmonary and/or neurological toxicity and a toxic mortality rate of 18%. The group from Lyon also used the two-drug combination of thiotepa and etoposide in 22 children and young adults with newly diagnosed, recurrent, or refractory high-grade gliomas (19). The dose of thiotepa was 300 mg/m2/day for three days with etoposide 500 mg/m2/day on the same three days. The overall response rate was 29% with 1 complete and 3 partial responses in 14 evaluable patients. Three patients were progression free survivors 54C to 65C months following autologous bone marrow rescue. All three of these patients were newly diagnosed (two patients in first partial remission and one patient with stable disease at the time of highdose chemotherapy), received irradiation prior to the high-dose chemotherapy, and had tumors that were not located in the brainstem. All three had mild to moderate disabilities and required special schooling. There were two toxic deaths.

Conclusions Responses have been seen using high-dose chemotherapy in patients newly diagnosed with high-grade gliomas as well as in those with recurrent disease. However, improvements in toxicity must be made in order for this approach to be beneficial.

558

Gardner and Dunkel

BRAINSTEM GLIOMAS Brainstem gliomas comprise approximately 10–20% of central nervous tumors in childhood, with 80% of these tumors consisting of diffuse intrinsic pontine tumors (20). These tumors remain one of the most difficult tumors to treat with no curative therapy available to date and median survival of less than one year (21–23). Dunkel et al. reported the Memorial Sloan-Kettering Cancer Center (MSKCC)/CCG experience using high-dose chemotherapy with autologous stem cell rescue in 16 patients with diffuse pontine tumors (24). Ten patients had recurrent or refractory disease, and six patients were newly diagnosed. All ten patients with recurrent disease had previously received irradiation. Patients with recurrent disease were treated with three different regimens. Six patients received thiotepa 300 mg/m2/day and etoposide 250 to 500 mg/m2/day daily for three days; two patients received carmustine 100 mg/m2 /dose q12 hours for three days, followed by thiotepa and etoposide; two patients received carboplatin 500 mg/m2/day for three days, followed by thiotepa and etoposide. The six patients with newly diagnosed diffuse pontine tumors received the carmustine/ thiotepa/etoposide regimen with hyperfractionated irradiation six weeks following autologous stem cell rescue. The toxic mortality rate was 13%. Median survival for patients with recurrent or refractory disease was 4.7 months and 11.4 months for newly diagnosed patients. Bouffet et al. reported the results of the French society of pediatric oncology using highdose chemotherapy with autologous stem cell rescue in children newly diagnosed with diffuse intrinsic pontine tumors (25). Thirty-five children received focal irradiation. Only 24 children went on to receive the high-dose chemotherapy two to three months following irradiation. Nine patients had progressive disease prior to chemotherapy, and two families refused the chemotherapy. The high-dose chemotherapy consisted of busulfan 150 mg/m2/day for four days and thiotepa 300 mg/m2/day for three days. Three patients died from treatment-related toxicity. Median survival was ten months.

Conclusions At the present time there does not appear to be a role for high-dose chemotherapy in the treatment of children with brainstem gliomas. The inability to achieve minimal residual disease prior to high-dose chemotherapy may be responsible for the poor results.

EPENDYMOMA Ependymomas account for 5% to 10% of pediatric central nervous system tumors (26). These tumors, which usually originate from the ependymal lining of the ventricular system, most commonly occur intracranially, with 60% of these lesions occurring in the posterior fossa (27). Complete surgical resection remains the key to curing these patients with irradiation increasing the five-year overall survival from 35% to 63% (28). Although several different chemotherapy regimens have been used, there has been no dramatic improvement in survival with this approach. There have been two studies published using high-dose chemotherapy with autologous stem cell rescue in children with recurrent ependymoma. Grill et al. reported the results of a phase II study using busulfan 150 mg/m2/d for four days and thiotepa 300 mg/m2/d for three days in 16 children with recurrent or refractory ependymoma (29). These patients had previously received surgery and conventional chemotherapy. Half of them had also received focal irradiation. There were no radiographic responses seen following the high-dose chemotherapy.

Pediatric Malignant Brain Tumors

559

Mason et al. reported the CCG experience using thiotepa 300 mg/m2/d for three days and etoposide 250 to 500 mg/m2/d for three days with or without carboplatin 500 mg/m2/d for an additional three days in children with intracranial ependymoma (30). Once again there were no responses. Furthermore, the toxic mortality rate was 33%.

Conclusions There is currently no effective high-dose chemotherapy regimen for patients with recurrent ependymoma.

MEDULLOBLASTOMA Medulloblastomas are tumors of neuroepithelial origin and comprise approximately 10% to 20% of all childhood brain tumors and 40% of tumors in the posterior fossa (31,32). Although the primary tumors are often surgically resectable, their propensity to undergo leptomeningeal as well as extraneural metastases precludes the use of surgery alone. Craniospinal irradiation has played an important role in the treatment of patients newly diagnosed with medulloblastoma (33). However chemotherapy has been used with increasing frequency because of the chemoresponsiveness of these tumors and the concern of long-term sequelae associated with irradiation (34–36). Moreover, for patients with recurrent disease, irradiation is often not an option, and standard dose chemotherapy has not improved upon their dismal outcome (37,38). Because of the chemosensitivity of many of these tumors as well as the recent advances in the area of hematopoietic stem cell rescue, several investigators have explored the use of high-dose chemotherapy with autologous stem cell rescue for these patients. One of the earliest studies published regarding the use of high-dose chemotherapy with autologous stem cell rescue in patients with medulloblastoma was reported by Kalifa et al. (39). Children with recurrent medulloblastoma/primitive neuroectodermal tumors (PNET) were treated with busulfan 150 mg/m2/day for four days and thiotepa 350 mg/m2/day for three days followed by autologous bone marrow rescue. The 13 oldest children had previously received surgery and irradiation, and the 29 youngest children had received surgery followed by standard dose chemotherapy. The response rate (complete and partial) was 75%. Four children with localized relapse were alive without disease 1C to 6C years following high-dose chemotherapy. Three of the four children received a radiation boost to the relapse site following the high-dose chemotherapy. There were four toxic deaths. Finlay et al. published results using etoposide 500 mg/m2/day and thiotepa 300 mg/m2/day on the same three days in patients with a wide variety of recurrent malignant brain tumors (16). There were two responses in the six patients with medulloblastoma who had measurable disease but no long-term survivors. Bulky disease at the time of the high-dose chemotherapy was a significant adverse prognostic factor. Mahoney et al. (14) published the POG pilot study involving a dose escalation of cyclophosphamide with a fixed dose of melphalan followed by ABMR for children with recurrent or progressive malignant brain tumors. The cyclophosphamide dose ranged from 750 mg/m2/day to 1200 mg/m2/day daily for four days, followed by melphalan 60 mg/m2/day for three days. Eight children with recurrent or refractory medulloblastoma were treated with this regimen. Although there were three toxic deaths, responses were seen in four children (one CR and three PR’s) with two children still alive at 24 and 25 months following autologous bone marrow rescue. Children with chemosensitive tumors and/or minimal disease at the time of the high-dose chemotherapy had longer intervals of progressionfree survival.

560

Gardner and Dunkel

In the study reported by Graham et al. 18 patients ranging in age from 1 to 32 years (median 12 years) had recurrent medulloblastoma (15). Three different high-dose chemotherapy regimens were used. Fifteen patients received cyclophosphamide 1.5 gm/m2/day for four days and melphalan 25 to 60 mg/m2/day for three days. Two patients received carboplatin 700 mg/m2/day for three days and etoposide 500 mg/m2/day for three days, and one patient received busulfan 37.5 mg/m2 every six hours for sixteen doses and melphalan 180 mg/m2. Four patients were free of disease 27 to 49 months following autologous bone marrow rescue. Once again, patients with chemosensitive tumors and localized disease at the time of relapse fared the best. Dunkel et al. reported the combined MSKCC/CCG experience using carboplatin 500 mg/m2/day (or area under the concentration curve of 7 mg/ml-min) for three days, followed by thiotepa 300 mg/m2/day and etoposide 250 mg/m2/day for three days with autologous stem cell rescue in 23 patients with recurrent medulloblastoma (40). All patients had received prior irradiation and/or chemotherapy. Twenty-one patients received therapy in addition to the high-dose chemotherapy including surgical resection (nZ7), conventional chemotherapy (nZ17), and external beam irradiation (nZ11). Three of 23 patients died from treatment related toxicity. Kaplan-Maier estimates of event-free and overall survival were 34% (C/K 10%) and 46% (C/K 11%), respectively, at 36 months following autologous stem cell rescue. Because of the encouraging results seen using this approach in patients with recurrent disease, investigators have recently reported results using high-dose chemotherapy with autologous stem cell rescue as part of the initial treatment of patients newly diagnosed with medulloblastoma, as well as the use of tandem courses of intensified standard dose chemotherapy with autologous stem cell rescue in an effort to rapidly administer repeated cycles of chemotherapy. Vassal et al. administered two courses of melphalan 100 mg/m2/day 21 days apart as part of the consolidation therapy for children newly diagnosed with central nervous system PNET (41). Thirteen of 16 children had the primary tumors located in the cerebellum, and 14 children had metastatic disease. The sequential melphalan followed two courses of standard dose etoposide and carboplatin. There were 11 partial remissions after two courses of high-dose melphalan in 14 patients with measurable disease. Strother et al. used sequential courses of intensified (but not myeloablative) chemotherapy with autologous stem cell rescue in patients newly diagnosed with medulloblastoma and supratentorial PNET (42). Nineteen patients had high-risk disease and 34 had standard risk disease. Patients with high-risk disease initially underwent surgical resection, followed by topotecan administered as a six-week phase II window therapy. They then received craniospinal irradiation followed by four cycles of high-dose chemotherapy with autologous stem cell rescue. Patients with standard risk disease did not receive the topotecan therapy. Following radiation therapy, 50 patients went on to receive four cycles of intensified chemotherapy consisting of cisplatin 75 mg/m2!1 day, vincristine 1.5 mg/m2!1 day, cyclophosphamide 2 gm/m2!2 days, followed by stem cell reinfusion. Of the remaining three patients, two patients progressed while receiving irradiation and received other treatment, and one did not meet eligibility criteria for high-dose chemotherapy. The doses of the chemotherapeutic agents had to be reduced in several patients secondary to toxicity. One further patient was removed from the study secondary to cardiac toxicity. Of the 53 original patients, five have died. The two-year progression-free survival from the start of treatment was 93.6%C/K 4.7% for the 34 average risk patients and 73.7%C/K 10.5% for the 19 high-risk patients.

Conclusions There appears to be a role for high-dose chemotherapy in some patients with medulloblastoma. Those who have chemosensitive disease and minimal residual disease prior to the high-dose chemotherapy have received the most benefit from this approach.

Pediatric Malignant Brain Tumors

561

OTHER PRIMITIVE NEUROECTODERMAL TUMORS Approximately 2% to 3% of childhood brain tumors are PNETs that occur outside the cerebellum (43). Although histologically these tumors resemble medulloblastomas, recent data using microarrays suggest that they are distinct tumors (44). The CCG achieved 45% three-year progression-free survival using surgery, standard dose chemotherapy and irradiation in children newly diagnosed with PNET (45). However many children less than 9 years of age at the time of irradiation suffered significant developmental delay. Furthermore, the survival for children with recurrent disease is dismal. Mahoney et al. included one patient with PNET in the report for the pediatric oncology group using high-dose melphalan and cyclophosphamide with autologous stem cell rescue in children with recurrent malignant brain tumors (14). The child had a minor response and survived eight months. Graham et al. included ten patients with high risk or recurrent PNET/pineoblastoma in their report using high-dose chemotherapy with autologous stem cell rescue (15). Two of seven patients with pineoblastoma had recurrent disease. Both of these patients developed recurrences shortly after stem cell rescue. Four of five children who received high-dose chemotherapy prior to recurrence survived disease free 28C to 49C months following stem cell infusion. Two of three patients with recurrent PNET outside the pineal region were also disease-free survivors 33C and 34C months following stem cell infusion. Broniscer et al. have compiled their results using high-dose chemotherapy and autologous stem cell rescue in patients with recurrent PNETs (46). Eight patients had pineal tumors, eight had other supratentorial tumors, and one patient had PNET in the cauda equina. Patient age ranged from 1.5 to 32.5 years (median 3.9 years). High-dose chemotherapy consisted of carboplatin 500 mg/m2/day or dosed using the Calvert formula with the area under the concentration curve of 7 mg/mL-min, whichever was the lower of the two. This was followed by thiotepa 300 mg/m2/day for three days and etoposide initially at a dose of 500 mg/m2/day and then reduced to 250 mg/m2/day on the same three days. The initial two patients received only thiotepa and etoposide. The five-year event-free survival for patients with recurrent pineoblastoma and other PNETs was 0% and 62.5%, respectively. There were two toxic deaths; one from septic shock and a second from multiorgan system failure and meningitis. Because of the poor prognosis associated with the diagnosis of supratentorial PNET, investigators at Duke have administered high-dose chemotherapy with autologous stem cell rescue following surgical resection, induction chemotherapy and irradiation in patients with newly diagnosed pineoblastoma (47). High-dose chemotherapy consisted of cyclophosphamide and melphalan in 11 patients and cyclophosphamide and busulfan in 1 patient. At the time of the report, 9 of 12 patients were alive without evidence of disease at a median of 62 months from diagnosis. This included three patients with metastatic disease and two infants who did not receive irradiation.

Conclusions The use of high-dose chemotherapy with autologous stem cell rescue merits further study particularly for nonpineal supratentorial PNETs.

GERM CELL TUMORS Germ cell tumors (GCT) comprise less than 2% of all pediatric central nervous system tumors but make up 40% to 60% of all tumors occurring in the pineal area (48). These tumors, which occur most often in the second decade of life, can be divided into two major

562

Gardner and Dunkel

categories: germinomas and nongerminomatous germ cell tumors (NGGCT). In contrast to most central nervous system tumors, complete surgical resection is not usually performed because of the potential morbidity associated with operating in this area. Furthermore germinomas in particular are sensitive to both irradiation and chemotherapy (49). Unfortunately, the therapeutic options for recurrent, central nervous system (CNS) GCT are limited. Therefore, investigators have been exploring the use of high-dose chemotherapy with autologous stem cell rescue. Modak et al. reported the results of highdose thiotepa-based regimens in 21 patients with recurrent or refractory CNS GCT: 9 with germinoma and 12 with NGGCT (50). Sixteen patients had disseminated CNS disease at relapse, including five patients with spinal disease. Seven of nine (78%) patients with germinoma were event-free survivors at a median of 48 months (range, 6–87 months). One patient died of disease four month following high-dose chemotherapy, and one patient died of pulmonary fibrosis without evidence of tumor 78 months following high-dose chemotherapy. Seven of 12 patients with NGGCT died of disease at a median of four months (range, 2–17 months) following high-dose chemotherapy. Four patients survived without disease a median of 33 months (range, 24–55 months) post high-dose chemotherapy, and a fifth patient was still surviving with a second relapse 31 months following high-dose chemotherapy. Bouffet et al. reported the French society of pediatric oncology experience using thiotepa 900 mg/m2 and etoposide 1500 mg/m2 in 20 patients with recurrent tumors: 7 patients with germinoma and 13 patients with NGGCT (51). All patients had previously received carboplatin based chemotherapy, and 15 patients had also received irradiation. Following high-dose chemotherapy, three patients underwent surgical resection, four patients received craniospinal irradiation, and three patients received focal irradiation. Thirteen patients (four germinoma, nine NGGCT) are alive without evidence of disease at a median of 29 months following autologous stem cell rescue. Because of the poor prognosis of NGGCT, Tada et al. explored the use of highdose chemotherapy with autologous stem cell rescue following surgical resection, induction chemotherapy and irradiation in six patients (three choriocarcinoma, two embryonal carcinoma, one yolk sac carcinoma) (52). All patients received prior radiation therapy and prior induction chemotherapy with cisplatin 20 mg/m2!5 days and etoposide 100 mg/m2!5 days until complete remission, and five patients received prior surgical removal. One patient had no mass to remove after induction chemotherapy and irradiation. Once in complete remission, the patients received cisplatin 40 mg/m2 for five days, etoposide 250 mg/m2 for five days, and ACNU 150 mg/m2 for one day. Two patients received carboplatin 160 mg/m2 for four days instead of cisplatin to avoid renal dysfunction. As of the report, all six patients were alive without evidence of disease 9–95 months following high-dose chemotherapy.

Conclusions There appears to be a role for the use of high-dose chemotherapy in patients with CNS GCT. Additional studies with longer follow-up are needed to confirm these preliminary results.

INFANTS Unfortunately even the youngest children are not spared the development of brain tumors, with 10% to15% of childhood brain tumors occurring in children younger than two years of age (53). The treatment of these very young children is particularly challenging because of the aggressive nature of their tumors and also because of the potential long-term sequelae associated with treating these patients at such a young age (54,55). In an attempt to avoid severe late effects associated with craniospinal irradiation in very young children with recurrent medulloblastoma, the French Society of Pediatric Oncology used

Pediatric Malignant Brain Tumors

563

a combination of busulfan 150 mg/m2/d for four days and thiotepa 300 mg/m2/d for three days with autologous bone marrow rescue (56). Children with local disease at the time of relapse also received focal irradiation to the posterior fossa. In the report by Dupuis-Girod et al. the eventfree survival was 50% in 20 children at a median time of 31 months post bone marrow rescue. Gururangan et al. also used high-dose chemotherapy with autologous bone marrow rescue in the treatment of young children with recurrent brain tumors (57). These children had several different types of brain tumors and were treated with one of three different regimens. High-dose chemotherapy consisted of thiotepa 300 mg/m2/day and etoposide 250 mg/m2/day on the same three days for three patients. Sixteen patients received the same two-drug regimen with the addition of carboplatin 500 mg/m2/day or area under the concentration curve of 7 mg/ml-min for three days prior to the thiotepa and etoposide. One patient received thiotepa 300 mg/m2/day and etoposide 500 mg/m2/day for three days, followed by carmustine 150 mg/m2/day for four days. Twelve children received radiation therapy six weeks following autologous bone marrow rescue. The event-free survival was 47%C/K 14% at a median of 37.9 months following autologous bone marrow rescue. The diagnoses of the ten children who were event-free survivors included medulloblastoma (three of five), supratentorial PNET (three of three), PNET of the pineal region (one of five), glioblastoma multiforme (two of four) and anaplastic astrocytoma (one of one). There was one patient each with ependymoma and choroid plexus carcinoma who died of disease. Based upon the encouraging results seen using high-dose chemotherapy in young children with recurrent brain tumors, several investigators have begun to use this approach in children newly diagnosed with malignant brain tumors. In the “Head Start I” approach, children younger than six years of age newly diagnosed with malignant brain tumors initially underwent maximal possible surgical resection (58). They then received five cycles of standard-dose induction chemotherapy, including cisplatin, etoposide and cyclophosphamide. Vincristine was given during the first three cycles. Patients who did not progress during induction underwent consolidation with carboplatin 500 mg/m2/day for three days with modification for renal function, based on the Calvert formula with an area under the concentration curve of 7 mg/mlmin, followed by thiotepa 10 mg/kg/day and etoposide 8.3 mg/kg/day daily for three days. Patients with residual disease at the time of consolidation received irradiation. The five-year event-free survival from diagnosis was 41, 39, and 22% for children with medulloblastoma, noncerebellar PNET and ependymoma, respectively. The five-year overall survival was 64, 43 and 42% for these same patients. Thirteen of 62 children were alive without tumor progression having not received irradiation.

Conclusions These pilot data suggest that high-dose chemotherapy may allow for reduced irradiation or in some cases complete elimination of radiation in young children. However, additional studies with randomization versus more traditional approaches are needed to confirm these findings.

FUTURE DIRECTIONS Several studies suggest a role for high-dose chemotherapy with autologous stem cell rescue in patients with recurrent medulloblastoma, recurrent central nervous system GCT, recurrent glioma, and young children newly diagnosed with malignant brain tumors. However, additional studies are needed to support these data. Several investigators have initiated trials using multiple courses of high-dose chemotherapy in order to achieve dose intensification. The use of peripheral blood rather than bone marrow stem cells has made the use of sequential high-dose chemotherapy feasible even in the smallest children. Vassal et al. used sequential high-dose melphalan 100 mg/m2 every

564

Gardner and Dunkel

21 days for two courses with autologous stem cell rescue in 16 children with cerebral PNET (59). Eleven of 14 children with measurable disease had partial responses. In a limited institution study, tandem high-dose thiotepa with ASCR was administered to patients with recurrent malignant brain tumors who were not eligible for a single course of multidrug high-dose chemotherapy because of measurable disease or organ toxicity (60). Thiotepa was administered at a dose of 200 mg/m2/day for three days/course. The median time between courses was 42 days. Twenty-five percent of the courses were administered totally in the outpatient setting. There were 2 toxic deaths amongst 43 patients. Five out of 26 patients with recurrent or refractory medulloblastoma/PNET were alive at a median of 35 months following the high-dose chemotherapy. The results were even more impressive in patients with recurrent CNS GCT, with four out of five patients alive 32, 60, 72 and 85 months following high-dose chemotherapy. Ozkaynak et al. further intensified therapy with the addition of carboplatin to thiotepa (61). Thiotepa was administered at a dose of 250 mg/m2/day for three days and carboplatin 400 mg/m2/day for three days per course. Four patients received only one course of therapy; two because of parental request and two because of tumor progression. Four patients received two courses of high-dose chemotherapy, and four patients received three courses of high-dose chemotherapy. Nearly all of the patients required parenteral nutrition and half had significant mucositis requiring intravenous narcotics; however, there were no toxic deaths. The pediatric blood and marrow transplant consortium used two courses of high-dose chemotherapy in patients with recurrent medulloblastoma (62). The first course consisted of thiotepa 300 mg/m2/day for three days and BCNU 100 mg/m2/day on the same three days. Thiotepa 300 mg/m2/day and carboplatin 1200 to 1500 mg/m2/day (or AUC 5.5 to 7 using the Calvert formula) were administered daily for three days during the second course. Nine of 12 patients were alive at a median of 15 months following the high-dose chemotherapy. However, pulmonary toxicity was significant with 5 of 12 patients requiring respiratory support. The Children’s Oncology Group recently closed two studies using multiple courses of high-dose chemotherapy with autologous stem cell rescue. The first study (CCG 99703), included children younger than three years of age newly diagnosed with malignant brain or spinal cord tumors. In this study, patients initially underwent surgical resection followed by three courses of induction chemotherapy with cisplatin, vincristine, cyclophosphamide, and etoposide. Autologous stem cells were collected during the induction chemotherapy. Patients who did not have progressive disease following induction chemotherapy proceeded to consolidation therapy with three courses of high-dose thiotepa and carboplatin with autologous stem cell reinfusion following each course. The thiotepa was administered in a phase I dose escalation fashion beginning at 5 mg/kg/day for two days/course, and carboplatin was given at a fixed dose of 17 mg/kg/day on the same two days as the thiotepa. Results of this study are presently being analyzed. The second CCG study (CCG 99702) involved sequential high-dose chemotherapy following craniospinal irradiation for children newly diagnosed with high-risk medulloblastoma or supratentorial PNET. High-risk disease was defined as posterior fossa PNET with O1.5 cm2 of residual tumor or with M1-3 metastatic disease, nonposterior fossa PNET (M0-3), medulloepithelioma and atypical teratoid tumors (M0-3). Patients underwent surgical resection followed by induction chemotherapy with cyclophosphamide and vincristine. Autologous stem cells were collected during induction chemotherapy. Patients then received craniospinal irradiation with weekly vincristine. Four to six weeks following irradiation, patients received three courses of high-dose chemotherapy with thiotepa, carboplatin, and vincristine during courses one and three and carboplatin, cyclophosphamide, and vincristine during course two. The thiotepa was dose escalated in a phase I fashion. Autologous stem cells were reinfused following each of the three courses of consolidation chemotherapy. Unfortunately, the study was closed prematurely because of dose limiting toxicity consisting of veno-occlusive disease. Other investigators are incorporating new chemotherapeutic agents, such as temozolomide, into the cytoreductive regimen (63). New chemotherapy agents or new dose schedules may further increase survival; however, significant advances will most likely be achieved through the

Pediatric Malignant Brain Tumors

565

use of other treatment modalities, either alone or in combination with high-dose chemotherapy. Fortunately, there are several different strategies currently being explored. Recent advances in understanding the molecular basis of tumorigenesis and tumor resistance are presently the focus of several novel therapeutic approaches to improve tumor kill and decrease toxicity to normal tissues (64,65). Possible strategies in the development of tumor specific therapy include targeting: (1) tumor specific antigens, (2) mechanisms of tumor resistance, (3) signaling pathways that are important for tumor growth and survival, and (4) pathways involved with blood vessel formation. Most of the initial studies targeting the signaling pathways and angiogenesis have involved the use of single agents. Although valuable insights into toxicity and feasibility have been learned, there has been little impact upon survival. Because of the redundancy found within tumors and their ability to develop resistance, these agents will most likely be more effective when multiple agents are combined or used with high-dose chemotherapy and/or irradiation.

REFERENCES 1. Duncan GG, Goodman GB, Ludgate CM, et al. The treatment of adult supratentorial high-grade astrocytoma. J Neurooncol 1992; 13:63–72. 2. Cohen ME, Duffner PK. Supratentorial hemispheric astrocytomas. In: Cohen ME, Duffner PK, eds. Brain Tumors In Children. New York: Raven Press, 1994:263–284. 3. Sposto R, Ertel IJ, Jenkin RD, et al. The effectiveness of chemotherapy for treatment of high-grade astrocytoma in children: results of a randomized trial. A report from the Children’s Cancer Study Group. J Neurooncol 1989; 7:165–177. 4. Hochberg FH, Parker LM, Takvorian T, et al. High-dose BCNU with autologous bone marrow rescue for recurrent glioblastoma multiforme. J Neurosurg 1981; 54:455–460. 5. Phillips GL, Wolff SN, Fay JW, et al. Intensive 1,3-bis (2-chloroethyl)-1-nitrosourea (bcnu) monochemotherapy and autologous marrow transplantation for malignant glioma. J Clin Oncol 1986; 4:639–645. 6. Johnson DB, Thompson JM, Corwin JA, et al. Prolongation of survival for high-grade malignant gliomas with adjuvant high-dose bcnu and autologous bone marrow transplantation. J Clin Oncol 1987; 5:783–789. 7. Abrams RA, Jr., Casper J, Kun L, et al. High-dose aziridinylbenzoquinone for patients with refractory central nervous system neoplasms: a preliminary analysis. In: Dicke KA, Apitzer G, Zander AR, Gorin NC, eds. Autologous Bone Marrow Transplantation: Proceedings of The First International Symposium. Houston, Texas: The University of Texas, M.D. Anderson Hospital and Tumor Institute, 1985:227–230. 8. Giannone L, Wolff SN. Phase II treatment of central nervous system gliomas with high-dose etoposide and autologous bone marrow transplantation. Cancer Treat Rep 1987; 71:759–761. 9. Leff RS, Thompson JM, Daly MB, et al. Acute neurologic dysfunction after high-dose etoposide therapy for malignant gliomas. Cancer 1988; 62:32–35. 10. Long J, Leff R, Daly MB, et al. Phase II trial of high-dose etoposide and autologous bone marrow transplantation for treatment of progressive glioma. Proc Ann Meet Am Soc Clin Oncol 1989; 8:92 (abstract). 11. Ahmed T, Feldman E, Helson L, et al. Phase 1-2 trial of high dose thiotepa with autologous bone marrow transplantation and localized radiotherapy for patients with astrocytoma grade III-IV. Proc Ann Meet Am Assoc Cancer Res 1990; 31:A1023 (abstract). 12. Heideman RL, Douglass EC, Krance RA, et al. High-dose chemotherapy and autologous bone marrow rescue followed by interstitial and external-beam radiotherapy in newly diagnosed pediatric malignant gliomas. J Clin Onc 1993; 11:1458–1465. 13. Kedar A, Maria BL, Graham-Pole J, et al. High-dose chemotherapy with marrow reinfusion and hyperfractionated irradiation for children with high-risk brain tumors. Med Ped Oncol 1994; 23:428–436. 14. Mahoney DH, Strother D, Camitta B, et al. High dose melphalan and cyclophosphamide with autologous bone marrow rescue for recurrent/progressive malignant brain tumors in children: a pilot pediatric oncology group study. J Clin Onc 1996; 14:382–388.

566

Gardner and Dunkel

15. Graham ML, Herndon JE, Casey JR, et al. High-dose chemotherapy with autologous stem-cell rescue in patients with recurrent and high-risk pediatric brain tumors. J Clin Onc 1997; 15:1814–1823. 16. Finlay JL, Goldman S, Wong MC, et al. Pilot study of high-dose thiotepa and etoposide with autologous bone marrow rescue in children and young adults with recurrent CNS tumors. J Clin Onc 1996; 14:2495–2503. 17. Papadakis V, Dunkel IJ, Cramer LD, et al. High-dose carmustine, thiotepa and etoposide followed by autologous bone marrow rescue for the treatment of high risk central nervous system tumors. Bone Marrow Trans 2000; 26:153–160. 18. Grovas AC, Boyett JM, Lindsley K, Rosenblum M, Yates AJ, Finlay JL. Regimen-related toxicity of myeloablative chemotherapy with BCNU, thiotepa, and etoposide followed by autologous stem cell rescue for children with newly diagnosed glioblastoma multiforme: report from the children’s cancer group. Med Pediatr Oncol 1999; 33:83–87. 19. Bouffet E, Motolese C, Jouvet A, et al. Etoposide and thiotepa followed by ABMT (autologous bone marrow transplantation) in children and young adults with high-grade gliomas. Eur J Cancer 1997; 33:91–95. 20. Farwell JR, Dohrmann GJ, Flannery JT. Central nervous system tumors in children. Cancer 1977; 40:3123–3132. 21. Packer RJ, Vezina G. Pediatric glial neoplasms including brain-stem gliomas. Semin Oncol 1994; 21:260–272. 22. Freeman CR, Krischer JP, Sanford RA, et al. Final results of a study of escalating doses of hyperfractionated radiotherapy in brain stem tumors in children: a pediatric oncology group study. Int J Radiat Oncol Biol Phy 1993; 27:197–206. 23. Packer RJ, Boyett JM, Zimmerman RA, et al. Outcome of children with brain stem gliomas after treatment with 7800 cGy of hyperfractionated radiotherapy: a children’s cancer group phase I/II trial. Cancer 1994; 74:1827–1834. 24. Dunkel IJ, Garvin JH, Goldman S, et al. High dose chemotherapy with autologous bone marrow rescue for children with diffuse pontine brain stem tumors. J Neuro-Onc 1998; 37:67–73. 25. Bouffet E, Raquin M, Doz F, et al. Radiotherapy followed by high dose busulfan and thiotepa: a prospective assessment of high dose chemotherapy in children with diffuse pontine gliomas. Cancer 2000; 88:685–692. 26. Ries LA, Kosary CL, Henkey BF, Miller BA, Edwards BK, editors. SEER Cancer statistics review 1973–1995. Bethesda, (MD): National Cancer Institute; 1998. 27. Evans AE, Anderson JR, Lefkoweitz-Boudreaux IB, et al. Adjuvant chemotherapy of childhood posterior fossa ependymomas: craniospinal irradiation with or without adjuvant CCNU, vincristine, and prednisone: A children’s cancer group study. Med Pediatr Oncol 1996; 27:8–14. 28. Perilongo G, Massimino M, Sotti G, et al. Analyses of prognostic factors in a retrospective review of 92 children with ependymoma: Italian Pediatric Neuro-oncology Group. Med Pediatr Oncol 1997; 29:79–85. 29. Grill J, Kalifa C, Doz F, et al. A high-dose busulfan-thiotepa combination followed by autologous bone marrow transplantation in childhood recurrent ependymoma. Pediatr Neurosurg 1996; 25:7–12. 30. Mason WP, Goldman S, Yates AJ, Boyett J, Li H, Finlay JL. Survival following intensive chemotherapy with bone marrow reconstitution for children with recurrent intracranial ependymoma. J Neurooncol 1998; 37:135–143. 31. Gurney JF, Severson RK, Davis S, Robison LL. Incidence of cancer in children in the United States. Cancer 1995; 75:2186. 32. Young J, Miller R. Incidence of malignant tumors in U.S. children. J Pediatr 1975; 86:254. 33. Jenkin D. The radiation treatment of medulloblastomas. J Neurooncol 1996; 29:45–54. 34. Cohen ME, Duffner PK, eds. Long-term clinical effects. 2nd ed. In: Brain Tumors in Children, New York: Raven Press, 1994:455–481. 35. Chin H, Maruyama Y. Age at treatment and long term performance results in medulloblastoma. Cancer 1984; 53:1952–1958. 36. Duffner PK, Horowitz ME, Krischer JP, et al. Post-operative chemotherapy and delayed radiation in children less than 3 years of age with malignant brain tumors. N Eng J Med 1993; 328:1725–1731. 37. Torres CF, Rebsamen S, Silber JH, et al. Surveillance scanning of children with medulloblastoma. N Engl J Med 1994; 330:892–895. 38. Belza MG, Donaldson SS, Steinberg GK, et al. Medulloblastoma: freedom from relapse longer than 8 years- a therapeutic cure? J Neurosurg 1991; 75:575–582.

