Allogeneic Stem Cell Transplantation (Second Edition)

Allogeneic Stem Cell Transplantation

C O N T E M P O R A R Y H E M AT O L O G Y

Judith E. Karp, Series Editor

For other titles published in the series, go to www.springer.com/7861

Allogeneic Stem Cell Transplantation Second Edition

Edited by

Hillard M. Lazarus University Hospitals Case Medical Center Cleveland, OH USA

Mary J. Laughlin Case Western Reserve University Cleveland, OH USA

Editors Hillard M. Lazarus University Hospitals Case Medical Center Cleveland, OH USA [email protected]

Mary J. Laughlin Case Western Reserve University Cleveland, OH USA [email protected]

ISBN 978-1-934115-33-6 e-ISBN 978-1-59745-478-0 DOI 10.1007/978-1-59745-478-0 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2009930362 © Springer Science+Business Media, LLC 2003, 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Dr. Hillard M. Lazarus dedicated his contributions to his wife Joan and his sons Adam and Jeffrey for their unwavering encouragement and support.

Preface

Allogeneic hematopoietic stem cell (HSC) transplantation has undergone fast-paced changes after our original publication of Allogeneic Stem Cell Transplantation: Clinical Research and Practice, first published more than 5 years ago. In this second edition, the editors have focused on topics relevant to evolving knowledge in the field in order to better guide clinicians in decisionmaking and management of their patients, as well as help lead laboratory investigators in new directions emanating from clinical observations. Some of the most respected clinicians and scientists in this discipline have responded in this second edition by providing state-of-the-art discussions addressing these topics. Important advances have been recognized in HLA disparity between HSC donor and recipient triggers for T-cell and NK-cell allorecognition; such may induce the graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects and may cause an engraftment failure. This text covers the scope of human genomic variation, the methods of HLA typing, and interpretation of high-resolution HLA results. Durable GVL responses may be the result of the elimination of leukemia stem cells or the establishment of a durable immune control on their progeny. Alternative sources of donor HSC continue to be used for transplantation at an increased frequency and include HLA-matched unrelated donor and umbilical cord blood; overall patient outcome has improved steadily using these diverse stem cell sources. The administration of reduced-intensity as well as non-myeloablative conditioning has also brought forth new concepts in the management of hematologic malignancies, thought to be of emerging importance in patients with lower grade malignant disorders such as chronic lymphocytic leukemia, multiple myeloma, and low-grade non-Hodgkin lymphoma. The elderly or those with comorbid conditions who have acute leukemia in complete remission also may benefit by using this lower-intensity therapy. The reduced toxicity of these novel conditioning regimens has also raised new possibilities in the application of allogeneic HSC transplantation for patients with non-malignant hematologic disorders such as sickle cell anemia and selected solid tumors such as renal cell carcinoma. Allogeneic SCT remains the only available curative therapy for hematologic malignancies and some inborn errors such as beta-thalassemia. Its application, however, may result in significant morbidity and mortality, predominantly as a consequence of opportunistic infections and GVHD. While differences in HLA between donor and recipient make a crucial contribution to the alloreactivity vii

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driving the donor-mediated GVL response, the cytokine milieu both promotes and regulates the allogeneic response after transplantation. As such, genetic studies correlating donor, host, or the combination of cytokine polymorphisms with disease outcomes have provided useful insight into disease pathogenesis, often confirming effects that have been determined in pre-clinical studies. It is now clear that the polymorphic expression of key cytokines (particularly tumor necrosis factor and interleukin 10) has a demonstrable effect on disease outcome and overall transplant-related mortality. Many challenges in allogeneic SCT remain and include the risk of graft failure, recurrent disease, acute GVHD, opportunistic infections and longterm sequelae such as chronic GVHD, increased risk of second malignancies, endocrinopathies, and iron overload. The editors hope that this new information, well summarized by the authors in this text, will be of significant benefit to clinicians and researchers in allogeneic HSC transplantation. We envision that the generation of further knowledge and clinical studies to be of ultimate benefit to our patients. Cleveland, Ohio, USA

Hillard M. Lazarus, MD Mary J. Laughlin, MD

Contents

  1 Allogeneic Stem Cell Transplantation: The Last Century................ John M. Goldman

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  2 Full Intensity and Reduced Intensity Allogeneic Transplantation in AML.................................................................... Charles Craddock

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  3 Allogeneic Stem Cell Transplantation for Adult Acute Lymphoblastic Leukemia (ALL)...................................................... Bella Patel, Anthony H. Goldstone, and Adele K. Fielding

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  4 Hematopoietic Progenitor Cell Transplantation for Treatment of Chronic Lymphocytic Leukemia........................... Leslie A. Andritsos, John C. Byrd, and Steven M. Devine

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  5 The Role of Allogeneic Hematopoietic Stem Cell Transplantation for Chronic Myelogenous Leukemia Patients in the Era of Tyrosine Kinase Inhibitors............................. Richard T. Maziarz   6 Allogeneic Transplantation for Hodgkin’s Lymphoma.................... William Broderick and Patrick Stiff   7 Myeloablative Allogeneic Stem Cell Transplantation for Non-Hodgkin’s Lymphoma......................................................... J. Kuruvilla, P. Mollee, and J.H. Lipton

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  8 Non-Hodgkin’s Lymphoma: Allogeneic Reduced Intensity Conditioning..................................................................................... 109 Sonali M. Smith and Ginna G. Laport   9 The Role of Allogeneic Transplantation for Multiple Myeloma in Older Adults................................................................. 127 Heidi D. Klepin and David D. Hurd

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10 Single Versus Tandem Autologous Hematopoietic Stem Cell Transplant in Multiple Myeloma..................................... 143 David H. Vesole 11 Treatment Strategies for Follicular Center Cell Non-Hodgkin’s Lymphoma.............................................................. 159 Frank Heinzelmann, Hellmut Ottinge, and Claus Belka 12 The Role of Transplantation in Favorable-Risk Acute Myeloid Leukemia............................................................................ 177 Mickey Liao and Gary J. Schiller 13 Allogeneic Stem Cell Transplantation for Acute Lymphoblastic Leukaemia in Adults................................................ 193 David I. Marks 14 Allogeneic Transplantation for Myelodysplastic Syndromes........... 203 Geoffrey L. Uy and John F. DiPersio 15 Allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Acute Lymphoblastic Leukemia.................................... 219 Adriana Balduzzi, Lucia Di Maio, Mary Eapen, and Vanderson Rocha 16 Allogeneic Transplantation for the Treatment of Multiple Myeloma........................................................................ 261 Rebecca L. Olin, Dan T. Vogl, and Edward A. Stadtmauer 17 Blood Vs. Marrow Allogeneic Stem Cell Transplantation............... 281 Brian McClune and Daniel Weisdorf 18 Hematopoietic Cell Transplantation from Partially HLA-Mismatched (HLA-Haploidentical) Related Donors.............. 299 Ephraim J. Fuchs and Heather J. Symons 19 Unrelated Donor Transplants............................................................ 345 Andrea Bacigalupo 20 Update on Umbilical Cord Blood Transplantation........................... 363 Karen Ballen 21 Selection of Cord Blood Unit(s) for Transplantation........................ 375 Donna A. Wall and Ka Wah Chan 22 Mobilization of Hematopoietic Cells Prior to Autologous or Allogeneic Transplantation........................................................... 387 Steven M. Devine

Contents 

23 Natural Killer-Cell Based Treatment in Hematopoetic Stem Cell Transplantation................................................................. 413 Martin Stern, Sandrine Meyer-Monard, Uwe Siegler, and Jakob R. Passweg 24 Cryopreservation of Allogeneic Stem Cell Products........................ 427 Noelle V. Frey and Steven C. Goldstein 25 Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic Stem Cell Transplantation....................... 441 Steven C. Goldstein and Selina Luger 26 Allogeneic Haematopoietic Stem Cell Transplantation and Natural Killer Cell Alloreactivity............................................... 459 Franco Aversa and Andrea Velardi 27 Therapeutic Potential of Mesenchymal Stem Cells in Hematopoietic Stem Cell Transplantation........................... 477 Luis A. Solchaga and Hillard M. Lazarus 28 Hematopoietic Stem Cell Transplantation for Thalassemia............. 491 Javid Gaziev and Guido Lucarelli 29 Viral Infections in Hematopoietic Stem Cell Transplant Recipients.......................................................................................... 505 Per Ljungman 30 Fungal Infections.............................................................................. 533 John R. Wingard 31 Immune Reconstitution and Implications for Immunotherapy Following Hematopoeitic Stem Cell Transplantation....................... 545 Kirsten M. Williams and Ronald E. Gress 32 Acute Graft Versus Host Disease: Prophylaxis................................. 565 Corey Cutler, Vincent T. Ho, and Joseph H. Antin 33 Chronic Graft-Versus-Host Disease.................................................. 577 Madan Jagasia and Steven Pavletic 34 Post-transplant Lymphoproliferative Disorder.................................. 597 Ran Reshef, Alicia K. Morgans, and Donald E. Tsai 35 Psychological Care of Adult Allogeneic Transplant Patients........... 619 Flora Hoodin, Felicity W.K. Harper, and Donna M. Posluszny 36 Second Allogeneic Transplantation: Outcomes and Indications.................................................................................. 657 Koen van Besien, Dan Pollyea, and Andrew Artz

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37 Minimal Residual Disease................................................................ 667 Mehmet Uzunel 38 Functional Assessment Tools and Co-morbidity Scoring in Hematopoietic Progenitor Cell Transplantation........................... 687 Sergio Giralt and Uday Popat 39 Unique Thrombotic and Hemostatic Complications Associated with Allogeneic Hematopoietic Stem Cell Transplantation................................................................. 695 Amber A. Petrolla, Hillard M. Lazarus, and Alvin H. Schmaier 40 How Much Isolation Is Enough for Allografts?................................ 717 Brandon Hayes-Lattin 41 Monoclonal Antibodies in Allogeneic Hematopoietic Stem Cell Transplantation for Hematologic Malignancies............... 733 Maria Corinna Palanca-Wessels and Oliver Press 42 Treatment of Acute Graft-vs-Host Disease....................................... 747 Steven C. Goldstein, Sophie D. Stein, and David L. Porter 43 The Importance of Non-Human Primate Models for Pre-clinical Studies in Hematopoiesis......................................... 767 Erzsebet Szilagyi, Nadim Mahmud, and Amelia Bartholomew 44 In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation.................................................................................. 789 Lisbeth A. Welniak and William J. Murphy 45 Dendritic Cells.................................................................................. 807 Jacalyn Rosenblatt and David Avigan 46 Augmentation of Hematopoietic Stem Cell Transplantation with Anti-cancer Vaccines................................................................ 855 Edward D. Ball and Peter R. Holman Erratum ................................................................................................... .

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Index......................................................................................................... 871

Contributors

Leslie A. Andritsos, MD Division of Hematology & Oncology, The Ohio State University Medical Center, Columbus, OH, USA Joseph H. Antin, MD Harvard Medical School and Dana Farber Cancer Institute, Boston, MA, USA Andrew Artz, MS, MD Section of Hematology and Oncology, Department of Medicine, University of Chicago, Chicago, IL, USA Franco Aversa, MD Section of Haematology and Clinical Immunology, Department of Clinical and Experimental Medicine, HSCT Unit, University of Perugia, Perugia, Italy David Avigan, MD Division of Hematological Malignancies/Bone Marrow Transplantation, Beth Israel Deaconess Medical Center, Boston, MA, USA Andrea Bacigalupo Ospedale San Martino, Genova, Italy Adriana Balduzzi, MD Hematopoeitic Transplant Unit, Clinica Pediatrica, Università degli Studi di Milano, Bicocca Ospedale, San Gerardo, Italy Edward D. Ball, MD Division of Blood and Marrow Transplantation, Department of Medicine and the Moores UCSD Cancer Center, University of California, San Diego, La Jolla, CA, USA Karen Ballen, MD Division of Hematology/Oncology, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA Amelia Bartholomew, MD Division of Transplant Surgery, Department of Surgery, University of Illinois at Chicago College of Medicine, Chicago, IL, USA

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Claus Belka, MD Department of Radiation Oncology, University of Tuebingen, Tuebingen, Germany William Broderick, MD Division of Hematology-Oncology, Department of Medicine, Bone Marrow Transplant Program, Loyola University Stritch School of Medicine, Maywood, IL, USA John C. Byrd, MD Division of Hematology & Oncology, The Ohio State University Medical Center, Columbus, OH, USA Ka Wah Chan, MD Pediatric Blood and Marrow Transplantation Program, Texas Transplant Institute, San Antonio, TX, USA Charles Craddock Centre for Clinical Haematology, Queen Elizabeth Hospital, Edgbaston, Birmingham, UK Corey Cutler, MD, MPH, FRCP Harvard Medical School and Dana Farber Cancer Institute, Boston, MA, USA Steven M. Devine, MD Division of Hematology & Oncology, The Ohio State University Medical Center, Columbus, OH, USA Lucia Di Maio, MD Hematopoeitic Transplant Unit, Clinica Pediatrica, Università degli Studi di Milano, Bicocca Ospedale, San Gerardo, Italy John F. DiPersio, MD, PhD. Section of BMT and Leukemia, Division of Oncology, Washington University School of Medicine, St. Louis, MO, USA Mary Eapen, MD Center for International Blood and Marrow Transplant Research, Medical College of Wisconsin, Milwaukee, WI, USA Adele K. Fielding, MD Department of Haematology, Royal Free and University College Medical School, London, UK Noelle V. Frey, MD Division of Hematology-Oncology and Abramson Cancer Center, University of Pennsylvania Medical Center, Philadelphia, PA, USA Ephraim J. Fuchs, MD Divisions of Pediatric Oncology, Cancer Immunology and Hematologic Malignancies, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA Javid Gaziev, MD International Centre for Transplantation in Thalassemia and Sickle Cell Anemia, Mediterranean Institute of Hematology, Rome, Italy

Contributors 

Sergio Giralt, MD Department of Stem Cell Transplant and Cellular Therapy, University of Texas MD Anderson Cancer Center, Houston, TX, USA John M. Goldman, MD Department of Hematology, Imperial College Faculty of Medicine and World Marrow Donor Association, London, UK Steven C. Goldstein, MD Division of Hematology-Oncology and Abramson Cancer Center, University of Pennsylvania Medical Center, Philadelphia, PA, USA Anthony H. Goldstone Department of Haematology, University College London Hospitals, London, UK Ronald E. Gress, MD Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Felicity W.K. Harper, PhD Communication and Behavioral Oncology Program, Barbara Ann Karmanos Cancer Institute and Department of Family Medicine and Public Health Sciences, Wayne State University School of Medicine, Detroit, MI, USA Brandon Hayes-Lattin, MD Center for Hematologic Malignancies, OHSU Cancer Institute, Oregon Health and Science University, Portland, OR, USA Frank Heinzelmann, MD Department of Radiation Oncology, University of Tuebingen, Tuebingen, Germany Vincent T. Ho, MD Harvard Medical School and Dana Farber Cancer Institute, Boston, MA, USA Peter R. Holman, MD Division of Blood and Marrow Transplantation, Department of Medicine and The Moores UCSD Cancer Center, University of California, San Diego, La Jolla, CA, USA Flora Hoodin, PhD Department of Psychology, Eastern Michigan University, Ypsilanti, MI, USA David D. Hurd, MD Section of Hematology-Oncology, Department of Internal Medicine, School of Medicine, Wake Forest University, Winston-Salem, NC, USA Madan Jagasia, MBBS, MS Division of Hematology-Oncology, Department of Medicine, Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA Heidi D. Klepin, MD Section of Hematology-Oncology, Department of Internal Medicine, School of Medicine, Wake Forest University, Winston-Salem, NC, USA

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Contributors

John Kuruvilla, MD Division of Medical Oncology and Hematology, Princess Margaret Hospital and University of Toronto, Toronto, ON, Canada Ginna G. Laport, MD Division of Blood and Marrow Transplantation, Stanford University Medical Center, Stanford, CA, USA Hillard M. Lazarus, MD Case Comprehensive Cancer Center, University Hospitals Case Medical Center, Cleveland, OH, USA Mickey Liao, MD Hematologic Malignancies Unit/Stem Cell Transplant Unit, University of California at Los Angeles, Los Angeles, CA, USA Jeffrey H. Lipton, MD Division of Medical Oncology and Hematology, Princess Margaret Hospital and University of Toronto, Toronto, ON, Canada Per Ljungman, MD Department of Hematology, Karolinska University Hospital, Stockholm, Sweden Guido Lucarelli, MD International Centre for Transplantation in Thalassemia and Sickle Cell Anemia, Mediterranean Institute of Hematology, Rome, Italy Selina Luger, MD Division of Hematology-Oncology and Abramson Cancer Center, University of Pennsylvania Medical Center, Philadelphia, PA, USA Nadim Mahmud, MD, PhD Division of Hematology-Oncology, University of Illinois at Chicago, Chicago, IL, USA David I. Marks, MD University Hospitals of Bristol, Oncology Day Beds, Bristol Children’s Hospital, Bristol, UK Richard T. Maziarz, MD Center for Hematologic Malignancies, Adult Bone Marrow Transplantation Program, Oregon Health Science Cancer Institute, Oregon Health & Science University, Portland, OR, USA Brian McClune, DO Blood and Marrow Transplantation Program, University of Minnesota, Minneapolis, MN, USA Keith McCrae, MD Division of Hematology and Oncology, Case Western Reserve University School of Medicine, Cleveland, OH, USA Sandrine Meyer-Monard, MD Division of Hematology, Basel University Hospital, Basel, Switzerland

Contributors 

Peter Mollee, MD Department of Haematology, Princess Alexandra Hospital and University of Queensland, Brisbane, QLD, Australia Alicia K. Morgans, MD Abramson Cancer Center, Hematologic Malignancies Program, University of Pennsylvania Medical Center, Philadelphia, PA, USA William J. Murphy, MD Department of Dermatology, University of California, Davis Sacramento, CA 95817 Rebecca L. Olin, MD Abramson Cancer Center, Bone Marrow and Stem Cell Transplant Program, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Hellmut Ottinger, MD Department of Bone Marrow Transplantation, University of Essen, Essen, Germany Maria Corinna Palanca-Wessels, MD Fred Hutchinson Cancer Research Center and Department of Medicine, University of Washington, Seattle, WA, USA Jakob R. Passweg, MD Division of Hematology, Geneva University Hospitals, Geneva, Switzerland Bella Patel Department of Haematology, Royal Free and University College Medical School, London, UK Steven Pavletic, MD Graft-versus-Host and Autoimmunity Unit, Experimental Transplantation and Autoimmunity Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Amber A. Petrolla, MD Department of Pathology, Case Western Reserve University and University Hospitals Case Medical Group, Cleveland, OH, USA Dan Pollyea, MD Divisions of Hematology and Oncology, Stanford University School of Medicine, Palo Alto, CA, USA Uday Popat, MD Department of Stem Cell Transplant and Cellular Therapy, University of Texas MD Anderson Cancer Center, Houston, TX, USA David L. Porter, MD Allogeneic Stem Cell Transplantation, University of Pennsylvania Medical Center, Philadelphia, PA, USA Donna M. Posluszny, PhD Department of Medicine, University of Pittsburgh School of Medicine and Behavioral Medicine Clinical Service, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

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Contributors

Oliver W. Press, MD Fred Hutchinson Cancer Research Center and University of Washington School of Medicine, Seattle, WA, USA Ran Reshef, MD Abramson Cancer Center, Hematologic Malignancies Program, University of Pennsylvania Medical Center, Philadelphia, PA, USA Vanderson Rocha, MD, PhD Acute Leukemia Working Party of the European Blood and Marrow Transplant Group, Hopital Saint Antoine and Hematopoeitic Transplant Unit and Eurocord Registry, Hopital Saint Louis, Assistance Publique des Hopitaux de Paris, University of Paris, Paris, France Jacalyn Rosenblatt, MD Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA Gary J. Schiller, MD Hematologic Malignancies Unit/Stem Cell Transplant Unit, University of California at Los Angeles, Los Angeles, CA, USA Alvin H. Schmaier, MD Division of Hematology and Oncology, Case Western Reserve University and University Hospital Case Medical Group, Cleveland, OH, USA Uwe Siegler, MD Division of Hematology, Basel University Hospital, Basel, Switzerland Sonali M. Smith, MD Section of Hematology/Oncology, The University of Chicago Medical Center, Chicago, IL, USA Luis A. Solchaga, PhD Case Comprehensive Cancer Center, University Hospitals Case Medical Center, Cleveland, OH, USA Edward A. Stadtmauer, MD Abramson Cancer Center, Bone Marrow and Stem Cell Transplant Program, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Sophie D. Stein, MD Department of Hematology-Oncology, University of Pennsylvania Medical Center, Philadelphia, PA, USA Martin Stern, MD Division of Hematology, Basel University Hospital, Basel, Switzerland Patrick Stiff, MD Division of Hematology-Oncology, Department of Medicine, Bone Marrow Transplant Program, Loyola University Stritch School of Medicine, Maywood, IL, USA Heather J. Symons, MD Divisions of Pediatric Oncology, Cancer Immunology and Hematologic Malignancies, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA

Contributors 

Erzsebet Szilagyi, MD Division of Hematology-Oncology, University of Illinois at Chicago, Chicago, IL, USA Donald E. Tsai, MD Abramson Cancer Center, Hematologic Malignancies Program, University of Pennsylvania Medical Center, Philadelphia, PA, USA Geoffrey L. Uy, MD Section of BMT and Leukemia, Division of Oncology, Washington University School of Medicine, St. Louis, MO, USA Mehmet Uzunel, PhD Karolinska University Hospital, Stockholm, Sweden Koen van Besien, MD Section of Hematology/Oncology, University of Chicago, Chicago, IL, USA Andrea Velardi, MD Section of Haematology and Clinical Immunology, HSCT Unit, Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy David H. Vesole, MD, PhD, FACP Attending Physician, St. Vincent’s Comprehensive Cancer Center, New York, NY, USA Dan T. Vogl, MD Abramson Cancer Center, Bone Marrow and Stem Cell Transplant Program, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Donna A. Wall, MD Cancer Care Manitoba, Winnipeg, MB, Canada Daniel Weisdorf, MD Blood and Marrow Transplantation Program, University of Minnesota, Minneapolis, MN, USA Lisbeth Welniak, PhD Department of Dermatology, University of California, Davis Sacramento, CA 95817 Kirsten M. Williams, MD Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA John R. Wingard, MD Division of Hematology-Oncology, Bone Marrow Transplant Program, University of Florida Shands Cancer Center, Gainesville, FL, USA

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Chapter 1 Allogeneic Stem Cell Transplantation: The Last Century John M. Goldman

Sporadic and always unsuccessful attempts to reconstitute bone marrow function by transfusion of blood, bone marrow or fetal liver cells collected from normal individuals were made in the nineteenth and first half of the twentieth centuries, but in practice the notion that hematopoietic stem cell transplantation could prove to be of clinical value proceeded only slowly in the last century [1]. One of the earlier important observations was made by a Dutch scientist in 1922, who noted that the expected thrombocytopenia and hemorrhage in guinea pigs following total body irradiation (TBI) could be prevented by shielding from irradiation the animal’s legs [2]. The studies were not pursued at that time, but in 1949, Jacobson and colleagues reported that mice exposed to lethal doses of irradiation could be protected by shielding the spleen, which functions as a hematopoietic organ in the mouse [3]. These workers went on to show that this protection could also be provided by transfusion of spleen cells into the mouse peritoneum [4]. In the same year, Lorenz and coworkers showed that “lethally” irradiated mice and guinea pigs could be protected by intravenous injections of bone marrow cells collected from syngeneic animals [5]. One possible interpretation of these findings was simply that a humoral factor transferred from the healthy animal was able to “stimulate regeneration” of hematopoiesis in the irradiated animal, but experiments using a variety of histochemical and genetic markers showed convincingly that this prevention of lethality after irradiation was due to transfer of donor cells rather than of components of the plasma [6–9]. In 1956, Ford introduced the term “radiation chimaera” to describe an animal whose hematopoiesis was derived from a donor animal after TBI [8]. Subsequently, it was shown that such chimerism could be established in animals whose own hematopoiesis was destroyed by combinations of cytotoxic drugs without any irradiation. Also, in 1956, Barnes and colleagues studied a lymphoid leukemia that could be transmitted in mice by passage of cells intravenously or subcutaneously [10, 11]; when these leukemic mice were subjected to 1,500 rad of TBI followed by transfusion of cells from a normal donor animal, the majority survived. This was the first convincing demonstration that leukemia cells could be killed by high dose irradiation and laid the foundation for the use of high dose cytotoxic drugs

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_1, © Springer Science + Business Media, LLC 2003, 2010

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treatment (with or without irradiation) followed by marrow infusion as therapy for leukemia in man.

1.  Early Clinical Studies Some of the earliest interpretable studies of stem cell transplantation in man involved autografting. In 1958, Kurnick and colleagues collected and cryopreserved bone marrow cells from two patients with metastatic malignant disease; the patients were then treated with high dose irradiation and their thawed marrow cells were infused intravenously [12]. Though the authors could not be certain that the subsequent marrow recovery was indisputably due to the autologous infusion of marrow cells, this seemed very probable. The following year Thomas and coworkers reported the results of treating two patients with acute leukemia, both of whom had identical twins, by high dose irradiation followed by transfusion of nucleated cells from their respective twins [13]. Both patients engrafted but in both cases the leukemia recurred and was the cause of death. Also, in 1959, Mathé reported the results of his attempts to treat six persons accidentally exposed to high dose irradiation in Vinca in Yugoslavia [14]; there was transient evidence of engraftment in some of the patients. Subsequently, his group in Paris reported the first case of complete engraftment with survival beyond 1 year; in the event the patient developed both acute and chronic graft-versus-host disease (GvHD) and died eventually of varicella encephalitis [15]. In 1968, Mathé summarized his experience in treating 21 patients by bone marrow transplantation, of which 6 had failed to engraft and 8 had sustained GvHD [16]. The first truly successful use of allogeneic hematopoietic cells in man was reported by Gatti and coworkers from Minneapolis in 1968 [17]. They treated a five-month-old male with a sexlinked lymphopenic immunological deficiency by transfusion of cells from blood buffy coat and bone marrow collected from an immunologically competent sibling donor. The patient was clinically well and the continued success of the procedure was confirmed by a follow-up report published 25 years later [18]. This was apparently the first patient cured by infusion of hematopoietic cells collected from an allogeneic donor, using of course contemporary HLA matching techniques. The first successful allograft for aplastic anemia was reported by Thomas and coworkers in 1972 [19]. In 1975, the status of syngeneic and allogeneic transplantation in man was well summarized by a two-part review published by Thomas and coworkers in the New England Journal of Medicine [20]. Techniques for conditioning the individual patient were discussed and methodology for collecting and transplanting bone marrow cells was described in detail. The experience with transplants performed for 37 patients with aplastic anemia and 73 patients with leukemia was summarized. Attention was drawn to the problems of GvHD, slow engraftment and opportunistic infection. This was followed by the first definitive paper describing the use of allogeneic stem cell transplantation to treat a large series of patients with “end-stage” acute leukemia in Seattle [21]; Thomas and colleagues reported that of 100 poor-risk patients, 13 had become very long-term leukemia-free survivors. It was clear that the risk of transplant-related mortality was substantial and some of those who survived the procedure relapsed with leukemia, but the notion that even a minority of

Chapter 1  Allogeneic Stem Cell Transplantation: The Last Century 

these poor-risk patients might be cured was seen by many as “exciting.” The workers reasoned that if some patients with advanced leukemia could be cured by allografting, the incidence of cure might be considerably higher if the transplants were performed in complete remission. This assumption proved subsequently to be correct.

2.  Allografting for Aplastic Anemia The first systematic approach to the use of allogeneic stem cell transplantation started in Seattle in the early 1970s. By 1974, the group was able to report results of transplanting 38 patients with aplastic anemia, most of whom had been conditioned with cyclophosphamide 50  mg/kg daily for 4  days [22], which was a modification of a regimen proposed originally by Santos in 1970. One major problem with these early transplants for aplastic anemia was failure of sustained engraftment, which was attributed to sensitization of the host to minor histocompatibility antigens present on donor cells. This could be overcome to some extent by increasing the intensity of the conditioning. In 1976, Camitta reported results of a randomized prospective study showing that allogeneic stem cell transplantation resulted in survival superior to that achieved with conventional nontransplant therapy [23].

3.  Allografting for Acute Leukemia In 1979, the Seattle group was able to report preliminary data on results of allogeneic stem cell transplant using HLA-identical siblings for 19 patients with “acute nonlymphoblastic leukemia” in remission [24]. A follow-up paper 4  years later reported that 10 of the first 19 patients were alive and free of leukemia at more than 5 years after their transplants [25]. These results led specialist groups on both sides of the Atlantic to initiate programs for transplanting adult patients with both acute myeloid and acute lymphoblastic leukemia (ALL) in remission. Clearly, children with ALL in first remission continued to be candidates for maintenance chemotherapy rather than allografting, but the notion of offering a transplant to a child in second remission of ALL gained support. A further important step in the use of allogeneic SCT for acute leukemia was the first successful use of an unrelated donor – as reported by Hansen and colleagues in 1980 [26]. Since that time it has been generally accepted that the best donor for a given patient is an HLA-matched sibling (or possibly a genetically identical twin), but HLA matched volunteer donors can in some series yield clinical results comparable to those achieved with sibling donors. For children cord blood stem cells, as originally demonstrated in 1988 by Gluckman and coworkers in Paris [27], have become an important alternative source of stem cells, in the absence of a matched sibling. The extent to which cells in the graft is depleted played an important role in control and eradication of leukemia was a matter of considerable debate in the 1970s. In the late 1979 and 1981, the Seattle group published two important papers that showed convincingly that relapse of acute leukemia was much rarer in patients who sustained acute or chronic GvHD compared with those who had little or no GvHD [28, 29]. This was impressive if indirect evidence that a

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graft-versus-leukemia (GvL) effect which segregated with a GvHD effect was important in suppressing or eradicating leukemia at least in some cases. The later demonstration that depletion of T-cells from the donor inoculum increased the incidence of leukemia relapse, most especially in chronic myeloid leukemia (CML) [30, 31], helped to establish the concept that a GvL effect did indeed play a crucial role in the cure of leukemia by allogeneic SCT. Final proof of the efficacy of the GvL effect came from the observation by Kolb and coworkers that relapse after allogeneic stem cell transplantation for CML could be readily reversed by transfusion of lymphocytes of donor origin [32].

4.  Allografting for Chronic Myeloid Leukemia It is difficult in the modern era to imagine that CML was generally regarded until the 1980s as an inexorably fatal disease for which no cure could usefully be contemplated. It was therefore very exciting when in 1979, Fefer and co­workers in Seattle published a report of four patients with CML in chronic phase that had been treated with high dose chemoradiotherapy followed by transplantation of marrow cells collected from their respective identical twins [33]. All four patients were well without evidence of Ph-positive marrow metaphases at follow-up periods from 22 to 31 months after transplantation. Though it was entirely possible that each of these patients would still relapse, it gave impetus to the notion that allogeneic stem cell transplantation performed with marrow cells from HLA-identical siblings might be a useful approach to managing and hopefully curing this form of leukemia. Transplant programs for CML using matched sibling donors were therefore initiated in Seattle and elsewhere [34–36]. Preliminary results with small numbers of patients confirmed that the principal risks were indeed GvHD and opportunistic infection, whereas relapse was rare in survivors. In the early 1980s, Prentice and colleagues in London showed that depletion of donor marrow cells by incubation with anti-T cells monoclonal antibodies prior to infusion to the patient very greatly reduced the incidence of GvHD [37]. Unfortunately, T-cell depletion was associated with an increased risk of nonengraftment and impaired immune reconstitution. It appeared also to abrogate the GvL effect, most prominently in CML, since the actuarial relapse for CML patients allografted in chronic phase who received sibling marrow cells treated with a pan-lymphoid monoclonal antibody (CD52, Campath, now known as alemtuzumab) approached 70%. Various methods of T-cell depletion have been explored subsequently and its use is undoubtedly valuable in selected cases, most especially where later relapse of leukemia is amenable to management with donor lymphocyte infusions.

5.  Graft-Versus-Host Disease In the 1950s, Barnes and Loutit reported that irradiated mice that received spleen cells from syngeneic donors engrafted and survived without significant problems, whereas irradiated mice transfused with spleen cells from a different murine strain died within 100 days of the transplant [38]. These observations were extended by Cohen and coworkers who noted that the affected animals had severe diarrhea, weight loss and skin lesions, and a syndrome that they

Chapter 1  Allogeneic Stem Cell Transplantation: The Last Century 

designated “secondary disease” [39]. Gradually it became clear that this secondary disease was probably caused by immunological incompatibility between donor lymphoid cells and specific organs in the recipient, a syndrome now designated “graft-versus-host disease.” The requirements for establishment of GvHD are: (1) that the graft consists of immunologically competent cells; (2) that the host cells express antigens that are absent on the donor cells; and (3) that the recipient is incapable of mounting an effective immunological reaction against the graft. The requirements can, of course, be met in patients who have not been subjected to conditioning prior to transplant, for example in the patient with severe combined immunodeficiency or the patient who has received extensive chemotherapy for Hodgkins’s disease. It is conventional today to ensure that all blood products administered to patients post transplant are irradiated in vitro to at least to 1,500 cGy to prevent allogeneic GvHD.

6.  Histocompatibility In the late 1930s, Gorer described alloantigens in the sera of mice which comprise part of a murine major histocompatibility complex (MHC), subsequently named H2. These alloantigens were shown to play a central role in tissue rejection [40, 41]. In 1958, Dausset described the first analogous human antigen, then named Mac and now known as HLA-A2 [42]. A number of scientific workshops were then convened at regular intervals and by 1968, it had become clear that human leukocyte antigens (HLA) A and B were closely linked on the short arm of human chromosome 6. HLA-C was identified in 1971. The HLA D locus which is defined in part by expression of alloreactivity in the mixed lymphocyte culture was characterized by Dupont in 1980 [43]. The term haplotype was introduced to describe a sequence of genes on one or other chromosome 6 that together comprise the major MHC in man [44]. In more recent years restriction fragment length polymorphisms were used to characterize more precisely the various HLA genes and this technique has, in turn, given way to a variety of molecular methods, which include direct sequencing of polymorphic regions and allele specific oligonucleotide PCR (Table 1-1).

7.  Evolution of Conditioning Regimens Therapeutic regimens were developed originally as immunosuppression to permit engraftment in patients with nonmalignant conditions (i.e., genetic diseases or aplastic anemia) and were at the time termed “conditioning regimens.” When the use of allogeneic stem cell transplantation was adapted for patients with leukemia, the regimen was intended to provide immunosuppression but also to eradicate residual leukemia cells, yet the term “conditioning regimen” was retained. Initially, the Seattle group concentrated on the use of TBI delivered from two opposing 60Cobalt sources while Santos in Baltimore performed transplants after administration of high dose cyclophosphamide. Because the leukemia relapse rate was relatively high with both approaches to conditioning, both were changed. The Seattle group adopted the regimen of high dose cyclophosphamide followed by 1,000 cGy of TBI (cyclo-TBI); initially the TBI was administered as a single dose but subsequently it was given as fractions over three consecutive days. Later, the use of gamma irradiation

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J.M. Goldman Table 1-1.  Chronology of selected important developments in hematopoietic stem cell transplantation in the last century. 1930s   Gorer lays the foundation for the major histocompatibility complex in mice 1950s   Description of the first “transplantation antigen” in man   Description of secondary disease, later termed “graft-versus-host disease” 1960s   Identification of the major histocompatibility complex in man   Syngeneic transplants for AA   Allografts for immune deficiency diseases 1970s   Technology refined for performed human stem cell allografts   SD allo-SCT to treatment AML in relapse   SD allo-SCT to treat AML in remission   SD-allo-SCT to treat CML in advanced phase   Syngeneic transplants to treat CML in chronic phase   Establishment of the first volunteer donor panel 1980s   Allo-SCT to treat CML in chronic phase   MUD-SCT to treat acute leukemia   Recognition that hematopoietic stem cells were present in the peripheral blood   Introduction of T-cell depletion with monoclonal antibodies   Identification of umbilical cord as source of hematopoietic stem cells 1990s   Demonstration of the efficacy of DLI, most notably in CML   First use of G-CSF to mobilize stem cell from peripheral blood   Introduction of reduced intensity conditioning allo-SCT (mini-transplants) SCT stem cell transplantation, AA aplastic anemia, AML acute myeloid leukemia, CML chronic myeloid leukemia, SD sibling donor, MUD matched unrelated donor, DLI donor lymphocyte infusions, G-CSF granulocyte colony-stimulating factor

was replaced by X-rays from a linear accelerator. Meanwhile the Baltimore group designed a combination of high dose busulfan with cyclophosphamide (BuCy) and despite many modifications tested subsequently by specialist groups on both sides of the Atlantic, most conditioning regimens for leukemia today still comprise either “cyclo-TBI” or “BuCy.”

8.  Donor Selection It is interesting to note that the progression in the choice of donors for the different diseases treated by allogeneic transplantation over the years has followed essentially the same sequence. Thus with some exceptions the first success was achieved with syngeneic donors, and this, in turn, set the scene

Chapter 1  Allogeneic Stem Cell Transplantation: The Last Century 

for studies with genetically HLA-identical sibling donors. It was for some while believed that human hematopoietic stem cell transplants could not be successful without this level of matching, but it appeared subsequently that one or even two antigens HLA mismatched family members could be used as transplant donors. The observation that matched unrelated donors could also be used with success (mentioned above) prepared the way for establishment of unrelated donor registries worldwide, such that today more than 13 million volunteers have been tissue typed with varying levels of resolution, each of whom could theoretically serve as a hematopoietic stem cell donor. Hematopoietic stem cells with marrow regenerating capacity can be collected either from the marrow or from the peripheral blood of selected donors. Cord blood stem cells are also valuable as source of stem cells in children and are now being used with increasing success also for adult patients. The use of stem cell transplantation has developed far over the last 40 years. There is every hope that this pace of development will be maintained, such that transplant-related mortality may fall below its current level and transplants may safely be offered on a more routine basis to patients with both malignant and nonmalignant conditions.

References   1. Santos GW (1983) History of bone marrow transplantation. Clin Haematol 12:611–639   2. Fabricius-Moeller J (1922) Experimental studies of hemorrhagic diathesis from X-ray sickness. Levin and Munksgaard, Copenhagen   3. Jacobson LO, Marks EK, Gaston EO, Zirkle RE (1949) Effect of spleen protection on mortality following X-irradiation. J Lab Clin Med 34:1538–1543   4. Jacobson LO, Simmons EL, Marks EK, Eldredge JH (1951) Recovery from irradiation injury. Science 113:510–511   5. Lorenz E, Uphoff DE, Reid TR, Shelton E (1951) Modification of irradiation injury in mice and guinea pigs by bone marrow injections. J Nat Cancer Inst 12:197–201   6. Lindsley DL, Odell TT, Tausche FG (1955) Implantation of functional erythropoietic elements following total body irradiation. Proc Soc Exp Biol Med 90:512–515   7. Nowell PC, Cells LJ, Habermeyer JG, Roan PL (1956) Growth and continued function of rat marrow cells in X-irradiated mice. Cancer Res 16:256–261   8. Ford CE, Hamerton JL, Barnes DWH, Loutit JF (1956) Cytological identification of radiation chimaeras. Nature 177:452–454   9. Mitchison NA (1956) The colonisation of irradiated tissue by transplanted spleen cells. Br J Exp Pathol 37:239–247 10. Barnes DWH, Corp MJ, Loutit JL et  al (1956) Treatment of murine leukaemias with x-rays and homologous bone marrow. Br Med J 2:626–627 11. Barnes DWH, Loutit JF (1957) Treatment of murine leukaemia with X-rays and homologous bone marrow. II. Br Med J 3:241–252 12. Kurnick NB, Montano A, Gerdes JC et al (1958) Preliminary observations on the treatment of postirradiation hematopoietic depression in man by the infusion of stored autogenous bone marrow. Ann Intern Med 49:973–986 13. Thomas ED, Lochte HL Jr, Cannon JH et al (1959) Supralethal whole body irradiation and isologous marrow transplantation in man. J Clin Invest 38:1709–1716 14. Mathé G, Amiel JL, Schwarzenberg L et al (1963) Hematopoietic chimera in man after allogeneic (homologous) bone marrow transplantation. BMJ 2:1633–1635 15. Mathé G, Jammet H, Pendic B et  al (1967) Transfusions et greffes de moelle osseuse homologue chez des humains irradiés à haute dose accidentellement. Rev Fr Etud Clin Biol 4:226–238

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J.M. Goldman 16. Mathé G (1968) Bone marrow transplantation. In: Rappaport FT, Dausset J (eds) transplantation. Grune and Stratton, New York, pp 284–303 17. Gatti RA, Meuwissen HJ, Allen HD (1968) Immunological reconstitution of sexlinked lymphopenic immunological deficiency. Lancet ii:1366–1369 18. Bortin MM, Bach FH, van Bekkum DW, Good RA, van Rood JJ (1994) 25th anniversary of the first successful bone marrow transplants. Blood 73:603–613 19. Thomas ED, Buckner CD, Storb R et al (1972) Aplastic anemia treated by marrow transplantation. Lancet 1:284–289 20. Thomas ED, Storb R, Clift RA et al (1975) Bone marrow transplantation. N Engl J Med 292:832–843 895–902 21. Thomas ED, Buckner CD, Banaji M et al (1977) One-hundred patients with acute leukemia treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation. Blood 49:511–533 22. Storb R, Thomas ED, Buckner CD et  al (1974) Allogeneic marrow grafting for treatment for aplastic anemia. Blood 43:157–180 23. Camitta BM, Thomas ED, Nathan DG et  al (1957) Severe aplastic anemia: a prospective study of the effect of early marrow transplantation on acute mortality. Blood 48:63–70 24. Thomas ED, Buckner CD, Clift RA et al (1979) Marrow transplantation for acute nonlymphoblastic leukemia in first remission. N Engl J Med 301:597–599 25. Thomas ED (1983) Marrow transplantation for acute nonlymphoblastic leukemia in first remission. N Engl J Med 309:1539 (letter) 26. Hansen JA, Cift RA, Thomas ED et al (1980) Transplantation of marrow from an unrelated donor to a patient with acute leukaemia. N Engl J Med 303:565–567 27. Gluckman E, Broxmeyer HE, Auerbach AD et al (1989) Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical-cord blood from an HLAidentical sibling. N Engl J Med 321:1174–1178 28. Weiden PL, Flournoy N, Thomas ED et  al (1979) Antileukemic effect of graftversus-host disease in human recipients of allogeneic grafts. N Engl J Med 300:1068–1073 29. Weiden PL, Sullivan KM, Flournoy N, Storb R, Thomas ED (1981) Antileukemic effect of chronic graft-versus-host disease : contribution to improved survival after allogeneic marrow transplantation. N Engl J Med 304:1529–1533 30. Apperley JF, Jones L, Hale G et al (1986) Bone marrow transplantation for chronic myeloid leukaemia: T-cell depletion reduces the risk of graft-versus-host disease but may increase the risk of leukaemic relapse. Bone Marrow Transplant 1:53–66 31. Goldman JM, Gale RP, Horowitz MM et al (1988) Bone marrow transplantation for chronic myelogenous leukemia in chronic phase: increased risk of relapse associated with T-cell depletion. Ann Intern Med 108:806–814 32. Kolb HJ, Mittermuller J, Clemm CH et  al (1990) Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 76:2462–2465 33. Fefer A, Cheever MA, Thomas ED et al (1979) Disappearance of the Ph1-positive cells in four patients with chronic granulocytic leukemia after chemotherapy, irradiation and marrow transplantation from an identical twin. N Engl J Med 300:333–337 34. Clift RA, Buckner CD, Thomas ED et al (1982) Treatment of chronic granulocytic leukaemia in chronic phase by allogeneic marrow transplantation. Lancet 2:621–623 35. Goldman JM, Baughan ASJ, McCarthy DM, Worsley AM et  al (1982) Marrow transplantation for patients in the chronic phase of chronic granulocytic leukaemia. Lancet 2:623–625 36. McGlave PB, Arthur DC, Kim TH et al (1982) Successful allogeneic bone-marrow transplantation for patients in the accelerated phase of chronic granulocytic leukaemia. Lancet 2:625–627

Chapter 1  Allogeneic Stem Cell Transplantation: The Last Century  37. Prentice HG, Blacklock HA, Janossy G et al (1984) Depletion of T lymphocytes in donor marrow prevents significant graft-versus-host disease in matched allogeneic leukaemic marrow transplant recipients. Lancet 1:472–476 38. Barnes DWH, Loutit JF (1955) Spleen protection: the cellular hypothesis. In: Bacq ZM (ed) Radiobiology Symposium Liege. Butterworths, London, pp 134–135 39. Cohen JA, Vos O, van Bekkum DW (1957) The present status of radiation protection by chemical and biological agents in mammals. In: de Hevesy GC, Forssberg AG, Abbott JD (eds) Advances in Radiobiology. Oliver & Boyd, Edinburgh, pp 134–144 40. Gorer RA (1936) The detection of antigenic differences in mouse erythrocytes by the employment of immune sera. Br J Exp Pathol 17:42–50 41. Gorer RA (1937) The genetic and antigenic basis of tumour transplantation. J Pathol Bacteriol 44:691–697 42. Dausset J (1958) Iso-leuko-anticorps. Acta Haematol 20:156–166 43. Dupont B (1980) HLA factors and bone marrow grafting. In: Burchenal JH, Oettgen HF (eds) Cancer: achievements, challenges and prospects for the 1980s. Grune & Stratton, New York, pp 683–693 44. Ceppellini R, van Rood JJ (1974) The HLA system. I. Genetic and molecular biology. Semin Hematol 11:233–251

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Chapter 2 Full Intensity and Reduced Intensity Allogeneic Transplantation in AML Charles Craddock

1.  Introduction Acute myeloid leukemia (AML) is now the commonest indication for allogeneic stem cell transplantation (SCT) in adults [1]. This reflects the continued inability of conventional chemotherapeutic regimens to deliver long-term disease-free survival in most adults -- a failure which is particularly marked in patients over the age of 50 years whose outcome has barely improved in the last three decades [2, 3]. Whilst it has been clear for a number of years that the allogeneic transplantation delivers a more potent anti-leukemic effect than chemotherapy, the toxicity of myeloablative conditioning regimens has precluded its use in precisely the group of patients who urgently need new therapeutic options. However, the recent demonstration that the use of reduced intensity conditioning regimens substantially reduces the transplant-related mortality (TRM) has provided the prospect of delivering a potentially curative graft-versus-leukemia (GVL) effect in patients in whom it was previously contraindicated [4, 5]. Importantly, this has provided a new treatment option for a group of patients whose outcome if treated with chemotherapy alone would be very poor.

2.  Biology of AML and Impact on Future Development of Therapeutic Strategies in AML The demonstration that AML originates from a population of mitotically quiescent leukemic stem cells (LSC) is likely to transform treatment strategies in the coming decade [6]. Since the markedly reduced sensitivity of LSC to a number of currently used chemotherapeutic agents is likely to underlie the high rate of disease relapse in AML, there is an urgent need to develop distinct therapeutic approaches which target this cellular compartment. The notable clinical activity of the tyrosine kinase inhibitor, imatinib, in patients with chronic myeloid leukemia (CML) led to a initial enthusiasm that targeted inhibition of dysregulated signaling pathways might be effective in AML [7]. However, this was not substantiated by the initial experience with drugs From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_2, © Springer Science + Business Media, LLC 2003,2010

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designed to inhibit constitutively active FLT3 and Ras pathways, and clinical responses to these agents in AML are rare and usually of short duration [8–10]. Furthermore, it is now clear from cytogenetic analysis and, more recently from microarray studies that AML is a highly heterogeneous disease at a molecular level whose pathogenesis is dependent on the acquisition of mutations in a number of different cellular pathways [11, 12]. This model is supported by data from transgeneic mouse models which demonstrate that mutations in at least two distinct cellular pathways are required for the pathogenesis of AML [13, 14]. As a consequence, it appears unlikely that a pharmacological strategy which targets a single dysregulated pathway will be of sustained therapeutic benefit. Attention is instead switching to the development of agents which can target the LSC and approaches are under investigation that include inhibitors of pathways mediating self-renewal and proliferation, such as NFkB, Wnt, and hox genes [15–17]. 2.1.  Emergence of Immunotherapeutic Strategies as an Important form of Targeted Therapy The challenges associated with the development of new drug therapies in AML provides a compelling case for the extension of immunotherapeutic approaches which target dysregulated cell surface antigens expressed on the surface of LSC rather than abnormalities in intracellular signaling pathways. The GVL effect in which the donor immune system targets “foreign” antigens expressed on the leukemic blast is the most widely exploited form of immunotherapy in clinical practice, but there is growing interest in the clinical benefit of strategies by which the patient’s own immune system can be manipulated to exert an anti-leukemic effect. A number of lines of evidence attest the presence of a potent GVL effect in patients allografted for AML. These include the demonstration that relapse risk is reduced in patients who develop GVHD and, a compelling observation by Bacigalupo that reduction in the level of post-transplant immunosuppression, achieved by reducing the cyclosporine dose to 1  mg/kg in the first 20 days post-transplant, markedly reduces the risk of disease relapse (Fig. 2-1) [18–20].

Fig.  2-1.  Impact of post-transplant cyclosporine (CyA) dose on disease relapse in patients allografted for AML in first CR [18]

Chapter 2  Full Intensity and Reduced Intensity Allogeneic Transplantation in AML 

The major factor limiting the effective exploitation of GVL effect in patients with high-risk AML is our continued inability to develop transplant protocols which effectively dissociate GVL from severe, potentially fatal, acute, and chronic GVHD. Putative targets of the GVL reaction include minor histocompatibility antigens such as HA-1, HA-2 and H-Y, and the leukemic-specific antigens WT1 and proteinase 3 [21–25]. However, attempts to target mHAgs such as HA-1 have been hampered by the rare frequency of this allele and its HLA restriction. In contrast, antigens such as WT1 represent an attractive option given its over-expression in up to 70% of patients with AML [26, 27]. It is clear, however, that further characterization of the immunogenicity of the AML stem cell will be vital if we are to improve the current immunotherapeutic strategies in high-risk AML [25]. The prospect of exploiting an autologous immune response for clinical benefit has been supported by the demonstration that gemtuzumab ozogamicin (GO), a monoclonal antibody to the putative LSC antigen CD33, can salvage patients with relapsed AML [28]. Based on this encouraging experience, humanized antibodies to a number of other antigens expressed on leukemic progenitors and LSC are currently under clinical trials in high-risk AML. Recent studies demonstrating the presence of an immune response to leukemiaspecific antigens such as WT1 and proteinase 3 has led to the development of strategies designed to induce an autologous T-cell response using either peptide vaccination or TCR gene transfer [29]. Early phase clinical trials studying the effect of peptide vaccination with immunodominant epitopes of WT1 or proteinase 3 confirm that it is possible to augment these responses in  vitro although compelling evidence of clinical benefit is awaited. In the future it will be important to define how such a strategy can be utilized to improve the outcome of allogeneic transplantation.

3.  Preparative Regimens in AML The clinical studies which, over the past three decades, have established a central role for allogeneic transplantation in AML therapy have been achieved using myeloablative conditioning regimens. It was initially unclear whether reduced intensity allografts would possess the capacity to deliver long-term disease-free survival in patients with AML. Whilst it is now evident that such regimens are also capable of delivering durable long-term remissions, a myeloablative conditioning regimen should continue to be viewed as the “gold standard” preparative regimen at least until the results of prospective studies of reduced intensity transplant regimens are available.

4.  Outcome in Patients with AML Transplanted Using a Myeloablative Conditioning Regimen 4.1.  Patients in First Complete Remission (CR1) Randomized controlled trials assessing the role of allogeneic transplantation in the management of patients in CR1 have been difficult to perform. Obstacles have not only included the randomization biases introduced because of the fixed perceptions of either physicians or patients, but also the very real problem

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that the allograft arm may include a smaller proportion of patients with highrisk disease, given the propensity of such patients to relapse before they reach transplant. The only effective approach which allows these biases to be removed is the use of a “donor versus no-donor” analysis which exploits the availability of an HLA identical sibling donor as a form of biological randomization. Although weakened by the fact that not all patients with an available donor will be transplanted, “donor versus no-donor” analyses have proved the only unbiased statistical methodology by which the benefit of a sibling allograft can be assessed [30]. A number of international co-operative groups have reported the results of “donor versus no-donor” analyses in the management of younger adults with AML in CR1 [31–35]. All but one study reported an improvement in the disease-free survival in patients with a donor (Tables 2.1 and 2.2). Although no individual study showed a statistically significant survival benefit – probably because a proportion of patients in the “no-donor” arm could be effectively salvaged by a transplant in CR2 – a recent meta-analysis has demonstrated both an improved disease-free and overall survival in the donor group [32]. Importantly, these studies demonstrate that presentation karyotype and patient age are powerful tools in identifying who will benefit from a sibling allograft. Allogeneic transplantation delivers a clear survival advantage in patients with intermediate or adverse risk cytogenetics [32]. Indeed the risk of relapse is so high in patients with adverse risk cytogenetics that allogeneic transplantation using an unrelated donor is indicated in all CR1 patients with an available donor [36]. In contrast, allogeneic transplantation should not be performed in CR1 patients with good risk cytogenetics whose outcome with chemotherapy can be predicted to be relatively good. This approach is also supported by the 90% salvage rate achievable in patients with good Table  2-1.  A donor-vs.-no-donor analysis of impact of sibling allogeneic transplantation on overall survival (OS) and disease-free survival (DFS) in patients with AML in CR1. OS(%)

DFS(%)

Study

Donor no-donor

p

Donor

no-donor

GOELAM [31]

53

53

NS

44

38

0.6

HOVON [32]

54

46

NS

48

37

<0.001

BGMT [33]

65

51

NS

66

42

<0.05

MRC [34]

56

50

NS

50

42

0.001

EORTC [35]

48

40

NS

46

33

0.01

Table  2-2.  Relapse rates and toxicity in “donor” and “no-donor” arms. Relapse rate

Toxic death

Donor

no-donor

Donor

no-donor

GOELAM [31]

37

54

22

 3

EORTC [35]

42

63

21

12

MRC [34]

36

52

19

 8

p

Chapter 2  Full Intensity and Reduced Intensity Allogeneic Transplantation in AML 

risk cytogenetics who relapse in contrast to salvage rates of 40% or less in patients with intermediate or adverse risk cytogenetics [37]. Age is also an important determinant of outcome since the TRM of a myeloablative allograft rises sharply above the age of 35–40 years [37]. In addition to defining patients who clearly benefit from allogeneic transplantation, these studies have also identified those in whom a myeloablative allograft in CR1 is not indicated. These include older patients (>40–45 years) and those with favorable cytogenetics in whom any reduction in relapse risk is offset by the toxicity of transplantation. Much work remains to be done in order to identify the patients with intermediate risk cytogenetics requiring an allograft. According to Cornelissen et al., the risk of disease relapse must be greater than 35% ,before a survival benefit is likely to be conferred by a sibling allograft in a patient under the age of 40 years [32]. Recently, molecular markers which can predict the relapse risk of patients with intermediate risk cytogenetics, who are treated with chemotherapy alone, have been predicted. The demonstration that acquired mutations of a number of genes such as NPM1 and FLT3 can be used to predict survival, has allowed the identification of different subgroups including a population of patients (NPM1 +ve, FLT3 ITD –ve) who may not benefit from transplantation [38, 39]. Immunophenotypic quantitation of minimal residual disease, after induction or consolidation chemotherapy, also predicts relapse risk allowing effective risk stratification, and future prospective trials will be vital for validating this additional method of risk stratification [40]. 4.2.  Patients Beyond CR1 Salvage chemotherapy can induce a second complete remission (CR2) in upward of 40% of patients who relapse after an initial treatment with chemotherapy, but durable responses are rare [41]. Whilst a proportion of patients with good risk cytogenetics who relapse appear to be cured using only chemotherapy, this is rarely the case in patients with intermediate or adverse risk cytogenetics [42]. In contrast, registry data demonstrates long-term disease-free survival rates of 40–50% for patients who undergo a myeloablative sibling or unrelated donor transplant in CR2 [36, 43, 44]. In these patients, additional courses of chemotherapy prior to transplantation, once a second remission has been achieved seems to have no benefit: rather it is important to ensure that patients should instead proceed as swiftly as possible for transplantation [45]. The role of allogeneic transplantation in the management of patients with primary refractory disease remains controversial. A number of centers report long-term disease-free survival rates in the range of 25–45% in patients with AML resistance to two or more lines of induction chemotherapy who have been transplanted using a sibling or unrelated donor [46–49]. The patients most likely to benefit include those with intermediate risk cytogenetics who are allografted using an HLA identical sibling donor. Prompt transplantation is critical if such an approach is to be effective, and therefore, tissue typing of siblings and an unrelated donor search is indicated in younger (<45 years) patients failing to enter remission with their first course of chemotherapy.

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5.  Unresolved Issues in Myeloablative Allogeneic Transplantation in AML The main causes of treatment failure in patients allografted for AML are transplant-related toxicity (principally, acute and chronic GVHD) and disease relapse. Both are susceptible to manipulation by judicious adjustment of the three most important components of the transplant protocol: the type of preparative regimen used, whether T-cell depletion is employed, and the choice of stem cell source. 5.1.  What is the Optimal Conditioning Regimen? The most widely used myeloablative conditioning regimens in AML combine either cyclophosphamide and TBI (Cy/TBI) or busulphan (Bu) and cyclophosphamide (Bu/Cy). A series of randomized studies in the 1990s compared these regimens in patients with myeloid malignancies undergoing a sibling allograft [50–53]. Although there was a broad equivalence of outcome in patients with early phase CML and AML in CR1, there was a survival advantage associated with the use of Cy/TBI in patients with advanced phase AML [54–56]. It is now clear that the pharmacokinetics of oral Bu are highly unpredictable. This variability significantly impacts the outcome of transplantation in myeloid malignancies as low Bu levels have been shown to be associated with an increased risk of relapse whilst high Bu levels are associated with excessive transplant toxicity – principally, veno-occlusive disease [57, 58]. It is, therefore, surprising that despite the unpredictable bioavailability of oral Bu, previous randomized studies have demonstrated broad equivalence in the outcome between patients transplanted using Cy/TBI or Bu/Cy regimens. This raised the possibility that Bu-based regimens might be superior to Cy/ TBI if methods could be developed allowing for more effective delivery of this very active anti-leukemic agent. In the past decade, two approaches which allow consistent Bu levels to be achieved were proposed. In the first, real-time pharmacokinetic calculations are used to make adjustments to the doses of oral Bu prescribed. This approach allows a well-tolerated Bu level to be achieved (targeted TBu/Cy) in the great majority of patients. Alternatively, a recently developed intravenous preparation of Bu can be used (iv Bu/Cy) to deliver consistent drug levels [59, 60]. Using a TBu/Cy regimen, the Seattle group reported markedly reduced transplant toxicity and relapse rates in patients allografted for CML and secondary AML [61]. Similar encouraging results have been reported in preliminary studies using an iv Bu/Cy regimen [62]. A randomized comparison of either or both of these regimens with Cy/TBI in patients transplanted for myeloid disease is therefore required. 5.2.  What is the Role of T-Cell Depletion Strategies in Myeloablative Allografts There is no consensus over the role of T-cell depletion in the design of myeloablative preparative regimens in AML. Enthusiasts of T-replete allografts cite the potency of the GVL effect in AML and the limited salvage options in patients who relapse post-transplant as justification for the use of unmanipulated stem cells. These arguments are countered by a school of thought which

Chapter 2  Full Intensity and Reduced Intensity Allogeneic Transplantation in AML 

highlights acute and chronic GVHD as the major causes of transplant toxicity and argues that the low rate of acute GVHD in T-depleted allografts permits intensification of the preparative regimen [63]. In the absence of any randomized studies, many units utilize T-replete strategies in sibling allografts and some form of T-cell depletion (using either anti-thymocyte globulin (ATG) or alemtuzumab) in unrelated donor transplants [36]. 5.3.  What is the Optimal Stem Cell Source? The use of peripheral blood stem cells (PBSC) compared with bone marrow (BM) harvested progenitors results in faster neutrophil and platelet engraftment, and appears to be associated with a lower TRM in patients undergoing a sibling allograft for advanced phase disease [64]. Accumulating evidence suggests that these benefits may be offset by an increased risk of chronic GVHD in patients with early phase disease who are transplanted using PBSC, and in children there is evidence that the use of mobilized progenitors prejudices outcome [65]. In unrelated donor transplants, the optimal stem cell source is unknown. The impact of the stem cell dose on the risk of disease relapse is of significance, and a number of papers identify a reduced relapse risk in patients receiving a stem cell inoculum over 4 × 106 CD34+ cells/kg. It has been postulated that this may be consequent to an increased GVL effect [66, 67]. Reports that the risk of chronic GVHD may be increased in recipients of >8 × 106 CD34+ cells/kg suggest, however, that there may be an upper limit to the optimal stem cell dose in the setting of T-replete allografts [68]. Improved tissue typing has made a major contribution to the development in the outcome of patients with AML undergoing an unrelated donor transplant. Disease-free survival rates in the range of 40–60% are achievable using a myeloablative conditioning regimen in CR1 patients [36, 44, 69]. A potentially important advance is the recent demonstration that the excellent results achievable in children using cord blood, are replicable in adults, provided the dose of nucleated cells transplanted is adequate [70]. It appears that this can reliably be achieved in many patients by the transplantation of two, rather than one, cord blood units [71]. Whilst GVHD and delayed immune reconstitution remain significant causes of mortality after cord blood transplantation, it now appears possible that this evolving transplant technology may have an important role to play in the treatment of patients with high-risk AML.

6.  The Emerging Role of Reduced Intensity Transplants in the Management of AML The notion, advanced in the late 1990s, that it might be possible to extend a potentially curative GVL effect to older patients by employing less toxic immunosuppressive preparative regimens has been triumphantly vindicated in the past decade. A number of different RIC regimens have been evaluated over this period. The two most commonly used classes of reduced intensity regimens incorporate either a non-myeloablative dose (200  cGy) of TBI or immunosuppressive doses of fludarabine in conjunction with conventional chemotherapy. Both classes of regimen clearly reduce the transplant toxicity substantially, allowing patients up to the age of 70 years to be viewed as potential allograft candidates [4, 5, 72]. It is only recently, however, that it has

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been possible to demonstrate that such regimens deliver durable long-term remissions in AML and prospective studies aimed at defining the precise role of RIC allografts in the management of both older and younger patients with AML are, therefore, now required. 6.1.  Outcome of Reduced Intensity Allografts in AML A number of reduced intensity conditioning regimens have been evaluated in adult AML. The most fundamental difference in approach involves whether T-cell depletion is employed as a central component of the preparative regimen or not. T-replete strategies have been shown to deliver DFS rates in excess of 50% in patients with high-risk AML in CR1 or CR2 with broadly similar results being reported using either a low dose TBI or fludarabine/melphalan regimen [73, 74]. The major limitation of a T-replete approach is the high risk of severe GVHD which not only represents a major cause of morbidity and mortality but also limits the potential for manipulating a GVL effect posttransplant. These concerns have resulted in the incorporation T-cell depleting antibodies, principally alemtuzumab and ATG into fludarabine-based reduced intensity protocols. A number of recent Phase II studies have demonstrated that such an approach significantly reduces the risk of both acute and chronic GVHD, without apparently compromising the ability of these regimens to deliver a potent, and durable, anti-leukemic activity in patients whose outlook with conventional chemotherapy would be extremely poor. Thus, both the French co-operative group, who use fludarabine, Bu and ATG [75, 76], and the UK collaborative group who use fludarabine, melphalan and alemtuzumab have demonstrated 3 year disease-free survival rates in excess of 50% in patients transplanted in CR1 or CR2 [74, 77, 78] (Fig. 2-2). In order to exclude potential biases favoring transplant, these results will require further confirmation in prospective trials using a “donor vs. no-donor” analysis. It is, however, noteworthy that the first of such studies has demonstrated a clear increase in both disease-free survival and overall survival in patients with a donor [79]. Further work is also required to identify more precisely, patients who will benefit from allogeneic transplantation. Importantly, both the T-replete and T-deplete strategies have shown that acquisition of morphological remission prior to transplant is critical if patients are to survive long term. It is also of interest that a procedure whose anti-leukemic potential is predicated on a T-cell mediated graft-versus-leukemic activity appears to remain effective despite T-cell depletion. This observation, confirmed by a number of groups, demonstrates the importance of further research into identifying both the target and the cellular effectors of the GVL reaction in AML.

7.  Strategies to Improve the Outcome of Reduced Intensity Allogeneic Transplantation in AML The major cause of treatment failure in patients transplanted using a RIC regimen is disease relapse. In patients transplanted in remission, the risk of relapse is in the range of 30–40%. Disease relapse occurs predominantly in the first year post-transplant -- in patients transplanted using an alemtuzumab containing regimen, 85% of those patients destined to relapse do so within the first year post-transplant [77] (Fig. 2-3). Therefore, approaches which reduce the

Chapter 2  Full Intensity and Reduced Intensity Allogeneic Transplantation in AML 

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Fig. 2-2.  Outcome of patients undergoing reduced intensity allogeneic transplantation for AML. Patients were transplanted using a fludarabine/melphalan/alemtuzumab containing reduced intensity regimen. Disease status during transplant is the most important predictor of outcome [77].

Fig. 2-3.  Kinetics of disease relapse after reduced intensity allogeneic transplantation using a fludarabine/melphalan/alemtuzumab regimen [77].

relapse risk must focus on either optimizing cytoreduction prior to transplant, increasing the anti-leukemic activity of the conditioning regimen or maximising the GVL effect in the first few weeks or months post-transplant.

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The observation that disease stage is an important predictor of disease relapse raises the possibility that minimal residual disease status immediately prior to transplant will be an important predictor of relapse risk. Whilst this hypothesis has not been formally tested, there is interest in the use of either further courses of chemotherapy as a way to reduce the risk of disease relapse. In this context it is of interest that the impressive results reported by the French Collaborative Group are achieved using a T-depleted regimen which incorporates a prior autograft. The use of intravenous preparations of Bu [80] or targeted radioimmunotherapy represent other possible approaches by which the anti-leukemic activity of the preparative regimen can be increased without a concomitant rise in transplant toxicity. The other strategy by which the risk of relapse might be reduced is the augmentation of the GVL effect post-transplant, particularly in recipients of T-cell depleted grafts. There is, therefore, interest in examining the impact on relapse risk of manipulating either the degree of T-cell depletion (by reducing the dose of ATG or alemtuzumab) or the intensity of post-transplant immunosuppression. Alternative approaches to augment a GVL response include the use of prophylactic donor lymphocyte infusions (DLI) in patients with mixed T-cell chimerism [81]. Such an approach is constrained by the risk of severe, potentially life-threatening, GVHD associated with DLI administration in the first 12 months post-transplant. This limits both the lymphocyte dose which can be safely administered and, in unrelated transplant recipients, represents a significant and largely unpredictable risk of morbidity and mortality in the first year post-transplant [82–84]. At present, there is no consensus concerning the role of DLI after RIC transplants and prospective studies examining dose, timing, and source of DLI (GCSF stimulated or not) are required [85]. Whether the use of peptide vaccination to candidate immunodominant epitopes implicated in a GVL reaction will reduce the relapse risk without the complication of GVHD remains to be seen. An alternative approach which needs to be explored involves the use of adjunctive leukemic-specific therapy post-transplant, such as a FLT3 inhibitor or a demethylating agent, in the hope that the kinetics of disease relapse can be manipulated so that the timing of DLI can be postponed to a time at which it can be administered with less toxicity. Two major barriers exist, preventing the wider use of reduced intensity allografts in AML: the lack of a suitable sibling or unrelated donor in a proportion of patients and the presence of refractory or resistant leukemia in many. The encouraging results reported by a number of groups using mismatched umbilical cord blood units are, therefore, extremely important as a method of increasing the donor availability and promising results have been reported in adults with high-risk AML who lack an unrelated donor [71, 86, 87]. The ground-breaking work of Kolb’s group who has shown that incorporation of a course of intensive chemotherapy several days before commencing the conditioning regimen followed by an aggressive post-transplant immunotherapy improves the outcome of patients with active disease, and offers the prospect of successfully transplanting even patients with resistant leukemia [88, 89].

8.  Conclusions Significant progress has been made in the last decade in defining and extending the role of allogeneic transplantation in the management of patients with

Chapter 2  Full Intensity and Reduced Intensity Allogeneic Transplantation in AML 

AML. As a result we can now identify, with some degree of precision, a sizeable population of patients whose outcome with chemotherapy is poor and who benefit from allogeneic transplantation. Equally importantly, patients who should not be exposed to the potential harm of an allograft can also now be identified. The ability of reduced intensity preparative regimens to decrease the transplant toxicity has allowed the extension of allogeneic transplantation to older adults with AML. However, disease relapse remains a significant cause of treatment failure in patients transplanted using a reduced intensity regimen. The major challenges for the next decade will, therefore, be the further refinement of our ability to identify patients whose prospect of long-term disease-free survival without transplantation is poor and the development of strategies which improve transplant outcome by reducing the relapse rate without increasing the risk of GVHD.

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C. Craddock 44. Tallman MS, Dewald GW, Gandham S, Logan BR, Keating A, Lazarus HM et al (2007) Impact of cytogenetics on outcome of matched unrelated donor hematopoietic stem cell transplantation for acute myeloid leukemia in first or second complete remission. Blood 110(1):409–417 45. Tallman MS, Rowlings PA, Milone G, Zhang MJ, Perez WS, Weisdorf D et  al (2000) Effect of postremission chemotherapy before human leukocyte antigenidentical sibling transplantation for acute myelogenous leukemia in first complete remission. Blood 96(4):1254–1258 46. Fung HC, Stein A, Slovak M, O’Donnell MR, Snyder DS, Cohen S et al (2003) A long-term follow-up report on allogeneic stem cell transplantation for patients with primary refractory acute myelogenous leukemia: Impact of cytogenetic characteristics on transplantation outcome. Biol Blood Marrow Transplant 9(12): 766–771 47. Oyekunle AA, Kroger N, Zabelina T, Ayuk F, Schieder H, Renges H et al (2006) Allogeneic stem-cell transplantation in patients with refractory acute leukemia: A long-term follow-up. Bone Marrow Transplant 37(1):45–50 48. Wong R, Shahjahan M, Wang X, Thall PF, De Lima M, Khouri I et  al (2005) Prognostic factors for outcomes of patients with refractory or relapsed acute myelogenous leukemia or myelodysplastic syndromes undergoing allogeneic progenitor cell transplantation. Biol Blood Marrow Transplant 11(2):108–114 49. Biggs JC, Horowitz MM, Gale RP, Ash RC, Atkinson K, Helbig W et al (1992) Bone marrow transplants may cure patients with acute leukemia never achieving remission with chemotherapy. Blood 80(4):1090–1093 50. Blaise D, Maraninchi D, Archimbaud E, Reiffers J, Devergie A, Jouet JP et  al (1992) 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 79(10):2578–2582 51. Clift RA, Buckner CD, Thomas ED, Bensinger WI, Bowden R, Bryant E et  al (1994) Marrow transplantation for chronic myeloid leukemia: A randomized study comparing cyclophosphamide and total body irradiation with busulfan and cyclophosphamide. Blood 84(6):2036–2043 52. Devergie A, Blaise D, Attal M, Tigaud JD, Jouet JP, Vernant JP et  al (1995) Allogeneic bone marrow transplantation for chronic myeloid leukemia in first chronic phase: A randomized trial of busulfan-cytoxan versus cytoxan-total body irradiation as preparative regimen: A report from the French Society of Bone Marrow. Blood 85(8):2263–2268 53. Ringden O, Ruutu T, Remberger M, Nikoskelainen J, Volin L, Vindelov L et  al (1994) 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 83(9):2723–2730 54. Socie G, Clift RA, Blaise D, Devergie A, Ringden O, Martin PJ et  al (2001) 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 98(13):3569–3574 55. Ringden O, Remberger M, Ruutu T, Nikoskelainen J, Volin L, Vindelov L et  al (1999) 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 93(7):2196–2201 56. Litzow MR, Perez WS, Klein JP, Bolwell BJ, Camitta B, Copelan EA et al (2002) Comparison of outcome following allogeneic bone marrow transplantation with cyclophosphamide-total body irradiation versus busulphan-cyclophosphamide conditioning regimens for acute myelogenous leukaemia in first remission. Br J Haematol 119(4):1115–1124

Chapter 2  Full Intensity and Reduced Intensity Allogeneic Transplantation in AML  57. Ljungman P, Hassan M, Bekassy AN, Ringden O, Oberg G (1997) High busulfan concentrations are associated with increased transplant-related mortality in allogeneic bone marrow transplant patients. Bone Marrow Transplant 20(11):909–913 58. Slattery JT, Clift RA, Buckner CD, Radich J, Storer B, Bensinger WI et al (1997) Marrow transplantation for chronic myeloid leukemia: The influence of plasma busulfan levels on the outcome of transplantation. Blood 89(8):3055–3060 59. Deeg HJ, Storer B, Slattery JT, Anasetti C, Doney KC, Hansen JA et  al (2002) Conditioning with targeted busulfan and cyclophosphamide for hemopoietic stem cell transplantation from related and unrelated donors in patients with myelodysplastic syndrome. Blood 100(4):1201–1207 60. Madden T, de Lima M, Thapar N, Nguyen J, Roberson S, Couriel D et al (2007) Pharmacokinetics of once-daily IV busulfan as part of pretransplantation preparative regimens: A comparison with an every 6-hour dosing schedule. Biol Blood Marrow Transplant 13(1):56–64 61. Chang C, Storer BE, Scott BL, Bryant EM, Shulman HM, Flowers ME et al (2007) Hematopoietic cell transplantation in patients with myelodysplastic syndrome or acute myeloid leukemia arising from myelodysplastic syndrome: Similar outcomes in patients with de novo disease and disease following prior therapy or antecedent hematologic disorders. Blood 110(4):1379–1387 62. Kim SE, Lee JH, Choi SJ, Ryu SG, Lee KH (2005) Morbidity and non-relapse mortality after allogeneic bone marrow transplantation in adult leukemia patients conditioned with busulfan plus cyclophosphamide: A retrospective comparison of oral versus intravenous busulfan. Haematologica 90(2):285–286 63. Papadopoulos EB, Carabasi MH, Castro-Malaspina H, Childs BH, Mackinnon S, Boulad F et  al (1998) T-cell-depleted allogeneic bone marrow transplantation as postremission therapy for acute myelogenous leukemia: Freedom from relapse in the absence of graft-versus-host disease. Blood 91(3):1083–1090 64. Bensinger WI, Martin PJ, Storer B, Clift R, Forman SJ, Negrin R et  al (2001) Transplantation of bone marrow as compared with peripheral-blood cells from HLAidentical relatives in patients with hematologic cancers. N Engl J Med 344(3):175–181 65. Eapen M, Horowitz MM, Klein JP, Champlin RE, Loberiza FR Jr, Ringden O et al (2004) 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 22(24):4872–4880 66. Bahceci E, Read EJ, Leitman S, Childs R, Dunbar C, Young NS et  al (2000) CD34+ cell dose predicts relapse and survival after T-cell-depleted HLA-identical haematopoietic stem cell transplantation (HSCT) for haematological malignancies. Br J Haematol 108(2):408–414 67. Nakamura R, Bahceci E, Read EJ, Leitman SF, Carter CS, Childs R et  al (2001) Transplant dose of CD34(+) and CD3(+) cells predicts outcome in patients with haematological malignancies undergoing T cell-depleted peripheral blood stem cell transplants with delayed donor lymphocyte add-back. Br J Haematol 115(1):95–104 68. Przepiorka D, Anderlini P, Saliba R, Cleary K, Mehra R, Khouri I et  al (2001) Chronic graft-versus-host disease after allogeneic blood stem cell transplantation. Blood 98(6):1695–1700 69. Sierra J, Storer B, Hansen JA, Martin PJ, Petersdorf EW, Woolfrey A et al (2000) Unrelated donor marrow transplantation for acute myeloid leukemia: An update of the Seattle experience. Bone Marrow Transplant 26(4):397–404 70. Rocha V, Labopin M, Sanz G, Arcese W, Schwerdtfeger R, Bosi A et  al (2004) Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 351(22):2276–2285 71. Barker JN, Weisdorf DJ, DeFor TE, Blazar BR, Miller JS, Wagner JE (2003) Rapid and complete donor chimerism in adult recipients of unrelated donor umbilical cord blood transplantation after reduced-intensity conditioning. Blood 102(5):1915–1919

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C. Craddock 72. Slavin S, Nagler A, Naparstek E, Kapelushnik Y, Aker M, Cividalli G et al (1998) 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 91(3):756–763 73. de Lima M, Anagnostopoulos A, Munsell M, Shahjahan M, Ueno N, Ippoliti C et al (2004) Nonablative versus reduced-intensity conditioning regimens in the treatment of acute myeloid leukemia and high-risk myelodysplastic syndrome: Dose is relevant for long-term disease control after allogeneic hematopoietic stem cell transplant. Blood 104(3):865–872 74. Hegenbart U, Niederwieser D, Sandmaier BM, Maris MB, Shizuru JA, Greinix H et al (2006) Treatment for acute myelogenous leukemia by low-dose, total-body, irradiation-based conditioning and hematopoietic cell transplantation from related and unrelated donors. J Clin Oncol 24(3):444–453 75. Blaise DP, Michel Boiron J, Faucher C, Mohty M, Bay JO, Bardoux VJ et  al (2005) Reduced intensity conditioning prior to allogeneic stem cell transplantation for patients with acute myeloblastic leukemia as a first-line treatment. Cancer 104(9):1931–1938 76. Mohty M, Bay JO, Faucher C, Choufi B, Bilger K, Tournilhac O et  al (2003) Graft-versus-host disease following allogeneic transplantation from HLA-identical sibling with antithymocyte globulin-based reduced-intensity preparative regimen. Blood 102(2):470–476 77. Tauro S, Craddock C, Peggs K, Begum G, Mahendra P, Cook G et  al (2005) Allogeneic stem-cell transplantation using a reduced-intensity conditioning regimen has the capacity to produce durable remissions and long-term disease-free survival in patients with high-risk acute myeloid leukemia and myelodysplasia. J Clin Oncol 23(36):9387–9393 78. van Besien K, Artz A, Smith S, Cao D, Rich S, Godley L et al (2005) Fludarabine, melphalan, and alemtuzumab conditioning in adults with standard-risk advanced acute myeloid leukemia and myelodysplastic syndrome. J Clin Oncol 23(24): 5728–5738 79. Mohty M, de Lavallade H, Ladaique P, Faucher C, Vey N, Coso D et  al (2005) The role of reduced intensity conditioning allogeneic stem cell transplantation in patients with acute myeloid leukemia: A donor vs no donor comparison. Leukemia 19(6):916–920 80. de Lima M, Couriel D, Thall PF, Wang X, Madden T, Jones R et al (2004) Oncedaily intravenous busulfan and fludarabine: Clinical and pharmacokinetic results of a myeloablative, reduced-toxicity conditioning regimen for allogeneic stem cell transplantation in AML and MDS. Blood 104(3):857–864 81. Mohty M, Avinens O, Faucher C, Viens P, Blaise D, Eliaou JF (2007) Predictive factors and impact of full donor T-cell chimerism after reduced intensity conditioning allogeneic stem cell transplantation. Haematologica 92(7):1004–1006 82. Dominietto A, Pozzi S, Miglino M, Albarracin F, Piaggio G, Bertolotti F et  al (2007) Donor lymphocyte infusions for the treatment of minimal residual disease in acute leukemia. Blood 109(11):5063–5064 83. Marks DI, Lush R, Cavenagh J, Milligan DW, Schey S, Parker A et al (2002) The toxicity and efficacy of donor lymphocyte infusions given after reduced-intensity conditioning allogeneic stem cell transplantation. Blood 100(9):3108–3114 84. Peggs KS, Thomson K, Hart DP, Geary J, Morris EC, Yong K et al (2004) Doseescalated donor lymphocyte infusions following reduced intensity transplantation: Toxicity, chimerism, and disease responses. Blood 103(4):1548–1556 85. Kolb HJ, Schmid C, Buhmann R, Tischer J, Ledderose G (2005) DLI: Where are we know? Hematology 10(Suppl 1):115–116 86. Tse WW, Zang SL, Bunting KD, Laughlin MJ (2008) Umbilical cord blood transplantation in adult myeloid leukemia. Bone Marrow Transplant 41(5):465–472

Chapter 2  Full Intensity and Reduced Intensity Allogeneic Transplantation in AML  87. Majhail NS, Brunstein CG, Tomblyn M, Thomas AJ, Miller JS, Arora M et  al (2008) Reduced-intensity allogeneic transplant in patients older than 55 years: Unrelated umbilical cord blood is safe and effective for patients without a matched related donor. Biol Blood Marrow Transplant 14(3):282–289 88. Schmid C, Schleuning M, Ledderose G, Tischer J, Kolb HJ (2005) Sequential regimen of chemotherapy, reduced-intensity conditioning for allogeneic stem-cell transplantation, and prophylactic donor lymphocyte transfusion in high-risk acute myeloid leukemia and myelodysplastic syndrome. J Clin Oncol 23(24):5675– 5687 89. Schmid C, Schleuning M, Schwerdtfeger R, Hertenstein B, Mischak-Weissinger E, Bunjes D et al (2006) Long-term survival in refractory acute myeloid leukemia after sequential treatment with chemotherapy and reduced-intensity conditioning for allogeneic stem cell transplantation. Blood 108(3):1092–1099

27

sdfsdf

Chapter 3 Allogeneic Stem Cell Transplantation for Adult Acute Lymphoblastic Leukemia (ALL) Bella Patel, Anthony H. Goldstone, and Adele K. Fielding

1.  Introduction Conventional treatment for adult acute lymphoblastic leukemia (ALL) consists of sequentially administered cycles of combination chemotherapy involving an induction, consolidation/intensification, and maintenance phase which is modeled on clinical protocols developed for the treatment of childhood ALL. With this approach, even though a majority of patients (80–92%) achieve complete remission, only one-third of patient or less are long-term survivors. The long-term survival has not changed for over a decade [1–7]. Thus, consolidation of remission is vital in achieving long-term survival in this disease. Intensive myeloablative therapy followed by allogeneic stem cell transplant is an alternative to conventional protracted consolidation/maintenance chemotherapy, offering the advantage of delivering a maximum dose intensive regime with the added benefit of a potential graft versus leukemia (GVL) effect. Thus, in an attempt to reduce the risk of relapse and improve the outcome of adult patients with ALL, numerous trials incorporating allo-HSCT into the treatment algorithm have been conducted. More recent studies have helped to define the subsets of adult patients who are likely to derive the most benefit from an intensive treatment strategy [8–10].

2.  Prognostic Factors in Adult ALL ALL is biologically heterogeneous. A number of features are associated with the failure of treatment (Table  3-1). Perhaps the best characterized and universally agreed adverse features are: patient age over 35 years, a high white blood count which for B lineage ALL is agreed as >30 × 109/l and for T lineage >100,000 × 109/l, and the presence of the Philadelphia chromosome t(9;22) or BCR/ABL fusion gene. In recent years, other cytogenetic subgroups associated with poor outcomes have been identified and include leukemia which harbor the t(4;11) or MLL/AF4 fusion gene, complex karyotypes, and

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_3, © Springer Science + Business Media, LLC 2003, 2010

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Table 3-1.  Prognostic factors in adult ALL. Age WBC

>35 years

  B-lineage

>30 × 109/l

  T-lineage

>100,000 × 109/l

Immunophenotype

B-lineage ALL Cortical T-ALL

Cytogenetics

t(9;22)/BCR/ABL t(4;11)/MLL/AF4 Low hypodiploidy/near triploidy Complex karyotype (>5 abnormalities)

Treatment response

Late achievement of CR: >4 weeks Minimal Residual Disease late into therapy

low hypodiploidy/near triploidy [10, 11]. Immunophenotypic subgroups such as early T-ALL and pro-B ALL are also regarded by some groups as poor prognostic features [8, 11]. The speed and magnitude of treatment response has also been shown to predict treatment outcome. Some studies showing a late achievement of CR >4 weeks identifies a subset of patients with a high risk of relapse, although the recent UKALLXII/ECOG2993 study did not show a worse outcome for patients who took two cycles of induction therapy to achieve remission [9]. The involvement of the central nervous system during diagnosis probably has a worse prognosis, although long-term disease free survival can be achieved in this setting [12]. More recently, the demonstration of residual disease or occult leukemia cells detected by sensitive methods at various times in therapy has been shown to be of prognostic value, predicting a worse outcome [13, 14]. Importantly, this assessment appears to be independent of age and WBC. In adult ALL, demonstration of residual disease at later time points in therapy (~16 weeks) appears to be most predictive. A summary of prognostic factors is given in Table 3-1. Due to the poor prognostic outcome of patients in whom the Philadelphia chromosme is detected, physicians have for long followed an aggressive approach with the use of allo-HSCT in this group. Hence, the main body of this chapter will focus on the use of allo-HSCT in Ph negative ALL. A smaller section at the end will comment specifically on the use of allo-HSCT in Ph pos ALL.

3.  Sibling Allogeneic Hematopoietic Stem Cell Transplantation in First Remission (CR1) In adult patients lacking the Ph chromosome, the role of allo-HSCT to consolidate remission is controversial and has been the focus of intense study. It is generally accepted that randomization between allogeneic HSCT and no HSCT is neither feasible nor desirable, so trials which seek to determine the role of sibling allo-HSCT have generally employed a so-called “biological randomization.” Sibling allo-HSCT is assigned to those with an HLA compatible family donor and where a donor is not available, allocation to either chemotherapy or autologous hematopoietic stem cell transplantation

Chapter 3  Allogeneic Stem Cell Transplantation for Adult Acute Lymphoblastic Leukemia (ALL) 

31

Table 3-2.  Definitions of “high risk” for stratification in adult ALL trials. Trial

High risk features

LALA 87 Pethema

Ph +/Common ALL with age >35/WBC > 30 × 109/l, CR >4/52, Null ALL Age 30–50,WBC ³ 25 × 109/l, t(4;11)/11q23 rearrangement, t(1,19)

GMALL

WBC > 30,000 × 109/l, CR > 4/52, immunophenotype

JALSG 93

Any patient with Ph+, age >30 or WBC over 30,000 × 109/l

LALA 94

Any patient with CNS disease at diagnosis, CR beyond first induction; B lineage ALL with 11q23 rearrangements, t(1;19), WBC > 30,000 × 109/l,or myeloid markers

GOELAL02

Ph+, t(4;11), t(1,19) WBC > 30 × 109/l. CR after first induction, age >35

UKALL XII/ECOG 2993 Ph+

(auto-HSCT) or a randomization between the latter two treatments is carried out. The results for such studies are compared in a “donor versus no-donor” intention-to-treat basis. A summary of the available trials, comparing the results of allo-SCT in first CR in patients lacking the Philadelphia chromosome are given in Table 3-2. Generally, results indicate that allo-HSCT provides the most potent antileukemic therapy, significantly reducing the risk of relapse to a greater magnitude than either conventional chemotherapy or autoSCT. However, an overall survival benefit is not always demonstrated due to the high incidence of toxicity associated with the procedure. In the LALA 87 study, patients under the age of 40 years with a donor received an HLA-identical sibling HSCT, those over 50 years were treated with chemotherapy, and the remaining patients were randomized between chemotherapy and auto-HSCT. At 10 years there was a significant survival benefit for allo-SCT in patients with high-risk features [15]. The BGMT study randomized patients in remission; those with an HLA matched sibling donor to allo-HSCT and the others to auto-HSCT. The 3 year disease free survival was significantly higher in allo-SCT recipients [16]. Recent prospective studies have focused on applying allo-HSCT to highrisk patients alone. Table 3-2 shows the features that the various studies have used to categorize “high-risk.” In the LALA 94 trial [17], high-risk patients (i.e., those falling outside the standard risk criteria or those with CNS disease at presentation) were assigned allo-HSCT if a HLA matched donor was available while others were randomized between auto-SCT and standard chemotherapy. Those receiving a sibling allo-HSCT had significantly better outcomes (5 year overall survival, 45 vs. 23%) [17]. In the Goelam-02 study [18], only high-risk patients under the age of 50 years were offered allo-SCT in first remission if a HLA-matched sibling donor was available, those lacking a donor were assigned to auto-HSCT. A clear survival advantage was observed in the allo-HSCT group at 6 years. Other studies have failed to demonstrate improved outcomes from allo-SCT in first remission. Two case-controlled studies performed by the IBMTR showed no overall advantage for transplant over chemotherapy even when stratifying for risk [19, 20] However, owing to the retrospective nature of these studies and inherent selection biases, it is difficult to arrive at any definitive conclusions from these studies. The largest donor vs. no-donor analysis comes from the UKALL12/ECOG2993 study, where almost 500 patients were included in

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the analysis. There was a clear and statistically significant survival advantage to having a matched sibling donor. Indeed, a number of patients with a donor did not receive allo-HSCT so the advantage may even have been underestimated. However, this study introduced a cautionary note in those patients who were older than 40 years;even though there was a considerable decrease in relapse risk among those who had a donor, there was no survival advantage, due to a very high treatment-related mortality, approaching 39% at 2 years. Thus, there is likely to be an upper age limit for patients who can benefit from myeloablative allo-HSCT, and this will continue to limit the applicability of the procedure. Overall, one can conclude that despite differences in the criteria applied for high-risk features between studies, there is a large body of data to support the use of allo-HSCT in first remission in adult ALL patients with high-risk features other than old age. However, the inclusion of patients with Ph + ALL in the overall results of many studies does not allow specific assessment of the value of allo-HSCT in patients with high-risk features outside this cytogenetic group. For patients with standard-risk disease, the role of allo-HSCT is less well studied. However, results from the largest study to compare post-remission therapies in adult ALL, the UKALL XII/ECOG 2993 study has helped to answer this question. Patients younger than 50 years (55 since 2004) in the first CR were assigned to allo-HSCT if a matched sibling donor was available. The other patients were randomized to auto-HSCT or standard chemotherapy. A survival advantage was observed for the allogeneic transplant group as a whole but this was largely due to the improved survival in standard risk patients; EFS was 59% with a donor, compared to 41% without a donor.

4.  Allo-HSCT Beyond CR1 Allogeneic stem cell transplantation has the best potential for long-term OS when applied in CR1 compared to CR2. However, in a proportion of patients with relapsed disease, long-term remission can be gained by receipt of an allogeneic stem cell transplant. Analysis based on IBMTR data showed that the probability of survival in patients achieving second remission is 30% at 5 years [21]. In the largest reported study of 609 patients, with recurring ALL, the overall estimated survival at 5 years was 7% [22]. Receipt of a hematopoietic stem cell transplantation from either a related or unrelated source in patients who had previously been treated with chemotherapy resulted in a better outcome compared to chemotherapy alone (23 vs. 4%). The same study showed the outcome after relapse was very poor and was not influenced by prior therapy. However, when treatment received post-relapse was evaluated in patients with an equal chance of receiving allo-HSCT (i.e., those alive at the median time to transplant and not having had allo-HSCT in CR1), a better outcome was observed in those receiving allo-HSCT than in those receiving chemotherapy alone [22]. Among the few published systematic studies examining factors responsible for achieving long-term remission following allogeneic stem cell transplantation for relapsed ALL, the receipt of a transplant

Chapter 3  Allogeneic Stem Cell Transplantation for Adult Acute Lymphoblastic Leukemia (ALL) 

at second remission compared to a sustained relapse, appears to be associated with better results [22, 23].

5.  Unrelated Donor Stem Cell Transplantation (UD-SCT) In Philadelphia chromosome positive disease UD-SCT in first remission is regarded as standard therapy in the absence of a matched sibling donor. The alternative for those lacking a sibling donor in this situation is an auto-HSCT. A retrospective comparison performed by the IBMTR of patients treated with either of these therapeutic modalities for ALL did not demonstrate a superior survival for UD-HSCT despite a demonstrated lower relapse risk due to an extremely high TRM which approached >40% [21]. With improvements in supportive care, HLA matching and GVHD prophylaxis, the TRM in UD-HSCT is improving. Three published studies have examined the role of UD-HSCT performed in the first remission in adult patients with ALL [24–26]. The results reported from these studies and from registry data are encouraging and do not show wide differences in outcomes from UD-HSCT (DFS ~40–50%) compared to sibling allogeneic transplantation. Sincethe analysis of these studies include Ph + cases, the role of UD-HSCT in first remission in patients outside this cytogenetic group have not been exclusively studied. As less than one-third of patients will have a HLA compatible sibling donor, UD-HSCT could pose a reasonable therapeutic option and is currently the subject of ongoing study.

6.  Other Stem Cell Sources Despite increasing size of donor pools, it is not possible to find an eight of eight allelic matched unrelated donor for all individuals. Using donors who are not fully matched or turning to alternative sources of stem cells are options which may be considered by transplant units. In children, a recent, and a very extensive retrospective study comparing the outcomes of HSCT using marrow or cord blood was carried out in patients receiving MUD or umbilical cord blood HSCT for leukemia [27]. Greater than 60% of those included had a diagnosis of ALL, mostly beyond CR1. TRM was statistically significantly higher after transplant of HLA-antigen mismatched umbilical cord blood compared to fully matched bone marrow stem cells (relative risk 2.31 for two antigen mismatch, 1.88 for one antigen mismatch), although relapse rates were lower after two-antigen HLA-mismatched umbilicalcord-blood transplants (RR 0.54, p = 0.0045). The authors conclude that the data support the use of HLA-matched and even one- or two-antigen HLAmismatched umbilical cord blood in children with acute leukemia who need transplantation. These data do not specifically address the issue of children with Ph + ALL in CR1, but the conclusion from this study may be relevant to this situation. The current pan-European trial in pediatric Ph + ALL allows the use of umbilical cord blood as a source of stem cells for HSCT and is recommended for all children with donors (Table 3-3).

33

Treatment compared

Chemo/auto-SCT

Auto-SCT

Chemo/auto-SCT

Chemo

Chemo

Chemo

Chemo

Chemo/auto-SCT

Group

LALAb 87 [15, 54, 55]

BGMTb [16]

PETHEMAb ALL 93 [56]

JALSG 1998 [20]

GMALL 1981 and 1984 [19]

Gupta et al. [57]

JALSGb 93 [5]

EORTCb ALL3 2004 [58]

68 vs. 116

34 vs. 108

48 vs. 39

234 vs. 484

214 vs. 76

84 vs. 98

43 vs. 77

116 vs. 141

N donor vs. no-donor

6 year RFS/OS 38 vs. 37%/41 vs. 39% p = not sig

6 year OS 40 vs. 46% p = 0.58

3 year RFS 40 vs. 39% p = 0.74

9 year RFS 34 vs. 32% p > 0.2 standard risk/high risk groups p³05

Age >30 years: 30 vs. 26% p = 0.70

Age < 30 years: 69 vs. 22%

Age <30 years: 53 vs. 30% p = 0.02

38 vs. 56% p = 0.001

Not reported

3 year RR 40 vs. 61% p = 0.07

9 year RR 30 vs. 66% p < 0.0001

Age >30 years: 70 vs. 32% p < 0.0001

5 year RR

62 vs. 51% p = 0.28

5 year RR

12 vs. 62%

34 vs. 60%

Relapse% donor vs. no-donor

5 year RFS

5 year RFS 37 vs. 46% p ³ 0.05

5 year OS 35 vs. 44% p = 0.35

3 year RFS 68 vs. 26% p < 0.001

High risk patients 10 year OS 44 vs. 11% p = 0.009

All patients 10 year OS 46 vs. 31% p = 0.04

Outcome donor vs. no-donor

Table 3-3.  Trials evaluating sibling allo HSCT in adult ALL in CR1.

23.5 vs. 6.9% p = 0.0004

Not reported

29 vs. 5% p = 0.004

9 year 53 vs. 5% p < 0.0001

Age >30 years: 57 vs. 13% p < 0.0001

Age <30 years: 32 vs. 3%

5 year

Not reported

12 vs. 2%

16 vs. 3%

TRM% donor vs. no-donor

34  B. Patel et al.

Auto-SCT

Chemo

GOELALM-02b [18]

UKALLb XII/ECOG 2993 (Rowe et al.)

384 vs. 418

41 vs. 115

100 vs. 159

b 

Outcome analysis may also include patients with Ph chromosome positive disease Prospective randomized studies

a 

Chemo/auto-SCT

LALAb 94 Trial [17]

High risk 36 vs. 63 p£0.05

High risk 41 vs. 35/38 vs. 31% (p > 0.5) Standard 64 vs. 51/59 vs. 47 p£0.05 standard risk

5 year RR

10 vs. 49%

5 year RR 36 vs. 62% p = 0.001

5 year OS/DFS

5 year OS 75 vs. 40% p = 0.0027

5 year RFS 45 vs. 23% p = 0.007

<35 years: 26 vs. 12%

>35 years: 45%

6 month 15.4 vs. 6.9%

5 year 18 vs. 7% p = 0.01

Chapter 3  Allogeneic Stem Cell Transplantation for Adult Acute Lymphoblastic Leukemia (ALL)  35

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In adults, due to the close relationship between cell dose and hematopoietic recovery/risk of TRM, a major limitation to the use of cord blood is the difficulty in obtaining sufficient numbers of hematopoietic precursor cells. An earlier report of umbilical cord blood HSCT in adults confirmed a higher TRM in adults compared with matched unrelated HSCT, although mismatched unrelated donor stem cells carried equivalent mortality to cord blood [28]. Technological advances and approaches to increasing cell dose by using, for example, dual-cord units may ultimately yield a technique which is safe enough for wider investigation of HSCT for ALL in adults. HSCT using haploidentical donors is also a clinical consideration in high-risk ALL when matched unrelated donors are not found. Just over 60 cases relating to adult ALL have been reported since 1993, 15 of these cases were Ph + , approximately half were beyond CR1. The data have been recently reviewed in detail [29]. Although the techniques have improved greatly, good results obtained by single centers have been almost impossible to replicate outside these very experienced settings due to the high TRM, stemming from both rejection and severe GVHD. Currently, it is not possible to recommend this approach for wider use.

7.  Preparative Regimes and T-Cell Depletion The most commonly used preparative regimes for allogeneic transplantation in ALL consist of cyclophosphamide or etoposide plus TBI. There have been few studies comparing different preparative regimes. A comparison of cyclophosphamide and TBI with etoposide and TBI as conditioning regimes for patients undergoing sibling allografting for ALL showed no differences in relapse, DFS, or survival [30]. However, for CR2 patients improved outcomes were observed with TBI and etoposide or cyclophosphamide with ³13 Gy [31]. Regarding the role of T-cell depletion, there are few data that directly address this issue in this disease. However, a general viewpoint would be that the lower TRM observed after T-cell depletion is likely compensated by a higher relapse rate. Marks et al. reported a relapse rate of 40% when using CD34 antibody selection or alemtuzumab for T-cell depletion [32]. Whether T-cell depletion can be “fine tuned,” for example an optimal dose of in vivo alemtuzumab, defined to balance the mortality caused by GVHD against the relapse risk remains to be seen.

8.  Reduced Intensity Conditioning Reduced intensity conditioning (RIC) for allografts is an interesting option, particularly in older patients or those with co-morbidities. It is clear that non-myeloablative or RIC regimens can deliver a powerful graft-versustumor effect in many diseases but the success of this approach likely relates to both disease burden and tempo of the underlying disease. A recent study of relapse risk in patients with various malignancies treated with RIC-HSCT attests the fact that the highest relapse risk is in those patients with advanced lymphoid and myeloid malignancies [33]. To date there are scant published

Chapter 3  Allogeneic Stem Cell Transplantation for Adult Acute Lymphoblastic Leukemia (ALL) 

data on the use of RIC-HSCT conditioning in ALL. A small study of 22 patients with ALL, 11 of whom had Ph + disease and most of whom were at very high risk, many having relapsed after previous myeloablative therapy, was reported [34]. Not surprisingly, the only leukemia-free survivors were among those individuals treated in CR. Martino et al. [35] reported another small retrospective series of 27 RIC-HSCT treated older patients (mean age 50), with high-risk ALL. Forty-one percent were Ph + and 85% were beyond CR1. In this high-risk group, 2 year TRM was 23% and 2 year OS was 31%. The low TRM and moderate OS suggest that there may have been some survival benefit to the procedure that outweighed the toxicity. For ALL in CR2, a series of 43 RIC-HSCT in both adults and children was recently published in Mexico [36]. The regimen was chosen above myeloablative HSCT, partly for economic reasons. Only two of the patients were Ph + . ALL patients became full donor chimeras, and the relapse rate was high, in the order of 70%. OS at 3 years was 31%. However, outcome after relapse is known to be poor [22, 37] and this study does not preclude benefit to the procedure in earlier stages of the disease. Data in abstract form were recently presented from the City of Hope transplant centre in which a fludarabine and melphalan RIC conditioning regimen was used in 21 patients with ALL in or beyond CR1 in whom the median age was 46, an age at which the toxicity of myeloablative conditioning would be expected to be considerable [38]. The 100 day non-relapse mortality was 10%. High rates of acute and chronic GVHD were observed, which may in part be responsible for the remarkably good DFS (1 year DFS 70.9%). However, since follow up is short, it is possible that the high rate of graft versus host disease may result in a higher late nonrelapse mortality. However, it remains a reasonable hypothesis that patients with ALL at high risk of relapsing as well as at very high risk of TRM, could benefit from RIC-SCT, especially if the disease is in “remission” at the time of HSCT. The definition of “remission” may be of crucial importance here. For example, it is clear in Ph + ALL that detection of MRD by BCR-ABL predicts relapse [39, 40]. However, it is not clear whether RIC-HSCT would be able to overcome the additional risk factor of having detectable MRD preHSCT. It will be interesting to observe if the depth of remission, i.e. being MRD negative as compared to being only in morphological CR or cytogenetic remission could alter the outcome. Whether donor lymphocyte infusions in combination with tyrosine kinase inhibitors in patients with Ph + ALL [41] might be of value to treat patients with residual MRD post-RIC HSCT is also not clear. A planned UK-USA collaborative trial will evaluate RIC HSCT in older patients with Philadelphia chromosome negative disease at highest risk of relapse in whom the predicted treatment-related mortality precludes myeloablative alloHSCT.

9.  Allo-HSCT in Philadelphia Chromosome Positive ALL Allogeneic stem cell transplantation in CR1 is the treatment of choice in Ph positive (Ph + ve) ALL where the overall outcomes with standard chemotherapy are uniformly dismal with less than 15% of patients surviving long-term. The use of allo-HSCT in this subset of patients has recently been comprehen-

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sively reviewed [42]. Although there are no randomized controlled trials comparing the results of allogeneic transplantation with standard chemotherapy, the reported OS for allo-HSCT in first or is 42–47% which is a superior to results obtained with intensive or auto-SCT [43–47]. Furthermore, the results of UD-SCT in this disease approach that observed in related allo-SCT. Taking into account the very low chance of cure with chemotherapy and auto-HSCT, UD-SCT in first remission in eligible patients lacking a related family donor is generally regarded as standard treatment. The introduction of the specific BCR/ABL tyrosine kinase inhibitor, Imatinib, has the potential to change the standard treatment for Ph + ALL. A number of studies have incorporated Imatinib into the treatment schedule for Ph + patients. Imatinib is commonly co-administered with induction drugs with a rationale of increasing the CR rate and therefore patients eligible for first CR allo-HSCT [48]. The most recently reported study of imatinib in induction from the French national group GRAAL, supports the notion that the addition of imatinib to induction can increase the number of patients reaching HSCT. Imatinib was added to induction (early poor responders) or to consolidation (good early responders), and maintained until HSCT. In 45 patients, CR rate was 96%. Each one of the 22 patients reaching CR who had a donor actually received allogeneic SCT in first CR. Follow up so far is short but is estimated at 65% overall survival at 18 months [49]. Imatinib may also play a role in post-HSCT maintenance. A study in which all patients with Ph + ALL, who became BCR/ABL positive after HSCT, were given imatinib show some long-term responses to those responding to imatinib in this setting [50].

10.  Improving Outcomes of Transplantation in Adult ALL The challenges in improving transplant outcomes in ALL focus on three factors: reducing toxicity,developing a rationalized approach for the application of therapies, and better treatment of post-transplant relapse. Novel drugs such as keratinocyte growth factor, an epithelial mitogen has been shown to significantly reduce both the incidence and duration of severe oral mucositis in patients receiving myeloablative chemoradiotherapy before autologous peripheral blood progenitor cell transplantation [51–53]. Better treatment and prevention of GVHD, and treatment of opportunistic viral infections may further help to abrograte the high TRM. A risk-adapted approach might further improve overall outcome; by reserving intensive treatments for those at high risk of relapse whilst avoiding this approach in those more likely to be cured by standard chemotherapy. Thus, a better assessment of risk is fundamental in improving the overall outcomes for adult patients with ALL. MRD assessment by molecular or immunological methods is an evolving risk stratification tool. Risk adapted therapy based on MRD assessment is currently the subject of numerous ongoing international studies. Furthermore, there are emerging data to suggest that detection of residual disease pre- and post-transplant predicts treatment failure. These observations could form a rationale for further cytoreduction prior to myeloablative therapy or in the post-transplant context guide the application of immunomodulatory strategies.

Chapter 3  Allogeneic Stem Cell Transplantation for Adult Acute Lymphoblastic Leukemia (ALL) 

References 1. Pui CH, Evans WE (2006) Treatment of acute lymphoblastic leukemia. N Engl J Med 354:166–178 2. Gokbuget N, Hoelzer D, Arnold R et al (2000) Treatment of adult ALL according to protocols of the German Multicenter Study Group for Adult ALL (GMALL). Hematol Oncol Clin North Am 14:1307–1325 ix 3. Linker C, Damon L, Ries C, Navarro W (2002) Intensified and shortened cyclical chemotherapy for adult acute lymphoblastic leukemia. J Clin Oncol 20:2464–2471 4. Annino L, Vegna ML, Camera A et al (2002) Treatment of adult acute lymphoblastic leukemia (ALL): Long-term follow-up of the GIMEMA ALL 0288 randomized study. Blood 99:863–871 5. Takeuchi J, Kyo T, Naito K et al (2002) Induction therapy by frequent administration of doxorubicin with four other drugs, followed by intensive consolidation and maintenance therapy for adult acute lymphoblastic leukemia: the JALSG-ALL93 study. Leukemia 16:1259–1266 6. Kantarjian H, Thomas D, O’Brien S et  al (2004) Long-term follow-up results of hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (Hyper-CVAD), a dose-intensive regimen, in adult acute lymphocytic leukemia. Cancer 101:2788–2801 7. Durrant IJ, Prentice HG, Richards SM (1997) Intensification of treatment for adults with acute lymphoblastic leukaemia: Results of UK Medical Research Council randomized trial UKALL XA. Medical Research Council Working Party on Leukaemia in Adults. Br J Haematol 99:84–92 8. Hoelzer D, Thiel E, Loffler H et al (1988) Prognostic factors in a multicenter study for treatment of acute lymphoblastic leukemia in adults. Blood 71:123–131 9. Rowe JM, Buck G, Burnett AK et al (2005) Induction therapy for adults with acute lymphoblastic leukemia: results of more than 1500 patients from the international ALL trial: MRC UKALL XII/ECOG E2993. Blood 106:3760–3767 10. Moorman AV, Harrison CJ, Buck GA et  al (2007) Karyotype is an independent prognostic factor in adult acute lymphoblastic leukemia (ALL): analysis of cytogenetic data from patients treated on the Medical Research Council (MRC) UKALLXII/Eastern Cooperative Oncology Group (ECOG) 2993 tria. Blood 109:3189–3197 11. Secker-Walker LM, Craig JM, Hawkins JM, Hoffbrand AV (1991) Philadelphia positive acute lymphoblastic leukemia in adults: Age distribution, BCR breakpoint and prognostic significance. Leukemia 5:196–199 12. Lazarus HM, Richards SM, Chopra R et al (2006) Central nervous system involvement in adult acute lymphoblastic leukemia at diagnosis: results from the international ALL trial MRC UKALL XII/ECOG E2993. Blood 108:465–472 13. Mortuza FY, Papaioannou M, Moreira IM et al (2002) Minimal residual disease tests provide an independent predictor of clinical outcome in adult acute lymphoblastic leukemia. J Clin Oncol 20:1094–1104 14. Bruggemann M, Raff T, Flohr T et  al (2006) Clinical significance of minimal residual disease quantification in adult patients with standard-risk acute lymphoblastic leukemia. Blood 107:1116–1123 15. Sebban C, Lepage E, Vernant JP et al (1994) Allogeneic bone marrow transplantation in adult acute lymphoblastic leukemia in first complete remission: a comparative study. French Group of Therapy of Adult Acute Lymphoblastic Leukemia. J Clin Oncol 12:2580–2587 16. Attal M, Blaise D, Marit G et al (1995) Consolidation treatment of adult acute lymphoblastic leukemia: a prospective, randomized trial comparing allogeneic versus autologous bone marrow transplantation and testing the impact of recombinant interleukin-2 after autologous bone marrow transplantation. BGMT Group. Blood 86:1619–1628

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B. Patel et al. 17. Thomas X, Boiron JM, Huguet F et  al (2004) Outcome of treatment in adults with acute lymphoblastic leukemia: Analysis of the LALA-94 trial. J Clin Oncol 22:4075–4086 18. Hunault M, Harousseau JL, Delain M et  al (2004) Better outcome of adult acute lymphoblastic leukemia after early genoidentical allogeneic bone marrow transplantation (BMT) than after late high-dose therapy and autologous BMT: A GOELAMS trial. Blood 104:3028–3037 19. Zhang MJ, Hoelzer D, Horowitz MM et al (1995) Long-term follow-up of adults with acute lymphoblastic leukemia in first remission treated with chemotherapy or bone marrow transplantation. The Acute Lymphoblastic Leukemia Working Committee. Ann Intern Med 123:428–431 20. Oh H, Gale RP, Zhang MJ et al (1998) Chemotherapy vs HLA-identical sibling bone marrow transplants for adults with acute lymphoblastic leukemia in first remission. Bone Marrow Transplant 22:253–257 21. Levine JE, Harris RE, Loberiza FR Jr et  al (2003) A comparison of allogeneic and autologous bone marrow transplantation for lymphoblastic lymphoma. Blood 101:2476–2482 22. Fielding AK, Richards SM, Chopra R et  al (2007) Outcome of 609 adults after relapse of acute lymphoblastic leukemia (ALL); an MRC UKALL12/ECOG 2993 study. Blood 109:944–950 23. Tavernier E, Boiron JM, Huguet F et  al (2007) Outcome of treatment after first relapse in adults with acute lymphoblastic leukemia initially treated by the LALA94 trial. Leukemia 21:1907–1914 24. Dahlke J, Kroger N, Zabelina T et al (2006) Comparable results in patients with acute lymphoblastic leukemia after related and unrelated stem cell transplantation. Bone Marrow Transplant 37:155–163 25. Kiehl MG, Kraut L, Schwerdtfeger R et  al (2004) Outcome of allogeneic hematopoietic stem-cell transplantation in adult patients with acute lymphoblastic leukemia: no difference in related compared with unrelated transplant in first complete remission. J Clin Oncol 22:2816–2825 26. Cornelissen JJ, Carston M, Kollman C et  al (2001) Unrelated marrow transplantation for adult patients with poor-risk acute lymphoblastic leukemia: strong graft-versus-leukemia effect and risk factors determining outcome. Blood 97:1572–1577 27. Eapen M, Rubinstein P, Zhang MJ et  al (2007) Outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukaemia: A comparison study. Lancet 369:1947–1954 28. Laughlin MJ, Eapen M, Rubinstein P et al (2004) Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia. N Engl J Med 351:2265–2275 29. Marks DI, Aversa F, Lazarus HM (2006) Alternative donor transplants for adult acute lymphoblastic leukaemia: A comparison of the three major options. Bone Marrow Transplant 38:467–475 30. Gassas A, Sung L, Saunders EF, Doyle JJ (2006) Comparative outcome of hematopoietic stem cell transplantation for pediatric acute lymphoblastic leukemia following cyclophosphamide and total body irradiation or VP16 and total body irradiation conditioning regimens. Bone Marrow Transplant 38:739–743 31. Marks DI, Forman SJ, Blume KG et al (2006) A comparison of cyclophosphamide and total body irradiation with etoposide and total body irradiation as conditioning regimens for patients undergoing sibling allografting for acute lymphoblastic leukemia in first or second complete remission. Biol Blood Marrow Transplant 12:438–453 32. Marks DI, Bird JM, Cornish JM et  al (1998) Unrelated donor bone marrow transplantation for children and adolescents with Philadelphia-positive acute lymphoblastic leukemia. J Clin Oncol 16:931–936

Chapter 3  Allogeneic Stem Cell Transplantation for Adult Acute Lymphoblastic Leukemia (ALL)  33. Kahl C, Storer BE, Sandmaier BM et  al (2007) Relapse risk in patients with malignant diseases given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning. Blood 110(7):2744–2748 34. Arnold R, Massenkeil G, Bornhauser M et al (2002) Nonmyeloablative stem cell transplantation in adults with high-risk ALL may be effective in early but not in advanced disease. Leukemia 16:2423–2428 35. Martino R, Giralt S, Caballero MD et  al (2003) Allogeneic hematopoietic stem cell transplantation with reduced-intensity conditioning in acute lymphoblastic leukemia: A feasibility study. Haematologica 88:555–560 36. Gutierrez-Aguirre CH, Gomez-Almaguer D, Cantu-Rodriguez OG et  al (2007) Non-myeloablative stem cell transplantation in patients with relapsed acute lymphoblastic leukemia: Results of a multicenter study. Bone Marrow Transplant 40(6):535–539 37. Tavernier E, Boiron JM, Huguet F et  al (2007) Outcome of treatment after first relapse in adults with acute lymphoblastic leukemia initially treated by the LALA94 trial. Leukemia 21(9):1907–1914 38. Stein A, O’Donnell M, Snyder DS et  al (2007) Reduced-Intensity Stem Cell Tansplantation for high-risk acute lymphoblastic leukaemia. Biol Blood Marrow Transplant 13:134 39. Preudhomme C, Henic N, Cazin B et al (1997) Good correlation between RT-PCR analysis and relapse in Philadelphia (Ph1)-positive acute lymphoblastic leukemia (ALL). Leukemia 11:294–298 40. Radich J, Gehly G, Lee A et  al (1997) Detection of bcr-abl transcripts in Philadelphia chromosome-positive acute lymphoblastic leukemia after marrow transplantation. Blood 89:2602–2609 41. Shimoni A, Kroger N, Zander AR et  al (2003) Imatinib mesylate (STI571) in preparation for allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusions in patients with Philadelphia-positive acute leukemias. Leukemia 17:290–297 42. Fielding AK, Goldstone AH (2007) Allogeneic haematopoietic stem cell transplant in Philadelphia-positive acute lymphoblastic leukaemia. Bone Marrow Transplant 41:447–453 43. Dombret H, Gabert J, Boiron JM et al (2002) Outcome of treatment in adults with Philadelphia chromosome-positive acute lymphoblastic leukemia-results of the prospective multicenter LALA-94 trial. Blood 100:2357–2366 44. Forman SJ, O’Donnell MR, Nademanee AP et al (1987) Bone marrow transplantation for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia. Blood 70:587–588 45. Chao NJ, Blume KG, Forman SJ, Snyder DS (1995) Long-term follow-up of allogeneic bone marrow recipients for Philadelphia chromosome-positive acute lymphoblastic leukemia. Blood 85:3353–3354 46. Barrett AJ, Horowitz MM, Ash RC et  al (1992) Bone marrow transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia. Blood 79:3067–3070 47. Snyder DS, Nademanee AP, O’Donnell MR et al (1999) Long-term follow-up of 23 patients with Philadelphia chromosome-positive acute lymphoblastic leukemia treated with allogeneic bone marrow transplant in first complete remission. Leukemia 13:2053–2058 48. Wassmann B, Pfeifer H, Scheuring U et al (2002) Therapy with imatinib mesylate (Glivec) preceding allogeneic stem cell transplantation (SCT) in relapsed or refractory Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL). Leukemia 16:2358–2365 49. de Labarthe A, Rousselot P, Huguet-Rigal F et al (2007) Imatinib combined with induction or consolidation chemotherapy in patients with de  novo Philadelphia

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B. Patel et al. chromosome-positive acute lymphoblastic leukemia: results of the GRAAPH-2003 study. Blood 109:1408–1413 50. Wassmann B, Pfeifer H, Stadler M et  al (2005) Early molecular response to posttransplantation imatinib determines outcome in MRD + Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL). Blood 106:458–463 51. Spielberger R, Stiff P, Bensinger W et al (2004) Palifermin for oral mucositis after intensive therapy for hematologic cancers. N Engl J Med 351:2590–2598 52. von Bultzingslowen I, Brennan MT, Spijkervet FK et al (2006) Growth factors and cytokines in the prevention and treatment of oral and gastrointestinal mucositis. Support Care Cancer 14:519–527 53. Worthington HV, Clarkson JE, Eden OB (2006) Interventions for preventing oral mucositis for patients with cancer receiving treatment. Cochrane Database Syst Rev:CD000978 54. Thiebaut A, Vernant JP, Degos L et al (2000) Adult acute lymphocytic leukemia study testing chemotherapy and autologous and allogeneic transplantation. A follow-up report of the French protocol LALA 87. Hematol Oncol Clin North Am 14:1353–1366 x 55. Fiere D, Lepage E, Sebban C et  al (1993) Adult acute lymphoblastic leukemia: a multicentric randomized trial testing bone marrow transplantation as postremission therapy. The French Group on Therapy for Adult Acute Lymphoblastic Leukemia. J Clin Oncol 11:1990–2001 56. Ribera JM, Ortega JJ, Oriol A et al (1998) Late intensification chemotherapy has not improved the results of intensive chemotherapy in adult acute lymphoblastic leukemia. Results of a prospective multicenter randomized trial (PETHEMA ALL89). Spanish Society of Hematology. Haematologica 83:222–230 57. Gupta V, Yi QL, Brandwein J et  al (2004) The role of allogeneic bone marrow transplantation in adult patients below the age of 55 years with acute lymphoblastic leukemia in first complete remission: A donor vs no donor comparison. Bone Marrow Transplant 33:397–404 58. Labar B, Suciu S, Zittoun R et al (2004) Allogeneic stem cell transplantation in acute lymphoblastic leukemia and non-Hodgkin’s lymphoma for patients £50 years old in first complete remission: results of the EORTC ALL-3 trial. Haematologica 89:809–817

Chapter 4 Hematopoietic Progenitor Cell Transplantation for Treatment of Chronic Lymphocytic Leukemia Leslie A. Andritsos, John C. Byrd, and Steven M. Devine

1.  Introduction: Chronic Lymphocytic Leukemia Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by a highly variable clinical course. It is the most common adult leukemia in Western countries, and although according to the SEER database the median age of diagnosis is 72, approximately 30% of newly diagnosed patients will be younger than age 65 [1]. Like other chronic lymphoproliferative disorders, CLL is not curable with chemotherapy alone. While standard chemo-immunotherapy with purine nucleoside analog-based therapies may result in excellent overall responses and complete remissions in a majority of patients [2–4], for patients with poor responses to initial therapy or those who exhibit early relapses (i.e., within 6–12 months of completing therapy), there are diminishing FDA-approved treatment options available for salvage therapy. Recent studies have suggested that the response rates of standard chemotherapies in patients who relapse following fludarabine-based regimens are on the order of 20% [5, 6]. The average survival for this group of patients is measured in months. Thus, despite encouraging advances in initial therapy for CLL, outcomes are frequently disappointing once this therapeutic option has failed.

2.  Risk Stratification in CLL Recent studies have demonstrated that patients with CLL can be subdivided into prognostic categories based on IgVH mutational status [7–9], ZAP-70 expression [10], CD38 expression [11], and metaphase and interphase cytogenetic analysis [12]. Perhaps the most powerful and widely available of these is cytogenetic analysis [13]. In 2000, Dohner et  al. described the clinical outcomes of patients with four commonly identified interphase cytogenetic abnormalities. This study clearly demonstrated the impact of adverse cytogenetic risk factors on time to first treatment and overall survival, with patients with 17p13.1 and 11q22.3 deletions having the worst outcomes [12]. One study evaluating genetic and molecular risk factors affecting patients randomized to From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_4, © Springer Science + Business Media, LLC 2003, 2010

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receive either fludarabine plus cyclophosphamide or fludarabine alone found that interphase cytogenetic features were the only risk factors that predicted progression-free survival [13]. Several later studies have demonstrated that del(11q22.3) when given hyperfractionated cyclophosphamide (days 1–3) with fludarabine may lose its prognostic factor in younger patients. These studies taken together, in addition to other recent studies evaluating the impact of cytogenetic analysis on patient outcomes, indicate that for select high risk patients with del(17p13.1), alternative strategies should be considered earlier in the disease course.

3.  Autologous Transplantation for Treatment of CLL High-dose chemotherapy with autologous stem cell support has been used extensively in the treatment of high-grade lymphomas, with a proportion of patients achieving long-term disease-free survival. The applicability of this approach to low-grade lymphomas has been less clear. The first study published in 1993 by Rabinowe et  al. was a pilot study assessing the feasibility of myeloablative conditioning with autologous stem cell support [14]. Twelve patients with chemosensitive disease underwent conditioning with cyclophosphamide and total body irradiation followed by reinfusion of purged autologous marrow products. Five of 12 patients achieved a complete clinical response without excess early toxicity. These promising results led to a number of studies evaluating the efficacy of autologous transplantation in CLL. Provan et al. looked specifically at the role of achievement of minimal residual disease (MRD) by polymerase chain reaction (PCR) in 21 patients who underwent myeloablative conditioning followed by purged marrow stem cell infusion. This study was the first to demonstrate that achievement of an MRD-negative state correlated with improved clinical outcomes, and was the basis for a number of studies evaluating disease status peri-transplant and the risk of relapse [15]. These results also confirmed the existence of a dose effect in CLL, with longer remissions seen in patients who received high-dose chemoradiotherapy with achievement of MRD, further stimulating interest in this approach. This pilot study was followed by a Phase II study of 137 patients with poor risk CLL which was reported by Gribben et  al. in 2005 [16]. This study found low 100 day mortality (4%), but no difference in overall survival between the autologous and allogeneic groups at 6.5 years. There was no significant difference in overall survival between the groups, and no demonstration of a plateau in progression-free survival in patients undergoing autologous transplantation. Importantly, 12% of patients who had undergone autologous transplantation developed myelodysplastic syndrome or AML by 8 years of follow-up. In our experience, this complication arising in the setting of CLL virtually always results in a fatal outcome. Balancing earlier benefit versus late toxicity is an important consideration in developing new strategies for a chronic disease such as CLL. No randomized studies have been performed comparing autologous transplantation to standard chemotherapy; however, Dreger et  al. performed a risk-matched analysis comparing 66 patients who underwent sequential high-dose therapy including autologous transplantation with 291 patients who received conventional chemotherapy [17]. The groups were matched for age,

Chapter 4  Hematopoietic Progenitor Cell Transplantation for Treatment of CLL 

lymphocyte count, Binet stage, and IgVH mutational status, with 44 patients matching all four variables. This study found a survival benefit in the autograft group, with a 4-year progression-free survival of 69% and a 4-year overall survival of 94%. No plateau in progression-free survival was demonstrated. A similar comparison was conducted by the European Bone Marrow Transplant Group (EBMT) database as part of the French CLL Cooperative Group study [18] which compared 621 patients who underwent autografting to 630 patients who received conventional therapy, which has been published in abstract form. This study found a benefit in patients who were autografted within 18 months of diagnosis but no benefit when patients were autografted more than 18 months after diagnosis. It is clear from the studies that have been performed to date that the greatest benefit from autografting is seen in patients who have chemosensitive disease at the time of transplant, are in remission with a low tumor burden, and are able to achieve MRD-negative status following transplant. In this setting, multiple studies have demonstrated prolonged progression-free survival, with retrospective studies showing improved overall survival over comparator groups who received standard chemotherapy. However, such assessment comes with significant bias. Despite conditioning regimens containing high-dose chemotherapy and total body irradiation, the reported early mortality has been in the range of 2–10%, which is comparable to other groups of patients undergoing autografting. However, patients with chemotherapy refractory disease or with advanced stage at the time of diagnosis are unlikely to benefit from autografting, and efforts at stem cell mobilization may be hampered by administration of prior fludarabine, which is now nearly universal in the treatment of CLL [19]. Furthermore, with the recent introduction of combination regimens of chemo-immunotherapy showing excellent overall responses and prolonged disease-free survival in a subset of patients [3, 20], it is unclear at this time whether autografting holds any advantage over the current standard of care. Rising concerns over the growing incidence of myelodysplasia and AML in patients who received TBI-containing regimens has further dampened enthusiasm for autografting in CLL. In addition, in the Dana-Farber series 19% of autografted patients developed subsequent secondary malignancies [21]. As there has been no demonstration of a plateau and therefore no potential for this as a curative procedure, more groups have turned to investigations of allogeneic transplantation because of the demonstrated graft-versus-leukemia effect and greater potential for long-term disease control. Few investigators at this time are pursuing autologous transplant for CLL as part of clinical trials with novel interventions. Autologous transplant is not a treatment approach we consider for CLL patients outside of a well-designed clinical trial.

4.  Allogeneic Transplantation Using Myeloablative Conditioning Given the demonstrated responsiveness of CLL to increasing doses of chemotherapy and/or radiotherapy, initial studies of allogeneic transplantation for CLL focused on high-dose chemotherapy and/or radiotherapy-based regimens. The first of these reported by Bandini et al. in 1991 was a retrospective review of IBMTR data and case reports [22]. Of 26 patients identified,

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12 (46%) died of treatment-related mortality. However, at between 5 and 48 months 11 patients were alive and in clinical remissions. A retrospective cohort study looking at data from the EBMT and IBMTR found 54 patients with CLL who had undergone myeloablative conditioning and matched related donor bone marrow transplantation between the years of 1984 and 1992 [23]. Initial results from this study found that 38 patients (70%) at some point achieved a hematologic remission, with a 3-year probability of survival of 46%. Treatment-related mortality was significant at 46%. Michallet et al. updated this series in 2003 [24], providing the longest follow-up to date of myeloablative conditioning in CLL. This update showed encouraging results, and found that despite high early rates of treatment-related mortality, at 10 years of follow-up there was an overall survival of 41% with a disease-free survival of 36%. This was the first study to suggest a significant graft-versus-leukemia effect in CLL, given that no studies of autologous transplantation using similar conditioning regimens demonstrated a plateau in survival. Pavlitec et al. next reported a series of 23 patients in 2000 who had undergone high-dose therapy with either bone marrow or peripheral blood stem cell transplantation [25]. This series found that 87% of patients achieved a complete remission following transplantation, and at a median follow-up of 26 months 14 (61%) were alive and free of disease. The incidence of grades II–IV acute GVHD was 54%, and the day 100 mortality was 17% with a projected 5-year overall survival of 62%. Larger studies soon followed, with the largest study to date published in 2000 by Horowitz et al. for the IBMTR [26]. This was a registry study of 242 patients who underwent allografting using myeloablative regimens between 1990 and 1999. The majority of recipients received marrow products from HLA-identical siblings. This group of patients was compared to 83 CLL patients who underwent autografting using a similar conditioning regimen during the same time period. The authors found that 3-year survival probabilities were significantly higher in patients undergoing autografting (49 ± 8% vs. 87 ± 9%); however, deaths from disease progression were lower in allografted patients. Three-year progression-free survival was 44% after allografting, with younger patients with less advanced disease faring better. Later studies addressed specific questions in myeloablative allogeneic transplantation for CLL, such as the risk of using unrelated versus related donors and whether transplantation could overcome poor risk features. In 2005, Pavletic et  al. reported a registry study of 36 evaluable patients with CLL who underwent myeloablative conditioning followed by unrelated donor marrow transplantation [27]. This was a particularly high risk group of patients, with 55% being chemotherapy refractory at the time of transplantation, a variable previously shown to adversely impact the success of transplantation [28]. Twenty-one (58%) patients achieved a complete remission; however, interestingly in three of these patients delayed CR was noted, with one patient ultimately achieving a CR at 48 months after transplantation without other intervention, adding further evidence of a graft-versus-leukemia effect. However, at a median follow-up of 6 years only 29% of patients were alive and disease free. The estimated 5-year overall survival was 38%, owing in part to a 3-year treatment-related mortality of 38%. As anticipated with unrelated donor transplantation, rates of graft-versus-host disease were high, with grades III–IV acute GVHD of 29% and rates of limited and extensive stage chronic GVHD of 85%.

Chapter 4  Hematopoietic Progenitor Cell Transplantation for Treatment of CLL 

Myeloablative regimens with allogeneic transplantation have therefore been shown to lead to long-term disease-free survival, with a plateau in survival not observed with autologous transplantation. Thus, with this therapy there is true hope that CLL may be controlled long term via the graft-versus-leukemia effect. However, the prohibitive early treatment-related mortality related to toxicity from preparative regimens in addition to the higher rates of GVHD seen with myeloablative conditioning have lead to investigations of nonmyeloablative preparative regimens for use in this hematologic malignancy that usually affects older patients who are often heavily pretreated and have comorbid conditions.

5.  Allogeneic Transplantation Using Non-myeloablative Conditioning The demonstration of the graft-versus-leukemia (GvL) effect in CLL as well as the toxicity of myeloablative conditioning has prompted recent studies in the use of non-myeloablative conditioning regimens [29–33]. Recent publications of both prospective and retrospective analyses have shown encouraging results. The first prospective study was published in 2003 by Schetelig et al. who reported the results of NMA conditioning using fludarabine, busulfan, and antithymocyte globulin in 30 patients with CLL [29]. At a median follow-up of 2 years, the overall survival was 72% with a progression-free survival of 67% and treatment-related mortality of 15%. A subset of patients achieved molecular remissions. A larger study by Sorror et al. in 2005 examined 64 patients with CLL undergoing NMA conditioning and found an overall response rate of 67%, with a CR rate of 50%, and 2-year non-relapse mortality of 22%, again with a proportion of patients achieving molecular remissions [30]. Three prospective studies followed. Brown et al. examined 46 high risk patients who underwent NMA conditioning [31]. While 2-year treatment-related mortality was low at 17%, this study found a somewhat higher incidence of relapse and a lower overall survival than the previous studies, with 48% of patients progressing at 2 years and an overall survival of 54% at 2 years. An analysis of risk factors for relapse in this study found that chemotherapy refractory disease at the time of transplant, increasing percentage of bone marrow involvement, and an increased number of prior therapies all contributed to diminished progressionfree and overall survival. Khouri et  al. performed a prospective study of 39 patients using NMA conditioning, including patients with high risk features such as Zap-70 positivity and chemotherapy refractory disease [34]. This study estimated a 4-year overall survival of 48% with 4-year progression-free survival of 44%. Again, chemotherapy refractory disease was found to be a risk factor for relapse, as was mixed T-cell chimerism at day 90; however, other biologic risk factors did not affect relapse rates. Finally, Delgado et al. evaluated outcomes in 41 patients undergoing NMA conditioning and found a 2-year overall survival of 51% with an event-free survival of 45%. The overall survival in this study was diminished by post-transplantation viral and invasive fungal infections [33]. While no studies have been performed that randomize patients to standard myeloablative versus NMA conditioning regimens, Dreger et al. performed a retrospective EBMT analysis of 73 patients who underwent NMA conditioning

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compared with 82 patients from the EBMT database who received MA conditioning during the same time period [35]. This study found a significant reduction in treatment-related mortality in the NMA group but a higher incidence of relapse (hazard ratio 2.65). There was no significant difference in overall survival. Therefore, while allogeneic transplantation using NMA conditioning has been shown to result in encouraging rates of complete remissions (including molecular remissions), with decreased toxicity and overall lower treatmentrelated mortality, it is clear that optimal outcomes are observed in patients with chemotherapy-sensitive disease who undergo transplantation in optimal remission. In the case of chemotherapy refractory disease, consideration for standard myeloablative conditioning may lead to improved outcomes in the subgroup of patients with few comorbidities and good performance status, although definitive randomized studies are not available versus newer therapies.

6.  Allogeneic Transplantation and Genomic High Risk CLL In light of the inferior outcomes observed in CLL patients with high risk features treated with standard therapies, one of the more important questions in allogeneic transplantation for CLL has been whether the GvL effect is seen in high risk disease subtypes such as unmutated IgVH status, Zap-70 positivity, or high risk cytogenetic features. Moreno et  al. performed a retrospective study of allogeneic transplants performed at two centers which examined 23 patients who were either IgVH mutated or unmutated, compared with a group of 27 mutated and unmutated patients who underwent autologous stem cell transplantation [36]. Seven of the allogeneic transplant patients received non-myeloablative transplants. This study found that the risk of relapse was significantly higher for all patients following autologous transplantation than for the allogeneic transplant group (61% at 5 years vs. 12% at 5 years, respectively). Furthermore, survival was significantly longer in patients who underwent allogeneic transplantation. The relapse rate was significantly lower in unmutated patients who underwent allogeneic transplantation; however, these patients were not separated by type of conditioning regimen. As described previously, the study by Khouri et al. which specifically examined the effect of Zap-70 positivity and risk of relapse following NMA conditioning found that allogeneic transplantation was able to overcome this high risk feature [34]. Recently, preliminary data were presented by the EBMT regarding outcomes in patients with 17p deletions [37]. This was a retrospective study which identified 44 patients with 17p deletions who had undergone reduced intensity conditioning allogeneic transplantation. This study found that 4-year overall survival was 48%, with progression-free survival of 37%. Relapse related mortality was 36%, with non-relapse mortality of 27%. Sorror et  al. examined the long-term outcomes of patients with CLL who underwent allogeneic transplantation using reduced intensity conditioning regimens, giving specific attention to patients with adverse cytogenetics [38]. This study found that at a median follow-up of 5 years, the 65 patients who were transplanted had an overall response rate of 70%, with 55% complete remissions. The 5-year overall survival was 50%, with 5-year progression-free survival of 39%. Importantly, adverse cytogenetic risk factors were not found to increase the risk of relapse post-transplantation.

Chapter 4  Hematopoietic Progenitor Cell Transplantation for Treatment of CLL 

7.  Indications for Allogeneic Transplantation in CLL The optimal timing of allogeneic transplantation in CLL and recommendation for application of this therapy remain controversial. In the United States, the only published evidence-based consensus guidelines currently are the National Comprehensive Cancer Network (NCCN) Physician Guidelines, which recommend strong consideration for allogeneic transplantation for patients with del (17p13.1) who achieve a complete or partial response with induction chemotherapy, or in patients who have progressed following initial standard therapy (http://www.nccn.org/professionals/physician_gls/PDF/nhl.pdf). Most transplant centers use center-specific transplant criteria and regimens. Additional formal recommendations regarding transplantation were recently published by the European Bone Marrow Transplant Group (EBMT), who performed a literature review of available data [39]. Their recommendation for European practitioners suggested that allogeneic stem cell transplantation should be considered in patients who fail to respond or relapse early (within 12 months) following a purine nucleoside analog containing regimen, who relapse within 24 months of receiving a purine nucleoside analog containing regimen, or who have (del 17p13.1) abnormality requiring treatment. A similar analysis was published by the Italian Society of Hematology, who performed a systematic review of the literature for publication of evidence-based guidelines [40]. This group recommended that patients with CLL be considered for allogeneic transplantation if unfavorable biological risk factors are present, or for patients who demonstrate non-response or early relapse following purine analog-containing regimens. Performance of this therapy in these CLL patient populations is becoming more accepted as one standard option given evidence that this produces stable remissions with an extended plateau concurrent with acceptable long-term toxicity.

8.  Special Issues in Transplantation for CLL Chronic immunosuppression: Patients with CLL may have complications from their underlying disease during the peri-transplant period which are not shared by other hematologic malignancies. In particular, a high percentage of CLL patients will at some point in their disease course suffer from an autoimmune phenomenon such as autoimmune hemolytic anemia, immune thrombocytopenia, or autoimmune rheumatologic disease. In addition, the majority of patients will enter the transplantation process following periods of long-term immunosuppression due to both the underlying disease and the prior administration of highly immunosuppressive therapy used to treat the disease (purine nucleoside analogs, alemtuzumab). The risk of infection is exacerbated by the use of in  vivo T-cell depletion strategies for prevention of graftversus-host disease and by post-transplant immunosuppression. Thus, the transplanted CLL patient may be at risk for developing infections related to chronic neutropenia, impaired cellular immunity, and chronic hypogammaglobulinemia. Patients with persistent disease at the time of transplantation will have the additional risk of CLL-related impaired cellular immunity. The judicious use of prophylactic antimicrobial therapy, regular screening for CMV and EBV reactivation (if applicable), prophylaxis against invasive fungal infections if steroids are required, and monitoring immunoglobulin levels

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and supplementing hypogammaglobulinemia if necessary all may help to improve outcomes peri-transplantation. Donor selection: Donor selection in transplantation for CLL typically follows the standard genetic randomization of most transplants, in that if patients have an HLA-identical sibling they will receive a matched related donor transplant product. However, in light of the high incidence of familial CLL (up to 10% of all CLL patients will have a first-degree relative with CLL), sibling donors should undergo screening for CLL as part of the transplant evaluation. Peripheral blood flow cytometry will in most cases detect a monoclonal population of B-lymphocytes with a typical immunophenotype of CLL, eliminating that sibling as a potential donor. In light of recent improvements in managing graft-versus-host disease and the diminishing differences in adverse outcomes between sibling and unrelated donors, in this case an unrelated donor would be preferable and in fact might improve chances for long-term disease-free survival [38]. For those patients without a related or unrelated donor product, umbilical cord blood transplantation may be considered. However, at the present time there is little published data regarding the outcomes using umbilical cord blood products for transplantation in CLL, and this procedure would best be performed as part of a clinical trial. Choice of conditioning regimen: The majority of CLL patients transplanted at the present time receive NMA conditioning as a result of unacceptable treatment-related mortality using myeloablative conditioning. However, this choice may lead to inferior outcomes in patients with chemotherapy refractory disease, who have developed Richter’s (large cell) transformation, or who have bulky lymph node disease or extensive bone marrow infiltration. A retrospective study by MD Anderson found that the only patients with Richter’s transformation who achieved long-term disease-free survival were patients with chemosensitive disease who underwent myeloablative allogeneic transplantation [41]. More aggressive approaches may be required in these instances.

References 1. Xie Y, Davies SM, Xiang Y, Robison LL, Ross JA (2003) Trends in leukemia incidence and survival in the United States (1973–1998). Cancer 97:2229–2235 2. Byrd JC, Rai K, Peterson BL et al (2005) Addition of rituximab to fludarabine may prolong progression-free survival and overall survival in patients with previously untreated chronic lymphocytic leukemia: an updated retrospective comparative analysis of CALGB 9712 and CALGB 9011. Blood 105:49–53 3. Wierda WG, Kipps TJ, Keating MJ (2005) Novel immune-based treatment strategies for chronic lymphocytic leukemia. J Clin Oncol 23:6325–6332 4. Lamanna N, Kalaycio M, Maslak P et al (2006) Pentostatin, cyclophosphamide, and rituximab is an active, well-tolerated regimen for patients with previously treated chronic lymphocytic leukemia. J Clin Oncol 24:1575–1581 5. Keating MJ, O’Brien S, Kontoyiannis D et  al (2002) Results of first salvage therapy for patients refractory to a fludarabine regimen in chronic lymphocytic leukemia. Leuk Lymphoma 43:1755–1762 6. Tam CS, O’Brien S, Lerner S et  al (2007) The natural history of fludarabinerefractory chronic lymphocytic leukemia patients who fail alemtuzumab or have bulky lymphadenopathy. Leuk Lymphoma 48:1931–1939 7. Damle RN, Wasil T, Fais F et  al (1999) Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 94:1840–1847

Chapter 4  Hematopoietic Progenitor Cell Transplantation for Treatment of CLL  8. Montillo M, Hamblin T, Hallek M, Montserrat E, Morra E (2005) Chronic lymphocytic leukemia: novel prognostic factors and their relevance for riskadapted therapeutic strategies. Haematologica 90:391–399 9. Maloum K, Davi F, Merle-Beral H et  al (2000) Expression of unmutated VH genes is a detrimental prognostic factor in chronic lymphocytic leukemia. Blood 96:377–379 10. Wiestner A, Rosenwald A, Barry TS et  al (2003) ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood 101:4944–4951 11. Del Poeta G, Maurillo L, Venditti A et  al (2001) Clinical significance of CD38 expression in chronic lymphocytic leukemia. Blood 98:2633–2639 12. Dohner H, Stilgenbauer S, Benner A et al (2000) Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 343:1910–1916 13. Grever MR, Lucas DM, Dewald GW et  al (2007) Comprehensive assessment of genetic and molecular features predicting outcome in patients with chronic lymphocytic leukemia: results from the US Intergroup Phase III Trial E2997. J Clin Oncol 25:799–804 14. Rabinowe SN, Soiffer RJ, Gribben JG et  al (1993) Autologous and allogeneic bone marrow transplantation for poor prognosis patients with B-cell chronic lymphocytic leukemia. Blood 82:1366–1376 15. Provan D, Bartlett-Pandite L, Zwicky C et  al (1996) Eradication of polymerase chain reaction-detectable chronic lymphocytic leukemia cells is associated with improved outcome after bone marrow transplantation. Blood 88:2228–2235 16. Gribben JG, Zahrieh D, Stephans K et  al (2005) Autologous and allogeneic stem cell transplantations for poor-risk chronic lymphocytic leukemia. Blood 106:4389–4396 17. Dreger P, Stilgenbauer S, Benner A et al (2004) The prognostic impact of autologous stem cell transplantation in patients with chronic lymphocytic leukemia: a riskmatched analysis based on the VH gene mutational status. Blood 103:2850–2858 18. Michallet M, Chevret S, Brand R. Comparative study between autologous or allogeneic transplantations and conventional chemotherapy in chronic lymphocytic leukemia: A European Blood and Marrow Transplantation (EBMT) and French CLL Cooperative Group Study [abstract]. Bone Marrow Transplant. 2003 19. Laszlo D, Galieni P, Raspadori D et  al (2000) Fludarabine containing-regimens may adversely affect peripheral blood stem cell collection in low-grade non Hodgkin lymphoma patients. Leuk Lymphoma 37:157–161 20. Wierda W, O’Brien S, Faderl S et al (2006) A retrospective comparison of three sequential groups of patients with Recurrent/Refractory chronic lymphocytic leukemia treated with fludarabine-based regimens. Cancer 106:337–345 21. Gribben JG (2007) Stem-cell transplantation in chronic lymphocytic leukaemia. Best Pract Res Clin Haematol 20:513–527 22. Bandini G, Michallet M, Rosti G, Tura S (1991) Bone marrow transplantation for chronic lymphocytic leukemia. Bone Marrow Transplant 7:251–253 23. Michallet M, Archimbaud E, Bandini G et al (1996) HLA-identical sibling bone marrow transplantation in younger patients with chronic lymphocytic leukemia. European Group for Blood and Marrow Transplantation and the International Bone Marrow Transplant Registry. Ann Intern Med 124:311–315 24. Michallet M, Michallet AS, Le Q (2003) Conventional HLA-identical sibling bone marrow transplantation is able to cure chronic lymphocytic leukemia. A study from the EBMT and IBMT Registries [abstract]. Blood 102:474a 25. Pavletic ZS, Arrowsmith ER, Bierman PJ et  al (2000) Outcome of allogeneic stem cell transplantation for B cell chronic lymphocytic leukemia. Bone Marrow Transplant 25:717–722 26. Horowitz M, Montserrat E, Sobocinski K (2000) Haematopoietic stem cell transplantation for chronic lymphocytic leukemia [abstract]. Blood 96:522a

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L.A. Andritsos et al. 27. Pavletic SZ, Khouri IF, Haagenson M et  al (2005) Unrelated donor marrow transplantation for B-cell chronic lymphocytic leukemia after using myeloablative conditioning: results from the Center for International Blood and Marrow Transplant research. J Clin Oncol 23:5788–5794 28. Khouri IF, Keating MJ, Saliba RM, Champlin RE (2002) Long-term followup of patients with CLL treated with allogeneic hematopoietic transplantation. Cytotherapy 4:217–221 29. Schetelig J, Thiede C, Bornhauser M et al (2003) Evidence of a graft-versus-leukemia effect in chronic lymphocytic leukemia after reduced-intensity conditioning and allogeneic stem-cell transplantation: the Cooperative German Transplant Study Group. J Clin Oncol 21:2747–2753 30. Sorror ML, Maris MB, Sandmaier BM et al (2005) Hematopoietic cell transplantation after nonmyeloablative conditioning for advanced chronic lymphocytic leukemia. J Clin Oncol 23:3819–3829 31. Brown JR, Kim HT, Li S et  al (2006) Predictors of improved progression-free survival after nonmyeloablative allogeneic stem cell transplantation for advanced chronic lymphocytic leukemia. Biol Blood Marrow Transplant 12:1056–1064 32. Khouri IF (2006) Reduced-intensity regimens in allogeneic stem-cell transplantation for non-hodgkin lymphoma and chronic lymphocytic leukemia. Hematology Am Soc Hematol Educ Program 390–397 33. Delgado J, Thomson K, Russell N et  al (2006) Results of alemtuzumab-based reduced-intensity allogeneic transplantation for chronic lymphocytic leukemia: a British Society of Blood and Marrow Transplantation Study. Blood 107:1724–1730 34. Khouri IF, Saliba RM, Admirand J et al (2007) Graft-versus-leukaemia effect after non-myeloablative haematopoietic transplantation can overcome the unfavourable expression of ZAP-70 in refractory chronic lymphocytic leukaemia. Br J Haematol 137:355–363 35. Dreger P, Brand R, Milligan D et al (2005) Reduced-intensity conditioning lowers treatment-related mortality of allogeneic stem cell transplantation for chronic lymphocytic leukemia: a population-matched analysis. Leukemia 19:1029–1033 36. Moreno C, Villamor N, Colomer D et al (2005) Allogeneic stem-cell transplantation may overcome the adverse prognosis of unmutated VH gene in patients with chronic lymphocytic leukemia. J Clin Oncol 23:3433–3438 37. Schetelig J, van Biezen A, Caballero D et  al (2007) Allogeneic Hematopoietic cell transplantation for chronic lymphocytic leukemia (CLL) with 17p deletion: a retrospective EBMT analysis. ASH Annual Meeting Abstracts 110:47 38. Sorror ML, Storer B, Sandmaier BM et al (2007) Long-term follow up of patients (pts) with high-risk chronic lymphocytic leukemia (CLL) given nonmyeloablative allogeneic hematopoietic cell transplantation (HCT). ASH Annual Meeting Abstracts 39. Dreger P, Corradini P, Kimby E et al (2007) Indications for allogeneic stem cell transplantation in chronic lymphocytic leukemia: the EBMT transplant consensus. Leukemia 21:12–17 40. Brugiatelli M, Bandini G, Barosi G et  al (2006) Management of chronic lymphocytic leukemia: practice guidelines from the Italian Society of Hematology, the Italian Society of Experimental Hematology and the Italian Group for Bone Marrow Transplantation. Haematologica 91:1662–1673 41. Tsimberidou AM, O’Brien S, Khouri I et  al (2006) Clinical outcomes and prognostic factors in patients with Richter’s syndrome treated with chemotherapy or chemoimmunotherapy with or without stem-cell transplantation. J Clin Oncol 24:2343–2351

Chapter 5 The Role of Allogeneic Hematopoietic Stem Cell Transplantation for Chronic Myelogenous Leukemia Patients in the Era of Tyrosine Kinase Inhibitors Richard T. Maziarz

When the history of hematopoietic stem cell transplantation (HSCT) for chronic myeloid leukemia (CML) is reviewed, it is observed that the improved outcomes were a consequence of slow and incremental change over the years, as a result of prospective clinical trials or retrospective institutional or registry observations and analyses. This gradual progress of therapeutic advancement was an excellent demonstration of Darwinian evolution. Gould et al. have developed a novel evolution theory of “punctuated equilibrium,” in which periods of incremental change can be disrupted by sudden and sometimes cataclysmic events [1]. In the management of CML, the relatively sudden introduction of tyrosine kinase inhibitors (TKIs) into the disease management has led to saltatory evolutionary changes in which major paradigm shifts have taken place in the treatment of CML patients. In this chapter, we will review the results of HSCT for CML and also review the impact on standard transplantation with the emergence of imatinib and other kinase inhibitors, and how they have now redefined a new natural history for this leukemia. More importantly, with the near universal exposure of patients with CML to TKIs, it will be most critical to define when transplantation options should be pursued and offered to maintain the therapeutic goal of the best long-term outcome for patients with this disease.

1.  CML Pathogenesis, Clinical Features and Response Criteria CML is a clonal hematopoietic cell disorder with an annual incidence of 1–2 cases per 100,000 per year with a median onset in the fifth or sixth decade of life [2, 3]. It has three distinct clinical phases: a stable or chronic phase, followed by advanced phase disease, characterized as either accelerated phase or blast crisis. The chronic phase is characterized by a massive expansion of myeloid cells, usually with normal maturation features. In the more advanced From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_5, © Springer Science + Business Media, LLC 2003, 2010

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CML monitoring options CML Bone Marrow

Fluorescence in situ hybridization (FISH) for BCR-ABL Fusion

Metaphase Chromosome (Karyotype)

Bcr-Abl Quantitative Real-Time RT-PCR (LightCycler) Unknowns BCR-ABL

Ph(+)

G6PDH

PCR cycle PCR cycle Standard Curves (Known Copies), Quantification

normal

Fig.  5-1.  Mechanisms by which the disease burden of CML can be assessed: (a) bone marrow analysis, (b) karyotypic analysis, (c) FISH, (d) molecular analysis by reverse transcriptase-polymerase chain reaction.

phases, the leukemic cells lose their capacity to differentiate and can result in acute leukemia, i.e., “blast crisis” which is often highly refractory to standard chemotherapy. There is a hallmark cytogenetic abnormality known as the Philadelphia (PH) chromosome (Fig.  5-1). This cytogenetic abnormality is a shortened chromosome 22 seen by karyotypic analysis, which results from the reciprocal translocation between the long arms of chromosome 9 and 22. As a molecular consequence, there is a fusion of the C-Abl oncogene from chromosome 9 with chromosome 22 sequences within the breakpoint cluster region (BCR) resulting in the fused BCR-ABL gene. The gene product is the BCRABL tyrosine kinase, a constituitively active kinase that is considered to be the etiologic basis for CML. The fusion product leads to deregulated cellular proliferation, inhibition of apoptosis, genetic instability, and perturbation of the interaction of CML cells and the bone marrow stroma. CML is a stem cell disorder and, therefore, no standard chemotherapy agents or combination of agents were ever capable of eradicating this disease. Rather, for years, non-transplantation therapy for CML was focused on suppressing the proliferative phase with the hope of delaying the otherwise inevitable transformation to the accelerated and blast phases. Thus, non-transplantation management with chemotherapy, alpha interferon, or any other agent was focused at reduction of the burden of disease and this was best defined by reduction of the Ph+ marrow cells. Bone marrow cytogenetics were used to measure the response and classically, as defined by the NCCN, complete response was defined as the absence of Ph+ cells (0% Ph+), partial response was a reduction to < 36% (1–35%), and minor response was a

Chapter 5  The Role of Allogeneic Hematopoietic Stem Cell Transplantation 

level of Ph+ cells between 36 and 95%. Persistence of >95% Ph+ cells was classified as no response [4]. Currently, molecular assessments with the quantitative polymerase chain reaction (qPCR) have become the standard monitoring tool for CML patients to determine levels of BCR-ABL transcripts with sensitivities of the assay several logs lower than assessable with cytogenetics. Molecular response measures include complete molecular response where BCR-ABL mRNA is undetectable by RT-PCR and major molecular response where there is a ³ 3 log reduction of BCR-ABL mRNA from baseline [4]. Ongoing monitoring is important to identify those patients who achieve the new goal of molecular remission as well as to identify those who develop early resistance [5].

2.  TKI and Other Targeted Therapy for CML There is no question that TKIs have emerged as the primary modality for treatment of CML patients [6–12]. The early phase I/II trials of imatinib mesylate (IM) demonstrated low toxicity despite the maximum tolerated dose never being reached with nearly 100% hematologic responses and over 50% cytogenetic remissions in patients with prior interferon resistance or intolerance. Advanced disease CML patients also demonstrated response although at lower rates compared to stable phase patients. In phase II trials of accelerated phase (CML-AP; n = 181), sustained hematologic responses and major cytogenetic responses to IM were found to be 69 and 24% respectively, with 17% complete cytogenetic responses identified [13]. In myeloid blast crisis CML (CML-BC; n = 229), IM responses were lower but still sustained hematologic responses in 31%, with major cytogenetic responses in 16%, of which 7% were complete [14]. However, only a small number of patients were available for long-term follow-up and only 18% of CML-AP and 3% of CML-BC patients maintained single agent IM on study. A clear dose--response effect emerged from these studies, with 600 mg showing superior response and survival advantage as opposed to 400 mg, the dose used in stable phase studies. However, IM at either dose was effective with low toxicity and served as a bridge to transplant for some and provided long-term remission to a small cohort of patients, particularly those in CML-AP. Overall survival estimates at 36 months for patients treated with 600 mg were 55% for CML-AP and 14% for CML-BC, and progression-free survival estimates were 40 and 7%, respectively [15]. These phase I and phase II studies rapidly led to phase III randomized, landmark study – International Randomized Study of Interferon vs. STI571 (IRIS) trial – comparing the interferon-based therapy with IM in the newly diagnosed chronic phase CML which proved that IM was the superior drug [9]. Recently, a 5-year update of the IRIS trial [8, 10] clearly demonstrated the ongoing superior survival in CML patients when they were treated with first-line IM, with an estimated 5-year survival in excess of 91% (even higher when scrutinized for CML-related deaths). What was quite unexpected was that the treatment response continued to improve with ongoing therapy. The likelihood of progression was highest in the first 2 years on TKI therapy, with the observed rate of progression per year lowest in years 4 and 5 (e.g., 7.5% rate of progression/year in year 2 vs. 1.5% in year 4) [10]. Additionally, for patients with a major molecular response at 12 months (currently, defined as > 3 log reduction in BCR-ABL from a standardized baseline), progression-free survival is 97% after 4.5 years, with no patients progressing to accelerated

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phase or blast crisis. Loss of response to IM could best predict the risk for relapse, and notably a good response to IM could override the stigma of having “higher risk” disease at the time of presentation, in contrast to patients treated with interferon [10, 16, 17]. The identification of patients with IM resistance, the elucidation of the resistance mechanisms, and the development of new targeted therapeutic agents have become the focus of intense CML investigation over the past half decade [11, 18–20]. Resistance is defined as primary (inability to achieve initial landmark responses) or secondary (those patients who achieved initial responses but subsequently lost hematologic or cytogenetic responses). In the latter condition, a large proportion of cases are demonstrated to have developed BCR-ABL kinase domain mutations, with clustering in the ATP binding loop (p-loop), the activation loop (a-loop), and at amino acid sites 315 and 351 of the molecule [11, 20]. The identification of these BCR-ABL mutations which confer resistance to IM have led to the development of a series of second-generation TKIs, including the recently FDA approved dasatinib and nilotinib [11, 21, 26]. Similar to IM, these agents have also rapidly demonstrated safety and efficacy in phase I and II trials, performed primarily in patients with imatinib resistance and intolerance with high percentages of major cytogenetic responses in patients with IM failure (although it is important to note that T315I mutations confer resistance to both of these agents) [27, 28]. Even major molecular responses (3-log or greater reduction in BCR-ABL transcripts) have been achieved in this IM-resistant CML patient population, although molecular remissions are more infrequent than seen in patients with primary disease. Research investigations continue to identify new small molecules with other molecular targets in CML patients that may ultimately be either substituted, for example the new aurora kinase inhibitors which appeared to target the mutated T315I BCR-ABL site, or potentially used in combination with the current group of TKI inhibitors [27]. However, in the background of these current investigations on BCR-ABL resistant CML, the potential application of allogeneic HSCT procedures remains relevant and critical to consider when one wishes to achieve the best long-term outcome for patients in this TKI era. To best appreciate the role of allogeneic HSCT for CML patients, it is appropriate to review its use in the past in the absence of available TKIs and as well as in the present, in this modern era of small molecule, targeted therapy.

3.  HSCT in the Pre-TKI Era HSCT had been the primary preferred target treatment for age-appropriate CML patients, based first and foremost on it being a highly curative modality. In contrast, only a minority of patients achieved long lasting responses with interferon-alpha, the main therapeutic agent in the era prior to the emergence of IM [29]. Some patients were rendered BCR-ABL negative, but few were able to maintain this status for years, and even in the best interferon responders, residual leukemic cells could be identified by RT-PCR. In contrast, using the immunotherapeutic maneuver of allografting, a great majority of patients gained and sustained molecular remission. Transplantation in the realm of conventional marrow ablative conditioning regimens was considered appropriate for young patients as well as for those who lacked medical co-morbidities. No clearly defined, absolute standard

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eligibility goals were ever demonstrated in clinical trials but many centers accepted human leukocyte antigen (HLA)-matched sibling donor HSCT for patients up to age 55–60 years with a slightly lower age being considered generally acceptable for unrelated HSCT. However, there was general recognition that these targets were influenced by additional factors such as co-morbid conditions, donor availability, disease stage (Fig.  5-2), sex and age shared between the donor and recipient, components of the “EBMT transplantation risk score” [29–34] (Table  5-1). Additionally, the traditional goal was to transplant patients within the first year of their disease, because single institutional

Fig.  5-2.  Probability of survival after unrelated HSCT for CML as determined by disease phase, 1998–2006. (Reproduced with permission of the CIBMTR and NMDP)

Table 5-1.  Overall survival according to EBMT transplantation risk score. Five-year overall survival (%) CIBMTR series Total risk score

EBMT series

All patients

ECP patients

0–1

72

69

70

2

62

63

67

3

48

44

50

4

40

26

29

5–7

22

11

25

All EBMT and CIBMTR patients were treated by conventional allo-HSCT procedures between 1989 and 1997. Leukemia-free survival (calculated only in the EBMT study) at 5 years was 61% for risk scores 0–1, 47% for risk score 2, 37% for risk score 3, 35% for risk score 4, and 19% for risk scores 5–7 This research was originally published in Blood. Ref. [90] © American Society of Hematology

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Fig.  5-3.  Probability of survival after related and unrelated HSCT for patients with CML in the first chronic phase, 1998–2004. (Reproduced with permission of the CIBMTR and NMDP)

studies as well as retrospective registry analyses provided evidence that early transplantation was associated with better outcomes [35] (Fig. 5-3). However, treatment related morbidity and mortality (TRM) associated with regimen related toxicity or graft versus host disease (GVHD) remained daunting and limiting for many patients. Additionally, it remained recognized that of all CML patients, only approximately 30% ever pursued sibling or unrelated HSCT; the other 70% were either too old or too infirm (45%), or had no suitable donors (25%) [36]. Regarding the history of CML transplantation, the first CML patient who underwent HSCT was one in the blast phase who underwent a single antigen mismatch, sibling donor HSCT. The patient engrafted, developed GVHD but unfortunately succumbed to CMV disease without evidence of leukemia [37]. This landmark event was the cornerstone that led to HSCT being the standard therapy for CML patients since the early 1980s, when the observations that the allograft procedure was associated with a graft versus leukemia (GVL) effect were made [38]. This was unfortunately and dramatically confirmed later in the 1980s when it was observed that CML patients experienced the highest relapse rates after T-cell depletion, confirming the critical importance of immune therapy in the management of this disease [39]. In 1991, Kolb et al. [40] confirmed the exquisite immune sensitivity of CML when they demonstrated that re-infusion of unstimulated donor T-cells in the absence of immune suppression was associated with both clinical responses and remissions in relapsed CML patients. Simultaneously, the Center for International Blood and Marrow Transplant Research (CIBMTR) performed a registry analysis that demonstrated that T-cell depleted HSCT actually led to lower PFS as syngeneic HSCT, as opposed to allogeneic HSCT, consistent with these other observations [39] (Table 5-2). Refinements in donor leukocyte infusions (DLI), either as sole therapy or in combination with TKI treatment, have continued to be made since this time [41–43]. In the 1990s, the

Chapter 5  The Role of Allogeneic Hematopoietic Stem Cell Transplantation 

59

Table 5-2.  Relative risk of relapse for early leukemia after HSCT. ALL first CR Study group

N

AML first CR

CML CP

All patients

RR

P

N

RR

P

N

RR

P

N

RR

P

Allogeneic, non-T-depleted No GVHD59a

  90

1.00

–

228

1.00

–

115

1.00

–

433

1.00

–

Acute GVHD only

141

0.36

0.004

330

0.78

0.26

267

1.15

0.75

738

0.68

0.03

Chronic GVHD only

  28

0.44

0.16

  54

0.48

0.12

  45

0.28

0.16

127

0.43

0.01

Acute and chronic GVHD

  84

0.38

0.02

237

0.34

0.0003

164

0.24

0.03

485

0.33

0.0001

  12

0.99

0.99

  34

2.58

0.008

  24

2.95

0.08

  70

2.09

0.005

Syngeneic

Allogeneic, T-depleted   All patients

  84

1.20

0.61

163

1.30

0.33

154

5.14

0.0001

401

1.76

0.002

   No GVHD

  43

1.48

0.33

  83

1.57

0.12

  74

6.91

0.0001

200

2.14

0.0001

  Acute and/ or chronic GVHD

  41

0.98

0.97

  80

0.80

0.60

  80

4.45

0.003

201

1.32

0.25

a Reference group Relative risks are derived from multivariate Cox regression adjusting for Leukocyte count at diagnosis, recipient age, organ impairment pre-transplant, donor-recipient sex-match, and drug used to prevent GVHD RR relative risk in comparison with reference group, CR complete remission, CP chronic phase This research was originally published in Blood. Ref. [39] © American Society of Hematology

busulfan/cyclophosphamide preparative regimen became the standard regimen for conventional myeloablative transplantation for CML, when relapse rates were shown to be similar whereas regimen-related morbidity and mortality were lower when this regimen was compared with cyclophosphamide and total body irradiation (TBI) [44, 45]. This observation was not subsequently confirmed, when a study from France comparing these regimens did not demonstrate a negative impact of radiation exposure [45]. However, outcomes could be improved by pharmacokinetic monitoring of busulfan delivery, which allowed determination of steady-state concentrations that could be used to guide individualized dosing for an individual patient [46]. Further refinements with pharmacokinetic targeting were achieved with the use of intravenous busulfan, owing to its more dependable delivery, with which some investigators have reported improved long-term outcomes with the least treatment-related mortality (TRM) [47]. These latter advances have cemented the role of this preparative regimen for the disease. In the latter half of the 1990s, molecular matching for unrelated HSCT donors became standard. As molecular matching improved, the outcomes of patients undergoing unrelated HSCT also improved in parallel, such that the outcomes mimicked those in patients who received sibling allografts [48]. Most recently, comparative analyses of therapeutic efficacy of blood vs. marrow HSCT have been performed, demonstrating no clear advantage of either product in terms of overall survival, disease-free survival, non-relapse mortality, or acute and chronic GVHD [49]. Given the increasing recognition of the prolonged necessity for immunosuppression for chronic GVHD patients [50, 51], many centers are reverting to bone marrow harvests for CML patients or exploring other stem cell sources, for example, cord blood HSCT for CML patients [52].

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4.  HSCT in the Peri-TKI Era: The Emergence of Reduced Intensity Conditioning Regimens HSCT has continued to evolve over the past decade. Leading the advance has been reduced intensity and non-myeloablative HSCT (for review [53]). These approaches were developed to decrease the risk for transplant-related morbidity/ mortality in procedures conducted in older patients or those with co-morbid clinical conditions and, for younger patients desiring to preserve fertility. Excellent outcomes were reported in the earliest single institutional transplant studies in patients with CML [54–59]. Acceptable outcome results have been observed, albeit with relatively short follow-up. The Hadassah University team reported that progression free overall survival of 85% was achieved with a median follow-up of 37 months utilizing Fludarabine (150  mg/m2), Busulfan (8 mg/kg), and rabbit ATG (40 mg) [57]. GVHD remained an issue since 75% of the patients experienced clinical grade II–IV acute GVHD, and 55% experienced chronic GVHD. Similar outcomes were noted within the Fred Hutchinson Cancer Research Center (FHCRC) consortium using single dose 200 cGy TBI +/− Fludarabine 90 mg/m2 reduced intensity regimen. In this study [59], 24 CML patients were treated and with 36 month median follow-up, 13 were found to be alive in complete remission. TRM was 21%, and acute GVHD, grades II–IV were found in 50% of the patients. Additional studies with small number of patients, shorter follow-up and using varying intensity preparative regimens demonstrated projected 5-year survival ranging between 50 and 70%. One long-term retrospective analysis has also been reported by the investigators from the M.D. Anderson Cancer Center using their fludarabine-based reduced intensity HSCT procedures [60]. In 64 patients with advanced phase CML (80% beyond CP1), with a median follow-up of 7 years, the 5-year overall survival and progression-free survival were reported at 33 and 20%, respectively with TRM reported at 33, 39, and 48% at 100 days, 2 years, and 5 years, respectively. These data confirm that the reduced intensity HSCT can provide prolonged disease control, even in advanced phase CML, but more optimal approaches are needed for the management of patients not suitable for conventional HSCT, to achieve better long-term survivals [61]. Many similar studies have since been published or presented, but the largest retrospective analysis is the study reported by Crawley et al. [62] conducted in patients reported to the EBMT (Fig. 5-4). The assessment included a total of 211 CML patients, who were undergoing reduced intensity HSCT. The CML patient population was markedly heterogeneous with varying presenting disease states, multiple preparative regimens, and varying graft sources. Survival was found to be markedly influenced by the disease state. Overall survival for patients in the first chronic phase was 69%, in the second chronic phase was 57%, in accelerated phase was 24%, and in blast crisis was only 8%, mirroring experience with conventional allografts or other long-term single center analyses [30, 35, 61]. TRM was 9% at 1 year. Grades II–IV acute GVHD was seen in 33% of patients but only 10% experienced grade III–IV. Rejection was found at rates of 11% for this population of patients. In this retrospective analysis, the busulfan/fludaribine/antithymocyte globulin preparative regimen was associated with the best outcomes, with reduction in the TRM and improvement noted in disease-free survival [62].

Chapter 5  The Role of Allogeneic Hematopoietic Stem Cell Transplantation 

Fig.  5-4.  Probability of overall and progression-free survival of patients with CML in all disease phases treated, undergoing sibling and unrelated donor reduced intensity HSCT. (This research was originally published in Blood. Ref. [62]. © The American Society of Hematology)

Based on these data, a proposal to the Blood and Marrow Transplant Clinical Trials Network for a randomized phase III trial of conventional myeloablative transplantation vs. reduced intensity transplantation in CML was submitted for consideration in patients aged 40–65 years (Maziarz and Druker, pers. comm.). The trial was not recommended for activation, however, because with the clear decline in allogeneic HSCT for CML in North America, it was believed that accrual targets would not be met in any reasonable time frame. In addition, despite excellent preliminary outcome data, there was a recognition that a longer follow up would be required before reduced intensity could truly emerge as a standard of care. Therefore, no randomized HSCT trial in CML is predicted to be forthcoming anytime soon; rather, we anticipate retrospective registry studies matching cohorts of CML patients receiving either reduced intensity or conventional HSCT will be performed [63]. These observations of a reduction in the HSCT procedures for CML were highlighted by the analysis of Giralt et  al. [64], who conducted a retrospective study of the CIBMTR database. Surprisingly, they demonstrated that the transplantation for CML was already declining before the US Food and Drug Administration approval of IM in 2001 (Fig. 5.5). At one time, CML was the number one malignancy for which unrelated transplants were performed; by 2005 it was the eighth most common hematologic disorder for which transplantation was offered, as indicated by the CIBMTR data and in the most recent updated CIBMTR statistics, only 2% of all unrelated transplantation procedures performed in 2007 was for CML patients. The analysis of the CIBMTR data pool conducted by Giralt et al. indicated that the greatest reduction in transplants was for CML patients in the first chronic phase, with the percentage change

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over 5 years ranging from 62 to 44% [64]. Additionally, 24% of patients were originally transplanted in the accelerated phase or second chronic phase, and this number had risen to 41% by 2005. HSCT for blast crisis remained relatively stable at 14–15%. Another trend noted was the marked increase in exposure to IM in CML patients undergoing HSCT, from 1% of CML patients registered in 1998 to 77% of patients registered with CIBMTR in 2003 (Fig. 5-6). It should be noted, however, that these changes in CML HSCT demographics may not be universal. The Latin American Cooperative Onco-Hematology Group [65] recently reported their experience in reduced intensity allogeneic HSCT, with over 95% survival in 24 patients at 28 months of follow up. Interestingly, they indicated that this may remain the optimal path for patients in their developing countries, because the median cost of allogeneic HSCT from days 0 through to 100 of transplantation was the same as 200 days of IM

Fig. 5-5.  The decline of HSCT procedures for CML in the peri-TKI era as documented by the CIBMTR. (Reproduced from [64]. © 2007 CIBMTR. © 2007 Blackwell Publishing Ltd)

Fig.  5-6.  Increased exposure to imatinib prior to HSCT. (Reproduced from [64]. © 2007 CIBMTR. © 2007 Blackwell Publishing Ltd)

Chapter 5  The Role of Allogeneic Hematopoietic Stem Cell Transplantation 

prescribed at 400 mg/day. A follow-up analysis, in Mexico, only compared 22 patients who underwent reduced intensity HSCT with a comparable group of 50 patients treated with TKI, primarily IM [66]. Six year overall survival after HSCT vs. TKI for PH + CML was 77 and 84%, respectively (p = NS), with the additional observation that freedom from progression to advance phases was similar in both groups. The authors highlight the fact that the median cost of each reduced intensity HSCT was US $18,000, an amount comparable to one hundred days of treatment of IM at 400 mg per day. This raises the concern that although the initial costs of allogeneic HSCT are higher, the lifetime treatment with expensive pharmaceuticals may actually place the greatest burden on resources. The same observation was also made in Europe, where it was suggested that the costs of reduced intensity HSCT became less expensive than IM within 2 years of the procedure [67]. These policy issues will continue to be highlighted, particularly in young patients in whom the greatest advantage of allogeneic transplantation can be gained as TRM is the lowest vs. potentially decades of continued drug therapy and its projected concomitant costs.

5.  HSCT in the Post-TKI Era: Does Early HSCT Provide Optimal Outcomes? Advances in allogeneic HSCT for CML continue with close scrutiny on where TKI and transplant therapies interact and intersect. Major questions that persist include assessing the impact on pre- and post-transplant TKI therapy, optimal donor source, monitoring, and decision-making regarding the management of relapse with either DLI, imatinib, or both. One of the most highly discussed issues currently is the management of the young patient with CML [68]. Despite the excellent data presented in the IRIS trial, some physicians still suggest that, in the absence of long-term data with IM, young patients with sibling donors should still consider proceeding to allogeneic HSCT. These recommendations have been based on prior retrospective analyses suggesting that HSCT during the first year of diagnosis was associated with better outcomes, and a lack of clear data, suggesting that pre-transplant IM would not enhance treatment-related morbidity and mortality. Hehlmann et al. [69] have now demonstrated that there is no benefit from early allogeneic HSCT with sibling donors in patients with CML. The German CML transplantation group conducted the first ever randomized prospective trial of early HSCT vs. best available therapy. This trial began in the interferon era in 1995, and all new, age-appropriate CML patients were enrolled (including children; youngest was age 11 years, accounting for about 4% of HSCT-eligible patients). If they had an HLA-matched sibling, they proceeded to early transplantation, but if not, then the patient received interferon-based therapy with unrelated HSCT used for salvage. This study clearly demonstrated a survival benefit for patients who were treated with interferon-based therapy over early transplantation. Additionally, the transplant risks were even more pronounced for CML patients with low risk disease on presentation with 4 year survival at 91% for the nontreatment arm vs. 66% for the HSCT arm. Clearly, TRM rates of 29% in the transplant arm influenced these data, but the conclusions were clear; HSCT was best utilized later in the CML disease course rather than within the first year of diagnosis.

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6.  HSCT in the Post-TKI Era: Does Pre-Transplant TKI Exposure Impact HSCT Outcomes? It is important to appreciate that to date there is no clear evidence to suggest that pre-transplant IM or other TKIs will negatively influence the outcome of patients undergoing future transplantation. Most studies reported on this issue included very small patient numbers [70–73]. For example, Zaucha et al. [70] reported 30 patients with PH+ leukemias who experienced no increased hepatic toxicity, graft-versus-host disease, or 100-day mortality when compared with historic control individuals. Deininger et al. presented the EBMT series of 70 patients with advanced PH+ disease [74], in which no impact on engraftment, TRM, graft-versus-host disease, or relapse was observed in patients pre-treated with IM. Most importantly, pre-transplant disease control was most predictive of superior outcomes. Finally, Lee et al. have reported the largest experience to date, mostly of patients who proceeded to HSCT after IM therapy administered mostly for interferon resistance and/or intolerance [75]. An analysis was performed on 409 CML patients transplanted either in the first chronic phase or beyond the first chronic phase, and was compared to 900 CML patients who had not received prior IM. Among patients in the first chronic phase, IM pre-HSCT therapy was associated with improved survival but no statistically significant difference in TRM, relapse, or leukemia-free survival (LFS) was observed. A matched pair analysis of this cohort was performed and supported the observation that there was a higher survival rate among first chronic phase patients. In patients with advanced disease, use of IM before HSCT was not associated with increased TRM, relapse, LFS, or overall survival. Similar observations are now being reported for HSCT after pre-transplant nilotinib and dasatinib exposure [73]. Thus, when all these data are taken together, there appears to be no clinical reason why CML patients should not receive upfront TKI therapy, even if they are HSCT eligible. Theoretically, TKI pre-treatment may decrease the tumor burden, thus allowing maximal time for the adaptation of the donor immune system and improvement of the ultimate goal of obtaining a graft vs. leukemia effect. Advanced phase patients remain at risk for worse outcomes, often due to higher relapse rates but as well, due to ongoing increased risks of transplant-related mortality [61, 74, 75]. For the non-advanced phase patients, it remains to be determined if worse outcomes are obtained by proceeding to HSCT after failure of TKI although at least one study makes the important observation that BCR-ABL mutation status does not confer resistance to the immune therapeutic manuever of an allograft [76, 77].

7.  HSCT in the Post-TKI Era: TKI Therapy in the Post Allograft Setting The use of TKIs after HSCT is also under scrutiny. Recent reports from the Hammersmith Hospital group have highlighted the need for ongoing molecular monitoring of CML patients. In 243 patients who underwent allogeneic HSCT for CML, long-term monitoring using RT-PCR (median follow up 84.3 months) revealed four categories of patients: (1) 129 patients with molecular or cytogenetic relapse, (2) 27 patients with persistently positive, low-level BCR -ABL signals but never more than three consecutive positive results, (3)

Chapter 5  The Role of Allogeneic Hematopoietic Stem Cell Transplantation 

51 patients with fluctuating positive, low-level signal with never more than two consecutive positive results, and (4) only 36 patients were characterized as persistently negative or with the single low-level positive result [78, 79]. Additionally, a CIBMTR analysis of long-term outcome of allogeneic HSCT for CML has been performed; 2234 patients were identified as alive in remission of 5 years post-allogeneic HSCT. It was identified after 5 years that there was a low but constant relapse rate that persisted for over 15 years and that 22% of the deaths occurring after 5 years from HSCT were CML-related deaths [80]. These long-term observations certainly support the efforts of CML transplant teams to further explore the post-allograft prophylaxis or therapeutic interventions for CML relapse with TKIs. In these circumstances, multiple groups have shown that IM for CML relapse after HSCT can successfully be used for treatment [81–85] and as well can be combined with therapeutic DLI (Fig. 5-5) [86, 87]. What is likely to become more widely incorporated will be the use of TKIs as post-HSCT adjuvant therapy for prophylaxis against relapse, while awaiting the establishment of full engraftment and full donor lymphoid chimerism. In this setting, several centers have shown that IM can be used safely from approximately 1 month after HSCT until beyond 1 year, with therapeutic efficacy in control of relapse [88, 89] (Fig. 5-7).

Fig. 5-7.  The use of imatinib may potentiate DLI in the management of CML relapse after allogeneic HSCT. (Reprinted by permission from Macmillian Publishers Ltd: Ref. [86], © 2005)

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8.  Who Should Receive Transplantation for CML in the IM Era? So which patients with CML should be considered for transplantation in 2008? It is expected that, primarily, patients with either advanced disease or with IM resistance will be considered for HSCT at least in those countries where TKI therapy is available (see Table  5-3 for definitions of TKI treatment failure and suboptimal primary response) [4, 11, 12, 18–20, 90]. Clearly, the degree of primary or secondary resistance that is encountered will influence patients as well as their treating physicians regarding how quickly to proceed. What is accepted universally is that the lack of complete hematologic response at 3 months, a lack of a major cytogenetic response at 12 months, or a lack of a complete cytogenetic response by 18 months are findings felt to be consistent with IM failure [4, 90]. Additionally, patients with cytogenetic progression or relapse and certainly those with hematologic relapse or even “sudden blast crisis” [91] while receiving IM, will be candidates for HSCT as well as for consideration for second-generation TKI therapy [4, 11, 12, 19–26, 90]. Longterm data on these agents are needed, however, to determine their ability to provide long-term salvage. Thus, given the unknown duration of response, these are the situations for CML patients when HSCT becomes relevant and should be considered. Not all patients will elect to pursue HSCT, being cognizant of experience with first-line IM and because of reluctance to accept the uncertainty of outcomes with HSCT, but defining the transplant options becomes important Table 5-3.  Operational definition of failure and suboptimal response for previously untreated patients in ECP CML who are treated with 400 mg IM daily. Time

Failure

Suboptimal response Warnings

Diagnosis

NA

NA

3 months after diagnosis

No HR (stable disease or disease progression)

Less than CHR

6 months after diagnosis

Less than CHR, no CgR (Ph+ > 95%)

Less than PCgR (Ph+ > 35%)

NA

12 months after diagnosis

Less than PCgR (Ph+ > 35%)

Less than CCgR

Less than MMolR

18 months after diagnosis

Less than CCgR

Less than MMolR

NA

Anytime

Loss of CHRa, loss of CCgRb, mutationc

ACA in Ph+ cellsd, loss of MMolRd, mutatione

Any rise in transcript level; other chromosome abnormalities in Ph- cells

a

High risk, del9q+, ACAs in Ph+ cells NA

To be confirmed on two occasions unless associated with progression to AP/BC To be confirmed on two occasions, unless associated with CHR loss or progression to AP/BC c High level of insensitivity to IM d To be confirmed on two occasions, unless associated with CHR or CCgR loss e Low level of insensitivity to IM Failure implies that the patient should be moved to other treatments whenever available. Suboptimal response implies that the patient may still have a substantial benefit from continuing IM treatment but that the long-term outcome is not likely to be optimal, so the patient becomes eligible for other treatments. Warnings imply that the patient should be monitored very carefully and may become eligible for other treatments. The same definitions can be used to define the response after IM dose escalation PCgR indicates partial CgR; and NA, not applicable This research was originally published in Blood. Ref. [90] © American Society of Hematology b

Chapter 5  The Role of Allogeneic Hematopoietic Stem Cell Transplantation 

Table 5-4.  An institutional perspective of CML patients and indications for hematopoietic stem cell transplantation: 2008. Phase

Clinical setting

CP1

Primary hematologic failure to IM therapy

CP1

Primary cytogenetic failure to IM therapy

CP1

Progression after primary imatinib therapy

CP1

Imatinib resistant, mutations at BCR-ABL 315 locus

CP1

Imatinib resistant, partial response to second-generation inhibitors

CP1

Intolerance to tyrosine kinase inhibitors

CP1

Clonal evolution, in setting of TKI resistance

CP1

Outgrowth of alternate Philadelphia-negative cytogenetically abnormal clones with myelodysplasia

AP

New diagnosis, after primary imatinib therapy, in patients with low EBMT score

AP

Imatinib responsive but later progression in patients with high EBMT score

BC

After induction with TKI ± combination chemotherapy

AP accelerated phase, BC blast crisis, CP1 first chronic phase, EBMT European Group for blood and marrow transplantation

[90]. In the first chronic phase, at our institution, potential HSCT scenarios are reviewed and considered for patients with primary hematologic or cytogenetic failure to IM therapy, as well in those with progressive disease (see Table 5-4). Described mechanisms of resistance such as tyrosine kinase mutations are more frequent in patients with secondary failure, and thus the concern is that over time such “unstable” clonal disease may evolve further and evade salvage options as well, thus warranting HSCT after re-establishing control in such patients [77]. A small number of patients are intolerant of IM and can be considered for second-generation TKI therapy with transplantation at progression. Clearly, patients who are resistant to IM and exhibiting a partial response only to second-generation TKI treatment must consider HSCT, particularly if they are young and have excellent matched options, either sibling or unrelated donor. Finally, there are some circumstances for which most patients are not monitored that would influence the decision to pursue HSCT. These include the identification of mutations at the 315 site of BCR-ABL, which are universally associated with resistance to the known first-generation and second-generation TKIs [27, 28]. Novel kinase inhibitors are being tested in clinical trials that target this particular mutant BCR-ABL site. Our policy, as in many centers, is to offer HSCT to CML patients with T315I mutations, either directly or after being treated in a clinical trial. Finally, there are relatively uncommon circumstances for consideration for HSCT when early clonal evolution is seen in patients failing IM [92] or when myelodysplasia is found in patients with TKI-induced PH– hematopoiesis [93]. For patients with advanced phase CML, early transplantation for accelerated phase patients is considered, but preferably for those with lower EBMT scores (see Table 5-2 for HSCT outcomes impacted by the EBMT score) [34]. For those patients who are in an accelerated phase with significant co-morbid conditions or with a high EBMT score, transplant options are outlined, but alternatively, one may choose to closely monitor the TKI failure before pursuing

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HSCT in those patients. For patients with blast crisis who are age eligible, HSCT is universally recommended after disease control is achieved, generally by induction chemotherapy in combination with IM or another TKI. The transplant community recognizes that these considerations will change. As new agents are developed, for instance those that may overcome the resistance associated with BCR/ABL T315I mutations, it will create circumstances where management algorithms will also evolve. Certainly, there are questions that remain that have attracted significant attention without clear answers or without clear agreement of the transplant community such as the role of transplantation in the very young patient with CML. We all hope and expect that the role of small molecule targeted therapy may impact and change the natural history of many malignancies. Certainly, there has been a dramatic alteration in the treatment strategies and improvement in clinical outcomes in adults with Ph+ ALL (rev in [94]). Early HSCT still remains the primary goal in this setting in adults, but IM therapy has entered all aspects of management from primary treatment to long-term maintenance, and appears to have changed the natural history with markedly improved PFS and OS reported. But, even in this clinical setting which had been universally considered a non-debatable indication for HSCT in the ageappropriate patient, novel trials are being considered within the United States to compare HSCT in CR1 to primary therapy with second-generation TKIs alone.

9.  Conclusions HSCT is a complex and morbid procedure that requires significant resources and technical skills in those whom perform it. The advent of molecular targeted therapy for CML has led to the dramatic decline in HSCT procedures, but the need remains for HSCT for patients who fail TKI therapies or who present with more aggressive disease. In the interim, ongoing advances in HSCT research may continue to improve outcomes for CML patients either by (a) decreasing transplant-related mortality, perhaps by the development of novel conditioning regimens [95] or developing technologies to identify those at highest risk of organ damage [96] or (b) by determining novel means to reduce relapse rates, for example by optimizing natural killer cell grafts [97] or by using selected tumor-specific, vaccine strategies [98]. Alternatively, autologous HSCT may reemerge in therapeutic algorithms, given the ability to collect large numbers of PH– peripheral blood stem cells [99–101]. Currently, one fact that we do know is that the future of HSCT for CML will change and will be determined by clinical investigations of novel therapeutics with close attention to the economics of cancer care, and it will be interesting to observe in this current age of cellular and regeneration therapy whether HSCT for CML takes the path of the Irish Elk toward extinction [102] or evolves dramatically and adapts to become more functional, similar to the development of the Panda’s thumb [103].

References 1. Gould SJ (2002) The structure of evolutionary theory. Harvard University Press, Cambridge 2. Hehlmann R, Hochhaus A, Baccarani M et al (2007) Chronic myeloid leukaemia. Lancet 370 (9584):342–350 3. Deininger M, Buchdunger E, Druker BJ (2005) The development of imatinib as a therapeutic agent for chronic myeloid leukemia. Blood 105(7):2640–2653

Chapter 5  The Role of Allogeneic Hematopoietic Stem Cell Transplantation  4. National Comprehensive Cancer Network (2008) "NCCN Clinical Practice Guidelines in Oncology," Chronic Myelogenous Leukemia. http://www.nccn.org/ professionals/physician_gls/PDF/cml.pdf Accessed July 14, 2008 5. Sahay T, Schiffer CA (2008) Monitoring minimal residual disease in patients with chronic myeloid leukemia after treatment with tyrosine kinase inhibitors. Curr Opin Hematol 15(2):134–139 6. Druker BJ, Talpaz M, Resta DJ et al (2001) Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Eng J Med 344(14):1031–1037 7. Kantarjian H, Sawyers C, Hochhaus A et al (2002) Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Eng J Med 346(9):645–652 8. Hochhaus A, Druker B, Sawyers C et al (2008) Favorable long-term follow-up results over six years for response, survival and safety with imatinib mesylate therapy in chronic phase chronic myeloid leukemia post failure of interferonalpha treatment. Blood 111(3):1039–1043 9. O'Brien SG, Guilhot F, Larson RA et al (2003) IRIS Investigators. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase myeloid leukemia. N Engl J Med 348(11):994–1004 10. Druker BJ, Guilhot F, O'Brien SE et al (2006) Five-year follow-up of imatinib therapy for newly diagnosed chronic leukemia in chronic-phase shows sustained responses and high overall survival. N Engl J Med 355(23): 2408–2417 11. Ramirez P, DiPersion JF (2008) Therapy options in Imatinib failures. The Oncologist 13(4):424–434 12. Mauro MJ, Heinrich MC (2008) Treatment of chronic myeloid leukemia with BCRABL kinase inhibitors. Innovative Leukemia and Lymphoma Therapy. Kaspers GJL, Coiffier B, Heinrich MC, Estety E (eds) Informa Healthcare, New York 13. Talpaz M, Silver RT, Druker BJ et al (2002) Imatinib induces hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase II study. Blood 99(6):1928–1937 14. Sawyers CL, Hochhaus A, Feldman E et al (2002) Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study. Blood 99(10):3530–3539 15. Talpaz M, Goldman JM, Sawyers CL et al (2003) High dose imatinib (ST1571, Gleevec) provides durable long-term outcomes for patients (pts) with chronic myeloid leukemia (CML) in accelerated phase (AP) or myeloid blast crisis (BC): Follow-up of the phase II studies. Blood 102(11):905a 16. Sokal JE, Cox EB, Baccarani M et al (1984) Prognostic discrimination in “goodrisk” chronic granulocytic leukemia. Blood 63(4):789–799 17. Hasford J, Pfirrmann M, Hehlmann R et al (1998) A new prognostic score for survival of patients with chronic myeloid leukemia treated with interferon-alpha. J Nat Cancer Inst 90(11):850–858 18. O'Hare T, Eide CA, Deininger MW (2007) BCR-ABL kinase domain mutations, drug resistance, and the road to a cure for chronic myeloid leukemia. Blood 110(7):2242–2249 19. Shah NP, Tran C, Lee FY et al (2004) Overriding imatinib resistance with a novel ABL kinase inhibitor. Science 305(5682):399–401 20. Mauro MJ (2006) Defining and managing imatinib resistance. Hematology Am Soc Hematol Educ Program 219–225 21. Kantarjian H, Pasquini R, Hamerschlak N et al (2007) Dasatinib or high-dose imatinib for chronic-phase myeloid leukemia after failure of first-line imatinib: a randomized phase II trial. Blood 109(7):5143–5150 22. Talpaz M, Shah NP, Kantarjian H et al (2006) Dasatinib in imatinib-resistant Philadelphia chromosome-positive leukemias. N Eng J Med 354(24):2531–2541

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R.T. Maziarz 23. Guilhot F, Apperley J, Kim DW et al (2007) Dasatinib induces significant hematologic and cytogenetic responses in patients with imatinib-resistant or intolerant chronic myeloid leukemia in accelerated phase. Blood 109(10):4143–4150 24. Cortes J, Rousselot P, Kim DW et al (2007) Dasatinib induces complete hematologic and cytogenetic responses in patients with imatinib-resistant or intolerant chronic myeloid leukemia in blast crisis. Blood 109(8)3207–3213 25. Kantarjian HM, Giles F, Gattermann N et al (2007) Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is effective in patients with Philadelphia chromosome-positive chronic myelogenous leukemia in chronic phase following imatinib resistance and intolerance. Blood 110(10):3540–3546 26. Hochhaus A, Kantarjian HM, Baccarani M et al (2007) Dasatinib induces notable hematologic and cytogenetic responses in chronic-phase chronic myeloid leukemia after failure of imatinib therapy. Blood 109(6):2303–2309 27. Giles FJ, Cortes J, Jones D et al (2007) MK-0457, a novel kinase inhibitor, is active in patients with chronic myeloid leukemia or acute lymphocytic leukemia with the T315I BCR-ABL mutation. Blood 109(2):500–502 28. Nicolini FE, Hayette S, Corm S et al (2007) Clinical outcome of 27 imatinib mesylate-resistant chronic myelogenous leukemia patients harboring at T315I BCR-ABL mutation. Haematologica 92(9):1238–1241 29. Chronic Myeloid Leukemia Trialists' Collaborative Group (1997) Interferon alpha versus chemotherapy for chronic myeloid leukemia: A meta-analysis of seven randomized trials. J Natl Cancer Inst 89(21):1616–1620 30. Apperely JF (2006) Managing the patient with chronic myeloid leukemia through and after allogeneic stem cell transplantation. Hematology Am Soc Hematol Educ Program 226–232 31. Gratwohl A, Brand R, Apperely J et al (2006) Allogeneic hematopoietic stem cell transplantation for chronic myeloid leukemia in Europe 2006: Transplant activity, long-term data and current results. An analysis by the Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT). Haematologica 91(4):513–521 32. Robin M, Guardiola P, Devergie A et al (2005) A 10-year median follow-up study after allogeneic stem cell transplantation for chronic myeloid leukemia in chronic phase from HLA-identical sibling donors. Leukemia 19(9):1613–1620 33. Goldman JM (2007) How I treat chronic myeloid leukemia in the imatinib era. Blood 110(8):2828–2837 34. Gratwohl A, Hermans J, Goldman JM et al (1998) Risk assessment for patients with chronic myeloid leukaemia before allogeneic blood or marrow transplantation. Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation. Lancet 352(9134):1087–1092 35. Horowitz MM, Rowlings PA, Passweg JR (1996) Allogeneic bone marrow transplantation for CML: A report from the International Bone Marrow Transplant Registry. Bone Marrow Transplant 17(Suppl 3):55–56 36. Goldman JM (2001) “Stem cell transplantation for CML: Its place in treatment algorithms 2001” in Chronic Myelogenous Leukemia." Hematology 103–112 37. Buckner CD, Epstein RB, Rudolph RH et al (1970) Allogeneic marrow engraftment following whole body irradiation in a patient with leukemia. Blood 35(6):741–750 38. Weiden PL, Flournoy N, Thomas ED et al (1979) Antileukemic effect of graftversus-host disease in human receipients of allogeneic-marrow grafts. N Eng J Med 300(19):1068–1073 39. Horowitz MM, Gale RP, Sondel PM et al (1990) Graft-versus-leukemia reactions after bone marrow transplantation. Blood 75(3):555–562 40. Kolb HJ, Mittermuller J, Clemm C et al (1990) Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 76(12):2462–2465

Chapter 5  The Role of Allogeneic Hematopoietic Stem Cell Transplantation  41. Guglielmi C, Arcese W, Dazzi F et al (2002) Donor lymphocyte infusion for relapsed chronic myelogenous leukemia: prognostic relevance of the initial cell dose. Blood 100(2):397–405 42. Weisser M, Tischer J, Schnittger S et al (2006) A comparison of donor leukocyte infusions or imatinib mesylate for patients with chronic myelogenous leukemia who relapsed after allogeneic stem cell transplantation. Haematologica 91(5):663–666 43. Traversari C, Marktel S, Magnani Z et al (2007) The potential immunogenicity of the TK suicide gene does not prevent full clinical benefit associated with the use of TK-transduced donor lymphocytes in HSCT for hematologic malignancies. Blood 109(11):4708–4715 44. Clift RA, Buckner CD, Thomas ED et al (1994) Marrow transplantation for chronic myeloid leukemia: A randomized study comparing cyclophosphamide and total body irradiation with busulfan and cyclophosphamide. Blood 84(6):2036–2043 45. Devergie A, Blaise D, Attal M et al (1995) Allogeneic bone marrow transplantation for chronic myeloid leukemia in first chronic phase: a randomized trial of busuifan-cytoxan versus cytoxan-total body irradiation as preparative regimen: a report from the French Society of Bone Marrow Graft (SGM). Blood 85(6):2263–2268 46. Slattery JT, Clift RA, Buckner CD et al (1997) Marrow transplantation for chronic myeloid leukemia: The influence of plasma busulfan levels on the outcome of transplantation. Blood 89(8):3055–3060 47. Thall PF, Champlin RE, andersson BS (2004) comparison of 100-day mortality rates associated with i.v. busulfan and cyclophosphamide vs other preparative regimens in allogeneic bone marrow transplantation for chronic myelogenous leukemia: Bayesian sensitivity analyses of confounded treatment and center effects. Bone Marrow Transplant 33(12):1191–1199 48. Hansen JA, Gooley TA, Martin PJ et al (1998) Bone marrow transplants from unrelated donors for patients with chronic myeloid leukemia. N Engl J Med 338(14):962–968 49. Oehler VG, Radich JP, Storer B et al (2005) Randomized trial of allogeneic related bone marrow transplantation versus peripheral blood stem cell transplantation for chronic myeloid leukemia. Bio Blood Marrow Transplant 11(2):85–92 50. Stewart BL, Storer B, Storek J et al (2004) Duration of immunosuppressive treatment for chronic graft-versus-host disease. Blood 104(12):3501–3506 51. Eapen M, Logan BR, Confer DL et al (2007) Peripheral blood grafts from unrelated donors are associated with increased acute and chronic graftversus-host disease without increased survival. Bio Blood Marrow Transplant 13(12):1461–1468 52. Nagamura-Inoue T, Kai S, Azuma H et al (2008) Unrelated cord blood transplantation in CML: Japan Cord Blood Bank Network analysis. Bone Marrow Transplant 42(4):241–251 53. Scott BL, Sandmeier BM (2006) Outcomes with myeloid malignancies. Hematology Am Soc Hematol Educ Program 381–389 54. Slavin S, Nagler A, Naparstek E et al (1998) 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 91(3):756–763 55. Bornhauser M, Kiehl M, Siegert W et al (2001) Dose-reduced conditioning for allografting in 44 patients with chronic myeloid leukemia: a retrospective ­analysis. Br J Haematol 115(1):119–124 56. Das M, Saikia TK, Advani SH et al (2003) Use of a reduced-intensity conditioning regimen for allogeneic transplantation in patients with chronic myeloid leukemia. Bone Marrow Transplant 32(2):125–129

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R.T. Maziarz 57. Or R, Shapira MY, Resnick I et al (2003) Nonmyeloablative allogeneic stem cell transplantation for the treatment of chronic myeloid leukemia in the first chronic phase. Blood 101(2):441–445 58. Okamoto S, Watanabe R, Takahaski S et al (2002) Long-term follow-up of allogeneic bone marrow transplantation after reduced-intensity conditioning in patients with chronic myelogenous leukemia in the chronic phase. Int J Hematol 75(7):493 59. Kerbauy FR, Storb R, Hegenbart U et al (2005) Hematopoietic cell transplantation from HLA-identical sibling donors after low-dose radiation-based conditioning for treatment of CML. Leukemia 19(6):990–997 60. Kebriaei P, Detry MA, Giralt S et al (2007) Long-term follow-up of allogeneic hematopoietic stem-cell transplantation with reduced-intensity conditioning for patients with chronic myeloid leukemia. Blood 110(9):3456–3462 61. Bornhauser M, Kroger N, Schwerdtfeger R et al (2006) Allogeneic haematopoietic cell transplantation for chronic myelogenous leukaemia in the era of imatinib: a retrospective multicentre study. Eur J Haematol 76(1):9–17 62. Crawley C, Szydlo R, Lalancette M et al (2005) Outcomes of reduced-intensity transplantation for chronic myeloid leukemia: an analysis of prognostic ­factors from the Chronic Leukemia Working Party of the EBMT. Blood 106(9): 2969–2976 63. Faber E, Koza V, Vitek A et al (2007) Reduced-intensity conditioning for allogeneic stem cell transplantation in patients with chronic myeloid leukemia is associated with better overall survival but inferior disease-free survival when compared with myeloablative conditioning – a retrospective study of the Czech National Hematopoietic Stem Cell Transplantation Registry. Neoplasma 54(5):443–444 64. Giralt SA, Arora M, Goldman JM et al (2007) Impact of imatinib therapy on the use of allogeneic haematopoietic progenitor cell transplantation for the treatment of chronic myeloid leukaemia. Br J Haematol 137(5):461–467 65. Ruiz-Arguelles GJ, Gomez-Almaguer D et al (2005) the early referral for reduced-intensity stem cell transplantation in patients with PH1(+) chronic myelogenous leukemia in chronic phase in the imatinib era: results of the Latin American Cooperative Oncohematolgoy Group (LACOHG) prospective, multicenter study. Bone Marrow Transplant 36(12):1043–1047 66. Ruiz-Arguelles GJ, Tarin-Arzaga LC, Gonzalez-Carrillo ML et al (2008) Therapeutic choices in patients with PH-positive CML living in Mexico in the tyrosine kinase inhibitor era: SCT or TKIs? Bone Marrow Transplant 42(1): 23–28 67. Krejci M, Mayer J, Doubek M et al (2006) Clinical outcomes and direct hospital costs of reduced-intensity allogeneic transplant 38(7):483–491 68. Millot F, Guilhot J, Nelken B et al Imatinib mesylate is effective in children with chronic myelogenous leukemia in late chronic and advanced phase and in relapse after stem cell transplantation. Leukemia 20(2):187–192 69. Hehlmann R, Berger U, Pfirrmann M et al (2007) Drug treatment is superior to allografting as first-line therapy in chronic myeloid leukemia. Blood 109(11):4686–4692 70. Zaucha JM, Prejzner W, Giebel S et al (2005) Imatinib therapy prior to myeloablative allogeneic stem cell transplantation. Bone Marrow Transplant 36(5): 417–424 71. Shimoni A, Kroger N, Zander AR et al (2003) Imatinib mesylate (STI571) in preparation for allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusions in patients with Philadelphia-positive acute leukemia. Leukemia 17(2):290–297 72. Oehler VG, Gooley T, Snyder DS et al (2007) The effects of imatinib mesylate treatment before allogeneic transplantation for chronic myeloid leukemia. Blood 109(4):1782–1789

Chapter 5  The Role of Allogeneic Hematopoietic Stem Cell Transplantation  73. Jabbour E, Cortes J, Kantarijian H et al (2007) Novel tyrosine kinase inhibitor therapy before allogeneic stem cell transplantation in patients with chronic myeloid leukemia: no evidence for increased transplant-related toxicity. Cancer 110(2):340–344 74. Deininger M, Schleuning M, Greinix H et al (2006) The effect of prior exposure to imatinib on transplant-related mortality. Haematologica 91(4):452–459 75. Lee SJ, Kukreja M, Wang T et al (2008) Impact of prior imatinib mesylate on the outcome of hematopoietic cell transplantation for chronic myeloid leukemia. Blood 112(8):3500–3507 76. Weisser M, Schmid C, Schoch C et al (2005) Resistance to pretransplant imatinib therapy may adversely affect the outcome of allogeneic stem cell transplantation in CML. Bone Marrow Transplant 36(11):1017–1018 77. Jabbour E, Cortes J, Kantarjian HM et al (2006) Allogeneic stem cell transplantation for patients with chronic myeloid leukemia and acute lymphocytic leukemia after BCR-ABL kinase mutation-related imatinib failure. Blood 108(4): 1421–1423 78. Marin D, Kaeda J, Szydlo R et al (2005) Monitoring patients in complete cytogenetic remission after treatment of CML in chronic phase with imatinib: patterns of residual leukaemia and prognostic factors for cytogenetic relapse. Leukemia 19(4):507–512 79. Kaeda J, O'Shea D, Szydlo RM et al (2006) Serial measurement of BCR-ABL transcripts in the peripheral blood after allogeneic stem cell transplantation for chronic myeloid leukemia: an attempt to define patients who may not require further therapy. Blood 107(10):4171–4176 80. Goldman JM, Sobocinski KA, Zhang MJ et al (2006) Long-term outcome after allogeneic hematopoietic cell transplantation (HCT) for CML. Bio Blood Marrow Transpl 12(2):17 81. Olavarria E, Ottmann OG, Deininger M et al (2003) Response to imatinib in patients who relapse after allogeneic stem cell transplantation for chronic myeloid leukemia. Leukemia 17(9):1707–1712 82. Kantarjian HM, O'Brien S, Cortes JE et al (2002) Imatinib mesylate therapy for relapse after allogeneic stem cell transplantation for chronic myelogenous leukemia. Blood 100(5):1590–1595 83. DeAngelo DJ, Hochberg EP, Alyea EP et al (2004) Extended follow-up of patients treated with imatinib mesylate (Gleevec) for chronic myelogenous leukemia relapse after allogeneic transplantation: during cytogenetic remission and conversion to complete donor chimerism without graft-versus-host disease. Clin Cancer Res 10(15):5065–5071 84. Ullmann AJ, Hess G, Kolbe K et al (2003) Current result of the use of imatinib mesylate in patients with relapsed Philadelphia chromosome positive leukemia after allogeneic or syngeneic hematopoietic stem cell transplantation. Keio J Med 52(3):182–188 85. Kim YJ, Kim DW, Lee S et al (2004) Cytogenetic clonal evolution alone in CML relapse post-transplantation does not adversely affect response to imatinib mesylate treatment. Bone Marrow Transplant 33(3):237–242 86. Savani BN, Montero A, Kurlander R et al (2005) Imatinib synergizes with donor lymphocyte infusions to achieve rapid molecular remission of CML relapsing after allogeneic stem cell transplantation. Bone Marrow Transplant 36(11): 1009–1015 87. Simula MP, Markel S, Fozza C et al (2007) Response to donor lymphocyte infusions for chronic myeloid leukemia is dose-dependent: the importance of escalating the cell dose to maximize therapeutic efficacy. Leukemia 21(9):943–948 88. Carpenter PA, Snyder DS, Flowers ME et al (2007) Prophylactic administration of imatinib after hematopoietic stem cell transplantation for high-risk Philadelphia chromosome-positive leukemia. Blood 109(7):2791–2793

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R.T. Maziarz 89. Olavarria E, Siddique S, Griffiths MJ et al (2007) Posttransplantation imatinib as a strategy to postpone the requirement for immunotherapy in patients undergoing reduced-intensity allografts for chronic myeloid leukemia. Blood 110(13):4614–4617 90. Baccarani M, Saglio G, Goldman J et al (2006) Evolving concepts in the management of chronic myeloid leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet. Blood 108(6):1809–1820 91. Jabbour E, Kantarjian H, O'Brien S et al (2006) Sudden blastic transformation in patients with chronic myeloid leukemia treated with imatinib mesylate. Blood 107(2):480–482 92. O'Dwyer ME, Mauro MJ, Blasdel C et al (2004) Clonal evolution and lack of cytogenetic response are adverse prognostic factors for hematologic relapse of chronic phase CML patients treated with imatinib mesylate. Blood 103(2):451–455 93. Deininger MW, Cortes J, Paquette R et al (2007) The prognosis for patients with chronic myeloid leukemia who have clonal cytogenetic abnormalities in Philadelphia chromosome-negative cells. Cancer 110(7):1509–1519 94. Kovacsovics T, Maziarz RT (2006) Philadelphia chromosome positive acute lymphoblastic leukemia: impact of imatinib treatment on remission induction and allogeneic stem cell transplantation. Curr Onc Rep 8:343–351 95. Holowiecki J, Giebel S, Wojnar J et al (2008) Treosulfan and fludarabine lowtoxicity conditioning for allogeneic haemotopoietic stem cell transplantation in chronic myeloid leukaemia. Br J Haematol 142(2):284–292 96. Mohty M, Szydlo RM, Yong AS et al (2008) Association between BMI-1 expression, acute graft-versus-host disease and outcome following allogeneic stem cell transplantation from HLA-identical siblings in chronic myeloid leukemia. Blood 112(5):2163–2166 97. Van der Meer A, Schaap NP, Schattenberg AV et al (2008) KIR2D55 is associated with leukemia free survival after HLA identical stem cell transplantation in chronic myeloid leukemia patients. Mol Immunol 45:3631–3638 98. Yong AS, Keyvanfar K, Eniafe R et al (2008) Hematopoietic stem cells and progenitors of chronic myeloid leukemia express leukemia-associated antigens: implications for the graft-versus-leukemia effect and peptide vaccine-based immunotherapy. Leukemia 22(9):1721–1727 99. Gordon MK, Sher D, Karrison T et al (2008) Successful autologous stem cell collection in patients with chronic myeloid leukemia in complete cytogenetic response, with quantitative measurement of BCR-ABL expression in blood, marrow, and apheresis products. Leuk Lymphoma 49(3):531–537 100. Olavarria E (2007) Autologous stem cell transplantation in chronic myeloid leukemia. Semin Hematol 44(4):252–258 101. CML Autograft Trials Collaboration (2007) Autologous stem cell transplantation in chronic myeloid leukemia: a meta-analysis of six randomized trials. Cancer Treat Rev 33(10):39–47 102. Gould SJ (1977) Ever since Darwin: Reflections in natural history. W.W. Norton & Company, New York 103. Gould SJ (1980) The Panda's thumb. W. W. Norton & Company, New York

Chapter 6 Allogeneic Transplantation for Hodgkin’s Lymphoma William Broderick and Patrick Stiff

1.  Introduction There are only approximately 7,500 cases of Hodgkin’s lymphoma diagnosed in the United States annually. While there is a bimodal distribution in incidence, the majority of cases occur in young adults 15–30 years of age [1]. Initial therapy is highly successful with progression-free survival (PFS) rates at 10 years of 70–90% for early stage disease and 60–70% for advanced disease. For those relapsing after initial therapy, 30–50% are long-term survivors after an autologous stem cell transplant, making this one of the most curable adult malignancies. However, a small minority of these typically young and otherwise healthy patients will relapse even after an autologous stem cell transplantation (ASCT) and it is for these rare patients that allogeneic transplantation has been increasingly considered. Initially discarded in the early 1990s as being too toxic utilizing myeloablative conditioning, allogeneic transplantation for this disease is increasing again based on the availability of reduced intensity regimens and therapies that have decreased mortality due to regimen-related toxicities, graft versus host disease (GVHD), and opportunistic infections. Whether or not this will be an effective approach for multiply resistant patients remains to be determined, making this one of the most controversial areas in 2008 in allogeneic transplantation. This chapter outlines modern therapy for this group of lymphomas and the potential utility of allogeneic transplantation in select patients.

2.  Conventional Therapy Initial therapy for patients with Hodgkin’s lymphoma is based primarily on stage, with early stage disease (stage I and II) without high risk features (B symptoms, bulky adenopathy, age >50 years) treated with radiation alone (stage I disease) or, short course chemotherapy combined with localized radiation therapy (stage II disease). Radiation alone in the past was usually reserved for patients with stage IA or IIA disease confirmed by laparotomy, however, From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_6, © Springer Science + Business Media, LLC 2003, 2010

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as PET scanning has recently replaced laparotomy as a staging tool, those with localized disease and a negative PET scan can receive mantle radiation with or without periaortic fields and expect a complete response (CR) >95%. Of these, approximately 70% of patients obtain a long-term remission and >90% of patients are alive 10 years from diagnosis [2]. To decrease relapses in early stage disease, the combination of abbreviated course chemotherapy with involved field radiation therapy (IFRT) is being increasingly used. Several studies have demonstrated that 2–4 cycles of ABVD chemotherapy followed by IFRT induces response rates >90% with 2–3 year follow up [2–4]. Patients presenting early stage disease with high risk features require systemic chemotherapy for optimal outcome. Patients with mediastinal masses >10 cm generally receive both full-course chemotherapy and IFRT. The addition of radiation to residual disease after chemotherapy, has also been shown to convert partial remissions (PR) in many to durable CRs with again a long-term survival of 70% [3, 4]. Patients with Stage III and IV disease require chemotherapy for optimal outcome. MOPP (nitrogen mustard, vincristine, procarbazine, prednisone) was developed for use in Hodgkin’s disease in the 1960s and achieved high response rates and long-term survival rates of 50–60%. ABVD (doxorubicin, bleomycin, vinblastine DTIC) demonstrated activity, first in MOPP resistant patients, then as a first-line regimen with less gonadal toxicity and a lower risk of secondary malignancies than MOPP. It still remains the standard regimen for advanced Hodgkin’s disease in adults with CR rates reaching 70% (Table  6-1.) [5–7]. Failure-Free Survival rates (FFS) of 65%, and overall survival (OS) reaching 89% at 3 years. There is general agreement that IFRT even for responders is needed in the 25–30% of patients who are present with mediastinal masses >1/3 of the chest diameter. Studies have also shown that IFRT is effective in consolidating PRs as well [8].

Table 6-1.  Selected results of standard therapy for newly diagnosed Hodgkin’s disease. Stage

Treatment

Freedom from treatment failure

Long-term survival (%)

Early stage [4]

EFRT alone

67% (7 years)

92

ABVD + EFRT

88% (7 years)

94

ABVD

81% (5 years)

90

ABVD + IFRT

86% (5 years)

97

ABVD + EFRT

91.4% (10 years)

90.4

EBVM + EFRT

80% (10 years)

90.3

MOPP + EFRT

62.8% (7 years)

67.9

ABVD + EFRT

82.8% (7 years)

77.4

ABVD

63% (5 years)

82

MOPP/ABV

66% (5 years)

81

IA–IIIA [3] IA–IIIB [6] Advanced stage [5] IIIA, IIIB, IV [7]

Early stage: stage I or II disease, no bulky disease Advanced stage: stage III or IV disease, bulky stage II disease EFRT extended field radiation

Chapter 6  Allogeneic Transplantation for Hodgkin’s Lymphoma 

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3.  High-Dose Therapy with Autologous Stem Cell Transplantation For those who relapse after an initial chemotherapy-based treatment regimen, ASCT is generally the considered standard of care, particularly for those who relapse early. Patients who relapse >12 months after an initial CR, however, may be treated with combination chemotherapy and if they achieve a second CR, 30–50% will remain disease-free at 4 years without any further therapy [9, 10]. Most relapsing patients do so less than 12 months after a CR, and for these patients and those without a CR after initial therapy, standard dose second-line therapy is not likely to lead to a durable remission. In fact, longterm PFS is 0% for this group (Table 6-2.) [6]. For these patients, high-dose chemotherapy with ASCT has emerged as the treatment of choice. Overall 30–50% of such patients are alive and disease-free for 5+ years suggesting superiority of this approach for otherwise eligible patients. Several randomized trials have been performed to validate the efficacy of this approach. In 1993, the British National Lymphoma Investigation (BNLI) group published a 3-year event-free survival rate of 53% compared to 10% in their standard combination chemotherapy arm [11]. There was no difference in OS, but the follow up appeared not to have been long enough to demonstrate a survival benefit. The German Hodgkin’s Lymphoma Study Group (GHSG) in conjunction with the European Bone Marrow Transplant Registry (EBMTR) randomized heavily treated patients with relapse to chemotherapy or high-dose chemotherapy with ASCT [12]. They demonstrated a 55%

Table 6-2.  Results of autologous bone marrow transplant in Hodgkin’s disease. Importance of risk factors. Outcome by # of high risk features Study

n

Reece [13]

  58

High risk features

0

1

2

3+

(1) B-symptoms

3 years PFS 100%

3 years PFS 81%

3 years PFS 40%

3 years PFS 0%

43 months EFS 27%

43 months EFS 10%

3 years FFP 41%

3 years FFP <20%

(2) Extranodal relapse (3) CR <1 year Moskowitz [14]

  65

(1) B-symptoms

43 months EFS 83%

(2) Extranodal relapse (3) CR <1 year

Horning [15] 119

(1) Systemic symptomatic 3 years FFP relapse 85%

3 years FFP 51%

(2) Pulmonary or bone marrow relapse (3) More than minimal disease at BMT Stiff [16]

  81

(1) >2 prior regimens

5 years OS 60%

5 years OS 38%

(2) Relapse in radiation field (3) Extranodal relapse PFS progression-free survival, EFS event-free survival, FFP freedom from progression, OS overall survival

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freedom from treatment failure rate in the ASCT group compared to 34% in the chemotherapy group. Again, at a short follow up, no survival benefit was demonstrated.

4.  Risk Factors for Autotransplant Outcome Given the dismal results with standard salvage chemotherapy for those who relapse after initial combination chemotherapy and in lieu of performing randomized Phase III trials of ASCT therapy in North America, phase II trials were performed to determine which patients with relapsed disease would benefit most (Table 6-2). In 1994, Reece et al. transplanted 58 patients who had relapsed after an initial chemotherapy regimen [13]. All underwent highdose chemotherapy with ASCT and had a 3-year PFS rate of 64%, seemingly higher than what would be obtained with any available salvage chemotherapy. Three independent prognostic factors were identified: B-symptoms at relapse, extranodal disease at relapse, and initial remission duration of less than 1 year. Patients with 0, 1, 2, and all 3 risk factors had a PFS of 100, 81, 40, and 0%, respectively. Similar results have been presented by Mososkowitz et al. [14] and from the group at Stanford who identified pulmonary or bone marrow disease at relapse, systemic symptoms at relapse, and more than minimal disease as poor prognostic factors [15]. Freedom from progression 3 years after ASCT was estimated to be 85, 57, 41, and <20% for patients with 0, 1, 2, and 3 risk factors, respectively. The first US phase II cooperative group study for this patient group also identified three adverse risk factors for autotransplant outcome: >2 prior regimens, relapse in a radiated field, and extranodal disease [16]. Five-year OS was 60% for those with 0–1 risk factors versus 38% for those with 2 or 3 factors. Those transplanted in CR had a 70% long-term DFS. Thus, autotransplants appear to produce long-term survival in many patients with low and intermediate risk disease at the time of relapse (Table 6-2). Based on risk factor analysis, it should be a standard of care for these patients. For high-risk patients, however, only a minority are long-term survivors and new approaches are needed.

5.  Strategies to Improve Outcome in High Risk Disease To improve survival after an autograft particularly in patients with multiple adverse risk factors, several approaches have been investigated. First tried was multi-course, sequential HD chemotherapy with ASCT. In 1997, a multicenter phase II trial was undertaken treating relapsed or refractory patients with two cycles of standard-dose salvage chemotherapy, followed by sequential highdose chemotherapy with a final myeloablative course and an ASCT [17]. The toxicity was tolerable with no treatment-related deaths. FFS/OS rates were 64%/87% for patients with early relapse, 68%/81% for patients with late relapse, 30%/58% for patients with progressive disease (at enrollment), and 55%/88% for patients with multiply relapsed disease. The authors concluded that, given the high response rate in relapsed patients, this three-phase treatment regimen warranted further study. Another approach utilized a tandem autologous transplantation approach as proven successful in patients with multiple myeloma. This is based on the

Chapter 6  Allogeneic Transplantation for Hodgkin’s Lymphoma 

data suggesting that autograft outcome is optimal in patients transplanted in CR. In 1997, Ahmed et al. reported the results of tandem autologous transplant in 45 patients with refractory Hodgkin’s disease [18]. Their median survival was 45 months. However, only 55% of the patients were able to undergo the second transplant. Patients were excluded from the second cycle due to toxicity or progression. Brice et  al. treated 43 patients who failed induction therapy or who have poor prognosis defined as a <12 month interval between the end of treatment and first progression, stage III or IV disease at relapse, or relapse occurred in a previously irradiated site [19]. Also included were patients in first relapse who did not achieve a response during chemotherapy given at relapse. There were two treatment associated deaths, one with venoocclusive disease and one with acute respiratory distress syndrome. Overall, 39 patients underwent the first transplant and 32 underwent the second. Three patients progressed after the first transplant and did not proceed to the second transplant. Survival was better for patients receiving both transplants than for patients receiving one or no transplant (74% vs. 40% vs. 0%) [12]. Fung et al. conducted a two institution pilot study between 1998 and 2000 in which 41 primary refractory or recurrent Hodgkin’s lymphoma patients with at least 1 of 3 poor prognostic factors: first CR <12 months, extra nodal disease, or B-symptoms at relapse underwent tandem autologous transplant [20]. After a median follow-up time of 5.3 years, the reported OS, PFS, and FFS were 54, 49, and 55%, respectively. This approach is currently being further investigated in the Southwest Oncology Group (SWOG) together with the US BMT Clinical Trials Network (CTN). Despite advances in technique and improvement in survival over conventional salvage therapy ASCT has its drawbacks. Even the best outcome data predict a high rate of treatment failure in patients with poor risk factors at relapse. Also, exposure to high-dose chemotherapy can result in the development of myelodysplastic syndrome (MDS) or secondary acute myelogenous leukemia (AML), despite ASCT. Akpek et  al. reported in 2001 that 3 of their 104 Hodgkin’s disease autologous transplant patients developed secondary AML/MDS at 3, 7, and 12 years post-autograft [21]. Other reports confirm that the incidence of AML/MDS after ASCT for Hodgkin’s disease ranges from 5% to 25% [22, 23]. Despite these issues, current strategies are to improve ASCT outcome and continue to reserve allogeneic transplantation for those who fail an autograft.

6.  Allogeneic Transplantation for Hodgkin’s Lymphoma Patients who relapse after an ASCT have a median survival less than 1 year [24], with rare exceptions being those who relapse late, although a report of 11 highly selected patients reported by the EBMT who had a survival of 50% after a second ASCT procedure [25]. With the exception of these occasional patients, until recently, all patients relapsing after an ASCT died of their disease. It is this group that has been the subject of several novel approaches using allogeneic transplantation based on the hypothesis that like other lymphomas there is a GVL effect in this disease. The other advantages for an allograft in this disease are similar to that for other lymphomas including a lack of contamination of donor stem cells with malignant cells, the elimination of the risk of treatment-related AML and MDS after transplant, the ability

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to offer transplant therapy to those with an existing MDS, or those failing to mobilize sufficient stem cells for an ASCT. Evidence of a potential benefit of GVL in this disease was first reported in 1991 by Jones et  al. [26]. They reported that for transplants performed between 1981 and 1988, patients undergoing an allogeneic transplant had a 24% lower risk of relapse than those undergoing an autologous transplant. Given no significant difference in patient or treatment characteristics between ASCT and allogeneic transplant patients, this difference was postulated to have been due to GVL. This report is cited as the basis of nearly all future investigations of allogeneic transplantation in this disease.

7.  Early Results of Allografting in Hodgkin’s Disease Unfortunately, in their 1991 publication, Jones et al. also reported that 47% of allogeneic transplant recipients died of treatment-related causes, compared to 21% of ASCT recipients. Acute GVHD developed in 63% and chronic GVHD developed in 32% of the allograft patients who survived 100 days. The majority of deaths in this group resulted from GVHD. This difference in procedurerelated mortality resulted in the lack of a difference in event-free survival between those receiving ASCT and allografts [26]. With other studies reported over the next several years reporting high rates of lethal complications there was a general abandonment of allografting for this disease (Table 6-3). In 1993, Anderson et  al. reported the Seattle experience with allogeneic, syngeneic, and autologous BMT in patients with Hodgkin’s Disease [27]. In comparing all allogeneic transplant recipients to autologous and syngeneic recipients combined, the event-free survival rates (EFS) did not differ significantly, but there was a trend toward decreased relapse rate (48% vs. 77%, p = 0.060) but again a significant increase in nonrelapse mortality (58% vs. 41%, p = 0.047) in the allogeneic group. When the comparison was limited to HLA-matched sibling transplants versus ASCT, there was a nonsignificant difference in EFS rates (26% vs. 14%, p = 0.6) and nonrelapse mortality rates (53% vs. 43%, p = 0.2), but a significantly lower relapse rate among the HLAidentical transplants (45% vs. 76%, p = 0.50). In 1996, Milpied et  al. from

Table  6-3.  Results of allogeneic bone marrow transplantation in Hodgkin’s disease using fully ablative regimens. Study

Patients

Transplant

TRM (%)

OS

EFS/PFS

Anderson [27]

  68

Auto

32

13% (5 years)

14%, 5 years EFS

   6

Syngeneic

NR

33% (5 years)

NR

  53

Allo

53

20% (5 years)

22%, 5 years EFS

Gajewski [29]

100

HLA-matched sibling

61

21% (3 years)

15%, 3 years DFS

Milpied [28]

  45

Allo

48

25% (4 years)

15%, 4 years PFS

  45

Auto

27

37% (4 years)

24%, 4 years PFS

104   53

Auto Allo

26 43

37% (10 years) 30% (10 years)

26%, 10 years EFS for each cohort

Akpek [21]

DFS disease-free survival, EFS event-free survival, OS overall survival, PFS progression-free survival, TRM transplant-related mortality

Chapter 6  Allogeneic Transplantation for Hodgkin’s Lymphoma 

Centre Hospitalier Universitaire, Nantes, France, reported a review of 1,200 patients with Hodgkin’s Disease who underwent a transplant from various donors to the EBMTR [28]. Of these, 49 patients had undergone allogeneic transplant, 45 of whom had adequate information to complete a review were compared to 45 matched ASCT recipients. They reported no significant difference in response rate (84% vs. 83%) or 4-year relapse rate (61% vs. 61.5%) for the allograft and ASCT groups, respectively. There was, however, an increased rate of procedure-related mortality in the allograft group compared to the ASCT group (48% vs. 27%, respectively). The same year, Gajewski et  al. [29] published a review of 100 consecutive patients with Hodgkin’s disease who underwent HLA-identical sibling bone marrow transplants reported to the International Bone Marrow Transplant Registry (IBMTR). They reported a 3 year probability of relapse of 65% and a 3 year disease-free survival rate of only 15% [22]. All of the above authors pointed out that the recipients of allografts in their reviews had been more heavily pre-treated and many had suffered multiple relapses. The Seattle group pointed out that there was a higher risk of relapse in their patients with bulky disease. They did not note a statistical difference in survival or relapse among the various myeloablative preparatory regimens except for Gajewsiki et al. who did note a higher rate of treatment failure and relapse in patients who received busulfan and cyclophosphamide as their preparatory regimen. The lack of improvement in OS and a high TRM reported in these publications, in particular the IBMTR report and the debate over the limited effect if any of a GVL effect ,resulted in a period of time during which allogeneic transplant was severely limited. In particular, these results suggested that allografts after failed ASCT would be lethal in particular therapies.

8.  Reduced Intensity Regimens Given the evidence for GVL demonstrated in early studies and subsequent data indicating a response to donor lymphocyte infusions (DLI) in some [30], and the fact that the high mortality rate was frequently attributed to regimen-related toxicity, acute GVHD, and infections, several groups have continue to explore allografting in this disease while attempting to limit the transplant-related mortality by reducing the intensity of the preparatory regimens, using peripheral blood stem cells (PBSC) and/or depleting the T-Cell content of the grafts. In theory, the reduced intensity or nonmyeloablative preparatory regimens should result in less regimen-related deaths and the use of PBSC, in fewer infections. Of course, the drawback to a reduced intensity preparatory regimen is, less cytotoxic effect on the lymphoma itself. This places greater importance on the GVL effect and given the modest if any effect in the early studies this could permit progression of disease before the GVL effect begins after day 100. Depleting the T-cell content of the graft slows the advent of GVL and, consequently, the graft’s ability to control the lymphoma. Attempts to counter this delay have been early withdrawal of immunosuppression and DLI. These trials are shown in Table  6-4. In 2000, Anderlini et  al. at MD Anderson Cancer Center reported the results of a pilot study in which six patients with advanced HD who had failed a median of six chemotherapy regimens, radiation therapy, and auto-SCT underwent HLA-matched sibling allogeneic transplants [31]. The patients were considered eligible for allografting if

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Table  6-4.  Results of allogeneic bone marrow transplantation in Hodgkin’s disease using reduced intensity preparative regimens. Study

TRM Preparative Patients Transplant (%) regimen

OS

Outcome

Cooney [33]

  10

Allo

0

BEAM

90% (1 year)

70%, 1 year PFS

Robinson [38]

311

Allo

27

Various

46% (2 years)

26%, 2 years PFS

Peggs [35]

  49

Allo

16.3

Fludarabine, melphalan, alemtuzumab

56% (4 years)

39%, 4 years PFS

Anderlini [34]

  40

Allo

22

Fludarabine based

60% (18 months)

32%, 18 months PFS

Alvarez [34]

  40

Allo

25

Fludarabine, melphelan

48% (2 years)

32%, 2 years PFS

Baron [37]

  35

Allo

–

TBI ± fludarabine

35% (3 years)

8%, 3 years PFS

Alvarez [36]

  62

Auto ³ Allo

21

Fludarabine or low-dose TBI

62% (2 years)

36%, 2 years PFS

BEAM = BCNU, Etoposide, Cytosine Arabinoside, Melphelan, OS overall survival, PFS progression-free survival, TBI total body irradiation, TRM transplant related mortality

they had chemosensitive or stable disease after salvage chemotherapy. The conditioning regimens chosen were “less intensive” fludarabine-cyclophosphamide-antithymocyte globulin (ATG) (n = 4), fludarabine-melphalan (n = 1), and fludarabine-cytarabine-idarubicin (n = 1). Bone marrow recovery was prompt and at 30 days 4/5 evaluable patients had 100% donor chimerism. Two of the six died before day 100; neither of the deaths were conditioning regimen related. Beyond 100 days, one patient relapsed and died 8 months after transplant. The remaining three patients were alive and progression free after 9 months of follow up. As none of the above regimens would be expected to result in more than a transient response, the authors speculated that the durability of these remissions was secondary to a graft-versus-Hodgkin’s effect. They also concluded that, in the absence of progressive disease, allogeneic transplantation with reduced-intensity fludarabine-based conditioning is feasible in high risk, heavily pretreated Hodgkin’s disease patients with acceptable engraftment, and regimen-related toxicity. Following the success of BCNU, etoposide, Ara-C, melphalan (BEAM) as a conditioning regimen in autotransplant for HD, and a single case report of BEAM in allo-transplant in the British Journal of Haematology [32], Cooney et al. reported in 2003 on the use of this regimen from our group in ten patients with HD who also had failed prior autografts [33]. This more intensive regimen was chosen to insure an optimal cytoreduction of disease until day 100. Prior to transplant all patients received re-induction chemotherapy. At transplant one patient was in CR, four were in PR, three were in resistant relapse, and one had primary refractory disease. Patients received matched-sibling bone marrow (5), single-locus mismatched-sibling bone marrow (1), or matchedunrelated bone marrow (4). All patients engrafted rapidly and there were no deaths by day 100. All ten patients responded, eight complete responses and two partial responses. Twelve months after allograft, 9/10 patients were alive, seven in remission. Anderlini et al. followed up their initial results in 2005 with a report of 40 patients with relapsed or refractory HD [34] who underwent reduced-intensity

Chapter 6  Allogeneic Transplantation for Hodgkin’s Lymphoma 

allografts from either an HLA-matched sibling (20) or matched unrelated donor (20). The regimens chosen were fludarabine-cyclophosphamide +/− ATG and fludarabine-melphalan. Engraftment was rapid and day 100 and cumulative mortality were 5 and 22%, respectively. At a median of 13 months follow up, 24 patients were alive (60%); however, only 14 were in complete remission (23%). Eight patients died of treatment-related causes, and eight died of progressive disease. Of note, the fludarabine-melphalan regimen, which was considered the more intensive regimen, resulted in better OS (73% vs. 39%, p = 0.03) and a trend toward better PFS (37 vs. 21%, p = 0.2), further evidence that more intensive regimens are needed in this disease. Also in 2005, Peggs et al. published the collective experience in England including 49 patients with multiply relapsed Hodgkin’s disease [35]. Most (90%) of their patients had progressive disease after autologous transplant. They undertook allogeneic transplant with a fludarabine-melphalan-alemtuzumab regimen. Alemtuzumab was added to reduce the incidence of acute GVHD. They included both matched related (31) and unrelated (18) donor sources. All of their patients engrafted. Unlike the other groups reported above, 16 patients (33%) received DLI for residual disease or progression, 9 of whom responded to the DLI (eight complete, one partial). Nonrelapse-related mortality was 16.3% at 730 days. Projected 4-year overall survival and PFS were 55.7 and 39%, respectively. Additional experience with the fludarabine and melphalan regimen was presented in 2005 by Alverez et al. from Spain [36]. They treated 40 patients of whom 21 patients had received more than 2 chemotherapy regimens, 23 patients had received previous radiation therapy, and 29 patients had failed prior autologous transplant and 20 were transplanted in chemoresistant relapse. The response rate reported was 67% (21 complete remissions, 6 partial remissions). The 1 year transplant-related mortality was 25%. DLI was administered to 11 for relapse or refractory disease with 6 responses (3 complete and 3 partial). OS and PFS were 48 and 32%, respectively at 2 years. Refractoriness to chemotherapy was the only adverse prognostic factor noted. Extensive chronic GVHD was noted to be associated with a trend toward reduced relapse rate, but this did not reach statistical significance (77% vs. 44%, p = 0.07). As part of a larger study on autograft failures, the Seattle group reported on the use of fludarabine with 2 GY of TBI patients with Hodgkin’s lymphoma [37]. They found in 35 patients only an 8% 3-year PFS. Together these data suggest that regimen intensity may play a role in this disease. A registry review of the use of reduced intensity regimens as reported to the EBMT registry was published by Robinson et al. [38]. A total of 311 patients from 127 centers were reported. They had undergone a median of two lines of chemotherapy and 45% had undergone prior high-dose chemotherapy with autologous transplant. At transplantation, 158 patients had chemosensitive disease, 100 had chemoresistant disease, and 53 had untested relapse. Transplant donors were matched related donors (221), matched unrelated donors (61), and mismatched donors (16). PBSC were used in 263 of the cases. With a median follow up of 1 year, 59% of patients remained alive. The projected OS and PFS at 2 years were 46 and 26%. The 100 day transplant-related mortality was 17%, but transplant-related mortality increased to 24% at 1 year and 27% at 2 years. Chemoresistant disease again was also the only factor that predicted for worse for both PFS and OS (p < 0.0001).

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Evidence of a GVL effect is strongly suggested in these studies and comes from the following: a clinical response to DLI for those with relapsed or persistent disease [30], long-term survivors in patients treated with preparative regimens less aggressive than those used for their failed autograft, and a suggestion of a better outcome in patients with chronic GVHD. However, as relapses do occur after DLI the importance of GVL in this disease has not been fully clarified. Indeed, like chronic myeloid leukemia, it may be more effective as suggested in patients with chemosensitive, low tumor burden disease. With improvements in the management of GVHD and other regimen-related toxicities, and the suggestion in these studies that the more intensive reduced intensity regimens are associated with the best outcome, a reconsideration of fully ablative regimens seems warranted. While no such direct comparison has been reported, the EBMT reported a registry study that compared myeloablative and nonmyeloablative conditioning in Hodgkin’s disease [39]. The authors compared 97 patients undergoing allogeneic transplant after reduced intensity regimens to 93 patients transplanted after full myeloablative regimens. Nonrelapse mortality was significantly reduced in the reduced intensity group (HR 2.43, 95% CI 1.48–3.98, p £ 0.001). There was a trend toward improvement in progression free survival (HR 1.28, 95% CI 0.92–1.78, p = 0.1) and a statistically significant improvement in OS (HR 1.62, 95% CI 1.15–2.28, p = 0.005) as well for this group. As it appears that regimen intensity is becoming more defined for these patients, other strategies are being considered to decrease the high relapse rate associated with this therapy. Unpublished emerging data from our group suggest that disease burden at transplant is critical for long-term PFS, so combining disease burden reduction affected by an ASCT with the potential of a GVL effect from an allograft, a tandem approach seems appropriate to investigate. Such was recently reported by Carella et al. who treated ten patients with relapsed or refractory HD with a tandem approach of an ASCT with BEAM conditioning and then at a median of 84 days, these patients then underwent an allograft after a fludarabine/cyclophosphamide conditioning regimen [40]. In this high risk group, three patients achieved CR after the ASCT, but two of the three relapsed prior to allografting. One of them was able to attain CR with further chemotherapy. The remaining seven patients attained a PR after ASCT. Overall seven patients were alive at 210–700 days post-transplant, six in CR; two died of progressive disease and one died in CR of aspergillosis of the brain [31]. While these results are promising, larger studies with longer follow up will be necessary to confirm the results. However, with effective new agents emerging for the treatment of refractory Hodgkin’s lymphoma, simpler strategies may be as effective in maximizing responses prior to an allograft.

9.  Early Allogeneic Transplant in High Risk Patients While high-dose chemotherapy with ASCT has become the standard in patients who have failed multiple conventional chemotherapy and/or radiation regimens, a significant number of patients will fail ASCT as well and require further therapy, including allogeneic transplant. If this subset of patients could be identified prior to ASCT, they could be spared the morbidity of an ASCT and proceed directly to allografting. As detailed above, various studies of ASCT have identified patients not likely to do well. While tandem autografts

Chapter 6  Allogeneic Transplantation for Hodgkin’s Lymphoma 

may decrease the number who relapse, early allogeneic transplantation may be more advantageous in these high risk patients. In 2005, a prognostic model for prolonged event-free survival after autologous or allogeneic transplant was proposed by Hahn et al. [41]. They used a multivariate model to measure time to relapse, progression, or death. In their study, significant predictors of shorter event free survival were chemotherapy resistant disease, Karnofsky Performance Status <90, and three or more prior treatment regimens. Patients with two or three adverse factors had significantly shorter event-free survival (58% vs. 11%, p < 0.001) not only in autologous transplant but also in allogeneic transplants of 38% vs. 0%, p = 0.0022. In subsequent correspondence, it was suggested that this prognostic model may not be applicable to reduced intensity allogeneic transplants [42, 43]. As part of the US SWOG/CTN tandem ASCT study another multivariate analysis for outcome is to be done with future plans to select a group for an early allograft approach.

10.  Conclusions With standard chemotherapy and radiation, a CR is obtained in >90% of patients with early stage disease and >80% of advanced disease patients. Unfortunately, a significant minority of these young patients will not respond or will relapse. Options for these patients include salvage chemotherapy, autologous or allogeneic transplantation. ASCT has been shown to lead to a 30–70% long-term survival. Tandem ASCT have demonstrated success, but in some studies, a significant fraction of patients are not able to receive the second transplant. Multiple studies have now shown that there are significant adverse risk factors that predispose patients to failure of salvage chemotherapy and ASCT transplant. Analogous to the more common large cell lymphoma, there appear to be two distinct patient groups in Hodgkin’s disease. Late relapse with indolent, chemosensitive disease portends a good chance of cure with salvage chemotherapy or autologous transplant. Early relapse, chemoresistant disease, and progressive disease consistently have disappointing results with standard salvage regimens and ASCT. Early experience with allogeneic transplantation in high risk patients was also disappointing. While there was a notable decrease in relapses, there was also a high rate of transplant-related mortality. Consequently, despite some evidence of a GVL effect in Hodgkin’s lymphoma, allogeneic transplantation has typically been reserved for patients with biopsy-proven relapse after autografting, patients with chemoresistant disease, or patients in whom adequate stem cells could not be harvested for autologous transplant. Early studies of allogeneic transplants using reduced intensity conditioning regimens have demonstrated the ability to decrease transplant-related mortality, but more long-term follow up is necessary to demonstrate improvement in OS in Hodgkin’s Lymphoma patients (Table 6-4). Clearly, there is room for continued improvement in the treatment of high-risk Hodgkin’s lymphoma patients. Consideration is being given to moving allogeneic transplant with reduced intensity earlier in the algorithm for the treatment of high-risk patients. The theory is that the disease will not have been exposed to multiple chemotherapy regimens and may be more chemosensitive. Also, patients will have less change for a regimen-related lethal event and be physically more able to tolerate an allogeneic transplant. The use of maintenance therapy after transplant, as with histone deacetylase inhibitors being studied

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at our center, is also being investigated. The role of allogeneic transplant in Hodgkin’s disease continues to evolve. As more data with reduced intensity conditioning regimens become available and other innovative treatment adjuncts are tested, its role will become more defined.

References 1. Freedman AS, Nadler LM (1998) Malignancies of lymphoid cells. In: Fauci AS (ed) Harrison’s principles of internal medicine, 14th edn. McGraw-Hill, New York, p 708 2. Densmore JJ, Williams ME (2006) Lymphoproliferative diseases. In: Williams ME, Kahn MJ (eds) American society of hematology self-assessment program. Blackwell Publishing, Washington, DC, pp 260–264 3. Straus D, Portlock C, Qin J, Myers J (2004) Results of a prospective randomized clinical trial of doxorubicin, bleomycin, Vinblastine, and dacarbazine (ABVD) followed by radiation therapy (RT) varsus ABVD alone for stages I, II, and IIIA non-bulky Hodgkin Disease. Blood 104:3483–3489 4. leMaignan C, Desablens B, Delwail V, Dib M, Berthou C (2004) Three cycles of adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) or epirubicin, bleomycin, vinblastine, and methotrexate (EBVM) plus extended field radiation therapy in early and intermediate Hodgkin disease: 10-year results of a randomized trial. Blood 103:58–66 5. Engert A, Franklin J, Eich H, Brillant C, Sehlen S, Cartoni C (2007) Two cycles of doxorubicin, bleomycin, vinblastine, and dacarbazine plus extended field radiotherapy is superior to radiotherapy alone in early favorable Hodgkin’s lymphoma: Final results of the GHSG HD7 trial. J Clin Oncol 25:3495–3502 6. Santoro A, Bonadonna G, Valagussa P, Zucali R, Vivani S, Villani F (1987) Longterm results of combined chemotherapy-radiotherapy approach in Hodgkin’s disease: superiority of ABVD plus radiotherapy versus MOPP plus radiotherapy. J Clin Oncol 5:27–37 7. Duggan D, Petroni G, Johnson J, Glick J, Fisher R, Connors J (2003) Randomized comparison of ABVD and MOPP/ABV Hybrid for the treatment of advanced Hodgkin’s disease: report of an intergroup trial. J Clin Oncol 21:607–614 8. Aisnberg AC (1999) Problems in Hodgkins disease management. Blood 93: 761–779 9. Josting A, Franklin J, May M, Koch P, Beykirch M, Heinz J (2002) New prognostic score based on treatment outcome of patients with relapsed Hodgkin’s lymphoma registered in the database of the german Hodgkin’s lymphoma study group. J Clin Oncol 20:221–230 10. Bonfonte V, Santoro A, Vivani S, Devizzi L, Balzarotti M, Soncini F (1997) Outcome of patients with Hodgkin’s disease failing after primary MOPP-ABVD. J Clin Oncol 15:528–534 11. Linch DC, Winfield D, Goldstone AH et  al (1993) Dose intensification with autologous bone marrow transplantation in relapsed and resistant Hodgkin’s disease: results of a BNLI randomised trial. Lancet 341:1051–1054 12. Josting A, Raemakers J, Diehl V, Engert A (2002) New concepts for relapsed Hodgkin’s disease. Ann Oncol 13:117–121 13. Reece D, Connors J, Spinelli J, Barnett M (1994) Intensive therapy with cyclophosphamide, carmustane, etoposide +/– cisplatin and autologous bone marrow transplantation for Hodgkin’s disease in first relapse after combination chemotherapy. Blood 83:1193–1199 14. Moskowitz C, Nimer S, Zelenetz A, Trippett T (2001) A 2-step comprehensive high-dose chemoradiotherapy second-line program for relapsed and refractory Hodgkin’s disease: Analysis by intent-to-treat and development of a prognostic model. Blood 97:616–623

Chapter 6  Allogeneic Transplantation for Hodgkin’s Lymphoma  15. Horning S, Chao N, Negrin R, Hoppe R (1997) High-dose therapy and autologous hematopoeitic progenitor cell transplantation for recurrent or refractory Hodgkin’s disease: analysis of the Stanford University results and prognostic indicies. Blood 89:801–813 16. Stiff P, Unger J, Forman S, McCall A (2003) The value of augmented preparative regimens combined with an autologous bone marrow transplant for the management of relapsed or refractory Hodgkin disease: A southwest oncology group phase II trial. Biol Blood Marrow Transplant 9:529–539 17. Josting A, Rudolph C, Mapara M (2005) Cologne high dose sequential chemotherapy in relapsed and refractory Hodgkin’s and aggressive non-Hodgkin’s lymphoma – results of a multi-center phase-II study. Ann Oncol 16(1):116–123 18. Ahmed T, Lake D, Beer M (1997) Single and double auto-transplants for relapsing/ refractory Hodgkin’s disease: Results of two consecutive trials. Bone Marrow Transplant 19:449–454 19. Brice P, Divine M, Simon D, Coiffier B (1999) Feasability of tandem autologous stem-cell transplantation (ASCT) in induction failure or very unfavorable relapse from Hodgkin’s disease. Ann Oncol 10:1485–1488 20. Fung H, Stiff P, Schriber J, Toor A (2007) Tandem autologous stem cell transplantation for patients with primary refractory or poor risk recurrent Hodgkin lymphoma. Biol Blood Marrow Transplant 13:594–600 21. Akpek G, Ambinder RF, Piantadosi S, Abrams R (2001) Long-term results of blood and marrow transplantation for Hodgkin’s lymphoma. J Clin Oncol 19:4314–4321 22. Pedersen-Bjergaard J, Anderson M, Christiansen D (2000) Therapy-related acute myelogenous leukemia and myelodysplasia after high-dose chemotherapy and stem cell transplantation. Blood 95:3273–3279 23. Miller J, Arthur D, Litz C, Neglia J (1994) Myelodysplastic syndrome after autologous bone marrow transplantation: an additional late complication of curative cancer therapy. Blood 83:3780–3786 24. Vose JM, Bierman PJ, Anderson JR et  al (1992) Progressive disease after highdose therapy and autologous transplantation for lymphoid malignancy: clinical course and patient follow-up. Blood 80:2142–2148 25. VandenBerghe E, Pearce R, Taghipour G, Fouilard L, Goldstone AH (1997) Role of second transplant in the management of poor-prognosis lymphomas: a report from the European blood and bone marrow registry. J Clin Oncol 15:1595–1600 26. Jones RJ, Ambinder RF, Piantadosi S, Santos GW (1991) Evidence of a graftversus-lymphoma effect associated with allogeneic bone marrow transplantation. Blood 77:649 27. Anderson JE, Litzow MR, Appelbaum FR, Schoch G (1993) Allogeneic, syngeneic, and autologous marrow transplantation for Hodgkin’s disease: the 21-year seattle experience. J Clin Oncol 11:2342–2350 28. Milpied N, Fielding A, Pearce R, Ernst P (1996) Alogeneic bone marrow transplantation is not better than autologous transplantation for patients with relapsed Hodgkin’s disease. J Clin Oncol 14:1291–1296 29. Gajewski JL, Phillips GL, Sobocinski KA, Armitage JO, Gale RP (1996) Bone marrow transplants from HLA-identical siblings in advanced Hodgkin’s disease. J Clin Oncol 14:572–578 30. Anderlini P, Acholonu S, Okoroji G-J, Andersson B (2004) Donor leukocyte infusions in relapsed Hodgkin’s lymphoma following allogeneic stem cell transplantation: CD3+ cell dose, GVHD, and disease response. Bone Marrow Transplant 34:511–514 31. Anderlini P, Giralt S, Andersson B, Ueno N (2000) Allogeneic stem cell transplantation with fludarabine-based, less intensive conditioning regimens as adoptive immunotherapy in advanced Hodgkin’s disease. Bone Marrow Transplant 26:615–620

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W. Broderick and P. Stiff 32. Lush R, Jones S, Haynes A (2001) Advanced-stage, chemorefractory lymphocyte-predominant Hodgkin’s disease: Long-term follow-up of allografting and monoclonal antobody therapy. Br J Haem 114:734–735 33. Cooney J, Stiff P, Toor A, Parthasarathy M (2003) BEAM allogeneic transplantation for patients with Hodgkin’s disease who relapse after autologous transplantation is safe and effective. Biol Blood Marrow Transplant 9:177–182 34. Anderlini P, Saliba R, Acholonu S, Okoroji G-J, Donato M (2005) Reducedintensity 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 35:943–951 35. Peggs K, Hunter A, Chopra R, Parker A (2005) Clinical evidence of a graft-versusHodgkin’s-lymphoma effect after reduced-intensity allogeneic transplantation. Lancet 365:1934–1941 36. Alvarez I, Sureda A, Caballero M, Urbano-Ispizua A (2005) Nonmyeloablative stem cell transplantation is an effective therapy for refaractory or relapsed Hodgkin lymphoma: results of a spanish prospective cooperative protocol. Biol Blood Marrow Transplant 12:172–183 37. Baron F, Storb R, Storer BE et al (2006) Factors associated with outcomes in allogeneic hematopoietic cell transplantation with nonmyeloablative conditioning after failed myeloablative hematopoietic cell transplantation. J Clin Oncol 25:4150–4157 38. Robinson S, Schmitz N, Taghipour G, Sureda A (2004) Reduced intensity allogeneic stem cell transplantation for hodgkin’s disease. Outcome depends primarily on disease status at the time of transplantation (abstract). Blood 104: Abstract 2322 39. Sureda A, Robinson S, Ruiz de Elvira C (2003) Non ablative allogeneic stem cell transplantation reduces transplant related mortality in comparison with conventional allogeneic transplantation in relapsed and refactory Hodgkin’s disease: results of the European group for blood and marrow transplantation. Blood 2003:692a 40. Carella A, Cavaliere M, Lerma E (2000) Autografting followed by nonmyeloablative immunosuppressive chemotherapy and allogeneic peripheral-blood hematopoietic stem-cell transplantation as treatment of resistant Hodgkin’s disease and NonHodgkin’s lymphoma. J Clin Oncol 18:3918–3924 41. Hahn T, Benekli M, Wong C, Moysich K (2005) A prognostic model for prolonged event-free survival after autologous or allogeneic blood or marrow transplantation for relapsed and refractory Hodgkin’s disease. Bone Marrow Transplant 25:557–566 42. Tey S, Butler J, Durrant S, Hill G, Morton J, Kennedy G (2005) Correspondence: a prognostic model for prolonged event-free survival after autologous or allogeneic blood or bone marrow transplantatin for relapsed and refractory Hodgkin’s disease. Bone Marrow Transplant 36:553–554 43. Hahn T, McCarthy P Jr (2005) Response to Tey et  al. Bone Marrow Transplant 36:554

Chapter 7 Myeloablative Allogeneic Stem Cell Transplantation for Non-Hodgkin’s Lymphoma J. Kuruvilla, P. Mollee, and J.H. Lipton

1.  Introduction The non-Hodgkin’s lymphomas (NHL) encompass a broad range of lymphoid malignancies that vary considerably in their underlying biology, natural history, and response to therapy. Classification schemas for NHL have evolved with the current World Health Organization (WHO) system attempting to identify biologically distinctive diseases that are usually associated with unique clinical presentation, natural history, and outcome. Conventional myeloablative allogeneic stem cell transplantation (allo-SCT) has long been studied in the treatment of acute leukemias and chronic myeloid leukemia. More recently allo-SCT has been increasingly applied to the management of NHL although the precise indications of allo-SCT in these disorders are not well defined. The role of allo-SCT in the treatment of NHL remains unclear. Randomized controlled trials have shown that autologous transplantation as part of secondline therapy appears to improve overall survival [1, 2], and several trials have evaluated autologous transplantation as consolidation compared to observation following primary chemotherapy for follicular lymphoma (FL) [3–5]. There are no randomized clinical trials of allo-SCT vs. autologous stem cell transplantation (ASCT) or conventional chemotherapy, nor do they seem likely. As the disease biology and thus the treatment approach (and particularly the availability of curative therapy) varies significantly between subtypes of NHL, few generalizations about allo-SCT can be made across all types of NHL. Determination of the optimal role and timing of allo-SCT in lymphoma requires an understanding of outcomes associated with allografting in each of the lymphoma subtypes as the outcome of the therapy can vary considerably between histologies as well as between different time points in the disease course. As such, the assessment of the role of allo-SCT in NHL is compromised by the paucity of quality trials in the literature. Most reports include highly heterogeneous patient cohorts with varying histologies (encompassing indolent and aggressive NHL as well as Hodgkin lymphoma), varying stages of disease (from patients in first complete remission to those in chemorefractory late relapse), and differing conditioning regimens (including reduced intensity From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_7, © Springer Science + Business Media, LLC 2003, 2010

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Table 7-1.  Conventional allogeneic stem cell transplantation for non-Hodgkin’s lymphoma. Advantages

Disadvantages

Normality of stem cells

Increased non-relapse mortality

Graft vs. lymphoma effect

Increased “allogeneic-related” morbidity

Avoidance of secondary AML/MDS

Age and donor restrictions

Absence of graft contamination by lymphoma

Increased costs

Alkylator and radiotherapy sensitive

Lack of randomized trials

regimens). There is a lack of central pathology review and most studies are affected by the many biases that come with retrospective cohort studies. Not surprisingly, the results of these studies are often inconsistent. 1.1.  Potential Advantages of Allo-SCT in NHL (Table 7-1) The lymphomas are generally sensitive, in a steep dose-response manner, to alkylating agents and radiation – agents that are typically employed in highdose conditioning regimens. Dose intensity (as demonstrated in the autologous setting) has been shown to improve response rates as well as potentially improving progression-free or overall survival [1, 2]. Evidence is accumulating (discussed below) that cure of lymphoma is not achieved purely via highdose therapy alone, but rather via the presence of a graft-vs-lymphoma effect (GVLy). There also appears to be a lower risk of secondary MDS/AML [6, 7] in comparison to ASCT [8]. The other advantage of using cells from an allogeneic donor is that they are not contaminated with lymphoma cells and the risk of infusing tumor cells is avoided. Similarly, a small population of patients who fail to obtain an autologous graft are obvious candidates for allo-SCT. 1.2.  Potential Disadvantages of Allo-SCT in NHL (Table 7-1) As seen with other transplant indications, any reduction in relapse achieved with allo-SCT is offset by mortality attributable to the treatment itself. The morbidity associated with allogeneic transplantation is also well recognized. Allo-SCT is limited by the availability of a suitably matched donor and has greater restrictions regarding recipient age, performance status, and organ function than ASCT. Finally, in comparison to ASCT, allo-SCT is an expensive way to deliver curative therapy to patients with malignant disease [9]. In this chapter, we summarize the evidence supporting myeloablative alloSCT in NHL on the basis of common individual disease histologies; follicular, mantle cell, diffuse large B cell (DLBC), and variants as well as lymphoblastic and Burkitt lymphoma.

2.  Allogeneic SCT in Indolent Lymphoma 2.1.  Introduction ASCT for FL has been evaluated in the front-line and relapsed setting and has resulted in an improved progression-free survival when compared to anthracycline-based chemotherapy [1, 3–5]. The randomized European CUP trial

Chapter 7  Myeloablative Allogeneic Stem Cell Transplantation for Non-Hodgkin’s Lymphoma 

was the only study to demonstrate an overall survival advantage for ASCT in a small group of patients with relapsed FL [1]. While ASCT has greater applicability in FL (compared to myeloablative allo-SCT) as it can be considered in patients up to age 65 or 70 years and may be performed with a relatively low treatment-related mortality (TRM), data fail to demonstrate a plateau in survival curves and there are concerns with secondary myelodysplasia and acute leukemia [1, 3–5]. Dose intensive chemotherapy has been demonstrated to improve progressionfree and overall survival in the relapsed lymphoma setting with the use of autografts to ameliorate the hematologic toxicity of high dose chemotherapy [1, 2]. Tumour graft contamination, as part of a SCT procedure, can be reduced through the use of purging strategies in the autologous setting (although the benefit of this remains a debatable point given the inconsistent published results) or eliminated through the use of syngeneic or allogeneic stem cells from a healthy donor [1, 10–13]. 2.2.  Is There a GVLy Effect in Indolent NHL? Donor-derived SCT offers the additional benefit of the GVLy effect that could provide sustained immune surveillance and a reduced rate of lymphoma recurrence in the recipient. The evidence of a GVLy effect has been indirectly suggested on the basis of a reduction in the rate of relapse in indolent NHL patients undergoing allogeneic SCT (allo-SCT) when compared to autologous SCT (ASCT), but the significance of this effect remains in question as this has never been tested in a large randomized trial [14–17]. Further direct evidence of a GVLy effect has been demonstrated through the use of donor lymphocyte infusion (DLI) that may induce remission in patients following allo-SCT [18–20]. 2.3.  Clinical Results in Indolent NHL Registry and single center reports have failed to clearly demonstrate the potential benefits of allo-SCT (particularly overall survival) and these data remain difficult to interpret because of sample size, patient selection, and study heterogeneity. Institutional studies are largely retrospective and include a mixture of indolent lymphoma subtypes although follicular lymphoma is the most indolent NHL subtype (Table  7-2). With a median follow-up of between 2 and 4 years, overall survival estimates range from 65 to 80% with non-relapse mortality of 20–35% [21–25]. The patients in these series are typically young (40–55 years of age) and may have a significant proportion of patients with refractory disease. Progression or event-free survival estimates range between 50 and 75% with few relapses reported. Larger series including registry series report comparative results of alloSCT with ASCT (Table 7-3). The smaller single institution series from MD Anderson and Utrecht are not matched with the allograft cohorts including more chemoresistant disease [26, 27]. However, the relapse rate appears higher in the ASCT arms of these retrospective series with a higher rate of non-relapse mortality in the allo-SCT cohorts although overall survival appears to favor the allograft patients. Patient selection remains a concern in

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Table 7-2. Single arm cohort studies detailing outcome of allo-SCT for ³20 patients with follicular lymphoma. N (all/ FL)

Refractory at alloSCT (%)

Toze (2000)

26/16

11/26

30% at 2 54% EFS years

58%

Median followup 2.4 years

Forrest [22]

24

1/22

21% at 2 78% PFS years

78%

Median followup of 2.3 years

33%

67%

Mandigers [23] 15/13 Kiss [24]

37/21

Yakoub-Agha [25]

16 (14 MA)

0

NRM

PFS/EFS

OS

Median Relapse follow-up

2/15

Median followup of 3 years

19%

70% RFS at 5 years

79% at 5 years

Median followup of survivors 4.2 years

5/16

55% EFS at 2 years

68% at 2 2 years

Median followup of 3.2 years

a Outcome includes all patients, not just those with intermediate-grade histology AlloSCT allogeneic stem cell transplantation, FL follicular lymphoma, NRM non-relapse mortality, PFS progression-free survival, EFS event-free survival, OS overall survival, MA myeloablative

the larger EBMT and IBMTR reviews but the registries have the advantage of larger patient numbers [10, 11]. The EBMT/IBMTR review included an analysis of patients with low grade NHL and compared outcomes of 199 allograft procedures (165 T-replete with 537 autografts; 394 unpurged and 143 purged) and 26 syngeneic transplants [10]. The relative risk of relapse was similar between syngeneic, T-cell replete and T-cell depleted allografts. Unpurged autografts had a statistically, significantly higher relative risk of relapse than syngeneic transplants suggesting that tumor cell contamination in autografting may be a larger factor in determining relapse than any putative GVLy effect present with an allograft. Overall survival was inferior in the T-cell replete allograft patients when compared to that in syngeneic recipients and this would be reflective of higher non-relapse mortality. A large study of patients with low grade NHL from the EBMT compared 231 allograft recipients with 2047 autograft patients [15]. The investigators also reported the results of a case control analysis (three matched ASCT patients for each allograft recipient) that was matched with status at SCT, year of SCT and age at diagnosis, SCT, and size of the largest lymphoma mass at SCT. A log-rank analysis demonstrated that overall survival and treatmentrelated mortality favored ASCT, progression-free survival was similar between allo-SCT and ASCT, and a reduced relapse rate was observed in the allo-SCT cohort. Overall, the data show that there appears to be a GVLy effect in indolent NHL as demonstrated by the demonstration of disease response with donor leukocyte infusion and series that show a reduced relapse rate in allograft recipients. Toxicity remains a significant concern and applicability remains an issue as myeloablative SCT is generally restricted to younger patients and TRM may be high (particularly when considering unrelated donor SCT).

10 18

Auto

14 697/2 047

Auto

Allo

1 185/231

Allo

68

Auto

597

Auto (Unpurged) 44

131

Auto (Purged)

Allo

176

26

143 (auto purged)

394 (auto unpurged)

34 (T-deplete)

165 (T-replete)

Allo

Syngeneic

Auto

Allo

0

3/10

NA

38% at 4 years

12%

34%

8%

14%

30%

NRM

22% at 2 years

68% at 2 years

NA

42.7% at 4 years

17% DFS

45% DFS

PFS/EFS

3/18

7/10

NA

51.1% at 4 years

34%

49%

55%

62%

51%

OS

83%

0

NA

NA

74%

19%

58%

43%

21%

Relapse

All chemosensitive

7/10 chemoresistant

Matched case control study showed better OS and NRM with ASCT, lower relapse with allo-SCT

Auto – 6% day 100 TRM, 6% TRM from t-MDS/AML

More chemoresistant in allo group

For OS, T-replete inferior to T-deplete and syngeneic SCT

Unpurged auto had higher RR for relapse than syngeneic

RR or relapse similar between allo and syngeneic, T-replete and T-deplete

Comment

Int-grade intermediate-grade non-hodgkin’s lymphoma, NRM non-relapse mortality, PFS progression-free survival, EFS event-free survival, OS overall survival, RR relapse rate

Verdonck

Peniket EBMT (2003)

Hosing MDACC (2003)

Van Besien IBMTR (2003)

Bierman IBMTR (2003)

N (all/FL)

Table 7-3.  Comparative analyses detailing outcome of transplantation for indolent histology non-Hodgkin’s lymphoma. Chapter 7  Myeloablative Allogeneic Stem Cell Transplantation for Non-Hodgkin’s Lymphoma  93

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2.4.  MUD BMT The role of matched unrelated donor (MUD) allo-SCT in indolent NHL has not been well studied. Institutional series report small numbers of MUD procedures but there are not many larger series. The largest series comes from the National Marrow Donor Program (NMDP) and reports the outcomes of 158 MUD recipients with NHL of whom 40 had indolent histology [28]. The 2-year PFS was 34% while actuarial mortality for the entire cohort at day 100 was 45%. The EBMT reported the outcomes of 56 MUD recipients with NHL of which six patients had indolent NHL [29], while the Japanese marrow donor program recently reported a series of MUD transplants including nine patients with FL [30]. Both series did not report specific outcomes for the indolent histology cases. 2.5.  When to Transplant? The optimal timing of allo-SCT in FL remains an open question; it is a disease with a typically indolent course that is frequently chemosensitive and patients may enjoy long remissions after therapies that may be intermittently applied over a number of years. While clinical factors such as those in the follicular lymphoma international prognostic index (FLIPI) may identify a high risk group of patients with a poor expected survival from diagnosis or at relapse, these prognostic factor analyses have not included therapy or remission duration as a factor [31]. Remission duration generally tends to shorten with the application of second-line or subsequent therapies, and therefore, multiple lines of therapy tend to be a significant factor in determining outcome as this is a marker of presumed chemoresistance. Allo-SCT is a therapy that will be used later in the disease course and given current clinical practice, patients will have received multiple applications of rituximab (with or without chemotherapy) and are likely judged to be “rituximab refractory” although this definition remains unclear. Results from carefully constructed prospective clinical trials that focus on discrete patients groups with a common histology will be required to allow allo-SCT to become one of the standard options that are available to patients with follicular lymphoma. Newer reduced intensity conditioning SCT (RICSCT) techniques are appealing as they increase the availability of allogeneic SCT procedures by increasing the upper age limit, expanding the limit of organ function and comorbidity and appear to reduce early TRM [20, 32–35]. RICSCT techniques improve the availability of allo-SCT to patients with FL in general; caution must be applied while interpreting results with short follow-up and consideration should be given to more proven strategies such as myeloablative allo-SCT in eligible patients.

3.  Allogeneic SCT in Mantle Cell Lymphoma Most patients with mantle cell lymphoma (MCL) present with advanced stage disease and while it is generally classified as an indolent lymphoma, its clinical behavior is usually one of an aggressive disease. Although conventional CHOP-like therapy, fludarabine-based therapy, and more recently, anti-CD20based therapies have shown increasing responses, the majority of patients will relapse and hence allo-SCT has been increasingly explored in this patient population.

Chapter 7  Myeloablative Allogeneic Stem Cell Transplantation for Non-Hodgkin’s Lymphoma 

3.1.  Is There a Graft-vs-Lymphoma Effect in MCL? Even though the literature in this area is limited, there is emerging evidence to support a GVLy effect in MCL. This includes reduced relapse rates after allografts, both in comparison to historic controls and to autografts [36–41]; disease response to withdrawal of immunosuppression [41]; delayed progressive post-transplant improvement in clinical or molecular remission status in the absence of anti-lymphoma therapy [36, 42, 43]; and clinical responses to DLI [19, 20, 36, 44–46]. In contrast, there is a substantial relapse rate postallo-SCT in an EBMT registry study [47] with similar relapse rates noted between auto- and allo-SCT in a report from the Johns Hopkins Cancer Center [48] and a lack of any plateau in the survival curves reported in an IBMTR registry study [38]. Thus while a GvLy effect clearly exists in MCL, questions remain regarding whether the GvLy effect is potent enough to overcome the almost inevitable risk of relapse in these patients. 3.2.  Clinical Results in MCL Several recent publications of myeloablative allo-SCT have expanded our understanding of allo-SCT in MCL (Table 7-4). The median age at transplant has been 45–50 years, several years less than the median age of patients with MCL, indicating the difficulty in broad application of conventional transplantation to this population. The small patient numbers and variety of conditioning regimens in these reports make interpretation of the data difficult. The largest experience comes from registry studies of the EBMT [49] and IBMTR [38], both having only been presented in abstract form. Vandenberghe [49] analyzed the outcomes of 22 patients with MCL and found a 2 year EFS and OS of 50 and 62%, respectively. The median follow-up was too short to establish any curative potential of allo-SCT. Armitage [38] presented the IBMTR experience of 212 patients (188 matched related donors and 28 unrelated or haploidentical donors). Sixteen percent of patients were transplanted in first remission, 38% in relapse or second remission and 46% were never in remission. With a median follow-up of 12 months (range, 3–106 months), EFS was extremely poor at 2 years with 2 year OS being ~40%. Disappointingly, there was no plateau in the EFS curve, even for patients transplanted in first remission. This registry data suggested that MCL remains incurable with conventional allo-SCT although follow-up is relatively short. Contrasting results are presented in the two small series with the longest follow-up. The City of Hope experience of 13 patients with a median follow-up of 4.75 years found a 3 year PFS of 53% with infrequent late relapses [39]. The Princess Margaret Hospital also detailed six patients with a median follow-up in excess of 4 years [37]. Remarkably, there was no TRM and no relapses in six patients, all with relapsed disease. One patient remains in remission 11 years post-transplant. Thus, for select patients, prolonged disease free survival and probable cure are achievable following allo-SCT. The importance of chemosensitivity in determining outcome after allo-SCT for MCL has been repeatedly demonstrated across most studies [36, 38, 49, 50]. This pattern is not dissimilar to that seen with ASCT, suggesting that there is only a limited role for allo-SCT to overcome chemoresistance. The importance of TBI in the conditioning regimen for mantle cell lymphoma remains uncertain. While an initial report in the autologous setting suggested that TBIbased conditioning was superior [51], this finding has not been supported by subsequent series [48, 52].

95

14 (+13 auto)

Laudi (2006)

48 years

47 years

29% at 5 years

50% at 5 years

19% at d100 44% at 5 years

Never in CR 46% Relapse/CR2 38% No refractory

28% refractory

N/A

N/A N/A

Relapse

Comment

49% at 5 years 21% at 5 years All patients chemosensitive No plateau in survival curve AlloSCT has increased NRM but lower relapse vs. autoSCT 49% at 5 years 1 Survival figures include 4 nonmyeloablative transplants

1 year FFP 90% vs. 44% for chemosensitive vs. refractory disease 42% at 3 years 37% at 3 years 20% at 3 years Refractory disease prognostic for worse EFS 1 late relapse at 51 months 100% at 100% at 0 1 patient in ongoing 4 years 4 years remission at 11 years 53% at 3 years 51% at 3 years 14% at 3 years 50% at 2 yr 62% at 2 yr N/A Refractory disease prognostic for EFS and OSa <20% at ~40% at N/A AlloSCT has increased 2 years 2 years NRM but lower relapse vs. autoSCT

OS

12% at 3 years 23% at 3 years N/A 55% at 3 years 55% at 3 years 1

CR1 16%

23% refractory N/A

0%

53% at 3 years

7 of 12 6 of 16

EFS/DFS

NRM non-relapse mortality, EFS event-free survival, DFS disease-free survival, OS overall survival, NA not available, FFP freedom from progression a  Both autologous stem cell transplant (autoSCT) and conventional allogeneic stem cell transplant (alloSCT)

17 (+80 auto)

Ganti [40]

212 (+553 auto) 50 years

47 years

No refractory

48.3 years

13 (+ 64 auto) 22 (+150 auto)

4% refractorya

55 yearsa

Berdeja [50] updated 19 (+ 38 auto) in Kasamon [48] and Kasamon (2007) Rifkind (2004) 6

Popplewell [39] Vandenberghe/ EBMT (2000) Armitage/IBMTR (2002)

25% refractory 5 refractory

47 years 52 years

12 (+ 16 auto) 16

Sohn (1998) Khouri [36]

a

a

Disease status at transplant NRM

N

Author

Median age

Table 7-4.  Selected studies (>5 patients) of conventional allogeneic stem cell transplantation in mantle cell lymphoma.

96  J. Kuruvilla et al.

Chapter 7  Myeloablative Allogeneic Stem Cell Transplantation for Non-Hodgkin’s Lymphoma 

Overall, conclusive evidence that conventional allo-SCT offers a cure for MCL is still lacking, although small numbers of patients with prolonged disease-free survival are appearing. The TRM associated with this procedure should preclude its use in first remission, but it may still play a role in patients in chemosensitive relapse.

4.  Allogeneic SCT in Aggressive Lymphomas 4.1.  Introduction Diffuse large B cell lymphoma (DLBCL) is the most common subtype of the aggressive or intermediate grade lymphomas; however, there are few reports of allo-SCT restricted to DLBCL. Most studies include a smattering of T-cell lymphomas and a variety of what is now considered to be indolent B cell lymphomas. Anthracycline-based chemotherapy with rituximab cures over 50% of patients with DLBCL [53], but for those with high-risk features at diagnosis or with relapsed disease, cure rates remain suboptimal. There is no randomized study demonstrating superiority of allo-SCT over other therapies as there is for ASCT [2], and as such, the use of allo-SCT in the management of aggressive histology lymphomas remains controversial and in many centers is somewhat unfashionable. 4.2.  Is there a Graft-vs-Lymphoma Effect in Aggressive NHL? Comparative trials of auto and allo-SCT have unfortunately revealed conflicting results regarding relapse rates with both positive [15, 54–56] and negative studies [57, 58]. Relapse is also neither consistently decreased in the presence of GVHD [15, 54, 55, 57, 59–62], nor increased after transplantation with T-cell depleted marrow [10, 63, 64]. Isolated but impressive cases of response to withdrawal of immunosuppression and to donor leukocyte infusions have also been reported in some [60, 65, 66], but not most cases [66, 67]. Biologically, differences in relapse between allo-SCT and ASCT may be due not only to GVLy, but potentially also to graft contamination by lymphoma cells or to greater efficacy of the TBI-based regimens generally used in alloSCT. To circumvent the issue of graft contamination in assessing relapse rates, Bierman recently reported a comparison of syngeneic transplants with auto and allo-SCT for lymphoma [10]. Compared to syngeneic transplants, there was no difference in relapse rates following allografts in the intermediate-grade lymphoma category. Thus, the evidence supporting a clinically meaningful graft-vs-tumor effect in intermediate-grade lymphoma is not compelling. It is possible that the slow acting GVLy effect is overridden by the rapidity of growth of the tumor. 4.3.  Clinical Results in Aggressive NHL Table 7-5 details select single arm cohort studies detailing specific outcomes of patients with aggressive non-Hodgkin’s lymphoma. While difficult to draw firm conclusions from these reports, they do demonstrate that long-term disease free survival is possible following allo-SCT even though transplantrelated mortality remains higher than that seen generally with autoSCT. Dhedin et  al [60] have published the largest detailed series of allo-SCT for

97

44/44

37/21

233/111

32/20

Doocey [68]

Juckett [64]

Kim [72]

Stein (2001)

44%

NA

29%

20%

37%

5 years PFS 40%

41% at 5 years

OS

5 years PFS 33%

NA 5 year EFS 11%a

N/A

42%a 17 of 32a

NA

NA

43% at 5 years

32% at 5 years

30% at 5 years

Relapse

Chemosensitivity most important prognostic factor

T cell depleted marrow. 5 years PFS 40% vs. 17% for chemosensitive vs. refractory disease

Durable remissions in chemorefractory patients

Subgroup of 22 patients with chemosensitive relapse had 60%PFS and 13% relapse

Comment

AlloSCT allogeneic stem cell transplantation, int-grade intermediate-grade non-Hodgkin’s lymphoma, NRM non-relapse mortality, PFS progression-free survival, EFS event-free survival, OS overall survival

15% at 5 year

35% at 5 years

N/A

25% at 1yr 5 years EFS 43% 48% at 5 years

32 of 73

PFS/EFS

 Outcome includes all patients, not just those with intermediate-grade histology

a

73/73

Dhedin [60]

Refractory at N (all/int-grade) alloSCT (%) NRM

Table 7-5.  Single arm cohort studies detailing outcome of alloSCT for ³20 patients with aggressive histology non-Hodgkin’s lymphoma.

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aggressive non-Hodgkin’s lymphoma (Burkitt’s and lymphoblastic lymphoma excluded). When the analysis was confined to 22 patients in chemosensitive relapse, the progression-free survival was 60%, a result that compares favorably with that in most ASCT series. The Vancouver group [68] noted a 5-year overall survival of 48% in 44 patients with intermediate-grade histology, some of whom were primary induction failures. The role of allo-SCT and ASCT for patients with aggressive NHL has been examined by a number of different comparative methodologies and is presented in Table 7-6. These reports, primarily from registry data, provide some guidance on the relative merits of the two approaches. A case-controlled study by the EBMT [57] matched 101 patients with lymphoma undergoing allo-SCT with 101 patients undergoing ASCT for disease status and stage at transplant, histological grade, transplant conditioning, age, sex, and time from diagnosis to transplant. Of these, 43 patients in each cohort had intermediate-grade lymphoma. Non-relapse mortality was less for ASCT patients (28% vs. 14%, p = 0.008), but there was no significant difference in PFS or relapse rates. The EBMT more recently compared allo-SCT and ASCT again using a matched case control design [15]. Of the 1185 patients undergoing allo-SCT 147 had intermediate-grade disease with resultant 42% non-relapse mortality, 35% PFS, and 38% overall survival at 4 years. In a matched (1:3) comparison with ASCT patients, relapse was reduced but overall survival was significantly worse as a result of the higher transplant-related mortality in the allo-SCT cohort. Bierman recently completed an analysis comparing syngeneic, auto-, and allo-SCT drawing patients from the EBMT, IBMTR, and ABMTR. [10] Five year disease-free survival was significantly worse for recipients of T-cell replete allogeneic grafts compared to those of syngeneic grafts (31% vs. 42%, p = 0.03), but there were no significant differences between syngeneic and autologous transplants. Similar findings were present with respect to overall survival. 4.4.  The Importance of Chemosensitivity The importance of chemosensitivity in determining outcome after allo-SCT for aggressive lymphoma has been repeatedly demonstrated across most studies and is the most consistent predictor of relapse, as well as event-free and overall survival [54, 55, 57–62, 64, 69–72]. 4.5.  MUD BMT With only 30%–40% of patients having a matched, related donor available, MUD transplants may increase the applicability of allo-SCT. Two registry reports describe outcomes for patients with aggressive histology lymphoma. The IBMTR [28] reported a series of 158 patients of whom 50 had intermediate-grade lymphoma. For the whole group, 100-day mortality was 45% and for the intermediate-grade group 2-year PFS was 35%. The EBMT has also reported their experience of 56 patients undergoing MUD BMT for lymphoma, 26 of whom were adults with aggressive non-Hodgkin’s lymphoma [29]. Transplant-related mortality was 42% and progression-free survival at 2 years was 16%. These reports indicate that while long-term survival is achievable for select patients, transplant-related mortality remains unacceptably high, limiting wider application of myeloablative MUD transplants.

99

Auto

49%

NA

35% at 4 years

42% at 5 years

34% at 5 years

31% at 5 years

PFS/EFS

24%

31% at 3 years

19% at 3 years

33% at 3 years

24% at 3 years

N/A

N/A

NA

38% at 4 years

48% at 5 years

39% at 5 years

32% at 5 years

OS

Comment

Matched case-control study of registry data

Matched case control study showed better OS and NRM with autoSCT, but increased relapse

Unusually high NRM in autoSCT

48% at 3 years Only diffuse large cell lymphoma

55% at 3 years Single centre retrospective study

35% (p = NS)

29%

NA

NA

29% at 5 years Syngeneic better OS and EFS than alloSCT, but not autoSCT

54% at 5 years

24% at 5 years No direct comparison of auto vs. alloSCT

Relapse

Int-grade intermediate-grade non-hodgkin’s lymphoma, NRM non-relapse mortality, PFS progression-free survival, EFS event-free survival, OS overall survival

51%

138/138

14%, Auto better 43% (p = NS) (p = 0.008)

28%

NA

45/45

101/43

14687/2863

Auto

Allo

1185/147

Allo

42% at 4 years

N/A

31/89

Syngeneic

N/A N/A

194/774

Auto (unpurged) 1417/2018

Allo (T-cell replete)

Aksentijevich Allo [71] Auto

Chopra [57]

Peniket [15]

Bierman [10]

N (all/int-grade) NRM

Table 7-6.  Comparative analyses detailing outcome of transplantation for aggressive histology non-Hodgkin’s lymphoma.