Pediatric Malignant Brain Tumors

567

39. Kalifa C, Hartmann O, Demeocq F, et al. High-dose busulfan and thiotepa with autologous bone marrow transplantation in childhood malignant brain tumors: a phase II study. Bone Marrow Trans 1992; 9:227–233. 40. Dunkel IJ, Boyett JM, Yates A, et al. High-dose carboplatin, thiotepa, and etoposide with autologous stem-cell rescue for patients with recurrent medulloblastoma. J Clin Onc 1998; 16:222–228. 41. Vassal G, Tranchand B, Valteau-Couanet D, et al. Pharmacodynamics of tandem high-dose melphalan with peripheral blood stem cell transplantation in children with neuroblastoma and medulloblastoma. Bone Marrow Transplant 2001; 27:471–477. 42. Strother D, Ashley D, Kellie SJ, et al. Feasibility of four consecutive high-dose chemotherapy cycles with stem-cell rescue for patients with newly diagnosed medulloblastoma or supratentorial primitive neuroectodermal tumor after craniospinal radiotherapy: results of a collaborative study. J Clin Onc 2001; 19:2696–2704. 43. Gaffney CC, Sloane JP, Bradley NJ, Bloom HJG. Primitive neuroectodermal tumors of the cerebrum. J Neurooncol 1985; 3:23–33. 44. Pomeroy SL, Tamayo P, Gaasenbeek M, et al. Prediction of central nervous system embryonal tumour outcome based on gene expression. Nature 2002; 415:436–442. 45. Cohen BH, Zeltzer PM, Boyett JM, et al. Prognostic factors and treatment results for supratentorial primitive neuroectodermal tumors in children using radiation and chemotherapy: a children’s cancer group randomized trial. J Clin Oncol 1995; 13:1687–1696. 46. Broniscer A, Nicolaides TP, Dunkel IJ, et al. High-dose chemotherapy with autologous stem cell rescue in the treatment of patients with recurrent non-cerebellar primitive neuroectodermal tumors. Pediatr Blood Cancer 2004; 42:261–267. 47. Gururangan S, McLauglin C, Quinn J, et al. High-dose chemotherapy with autologous stem-cell rescue in children and adults with newly diagnosed pineoblastomas. J Clin Oncol 2003; 21:2187–2191. 48. Heideman RL, Packer RJ, Albright LA, et al. Tumors of the central nervous system. In: Pizzo PA, Poplack DG, eds. Principles and Practice of Pediatric Oncology. 3rd ed. Philadelphia: LippincottRaven Publishers, 1997:674. 49. Shirato H, Nishio M, Sawamura Y, et al. Analysis of long-term treatment of intracranial germinoma. Int J Radiat Oncol Biol Phys 1997; 37:511–515. 50. Modak S, Gardner S, Dunkel IJ, et al. Thiotepa-base high-dose chemotherapy with autologous stemcell rescue in patients with recurrent or progressive CNS germ cell tumors. J Clin Oncol 2004; 22:1934–1943. 51. Bouffet E, Baranzelli MC, Patte C, et al. On behalf of the Societe Francaise d’Oncologie Pediatrique. High dose etoposide and thiotepa for refractory and recurrent malignant intracranial germ cell tumours (CNS-GCT). 9th International Symposium on Pediatric Neuro-Oncology, San Francisco, CA, June 11-14, 2000. 52. Tada T, Takizawa T, Nakazato F, et al. Treatment of intracranial nongerminomatous germ-cell tumor by high-dose chemotherapy and autologous stem-cell rescue. J Neuro-Onc 1999; 44:71–76. 53. Duffner PK, Cohen ME, Myers MH, et al. Survival of children with brain tumors: SEER program, 1973-1980. Neurology 1986; 36:597–601. 54. Farwell JR, Dohrmann GJ, Flannery JT. Intracranial neoplasms in infants. Arch Neurol 1978; 35:533–537. 55. Fessard C. Cerebral tumors in infancy. Am J Dis Child 1968; 115:302–308. 56. Dupuis-Girod S, Hartmann O, Benhamou E, et al. Will high dose chemotherapy followed by autologous bone marrow transplantation supplant cranio-spinal irradiation in young children treated for medulloblastoma? J Neuro-Onc 1996; 27:87–98. 57. Gururangan S, Dunkel IJ, Goldman S, et al. Myeloablative chemotherapy with autologous bone marrow rescue in young children with recurrent malignant brain tumors. J Clin Oncol 1998; 16:2486–2493. 58. Mason WP, Govas A, Halpern S, et al. Intensive chemotherapy and bone marrow rescue for young children with newly diagnosed malignant brain tumors. J Clin Oncol 1998; 16:210–221. 59. Vassal G, Tranchand B, Valteau-Couanet D, et al. Pharmacodynamics of tandem high-dose melphalan with peripheral blood stem cell transplantation in children with neuroblastoma and medulloblastoma. Bone Marrow Transplant 2001; 27:471–477. 60. Gardner S, Finlay J, Cook P, Kushner B, Lis E, Dunkel I. Sequential myeloablative thiotepa with autologous stem cell rescue (ASCR) for patients with recurrent tumors. Proc of ASCO 2003; 22:840.

568

Gardner and Dunkel

61. Ozkaynak MF, Sandoval C, Levendoglu-Tugal O, Jayabose. A pilot trial of tandem autologous peripheral blood progenitor cell transplantation following high-dose thiotepa and carboplatin in children with poor-risk central nervous system tumors. Pediatr Hematol Oncol 2004; 21:635–645. 62. Gilman A, Bunin N, Levine J, et al. Dual cycle high-dose chemotherapy and stem cell rescue for recurrent brain tumors. J Pediatr Hematol Oncol 2000; 22:382. 63. Gardner S, Belasco J, Phillips P, et al. Phase I dose escalation of temozolomide with thiotepa and carboplatin with autologous stem cell rescue (ASCR) in patients with recurrent/refractory central nervous system (CNS) tumors. 64. Gururangan S, Friedman H. Recent advances in the treatment of pediatric brain tumors. Oncology 2004; 18:1649–1661. 65. Jansen M, de Witt Hamer PC, Witmer AN, et al. Current perspective on antiangiogenesis strategies in the treatment of malignant gliomas. Brain Res Rev 2004; 45:143–163.

28 Hematopoietic Stem-Cell Transplantation for Pediatric Solid Tumors ˝ zkaynak M. Fevzi O Pediatric Blood and Marrow Transplantation, Division of Hematology/Oncology, Department of Pediatrics, New York Medical College, Valhalla, New York, U.S.A.

Marcio H. Malogolowkin Bone and Soft Tissue Tumor Program, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A.

EWING’S SARCOMA/PERIPHERAL PRIMITIVE NEUROECTODERMAL TUMORS Ewing’s sarcoma and peripheral primitive neuroectodermal tumors (ES/PNET) arise from bone or soft tissue and affect predominantly children and young adults. They share the same histochemical staining profile and a unique nonrandom chromosome 22 rearrangement. They are grouped as the ES family of tumors. Cure of patients with ES/PNET has improved with multimodality approach, including surgery, radiation, and chemotherapy. Patients with localized ES/PNET will achieve an event-free survival (EFS) of 60–65% with current treatment protocols (1). However, 20–30% of patients with ES/PNET present with metastatic disease. Sites of metastases appear to affect outcome in EWS. Up to one-third of patients whose metastatic disease is limited to pulmonary and/or pleural metastases may become long-term survivors, whereas this rate drops to 20% in patients with bone and/or bone marrow metastases (1). Recurrent ES/PNET carries an even worse prognosis, where the EFS is !10% (2). Because EWS/PNET is sensitive to conventional-dose chemotherapy, high-dose/myeloablative chemoradiotherapy with hematopoietic stem cell transplantation (HSCT) has become an attractive strategy when combined with other conventional multimodal treatment approaches for patients with metastatic ES/PNET either in remission or as part of a relapse regimen.

Ewing’s Sarcoma/Peripheral Primitive Neuroectodermal Tumors in First Remission As previously noted, patients with ES/PNET who present at the time of diagnosis with metastatic disease are considered to be a high-risk group. In addition, patients with bulky disease (O200 ml) are considered to have a worse prognosis than those with smaller primary tumors (!200 ml). Age greater than 15 is also considered to be a poor prognostic factor (1). The difficulty with analyzing published studies using HSCT is the lack of a uniform definition of high-risk ES/PNET and the absence of randomized studies. In addition, 569

570

˝ zkaynak and Malogolowkin O

high-dose/myeloablative chemoradiotherapy regimens have been highly variable. Some studies have reported results on only those patients who have made it to transplant, which makes it very difficult to draw any firm conclusions regarding the role of HSCT. The majority of published series have used autologous HSCT, with very limited data on allogeneic HSCT. Table 1 summarizes the results of the recently published large autologous HSCT reports. Burdach et al. treated seven patients with multifocal EWS with total body irradiation (TBI) (1200 cGy), melphalan (30–45 mg/m2/day for four days) and etoposide (40–60 mg/kg) with or without carboplatin (900–1500 mg/m2) (3). Some of the patients received IL-2 posttransplant. Three of seven patients were in remission at six years. Horowitz et al. treated 61 unselected metastatic or high-risk EWS/PNET patients with a common induction chemotherapy, and then those who achieved a complete remission (CR) became eligible for consolidation with HSCT (4). Twenty percent of patients failed to achieve a remission and were thus were unable to proceed to HSCT. The remaining 80% were conditioned using TBI (800 cGy), vincristine (2 mg/m2), adriamycin (35 mg/m2), and cyclophosphamide (1200 mg/m2). Only 10% of patients with metastases at diagnosis remained in remission at six years. Paulussen et al. reviewed the experience of the German Cooperative Group with megatherapy on 36 unselected metastatic patients (7). The myeloablative regimen varied depending on the judgment of the treating physician but contained melphalan and etoposide C/K carboplatin and C/K TBI. EFS at 4 years was 23% for 36 patients. Two of 36 developed secondary leukemia. Patients who had pulmonary and bony metastases who received megatherapy and TBI had an EFS of 27% at 4 years compared to no survivors in the group that did not undergo megatherapy. To date, one of the largest reported experiences has been derived from the European Bone Marrow Transplant Registry (EBMTR). Ladenstein et al. reported EBMTR results in 1995 with at least six different regimens during the period from 1982–1992 (5). Twenty-one of 32 patients had metastatic bone/bone marrow disease, and transplant was used as consolidation. Six of 21 (28%) were long-term survivors. TBI was associated with worse results. In a preliminary report on EBMTR data covering a longer period (1978–1997) published in 1999, Ladenstein et al. reported better results with busulfan-based regimens: 5-year overall survival (OS) for the whole group (128 patients with metastatic disease) was 30% (9). Patients with lung metastases treated with busulfan-based regimen (12 patients) had a 5-year OS of 66% versus 39% for those patients who were treated with a TBI-based regimen. Patients with non-pulmonary metastases also appeared to have a better outcome with busulfan-based regimens (18 patients), with an OS of 44% at 5 years versus 23% for patients on non-busulfan-based regimens (93 patients; pZ0.061). EFS rates were not given. The major criticism of this report is that it is retrospective, based on selected patients, using a variety of myeloablative regimens. Nevertheless, this report has been the impetus for the ongoing Euro-Ewing 99 protocol, which is the largest prospective trial to attempt to answer the role of a busulfan-based myeloablative regimen with autologous HSCT in the management of EWS/PNET. Others have reported their experience with busulfan-based regimens. Atra et al. reported eleven selected metastatic patients (ten of eleven with isolated pulmonary metastases) who were treated with oral busulfan (16 mg/kg or 600 mg/m2) and melphalan (160 mg/m2 or 140 mg/m2) (6). Six of eleven were event-free at two years. Prete et al. reported their experience with busulfan (16 mg/kg), etoposide (2400 mg/m2) and thiotepa (300 mg/m2) in 17 selected patients with high-risk EWS (17). Three of 17 patients did not have metastases but large primary tumors. The sites of metastatic disease were not reported. Two-year EFS was 63%. Perentesis et al. (10) and Davies et al. (16) also reported their experience with busulfan (12 mg/kg), melphalan (100 mg/m2), and thiotepa (500 mg/m2) in five and ten selected metastatic patients, with an EFS of 60% and 62%, respectively. Similarly, Diaz et al. showed 64% EFS in 11 selected metastatic patients who were treated with oral busulfan (16 mg/kg) and melphalan (140 mg/m2) (11). Other small reports with selected metastatic patients include Ozkaynak et al. (8) and Gamis et al. (15), who reported on five and eight patients in first remission, respectively. Ozkaynak et al. used a regimen of melphalan (200 mg/m2), carboplatin (1200 mg/m2), etoposide (800 mg/m2), and escalating doses of cyclophosphamide. Four of five patients were

1993 1993

1995

1997 1998

1998

1998

1999 1999

1999 2000 2001 2001 2001 2001

Burdach (3) Horowitz (4)

Ladenstein (5)

Atra (6) Paulussen (7)

Ozkaynak (8)

Prete (9)

Perentesis (10) Ladenstein (9)

Diaz (11) Burdach (12) Kushner (13) Meyers (14) Gamis (15) Davies (16)

11 17 21 23 8 10

5 128

17

5

11 36

32

7 61

No. of patients Outcome

Bu/Melp TBI/Melp/Etop C/K Carbo Melp/TBI or Thiotepa/Carbo TBI/Melp/Etop Thio/Etop/Cyclophosphamide Bu/Melp/Thiotepa

Bu/Melp/Thiotepa Various

Melp/Etop/CarboC/K Cyclophosphamide Bu/Thiotepa/Etop

3/5 EFS at 3 years 30% OS at 5 years (all sites) 44% OS at 5 years (Bu-based only); 23% OS at 5 years (non-Bu based regimens) 64% EFS at 4 years 25% EFS at 7 years 5% EFS at 3 years 24% EFS 2 years 17% EFS at 2 years 62% EFS at 3 years

63% EFS at 2 years

4/5 EFS at 3 years

Etop/Melp/TBI C/K Carbo, IL-2 3/7 EFS at 6 years TBI/Vincristine/Cyclosphosphamide/ 14% EFS for high-risk, 10% EFS for Doxorubicin metastatic patients at 6 years Melp. C other agents C/K TBI 21% EFS at 5 years; 6/21 with bone C/K BM (C) EFS at 3 years Bu/Melp 6/11 EFS at 2 years Melp/Etop C/K Carbo C/K TBI 23% EFS at 4 years (all); 27% EFS for lung/bone mets (C) pts

Regimen

Abbreviations: TBI, total body irradiation; EFS, event-free survival; HSCT, hematopoietic stem cell transplantation; OS, overall survival.

Year

Study

Yes, but select group No No No No Yes, but select group

Yes, but 3/17 had no mets but large pelvic tumor; sites of mets not given May be yes but select group Yes, Bu-based regimens

Yes, but 10/11 had lung mets only Yes for lung/bone (C) pts (27% for lung/bone versus 0% with no HSCT) May be yes, but select group

May be yes for Bu/Melp group

Yes No

Benefit

Table 1 High-Dose Therapy with Autologous Hematopoietic Stem-Cell Transplantation for High-Risk Ewing’s Sarcoma and Peripheral Primitive Neuroectodermal Tumors in First Remission

HSCT for Pediatric Solid Tumors 571

572

˝ zkaynak and Malogolowkin O

event-free survivors at three years. Gamis et al. administered a preparative regimen of thiotepa (900 mg/m2) etoposide (1500 mg/m2), and cyclophosphamide (200 mg/kg) with an EFS of 17% at 2 years (15). Burdach et al. updated their experience from the European Intergroup Study (EICESS) (12). Seventeen unselected metastatic patients were treated with TBI (1200 cGy), melphalan (120–180 mg/m2), and etoposide (40–60 mg/kg). In 28% of the patients, carboplatin (300–1000 mg/m2) was added. EFS was 25% at seven years. Two other prospective studies with unselected patients were recently reported. Kushner et al. reported the Memorial Sloan-Kettering Cancer Center (MSKCC) experience in 21 consecutive patients treated either with TBI (1500 cGy) and melphalan (180 mg/m2) or thiotepa (900 mg/m2) and carboplatin (1500 mg/m2) (13). EFS at five years was only 5%. Meyers et al. reported their experience in the Children’s Cancer Group, where 23 unselected patients were treated with a uniform induction regimen followed with a consolidation regimen of TBI (1200 cGy), melphalan (180 mg/m2), and etoposide (750 mg/m2) (14). EFS was 24% at 2 years. In summary, the results of high-dose chemotherapy with autologous HSCT in first remission have been disappointing. The only exception is the European data using busulfanbased conditioning regimens. Until the result of the Euro-Ewing 99 study is available, HSCT in patients in first remission remains an investigational approach. Allogeneic HSCT has also been investigated for EWS patients in first remission. Burdach et al. treated ten selected patients with TBI (1200 cGy), melphalan (120–180 mg/m2), and etoposide (40–60 mg/kg)C/K carboplatin (300–1000 mg/m2) (12). EFS was 20% at 7 years. Burdach compared the allogeneic experience with the autologous transplant patients with similar disease characteristics. Relapse rate was 40% in allogeneic and 39% in autologous transplant patients, arguing against the presence of any graft-versus-tumor effect. Toxic mortality was high at 40% for allogeneic patients, whereas it was 19% among autologous transplant patients. In short, based on experience with a very limited number of patients, allogeneic transplant did not seem to offer any benefit, either from an OS standpoint or from a decreased relapse rate. Because results with single HSCT have been disappointing, investigators are exploring the possibility of tandem transplants (18–21). Results of four published tandem transplant reports are summarized in Table 2. Because three of four reports (18–20) are pilot trials of the feasibility of the approach, with patients at varying stages of disease, no conclusions can be drawn on the role of tandem transplants in first remission patients.

Ewing’s Sarcoma/Peripheral Primitive Neuroectodermal Tumors in Second or Later Remission As in many diseases, patients who relapse early (defined as occurring within 24 months of diagnosis) have a worse prognosis than those who relapse at a later time (1). Patients who experienced an early relapse had a 4–8.5% 5-year survival as compared with a 23–35% 5-year survival in those who relapsed late (2,22). A recent publication from St. Jude Children’s Research Hospital reported on the outcome of 71 unselected patients who were treated for recurrent EWS/PNET (2). None of the patients received high-dose chemotherapy with autologous HSCT as part of salvage therapy. Recurrence R2 years after diagnosis predicted a better outcome (five years EFS 34.9%) compared with earlier recurrence (5%). Most importantly, this report can be used as a historical control in trials of autologous HSCT without a nontransplant arm. Table 3 summarizes the published results of several studies. Unfortunately, most of these studies suffer from small sample size and the lack of stratification for early-versus-late relapse. The best report is by Shankar et al. who followed the outcome of 64 relapsed patients (2). Only 7 of 64 patients made it to the transplant. Two of seven are event-free survivors at 15 and 24 months, following autologous HSCT with melphalan C/K TBI. OS for the whole group

1997

2000

2003 2005

Chan (18)

Hawkins (19)

Burdach (20) Barker (21)

4 of 13 single

28 13 of 55, 9 of 13 tandem

16

6

No. of patients

Abbreviations: EFS, event-free survival; NED, no evidence of disease.

Year

Study 1st Cyclophosphamide 2nd Thiotepa/Melp 1st Bu/Melp/Thiotepa 2nd total marrow irradiation 1st: Melp/Etop 2nd:Melp/Etop 1st Bu/Melp/Thiotepa 2nd total marrow Irradiation

Regimen

2/4 EFS

29% EFS at 68 months 6/9 EFS

36% EFS at 3 years

1/6 EFS at 3 years

Outcome

Yes, 8/12 in 2nd remission NED, but select group Unclear, select group Yes, but retrospective study, possible selection bias

No

Benefit

Table 2 Tandem High-Dose Therapy with Autologous Hematopoietic Stem-Cell Transplantation for High-Risk Ewing’s Sarcoma and Peripheral Primitive Neuroectodermal Tumors in First and Subsequent Remissions

HSCT for Pediatric Solid Tumors 573

1993

1998

1999

2000

2001

2003

Burdach (3)

˝ zkaynak O (8)

Perentesis (10)

Burdach (12)

Gamis (15)

Shankar (2)

Thiotepa/Etop Cyclophosphamide

TBI/Melp/Etop C/K Carbo

Bu/Melp/Thiotepa

Melp/Carbo/EtopC/K Cyclophosphamide

Etop/Melp/TBI C/K Carbo, IL-2

Regimen

Melp C/K TBI 7 of 64 unselected relapsed patients

8

19

5

9

10

No. of patients

Abbreviations: TBI, total body irradiation; EFS, event-free survival; OS, overall survival.

Year

Study

2/7 EFS at 15&42 months; OS 8% at 5 years for the 64 patients

21% EFS at 2 years

25% EFS at 7 years

2/5 EFS at 3 years

5/10 EFS follow-up not specified, one patient O6 years EFS 3/9 EFS at 3 years

Outcome

Yes, 6/10 relapsed !2 years from diagnosis Yes, but select group and no distinction between early-versuslate relapse Yes, but select group and no distinction between early-versuslate relapse Yes, but select group and no distinction between early-versuslate relapse Yes, but select group and no distinction between early-versuslate relapse Only 7/64 made it to transplant; may be yes for those who made it to transplant

Benefit

Table 3 High-Dose Therapy with Autologous Hematopoietic Stem-Cell Transplantation for High-Risk Ewing’s Sarcoma and Peripheral Primitive Neuroectodermal Tumors in Second or Subsequent Remissions

574 ˝ zkaynak and Malogolowkin O

HSCT for Pediatric Solid Tumors

575

was only 8% at five years. As reported earlier, patients who had a longer disease-free interval (DFI) prior to relapse had a better outcome (DFI ! one year, median survival of three months; DFI between one and two years, median survival of eight months; DFI O two years, median survival of 24 months). Similar to high-risk EWS in first remission, investigators are exploring the tolerability of tandem transplants in relapsed patients (18–21). Results of the four published tandem transplant are summarized in Table 2. As stated previously, because the first three reports (18–20) were feasibility trials, mixing patients at first and subsequent remissions, no conclusion can be drawn as to its efficacy in relapsed patients. Recently, Barker et al. reported the results of their retrospective review on 55 consecutive patients with relapsed EWS followed at their center (21). This report includes and expands on the 12 patients reported earlier by Hawkins et al. from the same center (19). Thirteen of 55 patients received HSCT. The initial clinical features of the 13 patients who received HSCT were similar to the whole study population. Twelve of 13 received autologous HSCT, whereas one patient underwent syngeneic HSCT. Nine of thirteen patients had tandem HSCT with the second HSCT being total marrow irradiation after recovery from the first HSCT with Busulfan/Melphalan/Thiotepa. Six of nine patients who had tandem HSCT are alive without recurrence. Two of four patients who received Busulfan/ Melphalan/ Thiotepa are enjoying relapse-free survival. Because all 13 patients who received HSCT also had chemoresponsive disease, a multivariate analysis was performed. HSCT as consolidation for relapsed EWS seemed to be associated with improved OS even after adjusting for earlyversus-late relapse (late O24 months) and response to second-line treatment. Although promising, this study also suffers from retrospective nature and possible selection bias for HSCT. In summary, current data makes it very difficult to establish the role of HSCT in relapsed EWS patients because most studies are small and on select patient populations. In addition, many of the studies lack stratification for early-versus-late relapse, which is critical for the correct interpretation of the results. Nevertheless, if busulfan-based regimens prove to be beneficial in first remission patients, the same regimen will likely be studied in relapsed patients either as single transplant or as part of a tandem transplant regimen. At this time, HSCT in relapsed EWS patients remains an investigational approach. Several investigators are exploring the role of allogeneic HSCT to exploit the graftversus-tumor effect if it exists—both in Europe and U.S.A. No results are available from these ongoing trials at this time.

Minimal Residual Disease Evaluation/Stem Cell Tumor Contamination There are several published studies addressing the issue of tumor cell contamination in stem cell collections. Fischmeister et al. applied reverse transcriptase polymerase chain reaction (RT-PCR) to evaluate the frequency of tumor cells in peripheral blood stem cell collection (PBSC) from 15 high-risk EWS patients who were treated according to EICESS 92 protocol (28). Tumor cell contamination of bone marrow detected by light microscopy was found in five cases and by RT-PCR in an additional eight cases. RT-PCR was serially performed on each PBSC sample at a sensitivity comparable to 20–100 EWS-Fli1 expressing tumor cells per ten ml of blood. Irrespective of whether or not bone marrow involvement was noted at diagnosis, all marrow samples obtained immediately prior to the harvest (which was after a mean of four cycles of chemotherapy) were RT-PCR negative. Thirty-five PBSC products were analyzed, and 12 were found to be RT-PCR positive on one occasion; however, only 1 sample was reproducibly positive for tumor contamination in independent determinations. In a similar report, Thomson et al. studied twelve patients [nine EWS, three alveolar rhabdomyosarcoma (RMS)] with RT-PCR performed on pretherapy bone marrow and peripheral blood samples, as well as repeat sampling at various times during chemotherapy treatment (29). In all patients, clearance of tumor cells as assessed by RT-PCR was documented

576

˝ zkaynak and Malogolowkin O

in peripheral blood and bone marrow by week nine of treatment. Tumor cell contamination was detected in 1/40 PBSC collections from 12 patients. In contrast to these two studies, Leung et al. and Yaniv et al. reported a higher rate of tumor contamination of the hematopoietic grafts by RT-PCR (30,31). Leung et al. studied five patients with EWS (30). For patients with bone marrow metastases, they only collected PBSC and bone marrow after their marrows were histologically negative for tumor following chemotherapy. They found that all five of the hematopoietic grafts, either PBSC or marrow or both, were positive for tumor contamination by RT-PCR for PGP 9.5, which has been shown to have a similar sensitivity to that of the RT-PCR assay for t(16,22) (q24;q12). Two of five patients did not make it to transplant. All three who did had gross residual disease. Yaniv et al. studied 27 harvests from 11 patients with EWS (31). All 11 patients had contaminating tumor cells in their hematopoietic grafts detected by quantitative RT-PCR for the EWS-Fli-1 fusion transcript. There may be several explanations for these conflicting results. First, the number of hematopoietic grafts studied in each report is very small, with the ensuing possibility of statistical variability. Second, the patient populations studied may be different with different chemotherapy treatment and mobilization protocols. Finally, the sensitivity of the RT-PCR tests may be different. Nevertheless, based on the above few reports with their small sample sizes, no conclusion can be reached with respect to the actual incidence of tumor contamination of hematopoietic grafts in EWS. There does not seem to be a consensus on how to purge these cells when they are detected. Hence, purging of PBSC and/or bone marrow has not been considered in most of the studies. Leung et al. performed immunomagnetic CD34C cell selection and documented depletion of tumor cells by a median of 3.0 logs for PBSC, and 2.6 logs for bone marrow harvests. Because only one of three patients who underwent transplant remained progression-free O20 months, it is impossible to assess the role of this method at this time.

WILMS TUMOR The outlook for children with Wilms’ tumors (WT) has improved dramatically with the advent of multimodal therapy, and survival rates currently approach 90% (32–34). Although the overall relapse rate for children with WT has decreased to less than 15%, the long-term survival for patients with recurrent disease remains less than 30% (35,36). Factors associated with a favorable outcome after relapse include initial stage I or II disease, treatment with vincristine and dactinomycin only, no prior radiotherapy, favorable histology, and relapse more than six months after initial diagnosis. All other patients have a poor outcome and a high risk of treatment failure (35,37). The poor outcome of these patients with recurrent WT has led to the investigation of the role of ifosfamide, etoposide, and platinum agents as single agents or in combination for the treatment of these patients (38–47). These studies have demonstrated response rates greater than 40%; however, the responses are transitory and the outcome has continued to be poor. High-dose chemotherapy followed by autologous stem-cell rescue has been used for the treatment of small numbers of patients with high-risk recurrent WT and preliminary results are encouraging (48,49). In a report for the European Bone Marrow transplantation (EBMT) Registry for Solid Tumors, Garaventa et al. reported on 25 children with resistant or relapsed WT who underwent treatment with high-dose chemotherapy followed by autologous HSCT between 1984 and 1991 (50). Although seven different high-dose chemotherapy regimens were used, most of them included high-dose melphalan. Of the 17 children treated after achieving CR with salvage therapy, eight remained in CR for a median of 34 months (range: 14 to 90 months). However, only one of the eight patients treated with measurable disease at the time of transplant remained

HSCT for Pediatric Solid Tumors

577

alive and free of disease at time of this report. Overall, this treatment was well tolerated, despite three early deaths as a result of pneumonitis. The authors concluded that the use of high-dose chemotherapy followed by autologous bone marrow transplant could be useful for relapsed patients in second CR. In a report for the French Society of Pediatric Oncology, Pein et al. reported on 29 patients with high-risk recurrent WT who received treatment with high-dose chemotherapy followed by autologous HSCT between 1988 and 1994 (51). All patients had chemosensitive disease defined as having achieved a complete or partial response with salvage chemotherapy. Twenty of the patients were entered onto this study after their first recurrence. Consolidation chemotherapy consisted of melphalan, etoposide, and carboplatin. Despite significant treatment related toxicity, 12 patients remain in continuous CR at a median of 48.5 months (range: 36 to 96 months); the disease-free survival (DFS) and OS at three years were 50G17% and 60G18%, respectively. Patients treated after their first recurrence (second CR or PR) had a significantly better outcome than the patients treated after their second recurrence, with three-year DFS of 63G20% versus 22G24%. From April 1992 to December 1998, 23 patients with relapsed WT received high-dose chemotherapy followed by autologous HSCT as part of the German Cooperative Wilms’ Tumor Study, as reported by Kremens et al. (52). Chemotherapy consisted of melphalan, etoposide, and carboplatin, similar to the French study previously mentioned. Ten of 13 patients transplanted in CR remained alive and free of disease, whereas only 2 of the 10 patients transplanted with a partial response remained alive and free of disease. After a median followup of 58 months (range: 37 to 116 months) the EFS is 48G14%, and the OS was 61G10%. Because the high-dose chemotherapy was given after unilateral nephrectomy in the majority of children entered into the French and German studies, the dose of carboplatin was adjusted to the individual glomerular filtration rate based on the Calvert formula (53). It is also important to note that in both the French and German studies, most of the survivors were children whose recurrent disease involved only the lungs, had chemosensitive disease, and were transplanted in complete response. Tannous et al. treated 66 patients with high-risk relapsed WT with chemotherapy consisting of two cycles cyclophosphamide and etoposide (CE) followed by two cycles of carboplatin and etoposide (PE). Patients who achieved a complete response received maintenance therapy with five cycles of CE alternating with PE, whereas those with partial response or stable disease received myeloablative chemotherapy followed by autologous HSCT. The three-year event-free survivals were 59G9% and 40G14% for the maintenance and HSCT subgroups respectively, whereas the three-year OSs were 64G8% and 42G14%, respectively (54). The major criticism of this study is that the complete responders were all nonrandomly assigned to the nontransplant group, thus potentially biasing the study against the role of HSCT in recurrent WT. Analysis of these studies suggests that high-dose chemotherapy followed by autologous HSCT may be beneficial for patients with recurrent WT who show a good response to reinduction therapy. Intensive chemotherapy trials that do not require HSCT have also shown positive results. Studies conducted by CCG and POG demonstrated that therapy with ifosfamide, carboplatin, and etoposide (ICE) is an effective retrieval strategy for children with advanced or relapsed WT. The response rate (CR C PR) to ICE was 91% and 71%, respectively (55,56). Similarly, Malogolowkin et al. treated 27 children with recurrent WT with alternating cycles of carboplatin/etoposide and ifosfamide/doxorubicin. This study demonstrated that the use of intensive, nontransplant, chemotherapy was safe and effective for the treatment of these children. The EFS and OS on this study were 58% at three years (57). In a review of 54 patients with recurrent WT who were treated according to consecutive nontransplant clinical trials at St. Jude Children’s Research Hospital between 1969 and 2000, Dome et al. reported a five-years OS of 48G16% for those patients with high-risk clinical features treated after 1984 (58).