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4.6.  NK and T-cell Lymphomas There remains a paucity of published data in regard to the role of allo-SCT in the management of NK and T-cell lymphomas. A number of small series demonstrate that reasonable survivals are achievable, especially in chemosensitive disease, although non-relapse mortality remains high [60, 73–75]. Further research outlining outcomes in relation to the specific lymphoma subtypes is required. 4.7.  When to Transplant? As a general rule, allo-SCT should be reserved until conventional chemotherapy is highly likely to fail, but before the patient experiences the effects of advanced stage disease. With scant data regarding allografting intermediategrade lymphomas in first remission, allo-SCT should not be routinely offered to these patients. ASCT is currently the preferred therapy for patients with chemosensitive relapsed aggressive NHL, but allo-SCT may be appropriate, especially if ASCT is precluded because of inadequate stem cell collection or bone marrow involvement. While results for patients who have relapsed after ASCT are sobering, allo-SCT may also benefit highly select patients, particularly those in remission with excellent performance status, younger age, and non-aggressive relapse [76–79].

5.  Allogeneic Transplantation in Burkitt’s and Lymphoblastic Lymphoma 5.1.  Introduction Both Burkitt lymphoma (BL) and Lymphoblastic lymphoma (LBL) are uncommon NHL and together account for <5% of all cases. LBL differs from acute lymphoblastic lymphoma in the WHO classification as by definition it must have less than 25% bone marrow involvement with disease. BL and LBL are similar in that primary therapy emphasizes the use of intensive multi-agent chemotherapy incorporating high dose methotrexate, fractionated cyclophosphamide, steroids, and anthracyclines (similar to pediatric ALL regimens). The role of SCT is not clearly defined for either disease. Autologous transplantation has been utilized to consolidate first remission in a number of cases with a randomized trial in LBL published. Allo-SCT has been typically reserved for patients with high risk features as part of primary treatment or as part of a salvage strategy in more advanced disease. 5.2.  Clinical Results 5.2.1.  Burkitt Lymphoma The role of allo-SCT in BL has not been evaluated in a prospective study and results are typically included with the outcomes of transplantation for other lymphoma subtypes. Registry data of allo-SCT are only available in one large series from the EBMT [15]. Seventy-one patients underwent allogeneic transplant with 62 from matched sibling donors. Of this group, 25 patients underwent transplant in CR1. Median survival from the date of transplant was 4.7 months

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and actuarial survival at four years was 37.1%. TRM and PFS at 4 years were 31 and 34.9%, respectively. The EBMT also reported a matched analysis in a 3:1 autologous to allo-SCT ratio. Overall and progression-free survival along with TRM favoured auto-SCT for BL while the relapse rate was the same. Of note, the presence of acute GVHD did not appear to reduce the relapse rate raising the question of the impact of GVLy in this group of patients. Unfortunately, there are few published series of myeloablative allo-SCT that include more than 5 BL patients. 5.2.2. Lymphoblastic Lymphoma Allografting in LBL has also not been studied in a published prospective study. A randomized EBMT and United Kingdom trial demonstrated that an event-free survival improved but there was no overall survival benefit for autologous SCT over conventional chemotherapy in first remission [80]. There is an IBMTR/AMBTR review of 204 autologous and allogeneic transplants performed between 1989 and 1998. [81] Allo-SCT recipients (n = 76) had a higher incidence of TRM at six months (18% vs. 3%, p = 0.002) and the higher incidence was maintained at both one and five years post-SCT. The relapse rate post-SCT was statistically lower in allo-SCT recipients (34% vs. 56% at 5 years, p = 0.004. Although autologous SCT recipients had a higher survival at six months (75% vs. 59%, p = 0.01), survival appeared similar at one (60% vs. 49%, p = 0.09) and 5 years (44% vs. 39%, p = 0.47) post transplant. The transplant groups differed in that autologous SCT patients were older, more likely to receive PBSC and to have undergone transplant before 1994, and were less likely to have been transplanted with a TBI-containing regimen. The EBMT review of NHL also reported outcomes of 314 patients with LBL that underwent allo-SCT. Transplantation was under gone by113 patients in CR1 and 268 patients received transplants from a matched sibling donor. A-GVHD appeared to reduced the relapse rate in LBL (HR = 0.50, 95% CI = 0.39–91) suggesting the potential impact of a GVLy effect. Median OS post transplant was one year with an actuarial 4-year OS of 42%. PFS and TRM at 4 years were 37.7 and 33.2%, respectively. In a 3:1 matched analysis comparing autografted and allografted patients, OS, TRM, and PFS favored auto-SCT while the relapse rate post-SCT was improved in allo-SCT recipients. There are few non-registry series of greater than five patients undergoing allogeneic transplant for LBL in CR1 [80, 82, 83]. These studies include between 7 and 12 patients and report DFS of 59–91%. Similarly there is a paucity of allo-SCT series in CR2 or beyond [84, 85]. Reported disease-free survival was <20%. The role of allogeneic SCT remains unclear on the basis of these data. The published series vary in how they define high risk disease and will have the selection bias inherent to single institution and registry series. Further prospective series that will be collaborative in order to report sufficient patient numbers will be needed.

6.  Conclusions The role of myeloablative allogeneic stem cell transplantation in NHL lacks some clarity because of a lack of any randomized studies along with the limitations of registry data, and retrospective institutional series. However, the

Chapter 7  Myeloablative Allogeneic Stem Cell Transplantation for Non-Hodgkin’s Lymphoma 

principles of delivering dose intensity, providing an uncontaminated stem cell source, and the potential benefit of a graft-vs-lymphoma effect make allo-SCT an appealing option. Registry and institutional series demonstrate feasibility and encouraging results in indolent (follicular lymphoma) and aggressive (diffuse large B cell lymphoma and variants) subtypes. The data in highly aggressive lymphomas (Burkitt and lymphoblastic lymphomas) are less clear because of a paucity of data and clearly further study is required. Dose intensity remains an important concept in NHL (on the basis of randomized trial results utilizing autologous transplants) and thus caution must be applied when considering reduced-intensity transplants for patients in which a fully myeloablative allograft may be offered safely. Further studies are needed to clarify the role of myeloablative transplants in this era, where reduced intensity transplants are being embraced and targeted novel therapies are changing the landscape of lymphoma treatment.

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J. Kuruvilla et al. 45. Escalon MP, Champlin RE, Saliba RM et al (2004) Nonmyeloablative allogeneic hematopoietic transplantation: A promising salvage therapy for patients with nonHodgkin’s lymphoma whose disease has failed a prior autologous transplantation. J Clin Oncol 22:2419–2423 46. Branson K, Chopra R, Kottaridis PD et al (2002) Role of nonmyeloablative allogeneic stem-cell transplantation after failure of autologous transplantation in patients with lymphoproliferative malignancies. J Clin Oncol 20:4022–4031 47. Robinson SP, Goldstone AH, Mackinnon S et al (2002) Chemoresistant or aggressive lymphoma predicts for a poor outcome following reduced-intensity allogeneic progenitor cell transplantation: An analysis from the lymphoma working party of the European group for blood and bone marrow transplant. Blood 100:4310–4316 48. Kasamon YL, Jones RJ, Diehl LF et  al (2005) Outcomes of autologous and allogeneic blood or marrow transplantation for mantle cell lymphoma. Biol Blood Marrow Transplant 11:39–46 49. Vandenberghe E, Ruiz de Elvira C, Isaacson P et  al (2000) Does transplantation improve outcome in mantle cell lymphoma (MCL)? A study from the EBMT. Blood 96:482a 50. Berdeja JG, Jones RJ, Zahurak ML et al (2001) Allogeneic bone marrow transplantation in patients with sensitive low-grade lymphoma or mantle cell lymphoma. Biol Blood Marrow Transplant 7:561–567 51. Milpied N, Gaillard F, Moreau P et  al (1998) High-dose therapy with stem cell transplantation for mantle cell lymphoma: Results and prognostic factors, a single center experience. Bone Marrow Transplant 22:645–650 52. Vandenberghe E, Ruiz de Elvira C, Loberiza FR et al (2003) Outcome of autologous transplantation for mantle cell lymphoma: A study by the European blood and bone marrow transplant and autologous blood and marrow transplant registries. Br J Haematol 120:793–800 53. Feugier P, Van Hoof A, Sebban C et  al (2005) Long-term results of the R-CHOP study in the treatment of elderly patients with diffuse large B-cell lymphoma: A study by the Groupe d’Etude des lymphomes de l’Adulte. J Clin Oncol 23:4117–4126 54. Jones RJ, Ambinder RF, Piantadosi S, Santos GW (1991) Evidence of a graftversus-lymphoma effect associated with allogeneic bone marrow transplantation. Blood 77:649–653 55. Ratanatharathorn V, Uberti J, Karanes C et al (1994) Prospective comparative trial of autologous versus allogeneic bone marrow transplantation in patients with nonHodgkin’s lymphoma. Blood 84:1050–1055 56. Schimmer AD, Jamal S, Messner H et  al (2000) Allogeneic or autologous bone marrow transplantation (BMT) for non- Hodgkin’s lymphoma (NHL): Results of a provincial strategy. Ontario BMT Network, Canada. Bone Marrow Transplant 26:859–864 57. Chopra R, Goldstone AH, Pearce R et  al (1992) 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 10:1690–1695 58. Appelbaum FR, Sullivan KM, Buckner CD et  al (1987) Treatment of malignant lymphoma in 100 patients with chemotherapy, total body irradiation, and marrow transplantation. J Clin Oncol 5:1340–1347 59. Nachbaur D, Oberaigner W, Fritsch E, Nussbaumer W, Gastl G (2001) Allogeneic or autologous stem cell transplantation (SCT) for relapsed and refractory Hodgkin’s disease and non-Hodgkin’s lymphoma: A single- centre experience. Eur J Haematol 66:43–49 60. Dhedin N, Giraudier S, Gaulard P et al (1999) Allogeneic bone marrow transplantation in aggressive non-Hodgkin’s lymphoma (excluding Burkitt and lymphoblastic lymphoma): A series of 73 patients from the SFGM database. Societ Francaise de Greffe de Moelle. Br J Haematol 107:154–161

Chapter 7  Myeloablative Allogeneic Stem Cell Transplantation for Non-Hodgkin’s Lymphoma  61. Mendoza E, Territo M, Schiller G, Lill M, Kunkel L, Wolin M (1995) Allogeneic bone marrow transplantation for Hodgkin’s and non-Hodgkin’s lymphoma. Bone Marrow Transplant 15:299–303 62. Mitterbauer M, Neumeister P, Kalhs P et al (2001) Long-term clinical and molecular remission after allogeneic stem cell transplantation (SCT) in patients with poor prognosis non-Hodgkin’s lymphoma. Leukemia 15:635–641 63. Soiffer RJ, Freedman AS, Neuberg D et  al (1998) CD6+ T cell-depleted allogeneic bone marrow transplantation for non- Hodgkin’s lymphoma. Bone Marrow Transplant 21:1177–1181 64. Juckett M, Rowlings P, Hessner M et  al (1998) T cell-depleted allogeneic bone marrow transplantation for high-risk non-Hodgkin’s lymphoma: Clinical and molecular follow-up. Bone Marrow Transplant 21:893–899 65. Bierman PJ (2000) Allogeneic bone marrow transplantation for lymphoma. Blood Rev 14:1–13 66. van Besien KW, de Lima M, Giralt SA et  al (1997) Management of lymphoma recurrence after allogeneic transplantation: The relevance of graft-versus-lymphoma effect. Bone Marrow Transplant 19:977–982 67. Collins RH Jr, Shpilberg O, Drobyski WR et al (1997) Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 15:433–444 68. Doocey RT, Toze CL, Connors JM et al (2005) Allogeneic haematopoietic stemcell transplantation for relapsed and refractory aggressive histology non-Hodgkin lymphoma. Br J Haematol 131:223–230 69. Dann EJ, Daugherty CK, Larson RA (1997) Allogeneic bone marrow transplantation for relapsed and refractory Hodgkin’s disease and non-Hodgkin’s lymphoma. Bone Marrow Transplant 20:369–374 70. Shepherd JD, Barnett MJ, Connors JM et al (1993) Allogeneic bone marrow transplantation for poor-prognosis non-Hodgkin’s lymphoma. Bone Marrow Transplant 12:591–596 71. Aksentijevich I, Jones RJ, Ambinder RF, Garrett-Mayer E, Flinn IW (2006) Clinical outcome following autologous and allogeneic blood and marrow transplantation for relapsed diffuse large-cell non-Hodgkin’s lymphoma. Biol Blood Marrow Transplant 12:965–972 72. Kim SW, Tanimoto TE, Hirabayashi N et  al (2006) Myeloablative allogeneic hematopoietic stem cell transplantation for non-Hodgkin lymphoma: A nationwide survey in Japan. Blood 108:382–389 73. Rodriguez J, Munsell M, Yazji S et al (2001) Impact of high-dose chemotherapy on peripheral T-cell lymphomas. J Clin Oncol 19:3766–3770 74. Feyler S, Prince HM, Pearce R et al (2007) The role of high-dose therapy and stem cell rescue in the management of T-cell malignant lymphomas: A BSBMT and ABMTRR study. Bone Marrow Transplant 40:443–450 75. Kahl C, Leithauser M, Wolff D et al (2002) Treatment of peripheral T-cell lymphomas (PTCL) with high-dose chemotherapy and autologous or allogeneic hematopoietic transplantation. Ann Hematol 81:646–650 76. de Lima M, van Besien KW, Giralt SA et al (1997) Bone marrow transplantation after failure of autologous transplant for non-Hodgkin’s lymphoma. Bone Marrow Transplant 19:121–127 77. Tsai T, Goodman S, Saez R et al (1997) Allogeneic bone marrow transplantation in patients who relapse after autologous transplantation. Bone Marrow Transplant 20:859–863 78. Vandenberghe E, Pearce R, Taghipour G, Fouillard L, Goldstone AH (1997) Role of a second transplant in the management of poor-prognosis lymphomas: A report from the European blood and bone marrow registry. J Clin Oncol 15:1595–1600 79. Freytes CO, Loberiza FR, Rizzo JD et  al (2004) Myeloablative allogeneic hematopoietic stem cell transplantation in patients who experience relapse after autologous

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J. Kuruvilla et al. stem cell transplantation for lymphoma: A report of the international bone marrow transplant registry. Blood 104:3797–3803 80. Sweetenham JW, Santini G, Qian W et  al (2001) High-dose therapy and autologous stem-cell transplantation versus conventional-dose consolidation/maintenance therapy as postremission therapy for adult patients with lymphoblastic lymphoma: Results of a randomized trial of the European group for blood and marrow transplantation and the United Kingdom lymphoma group. J Clin Oncol 19:2927–2936 81. Levine JE, Harris RE, Loberiza FR Jr et  al (2003) A comparison of allogeneic and autologous bone marrow transplantation for lymphoblastic lymphoma. Blood 101:2476–2482 82. Bouabdallah R, Xerri L, Bardou VJ et al (1998) Role of induction chemotherapy and bone marrow transplantation in adult lymphoblastic lymphoma: A report on 62 patients from a single center. Ann Oncol 9:619–625 83. Milpied N, Ifrah N, Kuentz M et  al (1989) Bone marrow transplantation for adult poor prognosis lymphoblastic lymphoma in first complete remission. Br J Haematol 73:82–87 84. Morel P, Lepage E, Brice P et al (1992) Prognosis and treatment of lymphoblastic lymphoma in adults: A report on 80 patients. J Clin Oncol 10:1078–1085 85. van Besien KW, Mehra RC, Giralt SA et al (1996) Allogeneic bone marrow transplantation for poor-prognosis lymphoma: Response, toxicity and survival depend on disease histology. Am J Med 100:299–307

Chapter 8 Non-Hodgkin’s Lymphoma: Allogeneic Reduced Intensity Conditioning Sonali M. Smith and Ginna G. Laport

1.  Introduction Non-Hodgkin’s lymphoma (NHL) is the fifth most common cancer among men and women in the United States [1]. High-dose chemotherapy with autoHCT offers curative potential to some patients who are not cured with frontline combination chemotherapy and/or radiotherapy. However, autoHCT is not the optimal therapy for patients with marrow involvement or for some patients with indolent disease. Conventional myeloablative allogeneic hematopoietic cell transplantation (HCT) has unequivocally demonstrated a reduction in relapse/progression compared with autoHCT in both indolent and aggressive NHL but is also associated with significant treatment-related mortality (TRM) [2, 3]. Over the last decade, the use of nonmyeloablative or reduced intensity conditioning (RIT) regimens has increased with an associated reduction in upfront toxicity in most published studies. RIT regimens broaden patient eligibility since older patients, patients with comorbid conditions, and heavily pretreated patients, including those who have failed a prior autoHCT can better tolerate such regimens. This approach incorporates immunosuppressive doses of chemotherapy and/or radiotherapy to achieve a mixed donor-host hematopoietic chimeric state, thus facilitating development of a donor immune-mediated graft-versus-lymphoma (GVL) effect. Limitations of RIT regimens include the relatively slow disease response as several weeks to a few months is required for a maximal GVL effect. Thus, the delayed immune responses will not occur rapidly enough in patients with a large tumor burden or rapidly proliferating disease. Another limitation of RIT is the variable susceptibility among hematologic malignancies to the GVL effect. Not surprisingly, the indolent NHL subtypes are most sensitive to the adoptive immunotherapy effect since these diseases demonstrate slower proliferation rates allowing time for the GVL effect to establish. In contrast, high-grade lymphomas appear to be the least sensitive to reduced intensity regimens. This chapter covers the most recent results involving the use of allogeneic RIT regimens for patients with indolent and aggressive NHL. Most of the larger studies published to date are retrospective in nature while smaller studies From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_8, © Springer Science + Business Media, LLC 2003, 2010

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originate from single institutions. Numerous conditioning regimens of varying dose intensities are being utilized, making it difficult to draw definitive comparisons across the various trials. The published data so far show high engraftment rates and sustained remissions, but graft versus host disease (GVHD) and infection remain the leading causes of TRM.

2.  Follicular Non-Hodgkin’s Lymphoma Follicular non-Hodgkin’s lymphoma (FL) is the second most frequent subtype of NHL with approximately 15,000 cases per year diagnosed in the United States. Median survival has historically ranged from 7 to 10 years, but newer agents such as monoclonal antibodies and radio immunoconjugates have improved response rates and early results show that overall survival (OS) may actually be affected [4, 5]. The definitive management of patients with advanced follicular NHL remains under considerable debate due to the numerous treatment options available. HCT has been offered to FL patients as an alternative approach, especially to younger patients, but typically was offered late in their course because of the long natural history inherent in this disease and the TRM associated with HCT. Refinements in HCT and improved supportive care have lowered TRM and thus may alter this paradigm. It is worth noting that most published transplant trials were initiated in the prerituximab era, which limits our current knowledge regarding the influence of rituximab on the outcomes of patients undergoing HCT [6, 7]. The addition of rituximab to frontline chemotherapy has improved the progression-free survival and OS for newly diagnosed FL patients and only longer follow-up will reveal the true impact of upfront chemoimmunotherapy [8, 9]. AlloHCT represents the only treatment modality with curative potential for patients with advanced FL. Although no randomized trials have been performed, several studies have consistently reported a lower risk of relapse compared to autoHCT. But the TRM associated with myeloablative alloHCT has invariably offset the benefit conferred by lower relapse rates. A Center for International Bone Marrow Transplant Research (CIBMTR) report compared the outcomes of 904 patients with FL who underwent either myeloablative alloHCT (n = 176), purged autoHCT (n = 131), or unpurged autoHCT (n = 597). The risk for relapse was 54% lower in the allogeneic recipients (p < 0.001) and 26% lower in recipients of purged autografts (p = 0.04) than in recipients of unpurged autografts [3]. However, the risk of TRM was 4.4 times higher after alloHCT than after autoHCT (p < 0.001), resulting in comparable 5-year probabilities of OS (52% after allogeneic, 62% after purged autologous, and 55% after unpurged autologous).

3.  Allogeneic Reduced Intensity Conditioning FL is a malignant disease that may be the most amenable to reduced intensity conditioning since (1) its indolent disease course allows establishment of the GVL effect and (2) the older median age of FL patients typically precludes them from receiving a myeloablative regimen. With follow-up ranging from 1 to 3 years, several studies utilizing various RIT regimens using matched related (MRD) and unrelated donors (URD) have reported disease-free survival (DFS) and OS ranging from 65% to 84% and 73% to 85%, respectively, with TRM reported from 10% to 18%. Table 8-1 lists

73

47

French registry [10] 2007

M.D. Anderson [12]

45

41

28

Japanese multicenter [65] 2005

UK multicenter [15] 2004

UK multicenter [14] 2004

46 (18–60)

48 (18–73)

48 (61–32)

54 (34–67)

53 yo

51 yo (33–66)

Median age (range)

Alemtuzumab BEAM

Alemtuzumab, flu Melphalan

Flu-based or low Dose TBI

Flu ± TBI

Flu, rituximab, cyclophosphamide

Flu-based

MRD URD

MRD URD

MRD URD

MRD URD

MRD URD

MRD URD

Stem Cell Preparative regimen source

69

65

83 sens 64 res

51

85

51

74

73

79

58

88

56

1.4

3

3

2

3

3

44 10

17a

 2

15

 8

10

13

11

18

34

19

32

TRM Relapse (%) (%)

15

49

60

11

34

Follow up Acute DFS/PFS (%) OS (%) (yrs) GVHD (%)

FHCRC Fred Hutchinson Cancer Research Center, flu fludarabine, PFS/EFS progression free survival/event free survival, OS overall survival, yrs years, UK United Kingdom, TBI total body irradiation, MRD matched related donor, URD, unrelated donor, BEAM carmustine, etoposide, cytarabine, melphalan, TRM treatment-related mortality, sens chemosensitive disease, res chemoresistant a  Grade 1–2 GVH only b  Includes all histologies

45

FHCRC [64] 2005

2005

n

Group/year

Table 8-1.  Allogeneic nonmyeloablative/reduced intensity transplantation for follicular NHL.

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these trials. Patient ages ranged from 18 to 73 years, and all studies included patients who had failed prior autoHCT and/or who had been heavily pretreated. The report with the largest number of patients comes from the French Registry, Societe Francaise de Greffe de Moelle Osseuse et de Therapie Cellulaire [10]. Seventy-three patients with indolent lymphoma received a fludarabine-based RIT regimen and received an allograft either from an MRD or from URD. Predictably, chemosensitivity significantly influenced event-free survival (EFS), OS, and TRM. The 3-year OS rates for patients in complete remission (CR), partial remission (PR) or with chemoresistant disease at the time of alloHCT were 66, 64, and 32%, respectively (p = 0.001). TRM rates were 32, 28, and 63%, respectively (p = 0.005). Relapse incidence was 10% for all patients with a median follow-up of 37 months. The most promising data are from the M.D. Anderson Cancer Center. Twenty patients with relapsed indolent NHL (including 18 follicular patients) who had matched sibling donors received RIT with fludarabine and cyclophosphamide ± high-dose rituximab [11]. Twelve patients were in second or greater CR at the time of transplantation. The CR rate was 100% after transplantation. DFS and OS at 2 years were both ~84%. The incidence of grade II–IV acute GVHD and chronic GVHD was 20 and 64%, respectively. Only one patient died from a treatment-related complication. These results were recently updated with a total accrual of 47 patients. With a median follow-up of 34 months, the 3-year DFS, OS, and risk of progression were 88, 85, and 3%, respectively [12]. This FCR regimen was notable for the use of high dose of rituximab at 1 g/m2 for three doses with a dual intent of upfront disease control and as part of GVHD prophylaxis [13]. These encouraging data support the existence of a GVL effect, although longer follow-up is necessary to confirm a true plateau in survival and confirmation in a multicenter setting is currently ongoing in the United States. Because GVHD remains a leading cause of TRM in the RIT setting, some investigators have incorporated alemtuzumab (anti CD-52 humanized monoclonal antibody) in the preparative regimen for in vivo donor T cell depletion. Graft rejection is also reduced since recipient T cells are also affected. Two separate groups from the United Kingdom have reported remarkably low acute GVHD rates with grade 3–4 acute GVHD being completely eliminated in the report by Faulkner et al. [14]. In this report, 65 patients including 28 patients with low-grade NHL received a BEAM-alemtuzumab conditioning regimen. Estimated 1-year TRM was only 8% with relapse/progression being the major cause of treatment failure. Relapse did not occur in any patient who developed acute or chronic GVHD although this association did not reach statistical significance. The other series from the United Kingdom that also utilized an alemtuzumab-containing regimen reported a 15% incidence of grade 2–4 acute GVHD [15]. However, this report demonstrated the highest rate of relapse compared to other comparable series suggesting that the in vivo T cell depletion induced by alemtuzumab may compromise the GVL effect. Further details regarding the use of alemtuzumab are further discussed in the Novel/ Emerging Therapies section of this chapter. A recent analysis from the CIBMTR confirmed the increasing utilization of RIT regimens for FL patients [16]. In 1997, <10% of matched sibling allogeneic transplants reported to the registry employed RIT regimens with this percentage increasing to 80% by 2002. The outcomes of 120 myeloablative recipients were compared to 85 RIT recipients. Interestingly, PFS, OS, and

Chapter 8  Non-Hodgkin’s Lymphoma: Allogeneic Reduced Intensity Conditioning 

TRM did not differ between the two groups. The 3-year PFS and OS were 65 and 55%, and 70 and 64%, respectively. However, relapse/progression was higher in the RIT group, 21 versus 9% (p = 0.03). It should be noted that nearly one-third of patients had chemotherapy-resistant disease and that the RIT recipients had an older median age (50 vs. 45 years) and a longer time from diagnosis to transplantation (34 vs. 24 months). Chemosensitivity and recipient performance status were better predictors of outcome than the conditioning regimen utilized. For patients with FL, RIT regimens have shown promising results and have broadened patient eligibility. However, patients with chemoresistant disease or who have received multiple prior regimens are not likely to benefit from this modality. The advent of prognostic indices such as the Follicular Lymphoma International Prognostic Index (FLIPI) and gene-expression profiling may allow earlier identification of patients who could benefit from alloHCT sooner rather than later [17, 18].

4.  Mantle Cell Lymphoma Mantle cell lymphoma (MCL) accounts for 5–10% of all lymphoma cases and is characterized by the t(11;14) translocation resulting in rearrangement of the bcl-1 locus and cyclin D1 protein overexpression. The median age at diagnosis is about 60-years old and, despite high initial response rates with conventional chemotherapy, the median PFS and OS are only 1 and 3 years, respectively [19]. AutoHCT in first remission has been shown to prolong EFS and possibly OS, but a definitive plateau in survival has not been demonstrated so far [9, 20, 21]. Myeloablative alloHCT can induce durable remissions in MCL patients with refractory or relapsed disease but is associated with high upfront mortality. As with FL patients, the median age of patients with MCL is ~60-year old, which deems such patients suboptimal candidates for myeloablative regimens. As seen with most other NHL subtypes, the existence of a GVL effect in MCL stems from findings of lower relapse rates after alloHCT compared to autologous HCT. Further support for GVL in MCL includes the delayed conversion of polymerase chain reaction (PCR) positivity to PCR negativity in bone marrow and the observation of disease regression following either withdrawal of immunosuppression or appearance of chronic GVHD [22, 23] (Table 8-2). The European Bone Marrow Transplant Group (EBMT) has reported the largest experience to date with 144 MCL patients who received various RIT regimens [24]. Many patients were heavily pretreated, and 42% had failed a prior autograft. One hundred patients had chemosensitive disease with the rest of the patients being either chemorefractory or untested. The 2-year relapse rate, PFS, and OS were 57, 26, and 31%, respectively. However, the TRM was a prohibitive 50% at 2 years. Patients with chemoresistant disease had a significantly lower PFS and higher TRM compared to patients with chemosensitive disease. In a recent separate study, the results of 66 MCL patients who were URD recipients were analyzed by the EBMT [25]. Forty-four patients (67%) received an RIT regimen. After a median follow-up of 15 months, the 2-year PFS and OS were 28 and 42%, respectively, for all patients. The RIT recipients did not have a lower nonrelapse mortality (NRM) or improved survival compared to the myeloablative patients but the RIT group was older and heavily pretreated.

113

  66

144

  33

  18

  10

EBMT [25] 2006

EBMT [66] 2005

FHCRC [26] 2004

M.D. Anderson [67] 2005

UK multicenter(15) 2004

FCR or Flu/cisp/araC Alemtuzumab, flu Melphalan

48b (18–73)

Flu + TBI

Various

33% ablative 67% RIT

57 (46–64)

54 (33–70)

49 yo (28–68)

50 yo (22–68)

MRD URD

MRD URD

MRD URD

MRD URD

URD

50

82

60

26

28

60

85

65

31

42

  3 years

26 months

  2 years

  9 months

15 months

Median age Stem cell DFS/PFS Follow up (range) Preparative regimen source (%) OS (%) (yrs)

15

17

57

36

35

52

17

 9

57

45

Acute GVHD Relapse (%) (%)

11b

  0a

  24

  50

  27

TRM (%)

FHCRC Fred Hutchinson Cancer Research Center, flu fludarabine, PFS/EFS progression free survival/event free survival, OS overall survival, yrs years, UK United Kingdom, TBI total body irradiation, MRD matched related donor, URD unrelated donor, BEAM carmustine, etoposide, cytarabine, melphalan, TRM treatment-related mortality, sens chemosensitive disease, res chemoresistant a  Day + 100 TRM b  Data for all 88 NHL patients

n

Group/year

Table 8-2.  Allogeneic nonmyeloablative/reduced intensity transplantation for mantle cell NHL.

114  S.M. Smith and G.G. Laport

Chapter 8  Non-Hodgkin’s Lymphoma: Allogeneic Reduced Intensity Conditioning 

Investigators from the Fred Hutchinson Cancer Research Center (FHCRC) utilized a fludarabine and low-dose total body irradiation (TBI) regimen with 33 MCL patients [26]. This group of patients was heavily pretreated as the median number of prior regimens was 4 and 14 patients had received a prior autoHCT. With a median follow-up of 24 months, the 2-year DFS and OS were 60 and 65%, respectively, with a 24% NRM. These results are particularly notable considering that 85% had failed front-line or salvage therapy and 36% had chemoresistant disease. The only pretransplantation factor predictive of relapse was number of prior regimens and, in contrast to the EBMT report, chemorefractory disease did not appear to affect relapse rate. The M.D. Anderson group described the outcomes of 18 MCL patients with chemosensitive disease who received predominantly the FCR regimen (fludarabine, cyclophosphamide, rituximab) [27]. The median age was 57-year old and most patients (89%) had chemosensitive disease with five patients who had failed a prior autoHCT. The 3-year EFS and OS were an impressive 82 and 85%, respectively with a 0% mortality at day + 100. Compared to the abovementioned FHCRC study and the EBMT series, the higher EFS and OS in this study may be explained by the better risk patient group since the majority patients were chemosensitive at the time of transplantation. A study from the United Kingdom with 188 NHL patients included 10 patients with MCL [15]. The 3-year PFS and OS were 50 and 60%, respectively with an actuarial relapse rate of 50% for the 10 MCL patients. The RIT regimens were alemtuzumab-based which was more immunosuppressive compared to other RIT regimens and may thus impair the GVL effect. In summary, the above data support the existence of graft versus lymphoma effects in patients with MCL. The use of allogeneic RIC is increasing and early results appear promising even for patients with MCL who have failed a prior autoHCT. However, longer follow-up is necessary to confirm a true plateau in survival.

5.  Aggressive B-cell Lymphomas Diffuse large B-cell lymphoma (DLCL) and other aggressive NHL subtypes are generally chemosensitive diseases with high initial response rates to combination chemotherapy. Nevertheless, almost half of patients with aggressive NHL eventually relapse and become candidates for further therapies. AutoHCT is the standard of care for relapsed chemosensitive aggressive lymphomas, but not all patients demonstrate chemosensitivity, and recurrence after autologous transplantation remains problematic [28, 29]. Myeloablative alloHCT transplants have demonstrated efficacy against relapsed DLCL but TRM rates of 25–41% dampen enthusiasm for this approach and less intensive regimens are of interest [30–33]. Currently, the role of reduced intensity allogeneic transplant remains least well-defined for aggressive NHL, and there is a paucity of studies focusing solely on this patient population. One concern is the lack of sufficient disease ablation with less intensive regimens. As an example, a retrospective comparison of 16 patients with aggressive NHL undergoing conventional myeloablative transplant to 12 patients undergoing reduced intensity regimens showed a higher 2-year relapse rate with less intensive conditioning (44 vs.

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12%, p = 0.02) [2]. In addition, most reports show a higher NRM and lower disease control for aggressive NHL as compared to indolent subtypes [14, 15, 24]. The EBMT reported 62 patients with aggressive NHL undergoing RIT in their series of which a majority had a matched sibling donor [24]. Although chemosensitivity was the only factor predictive of outcome by multivariate analysis, patients with aggressive subtypes had only a 32% 1-year PFS, which was inferior to patients with indolent histologies. A multicenter report from the United Kingdom showed similar results in a smaller group of patients; the relapse risk was higher for aggressive NHL compared to low-grade lymphomas (67.9 vs. 10%, p = 0.014) [14]. This translated into a dismal 16% EFS at 2 years. Finally, Morris and colleagues showed that patients with high-grade lymphomas fared much worse than other subtypes in terms of PFS (34 vs. 65%, p = 0.002), overall survival (34 vs. 73%, p < 0.001), and TRM (relative risk, 2.3; p = 0.02). Reasons for increased toxicity in the aggressive group were unexplained [15]. However, not all series have found aggressive histology to be a negative predictive factor. Dean and colleagues compared 13 patients with DLCL to 12 patients with follicular lymphoma (and 4 patients with mantle cell lymphoma), who received a uniform salvage regimen prior to RIT [34]. Chemosensitivity to the salvage regimen was the most important predictor of outcome, and histology did not correspond to either EFS or OS in this small study. The poorer results seen with aggressive NHL patients undergoing myeloablative alloHCT can be partially attributed to patient selection and intensive prior therapies. Most DLCL patients are offered autoHCT at the time of initial relapse due to its proven efficacy and low TRM (<5% in most series). Thus, by the time an allogeneic approach is considered, many patients have already failed a high-dose regimen. Myeloablative alloHCT in this setting is highly toxic and patients failing a prior autoHCT are typically not candidates for fully ablative regimens. In contrast, RIT may be a feasible salvage option. Studies evaluating RIT in patients with aggressive NHL failing a prior autoHCT, however, have shown mixed results. Escalon and colleagues from the M.D. Anderson Cancer Center presented pilot data on 20 patients with NHL relapsing after a prior autoHCT, including 10 with DLCL [35]. Most patients had minimal disease burden and a good performance status. Using fludarabine, cyclophosphamide, and rituximab, the authors showed an encouraging 95% 3-year PFS with a median follow-up of 25 months. These results are tempered, however, by two other studies of RIT of patients relapsing following autoHCT. Branson and colleagues used fludarabine, melphalan, and alemtuzumab conditioning in a heterogeneous group of NHL patients (10 with high-grade NHL) and demonstrated PFS and OS of 50 and 53%, respectively, with a median follow-up of 14 months [36]. The FHCRC reported a 3-year PFS and OS of 28 and 31%, respectively, with no plateau in terms of relapse in a group of 147 patients (including 24 with aggressive NHL) treated with fludarabine and low-dose TBI. The presence of acute GVHD increased toxicity but did not confer benefit, whereas chronic GVHD correlated with improved PFS. It should be emphasized that each of these studies offered RIT to patients relapsing after a prior autoHCT; others have proposed a “consolidative” RIT immediately following autoHCT which may have more optimistic results [37, 38]. We await mature data on these approaches.

Chapter 8  Non-Hodgkin’s Lymphoma: Allogeneic Reduced Intensity Conditioning 

Although inferior to results in patients with indolent histologies, some patients with aggressive lymphoma clearly benefit from RIT. A summary of current series, as discussed above, demonstrates that up to one-third of patients may enjoy long-term disease control. RIT for selected patients with aggressive lymphoma should be considered in patients who have failed a prior autoHCT, have an acceptable performance status, and have an appropriate matched sibling or matched URD. However, demonstration of chemosensitivity by either standard CT criteria or metabolic criteria prior to transplantation is essential [39].

6.  T-cell Lymphoma T-cell lymphomas (T-NHL) comprise approximately 10% of all NHL, and include a diverse group of clinicopathologic entities. The most common nodal subtype is peripheral T-cell lymphoma, unspecified, which accounts for nearly 50% of T-NHL and typically has an aggressive course. The remaining histologies include a diverse group of extranodal and nodal disorders, including cutaneous T-cell lymphoma/mycosis fungoides (CTCL/MF), angioimmunoblastic lymphoma (AILD), and anaplastic large cell lymphoma (ALCL). In general, T-NHL is characterized by drug resistance and inevitable relapse in the vast majority of subtypes. Much of the historical treatment approach for T-NHL has been extrapolated from data in B-cell NHL, but this is clearly suboptimal as the adverse significance of T-cell phenotype as compared to B-cell phenotype is well-established [40–42]. AutoHCT as part of front-line management is more promising than when offered at relapse as the long-term disease control rate is only ~30% in the salvage setting [3, 43, 44] (Table 8-3). There is now increasing evidence that T-NHL is susceptible to a GVL effect, and the literature is peppered with optimistic case reports and small series [45–48]. An early study by Rodriguez and colleagues from MDACC evaluated 29 patients undergoing autoHCT and 7 patients undergoing fully ablative alloHCT [43]. Although the OS was not different in the two groups, the main cause for treatment failure in the allogeneic group was toxicity and four of five patients died in CR. Reduced intensity approaches, as outlined above, may decrease the high TRM and capitalize on GVL effects. An Italian group recently reported on 17 patients with either relapsed or refractory T-NHL undergoing nonmyeloablative alloHCT [46]. All patients were heavily pretreated, and all had unfavorable histologies including alk-negative ALCL, angioimmunoblastic T-NHL, and peripheral T-NHL unspecified. This is one of the few prospective phase II studies on alloHCT for T-NHL and reported an impressive 3-year OS and PFS of 81 and 64%, respectively. This same group also retrospectively compared 11 patients with relapsed T-NHL undergoing autoHCT to 8 patients undergoing nonmyeloablative alloHCT; only one-third of autoHCT patients remained in remission at 3 years, whereas all 8 allogeneic patients remained in remission at 1.5 years [49]. Given the heterogeneity of T-NHL, it is likely that some subtypes will fare better than others in terms of GVL and outcome following RIT. One common subtype of T-NHL is cutaneous T-cell lymphoma, which includes mycosis fungoides/Sezary syndrome (MF/SS) and primary cutaneous ALCL. Investigators from the City of Hope reported on eight patients with heavily pretreated MF/ SS undergoing allogeneic stem cell transplant, including four patients receiving

117

26

4

3

7

Italian multicentera 2004, 2005 [46, 68]

City of Hopeb 2005 [47]

Italian multicenterc 2003 [69]

City of hoped 2006 [2]

Flu-mel

51 yo (20–67)

Flu-mel

NS

MRD URD

1 MRD 3 URD

57

67 (2/3)

75

51

DFS/PFS Stem cell source (%)

Flu-cy-thiotepa 22 MRD 3 MRD (mismatched) 1 URD

Preparative regimen

37, 47, 56 yo Flu-cy-TBI

46 yo (21–59)

45 yo (15–64)

Median age (range)

57

67 (2/3)

75

61

NS

1.5,2+

4.5+

2+

Follow up OS (%) (yrs)

  65

100

   0

  28

NS

NS

0

NS

Acute Relapse GVHD (%) (%)

28

33

25

13

TRM (%)

Flu fludarabine, PFS/EFS progression free survival/event free survival, OS overall survival, yrs years, TBI total body irradiation, MRD matched related donor, URD unrelated donor, TRM treatment-related mortality, NS not specified, GVHD graft-versus-host disease a  All patients had systemic T-NHL (either PTCL, AILD, or ALCL) b  All four patients had CTCL/MF c  All patients had CTCL/MF d  Four patients had CTCL/MF and three had PTCL

n

Group/year

Table 8-3.  Allogeneic nonmyeloablative/reduced intensity transplantation for T-NHL.

118  S.M. Smith and G.G. Laport

Chapter 8  Non-Hodgkin’s Lymphoma: Allogeneic Reduced Intensity Conditioning 

a reduced intensity preparative regimen. All patients achieved a CR and six remain in remission with nearly 5 years follow-up time, including three patients undergoing RIT. Of note, three of the four patients undergoing RIT had URD, and only one died of transplant-related complications [47]. The management of relapsed T-NHL remains problematic, but the available literature supports allogeneic approaches as a promising avenue. The heterogeneity and rarity of T-NHL will likely mandate a multicenter and collaborative effort to better understand the role of RIT. 6.1.  Novel/Emerging Therapies 6.1.1.  Alemtuzumab As with myeloablative regimens, there appears to be little consensus on the optimal conditioning regimen for reduced intensity transplants for NHL, but most groups combine fludarabine with alkylating agents, and occasionally use TBI. Alemtuzumab (Campath-1H), a monoclonal antibody directed against CD52, is a potent immunosuppressant that provides in vivo T-cell depletion. Alemtuzumab is increasingly being used in reduced intensity regimens due to early evidence of decreased acute GVHD and a potentially lower early TRM; however, an increase in CMV reactivation has been observed. This is usually managed with pre-emptive antiviral agents (i.e., valganciclovir) and close monitoring via CMV PCR [50]. Although less commonly seen in alemtuzumabcontaining versus anti-thymocyte globulin (ATG)-containing regimens, EBV reactivation may also occur [51, 52]. The main beneficial effect of alemtuzumab appears to be a decrease in acute GVHD. In a series of 44 patients with refractory hematologic malignancies who received fludarabine, melphalan, and alemtuzumab, no patient developed grade III or IV acute GVHD, and only 5 patients developed any GVHD [53]. Only one patient had chronic GVHD. A separate study of 38 patients by the same group focusing on malignant lymphomas likewise showed no cases of grade III or IV GVHD [36]. However, 18 of 24 CMV-seropositive patients (or those with CMV-seropositive donors) in this study had evidence of CMV reactivation by PCR techniques. Although only one of these patients developed clinical CMV infection, an increase in CMV-associated diseases due to alemtuzumab must be carefully considered. A multicenter comparison of two prospective trials using a nonmyeloablative regimen, one with alemtuzumab and one without alemtuzumab, confirmed these findings [54]. In this study of 129 patients with lymphoproliferative disorders, the incidence of acute and chronic GVHD was significantly lower in the alemtuzumab-containing regimen (21.7 vs. 45.1%, p = 0.006) whereas CMV reactivation almost entirely occurred in the patients receiving alemtuzumab (85 vs. 24%, p < 0.001). There was no difference in either DFS or OS between the two regimens, but relapse was a more frequent cause of death (60 vs. 20%) in patients receiving alemtuzumab. The impact of alemtuzumab on recurrence rates is an important outcome to keep in mind for ongoing and future evaluations of this compound in the transplant setting. Since alemtuzumab appears to significantly decrease the incidence and severity of acute GVHD, it is also being investigated in the URD transplantation setting where the risk of GVHD is prohibitive for many patients. A report of 47 patients, including 20 patients with malignant lymphomas, showed a notably

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low 6.4% rate of severe grade III GVHD; no patients had grade IV GVHD and no patients have died of GVHD with a median follow-up of almost 1 year [55].The overall rate of acute GVHD was 21%. More than 60% of patients had evidence of CMV reactivation, but only one patient died from CMV disease. Thus, the potent immunosuppressive effects of alemtuzumab may allow alternative donor stem cell sources to be more safely used and thereby broaden the applicability of alloHCT to patients without a matched sibling donor. In summary, alemtuzumab decreases early GVHD-related complications, but its impact on disease control remains to be established. Viral reactivation is common, but can be managed in most patients with close monitoring and preemptive therapy. Maribivir is an antiviral agent in clinical development that is a selective UL 97 viral protein-kinase inhibitor [56]. Unlike gancyclovir and foscarnet, it does not cause myelosuppression or nephrotoxicity which may help facilitate the use of alemtuzumab. 6.1.2.  Rituximab and Radioimmunotherapy Other monoclonal antibodies may also have utility as part of alloHCT in lymphoma. Rituximab, a chimeric monoclonal antibody directed against CD20, is nearly universally applied in patients with B-cell malignancies. Rituximab potentiates tumor control, is being explored in settings of minimal residual disease, and provides effective in vivo purging of malignant B-cells. Several investigators have proposed that B-cells are important in the pathogenesis of GVHD since rituximab therapy may be effective in this setting [57]. A small study of ten patients with refractory lymphoid malignancies undergoing alloHCT and treated with early post-transplant rituximab showed no GVHD and retention of efficacy suggesting that GVL effects were maintained [58]. Rituximab has also been directly used to treat refractory chronic GVHD in patients with a variety of hematologic malignancies with 65% of patients demonstrating symptomatic improvement [59]. A new humanized anti-CD20 monoclonal antibody, IMMU-106, has shown activity in relapsed NHL patients and is associated with less infusion reactions compared to rituximab [60]. Gopal and colleagues from the FHCRC found in vitro synergy between cytarabine, fludarabine, and 90Y-ibritumomab tiuxetan (Zevalin®), a radiolabeled monoclonal antibody against CD20 and are conducting an RIT trial with incorporation of this agent for patients with relapsed/refractory B-cell malignancies [61]. These observations of both disease and GVHD control are provocative and warrant ongoing investigations of anti-CD20 directed therapies in the post-allogeneic transplant setting, either as treatment of minimal residual disease or as immunomodulation of GVL and GVHD. Other agents in development include epratuzumab, a humanized anti-CD22 monoclonal antibody, that has induced responses in NHL patients as a single agent and in combination with chemotherapy [62]. CD22 is a B-cell restricted marker expressed late in the stages of differentiation and demonstrates >90% expression in both large B-cell lymphomas and follicular lymphomas. A novel class of targeted therapies are small modular immunopharmaceuticals (SMIP) with CD37-SMIP demonstrating impressive in  vitro cytotoxicity against B-cell leukemia/lymphoma cell and chronic lymphocytic leukemia lines [63]. CD37 is a lineage-specific B-cell antigen strongly expressed on B cells and transformed mature B-cell leukemia and lymphoma cells. In summary, several promising monoloconal antibodies with activity against B-cell NHL are in various phases of development and show great potential thus far.

Chapter 8  Non-Hodgkin’s Lymphoma: Allogeneic Reduced Intensity Conditioning 

7.  Conclusions Reduced intensity conditioning regimens followed by alloHCT are increasingly utilized in patients with relapsed NHL. RIT is feasible and the less intensive regimens, with or without alemtuzumab, have expanded the number of patients who may be considered for alloHCT. As seen in myeloablative HCT, patients with early stage, good-risk disease, and/or who demonstrate chemosensitivity stand to benefit the most from this treatment modality. Patients with rapidly proliferating NHL subtypes should be carefully selected as disease control prior to RIT is essential. Many questions remain, including (1) what is the optimal conditioning regimen (2) how can GVL be amplified while minimizing GVHD risk and (3) how can patient selection be optimized. In summary, RIT is a promising approach in both the frontline and salvage setting for a growing number of NHL patients and ongoing studies are warranted.

References 1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ (2007) Cancer statistics, 2007. CA Cancer J Clin 57:43–66 2. Rodriguez R, Nademanee A, Gruel N et al (2006) Comparison of reduced-intensity and conventional myeloablative regimens for allogeneic transplantation in nonHodgkin’s lymphoma. Biol Blood Marrow Transplant 12:1326–1334 3. van Besien K, Loberiza FR Jr, Bajorunaite R et al (2003) Comparison of autologous and allogeneic hematopoietic stem cell transplantation for follicular lymphoma. Blood 102:3521–3529 4. Fisher RI, LeBlanc M, Press OW, Maloney DG, Unger JM, Miller TP (2005) New treatment options have changed the survival of patients with follicular lymphoma. J Clin Oncol 23:8447–8452 5. Kaminski MS, Tuck M, Estes J et  al (2005) 131I-tositumomab therapy as initial treatment for follicular lymphoma. N Engl J Med 352:441–449 6. Rohatiner AZ, Nadler L, Davies AJ et al (2007) Myeloablative therapy with autologous bone marrow transplantation for follicular lymphoma at the time of second or subsequent remission: Long-term follow-up. J Clin Oncol 25:2554–2559 7. Schouten HC, Qian W, Kvaloy S et al (2003) High-dose therapy improves progression-free survival and survival in relapsed follicular non-Hodgkin’s lymphoma: Results from the randomized European CUP trial. J Clin Oncol 21:3918–3927 8. Forstpointner R, Dreyling M, Repp R et al (2004) The addition of rituximab to a combination of fludarabine, cyclophosphamide, mitoxantrone (FCM) significantly increases the response rate and prolongs survival as compared with FCM alone in patients with relapsed and refractory follicular and mantle cell lymphomas: results of a prospective randomized study of the German Low-Grade Lymphoma Study Group. Blood 104:3064–3071 9. Hiddemann W, Kneba M, Dreyling M et al (2005) Frontline therapy with rituximab added to the combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) significantly improves the outcome for patients with advancedstage follicular lymphoma compared with therapy with CHOP alone: results of a prospective randomized study of the German Low-Grade Lymphoma Study Group. Blood 106:3725–3732 10. Vigouroux S, Michallet M, Porcher R et  al (2007) Long-term outcomes after reduced-intensity conditioning allogeneic stem cell transplantation for low-grade lymphoma: a survey by the French Society of Bone Marrow Graft Transplantation and Cellular Therapy (SFGM-TC). Haematologica 92:627–634 11. Khouri IF, Saliba RM, Giralt SA et al (2001) Nonablative allogeneic hematopoietic transplantation as adoptive immunotherapy for indolent lymphoma: Low incidence

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Chapter 8  Non-Hodgkin’s Lymphoma: Allogeneic Reduced Intensity Conditioning  29. Caballero MD, Perez-Simon JA, Iriondo A et al (2003) High-dose therapy in diffuse large cell lymphoma: Results and prognostic factors in 452 patients from the GELTAMO Spanish Cooperative Group. Ann Oncol 14:140–151 30. Peniket AJ, Ruiz de Elvira MC, Taghipour G et  al (2003) An EBMT registry matched study of allogeneic stem cell transplants for lymphoma: Allogeneic transplantation is associated with a lower relapse rate but a higher procedurerelated mortality rate than autologous transplantation. Bone Marrow Transplant 31:667–678 31. van Besien K, Thall P, Korbling M et  al (1997) Allogeneic transplantation for recurrent or refractory non-Hodgkin’s lymphoma with poor prognostic features after conditioning with thiotepa, busulfan, and cyclophosphamide: Experience in 44 consecutive patients. Biol Blood Marrow Transplant 3:150–156 32. Dhedin N, Giraudier S, Gaulard P et al (1999) Allogeneic bone marrow transplantation in aggressive non-Hodgkin’s lymphoma (excluding Burkitt and lymphoblastic lymphoma): A series of 73 patients from the SFGM database. Societ Francaise de Greffe de Moelle. Br J Haematol 107:154–161 33. Doocey RT, Toze CL, Connors JM et al (2005) Allogeneic haematopoietic stemcell transplantation for relapsed and refractory aggressive histology non-Hodgkin lymphoma. Br J Haematol 131:223–230 34. Dean RM, Fowler DH, Wilson WH et  al (2005) Efficacy of reduced-intensity allogeneic stem cell transplantation in chemotherapy-refractory non-hodgkin lymphoma. Biol Blood Marrow Transplant 11:593–599 35. Escalon MP, Champlin RE, Saliba RM et al (2004) Nonmyeloablative allogeneic hematopoietic transplantation: A promising salvage therapy for patients with nonHodgkin’s lymphoma whose disease has failed a prior autologous transplantation. J Clin Oncol 22:2419–2423 36. Branson K, Chopra R, Kottaridis PD et al (2002) Role of nonmyeloablative allogeneic stem-cell transplantation after failure of autologous transplantation in patients with lymphoproliferative malignancies. J Clin Oncol 20:4022–4031 37. Carella AM, Cavaliere M, Lerma E et  al (2000) 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 18:3918–3924 38. Dey BR, McAfee S, Sackstein R et  al (2001) Successful allogeneic stem cell transplantation with nonmyeloablative conditioning in patients with relapsed hematologic malignancy following autologous stem cell transplantation. Biol Blood Marrow Transplant 7:604–612 39. Cheson BD, Pfistner B, Juweid ME et  al (2007) Revised response criteria for malignant lymphoma. J Clin Oncol 25:579–586 40. Melnyk A, Rodriguez A, Pugh WC, Cabannillas F (1997) Evaluation of the Revised European–American Lymphoma classification confirms the clinical relevance of immunophenotype in 560 cases of aggressive non-Hodgkin’s lymphoma. Blood 89:4514–4520 41. Gisselbrecht C, Gaulard P, Lepage E et  al (1998) Prognostic significance of T-cell phenotype in aggressive non-Hodgkin’s lymphomas. Groupe d’Etudes des Lymphomes de l’Adulte (GELA). Blood 92:76–82 42. Coiffier B, Brousse N, Peuchmaur M et  al (1990) Peripheral T-cell lymphomas have a worse prognosis than B-cell lymphomas: A prospective study of 361 immunophenotyped patients treated with the LNH-84 regimen. The GELA (Groupe d’Etude des Lymphomes Agressives). Ann Oncol 1:45–50 43. Rodriguez J, Munsell M, Yazji S et al (2001) Impact of high-dose chemotherapy on peripheral T-cell lymphomas. J Clin Oncol 19:3766–3770 44. Song KW, Mollee P, Keating A, Crump M (2003) Autologous stem cell transplant for relapsed and refractory peripheral T-cell lymphoma: Variable outcome according to pathological subtype. Br J Haematol 120:978–985

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S.M. Smith and G.G. Laport 45. Aoyama Y, Yamane T, Hino M et al (2001) Nodal gamma/delta T cell lymphoma in complete remission following allogeneic bone marrow transplantation from an HLA-matched unrelated donor. Acta Haematol 105:49–52 46. Corradini P, Dodero A, Zallio F et  al (2004) Graft-versus-lymphoma effect in relapsed peripheral T-cell non-Hodgkin’s lymphomas after reduced-intensity conditioning followed by allogeneic transplantation of hematopoietic cells. J Clin Oncol 22:2172–2176 47. Molina A, Zain J, Arber DA et al (2005) Durable clinical, cytogenetic, and molecular remissions after allogeneic hematopoietic cell transplantation for refractory Sezary syndrome and mycosis fungoides. J Clin Oncol 23:6163–6171 48. Mansour MR, Dogan A, Morris EC et  al (2005) Allogeneic transplantation for hepatosplenic alphabeta T-cell lymphoma. Bone Marrow Transplant 35:931–934 49. Corradini P, Dodero A, Zallio F et al (2002) Nonmyeloablative conditioning followed by allogeneic transplantation has a better outcome than high-dose chemotherapy plus autografting in non-Hodgkin’s lymphoma with T-cell histology. Blood 100:427a 50. Kline J, Pollyea DA, Stock W et  al (2006) Pre-transplant ganciclovir and post transplant high-dose valacyclovir reduce CMV infections after alemtuzumab-based conditioning. Bone Marrow Transplant 37:307–310 51. Cohen J, Gandhi M, Naik P et al (2005) Increased incidence of EBV-related disease following paediatric stem cell transplantation with reduced-intensity conditioning. Br J Haematol 129:229–239 52. Scheinberg P, Fischer SH, Li L et al (2007) Distinct EBV and CMV reactivation patterns following antibody-based immunosuppressive regimens in patients with severe aplastic anemia. Blood 109:3219–3224 53. Kottaridis PD, Milligan DW, Chopra R et  al (2000) In vivo CAMPATH-1H prevents graft-versus-host disease following nonmyeloablative stem cell transplantation. Blood 96:2419–2425 54. Perez-Simon JA, Kottaridis PD, Martino R et  al (2002) Nonmyeloablative transplantation with or without alemtuzumab: comparison between 2 prospective studies in patients with lymphoproliferative disorders. Blood 100:3121–3127 55. Chakraverty R, Peggs K, Chopra R et  al (2002) Limiting transplantation-related mortality following unrelated donor stem cell transplantation by using a nonmyeloablative conditioning regimen. Blood 99:1071–1078 56. Wade JC (2006) Viral infections in patients with hematological malignancies. Hematology Am Soc Hematol Educ Program 368–374 57. Ratanatharathorn V, Ayash L, Reynolds C et al (2003) Treatment of chronic graftversus-host disease with anti-CD20 chimeric monoclonal antibody. Biol Blood Marrow Transplant 9:505–511 58. Shimoni A, Hardan I, Avigdor A et al (2003) Rituximab reduces relapse risk after allogeneic and autologous stem cell transplantation in patients with high-risk aggressive non-Hodgkin’s lymphoma. Br J Haematol 122:457–464 59. Zaja F, Bacigalupo A, Patriarca F et  al (2007) Treatment of refractory chronic GVHD with Rituximab: A GITMO study. Bone Marrow Transplant 40:273–277 60. Morschhauser F, Leonard JP, Coiffier B et al (2005) Initial safety and efficacy of a second-generation humanized anti-CD20 antibody, IMMU-106 (hA20), in nonHodgkin’s lymphoma. Blood 106:683a 61. Gopal AK, Pagel JM, Rajendran JG et al (2006) Improving the efficacy of reduced intensity allogeneic transplantation for lymphoma using radioimmunotherapy. Biol Blood Marrow Transplant 12:697–702 62. Stein R, Qu Z, Chen S et al (2004) Characterization of a new humanized anti-CD20 monoclonal antibody, IMMU-106, and Its use in combination with the humanized anti-CD22 antibody, epratuzumab, for the therapy of non-Hodgkin’s lymphoma. Clin Cancer Res 10:2868–2878

Chapter 8  Non-Hodgkin’s Lymphoma: Allogeneic Reduced Intensity Conditioning  63. Zhao X, Lapalombella R, Joshi T et al (2007) Targeting CD37-positive lymphoid malignancies with a novel engineered small modular immunopharmaceutical. Blood 110:2569–2577 64. Maris MB, Sandmaier BM, Storer B et  al (2005) Allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning for relapsed or refractory follicular lymphoma. Blood 106:329a 65. Kusumi E, Kami M, Kanda Y et al (2005) Reduced-intensity hematopoietic stemcell transplantation for malignant lymphoma: A retrospective survey of 112 adult patients in Japan. Bone Marrow Transplant 36:205–213 66. Robinson SP, Schmitz N, Taghipour G, Sureda A (2004) Reduced intensity allogeneic stem cell transplantation for mantle cell lymphoma is associated with substantial late transplant related mortality and a poor outcome in patients with chemoresistant disease. Blood 104:865–872 67. Khouri IF, Lee MS, Saliba RM et al (2003) Nonablative allogeneic stem-cell transplantation for advanced/recurrent mantle-cell lymphoma. J Clin Oncol 21:4407–4412 68. Corradini P, Dodero A, Bregni M et al (2005) Reduced-intensity conditioning followed by allogeneic stem cell transplantation for relpapsed lymphohas: Impact of pre-transplantation factors on long-term outcome. Blood 106 69. Soligo D, Ibatici A, Berti E et al (2003) Treatment of advanced mycosis fungoides by allogeneic stem-cell transplantation with a nonmyeloablative regimen. Bone Marrow Transplant 31:663–666

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Chapter 9 The Role of Allogeneic Transplantation for Multiple Myeloma in Older Adults Heidi D. Klepin and David D. Hurd

1.  Introduction Multiple myeloma is a disease of the elderly. Survival outcomes remain unacceptably low in older adults with multiple myeloma. To date, no obvious difference in tumor biology has been elucidated to explain the survival disparity between older and younger patients. Multiple factors including comorbidity, performance status, decreased physiologic reserve, and potentially undertreatment contribute to poor outcomes in elderly patients with myeloma. Allogeneic stem cell transplantation remains the only curative treatment for multiple myeloma to date. Use of allogeneic transplantation in older adults remains limited due to high associated morbidity and mortality. Recently, traditional upper age limits for transplantation have been questioned, along with the definition of “elderly” itself. Multiple challenges will need to be met to advance the role of allogeneic transplantation into the treatment paradigm for older adults. These challenges include (1) optimizing conditioning regimens, (2) decreasing graft versus host disease (GVHD), (3) optimizing donor selection, and (4) optimizing patient selection.

2.  Myeloma and Age Multiple myeloma is a disease of the elderly with a median age of 70 years reported in the SEER database from 1997 to 2001 [1]. The incidence of multiple myeloma increases with age (Fig. 9-1). The number of elderly patients with myeloma is expected to increase with the aging of the US population translating into more complex and challenging treatment decisions in clinical practice. Age has a negative impact on prognosis in multiple myeloma in unselected populations. The SEER registry statistics from 1995 to 2001 report 5-year relative survival rates of 42.9 and 25.2% for patients <65 and ³65 years of age, respectively (SEER website). Survival is dramatically decreased in the ³75-year old population. In a retrospective review of 1,027 patients with newly diagnosed multiple myeloma evaluated at the Mayo Clinic, Kyle et al. reported a median age of 66 years with 38% of patients ³70 years of age [2]. From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_9, © Springer Science + Business Media, LLC 2003, 2010

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Fig. 9-1.  Age-specific SEER incidence rates for multiple myeloma 1998–2002. SEER surveillance epidemiology and end results. http://seer.cancer.gov/

The median duration of survival was 40.5 months for patients younger than 70 years and 26.4 months for those 70 years and older. In multivariate analysis, age remained one of the most important negative prognostic factors. To date, no clear differences in the biology of disease due to age have been elucidated in multiple myeloma. Several studies have reported no significant differences in older patients with regard to presenting biologic features including hematologic parameters, stage of disease, beta 2 microglobulin level, or renal function [3–5]. There is no evidence that clinically significant cytogenetic abnormalities are age dependent [6]. Available evidence, therefore, does not support the idea that older patients have more aggressive disease. The negative impact of age on prognosis was reviewed by Mileshkin et al. and related to a combination of patient-specific factors including decreased physiologic reserve, comorbidity, performance status, social support, referral bias, and potentially, undertreatment [7]. There are multiple physiologic changes associated with “normal” aging which can impact tolerance to the stress of tumor burden and treatment. These include altered immune function, changes in drug metabolism, and changes in organ function (i.e., decreased glomerular filtration rate, decreased cardiac output, decreased resting PAO2, decreased muscle mass). The degree to which any individual patient experiences age-related physiologic changes varies significantly and cannot accurately be measured by chronologic age alone.

Chapter 9  The Role of Allogeneic Transplantation for Multiple Myeloma in Older Adults 

In addition to subclinical changes in organ function, older adults are more likely to present with measurable comorbid disease. Significant concurrent comorbidity was recorded in 33.8% of patients in an unselected cohort aged 70 and above with newly diagnosed multiple myeloma [5] and likely plays a large role in treatment decisions and toxicity. Poor functional status (ECOG performance status score ³2) is more common in older patients with multiple myeloma and is independently associated with decreased survival [2, 5]. The impact of inadequate social support, referral bias, and treatment bias can be speculated but have not yet been studied in this population. Despite the negative impact of age on prognosis in unselected populations, there have been multiple small studies which have reported equivalent response to therapy and survival rates for selected older patients compared to younger patients treated with similar therapies including autologous stem cell transplantation [4, 8–11]. Most of these analyses were conducted on selected patients who were enrolled on clinical trials. These reports of equal benefit from equal treatment in a selected elderly population have resulted in increased interest in pursuing more aggressive therapies in older patients in an attempt to improve survival outcomes. In this context, an exploration of the role of potentially curative allogeneic transplantation becomes a reasonable question for older adults with myeloma. It is important, however, to carefully refine the definition of “older” in the transplant setting. The unacceptably high treatment-related toxicity (53%) documented for previously untreated myeloma patients £55 years of age who received myeloablative allogeneic transplantation on the US Intergroup Trial S9321 raises significant concerns that certain aggressive treatments will be prohibitively toxic in older adults [12]. It is unlikely that myeloablative allogeneic transplantation will be explored in adults >70 years of age. This study, however, did support the notion of curability with myeloablative allogeneic transplantation due to documented long-term survival. It is unclear whether treatment toxicity would be decreased if this trial were repeated with modern supportive care strategies. Recent advances including reduced intensity conditioning (RIC), use of peripheral blood stem cells, sophisticated GVHD prophylaxis regimens, and improved supportive care may pave the way for extension of allogeneic transplantation techniques in to the young–old population which has until recently been excluded.

3.  Trends in Allogeneic Transplantation by Age Allogeneic transplantation has traditionally been restricted in clinical trials and practice to younger adults due to concerns regarding increased toxicity with age. Many centers have used age limits of 50 or 55 years. In recent years upper age limits have been challenged. Worldwide the number of allogeneic transplantations being performed has increased. The number of allogeneic transplantations in adults over the age of 50 continues to increase as well (Fig. 9-2). According to the Center for International Blood and Marrow Transplant Research (CIBMTR), between the years 2002 and 2006 35% of allograft recipients were above the age of 50. These trends can be expected to continue as many of the common indications for allogeneic transplantation including acute myelogenous leukemia, myelodysplastic syndrome, chronic myelogenous leukemia, non-Hodgkin’s lymphoma, and multiple myeloma are most commonly diagnosed in older adults.

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H.D. Klepin and D.D. Hurd

100

80

Transplants, %

130 

≤ 20 yrs 21-40 yrs 41-50 yrs 51-60 yrs > 60 yrs

60

40

20

0

1987-1992

1993-1999

2000-2006

Fig.  9-2.  Trends in allogeneic transplantation by recipient age from 1987 to 2006.* Data acquired from the Center for International Blood and Marrow Transplant Research (CIBMTR).* Transplants for acute myelogenous leukemia, acute lymphocytic leukemia, and chronic myelogenous leukemia

4.  The Influence of Age on Myeloablative Allogeneic Transplantation Age is one of the most commonly used eligibility criteria for allogeneic transplantation. Multiple early studies have suggested a negative association between increasing age and outcomes with conventional myeloablative transplantation regimens used to treat various hematologic malignancies [13–15]. Many of these initial studies included patients younger than 20 raising suspicions that the improved outcomes associated with younger age were most attributed to inclusion of pediatric patients. The question of an upper age limit for transplantation has more recently been investigated in cohorts of adult patients using age cutoffs of 50 years or greater (Table 9-1). The majority of these studies are retrospective, single institution studies with relatively small numbers of patients >50 years of age represented. Of the single institution studies represented, 5 reported favorable outcomes in adults over 50 years of age while 4 reported increased treatment-related mortality (TRM) or decreased overall survival associated with age [16–24]. Two larger multi-institution retrospective studies have addressed this issue. Ringden et al. evaluated the outcome of 2,180 transplants for leukemia from 138 institutions reported to the International Bone Marrow Transplant Registry (IBMTR) [25]. Outcomes were analyzed by the following age groups: 30–39 years, 40–44 years, 45–49 years, and 50 years of age or older. The incidence of leukemia free survival, GVHD, and relapse were comparable between the age cohorts. TRM at 2 years was higher for those aged >45 years with advanced leukemia. The largest multi-center study to investigate the influence of age on myeloablative transplantation was reported by the Japan Society for Hematopoietic Stem Cell Transplantation [26]. In this study 5,127 patients

1986

1993

1993

1998

1998

2000

2002

2003

2004

2005

2006

Klingemann et al. [23]

Ringden et al. [25]

Clift et al. [16]

Du et al [20]

Hansen et al. [22]

Deeg et al. [18]

De la Camara et al. [17]

Farag et al. [21]

Yanada et al. [26]

Wallen et al. [24]

Ditschkowski et al. [19]

MSD

MSD

MSD/syngeneic

Donor

Various

Various

Various

Various

Various

MDS

CML

MSD/MUD

MSD

MSD/MUD

MSD

MSD

MSD/MUD

MUD

AML/MDS MSD/MUD

CML

Leukemia

Leukemia

Disease

215

52

5,147

313

129

50

196

436

33

2,180

63

Total N

215

  52

398

  52

  32

  50

  13

  59

  33

  80

  13

N > 50 years

Allogeneic transplantation is feasible in age >50

Allogeneic transplantation feasible in age >60

Age >50 was associated with increased TRM and decreased OS

Age >50 associated with increased TRM in patients with high risk disease only

No difference in GVHD, TRM, OS in age >50

Allogeneic transplantation feasible in age >55

Age >50 associated with decreased OS

No difference in OS and GVHD. Age >50 associated with increased TRM compared to age <40

Patients >50 had comparable survival to younger patients

No difference in OS, DFS, GVHD by age group. Patients >45 with advanced leukemia had increased TRM

Age >50 associated with increased GVHD, TRM, and decreased OS

Comments

CML chronic myelogenous leukemia, AML acute myelogenous leukemia, MDS myelodysplastic syndrome, MSD matched sibling donor, MUD matched unrelated donor, GVHD graft versus host disease, TRM treatment related mortality, OS overall survival, DFS disease free survival

Year

Author (reference)

Table 9-1.  Myeloablative allogeneic transplantation for patients above age 50.

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treated for various hematologic malignancies were analyzed, including 389 patients above age 50. Age above 50 was found to be independently associated with TRM and decreased overall survival. Estimated 1-year TRM was 34.7 and 22.7% in the older and younger groups, respectively. The probability of overall survival at 4 years was 35.6% for patients over 50 and 53.5% for those younger than 50.

5.  Potential Barriers to Allogeneic Transplantation in Older Adults There are multiple potential barriers to successful allogeneic transplantation in older adults. These include concerns relate to the toxicity of the preparative regimens, GVHD, donor availability, and patient selection. Optimizing the preparative regimens to maximize efficacy but minimize morbidity is critical to successful transplantation in older adults. There are no prospective studies comparing preparative regimens in older adults. Retrospective data suggest that regimens containing total body radiation may increase TRM in this population [26]. RIC regimens designed to minimize the toxicity of myeloablation have been explored in older adults and will be discussed in the next section. GVHD represents another potential barrier to transplantation in the elderly and has been identified as a major contributing factor to transplant-related mortality in older adults [19]. Increasing age has been identified as a risk factor for development of acute GVHD [27, 28]. Analysis of 2,036 recipients of HLA-identical sibling transplants from the IBMTR demonstrated an increased risk for acute GVHD in older patients. The age gradient was modest and the association was no longer significant after excluding female-to-male transplants [27]. Similar findings were reported by Weisdorf et  al. using an age cutoff of 18 while analyzing 469 patients with histocompatible sibling donors at a single institution [28]. The question of whether increasing age poses an incremental increased risk is unclear. One study supports an increased risk of acute GVHD in adults >50 years of age compared to younger adults [23] while others do not [20–22]. There is some evidence, however, that age is associated with increased severity of acute GVHD [29]. Several studies have also documented an increase in chronic GVHD associated with increasing age [30–32]. Analysis of 2,534 recipients of HLA-identical sibling transplants conducted by the IBMTR showed that the strongest risk factor for chronic GVHD was a history of acute GVHD [30]. When patients with a history of acute GVHD were excluded, age over 20 years became an independent risk factor. Again the question of incremental risk with increasing age remains unclear. There are conflicting results from single institution studies regarding the impact of age >50 years on risk of chronic GVHD [20–23]. A study from Aschan et  al. of 182 leukemia patients reported that adults >30 years benefited more significantly from double GVHD prophylaxis than younger patients with decreased chronic GVHD and improved survival [33]. Continued advances in GVHD prophylaxis will likely improve transplant outcomes in older adults. Donor availability represents another potential obstacle in allogeneic transplantation for older adults. The possibility of finding a matched family donor

Chapter 9  The Role of Allogeneic Transplantation for Multiple Myeloma in Older Adults 

tends to be lower in older adults due to aging of the family. Matched sibling donors are also likely to be older which is associated with an increased risk of developing acute GVHD and potentially increased TRM [34, 35]. Additional evidence regarding the significance of increased donor age comes from the National Marrow Donor Program. Analysis of 6,978 unrelated-donor marrow transplantations showed that increasing donor age was independently associated with increased risk of GVHD and decreased overall survival [36]. Optimal donor selection for older adults is unclear and may require investigation of the potential trade-off between use of older sibling grafts and younger matched unrelated grafts. Advances in HLA matching using high-resolution typing result in improved survival in unrelated donor transplantation [37] and may translate into improved options and outcomes for older adults. Finally, patient selection remains a major obstacle in myeloablative transplantation for older adults. There are two issues related to patient selection to consider in this context. The first relates to disease status and the second relates to the fitness of the individual patient. It is clear from retrospective studies that transplantation in earlier stages of disease results in superior outcomes for adults >50 years of age [19, 21]. One study of 215 adults >50 years of age who underwent myeloablative allogeneic transplantation for either early (41%) or advanced (59%) hematologic malignancies reported significantly decreased TRM and improved overall survival in the early stage group [19]. The timing of transplantation remains an unresolved issue particularly in the setting of multiple myeloma where multiple treatment options are now available. Finally, a systematic approach to the assessment of biologic age has not yet been developed making selection of “fit” older adults a challenge in the clinical setting. Older adults represent a heterogeneous population. They are more likely to present with diagnosed comorbid disease [5, 38, 39] or may have subclinical changes in organ function resulting in decreased physiologic reserve. Age-related changes in drug metabolism may also impact toxicity risk. The development of evidence-based patient selection algorithms to identify older adults who are most likely to tolerate and benefit from allogeneic transplantation is critical to successful application of this modality to an older population.

6.  Reduced Intensity Conditioning Transplantation RIC or non-myeloablative allogeneic transplantation approaches were designed to achieve engraftment without marrow ablation. This method may be efficacious in settings where the graft versus tumor effect is sufficient to eradicate or control underlying disease. Due to the non-myeloablative approach this method has been investigated in patients who would have been considered ineligible for traditional myeloablative transplantation including older adults. Data from the CIBMTR demonstrate that the majority of RIC transplants reported in 2005–2006 were performed in adults >50 years of age (Fig. 9-3). Multiple retrospective studies have compared outcomes between standard myeloablative conditioning and RIC regimens. Overall there appears to be a decrease in TRM and in the incidence of GVHD favoring the reduced intensity regimens despite increased comorbidity in many of the RIC patients [40–50]. The difference in overall survival appears less clear due in part to an increased risk of relapse seen with RIC particularly in the setting of leukemia [41, 44].

133

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H.D.Age Klepin and D.D.ofHurd Distribution Patients Receiving Allogeneic Transplants by Conditioning Regimen Intensity, 2005-2006

<20y

1,999

425

20-29y

1,130

30-39y

1,125

40-49y 50-59y

279

318

1,449

624

1,336

1,190

60-69y

1,004

213

£70y

3

2400 2000 1600 1200 800

Myeloablative

400

0

51 0

400

800

1200

1600

Reduced Intensity

Fig. 9-3.  Age distribution of patients receiving allogeneic transplants by conditioning regimen intensity, 2005– 2006. Data acquired from the Center for International Blood and Marrow Transplant Research (CIBMTR)

Reduced intensity regimens have been explored to a limited extent in adults over age 60. Multiple small series have reported favorable rates of engraftment, chimerism, TRM, and GVHD in this age group [39, 51–54]. Some of these studies reported no differences in outcome between related and matched unrelated transplants using RIC regimens [53, 54]. The impact of age on outcome was evaluated in two larger series using RIC transplantation [55, 56]. Gomez-Nunez et al. evaluated the results of 145 patients ineligible for myeloablative transplantation who underwent RIC transplantation from a matched sibling over a 3-year period [56]. The median age was 54. Multivariate analyzes found decreased overall survival and increased TRM in adults >60 years of age. Additional negative prognostic factors included prior autologous transplant and Eastern Cooperative Oncology Group Performance Score >1. Alternatively, Corradini et  al. compared outcomes of 90 patients younger than 55 years with 60 patients older than 60 years who were treated with RIC transplantation from a sibling donor [55]. There was no difference in TRM or overall survival between the two groups. However, in the subset of patients who had a prior autologous transplant, older age was associated with increased TRM. Overall, RIC regimens may offer a viable alternative to myeloablative transplantation in selected older adults.

7.  Improving Patient Selection Older patients with hematologic malignancies represent a very heterogeneous population requiring more detailed assessment of health status prior to treatment

Chapter 9  The Role of Allogeneic Transplantation for Multiple Myeloma in Older Adults 

135

determination. Eligibility criteria remain a central issue in future trials focused on older adults particularly with regard to allogeneic transplantation. Chronologic age alone is a poor surrogate marker for tolerance to therapy. Developing a set of measurable clinical characteristics which better reflect physiologic age is necessary to critically evaluate the role of allogeneic transplantation in the heterogeneous older adult population. Comorbidity and functional status represent measurable patient-specific characteristics that can refine evaluation of older adults in clinical trials and practice. Older adults are more likely to present with increased comorbid disease [5, 7]. Comorbidity assessment with the Charlson Comorbidity Index has been shown to be predictive of increased toxicity and mortality in allogeneic transplantation [49, 57]. Sorror et al. refined this index to improve sensitivity and documented the reliability and validity of this tool in allogeneic transplantation (Table 9-2) [58]. The new transplant-specific index showed better survival prediction than the Charlson Comorbidity Index in this population. Low scores on the transplant-specific comorbidity index appear predictive of improved survival in both myeloablative and non-myeloablative transplants and in patients with both low and high risk disease [59]. Similarly, Artz et al.

Table 9-2.  Hematopoietic cell transplantation (HCT)-specific comorbidity index (HCT–CI). Comorbidity

Definition

Score

Cardiac

Coronary artery disease, congestive heart failure, myocardial infarction, EF £ 50%

1

Arrhythmia

Atrial fibrillation or flutter, sick sinus or ventricular arrhythmia

1

Cerebrovascular disease

Transient ischemic attack or cerebrovascular accident

1

Diabetes

Requiring treatment with medication

1

Inflammatory bowel disease

Crohn’s disease or ulcerative colitis

1

Obesity

Body mass index >35 kg/m2

1

Infection

Requiring use of antimicrobial treatment

1

Psychiatric disturbance

Depression or anxiety requiring psychiatric consult or treatment

1

Peptic ulcer

Requiring treatment

2

Hepatic disease (mild)

Chronic hepatitis, bilirubin > ULN to 1.5 X the ULN, or AST/ ALT > ULN to 2.5 X ULN

2

Pulmonary (moderate)

DLCO and or FEV1 66–80% or dyspnea with slight activity

2

Rheumatologic

SLE, RA, polymyositis, mixed CTD, or polymyalgia rheumatica

2

Renal (moderate/severe)

Serum creatinine >2 mg/dL, on dialysis, or prior renal transplant

2

Prior solid tumor

Treated at any point in patient’s past history, excluding nonmelanoma

3

Hepatic (moderate/severe)

Liver cirrhosis, bilirubin >1.5X ULN, or AST/ALT > 2.5 X ULN

3

Heart valve disease

Except mitral valve prolapse

3

Severe pulmonary

DLCO and/or FEV1 £65% or dyspnea at rest or requiring oxygen

3 Total score

This table was adapted from Ref. [58] EF ejection fraction, ULN upper limit of normal, SLE systemic lupus erythmatosis, RA rheumatoid arthritis, CTD connective tissue disease, DLCO diffusion capacity of carbon monoxide

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reported that a simple scale combining the Kaplan–Feinstein Comorbidity Scale and the Eastern Cooperative Oncology Group Performance Status Scale enabled separation of high and low risk patients with 6-month cumulative incidences of TRM of 50 and 15%, respectively [60]. Prospective assessment of comorbidity using established or transplant-specific indices may provide important information for the development of evidence-based risk stratification in older adults evaluated for allogeneic transplantation. Comorbidity assessment alone may not provide sufficient information regarding the physiologic reserve of older patients. In clinical practice it is apparent that older patients with similar age and comorbidity may differ substantially with regard to functional status. Specific measures of functional status can provide more prognostic information than comorbidity alone in older adults [61]. In older cancer patients, Extermann et al. demonstrated that comorbidity and functional assessment were not well correlated and provided independent information [62]. Careful functional assessment during pretransplant evaluation may provide added information for risk stratification. It will be important to prospectively assess the predictive value of task-specific functional assessment tools such as activities of daily living [63] and instrumental activities of daily living [64]. These self-report measures are able to identify functional impairment in cancer patients with good performance scores on the Eastern Cooperative Oncology Group scale [65]. This added discriminatory capacity may be useful to detect subtle changes reflective of decreased functional reserve. Finally, objective measures of physical performance and cognition may be particularly useful in developing an evaluation protocol for older patients being considered for allogeneic transplantation. Physical performance measures such as walking speed and lower extremity function are predictive of future disability, hospitalizations, and mortality in the geriatric population [66–68]. These objective measures may be sensitive to subclinical disability which could be closely associated with morbidity and mortality outcomes in the setting of allogeneic transplantation.

8.  Applications in Multiple Myeloma Despite the increased use of dose-intensive therapy and autologous transplantation in older patients with multiple myeloma, there remains a dearth of information on the use of allogeneic transplantation in this patient population. Refined assessment of physiologic age may facilitate the evaluation of allogeneic transplantation techniques in the treatment of older adults with multiple myeloma. There is recent evidence which supports a potential role for RIC allogeneic transplantation in multiple myeloma treatment. A prospective study of 162 consecutive patients (median age 55; range 30–65 years) compared RIC allogeneic transplantation from a sibling donor with tandem autologous transplantation in newly diagnosed multiple myeloma [69]. All patients received vincristine, doxorubicin, and dexamethasone followed by high-dose melphalan with autologous stem cell rescue. After recovery, patients with an

Chapter 9  The Role of Allogeneic Transplantation for Multiple Myeloma in Older Adults 

HLA-identical sibling underwent non-myeloablative allogeneic transplantation. Patients without an HLA-matched sibling underwent a second autologous transplant. Overall survival was 80 months versus 54 months (p = 0.01) favoring the allograft group. Among patient who completed their assigned treatments, TRM was not significantly different between the two groups while disease-related mortality was higher in the double autologous transplant group. These findings suggest that allogeneic transplantation may offer longer term disease control for selected fit patients with multiple myeloma. However, this conclusion should be interpreted with caution due to the disproportionately poor outcomes seen in the tandem autologous transplant arm compared to other published studies. It is currently unclear which older adults might benefit from allogeneic transplantation and where transplantation fits in the sequencing of treatment options currently available. Additional clinical trials are underway evaluating tandem autologous transplantation followed by RIC transplantation for multiple myeloma. Similarly, the BMT Clinical Trials Network Protocol 0102 is a trial of tandem autologous stem cell transplants versus a single autologous stem cell transplant followed by a matched sibling non-myeloablative allogeneic stem cell transplant in patients up to the age of 70 (with a Karnofsky performance status of ³70). All autologous transplants utilize melphalan 200 mg/m2 while the RIC allogeneic transplant gives only a single fraction of 200  cGy of total body irradiation. GVHD prophylaxis is cyclosporine and mycophenolate mofetil. The primary objective of this study is to compare progression-free survival at 3 years between the two strategies. Approximately 600 subjects have been enrolled on this study, including 150 allografts, which should provide representative data to help define the role of RIC allogeneic transplantation for fit adults up to age 70 years with multiple myeloma. While these studies begin to address the role of RIC transplantation in the younger, older patients with multiple myeloma, the majority of patients with this disease will still be excluded secondary to age (no studies for patients >70 years of age) and the lack of a suitable sibling donor for the majority of patients.

9.  Future Directions Allogeneic transplantation may offer a potentially curative treatment option for selected older adults with hematologic malignancies including multiple myeloma. However, substantial concerns regarding treatment toxicity persist which will continue to limit application of this treatment modality in the true elderly or frail population. Future advances in this field will need to build upon a better understanding of the relationship between physiologic aging and allogeneic transplantation rather than relying on chronologic age cutoffs in research and practice. Future research goals include (1) prospective evaluation of elderly specific transplantation strategies; (2) development of refined patient selection criteria which incorporate comorbidity, physical function, and cognition; and (3) incorporation of additional outcome measures into clinical trials to evaluate transplantation in the context of its impact on quality of life and disability as well as overall survival (Table 9-3).

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Table 9-3.  Future research directions for allogeneic transplantation in older adults. Research goal

Topics for investigation

Development of elderly specific transplantation regimens

Reduced intensity conditioning regimens Optimal donor selection (sibling vs. younger matched unrelated) Optimal GVHD prophylaxis Optimal timing of transplantation

Development and validation of an elderly specific patient selection algorithm

Transplant-specific comorbidity assessment Predictive value of self-report functional status (e.g., Activities of daily living, instrumental activities of daily living) Predictive value of baseline physical performance measures (e.g., walking speed, lower extremity strength/balance) Evaluation of cognitive assessment

Incorporation of additional outcome measures into clinical trials

Quality of life measures Disability assessment

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H.D. Klepin and D.D. Hurd transplantation and prophylaxis with cyclosporine and methotrexate. Blood 80(7):1838–1845 30. Atkinson K, Horowitz MM, Gale RP, van Bekkum DW, Gluckman E, Good RA et al (1990) Risk factors for chronic graft-versus-host disease after HLA-identical sibling bone marrow transplantation. Blood 75(12):2459–2464 31. Carlens S, Ringden O, Remberger M, Lonnqvist B, Hagglund H, Klaesson S et al (1998) Risk factors for chronic graft-versus-host disease after bone marrow transplantation: A retrospective single centre analysis. Bone Marrow Transplant 22(8):755–761 32. Ochs LA, Miller WJ, Filipovich AH, Haake RJ, McGlave PB, Blazar BR et  al (1994) Predictive factors for chronic graft-versus-host disease after histocompatible sibling donor bone marrow transplantation. Bone Marrow Transplant 13(4):455–460 33. Aschan J, Ringden O (1994) Prognostic factors for long-term survival in leukemic marrow recipients with special emphasis on age and prophylaxis for graft-versushost disease. Clin Transplant 8(3 Pt 1):258–270 34. Doney K, Fisher LD, Appelbaum FR, Buckner CD, Storb R, Singer J et al (1991) Treatment of adult acute lymphoblastic leukemia with allogeneic bone marrow transplantation. Multivariate analysis of factors affecting acute graft-versus-host disease, relapse, and relapse-free survival. Bone Marrow Transplant 7(6):453–459 35. Mehta J, Gordon LI, Tallman MS, Winter JN, Evens AM, Frankfurt O et al (2006) Does younger donor age affect the outcome of reduced-intensity allogeneic hematopoietic stem cell transplantation for hematologic malignancies beneficially? Bone Marrow Transplant 38(2):95–100 36. Kollman C, Howe CW, Anasetti C, Antin JH, Davies SM, Filipovich AH et al (2001) Donor characteristics as risk factors in recipients after transplantation of bone marrow from unrelated donors: The effect of donor age. Blood 98(7):2043–2051 37. Lee SJ, Klein J, Haagenson M, Baxter-Lowe LA, Confer DL, Eapen M et al (2007) High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood 110(13):4576–4583 38. Popplewell LL, Forman SJ (2002) Is there an upper age limit for bone marrow transplantation? Bone Marrow Transplant 29(4):277–284 39. Shapira MY, Tsirigotis P, Resnick IB, Or R, Abdul-Hai A, Slavin S (2007) Allogeneic hematopoietic stem cell transplantation in the elderly. Crit Rev Oncol Hematol 64(1):49–63 40. Alyea EP, Kim HT, Ho V, Cutler C, Gribben J, DeAngelo DJ et  al (2005) Comparative outcome of nonmyeloablative and myeloablative allogeneic hematopoietic cell transplantation for patients older than 50 years of age. Blood 105(4):1810–1814 41. Aoudjhane M, Labopin M, Gorin NC, Shimoni A, Ruutu T, Kolb HJ et al (2005) Comparative outcome of reduced intensity and myeloablative conditioning regimen in HLA identical sibling allogeneic haematopoietic stem cell transplantation for patients older than 50 years of age with acute myeloblastic leukaemia: A retrospective survey from the Acute Leukemia Working Party (ALWP) of the European group for Blood and Marrow Transplantation (EBMT). Leukemia 19(12):2304–2312 42. Couriel DR, Saliba RM, Giralt S, Khouri I, Andersson B, de Lima M et al (2004) Acute and chronic graft-versus-host disease after ablative and nonmyeloablative conditioning for allogeneic hematopoietic transplantation. Biol Blood Marrow Transplant 10(3):178–185 43. Diaconescu R, Flowers CR, Storer B, Sorror ML, Maris MB, Maloney DG et  al (2004) Morbidity and mortality with nonmyeloablative compared with myeloablative conditioning before hematopoietic cell transplantation from HLA-matched related donors. Blood 104(5):1550–1558 44. Martino R, Iacobelli S, Brand R, Jansen T, van Biezen A, Finke J et  al (2006) Retrospective comparison of reduced-intensity conditioning and conventional

Chapter 9  The Role of Allogeneic Transplantation for Multiple Myeloma in Older Adults  high-dose conditioning for allogeneic hematopoietic stem cell transplantation using HLA-identical sibling donors in myelodysplastic syndromes. Blood 108(3):836–846 45. Massenkeil G, Nagy M, Neuburger S, Tamm I, Lutz C, le Coutre P et  al (2005) Survival after reduced-intensity conditioning is not inferior to standard high-dose conditioning before allogeneic haematopoietic cell transplantation in acute leukaemias. Bone Marrow Transplant 36(8):683–689 46. Mielcarek M, Martin PJ, Leisenring W, Flowers ME, Maloney DG, Sandmaier BM et al (2003) Graft-versus-host disease after nonmyeloablative versus conventional hematopoietic stem cell transplantation. Blood 102(2):756–762 47. Perez-Simon JA, Diez-Campelo M, Martino R, Brunet S, Urbano A, Caballero MD et al (2005) Influence of the intensity of the conditioning regimen on the characteristics of acute and chronic graft-versus-host disease after allogeneic transplantation. Br J Haematol 130(3):394–403 48. Shimoni A, Hardan I, Shem-Tov N, Yeshurun M, Yerushalmi R, Avigdor A et al (2006) Allogeneic hematopoietic stem-cell transplantation in AML and MDS using myeloablative versus reduced-intensity conditioning: The role of dose intensity. Leukemia 20(2):322–328 49. Sorror ML, Maris MB, Storer B, Sandmaier BM, Diaconescu R, Flowers C et al (2004) Comparing morbidity and mortality of HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative and myeloablative conditioning: Influence of pretransplantation comorbidities. Blood 104(4):961–968 50. Valcarcel D, Martino R, Sureda A, Canals C, Altes A, Briones J et  al (2005) Conventional versus reduced-intensity conditioning regimen for allogeneic stem cell transplantation in patients with hematological malignancies. Eur J Haematol 74(2):144–151 51. Bertz H, Potthoff K, Finke J (2003) Allogeneic stem-cell transplantation from related and unrelated donors in older patients with myeloid leukemia. J Clin Oncol 21(8):1480–1484 52. Falda M, Busca A, Baldi I, Mordini N, Bruno B, Allione B et  al (2007) Nonmyeloablative allogeneic stem cell transplantation in elderly patients with hematological malignancies: Results from the GITMO (Gruppo Italiano Trapianto Midollo Osseo) multicenter prospective clinical trial. Am J Hematol 82(10):863–866 53. Kroger N, Shimoni A, Zabelina T, Schieder H, Panse J, Ayuk F et  al (2006) Reduced-toxicity conditioning with treosulfan, fludarabine and ATG as preparative regimen for allogeneic stem cell transplantation (alloSCT) in elderly patients with secondary acute myeloid leukemia (sAML) or myelodysplastic syndrome (MDS). Bone Marrow Transplant 37(4):339–344 54. Tsirigotis P, Bitan RO, Resnick IB, Samuel S, Ackerstein A, Eladi S et al (2006) A non-myeloablative conditioning regimen in allogeneic stem cell transplantation from related and unrelated donors in elderly patients. Haematologica 91(6):852–855 55. Corradini P, Zallio F, Mariotti J, Farina L, Bregni M, Valagussa P et  al (2005) Effect of age and previous autologous transplantation on nonrelapse mortality and survival in patients treated with reduced-intensity conditioning and allografting for advanced hematologic malignancies. J Clin Oncol 23(27):6690–6698 56. Gomez-Nunez M, Martino R, Caballero MD, Perez-Simon JA, Canals C, Mateos MV et al (2004) Elderly age and prior autologous transplantation have a deleterious effect on survival following allogeneic peripheral blood stem cell transplantation with reduced-intensity conditioning: Results from the Spanish multicenter prospective trial. Bone Marrow Transplant 33(5):477–482 57. Alamo J, Shahjahan M, Lazarus HM, de Lima M, Giralt SA (2005) Comorbidity indices in hematopoietic stem cell transplantation: A new report card. Bone Marrow Transplant 36(6):475–479 58. Sorror ML, Maris MB, Storb R, Baron F, Sandmaier BM, Maloney DG et al (2005) Hematopoietic cell transplantation (HCT)-specific comorbidity index: A new tool for risk assessment before allogeneic HCT. Blood 106(8):2912–2919

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H.D. Klepin and D.D. Hurd 59. Sorror ML, Sandmaier BM, Storer BE, Maris MB, Baron F, Maloney DG et  al (2007) Comorbidity and disease status based risk stratification of outcomes among patients with acute myeloid leukemia or myelodysplasia receiving allogeneic hematopoietic cell transplantation. J Clin Oncol 25(27):4246–4254 60. Artz AS, Pollyea DA, Kocherginsky M, Stock W, Rich E, Odenike O et al (2006) Performance status and comorbidity predict transplant-related mortality after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 12(9):954–964 61. Inouye SK, Peduzzi PN, Robison JT, Hughes JS, Horwitz RI, Concato J (1998) Importance of functional measures in predicting mortality among older hospitalized patients. JAMA 279(15):1187–1193 62. Extermann M, Overcash J, Lyman GH, Parr J, Balducci L (1998) Comorbidity and functional status are independent in older cancer patients. J Clin Oncol 16(4):1582–1587 63. Katz S (1983) Assessing self-maintenance: Activities of daily living, mobility, and instrumental activities of daily living. J Am Geriatr Soc 31(12):721–727 64. Lawton MP, Brody EM (1969) Assessment of older people: Self-maintaining and instrumental activities of daily living. Gerontologist 9(3):179–186 65. Repetto L, Fratino L, Audisio RA, Venturino A, Gianni W, Vercelli M et al (2002) Comprehensive geriatric assessment adds information to Eastern Cooperative Oncology Group performance status in elderly cancer patients: An Italian Group for Geriatric Oncology Study. J Clin Oncol 20(2):494–502 66. Cesari M, Kritchevsky SB, Penninx BW, Nicklas BJ, Simonsick EM, Newman AB et al (2005) Prognostic value of usual gait speed in well-functioning older people – results from the Health, Aging and Body Composition Study. J Am Geriatr Soc 53(10):1675–1680 67. Guralnik JM, Ferrucci L, Simonsick EM, Salive ME, Wallace RB (1995) Lowerextremity function in persons over the age of 70 years as a predictor of subsequent disability. N Engl J Med 332(9):556–561 68. Studenski S, Perera S, Wallace D, Chandler JM, Duncan PW, Rooney E et  al (2003) Physical performance measures in the clinical setting. J Am Geriatr Soc 51(3):314–322 69. Bruno B, Rotta M, Patriarca F, Mordini N, Allione B, Carnevale-Schianca F et al (2007) A comparison of allografting with autografting for newly diagnosed myeloma. N Engl J Med 356(11):1110–1120

Chapter 10 Single Versus Tandem Autologous Hematopoietic Stem Cell Transplant in Multiple Myeloma David H. Vesole

1.  Single Autologous Transplantation It has been known for over 20 years, that high-dose therapy with melphalan can produce a profound anti-myeloma effect. Initially introduced to help overcome the native resistance of myeloma cells to conventional chemotherapy, highdose therapy requiring autologous hematopoietic stem cell support was first evaluated in patients with refractory disease [1]. This resulted in improved response rates and overall survival (OS). This approach was then extended to newly diagnosed patients. In 1996, the seminal randomized study on symptomatic stage II and III patients by the French Myeloma Intergroup, showed conclusively that high-dose therapy yielded a superior disease-free and OS outcome compared to conventional therapy [2]. The 5-year projected survival for the transplant group was 52 versus 12% for the standard therapy (SDT) group (Table 10-1). Whereas, complete responses were observed in only 5% of the conventional therapy group, 22% of the high-dose therapy group achieved complete remissions (CR). The transplant-related mortality was only 2.7% in the setting of bone marrow transplant without hematopoietic stem cell growth factors. Other randomized and nonrandomized comparisons have also demonstrated that high-dose therapy is superior to conventional chemotherapy [3–5]; this included a second large randomized trial by the Medical Research Council Myeloma VII trial also showing an improvement in median eventfree survival (EFS) and OS of approximately 12 months [3]. In an Arkansas study comparing tandem hematopoietic stem cell transplantation (HSCT) to conventional VAD chemotherapy, patients achieving complete response had a median disease-free survival of 50 months and median OS of more than 7 years [4]. Based on these results, multiple myeloma is currently the most common indication for HSCT in North America, with over 5,000 transplants performed yearly (Center for International Blood and Marrow Transplant Research [CIBMTR] estimates). Indeed, national and international guidelines consider upfront HSCT in transplant eligible patients as one of the standard treatment options for newly diagnosed myeloma (http://www.nccn.org).

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_10, © Springer Science + Business Media, LLC 2003, 2010

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Table 10-1.  Randomized trials of conventional chemotherapy compared to single ASCT as upfront therapy. Author

Group

N

Age years

CR% SDT vs. HSCT

p

Criteria for defining CR

OS benefit for HSCT

Comments

Attal et al. [2]

IFM90

200

£65

  5 vs. 22

S

Electrophoresis

Significant benefit

BMT. No GF. TRM 2.7%

Child et al. [3]

MRC7

401

£64

  8 vs. 44

S

EBMT– IBMTR (IF)

Significant benefit

TRM 3%

Blade et al. [6]

PETHEMA

216

£65

11 vs. 30

S

EBMT– IBMTR (IF)

No OS/EFS benefit

Only PR and CR included

Barlogie et al. [7]

USIG

899

<70

15 vs. 17

NS

EBMT– IBMTR (IF)

No OS benefit

34% delayed HSCT

SDT standard dose therapy, HSCT hematopoietic stem cell transplant, S statistically significant, NS not significant, IF immunofixation, BMT bone marrow transplant, GF growth factors, TRM transplant-related mortality

Furthermore, high-dose therapy with autologous transplantation and improved supportive care measures has resulted in improvement in myeloma survival rates over the past three decades [5]. In contrast, there are also randomized clinical trials that failed to show an improvement in event-free survival or OS. The Spanish PETHEMA trial included one hundred sixty-four patients who were randomly assigned: 83 to continued conventional chemotherapy and 81 to HSCT [7]. The CR rate was significantly higher with HSCT (30 vs. 11%; p  =  0.002). However, progressionfree survival (PFS) was not significantly different between HSCT and conventional therapy (median, 42 vs. 33 months; p  =  not significant), and OS was similar in both groups (median, 61 vs. 66 months). Survival after relapse was identical in the two arms (15.9 vs. 16.4 months). A large United States Intergroup trial (S9321) included 899 patients: with a median follow-up time of 76 months, no differences were observed in response rates between the two study arms (HSCT, n  =  261 patients; SDT, n  =  255 patients) [8]. Similarly, PFS and OS durations did not differ between the HSCT and SDT arms, with 7-year estimates of PFS of 17 and 16%, respectively, and OS of 37 and 42%, respectively.

2.  Differences in the Single HSCT Studies The United States Intergroup study and the Spanish PETHEMA study are not straightforward comparisons of conventional chemotherapy versus HSCT. In the PETHEMA trial only patients achieving PR or CR to initial chemotherapy were randamized to HSCT versus Continuing Conventional Chemotherapy [7]. Patients who were refractory to initial conventional chemotherapy were thus excluded. This suggests that the study was unable to demonstrate an increase in the EFS or OS between conventional chemotherapy and HSCT in patients responding to initial chemotherapy (34 vs. 42 months and 67 vs. 65 months for EFS and OS between conventional therapy and HSCT). Although there was no difference in EFS or OS, this study did demonstrate a higher CR rate in the HSCT arm. The findings of this study caution against accepting CR as a surrogate for event-free survival benefit in HSCT for MM. Also, these findings suggest that there may only be a small additional benefit to HSCT in patients who achieve CR to induction therapy.

Chapter 10  Single Versus Tandem Autologous Hematopoietic Stem Cell 

The United States Intergroup Study, S9321, is the largest of the studies comparing conventional chemotherapy to high-dose therapy [8]. All patients received 3–4 cycles of VAD chemotherapy followed by high-dose cyclophosphamide for stem cell mobilization. The patients were randomized to receive melphalan 140 mg/m2 with total body irradiation 1,200 cGy or VBMCP for 1 year. Thus, the study compared “maintenance” VBMCP chemotherapy versus HDT with HSCT for EFS. Responding patients in each arm underwent a second randomization to either observation or interferon maintenance. At the time of relapse in the VBMCP arm, patients were encouraged to undergo HSCT. Although not part of the original design, this was incorporated into the treatment later to compare early versus late transplant. The VBMCP and early HSCT groups were similar in terms of CR, EFS (21 vs. 25 months, p = 0.14) and OS (53 vs. 62 months, p = 0.87). Interpretation of the results for OS was murkier since 52% of patients in the VBMCP arm had a late transplant at relapse/progression. Interestingly, the CR rates were also not significantly different between conventional chemotherapy versus HDT (15 vs. 17%) possibly implying that the transplant regimen of melphalan 140 mg/m2 with TBI 1200 cGy may be an inferior regimen compared to melphalan 200 mg/m2 used in the Arkansas studies. Melphalan 140  mg/m2 plus TBI 800  cGy has been shown to have similar EFS but inferior OS (p = 0.05) compared to melphalan 200  mg/m2 in the randomized French study IFM95-02 [9] and an Arkansas retrospective study [10]. Finally, a recent meta-analysis of 9 randomized clinical trials with a minimum of 2 years of follow-up which included 2,411 patients demonstrated a superior PFS benefit (Fig.  10-1) but not OS benefit (Fig. 10-2) for HSCT [11]. There was an increase in treatment-related mortality in the transplant group. It should be noted that some of these trials

Fig. 10-1.  PFS benefit of upfront HDT/transplant vs. SDT

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Fig. 10-2.  Lack of OS benefit with upfront HDT/transplant vs. SDT

were completed in the early 1990s before the routine use of peripheral blood stem cells and hematopoietic growth factors, factors that may have impacted on transplant-related mortality. One question that commonly arises: should newly diagnosed patients be treated until they achieve a partial remission before proceeding to transplant? In patients who are initial responders to treatment (as noted in the PETHEMA study), HSCT may not add survival benefit over continuing conventional therapy. In initial nonresponders with minimally responsive or primarily refractory disease, HSCT remains the most effective means of achieving a response. Alexanian et  al. observed that patients with primary resistant disease who received intensification within 1 year achieved a high frequency of response (69%), a 16% CR rate, and a survival benefit [12]. HSCT beyond 1 year was less effective affirming the greater value of early intensive therapy for these patients. In the Royal Marsden experience 40% of nonresponders to initial CVAMP therapy achieved CR with melphalan 200 mg/m2 [13]. In the Mayo Clinic experience 20% patients in the primary refractory group achieved CRs following high-dose therapy compared to 35% in the chemosensitive group yet the groups had a similar PFS [14]. Therefore, the absence of response to initial induction does not preclude a good response to HSCT. A recent preliminary report by the PETHEMA group evaluated patients with primary refractory disease: those with stable disease (n = 50) versus those with progressive disease (n = 31) to induction therapy [15]. Both groups were compared to patients with chemotherapy-sensitive disease. They found comparable OS in patients with chemotherapy-sensitive disease and in patients with stable disease. Patients with progressive disease to induction therapy had a very poor prognosis.

Chapter 10  Single Versus Tandem Autologous Hematopoietic Stem Cell 

3.  Timing of Transplant Another issue that has been raised is the optimal timing of the transplant. Two randomized studies comparing early versus late transplant at relapse showed comparable OS [8, 16]. In the French study, they reported higher quality of life scores in the upfront transplant cohort suggesting a valid benefit for upfront HSCT [16]. Therefore, high-dose therapy with autologous stem cell transplantation has been well established as appropriate care for front-line therapy in many newly diagnosed multiple myeloma patients, particularly those younger than 70 years of age. 3.1.  Tandem Autologous Transplantation Sequential planned tandem HSCT was developed in an attempt to increase dose intensity and achieve deeper sustained remissions. Reported CR rates with single HSCT have been in the 25–35% range, whereas tandem autologous transplantation has an expected CR rate ranging from 35% to 50%. After the feasibility of double intensification with HSCT had been reported [17–19], the Arkansas group pioneered the tandem planned autologous transplant approach in their Total Therapy I program with noncross resistant induction chemotherapy regimens culminating in tandem autologous transplantation [20]. They observed impressive EFS and OS of 43 and 68 months, respectively, which were superior to historical controls. In a recent update of the original cohort of 231 patients, 33% of the patients were alive at 10 years and 17% at 15 years with 15 and 7%, respectively, in continuous remission [21]. The results of the Arkansas Total Therapy II trial which incorporates thalidomide into a tandem transplant approach have been reported [22]. They observed superior 5-year continuous CR (60%) and improved EFS (50% at 5 years) compared to Total Therapy I. The median follow-up of 2.5 years is not yet mature enough to compare the OS. The current Arkansas approach – Total Therapy III – includes bortezomib in the induction regimen and post-transplant consolidation [23] with further improvement in the depth and duration of responses. At 24 months, 83% had achieved near-complete remission, which was sustained in 88% at 2 years from its onset. With a median follow-up of 20 months, 2-year estimates of event-free survival and OS were 84 and 86%, respectively. Based upon the early results of Total Therapy I, the French Myeloma Intergroup initiated a randomized trial (IFM 94) comparing single and double HSCT [24]. Three hundred and ninety-nine previously untreated MM patients were randomly assigned to receive either a single HSCT using (Mel 140 mg/ m2 + TBI 8  Gy) or a double HSCT (Mel 140  mg/m2 followed by Mel 140  mg/m2 + TBI 8  Gy). Patients then received interferon-2a maintenance therapy. The projected 7-year EFS and OS benefits were significant for the double HSCT arm (10 vs. 20% for EFS and 21 vs. 42% for OS, respectively). Of note, in a subgroup analysis, the patients most likely to benefit from the second transplant were those who achieved less than very good PR (defined as >90% reduction in paraprotein) to the first transplant. Additional randomized clinical trials from Italy (Bologna 96), Netherlands (HOVON 24), Germany (GMMG-HD2), and France (MAG 95) have compared double intensive therapy to single intensive therapy (Table  10-2) [25–28].

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Table 10-2.  Randomized trials of single vs. double autologous ASCT. Age Conditioning regimen CR% single EFS% single OS% single limit first → second vs. double vs. double vs. double

Author

N

IFM 94 [23]

399 60

Mel140 + TBI → Mel 140 42 vs. 50

25 vs. 30*

48 vs. 58*

Follow-up 75M

HOVON 24 [25]

304 65

56M

Mel 140 → Cy 120 + TBI 13 vs. 32*

21 vs. 22*

55 vs. 50

GMMG HD2 [26] 261 65

Mel 200 → Mel 200

23 vs. 29*

No difference NR

MAG 95 [27]

227 55

Mel140 + CCNU/VP16/ 39 vs. 37 CY-TBI vs. MEL 140 → Mel 140 + TBI

31 vs. 33

49 vs. 73**

53M

Bologna 96 [24]

228 60

Mel 200 → Mel 120 + BU 12

35 vs. 48

22 vs. 35*

59 vs. 73

55M

Tunisia [28]

195 60

Mel 200 → Mel 200 vs. Mel 200 → *thal 100×6 mo

68 vs. 54*

85 vs. 57*

85 vs. 65*

33M

NR

*Statistically significant difference (p < 0.05) **OS difference statistically superior for non-CD34 selected tandem transplants only in subset analysis Mel melphalan (doses in mg/m2), CY cyclophosphamide, TBI total body irradiation, BU busulfan, NR not reported

The preliminary data show an EFS benefit for the tandem approach in three out of the four studies. OS benefit has thus far been shown only for the IFM 94 study. Even the MAG study, which had previously failed to show any meaningful outcome differences between the tandem and single transplant arms, has been re-analyzed recently [28]. In the subgroup of patients who received non-CD34+ selected stem cell products, there was a significant OS advantage to the tandem arm (30 vs. 40% at 7 years, p = 0.04) although the EFS was still similar. Recently, the Tunisian myeloma group reported a Phase III trial of tandem autologous transplant (without any maintenance) with a single transplant with thalidomide maintenance (100 mg) for 6 months post-HSCT [29]. At the time of progressive disease, the single HSCT arm would proceed to a second HSCT. A total of 202 newly diagnosed patients under age 60 were enrolled on the trial. They received thalidomide plus dexamethasone for induction therapy. The transplant regimen was melphalan 200 mg/m2. Thalidomide 100 mg daily was started 3 months post-transplant for 6 month duration. A second transplant was planned at the time of disease progression. In the tandem transplant arm, the patients were to receive thalidomide 200 mg daily at the time of disease progression. Over 80% received the assigned treatment in each arm. After second HSCT and 6 months of maintenance thalidomide, the CR + VGPR were 54 and 68%, respectively (p = 0.04). With a median follow-up of 33 months, 3-year PFS, and OS were 57 versus 85% (p = 0.02) and 65 versus 85% (p = 0.04), respectively. Prognostic factors showed that the benefit of the thalidomide maintenance was observed only in patients who did not achieve at least a VGPR after HSCT. Of note, however, is that the tandem transplant arm did not receive thalidomide. Also, only 18 of the patients received thalidomide as salvage therapy. Thus, the OS improvement is difficult to interpret since less than 50% of the patients received the thalidomide as salvage therapy. Further, only nine patients received a salvage second transplant. The Blood and Marrow Transplant Clinical Trials Network (BMT CTN) has recently completed a large Phase III clinical trial comparing tandem

Chapter 10  Single Versus Tandem Autologous Hematopoietic Stem Cell 

autologous HSCT to autologous transplant followed by a matched sibling nonmyeloablative allogeneic transplant. The proposed successor trial to this study will be a three arm study comparing single transplant with lenalidomide maintenance to a single transplant with bortezomib, lenalidomide, dexamethasone consolidation followed by lenalidomide maintenance to tandem autologous transplant with lenalidomide maintenance therapy. Table  10-2 is a summary of the randomized data in tandem autologous transplant.

4.  Benefits of Tandem Transplantation It was noted in the IFM 94 trial that the OS for patients undergoing tandem transplants diverged significantly from the single transplant arm only after the first 4 years of follow-up [24]. Furthermore, the results of this study show no significant increase in CR with the second transplant on an intentto-treat basis, although patients who actually HAD the second transplant did have higher CR rates compared to patients who only had a single transplant (63 vs. 49%, respectively). The authors suggest that this indicates that the difference in projected survival with double transplants does not correlate with improved response rates but longer duration of responses. This could also explain why differences in survival are not apparent with shorter follow-up in the other randomized studies of tandem transplantation. Also, the IFM results appear inferior to the Arkansas tandem transplant data in terms of crude rates of CR, EFS, and OS. These differences may be related to differences in patient selection, induction, and most likely the transplant conditioning therapy: the IFM 94 utilized Mel 140  mg/m2 followed by Mel 140 mg/m2 + TBI 8 Gy (total melphalan dose of 280 mg/m2) while the Arkansas regimen was Mel 200  mg/m2 followed by Mel 200  mg/m2 (total melphalan dose of 400 mg/m2). The patients who achieve the maximum benefit from the second planned HSCT are those who do not achieve a CR or a very good PR within 3 months after the first HSCT. In the IFM 94 trial, the probability of 7-year survival was 11% with single transplant versus 43% with the tandem approach [24]. The Bologna 96 data [25] also demonstrate that tandem HSCT was of particular benefit to patients who failed to respond to first-line therapy and/or were not in CR or near CR following the first transplant. Based on the IFM 94 and Bologna 96 evidence, a patient who fails to achieve a good PR after the first HSCT should be offered a tandem second transplant within 3–6 months of the first HSCT. Routine tandem transplants should otherwise only be considered in the context of well-designed clinical trials and not the standard of care at this time. The lack of OS benefit in any randomized study other than IFM 94 is striking. As discussed above, the explanations may include the differences in conditioning regimens, the failure to improve CR rates with second transplants, the lack of sufficiently lengthy follow-up, and the improving salvage options available to patients who relapse. Short follow-up and improvements in salvage therapy including late second transplants almost certainly account for the observed EFS benefits without an OS advantage for the tandem procedure.

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D.H. Vesole

5.  Second Transplants as Salvage Therapy The timing of a second HSCT in patients who have undergone a prior HDT with HSCT is an unanswered question. While the IFM 94 and Bologna 96 results suggest that for patients who do not achieve a good partial remission or near CR within 3 months after the first transplant, an immediate second transplant may be optimal, there are reports of the efficacy of salvage second transplants at relapse (31–35). Tricot et al. have shown that a salvage HSCT at relapse after prior auto transplant showed benefit for patients with >12 months remission duration and normal beta-2 microglobulin [30]. Unfortunately, there are no randomized studies that compare a salvage second transplant at relapse to a planned upfront tandem second transplant. The European Bone Marrow Transplant Registry (EBMT) published a registry-based analysis suggesting the performance of a second planned autologous transplant before relapse and within 6–12 months of the first transplant resulted in superior outcomes [36]. The Arkansas group performed serial landmark analyses to define the optimal timing of a planned second transplant in their original total therapy patient population [20]. EFS and OS both were longer among the patients who had received a second transplant within 13 months (85% of second transplants had been completed at that time) compared with the others receiving their second cycle of high-dose therapy later or not at all. A later analysis with a larger (1,000 patients) group undergoing melphalan-based tandem transplants suggested that timely application of a second transplant was significant for EFS and OS [37].

6.  Maintenance Therapy Following Transplant Interferon, a mainstay for maintenance therapy after transplantation, has been shown to be ineffective in the United States Intergroup S9321 trial [8]. Although corticosteroids have been shown to improve EFS following conventional therapy, there has not been a clinical trial to evaluate their efficacy post-HSCT [38, 39]. Other maintenance strategies are outlined in Table 10-3 [45–51]. Thalidomide, an effective agent in MM for relapsed disease and as induction therapy, has been evaluated as maintenance therapy post-transplant. Three randomized studies have demonstrated the benefit of thalidomide, either as a single agent or in combination, as maintenance therapy after autologous transplantation [22, 40, 41]. In a French trial, thalidomide maintenance improved the 3-year EFS compared to observation (52 vs. 36%) and 4-year OS (87 vs. 77%) [40]. Of note, only those patients who did not achieve at least a very good partial remission were the patients who benefited from thalidomide maintenance. Similar findings were observed in the Tunisian study [29]. An Arkansas trial showed superior CR rates (62 vs. 43%) and 5-year EFS with thalidomide (56 vs. 44%) but no improvement in OS [22]. The preliminary report by the Australian ALLG MM6 study of thalidomide plus prednisone for 12 months versus observations post-HSCT also showed an improvement in PFS (90 vs. 69%, p = 0.005) and OS (91 vs. 80%, p = 0.21) although this was observed until beyond the first year from transplant [41]. Recently, BMT CTN completed a large trial of over 500 autologous transplant recipients randomized to either thalidomide plus dexamethasone (for 1 year) or observation post-tandem HSCT. The results of these trials

Chapter 10  Single Versus Tandem Autologous Hematopoietic Stem Cell 

151

Table 10-3.  Maintenance after primary myeloma therapy. Study

N

Maintenance

PFS (months)

OS

US Intergroup Trial S9321 [7]

249

IFN

25 vs. 21 (p = 0.05)

58 vs. 53 (p = 0.8)

Cunningham [45]

  84

IFN

NSa

NSa

EBMT [46]

892

IFN

29 vs. 20 (p = 0.006)

78 vs. 47 (p = 0.007)

HOVON-50b [47]

128

IFN

13.5 vs. 8.5 (p = 0.04)

41 vs. 38.4 NSa

SWOGb [48]

125

Pred

14 vs. 5 (0.03)

37 vs. 26 (p = 0.05)

NCIC MY7b [49]

307

Dex

33 vs. 23

46.3 vs. 43.8

Arkansas TT2 vs. TT1 [50]

668 (TT2) 231 (TT1)

IFN

50 vs. 25 (p < 0.001)

70 vs. 60 (p = 0.06)

NCIC MY9 [51]

  67

Thal + Pred

42.2

91% at 1 year

IFM 99 02 [52]

588

Thal + Pam

56

EFS p < 0.009)

Pam

37

OS p < 0.04

Obs

34

a

Outcomes at 77 months Studies that include maintenance after a primary treatment other than transplant PFS progression-free survival, OS overall survival, IFN interferon-a, OR odds ratio, NS not statistically significant, Pred prednisone, Dex dexamethasone, TT1/TT2 total therapies one and two, Thal thalidomide, Pam pamidronate; NR not reported b

Table 10-4.  Post-transplant maintenance options and recommendations. Pro

Cons

Currently open trials

Agent/regimens

Dose

Dexamethasone

40 mg 4 d/mo Prolong PFS after SDT

Steroid side effects –

Thalidomide

50–100 mg

Prolong PFS/OS (IFM 99-02)

Neuropathy, DVT

Revlimid

10 mg

No available results

DVT, CALGB/ECOG myelosupression 100104

  Thal + Prednisone

200/50

  Thal + Dexamethasone

200/40

Good tolerability, Risk for compound ECOG/NCIC 76% of patients on side effects MY10 maintenance after 18   BMT CTN #0102 months (NCIC MY9)

Recommendations

(1) Clinical trial preferred whenever possible

HOVON/ GMMG-HD3

Combinations

(2) Thalidomide 50–100 mg daily and dexamethasone 40 mg × 4 days each month if the patient has not been treated with this regimen or responded to this regimen as induction therapy (3) Lenalidomide 10 mg days 1–21 or 1–28. Duration of treatment is still unknown, however most studies include 1–2 years post-transplant or until relapse or disease progression

are not anticipated until at least 2009. The Cancer and Leukemia Group B (CALGB) in collaboration with the Eastern Cooperative Oncology Group and the BMT CTN is currently accruing patients to single agent lenalidomide versus observation following a single HSCT. Upon completion of the CALGB trial, the proposed BMT CTN post-HSCT lenalidomide maintenance trial will be activated. Table  10-4 shows recently completed or currently active maintenance trials. Trials utilizing bortezomib maintenance therapy posttransplant will be initiated in the near future.

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7.  Novel Pre-transplant Regimens Until the recent introduction of novel agents, immunomodulatory drugs, and proteosome inhibitors, into our treatment paradigm for multiple myeloma, high-dose therapy with autologous HSCT provided the best opportunity for most patients to achieve a complete response and to prolong EFS. Novel anti-myeloma combinations are now revolutionizing the induction treatment and raising new questions in transplantation for MM. The most recently approved novel agents thalidomide, bortezomib, and lenalidomide in combination with dexamethasone have transformed the therapeutic options available to patients with relapsed MM and are now being integrated into induction treatment [42–44, 53–63]. Initial studies have been extremely promising with response rates close to 90% and with complete response rates close to 25% (Table 10-5). When these agents are combined with additional agents, the depth of response has improved further as shown in the Arkansas Total Therapy II and III trials (Fig. 3) [22, 23]. It must be noted, however, that the durability of responses induced by novel agent combinations is at this point unknown. If the sustainability of these responses is proven with longer follow-up, it is likely that such induction combinations might displace or delay high-dose treatment.

Table 10-5.  Impact of novel induction strategies on CR rates. Author

Regimen

Overall response rates (%)

CR or nCR + CR rates (%)

N

Palumbo [53]

MPT

76

28

255

Cavenagh [54]

PAD

95

29

  21

Harousseau [55]

VD

73

17

  18

Jagannath [56]

VD or V

88

25

  32

Wang [57]

VDT

92

19

  36

Richardson [58]

V

30

11

  46

Facon [59]

MPT

84

14

  95

Mateos [60]

VMP

85

28

  60

Barlogie [22]

VDT-PACE  +  tandem ASCT

Over 90

83% post-ASCT

303

Rajkumar [42]

Lenalidomide-D

91

38

  34

Rajkumar [41]

Thalidomide-D

63

CR

103

Palumbo [61]

Lenalidomide-MP

67

11

  24

Goldschmidt [62]

TAD followed by ASCT

80

7

406

Rosinol [63]

V alt D

65

12

  40

Caveat: The overall impact of these novel regimens on survival, long-term transplant outcomes, and post-transplant complications are not yet defined since these are pilot data PAD Bortezomib/Doxorubicin/Dexamethasone, MPT MEL/Prednisone/Thalidomide, VDT Bortezomib/Dexamethasone/ Thalidomide, VD Boretzomib/Dexamethasone, V alt D–alternating cycles, V–Bortezomib, TAD Thalidomide/ Adriamycin/Dexamethasone, VDT-PACE Bortezomib/Dexamethasone/Thalidomide/Cisplatin/Adriamycin/Cytoxan/Etoposide D Dexamethasone

Chapter 10  Single Versus Tandem Autologous Hematopoietic Stem Cell 

8.  Conclusion Randomized clinical trials have firmly established the role of upfront autologous transplantation in newly diagnosed MM. While novel agents in combination might result in comparable CR rates to single HSCT, there are insufficient data to recommend any one of the alternative induction strategies outside of clinical trials. With additional time, the newer agents may either supplant transplantation or change our current algorithm by delaying transplants until disease relapse. There is evidence that delayed transplantation is efficacious. Thus, it is doubtful that transplants will be replaced even by highly active newer therapies. Tandem autologous transplants have consistently shown better EFS compared to single autologous transplants but to date only the IFM 94 has shown an OS advantage. Given the improvement in salvage therapies, EFS not OS should be the primary objective of transplant trials. Therefore, the current recommendations for newly diagnosed MM are the following: collect sufficient autologous stem cells for at least 2 transplants and proceed to tandem autotransplants if the patient does not achieve at least a fairly good partial remission (defined as a decrease in paraprotein by >90% from diagnosis) after the first autologous transplant. This is one of the standards of care at this time. For those patients who do achieve a fairly good partial remission, a second transplant should be considered at the time of disease progression. The long-term benefit of maintenance therapy has not been clearly determined. Ideally, patients should be enrolled on clinical trials to assess this concept. In the absence of a clinical trial, it appears that immunomodulatory drugs provide superior EFS in patients who do not achieve a appreciable partial remission with HSCT. It is reasonable to treat these patients with an immunomodulatory agent. The dose and duration of treatment remains unknown.

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Chapter 10  Single Versus Tandem Autologous Hematopoietic Stem Cell  26. Sonneveld P, van der Holt B, Segeren CM et  al (2007) Intermediate-dose melphalan compared with myeloablative treatment in multiple myeloma: Longterm follow-up of the Dutch Cooperative Group HOVON 24 trial. Haematologica 92:928–935 27. Goldschmidt H (2005) Single vs. double high-dose therapy in multiple myeloma: Second analysis of the GMMG-HD2 Trial. Haematologica 90(Suppl 1):38 Abstract PL8.02 28. Fermand JP, Alberti C, Marolleau JP (2003) Single versus double high dose therapy supported with autologous blood stem cell transplantation using unselected or CD34 enriched ABSC: Results of a two by two designed randomized trial in 230 young patients with multiple myeloma. Hematol J 4(Suppl 1):S59 29. Abdelkefi A, Ladeb S, Torjman L et al (2007) Single autologous stem cell transplantation followed by maintenance therapy with thalidomide is superior to double autologous transplantation in multiple myeloma: Results of a multicenter randomized clinical trial. Blood 111(4):1805–1810 30. Tricot G, Jagannath S, Vesole DH et  al (1995) Relapse of multiple myeloma after autologous transplantation: Survival after salvage therapy. Bone Marrow Transplant 16:7–11 31. Mehta J, Tricot G, Jagannath S et  al (1998) Salvage autologous or allogeneic transplantation for multiple myeloma refractory to or relapsing after a first-line autograft. Bone Marrow Transplant 21:887–892 32. Kulkarni S, Powles R, Singhal S et al (1998) Second autografts for relapsed multiple myeloma: Is tandem autotransplantation better? Clinical care: recurrence, secondary neoplasia and late complications after transplantation. Blood 92(Suppl 1): 344b 33. Elice F, Raimondi R, Tosetto A et  al (2006) Prolonged overall survival with second ondemand autologous transplant in multiple myeloma. Am J Hematol 81:426–431 34. Alvares CL, Davies FE, Horton C et al (2006) The role of second autografts in the management of myeloma at first relapse. Haematologica 91:141–142 35. Qazilbash MH, Saliba R, De Lima M et al (2006) Second autologous or allogeneic transplantation after the failure of first autograft in patients with multiple myeloma. Cancer 106:1084–1089 36. Morris C, Iacobelli R, Brand B et al (2004) Benefit and timing of second transplantations in multiple myeloma: clinical findings and methodological limitations in a European group for blood and marrow transplantation registry study. J Clin Oncol 22:1674–1681 37. Desikan R, Barlogie B, Sawyer J et  al (2000) Results of high-dose therapy for 1000 patients with multiple myeloma: durable complete remissions and superior survival in the absence of chromosome 13 abnormalities. Blood 95:4008–4010 38. Berenson JR, Crowley JJ, Grogan TM et  al (2002) Maintenance therapy with alternate-day prednisone improves survival in multiple myeloma patients. Blood 99:3163–3168 39. Shustik C, Belch A, Robinson J et al (2007) A randomised comparison of melphalan with prednisone or dexamethasone as induction therapy and dexamethasone or observation as maintenance therapy in multiple myeloma: NCIC CTG MY.7. Br J Haematol 136:203–211 40. Attal M, Harousseau J, Leyvraz S et  al (2006) Maintenance therapy with thalidomide improves survival in patients with multiple myeloma. Blood 108:3289–3294 41. Spencer A, Prince M, Roberts A, et al (2006) First Analysis of the Australasian Leukaemia and Lymphoma Group (ALLG) Trial of thalidomide and alternate day prednisolone following autologous stem cell transplantation (ASCT) for patients with multiple myeloma (ALLG MM6). Blood 108, abstract 58

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Chapter 10  Single Versus Tandem Autologous Hematopoietic Stem Cell  57. Wang M, Giralt S, Delasalle K et  al (2007) Bortezomib in combination with thalidomide-dexamethasone for previously untreated multiple myeloma. Hematology 12:235–239 58. Richardson P, Chanan-Khan A, Schlossman R et  al (2005) A multicenter phase II trial of bortezomib in patients with previously untreated multiple myeloma: Efficacy with manageable toxicity in patients with unexpectedly high rates of baseline peripheral neuropathy. Blood 106:2548 59. Facon T, Mary JY, Hulin C et al (2007) Melphalan and prednisone plus thalidomide versus melphalan and prednisone alone or reduced-intensity autologous stem cell transplantation in elderly patients with multiple myeloma (IFM 99–06): A randomised trial. Lancet 370:1209–1218 60. Mateos MV, Hernandez JM, Hernandez MT et al (2006) Bortezomib plus melphalan and prednisone in elderly untreated patients with multiple myeloma: Results of a multicenter phase 1/2 study. Blood 108:2165–2172 61. Palumbo A, Falco P, Corradini P (2007) et al Melphalan, prednisone, and lenalidomide treatment for newly diagnosed myeloma: A report from the GIMEMA– Italian Multiple Myeloma Network. J Clin Oncol 25:4459–4465 62. Goldschmidt H, Sonneveld P, Breitkreuz I et al (2005) HOVON 50/GMMG-HD3Trial: Phase III study on the effect of thalidomide combined with high dose melphalan in myeloma patients up to 65 years. Blood 106:424 63. Rosinol L, Oriol A, Mateos MV et al (2007) Phase II PETHEMA trial of alternating bortezomib and dexamethasone as induction regimen before autologous stem-cell transplantation in younger patients with multiple myeloma: efficacy and clinical implications of tumor response kinetics. J Clin Oncol 25:4452–4458

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Chapter 11 Treatment Strategies for Follicular Center Cell Non-Hodgkin’s Lymphoma Frank Heinzelmann, Hellmut Ottinge, and Claus Belka

1.  Introduction Non-Hodgkin’s lymphomas (NHL), a heterogenous group of malignancies arising from lymphoid tissue, account for almost 5% of all cancers occurring in the United States [1]. With an estimated 63,190 new cases and 18,660 cancer deaths projected in 2007 in the United States, NHL is the fifth most common malignant neoplasm after the breast, prostate, lung, and colon [2]. The incidence and mortality rates were reported to have risen steadily in Western countries over the past decades and have doubled between 1950 and 1989, from 5.9 to 13.7 per 100,000 persons per year [3] but the incidence rate had slowed to 1–2% in the 1990s [4, 5]. Based on a large international study, follicular lymphoma (FL), a mature B-cell neoplasm, represents the second most common subtype of NHL with approximately 25% of cases; diffuse large B cell lymphoma (DLBCL) is more common (~30%) [6]. Rates for FL are two to three times greater among whites than among blacks [7]. FL is most commonly seen in middle-aged individuals and the elderly, with a median age of 59 years. Females (52%) are slightly more often affected than males (48%) [8]. The etiology is uncertain, but immunosuppression, specific autoimmune disorders, infectious agents, and environmental factors contribute [9]. FL is pathologically well characterized. Unlike other malignant lymphomas three different malignancy grades are defined according to the degree of centroblasts [10] with grade 3 being subdivided into grade 3a and 3b [11]. While it is broadly accepted that grade 3b FL behaves clinically more like DLBCL and should be treated accordingly, it remains questionable whether the discrimination of grades 1–3a is of clinical significance.

2.  Prognosis and General Treatment Approaches In general, for FL the aim of therapy is to maintain the best quality of life and to initiate treatment when patients develop symptomatic disease such as bulky lymphadenopathy, splenomegaly, risk of local compressive disease, marrow From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_11, © Springer Science + Business Media, LLC 2003, 2010

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compromise, or rapid disease progression. Any alteration of this approach, the so-called watchful waiting, requires demonstration of improved survival with early application of therapy or identification of criteria that define patients at high risk to initiate early therapy. In this regard, the Follicular Lymphoma International Prognostic Index (FLIPI) defines three prognostic risk groups with age ³60 years, stage III/IV disease, elevated LDH level, hemoglobin <12 g/dl, and a number of nodal sites ³5 as unfavorable factors [12]. For firstline treatment there are many therapies available with no clear consensus for an optimal first-line treatment. In patients with early stages I and II, representing 10–30% of the patients with FL [8], external beam radiotherapy (RT) using doses of 26–50 Gray (Gy) has a curative potential and is applied as involved/extended field or total lymphoid irradiation resulting in local control rate of 95% and longterm disease-free survival (DFS)/overall survival (OS) in 35–86% of cases [13–20]. Long-term follow-up studies from Stanford in 177 patients treated with RT demonstrated a median survival of 13.8 years and 20-year OS of 35% [14]. However, in selected stage I and II patients, deferred therapy is also an acceptable approach, as more than 50% of the patients remained untreated for a median of >6 years and OS was comparable to that seen in reports with immediate treatment [21]. Interestingly, a retrospective analysis from the M.D. Anderson Cancer Center provided evidence for a potential advantage of a combined chemotherapy/ RT approach compared to RT alone with a superior freedom from relapse at 5/10/15 years with no further relapses after 7.5 years [22]. Another phase II trial using a combined modality treatment (CMT) [cyclophosphamide, doxorubicin, vincristine, prednisolone (= CHOP)-Bleomycin/RT] in patients with poor prognostic factors documented a 10-year DFS and OS of 73% and 83%, respectively [23]. Taken together, in patients with stage I/II FL, external beam RT is recommended with curative potential. However, there is no clear consensus concerning the target volume and RT dose. The prospective randomized phase III trial by the German low-grade lymphoma study group comparing extended-field RT versus TLI has been closed in 2007, but results have not been published yet. Currently, a phase III randomized trial on low-dose total body irradiation (TBI) (1.5  Gy) and involved-field RT (26–40  Gy) versus involved-field RT alone is performed by the European Organization for research and treatment of cancer (EORTC). The role of additional chemotherapy and/or rituximab therapy to RT in limited stages I and II is being addressed in ongoing randomized trials. Noteworthy, in the majority of patients the disease is diagnosed at an advanced stage III or IV. In this situation a broad spectrum of therapeutic options are available including a watch and wait period until the disease becomes symptomatic, single agent to combination chemotherapy with or without additional irradiation, chemoimmunotherapy, use of conjugatedradiolabeled monoclonal antibody treatment, high-dose chemotherapy followed by autologous/allogeneic hematopoeitic stem cell transplantation (auto HSCT/allo HSCT), and reduced intensity conditioning (RIC) before allogeneic HSCT. For asymptomatic patients with low-bulk disease an expectant management can be recommended [24]. This approach is based on the results of three

Chapter 11  Treatment Strategies for Follicular Center Cell Non-Hodgkin’s Lymphoma 

randomized trials demonstrating no survival benefit of immediate compared with deferred treatment until time of progression in the era before rituximab [25–27]. For symptomatic patients, the aim of chemotherapy treatment is to achieve a durable remission. In spite of numerous efforts and the exploration of different conventional chemotherapy strategies resulting in 75–80% responses, the prognosis of FL remained unchanged in recent decades, with a median survival of 8–10 years [28]. In recent years, the introduction of monoclonal antibodies directed against CD20 in combination with chemotherapy (chemoimmuntherapy) as initial therapy has dramatically improved the treatment outcome of FL [29–32]. For example, adding rituximab (R) to CHOP significantly prolonged the time to treatment failure, enhanced overall response rate, prolonged duration of remission, and even translated to superior OS (median survival 12–14 years) when compared to CHOP alone [33, 34]. However, relapses seem to continue after immunotherapeutic treatment. Thus, the concept of maintenance therapy after successful induction therapy is an attempt to prevent the re-emergence of the disease. Maintenance therapy with interferon-a has been evaluated in several clinical trials and was shown to prolong remission duration and OS [35]. However, issues of toxicity coupled with the inconvenient administration have prevented its widespread uptake. Since rituximab couples a high antilymphoma activity with a favorable toxicity profile and was shown to enhance event-free survival (EFS) as maintenance therapy after induction with singleagent rituximab [36, 37] and after chemotherapy induction [38], the effect of rituximab maintenance therapy following first-line induction with rituximab plus chemotherapy is currently being evaluated in an international phase III randomized trial (PRIMA study). In this trial, patients with advanced FL successfully treated with rituximab, plus different chemotherapy schedules are randomized to rituximab maintenance versus observation. Another new treatment approach in patients with advanced FL was the introduction of radioimmunotherapy (complexing radioisotope to a monoclonal antibody) in the first-line treatment of advanced FL. In 90 patients, after 6 cycles of CHOP the additional application of tositumonab/iodine I-131 tositumonab resulted in a promising overall response rate of 91% resulting in an estimated 5-year OS of 87% and progression-free survival (PFS) of 67%, 23% better than CHOP alone [39]. Another single-center phase II-study in 69 patients using tositumonab/iodine I-131 tositumonab yielded in a similar 75% complete response rate resulting in a median PFS of 6.1 years with 40 patients remaining in remission for 4.3–7.7 years [40].

3.  Myeloablative Therapy with Autologous HSCT as Consolidation in First Remission The role of high-dose therapy followed by auto HSCT in patients with FL in first complete remission (CR) or partial remission (PR) has been explored in phase II and three phase III randomized trials. The rationale behind high-dose therapy for up-front treatment is that the intrinsic sensitivity of tumor cells is better early in the course of the disease than after acquirement of drug resistance in response to several cycles of cytotoxic therapy whereas the risks of

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stem cell mobilization and treatment-related toxicity should be lower. When compared to historical controls, phase II trials found extensively prolonged PFS and OS in a substantial proportion of patients receiving auto HSCT [41–45]. Therefore, several groups developed multi-center, randomized phase III trials comparing auto HSCT consolidation after induction chemotherapy versus interferon maintenance or observation. The German Low-grade Lymphoma Study Group (GLSG) recruited 307 previously untreated patients <60 years and patients who responded to induction chemotherapy were randomly assigned to auto HSCT consolidation or interferon-a maintenance. Among 240 evaluable patients, early mortality was below 2.5% in both study arms and the projected 5-year PFS after auto HSCT was 64.7% and only 33.3% in the interferon arm (p<0.0001) [46]. In the Groupe Ouest Est des Leucemies Aigues et autres Maladies du Sang (GOELAMS) study, patients treated with high-dose therapy had a higher response rate and a longer median PFS than patients treated with conventional chemotherapy/interferon-a but this did not translate into a better OS due to an excess of secondary neoplasms (8.5%) after transplantation [47] approved in a recently published analysis of the GLSG with a considerable increased risk of secondary hematologic malignancies after auto HSCT (3.8%) when compared to conventional chemotherapy (0%) [48]. A recently published phase III trial of the Groupe d’ Etude des Lymphomes de l’Adulte (GELA) showed no difference for OS and DFS after a median follow-up of 7.5 years between the standard chemotherapy regimen compared to the auto HSCT arm [49]. Taken together, despite the substantially improved PFS rate in comparison to conventional chemotherapy, long-term results demonstrated no statistically significant difference for OS in favor of first-line auto HSCT due to high recurrence rates and increased risk of secondary malignancies. It seems completely unclear whether relapses are prevented or only postponed after auto HSCT. Therefore, auto HSCT is probably not curative even if applied early in the course of the disease.

4.  Treatment of Relapsed Follicular Lymphoma The treatment options after relapse remain similar to those for first-line therapy. Patients with relapsed asymptomatic disease can be managed expectantly. Defining treatment of relapse should consider the aim of therapy (palliative vs. curative), performance status, age, previous therapy, response to initial therapy, and duration of response. Since, after first recurrence, the natural history of FL is a continuum of repeated chemosensitive relapses of progressively shorter duration and ultimately death from the underlying disease after a median survival of only 4.5 years from first relapse [50], new treatment modalities have been required. In this regard the role of rituximab has been evaluated for relapsed/resistant FL in several trials. In this regard Ghielmini et al. [37] and Hainsworth et al. [36] reported a beneficial effect of rituximab maintenance after response to initial single-agent rituximab treatment associated with little or almost no clinical side effects. A phase III randomized trial in patients with relapsed/ resistant FL evaluated the role of rituximab both in remission induction and in maintenance treatment. A total of 465 patients were randomly assigned to

Chapter 11  Treatment Strategies for Follicular Center Cell Non-Hodgkin’s Lymphoma 

receive 6 cycles of R-CHOP or CHOP alone. Those in CR/PR were secondly randomized to maintenance rituximab or observation. As a result, chemoimmunotherapy (R-CHOP) yielded in a superior PFS when compared to CHOP alone. Furthermore, rituximab maintenance considerably improved PFS not only after CHOP but also after R-CHOP induction [51].

5.  Myeloablative Therapy with Autologous HSCT in Relapsed/Refractory FL In patients with advanced FL, high-dose chemotherapy with subsequent auto HSCT represents an alternative treatment approach with several phase II studies suggesting a prolonged DFS [52]. Best results are seen when auto HSCT is performed early in the course of disease before patients become chemorefractory. For example, the largest series from Freedman and colleagues reported an estimated 8-year DFS and OS of 42%/66%, respectively [53]. In accordance, Brice et al. demonstrated that patients who underwent auto HSCT had a significantly increased 5-year DFS (42 vs. 16%) and OS (58 vs. 38%) as compared to those who received standard treatment alone [54]. Similar results have been obtained using different hematopoietic stem cell sources, purging methods, and conditioning regimens [55–59]. The only prospective randomized trial addressing the role of auto HSCT in 89 patients with recurrent FL revealed a superior PFS and OS at 2 years after transplant in patients randomized to auto HSCT (55%/71%) compared to those receiving conventional chemotherapy (26%/46%). However, considerable imbalances of clinical risk factors in the three respective subgroups limit the interpretation of these data [60].

6.  Myeloablative Therapy with Allogeneic HSCT The continuous pattern of relapses after auto HSCT without reaching a plateau and the fact that auto HSCT is not feasible when the marrow reserves are inadequate and is poorly effective for patients with refractory disease or extensive bone marrow involvement [61] led to an increased interest in exploring the possibility of an allo HSCT for the treatment of FL. The rationale for conventional allo HSCT is (1) to give maximal anti-tumor therapy to the recipient as part of the conditioning regimen; (2) to ablate the recipient hematopoietic stem cells; (3) to provide space for the incoming hematopoietic cells; (4) to suppress the recipient immune response directed toward the incoming allogeneic cells, thereby facilitating engraftment; and (5) to provide donor-derived tumorreactive T cells capable of mediating graft versus lymphoma (GvL) effects, which may be most effective in indolent lymphoma [62]. In addition, the risk of secondary myelodysplastic syndrome (MDS)/secondary acute myeloid leukemia (sAML) after allo HSCT is presumably negligible since the donorderived stem cells have not been exposed to chemotherapy. The first encouraging results have been published by van Besien in ten patients with refractory (n = 8) or recurrent (n = 2) disease. After predominant conditioning with TBI and use of HLA-identical siblings as donors, eight patients were in CR after a median follow-up of 2.5 years [63]. In 2000, van Besien and coworkers reported the long-term follow-up of this study: 6 years after allo HSCT, only one patient developed disease progression and was in

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Table 11-1.  Conventional conditioning prior to allogeneic HSCT. Author

No. of TBI-based Follow-up TRM Relapse PFS patients conditioning (%) Stem cell source (months) (%) rate (%) (%)

OS (%)

Van Besien [1995]

  10

80

80

BM

30

20

0

80

Van Besien [1998]

113

82

BM

25

40

16

49

49

Mandigers [1998]

  15

100

BM

36

33

13

67

67

Verdonck [1999]

  15

100

BM

25

27

0

70

70

Forrest [2002]

  24

8

BM 67% PB 33% 28

21

0

78

78

Kiss [2003]

  25

0

BM 89% PB 11% 36

NS

NS

79

87

Hosing [2003]

  44

55

BM 41% PB 59% 53

34

19

45

49

Van Besien [2003]

176

68

BM 77% PB 23% 36

24

21

45

51

Peniket [2003]

231

NS

NS

38

25

43

51

Toze [2004]

  29

93

BM

25

24

23

53

58

Yakoub-Agha [2002]   16

81

BM

39

31

0

55

68

60

BM bone marrow, PB peripheral blood, NS not stated

remission 4 years after a second allo HSCT [64]. Up to date, the exact value of allo HSCT can be assessed by analyzing a great amount of studies summarized in Table 11-1 [63, 65, 66, 62, 67–70, 61, 71, 72]. From these results, it appears that conventional allo HSCT was mainly offered to heavily pretreated patients with refractory/recurrent disease and extensive bone marrow involvement. For example, in an observational study in 113 patients with advanced lowgrade lymphoma including 93 patients with FL the 3-year probability of disease recurrence was only 16%, despite 38% of the patients had chemorefractory disease with a DFS and OS being 49% and 49%, respectively. Interestingly, there was only one recurrence among 33 patients monitored for more than 2 years. In this cohort, favorable prognostic factors were use of a TBI-containing pretransplant conditioning regimen, Karnofsky performance status (KPS)³90%, chemotherapy sensitive disease, and age less than 40 years [65]. In recent years, several studies compared the outcome of allogeneic with autologous transplantation. In a retrospective analysis, the International Bone Marrow Transplant Registry (IBMTR) analyzed the data of 904 patients with advanced FL. A total of 176 patients (19%) received allogeneic, 131 (14%) received purged autologous, and 597 (67%) received unpurged autologous transplants. A higher proportion of patients had poor KPS, advanced disease, increased lactate dehydrogenase, bone marrow involvement at transplantation, and chemotherapy resistant disease in the allograft recipients. As a result, the 5-year treatment-related mortality (TRM) was higher in the allo HSCT group (30%) due to infections and graft versus host disease (GvHD) compared with both the purged (14%) and unpurged auto HSCT (8%) group. In strong contrast, the 5-year recurrence rates were considerably lower in the allo HSCT group (21%) when compared to the purged auto HSCT (43%) and unpurged auto HSCT (58%) group providing evidence for a GvL effect resulting in similar estimated 5-year survival rates of 51, 62, and 55%. However, a continuous pattern of relapses was only observed after auto HSCT whereas after allo HSCT only few relapses were detected beyond one year post-transplantation [61].

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165

Taken together, the use of allo HSCT is able to cure patients with advanced FL. The very potent antitumoral effect of allo HSCT has led to encouraging PFS and long-term OS ranging from 49% to 80% accompanied by a low recurrence rate (0–20%) presumably due to a substantial GvL effect [73, 74] and/or lack of tumor contamination of the allogeneic graft [75]. Moreover, over the past decade, advances in supportive care and better patient selection have resulted in improved outcomes for allo HSCT particularly younger ones reducing TRM and severe GvHD [61].

7.  Nonmyeloablative Therapy with Allogeneic HSCT Myeloablative conditioning regimens prior to allogeneic transplantation represent an effective approach to cure patients with advanced FL. However, the use of myeloablative conditioning regimens to treat relapsed and refractory disease has been limited by unacceptable toxicities, particularly due to multiple lines of previous therapy, advanced age of patients, and concomitant diseases. Since FL is most commonly seen in patients with ~60 years, several groups have developed nonmyeloablative/reduced intensity conditioning (RIC) prior to allo HSCT. The aim of this pretransplant regimen is to maximize immunosuppression and to minimize organ toxicity while preserving an adequate antitumor effect by alloreactive donor T cells to ensure stable engraftment and elimination of residual hematopoietic cells of host origin [76]. Recipient immunosuppression is achieved in the main by the use of fludarabine (F) in combination with either low-dose TBI or an alkylating agent such as cyclophosphamide (C), melphalan (M), or busulfan (Bu). Some regimens incorporate additional T cell depletion via antithymocyte globulin (ATG) or alemtuzumab to reduce the risk of acute GvHD. The most important studies available addressing RIC in patients with advanced FL are summarized in Table 11-2 [77–88].

Table 11-2.  Reduced intensity conditioning prior to allogeneic HSCT. Author

No. of Preparative patients regimen

Stem cell source

Follow-up TRM Relapse PFS OS (months) (%) rate (%) (%) (%)

Khouri [2001]

20

FC

PB

21

10

 0

  84

  84

Robinson [2002]

52

Mainly F-based ± ATG/A; BEAM

Mainly PB

 9

22

21

  61

  73

Bertz [2002]

 3

F-based

PB

19

 0

 0

100

100

Ho [2003]

 5

BEAM-AR

PB

17

20

20

  60

  80

Tanimoto [2003]

 8

FC/FBu ± ATG

PB

14

 0

 0

100

100

Escalon [2004]   5

FCR

PB

25

 0

 0

100

100

Faulkner [2004]

28

BEAM-A

PB

17

15.8

10

  69

  69

Morris [2004]

41

FMA

NS

36

11

44

  65

  73

Maris [2005]

47

2Gy TBI ± F

PB

23.8

34

15

  51

  58

Mainly F-based

Mainly PB

23.9

18

NS

  77

  79

Kusumi [2005] 45

F fludarabine, C cyclophosphamide, ATG antithymocyte globulin, A alemtuzumab, BEAM carmustine, etoposide, cytarabine, melphalan, R rituximab, Bu busulfan, M melphalan, TBI total body irradiation, PB peripheral blood, NS not stated

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The first encouraging report of RIC prior to allo HSCT in patients with FL has been published in 1998. After pretransplant conditioning with FC both rapid engraftment and remissions in heavily pretreated, elderly patients could be achieved [89]. The same group reported on 20 patients (median age 51 years; range 31–68 years) who received an RIC regimen consisting of FC; 9 patients additionally received rituximab. All patients had rapid and sustained engraftment and TRM at day 100 was only 10%. The 2-year actuarial DFS/OS were 84%/84%; the rate of acute GvHD grade II–IV and the cumulative chronic GvHD were 20%/64%, respectively. These results are promising, but all patients in this small cohort had a chemosensitive disease, indicating a good-risk group. Moreover, the median observation time (21 months) was short [77]. A large series (n  =  52) of RIC prior to allo HSCT was reported by the European Group for Blood and Bone Marrow Transplantation Registry (EBMTR). After a median follow-up of 283 days, 1-year OS/PFS/TRM/probability of disease progression were 73%/61%/21%/21%, respectively. By multivariate analysis, only disease chemosensitivity at the time of transplant was significantly associated with better PFS. Noteworthy, higher TRM (30.9%) at 2 years highlights the potential toxicity of chronic GvHD and infectious complications after allo HSCT and underscores the need for longer follow-up [78]. In 2004, Morris et al. published results on intensively pretreated patients (n  =  29: FL; n  =  3: lymphoplasmocytoid NHL; n  =  9: CLL) using a T cell depleted graft. The OS at 3 years was 73% and the PFS rate was 65% including patients who achieved remission after DLI application for progressive or persistent disease. However, using a T-cell depleted graft, patients are still at risk of relapse posttransplant with an actuarial relapse rate of 44% at 3 years. Age >45 years and persistent disease status prior to transplant were predictive factors for subsequent relapse [84]. Kusumi et al. also demonstrated the efficacy of RIC in 45 patients with prior auto HSCT and/ or chemoresistant disease. The 3-year OS was 79%, and 3-year PFS were 83 and 64% for the chemosensitive and chemoresistant patients, respectively [86]. More recently, the largest series presenting the long-term outcomes in 73 patients with low-grade lymphoma (n = 61: FL) were reported by the French Society of Bone Marrow Graft Transplantation and Cellular Therapy (SFGM-TC). After a median follow-up of 37 months, 3-year OS/DFS rates were 56%/51%. Among patients in CR, PR, and chemoresistant disease, 3-year OS rates were 66%, 64%, and 32%, while the 3-year EFS rates were 66%, 52%, and 32%, respectively [88]. Taken together use of RIC regimens prior to allo HSCT has shifted some or all of the burden of tumor cell kill from the conditioning regimens to the graft versus tumor/GvL effect. These regimens are less toxic than conventional preparative regimens and allow the treatment of patients with pre-existing organ dysfunction or older patients with comorbid conditions. However, it should be taken into account that most studies of RIC published to date suffer from a relatively short follow-up, inclusion of a heterogeneity of conditioning regimens which makes the comparison difficult. However, all appear to permit durable donor engraftment and the invaluable GvL effect.

8.  Myeloablative Therapy Versus RIC with Allogeneic HSCT In recent years, an increased general use of RIC has been observed: RIC constituted fewer than 10% of the transplants reported in 1997, about 50% in 2000 and 80% in 2002. To evaluate the effect of RIC in FL the outcome

Chapter 11  Treatment Strategies for Follicular Center Cell Non-Hodgkin’s Lymphoma 

of HLA-identical sibling transplants in 205 patients reported to the IBMTR between 1997 and 2002 has been studied. Conditioning regimens were categorized as myeloablative (ABLAT; n  =  120) or reduced intensity (n  =  80; RIC). ABLAT was defined as TBI containing regimens with single fraction doses ³5 Gy, fractionated doses ³8 Gy, busulfan doses >9 mg/kg, or melphalan doses of >150 mg/m2. RIC were defined as TBI single dose <5 Gy, busulfan doses <9  mg/kg, melphalan doses £150  mg/m2, and fludarabine-based regimens. The median recipient age was 45 (range 28–70) years for ABLAT versus 50 (27–67) years for RIC. After a median follow-up of 49 months for ABLAT and 36 months for RIC, there were no significant differences in acute (37 vs. 40%) or chronic GvHD (44 vs. 53%), TRM (1 year: 23 vs. 20%; 3 years: 25 vs. 24%), PFS (1 year: 68 vs. 63%; 3 years: 65 vs. 55%), or OS (1 year: 72 vs. 72%; 3 years: 70 vs. 64%) between the two groups. However, there was a trend toward an increased risk of disease recurrence after RIC at 1 year (17 vs. 8%, p  =  0.09) reaching statistical significance at 3 years (21 vs. 9%, p  =  0.03) when compared to ABLAT. In multivariate analysis performance status and chemotherapy sensitivity were independently associated with TRM, OS, and PFS, but the type of the conditioning regimen was not [90]. These results have been confirmed in a recently published retrospective analysis in 88 patients including 34 patients with low-grade lymphoma [n  =  18: FL, n  =  16: small lymphocytic lymphoma (SLL)/chronic lymphocytic leukemia (CLL)] conditioned with conventional myeloablative regimens and an RIC regimen: no statistically significant differences in OS/PFS were detected for patients receiving conventional conditioning (52%/46%) versus RIC-based conditioning (53%/40%). However, RIC was associated with a significantly higher relapse rate (28 vs. 13%, p  =  0.05). These observations must be interpreted within the context of different patient characteristics between the cohorts such that the RIC group was of older age, previous auto HSCT and higher proportion of unrelated donor source [91].

9.  Transformed Follicular NHL Over the time a considerable percentage of FL patients develop disease transformation. Histologic transformation (HT) is defined as the conversion of an FL to intermediate or high-grade lymphoma independent of prior therapy occurring in up to 70% of patients during the course of their disease [92–95]. Despite the fact that a subgroup of patients with limited disease and no previous exposure to chemotherapy may have a relatively long-term survival [96], most series reported a poor prognosis with median survival durations of less than 1 year [94], [97]. Therefore, high-dose therapy followed by auto HSCT has been applied to these poor prognosis patients in an attempt to improve survival rates. In a report from the St. Bartholomew’s Hospital, 27 patients received TBI/cyclophosphamide as pretransplant regimen with autologous BM (n = 24) or peripheral blood stem cell (n = 3) support. With a median follow-up of 2.4 years, 14/27 remain alive and disease-free; 5 are alive and free of disease at more than 4 years with a median OS of 8.5 years [98]. The role of auto HSCT in the management of FL in transformation has been extensively investigated. Several studies found DFS/OS rates of 30–81%. Age >60 years, elevated LDH, chemorefractory disease, and HT beyond 18 months conferred a worse outcome [56], [59], [99–103].

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From these studies, it was concluded that especially younger patients who attain CR after high-dose therapy benefit from auto HSCT. Presently, it remains speculative whether these results could be further improved by the use of radioimmunotherapy [104], [105] or allo HSCT.

10.  Conclusion In general, for FL the goal of therapy is to maintain the best quality of life and to initiate treatment only when patients develop symptomatic disease. Despite the benign course of FL and any data demonstrating any benefit for early therapy, patients are generally treated precociously early in the course of their disease. Currently, for first-line treatment in early stages I and II, external beam RT is recommended with curative potential. The role of additional chemotherapy or rituximab therapy to RT in early stages I and II is being investigated in ongoing trials. In case of advanced disease, there is no universal agreement as to the optimal first-line or subsequent therapy. Up to date, in younger patients with good performance status, chemoimmunotherapy using R-CHOP with maintenance rituximab might be regarded as standard therapy based on data demonstrating higher response rates, longer durations of response, improved survival, lower rate of histological transformation, and high stem cell mobilization ability for potential later use for auto HSCT. However, there are promising results obtained by the early use of radioimmunoconjugates, too. Up to date there are no randomized trials comparing chemoimmunotherapy with radioimmunoconjugates as front-line therapy in advanced FL. In elderly patients with symptomatic disease, single agent chemotherapy with chlorambucil or rituximab might be appropriate. In case of relapse, asymptomatic disease is not necessarily an indication for treatment and patients can be managed expectantly. For symptomatic FL, single agent rituximab and chemoimmunotherapy followed by rituximab maintenance are widely used in this setting. For younger patients who are suitable candidates for transplantation high-dose therapy followed by auto HSCT continued to be offered in chemoresponsive relapsed disease. However, despite high response rates and improved PFS auto HSCT is associated with an increased risk of relapse. Results with myeloablative allo HSCT unequivocally demonstrated a reduction in relapse/disease progression but minimal late recurrences did not translate into an improved OS compared with auto HSCT yet due to high TRM. However, over the past decade, advances in supportive care and better patient selection have resulted in improved outcomes for allo HSCT reducing TRM. For patients with chemoresistant disease younger than 50 years, conventional allo HSCT is probably the best procedure if patients have an HLA-identical sibling. RIC has substantially reduced up-front toxicity and data seem highly encouraging with regard to efficacy. Nonmyeloablative treatment probably yields a similar cure rate with a lower TRM but higher relapse rate. Despite the fact that prospective randomized trials comparing this strategy with conventional allo HSCT are missing and most of the studies available lack a longer follow-up, RIC is actually the most common approach for allo HSCT in FL. Up to date, the best pretransplant conditioning regimen remains to be evaluated. On the one hand, TBI-based conditioning regimens were associated with a statistically significant decrease in disease recurrence

Chapter 11  Treatment Strategies for Follicular Center Cell Non-Hodgkin’s Lymphoma 

but on the other hand use of TBI was shown to increase TRM providing a rationale for the development of radiolabeled monoclonal antibodies as part of conditioning regimens. Peripheral blood stem cells should be recommended as stem cell source because of a faster neutrophil and platelet recovery and immune reconstitution when compared to BM-derived stem cells [106]. Acknowledgments  The authors highly acknowledge Marianne Engelhard (University of Essen, Department of Radiation Oncology, Hufelandstr. 55, D-45122 Essen, Germany) for helpful discussions and critical cross-reading of the chapter.

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F. Heinzelmann et al. 16. Sack H, Hoederath A, Stuschke M et al (1998) Radiotherapy of follicle center lymphoma. Results of a German multicenter and prospective study. Members of the Study Group “NHL-early stages”. Strahlenther Onkol 174:178–185 discussion 186 17. Neumann H, Blanck H, Koch R et al (2003) Follicle centre lymphoma: Treatment results for stage I and II. Strahlenther Onkol 179:840–846 18. Ott OJ, Rodel C, Gramatzki M et al (2003) Radiotherapy for stage I–III nodal lowgrade non-Hodgkin’s lymphoma. Strahlenther Onkol 179:694–701 19. Petersen PM, Gospodarowicz M, Tsang R et al (2004) Long-term outcome in stage I and II follicular lymphoma following treatment with involved field radiation therapy alone. J Clin Oncol 22(Suppl 14S):6521 20. Guadagnolo BA, Li S, Neuberg D et al (2006) Long-term outcome and mortality trends in early-stage, Grade 1–2 follicular lymphoma treated with radiation therapy. Int J Radiat Oncol Biol Phys 64:928–934 21. Advani R, Rosenberg SA, Horning SJ (2004) Stage I and II follicular nonHodgkin’s lymphoma: Long-term follow-up of no initial therapy. J Clin Oncol 22:1454–1459 22. Besa PC, McLaughlin PW, Cox JD et al (1995) Long term assessment of patterns of treatment failure and survival in patients with stage I or II follicular lymphoma. Cancer 75:2361–2367 23. Seymour JF, Pro B, Fuller LM et al (2003) Long-term follow-up of a prospective study of combined modality therapy for stage I-II indolent non-Hodgkin’s lymphoma. J Clin Oncol 21:2115–2122 24. Gribben JG (2007) How I treat indolent lymphoma. Blood 109:4617–4626 25. Young RC, Longo DL, Glatstein E et al (1988) The treatment of indolent lymphomas: Watchful waiting v aggressive combined modality treatment. Semin Hematol 25:11–16 26. Brice P, Bastion Y, Lepage E et al (1997) Comparison in low-tumor-burden follicular lymphomas between an initial no-treatment policy, prednimustine, or interferon alfa: A randomized study from the Groupe d’Etude des Lymphomes Folliculaires. Groupe d’Etude des Lymphomes de l’Adulte. J Clin Oncol 15:1110–1117 27. Ardeshna KM, Smith P, Norton A et  al (2003) Long-term effect of a watch and wait policy versus immediate systemic treatment for asymptomatic advanced-stage non-Hodgkin lymphoma: A randomised controlled trial. Lancet 362:516–522 28. Horning SJ (2000) Follicular lymphoma: Have we made any progress? Ann Oncol 11:23–27 29. Fisher RI, LeBlanc M, Press OW et al (2005) New treatment options have changed the survival of patients with follicular lymphoma. J Clin Oncol 23:8447–8452 30. Marcus R, Imrie K, Belch A et al (2005) CVP chemotherapy plus rituximab compared with CVP as first-line treatment for advanced follicular lymphoma. Blood 105:1417–1423 31. Herold M, Haas A, Srock S et al (2007) Rituximab added to first-line mitoxantrone, chlorambucil, and prednisolone chemotherapy followed by interferon maintenance prolongs survival in patients with advanced follicular lymphoma: An East German Study Group Hematology and Oncology Study. J Clin Oncol 25:1986–1992 32. Salles GA, Foussard C, Nicolas M et  al (2004) Rituximab added {alpha} IFN + CHVP improves the outcome of follicular lymphoma patients with a high tumor burden: To first analysis of the GELA-GOELAMS FL-2000 randomized trial in 359 patients, Blood (Ash Annual Meeting Abstracts), 104, abstract 160 33. Hiddemann W, Kneba M, Dreyling M et al (2005) Frontline therapy with rituximab added to the combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) significantly improves the outcome for patients with advancedstage follicular lymphoma compared with therapy with CHOP alone: Results of a prospective randomized study of the German Low-Grade Lymphoma Study Group. Blood 106:3725–3732

Chapter 11  Treatment Strategies for Follicular Center Cell Non-Hodgkin’s Lymphoma  34. Hiddemann W, Hoster E, Buske C et al (2006) Rituximab is the essential treatment modality that underlies the significant improvement in short and long term outcome of patients with advanced stage follicular lymphoma – A 10 year analysis of GLSG trials, Blood (Ash Annual Meeting Abstracts), 108, abstract 483 35. Rohatiner AZ, Gregory WM, Peterson B et al (2005) Meta-analysis to evaluate the role of interferon in follicular lymphoma. J Clin Oncol 23:2215–2223 36. Hainsworth JD, Litchy S, Shaffer DW et al (2005) Maximizing therapeutic benefit of rituximab: Maintenance therapy versus re-treatment at progression in patients with indolent non-Hodgkin’s lymphoma – a randomized phase II trial of the Minnie Pearl Cancer Research Network. J Clin Oncol 23:1088–1095 37. Ghielmini M, Schmitz SF, Cogliatti SB et  al (2004) Prolonged treatment with rituximab in patients with follicular lymphoma significantly increases event-free survival and response duration compared with the standard weekly × 4 schedule. Blood 103:4416–4423 38. Hochster HS, Weller E, Gascoyne RD et al (2005) Maintenance rituximab after CVP results in superior clinical outcome in advanced follicular lymphoma (FL): Results of the E1496 phase III trial from the Eastern Cooperative Oncology Group and the Cancer and Leukemia Group B, Blood (Ash Annual Meeting Abstracts), 106, abstract 349 39. Press OW, Unger JM, Braziel RM et al (2006) Phase II trial of CHOP chemotherapy followed by tositumomab/iodine I-131 tositumomab for previously untreated follicular non-Hodgkin’s lymphoma: Five-year follow-up of Southwest Oncology Group Protocol S9911. J Clin Oncol 24:4143–4149 40. Kaminski MS, Tuck M, Estes J et  al (2005) 131I-tositumomab therapy as initial treatment for follicular lymphoma. N Engl J Med 352:441–449 41. Freedman AS, Gribben JG, Neuberg D et al (1996) High-dose therapy and autologous bone marrow transplantation in patients with follicular lymphoma during first remission. Blood 88:2780–2786 42. Horning SJ, Negrin RS, Hoppe RT et al (2001) High-dose therapy and autologous bone marrow transplantation for follicular lymphoma in first complete or partial remission: Results of a phase II clinical trial. Blood 97:404–409 43. Seyfarth B, Kuse R, Sonnen R et al (2001) Autologous stem cell transplantation for follicular lymphoma: No benefit for early transplant? Ann Hematol 80:398–405 44. Voso MT, Martin S, Hohaus S et al (2000) Prognostic factors for the clinical outcome of patients with follicular lymphoma following high-dose therapy and peripheral blood stem cell transplantation (PBSCT). Bone Marrow Transplant 25:957–964 45. Ladetto M, Corradini P, Vallet S et al (2002) High rate of clinical and molecular remissions in follicular lymphoma patients receiving high-dose sequential chemotherapy and autografting at diagnosis: A multicenter, prospective study by the Gruppo Italiano Trapianto Midollo Osseo (GITMO). Blood 100:1559–1565 46. Lenz G, Dreyling M, Schiegnitz E et al (2004) Myeloablative radiochemotherapy followed by autologous stem cell transplantation in first remission prolongs progression-free survival in follicular lymphoma: Results of a prospective, randomized trial of the German Low-Grade Lymphoma Study Group. Blood 104:2667–2674 47. Deconinck E, Foussard C, Milpied N et al (2005) High-dose therapy followed by autologous purged stem-cell transplantation and doxorubicin-based chemotherapy in patients with advanced follicular lymphoma: A randomized multicenter study by GOELAMS. Blood 105:3817–3823 48. Lenz G, Dreyling M, Schiegnitz E et  al (2004) Moderate increase of secondary hematologic malignancies after myeloablative radiochemotherapy and autologous stem-cell transplantation in patients with indolent lymphoma: Results of a prospective randomized trial of the German Low Grade Lymphoma Study Group. J Clin Oncol 22:4926–4933 49. Sebban C, Mounier N, Brousse N et al (2006) Standard chemotherapy with interferon compared with CHOP followed by high-dose therapy with autologous stem

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F. Heinzelmann et al. cell transplantation in untreated patients with advanced follicular lymphoma: The GELF-94 randomized study from the Groupe d’Etude des Lymphomes de l’Adulte (GELA). Blood 108:2540–2544 50. Johnson PW, Rohatiner AZ, Whelan JS et al (1995) Patterns of survival in patients with recurrent follicular lymphoma: A 20-year study from a single center. J Clin Oncol 13:140–147 51. van Oers MH, Klasa R, Marcus RE et al (2006) Rituximab maintenance improves clinical outcome of relapsed/resistant follicular non-Hodgkin lymphoma in patients both with and without rituximab during induction: Results of a prospective randomized phase 3 intergroup trial. Blood 108:3295–3301 52. Tse WW, Lazarus HM, Van Besien K (2004) Stem cell transplantation in follicular lymphoma: Progress at last? Bone Marrow Transplant 34:929–938 53. Freedman AS, Neuberg D, Mauch P et al (1999) Long-term follow-up of autologous bone marrow transplantation in patients with relapsed follicular lymphoma. Blood 94:3325–3333 54. Brice P, Simon D, Bouabdallah R et al (2000) High-dose therapy with autologous stem-cell transplantation (ASCT) after first progression prolonged survival of follicular lymphoma patients included in the prospective GELF 86 protocol. Ann Oncol 11:1585–1590 55. Rohatiner AZ, Johnson PW, Price CG et  al (1994) Myeloablative therapy with autologous bone marrow transplantation as consolidation therapy for recurrent follicular lymphoma. J Clin Oncol 12:1177–1184 56. Bastion Y, Brice P, Haioun C et al (1995) Intensive therapy with peripheral blood progenitor cell transplantation in 60 patients with poor-prognosis follicular lymphoma. Blood 86:3257–3262 57. Bierman PJ, Vose JM, Anderson JR et al (1997) High-dose therapy with autologous hematopoietic rescue for follicular low-grade non-Hodgkin’s lymphoma. J Clin Oncol 15:445–450 58. Apostolidis J, Gupta RK, Grenzelias D et al (2000) High-dose therapy with autologous bone marrow support as consolidation of remission in follicular lymphoma: Long-term clinical and molecular follow-up. J Clin Oncol 18:527–536 59. Cao TM, Horning S, Negrin RS et  al (2001) High-dose therapy and autologous hematopoietic-cell transplantation for follicular lymphoma beyond first remission: The Stanford University experience. Biol Blood Marrow Transplant 7:294–301 60. Schouten HC, Qian W, Kvaloy S et al (2003) High-dose therapy improves progressionfree survival and survival in relapsed follicular non-Hodgkin’s lymphoma: Results from the randomized European CUP trial. J Clin Oncol 21:3918–3927 61. van Besien K, Loberiza FR Jr, Bajorunaite R et al (2003) Comparison of autologous and allogeneic hematopoietic stem cell transplantation for follicular lymphoma. Blood 102:3521–3529 62. Verdonck LF (1999) Allogeneic versus autologous bone marrow transplantation for refractory and recurrent low-grade non-Hodgkin’s lymphoma: Updated results of the Utrecht experience. Leuk Lymphoma 34:129–136 63. van Besien KW, Khouri IF, Giralt SA et al (1995) Allogeneic bone marrow transplantation for refractory and recurrent low-grade lymphoma: The case for aggressive management. J Clin Oncol 13:1096–1102 64. van Besien K, Champlin IK, McCarthy P (2000) Allogeneic transplantation for low-grade lymphoma: Long-term follow-up. J Clin Oncol 18:702–703 65. van Besien K, Sobocinski KA, Rowlings PA et al (1998) Allogeneic bone marrow transplantation for low-grade lymphoma. Blood 92:1832–1836 66. Mandigers CM, Raemaekers JM, Schattenberg AV et  al (1998) Allogeneic bone marrow transplantation with T-cell-depleted marrow grafts for patients with poorrisk relapsed low-grade non-Hodgkin’s lymphoma. Br J Haematol 100:198–206 67. Forrest DL, Thompson K, Nevill TJ et  al (2002) Allogeneic hematopoietic stem cell transplantation for progressive follicular lymphoma. Bone Marrow Transplant 29:973–978

Chapter 11  Treatment Strategies for Follicular Center Cell Non-Hodgkin’s Lymphoma  68. Yakoub-Agha I, Fawaz A, Folliot O et  al (2002) Allogeneic bone marrow transplantation in patients with follicular lymphoma: A single center study. Bone Marrow Transplant 30:229–234 69. Kiss TL, Panzarella T, Messner HA et al (2003) Busulfan and cyclophosphamide as a preparative regimen for allogeneic blood and marrow transplantation in patients with non-Hodgkin’s lymphoma. Bone Marrow Transplant 31:73–78 70. Hosing C, Saliba RM, McLaughlin P et al (2003) Long-term results favor allogeneic over autologous hematopoietic stem cell transplantation in patients with refractory or recurrent indolent non-Hodgkin’s lymphoma. Ann Oncol 14:737–744 71. Peniket AJ, Ruiz de Elvira MC, Taghipour G et al (2003) An EBMT registry matched study of allogeneic stem cell transplants for lymphoma: Allogeneic transplantation is associated with a lower relapse rate but a higher procedure-related mortality rate than autologous transplantation. Bone Marrow Transplant 31:667–678 72. Toze CL, Barnett MJ, Connors JM et al (2004) Long-term disease-free survival of patients with advanced follicular lymphoma after allogeneic bone marrow transplantation. Br J Haematol 127:311–321 73. van Besien KW, de Lima M, Giralt SA et  al (1997) Management of lymphoma recurrence after allogeneic transplantation: The relevance of graft-versus-lymphoma effect. Bone Marrow Transplant 19:977–982 74. Ratanatharathorn V, Uberti J, Karanes C et al (1994) Prospective comparative trial of autologous versus allogeneic bone marrow transplantation in patients with nonHodgkin’s lymphoma. Blood 84:1050–1055 75. Gribben JG, Freedman AS, Neuberg D et al (1991) Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B-cell lymphoma. N Engl J Med 325:1525–1533 76. Champlin R, Khouri I, Kornblau S et  al (1999) Reinventing bone marrow transplantation: Reducing toxicity using nonmyeloablative, preparative regimens and induction of graft-versus-malignancy. Curr Opin Oncol 11:87–95 77. Khouri IF, Saliba RM, Giralt SA et al (2001) Nonablative allogeneic hematopoietic transplantation as adoptive immunotherapy for indolent lymphoma: Low incidence of toxicity, acute graft-versus-host disease, and treatment-related mortality. Blood 98:3595–3599 78. Robinson SP, Goldstone AH, Mackinnon S et al (2002) Chemoresistant or aggressive lymphoma predicts for a poor outcome following reduced-intensity allogeneic progenitor cell transplantation: An analysis from the Lymphoma Working Party of the European Group for Blood and Bone Marrow Transplantation. Blood 100:4310–4316 79. Bertz H, Illerhaus G, Veelken H et  al (2002) Allogeneic hematopoetic stem-cell transplantation for patients with relapsed or refractory lymphomas: Comparison of high-dose conventional conditioning versus fludarabine-based reduced-intensity regimens. Ann Oncol 13:135–139 80. Ho AY, Devereux S, Mufti GJ et  al (2003) Reduced-intensity rituximab-BEAMCAMPATH allogeneic haematopoietic stem cell transplantation for follicular lymphoma is feasible and induces durable molecular remissions. Bone Marrow Transplant 31:551–557 81. Tanimoto TE, Kusumi E, Hamaki T et al (2003) High complete response rate after allogeneic hematopoietic stem cell transplantation with reduced-intensity conditioning regimens in advanced malignant lymphoma. Bone Marrow Transplant 32:131–137 82. Escalon MP, Champlin RE, Saliba RM et al (2004) Nonmyeloablative allogeneic hematopoietic transplantation: A promising salvage therapy for patients with nonHodgkin’s lymphoma whose disease has failed a prior autologous transplantation. J Clin Oncol 22:2419–2423 83. Faulkner RD, Craddock C, Byrne JL et al (2004) BEAM-alemtuzumab reducedintensity allogeneic stem cell transplantation for lymphoproliferative diseases: GVHD, toxicity, and survival in 65 patients. Blood 103:428–434

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F. Heinzelmann et al.   84. Morris E, Thomson K, Craddock C et al (2004) Outcomes after alemtuzumabcontaining reduced-intensity allogeneic transplantation regimen for relapsed and refractory non-Hodgkin lymphoma. Blood 104:3865–3871   85. Maris MB, Sandmaier BM, Storer B et  al (2005) Allogeneic hematopoietic cell transplantation (HCT) after nonmyeloablative conditioning for relapsed or refractory follicular lymphoma, Blood (Ash Annual Meeting Abstracts), 106, abstract 1130   86. Kusumi E, Kami M, Kanda Y et al (2005) Reduced-intensity hematopoietic stemcell transplantation for malignant lymphoma: A retrospective survey of 112 adult patients in Japan. Bone Marrow Transplant 36:205–213   87. Khouri IF, Saliba RM, Hosing CM et al (2005) Autologous stem cell (AUTO) vs non-myeloablative allogeneic transplantation (NMT) after high-dose rituximab (HD-R)-containing conditioning regimens for relapsed chemosensitve follicular lymphoma (FL), Blood (Ash Annual Meeting Abstracts), 106, abstract 48   88. Vigouroux S, Michallet M, Porcher R et  al (2007) Long-term outcomes after reduced-intensity conditioning allogeneic stem cell transplantation for low-grade lymphoma: A survey by the French Society of Bone Marrow Graft Transplantation and Cellular Therapy (SFGM-TC). Haematologica 92:627–634   89. Khouri IF, Keating M, Korbling M et  al (1998) 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 16:2817–2824   90. Van Besien K, Carreras J, Zhang M-J et al (2005) Reduced intensity vs myeloablative conditioning for HLA matched sibling transplantation in follicular lymphoma, Blood (Ash Annual Meeting Abstracts), 106, abstract 656   91. Rodriguez R, Nademanee A, Ruel N et al (2006) Comparison of reduced-intensity and conventional myeloablative regimens for allogeneic transplantation in non-Hodgkin’s lymphoma. Biol Blood Marrow Transplant 12:1326–1334   92. Acker B, Hoppe RT, Colby TV et  al (1983) Histologic conversion in the nonHodgkin’s lymphomas. J Clin Oncol 1:11–16   93. Horning SJ, Rosenberg SA (1984) The natural history of initially untreated lowgrade non-Hodgkin’s lymphomas. N Engl J Med 311:1471–1475   94. Schouten HC, Bierman PJ, Vaughan WP et al (1989) Autologous bone marrow transplantation in follicular non-Hodgkin’s lymphoma before and after histologic transformation. Blood 74:2579–2584   95. Horning SJ (1993) Natural history of and therapy for the indolent non-Hodgkin’s lymphomas. Semin Oncol 20:75–88   96. Yuen AR, Kamel OW, Halpern J et al (1995) Long-term survival after histologic transformation of low-grade follicular lymphoma. J Clin Oncol 13:1726–1733   97. Cullen MH, Lister TA, Brearley RI et  al (1979) Histological transformation of non-Hodgkin’s lymphoma: A prospective study. Cancer 44:645–651   98. Foran JM, Apostolidis J, Papamichael D et  al (1998) High-dose therapy with autologous haematopoietic support in patients with transformed follicular lymphoma: A study of 27 patients from a single centre. Ann Oncol 9:865–869   99. Friedberg JW, Neuberg D, Gribben JG et  al (1999) Autologous bone marrow transplantation after histologic transformation of indolent B cell malignancies. Biol Blood Marrow Transplant 5:262–268 100. Berglund A, Enblad G, Carlson K et al (2000) Long-term follow-up of autologous stem-cell transplantation for follicular and transformed follicular lymphoma. Eur J Haematol 65:17–22 101. Chen CI, Crump M, Tsang R et al (2001) Autotransplants for histologically transformed follicular non-Hodgkin’s lymphoma. Br J Haematol 113:202–208 102. Williams CD, Harrison CN, Lister TA et al (2001) High-dose therapy and autologous stem-cell support for chemosensitive transformed low-grade follicular nonHodgkin’s lymphoma: A case-matched study from the European Bone Marrow Transplant Registry. J Clin Oncol 19:727–735

Chapter 11  Treatment Strategies for Follicular Center Cell Non-Hodgkin’s Lymphoma  103. Corradini P, Ladetto M, Zallio F et  al (2004) Long-term follow-up of indolent lymphoma patients treated with high-dose sequential chemotherapy and autografting: Evidence that durable molecular and clinical remission frequently can be attained only in follicular subtypes. J Clin Oncol 22:1460–1468 104. Kaminski MS, Zelenetz AD, Press OW et al (2001) Pivotal study of iodine I 131 tositumomab for chemotherapy-refractory low-grade or transformed low-grade B-cell non-Hodgkin’s lymphomas. J Clin Oncol 19:3918–3928 105. Davies AJ, Rohatiner AZ, Howell S et al (2004) Tositumomab and iodine I 131 tositumomab for recurrent indolent and transformed B-cell non-Hodgkin’s lymphoma. J Clin Oncol 22:1469–1479 106. Korbling M, Anderlini P (2001) Peripheral blood stem cell versus bone marrow allotransplantation: Does the source of hematopoietic stem cells matter? Blood 98:2900–2908

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Chapter 12 The Role of Transplantation in Favorable-Risk Acute Myeloid Leukemia Mickey Liao and Gary J. Schiller

1.  Introduction Favorable-risk acute myeloid leukemia (AML) is a subgroup of AML that has been shown to have clinically unique behavior and improved response to therapy based on distinct molecular biology. Traditional post-remission therapies, including intensive consolidation chemotherapy as well as autologous or allogeneic transplantation, have been associated with improved clinical outcomes in this subpopulation. Still, the term favorable-risk incorrectly implies a high likelihood of long-term survival. In fact, most studies show that among patients in first complete remission (CR1) whose disease is characterized by favorable-risk features, long-term survival is achieved in only 40–60%. Notwithstanding that nearly half such patients may sustain relapse, there has been little enthusiasm for autologous or allogeneic stem cell transplantation as post-remission treatment for patients with otherwise favorable disease features on the basis of an excessively high rate of treatment-related morbidity and mortality. Given the remarkable advances in supportive care and immunoprophylaxis with stem cell transplantation over the last several decades and a shift toward risk-adapted therapy, standard post-remission strategies for favorable-risk AML in CR1 are being re-evaluated.

2.  Favorable-Risk AML Historically, AML has been broadly classified morphologically, distinguishing unique histological subtypes that are uniformly defined by an inappropriate increase in immature myeloid progenitor cells. Considering its molecular and cytogenetic complexity, AML is better defined according to these features that give insight into disease biology. Cytogenetic-risk categories are distinguished by response to conventional chemotherapeutic agents in terms of induction of complete remission (CR) and leukemia-free survival (LFS). The recent World Health Organization (WHO) Classification system emphasizes the cytogenetic and molecular diversity of AML, and oncology cooperative groups have

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_12, © Springer Science + Business Media, LLC 2003, 2010

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developed categories of disease based on cytogenetic profiles as poor-risk, intermediate-risk, and favorable-risk AML. Favorable-risk AML is defined by specific cytogenetic abnormalities including t(8;21); t(15;17); and inv(16). Numerous newly identified structural defects, including mutations of the FMSrelated tyrosine kinase 3 (FLT3) gene and nucleophosmin, member 1 (NMP1) gene, may also function as independent prognostic markers but are beyond the scope of this chapter [1]. In the early 1980s, a group of patients was identified in whom there was a distinct response to standard AML induction therapy. These patients were all subclassified as having acute myelomonocytic leukemia with abnormal bone marrow eosinophilia. A retrospective statistical analysis of this subgroup revealed a markedly improved response rate to cytarabine-/anthracyclinebased induction chemotherapy [2]. At the same time, a unique cytogenetic abnormality involving chromosome 16 was identified among those patients who were most likely to achieve a CR. A larger study evaluating over 30 patients with morphologic features consistent with acute myelomonocytic leukemia with abnormal eosinophils and association with inv(16) or t(16;16) cytogenetic abnormality would later corroborate these earlier findings [3]. Given the promising predictive value of cytogenetic analysis, a large retrospective study of previous acute leukemia trials from 1972 to 1980 was performed and again identified improved clinical outcomes in those patients whose leukemia was characterized by abnormalities involving chromosome 16. Interestingly, another subgroup for whom similarly impressive responses to both induction and consolidation therapy was identified – patients with t(8;21). This finding was later reproduced by a large prospective analysis from the Cancer and Leukemia Group B (CALGB) of over 1,000 patients with newly diagnosed AML [4]. In patients whose leukemia was characterized by abnormalities of chromosome 16 or t(8;21), a significantly improved prognosis, as compared to those with normal karyotype, was observed. In that review, an 85% CR rate was achieved in those with abnormalities of chromosome 16 and a 91% CR rate was reported for those with t(8;21), following standard induction chemotherapy [5]. Initial investigation of clinical outcomes in patients with AML characterized by t(15;17) cytogenetic abnormality produced variable results. However, since the introduction of all-trans retinoic acid (ATRA) into standard therapy for acute promyelocytic leukemia (APL), those patients with t(15;17) have similarly been identified as likely to achieve long-term LFS and are thus included among those with favorable-risk acute leukemia [6]. Several trials have now confirmed the clinical observation that cytogenetics play a major predictive role, not only in response to therapy, but in long-term survival as well. The CALGB demonstrated a significant increase in LFS and overall survival (OS), in patients whose disease was characterized by either inv(16) or t(8;21) chromosomal abnormalities, to single-agent cytarabine-based consolidation chemotherapy [7]. A large study from the United Kingdom, enrolling more than 1,600 child and adult patients with either de novo or secondary AML, concluded that age and cytogenetics were the most important predictors of relapse rate and OS [8]. Patients with favorable-risk cytogenetics who achieve a CR with induction therapy can typically expect a 5-year OS rate that approaches 60% [5]. Although this result is generally considered favorable, one must appreciate that nearly half of the patients with newly diagnosed, the so-called favorable-risk AML, will relapse of disease.

Chapter 12  The Role of Transplantation in Favorable-Risk Acute Myeloid Leukemia 

3.  Molecular Pathogenesis The predictive capacity of favorable cytogenetics suggests that a molecular abnormality is directly tied to the sensitivity to conventional therapeutic agents and consequently a clinical outcome better than that which can be achieved for other types of AML. In turn, understanding the genetic defects and associated mechanisms of action could potentially improve outcome further. Molecular studies of favorable-risk AML have identified multiple genes that encode either DNA-binding transcription factors or the regulatory components of transcriptional complexes [9]. The best characterized cytogenetic abnormality involves the t(15;17) translocation associated with APL. The t(15;17) translocation involves formation of the transgene PML-RARa and subsequent generation of a fusion protein which inhibits DNA binding of CCAAT/enhancer binding proteina (C/EBPa), a transcription factor involved in the regulation of myelopoiesis. ATRA has since been introduced as a therapeutic agent that targets the chimeric protein and allows for normal myeloid differentiation [10]. In addition to PML-RARa, other molecular subgroups to consider with favorable-risk AML include the core-binding factor (CBF) abnormalities. The CBF DNA binding target is in the promoter/enhancer region of many genes expressed in hematopoietic cells, hence the pathogenic role in leukemia. The associated transcription factor complex, AML1-CBFb, was originally discovered through cloning of the t(8;21) translocation and identification of the AML1 gene. The transcription factor complex regulates a number of hematopoiesis-specific genes as well as the normal development of the hematopoietic system through activation of transcription as well as recruitment of coactivators. The t(8;21) translocation joins the N-terminal part of the AML1 gene on chromosome 21 with the C-terminal portion of the ETO gene on chromosome 8. The resultant fusion protein retains the ability to bind AML1-targeted sequences through its interactions with CBFb, however, instead functions as a repressor of transcription through recruitment of a nuclear corepressor complex [11]. Furthermore, AML1-ETO may lead to down-regulation of C/EBPa [12]. Abnormalities with chromosome 16 involve the same transcription factor complex. The CBFb subunit on chromosome 16q22 is fused to the smooth muscle myosin heavy chain gene MYH11 on chromosome 16p13. The resultant chimeric product, CBFb-MYH11, directly represses AML1-mediated transcription by recruiting AML1 into functionally inactive complexes within the cytoplasm [13].

4.  Post-remission Therapy Patients whose AML is characterized by favorable-risk cytogenetic abnormalities represent not only a cohort of patients whose disease is more responsive to current induction regimens, but also a subgroup in whom intensive post-remission therapy may be increasingly curative. Although a variety of post-remission strategies have been utilized, studies have consistently demonstrated superior responses in the favorable-risk groups, as compared to the poor-risk groups, regardless of the therapy selected. Treatment of APL, in particular, has undergone some of the most remarkable changes. With CR rates of 80–90% and OS

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rates of 70–75% since the introduction of ATRA therapy to standard induction chemotherapy, the role of myeloablative therapy in CR1 has virtually been eliminated [14–16]. Even when APL is further risk stratified based on white blood cell count and platelet count, those at highest risk still have an OS rate of greater than 70% [17]. One could argue for more intensive chemotherapy in an attempt to further improve OS in the setting of APL. However, it would be difficult to demonstrate a superior result with myeloablative therapy for disease characterized by t(15;17) given the risk of some degree of transplant-related morbidity and mortality balanced against the remarkable survival data with standard induction chemotherapy plus ATRA. Furthermore, As2O3 has also already been shown to have significant activity in APL and is currently the treatment of choice for relapsed APL. Two studies have additionally demonstrated CR rates of over 80% when arsenic trioxide was used as a single agent in newly diagnosed APL [18,19]. However, in patients with t(8;21) or inv(16) favorable-risk cytogenetic abnormalities, long-term clinical outcomes are less impressive and the optimal post-remission strategy remains unclear. In a small study published in 1991, a single cycle of high-dose cytarabine/ daunorubicin consolidation therapy was found most likely to achieve a favorable response in those patients with favorable-risk cytogenetics [20]. In a much larger trial of older adults with AML from the United Kingdom, induction therapy followed by randomization to either no therapy or further consolidation chemotherapy, also showed a significantly improved LFS in patients with AML characterized by favorable-risk cytogenetics as compared to those with poor-risk features [8]. Finally, in a trial of four cycles of variably dosed cytarabine-based consolidation chemotherapy, the group with favorable-risk cytogenetics again was most likely to achieve an improved clinical outcome. In fact, the greatest improvement in LFS rate was identified in the group of patients with favorable-risk cytogenetics receiving the highest doses of cytarabine consolidation. Improved outcomes in favorable-risk groups are not only limited to dose-intensified consolidation chemotherapy, but can also be extended to myeloablative therapy followed by transplantation. In a study from France, patients with favorable-risk cytogenetic abnormalities achieved a superior clinical outcome with allogeneic or autologous transplantation as compared to those with poor-risk cytogenetics [21]. Thus, regardless of whether the postremission strategy included chemotherapy or transplantation, the best results were routinely identified in the favorable-risk group. Presently, allogeneic stem cell transplantation is the recommended approach only in those patients with features of poor-risk leukemia, for whom all strategies yield poor results. However, the role of transplantation in favorable-risk AML needs to be further investigated since it is conceivable that the most dose-intensive approach would be the most likely to achieve a favorable response.

5.  Stem Cell Transplantation Historically, adult patients with AML in CR1 have been treated with doseintense consolidation chemotherapy based on evidence documenting a superior median LFS when compared to less intensive post-remission therapy. However, the rate of sustained LFS can vary widely, from 15% to 60%, largely depending on the cytogenetic-risk group [22–26]. As a consequence,

Chapter 12  The Role of Transplantation in Favorable-Risk Acute Myeloid Leukemia 

a variety of other post-remission strategies have been implemented including myelosuppressive maintenance therapy, multiple and prolonged cycles of consolidation chemotherapy, as well as autologous or allogeneic transplantation. Transplantation has generally been reserved for those patients with poor-risk AML, including those patients with deletion 5q or 7q, trisomy 8, t(6;9), t(9;22), those with clinical features of adverse prognosis such as antecedent history of hematologic disease, or those patients with refractory or relapsed leukemia, in whom, given the dismal outcomes, transplantation affords the best potential for anti-leukemic effect despite the lack of impressive survival statistics. Meanwhile, patients whose disease is characterized by favorable-risk cytogenetics have generally been discouraged from pursuing myeloablative therapy given the favorable outcomes of intensive chemotherapy alone [27]. Although there are no data to support survival benefit, review of the literature consistently demonstrates a decreased relapse rate following stem cell transplantation as compared to intensive consolidation chemotherapy alone. Unfortunately, post-remission therapy with transplantation has historically been associated with an unacceptably high treatment-related mortality (TRM), with upwards of 20–30% TRM in allogeneic transplantation and 10–20% TRM in autologous transplantation, discouraging routine practice [28]. However, improved supportive care has increased the tolerability of transplantation over the past decades. These advancements have altered the risk–benefit ratio, and suggest a reappraisal of the role of stem cell transplantation in favorable-risk AML.

6.  Autologous Stem Cell Transplantation Autologous stem cell transplantation (ASCT) allows a larger, unselected population of patients, including potentially patients up to 70 years of age, to derive the benefits of a myeloablative preparative therapy without incurring the higher risk of transplant-related morbidity and mortality associated with allogeneic transplantation, including acute and chronic graft-versus-host disease (GVHD). ASCT was first utilized as a salvage therapy for relapsed disease in the late 1970s, when numerous studies showed it was capable of inducing a CR in most patients and was associated with a prolonged LFS in the range of 20–50% [29]. Given the encouraging results in relapsed leukemia, the role of ASCT for AML in CR1 was pursued. Numerous studies have since evaluated the safety and efficacy of autologous transplantation compared with intensive consolidation chemotherapy or no further therapy with AML in CR1 [22–24,30–33]. Although none of the studies have demonstrated a significant overall survival benefit, two large studies have shown a meaningful difference in LFS (see Tables 12-1 and 12-2). In 1986, the European Organization for Research and Treatment of Cancer (EORTC) in conjunction with the Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto (GIMEMA) Leukemia Cooperative Groups conducted a large prospective trial of three post-remission strategies to examine LFS and OS [22]. A total of 941 patients entered the trial; although, only 623 patients achieved a CR. Furthermore, there was a significant drop-out rate, leaving only 343 patients who received their intended post-remission therapy. In the intent-to-treat (ITT) analysis, LFS at 4 years was 48% in the ASCT group and 30% in the chemotherapy-alone group with relapse rates of 41 and 57%, respectively. Although the differences in results for LFS and relapse

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Table 12-1.  LFS with ASCT for AML in CR1. Study

ASCT (%)

Chemotherapy (%)

P-value

Zittoun et al. [22]   All patients – 4-year DFS

48

30

0.05

Burnett et al. [23]   All patients – 7-year DFS   Favorable risk

53 70

40 46

0.04 0.04

Cassileth et al.   All patients – 4 years [24]   Favorable risk [35]

35 71

35 35

NS 0.04

Harousseau et al. [32]   All patients – 4-year DFS

44

41

NS

Tsimberidou et al. [31]   All patients – 3-year DFS

42

33

NS

Breems et al. [33]   All patients – 5-year DFS

35

37

NS

NS non-significant

Table 12-2.  OS with ASCT for AML in CR1. Study

ASCT (%)

Chemotherapy (%)

P-value

Zittoun et al. [22]   All patients – 4-year OS

56

46

NS

Burnett et al. [23]   All patients – 7-year OS   Favorable risk

57 74

45 51

NS NS

Cassileth et al. [24]   All patients – 4-year OS [35]

43

52

0.05

Harousseau et al. [32]   All patients – 4-year OS

50

55

NS

Tsimberidou et al. [31]   All patients – 3-year DFS

58

46

NS

Breems et al. [33]   All patients – 5-year OS

45

55

NS

NS non-significant

rate were statistically significantly, there was no significant difference in OS. Unfortunately, these results were not further stratified by cytogenetic risk. The Medical Research Council Leukaemia Working Parties undertook a prospective trial (MRC AML 10), for which the principal aim was to assess the value of high-dose therapy supported by either ASCT or allogeneic stem cell transplantation (alloSCT) for AML in CR1. The outcomes of ASCT were first reported in 1998 with follow-up alloSCT results published in 2002 [23,34]. Unfortunately, there again was a high drop-out rate in the ASCT arm, with only 126 of 190 (66%) of the assigned patients receiving the intended therapy. Nevertheless, when evaluated based on an ITT analysis, LFS at 7 years was found to be 54% in the ASCT group versus 40% in the group assigned no

Chapter 12  The Role of Transplantation in Favorable-Risk Acute Myeloid Leukemia 

further therapy and the relapse rate was 37 and 58%, respectively. Both the differences in LFS and relapse rates were again statistically significant findings. Furthermore, a subgroup analysis of the data based on cytogenetic risk, revealed an added benefit in LFS and relapse rate. In favorable-risk patients, LFS improved to 70% with ASCT versus 48% in the group assigned no further therapy and relapse rate improved to 25% versus 49%, respectively. Interestingly, there was one study which demonstrated a modest survival benefit, albeit, surprisingly identified in the chemotherapy alone arm. In a large American Intergroup trial also reported in 1998, ASCT or alloSCT were compared to high-dose cytarabine in young adults with AML in CR1 in a manner similar to the previously described EORTC/GIMEMA schema [24,35]. In the ITT analysis, LFS at 4 years was not found to be statistically different. However, there was a trend toward a marginally improved OS. The OS rate at 4 years was 43% for the ASCT arm and 52% in the chemotherapy alone arm which was statistically significant. However, when risk-stratified by cytogenetics, LFS rates for favorable-risk AML were found to be 71% for ASCT and 35% for chemotherapy alone which was also significantly different, but in favor of ASCT. Unfortunately, overall survival data were not available stratified by cytogenetic risk. These large studies help support a potential role for ASCT in favorable-risk AML in CR1. When stratified by cytogenetic-risk group, the improvements in the relapse rate and LFS with ASCT as compared to chemotherapy alone are compelling. However, these results have to be interpreted with caution given the complex study designs inherent to transplantation trials, inconsistent patient populations including considerable patient withdrawal and treatment crossover, and perhaps most importantly, lack of survival benefit.

7.  Allogeneic Stem Cell Transplantation The mechanism behind the success of allogeneic peripheral stem cell transplantation (alloSCT) is multi-factorial by way of delivery of myeloablative conditioning therapy, similar to what is used in autologous transplantation, in combination with an additional element of immunologic stimulation associated with the allograft itself – that is, graft versus leukemia effect. Nevertheless, OS rates have not been demonstrated to be significantly superior compared to what is achieved with an initial strategy of intensive consolidation chemotherapy with the possibility of salvage alloSCT in relapse. Furthermore, alloSCT is associated with a significantly elevated TRM rate. Consequently, although AML remains the most common indication for alloSCT, its role in adults has largely been restricted to those patients with poor cytogenetic risk, refractory, and relapsed AML in whom all treatment options yield disappointing results. Although to date, there is no general recommendation for alloSCT in favorable-risk AML in CR1, improved transplantation technique and supportive care allow for reinvestigation. The impact of cytogenetics on outcomes of alloSCT was first described by Slovak et al. in 2000 [35]. Five-year estimates for overall OS in favorable-, intermediate-, and poor-risk cytogenetic groups were shown to be 63%, 52%, and 44%, respectively. Those results established

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the predictive role of karyotype on clinical outcome of alloSCT in AML. A study by Kim et  al. published in 2005 further illustrated that cytogenetic risk carried more discriminative power than even clinical disease status at time of presentation in predicting prognosis following alloSCT [36]. AlloSCT has additionally been associated with improved rates of relapse when compared to chemotherapy alone or ASCT. However, given the favorable outcomes associated with intensive consolidation chemotherapy alone, in combination with a historically significant TRM associated with alloSCT, data have been sparse with respect to the effect of alloSCT specifically in those with favorable-risk AML in CR1. Several large studies investigating the role of alloSCT as post-remission therapy have been published. Although, results have not routinely been stratified by cytogenetic risk (see Tables  12-3 and 12-4). When considering all cytogenetic-risk groups, no survival benefit has been demonstrated when comparing alloSCT to ASCT. However, several studies have shown a significant improvement in LFS. In the BGMT 87 study, Reiffers et al. report in 1996 on the outcomes of 240 previously untreated younger patients who were consolidated with alloSCT or, if an HLA-matched donor was not available, randomized to ASCT versus intensive consolidation chemotherapy [30]. LFS at 3 years was found to be 66% for the alloSCT group and 42% for the ASCT group, which was statistically significant. However, there was no significant difference in overall survival. The previously discussed MRC AML 10 trial also evaluated the value of alloSCT. When reviewed based on an ITT analysis, LFS at 7 years was found to be 50% in the alloSCT group versus 42% in the group assigned no further therapy with a relapse rate of 36 and 52%, Table 12-3.  LFS with AlloSCT for AML in CR1. Study

AlloSCT (%)

ASCT (%)

P-value

Zittoun et al. [22]   All patients – 4-year LFS

55

48

NS

50

42

0.001

53 63

66 52

NS NS

Cassileth et al. [24]   All patients – 4-year LFS

43

35

n/a

Suciu et al. [37]   All patients – 4-year LFS   Favorable risk (t(8;21)/inv 16)

52 62

42 66

0.04 NS

Jourdan et al. [42]   All patients – 10-year LFS   Favorable risk [t(8;21), inv16, t(15;17)]

n/a 51

n/a 68

n/a NS

Harousseau et al. [32]   All patients – 4-year LFS

44

44

NS

Reiffers et al. [30]   All patients – 3-year LFS

66

42

0.05

Tsimberidou et al. [31]   All patients – 3-year LFS

n/a

n/a

n/a

Burnett et al. [34]   All patients – 7-year LFS   Favorable risk   t(8;21)/inv(16)   t(15;17)

n/a not available; NS non-significant

Chapter 12  The Role of Transplantation in Favorable-Risk Acute Myeloid Leukemia 

Table 12-4.  OS with AlloSCT for AML in CR1. Study

AlloSCT (%)

ASCT (%)

P-value

Zittoun et al. [22]   All patients – 4-year OS

59

56

NS

56

50

NS

63 75

77 72

NS NS

Cassileth et al. [24]   All patients – 4-year OS

46

43

NS

Suciu et al. [37]   All patients – 4-year OS   Favorable risk [t(8;21)/inv16]

58 n/a

50 n/a

NS n/a

Jourdan et al. [42]   All patients   Favorable risk [t(8;21), inv16, t(15;17)]

51 51

43 74

NS NS

Harousseau et al. [32]   All patients – 4-year OS

53

50

NS

Reiffers et al. [30]   All patients – 3-year OS

n/a

n/a

n/a

Tsimberidou et al. [31]   All patients – 3-year OS

58

50

NS

Burnett et al. [34]   All patients – 7-year OS   Favorable risk   t(8;21)/inv(16)   t(15;17)

n/a not available; NS non-significant

respectively [34]. Both the differences in LFS and relapse rates were statistically significant findings while overall survival was again not significantly different. Finally, in 2003 Suciu et al. reported on the outcomes of over 2,100 patients who were assigned to alloSCT or ASCT consolidation therapy in CR1 based on the availability of an HLA-matched donor. ITT analysis again demonstrated a significantly decreased relapse rate and improved DFS in the alloSCT group versus ASCT although no survival benefit was seen [37]. Despite the statistical evidence for LFS benefit in several studies, the variability of results and overall lack of significant survival advantage among the available published data was described well in a large systematic analysis of consolidation therapies for adult AML conducted by Visani et al. in 2006 [39]. Finally, a large meta-analysis, conducted by Yanada et  al. in 2005, did collectively demonstrate a statistically significant OS benefit with alloSCT for AML in CR1 [40]. However, upon further stratification of the patients by cytogenetic-risk, survival benefit was limited only to those patients with intermediate- and poor-risk cytogenetic groups and not extended to the favorable-risk group. A recent study by the HOVON/SAKK collaborative study group in 2007 readdressed the question as to whether the use of alloSCT in all patients with AML in CR1 had a favorable impact on LFS as well as OS [41]. Over 1,000 patients, all less than 55 years of age, were recruited. LFS at 4 years was found to be 48% among patients with a donor and 37% for those without, while OS at 4 years was found to be 54 and 46%, respectively. Although there was a statistically significant difference in LFS, there was no significant difference in OS. When stratified by cytogenetic risk, significant LFS was again limited only to the intermediate- and poor-risk cytogenetic

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groups. A meta-analysis of the data from the HOVON/SAKK study and three previously published large studies from the EORTC, MRC, and BGMT was also prepared [34,37,42]. The cumulative results of donor versus no donor outcomes for AML in CR1 demonstrated a statistically significant LFS benefit observed particularly in younger patients and in patients without favorable-risk cytogenetics. Additionally, a significant survival advantage was again identified. But, further subgroup analysis similarly restricted the survival benefit to those patients without favorable-risk cytogenetics with improved outcomes most prominent in younger patients. Despite evidence for LFS and modest aggregate OS benefit with alloSCT for AML in CR1, when further stratified by cytogenetic risk, these advantages have not been reproduced within the favorable-risk group. However, there are a number of limitations within the available published data including a lack of randomization inherent to trials involving alloSCT. Additionally, given the low compliance rate often associated with the alloSCT treatment arms and an effort to analyze data based on ITT, one could also argue that the current data may under-represent the full benefit of alloSCT. Nevertheless, the sum of these studies gives credence to the value of early alloSCT from an HLA-identical sibling donor for patients with AML in CR1 in younger patients with intermediate- or poor-risk cytogenetics. Consolidation therapy with alloSCT in patients with AML in CR1 and favorable-risk cytogenetics, however, does not share the same LFS or survival benefits. Consequently, alloSCT remains an area of investigation in patients with AML in CR1 with favorable-risk cytogenetics and cannot currently be considered a part of standard care.

8.  Refractory Acute Myeloid Leukemia Despite therapeutic advances, up to 20–30% of AML patients will never be able to achieve a CR. The only curative option for those patients remains an allogeneic transplantation. Although transplantation is reasonable to consider in any patient with primary refractory AML, it is important to recognize that cytogenetic risk remains an important predictor of clinical outcome in a procedure that is traditionally associated with a high TRM as well as a high risk of relapse. Although publications are few, a review by Song et  al. in 2005 concluded that a number of predictive factors could be utilized, including karyotype, to weigh the risks and benefits of proceeding with alloSCT [43]. Given the unique underlying biology and associated improved clinical outcomes, transplantation should always be considered for younger patients with a matched sibling donor and favorable-risk refractory AML.

9.  Relapsed Acute Myeloid Leukemia Once patients have relapsed, myeloablative therapy with stem cell rescue remains the most durable treatment option regardless of cytogenetic-risk group. AlloSCT with a matched sibling donor is associated with a long-term LFS upwards of 30–40%. Although improved clinical outcomes have been shown in those with increased duration of CR1, superior data when stratified by

Chapter 12  The Role of Transplantation in Favorable-Risk Acute Myeloid Leukemia 

cytogenetic-risk are not available to support its use specifically in the favorablerisk group. Meanwhile, ASCT has been associated with a variable LFS rate of 25–40% [44–46]. However, here cytogenetic-risk group has been shown to be of some prognostic value. In a study by Linker et al. in 2002, the most significant prognostic factor for outcome in their study was cytogenetics, where all seven patients with t(15;17) undergoing ASCT had long-term LFS [47]. These findings were corroborated by a CALGB study of 50 patients with relapsed AML in CR2 who underwent ASCT, where patients with t(15;17) cytogenetic abnormality were found to have a significantly improved event-free survival (EFS) of 74% as compared to all other patients with an EFS of only 22% [48]. While transplantation is generally avoided with APL in CR1, these data support a substantial benefit with ASCT when applied in the salvage setting. Those patients with AML who relapse after myeloablative therapy have a poor prognosis independent of cytogenetic-risk group. The interval from the time of first transplant to relapse remains the most important predictor of outcome. Second transplantation in those with favorable-risk relapsed AML associated with an interval from first transplant to relapse of greater than 7 months could be considered in younger patients with good performance status. As one might expect, this subset represents a highly restricted group.

10.  Acute Myeloid Leukemia in the Elderly AML in the elderly has practically been defined as AML in those patients greater than or equal to 60 years of age, approximately 5–10 years less than the median age at presentation of all AML patients. Outcomes in this subpopulation are largely influenced by either TRM or therapeutic resistance. While increasing age certainly contributes to increased risk of TRM, therapeutic resistance is largely influenced by leukemia-cell cytogenetics [49]. Favorablerisk cytogenetics are uncommon in this patient population, however when present, continue to predict a more favorable outcome [50]. Nevertheless, given the particularly high TRM in this age group, alloSCT has been categorically excluded in the elderly. Although ASCT as a treatment option is not absolutely contraindicated, it would generally represent the exception rather than the rule. Current thinking suggests immunologically mediated graft-versus-leukemia (GVL) may outweigh the role of high-dose chemotherapy as the principal mechanism of an anti-leukemic effect in transplantation [51]. Based upon this philosophy, the use of reduced-intensity conditioning (RIC) SCT has gained increasing popularity. Given the decreased TRM in association with increased anti-leukemic effect, RIC SCT may serve a particular role in the older patient with good risk factors who is generally excluded from alloSCT. Although there remains a paucity of data on this subject, there are studies which endorse its use in the elderly population [52,53]. Mohty et al. reported on the first donor versus no donor analysis of RIC alloSCT in older patients with AML [54]. Although the study included only a limited number of patients, there was a suggestion of an improved LFS in the donor arm. A subsequent study from M.D. Anderson in 2007 also demonstrated a trend toward improved OS; however, this study was similarly limited by low patient enrollment [55]. Subgroup analysis by cytogenetic risk was not available for either study. Despite the observed reduction in TRM and suggestion of survival benefit

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with RIC SCT, one has to be cognizant of other associated morbidities with stem cell transplantation, including risk of GVHD and infection, which remain comparable to traditional allogeneic stem cell transplantation rates. Its use thus far has been focused on the geriatric population. However, it would be of value to evaluate its role specifically in favorable-risk AML as well.

11.  Conclusions The role of transplantation in AML has traditionally been limited to those patients with poor-risk cytogenetic features, clinical features of adverse prognosis including antecedent hematologic disease, and refractory or relapsed leukemia. Identification of karyotype as a prominent prognostic factor has led to re-evaluation of the optimal post-remission strategy, including in particular the role of myeloablative therapy followed by stem cell transplantation. The improved understanding of disease biology in AML has been followed by a shift toward a risk-adapted approach to therapy. Although transplantation in favorable-risk AML has repeatedly been shown to offer significant LFS benefits, these results have previously been tempered by the significant associated TRM. Modern day transplantation technique and supportive care have dramatically improved the TRM, shifting the balance of risk and benefit. Post-remission therapy with ASCT in those patients with favorable-risk AML can be expected to impart an improved rate of LFS superior to intensive consolidation chemotherapy alone. Although survival benefit has not yet been demonstrated, given the improvements in transplant care, this post-remission strategy may be appropriate with careful patient selection. Meanwhile, the data with alloSCT for favorable-risk AML in CR1 are lacking and require continued investigation. It should not be considered part of standard care at this time. An area of ongoing research includes the use of RIC SCT where early observations show a trend toward improved clinical outcomes in the elderly population. Stem cell transplantation has re-emerged as a therapeutic option for favorable-risk AML. Future studies should continue to be designed with an emphasis on the biological heterogeneity that defines AML to facilitate therapeutic advances.

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Chapter 12  The Role of Transplantation in Favorable-Risk Acute Myeloid Leukemia  36. Kim DH, Sohn SK, Kim JG et  al (2005) Parameters for predicting allogeneic PBSCT outcome of acute myeloid leukemia: cytogenetics as presentation versus disease status at transplantation. Ann Hematol 84:25–32 37. Suciu S, Mandeli F, De Witte T et  al (2003) For the EORTC and GIMEMA Leukemia Groups. Allogeneic compared with autologous stem cell transplantation in the treatment of patients younger than 46 years with acute myeloid leukemia (AML) in first complete remission (CR1): an intention-to-treat analysis of the EORTC/GIMEMA AML-10 trial. Blood 102:1232–1240 38. Keating S, de Witte T, Suciu S et  al (1998) The influence of HLA-matched sibling donor availability on treatment outcome for patients with AML: an analysis on the AML 8A study of the EORTC Leukaemia Cooperative Group and GIMEMA. European Organization for Research and Treatment of Cancer. Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto. Br J Haematol 102:1344–1353 39. Visani G, Olivieri A, Malagola M et  al (2006) Consolidation therapy for adult acute myeloid leukemia: a systematic analysis according to evidence based medicine. Leuk Lymphoma 47:1091–1102 40. Yanada M, Matsuo K, Emi N, Naoe T (2005) Efficacy of allogeneic hematopoietic stem cell transplantation depends on cytogenetic risk for acute myeloid leukemia in first disease remission. Cancer 103:1652–1658 41. Cornelissen JJ, van Putten WLJ, Verdonck LF et al (2007) Results of a HOVON/ SAKK donor versus no-donor analysis of myeloablative HLA-identical sibling stem cell transplantation in first remission acute myeloid leukemia in young and middle-aged adults: benefits for whom? Blood 109:3658–3666 42. Jourdan E, Boiron J-M, Dastugue N et al (2005) Early allogeneic stem-cell transplantation for young adults with acute myeloblastic leukemia in first complete remission: an intent-to-treat long-term analysis of the BGMT experience. J Clin Oncol 23:7676–7684 43. Song KW, Lipton J (2005) Is it appropriate to offer allogeneic hematopoietic stem cell transplantation to patients with primary refractory acute myeloid leukemia? Bone Marrow Transplant 36:183–191 44. Meloni G, Vignetti M, Avvisati G et  al (1996) BAVC regimen and autograft for acute myelogenous leukemia in second complete remission. Bone Marrow Transplant 18:693–698 45. Lioure B, Witz F, Deconninck E et  al (1997) Autologous stem cell transplantation (ASCT) represents the best consolidation therapy for acute myelogenous leukemia (AML) in second complete remission (CR): a retrospective study of the GOELAMS group. Blood 90(Suppl 1):384a (Abstr. 1710) 46. Califaretti N, Davidson M, Abraham R et  al (1998) Autologous bone marrow transplant (ABMT) for acute myelogenous leukemia (AML) in second or subsequent remission. Blood 92(Suppl 1):294a (Abstr. 1203) 47. Linker CA, Damon LE, Ries CA et al (2002) Autologous stem cell transplantation for advanced acute myeloid leukemia. Bone Marrow Transplant 29:297–301 48. Linker C, George S, Hurd D et al (2001) Autologous stem cell transplantation for acute myeloid leukemia in second remission-CALGB 9620. Blood 98(Suppl 1): 689a (Abstr. 2881) 49. Estey E (2007) Acute myeloid leukemia and myelodysplastic syndromes in older patients. J Clin Oncol 25:1908–1915 50. Estey E (2006) General approach to, and perspectives on clinical research in, older patients with newly diagnosed acute myeloid leukemia. Semin Hematol 43:89–95 51. Kolb HJ, Schattenberg A, Goldman JM et al (1995) Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients: European Group for Blood and Marrow Transplantation Working Party Chronic Leukemia. Blood 86:2041–2050

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M. Liao and G.J. Schiller 52. Niederwieser D, Gentilini C, Hegebart U et  al (2005) Allogeneic hematopoietic cell transplantation (HCT) following reduced-intensity conditioning in patients with acute leukemias. Crit Rev Oncol Hematol 56:275–281 53. Cornelissen JJ, Löwenberg B (2005) Role of allogeneic stem cell transplantation in current treatment of acute myeloid leukemia. Hematology Am Soc Hematol Educ Program 151–155 54. Mohty M, De Lavallade H, Ladaique P et al (2005) The role of reduced intensity conditioning allogeneic stem cell transplantation in patients with acute myeloid leukemia: a donor vs no donor comparison. Leukemia 19:916–920 55. Estey E, de Lima M, Tibes R et  al (2007) Prospective feasibility analysis of reduced intensity conditioning regimens for hematopoietic stem cell transplantation (HSCT) in elderly patients with acute myeloid leukemia and high-risk myelodysplasia syndrome. Blood 109:1395–1400

Chapter 13 Allogeneic Stem Cell Transplantation for Acute Lymphoblastic Leukaemia in Adults David I. Marks

1.  Introduction The results of all therapies for adults with acute lymphoblastic leukaemia remain disappointing. Five year survival of the 1929 intensively treated patients in the UKALL XII/ECOG 2993 study was 39% [1]. Chemotherapy is toxic and prolonged; this study reported a 12% 2-year non-relapse mortality in patients without donors. There is a feeling that chemotherapy cannot be pushed much further. Younger (<25–30 years) patients are being treated on more intensive “pediatric” protocols with more asparaginase but there are no mature multicenter data available to evaluate the efficacy of this approach. B cell antibodies are being pursued by a number of groups, nelarabine is being trialled upfront for T cell disease and the early experience with forodesine looks promising but increasing the doses or intensity of the standard drugs seems unlikely to produce significant survival benefits. There has been a better definition of adverse risk factors [2] in recent times, enabling us to target patients likely to fail chemotherapy with our most aggressive therapies. The 5-year overall survival of patients with t(4;11) low hypodiploidy/near triploidy or >5 abnormalities were 24, 22 and 28%, respectively making these patients valid targets for more aggressive approaches. In addition, a major German study of MRD at 9 time points in the first year of acute lymphoblastic leukaemia (ALL) therapy, has found that patients with molecular MRD detectable at week 16 have a very poor outcome (12 vs. 66%) [3] making these patients logical candidates for trials of different approaches including upfront allografting (Table 13-1).

2.  Rationale and the Graft-Versus-Leukaemia Effect Allogeneic stem cell transplantation allows considerably higher doses of chemoradiotherapy to be administered enabling a greater initial leukemic cell kill. However, careful examination of the (considerable) evidence reveals an important graft-versus-leukaemia effect. Given that this was the first disease in which a GvL effect was described, and given the multiple studies showing a

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_13, © Springer Science + Business Media, LLC 2003, 2010

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Table 13-1.  Studies comparing sibling allografting in CR1 acute lymphoblastic leukemia with chemotherapy and/or autologous SCT. Author

Patient no

Donor survival

No donor survival (%)

p value

Attal [20]

572

46% at 5 years

31

0.04

Hunaulta [21]

198

75% at 5 years

40

0.027

804

54% at 5 years

44

0.02

b

Goldstone  [1] a

Eligibility for randomisation: age >35, WCC >30, non-T cell, poor-risk cytogenetics, no CR at day 35 b Donor vs. No-donor analysis (chemotherapy only-this arm was superior to autograft)

reduced relapse rate in allograft patients, it is surprising that many haematologists and even transplanters deny or underrate this effect [29, 31]. Harnessing the allogeneic GvL effect and using the positive benefits of acute and chronic GVHD are essential for curing the high-risk patient or patients with positive MRD prior to transplant. Passweg and colleagues showed 10 years ago that patients with acute, chronic, or both acute and chronic GVHD had a 2.5fold reduction in relapse risk on multivariate analysis (RR = 0.40) [4]. Other studies have shown that chronic GVHD has more of an effect than acute GVHD. Some forms of T cell depletion prevent grade II–IV acute GVHD but there may still be a significant incidence of chronic GVHD; this may be a strategy worth further investigation. Further evidence will come from trials of RIC allografting where there is greater reliance on the GVL effect. There are no large-scale mature data available, but the CIBMTR will be analysing the outcome of >200 RIC allografts for ALL in 2009.

3.  Conditioning Regimens There are no randomised studies comparing conditioning regimens and many large studies have missed opportunities to compare regimens. There are very few data on non-TBI-containing regimens and the survival data does not recommend their use. The City of Hope and Stanford groups have a 20 year experience with etoposide (60 mg/kg) and 13.2 Gy of total body irradiation given in 9 fractions and have excellent survival data [5]. Marks and colleagues from the CIBMTR compared this regimen with standard cyclophosphamide and TBI. Cyclophosphamide and 12  Gy of TBI produced markedly inferior survival compared to etoposide-containing or higher-dose TBI regimens [6]. However, if >13 Gy TBI was given, etoposide/ TBI was not superior to cyclophosphamide/TBI. Transplant-related mortality was (perhaps surprisingly) not higher in the etoposide TBI arm although this is undoubtedly a more toxic regimen. The issue of mucositis will be discussed later in this chapter.

4.  Sibling Allografting in First Remission There are methodological difficulties establishing the efficacy of allograft in adults with ALL. A simple comparison of outcome in patients who received an allograft with those who did not would not suffice, as the allograft group

Chapter 13  Allogeneic Stem Cell Transplantation for Acute Lymphoblastic Leukaemia in Adults 

195

are on average fitter and had to survive a certain time in remission in order to have an allograft. To overcome these biases, donor vs. no-donor analyses were devised. These too are flawed in that they may underestimate the potential benefit of allograft as many patients with matched sibling donors do not proceed to allograft and indeed in many it was never the intention to do so. Nonetheless, they have become widely accepted and if they do show an advantage one can be confident that such a difference exists [7]. Sibling allografting has long been established as the therapy of choice in high risk ALL (Hoelzer criteria). Two French studies compared allografting with chemotherapy and autografting, and there were significant differences in overall survival [21]. A meta-analysis support this [30]. The results of the recent very large international ALL study have provided clarity. All patients <50 years, who were fit for transplant and had a matched sibling donor, were eligible to have an allograft. It was a donor vs. no-donor analysis, which is good at avoiding some known biases towards transplant but which may underestimate the positive effect of allograft. Survival was superior in the donor arm compared with patients without a donor. Subsequently, when chemotherapy was shown to be superior to autografting the comparison was with chemotherapy-treated patients but the donor arm was about 10% superior. However, this was not the case in the high-risk group, most of whom where >35 years. The donor arm is 6% better at 5 years but the difference was not significant. There was still excellent protection against relapse (35 vs. 67%) but survival was not improved because of a high non-relapse mortality (29% at 1 year and 39% at 2 years). The investigators concluded that if the allogeneic effect could be harnessed more safely in this group allograft in high-risk patients with ALLl in CR1 might be worth pursuing. The TRM in low-risk (younger) patients was a disappointing 20% at 2 years; improving this should also be the focus of research efforts.

5.  Unrelated Donor SCT in First Remission Encouraging results of sibling allografting and studies showing that UD SCT can produce similar results to sibling allografts for leukaemia have lead to many investigating the role of UD SCT in high-risk CR1 ALL (Table 13-2). Marks and colleagues from the CIBMTR recently described 169 adult patients with a median age of 33 years who underwent UD SCT. One hundred and fifty-seven were at highrisk and 93 had multiple high-risk factors. Overall survival was 40%. Multivariate analysis showed that the following factors affected survival: WCC at diagnosis, HLA mismatch, >8 weeks to CR1 and T cell depletion. This latter finding was a surprise and as there were only Table 13-2.  Studies of the outcome of unrelated donor stem cell transplantation for adult patients with acute lymphoblastic leukemia in CR1. Author

Patient no

Age (years)

Survival (%)

TRM

Grade 2–4 acute/chronic (%)

Marks [22]

169

33

39

42%

50/43

Dahlke [8]

  38

23

44

NK

36/23

Kiehl [7]

  45

29

45

NK

33/NK

NK not known or not specifically stated

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16 patients with T cell depletion (with a variety of techniques) it cannot be regarded as a definite finding. Patel and colleagues have analysed 55 patients (median age 25 years) who had T-depleted UD SCT for high-risk ALL in CR1 mainly using in vivo alemtuzamab (Patel et al. in press). About half of these patients were taken out of UKALL XII to have an “off-protocol” allograft. Survival was an excellent 59% at 3 years and there was a clear plateau with no events after 2 years. The incidences of grade II–IV, grade III–IV and chronic GVHD were 25, 7 and 22%, respectively. It is difficult to compare the two series but the UK series had a low TRM (19%) and good survival, albeit in a younger population. Kiehl and colleagues from Germany reported 97 patients, 87 of whom received TCD, who had a TRM of 31% and grades III–IV acute GVHD in only 15% [8]. On a similar note, Dahlke and colleagues compared sibling and unrelated donor allografts in 38 and 46 patients in CR1, respectively and found that survival was the same in the 2 groups (44 vs. 46%, p = NS) [9]. Unrelated donor SCT clearly has a growing role in this disease. The promising results from (albeit) limited data and the finding that survival is now similar to allografts with sibling donors make it reasonable to perform prospective trials of this therapeutic modality. However, patients should be entered in studies so that we learn how to optimise this procedure and the data do not support the use of unrelated donor SCT as standard therapy for ALL in CR1.

6.  The Role of RIC Allografting We have been slow to investigate this transplant modality in adult ALL. The mistaken notion that the GvL effect was less important in this disease and the view that conditioning regimens had to contain TBI may have led to this attitude. Consequently, it has initially been performed in patients who could not tolerate myeloablative conditioning because of comorbidity or in elderly patients who have little prospect of cure with chemotherapy (Table 13-3). There are no large scale prospective studies of RIC allografting in this disease. The data with us are relatively small retrospective series of patients with various stem cell sources and heterogeneous disease states. The largest series from the European Blood and Marrow Transplantation Group (EBMT), reported in 2008 by Mohty and colleagues describes 97 patients, 70 of whom had died at the time of analysis [10]. Two-thirds received stem cells from sibling Table 13-3.  Studies of the outcome of reduced intensity conditioning allogeneic stem cell transplantation for adult ALL. Author a

Patient no

Sibs/UDs (%)

CR1/CR2/other (%)

Regimens

Survival

Mohty  [9]

97

67/33

29/26/45

Various

21% at 2 year

Martino [23]

27

56/44

15/41/44

Various

31% at 2 year

Massenkeil [24]

 9

NK

NK

Flu/Bu/ATG

40% at 3 year

Forman [25]

22

33/67

48/19/33

Flu/Mel

77% at 1 year

Hamaki [26]

33

20/13

19/0/14

Flu/Bu/ATG

30% EFS 1 year

a

41% Philadelphia positive. Survival was 52% at 2 years if patient was in CR1 Reported transplant-related mortality ranged from 4 to 27% Flu fludarabine, Bu busulfan, ATG antithymocyte globulin, Mel melphalan, UD unrelated donor

Chapter 13  Allogeneic Stem Cell Transplantation for Acute Lymphoblastic Leukaemia in Adults 

donor and one-third from unrelated donors. Survival at 2 years was 21% in the whole group but promisingly exceeded 50% in those with high-risk disease. Patients in CR1 did significantly better with 52% surviving disease free at 2 years. Patients with chronic GVHD had superior OS (RR 0.4, p < 0.01). There were a variety of RIC regimens used (39% had ATG and 24% had a low-dose TBI-containing regimen) but these are not precisely described in the paper. A smaller series using a uniform protocol (fludarabine and melphalan) was described by the City of Hope group using sibling donors in and unrelated donors [11]. Given the disease status and risk status of the group, the TRM was low (10%) and survival of 70% at 1 year was promising. Longer followup is needed.

7.  Allografting for Relapsed Disease Selected series have produced some reasonable results for allografting in second complete remission using sibling or unrelated donors. However, the situation in a multicentre setting is less optimistic. Fielding and colleagues described the outcome of 607 adult ALL patients who relapsed and only 7% of these patients survive median term. Forty-two patients had matched sibling allografts and survival at 3 years was 23%. Even more disappointingly, 65 patients received stem cells from unrelated donors and only 16% survived at 3 years [12]. Only two-thirds of patients who relapse will achieve CR2 and many will relapse before an appropriate donor is found. I strongly recommend doing a preliminary unrelated donor search in all intensively treated adult ALL patients without sibling donors at diagnosis and referral of the patient to a specialist centre the day the patient is found to have relapsed. More studies need to focus on relapsed ALL but finding donors more rapidly is likely to improve outcome. The Fielding study also shows that allografting (our best antileukaemic therapy) cannot currently be reserved for relapse.

8.  Donor Lymphocyte Infusions Although there is a pronounced GvL effect for ALL, DLI have been relatively ineffective therapy when given for overt relapse of ALL post-allograft [28]. This is likely to be due to the rapid pace of the disease. Kolb and Mackinnon reported the follow-up results of an EBMT study. Only 2 out of 22 patients with relapsed ALL responded to DLI without prior chemotherapy [13]. In an American registry study, 2 of 15 patients obtained CR with DLI. The reported 10% response rate may be due to underreporting of unsuccessful cases and most transplanters do not give DLI as sole therapy for relapse. A recent comprehensive review by Tomblyn and Lazarus describes the data from various small studies [14] and lead the authors to conclude that no firm recommendations can be made on the basis of these (limited) data. However, further studies of DLI after induction chemotherapy or as part of a second transplant strategy are warranted [15]. A more promising pre-emptive approach has been followed by German investigators in paediatric patients. Bader and colleagues showed that in 163 patients, who had allografts from a variety of stem cell sources, the development of mixed chimerism (defined as a >5% shift in whole blood chimerism) was in the absence of intervention associated with universal relapse [16].

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Chimerism was monitored very intensively in their study, weekly in the first year. Their intervention was a program of escalating DLI which resulted in about a third of such patients surviving. Acute GVHD (of some grade) were seen in 31% of such patients. This approach deserves evaluation in an adult population. Some RIC regimens result in a high incidence of mixed chimerism and it is standard practice to correct this mixed chimerism with DLI but there are no data available to assess if this approach reduces relapse.

9.  Allografting for Refractory Disease About 10% of adult patients with ALL are refractory to primary chemotherapy and a very small percentage of these patients can be cured by allogeneic SCT either with a sibling or unrelated donor. Similarly, some patients who relapse fail to respond to reinduction chemotherapy. These patients are often extremely unwell due to prolonged neutropenia and severe infections. The author knows of few such patients who have survived and prefers to attempt to achieve remission with novel agents such as clofarabine, nelarabine or monoclonal antibodies prior to a curative allograft.

10.  Haploidentical and Cord Blood Transplantation for High Risk ALL Patients with ALL, who have a low chance of cure with chemotherapy but have no sibling or suitably matched unrelated donor, are candidates for allografts with haploidentical or cord blood stem cells. The data are small scale and heterogeneous, and come from a small number of centres of excellence. Aversa described 62 patients with ALL transplanted in remission who had 25% event-free survival [17]. TRM was substantial but there was no chronic GVHD, so longer-term quality of life was good. Survival data for patients purely with ALL cannot be gleaned from the reports from Henslee Downey’s group in South Carolina but TRM was seen in 15 of 49 patients and acute and chronic GVHD in about 15%. The data for cord blood transplant is limited. Many studies do not separate this disease from other diseases. Rocha on behalf of Eurocord reported 98 patients (34% in CR1) who achieved 36% 2 year survival and a 26% incidence of grade II–IV acute GVHD and 30% incidence of chronic GVHD [18]. The Minneapolis group have some excellent survival data in small number of adults with ALL but these data cannot be used to make decisions for individual patients. Further larger-scale studies are required, and infection and slow engraftment remain major hurdles.

11.  Other Issues and Supportive Care 11.1.  CNS Disease Patients with CNS disease at diagnosis have an inferior outcome to those without CNS disease (29 vs. 39% survival, p  =  0.03). However, they can achieve long-term DFS with a sibling allograft. In the Lazarus study, 11 of

Chapter 13  Allogeneic Stem Cell Transplantation for Acute Lymphoblastic Leukaemia in Adults 

25 such patients remain alive 21–102 months post-allograft. The issue of whether the dose of cranial radiotherapy that is part of TBI is sufficient for the control of CNS disease remains uncertain. CNS prophylaxis is a major issue for RIC allografting and additional therapy (such as post-transplant intrathecal injections) should be contemplated but there is no evidence to inform practice. 11.2.  Palifermin Grade 4 mucositis is a major problem with VP/TBI allografts with cyclosporin and minidose prophylaxis. Typical patients have severe symptoms requiring prolonged narcotic analgesia and frank bleeding from the mouth. This may prevent the delivery the four doses of methotrexate which, in turn, may affect the chance of acute and chronic GVHD. Some investigators have used mycophenolate mofetil but data showing this to be as effective as methotrexate are lacking. Palifermin (keratinocyte growth factor 1), which has level-one evidence after chemotherapy for autologous SCT has been the subject of phase I studies [19]. 11.3.  The Future Allografting for adults for ALL is currently too toxic and the TRM is too high. Exploration of reduced intensity conditioning in clinical trials will determine whether this is a viable approach and will answer the biologic question of how important conditioning is in obtaining a negative MRD status before the effects of an allogeneic GvL effect. It seems likely that patients with positive MRD prior to transplant and those with resistant disease will not be cured by less conditioning. TBI conditioning may be made less toxic by drugs such as palifermin and velafermin and this may improve the outcome. Selecting the right patients for allogeneic SCT is also a major issue. Gene profiling may add to our abilities to discriminate using the existing prognostic factors. Further trials are needed to determine if allografting can overcome the adverse prognostic impact of biologic factors such as a high WCC and adverse cytogenetics. If allografting can do this, the use of allografting will expand if unrelated donor allografting can be safely performed on a multicentre basis. If it can, then there will be exploration of the use of cord blood as a stem cell source but, again, this is mainly performed in certain specialist centres and there are no data to suggest that it can safely be “rolled out” to large number of transplant centres worldwide (Table 13-4).

Table 13-4.  Likely future developments. • Better selection of patients (who will benefit from allograft) • Increased role for reduced intensity allografting, particularly in older patients • Expanded role for alternative donor allografting including cord blood SCT • Recombinant keratinocyte growth factors to mitigate toxicity of TBI

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D. I. Marks

References 1. Goldstone AH, Richards SM, Lazarus HM, Tallman MS, Buck G, Fielding AK, Burnett AK, Chopra R, Wiernik PH, Foroni L, Paietta E, Litzow MR, Marks DI, Durrant J, McMillan A, Franklin IM, Luger S, Ciobanu N, Rowe JM (2008) In adults with standard-risk acute lymphoblastic leukemia, the greatest benefit is achieved from a matched sibling allogeneic transplantation in first complete remission, and an autologous transplantation is less effective than conventional consolidation/ maintenance chemotherapy in all patients: final results of the International ALL Trial (MRC UKALL XII/ECOG E2993). Blood 111:1827–1833 2. Moorman AV, Harrison CJ, Buck GA, Richards SM, Secker-Walker LM, Martineau M, Vance GH, Cherry AM, Higgins RR, Fielding AK, Foroni L, Paietta E, Tallman MS, Litzow MR, Wiernik PH, Rowe JM, Goldstone AH, Dewald GW, Adult Leukaemia Working Party, Medical Research Council/National Cancer Research Institute (2007) Karyotype is an independent prognostic factor in adult acute lymphoblastic leukemia (ALL): Analysis of cytogenetic data from patients treated on the Medical Research Council (MRC) UKALLXII/Eastern Cooperative Oncology Group (ECOG) 2993 trial. Blood 109:3189–3197 3. Brüggemann M, Raff T, Flohr T, Gökbuget N, Nakao M, Droese J, Lüschen S, Pott C, Ritgen M, Scheuring U, Horst HA, Thiel E, Hoelzer D, Bartram CR, Kneba M, German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (2006) Clinical significance of minimal residual disease quantification in adult patients with standard-risk acute lymphoblastic leukemia. Blood 107:1116–1123 4. Passweg JR, Cahn J-Y, Tiberghien P, Vowels MR, Camitta BM, Gale RP, Herzig RH, Hoelzer D, Horowitz MM, Ifrah N, Klein JP, Marks DI, Ramsey NKC, Rowlings PA, Weisdorf DJ, Zhang M-J, Barrett AJ (1998) Graft versus leukaemia effect in T-lineage and cALLa+ (B-lineage) acute lymphoblastic leukaemia. Bone Marrow Transplant 21:153–158 5. Snyder DS, Chao NJ, Amylon MD, Taguchi J, Long GD, Negrin RS, Nademanee AP, O'Donnell MR, Schmidt GM, Stein AS et al (1993) Fractionated total body irradiation and high-dose etoposide as a preparatory regimen for bone marrow transplantation for 99 patients with acute leukemia in first complete remission. Blood 82:2920–2928 6. Marks DI, Aversa F, Lazarus H (2006) Alternative donors transplants for adult acute lymphoblastic leukaemia: A comparison of the three major options. Bone Marrow Transplant 38:467–475 7. Frassoni F (2000) Randomised studies in acute myeloid leukaemia: The double truth. Bone Marrow Transplant 25:471–473 8. Kiehl MG, Kraut L, Schwerdtfeger R et al (2004) Outcome of allogeneic hematopoietic stem-cell transplantation in adult patients with acute lymphoblastic leukemia: No difference in related compared with unrelated transplant in first complete remission. J Clin Oncol 22:2816–2825 9. Dahlke J, Kröger N, Zabelina T, Ayuk F, Fehse N, Wolschke C, Waschke O, Schieder H, Renges H, Krüger W, Kruell A, Hinke A, Erttmann R, Kabisch H, Zander AR (2006) Comparable results in patients with acute lymphoblastic leukemia after related and unrelated stem cell transplantation. Bone Marrow Transplant 37:155–163 10. Mohty M, Labopin M, Tabrizzi R et  al (2008) Reduced intensity conditioning allogeneic stem cell transplantation for adults with acute lymphoblastic leukaemia: A retrospective study of the European BMT group. Haematologica 93:303–306 11. Stein A, O’Donnell M, Parker P, Nademanee A, Falk P, Rosenthal J, Palmer J, Tsai N, Forman S (2007) Reduced-intensity stem cell transplantation for high-risk acute lymphoblastic leukemia. Biol Blood Marrow Transplant 13:134

Chapter 13  Allogeneic Stem Cell Transplantation for Acute Lymphoblastic Leukaemia in Adults  12. Fielding AK, Richards SM, Chopra R, Lazarus HM, Litzow M, Buck G, Durrant IJ, Luger SM, Marks DI, McMillan AK, Tallman MS, Rowe JM, Goldstone AH (2006) Outcome of 609 adults after relapse of acute lymphoblastic leukaemia (ALL); an MRC UKALL12/ECOG 2993 study. Blood 113:4489–96, 2009 13. Kolb H-J, Mackinnon S (2004) Adoptive cellular immunotherapy for treatment or prevention of relapse of hematologic malignancy posttransplant. In: Atkinson K (ed) Chapter 65 in clinical bone marrow and blood stem cell transplantation, vol 3. Cambridge University Press, New York, pp 992–1008 14. Tomblyn M, Lazarus HM (2008) Donor lymphocyte infusions: The long and winding road: How should it be traveled? Bone Marrow Transplant 42:569–78, 2008 15. Shaw BE, Mufti GJ, Mackinnon S, Cavenagh JD, Pearce RM, Towlson KE, Apperley JF, Chakraverty R, Craddock CF, Kazmi MA, Littlewood TJ, Milligan DW, Pagliuca A, Thomson KJ, Marks DI, Russell NH (2008) Outcome of second allogeneic transplants using reduced-intensity conditioning following relapse of haematological malignancy after an initial allogeneic transplant. Bone Marrow Transplant 42:783–9, 2008 16. Bader P, Kreyenberg H, Hoelle W, Dueckers G, Handgretinger R, Lang P, Kremens B, Dilloo D, Sykora KW, Schrappe M, Niemeyer C, Von Stackelberg A, Gruhn B, Henze G, Greil J, Niethammer D, Dietz K, Beck JF, Klingebiel T (2004) 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 22:1696–1705 17. Aversa F (2008) Haploidentical haematopoietic stem cell transplantation for acute leukemia in adults: experience in Europe and the United States. Bone Marrow Transplant 41:473–481 18. Rocha V, Labopin M, Sanz G, Arcese W, Schwerdtfeger R, Bosi A, Jacobsen N, Ruutu T, de Lima M, Finke J, Frassoni F, Gluckman E (2004) Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 351:2276–2285 19. Spielberger R, Stiff P, Bensinger W, Gentile T, Weisdorf D, Kewalramani T, Shea T, Yanovich S, Hansen K, Noga S, McCarty J, LeMaistre CF, Sung EC, Blazar B R, Elhardt D, Chen MG, Emmanouilides C (2004) Palifermin for oral mucositis after intensive therapy for hematologic cancers. N Engl J Med 351:2590–2598 20. Attal M, Blaise D, Marit G, Payen C, Michallet M, Vernant JP, Sauvage C, Troussard X, Nedellec G, Pico J et al (2005) Consolidation treatment of adult acute lymphoblastic leukemia: A prospective, randomized trial comparing allogeneic versus autologous bone marrow transplantation and testing the impact of recombinant interleukin-2 after autologous bone marrow transplantation. BGMT Group. Blood 86:1619–1628 21. Hunault M, Harousseau JL, Delain M, Truchan-Graczyk M, Cahn JY, Witz F, Lamy T, Pignon B, Jouet JP, Garidi R, Caillot D, Berthou C, Guyotat D, Sadoun A, Sotto JJ, Lioure B, Casassus P, Solal-Celigny P, Stalnikiewicz L, Audhuy B, Blanchet O, Baranger L, Béné MC, Ifrah N, GOELAMS (Groupe Ouest-Est des Leucémies Airguës et Maladies du Sang) Group (2004) Better outcome of adult acute lymphoblastic leukemia after early genoidentical allogeneic bone marrow transplantation (BMT) than after late high-dose therapy and autologous BMT: A GOELAMS trial. Blood 104:3028–3037 22. Marks DI, Pérez WS, He W, Zhang MJ, Bishop MR, Bolwell BJ, Bredeson CN, Copelan EA, Gale RP, Gupta V, Hale GA, Isola LM, Jakubowski AA, Keating A, Klumpp TR, Lazarus HM, Liesveld JL, Maziarz RT, McCarthy PL, Sabloff M, Schiller G, Sierra J, Tallman MS, Waller EK, Wiernik PH, Weisdorf DJ (2008) Unrelated donor transplants in adults with Philadelphia-negative acute lymphoblastic leukemia in first complete remission. Blood 112:426–434

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D. I. Marks 23. Martino R, Giralt S, Caballero MD, Mackinnon S, Corradini P, Fernández-Avilés F et  al (2003) Allogeneic hematopoietic stem cell transplantation with reducedintensity conditioning in acute lymphoblastic leukemia: A feasibility study. Haematologica 88:555–560 24. Massenkeil G, Nagy M, Neuburger S, Tamm I, Lutz C et al (2005) Survival after reduced-intensity conditioning is not inferior to standard high-dose conditioning before allogeneic haematopoietic cell transplantation in acute leukemias. Bone Marrow Transplant 36:683–689 25. Marks DI, Forman SJ, Blume KG, Perez WS, Weisdorf DJ, Keating A, Gale RP, Cairo MS, Copelan EA, Horan JT, Lazarus HM, Litzow MR, McCarthy PL, Schultz KR, Smith DD, Trigg ME, Zhang MJ, Horowitz MM (2006) A comparison of cyclophosphamide and total body irradiation with etoposide and total body irradiation as conditioning regimens for patients undergoing sibling allografting for acute lymphoblastic leukemia in first or second complete remissi. Biol Blood Marrow Transplant 12:438–453 26. Hamaki T, Kami M, Kanda Y, Yuji K, Inamoto Y, Kishi Y et al (2005) Reduced intensity stem-cell transplantation for acute lymphoblastic leukemia: A retrospective study of 33 patients. Bone Marrow Tranplant 35:549–556 27. Collins RH, Shpilberg O, Drobyski WR et al (2000) Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 15:433–444 28. Horowitz M, Gale RP, Sondel PM, Goldman JM, Kersey J, Kolb HJ et al (1990) Graft versus leukemia reactions after bone marrow transplantation. Blood 75: 555–562 29. Yanada M, Matsuo K, Suzuki T, Naoe T (2006) Allogeneic hematopoietic stem cell transplantation as part of post-remission therapy improves survival for adult patients with high-risk acute lymphoblastic leukemia: A meta-analysis. Cancer 106:2657–2663 30. Weiden PL, Flournoy N, Thomas ED et  al (1979) Antileukemic effect of graftversus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 300:1068–1073

Chapter 14 Allogeneic Transplantation for Myelodysplastic Syndromes Geoffrey L. Uy and John F. DiPersio

1.  Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders characterized by peripheral cytopenias and marrow dysplasia with a variable propensity to evolve into acute myeloid leukemia. MDS is primarily a disease of the elderly with the median age of 76 years at diagnosis and 86% of patients diagnosed over the age of 60 [1]. Although the true incidence is unknown, reports from Germany and the United States estimate the incidence of MDS to be approximately 3 to 4 per 100,000 [1, 2]. While the majority of cases of MDS arise de novo, exposure to alkylating agents and ionizing radiation during treatment for other conditions are important etiological factors. Compared to de  novo MDS, secondary MDS is associated with higher rates of adverse cytogenetics abnormalities, poor treatment response, and a worse overall prognosis [3, 4]. Currently, allogeneic stem cell transplantation is considered the only curable treatment modality for MDS. However, the advanced age and the associated comorbidities typical for this patient population have limited the utility of this approach for most patients.

2.  Classification and Prognostic Factors in MDS The French–American–British classification of 1982, recognizes five distinct subgroups of MDS: (1) refractory anemia (RA), (2) refractory anemia with ringed sideroblasts (RARS), (3) refractory anemia with excess blasts (RAEB), (4) refractory anemia with excess blasts in transformation (RAEB-T), and (5) chronic myelomonocytic leukemia (CMML). These subtypes are based on morphological features in the bone marrow and peripheral blood including the presence of ringed sideroblasts, bone marrow blasts, and peripheral blood monocytes [5].

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_14, © Springer Science + Business Media, LLC 2003, 2010

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Table 14-1.  Comparison of French American British (FAB) and World Health Organization (WHO) classifications of MDS. FAB category

WHO category

Definition

Refractory anemia (RA)

Refractory anemia (RA)

Dysplasia in erythroid series, <5% blasts in marrow and <1% in blood

Refractory anemia with ringed sideroblasts (RARS)

Refractory anemia with ringed sideroblasts (RARS)

Anemia with ³15% erytroid precursors ringed sideroblasts

Refractory anemia with excess blasts (RAEB)

Refractory anemia with excess blasts-1 (RAEB-1)

5–10% myeloblasts in BM

Refractory anemia with excess blasts-2 (RAEB-2)

10–19% myeloblasts in BM

Refractory cytopenia with multilineage dysplasia (RCMD)

Bi- or pancytopenia with dysplastic changes in ³10% of cells in ³2 cell lines

Myelodysplastic syndrome associated with del(5q)

Isolated del(5q) cytogenetic abnormality, <5% blasts in blood and marrow

Myelodysplastic syndrome, unclassifiable (MDS-U)

No increase in blasts in blood and bone marrow, lacks finding appropriate for other categories

Refractory anemia with excess blasts in transformation (RAEB-t) Chronic myelomonocytic leukemia (CMML)

Reclassified from MDS to: Acute myeloid leukemia with multilineage dysplasia

³20% blasts in blood or marrow and dysplasia in ³2 cell lines

Myelodysplastic/myeloproliferative disease Chronic myelomonocytic leukemia (CMML)

<20% blasts in blood or marrow, peripheral blood monocytosis >1×109/L

Recognizing the importance of both bone marrow blast percentage and cytogenetics in MDS, the World Health Organization (WHO) classifi­ cation was devised in 1997 and has largely replaced the previous FAB scheme (Table  14-1). Important distinctions between the FAB and WHO classifications are the placement of CMML into a distinct MDS/MPD overlap subgroup and the lowering of the bone marrow blast percentage for AML to 20% in what was previously classified as RAEB-t. In addition, two additional categories of MDS were added: refractory cytopenias with multilineage dysplasia and MDS associated with an isolated del(5q) chromosome abnormality because of their unique morphologic and clinical characteristics[6]. The natural history of MDS is highly variable. Cytogenetics and the percentage of bone marrow blasts have been recognized as the principal prognostic factors [7]. A number of scoring systems have been developed to stratify the outcomes of patients [8–12]. The most widely adopted of these systems, the International Prognostic Scoring System (IPSS) incorporates cytogenetics, number of cytopenias, and bone marrow blast percentage to stratify patients into low, intermediate, and high risk groups both in terms of overall survival and evolution to AML (Table 14-2) [9].

Chapter 14  Allogeneic Transplantation for Myelodysplastic Syndromes 

205

Table 14-2.  International prognostic scoring system (IPSS) for MDS. Value Prognostic variable

0

0.5

1

1.5

2

BM blasts (%)

<5

5–10

–

11–20

21–30

Poor

a

Good

Intermediate

Cytopenias b

0/1

2/3

Risk group

IPSS score

Median survival (years)

25% AML evolution (years)

Low

0

5.7

9.4

Intermediate-1

0.5–1.0

3.5

3.3

Intermediate-2

1.5–2.5

1.2

1.1

High

>2.5

0.4

0.2

Karyotype

a

Good normal, –Y, del(5q), del(20q), Poor complex (³3 abnormalities) or chromosome 7 anomalies, Intermediate other abnormalities b  Hemoglobin <10 g/dL, absolute neutrophil count <1,800/mm3, or platelet count <100,000/mm3

Table 14-3.  Selected studies of bone marrow transplantation from HLA-matched siblings for MDS. Study

Year

O’Donnell et al. [16] 1987

Median age Median Preparative in years F/U in Patients regimen (range) months DFS

Relapse

TRM

  20

TBI based (75%)

36 (4–48)

35

35%

20%

45%

17

39%

Bu/Cy (25%) Longmore et al. [15]

1990

  23

Various

23 (3–46)

36

43%

Nevill et al. [18]

1992

  23

Bu/Cy

35 (18–55)

27

35% at 22% at 43% at 3 years 3 years 3 years

Sutton et al. [21]

1996

  71

Cy/TBI (37%)

37 (5–55)

72

32% at 78% at 39% at 7 years 7 years 7 years

Bu/Cy (24%) Runde et al. [22]

1998

131

TBI based (70%)

33 (2–55)

27

34% at 39% at 44% at 5 years 5 years 5 years

Sierra et al. [20]

2002

452

TBI based (44%)

38 (2–64)

49

40% at 23% at 37% at 3 years 3 years 3 years

3.  Early Trials of Hematopoietic Stem Cell Transplantation The potential curative role of allogeneic stem cell transplantation (alloSCT) in MDS was established by a series of single institutional studies conducted in the 1980s demonstrating long term disease free survival in patients following bone marrow transplantation from HLA-identical siblings [13–18]. These results have been confirmed by larger registry studies demonstrating 3 year disease free survivals of approximately 35–40% with a TRM of approximately 40% (Table 14-3) [19–23].

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4.  Impact of Age and Disease State Although MDS is primarily a disease of the elderly, until relatively recently alloSCT was rarely offered to individuals over the age of 55. In initial reports of transplantation for MDS, the median age of patients undergoing transplantation was typically between 30 and 40 years. Throughout these major studies, younger age was associated with improved OS due to decreased TRM [19–21, 24, 25]. In the EBMT series, these differences were most pronounced in patients under the age of 20 who had an improved OS (46% for age £ 20 vs. 33% for age > 40, P = 0.05) attributable to lower TRM (25 vs. 57%, P = 0.01) compared to older patients [19]. With improvements in conditioning regimens and supportive care, transplantation has been available to an increasing number of older patients. Deeg et al. reported the results of 50 older patients (55–66 years) undergoing alloSCT for MDS with a 3 year KM estimate of OS of 59% for RA patients and 46% for RAEB patients demonstrating the feasibility of this approach for older patients [26]. Similar to nontransplant studies of MDS, disease morphology and cytogenetics have been identified as important prognostic variables in the outcome of patients undergoing alloSCT. Patients with either low blast percentage (<5%) or RA/RARS subtypes were associated with improved OS post transplant because of a lower risk of relapse. In the IBMTR series, patients with 5–20% blasts at transplantation had a relative risk of relapse of 2.9 (95% CI 1.7–49, P < 0.001) compared those with <5% blasts [20]. In addition, cytogenetic abnormalities had been found to correlate with disease relapse [20, 27, 28]. Not surprisingly, in a study from the Seattle group of 109 patients, the IPSS score correlated with relapse and DFS with the 3 year DFS of 80% for patients with IPSS of 0 decreasing to 29% in those with IPSS > 2 (Fig. 14-1) [29].

5.  Optimal Timing of Transplantation While immediate transplantation is favored for patients with high risk IPSS scores, due to the high risk of transformation to AML and death, the optimal timing of transplantation for patients with lower risk disease is unknown. Retrospective studies have demonstrated that transplantation early in the disease course for MDS has been associated with improved transplant

Fig. 14-1.  Impact of IPSS score on clinical outcome post allogeneic stem cell transplant. From [29]

Chapter 14  Allogeneic Transplantation for Myelodysplastic Syndromes 

outcomes [22, 25] As outcomes are more favorable in patients with low blast counts, transplantation early in their disease course has been advocated prior to disease progression. In addition, earlier transplantation would allow for less alloimmunization and iron overload from blood transfusions. Armand et  al. studied patients with MDS undergoing myeloablative alloSCT and found that an elevated pre-transplantation serum ferritin level in the highest quartile of patients (>2,515  ng/ml) was strongly associated with lower OS and DFS in patients with MDS attributable to a significant increase in TRM (HR = 3.2, P = 0.02) [30]. However, it is difficult to subject younger patients with low or intermediate risk MDS to the risk of morbidity or mortality from stem cell transplantation as these individuals may also have a benign clinical course in the absence of transplantation [9]. To help answer this question, Cutler et  al. constructed a Markov model based on IPSS score to examine 3 transplantation strategies using HLAidentical sibling donors for newly diagnosed MDS: (1) transplantation at diagnosis, (2) transplantation at leukemic progression, or (3) transplantation during a fixed interval from diagnosis prior to progression. For low and int-1 IPSS groups, delayed transplantation maximized overall survival (9.70 (low), 9.68 years (int-1) vs. 5.17, 2.29 years for delayed vs. immediate transplantation) with the greatest differences predicted in the cohort of patients under age 40. For int-2 or high IPSS groups, transplantation at the time of diagnosis maximized overall survival [31]. In addition, no changes in the optimal transplant strategy were seen when adjustments for quality of life measures were incorporated (Fig.  14-2). Supporting this analysis, a report of the German MDS registry of 232 patients, younger than 50 years with MDS, indicates that survival was significantly longer in patients younger than 50 years (40 vs. 23 months, P < 0.005), primarily driven by the outcome of low, and int-1 risk patients with an overall survival rate of 86% at 20 years for low risk and a median survival 176 months for int-1 patients [32]. Although these studies are informative, they do not account for patients who experience life threatening infections or bleeding from their cytopenias and may benefit from early transplantation regardless of their IPSS score.

Fig. 14-2.  Net benefit or loss of overall discounted life expectancy for allogeneic transplantation in IPSS risk group. From [31]

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6.  Role of Induction Chemotherapy Although induction chemotherapy is universally administered in patients with AML prior to alloSCT, its role in MDS is controversial. Because the rate of relapse, post transplant, increases with increasing blast count, the use of induction chemotherapy prior to alloSCT has been attractive as a means to reduce relapse rates. Compared to de novo AML, aggressive antileukemic therapy in MDS results in lower remission rates and for those who fail induction, a poorer outcome following a subsequent allogeneic transplant [33–35]. In addition, induction chemotherapy may delay potentially curative therapy and any toxicities incurred during induction could preclude an individual from subsequent transplantation. The EBMT compared the outcome of unrelated donor transplantation in patients with MDS in CR1 (n = 136) vs. those who are untreated (n = 167). Overall survival at 3 years was superior in patients who were in CR1 (50 vs. 40%, P = 0.01 with improved 3 year DFS (44 vs. 34%, P = 0.03) and lower TRM [36]. Yakoub-Agha in an analysis of treatment related MDS and AML showed that induction chemotherapy before the graft tended to reduce the risk of relapse (HR, 0.44; P = 0.07) without significantly influencing the risk of TRM (HR, 0.60; P = 0.14). Furthermore, EFS and relapse rates were significantly better for patients who achieved CR, compared with those with active disease at transplant (P = 0.02 and P = 0.002), respectively [24]. In contrast, Sutton et  al. examined the role of induction chemotherapy in de novo MDS. They found that induction was associated with a low rate of CR (35%) and did not reduce the rate of relapse even in those achieving a CR and was associated with worse outcomes [21]. Scott et al. presented the Seattle data of 125 patients with MDS and tAML who received transplants from HLA-identical related or unrelated donors after conditioning with myeloablative conditioning regimens and found no benefit in posttransplant outcome associated with prior induction chemotherapy [37]. Due to their retrospective nature, none of these studies can account for patients who may have received induction chemotherapy but did not undergo subsequent transplant because of toxicity or death during induction. Although prospective randomized trials are required to compare the outcome of these two approaches, these trials are unlikely to be performed. With the introduction of the hypomethylating agents, decitabine and 5¢-azacytidine, responses may be seen in up to 40% of patients with minimal toxicity compared to standard antileukemic induction chemotherapy [38–40]. Preliminary data have suggested the feasibility of hypomethylating agents as induction therapy prior to transplant and may be a reasonable approach in symptomatic patients without an HLA-matched sibling in whom an URD transplant may require 3–4 months to coordinate[41].

7.  Donor and Stem Cell Source Approximately 70% of individuals being evaluated for alloSCT do not have a HLA-matched sibling donor and require an unrelated donor (URD) source of stem cells. Anderson et  al. reported on the outcome of 52 patients with MDS or MDS-related AML treated between 1987 and 1993 with unrelated donor marrow transplantation. The 2-year disease-free

Chapter 14  Allogeneic Transplantation for Myelodysplastic Syndromes 

survival, relapse, and non-relapse mortality rates were 38, 28 and 48%, respectively which compares favorably to series using HLA matched sibling donors [42]. The NMDP has reported outcomes of 510 patients with MDS undergoing URD transplant between 1988 and 1998 with a 2 year disease free survival of 26%. Two year incidence of relapse was 14% with 54% 2 year TRM. [43] The EBMT reported 118 patients who underwent alloBMT from URDs between 1986 and 1996 with an actuarial probability of survival at 2 year of 28%, disease-free survival of 28%, relapse risk of 35%, and transplant-related mortality of 58% [44]. Both the NMDP and EMBT indicate improving outcomes associated with transplantation in recent years suggesting that with improvements in HLA-typing and supportive care, the outcomes of fully matched URDs may approach those observed with related donors [19, 43]. More recently, Deeg et  al. reported the outcome of 109 patients with MDS transplanted from both related and unrelated donors who were conditioned with busulfan and cyclophosphamide and found that donor type had no significant impact on outcome [29]. Cytokine mobilized peripheral blood is increasingly used as an alternative stem cell source to bone marrow in alloSCT. Studies in acute and chronic leukemias have consistently demonstrated higher yields of CD34+ stem cells with improved hematopoietic recovery for PBSC compared to that for bone marrow. In several studies, rates of chronic GVHD appears to be increased in individuals receiving PBSCs which has been attributed to a higher T cell dose in the graft while differences in TRM, relapse risk, and OS are more variable [45–49]. Few studies have addressed the differences in stem cell source specifically for patients with MDS. In a retrospective analysis from the EBMT, in addition to faster hematopoietic recovery, the 2-year estimate EFS of 50 vs. 39% ,favored PBSCs due to a reduction in TRM (RR 0.33, 95% CI 0.15–0.73, P < 0.007) in non RA, and high risk cytogenetics patients [50]. In the only major randomized trial to include patients with MDS, 228 patients (36 with MDS) with myeloid malignancies received either G-CSF-mobilized PBSCs or bone marrow from an HLA-matched sibling following Bu/Cy conditioning. PBSC transplantation was associated with a significantly shorter time to engraftment of neutrophils (by 4 days) and platelets (by 6 days) with no difference observed in the rates of acute or chronic GVHD. Overall survival at 30 months was significantly improved for recipients of PBPCs (68 vs. 60 percent, hazard ratio 0.62, 95% CI: 0.39–0.97) due to a reduction in TRM. Although not powered for subgroup analysis, a trend toward improved OS was observed favoring the use of PBSCs for patients with MDS [49]. An recently completed BMT Clinical Trials Network (CTN) trial will help determine the utility of PBSC vs. bone marrow in URDs. For individuals with MDS without an HLA-matched adult donor, use of umbilical cord blood appears to provide a viable alternative [51–55]. Results of transplantation from haploidentical donors in MDS are currently insufficient to warrant its use outside of the context of a clinical trial.

8.  Conditioning Regimens Early studies of alloSCT primarily used Cy/TBI or Bu/Cy based conditioning regimens for allogeneic transplant. Although no direct comparisons have been made, in an analysis of the NMDP data of URD transplants, use of Bu/Cy

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conditioning regimen was associated with improved DFS with a lower incidence of severe acute GVHD and relapse [43]. Favorable outcomes have been observed in the Seattle data in which 109 patients with MDS (median age 46 years) were transplanted using pharmacokinetically targeted Bu/Cy from both HLAmatched and URDs. The nonrelapse mortality for the entire cohort was 31% at 3 years which compares favorably to studies using TBI based conditioning regimens [29]. A major limitation in the use of HSCT in MDS is the high rates of TRM observed using standard TBI or busulfan based conditioning regimens in older patients. Recognizing that the efficacy of alloHSCT is due in part to the immunologic graft vs. disease effect, reduced intensity and nomyeloablative conditioning regimens have become increasingly popular. These regimens attempt to minimize regimen related toxicity while providing enough immunosuppression to allow donor cell engraftment. A number of single arm phase 2 studies in myeloid malignancies have demonstrated the feasibility of this approach using a wide range of conditioning regimens (Table 14-4) [56–61]. Even among RIC regimens, major differences exist in treatment intensity. A comparison of the outcomes of a reduced intensity regimen of fludarabine and melphalan (FM) vs. a truly nonmyeloblative regimen of fludarabine, cytarabine, and idarubicin (FAI) illustrates these potential differences. FM was significantly associated with a higher degree of donor cell engraftment and lower relapse rates (30 vs. 61%) at the expense of higher treatment-related mortality [62]. The EBMT performed a retrospective comparison of RIC with conventional conditioning for alloHCT from HLA identical siblings in MDS. The 3-year probabilities of progression-free and overall survivals were similar in both groups (39% myeloablative vs. 33% in RIC; P = 0.9; and 45 vs. 41%, respectively; P = 0.8). A lower 3-year NRM was observed after RIC group (HR 0.61, 95% CI, 0.41–0.91, P = 0.015) which was encouraging as patients are frequently selected for RIC regimens on the basis of advanced age and comorbid conditions which preclude conventional myeloablative regimens. Supporting this notion, patients receiving RIC were older (age > 50 years in 73% RIC vs. 28%, P < 0.001) and had more adverse pretransplantation variables [63]. The use of RIC has expanded the pool of patients with MDS who are eligible for alloSCT by reducing the high rates of TRM observed with conventional conditioning regimens. These reductions in TRM, however, appear to come at the expense of increased risk of relapse with the optimal strategy to maximize patient outcomes still undefined.

9.  Transplantation of CMML Due to its inclusion as a subtype of MDS under the FAB classification, the outcomes of patients transplanted with CMML have generally been included in larger series of patients with MDS. A few small series have reported the outcome of patients with CMML separately [64–67]. The largest of these series is from the EBMT which reported the results of 50 allogeneic recipients. The 5-year OS was 21% (95% CI: 15–27%) and the 5-years DFS was 18%. Trends were seen favoring transplantation earlier in the disease course for improved DFS. In addition, a trend toward lower relapse rate was observed in patients with acute GVHD and non-T cell depleted grafts suggesting a graft vs. CMML effect [66].

2006

2004

2005

2003

2007

Hallemeier et al. [56]

Ho et al. [57]

Tauro et al. [58]

Maris et al. [61]

van Besien et al. [59]

52 (11)

89 (21)

76 (20)

62 (39)

51 (51)

CSA (+MTX in URD) CSA

CSA

CSA and MMF FK506

Flu (150 mg/m2), Busulfan, Alemtuzumab (100 mg)

Flu (150 mg/m2), Mel (140 mg/m2), Alemtuzumab (100 mg)

Flu (90 mg/m2), TBI (200 cGy)

Flu (150 mg/m2), Mel (140 mg/m2), Alemtuzumab (100 mg)

GVHD prophylaxis

Cy, TBI (550 cGy)

Preparative regimen

For MDS/MPD subgroup only

a

Flu fludarabine, TBI total body irradiation, Cy cyclophosphamide, mel melphalan

Year

Study

Patients (# with MDS)

52 (17–71)

53

52 (18–71)

53 (22–70)

44 (19–70)

Median age in years (range)

18

13

36

12

46

Median F/U in months

38% at 1 year

25% at 1 yeara

37% at 3 years

62% at 1 year

37%

PFS

Table 14-4.  Selected studies of reduced intensity and nonmyeloablative preparative regimens in MDS.

32%

NR

36%

NR

27%

Relapse

33% at 1 year

17%

19% at 1 year

15% at 1 year

37%

TRM

48% at 1 year

29% at 1 yeara

41% at 3 years

74% at 1 year

OS

Chapter 14  Allogeneic Transplantation for Myelodysplastic Syndromes  211

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10.  Transplantation of JMML Juvenile myelomonocytic leukemia is a rare hematopoietic disorder of early childhood characterized by proliferation of granulocytic and monocytic lineages with frequent erythroid and megakaryocytic abnormalities. JMML accounts for 2–3% of all pediatric leukemias, responds poorly to chemotherapy and can be rapidly fatal [68–72]. While allogeneic transplantation is considered the only curative treatment, published studies report limited number of patients with heterogeneous approaches [73–82]. The largest prospective trial for JMML was conducted by the European Working Group on Childhood MDS and the EBMT which treated 100 patients with alloHCT following conditioning with busulfan, cyclophosphamide, and melphalan. The 5-year EFS was 52% with the KM estimate of OS of 64% with a 13% TRM. Multivariate analysis showed that female sex and age > 4 years at diagnosis predicted for poorer outcomes [83].

11.  Conclusions Allogeneic stem cell transplantation is a potentially curative treatment option for patients with MDS. Patients at high risk for death and disease progression to AML based on IPSS score with high percentage of marrow blasts and adverse cytogenetic abnormalities should be transplanted early in their disease course. For patients who are transfusion independent with low risk disease, transplantation may be deferred until disease progression. Although controversial, either conventional antileukemic induction chemotherapy or the hypomethylating agents, decitabine and 5-azacytidine, may be considered prior to transplantation in patients with high marrow blasts percentages especially if there will be a significant delay until transplantation due to donor availability. Reduced intensity and nonmyeloablative treatment approaches have increased the number of patients who are eligible for transplantation with reduced rates of TRM at the expense of an increased relapse risk with no differences in overall survival.

References   1. Ma X et  al (2007) Myelodysplastic syndromes: Incidence and survival in the United States. Cancer 109(8):1536–1542   2. Aul C, Gattermann N, Schneider W (1992) Age-related incidence and other epidemiological aspects of myelodysplastic syndromes. Br J Haematol 82(2):358–367   3. Estey EH (1998) Prognosis and therapy of secondary myelodysplastic syndromes. Haematologica 83(6):543–549   4. Kantarjian HM et  al (1986) Therapy-related leukemia and myelodysplastic syndrome: clinical, cytogenetic, and prognostic features. J Clin Oncol 4(12):1748– 1757   5. Bennett JM et  al (1982) Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 51(2):189–199   6. Jaffe E et al (2001) World health organization classification of tumours: Pathology and genetics of tumours of haematopoietic and lymphoid tissues. IARC Press, Lyon, France   7. Jacobs RH et al (1986) Prognostic implications of morphology and karyotype in primary myelodysplastic syndromes. Blood 67(6):1765–1772

Chapter 14  Allogeneic Transplantation for Myelodysplastic Syndromes  8. Aul C et  al (1992) Primary myelodysplastic syndromes: analysis of prognostic factors in 235 patients and proposals for an improved scoring system. Leukemia 6(1):52–59 9. Greenberg P et al (1997) International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 89(6):2079–2088 10. Malcovati L et al (2007) Time-dependent prognostic scoring system for predicting survival and leukemic evolution in myelodysplastic syndromes. J Clin Oncol 25(23):3503–3510 11. Mufti GJ et al (1985) Myelodysplastic syndromes: A scoring system with prognostic significance. Br J Haematol 59(3):425–433 12. Sanz GF et al (1989) Two regression models and a scoring system for predicting survival and planning treatment in myelodysplastic syndromes: A multivariate analysis of prognostic factors in 370 patients. Blood 74(1):395–408 13. Appelbaum FR et  al (1987) Treatment of preleukemic syndromes with marrow transplantation. Blood 69(1):92–96 14. Belanger R et  al (1988) Bone marrow transplantation for myelodysplastic syndromes. Br J Haematol 69(1):29–33 15. Longmore G et  al (1990) Bone marrow transplantation for myelodysplasia and secondary acute nonlymphoblastic leukemia. J Clin Oncol 8(10):1707–1714 16. O'Donnell MR et al (1987) Bone marrow transplantation for myelodysplastic and myeloproliferative syndromes. J Clin Oncol 5(11):1822–1826 17. Ratanatharathorn V et  al (1993) Busulfan-based regimens and allogeneic bone marrow transplantation in patients with myelodysplastic syndromes. Blood 81(8):2194–2199 18. Nevill TJ et  al (1992) Treatment of myelodysplastic syndrome with busulfancyclophosphamide conditioning followed by allogeneic BMT. Bone Marrow Transplant 10(5):445–450 19. de Witte T et al (2000) Haematopoietic stem cell transplantation for patients with myelo-dysplastic syndromes and secondary acute myeloid leukaemias: A report on behalf of the Chronic Leukaemia Working Party of the European Group for Blood and Marrow Transplantati. Br J Haematol 110(3):620–630 20. Sierra J et al (2002) Bone marrow transplantation from HLA-identical siblings as treatment for myelodysplasia. Blood 100(6):1997–2004 21. Sutton L et  al (1996) 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 88(1):358–365 22. Runde V et al (1998) 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 21(3):255–261 23. Ratanatharathorn V et al (1992) Allogeneic bone marrow transplantation in highrisk myeloid disorders using busulfan, cytosine arabinoside and cyclophosphamide (BAC). Bone Marrow Transplant 9(1):49–55 24. Yakoub-Agha I et al (2000) Allogeneic bone marrow transplantation for therapyrelated 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 18(5):963–971 25. Anderson JE et al (1993) Allogeneic bone marrow transplantation for 93 patients with myelodysplastic syndrome. Blood 82(2):677–681 26. Deeg HJ et al (2000) Allogeneic and syngeneic marrow transplantation for myelodysplastic syndrome in patients 55 to 66 years of age. Blood 95(4):1188–1194 27. Nevill TJ et  al (1998) Cytogenetic abnormalities in primary myelodysplastic syndrome are highly predictive of outcome after allogeneic bone marrow transplantation. Blood 92(6):1910–1917

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Chapter 14  Allogeneic Transplantation for Myelodysplastic Syndromes  45. Schmitz N et  al (2002) Transplantation of mobilized peripheral blood cells to HLA-identical siblings with standard-risk leukemia. Blood 100(3):761–767 46. Gorin NC et al (2003) 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 102(8):3043–3051 47. Stem Cell Trialists' Collaborative Group (2005) Allogeneic peripheral blood stem-cell compared with bone marrow transplantation in the management of hematologic malignancies: An individual patient data meta-analysis of nine randomized trials. J Clin Oncol 23(22):5074–5087 48. Remberger M et  al (2005) Increased risk of extensive chronic graft-versus-host disease after allogeneic peripheral blood stem cell transplantation using unrelated donors. Blood 105(2):548–551 49. Couban S et  al (2002) A randomized multicenter comparison of bone marrow and peripheral blood in recipients of matched sibling allogeneic transplants for myeloid malignancies. Blood 100(5):1525–1531 50. Guardiola P et  al (2002) Retrospective comparison of bone marrow and granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells for allogeneic stem cell transplantation using HLA identical sibling donors in myelodysplastic syndromes. Blood 99(12):4370–4378 51. Brunstein CG et  al (2007) Umbilical cord blood transplantation after nonmyeloablative conditioning: impact on transplantation outcomes in 110 adults with hematologic disease. Blood 110(8):3064–3070 52. Ooi J et  al (2003) Unrelated cord blood transplantation for adult patients with advanced myelodysplastic syndrome. Blood 101(12):4711–4713 53. Ooi J et al (2001) Unrelated cord blood transplantation for adult patients with myelodysplastic syndrome-related secondary acute myeloid leukaemia. Br J Haematol 114(4):834–836 54. Ooi J et al (2005) Unrelated cord blood transplantation after myeloablative conditioning for adult patients with refractory anemia. Int J Hematol 81(5):424–427 55. Picardi A et al (2004) Unrelated cord blood transplantation for children with high risk myelodysplastic syndromes. Haematologica 89(5):ELT08 56. Hallemeier CL et al (2006) Long-term remissions in patients with myelodysplastic syndrome and secondary acute myelogenous leukemia undergoing allogeneic transplantation following a reduced intensity conditioning regimen of 550 cGy total body irradiation and cyclophos. Biol Blood Marrow Transplant 12(7):749–757 57. Ho AY et  al (2004) Reduced-intensity allogeneic hematopoietic stem cell transplantation for myelodysplastic syndrome and acute myeloid leukemia with multilineage dysplasia using fludarabine, busulphan, and alemtuzumab (FBC) conditioning. Blood 104(6):1616–1623 58. Tauro S et al (2005) Allogeneic stem-cell transplantation using a reduced-intensity conditioning regimen has the capacity to produce durable remissions and longterm disease-free survival in patients with high-risk acute myeloid leukemia and myelodysplasia. J Clin Oncol 23(36):9387–9393 59. van Besien K et al (2005) Fludarabine, melphalan, and alemtuzumab conditioning in adults with standard-risk advanced acute myeloid leukemia and myelodysplastic syndrome. J Clin Oncol 23(24):5728–5738 60. Parker JE et al (2002) Allogeneic stem cell transplantation in the myelodysplastic syndromes: Interim results of outcome following reduced-intensity conditioning compared with standard preparative regimens. Br J Haematol 119(1):144–154 61. Maris MB et  al (2003) HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative conditioning for patients with hematologic malignancies. Blood 102(6):2021–2030 62. de Lima M et  al (2004) Nonablative versus reduced-intensity conditioning regimens in the treatment of acute myeloid leukemia and high-risk myelodysplastic

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Chapter 14  Allogeneic Transplantation for Myelodysplastic Syndromes  82. Yusuf U et  al (2004) Allogeneic bone marrow transplantation in children with myelodysplastic syndrome or juvenile myelomonocytic leukemia: The Seattle experience. Bone Marrow Transplant 33(8):805–814 83. Locatelli F et al (2005) Hematopoietic stem cell transplantation (HSCT) in children with juvenile myelomonocytic leukemia (JMML): Results of the EWOGMDS/EBMT trial. Blood 105(1):410–419

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Chapter 15 Allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Acute Lymphoblastic Leukemia Adriana Balduzzi, Lucia Di Maio, Mary Eapen, and Vanderson Rocha

1.  Introduction Acute lymphoblastic leukemia (ALL) is the most common neoplastic disease in childhood with an incidence of four new diagnoses per year out of 100,000 children below 18 years of age [1–3]. Almost 40  years have passed since the first reports that childhood ALL was no longer incurable [4]. Its prognosis significantly improved over the last decades and important lessons about curing acute leukemia have been learned. The improved outcome of pediatric ALL is one of the greatest successes in modern medicine, with the cure rate having improved from 3% in the 1960s to 80% currently in developed countries [5]. Intensity of induction, rotational polychemotherapy, central nervous system directed treatment additional intensification and maintenance chemotherapy have led to remarkable improvements, despite the fact that the drugs available have not substantially changed over the last 50 years. Very early on, uniform therapy for all patients was replaced in the German Berlin-Frankfurt Munster (BFM) and the United States Clinical Oncology Group (COG) protocols in favor of treatment tailored to prognostic factors, which has allowed for the continued improvements in treatment outcome. These advances are the result of basic research, biologic investigations, large clinical trials and intensive collaboration of cooperative groups [6–8]. The COG recently reported event-free survival (EFS) rates in excess of 90% for standard risk patients defined as age less than 10 years at diagnosis and white blood cell count (WBC) count under 50 × 109/L. Overall, about three quarters of the affected children, at least in developed countries, should now achieve long-term remission and cure after front-line therapy. Unfortunately, the remaining 25% of children relapse despite appropriate treatment, leukemia relapse being the commonest cause of treatment failure [10].

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_15, © Springer Science + Business Media, LLC 2003, 2010

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1.1.  Transplantation: Where Are We Now? Indications for allogeneic hematopoietic stem cell transplantation (HSCT) for childhood ALL has evolved over time due to improvements in treatment outcome with chemotherapeutic regimens as well as better understanding of risk factors, including novel ways to detect minimal residual leukemia and the intensity of therapy tailored to the risk status of a given patient. Transplantation is recommended for only a minority of patients at presentation, commonly defined as “very high risk” (VHR) or “ultra high risk” (UHR) and for the majority of those who experience relapse. Eligibility criteria for HSCT vary according to different chemotherapy protocols; most current protocols identify about 10% of all childhood ALL population presenting with dismal prognostic factors [11]. Much of the philosophy of looking at prognostic factors in childhood leukemia has been aimed at reducing the intensity of therapy in children with low risk ALL and intensifying therapy in those at VHR [12]. For the relatively few VHR patients, only through international collaborations can the relevant therapeutic questions be asked in the context of randomized clinical trials [13]. Pediatric hematologists are now in the difficult position of identifying subsets of patients in whom the intensity of treatment can be lowered to reduce toxicity, and the patient groups who may benefit from intensifying current regimens to improve EFS [13]. HSCT is often used for children with ALL who fail conventional chemotherapy. Crucial roles in transplantation are played by the myeloablative effect of the conditioning regimen and the alloreactivity of donor cells, potentially eradicating residual leukemic cells in the patient. Defining the appropriate use of transplantation is a dynamic process, requiring constant assessment of the likelihood of cure with chemotherapy only, in order to identify the subsets of children who would benefit from transplantation. The role of HSCT is also influenced by changes in the technique of transplantation, mostly the increased availability of unrelated donors worldwide, the higher resolution of HLA typing, allowing better donor–recipient matching and, therefore, improved results and the encouraging results, of other alternative donors such as umbilical cord blood or fully haploidentical HSCT [14–17]. Though improved management of infection and graft-versus-host disease (GvHD) in the current era lessens the risk, death from HSCT-related complications remains higher than with chemotherapy alone. However, disease control appears to be superior after successful transplantation. There is a critical need for an international consensus on identifying those at greatest risk of recurrent disease who consequently benefit from HSCT and conversely those who are less likely to experience leukemia recurrence and for whom chemotherapy alone will achieve long-term cure [11, 15]. Randomized controlled clinical trials have progressively become the gold standard by which treatment choices are made. Each patient is randomized between a new treatment and the perceived standard treatment at trial design and initiation. Oncology, especially pediatric oncology, has par excellence been one of the specialties that have adopted the approach of randomized controlled trials. Faith in such an approach has been reinforced by the unique evidence in children in whom the outcome has been influenced by participation in trials. Even in hands of experienced physicians, it appears that following a protocol,

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation 

including treatment and supportive care, benefits the patient, regardless of the randomization question [13]. In fact, the question surrounding randomization of one treatment option versus another in a trial may not be the most important lesson learnt. The randomization between two doses of the same drug or two drugs may show no relevant differences, but often EFS and survival are superior to previous trials, implying systematic and consistent treatment also play an important role [13]. Nevertheless, to date, the evidence upon which a decision is made to transplant a child with ALL, either in first or in a subsequent remission, has rarely been based on data from randomized trials [13]. In the absence of randomized trials addressing the role of HSCT, there is reliance on data collected by observational databases (e.g., European Blood and Marrow Transplant group [EBMT]) to identify the role of HSCT [16]. Consequently, the co-operatives groups in several countries have established transplantation guidelines for frontline and relapse protocols, based on retrospective comparisons of treatment options (chemotherapy vs. HSCT). Seldom is the decision to offer transplantation at the discretion of the transplant center, often driven by the availability of a suitable donor [16]. In the last decade, results of allogeneic HSCT from unrelated donors has improved to the extent that there is increasing agreement that outcomes after related and matched unrelated donor HSCT in pediatric ALL are comparable [17]. Nevertheless, there is no demonstration that equipoise exists between the two treatments. As such, unrelated donor search activation and transplantation are not recommended for every child for whom an allogeneic transplantation is indicated. Disparities within donor and recipient pairs are progressively accepted as the risk profile of the patient worsens [18]. According to the concepts of the German-BFM, International-BFM (I-BFM) and the EBMT, pediatric patients with high risk frontline or relapsed ALL require additional strategies after polychemotherapy; HSCT shows promising results mainly due to the conditioning regimen and an immunological control by the graft-versus leukemia (GvL) effect, but transplant-related mortality remains an issue. To investigate the role of transplantation the I-BFM Study Group initiated a multicenter, controlled, prospective study enrolling all patients with ALL in first, second, or subsequent remission, at high risk of relapse defined by cytogenetics, biological characteristics, response to chemotherapy, time and site of relapse. The main objective of the study is to investigate whether transplant from a closely HLA-matched unrelated donor is equivalent to transplant from HLA-identical sibling donors [19].