578

˝ zkaynak and Malogolowkin O

The results of these intensive conventional chemotherapy regimens are not significantly different than the survival rates obtained in the studies using high-dose chemotherapy followed by HSCT described above. Therefore, a prospective study comparing the efficacy of intensive conventional chemotherapy to high-dose chemotherapy/HSCT will be necessary for definitive answers. Because patients with recurrent Wilms tumor are rare, an international study may be required to determine the comparative benefit of these two treatment strategies.

RHABDOMYOSARCOMA (RMS) The use of a multidisciplinary approach to the treatment of RMS has resulted in a significant improvement in survival rates, from approximately 25% in 1970 to greater than 70% with current therapies (59–62). However, the results continue to be dismal for patients with metastatic or recurrent disease, with survival rates between 20% to 30% (63,64). RMS is an extremely chemosensitive tumor, and RMS cells have been shown to be sensitive to the dose intensification of alkylating agents in vitro and in xenograft models (65,66). Therefore, the use of high-dose chemotherapy followed by HSCT is a very attractive strategy for the treatment of these high-risk patients. The use of high-dose chemotherapy followed by HSCT has been tested in multiple small studies. One of the first reports was published by Pinkerton et al. in 1991 and described the experience of the Royal Marsden Hospital. Forty-three patients with advanced stage RMS were treated with eight weeks of VAC, followed by high-dose melphalan and autologous bone marrow transplant. The three-year survival rate for patients with metastatic disease was 25% (67). Thirty-six patients with primary metastatic or recurrent RMS registered with the German/Austrian Pediatric Bone Marrow Transplantation Group were treated with high-dose chemotherapy consisting of melphalan, etoposide, and carboplatin with or without TBI, followed by HSCT between 1986 and 1994. Alveolar histology was present in 22 patients; embryonal in 13; and 1 patient had an undifferentiated sarcoma. Twenty-seven patients had primary metastatic, and nine recurrent disease. After a median follow-up of 57 months (range: 32 to 108 months), nine patients were alive and free of disease; four had relapsed disease; and five had primary metastatic disease (68). Boulad et al. reported on their experience treating 26 patients (21 with RMS, 3 with undifferentiated sarcoma, and 2 with extraosseous EWS) with a short course of multiagent chemotherapy, hyperfractionated radiotherapy, and surgery, followed by consolidation chemotherapy with high-dose chemotherapy and autologous HSCT. At the end of induction therapy 19 patients had achieved a complete or good partial response and proceeded to receive consolidation therapy and HSCT. After a median follow-up of 18.5 months (range: 2.8 to 54 months), 10 patients continued alive and free of disease. The two-year OS was 56% (69). Carli et al. reported on 52 patients with metastatic RMS treated according to the European Collaborative MMT4-91 trial. This prospective nonrandomized study evaluated the potential benefit of high-dose melphalan, followed by HSCT as consolidation for children in first CR after six courses of chemotherapy. The outcome of this group of patients was compared with 44 patients who were also in CR after identical chemotherapy but received further treatment with standard chemotherapy. The three-year EFS and OS rates were 30% and 40%, respectively, for those patients receiving consolidation with HSCT as compared to 19% and 28%, respectively, for those receiving standard chemotherapy. This difference was not statistically significant, and the authors concluded that their results did not support the use of HSCT for the treatment of patients with metastatic RMS (70). Malogolowkin et al. treated 23 patients with primary metastatic RMS with intensive chemoradiotherapy, followed by consolidation with high-dose melphalan, carboplatin, and etoposide followed by HSCT. After a mean follow-up of 38 months (range: 28 to 60 months), 6 of the 14 patients who underwent consolidation followed by HSCT are alive and free of disease. The two-year EFS was 44G12% (71).

HSCT for Pediatric Solid Tumors

579

Weigel et al. have published the results of 389 patients who underwent high-dose chemotherapy with HSCT identified by the meta-analysis of 22 published articles selected by a MEDLINE search between 1979 and 2000. The EFS at three to six years for those patients treated during first complete or partial remission ranged from 24% to 29%, and the OS at two to six years ranged from 20% to 40%. Patients with recurrent disease treated in their second or third CR experienced a much worse outcome with an estimated 3-year OS of 12%. Based on their analysis, the authors concluded that the use of high-dose chemotherapy followed by HSCT does not improve the outcome of patients with metastatic or recurrent RMS (72). Hara et al. reported on the use of a double-conditioning regimen with thiotepa, melphalan, and busulfan with HSCT for the treatment of children with solid tumors. Overall, this regimen was tolerable, with mucositis being the single most significant toxicity observed. Seven patients with RMS were treated on this study, four are alive and disease free from 15 to 32 months. Although, this is an encouraging result, because of the short follow-up period and the small number of patients, no conclusion can be drawn from this report (73). Further studies are necessary to determine if this approach improves the OS of patients with metastatic or recurrent RMS.

HEPATOBLASTOMA Primary tumors of the liver in infants and children are rare, comprising only 1.1% of malignancies for children younger than 20 years of age (74–76). In the United States, 100–150 children younger than 20 years are diagnosed with malignant hepatic tumors each year. Hepatoblastoma accounts for 66% of these tumors and hepatocellular carcinoma for most of the remainder. Cure of children with liver tumors is only possible when complete surgical excision is achieved. Unfortunately, complete excision is feasible in less than 60% of patients with hepatoblastoma at the time of diagnosis. Chemotherapy has become an important part of the therapy for patients with hepatic tumors. It has been used as adjuvant therapy for patients who undergo complete tumor resection at the time of diagnosis or to induce tumor shrinkage preoperatively in those tumors considered either unresectable or an extreme surgical hazard. Cisplatin based chemotherapeutic regimens have been used preoperatively in children with unresectable hepatoblastoma allowing for tumor resectability in more than 75% of patients with an OS of approximately 65% (77,78). However, for those patients who continue to have unresectable tumors after induction chemotherapy and for patients with primary metastatic disease, or for those with recurrent disease, survival rates continue to be dismal, with rates less than 20%. High-dose chemotherapy followed by HSCT has been reported in eight patients with hepatoblastoma. Yoshinari et al. reported on one patient with microscopic residual disease who has remained disease-free post high-dose chemotherapy and HSCT (79). Hara et al. reported on the use of a double-conditioning regimen with thiotepa, melphalan, and busulfan with HSCT for the treatment of children with solid tumors, including four children with hepatoblastoma. One patient with recurrent disease had a partial response to the high-dose chemotherapy and eventually died of disease; the other three patients had complete surgical excision of the tumor prior to high-dose therapy and have remained free of disease between 19 and 50 months (73). Katzenstein et al. reported on the use of high-dose chemotherapy followed by HSCT for three patients with hepatoblastoma (two with primary metastatic disease and one with recurrent disease). All three patients eventually experienced recurrences (80). Because the four patients with hepatoblastoma who survived after high-dose chemotherapy with HSCT had either microscopic residual disease or no tumor at the time of treatment, the role of this therapeutic approach for these patients remains unclear. However, the results suggest that high-dose chemotherapy followed by HSCT for patients with high-risk hepatoblastoma (primary metastatic or recurrent disease) should only be used in the context of a research study.

580

˝ zkaynak and Malogolowkin O

OSTEOSARCOMA The experience with autologous HSCT in patients with poor-risk osteosarcoma (OST) is very limited. The outcome of children and adolescents with OST has improved dramatically over the past two to three decades. However, the improvement has been mainly in patients with localized disease at favorable sites. Patients with metastatic disease at diagnosis and/or patients with larger tumors or tumors of the axial skeleton and patients who experience metastatic relapse after initial therapy have very poor prognosis. Increasing aggressiveness of chemotherapy combined with radical surgery has been responsible for the improved results seen in nonmetastatic patients, may be indicating a dose-response relationship. Therefore, high-dose chemotherapy with autologous HSCT seems to be a logical next step in the treatment of poor-risk OST patients. However, agents that are widely used and accepted as being efficacious in OST patients, such as doxorubicin, methotrexate, cisplatinum, and ifosfamide, all have unacceptable nonhematologic toxicities when used in high doses with HSCT. Hence, the main obstacle seems to be identifying agents with activity against OST that can be dose escalated with HSCT. Published reports of autologous HSCT in OST have been with the use high-dose melphalan, thiotepa, cyclophosphamide, etoposide, and carboplatin in various combinations. Table 4 summarizes the largest series of autologous HSCT in OST (23–27). There are no randomized trials. Fagioli et al. treated 32 patients with relapsed metastatic OST with two cycles of high-dose carboplatin and etoposide followed with HSCT (26). The OS and the disease-free survival at three years were 20% and 12%, respectively. Although the remission rates were high post HSCT, the duration of these remissions were very short, with a relapse rate of 85%. In the second largest study, Sauerbrey et al. reported on 15 children with relapsed OST, and a significant survival benefit could not be demonstrated—2/15 in continuous CR— compared to historical controls treated with conventional chemotherapy (25). All these trials have involved such small numbers of patients, which were at times not even uniformly treated, that it is very difficult to evaluate or assess the role of HSCT in poor-risk OST patients. Novel approaches are needed, such as the use of bone-seeking radiopharmaceutical samarium-153, which provides irradiation to osteoblastic bone metastases. In one recent phase I trial, escalating doses of samarium was administered with autologous HSCT (81). This agent has a dose-limiting toxicity of myelosuppression and has been approved for the palliative treatment of painful bone lesions. Anderson et al. has shown that reduction or elimination of opiates for pain is possible with high-dose samarium in patients with OST (81). Combination of samarium with high-dose chemotherapy with autologous HSCT may be attractive regimen to study in poor-risk OST patients.

EXTRACRANIAL GERM CELL TUMORS High-dose chemotherapy with autologous HSCT has been investigated mostly in adults either as salvage or first-line treatment. Most adult and pediatric patients with advanced extracranial germ cell tumors (GCT) are cured with cisplatin-based chemotherapy with or without surgical resection. However, 20–25% of patients with poor-risk GCT either relapse after initial complete response or fail to respond adequately to the first-line therapy. The definition of poor-risk GCT patients is: nonseminomatous GCT patients with mediastinal primary tumor or nonpulmonary visceral metastases or serum beta-human chorionic gonadotropin O50,000 IU/L or serum alphafetoprotein O10,000 ng/ml or LDH O10! upper limit of normal. In an attempt to improve the results, several studies have been performed in adults with high-dose chemotherapy with autologous HSCT. These can be divided into two groups: HSCT as salvage treatment or HSCT as part of the initial treatment of poor-risk GCT.

HSCT for Pediatric Solid Tumors

581

Table 4 High-Dose Chemotherapy with Autologous Hematopoietic Stem-Cell Transplantation in Osteosarcoma Author/year Miniero, 1998 (23)

Eligibility and number of patients 4 metastatic relapse patient

Regimen Carboplatin-etoposide

2 metastatic patient at diagnosis

Lucidarme, 1998 (24)

5/7 progressive disease

Thiotepa

2/7 in 1st PR

Sauerbrey, 2001 (25)

15 relapsed (8 after 1st, 6 after Melphalan, etoposide 2nd, 1 after 6th relapse) carboplatin, thiotepa C/K others

Fagioli, 2002 (26)

32 patients with metastatic relapse

Kasper, 2004 (27)

6 patients—3 in PD

6/15 had tandem (Double) transplants Carboplatin-etoposide (tandem): 28/32 had double, 4/32 single Melphalan C others

Comments 2/4 metastatic relapse alive and NED 1/4 metastatic relapse alive with PR 2/2 metastatic at diagnosis alive and NED 4/5 achieved PR 2/2 in PR remained with stable disease 1/7 had PD 2/15 in continuous complete remission

DFS 12% at 3 years, OS 20% at 3 years 1/6 alive and NED

2 in PR, 1 in CR—1 versus subsequent remission status unclear Abbreviations: NED, no evidence of disease; PR, partial remission; PD, progressive disease; DFS, disease-free survival; OS, overall survival.

Hematopoietic Stem-Cell Transplantation as Salvage Treatment in Adult Germ Cell Tumors The initial studies performed in the 1980s from Indiana University reported 3 patients remaining in continuous CR out of 33 with refractory GCT who underwent double autologous HSCT following high-dose carboplatin (doses ranged from 900 mg/m2 to 2000 mg/m2) and etoposide (1200 mg/m2) (82). A subsequent report from the same institution showed 52% EFS among 25 testicular GCT patients who had relapsed after one platinum-based regimen (83). They received one to two cycles of conventional dose salvage therapy, followed by tandem HSCT with carboplatin (2100 mg/m2) and etoposide (2250 mg/m2). Bhatia reported an additional 65 patients with relapsed testicular GCT, also from Indiana University, who received the same conditioning regimen, followed by HSCT with a 57% EFS (84). The EBMT reported their experience in 59 patients with relapsed mediastinal and retroperitoneal primary nonseminomatous GCT treated with high-dose carboplatin, etoposide C/K other agents (85). The

582

˝ zkaynak and Malogolowkin O

EFS for the whole group was 32%. None of these studies were randomized trials, and not all were prospective. European investigators have reported the only randomized study (86). Between 1994 and 2001, 280 poor-risk GCT patients who failed first-line platinum containing chemotherapy—relapsed or refractory—were randomly assigned to receive either four cycles of ifosfamide, cisplatin, and either etoposide or vinblastine or three cycles of the same regimen, followed by HSCT with renally adjusted high-dose carboplatin, etoposide (1800 mg/m2) and cyclophosphamide (6400 mg/m2) (CarboPEC). No significant improvement was observed with CarboPEC: 3-years EFS 42% with CarboPEC versus 35% with standard chemotherapy (PZ0.16). These results suggest that data from uncontrolled studies should be approached cautiously.

Hematopoietic Stem-Cell Transplantation as First-Line Treatment for Poor-Risk Adult Germ Cell Tumors Based on the early promising results from uncontrolled trials in relapsed or refractory patients, several investigators initiated studies where autologous HSCT was utilized in poor-risk GCT patients as part of the first-line treatment. Motzer et al. from MSKCC reported their experience in 28 previously untreated poor-risk GCT patients who received two cycles of VAB-6 (actinomycin-D, vinblastine, cyclophosphamide, bleomycin, and cisplatin), followed by autologous HSCT with carboplatin (1500 mg/m2) and etoposide (1200 mg/m2). The EFS at 3 years was 50% (87). The subsequent MSKCC experience was summarized by Morris and Bosl (88). Cyclophosphamide was added to carboplatin/etoposide and again an EFS of 50% was observed. This was considered an improvement compared with historical controls that underwent standard dose first-line chemotherapy, which resulted in an EFS of 17–31% at MSKCC. Decatris et al. reported the British experience in 20 poor-risk patients who received a standard induction with bleomycin, etoposide, and cisplatin, followed by an autologous HSCT with carboplatin (1800 mg/m2), etoposide (1800 mg/m2) and cyclophosphamide (140 mg/kg). The EFS at 4 years was 50% (89). Schmoll et al. from Germany has shown that the above results were reproducible and can be improved upon with sequential HSCTs (90). Two hundred and twenty-one poor-risk patients received one cycle of standard dose cisplatin, ifosfamide, and etoposide (VIP), followed by three to four sequential cycles of high-dose VIP with HSCT every three weeks, at six consecutive dose levels. Dose limiting toxicity occurred at level eight (cisplatin 100 mg/m2, etoposide 1750 mg/m2 and ifosfamide 12 g/m2). The EFS at five years was 68%. Rosti et al. reported the EBMT experience in 22 extragonadal poor-risk GCT patients who underwent HSCT following high-dose carboplatin, etoposide, and cyclophosphamide. The EFS at 50 months was 68% (91). Despite what appears to be improved EFS with HSCT single or tandem as part of the first-line therapy, it is obvious that historical controls cannot provide the definitive proof of the superiority of this approach. Several randomized prospective trials are ongoing in U.S.A. and Europe, which should be helpful in clarifying the role of HSCT in this setting.

Hematopoietic Stem-Cell Transplantation in Pediatric Germ Cell Tumors The adult HSCT experience dwarfs the pediatric one. The only report worth mentioning is from Europe. De Giorgi et al. reported the EBMT salvage high-dose chemotherapy/HSCT experience in 14 children with extragonadal, extracranial GCT, median age 12 years (range 1–20). Because this was a retrospective analysis, there was no uniformity in high-dose chemotherapy regimen. Regimens include Carboplatin/Etoposide/Cyclophosphamide, Carboplatin/Etoposide, Thiotepa/Etoposide and others. Eight of 14 patients (57%) remain disease-free at 66 months (92). In summary, high-dose chemotherapy with HSCT is a viable treatment option for adult and pediatric patients with either recurrent or refractory GCT or as first-line in patients with poor-risk features. The results of ongoing randomized phase III trials in adults are eagerly

HSCT for Pediatric Solid Tumors

583

awaited. Similar studies in pediatric and adolescent populations may be difficult to conduct given the large numbers required for randomization.

REFERENCES 1. Cotterill SJ, Ahrens S, Paulussen M, et al. Prognostic factors in Ewing’s tumor of bone: analysis of 975 patients from the European intergroup cooperative Ewing’s sarcoma study group. J Clin Oncol 2000; 18:3108–3114. 2. Shankar AG, Ashley S, Craft AW, et al. Outcome after relapse in an unselected cohort of children and adolescents with Ewing sarcoma. Med Pediatr Oncol 2003; 40:141–147. 3. Burdach S, Jurgens H, Peters C, et al. Myeloablative radiochemotherapy and hematopoietic stem cell rescue in poor-prognosis Ewing’s sarcoma. J Clin Oncol 1993; 11:1482–1488. 4. Horowitz ME, Kinsella TJ, Wexler LH, et al. Total body irradiation and autologous bone marrow transplant in the treatment of high-risk Ewing’s sarcoma and rhabdomyosarcoma. J Clin Oncol 1993; 11:1911–1918. 5. Ladenstein R, Lasset C, Pinkerton R, et al. Impact of megatherapy in children with high-risk Ewing’s tumors in complete remission: a report from the EBMT solid tumor registry. Bone Marrow Transplant 1995; 15:697–705. 6. Atra A, Whelan JS, Calvagna V, et al. High-dose busulphan/melphalan with autologous stem cell rescue in Ewing’s sarcoma. Bone Marrow Transplant 1997; 20:843–846. 7. Paulussen M, Ahrens S, Burdach S, et al. Primary metastatic (stage IV) Ewing tumor: survival analysis of 171 patients from the EICESS studies. European intergroup cooperative Ewing sarcoma studies. Ann Oncol 1998; 9:275–281. 8. Ozkaynak MF, Matthay K, Cairo M, et al. Double alkylator non-total-body irradiation regimen with autologous hematopoietic stem-cell transplantation in pediatric solid tumors. J Clin Oncol 1998; 16:937–944. 9. Ladenstein R, Hartman O, Pinkerton R, et al. A multivariate and matched pair analysis on high-risk Ewing tumor (ET) patients treated with megatherapy (MGT) and stem cell reinfusion (SCR) in Europe. Proc Am Soc Clin Oncol 1999; 18:2144 abstract. 10. Perentesis JP, Katsanis E, DeFor TE, et al. Autologous stem cell transplantation for high-risk pediatric solid tumors. Bone Marrow Transplant 1999; 24:609–615. 11. Diaz MA, Vicent MG, Madero L. High-dose busulfan/melphalan as conditioning for autologous PBPC transplantation in pediatric patients with solid tumors. Bone Marrow Transplant 1999; 24:1157–1159. 12. Burdach S, Van Kaick B, Laws HJ, et al. Allogeneic and autologous stem-cell transplantation in advanced Ewing tumors. An update after long-term follow-up from two centers of the European intergroup study EICESS. Ann Oncol 2000; 11:1451–1462. 13. Kushner BH, Meyers PA. How effective is dose-intensive/myeloablative therapy against Ewing’s sarcoma/primitive neuroectodermal tumor metastatic to bone or bone marrow? The Memorial SloanKettering experience and a literature review. J Clin Oncol 2001; 19:870–880. 14. Meyers PA, Krailo MD, Ladanyi M, et al. High-dose melphalan, etoposide, total-body irradiation and autologous stem-cell reconstitution as consolidation therapy for high-risk Ewing’s sarcoma does not improve prognosis. J Clin Oncol 2001; 19:2812–2820. 15. Gamis A, Wall D, Gordon B, et al. High dose chemotherapy with stem cell rescue for children with relapsed and high risk solid tumors; preliminary survival analyses—ONC9616, a pediatric blood and marrow transplant consortium trial. Blood 2001; 98:692a. 16. Davies SM, DeFor TE, Weigel BJ, et al. High dose busulfan-based chemotherapy with autologous stem cell transplantation (ASCT) for high risk Ewing’s sarcoma family tumors. Proc Am Soc Clin Oncol 2001; 19:364a abstract. 17. Prete A, Rosito P, Alvisi P, et al. G-CSF-primed peripheral blood progenitor (PBPC) support in highrisk Ewing sarcoma of childhood. Bone Marrow Transplant 1998; 22:S21–S23. 18. Chan KW, Petropoulos D, Choroszy M, et al. High-dose sequential chemotherapy and autologous stem cell reinfusion in advanced pediatric solid tumors. Bone Marrow Transplant 1997; 20:1039–1043. 19. Hawkins D, Barnett T, Bensinger W, et al. Busulfan, melphalan and thiotepa with or without total marrow irradiation with hematopoietic stem cell rescue for poor-risk Ewing-sarcoma-family tumors. Med Pediatr Oncol 2000; 34:328–337.

584

˝ zkaynak and Malogolowkin O

20. Burdach S, Meyer-Bahlburg A, Laws HJ, et al. High-dose therapy for patients with primary multifocal and early relapsed Ewing’s tumors: results of two consecutive regimens assessing the role of total-body irradiation. J Clin Oncol 2003; 21:3072–3078. 21. Barker LM, Pendergrass TW, Sanders JE, et al. Survival after recurrence of Ewing’s sarcoma family of tumors. J Clin Oncol 2005; 23:4354–4362. 22. Rodriguez-Galindo C, Billups CA, Kun LE, et al. Survival after recurrence of Ewing tumors: the St. Jude children’s research hospital experience, 1979–1999. Cancer 2002; 94:561–569. 23. Miniero R, Brach del Prever A, Vassalao E, et al. Feasibility of high-dose chemotherapy and autologous peripheral blood stem cell transplantation in children with high grade osteosarcoma. Bone Marrow Transplant 1998; 27:S37–S40. 24. Lucidarme N, Valteau-Couanet D, Oberlin O, et al. Phase II study of high-dose thiotepa and hematopoietic stem cell transplantation in children with solid tumors. Bone Marrow Transplant 1998; 22:535–540. 25. Sauerbrey A, Bielack S, Kempf-Bielack B, et al. High-dose chemotherapy (HDC) and autologous hematopoietic stem cell transplantation (ASCT) as salvage therapy for relapsed osteosarcoma. Bone Marrow Transplant 2001; 27:933–937. 26. Fagioli F, Aglietta M, Tienghi A, et al. High-dose chemotherapy in the treatment of relapsed osteosarcoma: an Italian sarcoma group study. J Clin Oncol 2002; 20:2150–2156. 27. Kasper B, Lehnert T, Bernd L, et al. High-dose chemotherapy with autologous peripheral blood stem cell transplantation for bone and soft-tissue sarcomas. Bone Marrow Transplant 2004; 34:37–41. 28. Fischmeister G, Zoubek A, Jugovic D, et al. Low incidence of molecular evidence for tumour in PBPC harvests from patients with high risk Ewing tumours. Bone Marrow Transplant 1999; 24:405–409. 29. Thomson B, Hawkins D, Felgenhauer J, et al. RT-PCR evaluation of peripheral blood, bone marrow and peripheral blood stem cells in children and adolescents undergoing VACIME chemotherapy for Ewing’s sarcoma and alveolar rhabdomyosarcoma. Bone Marrow Transplant 1999; 24:527–533. 30. Leung W, Chen AR, Klann RC, et al. Frequent detection of tumor cells in hematopoietic grafts in neuroblastoma and Ewing’s sarcoma. Bone Marrow Transplant 1998; 22:971–979. 31. Yaniv I, Cohen IJ, Stein J, et al. Tumor cells are present in stem cell harvests of Ewing’s sarcoma patients and their persistence following transplantation is associated with relapse. Pediatr Blood Cancer 2004; 42:404–409. 32. D’Angio GJ, Evans AE, Breslow N, et al. The treatment of Wilms’ tumor. Results of the national Wilms’ tumor study. Cancer 1976; 38:633–646. 33. D’Angio GJ, Evans AE, Breslow N, et al. The treatment of Wilms’ tumor. Results of the second national Wilms’ tumor study. Cancer 1981; 47:2302–2311. 34. Lemerle J, Voute PA, Tournade MF, et al. Effectiveness of preoperative chemotherapy in Wilms’ tumor: results of an international society of pediatric oncology (SIOP) clinical trial. J Clin Oncol 1983; 1:604–609. 35. Grundy P, Breslow N, Green DM, et al. Prognostic factors for children with recurrent Wilms’ tumor: results from the second and third national Wilms’ tumor study. J Clin Oncol 1989; 7:638–647. 36. Pinkerton CR, Groot-Loonen JJ, Morris-Jones PH, Pritchard J. Response rates in relapsed Wilms’ tumor. A need for new effective agents. Cancer 1991; 67:567–571. 37. Malogolowkin, MH, Bergman T, Seibel N, et al. Outcome and prognostic factors (PF) for patients with recurrent Wilms tumor (R-WT) national Wilms tumor study 3 and 4 (NWTS 3,4). Proceedings ASCO 2001, Vol. 20, abstract 3136. 38. Tournade MF, Lemerle J, Brunat-Mentigy M, et al. Ifosfamide is an active drug in Wilms’ tumor: a phase II study conducted by the French society of pediatric oncology. J Clin Oncol 1988; 6:793–796. 39. Pein F, Pinkerton R, Tournade MF, et al. Etoposide in relapsed Wilms’ tumor: a phase II study by the French society of pediatric oncology. J Clin Oncol 1993; 11:1478–1481. 40. Pein F, Tournade MF, Zucker JM, et al. Etoposide and carboplatin: a highly effective combination in relapsed or refractory Wilms’ tumor—a phase II study by the French society of pediatric oncology. J Clin Oncol 1994; 12:931–936. 41. Baum ES, Gaynon P, Greenberg L, et al. Phase II trial of cisplatin in refractory childhood cancers: children’s cancer study group report. Cancer Treat Rep 1981; 65:815–822. 42. Douglass EC, Wilimas JA, Sackey K, et al. Efficacy of combination cisplatin (DDP) and VP-16 in the treatment of recurrent and advanced Wilms’ tumors. Proc Am Soc Clin Oncol 1986; 5:201 abstract 789.

HSCT for Pediatric Solid Tumors

585

43. Loh W, Ortega JA, Wolff J, et al. Cisplatinum/VP-16 for the retrieval of Wilms’ tumor relapsing on chemotherapy. Proc Am Soc Clin Oncol 1987; 6:222 abstract 876. 44. De Camargo B, Melarango R, Saba e Silva N, et al. Phase II study of carboplatin as a single drug for relapsed Wilms’ tumor: experience of the Brazilian Wilms’ tumor study group. Med Pediatr Oncol 1994; 22:258–260. 45. Zoubek A, Kajtar P, Flucher-Wolfram B, et al. Response of untreated stage IV Wilms’ tumor to single dose carboplatin assessed by “up front” window therapy. Med Pediatr Oncol 1995; 25:8–11. 46. Miser J, Krailo M, Hammond GD. The combination of ifosfamide, etoposide, and mesna: a very active regimen in the treatment of recurrent Wilms’ tumor. Proc Am Soc Clin Oncol 1993; 12:417 abstract 1432. 47. Marina NM, Wilimas JA, Meyer WH, et al. Refining therapeutic strategies for patients with resistant Wilms’ tumor. Am J Pediatr Hematol Oncol 1994; 16:296–300. 48. Warkentin PI, Brochstein JA, Stranjord SE, et al. High dose therapy followed by autologous stem cell rescue for recurrent Wilms’ tumor. Proc Am Soc Clin Oncol 1993; 12:414 abstract 1418. 49. Kletzel M, Morgan E, Cohn S, et al. Autologous (AU) bone marrow (BM)/peripheral stem cells (PBSC) rescue in children with relapsed Wilms tumor (WT). Med Pediatr Oncol 1994; 23:201 abstract P47. 50. Garaventa A, Hartman O, Bernard JL, et al. Autologous bone marrow transplantation for pediatric Wilms tumor: the experience of the European bone marrow transplantation solid tumor registry. Med Pediatr Oncol 1994; 22:11–14. 51. Pein F, Michon J, Valteau-Couanet D, et al. High-dose melphalan, etoposide, and carboplatin followed by autologous stem-cell rescue in pediatric high-risk recurrent Wilms’ tumor: a French society of pediatric oncology study. J Clin Oncol 1998; 16:3295–3301. 52. Kremens B, Grihn B, Klingebiel T, et al. High-dose chemotherapy with autologous stem cell rescue in children with nephroblastoma. Bone Marrow Transplant 2000; 30:893–898. 53. Calvert AH, Newell DR, Gumbrell LH, et al. Carboplatin dosage: prospective evaluation of a simple formula based on renal function. J Clin Oncol 1989; 7:1748–1756. 54. Tannous R, Giller R, Holmes E, et al. Intensive therapy for high risk (HR) relapsed Wilms’ tumor (WT). A CCG-4921/POG-9445 study report. Proceedings ASCO 2000, Vol. 19, abstract 2315. 55. Kung FH, Bernstein ML, Camitta BM, et al. Ifosfamide/carboplatin/etoposide (ICE) in the treatment of advance, recurrent Wilms tumor. Proc Am Soc Clin Oncol 1999; 18:559a abstract 2156. 56. Abu-Gosh A, Goldman S, Krailo M, et al. Excellent response rate (91%) to ifosfamide, carboplatin, and etoposide (ICE) in children with advanced and/or relapsed Wilms’ tumor. Proc Am Soc Clin Oncol 1999; 18:559a abstract 2157. 57. Malogolowkin MH, Feusner J, Steele DA, Lavado MG, Ortega JA. Carboplatin (CBDCA)/etoposide (VP-16) for the treatment of children with high-risk or recurrent Wilms’ tumor. Proc ASCO 1994; 13:424. 58. Dome JS, Liu T, Krasin M, et al. Improved survival for patients with recurrent Wilms tumor: the experience at St. Jude children’s research hospital. J Pediatr Hematol Oncol 2002; 24:192–198. 59. Raney R. Disease patterns and survival rate in children with metastatic soft tissue sarcoma: a report from the intergroup rhabdomyosarcoma study (IRS)-I. Cancer 1988; 62:1257–1266. 60. Maurer H, Gehan E, Beltangady M, et al. The intergroup rhabdomyosarcoma study-II. Cancer 1993; 71:1904–1922. 61. Crist W, Gehan E, Ragab A, et al. The third intergroup rhabdomyosarcoma study. J Clin Oncol 1995; 13:610–630. 62. Arndt C, Tefft M, Gehan E, et al. A feasibility, toxicity, and early response study of etoposide, ifosfamide, and vincristine for the treatment of children with rhabdomyosarcoma: a report from the intergroup rhabdomyosarcoma study (IRS) IV pilot study. J Pediatr Hematol Oncol 1997; 17:331–337. 63. Raney R, Crist W, Maurer H, et al. Prognosis of children with soft tissue sarcoma who relapse after achieving a complete response. Cancer 1983; 52:44–50. 64. Pappo A, Anderson J, Crist W, et al. Survival after relapse in children and adolescents with rhabdomyosarcoma: a report from the intergroup rhabdomyosarcoma study group. J Clin Oncol 1999; 17:3487–3493. 65. Houghton JA, Cook RL, Lutz PJ, et al. Childhood rhabdomyosarcoma xenografts: responses to DNA-interacting agents and agents used in current clinical therapy. Eur J Cancer Clin Oncol 1984; 20:955–960.

586

˝ zkaynak and Malogolowkin O

66. Houghton JA, Cook RL, Lutz PJ, et al. Melphalan: a potential new agent in the treatment of childhood rhabdomyosarcoma. Cancer Treat Rep 1985; 69:91–96. 67. Pinkerton CR, Groot-Loonen J, Barret A, et al. Rapid VAC, high-dose melphalan regimen. A novel chemotherapy regimen for childhood soft tissue sarcomas. Br J Cancer 1991; 64:381–385. 68. Koscielniak E, Klingebiel TH, Peters C, et al. Do patients with metastatic and recurrent rhabdomyosarcoma benefit from high-dose therapy withhematopoietic rescue? Report of the German/Austrian pediatric bone marrow transplantation group. Bone Marrow Transplant 1997; 19:227–231. 69. Boulad F, Kerman NA, LaQuaglia MP, et al. High-dose induction chemotherapy followed by autologous bone marrow transplantation as consolidation therapy in rhabdomyosarcoma, extraosseous Ewing’s sarcoma, and undifferentiated sarcoma. J Clin Oncol 1998; 16:1697–1706. 70. Carli M, Colombatti R, Oberlin O, et al. High dose melphalan with autologous stem cell reserve in metastatic rhabdomyosarcoma. J Clin Oncol 1999; 17:2796–2803. 71. Malogolowkin MH, Sposto R, Grovas L, Breneman J, Womer R, Ortega JA. Lack of improvement in survival of children with metastatic rhabdomyosarcoma (RMS) treated with intensive therapy followed by stem cell transplant (SCT) for control of minimal residual disease. Proc Am Soc Clin Oncol 1999; 18:555a. 72. Weigel BJ, Breitfeld PP, Hawkins D, et al. Role of high-dose chemotherapy with hematopoietic stem cell rescue in the treatment of metastatic or recurrent rhabdomyosarcoma. J Pediatr Hematol Oncol 2001; 23:272–276. 73. Hara J, Osugi Y, Ohta H, et al. Double-conditioning regimens consisting of thiotepa, melphalan and busulfan with stem cell rescue for the treatment of pediatric solid tumors. Bone Marrow Transplant 1998; 22:7–12. 74. Bulterys M, Goodman MT, Smith MA, et al. Hepatic tumors. SEER pediatric monograph. National Cancer Institute. 1999:91–97. 75. Finegold MJ. In: Walker WA, Durie PR, Hamilton JR, Walker-Smith JA, Watkins JB, eds. Pediatric Gastrointestinal Disease. Philadelphia: BC Decker Inc., 1991:914–926. 76. Mann IR, Kasthuri N, Raafat F, et al. Malignant hepatic tumors in children: incidence, clinical features and etiology. Paediatr Perinat Epidemiol 1990; 4:276–289. 77. Ortega JA, Douglass EC, Feusner JH, et al. Randomized comparison of cisplatin/vincristine/fluorouracil and cisplatin/continuous infusion doxorubicin for treatment of pediatric hepatoblastoma: a report from the Children’s Cancer Group and the Pediatric Oncology Group. J Clin Oncol 2000; 18:2665–2675. 78. Pritchard J, Brown J, Shafford E, et al. Cisplatin, doxorubicin, and delayed surgery for childhood hepatoblastoma: a successful approach—results of the first prospective study of the International Society of Pediatric Oncology. J Clin Oncol 2000; 18:3819–3828. 79. Yoshinari M, Imaizumi M, Hayashi Y, et al. Peripheral blood stem cell transplantation for hepatoblastoma with microscopical residue: a therapeutic approach for incompletely resected tumor. Tohoko J Exp Med 1998; 184:247–254. 80. Katzenstein HM, Rigsby C, Shaw PH, et al. Novel therapeutic approaches in the treatment of children with hepatoblastoma. J Pediatr Hematol Oncol 2002; 24:751–755. 81. Anderson PM, Wiseman GA, Dispenzieri A, et al. High-dose samarium-153 ethylene diamine tetramethylene phosphonate: low toxicity of skeletal irradiation in patients with osteosarcoma and bone metastases. J Clin Oncol 2002; 20:189–196. 82. Nichols CR, Tricot G, Willimas SD, et al. Dose-intensive chemotherapy in refractory germ cell cancer—a phase I/II trial of high-dose carboplatin and etoposide with autologous bone marrow transplantation. J Clin Oncol 1989; 7:932–939. 83. Broun ER, Nichols CR, Gize G, et al. Tandem high-dose chemotherapy with autologous bone marrow transplantation for initial relapse of testicular germ cell cancer. Cancer 1997; 79:1605–1610. 84. Bhatia S, Abonour P, Porcu R, et al. High-dose chemotherapy as initial salvage chemotherapy in patients with relapsed testicular cancer. J Clin Oncol 2000; 18:3346–3351. 85. De Giorgi U, Demirer T, Wandt H, et al. Second-line high-dose chemotherapy in patients with mediastinal and retroperitoneal primary non-seminomatous germ cell tumors: the EBMT experience. Ann Oncol 2005; 16:146–151. 86. Pico JL, Rosti G, Kramar A, et al. A randomized trial of high-dose chemotherapy in the salvage treatment of patients failing first-line platinum chemotherapy for advanced germ cell tumours. Ann Oncol 2005; 16:1152–1159.

HSCT for Pediatric Solid Tumors

587

87. Motzer RJ, Mazumdar M, Gulati SC, et al. Phase II trial of high-dose carboplatin and etoposide with autologous bone marrow transplantation in first-line therapy for patients with poor-risk germ cell tumors. J Nat Cancer Inst 1993; 85:1828–1835. 88. Morris HJ, Bosl GJ. High-dose chemotherapy as primary treatment for poor-risk germ cell tumors: the memorial sloan-kettering experience (1988–1999). Int J Cancer 1999; 83:834–838. 89. Decartis MP, Wilkinson PM, Welch RS, et al. High-dose chemotherapy and autologous haematopoietic support in poor-risk non-seminomatous germ-cell tumors: an effective first-line therapy with minimal toxicity. Ann Oncol 2000; 11:427–434. 90. Schmoll HJ, Kollmannsberger C, Metzner B, et al. Long-term results of first-line sequential highdose etoposide, ifosfamide, and cisplatin chemotherapy plus autologous stem cell support for patients with advanced metastatic germ cell cancer: an extended phase I/II study of the German Testicular Cancer Study Group 2003; 21:4083–4091. 91. Rosti G, De Giorgi U, Wandt H, et al. First-line high-dose chemotherapy for patients with poor prognosis extragonadal germ cell tumors: the experience of the European Bone Marrow Transplantation (EBMT) solid tumors working party. Bone Marrow Transplant 2004; 34:1033–1037. 92. De Giorgi U, Rosti G, Slavin S, et al. Salvage high-dose chemotherapy for children with extragonadal germ-cell tumours. Br J Cancer 2005; 93:412–417.

29 Stem-Cell Transplantation in Neuroblastoma Stephan A. Grupp Division of Oncology and Department of Pathology, Stem Cell Biology, Children’s Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

INTRODUCTION The role of autologous stem-cell transplantation as consolidation therapy for malignancies has been debated, both in the pediatric as well as the adult setting. The use of this therapy has certain general design criteria: (1) a chemo-responsive tumor type, typically with a good response rate to induction therapy, but a poor long-term (i.e., three- or five-year) event-free survival (EFS) rate; (2) use of a conditioning (pretransplant chemotherapy) regimen that may be dose-escalated safely past marrow tolerance; (3) use of multiagent conditioning regimens that utilize agents active against the disease, ideally including agents not utilized in the induction chemotherapy; (4) use of optimal supportive care, especially as regards stem-cell source and processing techniques. In this chapter, we will consider the use of hematopoietic stem-cell transplantation (SCT) in neuroblastoma (NBL). The use of this treatment option, especially in the era of peripheral blood stem cell (PBSC) collection, has special challenges when applied to a disease where the median age is 3. Accordingly, we have examined in a previous chapter the practicalities of stem-cell collection when applied to small patients. We will consider the data supporting intensification of consolidation using multiple cycles of high-dose chemotherapy with stem-cell rescue, followed by an overview of the detection and potential importance of tumor cells contaminating an autologous stem-cell product. The use of metaiodobenzylguanidine (MIBG), both as an imaging and as a radiotherapeutic modality, is discussed. Finally, although the vast majority of transplant procedures for patients with NBL now utilize autologous PBSC as the stem-cell source, allogeneic transplant has been advocated by some investigators, so we will briefly explore this issue as well. The primary source of hematopoietic stem and progenitor cells for use in autologous and allogeneic transplantation has been, until recently, bone marrow. Over the past decade, there has been increasing use of peripheral blood containing mobilized stem and progenitor cells as a source of these cells for transplantation (1). This product is variously referred to as PBSC, peripheral blood progenitor cells, or given the shorthand designation “stem cells.” Although each cell source used for hematopoietic transplantation contains stem cells, when the term “stem cells” is used without a qualifier, it is usually referring to PBSC. 589

590

Grupp

AUTOLOGOUS TRANSPLANT IN NEUROBLASTOMA Commonly referred to as autologous transplant, the use of the patient’s own stem cells to support recovery from high-dose chemotherapy is more properly referred to as high-dose chemotherapy with stem-cell rescue (HDC/SCR). The HDC regimen used to prepare the patient is usually myeloablative, meaning that no BM recovery is possible without SCR. There are also submyeloablative HDC regimens, in which the SCR is used to speed recovery, decrease toxicity, and decrease treatment interval without being absolutely required for engraftment (2,3). Compared to autologous marrow, the use of PBSC provides faster hematopoietic recovery from HDC, which results in lower infection risk, more rapid resolution of mucositis, earlier discharge from the hospital, and less risk of prolonged need for transfusion, especially platelets. The use of PBSC, along with other advances in prophylaxis and supportive care, have decreased the treatment-related mortality (TRM) rate in autologous transplant to !5% in many studies. As a result, the use of autologous marrow to support HDC/SCR has very limited indications and has all but disappeared (4). In our own institution, no patient has received marrow in support of HDC/SCR since 1996. This improvement in TRM has properly returned the focus of clinical studies in autologous SCT to the issue of disease control. In the area of disease control, results have been more mixed. The general paradigm for HDC/SCR is this: in chemosensitive disease, many or most patients can be put into complete remission. However, in high-risk malignancies, the complete response rate, which can be high, does not translate into a high three- or five-year EFS rate. A good example of this is high-risk NBL, in which 80% or more of patients have responsive disease but !20% are long-term survivors with conventional chemotherapy. There are several well-established indications for autologous HDC/SCR. One is relapsed Hodgkin’s disease. The high risk of subsequent recurrence is seen even in the 70% of relapsed Hodgkin’s patients who successfully achieve a second complete remission. Use of HDC/SCR in relapsed Hodgkin’s disease increases EFS to approximately 40–60% (5). Achievement of a complete remission prior to HDC/SCR confers improved prognosis, although patients who are not in a complete remission prior to transplant but who achieve remission after transplant also benefit from the procedure. After years of clinical investigation, another established indication for HDC/SCR is highrisk NBL. A number of single-arm or retrospective studies indicated that autologous BM transplant might improve the EFS in children with this disease (6–8). In a large European Bone Marrow Transplant Registry (EBMT) retrospective analysis of 1070 HDC/SCR procedures for NBL, survival from transplant was noted to be 49% at two years. Relapses were noted to occur as late as seven years from transplant, although most of the events occurred within the first 18 months after HDC/SCR. Forty-eight of the 1070 procedures were performed after relapse, with no survivors among the group undergoing a second HDC/SCR procedure (9). In Table 1, EFS rates at or around three years are presented from several major studies: (1) the EBMT analysis (above), (2) the Children’s Cancer Group (CCG) phase III 3891 study (below), (3) the Boston/Philadelphia and Chicago tandem transplant studies (below), and (4) unpublished experience with a transplant regimen of carboplatin/etoposide/melphalan followed by postHDC/SCR local radiotherapy pioneered by Villablanca and colleagues that forms the backbone of the just-completed COG nationwide phase III study (Villablanca, J. unpublished data).

Children’s Cancer Group 3891 The Children’s Cancer Group 3891 study has provided the largest phase III experience in NBL to date. This study employed a 2!2 factorial design. Patients were randomized to consolidation with HDC/SCR versus continuation chemotherapy. Bone Marrow purged using an immunomagnetic method (see below) was the stem-cell source for HDC/SCR. The conditioning regimen employed carboplatin (1000 mg/m2), etoposide (640 mg/m2), melphalan (210 mg/m2), and total body irradiation (TBI) (1000 cGy). After completion of consolidation, patients in both groups

Stem-Cell Transplantation in Neuroblastoma

591

Table 1 Results from Large Studies of Autologous High-Dose Chemotherapy with Stem-Cell Rescue in High-Risk Neuroblastoma Group

N

EBMT

1070

Study type

EFS from

Retrospective

Transplant

CCG 3891

539

Phase III

Grupp et al.

97

Phase II

Estimated from diagnosis Diagnosis

Kletzel et al.

25

Phase II

Diagnosis

Villablanca et al.a

73

Phase II

Transplant

EFS 2 year 49% 5 year 33% 3.7 year 38% 3 year 5 year 7 year 3 year

55% 47% 45% 57%

3 year 49% 5 year 47%

Myeloablative regimen(s) Various CEM/TBI #1 CECtx #2 TBI—M #1 CE #2 CE #3 TCtx CEM

Study populations differed significantly in these five studies. The EBMT analysis included allogeneic transplants and transplants after relapse. Villablanca et al. included only stage four older than one year in the group presented. a Unpublished data, J. Villablanca. Abbreviations: EFS, event-free survival; C, carboplatin; E, etoposide; M, melphalan; Ctx, cyclophosphamide; T, thiotepa; TBI, total body irradiation; CCG, children’s cancer group; EBMT, European Bone Marrow Transplant Registry.

were then randomized to biological therapy with 13-cis retinoic acid or no further therapy (10). Patients randomized to bone marrow transplantation plus differentiation therapy with 13-cis retinoic acid had improved EFS compared to those treated with conventional chemotherapy without differentiation therapy. The authors concluded that the combination of HDC/SCR and 13-cis retinoic acid added 30% to overall survival (OS) when compared to chemotherapy alone. In the initial report, the authors estimated a 3.7-year EFS from diagnosis in the best group as being approximately 38% (10). This important study also showed the challenges of a 2!2 design and a complex treatment plan in a high-risk group of patients: of 579 eligible patients, 379 underwent the first randomization and 258 patients participated in the second randomization, leaving approximately 50 patients in each of the four treatment groups.

Metaiodobenzylguanidine (MIBG) Scans and MIBG Therapy Scanning with 123I-MIBG provides a useful way to assess disease burden and response in the approximately 90% of NBL patients whose tumors concentrate MIBG (11,12). Response as measured by MIBG can be scored for semiquantitative assessment; the scoring system has a good concordance rate among trained radiologists (13) and these scores have predictive value (14). Responses as assessed by dropping scores early in induction therapy correlate with improved EFS (14). Similarly, patients with a high score after induction therapy, especially in multiple osteomedullary sites, have an extremely poor outcome (15). This suggests that patients with a poor response to induction therapy form an ultra-high risk group that might reasonably be diverted from standard up-front therapy to earlier phase clinical trials. In addition to its use as a radionuclide for scanning purposes, MIBG can be used for therapy. Several groups have pioneered the use of therapeutic 131I-MIBG, a radiotherapeutic agent that can be given in lower or higher doses, and that in doses O12 mCi/kg and/or in multiple infusions may require PBSC support (16). The use of 131I-MIBG allows delivery of very high local radiation doses to tumor masses. The radiobiology of 131I-MIBG makes it an agent best delivered to tumor cells in bulk tumors or small aggregations: single cells (i.e., low-level

592

Grupp

BM disease) will take up the agent but are not exposed to the particles. In an extension of previous studies of myeloablative 131I-MIBG given as a single agent, newer studies have combined therapeutic doses of 131I-MIBG with high-dose chemotherapy with preliminary evidence of efficacy in high-risk patients (17,18). Recently, a phase I dose-escalation trial of 131I-MIBG followed by myeloablative carboplatin, etoposide, and melphalan was completed in patients with primary refractory NBL, reaching a maximum tolerated dose of 12 mCi/kg 131I-MIBG in combination with 1500 mg/m2 carboplatin, 1200 mg/m2 etoposide, and 210 mg/m2 melphalan (19). In this highly challenging group of 24 patients (22 were assessable for response), six experienced complete or partial responses, and 15 demonstrated mixed responses or stable disease, with an EFS of 31% at three years (from the time of MIBG treatment) and a median EFS time of 18 months. One patient experienced TRM. The combination of 131I-MIBG and high-dose chemotherapy may be promising for patients with primary refractory disease, ultra-high risk disease (as defined above), and the small subset of patients who experience progressive disease during high-risk induction who are able to achieve a second response. This treatment approach will continue to be developed both in Europe and in the United States. The urgency of further improving outcome for patients treated for high-risk NBL is highlighted by the poor outcomes seen in treatment of recurrent disease. Children who relapse soon after SCT are unlikely to have responsive disease and succumb rapidly, while a subset of children treated after a later relapse have initially responsive disease and can reenter remission, although still with very poor ultimate outcomes (20). Although some of these children have prolonged or multiple responses, even in this group of children with highly-responsive recurrent disease after HDC/SCR, there is very little prospect for cure. It is tempting to consider another HDC/SCR procedure for a child who has relapsed after high-risk therapy who has achieved an excellent second clinical response, but it is important to bear in mind that there is very little data supporting this approach. In the EBMT analysis, survival for children undergoing HDC/SCR after a relapse who had not undergone HDC/SCR previously was 15%, while no children were long-term survivors after the use of a salvage regimen including HDC/SCR if the relapse had occurred after a previous HDC/SCR (9,21). Therapies with lower toxicities and shorter hospital stays than intensive HDC/SCR, including phase I therapies, should be considered in this setting. Therapies that may require stem-cell support without HDC/SCR-level toxicities, such as high-dose 131I-MIBG, can also provide excellent disease control for a period of time and can be an important part of the overall package of therapies for recurrent disease.

Tandem High-Dose Chemotherapy with Stem-Cell Rescue The HDC/SCR concept has now been further extended, using the more rapid recovery and lower tumor burden afforded by PBSC, in studies which use sequential cycles of HDC/SCR. The approach is called tandem transplant and allows for greater dose intensification in the consolidation phase. This approach was initially tried using bone marrow as a stem-cell source, and seemed to be unfeasible due to a 24% TRM (22). However, the switch to PBSC has allowed more rapid recovery from HDC/SCR, and several groups have tested tandem HDC/SCR supported by PBSC in NBL (2,23–25). The largest of these studies was conducted over six years at four cooperating institutions [Fig. 1 (schema) and Fig. 2 (survival curve)]. Important characteristics of the study included pioneering the early collection of PBSC (generally after the third cycle of induction), use of CD34 selection of PBSC as a purging method, and use of two fully myeloablative consolidation regimens (carboplatin/etoposide/cyclophosphamide and melphalan/TBI). The 3 year EFS rate from diagnosis in this sequentially treated group of patients was initially reported at 56% (23,24). This encouraging EFS has been maintained in a recent update of the data in a cohort of 97 patients (Fig. 2) (26). Median follow up in this group of patients has almost reached six years, and five- and seven-year EFS, at 47% and 45%, respectively, suggest long-term disease control in a subset of patients. It is also useful, although unfortunate, to note that the OS curve

Stem-Cell Transplantation in Neuroblastoma

593

Induction

1

2

3

4

5

Adriamycin Cisplatin

Adriamycin Ifosfamide

Carboplatin Vincristine

Vincristine

PBSC Collection

Surgery

High-dose therapy with stem-cell rescue Etoposide 800 mg/m2 x 3d

Biological therapy 2

Melphalan 60 mg/m x 3d

Cyclophosphamide 1.8 g/m x 2d

D+90 to D+270 13-cis-RA

Carboplatin 667mg/m2 x 3d 2

Local XRT

TBI 1200 cGy over 3-4d

Figure 1 Schema of the Boston/Philadelphia tandem transplant study. Abbreviations: RA, retinoic acid; TBI, total body irradiation; PBSC, peripheral blood stem cell; XRT, X-ray therapy.

exceeds the EFS rate by almost 20% early on, suggesting that relapse is initially treatable in some patients. However, the curves converge over time and there is very little likelihood of long-term survival after a relapse in this setting. The use of PBSC to support tandem transplantation, in contrast to the use of marrow in the report of cited above (22), has made this approach feasible. Engraftment was very rapid with both transplants, reaching an ANC of 500 at a median of day 11 after the first procedure and day 12 after the second. No overt treatment-related AML has been observed, although one

Figure 2 Event-free survival and overall survival from diagnosis for patients enrolled on the Boston/Philadelphia tandem transplant study and treated with uniform induction chemotherapy and intensified consolidation. Median follow-up is 5.7 years in this cohort of 97 patients.

594

Grupp

patient has developed myelodysplasia with clonal trisomy 8 in the setting of normal peripheral blood counts 5.6 years after diagnosis. This encouraging finding may be a reflection of collecting PBSC fairly early in induction therapy, with correspondingly less chemotherapy exposure to the PBSC. TRM was 6% and VOD was infrequent, a primary feasibility concern during the early years of the protocol. Patients experiencing TRM included one who died of Epstein-Barr virus lymphoproliferative disease (EBV-LPD). EBV-LPD is described in the setting of autologous HDC/SCR but is an uncommon complication (27–30), although three cases were observed among these 97 patients. This indicates that the combination of tandem transplant plus the inherent T-cell depletion provided by CD34 selection is more immunosuppressive than single HDC/SCR using unselected PBSC (27,31). This also suggests that any study of cellular immunotherapy, best pursued in the status of minimal residual disease reached after HDC/SCR, might be significantly limited by the period of very low T-cell numbers and function that occurs for months after such procedures. In order to address this concern and to provide a foundation for antitumor vaccination in the setting of post-SCT minimal residual disease, we have now begun a study testing T-cell infusions after tandem SCT. This study uses the tandem transplant backbone and adds T-cell collection by pheresis at diagnosis. These T cells undergo costimulated expansion using the anti-CD3/anti-CD28 system developed by Carl June and colleagues (32–35). We have referred to this approach as T-cell augmentation. Our preliminary experience with T-cell augmentation shows a significant impact of co-stimulated T-cell infusion on CD4C T-cell reconstitution (Fig. 3). There is also preliminary indication that earlier T-cell augmentation (day 2 after PBSC infusion as opposed to day 12) may harness improved the homeostatic expansion in the profoundly lymphodepleted and cytokine-activated post-SCT milieu and result in even more robust CD4C T-cell reconstitution. If these findings are confirmed in the current pilot trial, and there is evidence of vaccine responses early after SCT in patients given T-cell augmentation (35), we have proposed to use this platform of tandem SCT with T-cell augmentation to test an antitumor vaccine. A similar HDC/SCR study was conducted by Kletzel and coworkers using three HDC/SCR regimens in sequence (“triple tandem”). Table 1 shows the details of this regimen,

Effect of T-cell augmentation 1600 SCT D+30-60

CD4+ cells/μl

1200

D+60-90 800

400

0 No T cells

T cells D+12

T cells D+2

Figure 3 Impact of costimulated T-cell infusions (T-cell augmentation) on CD4CT-cell recovery in children undergoing tandem SCT for neuroblastoma. Absolute number of CD4C cells in the peripheral blood were determined by flow cytometry at the days post-SCT indicated, except at the time of SCT, where absolute lymphocyte counts approaching 0 allowed measurement of CD4C cells in almost no patients. Abbreviation: SCT, stem-cell transplantation.

Stem-Cell Transplantation in Neuroblastoma

595

while Fig. 4 shows an updated EFS curve reflecting a larger number of patients accrued since the original report (Kletzel M, unpublished data). Among 25 patients in the published report, 19 completed HDC/SCR #2, 17 went on to HDC/SCR #3, and 1 late TRM was observed. Six of the patients received at least one course of anti-GD2 monoclonal antibody as well. EFS in this group of patients at three years was 57%.

Detection and Removal of Tumor from Stem-Cell Products One major concern in the use of autologous bone marrow and PBSC products from patients with a malignancy that metastasizes to the bone marrow or can be found in the peripheral blood is that tumor can inadvertently be collected with the stem-cell product. This concern has also been used to support the use of allogeneic transplant, in which there is no risk of tumor contamination of the graft. In absence of clear evidence of a benefit of graft-versus-host disease (GVHD) in NBL (see below), the approach to this concern has focused on (1) sensitive detection of tumor cells in autologous cell products used for transplantation, and (2) removal of tumor cells from the stem cell product (negative selection) or isolation and purification of the stem and progenitor cells away from all other cells, including any possible tumor cells (positive selection). Immunocytochemistry (ICC) is the current standard for NBL cell detection. Both positive selection and negative selection have been tested in NBL autografts. There are a variety of ways in which NBL can be detected in bone marrow or PBSC products. These include flow cytometry (36), ICC (37–40), immunofluorescence (41), and RT-PCR for one of several NBL-specific gene products such as tyrosine hydroxylase (42,43), GAGE (43–45) and PGP9.5 (46). The national COG A3973 trial employs ICC detection of NBL, a method with a sensitivity of 1 cell in 100,000 (10K5) to 10K6. At this point, PBSC have almost completely replaced bone marrow as a stem-cell source for autologous transplantation, largely because of the more rapid hematopoietic engraftment that results from the use of PBSC. In addition, the comparative experience in autologous bone marrow and PBSC harvesting has demonstrated that far fewer PBSC products collected from NBL patients are positive for tumor by ICC. Compared to bone marrow, PBSC from a patient receiving HDC/SCR for a malignancy

100

80

% surviving

OS

n = 46

60

40 EFS 20

0 0

24

48

72 96 Months from Diagnosis

120

144

Figure 4 Event-free survival and overall survival from diagnosis on the Chicago “triple tandem” transplant study. Source: From Ref. 2.

596

Grupp

are less likely to contain tumor cells (47) and, if they are present, have a lower content of tumor cells that are therefore more likely to be purged successfully of tumor cells (48). Interestingly, in our preliminary experience with pheresing 18 children with NBL for T cells, almost all at diagnosis and prior to any chemotherapy, only one of these mononuclear cell collections has had even low-level NBL contamination by ICC. These results support collection of PBSC much earlier in induction (cycle two or three) than bone marrow had previously been collected (usually the end of induction), and this has now become the standard. The very low rate of ICC-detectable NBL contamination of PBSC also indicates that low levels of bone marrow disease at the time of collection are not a contraindication to collection at that time. As long as the product is ICC-negative, it meets national standards for infusion, and it can be collected at the intended time and infused. Use of RT-PCR and real time RT-PCR detection of NBL cells in PBSC has also been studied. These techniques are tenfold more sensitive (10K6 to 10K7), and a significant fraction of products that are negative for ICC are positive by PCR. Unfortunately, there is no data to guide us as to the clinical significance of a PCRC/ICCK product. In a small study performed at our institution, Taqman quantitative RT-PCR was used to measure the number of copies of the GAGE gene product in PBSC products that subsequently underwent CD34 selection to remove potential tumor. We noted only one product that was ICCC at the level of one tumor cell in 105 PBSC. Although all others were ICC-, RT-PCR for GAGE was positive in 19/25 before CD34 selection and 14/25 after CD34 selection, with tumor depletion estimated by this method at approximately 3 logs (44). In this small cohort, there was no effect of PCR detection of disease either before or after CD34 selection on outcome. Two principal methods of removing tumor have been tested in NBL—negative selection and positive selection. To accomplish negative selection (removal of tumor cells), it is possible to deplete tumor using specific antitumor monoclonal antibodies, often followed by a magnetic depletion step. This approach has been proven to purge tumor cells from stem-cell products collected from patients with B-cell lymphomas (49). This technique has been the mostly widely used method to purge NBL (37,50,51). That tumor cells found in bone marrow may contribute to relapse was shown in gene marking studies in NBL patients undergoing autologous bone marrow transplantation. In these studies, a bone marrow aliquot was transfected with a marker gene and infused along with the remainder of the transplant. Tumor cells at sites of relapse were found to contain the marker gene, suggesting that clonogenic tumor had been infused with the graft (52). In follicular lymphoma, inability to detect tumor cells in the stem-cell product after purging is associated with improved outcome after autologous transplant (49), but no study has shown that purging itself improves outcome. The other approach to removal of tumor is purification of stem and progenitor cells from the other cells in the PBSC collection, which compose O97–99% of the cells and may include any contaminating tumor cells. This approach is called positive selection, and positive selection based on the stem/progenitor cell antigen CD34 is currently the only FDA-approved method of purging (53). CD34 selection is an automated, closed-system immunomagnetic approach that harvests stem cells rapidly from a PBSC product. Two devices are currently available in the United States to perform CD34 selection. The Isolex 300i is FDA-approved and the CliniMACS is in a number of clinical trials in stem-cell laboratories across the country. The Isolex device is limited to CD34 selection only, while the CliniMACS will allow for the use of many different antibodies recognizing antigens other than CD34, such as the stem-cell marker CD133 (54). The advantage of CD34 (or potentially CD133) selection is that it is automated and widely available. A potential disadvantage is that T cells are removed from the PBSC product along with other, nonstem cells. In the setting of tandem autologous HDC/SCR, this T-cell depletion may (31) or may not (55) impact on immune reconstitution or risk of postSCT viral diseases or other opportunistic infections. To better understand the issue of purging and tumor cell contamination of autologous PBSC, the Children’s Oncology Group is conducting the A3973 study, a phase III, randomized test of purged PBSC given in support of HDC/SCR in NBL. In A3973, patients with ICC- PBSC products are randomized to receive either an unpurged or purged PBSC product.

Stem-Cell Transplantation in Neuroblastoma

597

This study has just completed accrual. We will learn if purging has an impact on EFS, what the rate and extent of RT-PCR detection of contaminating tumor is, and the impact of purging on RT-PCR-detectable disease. We may discover that purging is important to prevent the reinfusion of cells into the patient which may cause a relapse, or we may discover that tumor in the PBSC product is a surrogate for residual/resistant disease in the patient, and the real challenge is to adequately purge such patients of their own disease burdens.