2.  Prognostic Factors in Pediatric Acute Lymphoblastic Leukemia Some children can be identified at diagnosis as being at a higher risk of relapse. The early identification of prognostic variables allow for tailored treatment such that children who are expected to have a very good outcome with modest therapy can be spared the more intensive and toxic treatment, whereas a more aggressive and thus more toxic, therapeutic approach, particularly HSCT, can be offered to those who have a lower probability of long-term survival with conventional therapy [20].

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Childhood ALL is a heterogeneous group of disorders, differing for only partially known molecular genetic abnormalities which lead to different clinical behavior. A number of clinical and laboratory features have demonstrated prognostic value and can be grouped in the following categories: · clinical and laboratory features at diagnosis: age, WBC count, extramedullary involvement · leukemic cell characteristics at diagnosis: morphology, immunophenotype, cytogenetic · response to initial treatment: clearance of leukemic cells in the peripheral blood (PB) or in the bone marrow (BM) (PB response to the 7-day steroid pro-phase, BM response at day 7 and 14, PB response to multiagent induction therapy, minimal residual disease (MRD) after 1- or 2-month induction chemotherapy). The treatment itself is regarded as the most important prognostic factor and consequently all other prognostic factors are treatment dependent. Therefore improvements in therapy may decrease or delete the significance of any of these presumed biologic prognostic factors. 2.1.  Clinical and Laboratory Features at Diagnosis 2.1.1.  Age Young children between 2 and 10 years of age have a better EFS than older children, adolescents, or infants, which is only partially explained by the more frequent occurrence of favorable cytogenetic features in leukemic blasts, hyperdiploidy or TEL-AML1 translocation and the less frequent occurrence of unfavorable features such as the MLL rearrangement (present in 80% of infants) or BCR/ABL translocations (present in 30% of adults) [21–23]. Infants with ALL have a particularly poor outcome, especially if they are younger than 6  months. Infants often present with a very high WBC count, CNS involvement and MLL gene rearrangement [24, 25]. Infant ALL blasts are typically CD10/cALLa negative, express myeloid antigens, high levels of FLT3 and show poor response to the prednisone prophase [26, 27]. The decision to offer HSCT as front-line therapy may be controversial, given these patients are subject to early toxicity from the chemotherapeutic regimen and HSCT and late effects after transplantation can be particularly severe. Induction failure, treatment-related mortality, higher rates of CNS relapse and early overall failure are common in infants with ALL. Overall 5-year EFS rates range between 25% and 40% in those who respond to steroids, as defined by the BFM. In the BFM 1986 study, 19 infants with good steroid response, defined as having less than 1,000 blasts/mm3 in peripheral blood after 7 days of steroids, reported EFS of 53%, compared with EFS of 14% in the 14 patients with poor steroid response. The importance of early response to steroids resulted in change of practice. HSCT as front-line therapy is not offered to infants who are good steroid responders in order to spare them from the higher early toxicity and long-term morbidity and mortality associated with the procedure [11]. Between 1999 and 2005, 22 European and American countries enrolled 482 infants with ALL on the Interfant-99 Protocol. The aims of this international collaboration were to assess the outcome of a hybrid treatment, based on high

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation 

risk BFM-like ALL protocols containing elements usually designed for AML, without irradiation, limited doses of anthracycline and alkylating agents, and to assess the impact of adding a late intensification course. The addition of the late intensification course was the randomized component of the trial. In Interfant-99, response to prednisone was the sole criteria for stratification and HSCT was recommended only for poor steroid responders. In the group of patients who were poor steroid responders, EFS rates in HSCT recipients and nonrecipients were similar after adjustment for time to transplant from remission [28]. The extremely good outcome (4-year EFS of 74%) in infants with germlineMLL has limited HSCT to infants with the MLL rearrangement (recorded in 89% of those for whom the MLL status was known), for whom an overall outcome of approximately 37% was reported, even though improved compared to previous reports [21, 25, 29]. Similarly a dismal prognosis was reported for infants 6-month-old or younger (4-year EFS 34%), with WBC counts 300 × 109/L or higher (4-year EFS 26%), and experiencing a poor response to prednisone prophase (4-year EFS 30%) [28]. The new international collaborative treatment protocol Interfant-06, for infants with ALL or biphenotipic leukemia, is ongoing. Infants presenting without MLL rearrangement are stratified as low risk; infants 6  months or younger presenting with MLL rearrangement and WBC ³300 × 109/L at the onset and/or poor prednisone response are considered high risk; all others are stratified as medium risk. HSCT using a matched related or unrelated donor is recommended to only those considered high risk and those achieving CR. Conditioning regimens consisting of chemotherapy only are indicated for infants, who should always be spared TBI. Busulfan is an established myeloablative alternative to TBI which has shown good results in AML. The recommended conditioning regimen for infants consists of busulfan (total dose 20 mg/kg; 1.25 mg/kg/dose at 6-h intervals for 4 consecutive days), cyclophosphamide (total dose 120 mg/kg; 60 mg/kg/dose per day for 2 consecutive days) and melphalan (140 mg/m2, single dose) [19]. Given the variability in absorption associated with oral administration of busulfan, monitoring blood levels has allowed for optimization of efficacy while reducing toxicity. Parenteral busulfan became available in the last decade. This has overcome problems with bioavailability, the discomfort of oral intake, and, in many cases, the placement of a feeding tube for busulfan administration. Among children, infants experience the worst sequelae in terms of growth after HSCT [25]. Tomizawa described growth-impairment in 59% of the MLL-rearranged group, with a highest incidence of transplant-related mortality [30]. 2.1.2.  Hyperleukocytosis Higher WBC counts represent an increased risk for treatment failure in patients with precursor-B ALL. WBC counts of 50 × 109/L in the United States and 100 × 109/L in Europe are adopted as operational cut-points between better and poorer prognosis strata, although the relationship between WBC count and prognosis is a continuous rather than a step function. Most current ­protocols do not consider WBC counts per se as a criterion for transplantation [20]. The National Cancer Institute (NCI)-risk classified ALL at initial presentation

223

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as standard-risk if age was 1–9 years and WBC count <50,000/ml, and high risk all others. 2.1.3.  Extramedullary Involvement Children with extramedullary disease at diagnosis, mostly in the CNS, occurring in approximately 10% of the childhood ALL population, are at higher risk of relapse, both within the CNS and systemically. Specific therapy directed at the involved extramedullary sites is part of current protocols. With more aggressive initial therapy, the prognostic significance of extramedullary involvement may be overcome. Involvement of the CNS at diagnosis per se is not an indication for transplantation in first complete remission, though an isolated CNS relapse is considered by some as an indication for HSCT (see indications in second CR). 2.1.4.  Down Syndrome Outcome for Down syndrome (DS) children with ALL, almost invariably characterized by B-cell precursor phenotype, and often TEL/AML1 negative, is generally inferior to children without DS [31]. DS per se is not an indication for HSCT. The lower EFS and overall survival (OS) in DS appear to be related to higher rates of treatment-related mortality (TRM), especially during induction therapy, and the absence of favorable biological features [32, 33]. The Children’s Cancer Group (CCG) reported significantly lower 10-year EFS (56% vs. 74%, p < 0.001) for 179 standard risk ALL patients with DS compared to 8,268 children without DS, despite similar prognostic factors in the two populations [34]. Among high risk DS and non-DS patients, EFS rates were similar suggesting intensification is beneficial for DS patients [34]. The Medical Research Council – United Kingdom ALL working party (MRC UKALL WP) – observed lower EFS in DS patients; treatment-related mortality rates were very high, regardless of stratification, and intensification of therapy in these patients may not always be feasible [35]. The Italian AIEOP (Associazione Italiana di Ematologia ed Oncologia Pediatrica) compared treatment outcomes in 120 patients with DS and 6,237 non-DS patients treated between 1982 and 2004. Induction death and leukemia relapse occurred more often than in the non-DS population and 10-year EFS was worse in the DS group but this improved overtime allowing 75% to survive [31]. 2.2.  Leukemic Cell Characteristics at Diagnosis 2.2.1.  Morphology The French-American-British (FAB) morphology classification is not associated with treatment outcome. 2.2.2.  Immunophenotype Precursor B-cell ALL, defined by the expression of cytoplasmic CD79a, CD19, HLA-DR and other B-associated antigens, represents 85% of childhood ALL. Approximately 80% of them express CD10 (cALLa) and have the best prognosis [36]. Two percent of the patients present with mature B-cell leukemia (surface Ig expression, generally with FAB L3 morphology and c-MYC gene translocation)

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation 

also called Burkitt-leukemia, which is a systemic manifestation of Burkitt-like non-Hodgkin lymphoma and should be treated accordingly. This is no longer considered as high risk leukemia. Use of short-term intensive protocols such as those used for B-lymphoblastic lymphoma of childhood achieve EFS of 85%, at least in patients who are t(8;14) (q24;q32) negative after the first chemotherapy cycle [37]. Patients with persistent t(8;14) (q24;q32) positivity may benefit from treatment with anti-CD20 monoclonal antibody and the role of HSCT unclear [38]. Approximately 15% of childhood ALL are precursor T, as defined by the expression of T-cell-associated antigens (cytoplasmic CD3, with CD7 plus CD2 or CD5). Precursor T-ALL is frequently associated with a constellation of clinical features, including male gender, older age, hyperleukocytosis and mediastinal mass. With appropriate intensive therapy, children with T-ALL have outcomes similar to that of children with B-lineage ALL. Recently, Schrauder reported overall 5-year EFS of 51% in 576 patients with T-ALL; 191 of these children had other VHR features which made them eligible for transplantation from a compatible related donor. Thirty-six of 191 underwent HSCT, 26 from an identical sibling and 13 from an alternative donor (8 unrelated and 5 haploidentical). Five-year DFS in the 191 VHR children with T-ALL were 42% after chemotherapy alone, 65% after HLAidentical sibling transplant and 69% after alternative donor transplant [39]. 2.2.3.  Cytogenetics The t(9;22) translocation is present in approximately 3–4% of children with ALL and commoner in older patients and/or those presenting with hyperleukocytosis. The t(9;22), detected either as Philadelphia chromosome at conventional cytogenetics or as BCR/ABL transcript at molecular analysis, confers unfavorable prognosis, especially when associated with either a high WBC count or slow early response to initial therapy [40]. Aricò reported 5-year disease-free survival (DFS) of 25% after chemotherapy alone and 65% after transplantation in 267 of 326 patients achieving CR1 and treated between 1986 and 1996 (recruited by investigators participating in the Ponte di Legno Group, a working group established in 1995 by the AIEOP, BFM, COG and St. Jude Research Hospital) [41]. Forty-two children with t(9;22) were enrolled in the UKALL trials between 1997 and 2002; 28 underwent HSCT. Three-year DFS was 52% after chemotherapy, 45% after HLA-identical sibling transplant and 68% after unrelated donor transplant [42]. In the prospective international study reported by Balduzzi, of the 83 patients presenting with t(9;22), 23 of the 75 patients in the no donor arm were alive in CCR (continuous complete remission) (DFS 26%) versus 4 of the 8 patients in the group with a donor available for HSCT. Of the 75 patients without a donor, 11 were also poor responders to prednisone and none of these patients were alive in CCR [43]. Talano reported a series of 29 children with t(9;22) who underwent transplantation from non-HLA-identical sibling donors with 65% of 5-year DFS [44]. Whether the addition of imatinib or other tyrosine kinase inhibitors to chemotherapy regimens prior to transplantation can further improve DFS is being investigated. Rearrangements involving the MLL (11q23) gene occur in approximately 2–5% of childhood ALL and are associated with increased risk for treatment

225

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failure [21, 25]. The t(4;11) is the most common translocation involving the MLL gene in children with ALL and occurs in approximately 2% of cases (see above) and is an indication for HSCT [29, 45]. 2.3.  Response to Initial Treatment Speed of response is increasingly emerging as the single most important treatment-related prognostic characteristic [46]. Various ways of evaluating the leukemia cell response to treatment have been utilized. · Prednisone poor response (PPR). Very early response after the first week of treatment is defined by peripheral blood response to steroid prophase, in protocols that adopt the steroid prophase, or by bone marrow or peripheral blood response after the weeks 1–2 of multiagent induction therapy. PPR is defined as the presence of 1 × 109 blasts/L or higher in peripheral blood after 7 days of prednisone monotherapy and a single dose of intrathecal methotrexate, as first described within the BFM. Its is presence per se defines patients as high risk for whom and intensified chemotherapy is planned, within BFM-based protocols. When associated with other high risk features, that is, hyperleukocytosis, age younger than 1 year, t(9;22) and T-immunophenotype these patients are defined as VHR. Aricò reported 4-year EFS of 57% in 198 PPR patients enrolled in the AIEOP trial, 176 of whom proceeded with chemotherapy alone and 22 with transplantation (15 from HLA-identical siblings and 12 from an unrelated donor) [47]. PPR per se was not considered an indication for transplantation, unless it was associated with hyperleukocytosis (WBC>100 × 106/L at the onset) or T-immunophenotype. The above criteria no longer hold unless associated with slow response to induction as defined by molecular analysis. · Early multidrug response. In the United States, the CCG and the Pediatric Oncology Group (POG) joined to form the Children’s Oncology Group (COG) in 2000. This merger allowed analysis of clinical, biologic and early response data predictive of EFS in ALL to develop a new classification system and treatment algorithm [9, 36]. Augmented treatment was adopted for high risk and standard risk patients with bone marrow blast count ³25% on day 14. Both standard and high risk patients with a slow early response to prednisone had a 10 point lower 5-year EFS compared to the rapid responders for the day-7 criterion and an 18 point lower 5-year EFS for the day-14 criterion [36]. · Minimal residual disease after induction therapy. In the last decade detection of minimal residual disease (MRD) turned out to be the most sensitive method to evaluate treatment response and one of the strongest predictors of outcome in childhood ALL, to the extent that it overcame most “biological” high risk features, which decrease their predictive value when MRD is included into survival analysis models [9, 48, 49]. The detection by PCR of leukemia-associated clonal rearrangements of T-cell receptor and immunoglobulin heavy chain genes at particular phases of treatment beyond induction is linked to poor prognosis.

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation 

MRD diagnostics using RQ-PCR analysis of immunoglobulin/T-cell receptor gene rearrangements is feasible in multicenter studies but requires standardization and strict guidelines for interpretation of RQ-PCR data along with regular laboratory quality controls. The European Study Group on MRD detection in ALL (ESG-MRD-ALL), consisting of 30 MRD-PCR laboratories worldwide, has developed guidelines for the interpretation of real-time quantitative PCR-based MRD data [50, 51]. Between 2000 and 2004, 3341 patients were enrolled in the multicenter trial AIEOP-BFM ALL 2000 (closed in 2006). They were stratified by MRD detection using quantitative PCR after induction (time point 1) and consolidation treatment (time point 2). MRD-based risk group assignment was feasible in 78% of the patients: 40% were classified as MRD-standard risk (two sensitive targets, MRD negativity at both time points), 8% as MRD-high risk (MRD ³10−3 at time point 2), and the remaining 52% as MRD-intermediate risk. In the previous pilot I-BFM-SG MRD 90 Study conducted on a small group, 10-year EFS were 93%, 74% and 16% in the three groups, respectively [52]. Preliminary analyses of the AIEOP-BFM ALL 2000 confirm the strong prognostic role of MRD; results of the 2000 study are very similar to those of the 90 study for MRD SR and IR groups, but are markedly improved in the HR group (Conter and Schrappe, personal communication). The prognostic role of MRD in day 8 blood or day 29 bone marrow and its relationship to other prognostic variables were assessed by COG. The presence of MRD above 10−3 at day 29 is associated with poor outcome, compared to negative MRD (5-year EFS: 59% vs. 88%). A plethora of studies have documented that gene expression profiling, using DNA microarrays for various types of hematological malignancies, provides novel information, which may have diagnostic and prognostic implications. Searching for genes related to treatment response across different regimens might provide clues about general mechanisms that regulate drug sensitivity in leukemia cells [53]. Whether genes that show independent relationships to treatment response can effectively be incorporated into the existing risk classification is yet to be assessed.

3.  Transplantation in Acute Lymphoblastic Leukemia in First Complete Remission 3.1.  Very High Risk ALL Frontline Treatment Only children carrying very dismal prognostic factors are eligible for HSCT in CR1. They are defined as the VHR or ultra-high risk subset [54, 55]. Patients who fall into the VHR category constitute approximately 8–12% of all children with ALL in most frontline protocols. These patients represent a therapeutic challenge, since remission rates are lower and relapse frequent. Definition of VHR ALL criteria vary across groups and different clinical trials (Table 15-1). Therefore, common efforts are concentrated in finding a consensus on VHR definitions, which is complicated by the fact that the impact of WBC, age, cytogenetics and early response are dependent on frontline chemotherapy protocols [11].

227

Group GATLA

BFM

PINDA

ALL-IC

NOPHO

Country or city

Argentina

Austria, Germany, Switzerland

Chile

Czech Rep., Chile, Uruguay, Hungary, Poland, Israel, Hong-Kong, Croatia, Serbia, Slovakia, Slovenia, Ukraine, Cuba, Moscow

Finland, Norway, Sweden

Next protocol: >5% marrow blasts at day 29, >10−3 MRD at day 79, WBC>200x109/L if no MRD marker, hypodiploid karyotype (<45)

WBC>200, no PR at day 29, 11q23, near-haploidy (modal number <34)

no PR at day 15

t(9;22)

√

√ √

√

√

√

PPR and T-lineage or CD10 negativity or WBC>100 × 109/L or 11q23 rearrangement

√

√

√

no CR at day 33

√

√

√

Infants and age <6 months or WBC >100 × 10 /L or 11q23 Rearrangement or no MRD available

√

√

√

No CR at day 33

√

√

√

PPR and t(9;22) 9

√

√

MRD ³10 at day +33 −2

PPR and T-lineage or CD10- or no PR at day 15 or WBC >100 × 109/L

√

t(9;22) and PPR

√

√

√

no CR at day 78

√

√

√

PPR and t(4;11)

√

√

√

t(9;22)

√

√

√

t(9;22)

√

√

√

MRD ³10−3 at day 78

√

t(4;11)

no PR at day 15 √

√

√

PPR and T-lineage or CD10 negativity or WBC >100 × 109/L or 11q23 rearrangement √

√

√

t(9;22)

MR

IS

Criteria for HSCT in CR1 no CR at day 33

√

√

CB

CB

CB

√

√

√

√

√

√

MUR

√

√

√

√

√

√

MM

Type of Donor Transplantation

Table 15-1.  Eligibility criteria for HSCT in first complete remission in childhood acute lymphoblastic leukemia according to main cooperative groups.

228  A. Balduzzi et al.

√ √ √ √ √ √ √

√ √ √ √ √ √ √

PPR and T or pro-B or WBC >100x10 /L t(4;11) MRD ³1 × 10 at day 78 t(9;22) t(4;11) and PPR no CR at day 33 MRD ³1 × 10 at day 78

COG

USA

√ √

√ √

MLL rearrangement and no CR at day 29 i Amp 21 and no PR at day 7 in HR or 15 in SR or MRD >10−4 at day 29[64]

√ √ √

√ √ √ √

t(9;22) with MRD >10−2 at day 29 Hypodiploid: <44 chromosomes no PR at day 29 −2

no CR or >10 MRD at day 43

√

√

t(9;22)

√

√

√

near haploid and no CR at day 29

√

√

√

no PR at day 29

infant and MLL rearranged and <6 months and WBC >300 or PPR

√

√

t(9;22)

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√*

√*

√*

√

√

√

√

√

√

√

According to center decision

a

IS HLA-identical sibling, MR matched related donor (³ 9/10 matched alleles – 4-digit typing at A,B,C,DRB1,DQB1 loci), MU matched unrelated donor (³ 9/10 matched alleles – 4-digit typing at A,B,C,DRB1,DQB1 loci), MM mismatched donor, includes haploidentical donor and/or partially mismatched unrelated donor (7 or 8/10 matched alleles) and cord blood donor, CB cord blood only, PPR prednisone poor response (³1 × 109/L peripheral blood blasts after 7 day steroid prephase), CR complete remission (M2 marrow: ³5% blasts), PR partial response (M3 marrow: ³25% blasts), MRD minimal residual disease, HR high risk, PB peripheral blood

DCLSG MRC

The Netherlands

United Kingdom

−2

−3

Same as BFM

√

√

MRD ³10−2 at day 35 (semi-quantitative fingerprinting method)

Same as BFM

√

√

Resistance at day 35 (no CR for B-lineage, no PR for T-lineage)

ISPHO

√

√

AUL

Israel

√

√

near haploidy or hypodiploidy

AIEOP

√

√

t(4;11) or any 11q23 rearrangement

Italy

√

√

t(9;22)

9

√

√

PPR and T-lineage or CD10 negativity or WBC>100x109/L

EORTC

France Belgium

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation  229

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A. Balduzzi et al.

3.2.  Eligibility Criteria According to Cooperative Groups At the beginning of the transplant era, prognostic factors in pediatric ALL included male gender, age <1  year or >10  years, high leukemic burden at diagnosis (WBC >50 or 100 WBC × 109/L) and extramedullary involvement (liver, spleen, mediastinum). Subsequently, prognosis has been defined by biological variables: T-immunophenotype, DNA index, chromosome numbers and cytogenetics. Additional features included morphological response after the first 7 days of steroid treatment (peripheral blasts less than 1.0 × 109/L) or marrow response after the first 7 or 14 days of induction treatment (marrow blasts less than 25%). Recently, flow cytometry with or without molecular analysis for MRD after the first 5 and 11 weeks of treatment are regarded to have prognostic significance and are considered for risk assignment. Gene profile microarray analyses are also performed now and may eventually be considered when assigning risk status. VHR childhood ALL, defined by biological characteristics at the onset (high WBC count, T-immunophenotype, t(9;22) and t(4;11) clonal abnormalities) or by resistance to treatment (poor prednisone response, induction failure), yields poor outcome [7, 56–62]. During the last decade, different definitions of HR criteria and different frontline protocols have produced a wide variety of results, with a cure rate ranging from 30% to 60% [56–61]. Eligibility criteria for HSCT as determined by the large cooperative groups are listed in Table 15-1. 3.3.  Results of Transplantation in First Remission Most reports on VHR ALL have compared the two performed treatments (chemotherapy alone or HSCT) in a nonrandomized manner. The data from these studies suggest transplantation from HLA-identical siblings improves the dismal prognosis of childhood VHR ALL, as compared to intensified chemotherapy regimens [64, 65]. In the absence of randomized trials, the results of retrospective reports comparing chemotherapy and transplantation are affected by major biases. First, the selection of patients to be transplanted is influenced not only by the availability of a donor, but also by the risk profile of the patient, as perceived by clinicians during the course of the disease. Second, the waitingtime to transplant causes the transplanted group not to include those patients who do not survive in CR1 long enough to undergo HCT [66]. So comparative studies require careful designs such that patients in both treatment groups are in CR1 and the start time for analysis of outcomes begins at the same time of CR1. It is also important to recognize other factors that may influence the choice of treatment when comparing nonrandomized strategies such as the preference of treating centers, their expertise with HSCT and donor availability [66]. A selection of studies comparing chemotherapy and transplantation outcomes in VHR ALL in CR1 is shown in Table 15-2. Additional data reporting outcome of transplantation in VHR ALL in CR1 are shown in Table 15-3. An international prospective randomized study conducted in seven countries within the International Berlin-Frankfurt-Muenster Study Group (I-BFM-SG) between 1995 and 2000 showed that VHR ALL children in CR1 benefit from related donor HSCT compared to chemotherapy and survival advantage between the treatment strategies increased as the risk profile of the patient worsened. Criteria for HSCT were homogeneously defined as the characteristics

1995–2000

I-BFM-SG Balduzzi [43]

1993–2002

1995-1998

Japan

29

42

326

55

106

191

357

473

Ph+

Ph+

Ph+

Infants

VHR

VHR T

VHR

VHR

 5

 3

 5

 3

 5

 5

 5

10

25

25

92 (MLL-)* 34 (MLL+)*

42

45

39*

(52)*

(46)

(51)

(41)

(38)*

Legend: VHR very high risk, Ph+ Philadelphia positive chromosome, DFS disease-free survival, ITT intention to treat Legenda: *: EFS; ^: any donor; #: autologous

Milwaukee Talano [44]

1987–2002

1986-1996

1997–2002

Roy [42]

I-BFM-SG Aricò [41]

UKALL

Isoyama [25]

PETHEMA Ribera [67]

Schrauder [39] 1990–2000

Wheeler [65]

UKALL

BFM

1985–1997

References

Group

Time point Recruitment Number of of estimaperiod patients Eligibility tion (years) Chemotherapy HSCT

65

45*

65

45

67

63

45* (72)

(57)

(45)*

68*

45

69

34

Identical sibling Unrelated

DFS (ITT)

Table 15-2.  Comparison of chemotherapy versus transplantation outcomes in childhood ALL in first CR.

51

11/19^ (MLL+ only)*

44 [#]

Alternative

10

3.5

7,3

6,5

6,5

 8

Follow-up (years)

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation  231

Klingebiel, unpublished data

Iori [68]

Eapen and Rocha, unpublished data

Rocha, unpublished data

ALWP-PDWP of EBMT

Roma

CIBMTR and EBMT

Eurocord

1998–2000

1988–2000

Rocha, unpublished data

Klingebiel

Locatelli [69]

Bunin [70]

Dini [71]

Eurocord

ALWP-PDWP of EBMT

AIEOP

NMDP

AIEOP

1989–1998

1995–2004

2000–2007

Eapen and Rocha, unpublished data

2000–2006

2000–2007

2000–2006

1995–2004

1993–1996

CIBMTR and EBMT

CR2

Satwani [54]

References

COG

CR1

Group

167

363

63

48

150

207

108

79

30

22

29

CR2

CR2

pre-, post-1998

CR2

CR2

CR2

CR2

25% younger than 2 years

VHR ( 80% Ph+ and t 4;11)

-

HR

3

5

3

2

2

2

2

2

9

2

20* vs. 67*

VHR

67*

59*

infants vs. no infants

5

32*

38; 36*

27, 58

40; 41*

45 (6 or 7/8 HLA, n = 104)

50 (8/8 HLA, n = 103)

56 (6 or 7/8 HLA, n = 37)

73 (8/8 HLA, n = 42)

46*, ˚

25*

26

34 (haploidentical)

43 (CB)

46 (CB)

30 (haploidentical)

DFS (ITT) after transplantation Time point of estimation (years) Identical sibling Unrelated Alternative

Ph+

VHR

Recruitment Number period of patients Eligibility

Table 15-3.  Outcome of HSCT in ALL.

>3.4

1,5-5,3

5

2

2

1.5

2

5

4

Follow-up (years)

Al-Kasim [79]

Gassas [80]

Eapen [81]

Sanders [82]

Houtenbos [83]

Canada

Canada

NMDP

Seattle

Columbia NY

1984–1994

1982–2003

1998–2006

1990–1998

2001–2005

154

2

6

43

rel

42

37 mm-BM

36 mm-CB-LD 45 mm-CB-HD 38 m-BM

60 m-CB;

41*

76

3

5

3

60*

47*

CR2/3

CR1

BM

282

40

CB

CR2

CR1

2 CR3

13 CR2 3

20

8 CR1

10 CR3

70

63*

55*

33*

46

3

3

3

5

8

4

34 CR2

10 CR1

CR3

CR3

Center experience

CR2

CNS

503

73

82

22

88

35

22

77

56

79

Legenda: *: EFS; §: OS; ~: cord blood; °: adjustment for waiting time to transplant and prognostic factors

Sedlacek [78]

Czech

1987–1999

1990–2002

Afify [76]

Woolfrey [77]

1994–2005

1990–1997

1989–1999

Gassas [75]

Seattle

Any phase

Utah

Canada

CR3

Leiden

Willemze [74]

Bleakley [73]

Australia

CR1 + CR2

Tsurusawa [72]

Japan

60*

63*

49*

78§

35*

32

9*

42*; ~

2

3,8

5,8

10

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which had identified those subsets of children reporting a 4-year EFS of 35% or lower after an intensive chemotherapy regimen based on the BFM backbone [7, 57]. These VHR criteria were (1) failure to achieve CR after the four-drug induction, (2) presence of t(9;22) or t(4;11) clonal abnormalities (conventional cytogenetic or molecular testing), (3) poor response to prednisone (PPR), or (4) WBC equal to or greater than 100 × 109/L. Children were assigned to receive chemotherapy in the absence of a matched related donor or HSCT when a matched related donor was available (biologic randomization). The study accrued 357 patients; 280 were randomized to receive chemotherapy alone and 77 HSCT (Fig. 15-1). The 5-year DFS of those without a matched related donor was 41% compared to 57% of those with such a donor (hazard ratio 0.67, p-value 0.02) [43]. However, 18% of patients deviated from their assigned arm; when analyzed by treatment received, after adjusting for waiting time to transplant, the 5-year DFS for the 259 patients who received chemotherapy was 45%, compared to 63% (p-value 0.08) for the 55 patients who received HSCT from a compatible related donor, and to 34% for the 43 undergoing HSCT from an alternative donor [43]. The novelty of this study was the prospective comparison, which relied on both an a-priori restrictive definition of VHR ALL (8% of the overall population), common to all participating groups, and the randomization by genetic chance, that was performed by assigning the treatment on the sole ground of the availability of a suitable donor, which allowed an unbiased analysis by intention to treat. Standard randomization was not feasible in this framework, as HSCT from a compatible

a

b

Fig.  15-1.  (a) Disease-free survival by donor availability (intention to treat analysis) of 357 children with very high risk acute lymphoblastic leukemia enrolled in the I-BFM-SG prospective study comparing intensive chemotherapy versus allogeneic transplantation from compatible related donor. The 5-year estimates are reported with their corresponding standard error in brackets. (b) Disease-free survival according to treatment performed (adjusted for waiting time to transplant) of 357 children with very high risk acute lymphoblastic leukemia enrolled in the I-BFM-SG prospective study comparing intensive chemotherapy versus allogeneic transplantation from compatible related donor. The 5-year estimates are reported with their corresponding standard error in brackets. CHEMO chemotherapy, HCT hematopoietic cell transplantation, RD related donor, AD alternative donor

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation 

related donor was widely regarded as more promising, although no evidence of this had ever been previously provided in a clinical trial. Ancillary analyses, focusing on the subsets defined by the VHR characteristics defined in the protocol, showed that patients with induction failure, regardless of other characteristics, benefited the most from an HSCT compared to other subgroups. When VHR was assigned solely on PPR, associated with T-immunophenotype or hyperleukocytosis, outcomes after chemotherapy alone were excellent. While some have observed DFS rates similar to the IBFM-SG [64, 67, 84], others [65] failed to detect an advantage after HSCT. Different definitions for VHR, relatively small numbers of patients limiting their ability to detect statistical significance, different conditioning regimens and delays between achieving CR and HSCT may explain the observed differences. None of these reports address eligibility for unrelated donor transplants as this option was not considered in the study design. Outcome of unrelated HSCT has markedly improved in recent years, mostly due to higher resolution HLA-typing and improved GVHD management [18, 85]. Donor-recipients pairs historically defined “5 out of 6 matched” or “one-locus mismatched” were likely to carry deeper incompatibilities, due to the lower resolution typing routinely adopted in the past. Current typing of A, B, C, DRB1 and DQB1 loci at the allelic level allowed recent experiences to suggest that “9 out of 10” matched transplants yield comparable outcomes to fully matched “10 out of 10” transplants in malignant diseases. As soon as very high risk features and patients’ eligibility are recognized and a compatible related donor is not available, an unrelated donor search should be promptly initiated within worldwide donor registries. Since donor searches require time, transplantation from unrelated donors may occur later than from a related donor [71]. Therefore, apart from eligibility per se, whenever transplantation is delayed either for lack of a suitably matched donor or for the duration of the search process, the risks and benefits of HSCT should be carefully considered. For example, “does HSCT offer an advantage over chemotherapy for a child with ALL who after induction failure subsequently achieved CR and has had sustained remission for 9 months. The impact on prognosis of the time elapsed in CR1, therefore the potential influence of the “waiting time to transplant,” was quantitatively assessed in the same cohort of children previously reported in the I-BFM-SG trial. The conditional 5-year DFS increased from 43% to 44%, 48%, 52% and 60% in patients who maintained their first remission for at least 3, 6, 9 and 12 months, respectively. The overall outcome was constantly better than that obtained with chemotherapy alone and estimates of DFS after HSCT were invariably 10–20% higher than DFS estimates of patients treated with chemotherapy, who had maintained their first remission for at least 9  months. Therefore the relative advantage of transplantation from matched related donors over chemotherapy in VHR ALL was consistent, regardless of the “waiting time,” which is mostly relevant in transplantation from unrelated donors [86–88]. Unpublished data from the CIBMTR and Acute Leukemia Working Party (ALWP) of EBMT have shown that in 79 ALL children transplanted in CR1, from 2000 to 2006, showed 2-year leukemia-free survival rates of 73% after HLA allele matched (HLA-A, -B, -C, -DRB1,) unrelated donor HSCT (n = 42) and 56% for those transplanted with one or more HLA allele mismatched unrelated donors (n = 37).

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3.4.  Outcomes of Umbilical Cord and Haploidentical HSCT Results of allogeneic HSCT with alternative donor/graft such as umbilical cord blood cells or fully haploidentical HSCT for children in CR1 ALL are scarce in the literature, since reports include children with other diseases and all disease phases. In the Eurocord database, 108 children with ALL in CR1 were transplanted with a single unrelated cord blood unit from 2000 to 2007. Majority of those children had VHR ALL [(82% had poor risk cytogenetics, mostly t(9;21) and t(4;11)]. Median age at UCBT was 5 years (0.2–16), (25% were younger than 2 years) and median follow-up was 17 months. TBI was used in 60%, and ATG/ALG in 80%. Majority of the cord blood graft had one or two HLA disparities. Overall 2-year LFS was 46%; 54% for children younger than 2 years and 42% for those who were older. In a recent unpublished survey of the Acute Leukemia and Pediatric Disease Working parties of EBMT results of fully haplo T-cell depleted peripheral blood (PB) HSCT for 102 children with ALL transplanted in CR1 from 1995 to 2004 showed that results of leukemia-free survival (LFS) were improved when the patients received a higher CD34 cell dose and were transplanted in centers with expertise in haplo transplantation. Two-years LFS was 30% for 22 children with VHR leukemia transplanted in CR1. Any comparison of these results should be done with extreme caution due to the heterogeneity of diseases, the small series and advances in the field of alternative donor transplantation. Importantly, the use of umbilical cord blood or a haploidentical donor ensure a donor will be available for virtually all children with VHR ALL in CR1 who meet criteria for allotransplantation. The choice of the donor will depend on factors related to graft acquisition, urgency of transplant, HLA, cell dose and transplant centre expertise (see donor choice).

4.  Acute Lymphoblastic Leukemia in Second Complete Remission 4.1.  Relapsed ALL Relapsed ALL is the fourth most common diagnosis in pediatric oncology and its outcome is dismal. The NCI-risk classification at initial presentation maintains its predictive value, but the main factors affecting outcome after relapsed ALL in children are site of relapse, duration of first remission and immunophenotype [20, 89–94]. Isolated extramedullary relapse (usually involving the CNS or testis), very late relapse, (i.e., occurring beyond 6–12 months after elective discontinuation of chemotherapy) and B-cell precursor immunophenotype ALL have a better prognosis than relapse involving the bone marrow, early relapse and T-immunophenotype ALL [92]. 4.2.  Eligibility Criteria According to Cooperative Groups The BFM stratifies patients into the four groups S1, S2, S3 and S4 as shown in Table 15-4. The S1 group includes patients with late extramedullary relapse, S2 includes an heterogeneous group of patients with early extramedullary relapse, non-T early combined medullary and extramedullary relapse and late medullary relapses, S3 includes B-lineage early medullary relapse and S4 very

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation 

237

Table 15-4.  Stratification of relapsed acute lymphoblastic leukemia according to the BFM. Non-T

T-lineage

Site of relapse/ Time point after CR1

Extra-BM

BM combined

BM isolated

Extra-BM

BM combined

BM isolated

Very early

S2

S4

S4

S2

S4

S4

Early

S2

S2

S3

S2

S4

S4

Late

S1

S2

S2

S1

S4

S4

BM: bone marrow

early medullary relapse and any T-lineage medullary relapse. A simplified stratification defines S1 patients as low risk, S2 as intermediate risk, and S3 and S4 as high risk. EFS according to the stratification above can be estimated as approximately 70%, 40–50%, 10–20% and 5–10% for S1, S2, S3 and S4, respectively. The BFM ALL Relapse Group (ALL BFM Rez) recently reported 15-year EFS of 30% in 207 with relapsed ALL diagnosed between 1987 and 1990. Five-years EFS of the more recently treated patients within the BFM (1990 and 2000), range between 33% and 49%, with better outcomes associated with strict treatment intensity, that is, shorter time interval between the first and the second induction block [95]. For high risk relapsed ALL the advantage of the immunological effect of an allogeneic graft over chemotherapy has been clearly demonstrated and all cooperative groups consider S3 and S4 patients at such dismal outcome that they are eligible for transplantation from any donor. For patients at low risk of relapse the excellent outcome with chemotherapy alone make them ineligible for HSCT. On the contrary, the treatment of intermediate risk relapsed ALL, that is, S2 patients, is still controversial [96–103]. MRD, a relevant prognostic factor in most frontline protocols, has also been shown to have prognostic significance in relapsed patients and treatment choices were re-directed accordingly [104–107]. In 2001 the retrospective evaluation of MRD in a cohort of 30 relapsed ALL patients, treated within the various ALL Rez BFM protocols, allowed the BFM Group to show the strong prognostic impact of early high-level MRD. Patients whose MRD was <10−3 at day 36 reported a 6-year EFS of 86%, while patients whose MRD was >10−3 at day 36 reported a 6-year EFS of 0%. Thereafter, the BFM group agreed to adopt a MRD-based stratification to direct treatment choices in S2 patients. Patients whose MRD rapidly improves are judged to have a very good outcome (above 65%) with chemotherapy associated with cranial radiotherapy, while when MRD does not promptly decrease after induction chemotherapy allogeneic HSCT (expected outcome <20%) is recommended instead of chemotherapy. In most ongoing relapse protocol, after the BFM, many cooperative groups adopted a MRD-based stratification, so that S2 patients are eligible for transplantation from a compatible donor according to their MRD level after the first month of treatment. The cut-off varies among groups between 10−3, adopted by BFM and COOPRALL, 10−2, adopted by the MRC, and 10−4, which is under investigation for the next European cooperative relapse protocol. Despite still in its planning phase, investigators are considering to enlarge

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eligibility to transplantation by adopting a lower cut-off of MRD (>10−4) after the second induction course to identify patients who may benefit of allogeneic transplantation. Current eligibility criteria for transplantation are reported in Table 15-5 for most cooperative groups. In summary, in the ongoing BFM relapse protocol S2 patients are eligible for transplantation from HLA-identical sibling donors, but transplantation from an unrelated donor is recommended only for those with a high MRD level (>10−3) after the second induction course. The Netherlands and Czech Republic adopt the same criteria as BFM. In the COOPRALL protocol transplantation from HLA-identical sibling is recommended for S2 patients with MRD >10−3 after induction and transplantation from unrelated donor is allowed in most early CNS relapse. In the ongoing AIEOP protocol S2 patients (medullary relapses beyond 48 months after diagnosis are excluded) are eligible for transplantation from either related or unrelated HLA-matched donors, but not from a partially mismatched related donor, unless a high level of MRD is detected after 3 months of treatment. In the United Kingdom early and very early CNS relapsed children are eligible for transplantation from any donor, while S2 patients overall are eligible for transplantation from either related or unrelated HLA-matched donors if the MRD level is >10−2 at day 35. In the United States, S2 patients are eligible for transplantation from HLA-identical sibling donors. Such lack of homogeneity discloses the lack of evidence of the optimal treatment, which would justify a randomized trial. Nevertheless randomization between chemotherapy and transplantation is hardly feasible and likely to fail, as previously demonstrated by other groups in the same or different settings. It is always difficult to counterbalance the advantage of higher efficacy with the drawbacks of early mortality and long-term toxicity. Generally, optimizing strategies aim at ensuring the best available treatment by sparing toxicity without jeopardizing ultimate outcome. It is difficult to find a consensus, particularly regarding treatment of S2 children, but possibly the next cooperative European relapse protocol will match the aim of re-defining the role of transplantation in S2 patients. HSCT sequelae may not be counterbalanced by a potential little improvement in MRD-low patients and its efficacy could even be not sufficient in MRD-high patients. Infertility and increased incidence of endocrinological problems, growth impairment, early cataracts, organ impairment, and second malignancies are to be expected after transplantation compared to chemotherapy, often associated with immunological complications, that is, graft-versus-host disease and infections, due to highly impaired immune-reconstitution. Lower complication rates are expected for not transplanted children, even if their treatment invariably includes cranial irradiation, which also increases the risk of brain tumors [9, 108–111]. 4.3.  Results of Transplantation in Second Remission Two major biases affect retrospective studies in relapsed ALL. First, the selection of patients for transplantation is not only dictated by the availability of a donor, but also influenced by the risk profile of the patient, as perceived by clinicians during the course of the disease. Second, patients not achieving second CR (CR2) or dying while waiting for HSCT are not taken into consideration in the HSCT studies [66].

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation 

239

Table 15-5.  Eligibility criteria for transplantation in second complete remission in childhood acute lymphoblastic leukemia according to main cooperative groups. Type of transplantation Country

Group

Criteria

Argentina

GATLA

Same as BFM

Austria, Germany, BFM Switzerland

IS

MR

MUR

MM

S1 S2 late medullary (isol or comb) with PB blasts <106/L and with MRD <10−3 at day 33 S2 extramedullary CNS or testis unilateral S2 late medullary (isol or comb) with PB blasts <106/L and no MRD at day 33 available

Ö

S2 late medullary (isol or comb) with PB blasts <106/L and with MRD <10−3 at day 33

Ö

Ö

Ö

S2 late medullary (isol or comb) with PB blasts <106/L and with MRD ≥10−3 at day 33

Ö

Ö

Ö

S2 late medullary (isol or comb) with PB blasts >10 × 109/L and no MRD at day 33 available

Ö

Ö

Ö

S2 extramedullary bilateral testis

Ö Ö

Ö Ö

Ö Ö

Ö Ö

S3/S4

Ö

Ö

Ö

Ö

S2 if MRD not available

Ö

Ö

ÖCB

Ö

S3/S4

Ö

Ö

ÖCB

Ö

S2 early combined and with MRD <10−3 at day 33

Chile

PINDA

Czech Rep.

CPH

Same as BFM

Israel

Israel SPHO

Same as BFM

Finland Norway Sweden

NOPHO

*

France

COOPRALL

Non-T CNS female

Belgium

S2 with MRD <10−3

Portugal

S1

Italy

AIEOP

S2 with MRD >10−3

Ö

T early or very early CNS

Ö*

Ö*

Ö*

Non-T very early CNS male, early CNS male >6 years

Ö*

Ö*

Ö*

All S3/S4, t(9;22)

Ö

Ö

Ö

S2 with CR1 duration >48 ms S2

The Netherlands

DCLSG

Ö

Ö

S2 with CR1 duration <48 ms

Ö

Ö

Ö

S2 with MRD >5 × 10−4 at 3 ms

Ö

Ö

Ö

Ö

S3/S4

Ö

Ö

Ö

Ö

Same as BFM (continued)

240 

A. Balduzzi et al.

Table 15-5.  (continued) Type of transplantation Country

Group

Poland

Criteria

IS

MR

B-lineage late medullary with persistent MRD

Ö

Ö

MUR

MM

combined without high MRD level

Ö

Ö

B-lineage early combined

Ö

Ö

Ö

Late medullary with persistent MRD (isolated or combined)

Ö

Ö

Ö

Late combined

Ö

Ö

Ö

T-ALL

Ö

Ö

Ö

Ö

B-lineage very early/early medullary (isolated or combined)

Ö

Ö

Ö

Ö

S2 and MRD >10−2 day 35 RS

Ö

Ö

Ö

S3/S4

Ö

Ö

Ö

Ö

t(9;22) with persistent MRD

United Kingdom

MRC

Ö

Early/very early CNS relapse USA

COG

B-lineage late medullary

Ö

Early isolated extramedullary Any T-lineage medullary

Ö

Ö

Ö

B-lineage early medullary

Ö

Ö

Ö

IS HLA-identical sibling, MR matched related donor (³ 9/10 matched alleles – 4-digit typing at A,B,C,DRB1,DQB1 loci), MU matched unrelated donor (³ 9/10 matched alleles – 4-digit typing at A,B,C,DRB1,DQB1 loci), MM mismatched donor, includes haploidentical donor and/or partially mismatched unrelated donor (7 or 8/10 matched alleles) and cord blood donor, CB cord blood only, PPR prednisone poor response (³1 × 109/L peripheral blood blasts after 7 day steroid prephase), CR complete remission (M2 marrow: ³5% blasts), PR partial response (M3 marrow: ³25% blasts), MRD minimal residual disease, HR high risk, PB peripheral blood * According to center decision

Some investigators retrospectively reported results after chemotherapy and transplantation in ALL in CR2, as listed in Table 15-6, attempting to overcome major biases by adjusting for the waiting time to transplant. Particularly in late relapses, the advantage of HSCT from matched related donors becomes more apparent with each successive year of follow-up, suggesting greater protection against late relapses than with chemotherapy alone in patients who survived the early toxic effects of treatment, so that a long follow-up is needed to account for chemotherapy failure, in a way “diluted overtime.” A selection of studies comparing outcome of chemotherapy and transplantation in ALL in CR2 is shown in Table 15-6. Borgmann and Eapen reported chemotherapy results after matching for most relevant known prognostic variables with the transplanted patients [88, 100].

1995–1998 214

Gaynon 98

COG

30* 26*

Overall

Overall  5 CR2 (76) Isol. BM

Matsuzaki112 1984–1996 117 Ped Blood Cancer, 2005

Rivera142 Cancer, 2005

Japan

St. Jude

 6

 5

22*

17*

(20)

(45)*

(44)*

38^

42*

59*

58

42

45*

30 without TBI (44)*, ˚

52* (31) *, ˚ 47 *; #

(29)

(46)*

(54)*

60 *

30 *

38 #

29 (21)

43*, #

(47)*, ˚

10

>10

9

8

6

5

8

9

60 with TBI

8

8 without TBI

2

Follow-up Alternative (years)

41 with TBI

46§

Unrelated

DFS (ITT) Transplantation Identical sibl.

Legenda: *: EFS; §: OS; ^: any donor; #: autologous; °: adjustment for waiting time to transplant and prognostic factors

1984–1994 106

Matched pair CR2

Borgmann100 1983–2001 162 Blood, 2003

30*

BFM

15

Overall

Einsiedel95 1987–1990 183 JCO, 2005

66

BFM

Matched  8 pair, CNS

Eapen96 Leuk, 2008

1990–2000 209

 5

 5

COG-CIBMTR

JCO, 2006

Overall

1982–1997 231

Harrison101 AnnOn, 2000

MRC CLWP

Early BM

UKALLR1

1991–1995 243 Lawson94 BJH, 2000

UKALL

48*

59

Late  5

23

Matched  8 pair, early

1991–1997 374

39§

Eapen97 Blood, 2006

10

COG-CIBMTR

Eligibility Overall

N

Saarinen141 1981–2001 854 JCO,2006

Period

Time point of estimate (years) Chemotherapy

NOPHO

Group

author journal/ publ year

Table 15-6.  Comparison of chemotherapy versus transplantation outcomes in childhood ALL in second CR.

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242 

A. Balduzzi et al.

Borgmann et  al., in a matched-pair analysis, compared children in CR2 receiving either chemotherapy or unrelated donor HSCT matching for site of relapse and immunophenotype, and, as closely as possible, duration of first remission, age, diagnosis date and peripheral blast cell count at relapse. The study could not show a significant difference in EFS between unrelated-HSCT and chemotherapy in 28 patient pairs with intermediate prognosis (39% vs. 49%, respectively) whereas EFS was significantly different in the 53 pairs with poor prognosis (44% vs. 0%, respectively), showing that unrelated-HSCT provides better EFS for patients with high risk relapsed ALL [100]. The Center of International Blood and Marrow Transplant registry (CIBMTR) and the COG compared outcomes in 188 patients in CR2 enrolled in chemotherapy trials and 186 HLA-matched sibling transplants, treated between 1991 and 1997. The relative efficacy of chemotherapy and transplantation depended on time from diagnosis to first relapse and the transplant conditioning regimen used. For children with early first relapse (<36 months), risk of a second relapse was significantly lower after total body irradiation (TBI)-containing transplant regimens than chemotherapy regimens. In contrast, for children with a late first relapse (>36  months), risks of second relapse were similar after TBI-containing regimens and chemotherapy, confirming the advantage of HLA-matched sibling donor transplantation using a TBI-containing regimen in CR2 for children with ALL after an early relapse [97]. The inclusion of pre-emptive CNS directed therapy as part of frontline treatment for ALL has led to significant decreases in meningeal relapse. Nevertheless, approximately 2–10% of children experience isolated CNS relapse. Outcome after chemotherapy is generally poor, particularly in older males with T-immunophenotype, and variable results are reported after HSCT. Though randomized trials are lacking, there is general agreement that when relapse occurs early (<18 months after diagnosis) the outcome of either treatment is dismal. Thus outcomes of 149 patients enrolled on two recent POG chemotherapy trials between 1990 and 2000 and 60 HLA-matched sibling transplant recipients reported to the CIBMTR during the same period were compared. The overall 5-year EFS was 46% and CR1 duration, site of relapse, age at diagnosis and gender emerged as factors of prognostic significance. The 8-year DFS adjusted for age and duration of CR1 were similar after chemotherapy with irradiation and transplantation (66% and 58%, respectively), suggesting that transplantation did not offer an advantage over an intensified chemotherapy approach with irradiation. A lack of graft-versus-leukemia effect in the CNS, higher transplant-related mortality after HSCT and the excellent EFS with an intensified chemotherapy and irradiation regimen explained why outcomes were similar in the two treatment groups [96]. The MRC reported the experience in the United Kingdom in relapsed ALL of the UKALLR1 study, running in 1991–1995. The overall 5-year EFS was 46% and CR1, site of relapse, age at diagnosis and gender emerged as prognostic factors. Five-year EFS was only 7% for very early medullary relapses, 35% for early medullary, 39% for very early extramedullary, 44% for early extramedullary, 57% for late medullary, and 77% for late extramedullary relapses (S1). For those receiving chemotherapy alone, the 5-year EFS was 48%, for autologous bone marrow transplantation 47%, for unrelated and related donor transplantation 52% and 45%. The groups, however, were not comparable with respect to risk factor profile, since patients were more likely

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation 

to be transplanted if they were at higher risk; therefore direct comparison of EFS is misleading. When adjustment for time to transplant and prognostic factors was used to reduce the effects of biases between treatment groups, no particular treatment resulted of benefit. The planned randomization between autologous transplantation and conventional chemotherapy for patients without a matched allogeneic sibling donor failed with only 9% of those eligible being randomized, which highlights the difficulties in running randomized trials in this setting [91]. The subsequent UKALL R2 MRC study, enrolling 150 relapsed children between 1995 and 2002, reported an overall 5-year EFS of 47%. The duration of first complete remission and immunophenotype, but not sites of relapse, was predictive for survival. According to the BFM relapse stratification, 5-year EFS for standard, intermediate and high risk groups were 92%, 51% and 15%, respectively. In the IR group, those with a very early isolated central nervous system relapse had a dismal outcome [94]. In the Australian analysis by intention to treat, the availability of a matched family donor improved 8-year EFS from 9% to 55% [73]. An AIEOP study retrospectively conducted “by intention to treat” between 1988 and 1998 reported the outcome of 167 consecutive children in CR2 ALL for whom an unrelated donor search was initiated at a median time of 2 months after relapse. A suitable donor was identified for 70 patients at 1 year after search activation before 1995 and 6.5 months after 1995. Further leukemia relapse occurred during the search in 94 children at a median of 4 months after search activation, 36 of whom underwent transplant from unrelated (14) or alternative (22) donors beyond CR2, while 58 died of disease progression. Of 78 patients not experiencing a second relapse, 64 underwent unrelated (46) or alternative (18) donor transplant, while of the 9 proceeding with chemotherapy, only 4 survived. The 3-year DFS for the 167 patients was 15%, and survival 25%, whereas after transplant for the 60 unrelated and 40 alternative donor transplanted children it was 31% and 25%, respectively. In conclusion, a 31% 3-year EFS was reported after unrelated transplant in CR2 ALL, but only 38% of the eligible patients could access the procedure, since relapse during the search jeopardized its feasibility [71]. In 163 patients with very early medullary relapse a 5-year DFS of 20% with chemotherapy and 42% with transplantation from HLA-identical sibling was reported by the COG [98]. In 93 patients experiencing very late relapse at >60 months after diagnosis the AIEOP reported a 40% 5-year DFS [93]. In multivariate analysis, the site of relapse was the only significant predictor of outcome, with patients with isolated bone marrow relapse with 5-year EFS of 25% compared with those with combined or isolated extramedullary relapse (5-year EFS 51% and 68%, respectively). All seven patients undergoing an allogeneic bone marrow transplantation from a matched related donor are alive in CR2 [93]. A Japanese study reported a 25% EFS in 90 relapsed patients achieving CR2. The significant prognostic factors identified by a multivariate analysis included the time of relapse (EFS: early 16%, intermediate 24%, late 35%) and the treatment after relapse (EFS after transplantation 30%, chemotherapy 22%) [112]. The NMDP retrospective study reviewed the outcomes of 363 children in CR2 ALL receiving unrelated donor transplant from 1988 to 2000. DFS at

243

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A. Balduzzi et al.

5 years was 36% overall and was significantly worse for patients 15 years or older. Acute GVHD developed in 71% of the patients, with 29% having grades III-IV and the incidence of chronic GVH disease was 39%; TRM was 42% and was associated with HLA mismatches, age 15 years and older, and early relapse [70]. The AIEOP described a significant improvement of outcome after unrelated donor transplant in ALL in CR2 from a 3-year EFS of 27% before 1998 to 58% after 1998, suggesting that outcome after transplantation from unrelated donors is becoming more and more similar to the outcome after HLA-identical siblings, as expected with higher resolution HLA typing and better donor recipient matching [69]. Recently, an unpublished collaborative survey on behalf of CIBMTR and EBMT has shown that in 207 ALL children transplanted in CR2, from 2000 to 2006, 2 years-LFS of HLA allele matched (HLA-A, -B, C-, DRB1, or 8/8) unrelated HSCT (n = 103) was 50% and it was 45% for those transplanted with one or two HLA allele mismatched unrelated HSCT (n = 104). (Table 15-3) 4.4.  Outcomes of Umbilical Cord and Haploidentical HSCT In the Eurocord database, 150 children with ALL in CR2 were transplanted with a single unrelated cord blood unit from 2000 to 2007. Two-year LFS was 43%. In a risk factor analysis the most important factor associated with LFS was the time from CR1 to CR2. The 2-year LFS was 45% for those patients relapsing 24 months after CR1 or later compared to 26% for those with an earlier relapse (unpublished data). In a recent survey of the Acute Leukemia and Pediatric Disease Working Parties of EBMT, 48 children with ALL were transplanted in CR2 with a fully haploidentical T-cell depleted PB. At 3 years, LFS, relapse and nonrelapse mortality were 34%, 36% and 60%, respectively. As already stated for patients in CR1, any attempt to compare outcomes between haploidentical and cord blood HSCT for patients in CR2 should be cautious given the heterogeneity of disease, the small series of patients, and developments in the field of alternative donor transplantation. The choice of the donor depends on factors not only related to the patients, but also to the expertise of each transplant centers (Table 15-7). 4.5.  Acute Lymphoblastic Leukemia in Advanced Phase All working groups agree on the eligibility for transplantation of all CR3 ALL from any donor, even though their risk of subsequent relapse is very high. Furthermore, transplantation in CR3 is associated with a high risk of transplant-related mortality, due to the pre-existing high cumulative organ toxicity [75–77]. Given the poor outcome of a second relapse, new strategies are required to identify those patients who will most benefit from stem cell transplantation already in CR2, particularly in the intermediate, most heterogeneous risk group. Findings in transplantation in CR3 ALL are reported in Table 15-3. The prognosis of relapse after transplant is severe, with the possibility to rescue fewer than 5–10%, as reported in limited series, mixed with adults and other diseases [113]. New experimental approaches and cell therapy may be applied for in these subset of patients [114].

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation 

Table 15-7.  Advantages and limitations of unrelated donor BMT, UCBT and haploidentical HSCT from a relative, and criteria to be considered for choosing an alternative donor for patients without an HLA-identical sibling. UBMT

UCBT

Haplo-HSCT

A, B, DRB1 molecular typing availability

16–56%

~80%

100%

Median search duration

3–6 months

<1 month

Nil

Unavailability of identified donors

20–30%

~1%

None

Rare haplotypes representation

2–10%

20%

None

Main limiting factor to graft acquisition

HLA compa­ tibility

Cell dose

Poor mobilization

Transplant date planning

Difficult

Easy

Easy

Potential for immunotherapy

Yes

No

Yes

Potential for viral transmission to recipient

Yes

No

Yes

Potential for unknown conge­ nital disease transmission to recipient

No

Yes

No

Risk for the donor

Very low

No

Very low

Main open clinical issues

GvHD

Graft failure, delayed immune recovery

Delayed immune recovery, lack of T-cell mediated GVL effect

*Modified from reference [17].

5.  Factors Related to HSCT Procedure 5.1.  Stem Cell Source: Bone Marrow, Peripheral Blood and Cord Blood The recommended source of stem cells is bone marrow and no randomized trials have been conducted in pediatric Acute Lymphoblastic Leukemia between bone marrow (BM) and G-CSF mobilized peripheral blood stem cells transplants (PBSCT) from HLA-identical sibling donors. However, in a retrospective-based registry analysis, Eapen, on behalf of CIBMTR, compared 143 children with acute leukemia receiving PBSCT with 630 BMT. Hematopoietic recovery was faster after PBSCT, risks of grade 2 to 4 acute GVHD were similar, but chronic GVHD risk was higher after PBSCT. In contrast to reports in adults, treatment-related mortality, treatment failure and mortality were higher after PBSCT. Risks of relapse were similar. These data suggest poorer outcomes after PBSC compared with BM transplantation, therefore in the absence of prospective trials, use of PBSC in this setting should be avoided [115]. In the unrelated transplant setting, only a recent unpublished collaborative study has addressed the question. The CIBMTR, in collaboration with the EBMT, has compared 385 unrelated BMT with 110 unrelated PBSCT in children with acute leukemia. Children transplanted with PBSC had higher risk of chronic GVHD but not different outcomes such as acute GVHD, NRM, relapse or LFS compared to BMT. In this study the follow-up was still short to detect an effect of PBSC and chronic GVHD on late outcomes. In order to

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circumvent the problem of the chronic GVHD with PBSC, one could suggest a graft T-cell depletion. However, there is a consensus that the graft should not undergo T-cell depletion, unless in case of mismatched/haploidentical donor [116, 117]. In the last decade, HLA-mismatched umbilical cord blood cells have been used as an alternative source of stem cells for allogeneic HSCT in the unrelated transplant setting for children and young adolescents (CIBMTR unpublished data). Outcomes of unrelated cord blood transplants (UCBT) have been compared to HLA antigen and allele matched bone marrow in many retrospective studies. In summary, neutrophils recovery is delayed, acute GVHD decreased and LFS not different from HLA-matched unrelated BMT if the cord blood graft contains higher numbers of nucleated cells and not more than two HLA disparities [17, 81]. Specific analyses of outcomes after UCBT in children with ALL are missing in the literature. Recently, the Eurocord group has analyzed 323 children with ALL receiving a single unit UCBT. Seventysix children were transplanted in CR1, 136 in CR2 and 111 in more advanced phases of the disease. Among these children poor cytogenetics were observed in 89% of children in CR1, 33% in CR2 and 42% in advanced phases. Twenty percent of children transplanted in advanced phases had been previously autografted. The median age was 6.5 years at UCBT, median cell dose infused was 4.1 × 107/kg and the median follow-up time was 22 months (3–96). The cord blood was HLA 5/6 in 46%, 4/6 in 39%. All children received myeloablative conditioning regimen (TBI in 66%). Cumulative incidence of neutrophil recovery at day 60, acute (grade II-IV) and chronic GVHD were 76%, 42%, 14%, respectively. Overall 2-year LFS was 42% for patients transplanted in CR1, 41% for those in CR2 and 24% for those transplanted in an advanced phase. In a multivariate analysis, only CR1 or CR2 were associated with better LFS (HR = 1.8; p < 0.0001). In a risk factor analysis for patients transplanted in CR2 only shorter time from diagnosis to first relapse unfavorably affected LFS, which was 45% for children relapsing after 2 years of treatment compared to 26% of those presenting with earlier relapse [118]. 5.2.  Donor Choice: Unrelated, Cord Blood or Haplo Hematopoietic Stem Cells? The use of alternative HSC donors has witnessed significant progress, mainly due to (1) better HLA matching at the allelic level between the unrelated donor and recipient translating into better patient outcome; (2) better donor choice and patient selection in unrelated, often HLA-mismatched, cord blood transplantation; and (3) new strategies of adoptive cell therapy, aimed at improving results of T-cell depleted haploidentical HSCT from a relative. Currently, it is possible to find a donor for virtually almost all children with an indication for allogeneic HSCT as long as stem cell source and degree of matching are considered appropriate for the risk profile of the patient. When an HLAidentical sibling is not available, as it occurs in 3 out of 4 children in need of a HSCT [43], one of the three options of HSCT from alternative donor/graft is available. Importantly, each of these options has advantages and limitations. Therefore, any physician considering alternative donor has to carefully evaluate all options, for each pediatric patient in need of an allograft, in order to choose the best donor, taking into account the urgency of transplantation,

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation 

donor characteristics and center experience. Advantages and limitations of each of the three options and main criteria to choose an alternative donor for pediatric patients lacking an HLA-identical sibling is not available are presented in Table 15-7 [17]. 5.3.  UCBT Compared to BMT from Unrelated Donors Three published reports, namely two single centre studies and a Eurocord registry analysis, have compared the outcome of UCBT and UBMT in children, most of them with acute leukemia [119–121]. Briefly, in these three studies, recipients of UCBT were transplanted earlier, had delayed neutrophil and platelet recovery and reduced incidence of acute GvHD as compared to children given a UBMT. Nevertheless, the overall survival (OS) probability was not significantly different in UCBT or UBMT pediatric recipients [119–121]. Recently, a meta-analysis combining these comparative studies was published [122]. A total of 161 children undergoing UCBT (mostly one or two antigenmismatched) and 316 children undergoing UBMT (HLA defined as low resolution typing of HLA-A and -B and high-resolution typing of HLA-DRB1) were analyzed; this study could detect no differences in 2-year OS between children given an unrelated UCBT or UBMT. In a more recent analysis, Eapen et al. [81] have compared results observed in 503 UCBT recipients with those of 282 UBMT recipients [116 were HLA allele-matched (HLA-A, -B, -C and DRB1, thus 8 out of 8) and 166 mismatched]. Of the UCBT recipients, 35 were matched at the HLA A, B (antigen-level) and DRB1 (allele-level), 201 were mismatched at 1-locus and 267 were mismatched at 2-loci. All patients (aged <16 years) had acute leukemia. In comparison with children given an allele-matched UBMT, patients transplanted with 1 or 2 HLA-disparate UCB unit had a similar 5-year DFS (45% for patients given a 1-antigen disparate UCBT with a cell dose greater than 3 × 107/kg nucleated cells, decreasing to 36% with a lower cell dose, and 33% for patients given a 2-antigen disparate UCB vs. 38% in allelic-matched UBMT), while an even possible better outcome was evident for the 35 children given HLA-matched UCBT, the 5-year DFS being 60%. TRM rates were higher after transplants of 2-antigen HLAmismatched UCB (relative risk 2.31, p = 0.0003) and possibly after 1-antigen HLA-mismatched UCB units containing less than 3 × 107/kg nucleated cells (1.88, p = 0.0455). By contrast, the incidence of relapse was lower after 2-antigen HLA-mismatched UCBT (0.54, p = 0.0045). These data support the use of HLA-matched and 1- or 2-loci HLA-mismatched UCB in children with acute leukemia when a good cell dose is available [123]. 5.4.  Comparison of Unrelated Cord Blood with Haploidentical Transplantation in Children with Acute Lymphoblastic Leukemia The Eurocord group, in collaboration with the ALWP and the PDWP of the EBMT, has compared the outcome of patients given either UCBT or Haplo-HSCT, by performing a retrospective comparison of pediatric patients (16 years or younger) with high risk ALL [124]. Children had received either Haplo-HSCT (n = 118) or UCBT (n = 341) in EBMT-Eurocord centers between 1998 and 2004. Haplo recipients tended to be older and to have CMV positive serology and t(9;22) more frequently. The median follow-up was 56 months and 24 months for Haplo-HSCT and UCBT patients, respectively. Failure of

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engraftment was significantly higher following UCBT than after Haplo-HSCT (23% vs. 11%, p = 0.007). In multivariate analysis, adjusted for differences between groups and prognostic factors, relapse incidence was higher in HaploHSCT recipients compared to UCBT (relative risk = 1.7, p = 0.01), but TRM and DFS were not significantly different. In conclusion, in pediatric patients with ALL, UCBT is associated with inferior rate of engraftment, higher incidence of grade II-IV acute GvHD and lower incidence of relapse compared to Haplo-HSCT; however, there was no difference in terms of TRM and DFS. Therefore, in the absence of an HLA-identical donor, both strategies are suitable options to treat a child with high risk ALL. 5.5.  Transplantation Procedure: Conditioning Regimen Fully myeloablative conditioning regimens are recommended, while reduced toxicity or reduced intensity conditionings are to be limited to patients undergoing second transplant or with contraindication to full-intensity conditionings due to comorbidities. The conditioning regimen has a significant impact on survival and incidence of relapse. While studies in acute myeloid leukaemia generally indicated comparable antileukemic efficacy of TBI and busulfan, specific ALL data showed a significant benefit of TBI-containing conditioning regimens, therefore TBI is highly recommended in ALL [125–127]. In 1991 in a retrospective study Dopfer showed the superiority of TBI associated with etoposide as a single drug to other conditioning regimens [99]. In the I-BFM transplantation prospective study, the transplantation is planned to be performed within a recommended time frame of at least 2  months after achievement of remission and within 5 months after diagnosis. GVHD prophylaxis consists of cyclosporine 3 mg/kg/day and the conditioning regimen consists of hyper-fractionated total body irradiation (2 Gy bid on days −6, −5, −4) and etoposide (60 mg/kg i.v. on day −3) for patients older than 2 years, and busulphan (5 mg/kg/day p.o. on days −8, −7, −6, −5), cyclophosphamide (60 mg/kg/day i.v. on days −3, −2) and melphalan (140 mg/mq i.v. on day −1) for patients younger than 2 years. The Nordic Bone Marrow Transplantation Group reported a randomized study comparing cyclophosphamide plus either busulfan or TBI and were able to show an advantage of TBI in terms of early toxicity and mortality reduction and a trend for a lower relapse-free survival (50% vs. 36%) [128]. Davies retrospectively compared two cohorts of ALL children transplanted from HLA-identical siblings reported to the IBMTR who received a TBI- or non-TBI-containing conditioning regimen. This latter may be associated with fewer late effects and is logistically easier to administer. The 451 children conditioned with cyclophosphamide and TBI versus the 176 conditioned with BU/CY at 3 years reported higher survival (55% vs. 40%,) and leukemia-free survival (50% vs. 35%), and lower TRM (15% vs. 23%) and a trend for a lower cumulative incidence of relapse (35% vs. 41%) [14]. Other conditionings containing TBI, either associated with cyclophosphamide as a single drug or in association with other drugs, are commonly adopted. The AIEOP reported good results with the conditioning consisting of TBI, thio-tepa and cyclophosphamide, yielding a 3-year DFS of 65% in 40 CR1 and CR2 ALL patients, with only one non-leukemic death and eight relapses [129].

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation 

One could expect that a previous TBI could jeopardize the feasibility of a second transplant; on the contrary many recent reports show that a reduced toxicity conditioning, often consisting of fludarabine and treosulfan, is feasible with very low toxicity [130]. 5.6.  Toxicity and Mortality The long-term effects of TBI are well documented, with growth impairment, cataracts, gonadal failure and hypothyroidism occurring in substantial numbers of patients and sterility almost invariably. The risks of secondary malignancy are also increased by TBI. Transplantation issues in pediatrics present unique challenges, since influences on growth and development are important factors in the evaluation of treatment outcomes [14]. All organs and tissues can be injured by the pretransplant conditioning, but only the hematopoietic bone marrow is replaced. Except for GVHD, autografting can result in similar sequelae from the transplant-conditioning regimen used. These complications occur in survivors cured of their leukemia, but can carry with them a significant burden of morbidity (legacy of growth, endocrine and neuropsychological disturbances, organ failure and high risk for second cancers) and in some cases, mortality unrelated to their leukemia [82, 131– 133]. Curing the child demand that the risk of adverse sequelae of treatment administered is carefully balanced with known therapeutic benefits [134]. 5.7.  MRD and Allogeneic Transplantation Chimerism analysis has become an important tool for transplant surveillance to monitor engraftment, graft rejection and can be used as an indicator for the recurrence of the underlying malignant disease [135–138]. Molecular analysis was adopted to assess the kinetics of detecting negative MRD after diagnosis, relapse, and after transplantation as well. The maintenance of molecular remission after transplantation in subsequent marrow aspirates sampled before HSCT and every 3 months up to 1 year after transplantation allowed some investigators to assess the prognostic role of MRD before and after transplant. Patients who are MRD positive at time of transplantation have an increased risk of relapse after transplant, suggesting that residual leukemia could not be eradicated either by the conditioning regimen or the immunological alloreactivity [107]. The adoption of additional treatment elements, as “FLA-DNXM,” or “AMSA-FLA” in patients who do not clear their MRD before transplant, aims to reduce leukemic burden and optimize the antileukemic effect of transplantation. Nevertheless MRD analyses at transplant and thereafter, adopted in recent years, represent crucial tools which may contribute to further improve results in VHR CR1 or CR2 patients. Since 1998, reports from three European centers have shown that MRD burden prior to conditioning is the strongest single predictive factor for relapse after HSCT [104, 106]. Recognition of the potential clinical impact of this test has led to the formation of a European Study Group on MRD detection in transplantation for ALL. Although formal meta-analysis is invalidated by differences in transplant protocol and MRD assay method, some clinically useful conclusions can be

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inferred by pooling results. First, it is clear that EFS for the MRD-negative group is uniformly good; second, in the MRC and Dutch studies, transplantation in children with high-level disease appears futile, whereas the BFM results suggest a minority of such children can be cured. It is intriguing to speculate whether differences in transplant protocol may explain the observed differences in outcome. A third observation common to these studies is the trend towards a high MRD level in children who relapse in the bone marrow after a short CR1, indicating that relative insensitivity to therapy underlies the high risk of relapse after transplant in such patients. It is important to acknowledge that current evidence regarding the clinical significance of MRD is based on retrospective studies of selected groups of patients. The small numbers of children treated at any one center argue strongly for both national and international cooperation. A major drawback of such studies in the past has been failure to agree to a common transplant protocol. Future common protocols should address two issues: (1) novel therapies for those with high-level MRD pretransplant and (2) definition of a low-risk group who can be cured without transplant. Many ongoing protocols manage high-level MRD patients by potentiating the graft-versus-leukemia effect. Thus, immunosuppression is tapered and discontinued early in children with MRD positivity before transplant and consecutive increasing doses of donor lymphocyte infusion may be added. The next European relapse protocol will contain a window therapy arm for those found to be high-level MRD positive prior to conditioning. Currently, a combination of fludarabine, high-dose cytosine and liposomal daunorubicin is being used in this window. If this approach failed, new drugs or monoclonal antibodies may be examined.

6.  Conclusions International collaboration among the large cooperative pediatric ALL working groups has allowed us to advance leukemia research [139]. The role of transplantation will evolve depending on chemotherapeutic regimens used. For any given patient the decision to allograft reflects a balance between the biology of the disease and the risk of transplant-related death and long-term sequelae. At present, information about the biology of the disease is based on known prognostic factors, including WBC, cytogenetics, and morphological and molecular assessment of response to therapy at various time-points. The risk of mortality is a function of HLA disparity between donor and recipient, performance and remission status [105]. Although the indications for transplantation in CR1 vary among the major treatment co-operatives, the procedure is strictly reserved for those with very high risk biologic features and a poor early response, however defined. There is a consensus that prognosis after relapse is dependent on the duration of first remission and the site of relapse. MRD-based stratification may further optimize the identification of patients who need the alloreactive effect of an allograft to be cured [105]. Pediatricians particularly recognize the need not only to increase the cure rate, beyond the 80% achieved with contemporary treatments, but also to improve the quality of life of leukemia survivors by limiting both acute and long-term sequelae [106].

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation 

Acknowledgments  We would like to thank the following colleagues who were asked and who provided eligibility criteria for transplantation on behalf of each cooperative group, as reported in Tables  15.1 and 15.5: Eduardo Dibar for GATLA (Grupo Argentino de Tratamiento de la Leucemia Aguda); Martin Schrappe, André Schrauder, Arend von Stackelberg and Christina Peters for BFM Group (Berlin-Frankfurt-Münster Group); Maria Campbell and Julia Palma for PINDA (Programa Infantil de Drogas Antineoplásicas de Chile); Jan Stary for Czech Republic and ALL-IC; Yves Benoit for EORTC (European Organization for Research and Treatment of Cancer); André Baruchel for FRALLE (French Acute Lymphoblastic Leukaemia Group); Pierre Rohrlich for COOPRALL (Coopération de Traitement des Rechutes de Leucémies Aigues Lymphoblastiques de l’enfant); Isaac Yaniv for ISPHO (Israeli Society for Pediatric Hematology and Oncology); Valentino Conter for AIEOP (Associazione Italiana Ematologia ed Oncologia Pediatrica); Kalman Nagy, Gergely Krivan and Gabor Kovacs for H-POG (Hungary Pediatric Oncology Group); Rob Pieters for DCLSG (Dutch Childhood Leukemia Study Group); Kim Vettenranta for NOPHO (Nordic Society for Pediatric Hematology and Oncology); Keizo Horibe for Japan; Jerzy Kowalczyk for Poland; Nick Goulden and Ajay Vora for MRC-ALL WP (Medical Research Council, Acute Lymphoblastic Leukaemia Working Party; Stephen Hunger for COG (Children’s Oncology Group); Stephen Sallan for Dana Farber.

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Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation  63. Moorman AV, Richards SM, Robinson HM, Strefford JC, Gibson BE, Kinsey SE, Eden TO, Vora AJ, Mitchell CD, Harrison CJ, UK Medical Research Council (MRC)/National Cancer Research Institute (NCRI) Childhood Leukaemia Working Party (CLWP) (2007) Prognosis of children with acute lymphoblastic leukemia (ALL) and intrachromosomal amplification of chromosome 21 (iAMP21). Blood 109(6):2327–30 64. Saarinen UM, Mellander L, Nysom K et  al (1996) Allogeneic bone marrow transplantation in first remission for children with very high-risk acute lymphoblastic leukemia: a retrospective case-control study in the Nordic countries. Nordic Society for Pediatric Hematology and Oncology (NOPHO). Bone Marrow Transplant 17(3):357–63 65. Wheeler KA, Richards SM, Bailey CC et al (2000) 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 96(7):2412–8 66. Gray R, Wheatley K (1991) How to avoid bias when comparing bone marrow transplantation with chemotherapy. Bone Marrow Transplant 7(Suppl 3):9–12 Review 67. Ribera JM, Ortega JJ, Oriol A et al (2007) Comparison of intensive chemotherapy, allogeneic, or autologous stem-cell transplantation as postremission treatment for children with very high risk acute lymphoblastic leukemia: PETHEMA ALL-93 Trial. J Clin Oncol 25(1):16–24 68. Iori AP, Arcese W, Milano F et  al (2007) Unrelated cord blood transplant in children with high-risk acute lymphoblastic leukemia: a long-term follow-up. Haematologica 92(8):1051–8 69. Locatelli F, Zecca M, Messina C et  al (2002) Improvement over time in outcome for children with acute lymphoblastic leukemia in second remission given hematopoietic stem cell transplantation from unrelated donors. Leukemia 16(11):2228–37 70. Bunin N, Carston M, Wall D et al (2002) Unrelated marrow transplantation for children with acute lymphoblastic leukemia in second remission. Blood 99(9):3151–7 71. Dini G, Valsecchi MG, Micalizzi C et al (2003) Impact of marrow unrelated donor search duration on outcome of children with acute lymphoblastic leukemia in second remission. Bone Marrow Transplant 32(3):325–31 72. Tsurusawa M, Yumura-Yagi K, Ohara A et  al (2007) Survival outcome after the first central nervous system relapse in children with acute lymphoblastic leukemia: a retrospective analysis of 79 patients in a joint program involving the experience of three Japanese study groups. Int J Hematol 85(1):36–40 73. Bleakley M, Shaw PJ, Nielsen JM (2002) 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 30(1):1–7 74. Willemze AJ, Geskus RB, Noordijk EM et al (2007) HLA-identical haematopoietic stem cell transplantation for acute leukaemia in children: less relapse with higher biologically effective dose of TBI. Bone Marrow Transplant 40(4):319–27 75. Gassas A, Ishaqi MK, Afzal S et al (2008) Outcome of haematopoietic stem cell transplantation for paediatric acute lymphoblastic leukaemia in third complete remission: a vital role for graft-versus-host-disease/ graft-versus-leukaemia effect in survival. Br J Haematol 140(1):86–9 76. Afify Z, Hunt L, Green A et al (2005) Factors affecting the outcome of stem cell transplantation from unrelated donors for childhood acute lymphoblastic leukemia in third remission. Bone Marrow Transplant 35(11):1041–7 77. Woolfrey AE, Anasetti C, Storer B et al (2002) Factors associated with outcome after unrelated marrow transplantation for treatment of acute lymphoblastic leukemia in children. Blood 99(6):2002–8 78. Sedláček P, Formanková R, Keslová P et al (2006) Low mortality of children undergoing hematopoietic stem cell transplantation from 7 to 8/10 human leukocyte

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A. Balduzzi et al. antigen allele-matched unrelated donors with the use of antithymocyte globulin. Bone Marrow Transplant 38(11):745–50 79. Al-Kasim FA, Thornley I, Rolland M et  al (2002) Single-centre experience with allogeneic bone marrow transplantation for acute lymphoblastic leukaemia in childhood: similar survival after matched-related and matched-unrelated donor transplants. Br J Haematol 116(2):483–90 80. Gassas A, Sung L, Saunders EF et al (2007) Graft-versus-leukemia effect in hematopoietic stem cell transplantation for pediatric acute lymphoblastic leukemia: significantly lower relapse rate in unrelated transplantations. Bone Marrow Transplant 40(10):951–5 81. Eapen M, Rubinstein P, Zhang MJ et  al (2007) Outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukaemia: a comparison study. Lancet 369(9577):1947–54 82. Sanders JE, Guthrie KA, Hoffmeister PA et  al (2005) Final adult height of patients who received hematopoietic cell transplantation in childhood. Blood 105(3):1348–54 83. Houtenbos I, Bracho F, Davenport V et  al (2001) Autologous bone marrow transplantation for childhood acute lymphoblastic leukemia: a novel combined approach consisting of ex vivo marrow purging, modulation of multi-drug resistance, induction of autograft vs leukemia effect, and post-transplant immuno- and chemotherapy (PTIC). Bone Marrow Transplant 27(2):145–53 84. Balduzzi A, Conter V, Uderzo C, Valsecchi MG (2007) Transplantation in childhood very high risk acute lymphoblastic leukemia in first complete remission: where are we now? J Clin Oncol 25(18):2625–6 85. Gustafsson Jernberg A, Remberger M, Ringdén O et al (2004) Risk factors in pediatric stem cell transplantation for leukaemia. Pediatr Transplant 8(5):464–74 86. Balduzzi A, De Lorenzo P, Schrauder A, Conter V, Uderzo C, Peters C, Klingebiel T, Stary J, Felice MS, Magyarosy E, Schrappe M, Dini G, Gadner H, Valsecchi MG (2008) Eligibility for allogeneic transplantation in very high risk childhood acute lymphoblastic leukemia: the impact of the waiting time. Haematologica 93:925–929 87. Gratwohl A, Baldomero H, Frauendorfer K et al (2007) Results of the EBMT activity survey 2005 on haematopoietic stem cell transplantation: focus on increasing use of unrelated donors. Bone Marrow Transplant 39(2):71–87 88. Eapen M, Rubinstein P, Zhang MJ et  al (2006) Comparable long-term survival after unrelated and HLA-matched sibling donor hematopoietic stem cell transplantations for acute leukemia in children younger than 18 months. J Clin Oncol 24(1):145–51 89. Quaranta BP, Halperin EC, Kurtzberg J et  al (2004) The incidence of testicular recurrence in boys with acute leukemia treated with total body and testicular irradiation and stem cell transplantation. Cancer 101(4):845–50 90. Malempati S, Gaynon PS, Sather H et al (2007) Outcome after relapse among children with standard-risk acute lymphoblastic leukemia: Children’s Oncology Group study CCG-1952. J Clin Oncol 25(36):5800–7 91. Lawson SE, Harrison G, Richards S et  al (2000) The UK experience in treating relapsed childhood acute lymphoblastic leukaemia: a report on the medical research council UKALLR1 study. Br J Haematol 108(3):531–43 92. Chessells JM, Veys P, Kempski H et  al (2003) Long-term follow-up of relapsed childhood acute lymphoblastic leukaemia. Br J Haematol 123(3):396–405 93. Rizzari C, Valsecchi MG, Aricò M et  al (2004) Outcome of very late relapse in children with acute lymphoblastic leukemia. Haematologica 89(4):427–34 94. Roy A, Cargill A, Love S et  al (2005) Outcome after first relapse in childhood acute lymphoblastic leukaemia – lessons from the United Kingdom R2 trial. Br J Haematol 130(1):67–75

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation    95. Einsiedel HG, von Stackelberg A, Hartmann R et al (2005) Long-term outcome in children with relapsed ALL by risk-stratified salvage therapy: results of trial acute lymphoblastic leukemia-relapse study of the Berlin-Frankfurt-Münster Group 87. J Clin Oncol 23(31):7942–50   96. Eapen M, Zhang MJ, Devidas M et  al (2008) Outcomes after HLA-matched sibling transplantation or chemotherapy in children with acute lymphoblastic leukemia in a second remission after an isolated central nervous system relapse: a collaborative study of the Children’s Oncology Group and the Center for International Blood and Marrow Transplant Research. Leukemia 22(2):281–6   97. Eapen M, Raetz E, Zhang MJ, Muehlenbein C, Devidas M, Abshire T, Billett A, Homans A, Camitta B, Carroll WL, Davies SM (2006) Outcomes after HLAmatched sibling transplantation or chemotherapy in children with B-precursor acute lymphoblastic leukemia in a second remission: a collaborative study of the Children’s Oncology Group and the Center for International Blood and Marrow Transplant Research. Blood 107(12):4961–7   98. Gaynon PS, Harris RE, Altman AJ et al (2006) Bone marrow transplantation versus prolonged intensive chemotherapy for children with acute lymphoblastic leukemia and an initial bone marrow relapse within 12 months of the completion of primary therapy: Children’s Oncology Group study CCG. J Clin Oncol 24(19):3150–6   99. Dopfer R, Henze G, Bender-Götze C et  al (1991) Allogeneic bone marrow transplantation for childhood acute lymphoblastic leukemia in second remission after intensive primary and relapse therapy according to the BFM- and CoALLprotocols: results of the German Cooperative Study. Blood 78(10):2780–4 100. Borgmann A, von Stackelberg A, Hartmann R et  al (2003) Unrelated donor stem cell transplantation compared with chemotherapy for children with acute lymphoblastic leukemia in a second remission: a matched-pair analysis. Blood 101(10):3835–9 101. Harrison G, Richards S, Lawson S et al (2000) 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 11(8):999–1006 102. von Stackelberg A, Hartmann R, Bührer C et al (2008) High-dose compared with intermediate-dose methotrexate in children with a first relapse of acute lymphoblastic leukemia. Blood 111(5):2573–80 103. Wheeler K, Richards S, Bailey C, Chessells J (1998) 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 101(1):94–103 104. Bader P, Hancock J, Kreyenberg H (2002) Minimal residual disease (MRD) status prior to allogeneic stem cell transplantation is a powerful predictor for posttransplant outcome in children with ALL. Leukemia 16(9):1668–72 105. Goulden N, Bader P, Van Der Velden V et  al (2003) Minimal residual disease prior to stem cell transplant for childhood acute lymphoblastic leukaemia. Br J Haematol 122(1):24–9 106. Knechtli CJ, Goulden NJ, Hancock JP et al (1998) 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 92(11):4072–9 107. Krejci O, van der Velden VH, Bader P et  al (2003) Level of minimal residual disease prior to haematopoietic stem cell transplantation predicts prognosis in paediatric patients with acute lymphoblastic leukaemia: a report of the Pre-BMT MRD Study Group. Bone Marrow Transplant 32(8):849–51 108. Dufourg MN, Landman-Parker J, Auclerc MF et  al (2007) Age and high-dose methotrexate are associated to clinical acute encephalopathy in FRALLE 93 trial for acute lymphoblastic leukemia in children. Leukemia 21(2):238–47

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A. Balduzzi et al. 109. Jansen NC, Kingma A, Schuitema A et al (2006) Post-treatment intellectual functioning in children treated for acute lymphoblastic leukaemia (ALL) with chemotherapy-only: a prospective, sibling-controlled study. Eur J Cancer 42(16):2765–72 110. Krappmann P, Paulides M, Stöhr W et al (2007) Almost normal cognitive function in patients during therapy for childhood acute lymphoblastic leukemia without cranial irradiation according to ALL-BFM 95 and COALL 06–97 protocols: results of an Austrian-German multicenter longitudinal study and implications for follow-up. Pediatr Hematol Oncol 24(2):101–9 111. Waber DP, Turek J, Catania L et  al (2007) Neuropsychological outcomes from a randomized trial of triple intrathecal chemotherapy compared with 18 Gy cranial radiation as CNS treatment in acute lymphoblastic leukemia: findings from Dana-Farber Cancer Institute ALL Consortium Protocol 95-01. J Clin Oncol 25(31):4914–21 112. Matsuzaki A, Nagatoshi Y, Inada H et al (2005) Prognostic factors for relapsed childhood acute lymphoblastic leukemia: impact of allogeneic stem cell transplantation – a report from the Kyushu-Yamaguchi Children’s Cancer Study Group. Pediatr Blood Cancer 45(2):111–20 113. Borgmann A, Baumgarten E, Schmid H et  al (1997) Allogeneic bone marrow transplantation for a subset of children with acute lymphoblastic leukemia in third remission: a conceivable alternative? Bone Marrow Transplant 20(11):939–44 114. Blair A, Goulden NJ, Libri NA et al (2005) Immunotherapeutic strategies in acute lymphoblastic leukaemia relapsing after stem cell transplantation. Blood Rev 19(6):289–300 115. Eapen M, Horowitz MM, Klein JP, Champlin RE, Loberiza FR Jr, Ringdén O, Wagner JE (2004) 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 22(24):4872–80 116. Marks DI, Khattry N, Cummins M et al (2006) Haploidentical stem cell transplantation for children with acute leukaemia. Br J Haematol 134(2):196–201 117. Ishaqi MK, Afzal S, Dupuis A et  al (2008) Early lymphocyte recovery postallogeneic hematopoietic stem cell transplantation is associated with significant graft-versus-leukemia effect without increase in graft-versus-host disease in pediatric acute lymphoblastic leukemia. Bone Marrow Transplant 41(3):245–52 118. Rocha V, Michel G, Kabbara N, et al (2005) Outcomes after unrelated cord blood transplantation in children with acute lymphoblastic leukemia. An EurocordNetcord survey. Blood abstract# 304, page 93a. 119. Rocha V, Cornish J, Sievers E, Filipovich A, Locatelli F, Peters C et  al (2001) Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood 97:2962–2971 120. Barker JN, Davies SM, DeFor T, Ramsay NK, Weisdorf DJ, Wagner JE (2001) 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 97:2957–2961 121. Dalle JH, Duval M, Moghrabi A, Wagner JE, Vachon MF, Barrette S et al (2004) Results of an unrelated transplant search strategy using partially HLA-mismatched cord blood as an immediate alternative to HLA-matched bone marrow. Bone Marrow Transplant 33:605–11 122. Hwang WY, Samuel M, Tan D, Koh LP, Lim W, Linn YC (2007) A meta-analysis of unrelated donor umbilical cord blood transplantation versus unrelated donor bone marrow transplantation in adult and pediatric patients. Biol Blood Marrow Transplant 13:444–53 123. Rocha V, Gluckman E (2007) Outcomes of transplantation in children with acute leukaemia. Lancet 369(9577):1906–8

Chapter 15  Allogeneic Hematopoietic Stem Cell Transplantation  124. Hough R, Labopin M, Michel G, Locatelli F, Klingebiel T, Arcese W et al (2007) Outcomes of fully haplo-identical haematopoietic stem cell transplantation compared to unrelated cord blood transplantation in children with acute lymphoblastic leukaemia. A retrospective analysis on behalf of Eurocord, PDWP and ALWP of EBMT. Bone Marrow Transplant 39(S1):S3 125. Bunin N, Aplenc R, Kamani N et al (2003) Randomized trial of busulfan vs total body irradiation containing conditioning regimens for children with acute lymphoblastic leukemia: a Pediatric Blood and Marrow Transplant Consortium study. Bone Marrow Transplant 32(6):543–8 126. Jamieson CH, Amylon MD, Wong RM et al (2003) Allogeneic hematopoietic cell transplantation for patients with high-risk acute lymphoblastic leukemia in first or second complete remission using fractionated total-body irradiation and high-dose etoposide: a 15-year experience. Exp Hematol 31(10):981–6 127. Marks DI, Forman SJ, Blume KG et al (2006) A comparison of cyclophosphamide and total body irradiation with etoposide and total body irradiation as conditioning regimens for patients undergoing sibling allografting for acute lymphoblastic leukemia in first or second complete remission. Biol Blood Marrow Transplant 12(4):438–53 128. Ringdén O, Ruutu T, Remberger M et  al (1994) 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 83(9):2723–30 129. Zecca M, Pession A, Messina C, Bonetti F, Favre C, Prete A, Cesaro S, Porta F, Mazzarino I, Georgiani G, Rondelli R, Locatelli F (1999) 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 17(6):1838–1846 130. Iravani M, Evazi MR, Mousavi SA et  al (2007) Fludarabine and busulfan as a myeloablative conditioning regimen for allogeneic stem cell transplantation in high- and standard-risk leukemic patients. Bone Marrow Transplant 40(2):105–10 131. Brydøy M, Fosså SD, Dahl O et al (2007) Gonadal dysfunction and fertility problems in cancer survivors. Acta Oncol 46(4):480–9 132. Chow EJ, Friedman DL, Yasui Y et al (2007) Decreased adult height in survivors of childhood acute lymphoblastic leukemia: a report from the Childhood Cancer Survivor Study. J Pediatr 150(4):370–5 375 133. Cohen IJ (2007) Comparison of long-term neurocognitive outcomes in young children with acute lymphatic leukemia treated with cranial radiation or high-dose or very high-dose intravenous methotrexate. J Clin Oncol 25(6):734–5 134. Löf CM, Forinder U, Winiarski J (2007) Risk factors for lower health-related QoL after allogeneic stem cell transplantation in children. Pediatr Transplant 11(2):145–51 135. Bader P, Niethammer D, Willasch A et al (2005) How and when should we monitor chimerism after allogeneic stem cell transplantation? Bone Marrow Transplant 35(2):107–19 136. Sànchez J, Serrano J, Gòmez P et  al (2002) Clinical value of immunological monitoring of minimal residual disease in acute lymphoblastic leukaemia after allogeneic transplantation. Br J Haematol 116(3):686–94 137. Uzunel M, Jaksch M, Mattsson J et al (2003) Minimal residual disease detection after allogeneic stem cell transplantation is correlated to relapse in patients with acute lymphoblastic leukaemia. Br J Haematol 122(5):788–94 138. Uzunel M, Mattsson J, Jakssch M et al (2001) The significance of graft-versushost disease and pretransplantation minimal residual disease status to outcome after allogeneic stem cell transplantation in patients with acute lymphoblastic leukemia. Blood 98(6):1982–4

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Chapter 16 Allogeneic Transplantation for the Treatment of Multiple Myeloma Rebecca L. Olin, Dan T. Vogl, and Edward A. Stadtmauer

1. Introduction Multiple myeloma is a hematologic malignancy characterized by the clonal proliferation of plasma cells. The annual incidence is four per 100,000 individuals. It was estimated that 19,900 new cases would be diagnosed in 2007, with 10,790 estimated deaths due to myeloma [1]. The median age at diagnosis is approximately 65 years, making it a disease that predominantly affects older patients. Although myeloma cells are chemosensitive, the median survival for patients with multiple myeloma remains less than 5 years. Melphalan and prednisone remain the chemotherapeutic agents of choice for older patients who are not transplant candidates, because long term outcomes have not shown any improvement with more intensive multi-agent chemotherapy regimens [2, 3]. The addition of novel agents such as thalidomide, bortezomib, and lenalidomide to melphalan and steroids may improve outcomes in this group of patients [4, 5]. The current standard of care for younger eligible patients is initial conventional dose therapy followed by high-dose melphalan and autologous hematopoietic stem cell transplant. Two randomized and several nonrandomized comparative studies show improved survival with transplant over standard chemotherapy [6, 7]. Recent investigations have shown promising results with tandem autologous transplants and incorporation of the newer agents into the first line of therapy. Unfortunately, despite these advances, almost all patients will ultimately relapse. Allogeneic transplantation is the only modality to have demonstrated curative potential in multiple myeloma. Early studies of allogeneic transplants for myeloma resulted in a small number of long-term survivors. Additional studies supported the existence of an immune mediated “graft-versus-myeloma” effect. However, these early studies were plagued by high transplant-related mortality (TRM), even in patients who were not heavily pre-treated. Because of this as well as the advanced age of most myeloma patients and the limited number of available donors, use of allogeneic transplants has been limited.

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_16, © Springer Science + Business Media, LLC 2003, 2010

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The focus of recent research has been to determine ways to decrease toxicity of transplantation while still achieving long-term disease control or cure. These studies will be reviewed here.

2. Background of Therapy Currently, multiple myeloma represents the most common indication for autologous stem cell transplantation in North America, with over four thousand transplants performed in 2003 [8]. Initial studies compared patients who received autologous transplants to historical controls and suggested that survival was prolonged [9, 10]. Subsequently, two prospective randomized trials have shown a survival advantage for autologous transplant over standard chemotherapy. In the French IFM study, 200 previously untreated patients were randomly assigned conventional chemotherapy or high-dose therapy with melphalan and total body irradiation (TBI) [6]. Overall response rates, progression-free survival (PFS), and overall survival (OS) were all significantly improved; the 5 year OS for patients receiving autologous transplant was 52 vs. 12% for conventional chemotherapy. The British MRC VII study, randomized 407 patients to conventional therapy or autologous transplantation using high-dose melphalan [7]. Median survival was extended from 42 months with chemotherapy to 54 months with transplantation. Some comparative trials have not shown a survival advantage for transplant over conventional chemotherapy [11–13]. These usually allowed for transplant at time of relapse for conventional dose treatment failures or the use of more toxic preparative regimens [14]. Given the palliative goal of autologous transplant, either an approach of initial cytoreduction followed by autologous transplant or use of transplant at time of first progression has become the common alternative. Recent studies have reported tandem autologous transplants as part of initial therapy for myeloma. The IFM94 randomized trial demonstrated an improved median OS with tandem autologous transplants when compared to a single transplant [15]; 7- year survival was 43% in the group randomized to tandem transplants vs. 11% in the single transplant group. A subgroup analysis suggested that the survival benefit was limited to the group of patients with a less than 90% disease response after the first transplant. Other studies have not confirmed this finding [16, 17]. In addition to high-dose melphalan and autologous stem cell transplantation, the outcomes of treatment for multiple myeloma have been improved by novel agents such as thalidomide, lenalidomide, and bortezomib [18]. Incorporation of these agents into both upfront, maintenance, and salvage therapy programs is currently under active investigation, but no study has yet shown the superiority of nontransplant approaches.

3. Rationale for Allogeneic Transplantation The anti-tumor effect of allogeneic transplantation results from administration of high doses of chemotherapy and radiation as well as an immune-mediated graft-versus-tumor effect in which donor immune cells recognize and eradicate residual disease. Clinical evidence, both direct and indirect, suggests that a true graft-versus-myeloma effect exists. Case reports have shown that for

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myeloma relapsing after allogeneic transplant, remission can be reestablished by withdrawing immunosuppression [19]. A number of reports also showed that donor lymphocyte infusion (DLI) could also result in clinical responses and achievement of prolonged remissions; [20–23] subsequent larger studies demonstrated the effectiveness of this approach [24, 25]. Withdrawal of immunosuppression and DLI have become accepted strategies for relapse or residual disease after allogeneic transplantation, in order to harness the graftversus-myeloma effect and induce disease responses. Finally, response to DLI has correlated with the development of acute and/or chronic graft-versus-host disease (GVHD) [25–27], suggesting that both are due to the immune activity of the donor cells.

4. Myeloablative Allogeneic Transplantation 4.1. Results: Transplant-Related Mortality and Overall Survival Most data for the use of allogeneic transplantation in multiple myeloma come from single-center trials and registry databases. Although heterogeneity among the trials limits comparisons, early studies of allogeneic transplant (performed in the 1980s and early 1990s) demonstrated high TRM but subsequent plateau of survival curves, with corresponding small but encouraging numbers of long-term disease-free survivors. Later studies have demonstrated somewhat lower TRM, which is thought to be due to decreased infection rates and earlier transplantation with less prior chemotherapy (Table 16-1). In 1991, Gahrton et  al. reported European registry data (EBMT) for 90 patients who received an allogeneic transplant between 1983 and 1989 from HLA-identical siblings [28]. Preparative regimens varied but the majority received cyclophosphamide/TBI (Cy/TBI) or melphalan/TBI (Mel/TBI). The overall complete response rate was 43%, and survival at 76 months was 40%. TRM, however, was 25%, due to GVHD, infections, and pneumonitis. The number of prior lines of treatment was inversely related to complete remission (CR). In a later review of EBMT data, median survival after allogeneic transplant was reported to be only 1.5 years and TRM approached 50% [29]. The long-term survival rate was 30%, and the relapse rate approached 70%, with

Table 16-1.  Major reports of myeloablative transplantation. Author

Source data

Dates of transplants

N

TRM (%)

OS

Gahrton [28]

EBMT

1983–1989

   90

25

40% (6.3 years)

Bjorkstrand [29]

EBMT

1983–2001

1,300+

50

Median 1.5 years

Bensinger [30]

Seattle

1987–1994

   80

57

24% (4.5 years)

Bensinger [31]

Seattle

1987–1999

  136

48

22% (5 years)

Barlogie [14]

Intergroup trial

1993–1994

   36

53

39% (7 years)

Gahrton [40]

EBMT

1983–1993

  334

46

40% (2 years), median 10 months

1994–1998

  356

30

57% (2 years), median 50 months

N number of patients, TRM transplant-related mortality, OS overall survival

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relapses occurring many years after transplantation. The TRM approached 50%, making allogeneic transplantation unattractive. The three-arm Intergroup trial S9321 compared early to late autotransplants and offered allogeneic transplants to patients who were younger than 56 years and had an HLA-identical sibling donor. The allogeneic arm of this trial was closed when a first-year TRM of 53% was observed after 36 eligible patients were enrolled [14]. The largest single-center published experience with allogeneic transplants for myeloma is from Seattle. In 1996, investigators there reported 80 patients who received transplants primarily from HLA-identical siblings between 1987 and 1994 [30]. The conditioning regimen used was busulfan/cyclophosphamide (Bu/Cy) with or without TBI. The CR rate was 36%, and actuarial survival was 20% at 4.5 years. Again, TRM was high at 57%, with 44% occurring in the first 100 days. Updated data published in 2001 for 136 patients again revealed a high TRM of 48% in the first 100 days [31]. However, OS was 22% at 5 years, and 12 patients remain alive and free of disease between 5 and 13 years post-transplant. Other early single-center reports have shown similar results [32–39]. Improvements in supportive care and GVHD prophylaxis may lead to reduced TRM in the current era. In 2001, an analysis of EBMT registry data was published comparing allogeneic transplants performed for multiple myeloma between 1983 and 1993 with those performed between 1994 and 1998 [40]. There was a highly significant difference between these two groups, with median OS of 10 months for patients transplanted in the earlier period, compared with 50 months for the later period. The improvement in survival was shown to be due to a reduction in TRM, which was 46% and 30% at 2 years for the early and late period, respectively. Significantly fewer cases of infections and interstitial pneumonitis were observed in the later group, and transplants were also performed sooner after diagnosis; rates of GVHD were similar. Indeed, single- and multi-center studies of allogeneic transplants performed in the 1990s do seem to support a somewhat lower TRM [41–51], the majority reporting a TRM in the range of 20–30%. 4.2. Comparison with Autologous Transplantation The relative benefits of autologous and allogeneic transplantation for multiple myeloma have been compared in several studies. (Table  16-2) The EBMT registry reported 189 patients who received allogeneic transplants compared to 189 case-matched controls who received autologous transplants [52]. Cases were matched for gender and for number of prior lines of treatment; 60% had at least two prior treatments. Median survival was significantly shorter for allogeneic transplant than autologous (18 vs. 34 months). This effect was consistent in all subgroups analyzed, and was due mainly to a difference in TRM, which was 41% for allogeneic and 13% for autologous transplants. When the data were analyzed using only those patients who survived >1 year, however, there was a trend toward improved survival in the allogeneic arm, with significantly longer PFS. Single-center studies have reported similar findings. Couban et  al. found that allogeneic transplantation, when compared to autologous, resulted in a higher 90-day mortality (27 vs. 5%), with a slightly improved 3-year PFS

Chapter 16  Allogeneic Transplantation for the Treatment of Multiple Myeloma 

265

Table 16-2.  Comparisons of allogeneic and autologous transplantation. Author

N

TRM (%)

PFS

OS

Bjorkstrand [52]

189 allo 189 auto

41* 13

Median 11 m† Median 20 m

Median 18 m* Median 34 m

Couban [33]

22 allo 40 auto

27 (90d)† 5 (90d)

22% 3 year† 17% 3 year

32% 3 year† 74% 3 year

Alyea [43]

66 allo 166 auto

24* 13

28% 2 year*, 18% 4 year 48% 2 year, 23% 4y

51% 2 year*, 39% 4 year 74% 2 year, 41% 4 year

Arora [48]

17 allo 70 auto

18 (100d)* 4 (100d)

58% 1 year, 32% 4 year 67% 1 year, 18% 4 year

64% 1 year, 64% 4 year 86% 1 year, 50% 4 year

Mehta [37]

42 allo 42 auto

43* 10

20% 3 year 25% 3 year

29% 3 year* 54% 3 year

Reynolds [42]

21 allo 35 auto

19 (100d) 9 (100d)

60% 2 year 30% 2 year

60% 2 year 42% 2 year

Kuruvilla [51]

72 allo 86 auto

22 14

33% 5 year, 31% 10 year 33% 5 year, 15% 10 year

48% 5 year, 40% 10 year 46% 5 year, 31% 10 year

N number of patients, TRM transplant-related mortality, PFS progression-free survival, OS overall survival. *Statistically different from autologous group. †Statistical significance not reported

(22 vs. 17%), but markedly worse 3-year OS (32 vs. 74%) [33]. Alyea et al. reported that at 2 years, OS was 51% for allogeneic vs. 74% for autologous transplantation, but by 4 years, OS was similar at 39 vs. 41% [43]. Relapses were less frequent in the allogeneic group; however, nonrelapse mortality was significantly higher. Arora et  al. also demonstrated that allogeneic transplant result in a higher TRM, but lower PFS than autologous transplant [48]. The survival curves crossed, with OS higher for autologous than for allogeneic transplant at 1 year (64 vs. 86%), but lower at 4 years (64 vs. 50%). Mehta et  al. compared 42 patients who received a salvage allogeneic transplant after a first autograft with 42 matched patients receiving a salvage autologous transplant [37]. Three-year survival was significantly lower after allogeneic transplant (29 vs. 54%), because of higher TRM (43 vs. 10%). Reynolds et  al. who controlled for treatment regimen by performing both allogeneic and autologous transplants with the same conditioning regimen of Bu/Cy/TBI, found no statistically significant difference in PFS (60 vs. 30% at 2 years) or OS (60 vs. 42%), although there was a trend toward improved outcomes in the allogeneic group [42]. Finally, Kuruvilla et al. recently reported 7-year follow-up data on 72 allogeneic and 86 autologous transplants; the estimated 10-year OS was 40 vs. 30% respectively, which was not significantly different [51]. As a group, these studies indicate that allogeneic transplantation results in higher TRM than autologous transplantation, although typically with decreased relapse rates and longer PFS. These factors exert opposite effects on OS, resulting in inferior OS soon after transplant and potentially similar or improved OS after longer follow-up. Interestingly, in the Intergroup study

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S9321, despite early closure of the allogeneic arm due to high TRM, the survival curve did subsequently plateau, indicating a fraction of patients possibly cured and resulting in similar 6-year PFS and OS for the allogeneic and autologous arms [14, 53]. 4.3. Syngeneic Transplantation Syngeneic transplantation from an identical twin offers the possibility of a tumorfree graft without the risk of GVHD. Bensinger et al. reported 11 patients who received syngeneic transplants using varying conditioning regimens. Two were long-term survivors: one disease-free at 9 years, and other with a persistent serum monoclonal protein 15 years post-transplant [54]. Gahrton et al. used EBMT registry data to compare 25 twin transplants with 125 case-matched patients who underwent autologous transplant and 125 who underwent allogeneic transplant [55]. Median OS and PFS both appeared to be better for syngeneic compared with autologous transplants (OS 73 vs. 44 months and PFS 72 vs. 25 months), though only the difference in PFS was statistically significant. Survival was significantly worse from allogeneic transplants (median survival 16 months); this was mainly due to high TRM. 4.4. Allogeneic Stem Cell Source and Alternative Donors Initial studies of allogeneic transplants utilized bone marrow stem cells (BMSC). More recently investigators have used peripheral blood stem cells (PBSC), which have been shown to decrease time to engraftment in a randomized trial of patients with diverse hematologic malignancies [56]. Majolino et al. demonstrated the feasibility of using PBSC in allogeneic transplants for myeloma in a retrospective analysis of 10 patients [36]. All patients engrafted, and all achieved CR. TRM was 20%, and OS was 80% at a median of 18.5 months of follow-up. In a later analysis of EBMT registry data, PBSC transplants were compared to BMSC transplants done for myeloma between 1994 and 1998 [40]. Use of PBSC was associated with earlier engraftment, but no survival advantage. There was a trend toward increased GVHD with PBSC (18% grade III/IV with PBSC vs. 11% with BMSC), but this was not statistically significant. Ballen et  al. retrospectively analyzed NMDP registry data for 70 patients who received a matched unrelated donor (MUD) allogeneic transplant for myeloma [57]. The 100-day TRM was 42%, and OS at 5 years was quite low at 9%. Survival was not significantly different when stratified for prior autologous transplant or for GVHD. 4.5. T-Cell Depletion GVHD remains the primary source of morbidity and mortality in allogeneic transplantation for multiple myeloma. Published rates of GVHD range from 20 to 80%, with a significant proportion of patients developing grades II–IV acute and chronic GVHD. A number of studies have investigated T-cell depleted grafts, in an attempt to decrease this toxicity. In a prospective study, Lokhorst et  al. treated 53 patients with multiple myeloma with allogeneic grafts that were partially T-cell depleted [46]. Cy/TBI conditioning was used for the majority of patients. Grade II–IV acute GVHD (aGVHD) was seen in 45%, and extensive chronic GVHD (cGVHD) in 30%.

Chapter 16  Allogeneic Transplantation for the Treatment of Multiple Myeloma 

TRM was 34%. The median OS was poor at 25 months, inferior to standard chemotherapy, which suggested loss of the graft-versus-myeloma effect. Using an alternative strategy, Kroger et al. used in vivo T-cell depletion with anti-thymocyte globulin (ATG) as part of a conditioning regimen also including Bu/Cy/TBI [45]. Grade II–IV GVHD was seen in 35%, and after a median of 41 months of follow-up, estimated OS at 6 years was 77%. Alyea et  al. utilized the combination of CD6 T-cell depleted grafts with subsequent prophylactic DLI at 6–9 months post-transplant in order to reduce TRM and maintain a graft-versus-myeloma effect [58]. T-cell depletion was the sole method of GVHD prophylaxis. Twenty-four patients were treated; grade II–IV aGVHD was seen in 21% after transplant, and 50% of patients who went on to receive DLI experienced acute or chronic GVHD. The 100day TRM was only 4%; however, estimated 2-year PFS was relatively low at 30%. Similarly, Huff et  al. report on the long-term follow-up of 51 patients who received T-cell depleted allogeneic transplants [44]. Only 14% had grade II–IV aGVHD after transplant, and 6% had cGVHD. However, TRM was 24%, and 31 of 39 patients had disease progression after transplant, suggesting that T-cell depleted grafts may yield lower TRM but also decreased PFS. 4.6. Prognostic Factors Factors predicting a good outcome with allogeneic transplantation are generally similar to those for autologous transplantation. Favorable prognostic factors include only one prior treatment regimen, responding disease at the time of transplant, and low b2-microglobulin at diagnosis [29, 59]. The most important post-transplant prognostic factor is achievement of CR. Gender of both donor and recipient arealso important, with the best OS seen in the combination of female patients with female donors [60]. The Seattle group reported adverse prognostic factors to include transplantation >1 year from diagnosis, b2-microglobulin >2.5 mg/L at transplant, female patient with male donor, >8 cycles of prior chemotherapy, and Durie stage 3 at transplant [30]. In a subsequent report with a larger number of patients, mortality within 100 days was also significantly influenced by pre-transplant serum albumin [31]. Donor gender did not affect PFS, because having a female donor resulted in a lower risk of relapse but also a higher TRM, with these two effects appearing to negate each other.

5. Nonmyeloablative Allogeneic Transplantation 5.1. Phase II Studies Nonmyeloablative allogeneic stem cell transplant to reduce TRM is particularly attractive for myeloma to reduce transplant related mortality. Several phase II studies of nonmyeloablative transplants in patients with myeloma have been reported [61–77]. These studies included between 10 and 120 patients. The most common conditioning regimens used were melphalan/fludarabine, other melphalan-based regimens, other fludarabine-based regimens, or TBIbased combinations. Many of the patients in these studies were very heavily pretreated: most had had at least one prior autograft, and some had many prior lines of therapy. TRM in these studies typically ranged from 0 to 25% with

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100 day post-transplant TRM not exceeding 19%, significantly lower than that for full-intensity allogeneic transplantation. No study reported difficulties with engraftment, and 80–100% of patients achieved full donor chimerism. While nonmyeloablative transplants may result in lower TRM, the risk of GVHD is still high. Rates of both grade II–IV aGVHD as well as cGVHD in phase II studies typically ranged from 30 to 70%. However, the variable GVHD prophylaxis regimens make direct comparison difficult. In a singlecenter study, Pérez-Simón et  al. analyzed 150 nonmyeloablative and 88 myeloablative transplant patients. All patients received the same GVHD prophylaxis and PBSC from an HLA-identical sibling donor [78]. The incidence of grade II–IV aGVHD was 29% for nonmyeloablative vs. 39% for myeloablative transplants, which was statistically significant. In a multivariate analysis, intensity of conditioning was the only variable to influence the incidence of aGVHD. Incidence of cGVHD was not significantly different for nonmyeloablative transplants (71 vs. 63%), although there was a higher incidence of limited, but not extensive, cGVHD. The percentage of patients on immunosuppression at 3 years post-transplant was also lower in the nonmyeloablative group (36 vs. 69%). Estimated PFS after nonmyeloablative transplants ranges from 15 to 50% at 1.5–4 years, and estimated OS ranges from 25 to 70%. Comparing these studies is difficult, however, given small numbers of patients, differences in patient populations, and differences in conditioning regimens. In the largest reported series, Kroger et al. describe 120 patients who received melphalan/fludarabine nonmyeloablative transplants [67]; 38 were done in tandem fashion after autologous transplant, 58 were done for relapse after autologous transplant, and 24 were done after chemotherapy alone. PFS of the entire group was 39% at 2 years, and OS was 59%. In a review of EBMT registry data, Crawley et al. report on 229 patients who received nonmyeloablative transplants; 169 had received at least one prior autograft. PFS was 21% at 3 years, and OS was 41% at 2 years [79]. MUD nonmyeloablative transplants have also been described in a single-center study of 24 patients with poor-risk myeloma; at 3 years, PFS was 33% and OS 61%, with TRM again only 21% [80]. EBMT data was used to compare patients who had received myeloablative allogeneic transplantation (n = 196) with those who had received nonmyeloablative transplants (n = 320) [81]. Patients who received nonmyeloablative transplants were older; they were also more likely to have received a prior autograft and to have progressive disease at the time of allogeneic transplant. TRM at 2 years was 24% for nonmyeloablative and 37% for myeloablative transplants. However, response rates were significantly lower in the nonmyeloablative group (CR in 34 vs. 53%) and PFS was also inferior (19 vs. 35% at 3 years). For this reason, OS was not significantly different between groups (38 vs. 50% at 3 years for nonmyeloablative and myeloablative, respectively). 5.2. Prognostic Factors Pre- and post-transplant prognostic factors have emerged from phase II studies; these have also provided insight into the mechanism of disease control in nonmyeloablative transplants. A number of studies have demonstrated, using multivariate analysis, that the presence of cGVHD after transplant improves both PFS [62, 64, 67, 68, 71, 73] and OS [71, 73]. Interestingly, aGVHD does

Chapter 16  Allogeneic Transplantation for the Treatment of Multiple Myeloma 

not improve PFS/OS [64], and in some cases may worsen OS via increased TRM [68, 82]. These findings support the theory that the immunologic activity of the graft against myeloma cells, when not associated with the high toxicity of severe GVHD, can reduce the risk of relapse and prolong survival. A pre-transplant prognostic factor, which has been shown to influence PFS and OS, is the presence of chemosensitive disease prior to nonmyeloablative transplant or, similarly, achievement of CR prior to transplant [62, 68, 79, 82]. Many studies have also shown that outcomes are improved if nonmyeloablative transplant is performed earlier in the course of disease treatment; this adverse prognostic factor has been expressed as >1 prior autologous transplant [61, 79], high number of prior cycles of chemotherapy [64, 83], prior relapse after an autograft [66, 67, 76], or a longer interval between prior autograft and nonmyeloablative transplant [73]. By contrast, when nonmyeloablative transplant was studied specifically as salvage therapy for relapse after prior autologous transplant, a shorter interval between transplants was shown to be an adverse prognostic factor [84]; however, this is likely an indicator of underlying chemoresistant disease. Additional adverse prognostic factors include chromosome arm 13q deletion [83] and the persistence of cytogenetic abnormalities after nonmyeloablative transplant [85]. 5.3. Tandem Autologous-Nonmyeloablative Transplantation The high relapse rate after nonmyeloablative allogeneic transplant for myeloma appears to be in part due to the inability of the graft vs. myeloma effect to eradicate a large burden of chemotherapy resistant disease. Outcomes from nonmyeloablative transplantation seem to be improved when carried out early in the course of myeloma treatment and a strategy of tandem autologous transplant followed by nonmyeloablative allogeneic transplant has been explored. This strategy pairs the cytoreductive effect of an autologous transplant with the immunologic benefits of a nonmyeloablative allogeneic transplant. A number of phase II studies have been performed using this approach [86–90]. TRM in these studies ranges from 0 to 20%, similar or lower than in studies of nonmyeloablative transplant alone in more heavily pretreated patients. The rates of acute and chronic GVHD are similar to those seen after nonmyeloablative transplant alone, ranging from 25 to 65%. PFS is approximately 50–60% at 2–3 years, and OS is 60–80% (Table 16-3; Fig. 16-1). Most notably, this approach has been tested in two randomized trials, with conflicting results. In 2000, the French IFM group began enrolling high- risk myeloma patients into one of two trials [91]. If an HLA-identical sibling were available, patients were offered tandem autologous-nonmyeloablative transplants (IFM 99-03, n = 65; melphalan 200 mg/m2 followed by fludarabine/ busulfan/ATG, with cyclosporine/methotrexate for GVHD prophylaxis). If patients did not have an HLA-identical sibling, they were offered tandem autologous transplants (IFM 99-04, n = 219; melphalan 200  mg/m2 followed by melphalan 220 mg/m2, dexamethasone, +/− anti-IL-6 monoclonal antibody). The results of these separate trials were then compared. Some selection bias was inevitable. Although patients were biologically assigned by the presence or absence of an HLA-identical sibling, 24 patients in the autologous study were not HLA typed. The two groups were also demographically different: patients in the autologous study were older (median age 58 vs. 54 years;

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Table 16-3.  Tandem autologous-nonmyeloablative transplantation Author

N

Nonmyeloablative regimen

TRM (%)

GVHD (% acute grades II-IV/% chronic)

Carella [86]

16

Flu/TBI

 6

43/55

62% 3 year

Galimberti [87]

20

Flu/Cy, Flu/TBI

20

45/30

58% 2 year

Kroger [88]

17

Mel/Flu/ATG

11 (100d)

38/40

74% 2 year

Maloney [89]

54

TBI

15

38/46

78% 2 year

Seok [90]

12

Mel/Flu

 0

33/50

100% with median 15 m follow-up

Garban [91]

65 auto-allo

Flu/Bu/ATG

11

24/43

Median 35 m

219 auto-auto

–

 5

–

Median 41 m

80 auto-allo

TBI

10

43/64

Median 80 m*

82 auto-auto

–

 2

–

Median 54 m

Bruno [92]

OS

N number of patients, TRM transplant-related mortality, GVHD graft-versus-host-disease, OS overall survival, Mel melphalan, Flu fludarabine, ATG anti-thymocyte globulin, TBI total body irradiation, Cy cyclophosphamide. *Statistically significant from autologous group

Fig. 16.1.  Cumulative incidence of acute and chronic GVHD after sequential autologousnonmyeloablative transplantation. (Reproduced from Maloney, 2003) [89]

P = 0.006) and had higher b2 microglobulin levels (median 4.9 vs. 4.1 mg/L; P = 0.049). When the results of these trials were compared, TRM was 11 vs. 5%, median PFS was 25 vs. 30 months, and median OS was 35 vs. 41 months, respectively. These were not significantly different, whether using intent-to-treat or per-protocol analysis (Fig. 16-2). A study recently published by an Italian group found a different result [92]. Two hundred and forty five consecutive patients with early responding myeloma were enrolled, and 162 underwent HLA-typing. Eighty patients had an HLA-identical sibling identified and 58 completed the sequential autologous followed by nonmyeloablative allogeneic transplant (“autograft– allograft”). Of the remaining 82 patients whose HLA-typed siblings were not matched, 46 completed tandem autologous transplant (“double-autologous”). The autograft–allograft group received melphalan 200 mg/m2 as conditioning

Chapter 16  Allogeneic Transplantation for the Treatment of Multiple Myeloma 

Fig. 16-2.  Overall (left) and event-free (right) survival in the French IFM 99-03 and 99-04 trials. (Reproduced from Garban, 2006) [91]

Fig. 16.3.  Overall (left) and event-free (right) survival in the Italian trial of sequential autologousnonmyeloablative allogeneic transplantation. (Reproduced from Bruno, 2007) [92]

for the first transplant, followed by 200  cGy TBI as sole conditioning for the second [93]. GVHD prophylaxis was cyclosporine and mycophenolate mofetil. The double-autologous group received melphalan 100–200  mg/m2 for each transplant. TRM was 10% in the autograft–allograft group and 2% in the double-autologous group. By intent-to-treat analysis, the presence of an HLA-identical sibling resulted in improved PFS and OS: 35 vs. 29 months, and 80 vs. 54 months, respectively (Fig. 16-3). In a per-protocol comparison of patients who actually received the assigned treatment, PFS remained superior for the auto-allograft patients. The results of these two trials are strikingly divergent. In the IFM study, median survival of the autograft-allograft group was 35 months, whereas in the Italian study median survival was 80 months. A number of factors may have contributed to this discrepancy. The patient population in the IFM trial was restricted to high-risk patients, who were less likely to respond to any treatment; the Italian study enrolled consecutive patients. Additionally, different conditioning regimens were used in the two trials: fludarabine-based vs. the potentially less toxic 200  cGy TBI. Finally, the conditioning used for the autologous transplants was also different. In the IFM study, relatively high doses of melphalan were used (200 followed by 220  mg/m2 in the doubleautologous arm) which might have improved results of tandem autografts, whereas in the Italian study, lower doses were used (100–200  mg/m2 in the double-autologous arm), which might have worsened results when compared to the autograft–allograft arm.

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Corroborative data regarding tandem autograft–allografts for myeloma will be provided by the larger North American BMT CTN 0102 trial, in which patients were biologically assigned to receive either two melphalan 200 mg/m2 autografts or an autograft–allograft strategy using 200  cGy TBI for the nonmyeloablative conditioning. This trial closed to accrual in March 2007, and preliminary results will be available in the next few years.

6.  Donor Lymphocyte Infusion and Post-Transplant Management DLI has been shown to induce responses for both persistent and recurrent disease after both myeloablative [25, 26] and nonmyeloablative [27, 94] allogeneic transplants for myeloma. In published series of 25–63 patients receiving DLI for persistent or recurrent disease, response rates range from 38 to 52%, with CR rates from 17 to 28%. Median survival in the larger series is approximately 2 years after DLI [25, 27], suggesting that DLI may not be curative in this setting. In one series of 54 patients given DLI after myeloablative transplant, the strongest predictor of response was the presence of acute or chronic GVHD [25]. GVHD also predicted response to DLI when given to 63 patients after nonmyeloablative transplant, although there was no effect on PFS or OS [27]. Novel agents, drugs such as thalidomide, have also been used for relapsed or persistent disease after an allograft. In one study, 31 patients received thalidomide for progression following allogeneic transplant: nine achieved a response, three with very good PR [95]. Five patients also developed GVHD, including the three who achieved very good PR, suggesting that thalidomide may work in these patients by modulating the donor graft. Thalidomide in combination with DLI has also been used effectively for patients not responding to DLI alone, as has been bortezomib [96, 97]. Molecular monitoring of disease status after transplantation is also promising. PCR reactions can be designed based on an individual’s clonal heavy chain rearrangement, and persistent PCR negativity is referred to as molecular remission. Molecular remissions are rarely achieved with autologous transplants (as virtually all reinfused stem cell products contained residual myeloma cells), whereas a higher proportion of patients achieve molecular remission after allogeneic transplant [98]. In a group of 48 patients who underwent allogeneic transplant, the risk of relapse was 0% for PCR-negative patients, 33% for PCR-variable patients, and 100% for PCR-positive patients [99]. Thus, it might be possible to target patients who do not achieve a molecular remission after allogeneic transplantation; these patients might selectively benefit from additional therapies before clinical relapse occurs.

7. Conclusions Multiple myeloma is a chronic bone marrow malignancy, and allogeneic hematopoietic stem cell transplantation holds great promise of eradicating disease while inducing a graft-versus-myeloma anti-tumor effect. Until recently,

Chapter 16  Allogeneic Transplantation for the Treatment of Multiple Myeloma 

the TRM associated with therapy has limited use of this treatment modality. Toxicity has been reduced with nonmyeloablative conditioning or T-cell depleted graft processing, but these approaches can result in an increased rate of relapse. The optimal setting in which to use these nonmyeloablative transplants has yet to be determined, but disease remission prior to transplant leads to improved outcome. The strategy of sequential autologous transplantation followed by nonmyeloablative allogeneic transplantation has acceptable toxicity, and evidence from at least one randomized trial shows improved OS relative to a tandem autologous transplant strategy. Additional corroborative evidence will certainly be required, however, and will be available from the larger North American BMT CTN 0102 trial. Agents with novel mechanisms of action (bortezomib, thalidomide, and lenalidomide) are now routinely incorporated into the early therapy of myeloma. These agents have been used alone or in combination, as part of initial induction therapy or as salvage therapy for relapse, with great success but without cure. Future research will determine the best combination and sequence of these therapies. Improved approaches to autologous and allogeneic transplant hold promise for improved outcomes for patients with multiple myeloma.

References 1. Jemal A, Siegel R, Ward E et al (2007) Cancer statistics, 2007. CA Cancer J Clin 57:43–66 2. Combination chemotherapy versus melphalan plus prednisone as treatment for multiple myeloma: An overview of 6,633 patients from 27 randomized trials. Myeloma Trialists’ Collaborative Group. J Clin Oncol 1998;16:3832–3842 3. Gregory WM, Richards MA, Malpas JS (1992) Combination chemotherapy versus melphalan and prednisolone in the treatment of multiple myeloma: An overview of published trials. J Clin Oncol 10:334–342 4. Facon T (2004) Randomized clinical trial comparing melphalan-prednisone (MP), MP-thalidomide (MP-THAL) and high-dose therapy using melphalan 100 MG/ M2 (MEL100) for newly diagnosed myeloma patients aged 65–75 years. Interim analysis of the IFM 99-06 trial on 350 patients. Blood 104, Abstract 206 5. Palumbo A, Bringhen S, Caravita T et al (2006) Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: Randomised controlled trial. Lancet 367:825–831 6. Attal M, Harousseau JL, Stoppa AM et  al (1996) A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. Intergroupe Francais du Myelome. N Engl J Med 335:91–97 7. Child JA, Morgan GJ, Davies FE et al (2003) High-dose chemotherapy with hematopoietic stem-cell rescue for multiple myeloma. N Engl J Med 348:1875–1883 8. Pasquini M (2006) Report on state of the art in blood and marrow transplantation. In: Center for international blood and marrow transplant research (CIBMTR) Newsletter 9. Lenhoff S, Hjorth M, Holmberg E et  al (2000) Impact on survival of high-dose therapy with autologous stem cell support in patients younger than 60 years with newly diagnosed multiple myeloma: A population-based study. Nordic Myeloma Study Group. Blood 95:7–11 10. Palumbo A, Triolo S, Argentino C et  al (1999) Dose-intensive melphalan with stem cell support (MEL100) is superior to standard treatment in elderly myeloma patients. Blood 94:1248–1253

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R.L. Olin et al. 11. Fermand JP, Ravaud P, Chevret S et al (1998) High-dose therapy and autologous peripheral blood stem cell transplantation in multiple myeloma: Up-front or rescue treatment? Results of a multicenter sequential randomized clinical trial. Blood 92:3131–3136 12. Fermand JP, Katsahian S, Divine M et al (2005) High-dose therapy and autologous blood stem-cell transplantation compared with conventional treatment in myeloma patients aged 55 to 65 years: Long-term results of a randomized control trial from the Group Myelome-Autogreffe. J Clin Oncol 23:9227–9233 13. Blade J, Rosinol L, Sureda A et  al (2005) High-dose therapy intensification compared with continued standard chemotherapy in multiple myeloma patients responding to the initial chemotherapy: Long-term results from a prospective randomized trial from the Spanish cooperative group PE. Blood 106:3755–3759 14. Barlogie B, Kyle RA, Anderson KC et al (2006) Standard chemotherapy compared with high-dose chemoradiotherapy for multiple myeloma: Final results of phase III US Intergroup Trial S9321. J Clin Oncol 24:929–936 15. Attal M, Harousseau JL, Facon T et  al (2003) Single versus double autologous stem-cell transplantation for multiple myeloma. N Engl J Med 349:2495–2502 16. Sonneveld P (2005) Intensive versus double intensive therapy in untreated multiple myeloma: Final analysis of the HOVON 24 trial. Blood 106, Abstract 2545 17. Cavo M, Tosi P, Zamagni E et al (2007) Prospective, randomized study of single compared with double autologous stem-cell transplantation for multiple myeloma: Bologna 96 clinical study. J Clin Oncol 25:2434–2441 18. Richardson PG, Schlossman R, Hideshima T, Anderson KC (2005) New treatments for multiple myeloma. Oncology (Williston Park) 19:1781–1792 discussion 1792, 1795–1797 19. Libura J, Hoffmann T, Passweg J et al (1999) Graft-versus-myeloma after withdrawal of immunosuppression following allogeneic peripheral stem cell transplantation. Bone Marrow Transplant 24:925–927 20. Tricot G, Vesole DH, Jagannath S et al (1996) Graft-versus-myeloma effect: Proof of principle. Blood 87:1196–1198 21. Aschan J, Lonnqvist B, Ringden O, Kumlien G, Gahrton G (1996) Graft-versusmyeloma effect. Lancet 348:346 22. Verdonck LF, Lokhorst HM, Dekker AW, Nieuwenhuis HK, Petersen EJ (1996) Graft-versus-myeloma effect in two cases. Lancet 347:800–801 23. Kroger N, Kruger W, Renges H et al (2001) Donor lymphocyte infusion enhances remission status in patients with persistent disease after allografting for multiple myeloma. Br J Haematol 112:421–423 24. Lokhorst HM, Schattenberg A, Cornelissen JJ et  al (2000) Donor lymphocyte infusions for relapsed multiple myeloma after allogeneic stem-cell transplantation: Predictive factors for response and long-term outcome. J Clin Oncol 18:3031–3037 25. Lokhorst HM, Wu K, Verdonck LF et al (2004) The occurrence of graft-versushost disease is the major predictive factor for response to donor lymphocyte infusions in multiple myeloma. Blood 103:4362–4364 26. Salama M, Nevill T, Marcellus D et al (2000) Donor leukocyte infusions for multiple myeloma. Bone Marrow Transplant 26:1179–1184 27. van de Donk NW, Kroger N, Hegenbart U et al (2006) Prognostic factors for donor lymphocyte infusions following non-myeloablative allogeneic stem cell transplantation in multiple myeloma. Bone Marrow Transplant 37:1135–1141 28. Gahrton G, Tura S, Ljungman P et al (1991) Allogeneic bone marrow transplantation in multiple myeloma. European Group for Bone Marrow Transplantation. N Engl J Med 325:1267–1273 29. Bjorkstrand B (2001) European Group for Blood and Marrow Transplantation Registry studies in multiple myeloma. Semin Hematol 38:219–225

Chapter 16  Allogeneic Transplantation for the Treatment of Multiple Myeloma  30. Bensinger WI, Buckner CD, Anasetti C et al (1996) Allogeneic marrow transplantation for multiple myeloma: An analysis of risk factors on outcome. Blood 88: 2787–2793 31. Bensinger WI, Maloney D, Storb R (2001) Allogeneic hematopoietic cell transplantation for multiple myeloma. Semin Hematol 38:243–249 32. Reece DE, Shepherd JD, Klingemann HG et  al (1995) Treatment of myeloma using intensive therapy and allogeneic bone marrow transplantation. Bone Marrow Transplant 15:117–123 33. Couban S, Stewart AK, Loach D, Panzarella T, Meharchand J (1997) Autologous and allogeneic transplantation for multiple myeloma at a single centre. Bone Marrow Transplant 19:783–789 34. Russell NH, Miflin G, Stainer C et al (1997) Allogeneic bone marrow transplant for multiple myeloma. Blood 89:2610–2611 35. Cavo M, Bandini G, Benni M et al (1998) High-dose busulfan and cyclophosphamide are an effective conditioning regimen for allogeneic bone marrow transplantation in chemosensitive multiple myeloma. Bone Marrow Transplant 22:27–32 36. Majolino I, Corradini P, Scime R et al (1998) Allogeneic transplantation of unmanipulated peripheral blood stem cells in patients with multiple myeloma. Bone Marrow Transplant 22:449–455 37. Mehta J, Tricot G, Jagannath S et  al (1998) Salvage autologous or allogeneic transplantation for multiple myeloma refractory to or relapsing after a first-line autograft? Bone Marrow Transplant 21:887–892 38. Kulkarni S, Powles RL, Treleaven JG et al (1999) Impact of previous high-dose therapy on outcome after allografting for multiple myeloma. Bone Marrow Transplant 23:675–680 39. Russell N, Bessell E, Stainer C et al (2000) Allogeneic haemopoietic stem cell transplantation for multiple myeloma or plasma cell leukaemia using fractionated total body radiation and high-dose melphalan conditioning. Acta Oncol 39:837–841 40. Gahrton G, Svensson H, Cavo M et al (2001) Progress in allogenic bone marrow and peripheral blood stem cell transplantation for multiple myeloma: A comparison between transplants performed 1983–93 and 1994–98 at European Group for Blood and Marrow Transplantation centres. Br J Haematol 113:209–216 41. Le Blanc R, Montminy-Metivier S, Belanger R et  al (2001) Allogeneic transplantation for multiple myeloma: Further evidence for a GVHD-associated graftversus-myeloma effect. Bone Marrow Transplant 28:841–848 42. Reynolds C, Ratanatharathorn V, Adams P et al (2001) Allogeneic stem cell transplantation reduces disease progression compared to autologous transplantation in patients with multiple myeloma. Bone Marrow Transplant 27:801–807 43. Alyea E, Weller E, Schlossman R et  al (2003) Outcome after autologous and allogeneic stem cell transplantation for patients with multiple myeloma: Impact of graft-versus-myeloma effect. Bone Marrow Transplant 32:1145–1151 44. Huff CA, Fuchs EJ, Noga SJ et  al (2003) Long-term follow-up of T celldepleted allogeneic bone marrow transplantation in refractory multiple myeloma: Importance of allogeneic T cells. Biol Blood Marrow Transplant 9:312–319 45. Kroger N, Einsele H, Wolff D et al. Myeloablative intensified conditioning regimen with in vivo T-cell depletion (ATG) followed by allografting in patients with advanced multiple myeloma. A phase I/II study of the German Study-group Multiple Myeloma (DSMM). Bone Marrow Transplant 2003;31:973–9. 46. Lokhorst HM, Segeren CM, Verdonck LF et  al (2003) Partially T-cell-depleted allogeneic stem-cell transplantation for first-line treatment of multiple myeloma: A prospective evaluation of patients treated in the phase III study HOVON 24 MM. J Clin Oncol 21:1728–1733

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R.L. Olin et al. 47. Rabitsch W, Prinz E, Ackermann J et al (2004) Long-term follow up of patients with multiple myeloma after high-dose chemotherapy and allogeneic stem cell transplantation. Eur J Haematol 72:26–31 48. Arora M, McGlave PB, Burns LJ et al (2005) Results of autologous and allogeneic hematopoietic cell transplant therapy for multiple myeloma. Bone Marrow Transplant 35:1133–1140 49. Hunter HM, Peggs K, Powles R et al (2005) Analysis of outcome following allogeneic haemopoietic stem cell transplantation for myeloma using myeloablative conditioning-evidence for a superior outcome using melphalan combined with total body irradiation. Br J Haematol 128:496–502 50. Kennedy GA, Butler J, Morton J et al (2006) Myeloablative allogeneic stem cell transplantation for advanced stage multiple myeloma: Very long-term follow up of a single center experience. Clin Lab Haematol 28:189–197 51. Kuruvilla J, Shepherd JD, Sutherland HJ et  al (2007) Long-term outcome of myeloablative allogeneic stem cell transplantation for multiple myeloma. Biol Blood Marrow Transplant 13:925–931 52. Bjorkstrand BB, Ljungman P, Svensson H et al (1996) Allogeneic bone marrow transplantation versus autologous stem cell transplantation in multiple myeloma: A retrospective case-matched study from the European Group for Blood and Marrow Transplantation. Blood 88:4711–4718 53. Barlogie B (2003) Comparable survival in multiple myeloma (MM) with high dose therapy (HDT) employing MEL 140  mg/m2 + TBI 12  Gy autotransplants versus standard dose therapy with VBMCP and no benefit from interferon (IFN) maintenance: Results of Intergroup Trial S9321, Blood 102, Abstract 135 54. Bensinger WI, Demirer T, Buckner CD et al (1996) Syngeneic marrow transplantation in patients with multiple myeloma. Bone Marrow Transplant 18:527–531 55. Gahrton G, Svensson H, Bjorkstrand B et al (1999) Syngeneic transplantation in multiple myeloma – a case-matched comparison with autologous and allogeneic transplantation. European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 24:741–745 56. Bensinger WI, Martin PJ, Storer B et al (2001) Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives in patients with hematologic cancers. N Engl J Med 344:175–181 57. Ballen KK, King R, Carston M et al (2005) Outcome of unrelated transplants in patients with multiple myeloma. Bone Marrow Transplant 35:675–681 58. Alyea E, Weller E, Schlossman R et  al (2001) T-cell-depleted allogeneic bone marrow transplantation followed by donor lymphocyte infusion in patients with multiple myeloma: Induction of graft-versus-myeloma effect. Blood 98:934–939 59. Gahrton G, Tura S, Ljungman P et al (1995) Prognostic factors in allogeneic bone marrow transplantation for multiple myeloma. J Clin Oncol 13:1312–1322 60. Gahrton G, Iacobelli S, Apperley J et al (2005) The impact of donor gender on outcome of allogeneic hematopoietic stem cell transplantation for multiple myeloma: Reduced relapse risk in female to male transplants. Bone Marrow Transplant 35:609–617 61. Badros A, Barlogie B, Siegel E et  al (2002) Improved outcome of allogeneic transplantation in high-risk multiple myeloma patients after nonmyeloablative conditioning. J Clin Oncol 20:1295–1303 62. Einsele H, Schafer HJ, Hebart H et al (2003) Follow-up of patients with progressive multiple myeloma undergoing allografts after reduced-intensity conditioning. Br J Haematol 121:411–418 63. Garban F, Attal M, Rossi JF et  al (2001) Immunotherapy by non-myeloablative allogeneic stem cell transplantation in multiple myeloma: Results of a pilot study as salvage therapy after autologous transplantation. Leukemia 15:642–646 64. Gerull S, Goerner M, Benner A et al (2005) Long-term outcome of nonmyeloablative allogeneic transplantation in patients with high-risk multiple myeloma. Bone Marrow Transplant 36:963–969

Chapter 16  Allogeneic Transplantation for the Treatment of Multiple Myeloma  65. Giralt S, Aleman A, Anagnostopoulos A et  al (2002) Fludarabine/melphalan conditioning for allogeneic transplantation in patients with multiple myeloma. Bone Marrow Transplant 30:367–373 66. Kroger N, Sayer HG, Schwerdtfeger R et al (2002) Unrelated stem cell transplantation in multiple myeloma after a reduced-intensity conditioning with pretransplantation antithymocyte globulin is highly effective with low transplantation-related mortality. Blood 100:3919–3924 67. Kroger N, Perez-Simon JA, Myint H et al (2004) Relapse to prior autograft and chronic graft-versus-host disease are the strongest prognostic factors for outcome of melphalan/fludarabine-based dose-reduced allogeneic stem cell transplantation in patients with multiple myeloma. Biol Blood Marrow Transplant 10:698–708 68. Lee CK, Badros A, Barlogie B et al (2003) Prognostic factors in allogeneic transplantation for patients with high-risk multiple myeloma after reduced intensity conditioning. Exp Hematol 31:73–80 69. Ma SY, Lie AK, Au WY et  al (2004) Non-myeloablative allogeneic peripheral stem cell transplantation for multiple myeloma. Hong Kong Med J 10:77–83 70. Majolino I, Davoli M, Carnevalli E et  al (2007) Reduced intensity conditioning with thiotepa, fludarabine, and melphalan is effective in advanced multiple myeloma. Leuk Lymphoma 48:759–766 71. Mohty M, Boiron JM, Damaj G et al (2004) Graft-versus-myeloma effect following antithymocyte globulin-based reduced intensity conditioning allogeneic stem cell transplantation. Bone Marrow Transplant 34:77–84 72. Peggs KS, Mackinnon S, Williams CD et al (2003) Reduced-intensity transplantation with in vivo T-cell depletion and adjuvant dose-escalating donor lymphocyte infusions for chemotherapy-sensitive myeloma: Limited efficacy of graft-versustumor activity. Biol Blood Marrow Transplant 9:257–265 73. Perez-Simon JA, Sureda A, Fernandez-Aviles F et  al (2006) Reduced-intensity conditioning allogeneic transplantation is associated with a high incidence of extramedullary relapses in multiple myeloma patients. Leukemia 20:542–545 74. Schmidt-Hieber M, Blau IW, Trenschel R et al (2007) Reduced-toxicity conditioning with fludarabine and treosulfan prior to allogeneic stem cell transplantation in multiple myeloma. Bone Marrow Transplant 39:389–396 75. Shimazaki C, Fujii H, Yoshida T et  al (2005) Reduced-intensity conditioning allogeneic stem cell transplantation for multiple myeloma: Results from the Japan Myeloma Study Group. Int J Hematol 81:342–348 76. van Dorp S, Meijer E, van de Donk NW et al (2007) Single-centre experience with nonmyeloablative allogeneic stem cell transplantation in patients with multiple myeloma: Prolonged remissions induced. Neth J Med 65:178–184 77. Zhang XH, Huang XJ, Liu KY et al (2007) Modified conditioning regimen busulfancyclophosphamide followed by allogeneic stem cell transplantation in patients with multiple myeloma. Chin Med J (Engl) 120:463–468 78. Perez-Simon JA, Diez-Campelo M, Martino R et al (2005) Influence of the intensity of the conditioning regimen on the characteristics of acute and chronic graftversus-host disease after allogeneic transplantation. Br J Haematol 130:394–403 79. Crawley C, Lalancette M, Szydlo R et al (2005) Outcomes for reduced-intensity allogeneic transplantation for multiple myeloma: An analysis of prognostic factors from the Chronic Leukaemia Working Party of the EBMT. Blood 105: 4532–4539 80. Georges GE, Maris MB, Maloney DG et  al (2007) Nonmyeloablative unrelated donor hematopoietic cell transplantation to treat patients with poor-risk, relapsed, or refractory multiple myeloma. Biol Blood Marrow Transplant 13:423–432 81. Crawley C, Iacobelli S, Bjorkstrand B et al (2007) Reduced-intensity conditioning for myeloma: Lower nonrelapse mortality but higher relapse rates compared with myeloablative conditioning. Blood 109:3588–3594

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R.L. Olin et al. 82. Perez-Simon JA, Martino R, Alegre A et  al (2003) Chronic but not acute graft-versus-host disease improves outcome in multiple myeloma patients after non-myeloablative allogeneic transplantation. Br J Haematol 121:104–108 83. Kroger N, Schilling G, Einsele H et al (2004) Deletion of chromosome band 13q14 as detected by fluorescence in situ hybridization is a prognostic factor in patients with multiple myeloma who are receiving allogeneic dose-reduced stem cell transplantation. Blood 103:4056–4061 84. Qazilbash MH, Saliba R, De Lima M et al (2006) Second autologous or allogeneic transplantation after the failure of first autograft in patients with multiple myeloma. Cancer 106:1084–1089 85. Lee CK, Zangari M, Fassas A et al (2006) Clonal cytogenetic changes and myeloma relapse after reduced intensity conditioning allogeneic transplantation. Bone Marrow Transplant 37:511–515 86. Carella AM, Beltrami G, Corsetti MT et al (2004) A reduced intensity conditioning regimen for allografting following autografting is feasible and has strong antimyeloma activity. Haematologica 89:1534–1536 87. Galimberti S, Benedetti E, Morabito F et  al (2005) Prognostic role of minimal residual disease in multiple myeloma patients after non-myeloablative allogeneic transplantation. Leuk Res 29:961–966 88. Kroger N, Schwerdtfeger R, Kiehl M et al (2002) Autologous stem cell transplantation followed by a dose-reduced allograft induces high complete remission rate in multiple myeloma. Blood 100:755–760 89. Maloney DG, Molina AJ, Sahebi F et al (2003) Allografting with nonmyeloablative conditioning following cytoreductive autografts for the treatment of patients with multiple myeloma. Blood 102:3447–3454 90. Seok L (2003) Autologous stem cell transplantation followed by nonmyeloablative allogeneic stem cell transplantation as a first-line therapy in patients with newly diagnosed multiple myeloma: A prospective phase 2 study. Blood 102, Abstract 91. Garban F, Attal M, Michallet M et al (2006) Prospective comparison of autologous stem cell transplantation followed by dose-reduced allograft (IFM99-03 trial) with tandem autologous stem cell transplantation (IFM99-04 trial) in high-risk de novo multiple myeloma. Blood 107:3474–3480 92. Bruno B, Rotta M, Patriarca F et  al (2007) A comparison of allografting with autografting for newly diagnosed myeloma. N Engl J Med 356:1110–1120 93. McSweeney PA, Niederwieser D, Shizuru JA et al (2001) Hematopoietic cell transplantation in older patients with hematologic malignancies: Replacing high-dose cytotoxic therapy with graft-versus-tumor effects. Blood 97:3390–3400 94. Ayuk F, Shimoni A, Nagler A et  al (2004) Efficacy and toxicity of low-dose escalating donor lymphocyte infusion given after reduced intensity conditioning allograft for multiple myeloma. Leukemia 18:659–662 95. Mohty M, Attal M, Marit G et al (2005) Thalidomide salvage therapy following allogeneic stem cell transplantation for multiple myeloma: A retrospective study from the Intergroupe Francophone du Myelome (IFM) and the Societe Francaise de Greffe de Moelle et Therapie Cellulaire (SFGM-TC). Bone Marrow Transplant 35:165–169 96. Kroger N, Shimoni A, Zagrivnaja M et al (2004) Low-dose thalidomide and donor lymphocyte infusion as adoptive immunotherapy after allogeneic stem cell transplantation in patients with multiple myeloma. Blood 104:3361–3363 97. van de Donk NW, Kroger N, Hegenbart U et  al (2006) Remarkable activity of novel agents bortezomib and thalidomide in patients not responding to donor lymphocyte infusions following nonmyeloablative allogeneic stem cell transplantation in multiple myeloma. Blood 107:3415–3416

Chapter 16  Allogeneic Transplantation for the Treatment of Multiple Myeloma  98. Corradini P, Voena C, Tarella C et al (1999) Molecular and clinical remissions in multiple myeloma: Role of autologous and allogeneic transplantation of hematopoietic cells. J Clin Oncol 17:208–215 99. Corradini P, Cavo M, Lokhorst H et al (2003) Molecular remission after myeloablative allogeneic stem cell transplantation predicts a better relapse-free survival in patients with multiple myeloma. Blood 102:1927–1929

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Chapter 17 Blood Vs. Marrow Allogeneic Stem Cell Transplantation Brian McClune and Daniel Weisdorf

1. Introduction Since first being reported in 1989 [1], peripheral blood stem and progenitor cells (PBPC) have become extensively used as a graft source in allogeneic transplantation. Case series demonstrating its feasibility in the allogeneic setting first appeared in the mid-1990s [2–4]. In 2007, data from the Center for International Blood Marrow Transplant Research (CIBMTR) indicated that nearly 80% of all allogeneic transplants performed between 2002 and 2006 used PBPC for those aged >20 years versus 1997–2001 when marrow grafts were used in nearly 60% of all adult transplants [5]. Contributing to this broadening use of peripheral blood stem cell transplants is the increased utilization of unrelated donors (URD) [6], more experience with the procedure, and the suggestion of superior outcomes in certain diseases and settings. Despite the rapid acceptance of PBPC for transplantation, only recently have data from randomized trials [7–20], large registry-based data [21, 22], and studies with some long term follow-up become available. This chapter will review the known biological and clinical characteristics of PBPC transplantation, with an emphasis on information obtained from randomized trials and large registry analyses.

2. Biology of Peripheral Blood Progenitor and Stem Cells Normally, very low numbers of hematopoietic progenitor cells circulate in the peripheral blood (0.06% of all nucleated cells) [23]. They are generally identified by surface marker expression of CD34 (stem cell antigen), though their progeny will also express CD34 in addition to lineage specific markers after commitment to myeloid, lymphoid, or erythroid lineages. However, within the CD34+ lineage-negative population are the hematopoietic stem cells capable of sustaining long-term engraftment. In recent years, other surrogates to identify and efnumerate hematopoietic stem and progenitors cells (HSC) have also been

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_17, © Springer Science + Business Media, LLC 2003, 2010

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identified. CD133 is a pentaspan molecule found on cells more primitive than CD34+ cells and is down-regulated early in differentiation [25]. The marker is also found on primitive endothelial and neuronal cells and thought to be involved in plasma membrane organization and structure. It was recognized that CD133 expression was restricted to CD34+ bright cells and that long-term hematopoietic reconstitution could be demonstrated not only in animals but also in humans with cells that were CD133+ [26]. Isidori et al. published their data on CD133+ selected grafts in 12 patients with chronic lymphocytic leukemia (CLL) undergoing autologous transplantation [26]. All patients exhibited rapid engraftment and platelet recovery that was independent of the number of CD133+ cells infused. Hicks et  al. described 20 myeloma patients after autologous transplantation and evaluated numbers of viable CD34+ and CD34+CD133+ cells infused along with engraftment kinetics [28]. Viable CD34+CD133+ cells correlated well with neutrophil recovery with more rapid engraftment accompanying higher CD34+ or CD34+CD133+ cell doses. No correlation with platelet engraftment was apparent suggesting that CD133+ determination may best be used in predicting neutrophil recovery. Two publications including a total of only 15 patients, report on the feasibility of CD133+ selection in the allogeneic setting and suggest that the repopulating potential of CD133+ selected cells is at least as good as CD34+ selection and provides less acute toxicities to patients from graft-versus-host disease (GVHD) likely because of a significant decrease in T cells with the allograft [26, 29]. Whether this is enough to supplant CD34+ selection will only be borne out by future studies. Administration of cytokines is generally needed to increase the numbers of HSC in circulation. This results not only in an increase in the total leukocyte count but also in a preferential increase in the concentrations of PBPC [24, 30]. Currently, only granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) are FDA approved for mobilization of stem cells with G-CSF being the most widely used. Earlier studies showed that GM-CSF is as effective as G-CSF in terms of CD34+ yield but may have an unfavorable side effect profile. These adverse effects may be balanced by the reduced occurrence of acute GVHD in the GM-CSF mobilized group although this has not been confirmed nor widely applied [30–33]. The mechanism of cytokine-mobilization is not well understood despite extensive study over the last 10  years. Adhesion molecules binding HSC to the marrow microenvironment and their interaction with these cytokines play a significant role in this process. Of particular importance is the CXC family which includes stromal-derived factor-1 (SDF-1). This factor interacts with CXCR4 to help regulate migration of CD34+ cells from the marrow space and has been recently exploited to further increase yields in stem cell mobilization (see below). The N-terminal portion of the cytokine GROb, multiple proteases that help break down adhesions in the marrow niche and even neuron-regulatory signals likely participate in movement of CD34+ cells into the circulation [35–37]. Other agents can mobilize stem cell populations including pegylated filgrastim (Neulasta), FLT-3 ligand in combination with G-CSF [38, 39], and a newer agent called AMD3100 (plerixafor). AMD3100 is a bicyclam derivative first identified as an agent with anti-HIV activity in the 1980s that induced sustained

Chapter 17  Blood Vs. Marrow Allogeneic Stem Cell Transplantation 

neutrophilia [40]. Its mechanism of action was eventually deduced to be reversible competitive inhibition of SDF-1 binding to the CXCR4 receptor, favoring mobilization of CD34+ cells into the circulation. Preclinical and animals trials demonstrated that AMD3100 could mobilize progenitor cells for engraftment. Investigations showed up to tenfold increases in PBPC following a single dose of 240 mcg/kg. Commonly reported side effects included erythema and pain at the injection site, nausea, perioral numbness, and headache [41]. Further study of the drug in the autologous transplant setting for myeloma, and nonHodgkins and Hodgkins lymphoma show that when added to G-CSF, collections have more CD34+ cells per leukopheresis, patients require fewer total collections, and that stable engraftment is successfully achieved in nearly all patients [42–44]. Subsequently, Devine et al. performed a pilot study examining the safety and efficacy of plerixafor in mobilization of HLA-matched related donors (MRD) [45]. The study evaluated mobilization with either one or two doses of AMD3100 at 240 mcg/kg and then after a 1 week washout period, mobilization with G-CSF alone. Eight of nine donors enrolled yielded at least 2.0 × 106 CD34+ cells/kg recipient weight after one or two collections. The grafts contained fewer CD34+ cells and greater numbers of T-,B-, and NK-cells compared to the G-CSF mobilized products. Seven of the patients then proceeded to transplant using the AMD3100 mobilized cells. Engraftment was comparable to G-CSF allografts as was the incidence of acute GVHD. Further trials to assess this new agent alone or in combination may be promising in limiting donor morbidity as long as recipient outcomes are carefully assessed as well.

3.  Clinical Aspects of Peripheral Blood Stem Cell Transplantation Knowledge of the clinical aspects of PBPC transplantation has increased dramatically in recent years with the proliferation of studies comparing it to marrow transplantation. While early results are clear, late outcomes continue to require further investigation and longer patient follow-up. 3.1. Graft Characteristics The evidence that greater CD34+ cell numbers are obtained using peripheral blood harvesting is strong. Seven of ten randomized trials published to date have harvested and infused greater numbers of CD34+ cells from peripheral blood donors than from marrow donors [7, 13–14, 16–19]. Three of these studies demonstrated a statistically significant increment in blood progenitors, with at least twice the number collected by apheresis as were collected from marrow [13, 14, 17]. Graft composition data were not uniformly available in a comparison of peripheral blood vs. marrow stem cell transplantation reported from the International Bone Marrow Transplant Registry (IBMTR) [21], but several smaller series comparing PBPC recipients with historical or nonrandomized controls have shown higher CD34+ cell concentrations in peripheral blood [23, 24]. The minimum number of cells needed for successful engraftment remains unclear. It has been estimated that approximately 2.5–5 × 106 CD34+ cells/kg is sufficient for consistently rapid engraftment [3], but no experimental

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evidence confirms this hypothesis. A higher CD34+ cell dose has been reported to facilitate more rapid platelet engraftment in allogeneic transplants [46], as was also shown in autologous transplants [47]. Data from clinical trials of PBPCs suggest that higher CD34+ counts promote more rapid neutrophil and platelet engraftment in the allogeneic setting as well, but other cell populations also affect both the quality of the graft and the likely occurrence of GVHD. 3.2. Engraftment Both neutrophil and platelet engraftment consistently occur more rapidly when using peripheral blood grafts as opposed to marrow grafts. All ten published randomized trials found a shorter time to both neutrophil and platelet engraftment (Table  17.1), as do virtually all smaller series with nonrandomized controls. In the ten published randomized trials, neutrophil engraftment occurred 1–6 days earlier in PBPC transplants than in marrow transplants. Differences were statistically significant in eight of the ten trials. In the larger IBMTR analysis, neutrophil engraftment occurred 5 days earlier in PBPC transplants than in marrow transplants [21]. Platelet engraftment also occurs more rapidly using peripheral blood allografts. All randomized trials found more rapid platelet recovery, with the median differences ranging from 4 to 13  days. The IBMTR analysis found that platelet engraftment occurred a median of 7  days earlier in the PBPC transplants than in traditional marrow transplants [21]. As with neutrophil engraftment, numerous smaller series have shown similar results. Seven of the ten randomized trials reported information about transfusions administered during the observational period. In the largest trial, the median days of platelet transfusions required in peripheral blood recipients was significantly less than that in marrow recipients (8 vs. 10 days; p = 0.0029); red blood cell (RBC) transfusion requirements were not reported [8]. One of the larger European trials found similar results in terms of platelet transfusions with the time to last platelet transfusion being less in the PBPC transplant arm (21 vs. 26 days, p = 0.03). Time to last red blood cell transfusion was also less (42 vs. 68 days, p < 0.005) [19]. There was no difference in the number of RBC transfusions between the marrow and blood groups in the Canadian trial [18]. A smaller trial found that fewer platelet transfusions were required in peripheral blood recipients (PBPC 12.5, BMT 17.5) but similar RBC transfusions in the two groups (PBPC 5, BMT 6) [7]. 3.3. Graft Versus Host Disease Since the earliest reports of peripheral blood allografting, there has been considerable concern that because such high numbers of lymphocytes are transplanted in peripheral blood grafts, GVHD would occur at high rates [1–4]. In clinical studies to date, however, little data has emerged suggesting differences in overall rates of acute GVHD [10, 13, 14, 17, 18, 20]. In contrast, chronic GVHD has been shown to occur more commonly in patients receiving peripheral blood allografts [21, 48]. Because of the longer observation time needed to ascertain the incidence of chronic GVHD, data are incomplete in many studies.

1998

1998

1999

2000

2000

2000

2001

2002

2003

2005

EBMT [7–9]

Brazil [10, 11]

Mahmoud [12]

France [13]

U.K. [14]

Hedal [15, 16]

U.S [17].

Canada [18]

Dutch [19]

Oehler [20]

32

56

109

81

31

20

48

15

18

163

PBSC

N

40

54

118

91

30

19

52

15

19

166

BMT

b

Indicates p < 0.05 for given result Reported as proportions, not estimation NR not reported

a

Year

Trial

CSA/Pred

CSA/MTX

GVHD prophylaxis

CSA/MTX

CSA/MTX

CML

AML,MDS, MM, lymphoma,

CML, AML, MDS

AML, ALL, NHL, HD, CML, MM, MDS,

CML, AML, ALL, MM

CSA/MTX

CSA

CSA/MTX

CSA/MTX

CSA/MTX

ALL, AML, CML, NHL, CSA/MTX MDS, MM

ALL, AML in first or second CRCML in first CP

AML, ALL, MDS, SAA

CML, AML, ALL, MDS, CSA/MTX NHL, MMEarly and advanced disease

AML, ALL in first or second remission, CML in first chronic phase

Major diagnoses

16 days

15 days

19 days

16 days

17 days

17 days

15 days

NR

17 daysa

20 daysa

BMT

15 days

11 days 13 days

13 days

16 days 25 days 14 days

21 daysa

23 daysa 23 daysa

21 daysa

23 daysa 20 daysa 21 daysa

21 days

38 days

22 daysa

19 daysa

21 daysa

18 daysa

26 daysa

Exact numbers NRa

12 days

15 days

15 daysa

18 days

PBSC

Platelet recovery

BMT

Exact numbers

16 days

12 days

PBSC

Neutrophil engraftment

Table 17-1.  Randomized clinical trials investigating PBPC vs. BMT.

82%

48%

68%

(p = 0.06)

66%

63%

70%

67%

NR

51%

58%

PBSC

84%

57%

60%a

54%

83%b

63%

65%

47%

65%

BMT

Overall survival

55

52

44

64

21

50

44

6.7

27

52

49

37

44

57

10

47

37%

40%

28%a

53%

55%a

BMT

59%

50%

85%

46%

50%

42%

69%

35%

(p = 0.03 for extensive cGVHD)

57%

44%

50%

NR

47a

42

71%

73%

PBSC

Chronic GVHD

19

39a

PBSC BMT (%) (%)

Acute GVHD (grade II–IV)

36 months

37 months

33 months

26 months

5 years

33 months

20 months

NR

>5 years

3 years

Median follow-up

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3.3.1. Acute Graft Versus Host Disease Overall rates of acute GVHD are generally similar in patients receiving peripheral blood or marrow transplants. Seven of ten randomized trials have found a 44–68% rate of grade II–IV acute GVHD in patients receiving peripheral blood transplants compared with a 37–57% rate in patients receiving marrow transplants [7, 13, 14, 17–20]. IBMTR analyses (Fig. 17.1) found that grade II–IV acute GVHD occurred in 40% of patients receiving peripheral blood transplants and 35% of patients receiving marrow transplants [21]. Only one study reported a significantly greater rate of acute GVHD using PBPC (47% vs. 7%, p = 0.013) [12]. One randomized trial, published only in abstract form, found similar rates of grade II–IV acute GVHD in peripheral blood and marrow transplant recipients (PBPC 54% vs. BMT 48%), but PBPC patients were more likely to develop steroid-dependent or refractory GVHD (PBPC 50% vs. BMT 14%, p = 0.003) [82]. In this trial, grade III–IV acute GVHD was also higher in patients receiving peripheral blood transplants (PBPC 46% vs. BMT 17%, p = 0.02). Rates of grade III–IV acute GVHD were found to be significantly worse with PBPC in the largest randomized trial (PBPC 28% vs. BMT 16%; p = 0.0088) [8]. 3.3.2. Chronic Graft Versus Host Disease A growing body of evidence suggests that chronic GVHD is more frequent in patients receiving peripheral blood allografts. Reported results from all randomized trials published show an increased risk of chronic GVHD [8–20], even though follow-up periods in these trials are generally short and may not

Fig. 17-1.  Probabilities of grades II–IV acute graft versus host disease (GVHD) after HLA-identical sibling blood stem cell transplantation compared with bone marrow transplantation for acute leukemia and CML. Probabilities were derived from multivariate Cox proportional hazards models and adjusted for effects of other significant covariates. Adapted from Champlin et al. Blood 2000

Chapter 17  Blood Vs. Marrow Allogeneic Stem Cell Transplantation 

fully represent the burden of disease in study participants. One trial designed to determine differences in GVHD on the basis of stem cell source closed enrollment early because the incidence of extensive chronic GVHD was significantly greater in patients receiving peripheral blood (77% vs. 27%) [80]. The IBMTR analysis (Fig. 17.2) also found significantly more chronic GVHD in PBPC transplants (65% vs. 53%, p = 0.05) [21], but the difference between groups did not become evident until 10–12 months after transplant. Moreover, rates of chronic GVHD did not plateau until 12–15  months after treatment, suggesting that follow-up times less than 2 years are inadequate for determining true incidence of chronic GVHD. The difference between the PBPC and BMT groups worsened in an updated analysis (PBPC 61% vs. BMT 45%; RR 1.65, p < 0.001) [22]. Another smaller cohort initially reported no difference in chronic GVHD at a median follow-up of less than 1  year [49], but more chronic GVHD was noted in peripheral blood recipients when follow-up was extended to 2 years [50]. Risk factors for developing chronic GVHD in the IBMTR analysis included gender, no GVHD prophylaxis, and age ³40 [22]. Two trials that reported an increased risk of chronic GVHD used limited methotrexate prophylactic regimens [10, 13] suggesting that prophylaxis with four doses of methotrexate is important. A meta-analysis done by the Stem Cell Trialists’ Collaborative Group confirmed a significant increase in the odds of developing chronic GVHD in patients undergoing PBPC transplantation (OR 1.43, p < 0.016); however, it did not confirm a statistical benefit from the day 11 dose of methotrexate [51]. In one report, chronic GVHD after PBPC transplant required treatment with more regimens and for longer periods suggesting that chronic GVHD related to PBPC is more difficult to control [52]. Follow up reports from other studies are awaited in order to better assess any differences in rates and severity of chronic GVHD.

Fig. 17-2.  Probabilities of chronic GVHD (limited or extensive disease) after HLAidentical sibling blood stem cell transplantation compared with bone marrow transplantation for acute leukemia and CML. Probabilities were derived from multivariate Cox proportional hazards models and adjusted for effects of other significant covariates. Champlin et al., Blood 2000. Used with permission

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3.4. Infections Some, though unconfirmed, data suggest that PBPC recipients have fewer infectious complications than marrow recipients. The Seattle report showed that culture-proven infections were 1.7 times more frequent in marrow recipients compared to PBPC recipients between days 30 and 365 of transplant (p = 0.001) [53]. The rate of infections requiring inpatient treatment was 2.4 times higher in marrow recipients (p = 0.002). The difference in infection rates was greatest for fungal infections, but was also apparent for bacterial and viral infections. Deaths from infections were slightly more frequent after BMT. Nucci reviewed infection rates as part of a larger randomized trial [54]. No differences in the frequency of fever, infection, use of antifungal therapy, or length of antibiotic pre-engraftment use were found. However, following engraftment, more extensive chronic GHVD in the PBPC group led to more immunosuppressant and antimicrobial use as well as more frequent infections although there was no increased need for hospitalization or survival difference (40% BMT group, 61% PBPC group, p = 0.27). In a multivariate model, only the duration of steroid use was associated with more infections (p < 0.001). Additional analyses including larger number of patients and carefully organized, prospective, clinical trials are required to probe infection morbidity more fully. 3.5. Survival Despite the popular shift to PBPC grafting, the available data do not demonstrate a clear survival benefit using PBPC. The largest EBMT uiuiu randomized trial demonstrated no overall survival difference between PBPC transplantation and BMT (58% vs. 65%, HR of death for PBPC 1.26, p = 0.19) [9]. Another trial showed a trend to modestly worse overall survival with PBPC [15]. The largest US, randomized trial published to date found a marginal benefit in 2 year survival rate (66% vs. 54%) with PBPC (p = 0.06) although longer follow-up has not confirmed this advantage [17]. A metaanalysis of individual patient data from nine randomized controlled trials also failed to show a survival benefit of PBPC transplantation (Fig. 17.3) [55]. Some studies reported a survival advantage in patients with advanced stage disease. However, updated IBMTR 6 year follow-up data showed lower transplant related mortality (TRM) with PBPC in advanced acute leukemias but equivalent survival rates for both early and advanced disease [22]. It is hypothesized that a more vigorous allogeneic response following PBPC transplantation and thus more potent GVL may reduce relapse rates. No consistent differences in causes of death have been reported. 3.6. Quality of Life Only a few studies have analyzed quality of life in survivors of peripheral blood transplants. Generally, data come from series comparing recent peripheral blood transplant recipients with historical controls who received marrow, limiting the validity of measures that may vary depending on length of follow-up. In one comparison with historical marrow controls 48% of surviving peripheral blood recipients had Karnofsky scores of £80 vs. only 5% of marrow

Chapter 17  Blood Vs. Marrow Allogeneic Stem Cell Transplantation  100

289

PBSCT

90

BMT__

80 Survival (%)

70

56.85%

60 50

55.76%

40

56.85%

53.15%

30 20 10 0

Chi2 Statistic: 1.84 P = 0.17495 Abs Diff at 5 yrs = 2.54%

0

1

2

3 Years

4

5

6

Death/Person-years: PBSCT : 142/455.10 42/351.86 13/292.90 5/174.03 5/99.11 0/45.85 BMT__ : 168/453.36 45/338.56 11/278.98 3/143.07 2/77.44 2/39.45

Fig. 17-3.  Overall survival of 1,111 patients undergoing peripheral blood stem cell (PBSCT) vs. marrow (BMT) transplantation; Abs diff, Absolute difference. (Reproduced and adapted with permission from Stem Cell Trialists’ Collaborative Group: Allogeneic peripheral blood stem-cell compared with bone marrow transplantation in the management of hematologic malignancies: an individual patient data meta-analysis of nine randomized trials. J Clin Oncol. 2005; 23(22):5084. © 2008 American Society of Clinical Oncology. All rights reserved

recipients [48]. Another uncontrolled series found that 64% of blood allograft survivors had Karnofsky scores between 70 and 80 [56]. Chronic GVHD is responsible for most of the excess morbidity accompanying allotransplants. No data on quality of life was included in other published trials. 3.7. Unrelated Peripheral Blood Transplants No randomized trials are reported comparing URD marrow versus peripheral blood allografting. Previous studies with adequate follow-up suggested that there was virtually no difference in rates of acute or chronic GVHD or overall survival in utilizing mobilized PBPC or marrow from URD (median follow-up 4.4, 5 years respectively) [57]. However, extensive chronic GVHD was significantly more common in the PBPC group. OS for both groups was 42%. The CIBMTR reported on URD transplants (2000–2003) including 331 PBPC and 586 BMTs for leukemia/MDS with a median 3  year follow-up [58]. Rates of grade II–IV acute GVHD and chronic GVHD were significantly higher with PBPC transplants (58% vs. 45%, p < 0.001; 56% vs. 42%, p < 0.001); TRM and relapse were similar as was leukemia free survival (30% PBPC vs. 32% BM). A continuing, prospective trial sponsored by the NHLBI/ NCI-funded Blood and Marrow Clinical Trials Network (BMT CTN), 0201, is testing BM versus filgrastim-primed PBPC from unrelated donors. This large trial (n = 550) will complete accrual in early 2009 and is critical to further inform graft choices for URD allografts.

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3.8. Cytokine-Primed Marrow Transplantation Some early data describes G-CSF primed marrow as an alternative source of stem cells. The rationale for marrow priming is on the basis of murine [59] and human [61, 62] studies demonstrating improved repopulating ability of G-CSF primed bone marrow, similar engraftment rates to PBPC transplant, and less acute and chronic GVHD [62, 63]. Cytokine primed marrow grafts generally include higher number of CD34+ cells than unprimed marrow and up to 1 log fewer T cells than PBPC allografts [64, 65]. Lowenthal et al. recently showed that 4 days vs. 6 days of G-CSF prior to marrow harvest allowed for the greatest yield of CD34+ cells/kg [64]. Several single-institution studies highlight the use and safety of primed marrow transplantation [65,57]. One report compared G-CSF mobilized marrow to mobilized PBPC autologous transplant for lymphoma. No significant differences were noted in neutrophil recovery, blood product support, febrile days, or antibiotic days. The PBPC led to slightly greater time to 50 × 109/L platelets (16 days BM vs. 14 days PBPC, p = 0.011). [67] Weisdorf et  al. studied autologous mobilized marrow and PBPC in NHL and Hodgkins patients randomly assigned to either GM-CSF or G-CSF. Graft composition analysis showed more long-term culture-initiating cells (LTC-IC) and committed precursors (CFU-GM) mobilized by either cytokine over unstimulated controls. G-CSF autografts contained more CFU-GM and LTC-IC than GM-CSF mobilized grafts. RBC and platelet but not neutrophil recovery was quicker in marrow mobilized with GM-CSF or PBPC mobilized with G-CSF (p = 0.01) resulting in faster hospital discharge. Similar relapsefree survival suggested that cytokine-primed marrow was as effective as PBPC for autologous transplantation [68, 69]. Morton et  al. randomized 57 patients to receive an allogeneic graft using G-CSF mobilized PBPC or marrow stratified according to risk of relapse [62]. Donors in the marrow arm received G-CSF (10 mcg/kg/day for 5 days) followed by marrow harvest or PBPC harvest. CD34 and CD3 doses were significantly lower in marrow versus PBPC. However, there were no significant differences in neutrophil or platelet engraftment or acute GVHD. Primed marrow led to significantly less chronic GVHD than the PBPC (47% vs. 90%, p < 0.02). Steroid-refractory GVHD was more common in the PBPC arm (47% vs. 18%; p < 0.02) as well. Stem cell source did not affect 18 month survival (67% marrow vs. 64% PBPC). Another series, comparing 26 primed bone marrow recipients to a historical cohort of 20 PBPC blood transplant recipients, found similar neutrophil recovery but slightly more rapid platelet recovery with PBPC. Marrow led to less chronic GVHD (PBPC 68%, primed BMT 37% p = 0.049) but 2 year survival was similar (PBPC 60%, primed BMT 54%, p = 0.9). Other small series [71–73] and a recent review [63] show similar findings, suggesting that further study of primed marrow is warranted.

4. Donor Considerations The safety for PBPC donors has not been well studied. Although short term effects of G-CSF in normal donors are mild [75], little is known about long term effects of G-CSF. Doses of G-CSF used in peripheral blood stem cell harvests range from 2 to 24 mg/kg/d, usually 10 to16 mg/kg/d [7, 10, 12–15, 17, 20, 75].

Chapter 17  Blood Vs. Marrow Allogeneic Stem Cell Transplantation 

The most common short-term adverse effects of G-CSF administration include bone pain, headache, fatigue, fever, and nausea [76]. Less commonly, non-cardiac chest pain, insomnia, night sweats, fluid retention, and dizziness have been reported [75]. Severe side effects requiring discontinuation of G-CSF are uncommon, occurring in 1–3% of donors [76]. Laboratory abnormalities, including transient elevations of alkaline phosphatase and lactate dehydrogenase and less commonly, electrolyte disturbances, have also been noted. Both thrombocytopenia and granulocytopenia have been reported after donation, but these findings result from the leukapheresis procedure itself, not from administration of G-CSF [76]. In general, adverse effects appear to be dose related. Bony pain occurs in 50–84% of donors. It is adequately treated with overthe-counter analgesics and generally subsides within a few days. Autoimmune conditions might be exacerbated by G-CSF administration, including thyroid dysfunction [78–80]. Severe adverse reactions are rare and include 11 cases of splenic rupture; four in healthy donors. Transient increases in spleen size in up to 10% of donors had previously been noted. Additionally, transient gas exchange abnormalities have been seen in healthy donors with one case report of acute lung injury seen by day 4 of G-CSF administration [81]. Rare reports describe acute myeloid leukemia and myelodysplasia arising after G-CSF administration; [76] three in healthy sibling donors, 14 months to 5 years after G-CSF use. Laboratory data from some small studies have shown transient gene expression changes, aneuploidy, and replication asynchrony in lymphocytic cell lines. These inferential data associating G-CSF with these later marrow disorders are weak as all three patients had siblings with malignant hematopoietic conditions. The NMDP/CIBMTR is conducting a prospective study to evaluate donor safety with regard to the possible leukemogenic potential of G-CSF.

5. Cost Only one randomized trial has included an economic analysis of peripheral blood transplants compared with marrow transplants [13]. It found that overall costs of peripheral blood transplants during the first 180 days were 16% less than costs of marrow transplants. The difference was primarily a result of lower room costs in the peripheral blood group, although cost was also reduced because of fewer platelet transfusions, low laboratory fees, and lower overall drug expenses. Graft collection costs were higher in peripheral blood donors. A retrospective analysis from the Netherlands looked at the costs of either a MRD marrow transplant (MRD BMT), a MUD marrow transplant (MUD BMT) or a MRD PBPC transplant performed from 1994–1999 across four transplant centers [84]. Average cost estimates included donor identification expenses and 2  years of follow-up for those who did and did not undergo transplantation. In this analysis, PBPC transplantation was equivalent to MRD BMT (€98,977 vs. €98,334) despite the documented advantage of shorter length of hospital stays, faster hematopoietic recovery, and less antibiotic administration. MUD BMT was particularly costly (€151,754) with nearly one-third of the cost of MUD BMT attributable to transplant donor searching. With the expansion of transplant techniques to include older patients and more indications for transplant being recognized, continued scrutiny of stem cell therapies and their inherent costs is required.

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6. Conclusion Our understanding of peripheral blood stem cell allogeneic transplantation has increased markedly during the past several years. Benefits of peripheral blood transplantation may include more rapid engraftment. More data are needed about long-term survival, late effects, and the use of peripheral blood grafts from both related and unrelated donors. While marrow is no longer the standard of care for most adult transplant allografts, the risks of chronic GVHD and its accompanying morbidity and mortality could shift that paradigm back in favor of BMT. This can only be confirmed by well-designed, long-term studies and follow-up.

References 1. Kessinger A, Smith DM, Strandjord SE, Landmark JD, Dooley DC, Law P et al (1989) Allogeneic transplantation of blood-derived, T cell-depleted hemopoietic stem cells after myeloablative treatment in a patient with acute lymphoblastic leukemia. Bone Marrow Transplant 4:643–646 2. Korbling M, Przepiorka D, Huh YO, Engel H, van Besien K, Giralt S et al (1995) Allogeneic blood stem cell transplantation for refractory leukemia and lymphoma: potential advantage of blood over marrow allografts. Blood 85:1659–1665 3. Bensinger WI, Weaver CH, Appelbaum FR, Rowley S, Demirer T, Sanders J et al (1995) Transplantation of allogeneic peripheral blood stem cells mobilized by re-combinant human granulocyte colony-stimulating factor. Blood 85:1655–1658 4. Schmitz N, Dreger P, Suttorp M, Rohwedder EB, Haferlach T, Loffler H et al (1995) Primary transplantation of allogeneic peripheral blood progenitor cells mobilized by filgrastim (granulocyte colony-stimulating factor). Blood 85:1666–1672 5. National Marrow Donor Program (2008) Outcomes and trends. http://www.marrow. org/PHYSICIAN/Outcomes_Data/index.html. Accessed Jan 2008 6. Ringden O, Remberger M, Runde V, Bornhauser M, Blau IW, Basara N et al (1999) Peripheral blood stem cell transplantation from unrelated donors: a comparison with marrow transplantation. Blood 94:455–464 7. Schmitz N, Bacigalupo A, Hasenclever D, Nagler A, Gluckman E, Clark P et al (1998) Allogeneic bone marrow transplantation vs. filgrastim-mobilised peripheral blood progenitor cell transplantation in patients with early leukaemia: first results of a randomised multicentre trial of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 21:995–1003 8. Schmitz N, Beksac M, Hasenclever D et  al (2002) Transplantation of mobilized peripheral blood cells to HLA-identical siblings with standard risk leukemia. Blood 100:761–767 9. Schmitz N, Beksac M, Bacigalupo A et al (2005) Filgrastim-mobilized peripheral blood progenitor cells versus bone marrow transplantation for treating leukemia: 3-year results from the EBMT randomized trial. Hematologica 90(5):643–648 10. Vigorito AC, Azevedo WM, Marques JF, Azevedo AM, Eid KA, Aranha FJ et al (1998) A randomised, prospective comparison of allogeneic bone marrow and peripheral blood progenitor cell transplantation in the treatment of haematological malignancies. Bone Marrow Transplant 22:1145–1151 11. Vigorito AC, Comenalli Marques JF, Penteado Aranha FJ et  al (2001) A randomized prospective comparison of allogeneic bone marrow and peripheral blood progenitor cell transplantation in the treatment of hematologic malignancies: an update. Haematologica 86:665–666 12. Mahmoud H, Fahmy O, Kamel A et al (1999) Peripheral blood vs. bone marrow as a source for allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant 24:355–358

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B. McClune and D. Weisdorf 29. Lang P, Bader P, Schumm M, Feuchtinger T (2004) Transplantation of a combination of CD133+ and CD34+ selected progenitor cells from alternative donors. Br J Haematol 124(1):72–79 30. Prosper F, Stroncek D, Verfaillie CM (1996) Phenotypic and functional characterization of long-term culture- initiating cells present in peripheral blood progenitor collections of normal donors treated with granulocyte colony-stimulating factor. Blood 88:2033–2042 31. Lane T, Law P, Maruyama M, Young D, Burgess J, Mullen M et  al (1995) Harvesting and enrichment of hematopoietic progenitor cells mobilized into the peripheral blood of normal donors by granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF: potential role in allogeneic marrow transplantation. Blood 85:275–282 32. Devine S, Brown R, Mathews V et  al (2005) Reduced risk of acute GVHD following mobilization of HLA-identical sibling donors with GM-CSF alone. Bone Marrow Transplant 36:531–538 33. Gazzitt Y (2002) Comparison between granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the mobilization of peripheral blood stem cells. Curr Opin Hematol 9:190–198 34. Weaver CH, Schulman KA, Wilson-Relyea B, Birch R, West W, Buckner CD (2000) Randomized trial of filgrastim, sargramostim, or sequential sargramostim and filgrastim after myelosuppressive chemotherapy for the harvesting of peripheralblood stem cells. J Clin Oncol 18:43–53 35. King AG, Horowitz D, Dillon SB, Levin R, Farese AM, MacVittie TJ et al (2001) Rapid mobilization of murine hematopoietic stem cells with enhanced engraftment properties and evaluation of hematopoietic progenitor cell mobilization in rhesus monkeys by a single injection of SB-251353, a specific truncated form of the human CXC chemokine GRO{beta}. Blood 97:1534–1542 36. Lapidot T, Petit I (2002) Current understanding of stem cell mobilization: the roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells. Exp Hematol 30:973–981 37. Katayama Y, Battista M, Kao WM, Hidalgo A, Peired AJ, Thomas SA et al (2006) Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow. Cell 124:407–421 38. Steid U, Fenk R, Bruns I et al (2005) Successful transplantation of peripheral blood stem cells mobilized by chemotherapy and a single dose of pegylated G-CSF in patients with multiple myeloma. Bone Marrow Transplant 35:33–36 39. Papayannopoulou T, Nakamoto B, Andrews RG et  al (1997) In vivo effects of Flt3/Flk2 ligand on mobilization of hematopoietic progenitors in primates and potent synergistic enhancement with granulocyte colony-stimulating factor. Blood 90:620–629 40. De Clercq E (2003) The bicyclam AMD3100 story. Nat Rev Drug Discov 2:581–587 41. Liles WC, Broxmeyer HE, Rodger E, Wood B, Hubel K, Cooper S et  al (2003) Mobilization of hematopoietic progenitor cells in healthy volunteers by AMD3100, a CXCR4 antagonist. Blood 102:2728–2730 42. Devine SM, Flomenberg N, Vesole DH et al (2004) Rapid mobilization of CD34+ cells following administration of the CXCR4 antagonist AMD3100 to patients with multiple myeloma and non-Hodgkin’s lymphoma. J Clin Oncol 22:1095–1102 43. Flomenberg N, Devine SM, DiPersio JF et al (2005) The use of AMD3100 plus G-CSF for autologous hematopoietic progenitor cell mobilization is superior to G-CSF alone. Blood 106:1867–1874 44. Cashen A, Devine S, Vij R, DiPersio J (2005) AMD3100 + G-CSF improves hematopoietic progenitor cell (HPC) collection in patients with Hodgkin’s disease (HD). Blood 106:1979 45. Devine SM, Andritsos L, Todt L et al (2005) A pilot study evaluating the safety and efficacy if AMD3100 for the mobilization and transplantation of HLA-matched

Chapter 17  Blood Vs. Marrow Allogeneic Stem Cell Transplantation  sibling donor hematopoietic stem cells in patients with advanced hematological malignancies. Blood 106:299 46. Lickliter JD, McGlave PB, DeFor TE, Miller JS, Ramsay NK, Verfaillie CM et al (2000) Matched-pair analysis of peripheral blood stem cells compared to marrow for allogeneic transplantation. Bone Marrow Transplant 26:723–728 47. Bensinger WI, Longin K, Appelbaum F, Rowley S, Weaver C, Lilleby K et  al (1994) 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 87:825–831 48. Russell JA, Larratt L, Brown C, Turner AR, Chaudhry A, Booth K et  al (1999) Allogeneic blood stem cell and bone marrow transplantation for acute myelogenous leukemia and myelodysplasia: influence of stem cell source on outcome. Bone Marrow Transplant 24:1177–1183 49. Bensinger WI, Clift R, Martin P, Appelbaum FR, Demirer T, Gooley T et al (1996) Allogeneic peripheral blood stem cell transplantation in patients with advanced hematologic malignancies: a retrospective comparison with marrow transplantation. Blood 88:2794–2800 50. Storek J, Gooley T, Siadak M, Bensinger WI, Maloney DG, Chauncey TR et  al (1997) Allogeneic peripheral blood stem cell transplantation may be associated with a high risk of chronic graft-versus-host disease. Blood 90:4705–4709 51. Bensinger W, Stem Cell Trialists’ Group (2006) Individual patient data meta-analysis of allogeneic peripheral blood stem cell transplant vs. bone marrow transplant in the management of hematological malignancies: indirect assessment of the effect of day 11 methotrexate administration. Bone Marrow Transplant 38(8):539–546 52. Flowers M, Parker P, Johnston L et al (2002) Comparison of chronic graft-versus-host disease after transplantation of peripheral blood stem cells versus bone marrow in allogeneic recipients: long-term follow-up of a randomized trial. Blood 100:415–419 53. Storek J, Dawson MA, Storer B, Stevens-Ayers T, Maloney DG, Marr KA et  al (2001) Immune reconstitution after allogeneic marrow transplantation compared with blood stem cell transplantation. Blood 97:3380–3389 54. Nucci M, Andrade F, Vigoriot A et al (2003) Infectious complication in patients randomized to receive allogeneic bone marrow or peripheral blood transplantation. Transpl Infect Dis 5(4):167–173 55. Stem Cell Trialists’ Collaborative Group (2005) Allogeneic peripheral blood stemcell compared with bone marrow transplantation in the management of hematologic malignancies: an individual patient data meta-analysis of nine randomized trials. J Clin Oncol 23(22):5074–5087 56. Brown RA, Adkins D, Khoury H, Vij R, Goodnough LT, Shenoy S et  al (1999) Long-term follow-up of high-risk allogeneic peripheral-blood stem-cell transplant recipients: graft-versus-host disease and transplant-related mortality. J Clin Oncol 17:806–812 57. Remberger M, Beelen D, Fauser A et al (2005) Increased risk of extensive chronic graft-versus-host disease after allogeneic peripheral blood stem cell transplantation using unrelated donors. Blood 105:548–551 58. Eapen M, Logan BR, Confer DL et al (2007) Peripheral blood grafts from unrelated donors are associated with increased acute and chronic graft-versus-host disease without improved survival. Biol Blood Marrow Transplant 13(12):1461–1468 59. Bodine DM, Seidel NE, Orlic D (1996) Bone marrow collected 14  days after in vivo administration of granulocyte colony-stimulating factor and stem cell factor to mice has 10-fold more repopulating ability than untreated bone marrow. Blood 88:89–97 60. Johnsen HE, Hansen PB, Plesner T, Jensen L, Gaarsdal E, Andersen H et al (1992) Increased yield of myeloid progenitor cells in bone marrow harvested for autologous transplantation by pretreatment with recombinant human granulocyte-colony stimulating factor. Bone Marrow Transplant 10:229–234

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B. McClune and D. Weisdorf 61. Martinez C, Urbano-Ispizua A, Rozman M, Rovira M, Marin P, Montfort N et al (1999) Effects of short-term administration of G-CSF (filgrastim) on bone marrow progenitor cells: analysis of serial marrow samples from normal donors. Bone Marrow Transplant 23:15–19 62. Morton J, Hutchins C, Durrant S (2001) 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 98:3186–3191 63. Elfenbein GJ, Sacktein R (2004) Primed marrow for autologous and allogeneic transplantation: a review comparing primed marrow to mobilized and steady-state marrow. Bone Marrow Transplant 32:327–339 64. Lowenthal RM, Ragg SJ, Anderson J et al (2007) A randomized controlled clinical trial to determine the optimum duration of G-CSF priming prior to BM stem cell harvesting. Cytotherapy 9(2):158–164 65. Ostronoff M, Ostronoff P, Souto M et al (2006) Pilot study of allogeneic G-CSF stimulated bone marrow transplantation: harvest, engraftment, and graft-versus-host disease. Bio Blood Marrow Transplant 12:729–733 66. Ji SQ, Chen HR, Wang HX et  al (2002) G-CSF-primed haploidentical marrow transplantation without ex vivo T cell depletion: an excellent alternative for highrisk leukemia. Bone Marrow Transplant 30(12):861–866 67. Damiani D, Fanin R, Silvestri F et  al (1997) Randomized trial of autologous filgrastim-primed bone marrow transplantation versus filgrastim-mobilized peripheral blood stem cell transplantation in lymphoma patients. Blood 90(1):36–42 68. Weisdorf D, Miller J, Verfaillie C et al (1997) Cytokine-primed bone marrow stem cells vs. peripheral blood stem cells for autologous transplantation: a randomized comparison of GM-CSF vs. G-CSF. Biol Blood Marrow Transplant 3(4):217–223 69. Dahl E, Burroughs T, DeFor T et al (2003) Progenitor content of autologous grafts: mobilized bone marrow vs. mobilized blood. Bone Marrow Transplant 32:575–580 70. Serody JS, Sparks SD, Lin Y, Capel EJ, Bigelow SH, Kirby SL et  al (2000) Comparison of granulocyte colony-stimulating factor (G-CSF)–mobilized peripheral blood progenitor cells and G-CSF–stimulated bone marrow as a source of stem cells in HLA-matched sibling transplantation. Biol Blood Marrow Transplant 6:434–440 71. Couban S, Messner HA, Andreou P, Egan B, Price S, Tinker L et al (2000) Bone marrow mobilized with granulocyte colony-stimulating factor in related allogeneic transplant recipients: a study of 29 patients. Biol Blood Marrow Transplant 6:422–427 72. Isola L, Scigliano E, Skerrett D et  al (1997) A pilot study of allogeneic bone marrow transplantation using related donors stimulated with G-CSF. Bone Marrow Transplant 20(12):1033–1037 73. Isola L, Scigliano E, Fruchtman S (2000) Long-term follow-up after allogeneic granulocyte colony-stimulating factor–primed bone marrow transplantation. Biol Blood Marrow Transplant 6:428–433 74. Anderlini P, Przepiorka D, Champlin R, Korbling M (1996) Biologic and clinical effects of granulocyte colony-stimulating factor in normal individuals. Blood 88:2819–2825 75. Anderlini P, Korbling M, Dale D, Gratwohl A, Schmitz N, Stroncek D et al (1997) Allogeneic blood stem cell transplantation: considerations for donors. Blood 90:903–908 76. Tigue CC, McKoy JM, Evens AM et al (2007) Granulocyte-colony stimulating factor administration to healthy individuals and persons with chronic neutropenia or cancer: an overview of safety considerations from the Research on Adverse Drug Events and Reports project. Bone Marrow Transplant 40:185–192 77. Stroncek DF, Clay ME, Petzoldt ML, Smith J, Jaszcz W, Oldham FB et al (1996) Treatment of normal individuals with granulocyte-colony-stimulating factor: donor

Chapter 17  Blood Vs. Marrow Allogeneic Stem Cell Transplantation  experiences and the effects on peripheral blood CD34+ cell counts and on the collection of peripheral blood stem cells. Transfusion 36:601–610 78. Hoekman K, von Blomberg-van der Flier BM, Wagstaff J et al (1991) Reversible thyroid dysfunction during treatment with GM-CSF. Lancet 338:541–542 79. Kroschinsky F, Hundertmark J, Mauersberger S et al (2004) Severe autoimmune hyperthyroidism after donation of growth factor-primed allogeneic peripheral blood progenitor cells. Haematologica 89:ECR05 80. de Luis DA, Romero E (1996) Reversible thyroid dysfunction with filgrastim. Lancet 348:1595–1596 81. Arimura K, Inoue H, Kukita T et al (2005) Acute lung injury in a healthy donor during mobilization of peripheral blood stem cells using granulocyte-colony stimulating factor alone. Haematologica 90:ECR10 82. Durrant S, Morton AJ (1999) A randomized trial of filgrastim (G-CSF) stimulated donor marrow (BM) versus peripheral blood (PBPC) for allogenic transplantation: increased extensive chronic graft versus host disease following PBPC transplantation. Blood 10:608a 83. Welch G, Larson E (1989) Cost-effectiveness of bone marrow transplantation in acute non-lymphocytic leukemia. New Engl J Med 32:807–812 84. van Agthoven M, Groot MT, Verdonck LF et  al (2002) Cost analysis of HLAidentical sibling and voluntary unrelated allogeneic bone marrow and peripheral blood stem cell transplantation in adults with acute myelocytic leukaemia or acute lymphoblastic leukaemia. Bone Marrow Transplant 30:243–251

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Chapter 18 Hematopoietic Cell Transplantation from Partially HLA-Mismatched (HLA-Haploidentical) Related Donors Ephraim J. Fuchs and Heather J. Symons

1.  Introduction Human Leukocyte Antigen-haploidentical stem cell transplantation, or HLA-haploidentical SCT, refers to the transplantation of blood or marrow from a donor into a recipient who is genotypically identical for one HLA haplotype and is variably mismatched for HLA alleles on the unshared haplotype. Typically, donors for HLA-haploidentical SCT are first-degree relatives, such as siblings, biological parents, or biological children, although aunts, uncles, cousins, and half-siblings may be HLA-haploidentical to the recipient. As compared to blood or marrow transplantation from an HLA-matched sibling, a distinguishing feature of HLA-haploidentical, or partially mismatched related donor (PMRD), SCT is intense, bidirectional T cell-mediated alloreactivity resulting in increased risks of graft failure, severe graft-versus-host disease, and nonrelapse mortality (NRM) [1–3]. The poor results of early trials of HLA-haploidentical SCT have motivated strategies to mitigate alloreactivity by depleting T cells from both the host and from the graft. While these strategies have reduced the risks of graft failure and GVHD, respectively, they have led to a compensatory increase in the risk of severe infectious complications from prolonged immunodeficiency, and possibly to an increased risk of disease relapse. More recent efforts have focused upon selective depletion of alloreactive T cells to enhance immune reconstitution without risking severe GVHD. Advances in the control of alloreactivity and in supportive care have combined to improve the results of HLA-haploidentical SCT. Current regimens for HLA-haploidentical SCT are associated with a 2-year nonrelapse mortality of 20 ± 5%, relapse of 35 ± 15%, and overall survival of 50 ± 20%. Another distinguishing feature of HLA-haploidentical SCT is the ready availability of donors. Only about a third of patients in need of allogeneic SCT have an HLA-matched sibling [4]. Even after extensive searching of unrelated donor registries, a suitably HLA-matched unrelated donor still can only be identified for about 40% of patients [5–9]. In contrast, nearly everyone has one or more HLA-haploidentical first-degree relatives. All biologic parents and children of an individual are HLA-haploidentical, and any sibling From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_18, © Springer Science + Business Media, LLC 2003, 2010

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or half sibling has a 50% likelihood of being HLA-haploidentical. Thus, most patients have more than one potential donor for PMRD SCT. The ability to choose between potential donors has motivated research into the demographic and biologic characteristics of HLA-haploidentical donors and grafts that are associated with favorable outcomes after allogeneic SCT. This research has identified the importance of donor age, histoincompatibility, natural killer cell alloreactivity, and tolerance of noninherited maternal antigens as being some of the key determinants of transplantation outcome. This chapter is organized into the following sections: (1) the immunobiology of HLA-haploidentical SCT, with a focus on T cell and NK cell alloreactions; (2) complications of HLA-haploidentical SCT; (3) principles of HLA typing and donor selection; (4) results of trials of HLA-haploidentical SCT; (5) approaches to improve the outcome of HLA-haploidentical SCT; and (6) a comparison of HLA-haploidentical and unrelated umbilical cord blood SCT, the two major alternatives to HLA-matched related or unrelated donor SCT.

2.  Immunobiology of HLA-Haploidentical SCT 2.1.  The Human T Cell Responses to Allogeneic HLA Molecules is Unusually Strong A cardinal function of HLA class I and class II molecules is to present peptide antigens for recognition by CD8+ and CD4+ T cells, respectively. However, these molecules also play a central role in the generation of histocompatibility reactions; thus the HLA locus is also referred to as the Major Histocompatibility Complex, or MHC. When donor and recipient are HLA-matched, graft rejection or graft-versus-host disease (GVHD) is ascribed to T cell responses to minor histocompatibility Ags (minor H Ags). Minor H Ags are naturally processed peptide fragments of allelically variable normal cellular proteins presented by MHC molecules [10]. They induce histocompatibility reactions because processing of the allelically variable portion of the protein generates a peptide that can be presented by MHC molecules and differs between donor and host. While histocompatibility reactions following HLA-matched SCT are due to minor H antigens, GVHD or graft rejection after HLA-haploidentical SCT can be triggered by minor H antigens, HLA antigens, or both. Compared to the T cell response to single or multiple minor H antigens, the human T cell response to allogeneic HLA molecules is unusually strong. Estimates of the precursor frequency of T cells responding to a single minor H antigen are in the range of 10−5 to 10−4. In contrast, estimates of the frequency of T cells responsive to a single MHC antigen or to a full MHC locus incompatibility are in the range of 1–10% [11–14]. At least three different hypotheses have been put forward to account for the strength of the allogeneic response. The first theory proposes that T cell receptor genes are biased toward recognition of MHC antigens, and that while self-MHC reactive T cells are eliminated by clonal deletion, T cells capable of reacting to allogeneic MHC molecules are not [15]. The second, or determinant density, hypothesis proposes that alloreactive T cells focus on determinants derived entirely from the allogeneic MHC molecule, with no contribution of the bound peptide to allorecognition [16]. The high frequency of alloreactive T cells is then a consequence of the high density of allogeneic MHC molecules on the surface of

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched 

a cell, as opposed to the relatively low frequency of a given peptide bound to a self MHC molecule. Finally, the “determinant frequency” hypothesis [17] proposes that the target of allorecognition is a composite determinant with contributions from both the allogeneic MHC molecule and its bound peptide, but since MHC molecules bind diverse peptides, an allogeneic cell presents a variety of unique determinants (allo + x1, allo + x2, allo + x3, etc., where xn is a unique peptide) for recognition by a diverse array of T cell receptors. Crystallographic and other evidence has provided support for both the determinant density and determinant frequency models; that is, there is evidence for T cells that focus on determinants provided exclusively by the allogeneic MHC molecule and for T cells that see a composite determinant with contributions from both allogeneic MHC and bound peptide [18]. Regardless of mechanism, the high frequency of HLA-reactive T cells in humans remains a hard fact that probably contributes to the elevated risk of graft failure and GVHD after partially HLA-mismatched SCT. The phenomenon of T cell cross-reactivity may also contribute to the strength of T cell responses to allogeneic MHC molecules. In mouse models, viral immunization generates cytotoxic effector T cells that also lyse unmodified allogeneic targets [19]. By analogy, exposure of the human immune system to pathogens, either naturally through infection or artificially through vaccination, may generate large numbers of memory and effector cells capable of anamnestic responses to allogeneic MHC molecules. Thus, while adult T cells and umbilical cord bloodT cells may not differ significantly with respect to the precursor frequency of T cells responsive to allogeneic HLA molecules, they may differ substantially in the proportion of alloreactive T cells that are in the memory or effector cell stages of differentiation. Thus, the phenomenon of T cell cross-reactivity may contribute to the higher risk of acute GVHD after haploidentical SCT from parental as compared to sibling donors [20], to the association between donor age and the risk of GVHD after unrelated donor SCT [21], and to the higher risk of acute GVHD after HLA-matched unrelated donor SCT as compared to HLA-matched umbilical cord blood SCT [22]. 2.2.  Limitations on HLA Mismatching in Allogeneic SCT To be an effective therapy of hematologic malignancies, allogeneic SCT must eradicate the patient’s disease while avoiding lethal GVHD and opportunistic infection. Since successful allogeneic SCT results in full donor hematopoietic chimerism, the donor’s immune system is ultimately responsible for the control of infection after the transplantation procedure. Prompt and adequate reconstitution of donor immunity is therefore critical to the avoidance of opportunistic infection after allogeneic SCT. Reconstitution of donor T cells after allogeneic SCT may derive from the homeostatic expansion of mature T cells contained within the graft or from the de  novo generation of T cells from donor-derived stem cells [23–25]. When mature T cells are depleted from the graft, immune reconstitution derives primarily from the de  novo generation of T cells from the thymus since there is no clinical evidence of extrathymic T cell maturation after allogeneic SCT [26]. Thymic maturation involves a series of differentiation steps termed positive and negative selections [27]. In positive selection, developing thymocytes are signaled to survive by the recognition of antigen presented

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by MHC molecules on thymic epithelium, whereas thymocytes that cannot recognize antigens in the context of thymic epithelial MHC die of neglect. This process ensures that only “useful” T cells, those that recognize antigens in the context of the MHC molecules expressed on peripheral (extra-thymic) antigen-presenting cells (APCs), are allowed to survive and emigrate from the thymus. Consider the situation, then, after the transplantation of a T cell-depleted graft from a completely MHC-incompatible donor. Donor thymocytes will be positively selected to recognize antigens in the context of thymic epithelial (host-type) MHC, but in the periphery antigens are presented exclusively in the context of donor MHC molecules on the surface of donor APCs. The complete mismatch between the MHC expressed on host thymic epithelium and on donor APCs would be predicted to result in immunoincompetence, which has been confirmed experimentally in some [28, 29] but not all [30] models. In contrast, after MHC-haploidentical, T cell-depleted SCT, T cells developing from transplanted donor stem cells would be able to recognize antigens presented in the context of the MHC antigens that are shared by host thymic epithelium and donor APCs. Thus, sharing of at least one haplotype between donor and recipient ensures the potential for functional reconstitution of T cell immunity through the de novo generation of donor T cells in the host thymus. 2.3.  Natural Killer Cell Alloreactions After Allogeneic SCT Natural killer, or NK, cells may play a significant role in inducing graftversus-leukemia (GVL) effects after haploidentical SCT in humans. NK cells probably represent an evolutionary intermediate between cells of the innate and adaptive immune systems. Unlike T and B cells, they do not express rearranging receptors for antigen, but like T cells, they do express receptors for HLA class I molecules including HLA-B and HLA-C. Moreover, like CD8+ T cells they secrete interferon gamma and kill target cells via granzymeand perforin-mediated cytotoxicity. A current consensus, the “missing self” hypothesis [31, 32], is that NK cells have evolved to detect and rapidly eliminate virally infected or tumor cells that have down-regulated cell surface expression of MHC class I molecules to evade the CD8+ T cell arm of the immune response. The molecular basis of NK cell alloreactivity is incompletely understood, but involves a dynamic balance of signals through activating as well as inhibitory receptors on the NK cell. The Killer Immunoglobulin-like Receptors, or KIRs, are encoded by a set of linked genes called the Leukocyte Receptor Complex (LRC) on human chromosome 19q13.4 [33]. KIRs contain two or three extracellular Ig-like domains and either a short (S) or a long (L) cytoplasmic tail, which mediates activating or inhibitory signals, respectively. The organization of the LRC is depicted in Fig. 18-1. The LRC is marked by significant inter-individual variation in KIR gene content as well as significant allelic variation in individual KIR genes [34]. As a consequence, the KIR locus rivals the HLA locus for polymorphism, and unrelated individuals are unlikely to share KIR genotypes. Distinct HLA class I molecules comprise the ligands for specific inhibitory KIRs (iKIRs). An organizing principle of NK cell biology is that NK cell self-tolerance is mediated by inhibitory signals delivered by self HLA class I

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Fig.  18-1.  Organization of the KIR locus in the Leukocyte Receptor Complex on chomrosome 19q13.4. The boundaries of the KIR locus on human chromosome region 19q13.4 are the KIR3DL3 gene on the telomeric end and the KIR3DL2 gene on the centromeric end. Between these two genes lie a variable number of between 7 and 12 additional KIR genes. Analysis of KIR loci in humans has identified two common haplotypes, group A and group B. The group A haplotype is distinguished by fewer genes and a single activating KIR gene, KIR2DS4, whereas the group B haplotype has more genes and as many as five activating KIR genes. In addition to interindividual differences in the number of inherited KIR genes, there is substantial allelic polymorphism, with the number of named alleles for each gene indicated in each box. As a consequence of variable gene content and allelic variation, unrelated individuals rarely have identical KIR genotypes [34]. Conserved genes are shown in brown. Genes that can be present in both group A and group B KIR haplotypes are shown in yellow, and genes and/or alleles that are specific to group B KIR haplotypes are shown in blue. Reprinted from Parham, with permission [225]

Fig.  18-2.  Interactions between inhibitory Killer Immunoglobulin-Like Receptors (iKIRs) and their HLA ligands of relevance to natural killer cell alloreactivity after allogeneic SCT. For convenience, a single NK cell expressing four distinct iKIRs is shown. Each NK cell need only express one molecular species of iKIR for functional maturation to occur. Individual members of the HLA-C1, -C2, and HLA-Bw4 groups are listed in Table 18.1. High resolution HLA typing is required to determine whether specific alleles of HLA-B and HLA-Cw are ligands of specific iKIRs. High resolution typing of HLA-B and -Cw loci are incorporated into the ligand incompatibility, receptor-ligand, and missing ligand models of natural killer cell alloreactivity (Fig. 18.3). Interactions between KIR3DL2 and HLA-A3 or -A11 are generally not considered in these models

molecules through iKIRs. The ontogeny of receptor expression on individual NK cells is poorly understood, but it is currently thought that each NK cells expresses at least one inhibitory receptor for a self HLA class I molecule [35]. There are four distinct categories of HLA ligands for iKIRs (Fig.  18-2 and

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Table 18-1.  Inhibitory killer immunoglobulin-like receptors (iKIRs) and their HLA ligands. Inhibitory KIR

Recognition motif

HLA ligand group

KIR2DL2/2DL3

Ser77, Asn80

C1

HLA-Cw1; Cw3 (except Cw*0307, 0310, 0315); Cw7 (except Cw*0707, 0709); Cw08; Cw12 (except Cw*1205, 12041/2); Cw13, Cw14 (except Cw*1404); Cw*1507; Cw16 (except Cw*1602)

KIR2DL1

Asn77, Lys80

C2

HLA-Cw2; -Cw*0307; Cw*0315; Cw4; Cw5; Cw6; Cw*0707; Cw*0709; Cw*1205; Cw*12041/2; Cw15 (except Cw*1507); Cw*1602; Cw17; Cw18

KIR3DL1

“Bw4 epitope” (residues 77–83)

Bw4

B5; B13; B17; B27; B37; B38; B44; B47; B49; B51; B52; B53; B58; B59; B63; B77; B*1513; B*1516, B*1517, B*1523; B*1524

KIR3DL2

Group members

HLA-A3, HLA-A11

Table 18-1). The C1 group of HLA ligands is characterized by the presence of an asparagine (Asn) residue at position 80 of the HLA-C molecule and is recognized by either KIR2DL2 or KIR2DL3. The complementary C2 group of HLA ligands is distinguished by a lysine (Lys) residue at position 80 of the HLA-C molecule and is recognized by KIR2DL1. The Bw4 serologic group is recognized by KIR3DL1. Finally, KIR3DL2 recognizes HLA-3 and HLA11 molecules. Recent studies have shown that developing NK cells undergo a host MHC class I-dependent functional maturation process, termed licensing [36, 37]. Licensing involves the same interaction between an iKIR and its autologous MHC class I ligand that later in the NK cell’s life would result in inhibition of effector function. This process helps achieve NK cell self-tolerance because the only NK cells that are licensed to kill are those that can be inhibited in the presence of an autologous MHC class I ligand. Since the genes comprising the Leukocyte Receptor Complex and Human Leukocyte Antigen locus are on chromosomes 19 and 6, respectively, KIR and HLA molecules are inherited independently and so there is the potential for an individual to inherit an inhibitory KIR for which there is no corresponding autologous HLA molecule. Presumably, NK cells whose sole inhibitory receptor is specific for an absent self HLA ligand are nonfunctional, or anergic, because they have not been licensed [38]. However, DNA damage resulting from transplantation conditioning or inflammatory conditions such as viral infection may be sufficient to induce stress ligands of NK cell activation receptors [39], break tolerance in unlicensed NK cells, and generate autoreactivity [40–42], including tumor regression. The potential for inflammation to break NK cell tolerance in the post-transplantation period has significant implications for models of natural killer cell alloreactivity, as discussed below. 2.4.  Models of NK Cell Reactivity After Stem Cell Transplantation Three methods have been employed to predict NK cell reactivity after autologous or allogeneic stem cell transplantation: (1) high resolution typing of HLA class I loci; (2) KIR gene typing and flow cytometry for KIR expression; and (3) cloning of alloreactive NK cells. These methods have generated four

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distinct classes of models of NK cell reactions after stem cell transplantation (Fig.  18.3): (1) the KIR ligand incompatibility model, also known as the “ligand–ligand” model [43–45]; (2) the “missing (HLA) ligand” model [46, 47]; (3) the “(KIR) receptor-(HLA) ligand” model [48]; and (4) the KIR gene– gene model [49, 50]. The first two models require only high resolution HLA typing for prediction of NK cell reactivity, whereas the last model requires only NK cell genotyping. The receptor-ligand model requires high resolution HLA typing, KIR genotyping, and assessment of KIR gene expression by flow cytometry. The KIR ligand incompatibility model [43, 44, 51] was first formulated by Ruggeri and colleagues in Perugia to account for NK alloreactivity after rigorously T cell-depleted, stem cell enriched HLA-haploidentical SCT [52, 53]. The model predicts NK cell alloreactivity when the donor expresses but the recipient lacks an HLA ligand (HLA group C1, C2, or Bw4) for an inhibitory KIR (Fig. 18.3a). In this situation, the donor is predicted to contain NK cells that have been licensed by and are self-tolerant of an HLA molecule that is present in the donor, but after HLA-haploidentical SCT these NK cells are

Fig. 18-3.  Models of natural killer cell alloreactivity after allogeneic SCT. (a) The ligand incompatibility model predicts NK cell alloreactivity in the GVH direction (depicted) when the recipient lacks expression of an iKIR ligand, in this case a member of the HLA-C1 group, that is present in the donor. The presence of functional donor NK cells expressing KIR2DL2, the receptor for HLA-C1 molecules, as their inhibitory receptor is assumed in this model. (b) The receptor-ligand model predicts NK cell alloreactivity in the GVH direction when the recipient lack an HLA ligand for donor inhibitory KIR, whose presence is verified by KIR genotyping and flow cytometry of donor NK cells. The HLA type of donor cells is irrelevant to this model. (c) The missing ligand model predicts NK cell alloreactivity in the GVH direction when recipient cells lacks expression of at least one of the HLA ligands (C1, C2, or -Bw4) for inhibitory KIR. (d) The KIR gene-gene model predicts NK alloreactivity when the donor and recipient are mismatched for KIR gene content. Inhibitory KIR genes are shown as unshaded boxes, whereas black boxes represent activating KIR genes. In the example shown, the recipients KIR genotype is said to be “included” in the donor’s KIR genotype [49]

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activated by recipient cells lacking expression of that HLA molecule. Support for the ligand incompatibility model was provided by the ability to generate donor alloreactive NK clones in all 51 ligand incompatible donor-recipient pairs but in none of the 61 donors who were KIR ligand matched with their recipients [51]. NK alloreactivity in the GVH direction was predicted to have three functional consequences [44]:(1) a graft-versus-leukemia effect arising from donor NK cytotoxicity against leukemia cells; (2) a decreased rate of graft rejection arising from donor NK cell killing of host T cells; and (3) a decreased rate of GVHD arising from donor NK cell elimination of host APCs such as dendritic cells, which are required to initiate graft-versus-host reactions [54]. Clinical trials from the Perugia group have consistently demonstrated a strong anti-tumor effect of KIR ligand incompatibility in acute myeloid leukemia (but not acute lymphocytic leukemia), but more recent studies from the group have failed to confirm a beneficial effect of NK cell alloreactivity in reducing the risk of graft rejection or GVHD [51]. Three features of the ligand incompatibility model are worth noting: (1) the model assumes the existence of licensed donor NK cells expressing an inhibitory receptor for the HLA molecule lacking in the recipient; (2) the model requires a mismatch between donor and recipient HLA-B or -C alleles, and therefore, does not predict NK cell alloreactions after either autologous or HLA-matched allogeneic SCT; and (3) since the model predicts NK cell alloreactivity solely on the basis of HLA differences between donor and recipient, the model can be applied retrospectively to the analysis of HLA-haploidentical transplant outcomes as long as high resolution HLA typing of donors and recipients was performed. The receptor-ligand model of Leung and colleagues [48] (Fig. 18.3b) predicts NK cell alloreactivity when the transplant recipient lacks expression of an HLA ligand for a verified donor inhibitory KIR. This model differs in three critical respects from the ligand incompatibility model. First, the receptorligand model requires verification, by KIR genotyping and flow cytometry of donor NK cells [55], of the presence of the receptor for the HLA ligand that is missing in the recipient. This is because some donors lack the gene for or surface expression of an inhibitory KIR for which the corresponding ligand is absent on recipient cells. Although most individuals encode inhibitory KIR genes for the HLA-C1, HLA-C2, and HLA-Bw4 groups of ligands [56], not all do so. For example, in a genetic analysis of a Caucasian population, 4.8% lacked KIR3DL1, the receptor for the HLA-Bw4 ligand group, and 8.4% lacked KIR2DL1, the receptor for the HLA-C2 group [57]. Moreover, surface expression of KIR2DL1 and KIR3DL1 proteins was undetectable in 7 and 15%, respectively, of individuals containing the corresponding genes [55]. Second, the receptor-ligand model does not require donor cell expression of the HLA ligand that is absent in the recipient for NK alloreactivity to occur. Thus, in Fig. 18.3b, the model predicts NK cell alloreactivity against a “missing” HLA-C1 group ligand even though it is also missing in the donor. The model of NK licensing discussed above would predict that such an NK cell would be anergic because the KIR2DL2 molecule fails to receive the licensing signal in the donor. However, as mentioned above, it is possible that unlicensed donor NK cells can be activated during the inflammation of the immediate posttransplantation period. A major consequence of this feature of the receptorligand model is that it allows for the occurrence of NK cell mediated anti-tumor

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched 

effects after HLA-matched allogeneic SCT or even after autologous SCT [58]. Finally, because the model requires flow cytometry of donor cells, it is not suited to the retrospective analysis of transplant outcomes from registry data containing only high resolution HLA typing of donors and recipients. The missing ligand model (Fig.  18.3c) is a modification of the receptorligand model that permits retrospective analysis of the effect of NK cell alloreactivity on transplantation outcomes. The model predicts NK-mediated GVH reactions when the transplant recipient is missing at least one of the three major classes of HLA ligands for inhibitory KIR. The missing ligand model differs from the ligand incompatibility model only in that it does not require the presence on donor cells of the HLA ligand that is missing in the recipient. Consequently, donors that are predicted by the ligand incompatibility model to contain alloreactive NK cells against their recipients are a subset of the donors that are predicted by the missing ligand model to contain anti-recipient alloreactive NK cells. For example, the ligand incompatibility model predicts NK alloreactivity in the situation depicted in Fig.  18.3a because the donor contains the HLA-C1 ligand that is absent in the recipient, but not in the situation depicted in Fig. 18.3c because the donor and recipient are HLA-C ligand compatible. The missing ligand model predicts NK cell alloreactivity in both situations because recipient cells are missing the HLA-C1 ligand. The KIR ligand incompatibility and missing KIR ligand models were compared for their ability to predict relapse after T cell-replete, unrelated donor SCT for hematologic malignancies [47]. Among recipients of HLAmismatched transplants, recipient homozygosity for HLA-B or -C KIR epitopes was used to define “missing” KIR ligand and was associated with a decreased hazard of relapse (hazard ratio, 0.61; 95% confidence interval, 0.43–0.85; p = 0.004). The effect was observed in patients with AML, CML, or ALL. The same effect was not observed in HLA-identical unrelated transplants. KIR ligand incompatibility was not associated with a decreased risk of relapse in recipients of either HLA-mismatched or HLA-matched grafts. Finally, the KIR gene–gene model characterizes the KIR genotype of both donor and recipient and asks whether differences in the expression of individual genes, of haplotypes, or in the number of KIR genes between donor and recipient have any effect on the outcome of allogeneic SCT [49]. KIR genes are inherited as haplotypes (Fig. 18.1), with the A haplotype containing fewer genes and only one stimulatory KIR gene (shaded box in Fig.  18.3d), while the B haplotype contains more genes and several stimulatory KIR genes. Since there are no KIR genes that are unique to the A haplotype, whereas several KIR genes are only present in the B haplotype, the KIR genes of the A haplotype are essentially “included” in the B haplotype, and individuals of Bx KIR type (AB or BB haplotypes) express more KIR genes than individuals expressing two A haplotypes (AA). When the recipient’s KIR genotype is “included” in the donor’s KIR genotype (presumably a Bx → AA transplant), there was a 100% risk of GVHD after unrelated donor SCT as compared to a 60% risk of GVHD with other combinations [49]. The adverse effect of a donor B haplotype on GVHD after T cell-replete BMT may relate to donor KIR2DS2 recognition of cognate HLA-C1 ligands on recipient cells [59, 60]. One study found no effect of KIR gene mismatching after T cell-replete, HLA-haploidentical SCT [50]; rather, the number of HLA ligands for inhibitory KIR was found to correlate with decreased NRM and improved overall and disease-free survival.

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2.5.  Interactions Between T and NK Cells After HLA-Haploidentical SCT Donor T cells may inhibit NK cell reconstitution and/or alloreactivity after HLA-haploidentical SCT. As a consequence, some of the discrepancies between studies reporting the effects of NK cell alloreativity on the outcome of haploidentical SCT may be attributed to differences in the method or extent of graft T cell depletion. The Perugia group has consistently shown a beneficial effect of ligand incompatibility in T cell-depleted transplants for AML, while studies from St. Judes Medical Center have shown a beneficial effect of receptor-ligand mismatch after CD34-selected, haploidentical SCT for both AML and ALL. While some studies have shown that ligand incompatibility reduces relapse [44, 51, 61, 62], GVHD [44]and NRM [61] improve overall survival [44, 51, 61], other studies have associated ligand incompatibility with increased GVHD [63], relapse [63–65], NRM [66–68], and worse overall survival [63, 66–70]. The benefit of NK cell alloreactions may be masked by residual alloreactive T cells causing GVHD, leading to poorer overall survival [69, 71, 72]. Furthermore, graft T cells may impair NK cell reconstitution [73]. Reconstituting NK cells after HLA-haploidentical SCT are characterized by functional immaturity [71], low expression of iKIRs, and increased expression of CD94/NKG2A [74], which turns off NK cells in response to HLA-E expressed on AML cells. When T cells are rigorously depleted, the benefit of NK cell alloreactions may be unopposed and therefore, improve survival due to decreased relapse. A better understanding of the mechanisms of self-tolerance in NK cells, the significance of allelic variation in KIRs, the ligands for NK cell activation, and NK cell reconstitution after allogeneic SCT is clearly needed before NK cell alloreactions can be effectively harnessed after HLA-haploidentical SCT.

3.  Complications of HLA-Haploidentical SCT Regardless of the immunologic disparity between donor and recipient, all patients undergoing allogeneic SCT are at risk for the same complications, namely conditioning regimen toxicity, graft failure, GVHD, infection, and relapse. However, HLA-haploidentical SCT is characterized by intense bi-directional alloreactions resulting in higher risks of both graft failure and acute GVHD compared to HLAmatched sibling SCT. Strategies employed to reduce the risk of one complication of haploidentical SCT have, to much frustration, resulted in an increased incidence of another serious complication. For example, T cell depletion of the donor graft reduces the risk of GVHD but unfortunately increases the risk of fatal graft failure [75, 76]. Consequently, T cell depletion has not been shown to improve the outcome of HLA-haploidentical SCT [76]. Increased conditioning regimen intensity reduces the risk of graft failure but all the same increases the risk of regimen-related toxicity [77] and also may increase the risk of GVHD [78]. Finally, low rates of graft rejection and GVHD can be achieved by giving rigorously T cell-depleted grafts to intensively conditioned recipients, but immune reconstitution is significantly delayed and NRM approaches or exceeds 40% [52, 53, 79, 80], much of it due to infection. The following section describes complications of allogeneic SCT whose incidence and/or severity may be affected by the immunologic disparity between donor and recipient.

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3.1.  Graft Failure Graft failure is a serious complication of allogeneic SCT and is nearly always fatal after myeloablative conditioning [81]. Graft failure may be primary, marked by the lack of initial engraftment (neutrophils >500/ml) and absence of donor hematopoietic chimerism, or it may be secondary, manifested as initial hematologic recovery followed by neutropenia and loss of donor chimerism. The primary cause of graft failure is immunologic rejection mediated by radioresistant host T and/or NK cells [82, 83]. Among patients receiving myeloablative conditioning, the incidence of either primary or secondary (late) graft failure was 2.0% in recipients of HLA-matched sibling marrow but was 12.3% in recipients of marrow from HLA-haploidentical related donors (p < 0.0001) [2]. The incidence of graft failure correlated with the degree of HLA incompatibility, occurring in 3 of 43 (7%) patients receiving haploidentical grafts mismatched for 0 HLA antigens (HLA-phenotypically matched grafts from a parent or child), 11 of 121 (9%) recipients of 1 HLA antigen-mismatched grafts, 18 of 86 (21%) recipients of 2 HLA antigen-mismatched grafts, and 1 of 19 recipients of 3 HLA antigen-mismatched grafts (p = 0.028). The effect of increasing HLA disparity on the risk of graft rejection after myeloablative SCT was also confirmed in an analysis performed by the IBMTR (Fig. 18-4a, c).

Fig. 18-4.  Effect of donor/recipient incompatibility and graft T cell depletion on the risk of graft rejection and GVHD after partially HLA-mismatched related donor BMT. Relative risks (a, b) and adjusted probabilities (c, d) of graft failure (a, c) and GVHD (b, d) among patients transplanted for leukemia. (a, b) Recipients of HLAmatched sibling transplants are the reference population (relative risk = 1.0) and are marked with an asterisk. p Values represent the comparison between the study population and the reference group. Number of patients per group is designated in each box. (c, d) Probabilities of graft failure and GVHD were adjusted for patient and disease characteristics. p Values refer to the comparison between T cell-depleted () and non-T cell-depleted () recipients. Adopted from Ash et al. [76], with permission

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In this study, the risk of graft rejection among recipients of grafts mismatched for 2–3 HLA antigens was approximately 6–8 times greater than among recipients of grafts from HLA-matched siblings. T cell depletion of the donor graft increased the risk of graft failure after HLA-mismatched as well as HLAmatched SCT (Fig.  18.4c). In the Seattle study, the risk of graft failure was increased in patients mismatched with the donor for both HLA-B and HLA-DR antigens, as well as in patients with a positive lymphocytotoxic crossmatch against donor cells [2]. The presence of a positive crossmatch predicted both graft failure and poor overall survival for patients receiving HLA-mismatched grafts [84]. Lymphocytotoxic crossmatching is strongly recommended as a step in determining donor eligibility prior to HLA-mismatched SCT. If the crossmatch is positive, further testing is recommended to assess for the presence, in the patient, of anti-donor HLA antibodies. While plasmapheresis or immunoadsorption has been used to clear anti-donor HLA antibodies and permit engraftment after HLA-haploidentical SCT [85], a search for alternative donors is strongly recommended. In addition to the degree of HLA disparity between donor and recipient, several other factors influence the risk of graft rejection after HLA-haploidentical BMT, including characteristics of the patient, the graft, the conditioning regimen, and post-transplantation immunoprophylaxis [86]. A competent host immune system is clearly required for allogeneic graft rejection, as the barrier to engraftment is lower in patients with severe combined immunodeficiency as compared to patients with hematologic malignancies [87]. Conversely, sensitization of immunocompetent recipients, for instance by blood transfusions, increases the risk of graft rejection following allogeneic SCT [88]. The dose of donor T cells and stem cells also has a powerful influence on donor cell engraftment after haploidentical SCT. Early studies clearly established that depletion of T cells from the graft significantly increases the risk of graft failure following HLA-haploidentical SCT [76]. In some series, the risk of graft failure following T cell depletion approached 50% [87]. However, the detrimental effects of T cell depletion on donor cell engraftment can be overcome by augmenting recipient immunosuppression and escalating the dose of transplanted stem cells [89, 90]. These studies ultimately led to the concept and practice of using “megadoses” of haploidentical, CD34+ stem cells (>107/kg of recipient body weight) obtained from G-CSF mobilized peripheral blood collections [91, 92]. Studies in mice suggest that megadoses of mismatched stem cells induce tolerance by the “veto” mechanism [93–95], in which the cytotoxicity of alloreactive donor cells is “vetoed” by recipient cells expressing the alloantigen [96, 97]. Transplantation of megadoses of stem cells into intensively conditioned recipients enables T cell depletion of haploidentical grafts to decrease the risk of acute GVHD without increasing the risk of fatal graft failure [52, 53]. Increasing the intensity of transplantation conditioning also lowers the risk of graft failure after PMRD SCT. For example, increased intensity of total body irradiation was inversely correlated to the rate of graft failure among leukemic patients receiving transplants from HLA-haploidentical donors [2]. Finally, post-transplantation pharmacologic immunosuppression is likely to decrease the risk of graft failure following PMRD SCT. While this has not been intensively studied in the HLA-haploidentical setting, the risk of graft failure after HLA-matched sibling BMT was significantly lower among patients receiving

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched 

post-transplantation prophylaxis with methotrexate, cyclosporine, or both compared to patients receiving no GVHD prophylaxis [75]. 3.2.  Graft-vs-Host Disease Severe acute GVHD was a major complication of early trials of T cell-replete, HLA-haploidentical SCT [3]. These early studies examined the relationship of serologic HLA incompatibility on the incidence and severity of GVHD. Among patients receiving methotrexate as sole post-transplantation prophylaxis, the cumulative incidence of acute GVHD by day 100 was 34% among recipients of phenotypically HLA-matched grafts, increasing to 84% among recipients of three locus incompatible marrow [1]. The vector of incompatibility has been found to influence the risk of GVHD. In a study conducted at the Fred Hutchinson Cancer Research Center (FHCRC) in Seattle, recipients of one HLA antigen-mismatched grafts were categorized according to the vector of incompatibility. The cumulative incidence of acute GVHD by day 100 was 18% when the incompatibility was solely in the host-versus-graft direction (homozygous recipient of a heterozygous graft; n = 17), but was >50% when there was incompatibility in the graft-versus-host direction (heterozygous recipients of a homozygous or heterozygous graft; n = 87; p = 0.03) [3] There was no significant difference in the incidence of acute GVHD between recipients of grafts mismatched for a single HLA class I antigen versus a single HLA class II antigen. In an IBMTR study [76], recipients of bone marrow mismatched for 2–3 HLA antigens had a three- to fivefold risk of GVHD compared to recipients of HLA-matched sibling grafts (Fig.  18.4b). T cell depletion of the graft significantly reduced the risk of GVHD after HLAmismatched as well as HLA-matched sibling BMT (Fig. 18.4d). The adverse effect of HLA mismatch on the incidence of acute GVHD was confirmed in a subsequent analysis by the IBMTR [98]. The incidence of grade II–IV acute GVHD was 29% among 1,176 recipients of HLA-matched sibling marrow, 44% among 223 recipients of one HLA antigen-mismatched related marrow (p < 0.001), and 56% among 86 recipients of 2 HLA antigenmismatched marrow (p < 0.001) [98]. The incidence of grades III–IV acute GVHD also differed significantly according to degree of incompatibility: 13% for HLA-matched siblings, 27% for 1 antigen-mismatched donors (p < 0.001) and 36% for 2 antigen-mismatched relatives (p < 0.001). Among patients surviving ³90 days with evidence of donor engraftment, the incidence of chronic GVHD by 2 years was 42% for recipients of HLA-matched sibling marrow, 52% for recipients of 1 HLA antigen-mismatched marrow (p < 0.001), and 60% for recipients of 2 HLA antigen-mismatched marrow (p = 0.02). An analysis of the outcomes of allogeneic stem cell transplants performed in Japan between 1991 and 2000 identified serologic HLA-mismatch, higher age, and high risk disease as independent risk factors for both short survival and the development of grades III–IV acute GVHD [99]. Importantly, the correlation between HLA mismatch and the risk of acute GVHD was preserved when the data were analyzed according to the degree of HLA allele mismatch, as determined by molecular typing methods. There was no significant difference in the risk of acute GVHD between patients mismatched for a single HLA class I vs. a single HLA class II allele. The risk of grades III–IV acute GVHD was significantly higher after 1 HLA antigen-mismatched SCT compared to HLA-matched unrelated donor BMT (30% vs. 16%; p = 0.0013). These data

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demonstrate that recipients of 1 HLA antigen-mismatched related grafts have a higher risk of severe, acute GVHD compared to recipients of HLA-matched sibling or unrelated grafts. The increased risk of acute GVHD with increasing HLA mismatch between donor and recipient has also been confirmed among recipients of HLA-haploidentical stem cells after reduced intensity conditioning [100]. The cumulative incidence of acute GVHD in this study was 39% (95% CI, 33–45%) in recipients with HLA-matched donors, 44% (95% CI, 30–57%) in those with one-locus-mismatched donors, and 50% (95% CI, 29–68%) in those with two- to three-loci-mismatched donors. In a multivariate analysis, patients who received a graft from a one-locus mismatched donor and a two- to three-loci-mismatched donor had a hazard ratio for acute GVHD of 1.83 (95% CI, 1.04–3.22; p = 0.035) and 2.44 (95% CI, 1.14–5.21; p = 0.021), respectively, when compared with those from an HLA-matched donor. There was no increased risk of chronic GVHD among recipients of partially HLA-mismatched grafts after reduced intensity conditioning as compared to recipients of HLA-matched sibling SCT. 3.3.  Impaired Immune Reconstitution and Infection Rapid reconstitution of innate and adaptive immunity is critical for resistance to opportunistic infections after allogeneic SCT. Patients who undergo HLAhaploidentical SCT have significantly impaired immune reconstitution as measured by delayed recovery of CD4+ T cells compared to recipients of HLA-matched sibling grafts [101]. A number of factors contribute to impaired immune reconstitution after HLA-haploidentical SCT. Damage to lymphoid tissues from conditioning may interfere with T cell homing and the generation of immunologic memory. The rigorous T-cell depletion necessary to prevent GVHD in the haploidentical setting results in profound post-transplantation immunodeficiency [102, 103]. Finally, acute and chronic GVHD both interfere with immune reconstitution [104, 105], in part through inhibition of thymopoiesis [106]. The result of delayed immune reconstitution after haploidentical SCT is an increased incidence of morbidity and NRM secondary to opportunistic infection. For example, among 101 patients receiving intensive conditioning and megadoses of rigorously T cell-depleted grafts in Perugia, Italy, 27 died of opportunistic infection [53]. Infection was the cause of death in 7 out of 29 hematologic malignancies patients receiving reduced intensity conditioning and haploidentical grafts depleted of CD3+ and CD19+ cells by magnetic cell sorting [107]. Infectious complications remain high even in the context of strategies to selectively deplete alloreactive T cells while sparing immunity to pathogens. In a study from the Dana-Farber Cancer Institute, haploidentical donor grafts were exposed to recipient cells in the presence of T costimulatory blockade, either CTLA-4Ig (n = 19) or a combination of antibodies to B7-1 and B7-2 (n = 5) to induce selective tolerance in alloreactive T cells prior to their transplantation into lethally conditioned recipients. Despite this maneuver, 12 of the 24 patients died of treatment-related causes, 6 of infection with or without GVHD [108]. Conditioning agents that deplete host and donor T cells, such as anti-thymocyte globulin or alemtuzumab (a monoclonal antibody against CD52 expressed on both B and T cells) may also increase the incidence of opportunistic infection after PMRD SCT. Among 49 patients receiving haploidentical SCT after a nonmyeloablative conditioning

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched 

regimen containing alemtuzumab, the rate of cytomegalovirus reactivation was 86%, and 11 patients (22%) died of opportunistic infections [109]. These results illustrate the general observation that strategies employed to reduce graft rejection and/or GVHD tend to increase the incidence and severity of opportunistic infections after haploidentical SCT.

4.  Principles of HLA Typing and Donor Selection for HLA-Haploidentical SCT Since allogeneic SCT from HLA-matched siblings has produced the best overall and event-free survivals [98], the search for a suitable donor always commences with HLA typing of the patient and all available siblings. An HLA-matched sibling can be identified for approximately 30% of patients. If the patient does not have an HLA-matched sibling, the treating physician may choose among a graft from a volunteer unrelated donor (VUD), unrelated umbilical cord blood (UCB), or a haploidentical first-degree relative [110]. Factors that may play a role in the choice of donor source include the urgency of the transplant (identification and mobilization of a VUD takes the longest [111]) and the size of the recipient, since the number of total and CD34+ cells in UCB collections are often insufficient for reliable engraftment of adults [112]. A major advantage of HLA-haploidentical SCT is the rapid availability of a donor for the vast majority of patients. When HLA typing is performed on all first degree relatives, an average of five HLA-haploidentical donors are identified for each patient (HJS and EJF, unpublished observations). The abundance of HLA-haploidentical first-degree relatives poses the challenge of identifying the most suitable donor for transplantation. Molecular methods of HLA typing have largely replaced serologic methods for donor identification in HLA-haploidentical SCT. Typing at the genomic level is capable of identifying allele level mismatches that are undetectable by serologic methods, and a study from Japan showed that survival was significantly worse for patients with standard risk leukemia who received grafts from a single allele-mismatched related donor as compared to those who received a transplant from an HLA-matched sibling [99]. This result raises the possibility that high resolution typing of first-degree relatives may outperform serologic typing for the detection of HLA mismatches that have clinical significance in the context of HLA-haploidentical SCT. Moreover, allele level typing of the HLA locus is required to discriminate allelic variations at the HLA-B and HLA-C loci of relevance to NK cell alloreactivity (Table 18.1). Figure 18-5a illustrates the nomenclature for HLA molecules, and Fig. 18.5b provides an example of allele level typing of a family comprising two parents and their five biological children, one of whom is the patient. HLA typing of the parents enables assignment of specific HLA alleles to either the maternal (designated “a” and “b”) or paternal HLA haplotypes (designated “c” and “d”). The patient is HLA-matched to sibling 1, HLA-haploidentical to siblings 2 and 3, and is completely HLA-mismatched to sibling 4. The patient and sibling 2 have both inherited the paternal “c” haplotype and so are mismatched for both inherited and noninherited maternal HLA antigens (NIMA). In contrast, the patient and sibling 3 have both inherited the maternal “a” haplotype and so are mismatched for inherited and noninherited paternal HLA antigens (NIPA). As is discussed later, donor tolerance of recipient NIMA may lessen the risk

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Fig. 18-5.  High resolution HLA typing in a representative family. (a) Nomenclature employed for high resolution typing. The HLA genetic locus is identified by the capital letter after the hyphen. The first two digits after the asterisk are the antigen designation, which facilitates comparisons of results of low or intermediate resolution vs. high resolution HLA typing. The four digit code designates allele level type by high resolution methods, such as sequence based typing (SBT) or a combination of sequence specific priming (SSP) and sequence-specific oligonucleotide probes (SSOP). (b) Family study. Results of high resolution HLA typing are shown for the parents. Based upon HLA typing of the children, membership of individual HLA alleles in haplotypes, designated “a”, “b”, “c”, and “d”, are assigned. The allele composition of the haplotypes are then used to assign haplotype inheritance in the children. The patient is genotypically HLA-matched to sibling 1 and is a completely mismatched for both HLA haplotypes with sibling 4. Siblings 2 and 3 are partially HLA-mismatched, or HLA-haploidentical, to the patient, and would be considered potential donors for allogeneic SCT only if sibling 1 is not a suitable or willing donor. (c) High resolution HLA typing of the patient is aligned with typing of the HLA-haploidentical siblings to facilitate analysis of the degree of mismatching in both the rejection (host-vs-graft) and graft-vs-host directions. Alleles of the shared haplotype are aligned in columns A1, B1, Cw1, DRB1-1, and DQB1-1. (d) Analysis of HLA mismatching at the antigen and allele levels. A mismatch occurs in the graft-vs-host direction when the recipient expresses an antigen and/or allele that is not expressed by the donor. A mismatch occurs in the rejection direction when the donor expresses an antigen and/or allele that is not expressed by the recipient. Allele level mismatching is always equal to or greater than antigen level mismatching. For example, the patient and sibling 3 are matched at the HLA-B locus at the antigen level but are bidirectionally mismatched at the allele level due to expression of HLA-B*1513 in the patient and HLA-B*1502 in sibling 3. (e) Analysis of natural killer cell alloreactivity by the ligand incompatibility and missing ligand models. The ligand incompatibility model predicts NK cell alloreactivity in the graft-vs-host direction when the patient lacks, but the donor expresses, and HLA ligand for inhibitory KIR. The missing ligand model predicts NK cell alloreactivity in the GVH direction when the patient lacks an HLA ligand for inhibitory KIR, regardless of expression in the donor. For example, the missing ligand model predicts that NK cells of sibling 3 will be alloreactive against the patient based upon a missing HLA-C2 ligand, but the ligand incompatibility model predicts no NK cell alloreactivity in the GVH direction for this pair

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched 

of GVHD after HLA-haploidentical SCT. In Fig. 18.5c, the HLA types of the patient and the HLA-haploidentical siblings are aligned to facilitate determination of the degree of HLA mismatch, which is listed in Fig. 18.5d. For example, T cells from sibling 2 would “see” HLA-B*0702, HLA-Cw*0701, HLA-DRB1*0401, and HLA-DQB1*0304 as being “non-self”; thus sibling 2 and the patient are mismatched for four HLA alleles in the GVH direction, or “vector”. Conversely, the patient’s T cells would see HLA-A*0101, HLAB*5701, HLA-Cw*0602, HLA-DRB1*0101, and HLA-DQB1*0601 as being “non-self”, indicating a five allele mismatch in the host-versus-graft (HVG) vector. This example illustrates the principle of the vector of HLA mismatch: since the patient is homozygous at the HLA-A locus, the donor does not see “non-self” at this locus, but the patient does. Thus, there is one fewer HLA allele mismatch in the GVH vector than in the HVG vector. In Fig.  18.5e, the HLA-B and HLA-Cw types of the patient and HLAhaploidentical siblings are translated into their corresponding KIR ligand groups to predict NK cell alloreactivity by the ligand incompatibility and missing ligand models. For example, the patient expresses but sibling 3 lacks expression of an HLA-Bw4 molecule (see Table  18.1), the ligand for KIR3DL1 (Fig. 18.2). The presence on recipient cells of a KIR ligand that is absent on donor cells predicts NK cell alloreactivity in the HVG direction in both the ligand incompatibility (Fig.  18.3a) and missing ligand (Fig.  18.3c) models. In contrast, the absence of the C2 ligand for KIR2DL1 on both donor and recipient cells predicts bidirectional NK cell alloreactivity in the missing ligand model but no alloreactivity by the ligand incompatibility model. The decision of how many first-degree relatives to type must balance the desire to find the “best” donor with feasibility of extended typing and the need to proceed to transplantation in a timely fashion. Typing all siblings, parents, and children is neither practical nor economically feasible. Aside from considering whether a specific relative is healthy and suitable to donate, the only strong contraindications to donation are prior blood transfusions from the same donor or a positive lymphocytotoxic crossmatch test of patient serum against donor cells. Conversely, the only strongly favorable donor characteristic is <2 HLA allele mismatches with the recipient. Since it is uncommon to find a first-degree relative that is a single allele mismatch with the recipient, an acceptable strategy for donor selection is to perform typing of siblings first in an effort to find an HLA-match and, if none is found, to select the HLAhaploidentical sibling with the least degree of mismatch as long as that sibling is suitable to donate and is “crossmatch negative” with the patient. If more than one HLA-haploidentical first-degree relative is available to donate, then the factors listed in Table 18-2 may be considered.

5.  Clinical Outcomes of HLA-Haploidentical SCT Table 18-3 lists some of the largest published studies of HLA-haploidentical SCT after either myeloablative or nonmyeloablative conditioning. The table illustrates the substantial progress that has been made in improving the safety, efficacy, and utility of the procedure for patients with hematologic malignancies.

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Table 18-2.  Donor-specific factors affecting outcome of allogeneic SCT. Characteristic

Favorable

Unfavorable

Unfavorable effect

Prior blood transfusion from donora

None

Yes

↑Graft rejection

Anti-donor HLA antibodies

Absent

Present

↑Graft rejection [2]

HLA mismatch with recipient

0–1 allele mismatch

³2 allele mismatch

↑GVHD, ↓OS, EFS [1, 76, 98]

a

Younger

Age

Sexa Relationship to recipient

Sibling

a

CMV serologic status

Older [21]

↑GVHD, ↓OS, EFS [21]

Female donor into male recipient

↑GVHD [221], ↑NRM [222]

Parent [20]

↑aGVHD, NRM ↓OS [20]

Positive donor for negative recipient

↑NRM [223]

NK cell alloreactivity

NK alloreactive

Non-alloreactive

↑Relapse in AML [224]

Non-inherited maternal HLA antigens

Unshared

Shared

↑GVHD, ↑NRM [20]

a

These characteristics have not been demonstrated to affect the outcome of HLA-haploidentical SCT

Table 18-3.  Selected published studies of HLA-haploidentical SCT

Authors

N

Graft-vs-host disease (%) Graft Median T failure Acute Acute Chronic NRM II–IV III–IV age depletion (%) (%)a

EventOverall free Relapse survival survival (%)a (%)a (%)a

Myeloablative conditioning Szydlo et al. 340 [98]

25

Ex vivo (49%)

  1 Ag MM   2 Ag MM O’Reilly 52 et al. [87] Mehta et al. [126]

Ex vivo

9

44

27b

52

50–57c 28–65

–

15–36

16

56

36b

60

55–67c 37–45

–

20–25

30d

9

3

–

>50c

–

20

–

201

23

In (71%)  2 + ex vivo

13

–

15

51c

31

18

19

Aversa et al. 104 [53]

33

Ex vivo

9

8

–

7

37

25

39

–

Lu et al. [136]

24

In vivo

1

40

16

55

22

18

71e

64e

135

Nonmyeloablative conditioning Rizzieri et al. [109]

49

48f

In vivo

14

16

–

14

31

–

31

–

Luznik et al. 68 [150]

48f

In vivo

13

34

6

22

15g

51

36

26

a Data on NRM (NRM) are for 1–2 years after transplantation. Data on relapse, overall survival, and event-free survival are for 1–2 years (all studies except Lu et al. [136]) or for 5 years after transplantation (Lu et al.). 1 Ag MM = 1 HLA Ag mismatch Boxes illustrate salient results: (a) Effect of HLA mismatch on severe GVHD after myeloablative, T cell-replete BMT; (b) Excessive NRM in early trials of haploidentical SCT; (c) Increased risk of graft failure with ex vivo graft TCD without intensive immunosuppressive conditioning; (d) Improved outcome of myeloablative SCT using in  vivo TCD; (e, f) Nonmyeloablative conditioning permits transplantation of older patients with reduced treatment-related mortality. See text for details of studies

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched 

Much of the progress in HLA-haploidentical SCT can be attributed to advances in supportive care such as monitoring and preemptive therapy against CMV [113] and EBV-related lymphoproliferative disease [114], and improved detection and treatment of invasive fungal infections [115]. 5.1.  Early Studies Employing T Cell Replete Grafts The early post-transplant complications of myeloablative Tcell replete haploidentical BMT were well described by Powles and colleagues [116]. Of 35 patients with advanced AML or acute lymphocytic leukemia (ALL) who received a one-to three-antigen mismatched BMT, 12 patients died of early complications characterized by pulmonary edema, seizures, intravascular hemolysis, and acute renal failure. Ten of the 35 had engraftment failure, requiring regrafting from the same donor, though 11 patients were alive at the time of reporting. Beatty et al. described the outcomes of HLA-matched vs. -mismatched donor bone marrow transplantation in patients with advanced hematologic malignancies who received myeloablative total body irradiation (TBI)-based conditioning [1]. The incidence of grades II–IV GVHD was higher after HLA-mismatched vs. matched donor BMT, however, overall survival was not worse, possibly due to an enhanced graft-versus-leukemia (GVL) effect. Additionally, overall survival was similar after HLA-matched and 1-antigen mismatched donor BMT for patients with acute leukemia in remission. While the number of patients who received 2- or 3-antigen mismatched BMT was too small to allow for definitive conclusions regarding overall survival, there was a very high, early transplant-related mortality. Taken together, these early results demonstrated that two or three HLA-antigen mismatched transplants were associated with a high risk of treatment-related death, at least in the setting of T-cell replete transplants using pharmacologic GVHD prophylaxis. Thus, the consequences of T-cell replete myeloablative BMT in which HLA barriers were crossed were readily apparent. 5.2.  Effect of T Cell Depletion Recognizing the prohibitive barriers of Tcell replete myeloablative BMT in which HLA-barriers were crossed, investigators looked towards haploBMT using T cell depleted grafts. Effective T cell depletion can completely prevent both acute and chronic GVHD even when using haploidentical parental bone marrow differing at three major HLA loci [75, 117, 118]. This remarkable potential of T cell depletion, based on numerous murine studies in the 1970s, was first demonstrated in the treatment of patients with severe combined immune deficiency (SCID) [119]. The problem of GVHD, which was almost uniformly fatal in such haploidentical transplants, was completely prevented by a 1000-fold, or “3 log”, depletion of T cells using soybean lectin and E-rosetting. Since this initial study, hundreds of transplants from HLAhaploidentical donors have been carried out worldwide for SCID patients with a high rate of long-term partial or complete immune reconstitution [120]. However, despite these encouraging results in SCID patients, patients with hematologic malignancies receiving supralethal chemoradiotherapy-based conditioning and T cell depleted mismatched transplants initially demonstrated graft rejection [82]. Enhancing immunosuppression and myeloablation with the addition of agents such as anti-T antibodies, cytosine arabinoside and

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thiotepa to the standard conditioning regimens still did not ensure engraftment of T-cell-depleted mismatched bone marrow [121–123]. Efforts to overcome the problems of GVHD following myeloablative haploidentical BMT were also complicated by recurrent malignancy [124] and EBV-related post transplant lymphoproliferative disease [125]. Mehta et al. employed in vitro T-cell depletion with MoAb T10B9 (n = 58) or OKT3 (n = 143) in 201 recipients of HLA-haploidentical SCT for advanced hematologic malignancy [126]. Patients received a preparative regimen comprising TBI, etoposide, cytarabine, and CY, with (n = 143) or without (n = 58) anti-thymocyte globulin. GVHD prophylaxis included methylprednisolone and CsA. Stable engraftment was achieved in 98% of patients, and acute grades II–IV GVHD and chronic GVHD occurred in 13 and 15% of evaluable patients, respectively. The cumulative incidences of relapse and NRM at 5 years after transplantation were 31 and 51%, respectively, resulting in 5-year actuarial overall and event-free survivals of 19 and 18%, respectively. Complications of infection, including EBV-related lymphoproliferative disease, accounted for 45 of 100 treatment-related deaths. These results illustrate that what is gained by suppressing graft rejection and GVHD is frequently lost to the complications of immunodeficiency and other causes of NRM. 5.3.  Megadose Stem Cell Transplantation: A Turning Point in HLA-Haploidentical SCT A turning point for haploidentical, T-cell depleted BMT came in 1993 [91] with the clinical application of an extensively T cell depleted megadose of stem cells, a concept pioneered in animal models by Reisner in the late 1980s [89, 92]. “Megadose” stem cell transplants, piloted by Aversa and colleagues, initially consisted of G-CSF mobilized peripheral blood stem cells and bone marrow cells, both depleted of T cells ex vivo by soybean agglutination and E-rosetting [91] and a conditioning regiment including TBI, cyclophosphamide, thiotepa, and anti-thymocyte globulin (ATG), with no additional pharmacologic immunosuppression after transplantation. The Perugia group subsequently modified this regimen extensively, with fludarabine replacing Cy in the TBI-based conditioning regimen after the observation in the mouse model that fludarabine and TBI provided equivalent immunosuppressive effect [127]. The substitution of fludarabine for Cy represented an attempt to reduce the conditioning regimen toxicity without jeopardizing its immunosuppressive effect. In addition, the total lung dose of radiation was decreased from 6 to 4 Gy. Other advances included implementation of a CD34+ selection device that provided a 4.5 log Tcell depletion, as well as the avoidance of granulocyte colony-stimulating factor (G-CSF) after transplant which was thought to impair dendritic cell production of IL-12 leading to abnormalities in antigen-presenting function and T cell reactivity [128, 129]. Over the past decade, the Perugia group has demonstrated that full haplotype mismatched transplants can be successful in acute leukemia patients in first or second complete remission when a megadose of stem cells is infused after an immunomyeloablative conditioning regimen [53]. Among 104 patients transplanted for acute leukemia, acute GVHD occurred in only 8 of 101 assessable patients and chronic GVHD developed in 5 of 70 evaluable patients. However, the megadose SCT regimen is associated with an increased rate of infectious morbidity and mortality, secondary to a prolonged time to immune reconstitution [130]. Early results showed a transplant-related

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched 

mortality risk of approximately 40% [52], with infection the leading cause of death. Somewhat improved immune reconstitution and fewer deaths secondary to infection occurred when G-CSF was discontinued. Other approaches using myeloablative conditioning and high dose CD34+ cell selected grafts described similarly favorable engraftment and GVHD rates, but unfortunately, recurrent malignancy and problems with infectiousrelated deaths were encountered. In a Canadian multi-center study, all 11 study patients engrafted without GVHD but 10 of 11 patients died of leukemic relapse or infection [79]. Waller et al. reported a 93% mortality rate in patients who received T cell-depleted, CD34+ enriched HLA-haploidentical SCT after an ATG-based regimen, with most deaths a result of infection or relapse [131]. In a retrospective analysis from Japan, severe infections occurred in 20 of 32 patients receiving CD34-selected PBSC from 2 to 3 HLA antigen-mismatched related donors [132]. Seventeen of 32 patients (53%) died of treatment-related causes, including 10 (31%) from infection, and 9 patients died of complications of progressive disease. These results suggest that transplantation of highly purified CD34+ PBSC from haploidentical donors is associated with a low incidence of GVHD but an increased risk of disease progression or fatal infection. Recently, methods of depleting CD3+ T cells and CD19+ B cells from megadose PBSC collections have been developed [133]. The Acute Leukemia Working Party of the European Blood and Marrow Transplant Group Registry has reported outcomes of 266 acute leukemia patients receiving myeloablative conditioning and CD34+-selected, “megadose” PBSC grafts from HLA-haploidentical related donors [134]. For the 119 patients who were not in remission at the time of transplantation, the cumulative incidence of transplant-related mortality was 66% for AML and 44% for AML, and the cumulative incidence of relapse was 32 and 49%, respectively. Only 5 patients were alive between 5 and 56 months after transplantation. Eighty-six patients with AML and 61 patients with ALL were transplanted in remission. Grade II–IV acute GVHD was observed in 4 (5%) AML patients and 11 (18%) ALL patients. Among patients who survived more than 100 days, chronic GVHD was observed in 6/56 (10%) and 7/39 (19%) of AML and ALL patients, respectively. For patients with AML, the cumulative incidence of NRM was 36 and 54% for patients transplanted in CR1 (n = 25) or CR ³ 2 (n = 61), respectively, and for patients with ALL, the corresponding numbers were 61% (n = 24) and 44% (n = 37) for patients in CR1 and CR ³2, respectively (Fig. 18-6). 5.4.  Blood vs. Marrow from G-CSF Primed Donors Treatment of bone marrow donors with granulocyte colony-stimulating factor (G-CSF) prior to donation increases marrow CD34+ and CFU-GM cells, reduces total lymphocytes and reverses the CD4+/CD8+ T cell ratio. In order to enhance engraftment by increasing the dose of transplanted HSCs, 15 patients with high risk leukemia received myeloablative conditioning with cytarabine, Cy, and 1000  cGy TBI, G-CSF-primed bone marrow from haploidentical donors, and GVHD prophylaxis with rabbit anti-thymocyte globulin (5  mg/ kg/day on days −4 → −1), CsA, MTX, and mycophenolate mofetil (MMF) [135]. All 15 patients had prompt trilineage hematopoietic engraftment, the cumulative incidence of GVHD was 33%, and 9 of 15 patients were alive at a median follow-up of 22 months (range 13–35 months) at the time of reporting.

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Fig. 18-6.  Outcomes of myeloablative conditioning and “megadose”, T cell-depleted SCT for acute leukemia in remission. Non-relapse mortality (a, b) and overall survival (c, d) for patients with acute myeloid leukemia (a, c) and acute lymphocytic leukemia (b, d). CR1, first complete remission; CR ³ 2, second or subsequent complete remission. This research was originally published in Blood. ©American Society of Hematology [134]

Based upon these results, Lu and colleagues at Peking University in Beijing, China compared the outcomes of 293 patients with leukemia receiving HLAmatched sibling (n = 158) or HLA-haploidentical related grafts (n = 135) from G-CSF-primed donors [136]. Patients undergoing haploidentical SCT were conditioned with cytarabine, oral busulfan, CY, and methyl-CCNU, received G-CSF primed bone marrow on day 0 (n = 134) and/or G-CSF primed peripheral blood on day 1 (n = 131), and GVHD prophylaxis with anti-thymocyte globulin 2.5 mg/kg/day on days −4 → −1, CsA, MTX, and MMF. All but two haploidentical SCT patients had sustained engraftment of donor neutrophils. The cumulative incidences of acute grades II–IV, grades III–IV, and chronic GVHD in recipients of matched vs. mismatched SCT were 32% vs. 40% (p = 0.13; Fig. 18-7a), 11% vs. 16% (no p value provided), and 56% vs. 55% (p = 0.90; Fig.  18-7b). Mismatched patients had a higher incidence of CMV antigenemia (65% vs. 39%; p < 0.001) and hemorrhagic cystitis (35% vs. 13%; p < 0.001) but not of CMV disease. Two-year rates of relapse and NRM were 13% vs. 18% (p = 0.40; Fig. 18.7c) and 14% vs. 22% (p = 0.10; Fig. 18.7d) for recipients of matched versus mismatched transplants, respectively. The 2-year probabilities of leukemia-free survival were 71% vs. 64% (Fig. 18.7e) and of overall survival were 72% vs. 71% (p = 0.72; Fig. 18.7f) in the matched and mismatched cohorts, respectively. In a follow-up report of 157 consecutive recipients of G-CSF-primed bone marrow plus peripheral blood from haploidentical related donors, recipients of CD3+ T cell doses higher than the median

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched 

321

Fig. 18-7.  HLA-haploidentical SCT from G-CSF-mobilized donors: comparison to recipients of HLA-matched sibling SCT. (a) acute GVHD; (b) chronic GVHD; (c) relapse; (d) non-relapse mortality; (e) leukemia-free survival; and (f) overall survival. This research was originally published in Blood. ©American Society of Hematology [136]

(1.77 × 108/kg) had a significantly lower NRM, better leukemia-free survival, and better overall survival [137]. The Beijing results are extremely encouraging and this regimen for haploidentical SCT needs to be evaluated at other centers. Novel aspects of the regimen that may contribute to the low rates of graft failure and GVHD may be the use of low-dose rabbit ATG [138], the use of G-CSF–mobilized bone marrow plus peripheral blood [128, 139] and the combination of CSP, MTX, and MMF. 5.5.  Nonmyeloablative HLA-Haploidentical SCT In an effort to reduce risk of treatment-related mortality while retaining the potential for a graft-versus-tumor (GVT) effect, several recent clinical trials have evaluated the efficacy of nonmyeloablative conditioning for haploidentical BMT. Based on murine models established by Sykes and colleagues [140, 141], clinical trials using nonmyeloablative conditioning (Cy ± flu) with invivo T cell depletion using polyclonal or monoclonal anti-T cell antibodies, pretransplant thymic irradiation, and most recently, ex vivo T cell depletion have been performed [142]. The rationale for this approach has included (1) the reduction of regimen-related toxicities with nonmyeloablative ­conditioning;

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(2) prevention of GVHD with in  vivo and ex vivo T cell depletion;and (3) the induction of a graft-versus-tumor effect with delayed donor lymphocyte infusions, when clinically indicated. Their current protocol includes Cy, Flu, MEDI-507 (a monoclonal CD2 antibody), and thymic irradiation which has resulted in a high incidence of mixed chimerism without early GVHD and the potential for conversion of Tcell chimerism with manageable or no GVHD. Recurrent malignancy and late infections have been the main reasons for treatment failure with this approach [143]. Based on the promising results from St. Judes Children’s Research Hospital in the pediatric population [133], Bethge et al. explored a new T cell depletion strategy in adult patients by negatively selecting T and B cells from peripheral blood stem cell collections with 3.5–4 log T cell depletion using anti-CD3 and anti-CD19 coated microbeads on a magnetic separation (CliniMACS; Miltenyi Biotec, Bergisch-Gladbach, Germany) device [144]. In contrast to the CD34+ selection strategy pioneered by the Perugia group, grafts harvested from this strategy contained not only CD34 stem cells but also CD34− progenitors and NK, dendritic, and graft facilitating cells. Ten adult patients were transplanted with a nonmyeloablative preparative regimen containing fludarabine, thiotepa, melphalan, and OKT3. Rapid engraftment was seen in all patients after 2 weeks, six patients developed Grade II acute GVHD and one patient, who received the highest T cell dose (4.4 × 105/kg) died from grade IV GVHD. NRM was 30% and OS was 50% with four patients in complete remission with a median follow-up of greater than 1 year. The fast engraftment seen with this regimen is another demonstration that successful haploidentical transplantation may be possible without megadoses of CD34+ stem cells. In an updated report, 29 patients with high risk hematologic malignancies received transplants with CD3/CD19 depleted grafts from haploidentical family donors [107]. All but one patient engrafted. T cell reconstitution was faster as compared with CD34 selected grafts, but was still slow overall. NRM in the first 100 days was 6/29 (21%). The incidence of grade II–IV GVHD was 48%. Three cases of limited chronic GVHD were observed to date. Twenty patients died, 12 due to relapse, 7 due to infections and 1 patient due to GVHD. Overall survival is 9/29 patients (31%) with a median follow-up of 241 days (range, 112–1271). The Kaplan–Meier estimate of event-free and overall survival is 35% at 12 months. In another recent study by Handgretinger and colleagues, 38 pediatric patients with high-risk hematologic malignancies (n = 35) or severe aplastic anemia (n = 3) receiving CD3/CD19-depleted haploidentical transplants after fludarabine 160 mg/m2, thiotepa 10 mg/kg, melphalan 140 mg/m2, and OKT3, demonstrated a primary engraftment rate of 83% and a 98% engraftment rate when the remaining patients with graft failure had a second transplant. Grade II–IV aGVHD occurred in only 27% of patients and NRM was low at 2.6%. There was a favorable event-free survival of 70% seen only in patients with nonmalignant diseases and those in remission at the time of transplant, indicating that disease relapse is still a major obstacle among patients with refractory malignancies [145]. Ogawa et  al. investigated the use of a nonmyeloablative conditioning regimen containing anti-thymocyte globulin [146], as previously reported by Slavin et  al. [147] Twenty six patients with advanced stage hematologic malignancies received haploidentical transplants with a preparative regimen of

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched 

fludarabine, busulfan, and anti-T-lymphocyte globulin and GVHD prophylaxis consisting of tacrolimus and methylprednisolone (1 mg/kg/day). All patients except for one achieved donor-type engraftment. Full donor chimerism was achieved by day 14. Only 5 of 20 evaluable patients (25%) developed grade II acute GVHD. Sixteen of the 26 patients were alive in complete remission at the time of reporting. Four died of transplantation-related causes, and six died of progressive disease. The event-free survival at 3 years was 55%. Kanda et al. evaluated the feasibility of haploidentical unmanipulated PBSC transplantation from two or three loci mismatched family member using in vivo alemtuzumab in 12 patients with high-risk hematologic malignancies [148]. Six patients received a TBI-based myeloablative regimen, whereas the remaining six patients older than 50 years received less intensive or nonmyeloablative fludarabine-based conditioning. Alemtuzumab was added on days −8 to −3 and CSP and MTX were used as GVHD prophylaxis. There was no graft rejection, and the incidence of grade III–IV aGVHD was only 9%. Nonrelapse mortality (NRM) was observed in only 2 of 12 patients. None of the patients died of infectious causes despite impaired T cells immune reconstitution during the first 2 months after transplantation. Rizzieri and colleagues at Duke University developed a nonmyeloablative conditioning regimen incorporating fludarabine, CY, and alemtuzumab for 49 hematologic malignancies patients receiving PBSC from HLA-haploidentical donors [44]. MMF, with (n = 25) or without CsA (n = 24), was used for posttransplantation GVHD prophylaxis. A total of seven patients (14%) experienced either primary or secondary graft failure, and the incidences of acute grades II–IV and chronic GVHD were 16 and 14%, respectively. Fifteen patients (31%) died of causes unrelated to disease progression. Twenty-five percent of patients experienced a severe infection, reactivation of cytomegalovirus (CMV) occurred in 86%, and CMV disease developed in 14%. Overall survival of patients 1 year after transplantation was 31%. Absence of GVHD was associated with improved recovery of CD4+ and CD8+ T cells and CD56+ NK cells following transplantation. 5.6.  Selective Allodepletion Using Cyclophosphamide-Induced Immunologic Tolerance Luznik, O’Donnell and colleagues exploited the protocol of drug-induced immunological tolerance, first described in 1959 by Schwartz and Dameshek [149], to achieve selective in  vivo depletion of alloreactive T cells after nonmyeloablative HLA-haploidentical bone marrow transplantation. In this protocol, in vivo exposure to antigen induces the proliferation of Ag-specific lymphocytes, which are then killed by the timely administration of a drug that is selectively toxic to proliferating over resting cells. Studies in mice established that high-dose, post-transplantation CY inhibits both graft rejection and GVHD after either MHC-matched or -mismatched SCT [46–49]. Based upon these studies, 68 patients with hematologic malignancies (n = 67) or paroxysmal nocturnal hemoglobinuria (n = 1) received CY 50 mg/kg on day 3 (n = 28) or days 3 and 4 (n = 40) after nonmyeloablative conditioning and transplantation of T cell-replete bone marrow from HLA-haploidentical related donors [150]. Graft failure occurred in 9 of 66 (13%) evaluable patients, and was fatal in one. The cumulative incidences of acute grades II–IV and grades

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III–IV GVHD were 34 and 6%, respectively, and of chronic GVHD was 22%. Serious infections were relatively infrequent: there were no cases of CMV disease and only five cases of invasive fungal infection, two of which were fatal. NRM and relapse at 1 year after transplantation was 15 and 51%, respectively. Actuarial overall and event-free survivals (EFS) at 2 years after transplantation were 36 and 26%, respectively. These results suggest that post-transplantation CY induces selective allodepletion in vivo, inhibiting fatal graft rejection and severe GVHD while sparing functional immunity to infection. Another novel approach to haploidentical BMT recently published by Amrolia et al. [151], described using an anti-CD25 immunotoxin to selectively deplete alloreactive lymphocytes with the goal of improving immune reconstitution while limiting GVHD. Allodepleted lymphocytes were infused at days 30, 60, and 90 after either myeloblative or nonmyeloablative haploidentical HSCT for high risk hematologic malignancies. Eight patients received 104 T cells/kg/dose, and eight patients received 105 T cells/kg/dose. Patients receiving 105 T cells/kg/dose showed significantly improved Tcell recovery at 3, 4, and 5 months after SCT compared with those receiving 104 T cells/kg/dose. The incidence of significant acute (2 of 16) and chronic GVHD (2 of 15) was low. These data demonstrate that allodepleted donor T cells may be safely used to improve T cell recovery after haploidentical SCT and may broaden the applicability of this approach. However, this selective allodepletion is potentially a time-consuming and expensive process, and the best technique to perform the allodepletion is uncertain.

6.  Strategies to Reduce the Complications of HLA-Haploidentical SCT 6.1.  Strategies to Mitigate GVHD As discussed above, nonselective depletion of grafted T cells significantly reduces the incidence and severity of GVHD after PMRD SCT but also increases the risk of graft failure and fatal opportunistic infection from prolonged immune compromise. Aside from selecting donors with the least degree of HLA mismatch with the patient, a number of additional strategies have been envisioned or employed to reduce the risk of GVHD without causing profound immune compromise. These strategies include: (1) selection of donors based upon the principle of tolerance to noninherited maternal Ags, or NIMA; (2) selective depletion of alloreactive T cells from the graft; (3) reconstitution of T cell-depleted grafts with T cells that protect against infection but do not cause GVHD; or (4) adding cells that suppress GVHD to T cell-replete grafts. 6.1.1.  Selection of Donors Tolerant to Non-inherited Maternal Antigens Exposure of the developing fetus to maternal cells, which occurs during pregnancy [152], can lead to either immunity or tolerance of non-inherited maternal HLA Ags NIMA and subsequently have an effect on transplant outcome. Two separate studies have demonstrated that approximately 50% of individuals with antibodies against a large number of HLA Ags do not have antibodies against NIMA [153, 154]. Reactivity against non-inherited paternal antigens (NIPA) is significantly higher. Siblings who are HLA-haploidentical to each other share either the paternal or the maternal HLA haplotype.

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched 

When siblings share the paternal HLA haplotype, they are mismatched for both inherited and non-inherited maternal antigens. Thus, HLA typing of both of the patient’s parents is required to determine whether a sibling is mismatched for NIMA or for NIPA. It has been speculated that there should be less GVHD and less graft rejection with NIMA- rather than with NIPAmismatched transplantations. Because graft failure and GVHD affect the outcome of HLA-haploidentical SCT, NRM and OS might also differ between NIMA- and NIPA-mismatched transplants. To date, several population studies have provided evidence in favor of the presence of the tolerogenic “NIMA” effect. Such evidence includes low rates of aGVHD in T cellreplete, HLA-haploidentical SCT from an NIMA-mismatched sibling [20, 155] as well as in unmanipulated marrow transplantation from fully HLAhaploidentical mothers using standard preparative regimens combined with peritransplantation ATG [156], and a significantly lower risk of cGVHD in recipients of non-T cell depleted maternal transplants vs. paternal transplants [20]. Several recent studies [157–160] have also demonstrated sustained remissions of chemorefractory hematologic malignancies with acceptable rates of GVHD after T cell-replete, HLA-haploidentical SCT from microchimeric NIMA-mismatched family members. Further studies are required to evaluate more precisely the effects of NIMA- or NIPA-specific allotolerance and to identify genetic factors associated with GVHD, NRM, and relapse free survival in a given NIMA-mismatched donor-recipient pair. 6.1.2.  Selective Graft Allodepletion Another strategy to reduce GVHD after HLA-haploidentical SCT is to induce tolerance, or “anergy”, in host-reactive T cells by exposing the graft ex vivo to host alloantigens (“signal 1”) while simultaneously blocking the delivery of T cell costimulatory signals (“signal 2”) [161]. Guinan and colleagues conducted two pilot trials of myeloablative, HLA-haploidentical SCT in which donor marrow was incubated with irradiated recipient mononuclear cells in the presence of CTLA-4-Ig, a fusion molecule that blocks interaction of the T cell costimulatory receptor CD28 with its ligands, B7-1 and B7-2, on APCs (n = 19), or with a combination of MoAbs against B7-1 and B7-2 (n = 5) [108]. GVHD developed in only nine of 21 evaluable patients (4 grade II, 4 grade III, 1 grade IV), and 8 patients were alive at a median of 8 years after transplantation. Ex vivo tolerance induction with the combination of antibodies against B7-1 and B7-2 resulted in a 99% reduction in T cells capable of proliferating to host Ags with no significant loss of reactivity to third- party alloantigens, viral Ags, or the WT-1 tumor Ag [162, 163]. These in vitro results correlated with the low incidence of late viral infections or of opportunistic infections requiring admission [164]. However, there were 12 early deaths due to bacterial or fungal infection and/or regimen-related toxicity. The investigators are currently studying the effects of administering allo-anergized T cells after CD34-selected haploidentical SCT, to determine the optimal dose for augmenting immune reconstitution without causing GVHD [108]. 6.1.3.  Graft T Cell Depletion Followed by Infusion of Allodepleted Lymphocytes An alternative to selectively depleting the stem cell graft of alloreactive lymphocytes is to administer a T cell-depleted stem cell graft followed by delayed infusion of mature lymphocytes selectively depleted of alloreactive cells.

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Selective allodepletion has been achieved by activating donor lymphocytes ex vivo with host APCs, followed by targeted removal based upon differential expression of surface-activation markers, proliferation, or retention of photoactive dyes. Methods of alloreactive cell elimination include treatment with immunotoxins [165, 166], immunomagnetic separation [167–171], activation of suicide genes [172, 173], activation-induced cell death [174], flow cytometric sorting [175, 176], or photodynamic purging [177, 178]. To achieve selective allodepletion, the group at Hôpital Necker in Paris, France co-cultured donor and irradiated host lymphocytes ex vivo, followed by addition of a ricin A chain-coupled MoAb against CD25, the a chain of the interleukin-2 receptor [179]. This procedure results in a >2 log depletion of host-reactive cells while sparing reactivity to viral and bacterial Ags as well as third-party alloantigens [165]. Allodepleted lymphocytes in doses ranging from 1 to 8 × 105 cells/kg were infused into 15 patients from 15 to 47 days after myeloablative conditioning and transplantation of CD34-selected stem cell grafts from HLA-haploidentical donors. Grade I–II acute GVHD occurred in four patients, correlating with anti-host residual proliferation above 1% in a mixed lymphocyte reaction, and limited chronic GVHD in one. Compared to controls, recipients of allodepleted T cells had a faster recovery of CD4+ and CD8+ T cells, and infections from EBV, CMV, and adenovirus were eliminated following infusion. At the time of reporting, 8 of 15 patients were alive and well at a median of 24 months of follow-up. Amrolia and colleagues conducted a clinical trial of infusing allodepleted lymphocytes, using the ricin A chain conjugated anti-CD25 moAb, into 16 recipients of T cell-depleted, haploidentical SCT [151]. Eight patients received a dose of 104 T cells/kg while another eight patients received 105 T cells/kg. Recipients of the higher dose demonstrated improved T cell recovery resulting from expansion of the effector memory population without evidence of new T cell generation in the thymus. In vitro T cell responses to CMV- and EBV-associated Ags were detected as early as 2–4 months after transplantation in four of six recipients of the higher T cell dose but not until 6–12 months after transplantation among recipients of the lower T cell dose. Acute and chronic GVHD occurred in only two patients each. More recently, the same group has found that immunomagnetic depletion of alloactivated lymphocytes expressing CD25 and/or CD71 is more effective at reducing alloreactivity than strategies based on depleting only CD25+ T cells [180]. This double depletion strategy may facilitate infusion of larger doses of T cells to promote immune reconstitution while avoiding GVHD. A potentially promising strategy for enhancing immune reconstitution and preserving GVL after TCD haploidentical SCT arises from the observation that effector memory (CD44+CD62L−) CD4+ T cells do not cause GVHD following their transfer into irradiated, MHC-mismatched recipients [181–184]. Their inability to cause GVHD stands in contrast to their ability to mediate GVL effects [183, 185] as well as protection from infection. These results suggest a strategy of augmenting immune reconstitution and GVL by infusing effector memory T cells into recipients of TCD haploidentical SCT. 6.1.4.  Adding T Cells that Suppress GVHD to T Cell Replete Grafts Mesenchymal stem cells (MSCs) and CD4+CD25+foxp3+ regulatory T cells (Tregs) are two types of cells that can inhibit T cell responses to alloantigens

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched 

and so could be infused with or after T cell-replete grafts to inhibit GVHD after haploidentical SCT. MSCs are bone marrow stromal cells that can differentiate into other cells derived from mesoderm, including chondrocytes, tenocytes, and myoblasts [186]. MSCs can be immunosuppressive, inhibiting the proliferation of human T cells stimulated by irradiated allogeneic PBMC [187–191]. Third-party MSCs have been co-transplanted with stem cell grafts to suppress GVHD after HLA-matched sibling SCT [192] and to treat established GVHD after HLA-matched or mismatched donor SCT [193]. In contrast to their effects on T cell responses to alloantigens, MSCs have little effect on established T cell responses to EBV and CMV [194]. Regulatory T cells have an established role in the maintenance of self-tolerance and the prevention of autoimmunity [195]. Addition of large numbers of donor Tregs to stem cell grafts suppresses the development of acute GVHD after MHC-mismatched allogeneic SCT in mice without impairing GVL activity [196–199]. Successful translation of these findings to haploidentical SCT in humans requires protocols to expand Tregs ex vivo to sufficient numbers to suppress alloreactivity in vivo. 6.2.  Strategies to Prevent or Treat Relapse 6.2.1.  Natural Killer Cells NK mediated lysis of a target cell is thought to occur when an inhibitory killer immunoglobulin receptor (KIR) is present on the NK cell while its corresponding MHC class I ligand is missing from the target cell, and an activating KIR is present on the NK cell with its corresponding ligand present on the target cell. In studies by the Perugia group, KIR ligand incompatibility (HLA ligand present in the donor but absent in the recipient; Fig. 18.3a) reduced the risk of relapse in 57 AML patients while improving engraftment and protecting against GVHD [44]. Their updated analysis of greater than 90 HLA-haploidentical transplants for high-risk AML showed that transplantation from NK alloreactive donors was associated with control of AML relapse and improved EFS, with a greater than 65% EFS of AML patients transplanted in remission from NK alloreactive donors, and a 30% EFS of chemoresistant AML patients. This was compared to an EFS of 18% in AML patients transplanted from non-NK alloreactive HLA-haploidentical donors [45]. Another updated analysis showed that 112 high-risk adult AML patients who received transplants from 1993 to 2006 demonstrated that transplantation from NK alloreactive donors did not cause GVHD and helped to control leukemia relapse in patients transplanted in remission [51] The “receptorligand” model (Fig. 18.2b) was better able to predict relapse in pediatric AML and ALL patients than the ligand incompatibility model [48]. Still, utilizing a third method where genotyping of inhibitory KIR was performed (Fig. 18.2d), patients with KIR gene mismatches (i.e., KIR gene present on donor but absent in the recipient, or vice versa) had a higher incidence of GVHD than those without mismatches [49]. Among patients receiving nonmyeloablative haploidentical SCT with high-dose post-transplantation CY, inhibitory KIR gene mismatches between donor and recipient was associated with improved overall survival and event-free survival [200]. Activating KIRs also deserve evaluation in HLA-haploidentical transplantation. Activating KIRs exhibit allelic polymorphisms in specific genes and

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extensive variation in gene number and content, which lead to heterogeneity within the general population and within diverse ethnic groups. In some studies, transplantation from donors carrying activating KIR genes was associated with improved control of leukemia relapse after HLA-identical transplantation [201], and improved survival after unrelated donor transplantation [202]. Other reports have shown that transplantation from donors carrying activating KIR genes adversely affected transplantation outcomes after partially TCD HLA-haploidentical transplants, mainly through an increased risk of GVHD [69]. Conversely, it has been shown that transplantation from donors carrying activating KIR genes (group B haplotype) did not cause GVHD but was surprisingly associated with less infectious mortality and better survival [45]. Since nontransformed tissues generally do not overexpress ligands for activating receptors on NK cells [203, 204], NK cell adoptive immunotherapy has the potential to induce GVL effects without causing GVHD [43, 44]. There are several strategies available to enhance the anti-tumor effects of NK cells in the context of HLA-haploidentical SCT. Since each patient has on an average five HLA-haploidentical first-degree relatives who are eligible to donate stem cells (HJS and EJF, unpublished observations), donors could be selected on the basis of optimal NK cell alloreactivity, as determined by models (Fig.  18.2) or by in  vitro assays. Chemotherapy can enhance the anti-tumor efficacy of subsequent NK cell infusions through multiple mechanisms, including the induction on tumor cell expression of stress ligands for NK cell activating receptors [205], sensitization of tumor cells to NK cellinduced apoptosis [206–208], or enhancement of the survival of adoptively transferred NK cells through lymphopenia-induced cytokines [209]. Finally, therapeutic MoAbs, such as rituximab, may enhance the tumoricidal activity of NK cells by engaging activating Fc receptors, such as FcgRIII (CD16) [210]. More work is clearly needed to define the optimal conditions and strategies for enhancing the anti-tumor effect of NK cells in the context of HLA-haploidentical SCT. The alloreactivity of NK cells has been used as a form of adoptive immunotherapy in patients with advanced or refractory hematologic malignancies. Miller et al. recently demonstrated the safety and potential efficacy of adoptive haploidentical related NK cell therapy without BMT following high-dose intensity conditioning. All NK cell donors were haploidentical family members; few with KIR ligand mismatches in the GVH direction. Twenty six percent of patients with AML achieved complete remission of their hematologic malignancy. A significantly higher complete remission rate was achieved when KIR-mismatched donors were used. Donor NK infusions were well tolerated without evidence of GVHD, and the study demonstrated in vivo donor-derived NK cell expansion in the majority of patients along with increased levels of endogenous IL-15 [209]. These findings demonstrate that haploidentical NK cells can persist and expand in  vivo and may have a role in treating select high-risk hematology malignancies. 6.2.2.  Donor T Cell Infusions The published literature on donor lymphocyte infusions (DLI) after HLAhaploidentical SCT is scanty. In a study from Israel, 28 patients received prophylactic (n = 6) or therapeutic DLI (n = 22) in doses ranging from 100 to 1.5 × 109 T cells/kg [211]. Of the six patients receiving prophylactic DLI, three

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched 

patients remain in remission, one relapsed, and two died of GVHD. Complete remission was achieved in only 4 of the 22 recipients of therapeutic DLI, and only one remains in CR. The group in Beijing administered G-CSF-primed DLI prophylactically to 29 patients [212] and therapeutically to 20 patients [213]. Two-year event-free survivals were 37.3 vs. 40% of recipients of prophylactic vs. therapeutic DLI, respectively. Severe GVHD occurred in six patients in each group. Further studies to define dose-response relationships for both GVHD and anti-tumor efficacy are clearly required before DLI can be routinely recommended for the prevention or treatment of relapse after HLA-haploidentical SCT. It is worth noting that the presence of T cells in allogeneic stem cell grafts affects NK cell reconstitution and function after unrelated [73] and HLAhaploidentical related donor SCT [214]. Cross-talk between T cells, NK cells, and dendritic cells occurs at the interface of innate and adaptive immunity [215], and these complicated interactions are only beginning to be explored in the context of allogeneic SCT. Both T cell and NK cell adoptive immunotherapies will benefit from an improved understanding of these cellular interactions. The ready availability of the original transplant donor for repeated lymphocyte donations is a distinct advantage of HLA-haploidentical related over unrelated donor SCT.