Allogeneic SCT for Neuroblastoma Another highly experimental approach to NBL transplant is the use of allogeneic transplant. A graft-versus-tumor effect, the positive counterpart of the allogeneic transplant reaction, GVHD, has been demonstrated in leukemias, especially chronic myelogenous leukemia (56). This effect has not as convincingly been demonstrated in the setting of solid tumors (57). Previous analyses of conventional allogeneic SCT for NBL (58) have failed to show any benefit over other therapies that do not entail the risk of GVHD. In 1994, Ladenstein et al. did a casecontrol analysis of the use of allogeneic transplant in NBL. Seventeen allogeneic cases were matched with 34 patients undergoing autologous transplant, and two year EFS was similar at 35% and 41%, respectively, a difference that was not statistically significantly different (59). Half of the allogeneic transplant patients developed GVHD, most not severe. Matthay and colleagues performed a study of myeloablative therapy for NBL using a uniform, TBI-based preparative regimen. Twenty patients for whom an HLA-matched sib was available underwent allogeneic transplant, while 36 patients without a matched sib received a purged bone marrow autograft. Four year progression-free survival trended lower in the allogeneic group at 25% compared to 49% in the autologous group, a result that just failed to reach statistical significance (pZ.051) (60). Relapse in the two groups was similar, tending not to support the assertion that reducing TRM or GVHD in allogeneic SCT would unmask a clearer benefit for an allogeneic effect in NBL (8). Neither of these analyses used a randomized study design, but neither provided support for conventional allogeneic SCT in NBL. At present, the support for an allogeneic (i.e., “graft-versus-NBL”) effect in NBL is modest and is based largely on case reports where patients experience resolution or response of disease after allogeneic SCT, in the setting of possible GVHD, and after any effect of the conditioning chemotherapy itself is presumed to have waned (61). With the advent of nonmyeloablative transplant regimens, there has again been increasing interest in the application of allogeneic transplant to NBL, with the hope that reduced intensity will reduce TRM and thus allow the detection of a therapeutic benefit. At this point, there is not data to justify this approach outside of the context of a carefully controlled clinical trial, especially in children undergoing their primary treatment for high-risk NBL.

Conclusion Currently, the standard treatment for high-risk NBL is based upon a package of therapy that includes multi-cycle induction, early collection of PBSC, testing of the PBSC product for evidence of NBL contamination, as complete a surgical resection as can be accomplished without organ sacrifice, HDC/SCR (without clear evidence of one conditioning regimen being superior to another), and local radiotherapy either before or after HDC/SCR. A small group of children with MYCN-nonamplified tumors in the 12–18 month age range, previously considered high-risk, may have a superior outcome and may not need as intensive treatment (62,63). The COG A3973 phase III trial, which has just completed accrual, will help answer the question of whether purging of PBSC results in improved outcome in NBL. The next COG phase III study (ANBL0532) will test single versus tandem HDC/SCR, using a tandem transplant regimen without TBI that was tested in the COG pilot study ANBL00P1 and a novel induction regimen including topotecan that was piloted in COG ANBL02P1. This COG randomized trial will provide us a better understanding of whether greater intensity in consolidation will further improve outcome over the current standard of care.

598

Grupp

REFERENCES 1. Kessinger A, Armitage JO, Landmark JD, Weisenburger DD. Reconstitution of human hematopoietic function with autologous cryopreserved circulating stem cells. Exp Hematol 1986; 14:192–196. 2. Kletzel M, Katzenstein HM, Haut PR, et al. Treatment of high-risk neuroblastoma with triple-tandem high-dose therapy and stem-cell rescue: results of the Chicago pilot II study. J Clin Oncol 2002; 20:2284–2292. 3. Kreissman SG, Rackoff W, Lee M, Breitfeld PP. High dose cyclophosphamide with carboplatin: a tolerable regimen suitable for dose intensification in children with solid tumors. J Pediatr Hematol Oncol 1997; 19:132–309. 4. Horowitz MM. Uses and growth of hematopoietic cell transplantation. In: Thomas ED, Blume KG, Forman SJ, eds. Hematopoietic Cell Transplantation. 2nd ed. Malden, MA: Blackwell, 1999:12–32. 5. Lazarus HM, Loberiza FR, Jr., Zhang MJ, et al. Autotransplants for Hodgkin’s disease in first relapse or second remission: a report from the autologous blood and marrow transplant registry (ABMTR). Bone Marrow Transplant. 2001; 27:387–396. 6. Matthay KK, Harris R, Reynolds CP, et al. Improved event-free survival for autologous bone marrow transplantation vs chemotherapy in neuroblastoma: a childrens cancer group study. Med Pediatr Oncol 1998; 31:191–207. 7. Matthay KK. Impact of myeloablative therapy with bone marrow transplantation in advanced neuroblastoma. Bone Marrow Transplant 1996; 18:S21–S24. 8. Matthay KK, O’Leary MC, Ramsay NK, et al. Role of myeloablative therapy in improved outcome for high risk neuroblastoma: review of recent children’s cancer group results. Eur J Cancer 1995; 31A:572–575. 9. Philip T, Ladenstein R, Lasset C, et al. 1070 myeloablative megatherapy procedures followed by stem cell rescue for neuroblastoma: 17 years of European experience and conclusions. European Group for Blood and Marrow Transplant Registry Solid Tumour Working Party. Eur J Cancer 1997; 33:2130–2135. 10. Matthay KK, Villablanca JG, Seeger RC, et al. Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. N Engl J Med 1999; 341:1165–1173. 11. Gordon I, Peters AM, Gutman A, Morony S, Dicks-Mireaux C, Pritchard J. Skeletal assessment in neuroblastoma—the pitfalls of iodine-123-MIBG scans. J Nucl Med 1990; 31:129–134. 12. Lastoria S, Maurea S, Caraco C, et al. Iodine-131 metaiodobenzylguanidine scintigraphy for localization of lesions in children with neuroblastoma: comparison with computed tomography and ultrasonography. Eur J Nucl Med 1993; 20:1161–1167. 13. Ady N, Zucker JM, Asselain B, et al. A new 123I-MIBG whole body scan scoring method— application to the prediction of the response of metastases to induction chemotherapy in stage IV neuroblastoma. Eur J Cancer 1995; 31A:256–261. 14. Matthay KK, Edeline V, Lumbroso J, et al. Correlation of early metastatic response by 123Imetaiodobenzylguanidine scintigraphy with overall response and event-free survival in stage IV neuroblastoma. J Clin Oncol 2003; 21:2486–2491. 15. Katzenstein HM, Cohn SL, Shore RM, et al. Scintigraphic response by 123I-metaiodobenzylguanidine scan correlates with event-free survival in high-risk neuroblastoma. J Clin Oncol 2004; 22:3909–3915. 16. Howard JP, Maris JM, Kersun LS, et al. Tumor response and toxicity with multiple infusions of high dose 131I-MIBG for refractory neuroblastoma. Pediatr Blood Cancer 2005; 44:232–239. 17. Klingebiel T, Bader P, Bares R, et al. Treatment of neuroblastoma stage 4 with 131I-meta-iodobenzylguanidine, high-dose chemotherapy and immunotherapy. A pilot study. Eur J Cancer 1998; 34:1398–1402. 18. Yanik GA, Levine JE, Matthay KK, et al. Pilot study of iodine-131-metaiodobenzylguanidine in combination with myeloablative chemotherapy and autologous stem-cell support for the treatment of neuroblastoma. J Clin Oncol 2002; 20:2142–2149. 19. Matthay KK, Tan JC, Villablanca JG, et al. Phase I dose escalation of iodine-131metaiodobenzylguanidine with myeloablative chemotherapy and autologous stem-cell transplantation in refractory neuroblastoma: a new approaches to neuroblastoma therapy consortium study. J Clin Oncol 2006; 24:500–506. 20. Lau L, Tai D, Weitzman S, Grant R, Baruchel S, Malkin D. Factors influencing survival in children with recurrent neuroblastoma. J Pediatr Hematol Oncol 2004; 26:227–232.

Stem-Cell Transplantation in Neuroblastoma

599

21. Ladenstein R, Lasset C, Hartmann O, et al. Impact of megatherapy on survival after relapse from Stage 4 neuroblastoma in patients over 1 year of age at diagnosis: a report from the European Group for Bone Marrow Transplantation. J Clin Oncol 1993; 11:2330–2341. 22. Philip T, Ladenstein R, Zucker JM, et al. Double megatherapy and autologous bone marrow transplantation for advanced neuroblastoma: the LMCE2 study. Br J Cancer 1993; 67:119–127. 23. Grupp SA, Stern JW, Bunin N, et al. Rapid-sequence tandem transplant for children with high-risk neuroblastoma. Med Pediatr Oncol 2000; 35:696–700. 24. Grupp SA, Stern JW, Bunin N, et al. Tandem high-dose therapy in rapid sequence for children with high-risk neuroblastoma. J Clin Oncol 2000; 18:2567–2575. 25. Sung KW, Yoo KH, Chung EH, et al. Successive double high-dose chemotherapy with peripheral blood stem cell rescue collected during a single leukapheresis round in patients with high-risk pediatric solid tumors: a pilot study in a single center. Bone Marrow Transplant 2003; 31:447–452. 26. George RE, Li S, Medeiros-Nancarrow C, et al. High risk neuroblastoma treated with tandem autologous peripheral blood stem cell-supported transplant: long-term survival update. J Clin Oncol, 2006 (In press). 27. Kanold J, Yakouben K, Tchirkov A, et al. Long-term results of CD34(C) cell transplantation in children with neuroblastoma. Med Pediatr Oncol 2000; 35:1–7. 28. Peniket AJ, Perry AR, Williams CD, et al. A case of EBV-associated lymphoproliferative disease following high-dose therapy and CD34-purified autologous peripheral blood progenitor cell transplantation. Bone Marrow Transplant 1998; 22:307–309. 29. Heath JA, Broxson EH, Jr., Dole MG, et al. Epstein-Barr virus-associated lymphoma in a child undergoing an autologous stem cell rescue. J Pediatr Hematol Oncol 2002; 24:160–163. 30. Lones MA, Kirov I, Said JW, Shintaku IP, Neudorf S. Post-transplant lymphoproliferative disorder after autologous peripheral stem cell transplantation in a pediatric patient. Bone Marrow Transplant 2000; 26:1021–1024. 31. Powell JL, Bunin NJ, Callahan C, Aplenc R, Griffin G, Grupp SA. An unexpectedly high incidence of Epstein-Barr virus lymphoproliferative disease after CD34C selected autologous peripheral blood stem cell transplant in neuroblastoma. Bone Marrow Transplant 2004; 33:651–657. 32. Levine BL, Bernstein WB, Aronson NE, et al. Adoptive transfer of costimulated CD4CT cells induces expansion of peripheral T cells and decreased CCR5 expression in HIV infection. Nat Med 2002; 8:47–53. 33. Laport GG, Levine BL, Stadtmauer EA, et al. Adoptive transfer of costimulated T cells induces lymphocytosis in patients with relapsed/refractory non-Hodgkin lymphoma following CD34Cselected hematopoietic cell transplantation. Blood 2003; 102:2004–2013. 34. Porter DL, Levine BL, Bunin N, et al. A phase 1 trial of donor lymphocyte infusions expanded and activated ex vivo via CD3/CD28 costimulation. Blood 2006; 107:1325–1331. 35. Rapoport AP, Stadtmauer EA, Aqui N, et al. Restoration of immunity in lymphopenic individuals with cancer by vaccination and adoptive T-cell transfer. Nat Med 2005; 11:1230–1237. 36. Tsang KS, Li CK, Tsoi WC, et al. Detection of micrometastasis of neuroblastoma to bone marrow and tumor dissemination to hematopoietic autografts using flow cytometry and reverse transcriptasepolymerize chain reaction. Cancer 2003; 97:2887–2897. 37. Reynolds CP. Detection and treatment of minimal residual disease in high-risk neuroblastoma. Pediatr Transplant 2004; 8:56–66. 38. Reynolds CP, Black AT, Woody JN. Sensitive method for detecting viable cells seeded into bone marrow. Cancer Res 1986; 46:5878–5881. 39. Seeger RC, Reynolds CP, Gallego R, Stram DO, Gerbing RB, Matthay KK. Quantitative tumor cell content of bone marrow and blood as a predictor of outcome in stage IV neuroblastoma: a Children’s Cancer Group study. J Clin Oncol 2000; 18:4067–4076. 40. Moss TJ, Reynolds CP, Sather HN, Romansky SG, Hammond GD, Seeger RC. Prognostic value of immunocytologic detection of bone marrow metastases in neuroblastoma. N Engl J Med 1991; 324:219–226. 41. Mehes G, Luegmayr A, Kornmuller R, et al. Detection of disseminated tumor cells in neuroblastoma: 3 log improvement in sensitivity by automatic immunofluorescence plus FISH (AIPF) analysis compared with classical bone marrow cytology. Am J Pathol 2003; 163:393–399. 42. Naito H, Kuzumaki N, Uchino J, et al. Detection of tyrosine hydroxylase mRNA and minimal neuroblastoma cells by the reverse transcription-polymerase chain reaction. Eur J Cancer 1991; 27:762–765. 43. Cheung IY, Cheung NK. Detection of microscopic disease: comparing histology, immunocytology, and RT-PCR of tyrosine hydroxylase, GAGE, and MAGE. Med Pediatr Oncol 2001; 36:210–212.

600

Grupp

44. Donovan J, Temel J, Zuckerman A, et al. CD34 selection as a stem cell purging strategy for neuroblastoma: preclinical and clinical studies. Med Pediatr Oncol 2000; 35:677–682. 45. Cheung IY, Cheung N-KV. Molecular detection of GAGE expression in peripheral blood and bone marrow: utility as a tumor marker for neuroblastoma. Clin Cancer Res 1997; 3:821–826. 46. Mattano LA, Jr., Moss TJ, Emerson SG. Sensitive detection of rare circulating neuroblastoma cells by the reverse transcriptase-polymerase chain reaction. Cancer Res 1992; 52:4701–4705. 47. Ladetto M, Omede P, Sametti S, et al. Real-time polymerize chain reaction in multiple myeloma. Quantitative analysis of tumor contamination of stem cell harvests. Exp Hematol 2002; 30:529–536. 48. Ladetto M, Sametti S, Donovan JW, et al. A validated real-time quantitative PCR approach shows a correlation between tumor burden and successful ex vivo purging in follicular lymphoma patients. Exp Hematol 2001; 29:183–193. 49. Freedman AS, Neuberg D, Mauch P, et al. Long-term follow-up of autologous bone marrow transplantation in patients with relapsed follicular lymphoma. Blood 1999; 94:3325–3333. 50. Reynolds CP, Seeger RC, Vo DD, Black AT, Wells J, Ugelstad J. Model system for removing neuroblastoma cells from bone marrow using monoclonal antibodies and magnetic immunobeads. Cancer Res 1986; 46:5882–5886. 51. Seeger RC, Vo DD, Ugelstad J, Reynolds CP. Removal of neuroblastoma cells from bone marrow with monoclonal antibodies and magnetic immunobeads. Prog Clin Biol Res 1986; 211:285–293. 52. Rill DR, Santana VM, Roberts WM, et al. Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells. Blood 1994; 84:380–383. 53. Shpall EJ, Jones RB, Bearman SI. Transplantation of enriched CD34-positive autologous marrow into breast cancer patients following high-dose chemotherapy: influence of CD34-positive peripheral-blood progenitors and growth factors on engraftment. J Clin Onc 1994; 12:28–36. 54. Gordon PR, Leimig T, Babarin-Dorner A, et al. Large-scale isolation of CD133C progenitor cells from G-CSF mobilized peripheral blood stem cells. Bone Marrow Transplant 2003; 31:17–22. 55. Mackall CL, Stein D, Fleisher TA, et al. Prolonged CD4 depletion after sequential autologous peripheral blood progenitor cell infusions in children and young adults. Blood 2000; 96:754–762. 56. Molldrem JJ, Lee PP, Wang C, et al. Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Nat Med 2000; 6:1018–1023. 57. Srinivasan R, Barrett J, Childs R. Allogeneic stem cell transplantation as immunotherapy for nonhematological cancers. Semin Oncol 2004; 31:47–55. 58. August CS, Serota FT, Koch PA, et al. Treatment of advanced neuroblastoma with supralethal chemotherapy, radiation, and allogeneic or autologous marrow reconstitution. J Clin Oncol 1984; 2:609–616. 59. Ladenstein R, Lasset C, Hartmann O, et al. Comparison of auto versus allografting as consolidation of primary treatments in advanced neuroblastoma over one year of age at diagnosis: report from the European group for bone marrow transplantation. Bone Marrow Transplant 1994; 14:37–46. 60. Matthay KK, Seeger RC, Reynolds CP, et al. Allogeneic versus autologous purged bone marrow transplantation for neuroblastoma: a report from the Children’s Cancer Group. J Clin Oncol 1994; 12:2382–2389. 61. Inoue M, Nakano T, Yoneda A, et al. Graft-versus-tumor effect in a patient with advanced neuroblastoma who received HLA haplo-identical bone marrow transplantation. Bone Marrow Transplant 2003; 32:103–106. 62. London WB, Castleberry RP, Matthay KK, et al. Evidence for an age cutoff greater than 365 days for neuroblastoma risk group stratification in the Children’s Oncology Group. J Clin Oncol 2005; 23:6459–6465. 63. Schmidt ML, Lal A, Seeger RC, et al. Favorable prognosis for patients 12 to 18 months of age with stage 4 non-amplified MYCN neuroblastoma: a Children’s Cancer Group study. J Clin Oncol 2005; 23:6474–6480.

Appendix A Brief Overview of Hematopoietic Stem-Cell Transplantation Vicki L. Fisher Pediatric BMT Program, Rainbow Babies and Children’s Hospital, Cleveland, Ohio, U.S.A.

Linda Z. Abramovitz Pediatric Bone Marrow Transplant, Children’s Hospital at the University of California, San Francisco, California, U.S.A.

INTRODUCTION Hematopoietic Stem Cell Transplantation (HSCT) enables patients to receive potentially lethal doses of chemotherapy and/or radiation therapy followed by rescue with hematopoietic stem cells (HSC). In recent years there has been a dramatic increase in the number of transplants and transplant centers. According to the International Blood and Marrow Transplant Registry (IBMTR) in 2000 more than 30,000 transplants were performed worldwide in more than 350 centers. The treatment processes of HSCT vary somewhat, but the common goal for most diseases is cure. The role of transplantation in non-malignant diseases is to replace defective marrow; in transplant for solid tumors the goal of stem cell infusion is to rescue the marrow from irreversible aplasia after the patient has received toxic doses of myelosuppressive therapy aimed at eradicating the underlying disease. For leukemias, HSCT cures in three ways. First, by allowing the administration of high doses of chemotherapy, second by replacing malignant marrow, and finally, (in the allogeneic setting) by stimulating a Graft versus Leukemia (GVL) effect. The concept of transplanting blood and marrow stem cells seems simple in theory, however life threatening side effects and toxicities make care for the transplant patient extremely complex. The patients require sophisticated technology and procedures, a highly specialized team of health care workers, an adequately supportive environment, and many additional resources. Transplantation is defined as the transfer of living tissues or organs from one part of the body to another, or from one individual to another. There are three major types of HSCT: autologous, allogeneic, and syngeneic. Their names indicate the source of HSC that are infused into the recipient.

Autologous Hematopoietic Stem-Cell Transplantation Autologous HSCT involves the removal, storage, and re-infusion of the patient’s own healthy HSC. In essence, the autologous patient is his or her own donor. In autologous HSCT, the stem 601

602

Fisher and Abramovitz

cells are harvested from the patient weeks to months before the start of the preparative regimen and are cryopreserved (frozen) until “day 0.” Autologous stem cells can be harvested by peripheral apheresis or by a bone marrow harvest. High dose chemotherapy followed by autologous HSCT provides the opportunity to cure malignancies in situations where marrow toxicity prevents the adequate use of chemotherapy needed to cure these diseases. The failure of early studies was due primarily to disease re-occurrence or inadequate doses of chemotherapy. During the last decade, interest in autologous transplantation has escalated as a result of new technology for marrow storage, cryopreservation, and purging as well as improvements in supportive care. The major challenges still confronting autologous HSCT are major organ, nonhematologic, dose limiting toxicities, and relapse of the disease. Two theories as to the cause of relapse following autologous transplant include tumor contamination of the hematopoietic graft and inadequate treatment of minimal residual disease in the patient.

Allogeneic Hematopoietic Stem-Cell Transplantation In allogeneic transplantation, HSC are removed either by bone marrow harvest, peripheral apheresis, or are collected from the placenta and infused into the patient (recipient). The donor can be related (other than an identical twin) or unrelated. The ideal donor is human leukocyte antigen (HLA) identical to the patient. Commonly, the marrow is donated by a fully HLA matched sibling. Because only about 1 in 3 patients eligible for transplant have an HLA identical sibling, partially matched family members and unrelated donors from volunteer registries may be utilized as donors. The National Marrow Donor Program (NMDP) is the largest unrelated donor service in the world. The NMDP was established in 1986 and is an international leader in facilitating unrelated transplants. The median time from initiation of a preliminary search to transplantation is four months. HLA typing involves testing leukocytes to identify genetically inherited antigens common to both the donor and the patient. It is important to obtain a full antigen match for a hematopoietic transplant whenever possible to prevent the donor marrow (specifically the T-lymphocytes) from recognizing the recipient as foreign, leading to Graft-versus-Host disease (GVHD). GVHD is a unique complication of allogeneic transplantation that can be a major impediment to successful transplantation. Alternatively, the patient’s immune system can destroy the new bone marrow. This is referred to as graft rejection. In the last decade, advancements in biotechnology related to clinical HSCT have provided new therapies for the prevention and treatment of GVHD with monoclonal antibodies, and the use of hematopoietic growth factors that promote hematopoietic and immune reconstitution. The major challenges still facing transplant research are to reduce the mortality rate due to GVHD, to develop more effective conditioning regimens, and to safely transplant stem cells across major histocompatibility barriers.

Syngeneic Hematopoietic Stem-Cell Transplantation Syngeneic HSCT involves harvesting stem cells from one identical twin and infusing the cells into the other. Identical twins have identical genotypes and are considered a perfect match. Syngeneic transplants were first attempted in the early 1960s to treat Severe Aplastic Anemia and allowed investigators to prove for the first time that the hematopoietic system in humans could be replaced by that of a genetically identical donor. This type of transplant has become relatively routine with few complications. A higher incidence of leukemic relapse has been reported in syngeneic HSCT than allogeneic marrow recipients because of the demonstrated anti-leukemic effect of GVHD, a term known as the GVL.

Appendix

603

PRETRANSPLANT CONSIDERATONS Prehematopoietic Stem-Cell Transplantation Evaluation In some instances, patients are referred to a transplant center, since not all institutions perform blood and marrow transplants. In a majority of situations, however, the patients are identified as needing a transplant while on the hematology/oncology service. They are then referred to the transplant service within their own institution. Communication is vital to a smooth transition, regardless of whether or not the patient is an external or internal referral. The patient and family should receive a variety of educational materials: pamphlets, notebooks, videos, and computer programs that describe the transplant process. It is helpful if a portion of the material is sent or given to the patient prior to the first meeting. The initial consultation can be quite overwhelming for the patient and his family. During the initial consultation, a detailed explanation of the transplant process, potential risks and benefits of transplant and other questions are answered. A professional similar to a transplant coordinator or case manager who understands the patient’s history, the transplant process, and insurance terminology can assist with this process. The patient’s eligibility for transplantation includes an extensive medical work up, which includes a variety of physiological and psychological assessments that determine disease status, organ function and emotional status. Also assessed is the family’s ability to emotionally and physically care for this child through the trajectory of transplantation. Table 1 outlines the evaluation required for HSCT. During the pre-transplant phase; which may last several days to one week; the patient and family continue to learn about the transplant process. The education and preparation of the patient and family before transplantation are critical to the subsequent adjustment and adaptation during and after the transplant process. Table 2 outlines the necessary components of the transplant education plan.

Donor Issues When there is not a sibling or family donor that is suitable, a search is initiated through the national and international unrelated registries. Table 3 reviews the numerous unrelated banks. A preliminary search is initiated through the registries and results are quickly obtained. The information available on the potential donors is variable in detail in terms of the degree of HLA typing, stem cell numbers and cytomegalovirus (CMV) status. There are numerous factors that Table 1

Evaluation of the Pediatric Blood and Marrow Transplant Patient

Comprehensive history and physical Comprehensive organ function assessment CBC with differential Chemistry panel Liver function tests Urine/serum creatinine clearance or glomerular filtration rate Hepatitis, cytomegalovirus, herpes virus, varicella titers, EBV, HIV testing Cardiac function: Echocardiogram and EKG Pulmonary function: PFTs, VBG and pulse oximetry Chest X-ray Dental assessment Audiogram Nutritional assessment Neuropsychological testing and psychosocial assessment Tumor/disease status Abbreviations: CBC, complete blood count; EBV, Epstein-Barr virus; HIV, human immunodeficiency virus; EKG, electrocardiogram, PFT, pulmonary function test, VBG, venous blood gas.

604 Table 2

Fisher and Abramovitz Transplant Education Plan

Materials may be written, computer based and audiovisual Orientation to transplant unit Role and rationale of blood and marrow transplantation Type of transplant Preparative/conditioning regimen Pretransplant evaluation PBSCs versus bone marrow Apheresis versus harvest Donor evaluation Transplant related routines/restrictions Stem cell infusion Peri-transplant toxicities/complications Intensive care issues Advance directives Graft-versus-Host Disease Immunosuppressive therapy Discharge guidelines/planning Home care management Posttransplant complications Posttransplant aftercare Posttransplant follow-up Family/sibling issues Financial issues Lodging/transportation resources Late effects/survivorship issues Community and hospital based resources Relapse Physical changes during transplantation

influence the selection of the donor: diagnosis, type of transplant planned, the weight of the recipient (especially when umbilical cord blood transplant is being considered). Eventually, potential donors will be identified and confirmatory typing will be performed. The confirmatory typing involves repeating the HLA typing at the transplant center. The process of obtaining the confirmatory typing can be time consuming, so usually a couple of donors are processed at the same time. Once the transplant center has the confirmatory typing a decision is made on the suitability of various donors (assuming there is more than one). Factors that are considered Table 3

Major National Unrelated Donor Registries Organization National Marrow Donor Program (NMDP) coordinating center for national bone marrow/cord blood donor searches Caitlin Raymond International Registry Coordinating Center for international bone marrow and cord blood searches Gift of Life Donor recruitment, patient advocacy, search correlation, family studies/genealogy. Complied of entirely Of Jewish Volunteer donors American Red Cross Cord Blood Program Lists cord blood units from multiple Red cross sites American Bone Marrow Donor Registry Coordinated by Caitlin Raymond International Registry

Appendix

605

include the age and sex of the donor, number or pregnancies in female donors, CMV status, and ABO incompatibility.

Donor Evaluation Once a donor has been identified; a pre donation evaluation is preformed, which is similar to the recipient’s evaluation process. Regardless of whether the cells are collected by peripheral apheresis or bone marrow harvest, emotional preparation and teaching are essential for the donor since both forms of collection can be frightening to the donor. Children can safely donate peripheral blood stem cells and bone marrow with parental consent.

The Collection of Stem Cells Stem cells can be collected via peripheral apheresis or bone marrow harvest. Both types of products can be cryopreserved for future use. All products are tested for cell numbers and sterility.

Bone Marrow Harvest Harvesting is performed in the operating suite utilizing sterile technique. The bone marrow is generally harvested through the posterior iliac crests. The bone marrow aspirating needles are inserted 50–100 times on each side to aspirate the desired amount of stem cells. In a large majority of the cases, there are only two skin entry points, despite the numerous bone marrow aspiration sites, with the aspiration needles being angled at different trajectories through the same skin entry point. The harvested cells are placed in a heparinized medium to prevent clotting and then filtered to remove bone spicules, fat and thrombi. Once the procedure is completed, the area is cleansed, and a pressure dressing is applied. The parents and donor are instructed to keep the dressing clean and dry to prevent infection. There is mild to moderate pain associated with a bone marrow harvest, which can be alleviated with a non-steroidal agent or acetaminophen. The requisite number of cells required for transplantation is 2–5!108 nucleated cells/kilogram of recipient’s weight. The usual volume collected is 10–20 ml/kg of the donor’s weight. In the allogeneic transplant process, the bone marrow is given to the recipient on the same day. The bone marrow harvest procedure is essentially the same for the autologous stem cell transplant donor. It is often necessary to collect an additional amount of bone marrow to compensate for a lower concentration of cells in a marrow that has been compromised secondary to chemotherapy and/or radiation. Autologous marrow is cryopreserved, or frozen until the patient’s “day 0.” The cryopreservation process involves the use of a cryoprotectant such as dimethyl sulfoxide (DMSO); which protects the stem cells from lysis caused by the formation of microscopic ice crystals at low temperatures.

Leukopheresis Hematopoietic progenitor cells reside in the bone marrow and can be detected in small numbers in the peripheral blood. In order to collect adequate stem cells by leukopheresis, the cells must move from the marrow space to the peripheral circulation. This process is called “mobilization” and occurs in autologous patients during the recovery phase following myelosuppressive therapy. It is greatly enhance by the simultaneous administration of growth factors such as granulocyte colony stimulating factor (G-CSF). In the allogeneic transplant setting, the “normal donor” is given G-CSF for four days prior to the first day of apheresis. The dose of G-CSF is generally doubled for those four days to 10 mcg/kg. The collection (leukopheresis) refers to the day of the procedure and the volume collected. The mechanics of the apheresis machine is

606 Table 4

Fisher and Abramovitz Special Considerations for Pediatric Leukapheresis

Venous access

Hypocalcemia

Hypotension

Safety

Temporary or permanent central venous catheters Large bore: 2.0 mm Catheter recommendations in !25 kg patients: 10 Fr Hickman/Broviac or 7Fr double lumen apheresis catheter Occurs due to citrate toxicity Watch closely for clinical signs: tingling of extremities, lip smacking in infants, muscle cramps, chills, vertigo, and nausea Have child eat/drink calcium rich diet May use prophylactic oral calcium supplements Secondary to fluid shifts during apheresis procedure Baseline hematocrit Blood prime of apheresis machine in small children Watch closely for fluid shifts Emergency equipment and procedures in place at site of apheresis Trained personnel Psychological support is necessary Distraction for the duration of the procedure Consultation of child life specialist

similar to that of the dialysis machine. The patient requires two venous lines, one line that draws blood out, and one that returns it to the patient. The apheresis machine centrifuges blood drawn from the patient, separating different blood components based on density, with the leukocyte layer being removed into a collection bag and the remainder returned to the patient. The timing of the stem cell collection, particularly in the autologous patient weighing less than 25 kilograms requires considerable organization, coordination and proactive planning. The WBC and CD34C cell count is monitored to determine the correct timing of apheresis. In the autologous patient it is important to identify the point when the patient is recovering from their chemotherapy nadir in order to ensure collection of the largest numbers of stem cells in the least number of collections. Table 4 outlines the special needs of the pediatric patient during apheresis.