7.  Unrelated Donor Umbilical Cord Blood vs. HLA-Haploidentical Related Donor SCT Patients who lack suitably HLA-matched related or unrelated donors have a choice between two sources of alternative donor stem cells: unrelated umbilical cord blood (UCB) or HLA-haploidentical related stem cells. Is there any rational or empirical basis for choosing between these two alternatives? Unrelated UCB has an established track record in the treatment of hematologic malignancies of children. A retrospective analysis compared the outcomes of unrelated UCB transplantation (UCBT; n = 503) vs. 8/8 HLA allele (HLA-A, -B, -C, and -DRB1)-matched unrelated donor marrow transplantation (n = 116) for children under the age of 16 with leukemia [22]. Typing of the UCB grafts was performed at low resolution (antigen level) for HLA-A and -B and at high resolution (allele level) for HLA-DRB1, and results for 1 HLA locus mismatch grafts were analyzed according to cell dose (>3.0 × 107 nucleated cells/kg vs. £3.0 × 107 nucleated cells/kg). The results of Table 18.4 show that, at the very least, HLA-matched and high-dose, single locus-mismatched UCB grafts produce overall and leukemia-free survivals that are at least as good as is seen after 8/8 allele-matched unrelated bone marrow transplants. Leukemia-free survival after 1 or 2 HLA locus-mismatched UCBT was not significantly worse than after HLA-matched unrelated donor SCT. These results establish 4–6/6 HLA Ag-matched UCB as a viable alternative to the use of HLA-matched unrelated donor bone marrow for the transplantation of children with acute leukemia. Further, the results suggest that HLA-matched UCB is the new “gold standard” among alternative graft sources for allogeneic SCT in childhood leukemia. There are not enough data at present to make statistically valid comparisons of the outcomes of HLA-haploidentical related versus HLA-matched unrelated

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Table 18-4.  Clinical outcomes of unrelated adult marrow versus cord blood transplantation for leukemia in children. Graft

TRM (%)

Relapse (%)

LFS (%)

OS (%)

BM, allele matched at HLA-A, -B, -C, -DRB1

19

41

38

45

CB, A, B, antigen-matched, DRB1 allele matched

6

34

60

63

CB, 1-locus mismatched, high cell dose

29

31

41

45

CB, 1-locus mismatched, low cell dose

43

21

37

36

CB, 2-loci mismatched any cell dose

49

20

33

33

BM bone marrow, CB cord blood, TRM treatment-related mortality, OS overall survival, LFS leukemia-free survival. Low cell dose, £3.0 × 107 nucleated cells/kg; High cell dose, >3.0 × 107 nucleated cells/kg. From Eapen et al. [22]

donor SCT in the treatment of childhood leukemia. Therefore, HLAhaploidentical related donor SCT for childhood leukemia should only be conducted in the context of carefully designed clinical trials. For adult patients, cell dose is a major limitation in the use of UCBT. Most single UCB units simply do not contain enough hematopoietic stem cells to guarantee reliable engraftment in older adults. In the first series of U.S. adults receiving UCBT, median UCB graft cell dose was tenfold lower than among recipients of HLA-matched or mismatched marrow (0.22 vs. 2.4 and 2.2 × 108 cells/kg, respectively), sustained neutrophil engraftment occurred in <70% of UCBT recipients, and NRM occurred in 95 of 150 patients, many due to infection within the first 100 days after transplantation [216]. HLA matching is also a significant limitation of UCBT. In an analysis of 1,511 recipients of single cord blood units from the New York Blood Center National Cord Blood Program, the degree of HLA mismatch was found to correlate adversely with engraftment, GVHD, relapse, TRM, and overall survival [217]. While cell dose did not affect the outcome of fully HLA-matched UCB transplants, a twofold increase in the cell dose was required to overcome differences in treatment-related mortality and survival for 2 vs. 1 HLA Ag-mismatched grafts. These findings are significant because the likelihood of finding a single cord blood unit that is mismatched for at most 1 HLA Ag and that contains >3 × 107 nucleated cells/kg for an adult recipient is low. Recently, adult transplantation protocols incorporating the infusion of two UCB units, each containing ³1.5 × 107 nucleated cells/kg, have been a major step toward overcoming the limitations of inadequate cell dose in individual units. Double unit UBCT after myeloablative conditioning was associated with improved engraftment and lower NRM compared to historical controls receiving a single unit, and 1-year disease-free survival among 23 patients was 57% [218]. In that study, the median total nucleated cell dose was 4.8 × 107/ kg, and 13 patients received at least one unit that was matched to the patient at 5–6/6 HLA Ags. Two recent studies have demonstrated the feasibility of double unit UCBT after nonmyeloablative conditioning in adults [219, 220]. Among 110 patients studied by Brunstein et al. [220], 93 (85%) required two

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched 

units to achieve the target nucleated cell dose of 3 × 107/kg. Fifty-three (57%) of these patients received at least one unit matched for 5–6/6 HLA Ags, while the remainder received two units matched to the patient and each other at 4/6 HLA Ags. Among the total group of patients, neutrophil engraftment occurred in 92%, TRM was 19% at 180 days and 26% at 3 years, and overall and eventfree survivals at 3 years after transplantation were 45 and 38%, respectively. Importantly, receipt of double UCBT was associated with a favorable eventfree survival. In summary, UCB transplantation is an acceptable therapy for children with leukemia who lack an HLA-matched sibling donor. In the light of the availability of 4–6/6 HLA-matched unrelated cord blood units, haploidentical SCT in children should only be performed in the context of clinical trials. In adults, unrelated double cord blood or haploidentical-related donor SCT is a reasonable therapeutic option for patients who lack an HLA-matched sibling or a 10/10 HLA allele-matched unrelated donor. Nonmyeloablative SCT using UCB or haploidentical marrow can provide long-term disease-free survival for hematologic malignancies patients who are ineligible for intensive conditioning and who lack an HLA-matched donor. The relative merits of these two graft sources will be evaluated in multicenter clinical trials.

8.  Conclusions Results of HLA-haploidentical SCT have improved steadily since their inception. The problems of excessive graft rejection and severe GVHD have been addressed by transplanting megadoses of Tcell-depleted stem cells into intensively conditioned recipients or by selective allodepletion techniques. Nonmyeloablative conditioning safeguards against the possibility of fatal graft rejection and has extended the application of haploidentical SCT to older or more infirm patients and to those who have failed a prior autologous SCT procedure. The main developmental challenges for the future are to enhance immune reconstitution and to prevent relapse after haploidentical SCT. The respective contributions of UCB vs. haploidentical-related donor SCT for adult patients lacking HLA-matched donors need to be defined. Both of these graft sources offer the advantages of rapid and easy availability for nearly all patients in need of transplantation. Going forward, no patient should be denied access to hematopoietic SCT for lack of an available donor.

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Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched  unmanipulated haploidentical blood and marrow transplantation after conditioning including antithymocyte globulin. Biol Blood Marrow Transplant 13:1515–1524 138. Nachbaur D, Eibl B, Kropshofer G et al (2002) In vivo T cell depletion with lowdose rabbit antithymocyte globulin results in low transplant-related mortality and low relapse incidence following unrelated hematopoietic stem cell transplantation. J Hematother Stem Cell Res 11:731–737 139. Morton J, Hutchins C, Durrant S (2001) 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 98:3186–3191 140. Pelot MR, Pearson DA, Swenson K et  al (1999) Lymphohematopoietic graftvs.-host reactions can be induced without graft-vs.-host disease in murine mixed chimeras established with a cyclophosphamide-based nonmyeloablative conditioning regimen. Biol Blood Marrow Transplant 5:133–143 141. Mapara MY, Pelot M, Zhao G et  al (2001) Induction of stable long-term mixed hematopoietic chimerism following nonmyeloablative conditioning with T celldepleting antibodies, cyclophosphamide, and thymic irradiation leads to donorspecific in vitro and in vivo tolerance. Biol Blood Marrow Transplant 7:646–655 142. Sykes M, Preffer F, McAfee S et al (1999) Mixed lymphohaemopoietic chimerism and graft-versus-lymphoma effects after non-myeloablative therapy and HLAmismatched bone-marrow transplantation [see comments]. Lancet 353:1755–1759 143. Spitzer TR (2005) Haploidentical stem cell transplantation: The always present but overlooked donor. Hematology 2005:390–395 144. Bethge WA, Haegele M, Faul C et al (2006) Haploidentical allogeneic hematopoietic cell transplantation in adults with reduced-intensity conditioning and CD3/ CD19 depletion: Fast engraftment and low toxicity. Exp Hematol 34:1746–1752 145. Handgretinger R, Chen X, Pfeiffer M et  al (2007) Feasability and outcome of reduced intensity conditioning in haploidentical transplantation. Ann NY Acad Sci 1106:279–289 146. Ogawa H, Ikegame K, Yoshihara S et al (2006) Unmanipulated HLA 2–3 antigenmismatched (haploidentical) stem cell transplantation using nonmyeloablative conditioning. Biol Blood Marrow Transplant 12:1073–1084 147. Slavin S, Nagler A, Naparstek E et  al (1998) 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 91:756–763 148. Kanda Y, Oshima K, Sano-Mori Y et al (2005) In vivo alemtuzumab enables haploidentical human leukocyte antigen-mismatched hematopoietic stem-cell transplantation without ex vivo graft manipulation. Transplantation 79:1351–1357 149. Schwartz R, Dameshek W (1959) Drug-induced immunological tolerance. Nature 183:1682–1683 150. Luznik L, O’Donnell PV, Symons HJ et  al (2008) HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide. Biol Blood Marrow Transplant 14:641–650 151. Amrolia PJ, Muccioli-Casadei G, Huls H et al (2006) Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation. Blood 108:1797–1808 152. Ichinohe T, Maruya E, Saji H (2002) Long-term feto-maternal microchimerism: Nature’s hidden clue for alternative donor hematopoietic cell transplantation? Int J Hematol 76:229–237 153. Claas FH, Gijbels Y, van der Velden-de Munck J, van der Velden-de Munck J, Van Rood JJ (1988) Induction of B cell unresponsiveness to noninherited maternal HLA antigens during fetal life. Science 241:1815–1817

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E.J. Fuchs and H.J. Symons 154. Van Rood JJ, Zhang L, van LA, Claas FH (1989) Neonatal tolerance revisited. Immunol Lett 21:51–54 155. Cairo MS, Wagner JE (1997) Placental and/or umbilical cord blood: An alternative source of hematopoietic stem cells for transplantation. Blood 90:4665–4678 156. Polchi P, Lucarelli G, Galimberti M et  al (1995) Haploidentical bone marrow transplantation from mother to child with advanced leukemia. Bone Marrow Transplant 16:529–535 157. Ichinohe T, Uchiyama T, Shimazaki C et  al (2004) Feasibility of HLAhaploidentical hematopoietic stem cell transplantation between noninherited maternal antigen (NIMA)-mismatched family members linked with long-term fetomaternal microchimerism. Blood 104:3821–3828 158. Umeda K, Adachi S, Ishihara H et al (2003) 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 31:1061–1063 159. Narimatsu H, Morishita Y, Saito S et al (2004) Conditioning regimen of melphalan, fludarabine and total body irradiation in unmanipulated HLA haploidentical stem cell transplantation based on feto-maternal tolerance. Intern Med 43:1063–1067 160. Obama K, Utsunomiya A, Takatsuka Y, Takemoto Y (2004) Reduced-intensity nonT-cell depleted HLA-haploidentical stem cell transplantation for older patients based on the concept of feto-maternal tolerance. Bone Marrow Transplant 34:897–899 161. Guinan EC, Boussiotis VA, Neuberg D et  al (1999) Transplantation of anergic histoincompatible bone marrow allografts [see comments]. N Engl J Med 340: 1704–1714 162. Davies JK, Gorgun G, Nadler LM, Guinan EC (2006) Effective control of mismatched alloreactivity via ex vivo alloantigen-specific co-stimulatory blockade does not significantly impact pathogen-specific immunity. ASH Annu Meet Abstr 108:3177 163. Davies J, Yuk D, Nadler L, Guinan E (2007) Donor-derived T cells can be rendered hyporesponsive to alloantigen without loss of pathogen or tumor immune responses. ASH Annu Meet Abstr 110:771 164. Guinan EC, Gribben JG, Brennan LL, Nadler LM (2005) Patients (pts) surviving haploidentical stem cell transplantation (SCT) after ex vivo costimulatory blockade to induce anergy experience few long-term complications. ASH Annu Meet Abstr 106:599 165. Montagna D, Yvon E, Calcaterra V et al (1999) Depletion of alloreactive T cells by a specific anti-interleukin-2 receptor p55 chain immunotoxin does not impair in vitro antileukemia and antiviral activity. Blood 93:3550–3557 166. Mavroudis DA, Jiang YZ, Hensel N et al (1996) Specific depletion of alloreactivity against haplotype mismatched related individuals by a recombinant immunotoxin: A new approach to graft-versus-host disease prophylaxis in haploidentical bone marrow transplantation. Bone Marrow Transplant 17:793–799 167. Koh MB, Prentice HG, Lowdell MW (1999) Selective removal of alloreactive cells from haematopoietic stem cell grafts: Graft engineering for GVHD prophylaxis. Bone Marrow Transplant 23:1071–1079 168. van Dijk AMC, Kessler FL, Stadhouders-Keet SAE et al (1999) Selective depletion of major and minor histocompatibility antigen reactive T cells: Towards prevention of acute graft-versus-host disease. Br J Haematol 107:169–175 169. Davies JK, Koh MBC, Lowdell MW (2004) Antiviral immunity and T-regulatory cell function are retained after selective alloreactive T-cell depletion in both the HLA-identical and HLA-mismatched settings. Biol Blood Marrow Transplant 10:259–268 170. Ge X, Brown J, Sykes M, Boussiotis VA (2008) CD134-allodepletion allows selective elimination of alloreactive human T cells without loss of virus-specific and leukemia-specific effectors. Biol Blood Marrow Transplant 14:518–530

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched  171. Wehler TC, Nonn M, Brandt B et  al (2007) Targeting the activation-induced antigen CD137 can selectively deplete alloreactive T cells from antileukemic and antitumor donor T-cell lines. Blood 109:365–373 172. Bonini C, Ciceri F, Marktel S, Bordignon C (1998) Suicide-gene-transduced T-cells for the regulation of the graft-versus-leukemia effect [Review] [7 refs]. Vox Sang 74(Suppl 2) 341–343 173. Gendelman M, Yassai M, Tivol E et al (2003) Selective elimination of alloreactive donor T cells attenuates graft-versus-host disease and enhances T-cell reconstitution. Biol Blood Marrow Transplant 9:742–752 174. Hartwig UF, Nonn M, Khan S et al (2008) Depletion of alloreactive donor T lymphocytes by CD95-mediated activation-induced cell death retains antileukemic, antiviral, and immunoregulatory T cell immunity. Biol Blood Marrow Transplant 14:99–109 175. Godfrey WR, Krampf MR, Taylor PA, Blazar BR (2004) Ex vivo depletion of alloreactive cells based on CFSE dye dilution, activation antigen selection, and dendritic cell stimulation. Blood 103:1158–1165 176. Martins SLR, Martins SLR, St John LS, Champlin RE et  al (2004) Functional assessment and specific depletion of alloreactive human T cells using flow cytometry. Blood 104:3429–3436 177. Mielke S, Nunes R, Rezvani K et al (2008) A clinical-scale selective allodepletion approach for the treatment of HLA-mismatched and matched donor-recipient pairs using expanded T lymphocytes as antigen-presenting cells and a TH9402based photodepletion technique. Blood 111:4392–4402 178. Roy DC, Cohen S, Busque L et  al (2007) Phase I clinical trial of haplotype mismatched myeloablative stem cell transplantation: Higher doses of donor lymphocyte infusions depleted of alloreactive cells using ATIR may improve outcome without causing GVHD. ASH Annu Meet Abstr 110:2976 179. Schmutz I, Le Deist F, Hacein-Bey-Abina S et al (2002) Immune reconstitution without graft-versus-host disease after haemopoietic stem-cell transplantation: A phase 1/2 study. Lancet 360:130–137 180. Samarasinghe SR, Nawroly N, Karlsson H et al (2007) Functional characterisation of alloreactive T-cells identifies CD25 and CD71 as the optimal targets for allodepletion strategies. ASH Annu Meet Abstr 110:2183 181. Anderson BE, McNiff J, Yan J et al (2003) Memory CD4+ T cells do not induce graft-versus-host disease. J Clin Invest 112:101–108 182. Chen BJ, Cui X, Sempowski GD, Liu C, Chao NJ (2004) Transfer of allogeneic CD62L- memory T cells without graft-versus-host disease. Blood 103:1534–1541 183. Zhang Y, Joe G, Zhu J et al (2004) Dendritic cell-activated CD44hiCD8+ T cells are defective in mediating acute graft-versus-host disease but retain graft-versusleukemia activity. Blood 103:3970–3978 184. Beilhack A, Schulz S, Baker J et al (2005) In vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of T-cell subsets. Blood 106:1113–1122 185. Zheng H, Matte-Martone C, Li H et  al (2008) Effector memory CD4+ T cells mediate graft-versus-leukemia without inducing graft-versus-host disease. Blood 111:2476–2484 186. Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276:71–74 187. Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O (2003) Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 57:11–20 188. Potian JA, Aviv H, Ponzio NM, Harrison JS, Rameshwar P (2003) Veto-like activity of mesenchymal stem cells: Functional discrimination between cellular responses to alloantigens and recall antigens. J Immunol 171:3426–3434

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E.J. Fuchs and H.J. Symons 189. Tse WT, Pendleton JD, Beyer WM, Egalka MC, Guinan EC (2003) Suppression of allogeneic T-cell proliferation by human marrow stromal cells: Implications in transplantation. Transplantation 75:389–397 190. Maitra B, Szekely E, Gjini K et al (2004) Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation. Bone Marrow Transplant 33:597–604 191. Di Nicola M, Carlo-Stella C, Magni M et al (2002) Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 99:3838–3843 192. Lazarus HM, Koc ON, Devine SM et  al (2005) Cotransplantation of HLAidentical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant 11:389–398 193. Ringden O, Uzunel M, Rasmusson I et  al (2006) Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease. Transplantation 81: 1390–1397 194. Karlsson H, Samarasinghe S, Ball LM et  al (2008) Mesenchymal stem cells exert differential effects on alloantigen and virus-specific T cell responses. Blood 112(3):532–541 195. Sakaguchi S, Ono M, Setoguchi R et al (2006) Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol Rev 212:8–27 196. Cohen JL, Trenado A, Vasey D, Klatzmann D, Salomon BL (2002) CD4(+) CD25(+) immunoregulatory T Cells: New therapeutics for graft-versus-host disease. J Exp Med 196:401–406 197. Taylor PA, Lees CJ, Blazar BR (2002) The infusion of ex vivo activated and expanded CD4(+)CD25(+) immune regulatory cells inhibits graft-versus-host disease lethality. Blood 99:3493–3499 198. Edinger M, Hoffmann P, Ermann J et al (2003) CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nat Med 9:1144–1150 199. Hoffmann P, Ermann J, Edinger M, Fathman CG, Strober S (2002) Donor-type CD4+CD25+ regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. J Exp Med 196:389 200. Symons HJ, Leffell MS, Rossiter ND et al (2006) Impact of killer immunoglobulin receptor (KIR) ligand incompatibility in nonmyeloablative bone marrow transplantation (BMT) from haploidentical donors. ASH Annu Meet Abstr 108:604 201. Verheyden S, Schots R, Duquet W, Demanet C (2005) A defined donor activating natural killer cell receptor genotype protects against leukemic relapse after related HLA-identical hematopoietic stem cell transplantation. Leukemia 19:1446–1451 202. De Santis D, Bishara A, Witt CS et al (2005) Natural killer cell HLA-C epitopes and killer cell immunoglobulin-like receptors both influence outcome of mismatched unrelated donor bone marrow transplants. Tissue Antigens 65:519–528 203. Moretta L, Bottino C, Pende D et al (2006) Surface NK receptors and their ligands on tumor cells. Semin Immunol 18:151–158 204. Gasser S, Raulet D (2006) The DNA damage response, immunity and cancer. Semin Cancer Biol 16:344–347 205. Gasser S, Raulet DH (2006) The DNA damage response arouses the immune system. Cancer Res 66:3959–3962 206. Lundqvist A, Abrams SI, Schrump DS et al (2006) Bortezomib and depsipeptide sensitize tumors to tumor necrosis factor-related apoptosis-inducing ligand: A novel method to potentiate natural killer cell tumor cytotoxicity. Cancer Res 66:7317–7325 207. Hallett WHD, Ames E, Motarjemi M et al (2008) Sensitization of tumor cells to NK cell-mediated killing by proteasome inhibition. J Immunol 180:163–170

Chapter 18  Hematopoietic Cell Transplantation from Partially HLA-Mismatched  208. Shi J, Tricot GJ, Garg TK et al (2008) Bortezomib down-regulates the cell-surface expression of HLA Class I and enhances natural killer cell-mediated lysis of myeloma. Blood 111:1309–1317 209. Miller JS, Soignier Y, Panoskaltsis-Mortari A et  al (2005) Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in cancer patients. Blood 105(8):3051–3057 210. Clynes RA, Towers TL, Presta LG, Ravetch JV (2000) Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med 6:443–446 211. Or R, Hadar E, Bitan M et  al (2006) Safety and efficacy of donor lymphocyte infusions following mismatched stem cell transplantation. Biol Blood Marrow Transplant 12:1295–1301 212. Huang Xj, Liu Dh, Liu Ky et al (2008) Modified donor lymphocyte infusion after HLA-mismatched/haploidentical T cell-replete hematopoietic stem cell transplantation for prophylaxis of relapse of leukemia in patients with advanced leukemia. J Clin Immunol 28:276–283 213. Huang Xj, Liu Dh, Liu Ky et al (2007) Donor lymphocyte infusion for the treatment of leukemia relapse after HLA-mismatched/haploidentical T-cell-replete hematopoietic stem cell transplantation. Haematologica 92:414–417 214. Nguyen S, Kuentz M, Vernant JP et  al (2007) Involvement of mature donor T cells in the NK cell reconstitution after haploidentical hematopoietic stem-cell transplantation. Leukemia 22:344–352 215. Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S (2008) Functions of natural killer cells. Nat Immunol 9:503–510 216. Laughlin MJ, Eapen M, Rubinstein P et al (2004) Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia. N Engl J Med 351:2265–2275 217. Stevens CE, Rubinstein P, Scaradavou A (2006) HLA matching in cord blood transplantion: Clinical outcome and implications for cord blood unit selection and inventory size and ethnic composition. ASH Annu Meet Abstr 108:3104 218. Barker JN, Weisdorf DJ, Defor TE et  al (2005) Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 105:1343–1347 219. Ballen KK, Spitzer TR, Yeap BY et al (2007) Double unrelated reduced-intensity umbilical cord blood transplantation in adults. Biol Blood Marrow Transplant 13:82–89 220. Brunstein CG, Barker JN, Weisdorf DJ et al (2007) Umbilical cord blood transplantation after nonmyeloablative conditioning: Impact on transplantation outcomes in 110 adults with hematologic disease. Blood 110:3064–3070 221. Randolph SSB, Gooley TA, Warren EH, Appelbaum FR, Riddell SR (2004) Female donors contribute to a selective graft-versus-leukemia effect in male recipients of HLA-matched, related hematopoietic stem cell transplants. Blood 103:347–352 222. Frassoni F, Labopin M, Gluckman E et al (1996) Results of allogeneic bone marrow transplantation for acute leukemia have improved in Europe with time – a report of the acute leukemia working party of the European group for blood and marrow transplantation (EBMT). Bone Marrow Transplant 17:13–18 223. Nichols WG, Corey L, Gooley T, Davis C, Boeckh M (2002) High risk of death due to bacterial and fungal infection among cytomegalovirus (CMV)-seronegative recipients of stem cell transplants from seropositive donors: Evidence for indirect effects of primary CMV infection. J Infect Dis 185:273–282 224. Ruggeri L, Capanni M, Urbani E et  al (2002) Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 295:2097–2100 225. Parham P (2005) MHC Class I molecules and kirs in human history, health and survival. Nat Rev Immunol 5:201–214

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Chapter 19 Unrelated Donor Transplants A Bacigalupo, Ospedale San Martino, Genova, Italy

Abbreviations: UD unrelated donors EBMT European group for Blood and Marrow Transplantation HSCT hemopoietic stem cell transplants HLA human leukocyte antigen BMT bone marrow transplantation m-HA minor histocompatibility antigens NK natural killer cells NMDP National Marrow Donor Program JMDP Japanese marrow donor program BMDWW Bone Marrow Donors World Wide HSC hemopoietic stem cells FACT Foundation for the Accreditation of Cell Therapy ISCT Internation Society for Cell Therapy JACIE Joint Accrediation Committee of the IST and EBMT EUSTITE European Union Standards and Training in the Inspection of Tissue Establishements. WMDA World Marrow Donor Association BM bone marrow PB peripheral blood G-CSF granulocyte colony stimulating factor CML chronic myeloid leukemia CP1 first chronic phase IHWG International HLA Working Group AML acute myeloid leukemia ALL acute lymphoblastic leukemia MDS myelodisplastic syndromes TRM transplant related mortality CsA cyclosporin

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_19, © Springer Science + Business Media, LLC 2003, 2010

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MTX FK MMF ATG GvHD RIC FLU-CY LFS CMV EBV

methotrexate tacrolimus mycofenolate mofetil anti-thymocyte globulin graft versus host disease reduced intensity conditioning fludarabine/cyclophosphamide leukemia free survival cytomegalovirus epstein barr virus

1  Introduction Unrelated donor (UD) hemopoietic stem cell transplants (HSCT)are being increasingly used, as shown by the annual survey of the European group for Blood and Marrow Transplantation (EBMT): in Europe the number of recorded UD transplants was 752 in 1995 and it was 3617 after 10 years in 2005 (Fig. 19.1), suggesting that most transplant Centres are becoming more confident with the procedure, and that clinical results justify the investment in clinical care and research.Several factors have contributed to this very significant increase in activity: improved human leukocyte antigen (HLA) typing technology, better matching criteria, increased size of the world wide donor pool and improved transplant programs. We will briefly outline each of these different factors. We will not discuss in this chapter transplants from unrelated cord blood, since this is a procedure with specific characteristics; we will restrict the discussion to transplants from unrelated donors who are filed in the National Donor Registries and then in the World Wide File.

UD HSCT in Europe 1995-2005 3000 2495

2500 2054

2000

1685

1500 1000

1161 752

500 0 1995

1997

1999

2001

2003

2005

Fig. 19.1.  Numbers of unrelated donor transplants in Europe 1995-2005

Chapter 19  Unrelated Donor Transplants 

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1.1  Bone Marrow Donors World Wide Potential healthy volunteer donors agree to have a small aliquot of venous blood drawn for HLA typing, and agree to have their HLA typing stored in a file, together with identification of the donor itself. Each Nation has one or more of these donor based data bases. Bone Marrow Donors World Wide (BMDWW) compiles different National Data Bases (Registries) in one single file, residing in Leiden University, Holland. It was Jan van Rood, who strongly argued in favour of this project in the late eighties: in those days, each Nation had a Donor Registry, and patient searches would start in one Nation, to be then extended to an other Nation, and sometimes come back to the first: this was time consuming, costly and very inefficient. It was obvious that creating one single file would have allowed immediate access to the entire data base. The initial resistance was won by the scientific stature of the founder: today BMDWW is a potent, fast and updated tool, compiling over 10.000.000 volunteer donors world wide. It has proved to be clinically useful (a preliminary search is done on line in “real time”), but also scientifically relevant, since the large number of individuals represented, allows for genetic insight in different populations. Finding a donor. Once a potential donor is identified through BMDWW a formal search is activated. The time to identify a donor depends on the HLA of the patients (a common HLA will lead to larger number of potential donors), whether there is a fast lane for a specific disease (acute leukemia) or disease phase, and whether the transplant Center is willing to accept a donor which is less than a match. The time to identify a donor will go from as little as 30 days to as long as many months. An unrelated donor transplant should ideally be considered early in the course of the disease: for example in a patient with high risk AML (unfavourable chromosomal abnormalities) if the HLA typing is performed at diagnosis, there is time to search for an appropriate donor, while the patient undergoes induction chemotherapy. If the patient fails induction, or if the patient has an early relapse, a donor may have already been identified and the patient can proceed to transplant. If one starts HLA typing and a donor search at the time of failure (refractoriness or relpase) the patient may not live long enough to come to transplant, or may be too sick to have a good chance of cure. 1.2  HLA and matching criteria The issue of matching between donor and recipient has been crucial from the origin of clinical transplant activity, but the concept has evolved during the years, with an increasing level of sophistication (Table 19.1). The methods of HLA typing have changed from serological indetification (making use of human antibodies identifying specific antigens), to molecular Table 19.1.  Matching criteria during different time periods (intervals are artificially set at decades)  

<1990

1990-1999

2000-2006

=>2007

HLA LOCI considered HLA typing Best match

A,B,DR

A,B,C,DRb1

Serology 6/6 antigens

Serology+molecular biol 8/8 antigens DRb1 allele

A,B,C,DRb1, DRb2, DRb3, DQ,DP +molecular biol 10/10 alleles 12/12 alleles

A,B,C,DRb1, DRb2, DRb3, DQ,DP Haplotype Algorithm Haplotype matched

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biology (identifying genes coding for specific alleles). Thus the definition of a matched donor a decade ago is different from the definition of a matched donor today. Ten years ago, we were matching for the A,B,DR locus at the antigenic, or serologically determined, level, and a 6/6 match would have identified a donor matched with his recipient for 6 antigens (2 on each of the A,B,DR loci). We then started to use molecular biology for the DR, but were still calling this a 6/6 match. A 5/6 match would have been a donor with one of the antigens mismatched. Currently we are matching for A,B,C,DRB1,DQ at the allelic level, that is with molecular subtyping of the antigenes, looking for 10/10 matched donors (Table 19.1). Some Centres are typing also for DP looking for a 12/12 matched donor. Most recently E Petersdorf has shown in an elegant paper that beyond allelic matching, one can also match for haplotypes (Petersdorf E et al, 2007) A donor and recipient can be matched by phenotype (presence of the same antigens on locus A,B,C,DR) but mismatched by haplotypes. Petersdorf and coworkers could show that matching for haplotypes significantly reduces the risk of GvHD, and this is probably due to the fact that haplotype matching indicates that a greater part of chromosome 6 is matched between donor and recipient. Finally, mismatching can be permissive or non permissive: some studies have shown that mismatching at specific aminoacidic positions may be conductive to more GvHD (Ferrara GB et al, 2001; Takakazu K et al, 2007) 1.3  HLA matching and clinical outcome In a study by the Seattle group (Petersdorf E et al, 2004) in chronic myeloid leukemia (CML) in first chronic phase (CP1), patients with a 10/10 allelic HLA match, had significantly improved survival (70%) as compared to patients with a 9/10 match (40%). The effect was not seen in patients with CML in accelerated or blastic phase. A mismatch for one single C allele had the greatest impact on survival (RR 3.18). (Table 19.2) The CIBMTR-NMDP paper analyzed 3857 allelic typed transplants, including acute myeloid leukemia (AML) acute lymphoblastic leukemia (ALL), Table 19.2.  The effect of HLA mismatch in unrelated donor transplants: representative studies Study

Seattlle  

NMDP CIBMTR

JMDP

Seattle

Reference

Petersdorf Blood 2004; 104: 2976

Lee

Takakazu

Petersdorf

Blood 2007

Blood 2007

Plos Med 2007;

110; 2235 5210 Malignant and non malignant All patients

4: 59 246 Leukemia

C locus

Haplotype

Non permissive mismatches positions 9, 77, 80, 99, 116, 156  

Mismatched

N.patients Diagnosis

1249 Leukemia

Survival disadvantage in mismatched pairs Type of mismatch

Early dis

Online sep4 3860 AML ALL MDS CML Early dis

C locus

A or DRb1

One should avoid

All patients

Chapter 19  Unrelated Donor Transplants 

myelodisplastic syndromes (MDS) and chronic myeloid leukemia (CML) (Lee S et al, 2007 ): again the impact of a single allelic mismatch over a full allelic match was greater in patients with early disease (Table 19.2). Actuarial 5 year survival in patients with early disease was 50% for 8/8 matched pairs, decreasing to 39% for 7/8 matched pairs and 28% for 6/8 matched pairs. In patients with intermediate disease these 3 figures were respectively 32% 27% 15% and in patients with advanced disease 17% 15% 10%. Again an effect of HLA mismatching is shown, but most prominent in patients with early disease. A study from the Japanese Donor Registry (JMDP) on 5210 patients, found 6 specific amino acidic substitution positions in HLA class I, associated with severe acute GvHD: the Authors refer to these as “non permissive mismatches”. The positions are 9,77,80,99,116,156. (Table 19.2). One of these (116) had already been reported as risk factor for GvHD (Ferrara GB et al, 2001). Patients with a full matched donor (n=712) have the same risk of GvHD as patients with zero non permissive mismatched (n=2670), but significantly less GvHD as compared to patients with one non permissive (n= 602) or two non permissive (n=66) mismatched donors. Finally a recent paper (Petersdorf E et al, 2007) shows that matching donor and recipient for haplotypes reduces GvHD and TRM (Table 19.2). In keeping with this result, survival in excess of 90% has been reported for thalassemia patients, prospectively assigned to receive transplants from UD, matched by ancestral haplotypes: this study was possible because the patients were from the island Sardinia, known to be homogeneous by HLA typing, and ancestral haplotypes are common in the population (La Nasa G et al, 2005). Because of the protective effect of GvHD on leukemia relapse, haplotype matched pairs experienced more leukemia recurrence and survival was overall similar in matched/mismatched pairs (Petersdorf E et al, 2007): however in patients with early disease, or in patients with non malignant disorders, one may wish to transplant from haplotype matched donors with little risk of GvHD and TRM. In patients with advanced disease a partially mismatched donor may also be acceptable, or perhaps preferable. From these studies we can derive the following conclusions on HLA matching and outcome: First: better matching leads to better outcome. Second: the effect may be different according to the disease phase (greater effect in early disease) and different ethnic origin. Third: some specific mismatches can be considered as non “permissive” as compared to others, considered “permissive”. 1.4  Donation of hemopoietic stem cells (HSC) There are two good reasons to trace HSC donations: the first is donor safety, the second is quality of the HSC collection. Although not unique to UD transplants, the issue of safety in HSC donation has gained momentum in the past decade: this is because with the increasing number of donations, probably exceeding 200.000 world wide, potential risks for the donor have become evident. Several international regulatory agencies have addressed this issue: FACT and JACIE have issued the third edition of International Standards for Cellular Therapy Product Collection, Processing and Admninistration: this is a concerted action of Europe and USA and

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includes a dedicated section for Adverse Events (AE). The European Union has recently created the project named EUSTITE, aimed at regulating tracebility, requirements, and notification of serious adverse events concerning the processing, preservation and distribution of cell products, from so called Tissue Establishements. The World Marrow Donor Association (WMDA), a consortium of all existing Donor Registries, has established a Registry of Serious Adverse Donor Events: this Registry has specific and detailed questions relating to the type, timing and consequences of the adverse event, as well as detailed informations on the procedures used to collect HSC. Because collection can be from the bone marrow (BM) or from the peripheral blood (PB), the procedures and the associated risks vary considerably. BM donation is mainly, but not exclusively, associated with the risk of general anesthesia, whereas PB donation carries the risk of vascular complications associated with leukocytosis induced by growth factor (usually granulocyte colony stimulating factor G-CSF) and stem cell pheresis procedures. Additional adverse events of G-CSF administration include pulmonary hemorrhage (Kopp GC et al, JCO 2007; Arimura K et al, 2005), splenic ruptures (Nuamah NM et al, 2006) and a recently reported risk of developing leukemia (Tigue CC, et al, 2007) In a prospective study involving over 300 donors, randomized to undergoe BM or PB collections, the total proportion of AE was similar in the two groups (57% vs 66%): skeletal pain was more frequent in PB collections (2% vs 26%) whereas pain at the site of collection was more frequent in BM donors (23% vs 1%) (Schmitz N, et al, 2002). The number of days with restricted activity was greater in BM donors (6 vs 2 days). Death of the donor has been reported: in a survey of the EBMT on 44566 donations, there were 4 deaths (Gratwohl A 2004, personal communication), giving a risk of 1.18 deaths/100.000 donation for PB and 0.35 deaths /100.000 donations for BM. These data support the current efforts of Regulatory Agencies to trace adverse events, and suggest that great care should be given to the identification of donors suitable for donation. Quality of HSC collection is also a relevant issue: when the source of stem cells is bone marrow, the number of cells collected will impact on the outcome of the transplant. It has been shown that different techniques of collecting marrow can lead to different yields in nucleated cell counts: aspiration of a small volume of marrow blood ( 2ml), at each aspiration, is significantly superior compared to aspiration of a larger volume (20 ml), both in terms of nucleated cells and hemopoietic progenitors (Bacigalupo A et al, 1992) Harvesting marrow is cumbersome and time consuming, and collecting centers may harvest from 10 ml/kg of donor body weight up to 25 ml/kg od donor body weight. Given the strong impact of marrow cell dose on outcome, it seems justified to harvest marrow with great care: indeed if the dose of nucleated cells transplanted is greater than 3x10^8/kg, survival of the patient is significantly improved (Sierra J et al, 1997). When the stem cell source is peripheral blood, the dose given is less important, and equivalent engraftment has been observed in patients receiving cell doses from 2 to over 10x10^8/kg. 1.5  Patient selection and indications Eligibility for an UD transplants includes patient related and disease related variables. The age of the patients in a pediatric Centres will go from newborn to 18 years, in a Centre transplanting adults it will range from 18 up to 70.

Chapter 19  Unrelated Donor Transplants 

There may be some programs designed for patients above the age of 70, but it would involve a small minority of patients. Patients of course come to transplant in different clinical conditions, depending on their age, disease phase, number of courses of chemo and/or radiotherapy and with a variable number of co-morbidities, some of them age dependent, like hypertension, heart failure, chronic lung disorders. Ideally we would like to have patients in the early phase of the disease, having received little chemotherapy, with no co-morbidities and a 10/10 matched donor. One is often confronted with 9/10 or 8/10 donors, in patients with evidence of disease, older age, and co-morbidities. A recent study from the Seattle group (Sorror M et al, 2009) has identified a scoring system which allows to quantitate co-morbidities: patients with a low score have a signikficantly lower transplant related mortality (TRM). Patient eligibility also raises regulatory and insurance issues: the high cost of an UD transplant may not be covered in certain advanced disease phases, because of the very low chance of success. This may also be true for some disease in which evidence of efficacy of HSCT is lacking (such as solid tumours). The EBMT has attempted to categorize indications for allogeneic sibling and unrelated transplants in different diseases and disease states (Urbano Ispuiza A et al, 2002) The decision to proceed with an UD HSCT resides ultimately with the patient, his hematologist and the transplanter. The side effects and toxicities are usually weighed against the risk of the underlying disease: a 50 year old patient with AML and a –7 chromosomal abnormality, in second remission, has only one chance: an UD HSCT. But a patient with Hodgkin’s disease, with a relapse within one year from his initial chemotherapy, has several options, and, although none of them is probably curative, an UD transplant is not necessarily the best one. The current distribution of diagnosis in patients transplanted from UD facilitated by the Italian Bone Marrow Donor Program are as follows: AML 27%, ALL 256, lymphoma 15%, MDS 7%, immune deficiency 9%, Myeloma 8%, CML 4%, aplastic anemia 2% (Fig. 19.2). Therefore over 50% of all patients undergoing an UD HSCT are acute leukemias.

40 35 30

27

26

25 20

15

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7

9

8 4

5

L M C

IE

M M

M

D

S

a om

LL

Ly m ph

A

A

M

L

0

Fig. 19.2  Proportion of patients undergoing an UD transplant in 2006, stratified by diagnosis. Over 50% are patients with acute leukemia.

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2  Prophylaxis of acute Graft vs Host Disease (GvHD) The conventional prophylaxis for graft versus host disease (GvHD) is cyclosporin (CsA) and methotrexate (MTX) or tacrolimus (FK) and MTX. With a two drug combination acute GvHD grade III-IV is seen in 10-30% patients. Recently the Boston group has reported an incidence of grade III-IV acute GvHD of less than 5%, using the combination tacrolimus and sirolimus, in a relatively large group of related and unrelated transplants (Cutler C et al, 2007). The addition of a polyclonal antibody, such as anti-thymocyte globulin (ATG) (Bacigalupo A et al, 2001) or the use of a monoclonal antibody such as alemtuzumab (Byrne JL et al, 2000) will also significantly reduce acute GvHD. The prophylaxis of GvHD should be designed acording to the Centers experience and ongoing protocols. It is unlikely that a given regimen will be totally successfull, especially because the incidence and severity of acute GvHD, depend not only on the in vivo prophylaxis, but also on factors such as disease phase, patient age, intensity of the conditioning regimen, stem cell source and graft composition. Agents or manouvres which will reduce the incidence of GvHD, have been shown in the past to increase the risk of relapse, and this must be factored in the choice of a given combination. 2.1  Prophylaxis of chronic Graft vs Host Disease (GvHD) The strongest predictor of chronic GvHD, is the presence of acute GvHD. The most efficient way of preventing both acute and chronic GvHD is physical removal of T cells from the graft, or ex-vivo T cell depletion (TCD) (Reisner Y et al, 1980; Janossy G et al, 1984): this suggests that chronic GvHD, has its pathogenesis in the first days of transplant, although it may become clinically manifest months or years later. In keeping with this hypothesis, also the addition of ATG in the conditioning regimen, so called in-vivo T cell depletion, has shown, in a randomized trial to reduce the incidence and severity of chronic GvHD, together with its long term negative sequelae (Bacigalupo A et al, 2006). Not all brands of ATG may have the same effect on chronic GvHD, as shown in a recent study on 155 UD transplants for AML (Basara N et al, 2005). Therefore manouvres or treatments given at the time of, or in the immediate proximity of the infusion of the graft, may impact on the occurrence of chronic GvHD, and indirectly on the risk of leukemia relapse, given the strong protective effect of the former on the latter.

3  Conditioning regimens The conventional conditioning regimen for a patient under the age of 50, undergoing an UD HSCT is cyclophosphamide 60 mg/kgx2 followed by total body irradiation (TBI) 2 Gy twice daily on each of 3 consecutive days, for a total of 12 Gy. As already pointed out results may be quite different using the same conditioning regimen, due to the variability and somehow unknown level of donor/recipient matching. Above the age of 55 toxity of TBI becomes more evident and many Centres have explored reduced intensity conditioniong (RIC) regimens in the last decade. the rationale behind this being that one would reduce the toxicity, maintaining the immune effect that cures leukemia.

Chapter 19  Unrelated Donor Transplants 

Conventional and reduced intensity conditioniong (RIC) have been compared in the setting of HLA identical sibling transplants. Several studies have now appeared also in the unrelated transplants: in the first place it is now ascertained that an UD HSCT can be safely performed with RIC regimens. Results in some disorders like AML, can actually be superior when using an UD as compared to a matched sibling (Hegenbart U et al, 2006). This has also been the experience with the non-myeloablative regimens involving a very small dose of TBI (2 Gy) (Storb R et al, 1999) Direct comparisons on the efficacy of this procedure with conventional regimens are scarse: in a study on patients with AML, RIC regimens were associated with significantly increased risk of relapse, as compared to the conventional regimens, in patients with advanced disease, and similar leukemia free survival in patients with early disease (Ringden O et al, 2007, personal communication). It is difficult to draw conclusions or generalize: trials are ongoing and it is best for patients to enter such trials. The German group is comparing two different regimens 12 Gy vs 8 Gy, in patients with AML and this may give an answer for this disease and for these levels of intensity of the preparative regimen. For other disorders, and other regimens one needs to refer to a specific trial. In thalassemia patients, a conditioning regimen including thiotepa, fludarabine and treosulfan, has recently been reported to produce survival in excess of 90% (La Nasa G et al, 2005). This brilliant result is due to the strong immunosuppressive and myeloablative effect of the conditioning, but also to the selection of donors based on ancenstral haplotypes. In patients with aplastic anemia, an other non malignant disease which was not a routine indication for UD HSCT 10 years ago, the preferred combination is currently the association of fludarabine/cyclophosphamide (FLU-CY) combinaed with low dose (2 Gy ) TBI: encouraging results have been produced in the US, Japan, and Europe (Deeg HJ et al, 2006; Kojima S et al, 2002; Bacigalupo A et al, 2005 ). Therefore modification of conditioning regimens, based on patient age, co-morbidity index, and specific diagnosis, has lead to improved results in some disorders, although it would be difficult to generalize and the original combination CY-TBI remains the standard regimen 30 years after it was first reported (Thomas ED et al, 1975).

4  Stem cell source There is growing use of peripheral blood (PB) as a source of stem cells for transplantation. In the 5 year period 2000-2004 the ratio of PB to BM in unrelated transplants has gone from 763/1191 (0.6) to 1918/919 (2.0) (Fig. 19.3). It is unclear why this has happened: from the donor’s perspective, the side effects of PB donation, are not less worrying than BM donation, as we have seen; PB collections certainly reduce the burden of marrow harvest in the operating room (at least 3 full hours each), and for a Unit performing 100 transplants/year, this could mean saving several hundred person days. PB collections do increase the work load of the blood bank. For the patient, PB grafts give faster hematologic and immune recovery, this is well established. In the long term, however, there does not seem to be a significant benefit: the CIBMTR has compared 331 PB and 561 BM unrelated transplants performed

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UD HSCT in Europe 2000-2004 UD BMT

3000

UD PBSC

2500 1918

2000 1500 1000

1191 763

901 870

1063

1244

1448 1047

919

500 0

2000

2001

2002

2003

2004

Fig. 19.3.  Numbers of transplants performed with bone marrow (BM) or peripheral blood (PB) from unrelated donors in the years 2000-2004

between 2003 and 2006. Engraftment was faster with PB, but PB recipients had more acute and chronic GvHD. Transplant mortality (TRM), relapse and survival were identical: late TRM was probably increased in patients with early disease. A retrospective study of EBMT compared 388 ALL patients CR1+CR2, receiving UD BM and 337 receiving UD PB: overall leukemia free survival (LFS) was significantly superior for the BM (45%) vs the PB group (36%); this was due to a lower risk of relapse after BMT (Basara N et al, 2005, EBMT data unpublished). A prospective randomized trial is well under way within the NMDP comparing BM vs PB for first UD grafrts. Untill data from that trial are available it would seem reasonable to continue using marrow for patients with early disease, and peripheral blood for patients with advanced disease. The choice is not always straightforward, because donors may have their preference: usually the transplant center asks for a given source (BM or PB), declares whether only one source, or both, will be accepted as suitable, and then the donor must have the last choice.

5  Complications of UD HSCT The problems of UD HSCT are not different from any other allogeneic transplant - GvHD, infections, organ toxicity- they are however more frequent and more severe, because the level of HLA mismatching in UD transplants will always be greater as compared to HLA identical sibling donors. UD HSCT exhibit delayed immune reconstitution, which is worsened by the frequent use of T cell antibodies, given to prevent GvHD: prolonged immunedeficiency is associated with repeated infections, which cause significant morbidity and mortality. The increased severity of infections, poses specific problems of monitoring and pre-emptive therapy: patients should be monitored for viral infections, such as cytomegalovirus (CMV) and Epstein Barr Virus (EBV). Monitoring is usually performed by PCR and patients who prove positive can be placed

Chapter 19  Unrelated Donor Transplants 

Table 19.3.  Causes of death in 291 consecutive unrelated transplants  

CR1 MA

CR1 RIC

>CR1 MA

>CR1 RIC

Total n.patients Rejection Acute GvHD Infections Chronic GvHGD Interstitial pneumonia MOF Relapse Alive

90   1%   6% 14% 10%   3%   4%   3% 58%

14   0%   0% 21%   0%   0%   7%   7% 64%

107   1%   9% 19%   5%   2%   8% 18% 38%

80   4% 13% 18%   0%   3% 14% 15% 35%

on so-called pre-emptive therapy, with gancyclovir or foscarnet (for CMV) and with rituximab (for EBV). Monitoring for invasive fungal infections (IFI) is also recommended with aspergillus antigenemia, and early treatment with active agents (such as voriconazole, lyposomal amphotericin or caspofungin). Organ toxicity can be anticipated by using co-morbidity scores (Sorror M et al, 2009) and conditioning regimens can be tailored accordingly. As a general rule young patients (<50 years) will be prepared with a myeloablative regimen, whereas older patients, or patients with significant co-morbidity, will receive a reduced intensity regimen. Causes of death in 291 consecutive unrelated transplants (Genova, San Martino, unpublished) are outlined in Table 19.3: Patients are stratified according to disease phase (1st CR or beyond) and intensity of the conditioning regimen (myeloablative/ RIC). Infections are the major cause of death in all 4 groups, followed closely by GvHD (in its acute and chronic form). (Table 19.3) Relapse related death is a problem mainly in patients with disease beyond first remission. Rejection is an unfrequent cause of death, and is seen in more than 1% of patients only with advanced disease prepared with RIC regimen. 5.1  Hematologic recovery Although primary acute rejection is rare, hematologic recovery can be unsatisfactory in a proportion of patients. Platelet counts are a sensitive indicator of graft function post-transplant. In a study on 342 patients prepared with a conventional CY-TBI regimen (Dominietto A et al, 2001), the median platelet count on day +50 after HSCT, was 85 x 109/L (range 1-298). Of the 78 patients with <=50x109/L platelets on day +50, 54 (69%) were still thrombocytopenic on day +100. Of the 244 patients with >50x109/L platelets on day +50, 32 (13%) dropped their counts on day +100. Thus, on day +100, 27% of patients had less than 50x10^9/L platelets Unrelated donor transplants had significantly lower platelet counts as compared to HLA matched sibling donor transplants. Other factors influencing graft function were the severity of acute GvHD, CMV infections and number of cells infused at transplant (Dominietto A et al, 2001). Therefore cytopenia can occur as a consequence of multiple factors, including the stem cell dose, the donor/recipient immune reaction, and infections. Thrombocytopenia can be prolonged, and is an indicator of poor graft function: a second infusion of stem cells, without conditioning, possibly CD34 selected

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(in order to remove T cells), can improve graft function in over 75% of patients (Larocca A et al, 2006) 5.2  Current results in Acute Leukemia Despite the great variability of donor matching criteria, of conditioning regimens, of stem cell sources and of GvHD prophylaxis regimen, two variables maintain their prognostic relevance: age and phase of the disease. Fig. 19.4 outlines the outcome of 1993 children (under the age of 18) and of 5917 adults (18 years and older) undergoing an UD HSCT in Europe between 1994 and 2007. The graph is compiled from the registry of the acute leukemia working party (courtesy of Rocha V, Paris). The effect of remission phase is very strong: patients in CR1 have significantly superior survival as compared to patients in CR2 or more advanced phase. The effect of age is also evident: children have superior survival in all phases of disease, although the greatest advantage seems to be in CR2; it should also be noted that CR2 is the largest group of patients in the pediatric age, this being the major indication, whereas in adults there are more patients in CR1. All curves show a steep decline in the first 2 years and then they level off, with few events occurring beyond 5 years. This shows the durability of remission in patients with acute leukemia after an allogeneic unrelated HSCT.

6  Current results in patients with myelodysplastic syndromes Apart from acute leukemia, the other major indication for unrelated transplants is myelodysplastic syndromes (MDS). This is because chronic myeloid leukemia (CML), the major indication ofr UD HSCT ten years

ACUTE LEUKAEMIA REGISTRY : 1ST & 2ND TRANSPLANT JANUARY 1994 - FEBRUARY 2007 (n=45283) CHILDREN (n=1993)

ADULTS (n=5917)

1,0

1,0

,8

,8

,6

55 ± 7

CR1 n=525

47 ± 2

CR2 n=882

27 ± 3

ADVANCED n=425

,6

49 ± 1

CR1 n=1977

37 ± 2

CR2 n=1520

18 ± 1

ADVANCED n=1802

,4

,4 ,2

,2

0,0

0,0

0

1

2

3

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9 10

0

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2

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4

5

6

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9 10

MUD ALLOGENEIC STEM CELL TRANSPLANT OVERALL SURVIVAL AT 5 YEARS Lyon, March 2007

Fig. 19.4.  Actuarial leukemia free survival (LFS) of 1993 children and 5917 adults with acute leukemia undergoing an UD HSCT between 1994 and 2007 (EBMT data 2007).

Chapter 19  Unrelated Donor Transplants 

Fig. 19.5.  Actuarial disease free survival (DFS) of 552 patients with myelodysplastic syndromes undergoing an UD HSCT between 1988 and 1998, facilitated by the National Marrow Donor Program (NMDP).

ago, is currently being treated with imatinib, with very high and prolonged complete cytogenetic responses. MDS is a very heterogeneous group of diseases, ranging from chronic anemia to agressive acute leukemia. Fig. 19.5 outlines the survival of 552 MDS patients undergoing an UD HSCT, based on their remission status: best results are seen in the low risk patients (refractory anemia, RA) or in patients who had progressed to leukemia but were in remission. Worst results are seen in patients with MDS in transformation to acute leukemia (RAEB-t, now classified as acute leukemia), and in the secondary AML (following MDS). This graph represents results of the nineties, and current results may be superior: however it outlines the strong impact of differrent forms of MDS and different disease phases on long term cure.

7  Current results in patients with aplastic anemia Aplastic anemia (AA) has been a strong indication for allogeneic transplants since the early seventies. The problem of these patients is that they come to transplant with significant problems, such as hemorrhages and infections, often invasive fungal infections (due to prolonged neutropenia) or sepsis. Therefore conditioning regimens need to deliver strong immunosuppression (to prevent rejection) but at the same time one must consider the fragile clinical condition of the patients. The advent of reduced intensity regimens, and the widespread use of the combination fludarabine and cyclophosphamide, has found a successful application in AA patients. Fig. 19.6 outlines to improvement of survival seen in Europe, for patients with AA undergoing an UD HSCT before and after 1998: survival was 32% at 5 years and is currently 57%. Our current regimens include the combination FLU-CY-ATG and for adults also the addition of low dose (2 Gy) TBI. Results are very promising.

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Fig. 19.6.  Improved survival for patients with AA, undergoing an UD HSCT before and after 1998: survival was 32% at 5 years and is currently 57%.

8  Conclusions Unrelated transplants should be considered a therapeutic option in patients with both malignant and non malignant hematologic disorders. The choice of using an UD should be considered early in the course of the disease. This is true especially because search for a suitable donor may require weeks and sometimes months: the average interval between activation of a formal search, and the identification of a donor is between 30 and 150 days. The success of the transplant will depend on a mixture of different factors: donor matching, donor age, conditioning regimen, phase of the disease, recipient age, prophylaxis of GvHD, careful monitoring for infections and expertise of the transplant center. The increasing numbers of UD transplants performed, suggest continuing success and growing confidence in the scientific community.

References   1. Arimura K, Inoue H, Kukita T, Matsushita K, Akimot M, Kawamata N, Yamaguchi A, Kawada H, Ozak A, Arima N, Te C. Acute lung Injury in a healthy donor during mobilization of peripheral blood stem cells using granulocyte-colony stimulating factor alone. Haematologica, 2005 Mar; 90: ECR10. 2. Bacigalupo A, Lamparelli T, Barisione G, Bruzzi P, Guidi S, Alessandrino PE, di Bartolomeo P, Oneto R, Bruno B, Sacchi N, van Lint MT, Bosi A; Gruppo Italiano Trapianti Midollo Osseo (GITMO).  Thymoglobulin prevents chronic graft-versushost disease, chronic lung dysfunction, and late transplant-related mortality: long-term follow-up of a randomized trial in patients undergoing unrelated donor transplantation. Biol Blood Marrow Transplant. 2006 May;12(5):560-5. 3. Bacigalupo A, Tong J, Podestà M, Piaggio G, Figari O, Colombo P, Sogno G, Tedone E, Moro F, van Lint MT, Frassoni F, Occhini D, Gualandi F, Lamparelli T, Marmont M: Bone marrow harvest for marrow transplantation: effect of multiple small (2 ml) or large (20 ml) aspirates. Bone Marrow Transplant 1992; 9; 467-470.

Chapter 19  Unrelated Donor Transplants  4. Bacigalupo A, Locatelli F, Lanino E, Marsh J, Socie G, Maury S, Prete A, Locasciulli A, Cesaro S, Passweg J; Severe Aplastic Anemia Working Party of the European Group for Blood and Marrow Transplantation.  Fludarabine, cyclophosphamide and anti-thymocyte globulin for alternative donor transplants in acquired severe aplastic anemia: a report from the EBMT-SAA Working Party. Bone Marrow Transplant. 2005 Dec;36(11):947-50. 5. Bacigalupo A, Lamparelli T, Bruzzi P, Guidi S, Alessandrino PE, di Bartolomeo P, Oneto R, Bruno B, Barbanti M, Sacchi N, Van Lint MT, Bosi A.  Antithymocyte globulin for graft-versus-host disease prophylaxis in transplants from unrelated donors: 2 randomized studies from Gruppo Italiano Trapianti Midollo Osseo (GITMO). Blood. 2001 Nov 15;98(10):2942-7. 6. Basara N, Baurmann H, Kolbe K, Yaman A, Labopin M, Burchardt A, Huber C, Fauser AA, Schwerdtfeger R. Antithymocyte globulin for the prevention of graftversus-host disease after unrelated hematopoietic stem cell transplantation for acute myeloid leukemia: results from the multicenter German cooperative study group. Bone Marrow Transplant. 2005 May;35(10):1011-8. 7. Byrne JL, Stainer C, Cull G, Haynes AP, Bessell EM, Hale G, Waldmann H: The effect of the serotherapy regimen used and the marrow cell dose received on rejection, graft-versus-host disease and outcome following unrelated donor bone marrow transplantation for leukaemia. Bone Marrow Transplant 2000; 25: 411-417. 8. Cutler C, Li S, Ho VT, Koreth J, Alyea E, Soiffer RJ, and Antin JH. Extended follow-up of methotrexate-free immunosuppression using sirolimus and tacrolimus in related and unrelated donor peripheral blood stem cell transplantation. Blood 2007 109: 3108-3114. 9. Deeg HJ, O’Donnell M, Tolar J, et al. Optimization of conditioning for marrow transplantation from unrelated donors for patients with aplastic anemia after failure of immunosuppressive therapy. Blood 2006;108:1485-1491. 10. Dominietto A, Raiola AM, van Lint MT, Lamparelli T, Gualandi F, Berisso G, Bregante S, Frassoni F, Casarino L, Verdiani S, Bacigalupo A: Factors influencing hematologic recovery after allogeneic hemopoietic stem cells transplant (HSCT): graft versus host disease, donor type, cytomegalovirus infections and cell dose. Br J Haematol, 2001, 112; 219-227. 11. Ferrara GB, Bacigalupo A, Lamparelli T, Lanini E, Delfino L, Morabito A, Parodi AM, Pera C, Pozzi S, Sormani MP, Bruzzi P, Bordo D, Bolognesi M, Bandini G, Bontadini A, Barbanti M, Frumento G. Bone marrow transplantation from unrelated donors: the impact of mismatches with substitutions at position 116 of the human lukocyte antigen class I heavy chain. Blood 2001; 98 (10): 3150-3155. 12. Hegenbart U, Niederwieser D, Sandmaier BM, Maris MB, Shizuru JA, Greinix H, Cordonnier C, Rio B, Gratwohl A, Lange T, Al-Ali H, Storer B, Maloney D, McSweeney P Chauncey T, Agura E, Bruno B, Maziarz RT, Petersen F, Storb R. Treatment for acute myelogenous leukemia by low-dose, total-body, irradiation-based conditioning and hematopoietic cell transplantation from related and unrelated donors. J Clin Oncol. 2006 Jan 20;24(3):444-53. Epub 2005 Dec 12. 13. Janossy G, Prentice HG, Hoffbrand AV, Blacklock HA, Ivory K, Gilmore MJ. The role of monoclonal antibodies in the prevention of graft versus host disease. Med Oncol Tumor Pharmacother. 1984;1(4):279-84. 14. Kojima S, Matsuyama T, Kato S, Kigasawa H, Kobayashi R, Kikuta A, Sakamaki H, Ikuta K, Tsuchida M, Hoshi Y, Morishima Y, Kodera Y.   Outcome of 154 patients with severe aplastic anemia who received transplants from unrelated donors: the Japan Marrow Donor Program. Blood. 2002 Aug 1;100(3):799-803. 15. Kopp HG, Horger M, Faul C, Hartmann JT, Kanz L, Lang P, Vogel W. Granulocyte Colony-Stimulating Factor–Induced Pulmonary Hemorrhage in a Healthy Stem Cell Donor. JCO 2007; 25: 3174-3175

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A. Bacigalupo 16. La Nasa G, Caocci G, Argiolu F, Giardini C, Locatelli F, Vacca A, Orofino MG,Piras E, Addari MC, Ledda A, Contu L. Unrelated donor stem cell transplantation in adult patients with thalassemia. Bone Marrow Transplant. 2005 ;36:971-5. 17. Larocca A, Piaggio G, Podesta M, Pitto A, Bruno B, Di Grazia C, Gualandi F, Occhini D, Raiola AM, Dominietto A, Bregante S, Lamparelli T, Tedone E, Oneto R, Frassoni F, Van Lint MT, Pogliani E, Bacigalupo A.  Boost of CD34+-selected peripheral blood cells without further conditioning in patients with poor graft function following allogeneic stem cell transplantation. Haematologica. 2006 Jul;91(7):935-40. 18. . Lee SJ, Klein J, Haagenson M, Baxter-Lowe LA, Confer DL, Eapen M, Fernandez-Vina M, Flomenberg N, Horowitz M, Hurley CK, Noreen H, Oudshoorn M, Petersdorf E, Setterholm M, Spellman S, Weisdorf D, Williams TM, Anasetti C. High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation.Blood. 2007 Sep 4; [Epub ahead of print] 19. . Nuamah NM, Goker H, Kilic YA, Dagmoura K,, Cakmak A, Spontaneous splenic rupture in a healthy donor of peripheral blood stem cells, following the administration of granulocyte colony stimulating factor. A case report and review of the literature. Haematologica 2006; 91: ECR08. 20. Petersdorf EW, Anasetti C, Martin PJ, Gooley T, Radich J, Malkki M, Woolfrey A, Smith A, Mickelson E, and Hansen JA. Limits of HLA mismatching in unrelated hematopoietic cell transplantation. Blood 2004;104(9):2976-80. 21. Petersdorf EW, Malkki M, Gooley TA, Martin PJ, and Guo Z.. MHC haplotype matching for unrelated hematopoietic cell transplantation. Plos Med 2007; 4(1): 59-68. 22. Sierra J, Storer B, Hansen JA, Bjerke JW, Martin PJ, Petersdorf EW, Appelbaum FR, Bryant E, Chauncey TR, Sale G, Sanders JE, Storb R, Sullivan KM, Anasetti C: Transplantation of marrow cells from unrelated donors for treatment of highrisk acute leukemia: the effect of leukemic burden, donor HLA-matching and marrow cell dose. Blood 89: 4226, 1997 23. Reisner Y, Kapoor N, O'Reilly RJ, Good RA. Allogeneic bone marrow transplantation using stem cells fractionated by lectins: VI, in vitro analysis of human and monkey bone marrow cells fractionated by sheep red blood cells and soybean agglutinin Lancet. 1980 Dec 20-27;2(8208-8209):1320-4. 24. Schmitz N, Beksac M, Hasenclever D, Bacigalupo A, Ruutu T, Nagler A, Gluckman E, Russell N, Apperley JF, Gorin NC, Szer J, Bradstock K, Buzyn A, Clark P, Borkett K, Gratwohl A for the European Group for Blood and Marrow Transplantation. Transplantation of mobilized peripheral blood cells to HLA identical siblings with standard risk leukaemia. Blood 2002; 100: 761-767. 25. . Sorror ML, Storer B, Storb RF. Validation of the hematopoietic cell transplantation-specific comorbidity index (HCT-CI) in single and multiple institutions: limitations and inferences. Biol Blood Marrow Transplant. 2009 26. Storb R, Yu C, Sanmeier BM,Mc Sweeney PA, Georges G, Nash RA, Woolfrey A. Mixed hemopoietic chimerism after marrow allografts. Transplantation in the ambulatory care setting. Ann N.Y. Acad Sci 1999; 872: 372-5 27. Takakazu K, Yasuo M, Keitaro M, Koichi K, Hidetoshi I, Hiroh S, Shunichi K, Takeo J, Yoshihisa K, and Takehiko S, for The Japan Marrow Donor Program. High-risk HLA allele mismatch combinations responsible for severe acute graft-versus-host disease and implication for its molecular mechanism. Blood 2007; 110: 2235-41. 28. Tigue CC, McKoy JM, Evens AM, Trifilio SM, Tallman MS, Bennett CL. Granulocyte-colony stimulating factor administration to healthy individuals and persons with chronic neutropenia or cancer: an overview of safety considerations from the Research on Adverse Drug Events and Reports project. Bone Marrow Transplant. 2007 Aug;40(3):185-92. Epub 2007 Jun 11.

Chapter 19  Unrelated Donor Transplants  29. Thomas ED, Storb R, Clift RA, Fefer A, Johnson L, Neiman PE, Lerner KG, Glucksberg H, Buckner CD.  Bone-marrow transplantation (second of two parts). N Engl J Med. 1975 Apr 24;292(17):895-902. Review. 30. Urbano-Ispizua A, Schmitz N, de Witte T, Frassoni F, Rosti G, Schrezenmeier H, Gluckman E, Friedrich W, Cordonnier C, Socie G, Tyndall A, Niethammer D, Ljungman P, Gratwohl A, Apperley J, Niederwieser D, Bacigalupo A; European Group for Blood and Marrow Transplantation.   Allogeneic and autologous transplantation for haematological diseases, solid tumours and immune disorders: definitions and current practice in Europe. Bone Marrow Transplant. 2002 Apr;29(8):639-46.

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Chapter 20 Update on Umbilical Cord Blood Transplantation Karen Ballen

1.  Pre-Clinical Background of Umbilical Cord Blood Transplantation The initial observations on the potential of umbilical cord blood came from the laboratories of Broxmeyer, Knudtzon, Lansford, Metcalf and others, demonstrating that the cord blood contained a high percentage of granulocyte-macrophage progenitor cells [1–3]. These initial experiments led to work in a murine model, showing that neonatal mice contain sufficient progenitor cells that repopulate the lethally irradiated mice. Sufficient progenitor cells were also found in the human cord blood, and was used to promote engraftment, leading to the first successful cord blood transplant in 1988 [2, 4]. Further investigation, subsequent to the initial clinical studies, revealed the unique properties of umbilical cord blood grafts. CD34+ cord blood cells increase faster than adult bone marrow cells in culture, allowing a 10-fold lower CD34+ cell dose to be used in cord blood transplants [3, 5]. The immunologic characteristics of cord blood cells differ from those of the adult bone marrow or peripheral blood stem cells, which explains the decreased incidence of graft vs. host disease in cord blood transplants, even in those that are mismatched at two alleles. Cord blood cells contain a high proportion of “naïve” T cells that express the CD45RA+/CD45RO−, CD62L+ phenotype and low cytotoxic activity [6, 7]. Compared with adult peripheral blood T cells, more T cells from cord blood progress through cell cycle and enter apoptosis. Cord blood T cells have decreased expression of granzyme and perforin, which help to eradicate viral infection, and these differences may explain the increased propensity of cord blood recipients for viral infections [8].

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_20, © Springer Science + Business Media, LLC 2003, 2010

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2. Cord Blood Banking The initial success of the first cord blood transplant led to the creation of cord blood banks to collect, process, and store donated cord blood units. Cord blood banks may be public (donated cord blood units used for unrelated patients in need) or private (cord blood units saved for the use of the donating family). Currently, there are approximately 250,000 cord blood units stored worldwide for public use and 700,000 cord blood units stored worldwide for private use [9]. A few cord blood banks, such as the Directed Cord Blood Banking Program for England and Wales and the Sibling Cord Banking Program in California use federal funds to store cord blood for families with hemoglobinopathies and other diseases amenable to transplantation [10]. One of the goals of the cord blood banks is to increase the number of donations from the non-Caucasian donors, as the non-Caucasian patients have greater difficulty in finding appropriately matched donors through the National Marrow Donor Program (NMDP) and other international registries. The American Red Cross studied the cord blood donor population in a concerted effort to collect cord blood units at different sites throughout the country and revealed a diverse donor population, with 64% Caucasian, 16% African American, 12% Hispanic, 4% Asian, 1% Native American, and 3% others [11]. Several cord blood banks have reported a lower CD34+ count in African American donors, suggesting that more resources need to be devoted to collecting suitable units from this donor population [12]. Current issues in cord blood banking include pre-donation screening, collection, processing, and thawing techniques. Cord blood can be collected in utero by an obstetrical staff before the delivery of the placenta, or ex utero by trained collection staff. With either technique, a needle is inserted into the umbilical vein, and the cord blood flows by gravity into a citrate-phosphate dextrose (CPD) blood donor sterile bag. The ex utero method is less invasive and there is better regulatory control over this technique, but it may be less cost effective, given the need for an extra personnel. One study found that no difference existed in cell counts or CD34+ counts with either method [13]. Recently, the necessity of washing the cord blood unit prior to infusion has become an issue of controversy. Early studies reported that a post-thaw cord blood dilution to decrease the osmolality, followed by a dextran albumin-based washing, was important to maintain the cord blood progenitor cell viability and engraftment speed. However, recent clinical studies show that the postthaw cord blood dilution without washing, or even direct infusion of thawed cord blood is associated with an equally reliable engraftment that is free from side effects [14]. An advantage of cord blood is the speed of the search process, as there is no living donor to locate, retest, and reconsent [15]. Recently, 79 million dollars in US federal funds has been allocated to expand the cord blood banking program in the United States. The “Stem Cell Therapeutic and Research Act of 2005” supports the creation of National Cord Blood Program and the collection of 150,000 additional cord blood units. The goals of this effort are to ease the cord blood search and acquisition process, and to provide more high quality units, particularly for minority patients [16].

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3.  Ethical Issues in Cord Blood Banking and Transplantation Personal or private storage of cord blood has expanded over the last 5 years, due to parental concern over providing the “biologic insurance” in case a family member develops a disease that could be treated with cord blood transplantation, and due to aggressive marketing techniques by “for-profit banks” offering private collection. In private cord blood banking, a fee is charged upon receipt of the cord blood unit, whereas in public banking, financial investment is returned to the cord blood bank only when the cord blood unit is selected for transplant. The American Academy of Pediatrics, the American College of Obstetrics and Gynecology, and the American Society of Bone Marrow Transplantation have all issued position papers on this issue [17–19]. All three organizations encourage prospective parents to donate their child’s cord blood to a public bank, if available. They also suggest storing the cord blood for family use if the sibling of the expected child has a disease that can be cured by the allogeneic transplantation, and discourage private storage of cord blood if there is no such affected family member.

4. Pediatric Cord Blood Transplantation The first cord blood transplants were performed in children; the first cord blood transplant was a related donor pediatric transplant in a child with Fanconi’s anemia [20]. Locatelli and colleagues reported on 42 children receiving a related donor cord blood transplant for leukemia, with a 2-year event free survival of 41% [21]. This Eurocord group transplanted cord blood in 44 children with sickle cell anemia and thalassemia, from related cord blood donors, with event-free survival of 79% for thalassemia and 90% for sickle cell anemia [22]. The establishment of the Placental Banking Program at the New York Blood Center and other international cord blood banks facilitated the initial experience with unrelated cord blood transplantation in children. Table 20.1 outlines

Table 20-1.  Results of pediatric unrelated cord blood transplantation. Investigator (citation)

Number of patients

Kurtzberg et al. [23]

  25

AML, ALL, MDS, Fanconi, 13 metabolic

48%

Wagner et al. [59]

102

AML, ALL, CML, lym32 phoma, Fanconi, metabolic

47%

Staba et al. [26]

  20

Hurler syndrome

30

85%

Eapen et al. [24]

503

AML

44

60% (6/6 matched)

Gluckman et al. [29]

  93

Fanconi

22

Diseases

Median followup (months)

Disease-free survival (%)

38% (5/6,4/6) 40%

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K. Ballen

some of these studies in pediatric unrelated cord blood transplantation. In 1996, Kurtzberg et  al. analyzed the outcomes of the first 25 unrelated cord blood transplants. The event-free survival was 48% and the results suggested that engraftment was possible even with two antigen mismatched cord blood units [23]. A randomized comparison of the unrelated cord blood transplants with fully matched sibling donor transplant or unrelated bone marrow/peripheral blood stem cell transplantation has not been performed. Given the expense and logistics of such a study, this prospective comparison may never be performed. However, several retrospective comparisons of cord blood patients with historically matched controls have been completed. Eapen et al. compared 4-6/6 HLA A, B antigen, and DRB1 allele matched single unit myeloablative cord blood transplantation with 8/8 allele level matched unrelated donor bone marrow transplantation in children with acute leukemia [24]. Disease-free survival was found to be the best for those patients who received a 6/6 matched cord blood (60% vs. 38%) and comparable for those patients who received a 8/8 matched unrelated bone marrow or 4/6 or 5/6 matched cord blood units. A meta analysis compared outcomes of 161 children undergoing cord blood transplantation with 316 children undergoing unrelated bone marrow transplantation [25]. The risk of chronic GVHD, but not acute GVHD, was lower in the cord blood recipients. There was, however, no difference in the overall survival. Cord blood has been shown to be a highly effective treatment for non-malignant disorders. The low incidence of GVHD after cord blood transplantation and the ease of finding a unit with sufficient cell dose for young children with metabolic storage disorders make the cord blood a favorable stem cell source [26–28]. Gluckman and colleagues transplanted 93 children with Fanconi’s anemia using unrelated cord blood grafts. Overall survival was 40%; and a higher cell dose and use of fludarabine in the conditioning regimen correlated with improved survival [29]. Current controversies in pediatric unrelated cord blood transplantation include the optimal conditioning regimen and the choice of cord blood unit [30]. The Clinical Trials Network (CTN) performed a randomized study of single vs. double cord blood transplantation in pediatric patients to address the importance of double cord blood unit transplantation in a given patient population.

5. Adult Cord Blood Transplantation Based on the encouraging results in children, a number of investigators attempted unrelated cord blood transplantation in adults in the 1990s. The initial transplants were single cord blood transplants using a myeloablative regimen. Laughlin et  al. analyzed the results for 68 adults treated with unrelated cord blood transplantation [31]. Transplant-related mortality at 100 days was high at 47%, and the disease-free survival was 26%. An important finding was that a higher CD34+ cell count in the cord blood graft predicted better survival rate. There have been no randomized studies comparing the unrelated cord blood and bone marrow or peripheral blood stem cell transplantation. The Center for International Bone Marrow Transplant Registry performed a retrospective comparison of 116 patients with leukemia receiving unrelated cord blood transplantation, with 367 adults receiving fully matched unrelated bone marrow transplants, and 83 patients receiving a single antigen mismatched

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bone marrow transplant [32]. All patients received a myeloablative conditioning regimen and single unit cord blood transplantation. The results showed a 3-year leukemia-free survival of 33% for fully matched bone marrow, 23% for cord blood, and 19% for single antigen mismatched bone marrow, suggesting that the cord blood transplantation is comparable to single antigen mismatched bone marrow. A similar study from the Eurocord group indicated comparable survival between recipients of cord blood transplants and fully matched unrelated donor bone marrow transplants [33]. The Japanese study has impressive results with a 3-year disease free survival of 70% after cord blood transplant, compared to 60% after related bone marrow or peripheral blood stem cell transplant [34]. The results of these single unit myeloablative studies are shown in Table 20.2. In general, the risk of graft vs. host disease is reduced, but engraftment is delayed after the cord blood transplantation. With the exception of the Japanese experience, however, survival has been disappointing, with about one-third of patients surviving. Several modalities have been used to improve the results for adult cord blood transplantation. These include the use of multiunit grafts (double cord), cord blood expansion, reduced intensity conditioning regimens, and infection prophylaxis; these strategies will be discussed below. Non-myeloablative or reduced intensity transplant regimens have been used extensively over the last 7 years, in the related and unrelated donor setting, to reduce the transplant-related mortality and allow successful transplant in an older group of patients [35, 36]. As outlined in Table 20.3, the Japanese group treated

Table 20-2.  Adult cord blood transplantation – single unit ablative regimen. Investigator (citation)

N

Conditioning regimen

Diseases

Laughlin et al. [31]

Median follow-up Disease-free (months) survival (%)

  68

Cytoxan/TBI or Bu/Cy/ATG

CML, AML, CLL, ALL, lymphoma

22

26

Laughlin et al. [32]

116

Multiple

CML, AML, MDS, ALL

40

23

Rocha et al. [33]

  98

Multiple

AML, ALL

27

33

Takahashi et al. [34]

100

Cytoxan/TBI or TBI/ARA-C

AML, ALL, MDS, CML,

25

70

Table 20-3.  Adult cord blood transplant – single unit – reduced intensity regimen. Investigator (citation)

N

Conditioning

Diseases

Median follow-up Disease-free (months) survival (%)

Yuji et al. [37]

20

Fludarabine/Melphalan/ low dose TBI

Lymphoma

12

50

Chao et al. [39]

13

Fludarabine/ Cyclophosphamide/ ATG

AML, ALL, MDS, lymphoma

20

24

Miyakoshi et al. [38]

34

Fludarabine/melphalan/ low dose TBI

AML, ALL, MDS, lymphoma,

12

55

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K. Ballen

20 patients suffering from refractory lymphoma with a single cord blood unit and a reduced intensity conditioning regimen of low dose total body radiation, fludarabine, and melphalan. Transplant-related mortality continued to be high at 41%, similar to a myeloablative regimen [37]. A follow-up study using tacrolimus for GVHD prophylaxis produced a lower transplant-related mortality at 12% at 100 days and 1-year disease-free survival of 55% [38]. The Duke group used a reduced intensity conditioning regimen of fludarabine, cyclophosphamide, and horse antithymocyte globulin followed by single cord blood transplantation in 13 refractory hematologic malignancy patients. Treatment-related mortality was low – there was only one death – but the 1-year event free survival was 43% [39]. These studies show that the transplant-related mortality is often high in adult patients receiving single cord blood units. Most of the deaths, regardless of the conditioning regimen, have been related to infection. The cell dose correlated with engraftment and outcome in many studies. Thus, increasing the cell dose by infusing two partially matched cord blood units is one strategy to improve survival. The University of Minnesota has pioneered the double cord blood approach in both the ablative and reduced intensity setting. Barker and colleagues treated 23 adults with a myeloablative preparative regimen of cyclophosphamide, total body radiation, and fludarabine, followed by infusion of cord blood units, each unit a 4/6 match to the patient and to each other [40]. All patients engrafted, with a median time to neutrophil engraftment of 23 days. The risk of Grades III–IV GVHD was 13% and the 1-year survival was 57%. The double cord blood approach has been extended to the reduced intensity setting, as illustrated in Table 20.4. Brunstein and colleagues treated 110 patients with a conditioning regimen of fludarabine, cyclophosphamide, and low dose total body radiation, followed by double cord blood transplantation in 93 patients [41]. Neutrophil engraftment occurred at a median of 12 days, and acute GVHD Grades III–IV occurred in 22% patients. Transplantation-related mortality was 26% at 3 years. Disease-free survival at 3 years was 38%. Fifty-three patients were treated in Boston with the reduced intensity conditioning regimen of fludarabine, melphalan, and thymoglobulin, followed by double cord blood transplantation [42, 43]. Two cord blood units, achieving a minimum combined pre-freeze cell dose of >3.7 × 10(7) NC/kg, and matching at 4/6 or more HLA loci with each other and with the patient, were infused on the same day. Twenty-one patients received the GVHD prophylaxis of

Table 20-4.  Reduced intensity double cord blood transplantation in adults. Investigator (citation)

N

Conditioning regimen

Diseases

Median follow-up Disease-free (months) survival (%)

Brunstein et al. [41]

110

Fludarabine, cyclophospha- AML, ALL, MDS, mide, low dose TBI lymphoma

19

38

Ballen et al. [43]

  21

Fludarabine, melphalan, thymoglobulin

AML, ALL, MDS, lymphoma

18

55

Cutler et al. [42]

  32

Fludarabine, melphalan, thymoglobulin

AML, ALL, MDS, lymphoma

15

54

Chapter 20  Update on Umbilical Cord Blood Transplantation 

cyclosporine and mycophenolate mofetil, and 32 patients received sirolimus and tacrolimus. The median days to neutrophil engraftment were 21 days and median days to platelet engraftment to 20 × 109/l were 42 days. The 100-day transplant-related mortality was 12%. The risk of acute GVHD was less in the sirolimus/tacrolimus patients (10% vs. 40%). With a median follow-up of 20 months, the 1-year overall and disease-free survival was 74% and 59%, respectively. Double cord blood transplantation offers unique challenges in the interpretation of post transplantation chimerism, or the contribution of each cord blood donor and recipient to hematopoiesis. Brunstein reported the presence of only one unit contributing to hematopoiesis [41]. In our Boston experience, by Day +100, 72% of patients had hematopoiesis derived from a single cord blood unit [44]. A higher post-thaw nucleated cell count and CD34+ cell dose were associated with cord predominance; in 68% patients, the predominant cord blood unit was the first unit infused. The post-thaw CD34+ dose of the predominant unit predicted time to neutrophil and platelet engraftment. However, other programs have not reported an association between the order of infusion and predominant unit; the issues affecting the cord unit predominance remain unclear. The presence of only one cord contributing to long-term hematopoiesis raises the controversy over the importance of the second cord blood unit, and the benefit of double cord blood transplants in general. Verneris et  al. reported a lower relapse rate in patients who received double, rather than single, cord blood unit transplants, perhaps related to a stronger immunologic attack [45]. These questions have not been fully answered but the superior results of double cord blood transplantation, in comparison to historical controls receiving single cord blood units, have fostered the growth of double cord blood transplants, particularly in the heavier US population.

6. Other Strategies for Adult Cord Blood Transplantation Ex vivo expansion is another strategy to improve the infused progenitor cell doses, and decrease the risk of graft failure. Early attempts of ex vivo expansion failed to show improvement in engraftment, probably due to the expansion of mature cells. Shpall and colleagues expanded CD34+ cord blood cells with a mixture of stem cell factor, G-CSF, and megakaryocyte growth and differentiation factor [46]. The median time to neutrophil engraftment was 28 days and the overall survival was 37%, with a median follow-up of 30 months. Current strategies for expansion include targeting the Notch signaling pathway that is involved in cellular differentiation and proliferation; this approach was used by the Seattle group [47, 48]. MD Anderson has initiated a trial comparing two unmanipulated cord blood units with one unmanipulated and one expanded cord blood unit. The expanded cells are prepared from the CD133+ fraction and incubated with G-CSF, stem cell factor, and thrombopoietin [49]. The Spanish group had adopted a novel approach, infusing haploidentical peripheral blood stem cells and a single cord blood unit [50]. Twenty-seven patients received a myeloablative conditioning regimen followed by co-infusion of a cord blood unit and peripheral blood stem cells from a third party. Neutrophil engraftment occurred at a median of 10 days, usually from the stem cells, but hematopoiesis

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converted to cord blood origin in all patients. The incidence of acute Grades II–IV graft vs. host disease was 15%. The 4-year overall survival was 69%.

7. Unique Challenges of Cord Blood Transplantation The emergence of better expansion techniques may help to improve engraftment after adult cord blood transplantation, but infection and poor immune reconstitution remain significant concerns even with expanded cells or double cord blood transplantation. A review of infections in 100 recipients of pediatric and adult cord blood transplants revealed 221 infections in the first 100 days post transplant [51]. These infections included 22 fungal infections, 105 bacterial infections, and 62 viral infections, including adenovirus, respiratory synctial virus, and influenzae. There were four cases of CMV end organ disease. Late infections included CMV reactivation, varicella zoster, staphylococcal and pseudomonas bacteremia, aspergillus, and mycobacterium. A survey of 128 cord blood recipients revealed 14 cases of invasive fungal infection, 13 related to aspergillus, with a mortality rate of 86% [52].

8. Future Trends in Cord Blood Transplantation During the next 5–10  years, the availability and applications of cord blood transplantation will continue to increase. Future trials are needed to determine the best donor choice for those patients without a matched sibling donor. There may be indications for transplantation in situations where graft vs. host disease is intolerable or unnecessary, such as with older patients or patients with nonmalignant disease. There are potential exciting avenues for cord blood for non-hematopoietic diseases. Cord blood cells are a more primitive population than adult marrow cells, and have increased capacity for multi-lineage differentiation [53]. Cord blood cells have been shown to improve the neurologic recovery in rats with strokes [54]. Preliminary investigation has analyzed the use of autologous CB for autoimmune disease, particularly childhood diabetes. Haller and colleagues infused seven children with Type I diabetes with autologous CB; these children had lower hemoglobin A1c and fewer insulin requirement than a randomly selected control population of severe diabetic children [55]. Repair of damaged cardiac tissue is another exciting avenue for cord blood stem cells. In a mouse model, cord blood cells injected into the tail vein migrated to infarcted myocardial tissue and reduced the infarct size [56]. Cord blood cells express connexin and stromal cell-derived factor (SDF)-1 alpha, which are proteins that are important for cardiovascular regeneration [57]. Cord blood cells were injected into a rat myocardial infarction model. Apoptotic cells were decreased and cardiac function improved in rats that received cord blood as opposed to rats that received a mock injection [58]. The next 10 years should prove exciting for the field of cord blood transplantation. Improved results in adult transplantation and strategies for advancing immune reconstitution will extend the application of cord blood in the oncology setting. The use of cord blood for indications outside of oncology will likely develop as we learn more about the multiple applications of umbilical cord blood.

Chapter 20  Update on Umbilical Cord Blood Transplantation 

References 1. Knudtzon S (1974) In vitro growth of granulocytic colonies from circulating cells in human cord blood. Blood 43:357–361 2. Broxmeyer HE, Kurtzberg J, Gluckman E et al (1991) Umbilical cord blood hematopoietic stem and repopulating cells in human clinical transplantation. Blood Cells 17:313–329 3. Lansdorp PM, Dragowska W, Mayani H (1993) Ontogeny-related changes in proliferative potential of human hematopoietic cells. J Exp Med 178:787–791 4. Gluckman E, Broxmeyer HA, Auerbach AD (1989) Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical cord blood from an HLA identical sibling. N Engl J Med 321:1174–1178 5. Vaziri H, Dragowska W, Allsopp RC, Thomas TE, Harley CB, Lansdorp PM (1994) Evidence for a mitotic clock in human hematopoeitic stem cells: Loss of telomeric DNA with age. Proc Natl Acad Sci USA 91:9857–9860 6. Szabolcs P, Park KD, Reese M, Marti L, Broadwater G, Kurtzberg J (2003) Coexistant naïve phenotype and higher cycling rate of cord blood T cells compared to adult peripheral blood. Exp Hematol 31:708–714 7. Kim YJ, Brutkiewicz PR, Broxmeyer HE (2002) Role of 4–1BB (CD137) in the functional activation of cord blood CD28-CD8+ T cells. Blood 100:3253–3260 8. Berthou C, Legros-Maida S, Soulie A et  al (2003) Cord blood T lymphocytes lack constitutive perforin expression in contrast to adult peripheral blood T lymphocytes. Blood 102:4608 9. Bone Marrow Donors Worldwide, Annual Report 2006 10. Smythe J, Armitage S, McDonald D, Pamphilon D, Guttridge M, Brown J et  al (2007) Directed sibling cord blood banking for transplantation: The 10-year experience in the national blood service in england. Stem Cells 25:2087–2093 11. Ballen KK, Kurtzberg J, Lane TA, Lindgren BR, Miller JP, Nagan D et al (2004) Racial diversity with high nucleated cell counts and CD34 counts achieved in a national network of cord blood banks. Biol Blood Marrow Transplant 10:269–275 12. Cairo MS, Wagner EL, Fraser J et al (2005) Characterization of banked umbilical cord blood hematopoietic progenitor cells and lymphocyte subsets and correlation with ethnicity, birth weight, sex, and type of delivery: A cord blood transplantation (COBLT) study report. Transfusion 45:856–866 13. Laskey LC, Lane TA, Miller JR, Lindgren B, Patterson H, Haley NR et al (2002) In utero or ex utero cord blood collection: Which is better?. Transfusion 42:1261–1267 14. Chow R, Nademanee A, Rosenthal J, Karanes C, Jaing TH, Graham ML (2007) Analysis of hematopoietic cell transplants using plasma-depleted cord blood products that are not red blood cell reduced. Biol Blood Marrow Transplant 13:1346–1357 15. Barker JN, Krepski TP, DeFor TE, Davies SM, Wagner JE, Weisdorf DJ (2002) Searching for unrelated donor hematopoietic stem cells: Availability and speed of umbilical cord blood versus bone marrow. Biol Blood Marrow Transplant 8:257–260 16. Atlas LD (2006) The national marrow donor program in 2006: Constants and challenges. Transfusion 46:1080–1084 17. American Academy of Pediatrics Section of Hematology/Oncology (2007) Cord blood banking for potential future transplantation. Pediatrics 119:165–170 18. American College of Obstetricians and Gynecology (1997) Routine storage of umbilical cord blood for potential future transplantation. ACOG Comm Opin 183:1–3 19. Ballen KK, Barker JN, Stewart SK, Greene MF, Lane TA (2008) Collection and preservation of cord blood for personal use. Biol Blood Marrow Transplant 14:356–363 20. Gluckman E, Auerbach BHA, AD FHS, Douglas GW, Devergie A et  al (1989) Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilicalcord blood from an HLA-identical sibling. N Engl J Med 321:1174–1178

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K. Ballen 21. Locatelli F, Rocha V, Chastang C et  al (1999) Factors associated with outcome after cord blood transplantation in children with acute leukemia: Eurocord-cord blood transplant group. Blood 93:3662–3671 22. Locatelli F, Rocha V, Reed W et al (2003) Related umbilical cord blood transplantation in patients with thalassemia and sickle cell disease. Blood 101:2137–2143 23. Kurtzberg J, Laughlin M, Graham ML et al (1996) Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 335:157–166 24. Eapen M, Rubinstein P, Zhang MJ, Stevens C, Kurtzberg J, Scaradavou A et  al (2007) Outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukemia: A comparison study. Lancet 369:1947–1954 25. Hwang WY, Samuel M, Tan D, Koh LP, Lim W, Lim YC (2007) A meta-analysis of unrelated donor umbilical cord blood transplantation versus unrelated donor bone marrow transplantation in adult and pediatric patients. Biol Blood Marrrow Transplant 13:444–453 26. Staba SL, Escolar ML, Poe M, Kim Y, Martin PL, Szabolcs P et  al (2004) Cord-blood transplants from unrelated donors in patients with Hurler’s syndrome. N Engl J Med 350:1960–1969 27. Escolar ML, Poe MD, Provenzale JM et al (2005) Transplantation of umbilical-cord blood in babies with infantile Krabbe’s disease. N Engl J Med 352:2069–2081 28. Kobayashi R, Ariga T, Nonoyama S et al (2006) Outcome in patients with WiskottAldrich syndrome following stem cell transplantation: An analysis of 57 patients in Japan. Br J Hematol 135:362–366 29. Gluckman E, Rocha V, Ionescu I, Bierings M, Harris RE, Wagner J et  al (2007) Results of unrelated cord blood transplant in Fanconi anemia patients: Risk factor analysis for engraftment and survival. Biol Blood Marrow Transplant 13:1073–1082 30. Wall DA, Chan KW (2008) Selection of cord blood unit (s) for transplantation. Bone Marrow Transplant 42:1–7 31. Laughlin MJ, Barker J, Bambach B et  al (2001) Hematpoietic engraftment and survival in adult recipients on umbilical-cord blood from unrelated donors. N Engl J Med 344:1815–1822 32. Laughlin MJ, Eapen M, Rubinstein P, Wagner JE, Zhang MJ, Champlin RE et al (2004) Outcomes after transplantation of cord blood or bone marrow from unrelated donors in patients with leukemia. N Engl J Med 351:2265–2275 33. Rocha V, Labopin M, Sanz G, Arcese W, Schwerdtfeger R, Bosi A et al (2004) Transplants of umbilical cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 351:2276–2285 34. Takahashi S, Ooi J, Tomonari A, Konuma T, Tsukada N, Monna MO et al (2007) Comparative single-institute analysis of cord blood transplantation from unrelated donors with bone marrow or peripheral blood stem-cell transplants from related donors in adult patients with hematologic malignancies after myeloablative conditioning regimen. Blood 109:1322–1330 35. Khouri IF, Saliba RM, Giralt SA et al (2001) Nonablative allogeneic hematopoietic transplantation as adoptive immunotherapy for indolent lymphoma: Low incidence of toxicity, acute graft vs host disease, and treatment-related mortality. Blood 98:3595–3599 36. Daly A, McAfee S, Dey B, Colby C, Schulte L, Yeap B et al (2003) Nonmyeloablative bone marrow transplantation: Infectious complications in 65 recipients of HLA-identical and mismatched transplants. Biol Blood Marrow Transplant 9:373–382 37. Yuji K, Miyakoshi S, Kato D et  al (2005) Reduced-intensity unrelated cord blood transplantation for patients with advanced lymphoma. Biol Blood Marrow Transplant 11:314–318

Chapter 20  Update on Umbilical Cord Blood Transplantation  38. Miyakoshi S, Kami M, Tanimoto T, Yamguchi T, Narimatsu H, Kusumi E et  al (2007) Tacrolimus as prophylaxis for acute graft versus host disease in reduced intensity cord blood transplantation for adult patients with advanced hematologic diseases. Transplantation 84:316–322 39. Chao NJ, Koh LP, Long GD et al (2004) Adult recipients of umbilical cord blood transplants after nonmyeloablative preparative regimens. Biol Blood Marrow Transplant 10:569–575 40. Barker JN, Weisdorf DJ, Defor TE, Blazar BR, McGlave PB, Miller JS et al (2005) Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 105:1343–1347 41. Brunstein CG, Barker JN, Weisdorf DJ, DeFor TE, Miller JS, Blazar BR et  al (2007) Umbilical cord blood transplantation after nonmyeloablative conditioning: Impact of transplantation outcomes in 110 adults with hematologic disease. Blood 110:3064–3070 42. Cutler C, Mitrovich R, Kao G, Ho V, Alyea E, Koreth J et al (2007) Double umbilical cord blood transplantation with reduced intensity conditioning and sirolimus-based GVHD prophylaxis. Blood 118:600a Abstract 43. Ballen KK, Spitzer TR, Yeap B, McAfee S, Dey BR, Attar E et al (2007) Double unrelated reduced-intensity umbilical cord blood transplantation in adults. Biol Blood Marrow Transplant 13:82–89 44. Haspel RL, Kao G, Yeap BY, Cutler C, Soiffer RJ, Alyea EP et  al (2008) Preinfusion variables predict the predominant unit in the setting of reduced intensity double cord blood transplantation. Bone Marrow Transplant 41:523–529 45. Verneris MR, Brunstein C, DeFor TE, Barker J, Weisdorf DJ, Blazar BR et al (2005) Risk of relapse after umbilical cord blood transplantation in patients with acute leukemia: Marked reduction in recipients of two units. Blood 106:305a Abstract 46. Shpall EJ, Quinones R, Giller R, Zeng C, Baron AE, Jones RB et  al (2002) Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant 8:368–376 47. Hofmeister CC, Zhang J, Knight KL, Le P, Stiff PJ (2007) Ex vivo expansion of umbilical cord blood stem cells for transplantation: Growing knowledge from the hematopoietic niche. Bone Marrow Transplant 39:11–23 48. Delaney C, Varnum-Finney B, Aoyama K, Brashem-Stein C, Bernstein ID (2005) Dose-dependent effects of the Notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells. Blood 106:2693–2699 49. Shpall EJ, De Lima M, McManis JD, Robinson S, McNiece IK, Champlin RE (2005) Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant 11:932–935 50. Magro E, Regidor C, Cabrera R, Sanjuan I, Fores R, Garcia-Marco JA et al (2006) Early hematopoietic recovery after single unit unrelated cord blood transplantation in adults supported by co-infusion of mobilized stem cells from a third party donor. Hematologica 91:640–648 51. Safdar A, Rodriguez G, DeLima M, Petropoulos D, Chemaly R, Worth L et  al (2007) Infections in 100 cord blood transplantations: Spectrum of early and late posttransplant infections in adult and pediatric patients 1996–2005. Medicine 86:324–333 52. Miyakoshi S, Kusumi E, Matsumura T, Hori A, Murashige N, Hamaki T et  al (2007) Invasive fungal infection following reduced-intensity cord blood transplantation for adult patients with hematologic diseases. Biol Blood Marrow Transplant 13:771–777 53. Goodwin HS, Bicknese AR, Chien SN, Bogucki BD, Quinn CO, Wall DA (2001) Multilineage differentiation activity by cells isolated from umbilical cord blood: Expression of bone, fat, and neural markers. Biol Blood Marrow Transplant 7:581–588

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K. Ballen 54. Vendrame M, Cassady J, Newcomb J et al (2004) Infusion of human umbilical cord blood cells in a rat model of stroke dose-dependently rescues behavioral deficits and reduces infarct volume. Stroke 35:2390–2395 55. Viener HL, Brusko T, Wasserfall C et  al (2007) Changes in regulatory T cells following autologous umbilical cord blood transfusion in children with type I diabetes. J Am Diab Assoc 7:0314 Abstract 56. Ma N, Stamm C, Kaminski A et  al (2005) Human cord blood cells induce angiogenesis following myocardial infarction in NOD/scid-mice. Cardiovasc Res 5:45–54 57. Prat-Vidal C, Roura S, Farre J, Galvez C, Llach A, Molina CE et  al (2007) Umbilical cord blood-derived stem cells spontaneously express cardiomyogenic traits. Transplant Proc 39:2434–2437 58. Wu KH, Zhou B, Yu CT, Cui B, Lu SH, Han ZC, Liu Y (2007) Therapeutic potential of human umbilical cord derived stem cells in a rat myocardial infarction model. Ann Thorac Surg 83:1491–1500 59. Wagner JE, Barker JN, DeFor TE et al (2002) Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: Influence of CD34 cell dose and HLA disparity n treatment-related mortality and survival. Blood 100:1611–1618

Chapter 21 Selection of Cord Blood Unit(s) for Transplantation Donna A. Wall and Ka Wah Chan

1. Introduction Over the past decade, cord blood (CB) has been established as an alternative source of donor cells for allogeneic hematopoietic stem cell transplantation. The outcome of CB transplants, particularly in children, is similar to the unrelated donor transplants using bone marrow cell or mobilized peripheral blood progenitor cells [1–4]. Early experience in adult CB transplantation was hampered by poor engraftment and immune recovery [5–7]. Recent experiences with better risk patients, double CB unit transplants, and submyeloablative preparative regimens have been more encouraging [8–10]. There are several important differences between bone marrow or GCSFmobilized peripheral blood progenitor cells and cord blood, that have to be taken into account when selecting donors/products for transplantation. Cord blood transplants are being performed with approximately a lot fewer hematopoietic progenitors than other stem cell sources [1]. The adult donor grafts deliver cell numbers well above the engraftment threshold such that a loss of even half of the product would not have a major impact on the transplant. CB as a donor source is not as tolerant. In general, clinical series have shown that the CB transplantation is associated with a higher incidence of graft failure and delayed count recovery [6, 11]. However, these risks are offset by a lower risk for acute and chronic GVHD despite major HLA disparity. This is due in part to the lower number of mature T-cells in the graft (functionally, CB is a partially T-cell depleted graft) and to the nature of the cord blood T-cell responsiveness to the allogenic stimulus [12–19]. The question that arises is how to apply these observations to one’s strategy for cord blood unit selection, especially when there are competing variables. In this review, we summarize the literature on selection strategy, comparing unrelated adult donor to CB searches, and provide our personal preferences on this issue.

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_21, © Springer Science + Business Media, LLC 2003, 2010

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1.1.  Selection of Unrelated Adult Donor HSC for Transplantation: General Principles The two critical components in selecting adult donors for transplantation are HLA compatibility and the ability to harvest hematopoietic progenitors (i.e., the availability of the donor). By and large, cell dose is not an issue. The numbers of hematopoietic progenitors delivered in adult donor HSC transplantation are well above the minimum threshold for engraftment and the size differential between donor and recipient is rarely greater than two-fold. It has been shown that a larger graft improves the outcome of transplants in bone marrow transplantation [20, 21]. Higher numbers of cells are required when there is a graft manipulation (such as T-cell depletion, CD34 selection) or when submyeloablative preparative regimens are planned. With adult unrelated donors, it is the HLA matching that is generally the most significant challenge. Current search algorithm recommends matching at least seven of eight high resolution at HLA-A, HLA-B, HLA-C, and HLADRB1 loci [22]. Recent data support that allelic (high resolution) mismatches are as significant as broad antigen mismatches. A recent National Marrow Donor Program review of 3,857 US transplantations performed from 1988 to 2003 with patient-donor pairs fully typed for HLA-A, B, C, DRB1, DQB1, DQA1, DPB1, and DPA1 alleles, demonstrated that high resolution DNA matching for HLA-A, B, C, and DRB1 [8/8 match] was the minimum level of matching associated with the highest survival [23]. A single mismatch detected by low or high resolution DNA testing at HLA-A, B, C, or DRB1 [7/8 match] was associated with higher mortality (relative risk 1.25, 95% CI 1.13–1.38, p < 0.0001) and 1-year survival of 43% compared to 52% for 8/8 matched pairs. In this series, single mismatches at HLA-B or HLA-C appear to be better tolerated than mismatches at HLA-A or HLA-DRB1. Mismatching at two or more loci compounded the risk. Mismatching at HLA-DP or DQ loci and donor factors other than HLA type were not associated with survival. When choosing between HLA mismatches with multiple donor options, there is no consensus as to which HLA locus mismatches are better tolerated [23–25]. Other factors that impact transplant outcome are CMV negative recipient receiving cells from a CMV positive donor, ABO mismatching, and parity. Ethnic differences may also be an important factor [26]. In general, GVHD risk is reported to be lower in unrelated adult donor within more homogenous populations. The Japanese NMDP, reporting on a very homogenous population, reported an overall lower rate of GVHD with an increase of GVHD in the HLA mismatch setting (HLA-A, and -C mismatch) and a lower survival with HLA-A disparity [27]. 1.2.  Selection of Unrelated Donor CB for Transplantation: The Product in General A typical, successful CB collection is on an average 120 ml (range 60–300 ml) and contains 0.8 to 3 × 109 total nucleated cells (TNC). Processing and testing generally result in a loss of 10–20% of the initially harvested blood. The cell dose that is listed on the registries is the TNC at the time of freezing, after processing and testing has occurred. Historically, this number has been similar between the different banks for similar products. However, recent evolution in banking practices have resulted in different TNC for a given ­hematopoietic

Chapter 21  Selection of Cord Blood Unit(s) for Transplantation 

progenitor content (CD34 or CFU) due to greater or lesser removal of neutrophils during processing (some automated processors will remove more neutrophils during processing while other banks will store CB without red cell/neutrophil depletion). This makes interpretation of hematopoietic potential difficult to compare between banks. In general, as CB is a peripheral blood product there is a fairly close correlation between TNC and hematopoietic progenitor in the “conventional” red cell and plasma depleted product [28–31]. As yet, no correlative tools have been developed to interpret the differences between the TNC reported by the different banks. Unfortunately, multiple attempts to standardize CD34 and CFU quantization between the laboratories have not been successful. Thus, despite the limitations of the TNC, it is the most commonly used measurement in the CB unit selection. The transplant program is fully dependent on the bank for the quality of the CB unit. In unrelated adult donors, collections are performed by the remote collection site but all subsequent processing and storage is performed at the transplant center. In contrast with CB collection, screening, testing, processing, freezing, and storage are performed at the bank. Additionally, CB units may have been collected years prior to use so that the current bank quality measures may not be applicable to those that were operational at the time of banking. This becomes critically important given that CB transplants are performed with cell doses near the threshold of reliable engraftment; modest loss of potency of a product could have major impact on engraftment. There are many organizations (FDA, NMDP, FACT/Eurocord and other national regulatory bodies) that are working with the cord blood banking community to ensure the quality of the CB units listed available for transplant. At the time of consideration of a CB unit for transplantation, it is reasonable to ask the bank for details of their processing, storage, and transplant outcomes in general as they apply to the specific cord blood unit. Requalification testing prior to release of a unit utilizing a contiguous segment of the CB unit is being developed to confirm the unit identity and growth of hematopoietic progenitors prior to transplantation [31]. There has been no correlation between length of storage and transplant outcome measurements. Cord blood units that have been stored for over 10 years are now being used successfully in transplant. There is no consensus as to the shelf life of CB units that are properly stored in liquid nitrogen. 1.3.  Selection of Unrelated Donor CB for Transplantation: Cell Dose While most CB units are analyzed for TNC, CD34, and colony forming unit content at the time of cryopreservation, the TNC is the most standardized between the CB banks and hence, is the hematopoietic measure that is used for CB unit selection (see caveat above). Table  21.1 summarizes reported minimum cell dose thresholds and impact of cell dose on transplant outcomes. In general, more is better. For consistent engraftment, a TNC dose >3.7 × 107/kg was required. Gluckman et  al. reported a log linear relation between cell dose and the probability of engraftment [32]. Rubinstein et  al. also noted a step-wise increase in cell dose correlated with the speed of myeloid recovery [33]. Many pediatric centers accept a minimum cell dose of 2 × 107 TNC/kg but most target cell doses above 5 × 107/kg with no upper cell dose limit. This approach does not work for adults where the cell doses achievable are rarely above 3 × 107/kg with

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Table 21-1.  Minimum cell doses based on hematopoietic measurement and the effect of higher cell dose on transplant outcomes for unrelated donor CB transplant. Author

Engraftment rate

Cell dose (/kg) recommended

Effect of higher cell dose noted on

Rubinstein [33] (n = 861)

93%

>2.5 × 107 TNC

↑Engraftment, ↓TRM

Wagner [34](n = 102)

88%

>1.7 × 10 CD34

↑Engraftment, ↓TRM,↑ survival

Migliaccio [28] (n = 204)

NA

>5 × 104 CFC

↑Engraftment, ↓TRM

Barker [35] (n = 608)

NA

5

7

>2.5 × 10 TNC

↓TRM

TRM transplant-related events/mortality; NA not reported

s­ ingle CB units. Only a small fraction of CB inventories have adequate cell numbers to support adult transplants. The institutional cellular therapy laboratory plays a critical role in the CB transplant process. Approximately 20% of CB cells may be lost in the thaw process – due to cell death arising out of thaw, institutional testing, loss in manipulation. Laboratories need to develop operating procedures that minimize the cell loss on thaw. Convention is to use the pre-cryopreservation cell dose and not the post-thaw cell dose in reporting transplant outcomes. Other measures of graft progenitor content, such as CD34 and hematopoietic colony-forming cell (CFC) enumeration, are likely to be equal or better predictors of successful engraftment (Table 21.1). Unique to CB is the high percentage of nucleated red blood cells (NRBC) in the cell product. The NRBC are included in the TNC. Our experience and that of the National Cord Blood Program support that the increased number of primitive progenitors that accompany higher NRBC offset any difference in cell content (i.e., we do not adjust the TNC for NRBC content [30]). For search situations where there are several CB units with comparable TNC and HLA matching, some authors advocated selecting units of higher CD34 or CFC [28, 34]. Given the variability of these results between banks, we use the CD34 or CFU assessments to screen the units that may have poor hematopoietic potential – avoiding selection of units with very low CFU/CD34 content. Similarly, if available, CB CD3 content may be used to screen units to avoid subsequent severe immunodeficiency syndromes. 1.4. Selection of Untreated Donor CB for Transplantation: HLA Matching CB inventories are only a small fraction of the 9 million HSC donors registered around the world. Convention is to define the HLA matching for CB transplantation as low resolution HLA-A and B matching and high resolution HLA-DR matching – a huge difference from the 8/8 high resolution HLA matching used in adult unrelated donor matching. Multiple HLA mismatches are tolerated with CB grafting. When high resolution matching of 10 alleles is looked at in the units selected for transplant, there are frequently many more mismatches present [11].

Chapter 21  Selection of Cord Blood Unit(s) for Transplantation 

Over half of our transplants are performed with 4/6 or less HLA matching. In practice, there is no difference in survival based on degree of HLA matching. The reason for this is that in a primarily pediatric population we usually achieve high cell doses, and treat many patients with high risk leukemia. A distinction needs to be made between adequate and ideal matching. Higher degrees of HLA matching were associated with improved engraftment and transplant outcome compared to 5/6, or 4/6 matches (Table  21-2) but the impact is relatively small [1, 33, 34]. This is because of the confounding impact of cell dose on engraftment and HLA mismatch on graft vs. leukemia effect. As yet, there is no consensus as to which specific HLA mismatches are better tolerated. Given that 90% of transplants are performed with at least one major HLA mismatch, it has not been possible to isolate the impact of HLA C or DQ mismatching on CB transplant outcomes. Recipients of two HLA mismatched grafts have fared surprisingly well and the limited data on 3/6 matches is surprisingly good. In general, 3/6 matching is reserved for small children with no other options [11]. Rubinstein and colleagues observed that any HLA disparity adversely affected the engraftment rate and increased the risk of acute GVHD; but there was no additive effect with increasing incompatibility [33]. There was, however, a step-wise increase in the incidence of transplant-related complications with increasing number of HLA mismatches. Gluckman et al. noted that co-existence of class I and class II disparities was associated with a higher incidence of severe GVHD and failure of platelet engraftment [32]. The effect of HLA mismatch is most apparent when the cell dose is low [35]. An important question is how much of the adverse effect of HLA disparity can be overcome by a higher cell dose. In malignant diseases, data from the Eurocord registry has demonstrated that with 2–4 HLA differences, the negative effect of delayed engraftment was abrogated by a higher cell dose [36]. However, a threshold of cell dose to overcome HLA disparities could not be defined. Based on the immunobiologic fundamentals, it seems logical to pick a unit with the most HLA alleles matching with the recipient for transplant. However, data to support this approach are scarce. Using allelic typing for HLA-A, -B, -C, -DR and -DQ loci, Kogler et al. showed retrospectively that three-quarter of CB transplants had three or more mismatches. Surprisingly, there was no improved survival in the subset of children receiving 10/10 allelematched CB units [37]. In children with leukemia, when an adequate cell dose could be administered, high-resolution HLA-A, -B, and -DRB1 matching was not found to improve survival [11]. There is some evidence that class II mismatching is less well tolerated [38]. However, this has not been a universal finding. In fact the reviews from the National Cord Blood Bank do not find an impact of location of mismatch and outcome in single major HLA antigen mismatched (5/6) transplants [35]. 1.5.  Selection of Untreated Donor CB for Transplantation: Non-inherited Maternal Allele Matching One area that needs further exploration and which is unique to CB is the potential exploitation of the non-inherited maternal (NIMA) and paternal (NIPA) alleles [39]. A fetus is a haplotype match with the mother. In utero

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Table 21-2.  Impact of HLA disparity on outcomes following unrelated donor CB transplant. Effect of ≠ mismatch noted on

Authors

HLA disparity

No effect noted on

Rubinstein [33] (n = 861)

0 vs. ³1

↓Engraftment, Relapse ↑aGVHD, ↑TRM, ↓event-free survival

Wagner [34] (n = 102)

0–1 vs. 2

None

Gibbons [55] (n = 755)

0 vs. 1 vs. 2

↓Engraftment, ↓survival

Gluckman [32] (n = 550)

0 vs. 1 vs. 2 vs. 3–4

↓Engraftment, aGVHD (II–IV), ↑aGVHD (III–IV), TRM, survival ↓ relapse

Barker [35] (n = 608)

0 vs. 1 vs. 2 vs. 3

↑TRM

GVHD, TRM, relapse, survival

the fetal lymphocytes, which are immunocompetent from 18-weeks gestation, are kept in a state of non-responsiveness to the mismatching maternal antigens. As most cord blood transplants are being performed with major HLA mismatches, a question that arises is whether matching the HLA mismatch to the NIMA would result in a transplant with less GVHD. There is evidence in the renal transplantation and partially HLA matched family member marrow transplants that there is less graft rejection or acute GVHD, respectively, if the HLA mismatch is a NIMA [40–42]. In fact in a small series of haploidentical sibling CB transplants, the haplotype mismatch was disparate at the NIMA, 0/10 recipients. However, grII-IV GVHD was observed in 4/5 transplants with the NIPA mismatch [43]. It is possible that targeting the mismatch in an unrelated donor setting to the NIMA may result in less GVHD – admittedly this will be difficult to test. Following this logic, one would postulate that transplanting CB from the donor infant to its mother should also have a lower risk of GVHD, given that those CB immune cells have been exposed to the mismatching maternal haplotype while in utero. Most banks store samples of maternal DNA so NIMA testing is feasible. 1.6.  Selection of Unrelated Donor CB for Transplantation: Other Factors As only a small fraction of CB transplants are performed with fully HLA matched CB units, it is difficult to tease out the impact of other factors conventionally associated with transplant outcome – gender or ABO mismatch or CMV status. Most CB units are CMV naive due to the placental barrier. There has repeatedly been an association between CMV positivity in the recipient and poorer transplant outcome which may be due to the lack of prior exposure of CB cells to CMV [11, 44, 45]. ABO mismatches have been associated with delays in red cell and platelet transfusion independence. In a study of 95 adults who underwent unrelated cord blood transplantation (CBT) (27 ABO-identical, 29 minor, 21 major, and 18

Chapter 21  Selection of Cord Blood Unit(s) for Transplantation 

bidirectional ABO-incompatible recipients), Tomonari and colleagues reported that neutrophil and red cell engraftment did not differ but that the cumulative incidence of platelet engraftment in ABO-identical/minor ABO-incompatible recipients was higher than in major/bidirectional ABO-incompatible recipients (HR 1.88, p = 0.013). In addition, fewer platelet and red cell transfusions were required in ABO-identical/minor ABO-incompatible recipients (HR 0.80, p = 0.040) [46]. 1.7. Selection of Unrelated Donor CB for Transplantation: Effect of the Underlying Diagnosis In analyzing the data from the Eurocord registry, Gluckman et al. showed that underlying disease affects the cord blood selection criteria [36, 47]. Transplant for malignant diseases can be successfully performed with a lower cell dose (down to 2 × 107 NC/kg infused) and that with HLA mismatching relapse was less frequent. Therefore, in the high-risk cases, a larger unit with greater HLA mismatch may actually be the preferred CB graft. In the setting of unrelated CBT for non-malignant disorders the needs are very different. It is in this population, where GVHD is not beneficial, one would expect to see the greatest impact of HLA matching. In a recent analysis of CBT for Fanconi’s anemia, Gluckman and colleagues retrospectively analyzed results of unrelated CB transplantation in 93 Fanconi anemia patients [45]. In this series, HLA mismatches were associated with poorer survival. 1.8. Selection of Unrelated Donor CB for Transplantation: Double Cord Blood Units To overcome the cell dose restriction, infusion of two separate cord blood units (double CB) have been used with encouraging results [8, 9, 48, 49]. When two or more CB units are used as the HPC source, there is only one unit contributing to hematopoiesis within 1 month of post transplant. Neither the total nucleated, CD34, and CD3 cell doses, HLA matching, nucleated cell viability, ABO typing, gender match, or order of unit infusion was predictive of which unit eventually dominated [9]. The potential benefits of the two units are transient hematopoiesis from the second unit ameliorating toxicity during the time of early post transplant period and immunologic synergy between the two units during the early post transplant period which is known to be important in the development of alloreactivity and possibly speeding hematopoietic recovery. The potential risks with double CBT are negative graft-graft interactions and possibly increased risk of chronic GVHD. Given the difficulty in determining tolerable mismatches in the single CBT setting, it is hard to be dogmatic about HLA matching in the double CBT setting. As a generalization, higher cell doses in at least one of the CB units is important (ideally >2.5 × 107/kg) and it is thought that there should be some matching between both patient-cord and cord-cord. A comparison between single vs. double CBT is being tested in a phase III trial in children with acute leukemia (BMT CTN 0501). In an extension of this approach in a small pilot trial, Lister and colleagues infused multiple CB units (5–7 units) at the time of transplant and found that in the patients evaluable there was always one unit becoming the sole source

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for long-term hematopoiesis [50]. In one long-term survivor, the CB source was only a single antigen match with the recipient. 1.9.  Selection of Unrelated Donor CB for Transplantation: Texas Transplant Institute Perspective The transplant population in South Texas is highly represented by Hispanic recipients, and as such HLA matching is frequently a challenge. Given the rapid availability of CB and the ability to use partially matched donors, most unrelated donor transplants in the pediatric program utilize CB as the donor source. The independent influences of HLA matching and cell dose on the outcome of CBT has driven our CB selection approach. Over the past 5  years, we have developed an algorithm for selection of CB units for transplant. As outlined in Table 21-3, we target a maximum cell dose, even at the expense of HLA matching. Only 10% of CB transplants at our center are performed with 6/6 antigen matched units. A single unit CB is used if there are more than 2 × 107 TNC/kg pre-cryopreservation with 6/6 antigen matched CB, and a minimum of 3 × 107 TNC/kg if there is 4/6 HLA-matching. High resolution HLA-A, -B, and DR typing is obtained on both donor and recipient. While we start with matching at low-antigen level for class I and high resolution for class II, if a 4/6 or 3/6 matched unit is being considered we would like to have the matches to be allelic matches. Matching at both HLA-DRB1 alleles is preferred, especially if there are multiple mismatches (i.e., 4/6 match). Mismatches at both class II (either antigen or allele) loci are not accepted. When several large CB units are available, and the cell dose is at least 3–5 × 107 TNC/kg, we would choose the unit with best HLA match. In situations of a small, well matched unit and a much larger, less well matched unit we will utilize the larger unit preferentially, especially when treating malignancies. In larger units, when the cell dose threshold is not met, we use two CB units for transplantation. This functionally includes all adult transplants. In young children with no other options, we will utilize 3/6 HLA matched units, with at least one match at HLA-A, B, and DR. We have not noticed a difference in engraftment, GVHD or survival with these less well matched units, but

Table 21-3.  Texas Transplant Institute algorithm for CB unit selection. HLA match 6/6

Cell dose (TNC/kg of recipient weight)

Location of mismatch

7

N/A

7

Class I (A or B) mismatch preferred over DR mismatch

>2 × 10

5/6

>3 ×10

4/6

>3 × 107

3/6

7

>5 × 10

A + B, A/B + DRB1 At least one DRB1 match Younger recipients

7

Target cell dose: TNC >5 × 10 nucleated cells/kg (no upper limit on cell dose) Acceptable cell dose is dependent on degree of HLA matching If no CB unit is identified that meets these criteria we consider double cord blood grafts or ex vivo expansion

Chapter 21  Selection of Cord Blood Unit(s) for Transplantation 

our numbers are small and these are a selected group of young children for whom we can generally achieve a very large cell dose. Up to 10% of CB transplants are complicated by primary graft failure. We monitor engraftment closely in the first few months post transplant and will proceed to an early second transplant utilizing a submyeloablative preparative regimen [51]. In these situations we target the largest CB unit(s) with a minimum of 4/6 HLA matches, accepting a DRB1 allele mismatch if necessary. Given the real risk for graft failure, it is advised to have an alternative stem cell source identified prior to start of transplant. At our center we have a second CB unit on reserve at the bank.

2. Summary Selection of CB units for transplantation involves combining both cell dose and HLA matching as independent yet overlapping variables. Cell dose and cell yield at the time of transplant are critical given that the transplants are being performed with minimal cells for reliable engraftment. In transplants for malignant disorders, the greater allogeneicity and lower relapse rate associated with the less well matched units balances any benefit that better HLA matching may have on transplant-related morbidity/mortality. The only factor that has repeatedly been associated with improved outcome post CB transplant is cell dose. The CB inventories are rapidly increasing in size and the quality of the CB units being banked (larger, better characterized) is improving. With this some of our current limitations in CB availability will soon become moot. Explorations into the CB expansion and multiple CB unit transplants address the limited cell doses attainable with a single CB collection [49, 52–54]. At this point one must conclude that bigger is better when selecting CB units for transplantation.

References 1. Grewal SS, Barker JN, Davies SM, Wagner JE (2003) Unrelated donor hematopoietic cell transplantation: marrow or umbilical cord blood? Blood 101: 4233–4244 2. Eapen M, Rubinstein P, Zhang MJ et al (2006) Comparable long-term survival after unrelated and HLA-matched sibling donor hematopoietic stem cell transplantations for acute leukemia in children younger than 18 months. J Clin Oncol 24:145–151 3. Bunin N, Carston M, Wall D et al (2002) Unrelated marrow transplantation for children with acute lymphoblastic leukemia in second remission. Blood 99:3151–3157 4. Hwang WY, Samuel M, Tan D, Koh LP, Lim W, Linn YC (2007) A meta-analysis of unrelated donor umbilical cord blood transplantation versus unrelated donor bone marrow transplantation in adult and pediatric patients. Biol Blood Marrow Transplant 13:444–453 5. Chao NJ, Liu CX, Rooney B et  al (2002) Nonmyeloablative regimen preserves "niches" allowing for peripheral expansion of donor T-cells. Biol Blood Marrow Transplant 8:249–256 6. Cornetta K, Laughlin M, Carter S et al (2005) Umbilical cord blood transplantation in adults: results of the prospective cord blood transplantation (COBLT). Biol Blood Marrow Transplant 11:149–160

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D.A. Wall and K.W. Chan 7. Laughlin MJ, Eapen M, Rubinstein P et al (2004) Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia. N Engl J Med 351:2265–2275 8. Ballen KK, Spitzer TR, Yeap BY et al (2007) Double unrelated reduced-intensity umbilical cord blood transplantation in adults. Biol Blood Marrow Transplant 13:82–89 9. Brunstein CG, Barker JN, Weisdorf DJ et al (2007) Umbilical cord blood transplantation after nonmyeloablative conditioning: impact on transplantation outcomes in 110 adults with hematologic disease. Blood 110:3064–3070 10. Takahashi S, Iseki T, Ooi J et  al (2004) Single-institute comparative analysis of unrelated bone marrow transplantation and cord blood transplantation for adult patients with hematologic malignancies. Blood 104:3813–3820 11. Kernan N, Carter S, Wagner J, Baxter-Lowe L, Wall D, Kapoor N (2006) Umbilical cord blood transplantation in pediatric patients: results of the prospective, multiinstitutional cord blood transplantation study (COBLT). Biol Blood Marrow Transplant 12:14 (abst 33) 12. Roncarolo MG, Bigler M, Martino S, Ciuti E, Tovo PA, Wagner J (1996) Immune functions of cord blood cells before and after transplantation. J Hematother 5:1 57–160 13. Han P, Hodge G (1999) Intracellular cytokine production and cytokine receptor interaction of cord mononuclear cells: relevance to cord blood transplantation. Br J Haematol 107:450–457 14. Rainsford E, Reen DJ (2002) Interleukin 10, produced in abundance by human newborn T cells, may be the regulator of increased tolerance associated with cord blood stem cell transplantation. Br J Haematol 116:702–709 15. Gardiner CM, Meara AO, Reen DJ (1998) Differential cytotoxicity of cord blood and bone marrow-derived natural killer cells. Blood 91:207–213 16. Joshi SS, Tarantolo SR, Kuszynski CA, Kessinger A (2000) Antitumor therapeutic potential of activated human umbilical cord blood cells against leukemia and breast cancer. Clin Cancer Res 6:4351–4358 17. El Marsafy S, Dosquet C, Coudert MC, Bensussan A, Carosella E, Gluckman E (2001) Study of cord blood natural killer cell suppressor activity. Eur J Haematol 66:215–220 18. Hodge S, Hodge G, Flower R, Han P (2001) Cord blood leucocyte expression of functionally significant molecules involved in the regulation of cellular immunity. Scand J Immunol 53:72–78 19. Nomura A, Takada H, Jin CH, Tanaka T, Ohga S, Hara T (2001) Functional analyses of cord blood natural killer cells and T cells: a distinctive interleukin-18 response. Exp Hematol 29:1169–1176 20. Gorin NC, Labopin M, Rocha V et al (2003) 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 102:3043–3051 21. Sierra J, Storer B, Hanson J, Bjerke J, Martin P, Petersdorf E (1997) Transplantation of marrow cells from unrelated donor for the treatment of high-risk acute leukemia: the effect of leukemia burden, donor HLA matching, and marrow cell dose. Blood 89:4226–4235 22. Hurley C, Baxter-Lowe L, Logan B, Karanes C, Anasetti C, Weisdorf D (2003) National Marrow Donor Program HLA-matching guidelines for unrelated marrow transplants. Biol Blood Marrow Transplant 9:610–615 23. Lee SJ, Klein J, Haagenson M et al (2007) High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood 110:4576–4583 24. Peterdorf E, Kollman C, Hurley C, Dupont P, Nademannee A, Bogovich A (2001) Effect of HLA class II gene disparity on clinical outcome in unrelated donor

Chapter 21  Selection of Cord Blood Unit(s) for Transplantation  hematopoietic cell transplantation for chronic myeloid leukemia: the US national marrow donor program experience. Blood 98:2922–2929 25. Flomenberg N, Baxter-Lowe L, Confer D, Fernandez-Vina M, Filipovich A, Horowitz M (2004) 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 104:1923–1930 26. Serna DS, Lee SJ, Zhang MJ et al (2003) Trends in survival rates after allogeneic hematopoietic stem-cell transplantation for acute and chronic leukemia by ethnicity in the United States and Canada. J Clin Oncol 21:3754–3760 27. Sasazuki T, Juji T, Morishima Y, Kinukawa N, Kashiwabara H, Inoko H (1998) Effect of matching class I alleles on clinical outcome after transplantation of hematopoietic stem cells from an unrelated donor. N Engl J Med 339:1177–1185 28. Migliaccio AR, Adamson JW, Stevens CE, Dobrila NL, Carrier CM, Rubinstein P (2000) 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 96:2717–2722 29. Wall DA, Noffsinger JM, Mueckl KA et al (1997) Feasibility of an obstetricianbased cord blood collection network for unrelated donor umbilical cord blood banking. J Matern Fetal Med 6:320–323 30. Stevens CE, Gladstone J, Taylor PE et  al (2002) Placental/umbilical cord blood for unrelated-donor bone marrow reconstitution: relevance of nucleated red blood cells. Blood 100:2662–2664 31. Goodwin HS, Grunzinger LM, Regan DM et al (2003) Long term cryostorage of UC blood units: ability of the integral segment to confirm both identity and hematopoietic potential. Cytotherapy 5:80–86 32. Gluckman E, Rocha V, Arcese W et  al (2004) Factors associated with outcomes of unrelated cord blood transplant: guidelines for donor choice. Exp Hematol 32:397–407 33. Rubinstein P, Stevens CE (2000) Placental blood for bone marrow replacement: the New York Blood Center's program and clinical results. Baillieres Best Pract Res Clin Haematol 13:565–584 34. Wagner JE, Barker JN, DeFor TE et al (2002) 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 100:1611–1618 35. Barker J, Scaradavou A, Stevens C, Rubinstein P (2005) Analysis of 608 umbilical cord blood transplants: HLA-match is a critical determinant of transplant-related mortality in the post-engraftment period even in the absence of acute graft-versushost disease. Blood 106:abst 303 36. Gluckman E, Rocha V (2006) Donor selection for unrelated cord blood transplants. Curr Opin Immunol 18:565–570 37. Kogler G, Enczmann J, Rocha V, Gluckman E, Wernet P (2005) High-resolution HLA typing by sequencing for HLA-A, -B, -C, -DR, -DQ in 122 unrelated cord blood/patient pair transplants hardly improves long-term clinical outcome. Bone Marrow Transplant 36:1033–1041 38. van Heeckeren WJ, Fanning LR, MH J et al. Influence of HLA disparity and graft lymphocytes on allogeneic engraftment and survival after umbilical cord blood transplants in adults. Leukemia (in press) 39. Moretta A, Locatelli F, Mingrat G et al (1999) Characterisation of CTL directed towards non-inherited maternal alloantigens in human cord blood. Bone Marrow Transplant 24:1161–1166 40. van Rood JJ (2000) Double role of HLA in organ transplantation. World J Surg 24:823–827

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D.A. Wall and K.W. Chan 41. van Rood JJ, Roelen DL, Claas FH (2005) The effect of noninherited maternal antigens in allogeneic transplantation. Semin Hematol 42:104–111 42. van Rood JJ, Loberiza FR Jr, Zhang MJ et al (2002) Effect of tolerance to noninherited maternal antigens on the occurrence of graft-versus-host disease after bone marrow transplantation from a parent or an HLA-haploidentical sibling. Blood 99:1572–1577 43. Wagner JE, Kurtzberg J (1998) Allogeneic umbilical cord blood transplantation. In: Broxmeyer HE, Broxmeyer HE, Broxmeyer HE (eds) Cellular characteristics of cord blood and cord blood transplantation. AABB, Bethesda, MD, pp 113–145 44. Rocha V, Chastang C, Souillet G, Rocha V, Chastang C, Souillet G et  al (1998) Related cord blood transplants: the Eurocord experience from 78 transplants. Eurocord transplant group. Bone Marrow Transplant 21(Suppl 3):S59–S62 45. Gluckman E, Rocha V, Ionescu I et al (2007) Results of unrelated cord blood transplant in fanconi anemia patients: risk factor analysis for engraftment and survival. Biol Blood Marrow Transplant 13:1073–1082 46. Tomonari A, Takahashi S, Ooi J et  al (2007) Impact of ABO incompatibility on engraftment and transfusion requirement after unrelated cord blood transplantation: a single institute experience in Japan. Bone Marrow Transplant 40:523–528 47. Gluckman E (2006) Cord blood transplantation. Biol Blood Marrow Transplant 12:808–812 48. Barker JN, Weisdorf DJ, Defor TE et  al (2005) Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 105:1343–1347 49. Majhail N, Brunstein C, Wagner J (2006) Double umbilical cord blood transplantation. Curr Opin Immunol 18:571–575 50. Lister J, Gryn JF, McQueen KL, Harris DT, Rossetti JM, Shadduck RK (2007) Multiple unit HLA-unmatched sex-mismatched umbilical cord blood transplantation for advanced hematological malignancy. Stem Cells Dev 16:177–186 51. Chan K, Grimley M, Taylor C, Wall D (2006) Primary graft failure after unrelated donor cord blood transplant: risk factors and management. Blood 108:abst 44 52. Shpall EJ, McNiece IK, De Lima M et  al (2004) Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant 10:738 53. Shpall EJ, Quinones R, Giller R et al (2002) Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant 8:368–376 54. Peled T, Landau E, Mandel J et al (2004) Linear polyamine copper chelator tetraethylenepentamine augments long-term ex vivo expansion of cord blood-derived CD34+ cells and increases their engraftment potential in NOD/SCID mice. Exp Hematol 32:547–555 55. Gibbons R (2005) Appendix G: statistical report. Cord blood: establishing a national hematopoietic stem cell bank program. National Academy of Sciences, Washington, DC

Chapter 22 Mobilization of Hematopoietic Cells Prior to Autologous or Allogeneic Transplantation Steven M. Devine

1. Introduction The recruitment of hematopoietic progenitor/stem cells (HPCs) from the marrow into the peripheral blood is termed stem cell mobilization [1, 2]. These stem cells are capable of homing to the marrow cavity and regenerating a full array of hematopoietic cell lineages in a timely fashion after ablative and nonmyeloablative conditioning. Pioneering studies performed over 40 years ago, demonstrated that hematopoietic stem cells (HSC) circulate in the peripheral blood at low frequency [3]. In the 1980s, investigators demonstrated that the frequency of circulating hematopoietic stem and progenitor cells was greatly enhanced (10- to 100-fold) following recovery from myelosuppressive chemotherapy through a process termed mobilization [4–7]. These hematopoietic progenitor cells (HPC) could be collected by leukapheresis (LP) in sufficient quantities to promote hematopoietic reconstitution following myeloablative therapy. These seminal observations have revolutionized clinical stem cell transplantation. In current practice, mobilized peripheral blood (MPB) has essentially replaced BM (BM) as a source of autologous cells for hematopoietic rescue in patients undergoing high-dose chemotherapy/radiotherapy due to improved neutrophil and platelet engraftment, shortened hospital stay, and lower cost [8–11]. More recently, recipients of allogeneic HSC have been administered cytokine MPB in preference to BM based on recent randomized studies that have clearly demonstrated improved kinetics of neutrophil and platelet engraftment, somewhat higher rates of acute and chronic graft-versus-host disease (GVHD), and similar to improved overall survival rates following the use of MPB compared with BM [12–19]. Despite the fact that virtually all autologous transplants are now performed using MPB, the optimal method to mobilize HPC remains the subject of debate. While chemotherapy-based mobilization typically results in collection of greater numbers of CD34+ cells compared to granulocyte colony-stimulating factor

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_22, © Springer Science + Business Media, LLC 2003, 2010

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(G-CSF) alone, as a strategy, chemotherapy-based mobilization is associated with greater morbidity due to infectious complications and has not decisively improved outcomes following transplantation [20–23]. Efforts to improve the yield of CD34+ cells following G-CSF-based mobilization through combination with other hematopoietic cytokines, have met with limited success due either to lack of efficacy or unacceptable toxicity. Novel strategies continue to be sought given that a substantial proportion of patients who have been heavily pretreated have poor stem cell mobilization with current approaches. The development of innovative strategies has recently accelerated due to a more complete understanding of the mechanism underlying stem/progenitor cell mobilization.