THE TRANSPLANT PROCESS The Preparative Regimen The ideal preparative regimen is capable of eradicating malignancy, has tolerable morbidity without mortality, and has a sufficient immunosuppressive effect on the recipient marrow (in the allogeneic setting) to avoid graft rejection. Preparative regimens vary according to disease, the medical condition of the patient, and institutional protocols and biases. Table 5 lists the ideal properties of a potential preparative regimen. Total body irradiation (TBI), total lymphoid irradiation, or total abdominal irradiation can be used both for immunosuppression of patients and to eradicate disease. In addition, localized radiation may be used to focus treatment on presumed areas of local disease. TBI may be given in one dose or in multiple doses over the course of several days (fractionated radiation therapy). Fractionated dosing schedules appear to minimize adverse effects and are generally preferred over single doses. The child receiving TBI may require sedation or general anesthesia for each radiation session. The treatments, which may be once or twice daily, will require that the child be NPO for several hours prior to each radiation session. The preparative regimen may take place in the inpatient or outpatient setting. In the majority of cases, the patient will be admitted to a room on a unit designated for transplant patients. Isolation techniques vary among transplant institutions; they include reverse isolation

Appendix Table 5

607

Ideal Properties of Blood and Marrow Transplant Preparative Regimens Allogeneic for malignant disease

Allogeneic for nonmalignant disease

Autologous for malignant disease

Yes

Yes

No

Yes

No

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Immunosuppression (antigraft rejection) Eradicate malignancy (antitumor) Make space for new marrow (ablative) Avoid/minimize overlapping toxicity

or reverse isolation with special air handling systems, including high efficiency particulate air filters. The actual transplant process begins with the administration of the preparative regimen, also known as the conditioning regimen. The scheduled days of the preparative regimen are generally referred to in negative numbers, counting down to day 0, which is the day that the HSC are infused. Central venous access is required in pediatric transplant patients. The size of the catheter is dependent on the size of the transplant recipient. The catheter is used to deliver IV fluids, mediations, blood products, and stem cells and to obtain blood specimens. Table 6 reviews chemotherapy agents commonly used in preparative regimens.

Stem-Cell Infusion Prior to the infusion of stem cells, the patient is usually premedicated to lessen the side effects associated with the infusion. Most pre-medication regimens include anti-emetic and antihistamine medications. Table 7 lists the different medications and doses used as premedications. The patient is usually hydrated prior to and following the infusion to allow the renal clearance of cellular debris associated with the freeze/thaw process, or with stem cell collection. The stem cells are thawed in a water bath at approximately 37 degrees Celsius until the product is in the liquid phase. The stem cells are then quickly infused via a central venous catheter using a syringe or a gravity infusion method. Peristaltic pumps are not used because of possible damage to the cells from the pumps. During the infusion, and for several hours following, the patient may experience side effects associated with the DMSO. The common side effects of stem cell infusion are listed in Table 8. It has been reported that patients who received larger volumes of stem cells, containing a greater number of red cells, had more frequent and severe side effects.

Acute Toxicities of Transplantation The time between stem cell infusion and marrow recovery (engraftment) is a critical time, with the patient at risk for many life-threatening toxicities. The acute complications that children experience post transplant are similar to those experienced by adults. Table 9 provides an overview of the toxicities seen in the post transplant setting.

Hematologic Toxicities The most common toxicity seen post transplant is hematologic; which includes neutropenia, thrombocytopenia, anemia, delayed engraftment and graft failure. In most cases the causative

Alkylating agent

Alkylating agent

Alkylating agent

Alkylating agent

Busulfan

Carmustine

Carboplatin

Classification

Special considerations/management It is administered intravenously (IV), generally used with total body irradiation (TBI) or busulfan. Hemorrhagic cystitis is the most common toxicity associated with high dose cytoxan. Mesna and hyperhydration are given to protect the bladder during cytoxan administration. Nausea and vomiting: aggressive anti-emetic therapy and IV hydration. Syndrome of inappropriate antidiuretic hormone (SIADH) is characterized by hyponatremia and plasma hypo-osmolality induced water retention caused by persistent antidiuretic hormone (ADH) release. Cardiac monitoring may be indicated; guidelines are institution-specific Administered orally or IV, commonly given with Cyclophosphamide. There are specific guidelines (protocol and industry recommended) for administration and re-administration of the oral preparation of busulfan. Seizures are associated with the administration of Busulfan. Anti-convulsants are recommended. Pharmacokinetics studies are also recommended in pediatric patients to monitor the variable absorption and metabolism of the drug that can be variable. If the patient does not receive an adequate drug level, the desired effect of the drug may not be achieved. Conversely if the patient receives an excessive dose of the drug, hepatotoxicity may occur. Skin changes resulting from Busulfan include dermatitis and hyperpigmentation Used in Hodgkin’s Disease or brain tumors conditioning regimens The most common toxicity is pulmonary. The use of steroids during the conditioning regimen is institution and protocol specific. When Carmustine is used in conjunction with cyclophosphamide and pulmonary radiation, the patient may experience severe pulmonary toxicity. Nausea and vomiting; aggressive anti-emetic therapy Used in solid tumor conditioning regimens, such as neuroblastoma and brain tumors. It is administered IV. A glomerular filtration rate should be determined to be adequate prior to starting the preparative regimen; the dose should be adjusted accordingly. Nausea and vomiting; aggressive anti-emetic therapy. Ototoxicity. A baseline audiogram should be obtained and hearing subsequently monitored post BMT. Peripheral neuropathy and paresthesias occur from high dose carboplatin, monitor patient for symptoms and safety issues should be addressed

Common Chemotherapy Agents Used During Blood and Marrow Transplant

Cyclophosphamide

Agent

Table 6

608 Fisher and Abramovitz

Antimetabolite

Antimetabolite

Plant alkaloid/irritant

Alkylating agent

Nitrosourea

Fludarabine

Cytosine Arabinoside

Etoposide

Thiotepa

Melphalan

Used in reduced intensity regimens. It is administered IV. Nausea and vomiting; aggressive anti-emetic therapy. Fludarabine can cause pulmonary fibrosis. GI toxicity is seen with fludarabine with the patient experiencing severe mucositis. Hypersensitivity reactions are common with the administration of Fludarabine. The recipient may experience fever, chills and anaphylaxis. The pre-medications utilized with the administration of Fludarabine are protocol and institution-specific Used in hematologic malignancies, cytarabine is often used in combination with cyclophosphamide, fludarabine or TBI. It is administered IV. Nausea and vomiting; aggressive anti-emetic therapy. Chemical conjunctivitis is a major toxicity of cytarabine. Prophylaxis with steroid eye drops is strongly recommended to start 12 hours before the start of cytarabine and for 48 hours after the medication stops. Flu-like symptoms and fevers are commonly seen with the administration of high dose cytarabine Used in numerous BMT conditioning regimens for hematologic malignancies and solid tumors. It is administered IV. Nausea and vomiting; aggressive anti-emetic therapy. Hypersensitivity reactions are common with the administration of high dose etoposide. The recipient may experience fever, hypotension and anaphylaxis. It is desirable to have emergency medications readily available according to institution-guidelines. High dose etoposide is toxic to the liver; close monitoring of liver function tests is recommended. Peripheral neuropathy and paresthesias are associated with high dose etoposide; monitor for symptoms and safety issues should be addressed. Dilution of etoposide is protocol and institutional specific. The medication precipitates easily Used in solid tumors. It is administered IV. Is excreted through the skin to a degree that skin irritation, rash or sloughing may occur. Bathing/skin care protocols are institution-specific. Patients receiving thiotepa require aggressive IV hydration, accurate intake and output recording and close monitoring of renal function. Nausea and vomiting; aggressive antiemetic therapy Used in hematologic malignancies in neuroblastoma. Melphalan causes excessive GI toxicity with the patient experiencing severe mucositis. The patient receiving melphalan requires aggressive oral care and close observation of the airway due to severe inflammation of the mucosal tissues. Skin changes resulting from melphalan include dermatitis and hyperpigmentation also known as the “melphalan tan.” The dermatitis may be limited and improved with lotions and meticulous skin care. The hyperpigmentation is self-limiting; it resolves several months after transplantation. Nausea and vomiting; aggressive anti-emetic therapy

Appendix 609

610 Table 7

Fisher and Abramovitz Premedications for Stem-Cell Infusion

Medication Acetaminophen Diphenhydramine hydrochloride Furosemide Granisetron Lorazepam Mannitol Methylprednisolone Ondansetron

Suggested pediatric dosing children !50 kg 10–15 mg/kg Q 4–6 hours 0.5–1 mg/kg Q 4–6 hours

Suggested pediatric dosing for children O50 kg 650 mg Q 4–6 hours 25 mg–50 mg Q 4–6 hours

0.5–2 mg/kg Q 6–12 hours 10–20 mg/kg Q 6–8 hours 0.04–0.08 mg/kg Q 6 hours 0.2–0.5 g/kg Q 4–6 hours 0.5–1.7 mg/kg Q 6–12 hours 0.15 mg/kg Q 4–6 hours

20–40 mg 1–2 mg/daily 1–2 mg Q 2–4 hours 12.5–25 g Q 1–2 hours 100 mg Q 4–6 hours 8–10 mg Q 8 hours

agent is the preparative regimen, however, other etiologic factors may also play a role. Infused stem cell dose, infection, and pharmacologic agents can all impact the time to engraftment.

Neutropenia Infection is the major cause of morbidity and mortality in the clinical transplant setting until neutropenia resolves. Neutropenia is defined as a decrease in the number of circulating neutrophils and is the most severe consequence of bone marrow suppression, placing the patient at increased risk of severe and life-threatening infections. Prolonged periods of neutropenia, combined with the various other toxicities of transplant, including mucositis, impaired skin integrity, GVHD, immunosuppressive therapy, and malnutrition all increase the patient’s risk of morbidity and mortality during HSCT. The duration of neutropenia in the transplant setting is dependent on several factors: type of cells infused (marrow or peripheral stem cells), number of cells infused, previous therapy (which impacts upon the quality of the stem cells and the marrow microenvironment), use of colony stimulating factors, and post transplant complications. The use of colony stimulating factors after transplant has decreased the duration of neutropenia and improved outcomes following autologous stem cell transplantation. The more prolonged the period of neutropenia, the greater the risk of infection; therefore decreasing the time interval between stem cell infusion and neutrophil engraftment directly impact’s upon the patient’s outcome. The infectious risks during HSCT are divided into three phases: pre-engraftment, post engraftment and late post transplant. Table 10 outlines the different infectious organisms commonly seen, and methods of surveillance. Table 8

Potential Side Effects of Stem-Cell Infusion

Cardiac Bradycardia, tachycardia, hypotension, hypertension, circulatory overload Pulmonary chest pain, cough, dyspnea Gastrointestinal Nausea, vomiting, abdominal cramps Renal Fluid overload, hemoglobinuria Immunologic Anaphylaxis, fever, chills, allergic reaction, flushing, rash, erythema Neurologic Headache

Appendix Table 9

611

Common Toxicities During Transplant

Hematologic Neutropenia Anemia Thrombocytopenia Infectious Bacterial infections Viral Infections Fungal Infections GI Mucositis Nausea/vomiting Diarrhea Malnutrition Hepatic Veno-occlusive disease Chemical hepatitis Renal Insufficiency Urologic Hemorrhagic cystitis Pulmonary Pneumonia Interstitial pneumonitis Idiopathic pneumonia syndrome (IPS) Cardiac Pericardial effusions Pericarditis Integumentary Infection Radiation recall

In addition to infectious risks due to severe neutropenia, the risk of infection is also increased by an alteration in the patient’s mucosal barriers (mucositis) as well as the interruption of skin integrity from venous access lines. Previous exposure to infections such as herpes simplex virus (HSV) and CMV are also risks because of the possibility of reactivation during this time of extreme stress and immune suppression. Nevertheless, bacteria are the most common cause of infection during the pre-engraftment period. A thorough assessment of any febrile child during the transplant process is a necessity. The evaluation includes monitoring for hemodynamic instability, assessing the patient’s skin for abnormal integrity, erythema, swelling or drainage, particularly the central venous catheter site, as well as monitoring the perineum for peri-rectal tenderness, abscess or fissures. Treatment of fever and neutropenia with intravenous antibiotic therapy is imperative. Empiric antifungal therapy is often added if the fever persists.

Thrombocytopenia Thrombocytopenia is defined as having fewer than 100,000/mm3 circulating platelets. This can be caused by marrow suppression from the preparative regimen, or increased platelet consumption from toxicities such as disseminated intravascular coagulation or veno-occlusive disease (VOD). Fever and other conditions may also exacerbate thrombocytopenia from increased consumption. Spontaneous bleeding is generally associated with a platelet count of !10,000/mm3. Children may present with petechiae, increased bruising, epistaxis, or other

612 Table 10

Fisher and Abramovitz Common Infectious Organisms Seen During Blood and Marrow Transplant

General classification

Specific type of organism

Bacterial

Gram positive Staphylococcus epidermidis Corynebacterium Coagulase negative staphylococci Enterococcus Streptococcus viridians Staphylococcus aureus Gram negative Actinobacter Escherichia coli Pseudomonas species Enterobacter species Klebsiella species Cytomegalovirus Human herpes viruses Community respiratory viruses Human polymaviruses Candida species Aspergillus species Pneumocystis carinii Toxoplasmosis gondii

Viral

Fungal Protozoal

mucosal bleeding. Clearly platelet transfusions are indicated when the child is bleeding. They are also generally given “prophylactically” when the child’s platelet count is 10,000– 20,000 cells/mm3. HLA matched platelets may be necessary for patients who become refractory to random platelet transfusions.

Anemia Anemia is defined as a decrease in red cell mass and in the transplant setting is usually the result of a combination of myelosuppressive chemotherapy, radiation, blood loss, viral suppression or the effects of chronic disease. All transfused blood products should be irradiated to prevent transfusion associated GVHD caused by the ability of leukocytes from the transfusion to recognize and proliferate in response to allo-antigens expressed by the recipient, combined with the immunosuppressed recipient’s inability to reject these cells. Leukocyte reduction filters can remove most, but not all, WBC from the product, thereby decreasing the number of transfusion reactions, as well as reducing the likelihood of CMV transmission.

Infectious Complications Bacterial infections are the most common etiology of infection in the clinical transplant setting. Normal bacterial flora that colonizes all patients can result in infection when the patient is immunocompromised and mucosal barriers are altered. Mucositis caused by high dose therapy makes the gastrointestinal (GI) tract one of the most common sources of infection. Fungi are another important cause of severe infections. HSCT treatment measures, including antibiotics, catheters, and total parental nutrition can all contribute to the development of fungal infections. Environmental factors such as nearby construction work can also contribute to the development of fungal infections, especially aspergillus. Detecting fungal infections can be very difficult, and anti-fungal therapy is often started empirically when

Appendix

613

fever persists in spite of the use of broad-spectrum antibiotics. Many centers use prophylactic anti-fungal agents in an attempt to decrease fungal infections. Oropharyngeal mucositis is a common complication and colonization by gram positive or gram-negative organisms particularly Pseudomonas species, Klebsiella, and Enterobacter species frequently occurs. Spread to the lungs may occur by direct aspiration or indirectly as a result of bactiremia leading to lower respiratory tract infections. Empiric treatment is usually initiated when the patients become febrile, and most antibiotic regimens are designed to treat both gram positive and gram-negative infections. Early intervention results in the resolution of most bacterial infections. If the patient does not respond to antibiotic therapy and exhibits signs of pulmonary infection, a bronchoscopy with a bronchoalveolar lavage may be performed in an attempt to identify the causative organism. HSV and CMV account for the majority of post transplant viral infections. Other respiratory viruses that have been implicated in episodes of post transplant pneumonitis include respiratory syncytial virus, parainfluenza virus, and adenovirus.

Gastrointestinal Complications The GI system is significantly affected by myeloablative therapy. The most common GI toxicities include mucositis, nausea, vomiting, diarrhea, and malnutrition. Neutropenic colitis (e.g., Typhlitiis) is a life threatening GI complication that may also occur in these patients

Mucositis Inflammation of the oral mucosa known, as mucositis or stomatitis is a common toxicity caused by the preparative regimen. Cell destruction by chemotherapy or radiation, and inadequate production of new epithelial cells results in a loss of mucosal integrity. The consequences of mucositis include painful lesions that often result in decreased oral intake and poor nutrition. Often the patient requires parental narcotics for pain management. The established use of the Wong-Baker Faces Pain Rating Scale is important during transplantation in order to assess the amount of pain the child is experiencing. The compromised oral mucosa is of the utmost concern because it provides a portal of entry for infection in the immunocompromised patient. The onset, duration, and severity of mucositis is related to the agents used during the preparative regimen. Stomatitis/mucositis can range from mild erythema of the oral mucosa to consolidative ulcerations. Other signs and symptoms include pain, difficulty swallowing, thick oral secretions, white patches, cracked/dried lips and inability to handle secretions (e.g., drooling). Fungal infections of the oral cavity are commonly caused by candida albicans; which presents as white plaques with indurated borders. The tongue is often swollen, dried and cracked. HSV can present as painful blisters on the lips or anywhere in the oral cavity, accompanied by a yellowish brown membrane. Airway obstruction may result from edema associated with severe mucositis. This is of particular concern in smaller children as they have considerably smaller airways. Maintaining adequate nutrition and hydration is a challenge for the child and young adult with moderate to severe mucositis. A soft diet with cool and bland foods may be most widely accepted form of nutrition. Palifermin (Kepivance e), which is a recombinant keratinocyte growth factor, has recently been approved for the prevention of mucositis. At this time, clinical experience with it is limited. The use of Glutamine, an essential amino acid, can also be helpful in decreasing the severity of mucositis. Topical antiseptic rinses with chlorhexidene (Peridex) or Biotene are commonly used to suppress oral flora and prevent mucositis. Recovery from mucositis often occurs at the same time as engraftment, as circulating neutrophils become available to promote healing, and epithelial proliferation resumes.

614

Fisher and Abramovitz

Nausea and Vomiting Nausea and vomiting are common side effects of cancer treatment. It may be acute, delayed, or anticipatory. There are a variety of techniques that are used to control or prevent nausea and vomiting. The most common is the administration of anti-emetics. There are numerous categories of anti-emetics that are used in children and young adults undergoing transplantation. They include serotonin antagonists (ondansetron, granisetron and dolasetron); phenothiazines (chlorpromazine, prochlorperazine); corticosteroids (dexamethasone and methylprednisolone), benzodiazepines (lorazepam); anti-histamines (diphenhydramine and hydroxyzine), procainamide derivatives (metoclopramide), and cannabinoid (THC). Nonpharmacologic techniques can be combined with anti-emetic therapies depending on the patient’s age and preferences.

Diarrhea Diarrhea is defined as an abnormal increase in the quantity, frequency and liquidity of stool. Diarrhea in the pediatric transplant patient can be particularly debilitating and can quickly lead to severe dehydration, renal insufficiency, electrolyte imbalances, and impaired skin integrity. Determining the etiology of diarrhea is of great importance in identifying the proper treatment course. In the pediatric transplant patient the most common cause of diarrhea is from TBI, which can causes radiation enteritis. The clinical presentation of diarrheal illness depends largely on the type and cause. Patients may have stool varying in consistency, water content, quantity and frequency. They may also experience dehydration, electrolyte imbalances, abdominal cramping, rectal excoriation or ulceration, and blood or mucous discharge in the stools. The goal of diarrhea management is to restore normal bowel habits, maintain adequate nutrition, restore fluid and electrolyte balance, protect skin integrity, enable the patient to achieve comfort and maintain dignity, and to support the patient in resuming the activities of daily living. The management of diarrhea is directly related to its cause and therefore establishing the underlying cause is the first step in effective treatment. Extensive diarrhea is very debilitating for the child undergoing transplant and young children who may have been toilet trained may regress during transplantation. Protection of the peri-rectal area can be accomplished with a barrier cream, and/or sitz baths.

Malnutrition Nutritional support is an integral component of transplant care that can directly affect clinical outcomes. Adequate nutritional intake is critical because the maintenance of a positive nitrogen balance supports wound healing, and enhances the immune response. TBI can affect the tissues of the salivary glands and oral mucosa, resulting in xerostomia, stomatitis, dysphagia, and taste alteration. Anorexia is frequently seen in pediatric transplant patients, and is a complex and multifactorial problem. Contributing factors include nausea, vomiting, taste alteration, early satiety, anxiety, depression, and environmental changes. Clinical features include muscle wasting, weakness, anemia, and metabolic deficit. The primary goals of nutritional intervention are to restore and promote growth and development and to minimize the side effects of therapy. The pre-transplant assessment of the child’s nutritional status provides the basis for developing a supportive nutritional plan. To prevent malnutrition and subsequent treatment complications, nutritional interventions are initiated early. Nutritional management may include diet modifications, oral supplements, enteral feedings, and parental nutrition. Controversy often exists over the choice of enteral feeds versus parental nutrition. Unless contraindicated, the enteral approach to nutritional support is more advantageous because continued stimulation of the intestine prevents atrophy of the intestinal villi and minimizes the change in bacterial flora and the risk of sepsis.

Appendix

615

Hepatic Complications Side effects following HSCT that involve the liver include VOD and hepatic injury caused by medications and parental nutrition.

Veno-occlusive Disease VOD of the liver is one of the major concerns during transplantation. Its incidence ranges from 2–54% of pediatric stem cell transplant patients. Clinical presentation includes hyperbilirubinemia, hepatomegaly and tenderness, and fluid retention, including edema and ascites. There is no proven method for preventing or treating VOD. VOD is caused by damage to the endothelial cells in the hepatic venules and sinusoids as a result of activation of the coagulation cascade. Damaged endothelial cells and fibrin occlude the sinusoidal pores, causing obstruction to venous blood flow. The result is leakage of protein rich fluid from the liver sinusoids into the extravascular spaces, resulting in ascites. No laboratory tests are specific for VOD and the diagnosis of VOD is a clinical one. The Fred Hutchinson transplant program in Seattle has developed criteria for the diagnosis of the VOD. They are jaundice, painful hepatomegaly and fluid retention. Two of three criteria are required by the 20th day after transplant for the definitive diagnosis of VOD. Risk factors for the development of VOD include a previous stem cell transplant, elevated liver transaminases prior to the start of ablative therapy, prior hepatic area radiation, and bacterial or viral treatment during the preparative regimen. Severity can range from mild to severe, including fatal. Supportive care is the hallmark of treatment, which includes maintaining intravascular volume status in order to optimize intrahepatic and intrarenal perfusion. Defibrotide is a new agent that has shown promise in treating VOD.

Chemical Hepatitis Chemical hepatitis is an inflammation of the liver that can be caused by exposure to therapeutic agents such as chemotherapy and radiation. The liver plays a central role in the metabolism of many chemotherapy agents and is therefore a target for drug toxicity. The clinical presentation of chemical hepatitis varies, as does the extent of hepatic injury. Chemical hepatitis is usually identified first by an elevation in liver transaminases (e.g., AST, ALT). This results from the lysis of hepatocytes, which release these proteins into the circulation. The patient may also exhibit clinical signs and symptoms of hepatitis including jaundice, right upper quadrant pain, fever, diaphoresis, malaise, flu-like symptoms, nausea, vomiting, anorexia, bruising or bleeding. Because there is no specific test for chemical or drug induced hepatitis, the diagnosis requires a careful medication history, precise documentation as to the onset of symptoms, the exclusion of infectious forms of hepatitis and the exclusion of other disorders. Treatment for the patient experiencing chemical hepatitis is directed to identifying the cause of hepatic injury and modifying or removing the offending agent to decrease toxicity.

Renal Insufficiency Both the conditioning therapy used as part of transplant and other nephrotoxic medications, such as antibiotics, can contribute to renal insufficiency by causing tubular damage. Infection can also play a role in renal injury. This damage may lead to excessive wasting of electrolytes, particularly magnesium, potassium, bicarbonate and phosphorus. The patient experiencing tubular damage may exhibit clinical signs and symptoms related to electrolyte imbalance and wasting of amino acids and glucose in the urine. The patient may also have metabolic acidosis from excessive urinary bicarbonate losses or fluid disturbances. Adequate hydration, the maintenance of intravascular fluid balance, and the use of diuretics (only after adequate intravascular is achieved) are all necessary supportive care measures. If electrolyte imbalances occur, correction with oral or intravenous supplementation may be necessary. Hemodialysis or continuous renal ultrafiltration therapy may be required to help support the patient until the renal injury resolves.

616

Fisher and Abramovitz

Urologic Complications Hemorrhagic cystitis is defined as dysuria from bleeding and inflammation in the bladder with the presence of leukocytes, erythrocytes, and thrombi in the urine. In the pediatric HSCT patient, hemorrhagic cystitis is frequently seen as a complication of cyclophosphamide therapy. One of the metabolites of cyclophosphamide is acrolein, which is excreted in the urine and can cause irritation necrosis of the bladder lining. Necrosis subsequently expose the submucosal blood vessels of the bladder which can rupture, causing hematuria ranging from microscopic to grossly visible. The clinical signs of hemorrhagic cystitis include mild to severe dysuria and hematuria. Ultrasonography may reveal an edematous hemorrhagic bladder. Hemorrhagic cystitis can be prevented by the use of Mesna (2-Mercaptoethane Sulfonate Sodium), which is an intravenous drug that binds acrolein in the bladder and prevents damage to the bladder wall. It is generally given at the same time as cyclophosphamide, with several subsequent doses given over the next twelve hours. Patients who receive Mesna but still develop hemorrhagic cystitis may receive aggressive intravenous hydration and/or bladder irrigation, as well as the possible placement of a Foley catheter to improve rapid bladder emptying. Bleeding may be exacerbated by thrombocytopenia, so platelet transfusions may be required to decrease bleeding. Infection may also cause bladder toxicity. Viruses such as adenovirus and BK virus are two common causes of bladder infection leading to bleeding, dysuria, and bladder pain. Medications such as oxybutynin (Ditropane) may be used to control bladder spasms.

Pulmonary Complications Pulmonary complications following HSCT are a major cause of morbidity and mortality, affecting between 40–60% of the patients. Spontaneous pneumothorax may occur in the acute phase of transplant, with predisposing factors including high dose steroids, TBI, and poor nutrition with recent weight loss. Pulmonary edema can also be seen in the first few days following HSCT and is due to capillary leakage from the toxicity of the myeloablative therapy. Previous exposure to anthracyclines and the use of cyclophosphamide and TBI as part of the conditioning therapy can exacerbate this complication. Pulmonary hemorrhage is generally associated with infection and thrombocytopenia, but an often-fatal form can occur as a rare complication of high dose cyclosporine.

Pneumonia Pneumonia can occur in 20–50% of individuals during the acute neutropenic phase following transplant. There may be few clinical or radiographic signs because of the inability to mount an inflammatory reaction in a neutropenic patient. Bacteria, fungi, and viruses can all be the etiologic agent in pneumonia.

Interstitial Pneumonitis Interstitial pneumonitis (IPN) is the most common pulmonary complication following HSCT. It occurs in 40% of all transplant cases and is fatal in 60% of the cases. IPN is the leading cause of pulmonary death in transplantation.

Idiopathic Pneumonia Syndrome Idiopathic pneumonia syndrome (IPS) is a noninfectious interstitial pneumonia, which generally occurs during engraftment. IPS is more common in the allograft setting than the autograft setting. This fact suggests that there is a strong immunologic component to this syndrome, perhaps in combination with toxicity from the conditioning regimen. There is some evidence that T lymphocytes play a key role in the initial immunologic event and there is

Appendix

617

a correlation between the onset of IPS and GVHD. Preliminary studies have shown that Etanercept (Enbrele) can modify this disease and improve the outcome.

Cardiovascular Complications Cardiac complications related to therapy may be categorized as acute and chronic. They are also categorized according to their cardiac effect. These include decreased left ventricular function, ischemia, and conduction problems resulting in dysrhythmias. Complications may arise as a direct result of chemotherapy (e.g., anthracyclines, cyclophosphamide), radiation to the mediastinum, or direct tumor invasion into the heart. Anthracyclines cause both and acute and long-term cardiotoxic events. Reports in the adult literature indicate that these incidents increase rapidly in patients who receive a cumulative dose O450 mg/m2 for both doxorubicin and daunorubicin, and 125 mg/m2 for idarubicin. However there are reports of toxicity occurring in cumulative doses as low as 200 mg/m2. Chemotherapy induced cardiotoxicity generally manifests itself as congestive heart failure with shortness of breath, exercise/activity intolerance, chronic cough, weight gain, pulmonary edema, and/or frequent respiratory infections.

Pericardial Effusions The pericardial membranous sac surrounding the heart normally contains !50 cc of fluid. Excessive accumulation of fluid between the pericardium and the heart muscle itself may result in constriction of the heart. Accumulation may be gradual, allowing for compensation, or acute resulting in cardiac tamponade. Pericardial effusions can be associated with solid tumors or hematologic malignancies. Patients who receive radiation to the pericardium may be at higher risk. Signs and symptoms are related to the degree of cardiac compromise and may include chest pain, muffled heart sounds, jugular venous distention, pulsus paradoxis, and hypotension.

Pericarditis Pericarditis is an inflammation of the pericardium. It may be the results of radiation to the mediastinum or to the underlying malignant process. Pericarditis results in edema, thrombosis, destruction of peripheral capillaries, and fibrosis. The symptoms of pericarditis may include fever and pleuritic chest pain. The patient may be more comfortable in a forward leaning setting position. A pericardial friction rub may be heard on auscultation. Diagnosis is based on physical examination, EKG, and echocardiogram.

Hypertension Hypertension in the pediatric BMT patient may be the result of renal insufficiency, fluid overload, and nephrotoxic drugs such as aminoglycosides, cyclosporine, tacrolimus, and corticosteroids.

Integumentary System The integumentary system comprising the skin and hair follicles is the first line of defense against infection. Insults to this system in the transplant patient can be multifactorial since the skin is susceptible to a wide range of treatment related side effects. Disruption of skin integrity alters an important protective barrier and increases the risk for opportunistic infection. Alterations may also result in pain and changes in body image and self-esteem. Clinical presentations of alterations in the integumentary system occur with a tremendous amount of

618

Fisher and Abramovitz

variation depending on the underlying cause. Radiation effects on the skin can manifest as mild erythema and tenderness, dry desquamation, or weeping desquamation. Radiation recall can also affect skin integrity, resulting in changes that vary from warmth and erythema at the site to severe desquamation and ulceration. Protection of the skin is essential, particularly in the care of the immunocompromised patient. Thorough assessment and early intervention are imperative to maintain the barrier to infection. Treatment strategies include good daily hygiene, encouragement of mobility, application of antibiotic or moisture barrier ointments as indicated, and a wound and/or skin care plan specific for the patient’s clinical status. Some chemotherapy agents such as Thiotepa are associated with cutaneous toxicity that can be minimized by frequent bathing of the patient during infusions to remove the thiotepa excreted through the skin.

Engraftment The time of engraftment is dependent on the type of transplant and stem cell source. During the actual stem cell transplant, a large variety of cells with different functions and life spans are infused. Durable engraftment that provides lasting immune reconstitution and hematopoiesis is the ultimate goal of transplantation. Engraftment is often defined, for the purposes of transplantation, as the first day that the absolute neutrophil count (ANC) exceeds a certain level; or commonly as the first of three consecutive days that it exceeds a certain level. This latter criterion is designed to account for temporary, short term increases in the ANC that do not represent true engraftment. In the allogeneic transplant setting, engraftment refers not only to the ANC, but also to whether hematopoietic function is derived from donor cells. The two tests that are most commonly used to measure engraftment are fluorescent in situ hybridization (FISH) and variable number tandem repeat (VNTR) polymorphisms assessed by DNA amplification. FISH is used to differentiate male from female cells in a sex-mismatched transplant by using fluorescent probes of different colors that bind to either the X or Y chromosome. The presence of female donor cells in a male recipient is evidence of engraftment, as is the presence of male donor cells in a female recipient. VNTR is used to detect donor cells by assessing DNA differences in highly polymorphic areas of the human genome. Engraftment is assessed by analyzing whether the pattern of DNA is similar to donor, recipient, or a combination of both (e.g., mixed chimera). In order for this to be done, a sample from the recipient prior to transplant must be available. When HLA mismatched transplants occur, engraftment can be assessed through HLA typing.

Graft-Versus-Host Disease Allogeneic transplantation can be complicated by GVHD and continues to be one of the most serious complications of this type of HSCT. GVHD is defined as an immune response of the donor cells to the non-self antigens that are present in the recipient’s body. Acute GVHD is defined as occurring within the first 100 days after transplant, chronic GVHD after 100 days. There may be some chronological overlap between these two variations of GVHD, although the two diseases are clinically different, with different presentations.

Acute Graft-Versus-Host Disease Acute GVHD is an inflammatory disease that involves the skin, liver and GI tract. Risk factors include HLA disparity, older donor age, female donor (into a male recipient), positive CMV serology, and female donors with a previous history of pregnancy or blood transfusion. The overall grade of GVHD is based on involvement of the three-targeted organs. Table 11 reviews the clinical grading for Acute GVHD.

Appendix Table 11

619 Clinical Graft-Versus-Host Disease Staging and Grading

Organ Skin

Gut

Liver

Stage 1 2 3 4 1 2 3 4 1 2 3 4

Grading of GVHD Grade I II III IV

Involvement Maculopapular rash !25% body Maculopapular rash 25–50% body Generalized erythroderma Generalized erythroderma with bullous formation DiarrheaO30 ml/kg or O500 ml/day DiarrheaO60 ml/kg or O1000 ml/day DiarrheaO90 ml/kg or 1500 ml/day DiarrheaO90 ml/kg or O2000 ml/day; severe abdominal pain and bleeding with or without ileus Bilirubin 2.0–3.0 mg/dl Bilirubin 3.1–6.0 mg/dl Bilirubin 6.1–15.0 mg/dl BilirubinO15.0 mg/dl Skin

Liver

Gut

1 to 2 1 to 3 2 to 3 2 to 4

0 1 2 to 4 2 to 4

0 1 2 to 3 2 to 4

Skin Skin is the organ most commonly affected by acute GVHD. The presentation is generally in the form of a maculopapular skin rash, which usually involves the ears, neck, shoulders, torso, palms and soles. In early stages, the rash may be pruritic or painful, and it is often described as sunburn. The rash may spread, becoming more confluent and involving the whole body. In severe cases, the rash can evolve into erythroderma and bullous lesions. It is important to different GVHD from chemotherapy and radiation effects, viral infections, or drug hypersensitivity. A skin biopsy is commonly helpful in making the diagnosis. Pathology will show mononuclear and/or lymphocytic infiltration, dyskeratotic cells, and follicular involvement. If done too early in the process, the biopsy may be inclusive.

Liver The liver can also be involved in acute GVHD. Liver involvement without skin involvement is unusual, though possible. Initially, there is a rise in the conjugated bilirubin, alkaline phosphatase, and gamma glutamyl transferase (GGT) caused by damage and destruction of the bile canaliculi. Unfortunately the timing of the rise in bilirubin, alkaline phosphatase and GGT can often overlap in the clinical period when VOD is a concern, making diagnosis difficult. Drugs such as cyclosporine, tacrolimus, and oral contraceptives (used to prevent menstrual bleeding during transplant), as well as parenteral nutrition can also cause a rise in the conjugated bilirubin. The definitive procedure to make the diagnosis is a liver biopsy, which can be a high-risk procedure in a transplant patient, and may not be clinically feasible. Pathology shows bile duct atypia and degeneration if hepatic GVHD is present.

Gastrointestinal Tract Gastrointestinal (GI) GVHD is often the most severe form of GVHD, and the most difficult to treat. Clinical manifestations of GI GVHD are a hemorrhagic and secretory diarrhea and

620

Fisher and Abramovitz

abdominal cramping. The diarrhea can be severe, making it a challenge to maintain an acceptable hemoglobin, as well as to maintain fluid and electrolyte status. It is important to consider other etiologies of diarrhea in this clinical setting, including infection (especially CMV enteritis), mucositis, and radiation induced enteritis. The definitive diagnosis is made by intestinal biopsy, usually during a colonoscopy.

Management of Graft-Versus-Host Disease Prophylaxis of GVHD is the most important aspect of acute GVHD management. Patients receive cyclosporine, tacrolimus, methotrexate or steroids alone or in combinations to prevent GVHD. First line treatment of acute GVHD usually involves steroids. Multiple other agents have become available recently, although there is no consensus at this time as to the priority or timing of their use. These new agents include sirolimus (Rapamunee), mycophenolate mofetil (Cellcepte), and daclizumab (Zenapaxe).

Late Effects Although long-term survivors of HSCT should have the opportunity to be mainstream members of society, they often have complex psychosocial and physical problems that preclude that activity. These problems can preclude the patient’s right to a normal life. Understanding the long-term complications or delayed effects that can occur after HSCT is important in determining the appropriate evaluations and medical treatment for the involved patient. Delayed effects of HSCT are caused by either the preparative regimen (which includes high dose chemotherapy and/or radiation therapy), GVHD, or the underlying disease process. Almost all major organ systems can be adversely affected by HSCT and it is important to take note of therapy received prior to transplant, as well as the conditioning regimen when assessing the long-term effects of treatment.

Ocular Complications The incidence of ocular chronic GVHD is approximately 80–90%. Dry eye syndrome or keratoconjunctivitis sicca syndrome is a manifestation of chronic GVHD. It may manifest itself as early as day 100 as the patient develops manifestations of chronic GVHD. Symptoms include burning, grittiness, and photophobia. The diagnosis of dry eye syndrome can be confirmed by a Schirmer’s test. A positive test has !10 mm of wetness. The use of prophylactic preservativefree artificial tears in patients who have acute GVHD is strongly recommended since ocular damage usually occurs before patient become symptomatic. Surveillance for sicca syndrome includes regular visits to the ophthalmologist, as well as clinical exams by the primary care group. Soft contacts and punctural ligation for tear duct outflow is helpful. Sunglasses can be of benefit in patients with photophobia. Cataracts are a late ocular complication of transplantation. Originally, early studies determined that TBI was the primary cause of cataract formation. The incidence of cataract formation is O50% for patients who receive single dose TBI and !50% for patients who have received fractionated dose TBI. Patients who have received glucocorticoid therapy for GVHD or as part of their primary disease treatment prior to HSCT are also at risk. Typically, patients present with cloudy vision anywhere from 1.5 to 5 years post transplant. Yearly surveillance should include an ophthalmology exam particularly for patients who received TBI, or who have experienced extensive acute GVHD. The treatment is surgical removal and interocular lens replacement.

Pulmonary Complications Pulmonary complications continue to be a major source of morbidity and mortality for children who undergo HSCT. The pulmonary tissue is very sensitive to various cytotoxic agents and

Appendix

621

irradiation, and its responses to injury are particularly prominent in the interstitium. Unfortunately as a result, the repair process may result in the formation of scar tissue in the interstitial space, which in turn will then interfere with effective gas exchange and respiratory kinetics. The lungs are also a primary target for viral, bacterial and fungal infections that, in association with an already altered interstitium, can further impair pulmonary function. Lastly, the bronchopulmonary tree is also a target for GVHD, which can further result in severe structural and functional damage. As a result, pulmonary complications occur in approximately 10–15% of all pediatric patients following HSCT. IPN accounts for approximately 40% of all transplant related deaths. Pulmonary IPN is a general term that refers to an inflammatory pulmonary process involving the interstitium of the lungs. There are multiple factors that contribute to the development of IPN. The three predisposing factors include previous lung damage, the presence of an opportunistic infection, and an immunocompromised host. IPN typically occurs in the first hundred days after transplant, with the most common time of occurrence at approximately 85 days post transplant, after many patients have left the transplant center. The most common organism identified is CMV. Agents that are associated with IPN include: busulfan, methotrexate, BCNU, cyclophosphamide, bleomycin, and melphalan.

Restrictive Disease Most patients who undergo transplantation have some degree of restrictive defect in their pulmonary function tests (PFT) afterwards. A study performed at the Fred Hutchinson Cancer Research Center in patients who were considered healthy long-term survivors, showed a 20% loss in total lung and diffusion capacity. In this study there was no correlation between pulmonary function and any particular conditioning regimen or chronic GVHD. Three to four years post transplant, the restrictive pulmonary changes were no longer evident. In a more recent comprehensive study comparing the results between autologous and allogeneic transplant patients without GVHD, both sets of transplant patients experienced the same restrictive changes in pulmonary function.

Obstructive Disease The etiology of obstructive lung disease in HSCT patients is unknown. It is thought to occur secondary to extensive restrictive changes in the small airways of the pulmonary tree. Obstructive pulmonary defects occur in 10–15% of all patients with chronic GVHD. As a consequence of the immunosuppression used to treat their GVHD, these patients are predisposed to lung infections. Additionally, chronic GVHD has a direct effect on the pulmonary epithelium. The sicca syndrome of GVHD, which exerts its effects on other target organs in the body, also exerts its effect on the pulmonary system. One of the major subclasses of obstructive lung disease is bronchiolitis obliterans. Bronchiolitis obliterans affects approximately 10% of all patients with chronic GVHD. HSCT patients with bronchiolitis obliterans have been compared to heart and lung transplant patients with the same disease. The mechanisms of injury are thought to be similar. Bronchiolitis obliterans is characterized by tissue plugs that resemble granulation tissue in the small airways, which often extend into the alveolar duct causing an obstructive picture on pulmonary function testing. The upper airway tissue remains normal. The granulomas may also obstruct the alveolar spaces secondary to the effects of sicca syndrome. The clinical course of bronchiolitis obliterans can vary from being very mild, with slow deterioration, to diffuse necrotizing fatal bronchiolitis of the small airways. Bronchiolitis obliterans has been seen as early as three months post transplant but generally appears between one and two years post HSCT. Clinically the patient rapidly develops shortness of breath, inspiratory rales, a nonproductive cough, and airway obstruction. CXR classically shows hyperinflation of the lungs and flattening of the diaphragm. Recurrent pneumothorases have been reported as well as pneumomediastinum.

622

Fisher and Abramovitz

PFT show a marked reduction in expiratory flow and a severe reduction in forced vital capacity, as well as a decrease in the diffusion capacity. The treatment of bronchiolitis obliterans is difficult. Unfortunately, the obstruction is irreversible and usually unresponsive to immunosuppression, although there are some patients for whom immunosuppression has been helpful. Bronchodilators can also help on some occasions. Bronchiolitis obliterans organizing pneumonia (BOOP) has been reported after HSCT in the past few years. The term BOOP has led to confusion between bronchiolitis obliterans and BOOP. BOOP is a completely different disease as far as overall prognosis. An increased incidence of BOOP has been reported in patients with chronic GVHD. BOOP usually occurs two to four months after transplant and usually presents with dyspnea, which has been preceded by a flu-like syndrome. PFT show a restrictive defect as opposed to the obstructive defect seen in bronchiolitis obliterans. A chest radiograph or CT scan of the chest will reveal patchy peripheral infiltrates. The treatment is high-dose steroids and antibiotics after a microorganism has been identified by bronchial alveolar lavage. In most cases there is a rapid resolution of BOOP after treatment.

Cardiac Complications Cardiac difficulties post transplant are not uncommon but are rarely the cause of death in these patients. The overall incidence of cardiac complications after HSCT is approximately 20%. Most cardiac complications have been reported in the autologous population of patients, and there is a direct relation between the amount of anthracycline and cyclophosphamide that patients have received and cardiac complications. Risk factors for the development of cardiac complications include: a history of previous anthracycline administration, a history of radiation to the chest, a total cyclophosphamide dose O150 mg/kg, cyclophosphamide as a pretransplant conditioning agent especially if the patient has received previous Ara-C or 6-TG, cyclophosphamide and TBI as part of the ablative regimen, sepsis, a history of mitral valve disease, a pre-transplant ejection fraction of !50%, a diagnosis of Hurler’s syndrome or thalassemia. Anthracycline induced cardiac damage is known to occur at doses exceeding 450 mg/m2. The damage caused by anthracyclines affects myocardial fibers, which in turn causes mitochondrial changes and then cellular degeneration. The cardiac damage associated with anthracyclines is generally irreversible. Cardiotoxicity from anthracyclines can occur very early in the pre-transplant course. The interval from myeloablation to engraftment places the patient under extreme physical stress. If during that time period, the patient develops pulmonary complications, this can put an additional stress on the heart and may send the patient into heart failure. Cardiac complications that are associated with cyclophosphamide use have been well documented. Most transplant centers use cyclophosphamide as part of their preparative regimens. Recent data has demonstrated late cardiac abnormalities in children following HSCT, and that there was a clear need for continued and serial long-term cardiac evaluations in transplant survivors.

Chronic Graft-Versus-Host Disease Chronic GVHD is a significant complication of allogeneic HSCT, and despite progress in immunosuppressive drug therapy, it continues to be a major contributing factor to transplant related deaths. Chronic GVHD is a syndrome that occurs more than 100 days post HSCT, and is caused by donor immunocompetent T lymphocytes recognizing and mounting an immune response against host cells. Chronic GVHD can be classified into three forms; progressive, quiescent, and de Novo. Progressive GVHD is a continuation of acute GVHD and it is associated with the highest mortality rate. Quiescent chronic GVHD occurs following the resolution of a previous episode

Appendix

623

of acute GVHD. De Novo chronic GVHD refers to the development of chronic GVHD without any preceding episode of acute GVHD. De Novo GVHD has the most favorable prognosis, but occurs less frequently than quiescent and progressive GVHD. Chronic GVHD can be clinically differentiated by the degree of organ involvement. It is classified as limited if there is skin and/or liver involvement requiring very little medical intervention. Extensive chronic GVHD refers to multi-organ system involvement with the disease and has a worse prognosis. Chronic GVHD is a very complex syndrome whose primary clinical manifestations resemble a collagen vascular or an autoimmune disorder. There are numerous clinical manifestations in multiple organs that can occur in chronic GVHD. The skin is the primary organ affected in 80% of patients with chronic GVHD. Pruritus is often the first symptom, which is followed by an erythematous, lichenoid papular rash that can be either hypo or hyperpigmented. As the disease progresses, fibrosis occurs in the subcutaneous and cutaneous tissues. Fibrosis causes impaired blood flow to the tissues, and with trauma may cause ulcerated plaque lesions that can affect the skin’s integrity. Contractures or a tightening of the tendons and joints may occur that limits range of motion. Immunosuppressive agents such as cyclosporine, tacrolimus, and steroids are used to prevent and treat chronic GVHD. In addition, emollient lotions can help to maintain the skin’s elasticity by keeping it moist and supple. Steroid creams can minimize itching and inflammation. Other modalities, specifically for cutaneous GVHD, include the use of oral psoralen in combination with ultraviolet light (PUVA) for patients who have not responded to initial therapy. Psoralen is taken up in an inactive form by lymphocytes. When the lymphocytes in the skin are exposed to ultraviolet light, the psoralen becomes activated, binding to DNA, and causing cell death. The selective destruction of these lymphocytes has proven to be an effective, and relatively non-toxic treatment for cutaneous chronic GVHD.

Leukoencephalopathy Leukoencephalopathy is a degenerative lesion occurring in the white matter of the CNS. The onset may occur within days or months of BMT. Leukoencephalopathy is characterized by severe neurological degeneration resulting in permanent neurological disability or death. Clinically, the patient presents with lethargy, slurred speech, ataxia, seizures, confusion, dysphasia, spasticity, decerebrate posturing and coma. The development of leukoencephalopathy is most closely associated with prophylactic or therapeutic CNS treatment, both before and after transplant. The treatment usually involves cranial irradiation in combination with IT Methotrexate or high-dose IV methotrexate. The administration of methotrexate after TBI directly contributes to the development of leukoencephalopathy.

LEARNING DISABILITIES Central nervous system effects are subtle and often difficult to discern, but they can influence the long-term physical and psychological welfare of the patient. Late cognitive effects often compromise school and work performance. Children may manifest learning disabilities associated with visual motor coordination, abstract thinking, spacial processing, and behavioral and language performance. A preliminary study performed at the Fred Hutchinson Cancer Institute demonstrated that pediatric transplant patients, when tested against controls, scored lower on tests of intelligence within 90 days after treatment. Patients and their families need to be aware of potential problems related to concentration, memory, and processing complex tasks. Pediatric transplant patients may require educational monitoring and special job training to minimize the adverse outcomes related to CNS toxicities.

624

Fisher and Abramovitz

CONCLUSION Long-term survivors of HSCT are increasing in numbers, and there is increasing success in transplantation for an expanding number of diseases and disorders. Greater attention must be given to delayed effects of HSCT, especially those that do not appear for years after the transplant procedure,

Index

ABO incompatibility hemolytic transfusion reactions, 14–15 transfusion and, 14 transfusion associated lung injury, 15 viral infection transfusion transmission, 15–16 Acquired aplastic anemia, 369–379 alternative donor transplant, 377 clinical features, 371 epidemiology, 371 GVHD, 374–375 acute, 374–375 chronic, 375 HLA-nonidentical related donors, 378 HSCT, 372–374 allogeneic transplant, 373–374 graft failure, 374 syngeneic transplant, 373 immunosuppressive therapy, 371–372 vs. HSCT treatment, 375–376 pathophysiology, 370 survival rates, 375 unrelated donor transplants, 377–378 Acute graft-versus-host disease. See aGVHD. Acute lymphoblastic leukemia immune subversion by, 487–488 unrelated hematopoietic stem-cell transplantation and, 152–153 Acute myelogenous leukemia, unrelated hematopoietic stem-cell transplantation and, 153 Acute myeloid leukemia, bone marrow transplantation and, 497–518 Acute toxicities common types, 611 hematologic, 607–610 transplant process and, 606–607 Adenovirus viral infection, 50–51

Adoptive immunotherapy, immune reconstitution and, 280–281 aGVHD acquired aplastic anemia and, 374–375 acute graft-versus-host disease, 65–77 pathophysiology, 65–66 post-autologous stem-cell transplantation, 67–68 prophylaxis, 71–75 agents, 71–74 antibody therapy, 73–74 nonselective, 71 T-cell inhibitor, 71 combined-agent, 74–75 single-agent, 74–75 vs. non-prophylaxis treatment, 74–75 risk factors, 68–71 conditioning regime, 71 donor characteristics, 70 human leukocyte antigen matching, 70 stem cell sources, 68–69 bone marrow, 69 cord blood, 69 peripheral blood stem cells, 69 staging/clinical description, 66–67 treatment, 71–75 antibody therapy, 76–77 mycophenolate mofetil, 76 photopheresis, 77 sirolimus, 76 steroids, 76 umbilical cord blood transplantation and, 166–169 Alemtuzumab, 74 Allogeneic BMT, neuroblastoma and, 597 Allogeneic hematopoeitic stem-cell transplantation, 467–468, 602 Hodgkin’s disease and, 546 625

626 Allogeneic hematopoietic stem cells, sources of, sickle cell disease and, 407–408 Allogeneic stem-cell transplantation beta thalassemia and, 388–389 nonmyeloablative cytoreductive regimens, 390 T-cell depletion, 389 Allogeneic transplantation, 373–374 alternative donor, 509–510 bone marrow, 506–507 colony-stimulating factors and, 18 cytokines, 508–509 donor sources, 507–508 effect of, 506–507 natural killer cells, 507 peripheral blood stem cell vs. bone marrow, 508 T-cell depletion vs. immune suppression, 509 Alloreactivity, mismatched related donor transplantation and, 202–203 a-L Iduronidase deficiency, mucopolysaccharidoses I and, 420–424 Alpha-mannosidosis, 434–435 Alternative donor transplant, acquired aplastic anemia and, 377 Alternative donors, beta thalassemia treatment and, 386–387 Alternative stem cell sources, beta thalassemia treatment and, 386–388 Anemia, 612 Anergy induction, graft manipulation and, 207 Animal models, autoimmune diseases and, 450–451 Antibacterial treatment, 32–33 Antibiotic prophylaxis, bacterial infections and, 30–31 Antibody therapy aGVHD prophylaxis, agents and, 73–74 aGVHD treatment and, 76–77 alemtuzumab, 74 antithymoctye globulin, 73–74 daclizumab, 74 infliximab, 74 Antigen-presenting cells, chronic GVHD and, 88–89 Antigens, associated with chronic GHVD, 89–90 Antigen-specific responses immune reconstitution and, 278–280 vaccine responses, 279–280 Antithymoctye globulin, 73–74 immunosuppressive therapy and, 371–372 Aspartylglucosaminuria, 435

Index Attempted induction vs. untreated relapse, bone marrow transplantation and, 515–516 Autoimmune diseases allogeneic hematopoeitic stem-cell transplantation, 467–468 animal models, 450–451 Crohn’s disease, 458–461 ex vivo stem cell selection, 452–453 hematopoietic stem-cell transplantation and, 449–489 immunologic mechanisms, 467 juvenile dermatomyositis, 466–467 juvenile idiopathic arthritis, 454–458 regimen designs, 453 stem cell mobilization, 452 systemic lupus erythematosus, 461–464 thyroid disease, 291 type I diabetes, 465–466 Autologous donor sources, childhood acute lymphoblastic leukemia and, 483–484 Autologous hematopoietic stem-cell transplantation, 601–602 aGVHD and, 67–68 Hodgkin’s disease and, 540, 544 tumor cell purging, 122–126 Autologous transplantation allogeneic BMT, 597 bone marrow and, 510–513 Children’s Cancer Group 3891 study, 590–591 colony-stimulating factors and, 17–18 metaiodobenzylguanidine scans and therapy, 591–592 tandem high-dose chemotherapy, 592–595 tumor removal, 595–597 B-cell reconstitution, immune reconstitution and, 277–278 Bacterial infections antibacterial treatment, 32–33 epidemiology, 27–28 infectious disease and, 27–34 patient risk factors, 28–30 prevention and treatment antibiotic prophylaxis, 30–31 environmental protection, 30–34 fever and neutropenia, 31–32 supplementary therapies, 33–34 vaccinations, 34 Beta thalassemia treatment allogeneic stem-cell transplantation, alternative approaches, 388–389 alternative donors, 386–388 ex-thalassemic patients, 390–392

Index [Beta thalassemia treatment] hematopoietic stem-cell transplantation and, 383–393 alternative donors, 386–388 alternative stem cell sources, 386–388 human leukocyte antigen closely-matched unrelated donors, 387 matched siblings, 384–386 mismatched related donors, 386–387 mixed chimerism, 388–389 other treatment groups, 386–387 Pesaro transplant group, 384–386 BK virus, 52 Blood product irradiation, transfusion and, 13 Blood units, umbilical cord blood transplantation and, 180 BMT. See Bone marrow transplantation. Bone genetic disorders, bone marrow transplantation and, 306–310 Bone marrow aGVHD risk factors and, 69 cell therapy, principles of, 299–302 childhood acute lymphoblastic leukemia and, 485 chronic GHVD and immunological differences, 90–91 harvesting, hematopoietic stem-cell transplantations, growth factor support and, 16–17 pretransplant considerations and, 605 peripheral blood stem cell, allogeneic transplantation and, 508 Bone marrow transplantation (BMT) acute myeloid leukemia and, 497–518 allogeneic, 506–507 autologous, 510–513 development of, 305 future directions of, 299–315, 514–518 gene therapy principles, 301–304 genetic disorder treatment, 306–310 immunodeficiency disorder therapy, 310–311 inborn errors of metabolism, marrow mesenchymal stem cell therapy for, 311 mesenchymal stem cells, 304–305 GVHD treatment, 312 hematopoietic stem cell engraftment treatment, 312–313 metabolic liver disease, 313–314 metabolic storage disease, 313 muscular dystrophy, 314 nonhematopoietic disorders metabolic storage diseases, 313

627 [Bone marrow transplantation (BMT) nonhematopoietic disorders] metabolic liver diseases, 313–314 muscular dystrophy, 314–315 patient-based research, 305–306 preemptive immunotherapy, 517–518 preparative regimens, 501–506 busulfan-based, 502 nonmyeloablative, 506 pharmacokinetic targeting, 502–503 regimen intensification, 505 total body irradiation, 501–502 vs. busulfan, 503–504 prognosis factors, 498–501 transplanted patients, 499–501 quality of life issues, 513–514 relapse therapy, 516–517 risk groups, 498 sickle cell disease and, 397–399 untreated relapse vs. attempted induction, 515–516 Busulfan-based regimens, bone marrow transplantation and, 502 Busulfan vs. total body irradiation, bone marrow transplantation and, 503–504 Cardiac complications, 622–623 Cardiovascular complications, 617 CD34C selection, graft manipulation and, 207 Cell dose, umbilical cord blood transplantation and, 180 Cerebral X-adrenoleukodystrophy, 429–431 Chediak-Higashi syndrome, 328–329 Chemical hepatitis, 615 Chemotherapy agents, hematopoietic stem-cell transplantations and, 608–609 endocrine complications and, 287–288 gonadal dysfunction and, 291–292 Hodgkin’s disease and, 539–540 Childhood acute lymphoblastic leukemia disease control pretransplant, 477–478 graft-versus-leukemia acute lymphoblastic leukemia, 487–488 donor lymphocyte infusions, 487 mechanisms of, 487 hematopoietic stem-cell transplantation and, 477–489 stem cell source, 483–486 autologous donor sources, 483–484 peripheral blood progenitor cells, 484 bone marrow, 485

628 [Childhood acute lymphoblastic leukemia] hematopoietic stem-cell transplantation and stem cell source] umbilical cord blood, 485 haploidentical donor source, 486 immunity response enhancers to, 488–489 cytokines, 488 effector cell retargeting, 488 specific T-cell generation, 489 vaccination, 489 transplantation timing, 478–482 first remission, 478–480 relapse, 482 second remission, 480–482 Children’s Cancer Group 3891 study, neuroblastoma and, 590–591 Chimerism, mixed, sickle cell disease and, 404–407 Chronic GHVD in children, 85–100 acquired aplastic anemia and, 375 classification of, 93–94 clinical manifestations of, 94–98 liver, 96 cutaneous, 94 dermal appendages, 94 eyes, 94–95 gastrointestinal tract, 96 hematopoietic system, 97 immune system, 97–98 mouth, 95–96 musculoskeletal system, 94–95 respiratory tract, 96–97 donor impact, 91 immunological differences, bone marrow, peripheral blood, 90–91 impact of recipient age, 91 incidence in children, 91–93 overview, 85–91 associated antigens, 89–90 antigen-presenting cells, 88–89 cytokine polymorphisms, 88 T cells role in, 85–86 cytokines profile of, 86–88 risk factors, 91–93 treatment of, 98 salvage regimens, 98–100 standard therapy, 98 Chronic granulomatous disease, 329 Chronic myelogenous leukemia, unrelated hematopoietic stem-cell transplantation and, 154 Colony-stimulating factors allogeneic transplantation, 18 autologous transplantation, 17–18

Index [Colony-stimulating factors] engraftment failure management, 18 hematopoietic growth factor support and, 17–18 umbilical cord blood transplantation, 18 Combined agent prophylaxis, aGVHD and, 74–75 Complications graft failure, unrelated, hematopoietic stem-cell transplantation, 151–152 graft-versus-host disease, unrelated, hematopoietic stem-cell transplantation, 151-152 hematopoietic stem-cell transplant (HSCT) and, 1–6 infections, unrelated, hematopoietic stem-cell transplantation, 151–152 Conditioning regime aGVHD risk factors and, 71 immunodeficiency diseases and, 324–325 Conflicts of interest, ethical considerations and, 262–266 Congential amegakaryocytic thrombocytopenia, 357–359 stem-cell transplants, 358 Consent pediatric ethical considerations and, 253–256 hematopoietic stem-cell transplantation and, 256–257 process advances in, 257–258 Coping psychological adjustments to HSCT, 242 psychosocial intervention and, 244 Cord blood, aGVHD risk factors and, Cord blood donation, ethical considerations and, 261–262 Corticotropin, 290 Crohn’s disease, hematopoietic stem-cell transplantation and, 458–461 Cross-correction principal, metabolic diseases and, 418 Cryoprecipitate, transfusion and, 13 Cutaneous manifestations, chronic GHVD and, 94 Cyclosporine, 71–73 immunosuppressive therapy and, 371–372 Cytokine polymorphisms, chronic GVHD and, 88 Cytokines, allogeneic transplantation and, 508–509

Index Cytokines childhood acute lymphoblastic leukemia and, 488 chronic GVHD and, 86–88 Cytomegalovirus, viral infections and, 47–49 Cytotoxic agents. See Nonselective agents. Daclizumab, 74 Dermal appendages, chronic GHVD and, 94 Diabetes, hematopoietic stem-cell transplantation and, 465–466 Diamond blackfan anemia, 347–348 Diarrhea, 614 Donor beta thalassemia treatment and, 386–388 characteristics, aGVHD risk factors and, 70 closely-matched unrelated, human leukocyte antigen and, 387 cord blood donation, 261–262 ethical considerations and, 258–262 evaluation, hematopoietic graft and, 116 pretransplant considerations and, 603, 605 graft selection, umbilical cord blood transplantation and, 179–180 haploidentical, 262 impact on GHVD and, 91 hematopoietic stem-cell transplantations, pre-transplant considerations and, 603, 605 leukocyte infusion, 192–194 outcome studies, 193 childhood acute lymphoblastic leukemia and, 487 matched human leukocyte antigen and, matches, 386–387 unrelated donor, 258–260 mismatched related, human leukocyte antigen and, 386–387 registries, unrelated, 147 selection, mismatched related donor transplantation and, 204–205 unrelated hematopoietic stem-cell transplantation and, 149–150 siblings, 260–261 transplant alternative, acquired aplastic anemia and, 377 unrelated, acquired aplastic anemia and, 377–378

629 Dsykeratosis congenita, 342–344 stem-cell transplants for, 345 Educational accommodations, psychosocial intervention and, 245–246 Effector cell retargeting, childhood acute lymphoblastic leukemia and, 488 Endocrine complications glucose homeostasis disorders and, 293–294 gonadal dysfunction, 291–292 growth hormone deficiency, 289 growth impairment, 287–290 risk factors, 287–289 GVHD, 289 hematopoietic stem-cell transplantation and, 287–294 hypothalamic-pituitary disturbances, 290 osteoporosis, 293 reproductive dysfunction and, 291–293 risk factors chemotherapy, 287–288 radiotherapy, 288–289 thyroid dysfunction, 291 Endocrine dysfunction, ex-thalassemic patients and, 391–392 End-of-life issues, ethical considerations and, 266 Engraftment, 618 failure management, colony-stimulating factors and, 18 mismatched related donor transplantation and, 208–209 umbilical cord blood transplantation and, 166–169 Environmental protection, bacterial infections and, 29–34 Epstein-Barr virus infections, 48–49 Erythropoietin, hematopoietic growth factor support and, 18–19 Ethical considerations conflicts of interest, 262–266 donor, 258–262 end-of-life issues, 266 foundational concepts, 252–253 hematopoietic stem-cell transplantation and, 251–267 pediatric consent, 253–256 quality of life, 266 Ewing’s sarcoma and peripheral primitive neuroectodermal tumors, 569–577 first remission, 569–572 second remission, 572–575 tumor cell contamination, 575–576

630 Ex vivo stem cell selection, hematopoietic stem-cell transplantation and, 452–453 Ex-thalassemic patients, 390–392 endocrine dysfunction, 391–392 liver issues, 390 hepatitis, 391 iron, 390 organ functions, 391 Extracranial germ cell tumors, 580 hematopoietic stem-cell transplants, 581–583 Eyes, chronic GHVD and, 94–95 Fabry disease, 435–436 Family influence of, psychological adjustments to HSCT pediatric patient and, 242–243 psychosocial intervention and, 245 Fanconi anemia, 339–340 Fever, bacterial infections and, 31–32 First remission, Ewing’s sarcoma and peripheral primitive neuroectodermal tumors, 569–572 Fucosidosis, 433

Gangliosidoses, 436 Gastrointestinal complications, 613 Gastrointestinal tract, 619–620 chronic GHVD and, 96 Gaucher disease, 433–434 Gene therapy, principles of, bone marrow transplantation and, 301–304 Germ cell tumors, 580 Globoid-cell leukodystrophy, 431–432 Glucose homeostasis disorders, endocrine complications and, 293–294 Glycoprotein metabolic diseases, 433–435 alpha-mannosidosis, 434–435 aspartylglucosaminuria, 435 fucosidosis, 433 Gaucher disease, 433–434 Gonadal dysfunction chemotherapy and, 291–292 endocrine complications and, 291–292 total body irradiation, 292 Gonadotropin secretion disorder, 290 Graft failure acquired aplastic anemia and, 374 unrelated hematopoietic stem-cell transplantation complications and, 152 Graft manipulation anergy induction, 207 CD34C selection, 207

Index [Graft manipulation] mismatched related donor transplantation and, 205–207 T-cell depletion, 205, 207 Graft-versus-host disease. See GVHD. Graft-versus-leukemia acute lymphoblastic leukemia, immune subversion by, 487–488 childhood acute lymphoblastic leukemia and, 486–488 donor lymphocyte infusions, 487 Granulocytes, transfusion and, 12–13 Granulomatous disease, 329 Grief and loss, psychological aspects of HSCT and, 246 Growth hormone deficiency, endocrine complications and, 289 Growth impairment, endocrine complications and, 287–290 GVHD acquired aplastic anemia, 374–375 acute, acquired aplastic anemia and, 374–375 chronic acquired aplastic anemia and, 375 in children 85–100 endocrine complications and, 289 outcome of, mismatched related donor transplantation and, 209–211 prevention, T-cell depletion, 119–122 transplant process and, 618–619, 620, 623 treatment, mesenchymal stem cells and, 312 Haploidentical donor source childhood acute lymphoblastic leukemia and, 486 ethical considerations and, 262 Hematologic toxicities, 607–610 Hematopoietic graft cellular engineering, 111–128 collection of, 114–115 definitions, 111–118 donor evaluation, 116 GVHD prevention, T-cell depletion, 119–122 manipulation of, 118 nomenclature, 112 processing of, 115–118 regulation of, 127–128 Hematopoietic growth factor support, 16–19 bone marrow harvesting, 16–17

Index [Hematopoietic growth factor support] colony-stimulating factors, 17–18 erythropoietin, 18–19 peripheral blood stem cells, 17 thrombopoietic agents, 19 Hematopoietic stem cell (HSC) autologous donor sources, childhood acute lymphoblastic leukemia and, 483–484 engraftment, mesenchymal stem cell treatment and, 312–313 graft, 111–128 source bone marrow, 485 childhood acute lymphoblastic leukemia and, 483–486 haploidentical donor source, 486 peripheral blood progenitor cells, 484 umbilical cord blood, 485 Hematopoietic stem-cell transplantation (HSCT) acquired aplastic anemia, 369–379 allogeneic, 602 transplant, acquired aplastic anemia and, 373–374 autoimmune diseases, 449–489 allogeneic, 467–468 animal models, 450–451 Crohn’s disease, 458–461 ex vivo stem cell selection, 452–453 immunologic mechansisms, 467 juvenile idiopathic arthritis, 454–458 dermatomyositis, 466–467 regimen designs, 453 stem cell mobilization, 452 systemic lupus erythematosus, 461–464 type I diabetes, 465–466 autologous, 601–602 beta thalassemia treatment, 383–392 alternative donors, 386–388 alternative stem cell sources, 386–388 childhood acute lymphoblastic leukemia, 477–489 endocrine complications, 287–294 ethical considerations, 251–267 foundational concepts, 252–253 pediatric consent, 253–256 extracranial germ cell tumors and, 580–583 future possibilities, 126 graft failure, 374 growth factor support, 16–19 Hodgkin’s disease, 539–548 new approaches, 546–547 immune reconstitution, 271–281

631 [Hematopoietic stem-cell transplantation (HSCT)] inherited bone marrow failure syndromes, 337–360 learning disabilities, 624 liver dysfunction from, 4 metabolic diseases and, 418–419 neuroblastoma, 589–598 neurologic complications, 9–11 non-Hodgkin’s disease, 529–539 noninfectious complications, 1–6 hemorrhagic cystitis, 7 hepatic veno-occlusive disease, 2–6 mucositis, 1–2 nutrition support, 6–7 renal disease, 7–8 nursing care, 223–232 obstructive disease, 621–622 overview of, 601–624 pancytopenia and, 340–342, 344 pediatric consent, 256–257 transplant patient, 1–19 pretransplant considerations, 603–606 bone marrow harvest, 605 donor evaluation, 603, 605 donor issues, 603, 605 leukopheresis, 605–606 stem cell collection, 605 psychological aspects of, 235–247 adjustments, 241–244 grief and loss, 246 healthcare professional, 247 stress on child and family, 235–237 psychosocial intervention, 244–246 outcomes, 237–241 reduced intensity conditioning, Hodgkin’s disease and, 547–548 sickle cell disease, 397–409 acute toxicities, 399–400 single cytopenias and, 348–349, 351–352, 353–354, 357 solid tumors, 569–583 syngeneic, 602 transplant, acquired aplastic anemia and, 373 transfusion support, 11–13 transplant process, 606–624 acute toxicities, 607 anemia, 612 cardiovascular complications, 617 cardiac complications, 622 chemotherapy agents, 608–609

632 [Hematopoietic stem-cell transplantation (HSCT) transplant process] chemical hepatitis, 615 diarrhea, 614 engraftment, 618 gastrointestinal complications, 613 tract, 619–620 GVHD, 618–619, 620, 623 hepatic complications, 615 hypertension, 617 idiopathic pneumonia syndrome, 616–617 infectious complications, 612–613 interstitial pneumonitis, 616 integumentary system, 617–618 late effects, 620 leukoencephalopathy, 623 liver, 619 malnutrition, 614 mucositis, 613 neutropenia, 610–611 ocular complications, 620 pericardial effusions, 617 pericarditis, 617 pneumonia, 616 preparative regimen, 606–607 pulmonary complications, 616, 621 pulmonary function restrictions, 621 renal insufficiency, 615 skin, 619 stem cell infusion, 607 thrombocytopenia, 611–612 urologic complications, 616 veno-occlusive disease, 615 treatment, immunosuppressive treatment, acquired aplastic anemia and, 375–376 tumor cell purging, 122–126 unrelated donor, 147–155 complications, 151–152 histocompatibility and, 148–149 HLA typing and, 149 preparative regimens, 151 registries and, 147, 149–150 role of, 152–155 acute lymphoblastic leukemia, 152–153 acute myelogenous leukemia, 153 chronic myelogenous leukemia, 154 hurler syndrome, 155 myelodysplastic syndrome, 153–154 severe aplastic anemia, 154–155 Wiskott-Aldrich syndrome, 155 selection of, 149–150 Hematopoietic system, chronic GHVD and, 97

Index Hemolytic transfusion reactions, ABO incompatibility and, 14–15 Hemolytic uremic syndrome, 9 Hemophagocytic lymphohistiocytosis, 330 Hemorrhagic cystitis, hematopoietic stem-cell transplant (HSCT) and, 7 Hepatic complications, 615 Hepatic veno-occlusive disease diagnostic criteria of, 5 hematopoietic stem-cell transplant (HSCT) and, 2–6 Hepatitis, ex-thalassemic patients and, 391 Hepatoblastoma, 580 Herpes simplex viral infection, 55 High-dose therapy, non-Hodgkin’s disease, 530 Histocompatibility, unrelated hematopoietic stem-cell transplantation and, 148–149 HLA typing, unrelated hematopoietic stem-cell transplantation and,149 HLA-nonidentical related donors, acquired aplastic anemia and, 378 Hodgkin’s disease adult/pediatric patient comparison, 545–546 allogeneic hematopoietic cell transplant, 546 autologous hematopoietic cell transplant, 540, 544 available literature on, 541–543 chemotherapy approaches, 539–540 hematopoietic-cell transplantation, 539–548 new approaches, 546–547 reduced intensity conditioning, 547–548 late effects, 548 preparative regimens, 544 stem cell source, 544 survival prognostic factors, 544–545 HSCT. See Hematopoietic stem-cell transplantation. Human herpes virus type 6, 51–52 Human leukocyte antigen barriers, suppression of, mismatched related donor transplantation, 205 closely-matched unrelated donors, beta thalassemia treatment and, 386 match aGVHD and, 70 umbilical cord blood transplantation and, 180 mismatch beta thalassemia treatment and, 386–388 related donor transplantation and, 203 Hunter syndrome, 426 mucopolysaccharidoses I and, 420–424 unrelated hematopoietic stem-cell transplantation and, 155

Index Hurler-Scheie syndromes, 425–426 Hypertension, 617 Hypothalamic-pituitary disturbances corticotrophin, 290 gonadotropin secretion disorder, 290 thyrotropin deficiency, 290 Idiopathic pneumonia syndrome, 616–617 Immunodeficiency diseases, donor selection, 323–324 Illness stressors, psychological adjustments and, 241–242 Immune reconstitution adoptive immunotherapy, 280–281 antigen-specific responses, 278–280 B cell reconstitution, 277–278 hematopoietic stem-cell transplantation and, 271–281 immunomodulatory factors, 281 mismatched related donor transplantation and, 211–212 natural killer cells, 272–273 T-cell reconstitution, 273–277 Immune suppression vs. T-cell depletion, allogeneic transplantation and, 509 Immune system, chronic GHVD and, 97–98 Immunity childhood acute lymphoblastic leukemia and, 488–489 response enhancers, 488–489 subversion by, 487–488 Immunodeficiency diseases, 321–332 clinical results, 325–328 conditioning regimes, 324–325 lymphoid disorders, 326–328 maternal engraftment, 323 myeloid disorders, 328–330 pretransplant infections, 322–323 stem-cell transplantation, 321–322 Immunodeficiency disorder therapy, bone marrow transplantation and, 310–311 Immunoglobulin-like receptors, killer, 203 Immunologic mechanisms, hematopoietic stem-cell transplantation and, 467 Immunological phenotype, umbilical cord blood transplantation and, 163 Immunomodulatory factors, immune reconstitution and, 281 Immunomodulatory therapy for aplastic anemia. See Immunosuppressive therapy. Immunosuppressive therapy antithymocyte globulin, 371–372 cyclosporine, 371–372

633 [Immunosuppressive therapy] HSCT treatment vs., 375–376 other types, 372 Immunotherapy, reduced intensity conditioning and, 189–192 Inborn errors of metabolism, mesenchymal stem cell therapy for, 311 Infections mismatched related donor transplantation and, 211–212 unrelated hematopoietic stem-cell transplantation complications and, 151–152 Infectious disease bacterial infections, 27–35 complications from, 612–613 invasive fungal infections, 35–43 prevention of, 27–55 treatment of, 27–55 viral infections, 43–55 Infliximab, 74 Inherited bone marrow failure syndromes genes, known and presumed, 338 hematopoietic stem-cell transplant and, 337–360 pancytopenia, 339–346 single cytopenias, 347–359 Integumentary system, 617–618 Interstitial pneumonitis, 616 Invasive fungal infections diagnosis and detection, 36–38 epidemiology of, patient risk factors, 35–36 infectious disease and, 35–43 new treatments for, comparisons of, 44–47 prevention and treatment, 38–43 empirical therapy, 40–41 invasive mold, 42–43 invasive yeast, 41–42 new treatments, 43 other, 43 yeast organism prophylaxis, 39 Iron, ex-thalassemic patients and, 390–391 Irradiation, blood product, 13 Juvenile dermatomyositis, hematopoietic stem-cell transplantation and, 466–467 Juvenile idiopathic arthritis, hematopoietic stem-cell transplantation and, 454–458 Killer immunoglobulin-like receptors, 203 Kostmann syndrome, 349–351

634 Large cell lymphoma, non-Hodgkin’s disease and, 537–538 Learning disabilities, hematopoietic stem-cell transplantation and, 624 Leukemia childhood acute lymphoblastic leukemia, 477–489 graft vs., 486–488 umbilical cord blood transplantation and, risk of, 177 Leukocyte adhesion deficiency type, 329–330 Leukocyte antigen-matched siblings, beta thalassemia treatment and, 383–386 Leukocyte antigen-matched siblings, Pesaro transplant group, 383–386 Leukocyte depletion, transfusion and, 13 Leukocyte infusion, donor, 192–194 Leukodystrophies, 429–433 cerebral X-adrenoleukodystrophy, 429–431 globoid-cell, 431–432 metachromatic, 432–433 other types, 433 Leukoencephalopathy, 623 Leukopheresis, hematopoietic stem-cell transplantations, pre-transplant considerations and, 605–606 Liver chronic GHVD and, 96 ex-thalassemic patients and, 390–391 transplant process and, 619–620 Lymphoblastic lymphomas, non-Hodgkin’s disease and, 529–530, 537 Lymphoid disorders immunodeficiency diseases and, 326–328 major histocompatibility complex class II deficiency, 327 other types, 328 severe combined immunodeficiency syndrome, 326–327 Wiskott-Aldrich syndrome, 327 X-linked hyper IgM syndrome, 327–328 Lyososmes gene coding, 417 metabolic diseases and, 415–416 Major histocompatibility complex class II deficiency, 327 Malnutrition, 614 Maroteaux-Lamy syndrome, 427–428 Marrow infusion-associated acute renal failure, 8–9 Marrow stimulation, mismatched related donor transplantation and, 207

Index Marrow transplant vs. umbilical cord blood transplantation, unrelated donor, 179–180 Matching unrelated donor, ethical considerations and, 258–260 Maternal engraftment, immunodeficiency diseases and, 323 Methotrexate, 71 Mesenchymal stem cells bone marrow transplantation and, 304–305 GVHD treatment, 312 hematopoietic stem cell engraftment treatment, 312–313 inborn errors of metabolism, 311 Metabolic diseases, 415–439 causes, 415–418 clinical evaluation, 416, 417 genetics, 416 laboratory evaluation of, 418 lysosomes, 415–416 pathogenesis, 416 peroxisomes, 415–416 cross-correction principal, 418 developing therapies, 437–438 Faber disease, 437 Fabry disease, 435–436 gangliosidoses, 436 glycoprotein, 433–435 hematopoietic cell transplantation, 418–419 leukodystrophies, 429–433 liver disease, 313–314 mucolipidosis, 436 mucopolysaccharidoses, 420–428 Neimann-Pick disease, 436 neuronal ceroid lipofuscinoses, 435 osteopretrosis, 437 prevention, 418 storage, bone marrow transplantation and, 313 treatment, 418 Wolman disease, 437 Metachromatic leukodystrophy, 432–433 Metaiodobenzylguanidine scans and therapy, neuroblastoma and, 591–592 Mismatched related donor transplantation, 201–215 alloreactivity, 202–203 alternative donors, pediatric studies of, 213–214 clinical outcomes of, 210 donor selection criteria, 204–205 donor transplants, comparisons of, 212–215 engraftment, 208–209 graft manipulation, 205–207 GVHD, outcome of, 209–211

Index [Mismatched related donor transplantation] human leukocyte antigen, 203 barriers, suppression of, 205, 206 immune reconstitution, 211–212 infection, 211–212 killer immunoglobulin-like receptors, 203 marrow stimulation, 207 pediatric patients, clinical outcomes of, 211 recipient conditioning, 208 related donor, terminology of, 202 Mixed chimerism beta thalassemia treatment, 388–389 sickle cell disease and, 404–407 Mold, invasive, 42–43 Morquio syndrome, 427 Mouth, chronic GHVD and, 95–96 Mucolipidosis, 436 Mucopolysaccharidoses, metabolic diseases and, 420–428 mucopolysaccharidoses I, 420–426 Hurler syndrome, 420–424 Hurler-Scheie syndromes, 425–426 Scheie syndromes, 425–426 a-L Iduronidase deficiency, 420–424 mucopolysaccharidoses II, Hunter syndrome, 426 mucopolysaccharidoses III, Sanfilippo syndrome, 426–427 mucopolysaccharidoses IV, 427 Maroteaux-Lamy syndrome, 427–428 Morquio syndrome, 427 mucopolysaccharidoses VI, 427–428 mucopolysaccharidoses VII, Sly syndrome, 428 multiple sulfatase deficiency, 428 Mucositis, 613 hematopoietic stem-cell transplant (HSCT) and, 1–2 toxicity grading of, 3 Multiple sulfatase deficiency, 428 Muscular dystrophy, bone marrow transplantation and, 314 Musculoskeletal system, chronic GHVD and, 94–95 Mycophenolate mofetil, 73 aGVHD treatment and, 76 Myelodysplastic syndrome, unrelated hematopoietic stem-cell transplantation and, 153–154 Myeloid disorders Chediak-Higashi syndrome, 328–329 chronic granulomatous disease, 329 hemophagocytic lymphohistiocytosis, 330 immunodeficiency diseases and, 328–330

635 [Myeloid disorders] leukocyte adhesion deficiency type, 329–330 other types, 330 Natural killer cells allogeneic transplantation and, 507 immune reconstitution and, 272–273 Neimann-Pick disease, 436 Nephrotoxic drugs, 8 Neuroblastoma, 589–598 transplant, 590–598 allogeneic BMT, 597 Children’s Cancer Group 3891 study, 590–591 metaiodobenzylguanidine scans, 591–592 tandem high-dose chemotherapy, 592–595 tumor removal, 595–597 Neurocognitive impact, psychosocial outcomes of HSCT and, 237–239 Neurocognitive interventions, psychosocial intervention and, 246 Neurologic complications, hematopoietic stem-cell transplant (HSCT) and, 9–11 Neurologic effects, sickle cell disease and, 402–403 Neuronal ceroid lipofuscinoses, 435 Neutropenia, 610–611 bacterial infections and, 31–32 Non-Hodgkin’s disease adult studies, 531–532 childhood studies, 531–532 hematopoietic stem-cell transplantation and, 529–539 high-dose therapy, 530 large cell lymphoma, 530, 537–538 lymphoblastic lymphoma, 529–530, 537 small noncleaved cell lymphoma, 530 Nonmyeloablative cytoreductive regimens, allogeneic stem-cell transplantation and, 389–390 Nonmyeloablative preparative regimens, bone marrow transplantation and, 506 Nonmyeloablative transplant, reduced intensity conditioning and, 194–196 Nonselective agents aGVHD prophylaxis/treatment and, 71 methotrexate, 71 pentostatin, 71 steroids, 71 Nursing care education, 225–226 hematopoietic stem-cell transplantation and, 223–232

636 [Nursing care] patient education, 227–228, 229–231 practice settings, 223–225 professional organizations, 226–227 roles of, 223–225 special programs, 228, 232 Nutrition support, hematopoietic stem-cell transplant (HSCT) and, 6–7 Obstructive disease, 621–622 Ocular complications, 620 Osteonecrosis, sickle cell disease and, 401 Osteoporosis, 437 endocrine complications and, 293 Osteosarcoma, 580 Pancytopenia, 339–346 dyskeratosis congenita, 342–344 fanconi anemia, 339–340 hematopoietic stem-cell transplantation, 340–342, 344 Patient risk factors bacterial infections and, 28–39 invasive fungal infections and, 35–36 Pediatric consent, ethical considerations and, 253–256 Pediatric growth, sickle cell disease and, 401–402 Pediatric hematopoietic stem-cell transplantation, consent, 256–257 Pediatric peripheral blood stem cells, 137–143 Pediatric transplant patient, hematopoietic stem-cell transplant (HSCT) and, 1–19 Pentostatin, 71 Pericardial effusions, 617 Pericarditis, 617 Peripheral blood, chronic GHVD and immunological differences, 90–91 Peripheral blood progenitor cells. See Peripheral blood stem cells. Peripheral blood stem cells aGVHD risk factors and, 69 bone marrow vs., allogeneic transplantation and, 508 childhood acute lymphoblastic leukemia and, 484 collection of, 137–143 hematopoietic growth factor support and, 17 infusion target dose, 140–142 mobilization techniques, 139–140 pheresis, 137–138 processing of, 142 sickle cell disease and, 408–409

Index [Peripheral blood stem cells] storage of, 142, 143 tumor cell purging, 142–143 various sources of, 138 vascular access, 137–138 Peroxisomes gene coding, 417 metabolic diseases and, 415–416 Personality, psychological adjustments to HSCT pediatric patient and, 243 Pesaro transplant group, 384–386 leukocyte antigen-matched siblings, 383–386 risk classification, 385 Pharmacokinetic targeting, bone marrow transplantation and, 502–503 Pheresis, peripheral blood stem cells and, 137–138 Photopheresis, aGVHD treatment and, 77 Plasma, transfusion and, 13 Platelets, transfusion and, 11–12 Pneumonia, 616 Preemptive immunotherapy, bone marrow transplantation and, 517–518 Premedications, stem-cell infusion and, 10 Preparative regimens bone marrow transplantation and, 501–506 busulfan-based, 502 hematopoietic stem-cell transplantations and, 606–607 intensification of, 505 unrelated, 151 pharmacokinetic targeting, 502–503 total body irradiation, 501–502 vs. busulfan, 503–504 Pretransplant infections, immunodeficiency diseases and, 322–323 Progenitor cell function, umbilical cord blood transplantation and, 161–162 Prophylaxis, aGVHD and, 74–75 combined agent, aGVHD and, 74–75 non-use of prophylaxis, 74–75 single agent, aGVHD and, 74–75 Psychological effects, pediatric hematopoietic stem-cell transplantation and, 235–247 adherence, 245 coping, 242, 244 educational accommodations, 245–246 family influence, 239–240, 242–243, 245 grief and loss, 246 health-care professional, 247 illness stressors, 241–242 individual, 239 neurocognitive, 237–239, 246

Index [Psychological effects, pediatric hematopoietic stem-cell transplantation and] peers, 240–241 personality, 243 psychosocial intervention, 244–246 outcomes, 237–241 sociocultural factors, 243–244 stress on child and family, 235–237 temperament, 243 Pulmonary complications, 616, 621 Pulmonary effects, sickle cell disease and, 403 Pulmonary function, restrictions, 621 Quality of life ethical considerations and, 266 sickle cell disease and, 403

Radiotherapy, endocrine complications and, 288–289 Rapamycin. See Sirolimus. Recipient age, impact of on chronic GHVD and, 91 conditioning, mismatched related donor transplantation and, 208 Red blood cells, transfusion and, 11–12 Reduced intensity conditioning, 189–196 donor leukocyte infusion, 192–194 immunotherapy, 189–192 nonmyeloablative transplant, 194–196 Regimen designs, hematopoietic stem-cell transplantation, and, 453 Registries, unrelated hematopoietic stem-cell transplantation and, 149–150 Relapse childhood acute lymphoblastic leukemia and, 482 therapy, bone marrow transplantation and, 516–517 Related donor mismatched donor transplantation and, 201–215 umbilical cord blood transplantation and, 163–165, 170–171 Remission childhood acute lymphoblastic leukemia and, 478–482 first, Ewing’s sarcoma and peripheral primitive neuroectodermal tumors, 569–572 second, Ewing’s sarcoma and peripheral primitive neuroectodermal tumors, 572–575

637 Renal disease causes of early dysfunction, 8 hematopoietic stem-cell transplant (HSCT) and, 7–8 hemolytic uremic syndrome, 9 long term complications from, 9 marrow infusion-associated acute renal failure, 8–9 nephrotoxic drugs, 8 tumor lysis syndrome, 8 Renal insufficiency, 615 Reproductive dysfunction, endocrine complications and, 291–293 Respiratory syncytial virus, 52–54 prevention of, 53 treatment of, 53–54 Respiratory tract, chronic GHVD and, 96–97 Rhabdomyosarcoma, 578–579 Risk factors, aGVHD and, 68–71 Salvage regimens, chronic GHVD and, 98–100 Sanfilippo syndrome, 426–427 Scheie syndromes, mucopolysaccharidoses I and, 425–426 Schwachman-Diamond syndrome, 352–353 stem-cell transplants, 355–356 Second remission, Ewing’s sarcoma and peripheral primitive neuroectodermal tumors, 572–576 Severe aplastic anemia, unrelated hematopoietic stem-cell transplantation and, 154–155 Severe combined immunodeficiency syndrome, lymphoid disorders and, 326–327 Sibling donors, ethical considerations and, 260–261 Sickle cell disease bone marrow transplantation, 397–399 effects hematologic, 400–401 osteonecrosis, 401 pediatric growth, 401–402 splenic function, 401 hematopoietic stem-cell transplantation, 397–409 acute toxicities from, 399–400 allogeneic hematopoietic stem cells, sources of, 407–408 challenges of, 403–404 chimerism, mixed, 404–407 effects on organs, 401–403 peripheral blood stem cells, 408–409

638 [Sickle cell disease hematopoietic stem-cell transplantation] neurologic effects, 402–403 pulmonary effects, 403 quality of life, 403 umbilical cord blood transplantation, 399 Side effects, stem cell infusion and, 10 Single agent prophylaxis, 74–75 Single cytopenias, 347–359 amegakaryocytic thrombocytopenia, 357–359 diamond blackfan anemia, 347–348 hematopoietic stem-cell transplantation, 348–349, 351–352, 353–354, 357 Kostmann syndrome, 349–351 Shwachman-Diamond syndrome, 352–353 Sirolimus, 73 aGVHD treatment and, 76 Skin, 619 Sly syndrome, 428 Small noncleaved cell lymphoma, Non-Hodgkin’s disease and, 530 Sociocultural factors, psychological adjustments to HSCT pediatric patient and, 243–244 Solid tumors Ewing’s sarcoma and peripheral primitive neuroectodermal tumors, 569–577 extracranial germ cell tumors, 581 hematopoietic stem-cell transplantation and, 569–583 hepatoblastoma, 580 osteosarcoma, 580–581 rhabdomyosarcoma, 578–580 Wilms tumor, 577–578 Splenic function, sickle cell disease and, 401 Stem-cell collection, hematopoietic stem-cell transplantations, pretransplant considerations and, 605 Stem-cell infusion hematopoietic stem-cell transplantations and, 606 potential side effects, 10 premedications, 10 Stem-cell mobilization, autoimmune diseases and, 452 Stem cell sources, alternative, beta thalassemia treatment and, 386–388 Stem-cell transplants congential amegakaryocytic thrombocytopenia and, 358 Schwachman-Diamond syndrome and, 355–356

Index Stem cells, sources of aGVHD risk factors and, 68–79 bone marrow, aGVHD risk factors and, 69 cord blood, aGVHD risk factors and, 69 peripheral blood stem cells, aGVHD risk factors and, 69 Stem cell transplantation, immunodeficiency diseases and, 321–322 Steroids, aGVHD treatment and, 76 Syngeneic hematopoietic stem-cell transplantation, 602 Syngeneic transplant, 373 Systemic lupus erythematosus, hematopoietic stem-cell transplantation and, 461–464 Tacrolimus, 73 Tandem high-dose chemotherapy, neuroblastoma and, 592–595 T-cell depletion allogeneic stem-cell transplantation and, 388 graft manipulation and, 205, 207 GVHD prevention and, 119–122 immune suppression vs., allogeneic transplantation and, 509 methodologies, 120 T-cell generation, specific, childhood acute lymphoblastic leukemia and, 489 T-cell inhibitory agents aGVHD prophylaxis/treatment and, 71–73 cyclosporine, 71, 73 mycophenolate mofetil, 73 tacrolimus, 73 T-cell reconstitution, immune reconstitution and, 273–277 T cells chronic GVHD and, 85–86 cytokines profile, 86–88 Temperament, psychological adjustments to HSCT pediatric patient and, 243 Therapy-induced primary hypothyroidism, thyroid dysfunction and, 290 Thrombocytopenia, 611–612 Thrombopoietic agents, hematopoietic growth factor support and, 19 Thymoglobulin. See Antithymoctye globulin. Thyroid carcinoma, thyroid dysfunction and, 291 Thyroid dysfunction autoimmune thyroid disease, 291 carcinoma, 291 therapy-induced primary hypothyroidism, 290 Thyrotropin deficiency, 290

Index Total body irradiation acute myeloid leukemia and, 501–502 busulfan vs., bone marrow transplantation and, 503–504 gonadal dysfunction and, 292 TRALI. See Transfusion associated lung injury. Transfusion ABO incompatibility, 14 transfusion associated lung injury (TRALI) and, 15 blood product irradiation, 13 components cryoprecipitate, 13 fresh frozen plasma, 13 granulocytes, 12–13 platelets, 11–12 processing, 13 red blood cells, 11–12 leukocyte depletion, 13 support components, 11–13 hematopoietic stem-cell transplant (HSCT) and, 11–13 therapy complications, 14–16 viral infection transmission and, 15–16 Transfusion associated lung injury (TRALI), 15 Transplant patient, pediatric, hematopoietic stem-cell transplant (HSCT), 1–19 Transplant process acute toxicities, 607 anemia, 612 cardiac complications, 622 cardiovascular complications, 617 chemical hepatitis, 615 chemotherapy agents, 608–609 diarrhea, 614 engraftment, 618 gastrointestinal complications, 612 tract, 619 GVHD, 618–620, 623 hematopoietic stem-cell transplantations and, 606–623 hepatic complications, 615 hypertension, 617 idiopathic pneumonia syndrome, 616–617 infectious complications, 612–613 integumentary system, 617–618 interstitial pneumonitis, 616 late effects, 620 leukoencephalopathy, 623 liver, 619 malnutrition, 614

639 [Transplant process] mucositis, 613 neutropenia, 610–611 obstructive disease, 621–622 ocular complications, 620 pericardial effusions, 617 pericarditis, 617 pneumonia, 616 preparative regimen, 606–607 pulmonary complications, 616, 620 pulmonary function restrictions, 621 renal insufficiency, 615–616 skin, 619 stem-cell infusion, 607 thrombocytopenia, 611–612 urologic complications, 616 veno-occlusive disease, 615 Tumor cell contamination, Ewing’s sarcoma and peripheral primitive neuroectodermal tumors, 576–577 Tumor cell purging autologous hematopoietic stem cell and, 122–126 methodologies, 124 peripheral blood stem cells and, 142–143 Tumor lysis syndrome, 8 Tumor removal, neuroblastoma and, 595–597 Type I diabetes, hematopoietic stem-cell transplantation and, 465–466 Umbilical cord blood transplantation, 161–181 aGVHD, 166–169 biological features, 161–163 immunological phenotype, 163 progenitor cell function, 161–162 childhood acute lymphoblastic leukemia and, 485 clinical experience, 163–178 related donor, 163–165, 170–171 unrelated donor, 171–178 colony-stimulating factors and, 18 donor graft selection, blood units, 180 cell dose, 180 considerations for, 178–180 human leukocyte antigen-match, 180 unrelated, 179–180 engraftment, 166–169 larger sized recipients, 181 risk of leukemia, 177 sickle cell disease and, 399 unrelated donor, marrow vs. umbilical, 179–180

640 Unrelated donor registries, hematopoietic stem-cell transplantation and, 147 Unrelated donor transplants, acquired aplastic anemia and, 377–378 Unrelated donor, umbilical cord blood transplantation and, 171–178 graft selection and, 179–180 marrow vs. umbilical, 179–180 Unrelated, hematopoietic stem-cell transplantation, 147–155 complications, 151–152 infections, 151 graft-versus-host disease, 151–152 graft failure, 152 donor selection, 149–150 histocompatibility, 148–149 HLA typing, 149 preparative regimens, 151 registries, 149–150 role of, 152–155 acute lymphoblastic leukemia, 152–153 acute myelogenous leukemia, 153 chronic myelogenous leukemia, 154 Hurler syndrome, 155 myelodysplastic syndrome, 153–154 severe aplastic anemia, 154–155 Wiskott-Aldrich syndrome, 155 Untreated relapse vs. attempted induction, bone marrow transplantation and, 515–516 Urologic complications, 616

Index Vaccinations, 34 childhood acute lymphoblastic leukemia and, 489 responses, antigen-specific responses and, 279–280 Vascular access, peripheral blood stem cells and, 137–138 Veno-occlusive disease (VOD), 2–6, 615 Viral infections adenovirus, 50–51 BK virus, 52–53 cytomegalovirus, 47–50 Epstein-Barr, 50–51 herpes simplex, 55 human herpes virus type 6, 51 respiratory syncytial virus, 52–55 transfusion transmission and, 15–16 VOD. See Veno-occlusive disease.

Wilms tumor, 576–578 Wiskott-Aldrich syndrome, 327 unrelated hematopoietic stem-cell transplantation and, 155 Wolman disease, 437

X-linked hyper IgM syndrome, 327–328

Yeast, invasive, 41 Yeast organism prophylaxis, invasive fungal infections and, 38–39