2. The Mechanisms of Stem Cell Mobilization Several adhesion molecules, including LFA-1, VLA-4, CXCR4, c-kit, CD44, and Mac-1, are known to anchor stem cells to the bone marrow microenvironment, and disruption of the interactions between these adhesion molecules and their ligands by both chemotherapy and cytokines may promote stem and progenitor cell egress into the peripheral circulation. For instance, hematopoietic progenitor cells (HPCs) are mobilized after exposure to antibodies which interrupt the interaction of the b1 integrin VLA-4, expressed on HPCs, and its ligand, VCAM-1, expressed on endothelial and stromal cells [24, 25]. Chemokines have recently been identified as key regulators of HPC mobilization, particularly members of the CXC chemokine family, including stromalderived factor 1 (SDF-1, also known as CXCL-12), and GROb. The chemokine SDF-1 is constitutively expressed by bone marrow stromal cells, and its receptor CXCR4 is a transmembrane G-protein-coupled receptor expressed on CD34+ cells. Interaction between SDF-1 and CXCR4 critically regulates migration of bone marrow, umbilical cord blood, and G-CSF-mobilized peripheral blood cells transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice [26], and neutralizing antibodies to CXCR4 or SDF-1 significantly reduce HPC homing, migration, and mobilization [27]. As discussed below, a direct inhibitor of the SDF-1/CXCR4 interaction, AMD3100, is an effective mobilizing agent currently being tested in clinical trials. Binding of the chemokine GROb to its receptor CXCR2 also plays an important role in stem cell localization in bone marrow. The N-terminal truncated variant of GROb, SB-251353 (GROb-T) can rapidly mobilize HPCs in mice and monkeys, an effect that is enhanced by combination with G-CSF. A single injection of SB-251353 combined with 4 days of G-CSF results in fivefold greater HPC mobilization than G-CSF alone [28, 29]. These promising results in animal models suggest that the efficacy of SB-251353 for stem cell mobilization might be exploited clinically if this agent was well tolerated, but the fact that this agent may activate neutrophils is a real concern. The mechanisms by which cytokines trigger stem cell mobilization remain incompletely understood. Liu et al. have shown that G-CSF receptor expression is not required on HPCs for their mobilization by G-CSF, suggesting that G-CSF acts indirectly on HPCs [30]. Proteases have been implicated as the secondary signals which lead to HPC mobilization induced by G-CSF. G-CSF may activate neutrophils and other target cells, leading to the release of proteases which cleave the adhesive interactions between HPCs and the bone marrow microenvironment [31, 32]. In support of this model, Levasque et al.

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identified two serine proteases, neutrophil elastase and cathepsin G, which increase in concentration during G-CSF mobilization and are directly able to cleave VCAM-1 [33]. In addition, the activity of IL-8, a CXC chemokine that induces rapid mobilization of HPCs [34], appears to be mediated by neutrophil activation and subsequent release of matrix metalloproteinase (MMP-9) [35]. In rhesus monkeys, IL-8-induced mobilization is completely inhibited by antibodies that block MMP-9 activity [36]. GROb-induced mobilization also may be mediated by MMP-9 [37]. Regulation of the SDF-1/CXCR4 interaction may occur via proteolytic cleavage of SDF-1 or CXCR4 [2, 38, 39]. This mechanism may be operational whether mobilization is induced by G-CSF alone or when combined with the chemotherapeutic agent cyclophosphamide [40]. However, a recent study suggests that cyclophosphamide-based mobilization may differ fundamentally from G-CSF alone. Using various murine systems, Mayack and Wagers demonstrated that treatment with cyclophosphamide and G-CSF enhances first osteoblast proliferation followed by HSC proliferation which is mediated at least in part by the function of the ataxia telangiectasia mutated (ATM), the product of the ARM gene, which is induced in response to DNA damage and oxidative stress. These potential differences require further study [41]. Recent evidence has identified CD26, a membrane-bound extracellular peptidase, as the prime protease that cleaves SDF-1 within the bone marrow [42]. Of note, G-CSF-induced mobilization is inhibited in mice deficient in CD26 [43, 44]. Importantly, inhibition of this molecule may enhance the homing of HPC/HSC to the bone marrow and provide a means to enhance engraftment [42]. This could become a novel strategy to enhance engraftment of umbilical cord blood cells, for instance. Despite this evidence, important studies in protease-deficient mice may contradict a central role for proteases in cytokine-induced stem cell mobilization. Three studies have shown that MMP-9-deficient mice have a similar increase in peripheral HPCs following treatment with IL-8 or G-CSF as wild-type mice [45]. Similarly, HPC mobilization with IL-8 or G-CSF was intact in neutrophil elastase- cathepsin G-deficient mice, and SDF-1 protein levels were decreased in the protease-deficient mice, as is observed in wild-type mice [37, 46]. These studies suggest that nonproteolytic mechanisms may play a fundamental role in stem cell mobilization. One likely mechanism is transcriptional regulation. Semerad et al. have found that SDF-1 mRNA in bone marrow cells is significantly reduced during G-CSF mobilization, suggesting that SDF-1 expression is regulated at the mRNA level [47].

3.  Regulation of Stem Cell Mobilization by Neural and Osteolineage Derived Cells Recent studies have shed light on another pathway in which G-CSF may indirectly exert its effects on SDF-1/CXCR4 signaling. UDP-galactose ceramide galactosyltransferase-deficient (Cgt(−/−)) mice exhibit aberrant nerve conduction and display virtually no PBSC egress from BM following G-CSF or fucoidan administration. Using mice lacking Cgt, a known neurotransmitter, Katayama and colleagues demonstrated that G-CSF-dependent stem cell mobilization may be governed by neural signals such as those transmitted by norepinephrine (NE) [48]. Stem cell mobilization was deficient in mice lacking

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Cgt, and could also be diminished using beta-blockers. Beta-adrenergic drugs, on the other hand, stimulated mobilization. These data suggest a heretofore unknown neural control over stem cell trafficking and raise the intriguing possibility that currently available agents (e.g., beta agonists) may be exploited to enhance mobilization in certain subsets of patients. This concept of neural control of stem/progenitor cell trafficking was further developed in an intriguing study in which mice were observed to exhibit circadian secretion of noradrenaline by sympathetic nerves contained within the bone marrow. These adrenergic signals were transmitted to stromal cells within the marrow by beta-adrenergic receptors, leading ultimately to downregulation of CXCL12 levels through decreases in transcription factors regulating CXCL12 production [49]. These findings support the concept of an optimal time to mobilize and collect HPC for clinical uses. The hematopoietic stem cell niche is a complex multidimensional environment where HSC/HPC communicate with a variety of supporting elements such as osteoblasts, endothelial cells, and extracellular matrix, which collectively sustain stem cell function [50]. Osteoblasts, in particular, have been shown to provide critical signals (e.g. via notch) that can either maintain stem cell quiescence or direct self-renewal, differentiation, or both. Until recently, osteoclasts (OCLs) were not recognized to have an important role in regulating stem cell behavior, including mobilization. However, Kollet et al. demonstrated that specific stimulation of OCLs with RANK ligand (RANKL) recruited immature progenitors to the circulation in a CXCR4- and MMP-9dependent manner [51]. RANKL did not induce mobilization in mice with defective OCL bone adhesion and resorption. Inhibition of OCLs with calcitonin reduced progenitor egress during homeostasis, and following G-CSF stimulation. RANKL-stimulated bone-resorbing OCLs also reduced the stem cell niche components SDF-1, stem cell factor (SCF), and osteopontin, which were associated with progenitor mobilization. These findings indicate a potential involvement of OCLs in selective progenitor recruitment as part of homeostasis and host defense. Osteoclasts may therefore be an important mediator of mobilization induced by G-CSF and other cytokines, creating a possible link between bone remodeling and regulation of hematopoiesis. Taken together, recent insights seem to implicate the CXCR4/SDF-1 pathway as a critical determinant of HSC/HPC mobilization. This interaction therefore has become a logical target for manipulations designed to enhance mobilization or, conversely, improve homing of transplanted cells back into the hematopoietic microenvironment. A more complete understanding of the interactions between HPCs and the bone marrow microenvironment is needed to identify additional targets for the stimulation of HPC mobilization.

4.  Novel Agents Capable of Inducing Hematopoietic Stem/Progenitor Cell Mobilization HPC mobilization has been induced clinically in humans or experimentally in mouse models using variety of approaches including: chemotherapeutic agents such as cyclophosphamide or paclitaxel; cytokines such as G-CSF, GM-CSF, IL-7, IL-3, IL-12, SCF, and Flt-3 ligand, and chemokines such as SDF-1, IL-8, or GROb. Below, we review several different strategies used recently in patients to enhance HPC mobilization.

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4.1. Stem Cell Factor Recombinant human SCF (rHuSCF) is a cytokine that stimulates pre-lineagecommitted HPC. Most clinical studies of (SCF) report the use of this agent with other cytokines. Limited reports of SCF by itself are available and this cytokine appears to result in a dose-dependent 6- to 10-fold mobilization of CFU-GM [52]. One phase 2 study demonstrated enhanced mobilization when SCF was used in conjunction with G-CSF to mobilize stem cells from lymphoma patient undergoing auto-SCT [53]. Recently, rHuSCF (20 mg/kg/day) when combined with G-CSF (10 mg/kg/day) was shown to enhance mobilization of HPC in heavily pretreated patients who have failed a previous attempt with G-CSF alone [54]. In this study, 29/48 (60%) achieved a cumulative total of >2.0 × 106 CD34+ cells/kg following remobilization with SCF and G-CSF after initial failure with G-CSF alone. Owing to occasional anaphylactoid reactions to SCF, including angioedema, urticaria, pruritus, and laryngospasm [55], the FDA decided not to approve the agent for use as an agent to enhance autologous stem cell mobilization in the United States. SCF is approved for use in Canada and New Zealand. 4.2.  Recombinant Human Growth Hormone Growth hormone is a pleiotropic cytokine targeting a variety of nonhematopoietic and hematopoietic cells by binding to its specific receptor [56]. In vitro, recombinant human growth hormone (rhGH) increases colony formation by HPC (CFU-GM and BFU-E) [57]. In vivo, a 7-day course of rhGH in mice significantly induces HPC mobilization into peripheral blood [58]. Carlo-Stella and colleagues investigated rhGH administration associated with chemotherapy plus G-CSF (5 mg/kg/day × 5 days) for enhancing stem cell mobilization in 16 patients with relapsed or refractory hematological malignancies who had failed a first mobilization attempt with chemotherapy plus G-CSF [59]. Patients were then re-mobilized with chemotherapy, G-CSF (5 mg/kg/day × 5 days) and rhGH (100 mg/kg/day, maximum daily dose of 6  mg). This combination resulted in efficient mobilization and collection of ³5 × 106 CD34+ cells/kg in 87% of these poor mobilizers with a median of three leukapheresis (i.e., from 1.1 × 106/kg up to 6 × 106/kg). The exact mechanism by which rhGH restores stem cell mobilization capacity in heavily pretreated patients with relapsed or refractory hematological malignancies is not clear, but is probably related to the expansion of HSC or HPC which become susceptible to be released upon a subsequent or concomitant stimulus, such as G-CSF. No further clinical trials have been reported, however, possibly because of regulatory issues or safety concerns in using this molecule in cancer patients. 4.3.  Pegfilgrastim Polyethylene glycosolated filgrastim (Pegfilgrastim;Neulasta®) differs from filgrastim only by the addition of a 20-kDa polyethylene glycol (PEG) molecule covalently bound to the N-terminal methionyl residue [60]. PEG modification of proteins has been demonstrated to sustain the duration of action by reducing renal clearance of the protein and decreasing rates of cellular uptake and proteolysis. In contrast to filgrastim, it appears that the kidney does not play a significant role in the elimination of pegfilgrastim, which appears to be

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primarily cleared by neutrophils and neutrophil precursors. Because pegfilgrastim directly stimulates the production of neutrophils, it effectively regulates its own clearance from the body. In clinical trials, it was demonstrated that a single dose of pegfilgrastim is as safe and effective as daily injections of filgrastim for the prevention and treatment of chemotherapy-induced neutropenia in a phase 2 study [61]. On the basis of this and other trials, it received approval by the FDA to decrease the incidence of infection in patients with nonmyeloid malignancies receiving myelosuppressive chemotherapy. It has been compared to its parent compound, filgrastim, to determine its efficacy in the stimulation and collection of progenitor cells for autologous transplantation. In mice, higher peak numbers of progenitor cells were mobilized into the peripheral blood following pegfilgrastim (300-fold over baseline) compared to filgrastim (100-fold over baseline), and occurred more rapidly, with a peak occurring over days 2–4, as compared with days 4 and 5 for filgrastim [60]. In a trial of chemotherapy-naive subjects with nonmall cell lung cancer, 13 patients were randomized to receive daily filgrastim 5 mg/kg or a single injection of pegfilgrastim 30, 100, or 300 mg/kg 2 weeks before chemotherapy, and again 24  h after administration of carboplatin and paclitaxel. In the prechemotherapy cycle, the median peak CD34+ count was similar in the filgrastim and pegfilgrastim 30 mg/kg cohorts, with higher median peaks observed in the 100 and 300 mg/kg pegfilgrastim cohorts. Adverse events attributed to study drug were mild-to-moderate bone pain, and were similar for those receiving pegfilgrastim or filgrastim [62]. A multicenter trial in patients with Hodgkin’s disease and non-Hodgkins lymphoma eligible for autologous transplantation was performed comparing the safety and efficacy of pegfilgrastim to filgrastim for CD34+ cell mobilization. In this randomized, double-blinded phase 2 trial, patients received either daily filgrastim at the standard mobilization dose of 10 mg/kg or one of two fixed doses (6 or 12 mg) of pegfilgrastim. Safety and capacity to mobilize CD34+ cells were the primary endpoints. The trial was halted when it was demonstrated that in this setting G-CSF was more effective than pegfilgrastim in mobilizing CD34+ cells (Amgen database). This may have been due to the clearance of pegfilgrastim, given that the patients had normal neutrophil counts at the start of mobilization. This hypothesis is further substantiated by the data that demonstrate pegfilgrastim can be used effectively to mobilize HPC following myelosuppressive chemotherapy [63–65]. Some groups continue to use this agent following chemotherapy since it can be given as a single injection following chemotherapy. 4.4.  Thrombopoietin Thrombopoietin (TPO) is a cytokine that regulates megakaryocytopoiesis. Some studies have showed that this also induces mobilization of CD34+ [66], and it synergizes with G-CSF to enhance stem cell mobilization [67]. Currently, no thrombopoietins have been approved by the FDA for stem cell mobilization and there are no data currently available to determine whether the thrombopoiesis-stimulating peptibodies currently being evaluated in patients with immune thrombocytopenic purpura will have any mobilizing activity.

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4.5.  Parathyroid Hormone (hrPTH) Calvi and colleagues showed that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow [68]. They showed that PTH/PTHrP receptors-stimulated osteoblastic cells that are increased in number, produce high levels of the Notch ligand, Jagged-1, and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in  vivo. Furthermore, liganddependent activation of PTH/PTHrP receptors with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by gammasecretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, they showed that osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function. Niche constituent cells or signaling pathways provide pharmacological targets with therapeutic potential for stem cell- based therapies. A clinical trial reported by Ballen and colleagues demonstrated a modest increase in CD34+ cell mobilization when combined with G-CSF in a phase 1 trial but no larger studies of this combination have been reported [69]. The major drawback of the 10–14-day time period for maximal effect with PTH remains, but further study to potentially exploit this pathway seems warranted [70]. 4.6.  CXCR4 Peptide CTCE-0021 is a novel cyclized CXCR4 agonist peptide (SDF-1a analog) developed to stabilize the SDF-1 a-helix to increase their bioactivity, and terminating the C-terminus as an amide to reduce its immunogenicity [29, 71]. This compound retains comparable CXCR4 receptor agonist activity. In mice, a single bolus administration of CTCE-0021 demonstrated a rapid dose-dependent mobilization of HPC between 5 min and 4 h post-dosing, with an increase in WBC resulting from an increase in granulocytes within 5 min post-dosing that persisted for approximately 24  h. The mechanisms involved in this CXCR4 agonist peptide mobilization remains unknown, but Pelus et al. suggested that CTCE-0021 mobilization is associated with downregulation of CXCR4 on HPC, and alteration in the plasma to marrow SDF-1 gradient [29, 37]. CTCE0021 is an efficient and rapid mobilizer of PMN and HPC when used alone and shows synergistic activity when used in combination with G-CSF. No clinical trials have been reported. 4.7.  GROb GRO is a member of the CXC chemokine family, which includes the related ligands GRO, GRO, ENA78, NAP-2, GCP-2, IP10, and interleukin-8 (IL-8), and it has biological activities related to specific binding to the CXCR2 receptor [29, 71]. SB-251353 is a recombinant N-terminal 4-amino acid truncated form of the human chemokine GRO specifically binding only to CXCR2 and with greater potency than full-length GRO [72]. The human CXCR2 selective ligand SB-251353 induces rapid mobilization of hematopoietic stem and progenitor cells in mice and monkeys and synergizes with G-CSF [28].

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Initially, chemokine administration is associated with a leukopenia within 5 min of injection followed by a period of neutrophilia 30–45 min later. The combination of SB-251353 with G-CSF resulted in augmented stem cell mobilization compared with the use of G-CSF alone. The mechanism of action of SB-251353-induced stem and progenitor cell mobilization appears similar to IL-8, which involves upregulation of MMP-9 activity [29]. Concerns that GRO-b may also activate neutrophils have dampened enthusiasm for its clinical development in this setting. 4.8.  AMD3100 AMD3100 is a bicyclam derivative that reversibly inhibits the binding of SDF-1 to its receptor CXCR4, promoting mobilization of CD34+ cells to the peripheral circulation [73]. Preclinical work in murine, canine, and nonhuman primate systems have suggested that AMD3100 alone can rapidly mobilize hematopoietic cells possessing both short- and long-term term repopulating capacity [74–76]. Broxmeyer and colleagues compared the SCID-repopulating capacity (SRC) of human hematopoietic cells mobilized by G-CSF to AMD3100-mobilized cells and found a greater frequency of SRC among the population of cells mobilized by AMD3100 [74]. Later, Burroughs used a well-described canine allogeneic transplant model to demonstrate the long-term repopulating capacity of cells contained within apheresis products collected from donors treated just 6  h previously with a single injection of AMD3100 [75]. The cells were able to maintain trilineage hematopoiesis and promote a full-donor chimeric state in three lethally irradiated recipients for up to 32 months. Further, Larochelle and colleagues collected CD34+ cells from rhesus macaques mobilized following either 5 days of G-CSF or a single dose of AMD3100 [76]. The CD34+ cells were retrovirally marked with the neomycin resistance gene (NeoR) and subsequently transplanted back into autologous recipients following myeloablative conditioning. AMD3100-mobilized cells engrafted gene marked myeloid and lymphoid cells up to 32 months following transplantation. The AMD3100mobilized CD34+ population contained a higher frequency of cells in the G1 phase of the cell cycle, with greater expression of CXCR4 and VLA-4 compared to G-CSF mobilized cells. Two recent studies revealed that G-CSF MPB CD34+ cells have higher levels of the pro-apoptotic genes caspase 3, 4 and 8 and reduction in inhibitors of apoptosis, such as anti-proteinase-2 compared to BM CD34+ cells [77, 78]. These data support the recent studies of Abkowitz in parabiotic mice which suggest that release of HSC into the circulation may also serve as an apoptotic pathway at steady state or following stress signals such as G-CSF stimulation [79]. DNA array technology will be useful to compare the gene expression profile between CD34+ cells mobilized by G-CSF with AMD3100. Together, these preclinical studies suggest fundamental differences in the characteristics of HSC/HPC mobilized by AMD3100 versus G-CSF and set the stage for initial clinical trials to evaluate the effects of transplanting cells mobilized by AMD3100 into humans. Recently, the repopulating capacity of CD34+ cells mobilized with AMD3100 was more fully characterized. Using leukapheresis products collected from 7 sibling donors treated on a trial of AMD3100 mobilization, Hess et al. compared the NOD/SCID-repopulating activity of the total mononuclear cell (MNC) fraction and purified CD34+ cells mobilized from each donor by

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AMD3100 or G-CSF [80]. Comparison of paired samples from each patient eliminated inter-patient variability in the analysis. Bone marrow repopulation was found to be threefold greater with AMD3100-mobilized MNCs than with G-CSF-mobilized MNCs. Purified AMD3100-mobilized CD34+ cells also possessed strong repopulating capacity, which was still superior to that of G-CSF-mobilized CD34+ cells in most patients. These results demonstrate potentially important qualitative differences in the repopulating capacity of grafts mobilized by the two agents. Initial clinical trials of AMD3100 in healthy volunteers demonstrated a more than tenfold increase in PBSCs beginning at1  h and peaking at 9  h after subcutaneous injection of AMD3100 [81]. The addition of AMD3100 to G-CSF results in even greater increases in circulating CD34+ cells [82]. AMD3100 can mobilize PBSCs in patients who have received prior chemotherapy as well. In a phase 1 study, patients with multiple myeloma or nonHodgkin lymphoma had a sevenfold increase in circulating CD34+ cells 6 h after a single dose of AMD3100 240 mg/kg [83]. In autologous stem cell collection trials, AMD3100 160–240 mg/kg has been added to G-CSF on day 4, 6–12 h prior to pheresis. Flomenberg et al. reported use of this combination in 25 multiple myeloma and non-Hodgkin lymphoma patients each of whom underwent two mobilizations, one using G-CSF alone and the other with G-CSF + AMD3100 [84]. Given as either the first or second mobilization regimen, G-CSF + AMD3100 mobilized more CD34+ cells per leukapheresis. In addition, patients underwent fewer leukaphereses, and more patients attained the target collection of 5 × 106 CD34+ cells/kg with the combination of G-CSF and AMD3100. Eighteen of 19 patients undergoing transplant with the G-CSF/AMD3100-mobilized product had early, stable engraftment. Mobilization with G-CSF + AMD3100 is also efficacious in patients with Hodgkin disease (HD). Cashen et al. reported on ten HD patients mobilized with AMD3100 and G-CSF [85]. All patients collected the minimum 2 × 106 CD34+ cells/kg, and 60% of patients collected more than 5 × 106 CD34+ cells/kg, a significantly higher percentage than a historic control group mobilized with G-CSF alone. All eight patients who had been transplanted with AMD3100 + G-CSF mobilized grafts had stable engraftment. This proof-of-principle study paved the way for phase 3 trials. Two randomized phase 3 clinical trials with AMD3100 plus G-CSF in MM and NHL patients have recently been successfully completed. An initial analysis of the data indicates that the prospectively defined clinical endpoints were exceeded [86, 87]. A Compassionate Use Program (CUP) has allowed patients, who have previously failed mobilization with regimens such as cytokine or chemotherapy treatment, to be given access to AMD3100. An analysis of 115 patients with either nonHodgkin’s lymphoma, Hodgkin’s disease or multiple myeloma, who had been unable to collect enough HSC for transplant and were eligible for CUP, showed an overall >66% success of collecting ³2 × 106 CD34+ cells/kg with G-CSF plus AMD3100 [88]. Similarly, patients who failed to collect enough cells for transplant in the phase 3 non-Hodgkin’s lymphoma trial were eligible for rescue by AMD3100 plus G-CSF. Thirty-three of 52 patients who failed on the G-CSF plus placebo arm successfully mobilized with G-CSF plus AMD3100 [89]. These results support the initial observation in the phase 2 trial that AMD3100 treatment can enhance mobilization of HSC in the poor mobilizer patient population. Table 22-1 lists molecules implicated in stem/progenitor cell mobilization.

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Table 22-1.  Molecules implicated in hematopoietic stem/progenitor cell mobilization. Cytokines/chemokines

Adhesion molecules

Receptors

Proteases

Agents used/tested clinically

G-CSF

VLA-4/VCAM-1

cKit

NE

G-CSF

CXCR4

CG

GM-CSF

G-CSF

MMP-9

SCF

GM-CSF

CD26

PTH

GM-CSF CD44 SDF-1/CXCL12 IL-8

Il-3

GH

SCF

Flt-3

Pegfilgrastim

TPO

FL

FL

TPO

Gro-b

IL-3/GM-CSF (pixy321)

CTCE-0021 IL-3 agonist (daniplestim) Il-3/G-CSF (leridistim) G-CSF granulocyte-colony stimulating factor, GM-CSF granulocyte-macrophage colony stimulating factor, SDF-1 stromal derived factor-1, IL-8 interluekin-8, SCF stem cell factor, TPO thrombopoietin, FL fms-like tyrosine kinase ligand, VLA-4 very late antigen 4, VCAM-1 vascular cell adhesion molecule-1, Flt-3 fms-like tyrosine kinase 3 receptor, NE neutrophil elastase, CG cathepsin-G, MMP-9 matrix metalloproteinase-9, CD26 dipeptidylpeptidase IV, PTH parathyroid hormone, GH growth hormone, IL-3 interleukin-3

5.  Choosing a Regimen to Mobilize Autologous Stem/Progenitor Cells There are several factors which may determine the success of HPC ­mobilization, which include extent of prior cytotoxic chemotherapy, especially ­treatments with certain drugs such as alkylating agents (melphalan, carmustine) or fludarabine, radiotherapy, advanced age, and certain diseases such as Hodgkin’s disease, non Hodgkin’s lymphoma, and myelodysplasia [23, 90]. The mobilization capacity of patients with hematological malignancies is, in general, lower than in patients with solid tumors such as breast or testicular cancer [90]. Some authors reported higher probabilities of mobilization failure in woman than in men [91], but this could be related more to differences in ideal body weight between men and woman. The differential expression of diverse adhesion molecules and their cognate receptors, probably impact the characteristics of a specific mobilization. Consistent with this hypothesis, “good mobilizers” showed significantly lower CXCR4, SDF-1, and VLA-4 expression than “poor mobilizers” [92, 93]. SDF-1 gene polymorphism has been proposed as a conditional factor for CD34+ cell mobilization [94]. Autologous HPC/HSC may be mobilized by the administration of chemotherapy followed by hematopoietic growth factors (HGF) or by HGF alone, either as single agents or in combination. While it is generally believed that the combination of chemotherapy followed by HGF results in greater yields of HPC, not all data are in agreement [20–23]. G-CSF is the most commonly used HGF for HPC mobilization and has been demonstrated to be superior to GM-CSF when used as a single agent to mobilize HPC [95]. A number of variables may impact the decision to use a chemotherapy-based or a hematopoietic

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growth factor only mobilization regimen and these include underlying disease and status of disease prior to transplantation, extent of prior chemotherapy and/ or radiation, anticipated morbidity to the patient, target CD34 dose intended for transplantation, intent to manipulate the HPC product, cost and resource utilization, and transplant center experience. Mobilization of HPC by G-CSF alone may be preferable to other techniques due to the ease of administration, decreased cost and morbidity to the patient, and the greater ability to predict the kinetics of mobilization. If G-CSF alone could consistently affect the mobilization of the required number of HPC to be used in patients treated for malignant diseases, it would likely be used preferentially in most circumstances because of its predictability and safety, but concerns remain regarding high rates of failure when used alone to mobilize CD34+ cells. Although some controversy exists regarding optimal number of CD34+ cells required to fully reconstitute hematopoiesis following autologous transplantation, the general consensus is that reinfusion of greater than 5 × 106 CD34+ cells/kg recipient weight will promote prompt trilineage reconstitution [23, 96–98]. Lesser numbers (2.5 to 4.9 × 106 CD34+ cells/kg) infused results in prompt neutrophil recovery, but platelet recovery and hospital stays may be prolonged. Below 2.0–2.5 × 106 CD34+ cells/kg re-infused, meaningful delays in platelet recovery may be observed. A recent study suggests that expression of aldehyde dehydrogenase (ALDH) in mobilized cells may serve as a functional marker for engrafting cells (both CD34+ and CD34 cells can be ALDH+) and correlates with engraftment kinetics better than CD34 [99]. Since ALDH is expressed in cells enriched with engraftment capacity, it may serve as a better marker than CD34 to designate the functional capacity of mobilized cells but this will need to be confirmed by other groups [100]. At any rate, current trends favor strategies designed to mobilize the greatest number of HPC in the fewest number of collections. Unfortunately, recent data suggest that a significant proportion of patients who have received prior chemotherapy fail to mobilize >5.0 × 106 CD34+ cells/kg with G-CSF alone [23, 87, 101]. In one large trial involving breast cancer patients, only one-third of patients given G-CSF as a single mobilizing agent achieved a 5.0 × 106 CD34+ cell dose per kilogram level [101]. In the recently completed phase 3 trial comparing G-CSF plus placebo to G-CSF plus AMD3100 (Optimize 1), a startling 53% of patients with NHL on the placebo-controlled arm failed to achieve a minimum CD34+ cell dose of 2.0 × 106/kg after four collections [87]. Thus, in previously treated patients with NHL (and probably HD) it is likely that G-CSF will need to be combined with other methods (additional HGF, chemotherapy, novel mobilizing agents such as AMD3100) in order to consistently induce mobilization of large number of HPC for transplantation. Ultimately, the development of predictive models to estimate the likelihood of an efficient HPC mobilization for any given mobilization strategy seems the most rational way to determine the likelihood of success as well as the need for novel approaches. Finally, some have argued that the use of chemotherapybased mobilization strategies are required since this additional chemotherapy will result in a lower risk of relapse during or after HPC mobilization. The randomized trials performed have not substantiated this claim and a recent French study even suggested that in multiple myeloma patients cyclophosphamide-based mobilization may skew the collection toward increased number of regulatory T-cells

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which emerge upon recovery from chemotherapy, which paradoxically when reinfused could raise the risk of disease recurrence [102]. This observation in a small number of patients requires further study by others. Studies have been conflicting on whether there are differences, particularly clinically relevant ones, in the mobilization of contaminating tumors cells based on the type of mobilization strategy employed but in general this if difficult to prove one way or the other. Many centers use chemotherapy-based mobilization simply because they believe that it will result in a higher yield of CD34+ cells mobilized compared to G-CSF alone, and this may indeed be the case. So, despite the potential drawbacks chemotherapy-based mobilization has its merits. The addition of AMD3100 to the list of agents capable of inducing mobilization may change the landscape toward a cytokine-chemokine antagonist approach but there are still concerns that the need to administer AMD3100 8–10 h prior to leukapheresis, to achieve optimal mobilization, will prove impractical and clearly further studies aimed at evaluating alternative schedules will be necessary before this agent is widely adopted. One practical approach may be to base the use of AMD3100 on a peripheral blood CD34+ cell count obtained on the fourth or fifth day of mobilization with G-CSF alone and to add it to G-CSF only in those individuals who do not appear to be mobilizing well.

6.  Mobilization of Stem Cells from Normal Donors In the allogeneic transplantation setting, the need for novel strategies to collect HPC from normal donors is less clear than in patients with hematological malignancies. In the vast majority of donors, G-CSF safely and effectively mobilizes HPC for transplantation. Seven randomized trials comparing HLAidentical BM to MPB have been published and uniformly confirm that MPB results in more rapid hematopoietic engraftment compared to BM [12, 13, 15–19, 103]. Increasingly, G-CSF is used for mobilization of HPC in unrelated donor transplantation and will be compared to BM in an upcoming randomized clinical trial supported by the NIH-sponsored Blood and Marrow Transplant Clinical Trials Network. Nevertheless, meta-analyses suggest a higher risk of either acute or chronic GVHD following transplantation of MPB [12, 16]. Further, a recent retrospective study in recipients of unrelated donor cells did not demonstrate an advantage to mobilized blood over marrow due to more GVHD [104]. Controversy exists regarding the optimal CD34+ cell dose to be transplanted. The general consensus is that cell doses greater than 4 × 106 CD34+ cells are necessary for prompt and durable engraftment [105, 106]. However, a small proportion of donors will not mobilize a graft containing at least 4.0 × 106 CD34+ cells/kg following G-CSF alone. In our institution, about 10–15% of donors given G-CSF at 10 mg/kg/daily mobilize a graft containing <4.0 × 106CD34+ cells/kg (Steven Devine, unpublished observations). At the other end of the spectrum, recent data suggest that there may be a limit to the number of CD34+ cells that can be safely transplanted and that very high doses of CD34+ cells may be deleterious in terms of a greater risk of both acute and chronic GVHD [107–109]. This has been demonstrated in a number of recent studies including one showing a higher risk of chronic GVHD and worsening survival in certain subsets of patients receiving very high CD34+ cell doses [107].

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Given the data suggesting an influence of graft composition on clinical outcomes following HLA-identical transplantation, there has been interest in the role of an alternative mobilizing agent, GM-CSF, either as a single agent or in combination with G-CSF. The group at Washington University recently reviewed their experience with grafts mobilized from 230 HLA-matched sibling donors using G-CSF alone at 10 mg/kg/day, G-CSF plus GM-CSF at mg/kg/day, or GM-CSF alone at 10 or 15 mg/kg/day [110]. Grafts mobilized with GM-CSF alone contained significantly fewer CD34+ cells, but sufficient numbers to promote engraftment. More leukapheresis procedures were required following GM-CSF alone. Grafts obtained following G plus GM or GM-CSF alone contained significantly fewer T and NK cells compared to G-CSF alone. Following transplantation, all recipients of PBSC mobilized following GM-CSF alone engrafted with kinetics similar to recipients of PBSC mobilized following G-CSF alone, although neutrophil recovery was about 1 day longer. Interestingly, compared to G-CSF alone, the GM-CSF group had a statistically significantly lower risk of grades 2–4 acute GVHD. No patients in the GM-CSF alone group experienced grade 3 or 4 GVHD. In a multivariable analysis which included patient/donor age, sex, mismatching, conditioning regimen, CD34+ dose, and CD3+ dose, only receipt of PBSC mobilized with GM-CSF alone correlated with a lower risk of grades 2–4 acute GVHD. Whether the differences in the risk of GVHD observed were due to chance or to real qualitative changes in the T cells, dendritic cells, or CD34 cells mobilized by GM-CSF alone or due to the lower doses of CD34+ and CD3+ cells contained within these allografts is unknown. A recent publication suggested that GM-CSF stimulates cellular signaling that might favor the emergence of regulatory T-cells and this could be one explanation for the lower rates of GVHD observed [111]. Further studies to determining the relevance of this observation seem warranted. G-CSF is commonly used at a dose of 10 mg/kg/day subcutaneously to mobilize PBSCs from healthy donors. Mobilization of cells expressing CD34 antigen peaks in the peripheral blood between days 4 and 5 of G-CSF dosing [112, 113]. Thus, leukapheresis is typically commenced on day 4 or 5 of G-CSF treatment and is repeated until a target number of CD34+ cells are collected (usually ³4 × 106/kg). Compared to the graft composition of bone marrow harvests, G-CSF-mobilized products contain three- to fourfold higher CD34+ doses and roughly a 10- to 20-fold increase in CD3+ T-cells [106]. While many factors including the patient’s age, diagnosis, and amount of prior therapy have been shown to predict the likelihood of collecting a sufficient number of autologous repopulating cells, factors affecting the capacity of normal donors to mobilize CD34+ cells following G-CSF are less well established. Reports have indicated in some cases wide inter-individual variation among normal subjects given G-CSF for PBSC mobilization [114–118]. In an IBMTR/EBMT analysis, 60% of donors required more than one leukapheresis procedure to collect the target number of CD34+ cells, and 15% required three or more [119]. de la Rubia and colleagues evaluated 261 donors and identified donor age >38 years and use of a single rather than multiple daily G-CSF doses as factors associated with a low CD34 yield [120] Suzuzya et  al. [118] and Lysak et  al. [117] also found increased age to be a negative predictor for successful mobilization. Another study found

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that donors with higher baseline peripheral blood CD34+ cell levels prior to G-CSF mobilized better than those with lower levels [121]. Thus, donor age, steady-state CD34 level, and both the total dose and schedule of G-CSF may impact CD34+ cell mobilization. In addition to causing CD34+ cell mobilization by its indirect effects on CXCR4/SDF-1, G-CSF has pleiotropic effects on other important allograft constituents including T-, B-, NK-, and dendritic cells. G-CSF administration has been associated with a polarization of T-cells to a Th2 phenotype [122–124]. G-CSF effects circulating monocytes, leading to an increase in IL-10 secretion. Also, G-CSF administration may induce a plasmacytoid differentiation effect on donor dendritic cells [125–128]. Together, these immunomodulatory effects of G-CSF have been cited to explain the observation that rates of acute GVHD are not substantially different using mobilized cells compared to bone marrow despite the transplantation of about tenfold greater numbers of T-cells. In an effort to further understand and possibly exploit these immunomodulatory effects, G-CSF analogs have recently been studied to determine their influence on stem cell mobilization, GVHD, and graft versus leukemia (GVL). Pegylated filgrastim (Neulasta, Amgen) has a much longer half-life compared to native filgrastim and a single dose results in the mobilization of sufficient number of CD34+ cells to promote engraftment in allogeneic recipients following myeloablative conditioning [129]. One study in HLA-identical sibling donors suggested that a 12 mg dose may be superior to 6 mg in terms of CD34+ cell dose mobilized [129]. The allografts mobilized by pegfilgrastim appeared to result in similar kinetics of engraftment as well as comparable rates of GVHD compared to native filgrastim. The kinetics of mobilization following a single dose of pegylated filgrastim were roughly the same as that observed following daily G-CSF administration with peak CD34+ cell mobilization occurring by day 5–6. Recently, a chimeric molecule containing ligands for both G-CSF and Flt-3 has been shown to limit GVHD while retaining the capacity to induce GVL in murine models, possibly through the expansion of regulatory NK/T type cells [130, 131]. Further work is needed to validate these effects. It is unclear whether chimeric molecules will be further developed for clinical use, particularly in volunteer donors, due to concerns regarding enhanced toxicity associated with stimulating multiple receptors.

7.  Safety and Toxicity of G-CSF in Normal Donors G-CSF-based PBSC mobilization is generally well tolerated, and essentially all donors complete the mobilization and collection procedures. However, retrospective and prospective studies have identified transient, but not insignificant, morbidities experienced by G-CSF-mobilized donors. The most common symptoms are bone pain, headache, fatigue, and nausea, and the incidence of pain and anxiety are similar to that observed with bone marrow donation [119, 121, 132–135]. In a retrospective analysis of >1,300 donors registered with the International Bone Marrow Transplant Registry (IBMTR) or European Blood and Marrow Transplant Group (EBMT), the rate of serious complications from G-CSF mobilization and PBSC collection was 1.1%, as compared to 0.5% following bone marrow collection [132, 136].

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Many of these complications were associated with central venous catheters, which were inserted in 20% of donors. More serious side effects, while rare, nonetheless can occur and represent a major issue when they affect normal individuals. Stroncek et  al. reported that 19 of 20 adult allogeneic blood donors given G-CSF 10 mg/kg/day had transient increases in spleen length by an average of 17% [137]. A prospective trial published by Platzbecker et  al. in 91 healthy allogeneic donors treated with G-CSF 7.5 mg/kg/day for 5 days evaluated changes in spleen size using serial ultrasound examinations [138]. Donors exhibited a mean 110% transient increase in spleen length; 20% had a 1.9 cm increase in length and 0.9  cm increase in spleen thickness. A similar study design communicated by Stroncek et  al. in 18 healthy subjects given G-CSF 10 mg/kg/day for 5 days revealed a transient mean enlargement of spleen length from 10.7 to 12.1 cm that returned to baseline 10 days after completion of apheresis [139]. Falzetti et  al. reported the development of splenomegaly and spontaneous splenic rupture in a 33-year-old male allogeneic blood stem cell donor given G-CSF 16 mg/kg/day for 6 days [140]. He recovered fully after an emergency splenectomy that revealed a 445  g spleen and a capsular tear with massive extra-medullary myelopoiesis. Becker et al. reported a 22-year-old man who donated bone marrow for a first cousin and 4 months later was mobilized with G-CSF 10 mg/kg/day for 6 days as a planned blood progenitor cell collection for relapse [141]. Four days after completion of collection, he underwent an emergency splenectomy for spontaneous splenic rupture. G-CSF therapy was not unequivocally the etiology as the donor has serologic findings consistent with convalescence after Epstein-Barr infection. Kröger et al. retrospectively reviewed data obtained from 90 healthy allogeneic donors given G-CSF 5 or 8 mg/kg twice daily [142]. One young subject had a nonfatal traumatic splenic rupture after 5 days of the G-CSF 5 mg/kg twice daily dosing, but this serious adverse event fully resolved without surgical intervention. Dincer and coworkers reported a 43-year-old man who received the same treatment experienced a spontaneous splenic rupture on the fifth day of therapy that resolved without surgical intervention [143]. Finally, Balaguer et al. reported a case of a 51-year-old man who developed spontaneous splenic rupture after G-CSF treatment for stem cell mobilization for his HLA-identical sibling [144]; he fully recovered after undergoing an emergency splenectomy. The Spanish National Donor Registry reported this case to be the only such occurrence in 1,240 registered PBSC donors [145]. Another potentially serious toxicity may be related to the procoagulant effects of G-CSF and include the risk of precipitating a myocardial infarction or causing cerebral ischemia in high-risk individuals. Both adverse events have been reported and therefore all donors treated with G-CSF should be carefully screened for any history of coronary artery or cerebrovacular disease [146– 149]. Owing to its potential to precipitate acute sickle crisis, G-CSF is contraindicated in patients with sickle cell disease; further, in donors with sickle cell trait, G-CSF has been associated with precipitation of sickle crisis [150–152]. Patients with underlying autoimmune disease have been treated with G-CSF alone to mobilize cells prior to planned autologous transplantation. In this setting, its use has been associated with flares of disease in patients with rheumatoid arthritis, systemic lupus erythematosis, and multiple sclerosis [153–156]. Therefore, its use in donors with these disorders also is not recommended.

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Even short-term stimulation by G-CSF results initiation of the cell cycle by at least a fraction of primitive hematopoietic cells [157]. The effects of short-term G-CSF stimulation on genomic stability or chromatin remodeling are unclear [158–160]. A few studies raise concern that even short-term G-CSF treatment may affect genomic stability [158, 159]. The long-term ramifications of these changes, if any, are unknown and concerns remain largely speculative. That said, there has always been a theoretical concern that exogenous stimulation of hematopoiesis in a first-degree relative of a patient with a hematopoietic malignancy, particularly acute myeloid leukemia, may put the donor at risk for later development of leukemia. In fact, acute leukemia has been noted in G-CSF-stimulated siblings, including two cases of AML occurring 5–6 years after G-CSF administration to mobilize cells for transplant into recipients with AML [161, 162]. Given that the cases of leukemia may have just as easily occurred by chance and there is a higher risk of leukemia in first-degree relatives of patients with acute leukemia, the role that G-CSF short-term administration may have played in these cases is speculative [132, 163–165]. Clearly, such occurrences raise concern and justify the call for continued long-term follow-up of G-CSF stimulated donors. The use of G-CSF in pediatric siblings donating for individuals with malignant or non-malignant disorders also raises concern as to the long-term impact of this maneuver on the health in a young individual [166]. Further, this concern raises several ethical issues related to obtaining both parental consent and assent of the adolescent donor. In summary, collection of G-CSF-mobilized blood is associated with morbidity comparable to that experienced after bone marrow donation, and many donors must undergo more than one leukapheresis procedure. Donors may benefit from new mobilization strategies that minimize exposure to G-CSF injections and reduce the number of leukapheresis sessions. In addition, recipient outcomes could be improved with grafts that provide faster count recovery or that reduce the incidence of GVHD.

8.  Mobilizing Donor Cells without Using Cytokines Although G-CSF is an effective agent for the mobilization of stem cells from normal donors, such altruistic individuals could benefit from new mobilizing strategies which are more effective and/or less toxic. The agents currently in use to mobilize stem cells in donors have both unique and overlapping toxicities. Each has its own potential advantages and disadvantage. Notably, each product results in the mobilization of an allograft with unique cellular compositions that may alter the likelihood of graft failure, GVHD, immune reconstitution, and possibly relapse. The search for new mobilizing agents currently is driven by pre-clinical research which is elucidating the mechanisms of stem cell localization within the bone marrow and mobilization in response to cytokine signals. As our understanding of the interactions between stem cells and the bone marrow microenvironment improves, new mobilizing agents can be designed rationally. Given the promising results in autologous mobilization, AMD3100 is now being investigated for stem cell mobilization and transplantation from healthy donors. Devine et  al. have reported the preliminary results of a pilot study evaluating the safety and efficacy of AMD3100 mobilization and transplantation

Chapter 22  Mobilization of Hematopoietic Cells Prior to Autologous  403

Table 22-2.  Agents used to mobilize stem/progenitor cells in normal donors. Agent

Results

Unique aspects

G-CSF

Effective as single agent

Bone pain, requires 5 days for maximal effect

GM-CSF

Less effective than G-CSF and possibly more toxic

Causes fever, edema, bone pain; may be associated with less acute GVHD

Pegfilgrastim

Similar ability to mobilize compared to G-CSF

Same toxicity as G-CSF; requires only one dose; same kinetics as G-CSF

AMD3100

Mobilized functional cells capable of long-term engraftment

Mobilized functional cells in only 4 h; lower CD34+ cell doses mobilized compared to G-CSF; possibly less toxic than G-CSF

G-CSF granulocyte-colony stimulating factor, GM-CSF granulocyte-macrophage colony stimulating factor

in HLA-matched sibling donors [167]. Allografts from HLA-matched sibling donors were mobilized and collected without G-CSF using AMD3100. Donors (N = 25) were treated with AMD3100 at a dose of 240 mg/kg by subcutaneous injection and leukapheresis was then initiated just 4 h later. Two-thirds of the donors collected an allograft with a CD34+ cell dose, sufficient for transplantation after just one dose of AMD3100. No donor experienced more than grade one toxicity. The allografts collected after AMD3100 contained higher CD4+ T-cell doses compared to G-CSF mobilized grafts. Following a myeloablative regimen, twenty patients with hematological malignancies received allografts collected after AMD3100 alone. All patients engrafted neutrophils (median day +10) and platelets (median day +12) promptly. Acute GVHD grades 2–4 occurred in 35% of patients. Therefore, despite the infusion of higher T-cell doses, there was no appreciable increase in GVHD in comparison to G-CSFmobilized grafts. One patient died due to complications related to acute GVHD. No unexpected adverse events were observed in any of the recipients. All 14 patients surviving in remission had robust trilineage hematopoiesis and were transfusion-free with a median follow-up of 277 days (range 139–964 days). This small study suggests that direct antagonism of CXCR4 by AMD3100 may provide a more rapid and possibly less-toxic and cumbersome alternative to traditional G-CSF-based mobilization in normal donors and appears worthy of further pursuit in larger multi-center trials. Table 22-2 lists unique aspects of agents used to mobilize stem/progenitor cells in normal donors.

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S.M. Devine 111. Kared H, Leforban B, Montandon R et al (2008) Role of GM-CSF in tolerance induction by mobilized hematopoietic progenitors. Blood 112:2575–2578 112. Fischmeister G, Kurz M, Haas OA et  al (1999) G-CSF versus GM-CSF for stimulation of peripheral blood progenitor cells (PBPC) and leukocytes in healthy volunteers: Comparison of efficacy and tolerability. Ann Hematol 78:117–123 113. Korbling M, Huh Y, Durett A et al (1995) Allogeneic blood stem cell transplantation: Peripheralization and yield of donor-derived primitive hematopoietic progenitor cells (CD34+ Thy- 1dim) and lymphoid subsets, and possible predictors of engraftment and graft-versus-host disease. Blood 86:2842–2848 114. Anderlini P, Przepiorka D, Seong C et al (1997) Factors affecting mobilization of CD34+ cells in normal donors treated with filgrastim. Transfusion 37:507–512 115. Grigg A, Roberts A, Raunow H et al (1995) Optimizing dose and scheduling of filgrastim (granulocyte colony- stimulating factor) for mobilization and collection of peripheral blood progenitor cells in normal volunteers [see comments]. Blood 86:4437–4445 116. Holm M (1998) Not all healthy donors mobilize hematopoietic progenitor cells sufficiently after G-CSF administration to allow for subsequent CD34 purification of the leukapheresis product. J Hematother 7:111–113 117. Lysak D, Koza V, Jindra P (2005) Factors affecting PBSC mobilization and collection in healthy donors. Transfus Apher Sci 33:275–283 118. Suzuya H, Watanabe T, Nakagawa R et al (2005) Factors associated with granulocyte colony-stimulating factor-induced peripheral blood stem cell yield in healthy donors. Vox Sang 89:229–235 119. Anderlini P, Rizzo JD, Nugent ML, Schmitz N, Champlin RE, Horowitz MM (2001) Peripheral blood stem cell donation: An analysis from the international bone marrow transplant registry (IBMTR) and european group for blood and marrow transplant (EBMT) databases. Bone Marrow Transplant 27:689–692 120. de la Rubia J, Arbona C, de Arriba F et al (2002) Analysis of factors associated with low peripheral blood progenitor cell collection in normal donors. Transfusion 42:4–9 121. Anderlini P, Przepiorka D, Champlin R, Korbling M (1996) Biologic and clinical effects of granulocyte colony-stimulating factor in normal individuals. Blood 88:2819–2825 122. Arpinati M, Green CL, Heimfeld S, Heuser JE, Anasetti C (2000) Granulocytecolony stimulating factor mobilizes T helper 2-inducing dendritic cells. Blood 95:2484–2490 123. Liu Y-J, Blom B (2000) Introduction: TH2-inducing DC2 for immunotherapy. Blood 95:2482–2483 124. Pan L, Delmonte J Jr, Jalonen CK, Ferrara JL (1995) Pretreatment of donor mice with granulocyte colony-stimulating factor polarizes donor T lymphocytes toward type-2 cytokine production and reduces severity of experimental graft-versus-host disease. Blood 86:4422–4429 125. Pulendran B, Banchereau J, Burkeholder S et  al (2000) Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo. J Immunol 165:566–572 126. Reddy V (2000) Granulocyte colony-stimulating factor mobilization alters dendritic cell cytokine production and initiates T helper 2 polarization prior to host alloantigen presentation. Blood 96:2635 127. Rondelli D, Raspadori D, Anasetti C et al (1998) Alloantigen presenting capacity, T cell alloreactivity and NK function of G-CSF-mobilized peripheral blood cells. Bone Marrow Transplant 22:631–637 128. Vasconcelos ZFM, dos Santos BM, Farache J et al (2006) G-CSF-treated granulocytes inhibit acute graft-versus-host disease. Blood 107:2192–2199 129. Hill GR, Morris ES, Fuery M et al (2006) Allogeneic stem cell transplantation with peripheral blood stem cells mobilized by pegylated G-CSF. Biol Blood Marrow Transplant 12:603–607

Chapter 22  Mobilization of Hematopoietic Cells Prior to Autologous  411 130. MacDonald KPA, Rowe V, Filippich C et  al (2003) Donor pretreatment with progenipoietin-1 is superior to granulocyte colony-stimulating factor in preventing graft-versus-host disease after allogeneic stem cell transplantation. Blood 101: 2033–2042 131. Morris ES, MacDonald KPA, Hill GR (2006) Stem cell mobilization with G-CSF analogs: A rational approach to separate GVHD and GVL? Blood 107:3430–3435 132. Anderlini P, Champlin RE (2008) Biologic and molecular effects of granulocyte colony-stimulating factor in healthy individuals: Recent findings and current challenges. Blood 111:1767–1772 133. Fortanier C, Kuentz M, Sutton L et al (2002) Healthy sibling donor anxiety and pain during bone marrow or peripheral blood stem cell harvesting for allogeneic transplantation: Results of a randomised study. Bone Marrow Transplant 29:145–149 134. Murata M, Harada M, Kato S et al (1999) Peripheral blood stem cell mobilization and apheresis: Analysis of adverse events in 94 normal donors. Bone Marrow Transplant 24:1065–1071 135. Rowley SD, Donaldson G, Lilleby K, Bensinger WI, Appelbaum FR (2001) Experiences of donors enrolled in a randomized study of allogeneic bone marrow or peripheral blood stem cell transplantation. Blood 97:2541–2548 10.1182/ blood.V97.9.2541 136. Anderlini P, Przepiorka D, Korbling M, Champlin R (1998) Blood stem cell procurement: Donor safety issues. Bone Marrow Transplant 21:S35–S39 137. Stroncek D, Shawker T, Follmann D, Leitman SF (2003) G-CSF-induced spleen size changes in peripheral blood progenitor cell donors. Transfusion 43:609–613 138. Platzbecker U, Prange-Krex G, Bornhauser M et al (2001) Spleen enlargement in healthy donors during G-CSF mobilization of PBPCs. Transfusion 41:184–189 139. Stroncek D, Dittmar K, Shawker T, Heatherman A, Leitman S (2004) Transient spleen enlargement in peripheral blood progenitor cell donors given G-CSF. J Transl Med 2:25 140. Falzetti F, Aversa F, Minelli O, Tabilio A (1999) Spontaneous rupture of spleen during peripheral blood stem-cell mobilisation in a healthy donor. The Lancet 353:555 141. Becker P, Wagle M, Matous S et al (1997) Spontaneous plenic rupture following administration of granulocyte colony-stimulating factor (G-CSF): Occurrence in an allogeneic donor of peripheral blood stem cells. Biol Blood and Marrow Transplant 3:45–49 142. Kröger N, Renges H, Sonnenberg S et  al (2002) Stem cell mobilisation with 16 mg/kg vs 10 mg/kg of G-CSF for allogeneic transplantation in healthy donors. Bone Marrow Transplant 29:727–730 143. Dincer AP, Gottschall J, Margolis DA (2004) Splenic rupture in a parental donor undergoing peripheral blood progenitor cell mobilization. J Pediatr Hematol Oncol 26:761–763 144. Balaguer H, Galmes A, Ventayol G, Bargay J, Besalduch J (2004) Splenic rupture after granulocyte-colony-stimulating factor mobilization in a peripheral blood progenitor cell donor. Transfusion 44:1260–1261 145. de la Rubia J, Martínez C, Solano C et al (1999) Administration of recombinant human granulocyte colony-stimulating factor to normal donors: Results of the Spanish national donor registry. Bone Marrow Transplant 24:723–728 146. Dagia NM, Gadhoum SZ, Knoblauch CA et al (2006) G-CSF induces E-selectin ligand expression on human myeloid cells. Nat Med 12:1185–1190 147. Fukumoto Y, Miyamoto T, Okamura T et  al (1997) Angina pectoris occurring during granulocyte colony-stimulating factor-combined preparatory regimen for autologous peripheral blood stem cell transplantation in a patient with acute myelogenous leukaemia. Br J Haematol 97:666–668 148. Hill JM, Syed MA, Arai AE et  al (2005) Outcomes and risks of granulocyte colony-stimulating factor in patients with coronary artery disease. J Am Coll Cardiol 46:1643–1648

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S.M. Devine 149. Lindemann A, Rumberger B (1993) Vascular complications in patients treated with granulocyte colony-stimulating factor (G-CSF). Eur J Cancer 29:2338–2339 150. Adler BK, Salzman DE, Carabasi MH, Vaughan WP, Reddy VVB, Prchal JT (2001) Fatal sickle cell crisis after granulocyte colony-stimulating factor administration. Blood 97:3313–3314 151. Horowitz MM, Confer DL (2005) Evaluation of hematopoietic stem cell donors. Hematology 2005:469–475 152. Kang EM, Areman EM, David-Ocampo V et al (2002) Mobilization, collection, and processing of peripheral blood stem cells in individuals with sickle cell trait. Blood 99:850–855 153. Burt RK, Fassas A, Snowden J et al (2001) Collection of hematopoietic stem cells from patients with autoimmune diseases. Bone Marrow Transplant 28:1–12 154. Gottenberg JE, Roux S, Desmoulins F, Clerc D, Mariette X (2001) Granulocyte colony-stimulating factor therapy resulting in a flare of systemic lupus erythematosus: Comment on the article by Yang and Hamilton. Arthritis Rheum 44:2458–2460 155. Nash RA, Bowen JD, McSweeney PA et al (2003) High-dose immunosuppressive therapy and autologous peripheral blood stem cell transplantation for severe multiple sclerosis. Blood 102:2364–2372 156. Stricker RB, Goldberg B (1996) G-CSF and exacerbation of rheumatoid arthritis. Am J Med 100:665–666 157. Mahmud N, Devine SM, Weller KP et al (2001) The relative quiescence of hematopoietic stem cells in nonhuman primates. Blood 97:3061–3068 158. Hernandez JM, Castilla C, Gutierrez NC et al (2005) Mobilisation with G-CSF in healthy donors promotes a high but temporal deregulation of genes. Leukemia 19:1088–1091 159. Nagler A, Korenstein-Ilan A, Amiel A, Avivi L (2004) Granulocyte colonystimulating factor generates epigenetic and genetic alterations in lymphocytes of normal volunteer donors of stem cells. Exp Hematol 32:122–130 160. Pamphilon D, Mackinnon S, Nacheva E et  al (2006) The use of granulocyte colony-stimulating factor in volunteer blood and marrow registry donors. Bone Marrow Transplant 38:699–700 161. Bennett CL, Evens AM, Andritsos LA et al (2006) Haematological malignancies developing in previously healthy individuals who received haematopoietic growth factors: Report from the research on adverse drug events and reports (RADAR) project. Br J Haematol 135:642–650 162. Makita K, Ohta K, Mugitani A et  al (2004) Acute myelogenous leukemia in a donor after granulocyte colony-stimulating factor-primed peripheral blood stem cell harvest. Bone Marrow Transplant 33:661–665 163. Hasenclever D, Sextro M (1996) Safety of AlloPBPCT donors: Biometrical considerations on monitoring long term risks. Bone Marrow Transplant 17(Suppl 2) :S28–S30 164. Rauscher GH, Sandler DP, Poole C et  al (2002) Family history of cancer and incidence of acute leukemia in adults. Am J Epidemiol 156:517–526 165. Shpilberg O, Modan M, Modan B, Chetrit A, Fuchs Z, Ramot B (1994) Familial aggregation of haematological neoplasms: A controlled study. Br J Haematol 87:75–80 166. Pulsipher MA, Nagler A, Iannone R, Nelson RM (2006) Weighing the risks of G-CSF administration, leukopheresis, and standard marrow harvest: Ethical and safety considerations for normal pediatric hematopoietic cell donors. Pediatr Blood Cancer 46:422–433 167. Devine SM, Vij R, Rettig M et al (2008) Rapid mobilization of functional donor hematopoietic cells without G-CSF using plerixafor, an antagonist of the CXCR4/ SDF-1 interaction. Blood 112:990–998

Chapter 23 Natural Killer-Cell Based Treatment in Hematopoetic Stem Cell Transplantation Martin Stern, Sandrine Meyer-Monard, Uwe Siegler, and Jakob R. Passweg

1. Background Natural killer cells (NK) reside in the bone marrow, spleen, and peripheral blood where they form approximately 10% of peripheral blood lymphocytes [1]. Unlike the B- and T-lymphocytes, NK cells do not express clonally rearranged receptors to detect antigens. Instead, activation is regulated by the integration of signaling from germline-encoded activating and inhibitory cell surface receptors [2]. These include inhibitory receptors for HLA class I antigens and activating receptors such as DNAM-1, NKG2D, and natural cytotoxicity receptors (NCRs) [3]. Inhibitory receptors for self HLA include Killer cell Immunoglobulin-like Receptors (KIR), the lectin-like receptor NKG2A, and LIR1/ILT-2 [4] (Table 23-1). Upon interaction with target cells expressing activating ligands, lack of involvement of inhibitory receptors results in predominance of activating signaling and target cell lysis. These systems form the basis of the “missing self” recognition and exemplify the mechanisms of the immune system to counteract the HLA downregulation induced by tumors and viral infection to escape the T-cell recognition. While initial data derived from clonally expanded NK cells had suggested that every NK cell expresses at least one inhibitory receptor for self MHC [5], more recent analyses in mice and humans have shown that subsets of NK cells do not express inhibitory receptors for self HLA [6–8]. The mechanism of tolerance in this subset is not completely understood. However, growing evidence exists for the role of KIR-HLA interactions in “licensing” of NK cells in a manner that only NK cells expressing inhibitory receptors for self HLA acquire full functional competence. The ligands for inhibitory KIRs are HLA class I antigens. The main inhibitory KIR/HLA pairs are KIR2DL1 recognizing HLA-C antigens with a lysine at position 80 (e.g. HLA-C 2, 4, 5, 6); KIR2DL2 and KIR2DL3 recognizing HLA-C antigens with asparagine at position 80 (e.g. HLA-C 1, 3, 7, 8), and KIR3DL1 recognizing HLA-B antigens with Bw4 specificity (e.g. HLA B5, 13, 17, 27). KIR3DL2 has been shown to recognize HLA-A3 and A11

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_23, © Springer Science + Business Media, LLC 2003, 2010

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Table 23-1.  Main natural killer cell surface receptors and their ligands. Inhibitory receptors

Ligands

Inhibitory KIR

HLA-A/B/C

KIR2DL1

HLA C group 2 (e.g. C2,4,5,6)

KIR2DL2/3

HLA C group 1 (e.g. C1,3,7,8)

KIR3DL1

HLA Bw4 (e.g. B5, 13, 17, 27)

KIR3DL2

HLA A3/A11

NKG2A

HLA-E

Activating receptors

Ligands

Natural cytotoxicity receptors (NKp30, NKp44, NKp46)

Unknown

Activating KIR

Unknown (HLA class I ?)

NKG2D

MIC-A/B, ULBPs

DNAM-1

PVR, Nectin-2

CD16

IgG

2B4

CD48

KIR killer cell immunoglobulin-like receptors, HLA human leukocyte antigen, NKG2A/D natural killer cell group 2A/D, MIC major histocompatibility complex class I chain-related, ULBP UL16 binding proteins, DNAM-1 DNAX accessory molecule 1, PVR polio virus receptor, IgG immunoglobulin G

expressed on target cells in  vitro depending on the peptide presented; its significance in  vivo remains unclear [9]. KIR2DL4 recognizes HLA-G, an atypical class I antigen expressed on decidual cells, and is implicated in maintaining the tolerance against the fetal-derived placental tissue [10]. KIR3DL3 and KIR2DL5 are still orphan receptors [11]. Approximately 30% of a Caucasian population carries the gene for a single activating KIR (KIR2DS4), whereas the rest carry genes for between one and six activating KIRs. Ligands for activating KIRs have not been defined. Extensive homology exists in the extracellular domains of activating and inhibitory KIRs suggesting that they might share ligands; functional studies have, however, shown only very weak affinities between activating KIRs and HLA-class I antigens, indicating that HLA class I antigens may not be the true ligand for activating KIR [12]. While activating KIR appears to be implicated in host defense in immunosuppressed patients [13], their possible role in allorecognition remains controversial. Phenotypically, NK cells are defined by an expression of CD56 and lack of the T-cell receptor-associated antigen CD3. NK cells respond to cytokines, their in vitro killing activity can be greatly enhanced by culture in IL-2, and some studies suggest that adoptive transfer of NK cell subsets in an activated state (i.e. after stimulation with IL-2, IL-12, IL-15, or combinations thereof), or in vivo activation (e.g. by administration of IL-2 after infusion of NK cells) may be required for optimal efficacy.

Chapter 23  Natural Killer-Cell Based Treatment in Hematopoetic Stem Cell Transplantation 

1.1. Transplants from Haploidentical Donors Haploidentical stem cell transplantation (HSCT) from mismatched family donors is a treatment option for patients lacking an HLA-identical sibling, a matched unrelated donor, and a suitable cord blood. As HLA antigens are inherited as two haplotypes, most patients lacking an HLA-identical sibling will have a haploidentical donor, i.e. a sibling or parent, or other relative sharing at least one HLA-haplotype. Haploidentical HSCT remains difficult, even though progress has been made using large doses of highly purified stem cells. Graft-versus-host disease is effectively prevented by extensive T-cell depletion, however, immune reconstitution is slow with a high incidence of infectious complications. Because of T-cell depletion, T-lymphocyte mediated graft-versus-leukemia reactions are limited and many patients relapse. Lymphocyte, especially CD4 counts, remain suppressed for many months after haploidentical HSCT, the first lymphoid population to recover are NK cells. Recent data have shown that these early reconstituting NK cells are immature with impaired cytotoxicity [14]. Such patients are, therefore, candidates for adoptive immunotherapy to enhance immune reconstitution and graft versus leukemia effects. 1.2. NK Cell Alloreactivity As inhibitory signals from self HLA-receptors usually override signals from activating receptors, early trials using autologous NK cells in the 1980s were largely unsuccessful [15], and the focus of NK therapy shifted to the use of allogeneic NK cells. NK cell alloreactivity could be broadly defined as any NK cell effect against cells involving some form of allorecognition. Based on the HLA class I typing of recipient and donor, NK cell alloreactivity, i.e. a lack of inhibition of donor NK cells and hence killing activity can be expected if functional donor NK cells expressing a given KIR encounter recipient cells that lack the corresponding KIR ligand (i.e. HLA class I molecule). Of the relevant inhibitory KIRs with defined ligand specificity, either KIR2DL2 or KIR2DL3 is expressed in all donors, and both KIR2DL1 and KIR3DL1 are found in >90% of donors [16, 17]. Therefore, assessing only KIR ligands will provide a reasonable approximation of potential KIR/KIR-ligand mismatches in most cases; KIR genotyping as well as flowcytometric studies may be performed to document the presence of a KIR in the donor and estimate the magnitude of the potentially alloreactive NK cell subset. NK cells may exert alloreactivity either in the graft versus host/tumor or in the host versus graft direction. NK cell alloreactivity in the host versus graft direction was first described as the phenomenon of “hybrid resistance” in a mouse transplantation model in the 1960s. In the clinical setting of HSCT after myeloablative conditioning, it rarely has any measurable effects due to the intense nature of the conditioning regimen effectively ablating host NK cells before transplantation. NK cell alloreactivity in the graft versus host direction is of specific interest as NK cells may mediate graft versus leukemia effects. There is evidence in animal studies of a multitude of potentially beneficial effects including NK versus leukemia activity, reducing relapse risks; NK-versus residual host T-cell activity, reducing graft rejection risks; and NK versus host antigen presenting cell activity [18], potentially associated with reduced GVHD risks

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as host antigen presenting cells have been implicated in the initiation of acute graft versus host disease [19]. In the setting of human haploidentical HSCT, NK cells take 2–3 weeks to mature from transplanted CD34+ cells; graftderived NK cells may, therefore, emerge too late to have a significant impact on the risk of rejection or GVHD, providing a rationale for infusion of mature donor NK cells along with the graft. Importantly, due to apparent restriction of NK cell alloreactivity to hematopoetic cells, large doses of NK cells may be infused without causing GVHD. In human studies, a positive outcome of KIR/HLA disparity has been demonstrated in the haploidentical HSCT setting [20]. A lower than expected rate of leukemia relapse was noted in patients with AML when the haploidentical donor possessed inhibitory KIRs with their corresponding ligand missing in the recipient (KIR-ligand mismatch). Alloreactive NK clones that killed recipient hematopoeitic cells including leukemic blasts in vitro were isolated from recipients following HSCT [21]. While this translated into a reduction of relapse risk in patients transplanted for an acute myeloid leukemia, no beneficial effect concerning relapse was noted in patients transplanted in chemoresistant relapse (perhaps due to unfavorable effector:target cell ratios) [22] or for adult patients transplanted for B-cell acute lymphoblastic leukemia [18]. Clinical results correlated with in vitro alloreactivity: acute myeloid leukemia blast cells were almost universally killed by alloreactive NK cells, whereas in adult acute lymphoblastic leukemia blast cells were in vitro resistant to NK alloreactivity due to lack of expression of activating NK ligands [23]. In contrast, both in vitro studies [24] and transplant outcome of pediatric patients [25] provide evidence that in pediatric ALL, blast cells are sensitive to NK alloreactivity. 1.3. Adoptive Immunotherapy/Donor Lymphocyte Infusion Adoptive immunotherapy using donor lymphocyte infusion (DLI) has become a standard practice in patients relapsing after HSCT, since the initial description in patients with CML in 1990 [26]. DLI may be administered in bulk doses or in a graded incremental fashion which may be beneficial because of less GVHD [27]. DLI appears to be highly effective in slowly progressing diseases such as CML and less so in diseases with rapid proliferation. This is likely due to the fact that graft versus leukemia activity of unfractionated DLI is mainly exerted by T-lymphocytes: as frequencies of alloreactive T-cells in an HLA matched setting are minute and as antigen-driven expansion requires time, responses take weeks and months to develop. The major risk of DLI is GVHD. Unfractionated DLI have been rarely used in recipients of haploidentical HSCT, mainly because of GVHD risks. Some investigators have been using very small doses of DLI to stabilize the graft and to promote the immune reconstitution; however, GVHD remains a significant problem [28]. DLI with highly selected NK cells in recipients of haploidentical HSCT provide a model to study the effects of NK cells and to elucidate mechanisms of NK cell alloreactivity without carrying a risk for GVHD.

2.  NK Cell DLI Based on the above, several groups have investigated – in the context of allogeneic HSCT – the preparation and infusion of purified, T-cell depleted, donor

Chapter 23  Natural Killer-Cell Based Treatment in Hematopoetic Stem Cell Transplantation 

NK lymphocytes (NK DLI) with the aim to (a) consolidate engraftment and (b) to induce graft versus leukemia effects. Most of these studies include small numbers of patients, and have been published as abstracts only.

3. Technical Aspects 3.1. Natural Killer Cell Product The aim of NK cell engineering in haploidentical HSCT is to obtain a product with a high number of functionally active CD56+/CD3− NK cells, depleted of CD3+ T lymphocytes. The choice of the target cell number for adoptive immunotherapy is based on the capacity to harvest NK cells, and on the experience with T-cell collection for DLI, where usually between 1.0 × 10×6/ kg and 1.0 × 108/kg CD3+ cells are infused. Therefore, the target NK cell dose for NK-DLI products is often fixed at ³1.0 × 107/kg body weight. However, there are no experimental or clinical data to help define an adequate cell dose. The prerequisite for an NK cell product in the haploidentical setting is T-cell depletion with a target CD3+ T-cell contamination of less than 0.5–1.0 × 105/ kg, the threshold dose which can be administered without risk of causing graftversus-host disease. 3.2. Harvesting of NK Cell Product The efficacy of NK cell collection from a healthy donor is related to the number of NK cells in the peripheral blood at the time of harvest, and the blood volume processed during leukapheresis. The pre-leukapheresis peripheral blood values correlate with the yields, therefore efficacy of NK cell collection can be predicted from the peripheral cell counts of the donor [29]. In our experience of haploidentical donors (leukapheresis volume 10–12  L), the number of collected NK cells ranged between 1.7 and 30 × 108, with contaminating T-cells between 28 and 155 × 108 [30, 31]. The Memphis group reported on 12 leukapheresis products after processing twice the blood volume in healthy adult volunteers. Products contained a median of 65 × 108 mononuclear cells (range, 20–137 × 108), with a median number of NK cell count of 4.4 × 108 (range, 0.58–22.3 × 108) [32]. Finally, the Minneapolis group reported on 70 leukaphereses of 15  L each: mean nucleated cell count after apheresis was 197 × 108, with a 10.7% fraction of NK cells [33].

4. NK Cell Product Engineering The aim of mononuclear cell engineering is to obtain a highly purified NK cell fraction with minimal T-cell contamination and conserved natural cytotoxicity. The NK purification steps should lead to an optimal NK cell yield with minimal loss of the target CD3−/CD56+ cell population. For clinical application, most commonly a large-scale purification method allowing automated, efficient, and relatively rapid isolation of human NK cells is used. This NK selection is based on a two-step immunomagnetic method, with first a CD3+ cell depletion followed by a CD56+ cell enrichment. Using this purification method, NK purity of more than 90% is obtained, with an efficient T-cell depletion of 3–5 logs, allowing an infusion of less than 0.5–1.0 × 105/kg

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CD3+ cells. The high NK cell purity and an extensive T-cell depletion are at the expense of a considerable loss of NK cells during engineering. The final recovery of CD3−/CD56+ NK cells ranges between 30% and 70%, with an inverse correlation between NK recovery and NK purity. Natural cytotoxicity of the purified cells is increased by approximately five-fold as compared to the unpurified mononuclear cells, and may be increased further by stimulation with different cytokines in vitro. The overall processing time is about 8–10 h. Preliminary data using a single-step positive selection of NK cells using antibodies directed against the NK cell-specific NKp46 antigen are promising and this procedure may allow a more rapid and cost effective isolation of NK cells in the future [34]. Enriched NK cells can be infused without any additional manipulation, after overnight incubation in high dose IL-2 or after cytokine driven in vitro expansion. Expansion has two aims: to activate the freshly selected CD56+ cells and to increase the total number of NK cells. Using CD69 as an activation marker, activation of NK cells occurs within 24 h of incubation with IL-2 [35]. When enriched CD56+ NK cells are cultured with either IL-2 alone or IL-2 combined with IL-15, a significant expansion can be observed. However, there is a lag of 1 week before NK cells start to proliferate. During the second week, the expansion occurs, leading to a five- to 20-fold increase of CD56+ NK cells at the end of two weeks of culture [36]. With a protocol that enables the generation of NK cells on a clinical scale using a closed system that allows good manufacturing practice (GMP) conformity, the expanded NK cells are highly cytotoxic against different malignant target cells [35]. As infused cell number appears to be critical, alternative expansion protocols are currently being developed with the aim to augment NK expansion such as co-culture with cytokine producing feeder cells [37] or expansion of NK cells derived from umbilical cord blood units [38, 39]. Clinical scale collection, enrichment, activation, and expansion of purified NK cells are feasible. Most of the technical aspects for adoptive NK cell therapy are mature and ready for clinical application. However, the laboratory procedures involved are time consuming and expensive, need specific skills, and must be performed according to a GMP-compliant protocol. 4.1. NK Cell Infusion Several groups have worked on pilot projects investigating feasibility and effects of adoptive immunotherapy using NK cells. Different approaches have been tested (summarized in Table 23-2). Fifteen recipients of haploidentical HSCT with AML (N = 7) or ALL (N = 5), or other hematological malignancy (N = 3) were selected for the Basel/ Frankfurt pilot protocol [31]. Donors were parents (N = 12) or mismatched siblings (N = 3). NK-DLI were given as an outpatient procedure, several weeks or months after the transplant, either immediately after processing or as cryopreserved NK-units, thawed at the bedside and infused rapidly. During infusion of fresh or thawed NK-products, no immediate adverse reactions were observed. NK cell collection and ex vivo purification was successful in all donors. One patient in each center developed severe (grade III/grade IV) GVHD. These two patients had received the highest T-cell dose. The remaining patients tolerated NK-DLI and did not develop GVHD. Eight patients were alive during the last follow-up [40].

Preemptive on day+2

Preemptive with transplant

DLI for relapse or imminent relapse

Part of conditioning regimen

17

3

8

3

AML 3

AL 3, MDS 2, NHL 2, HD 1

AML 1, ALL 2

AML 10, ALL 3, other 3

AML 7, ALL 5, other 3

Disease

UCB

Haploidentical 3, unrelated 1, sibling 4

Haploidentical

Haploidentical

Haploidentical

Donor

CD3 depleted/IL-2 activated UCB

IL-2 expanded, then CD56 selected

CD3 depleted, CD56 selected and IL-2 expanded

Waste of CD34 selection after CD3 depletion/CD56 selection. Overnight IL-2 activation in 7 patients.

CD3 depletion, CD56 selected

Selection/activation

0

0

0

Grade ³ II a GVHD 7/10 (without IL-2); and 1/7 (with IL-2)

1 Grade III, 1 grade IV

GVHD after NK cells

44

43

30

42

31

References

This table includes all reported data known to the authors of the clinical use of adoptive immunotherapy with purified NK-cells in HSCT recipients AML acute myeloid leukemia, AL acute leukemia, ALL acute lymphoblastic leukemia, MDS myelodysplastic syndrome, NHL non-Hodgkin lymphoma, HD Hodgkin disease, IL-2 interleukin 2, UCB umbilical cord blood

Decreasing chimerism/ incipient relapse/ preemptive

Indication for NK-cell infusion

15

N patients

Table 23-2.  Clinical application of purified NK cells in patients in the context of HSCT.

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Uharek et al. reported on 17 recipients of haploidentical CD34+ cell grafts receiving an add-back of 0.8 × 107 CD56+CD3− NK cells/kg at day +2 after transplant. Ten patients received unstimulated NK products, in the remaining seven patients NK cells were incubated in high-dose IL-2 for 16 h. No severe acute toxicity attributable to NK cell infusion was observed in both groups of patients. Whereas only one patient developed GVHD ³grade II after treatment with IL-2 activated NK cells, seven out of 10 patients showed GVHD ³grade II after transfer of non-activated NK cells. In a parallel study, 18 patients received CD3/CD19 depleted grafts (i.e. grafts depleted of B- and T-cells but containing large numbers of NK cells). Compared to the patients receiving NK-DLI, recovery of NK counts was faster and more sustained in patients receiving CD3/CD19 depleted grafts [41, 42]. Slavin et  al. used IL-2 activated NK cells (CD 56+ selected) following transplantation from haploidentical (3) sibling (4) or unrelated (1) donors. Patients with hematological malignancies (including acute leukemia and lymphoid malignancies), age 4–63 (median 25) years, had relapsed or were at very high risk. Donor lymphocytes were incubated for 4 days with IL-2 and then positively selected for CD56+. Purity of CD56+ was 39 (30–71)% and CD3+ was 3 (2–21)%. The number of CD56+ cells was 120 (10–600) × 106 cells. Cell infusion was uneventful, and no GVHD was observed. One patient with relapsed ALL and a patient with MDS transplanted from a KIR ligand matched mother achieved CR. Four patients are alive; one with disease; three with no evidence of disease at 9–22 months post HSCT [43]. Koehl et al. reported on three pediatric patients with multiply relapsed ALL (2) and AML (1) treated with repeated transfusions of IL-2 activated NK cells post haploidentical HSCT from parental donors (single dose: 3–34 × 106 CD56+CD3−/kg). Blast persistence (37–97%) was demonstrated in pretransplant bone marrow in all three patients. KIR ligand mismatches in GVH direction were demonstrated in all donor:recipient pairs. All patients achieved complete remission 4 weeks post HSCT, which was accompanied by complete donor chimerism. NK cell infusion was well tolerated, two patients died of transplant-related complications, while one patient died of relapse [30]. Miller et  al. reported on three patients with refractory AML treated with a triple umbilical cord blood (UCB) transplantation strategy. UCB unit 1 was immunomagnetically depleted of T-cells, ex vivo treated with high-dose interleukin-2, and infused in patients on day 12 after completion of myeloablative conditioning. NK cells were then expanded in  vivo by administration of subcutaneous interleukin-2 until day 0, when UCB units 2 and 3 were transplanted for hematopoeitic rescue. Unexpectedly, two patients showed neutrophil engraftment on days +3 and +7 from UCB unit 1. NK infusion was tolerated without toxicity and all patients were leukemia-free at the time of engraftment [44]. Other investigators have presented data on CD3/CD19 depletion for haploidentical HSCT which corresponds to selecting CD34+ cells along with, what they call: CD34-progenitors, natural killer, graft-facilitating, and dendritic cells. A recent report describes 29 patients receiving a peripheral stem cell graft containing 7.2 × 107/kg CD56+ cells after a conditioning of reduced intensity with engraftment in all patients and survival in 9/29 patients [45]. Cumulative incidence of grade II–IV acute GVHD was considerable (48%), however, infused T-cell doses with this approach are higher than those achieved

Chapter 23  Natural Killer-Cell Based Treatment in Hematopoetic Stem Cell Transplantation 

by CD34+ selection (median, 0.44 × 10e5/kg). A retrospective comparison of patients undergoing haploidentical HSCT using CD34+ selection versus CD3+ CD19+ depletion showed faster engraftment and faster NK cell reconstitution with the latter method [46]. Patients were, however, not entirely comparable and there were also differences in the conditioning regimens used. Data available so far show that ex vivo purification of donor NK cells from leukapheresis products is technically feasible, and large numbers of CD56positive, highly CD3-depleted cells can be obtained using the CliniMACS® two-step procedure of T-cell depletion and NK cell enrichment. NK cells are infused without immediate adverse events and possibly without inducing GVHD. Several cases of GVHD occurring after NK cell infusion have been described. In some instances, this has been associated with a less efficient T-cell depletion. Whether GVHD is attributable to contamination by T-cells or is due to the effects of NK cells cannot be determined based on this clinical data. The fact that at least in some cases the T-cell content was highest in patients developing GVHD is in favor of a T-cell effect. Clinical data on efficacy are very limited at this point in time. In addition, it is difficult to separate NK cell effects from effects of small number of residual T-cells in the product. Another question to be resolved is the importance of cytokine activation. While infusion of haploidentical donor NK in a non-transplant setting has only led to in vivo expansion of infused NK cells if patients received concomitant treatment with interleukin-2 [47], most investigators have refrained from treating patients with cytokines after NK infusion, due to concerns that this might trigger graft-versus-host disease. Infusion of ex vivo cytokine activated NK cells may combine the benefits of infusing highly cytotoxic NK cells without activating potentially alloreactive T-lymphocytes in vivo. Finally, the question arises whether allogeneic transplantation is required as a prerequisite for successful therapy with allogeneic NK cells. Interesting data from the Minneapolis group have shown that infusion of a purified NK cell product into patients treated with cyclophosphamide, fludarabine, and IL-2 may lead to transient engraftment of transfused NK cells and induction of remission in refractory AML patients [47]. NK cell chimerism was detectable for up to several months after infusion, and ultimately all patients rejected transferred NK cells and relapsed. However, the data shows the powerful effects of NK cells in vivo and may argue for a combination of (haploidentical) allogeneic transplantation and NK DLI, as a transplant preceding NK infusion will prevent rejection of NK cells and ultimately serve as a source of alloreactive NK cells on its own.

5.  Outlook Future studies will have to determine the usefulness of NK cells as an adoptive immunotherapy in recipients of HSCT from haploidentical and other donors. Open issues include NK cell doses, timing, and appropriate selection of donor and recipients amongst others (see Table  23-3). Whether NK DLI should be used preemptively or as a salvage treatment is unknown. Timing may be crucial. NK cells infused simultaneously with the transplant have the benefit of being administered at a time of minimal tumor load. Conditioning regimen induced aplasia and also upregulation of growth factors important for NK cell survival.

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Table  23-3.  Issues in harvesting, purification, and administration of natural killer (NK) cells. Production issues Mobilizing NK cells

Increased NK-cell numbers and lower adhesion molecule expression with epinephrine and exercise

Positive NK-cell selection

CD3 depletion followed by CD56+ selection

Negative “NK”-cell selection

CD3 (T-cell) depletion and CD19 (B-cell) depletion, may result in similar numbers but different purity of NK cells

In vitro activation of NK cells

IL-2 short- or median-time culture, other cytokines (e.g. IL-12, IL-15); is known to increase NK-cell killing in vitro

In-vitro expansion

Activation of NK cells and expansion of their number without increasing the number of T-cells

Infusion issues Infusion at the time of transplant

Purification from waste after CD34+ selection, potential impact of G-CSF mobilization on NK cells; this will not provide information on safety or on NK-cell effects

Infusion during post-transplant course but pre-emptive

Information on safety, but no information on NK-cell effects to be expected

Infusion with relapse or with falling chimerism

Information on safety and on NK-cell effects to be expected

Patient selection issues AML versus other diseases

NK-alloreactivity has been shown to be strongest in myeloid leukemia and possibly absent in other diseases, but this has not been studied prospectively and not using adoptive immunotherapy protocols

Donor selection issues Any suitable haploidentical donor versus donor with known NK-alloreactivity

The skeptical approach of not restricting inclusion for adoptive immunotherapy protocols to donors with known NK-alloreactivity will allow for testing of NK-cell effects in donor/ recipient pairs with or without predicted or measured NK-cell alloreactivity

Other allogeneic donors or autologous NK cells

There is little or no information available

IL interleukin, G-CSF granulocyte colony stimulating factor, AML acute myelogenous leukemia

However, effects will be much harder to measure than if NK cells were administered later in the course. Some groups have used NK cells purified from the stem cell product, and the impact of mobilization on NK cells needs to be studied. Other groups have reported the enrichment of NK cells by depletion of T- and B-cells, which is attractive in both financial and practical aspects, as a product containing both progenitor cells and high numbers of NK cells that can be produced in one step.

Chapter 23  Natural Killer-Cell Based Treatment in Hematopoetic Stem Cell Transplantation 

Whether NK cells will be used without additional manipulation, or whether stimulation and culture either in the short- or medium-term with cytokines such as IL-2 will prove to be more effective awaits further studies. Purging and enrichment technology using magnetic beads for clinical application is technologically fascinating but expensive, in direct costs of antibodies and columns and the time of the laboratory personnel. The NK cell enrichment technology will require some improvement for broad application. Future studies may, therefore, include intervention on the part of NK cells by selecting donors with appropriate NK receptor profiles and possibly activating the cells while promoting ligand expression on the blasts to enhance killing. The burden of proof of principle and of usefulness in clinical practice lies, therefore, with those who want to apply this technology. Multi-institutional phase III trials in recipients of haploidentical HSCT, comparing standard transplant to transplant plus NK-DLI could be instrumental in establishing the clinical role, if any, of adoptive NK-cell therapy.

References 1. Farag SS, Caligiuri MA (2006) Human natural killer cell development and biology. Blood Rev 20:123–137 2. Farag SS, Fehniger TA, Ruggeri L, Velardi A, Caligiuri MA (2002) Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect. Blood 100:1935–1947 3. Bottino C, Moretta L, Moretta A (2006) NK cell activating receptors and tumor recognition in humans. Curr Top Microbiol Immunol 298:175–182 4. Biassoni R, Cantoni C, Pende D et al (2001) Human natural killer cell receptors and co-receptors. Immunol Rev 181:203–214 5. Valiante NM, Uhrberg M, Shilling HG et al (1997) Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity 7:739–751 6. Kim S, Poursine-Laurent J, Truscott SM et  al (2005) Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature 436: 709–713 7. Fernandez NC, Treiner E, Vance RE, Jamieson AM, Lemieux S, Raulet DH (2005) A subset of natural killer cells achieves self-tolerance without expressing inhibitory receptors specific for self-MHC molecules. Blood 105:4416–4423 8. Anfossi N, Andre P, Guia S et al (2006) Human NK cell education by inhibitory receptors for MHC class I. Immunity 25:331–342 9. Hansasuta P, Dong T, Thananchai H et  al (2004) Recognition of HLA-A3 and HLA-A11 by KIR3DL2 is peptide-specific. Eur J Immunol 34:1673–1679 10. Rajagopalan S, Long EO (1999) A human histocompatibility leukocyte antigen (HLA)-G-specific receptor expressed on all natural killer cells. J Exp Med 189:1093–1100 11. Estefania E, Flores R, Gomez-Lozano N, Aguilar H, Lopez-Botet M, Vilches C (2007) Human KIR2DL5 is an inhibitory receptor expressed on the surface of NK and T lymphocyte subsets. J Immunol 178:4402–4410 12. Vales-Gomez M, Reyburn HT, Erskine RA, Strominger J (1998) Differential binding to HLA-C of p50-activating and p58-inhibitory natural killer cell receptors. Proc Natl Acad Sci USA 95:14326–14331 13. Cook M, Briggs D, Craddock C et al (2006) Donor KIR genotype has a major influence on the rate of cytomegalovirus reactivation following T-cell replete stem cell transplantation. Blood 107:1230–1232

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M. Stern et al. 14. Nguyen S, Dhedin N, Vernant JP et  al (2005) NK-cell reconstitution after ­haploidentical hematopoietic stem-cell transplantations: immaturity of NK cells and inhibitory effect of NKG2A override GvL effect. Blood 105:4135–4142 15. Rosenberg SA, Lotze MT, Muul LM et al (1987) A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. N Engl J Med 316:889–897 16. Uhrberg M, Valiante NM, Shum BP et  al (1997) Human diversity in killer cell inhibitory receptor genes. Immunity 7:753–763 17. Uhrberg M, Parham P, Wernet P (2002) Definition of gene content for nine common group B haplotypes of the Caucasoid population: KIR haplotypes contain between seven and eleven KIR genes. Immunogenetics 54:221–229 18. Ruggeri L, Capanni M, Urbani E et  al (2002) Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 295: 2097–2100 19. Shlomchik WD, Couzens MS, Tang CB et al (1999) Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science 285:412–415 20. Ruggeri L, Mancusi A, Burchielli E et al (2008) NK cell alloreactivity and allogeneic hematopoietic stem cell transplantation. Blood Cells Mol Dis 40:84–90 21. Ruggeri L, Capanni M, Casucci M et  al (1999) Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation. Blood 94:333–339 22. Ruggeri L, Mancusi A, Capanni M et al (2007) Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value. Blood 110:433–440 23. Pende D, Spaggiari GM, Marcenaro S et al (2005) Analysis of the receptor-ligand interactions in the natural killer-mediated lysis of freshly isolated myeloid or lymphoblastic leukemias: evidence for the involvement of the Poliovirus receptor (CD155) and Nectin-2 (CD112). Blood 105:2066–2073 24. Pfeiffer M, Schumm M, Feuchtinger T, Dietz K, Handgretinger R, Lang P (2007) Intensity of HLA class I expression and KIR-mismatch determine NK-cell mediated lysis of leukaemic blasts from children with acute lymphatic leukaemia. Br J Haematol 138:97–100 25. Leung W, Iyengar R, Turner V et al (2004) Determinants of antileukemia effects of allogeneic NK cells. J Immunol 172:644–650 26. Kolb HJ, Mittermuller J, Clemm C et  al (1990) Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 76:2462–2465 27. Dazzi F, Szydlo RM, Craddock C et  al (2000) Comparison of single-dose and escalating-dose regimens of donor lymphocyte infusion for relapse after allografting for chronic myeloid leukemia. Blood 95:67–71 28. Lewalle P, Triffet A, Delforge A et  al (2003) Donor lymphocyte infusions in adult haploidentical transplant: a dose finding study. Bone Marrow Transplant 31:39–44 29. Wolf CE, Meyer M, Riggert J (2005) Leukapheresis for the extraction of monocytes and various lymphocyte subpopulations from peripheral blood: product quality and prediction of the yield using different harvest procedures. Vox Sang 88:249–255 30. Koehl U, Sorensen J, Esser R et al (2004) IL-2 activated NK cell immunotherapy of three children after haploidentical stem cell transplantation. Blood Cells Mol Dis 33:261–266 31. Passweg JR, Tichelli A, Meyer-Monard S et al (2004) Purified donor NK-lymphocyte infusion to consolidate engraftment after haploidentical stem cell transplantation. Leukemia 18:1835–1838 32. Iyengar R, Handgretinger R, Babarin-Dorner A et al (2003) Purification of human natural killer cells using a clinical-scale immunomagnetic method. Cytotherapy 5:479–484

Chapter 23  Natural Killer-Cell Based Treatment in Hematopoetic Stem Cell Transplantation  33. McKenna DH Jr, Sumstad D, Bostrom N et al (2007) Good manufacturing practices production of natural killer cells for immunotherapy: a six-year single-institution experience. Transfusion 47:520–528 34. Bondzio I, Schmitz J, Huppert V (2007) CliniMACS cell enrichment using NKp46. A large scale, single-step NK cell isolation method. Blood 110:abstract 3877 35. Koehl U, Esser R, Zimmermann S et al (2005) Ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children. Klin Padiatr 217:345–350 36. Klingemann HG, Martinson J (2004) Ex vivo expansion of natural killer cells for clinical applications. Cytotherapy 6:15–22 37. Fujisaki H, Kakuda H, Lockey T, Eldridge PW, Leung W, Campana D (2007) Expanded natural killer cells for cellular therapy of acute myeloid leukemia. Blood 110:abstract 2743 38. Xing D, Fang W, Decker WK, et al (2007) Ex vivo expansion of cord blood NK cell have in  vivo efficacy against leukemia. ASH Annu Meet Abstr 110(11): abstract 2741 39. Ayello J, Nemiroff J, Satwani P, et al (2006) Enhanced NK cell activation, cytotoxicity and ex-vivo expansion (EvE) of cryopreserved cord blood (CB) natural killer (NK) cells: potential role for CB NK cells in adoptive cellular immunotherapy (ACI). ASH Annu Meet Abstr 108:726 40. Passweg JR, Koehl U, Stern M, et  al (2006) Preemptive immunotherapy with highly purified CD56+/CD3− natural killer cells after haploidentical stem cell transplantation. A prospective phase II study in 2 centers. ASH Annu Meet Abstr 108(11):abstract 411 41. Gentilini C, Haegele M, Muessig A, et al. (2007) NK-Cell recovery and immune reconstitution after haploidentical hematopoietic cell transplantation using either CD34 selected grafts and adoptive NK-Cell transfer or CD3/CD19 depleted grafts: comparison of two strategies for NK cell based immunotherapy. ASH Annu Meet Abstr 110(11):abstract 2988 42. Gentilini C, Hilbers U, Huppert V, et al (2007) Patients Receiving IL-2 Activated Donor NK Cells Show Lower Incidence of Severe GvHD after Haploidentical SCT. ASH Annu Meet Abstr 110(11):abstract #354 43. Slavin S, Morecki S, Shapira M, Samuel S, Ackerstein A, Gelfand Y (2004) Immunotherapy using rIL-2 activated mismatched donor lymphocytes positively selected for the treatment of resistand haematologic malignancies after stem cell transplantation. Bone Marrow Transplant 37(S1) 44. Miller JS, Brunstein CG, Cooley S, et al (2006) A novel triple umbilical cord blood transplant (UCBT) Strategy to promote NK cell immunotherapy (Unit 1) with a fully ablative preparative regimen followed by a double UCBT in patients with refractory AML. ASH Annu Meet Abstr 108:abstract 3111 45. Bethge WA, Faul C, Bornhauser M et al (2008) Haploidentical allogeneic hematopoietic cell transplantation in adults using CD3/CD19 depletion and reduced intensity conditioning: an update. Blood Cells Mol Dis 40:13–19 46. Bethge WA, Haegele M, Faul C et al (2006) Haploidentical allogeneic hematopoietic cell transplantation in adults with reduced-intensity conditioning and CD3/ CD19 depletion: fast engraftment and low toxicity. Exp Hematol 34:1746–1752 47. Miller JS, Soignier Y, Panoskaltsis-Mortari A et  al (2005) Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 105:3051–3057

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Chapter 24 Cryopreservation of Allogeneic Stem Cell Products Noelle V. Frey and Steven C. Goldstein

1. Introduction Donor stem cells for allogeneic transplant are traditionally collected and transfused “fresh” into the recipient on the day of transplant. Alternatively, donor stem cells can be collected in advance and cryopreserved until needed. Due to historical momentum and concerns that the cryopreservation and thawing process may damage the graft and worsen clinical outcomes, most institutions favor the former approach. The use of cryopreserved grafts has, therefore, traditionally been reserved for extreme circumstances of questionable donor reliability or availability. This trend is, however, slowly changing as some individual centers are favoring the use of frozen grafts in their related donor transplants due to increased ease of transplant coordination. Similarly, The National Marrow Donor Program (NMDP), which authorizes the collection of all cryopreserved unrelated grafts, has noticed an increasing trend in the use of frozen stem cell products. The total of cryopreserved stem cell grafts, however, still represents less than 2% of all unrelated products (R King; NMDP, personal communication). The paramount question when considering using a fresh vs. a frozen stem cell allograft is whether the cryopreservation and thawing processes alter the viability or activity of individual mononuclear cell (MNC) subsets in the graft. The next question is whether these alterations in stem cell graft content correlate with clinically meaningful disparate outcomes between cryopreserved graft recipients and fresh graft recipients. Of specific concern is the impact of cryopreservation on T-cell subsets which are important mediators of engraftment, graft vs. tumor (GVT), and graft vs. host disease (GVHD). This data cannot be extrapolated from the autologous literature where cryopreserved products are the mainstays of therapy. In this chapter, we will review the sparse clinical data which evaluates the effects of the cryopreservation and thawing processes on the allogeneic stem cell graft and transplant clinical outcomes. We will also examine the potential advantages and disadvantages of using a cryopreserved allograft [1].

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_24, © Springer Science + Business Media, LLC 2003, 2010

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2. Cryopreservation 2.1. Methodology Hematopoietic stem cells are progressively lost during storage at room ­temperature or 4°C. Peripheral blood (PB) and bone marrow (BM) stem cell grafts are, therefore, cryopreserved if storage for longer than 3–4 days is anticipated. In addition, cryopreservation serves as the primary method of storage for cord blood grafts [2]. There are several aspects of the freeze-thaw process that are designed to minimize the damage to stem cells and other MNCs. To avoid mechanical and osmotic damage to cells from ice crystal formation during the freeze process, colligative cryoprotectants are added to the product prior to freezing. Dimethylsulfoxide (DMSO) at a final concentration of 10% is a commonly used cryoprotectant and its breakdown into dimethylsulfide (DMS) accounts for the characteristic sulfur smell originating from patients after re-infusion. For further optimization of cell survival, the cells are subsequently suspended in a solution containing various protein and solute concentrations, and the graft is frozen at a slow rate (commonly 1–2°C/ min) and ultimately stored at a temperature colder than −80°C. Thawing can safely occur more quickly, often with use of a water bath at the bedside [3]. It should be noted that individual center’s freeze-thaw techniques are variable, which complicates the interpretation and applicability of single center data reporting outcomes of cryopreserved grafts. It is also important to emphasize that in the unrelated setting, stem cell products are collected at the donor center and subsequently transported at 4°C until they are cryopreserved at the recipient center. Transport time has been shown to affect the outcomes in unrelated transplants and is therefore another factor which could influence the cryopreservation outcomes in the unrelated setting [4]. 2.2. Effect of Cryopreservation on Graft content It is important to recognize that a donor graft is composed of several different MNC subtypes including CD34+ stem cells, dendritic cells, T-cells, and NK cells which are variably important for effecting certain transplant outcomes such as engraftment, GVT, and GVHD. These MNC subtypes are also differentially affected by the freeze-thaw process and have different optimal conditions for survival. It is, therefore, reasonable to suspect that cryopreservation may alter a donor graft to such a degree that transplant outcomes are altered. For example in the allogeneic setting, it is known that CD34+ cells, T-cells, and NK cells are important for engraftment and that GVHD is T-cell dose dependent [5–8]. The cryopreservation process could potentially affect the engraftment and GVHD outcomes if any of these subsets were significantly damaged by the freeze-thaw process. The idea that differences in graft content can alter transplant outcomes is best illustrated by reports comparing recipients of PB or BM allografts. Recipients of PB grafts, which have a higher T-cell content, have shortened engraftment times and increased incidence of GVHD compared to recipients of BM grafts [9, 10]. The impact of cryopreservation on graft content and function is best described for CD34+ stem cells, burst forming units-erythroid (BFU-E), and colony forming units granulocyte-macrophage (CFU-GM). As cryopreservation is the mainstay of autologous graft storage, most studies have been

Chapter 24  Cryopreservation of Allogeneic Stem Cell Products 

performed on autologous grafts and have shown a strong association with CD34+ number and time to platelet and neutrophil reconstitution [11–14]. Of interest however, engraftment outcomes in these studies are often correlated with pre-frozen CD34+ numbers with no post-thaw comparison. An interesting study compared pre- and post-thaw MNC, CD34+ cells, and CFU-GM in 83 PB and 43 BM autografts. Pre- and post-thaw CD34+ cells were well correlated with each other while pre- and post-thaw MNC and CFU-GM were less well correlated. In this study, the total CD34+ cells infused were found to be the only factor predictive of engraftment outcomes [14]. A recent large report describing outcomes for 105 recipients of cryopreserved PB allografts reported CFU-GM, BFU-E, and colony forming unit megakaryocyte (CFUMEG) in donor grafts before and after cryopreservation. In this study, the viability of MNCs after cryopreservation was globally assessed by tryptan blue exclusion which showed a median recovery rate of 71%. CFU-GM, BFU-E, and CFU-MEG growth rates were reduced by 25%, 30.5%, and 61%, respectively (see Fig. 2-1). These findings, as discussed in more detail below, did not translate into statistically significant reduction in time to engraftment when compared to recipients of fresh PB allografts [15]. The reported experience regarding the impact of cryopreservation on the viability and functionality of other MNCs is quite limited. Early studies using crude estimates of T-cell number and function suggested no significant reduction in T-cell percentage or function after the cryopreservation and thaw process [16, 17]. A recent investigation incorporating flow cytometry and more sophisticated functional analysis has showed a small but statistically significant reduction in the percentage of CD3+, CD4+, and CD8+ cells after cryopreservation and 3 months of storage [18]. The full impact of donor derived antigen presenting dendritic cells (DCs) in effecting GVT and GVHD responses is yet to be determined [19]. Several studies have suggested that in G-CSF mobilized blood, the freeze-thaw process does not affect the phenotype, viability, or biological activity of immature and mature DCs [20, 21].

Fig. 24-1.  Frequency of clonogenic hematopoietic progenitors in PB allograft before and after cryopreservation (reprinted from [15])

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The assessment of functionality of mononuclear cell subsets after cryopreservation deserves further investigation. Methods to optimize the viability of MNCs in cryopreserved cord blood products may contribute to optimizing the freeze-thaw processes in PB and BM allografts [22]. 2.3.  Effect of Cryopreservation on Transfusion Reactions In addition to potentially altering the graft content, the cryopreservation and thawing processes result in an increased risk of transfusion reactions due to the presence of DMSO as a cryoprotectant. These symptoms are for the most part transient, well tolerated, and are generally not associated with more clinically significant adverse outcomes. One study compared transfusion reactions between 134 recipients of autologous cryopreserved BM and 71 recipients of fresh allogeneic BM [23]. A statistically significant increase in the incidence of nausea (45 vs. 14%; p < 0.0005), vomiting (24 vs. 9%; p < 0.01), and fever (18 vs. 0%; p < 0.005) were found in the patients receiving cryopreserved products. No differences were found, however, in serious transfusion reactions marked by hemodynamic instability or pulmonary compromise. Kim and colleagues found that transfusion-related toxicities in 105 recipients of frozen PB allografts were generally well tolerated with a 40% incidence of nausea and vomiting [15]. 2.4. Effect of Cryopreservation on Bacterial Contamination The incidence of bacterial contamination in cryopreserved autografts can range from 6 to 17%. While bacterial graft contamination can result in febrile reactions and bacteremia in recipients, it is unlikely to correlate with more serious adverse outcomes [23–26]. The increased rate of bacterial contamination is due to the increased manipulation of a cryopreserved product as compared with a fresh product and the use of a water bath to thaw samples. This finding is supported by a large study in which investigators performed microbial cultures on 194 bone marrow grafts in different stages of processing and showed an increased number of positive bacterial cultures after certain cryopreservation and thawing procedures [24]. Of note, there is a lower incidence of bacterial contamination with the use of PB stem cell products compared with BM (6 vs. 0.5% in one study) due to the use of a closed apheresis system to collect cells and the avoidance of multiple punctures through the skin [26]. Also of note, cryopreservation storage bags are structurally improved leading to less breakage and subsequent contamination during thawing procedures. In fact in a recent report of 105 recipients of frozen PB allografts, no bacterial contamination was identified [15].

3.  Clinical Outcomes No prospective, randomized trials comparing outcomes of patients receiving cryopreserved donor stem cells to patients receiving fresh donor stem cells for allogeneic stem cell transplant have been performed. The reported experience is limited to retrospective cohort studies and case series from individual institutions (see Table 24-1) [15, 27–32]. The largest series to date reporting outcomes of cryopreserved allograft recipients comes from Kim and colleagues at the University of Toronto. In this well-designed retrospective study,

6

NR

Fresh

33

Frozen

10

Fresh

NR

Fresh

Frozen

10

Frozen

40

Fresh

NR

Related

Related

Related

NR

Unrelated

Related

Related

Related

Related

Donor

NR

BM

BM

BM

NR

BM

BM

BM

PBSC

PBSC

HSC source

NR

21.3

16

19

NR

22.6

17.5

17

18

17

Days to ANC >0.5

NR

NR

23

28(plt > 50)

NR

NR

20

21(plt > 20)

13

13

Days to Plt engraftment

a

NR

57.5%

20%

NR

75%

60%

61%

81.2 (p = 0.113)

78.2

aGVHD ³gr II

50%

61%

70%

NR

55%

72%

82%

No difference in OS

Day 100 survival

a Statistically significant difference. NR not reported, HSC hematopoietic stem cell source, ANC absolute neutrophil count, Plt platelet, aGVHD acute Graft-vs.-host disease, OS overall survival

[28]

[27]

[31]

40

Frozen

106

Fresh

[29]

105

Frozen

[15]

N

Storage

Reference

Table 24-1.  Summary of engraftment and outcome data for selected reports of allogeneic stem cell transplantation using cryopreserved grafts.

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the authors compare outcomes of 105 consecutive related cryopreserved PB allograft recipients with 106 historical recipients of related fresh PB allografts. The groups were well matched with regard to the recipient age, donor age, disease type, conditioning regimens, and GVHD prophylaxis. Donor grafts were cryopreserved using 10% DMSO, stored at −86°C and thawed at the bedside in a 40°C water bath. The transfusions were overall well tolerated and no grafts had bacterial contamination. Grafts were stored for a median of 15 days (range 5–238 days) [15]. In multivariate analyses, no statistically significant differences were found between fresh and frozen allograft recipients in terms of times to platelet and neutrophil engraftment, incidence of acute and chronic GVHD, relapse rates, or overall survival. Due to concerns that T-cells may be more deleteriously affected by the cryopreservation process, the authors also compared lymphocyte recovery over time between the two groups and found no difference. In the recipients of cryopreserved allografts, the median time to neutrophil (ANC >0.5 × 109/L for 2 consecutive days) and platelet engraftment (platelets >20 × 109/L for 3 consecutive days) were 17 and 21 days, respectively. Similar engraftment kinetics were also observed in recipients of fresh allografts. Only one subject who received a cryopreserved allograft failed to engraft, compared to five recipients of fresh allografts. The incidence of Grade II–IV acute GVHD was 78.2% in the cryopreserved graft recipients and 81.2% in the fresh graft recipients (p = 0.113) (see Fig. 24-2). Chronic GVHD affected 83.8% of cryopreserved graft recipients and 90.1% of fresh graft recipients (p = 0.673). While no statistically significant difference in acute GVHD was found between fresh and cryopreserved recipients, a trend towards less GVHD from frozen allografts is noted [15]. While well designed, this study is underpowered to detect a 5 or 10% difference in GVHD. As discussed earlier, viability studies on graft content showed a 25–30% reduction of CFU-GM, BFU-E, and MNCs. The authors interestingly noted a significant 61% reduction in CFU-MEG which did not translate into slower platelet engraftment times in recipient’s cryopreserved products. To further evaluate the impact of CFU-MEG on platelet engraftment kinetics, the authors divided patients into quartiles based on their infused CFU-MEG and found a delayed time to platelet recovery (29 vs. 18 days) in the bottom quartile of the cryopreserved graft recipients [15]. The above study by Kim and colleagues is limited to related PB allograft recipients. In an earlier study, Stockshlader and colleagues retrospectively compared 40 patients who received cryopreserved related BM allografts with 40 patients matched for age, disease, and disease stage who received fresh BM allografts [29]. Information on donors was not reported. Indications for cryopreservation appeared to be independent of disease status and included concerns regarding donor age, reliability, scheduling, and operating room availability. The cryoprotectant was DMSO (final concentration 10%) and the median time of graft storage was 17.5 days (range 3–455 days). All patients with two exceptions received the same GVHD prophylaxis with cyclosporine (CSA) and methotrexate (MTX). Conditioning regimens were similar. No statistically significant differences were found in time to neutrophil and platelet engraftment, day 100 survival, or incidence of GVHD between the two groups [29]. This group compared the total number of BFU and CFU-GM infused into recipients of fresh vs. frozen allografts and found no statistically significant difference. Prior published results from a subset of 19 patients who received a

Chapter 24  Cryopreservation of Allogeneic Stem Cell Products 

Fig. 24-2.  Incidence of GVHD in cryopreserved and fresh allograft recipients (reprinted from [15])

cryopreserved product directly measured the impact of cryopreservation and thawing on graft content by comparing CFU-GM and MNC numbers before and after cryopreservation. This analysis revealed no statistically significant difference in the pre-frozen and post-thaw numbers of CFU-GM and MNC [30]. Stockshlader and colleagues also reported their limited experience in using cryopreserved BM for 10 patients undergoing unrelated allogeneic transplantation [31]. Outcomes for these patients were not directly compared to matched or historical controls, and are reported in Table 24-1. Two other small series from the early 1990s also showed no significant reduction in time to platelet or neutrophil engraftment and overall survival in cryopreserved allograft recipients when compared to institutional or historic controls [27, 28].

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One of these studies, however, reported a statistically significant reduction in the incidence of GVHD for 10 recipients of cryopreserved BM compared with 33 unmatched institutional controls [27]. In this study, recipients of cryopreserved products had a 20% incidence of acute GVHD compared to a 56% incidence in the control group. There were, however, significant differences between the two recipient groups with regards to age, disease type, disease stage, and type of GVHD prophylaxis. Attempts to account for these differences with a multivariate analysis failed to identify cryopreservation as an independent predictor of GVHD [27].

4. Donor Lymphocyte Infusion Donor lymphocyte infusion (DLI) is increasingly being used after both myeloablative and non-myeloablative stem cell transplantation to treat and prevent relapse, to establish full donor chimerism, and to treat and prevent infections. It is feasible to collect and cryopreserve DLI products at the time of original donor stem cell collection [33, 34]. The potential benefits of having a readily available DLI product (especially in the unrelated donor setting) needs to be weighed against the potential deleterious effects of cryopreservation and storage of this lymphocyte-rich product as well as the potential cost of long-term storage of DLI products that may never be utilized. It is important to note that due to differences in cell content and timing of administration between stem cell grafts and DLI products, cryopreservation may differentially affect clinical outcomes in recipients. Unfortunately, the data describing outcomes of recipients of cryopreserved DLI products is very limited. Sohn et  al. reported their experience with 17 patients at high risk for relapse whose donors underwent donor lymphocyte collection with cryopreservation at the time of original harvest [34]. DLI was given to transplant recipients without GVHD at pre-specified time points with the goal of preventing recurrent disease (i.e., prophylactic DLI). The incidence of GVHD after DLI was ~60%, a finding consistent with other reports of GVHD incidence after DLI [35, 36]. While this study was not designed to assess the efficacy of a cryopreserved vs. a fresh DLI product, it suggests that cryopreservation of donor lymphocytes at the time of collection for subsequent re-infusion in high risk patients may be a reasonable strategy to avoid delay of DLI in the event of relapse [34]. Lane et al. reported the outcomes of 19 subjects who received cryopreserved DLI for either relapsed disease or low donor chimerism after non-myeloablative transplantation. The infusions were well tolerated. Three of 15 patients who were treated for relapse developed a complete response and donor chimerism improved by a mean of 16% (range 0–50%). GVHD outcomes were not reported [33]. Further investigation of the impact of cryopreservation on DLI outcomes is warranted.

5. Logistics It is logistically challenging to coordinate a patient’s conditioning regimen with donor stem cell collection when a fresh product is used. Securing operating rooms, apheresis time, and total body irradiation (TBI) slots are sometimes difficult. The donor, who must be available on day 0, is often asked to be available at

Chapter 24  Cryopreservation of Allogeneic Stem Cell Products 

times which may not be convenient. These coordinating challenges are intensified in the unrelated setting in which the NMDP helps to organize these events between two different centers. The use of a cryopreserved product introduces a greater amount of flexibility into the system and would inherently be more convenient for the donor and the hospital. Streamlining the donor experience could also help relieve potential recipient guilt over causing inconvenience to family members. An increased cost would be incurred with cryopreservation and storage of stem cell products but these may be balanced by potential “hidden” savings of streamlining bed utilization, and optimal scheduling of stem cell laboratory and radiation oncology personnel. While a rare event, there is always a possibility that a donor will become unavailable for unforeseen circumstances after the conditioning regimen has been initiated. There is also a 2–5% inherent risk that a donor undergoing peripheral blood stem cell collection will be a poor-mobilizer, a disconcerting finding if discovered on day 0 of transplant [37, 38]. In fact, in the report by Kim and colleagues 14% of PB donors required collection over two or more days [15]. While this issue may become less important with the mobilization success rates of AMD-3100 (a CXCR-4 antagonist), the collection of certain donors at high risk for mobilization-failure ahead of time may be appropriate [39–42].

6. Ethical Concerns The increased use of cryopreserved products raises the possibility of an increase of collected, but not utilized grafts. This raises ethical concerns about subjecting some donors to unnecessary, time-consuming, and potentially harmful harvesting procedures. If collection of stem cells were undertaken closer in time to the transplant (e.g., within 30 days), the incidence of unnecessary harvesting procedures would be minimized while maintaining many of the benefits mentioned above. The practicality of this approach is supported by the reported experience of Kim and Stockschlader who noted a median time for graft storage of 15 and 17.5 days, respectively [15, 29]. Of note, the manuscripts discussed above do not comment on how many grafts were collected by the individual centers and never utilized.

7. Summary and Conclusions We have reviewed the potential advantages and disadvantages of using cryopreserved grafts in the allogeneic stem cell transplant setting (Table  24.2). Cryopreserved grafts are associated with a higher incidence of transfusion reactions and bacterial contamination but these are rarely associated with significant morbidity. The limited literature on this subject to date shows no significant difference in overall survival, relapse rates, GVHD, or time to engraftment between recipients of fresh or frozen stem cell products. However, one should be cautious in making firm conclusions based on the available literature as it is quite limited. The studies published to date are retrospective and insufficiently powered to assess for 5–10% differences in GVHD or relapse rates. In fact, two studies reported thus far suggest that there may be a trend towards less GVHD in recipients of cryopreserved products [15, 27].

435

Comments The limited published experience shows no significant delay in time to platelet or neutrophil engraftment. Further, more appropriately powered studies with laboratory correlates are needed Cryopreserved products are associated with more transfusion related nausea, vomiting, and fever but not necessarily more serious events such as hemodynamic instability or pulmonary compromise Higher rates of bacterial contamination do not translate into significantly higher rates of bacteremia and sepsis in recipients. Peripheral blood grafts correlate with a lower incidence of contamination Timing of collection within 1–3 weeks of transplant would minimize this inevitable outcome Comments Coordinating donor collection on Day 0 of transplant is challenging. Cryopreservation introduces greater flexibility into the system Greater flexibility for donor to schedule collection in setting of other obligations Encouraging mobilization results of AMD-3100 may decrease current 2–5% poor mobilization rates among healthy peripheral blood stem cell donors While rare, cryopreservation would allay this commonly reported fear among recipients Most studies to date do not support this hypothesis. Adequately powered clinical studies with laboratory correlates are needed Limited data on effect of cryopreservation on DLI. Prophylactic collection of DLI could increase the number of products that are not infused (concern for costeffectiveness and donor safety)

Potential disadvantages

Concern over delay of neutrophil and platelet engraftment due to damage of the graft during cryopreservation

Increased incidence of transfusion reactions due to the presence of DMSO as a cryoprotectant

Increased incidence of bacterial contamination of the graft due to increased handling in the freeze-thaw process

Increased incidence of collecting grafts which are never utilized, putting the donor through an unnecessary harvesting procedure

Potential Advantages

Decreased stress on the healthcare system

Decreased stress on the donor

Identification of donors who are poor mobilizers before day 0 of transplant

Ensured availability of donor graft in the event of donor death or unavailability

Decreased incidence of GVHD due to preferential destruction of T-cell subsets in the cryopreservation process

Collection and cryopreservation of DLI at the time of original stem cell procedure allows for readily available DLI

Table 24-2.  Potential advantages and disadvantages of using cryopreserved stem cell products over fresh stem cell products for allogeneic stem cell transplantation.

436  N.V. Frey and S.C. Goldstein

Chapter 24  Cryopreservation of Allogeneic Stem Cell Products 

There is also a paucity of data on the impact of cryopreservation on outcomes with unrelated donor transplants and DLI, both products which logistically would benefit greatly from cryopreservation. We, in conjunction with the NMDP, are performing a retrospective cohort study comparing outcomes of over 300 recipients of unrelated cryopreserved grafts with matched controls to further shed light on this issue. If further, more appropriately powered studies are performed yielding no significant difference between recipients of cryopreserved and fresh allografts, it may be that the noted trend by individual centers to use cryopreserved products is appropriate. The increased ease of scheduling collections and transplants in conjunction with ensured availability of a suitable donor graft on day 0 may financially and logistically justify the increased cost of cryopreservation. It is important to continue to monitor this practice with an eye on donor safety to ensure that an unacceptable number of unnecessary harvests are not performed.

References 1. Frey NV, Lazarus HM, Goldstein SC (2006) Has allogeneic stem cell cryopreservation been given the ‘cold shoulder’? An analysis of the pros and cons of using frozen versus fresh stem cell products in allogeneic stem cell transplantation. Bone Marrow Transplant 38(6):399–405 2. Harris DT, Schumacher MJ, Rychlik S et  al (1994) Collection, separation and cryopreservation of umbilical cord blood for use in transplantation. Bone Marrow Transplant 13(2):135–143 3. Rowley S (2004) Cryopreservation of hematopoietic Cells. In: Blum K (ed) Thomas’ hematopoitic cell transplantation. Malden, MA, Blackwell, pp 599–612 4. Lazarus HM, Kan F, Tarima S et  al (2007) Rapid Transport and Infusion of Hematopoietic Stem Cells Can Improve Outcome after Unrelated Donor Transplant. In 2007, p. 3063 5. Manilay JO, Sykes M (1998) Natural killer cells and their role in graft rejection. Curr Opin Immunol 10(5):532–538 6. Martin PJ (1993) Donor CD8 cells prevent allogeneic marrow graft rejection in mice: Potential implications for marrow transplantation in humans. J Exp Med 178(2):703–712 7. Marmont AM, Horowitz MM, Gale RP et  al (1991) T-cell depletion of HLAidentical transplants in leukemia. Blood 78(8):2120–2130 8. Kernan NA, Collins NH, Juliano L et  al (1986) Clonable T lymphocytes in T cell-depleted bone marrow transplants correlate with development of graft-v-host disease. Blood 68(3):770–773 9. Group SCTC (2005) Allogeneic peripheral blood stem-cell compared with bone marrow transplantation in the management of hematologic malignancies: An individual patient data meta-analysis of nine randomized trials. J Clin Oncol 23(22):5074–5087 10. Schmitz N, Eapen M, Horowitz MM et al (2006) Long-term outcome of patients given transplants of mobilized blood or bone marrow: A report from the international bone marrow transplant registry and the european group for blood and marrow transplantation. Blood 108(13):4288–4290 11. Robinson SN, Freedman AS, Neuberg DS et  al (2000) Loss of marrow reserve from dose-intensified chemotherapy results in impaired hematopoietic reconstitution after autologous transplantation: CD34(+), CD34(+)38(−), and week-6 CAFC assays predict poor engraftment. Exp Hematol 28(12):1325–1333 12. Tricot G, Jagannath S, Vesole D et al (1995) Peripheral blood stem cell transplants for multiple myeloma: Identification of favorable variables for rapid engraftment in 225 patients. Blood 85(2):588–596

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N.V. Frey and S.C. Goldstein 13. Weaver CH, Hazelton B, Birch R et al (1995) An analysis of engraftment kinetics as a function of the CD34 content of peripheral blood progenitor cell collections in 692 patients after the administration of myeloablative chemotherapy. Blood 86(10):3961–3969 14. Feugier P, Bensoussan D, Girard F et al (2003) Hematologic recovery after autologous PBPC transplantation: Importance of the number of postthaw CD34+ cells. Transfusion 43(7):878–884 15. Kim DH, Jamal N, Saragosa R et  al (2007) Similar outcomes of cryopreserved allogeneic peripheral stem cell transplants (PBSCT) compared to fresh allografts. Biol Blood Marrow Transplant 13(10):1233–1243 16. Ludgate ME, Dryden PR, Weetman AP, McGregor AM (1983) T-cell subset analysis of cryopreserved human peripheral blood mononuclear cells. Immunol Lett 7(3):119–122 17. Jones HP, Hughes P, Kirk P, Hoy T (1986) T-cell subsets: Effects of cryopreservation, paraformaldehyde fixation, incubation regime and choice of fluoresceinconjugated anti-mouse IgG on the percentage positive cells stained with monoclonal antibodies. J Immunol Methods 92(2):195–200 18. Tollerud DJ, Brown LM, Clark JW et  al (1991) Cryopreservation and long-term liquid nitrogen storage of peripheral blood mononuclear cells for flow cytometry analysis: Effects on cell subset proportions and fluorescence intensity. J Clin Lab Anal 5(4):255–261 19. Shlomchik WD (2007) Graft-versus-host disease. Nat Rev Immunol 7(5):340–352 20. Celluzzi CM, Welbon C (2003) A simple cryopreservation method for dendritic cells and cells used in their derivation and functional assessment. Transfusion 43(4):488–494 21. Hori S, Heike Y, Takei M et al (2004) Freeze-thawing procedures have no influence on the phenotypic and functional development of dendritic cells generated from peripheral blood CD14+ monocytes. J Immunother 27(1):27–35 22. Woods EJ, Liu J, Derrow CW et al (2000) Osmometric and permeability characteristics of human placental/umbilical cord blood CD34+ cells and their application to cryopreservation. J Hematother Stem Cell Res 9(2):161–173 23. Stroncek DF, Fautsch SK, Lasky LC et  al (1991) Adverse reactions in patients transfused with cryopreserved marrow. Transfusion 31(6):521–526 24. Lazarus HM, Magalhaes-Silverman M, Fox RM et al (1991) Contamination during in vitro processing of bone marrow for transplantation: Clinical significance. Bone Marrow Transplant 7(3):241–246 25. Rowley SD, Davis J, Dick J et  al (1988) Bacterial contamination of bone marrow grafts intended for autologous and allogeneic bone marrow transplantation. Incidence and clinical significance. Transfusion 28(2):109–112 26. Padley D, Koontz F, Trigg ME et al (1996) Bacterial contamination rates following processing of bone marrow and peripheral blood progenitor cell preparations. Transfusion 36(1):53–56 27. Eckardt JR, Roodman GD, Boldt DH et al (1993) Comparison of engraftment and acute GVHD in patients undergoing cryopreserved or fresh allogeneic BMT. Bone Marrow Transplant 11(2):125–131 28. Lasky LC, Van Buren N, Weisdorf DJ et al (1989) Successful allogeneic cryopreserved marrow transplantation. Transfusion 29(2):182–184 29. Stockschlader M, Hassan HT, Krog C et al (1997) Long-term follow-up of leukaemia patients after related cryopreserved allogeneic bone marrow transplantation. Br J Haematol 96(2):382–386 30. Stockschlader M, Kruger W, Kroschke G et al (1995) Use of cryopreserved bone marrow in allogeneic bone marrow transplantation. Bone Marrow Transplant 15(4):569–572 31. Stockschlader M, Kruger W, tom Dieck A et al (1996) Use of cryopreserved bone marrow in unrelated allogeneic transplantation. Bone Marrow Transplant 17(2):197–199

Chapter 24  Cryopreservation of Allogeneic Stem Cell Products  32. Shinkoda Y, Ijichi O, Tanabe T et  al (2004) Identical reconstitution after bone marrow transplantation in twins who received fresh and cryopreserved grafts harvested at the same time from their older brother. Clin Transplant 18(6):743–747 33. Lane TA, Medina B, Bashey A et  al (2007) Clinical efficacy of cryopreserved donor lymphocytes for infusion (DLI). Biol Blood Marrow Transplant 13:352 34. Sohn SK, Jung JT, Kim DH et al (2002) Prophylactic growth factor-primed donor lymphocyte infusion using cells reserved at the time of transplantation after allogeneic peripheral blood stem cell transplantation in patients with high-risk hematologic malignancies. Cancer 94(1):18–24 35. Collins RH Jr, Shpilberg O, Drobyski WR et al (1997) Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 15(2):433–444 36. Kolb HJ, Schattenberg A, Goldman JM et al (1995) Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood 86(5): 2041–2050 37. Suzuya H, Watanabe T, Nakagawa R et al (2005) Factors associated with granulocyte colony-stimulating factor-induced peripheral blood stem cell yield in healthy donors. Vox Sang 89(4):229–235 38. Anderlini P, Donato M, Chan KW et  al (1999) Allogeneic blood progenitor cell collection in normal donors after mobilization with filgrastim: The M.D. Anderson Cancer Center experience. Transfusion 39(6):555–560 39. Devine SM, Andritsos L, Todt L et al (2005) A pilot study evaluating the safety and efficacy of AMD3100 for the mobilization and transplantation of HLA-matched sibling donor hematopoietic stem cells in patients with advanced hematological malignancies. ASH Annu Meet Abstr 106(11):299 40. Devine SM, Flomenberg N, Vesole DH et al (2004) Rapid mobilization of CD34+ cells following administration of the CXCR4 antagonist AMD3100 to patients with multiple myeloma and non-Hodgkin’s lymphoma. J Clin Oncol 22(6):1095–1102 41. Flomenberg N, Devine SM, Dipersio JF et al (2005) The use of AMD3100 plus G-CSF for autologous hematopoietic progenitor cell mobilization is superior to G-CSF alone. Blood 106(5):1867–1874 42. Liles WC, Rodger E, Broxmeyer HE et  al (2005) Augmented mobilization and collection of CD34+ hematopoietic cells from normal human volunteers stimulated with granulocyte-colony-stimulating factor by single-dose administration of AMD3100, a CXCR4 antagonist. Transfusion 45(3):295–300

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sdfsdf

Chapter 25 Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic Stem Cell Transplantation Steven C. Goldstein and Selina Luger

The number of reduced-intensity conditioning (RIC)/nonmyeloablative transplants (NMT) has risen steadily over the last 10 years, now comprising approximately 30% of all allogeneic transplants performed annually [1]. Despite the rapid rise in its application, we have much to learn in terms of optimizing conditioning regimens, GvHD prophylaxis, identifying appropriate patient cohorts, and disease states, thus balancing the critical endpoints of chimerism, GvHD, relapse, and toxicity for the optimal utilization of this strategy. Building on the landmark work by Storb et al. [2, 3] in the canine model, the paradigm requiring myeloablation of the host immunohematopoietic system for successful long-term donor hematopoietic engraftment, has been replaced by the view that nonmyeloablative allogeneic transplantation is at its essence, the truest form of cellular immunotherapy. At its inception approximately a decade ago, the initial goal was to offer potentially curative treatment to patients previously excluded from consideration for standard allotransplantation secondary to age and/or other comorbid conditions. Early papers in RIC/ NMT focused on the critical goals of establishing donor hematopoiesis with low early treatment-related mortality; notably absent was the demonstration of long-term disease control [4]. Whether NMT/RIC can improve on the disease outcomes of standard transplantation as opposed to simply broadening the pool of potential candidates for allotransplantation remains an area of active investigation.

1. Defining Dose Intensity Although definitions of truly nonmyeloablative (e.g., TBI 200, fludarabine/ Cyclophosphamide as “immunosuppressive only”) regimens achieve wide consensus among BMT physicians, there remains a gray area for regimens in

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_25, © Springer Science + Business Media, LLC 2003, 2010

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Table 25-1.  Characteristics of NMT versus RIC conditioning. NMT

TBI 200 cGy +/− Fludarabine

RIC

Fludarabine plus

£ 500 cGy TBI £9 mg/kg total busulfan dose £140 mg/m2 total melphalan dose £10 mg/kg total thiotepa dose

NMT nonmyeloablative transplantation, RIC reduced-intensity conditioning, TBI total-body irradiation

which the dosages of fully ablative regimens have been attenuated to decrease early treatment-related morbidity and mortality, but still likely to require hematopoietic progenitor cell support (i.e., reduced intensity conditioning, RIC). Although scientifically interesting, studies to define the exact dosages at which stem cell support is required are not feasible and remain hypothetical in practice. Therefore, there is a spectrum of regimens which are deemed as RIC, but may or may not require stem cell rescue. The distinction between myeloablative, RIC, and NMT strategies goes beyond mere stratification by dose; the underlying principles of balancing treatment-related morbidity and mortality, graft-versus-host disease, and relapse are critical in defining the optimal strategy for an individual patient. In RIC, cytotoxic and immunosuppressive conditioning is combined to provide disease control and suppression of the host-versus-graft reaction, anticipating the several month interim required for the establishment of a graft-versus-tumor (GvT) effect by donor immunohematopoiesis; whereas the noncytotoxic, “immunosuppressive-only” conditioning employed in NMT via aggressive post-grafting immunosuppression is designed to minimize early toxicity with the goal of establishing adequate immunosurveillance and GvT with inherently less emphasis on early disease control. Although often used interchangeably, consensus definitions have been adopted to allow for more consistent characterization of transplant outcomes across standard, RIC, and NMT, as outlined in Table 25-1.

2. Does Dose Intensity Matter? 2.1. Dose Intensity in Standard Transplantation Although the rationale of high dose, myeloablative therapy in optimizing disease control for patients undergoing standard conditioning with allogeneic or autologous stem cell rescue has long been accepted as an important attribute in their curative potential, data demonstrating a significant disease-free or overall survival benefit of increasing dose intensity between myeloablative regimens is lacking. Although a meta-analysis by Hartman et al. [5] suggested a possible trend toward improved survival for patients receiving TBI-containing regimens compared to busulfan/cyclophosphamide, a subsequent study by Socie et al. [6] retrospectively compared the long-term outcomes for patients with myeloid leukemia (CML and AML) receiving busulfan/cyclophosphamide (BuCy2) vs. the more intensive cyclophosphamide/total-body irradiation (Cy/ TBI) across four randomized trials [7–10]; no statistically significant difference in survival was identified. In the report from the IBMTR, summarizing

Chapter 25  Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic 

the outcomes of AML patients in CR1 who underwent matched sibling bone marrow transplantation after BuCy2 (n = 318) or Cy/TBI (n = 200) between 1988 and 1996, Litzow et  al. [11] also noted no significant differences in treatment-related mortality, disease-free survivalor overall survival. Studies such as these require large number of patients and many years to complete; thus their extrapolation to current practice is potentially confounded by the subsequent implementation of busulfan dose-targeting and use of alternate stem cell sources. As noted above, despite their inherent limitations, most series published to date demonstrate a similar survival after myeloablative conditioning regardless of dose intensity. Recent investigations into novel, myeloablative regimens modified to decrease dose-related toxicity of conditioning (i.e., often referred to as RIC) have been associated with lower TRM without significant increase in relapse or decrease in overall survival, even among patients traditionally excluded by standard transplantation based on age or comorbidities [12–15] (see Table  25-2). These efforts warrant further investigation as they pursue many of the same goals of truly nonmyeloablative transplantation, specifically to expand the potential cohort of patients that might be treated with curative intent without undue toxicity. 2.2. Dose Intensity within RIC/NMT One must be cautious in extrapolating the lack of dose/conditioning effect in myeloablative conditioning to the realm of NMT/RIC where the kinetics of disease control must take into account the time it takes to establish donor immunosurveillance and withdrawal of immunosuppression to maximize GVL responses. In essence, the “intensity” of the various RIC/NMT regimens that may be critical for success is of a different quality; the intensity of immunosuppression, rather than cytotoxicity or myelosuppression. Whereas efforts to maximize dose intensity within standard transplantation have not been definitively proven to improve the outcome, variance between NMT/RIC conditioning regimens are much more likely to be relevant not only to overall outcomes, but will likely be closely linked in terms of tumor burden, disease activity, pace of disease progression, and inherent disease immunogenicity (i.e., susceptibility to GvT effect) at the time of transplantation. 2.2.1. Relevance of Tumor Burden/Disease Activity in NMT In addition to the two most common indications for pursuing NMT over standard transplantation, age and comorbidity, several studies have explored the impact of tumor burden, disease status, and rate of disease progression prior to NMT on outcomes after NMT and their relevance to the decision on whether to pursue NMT and/or choice of NMT regimen. A consistent message that can be drawn from both registry and single-center data is that active disease at the time of NMT/RIC, particularly in patients with AML and aggressive NHL, predicts for a significantly worse outcome as compared to patients transplanted in a minimal residual disease state [16–18]. Of note, however, is that even among leukemic patients with active disease (i.e., >5–10% blasts in marrow or periphery) there is a small subset of long-term disease free survivors after RIC (as opposed to strictly immunosuppressive therapy in NMT), suggesting that dose intensity is relevant for this high-risk cohort [19]. In their report of 102 AML patients receiving reduced intensity conditioning with

443

Bu 1 mg/kg × 10, flu 150/m2

Bu 130 mg/m2, then targeted to AUC 4500–5600; flu 160 mg/m2

Bu 1.0 mg/kg × 16 doses, flu 120 mg/ m2

Bu 130 mg/m /d × 4, flu 160/m2

10 mg/kg po

16 mg/kg po

520 mg/m IV

2

19

NR

54

25

43

NR

49

55

65 ± 11%

65% PFS

OS NR

51% EFS (1 year)

61% OS (1 year)

35% DFS (1.5 years)

42% OS (1.5 years)

52% EFS (1 year)

65% OS (1 year)

non-AML 26 ± 11%

74 ± 8%

4%

24%

0

8%

2%

AML

D100 High risk

Low risk

High risk

DFS % (2 years)

38

12.8 mg/kg IV

8

NRRM

GvHD% Total BU dose Acute Chronic

5% 1 year

17%

1 year

NR

19%

5%

2 years

URD

rel

Flu fludarabine, Bu busulfan, REL related donor, URD unrelated donor, ATG anti-thymocyte globulin, GvHD graft-versus-host disease, OS overall survival, NR not reported, EFS event-free survival, PFS progression-free survival, NRRM non-relapse-related mortality

N = 37 (sib)

Martino [75]

[33 rel, 36 URD]

N = 69

Field et al. [15]

[16 rel, 26 URD]

N = 42

Bornhauser et al. [14]

[60 rel, 36 URD]

N = 96

deLima et al. [13]

[49 rel, 21 URD]

2

Bu 3.2 mg/kg/D × 4 IV, flu 250 mg/m2, ATG

Russell et al. [12]

N = 70

Dose/schedule

N

Reference

Table 25-2.  Novel myeloablative regimens (selected references).

444  S.C. Goldstein and S. Luger

Chapter 25  Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic 

fludarabine/busulfan, Sayer et al. described an EFS of 49% for patients with less than 5% blasts, 24% for patients with 5–20% blasts, and 14% for patients with >20% blasts at 12 months of median follow-up [18]. It should be noted that the correlation between tumor burden and adverse outcome after transplant is not unique to NMT/RIC strategies, a similar finding has been noted for myeloablative approaches for acute leukemia as well [20]. In contrast to Sayer et al. [18], in which a small subset of patients with active disease could be salvaged via RIC, Shimoni et al. concluded that only patients undergoing myeloablative conditioning could be salvaged if they had active disease at the time of transplant [21]. 2.3. Myeloablative Conditioning vs. NMT/RIC The growing list of reports demonstrating a similar overall outcome for patients with acute leukemia and non-Hodgkin lymphoma who have undergone standard, myeloablative conditioning vs. RIC/NMT must be interpreted cautiously in light of the selection bias of the two approaches. These cohorts are inherently unbalanced, as in almost all circumstances patients undergoing RIC/NMT were deemed ineligible to undergo standard allotransplantation. Of course, achieving a similar outcome in a much higher-risk cohort of patients is a critical first step in the broader application of this strategy but, to date, there has been no conclusive data demonstrating a benefit of RIC/NMT in patients eligible to receive standard conditioning. As regimens evolve that are myeloablative, but clearly reduced in toxicity, the debate as to whether to maximize dose with the least toxicity vs. minimizing dose with the most immunotherapeutic potential, and in which circumstance, will continue unabated. Indeed, prospective trials comparing myeloablative to nonmyeloablative conditioning in the same cohort of patients are underway or in development in the United States and Europe. Until these studies are completed, we have only retrospective series with their inherent inadequacies on which to base our decision-making. Despite (or because of) the vagaries in comparing cohorts using different strategies over time, the results of most of the retrospective reports are remarkably similar; each has demonstrated that the overall survival outcomes after fully ablative vs. nonmyleloablative/RIC are nearly identical [21–29] (see Table 25-3).

3.  Does Dose Intensity Impact on Graft-Versus-Host Disease? 3.1. Acute GvHD after RIC/NMT Extrapolating from the initial GvHD paradigm [30] relating the cytokine storm triggered by tissue damage from conditioning regimens as a critical component of the acute pathophysiology of acute GvHD, one would predict that the incidence and severity of acute GvHD after RIC/NMT should be lower than that seen after standard conditioning, a trend often alluded to in small case series [31–33]. However, retrospective studies must be interpreted carefully before drawing this conclusion. Initial reports were in patients receiving TBI-based conditioning. There is remarkably little data available to correlate dose intensity with acute GvHD and whether it is relevant to non-TBI-based conditioning; a

445

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Table 25-3.  Outcomes after NMT/RIC versus Myeloablative (selected references).

Alyea et al. [28]

71 RIC Flu/bu OS at 1 year, 2 years AML 21, ALL1, CML 9, CLL 13, MDS 15, NHL 9, CMML 3 81 std bucy/cy-tbi

Aoudjhane et al. [22]

Valcarcel et al. [23]

NMT/RIC Myeloablative (%) (%) P-value 51, 39 38, 29 0.06

27, 25

0.24

AML 13, ALL 3, CML 33, CLL2, NRRM cum MDS17, NHL 10

PFS at 1 year, 2 years 40, 36 32

50

0.01

315 RIC

OS at 2 years

47

46

0.43

407 std

LFS at 2 years

40

44

0.8

AML CR1 245, AML CR2 52, AML adv 110

TRM at 2 years

18

32

<0.0001

57 RIC

OS at 2 years

59

52

TRM at 2 years

22

30

NS

OS at 3 years

41

45

0.8

621 std

PFS at 3 years

33

41

0.9

MDS 621

NRRM cum

22

32

41 RIC (flu/bu2)

OS at 2 years

47

50

NS

NRRM cum

8

22

0.05

OS at 3 years

62

71

NS

120 std

PFS at 3 years

55

67

NS

Follicular NHL 120

TRM at 3 years

28

25

NS

AML CR1 171, AML CR2 52, AML adv 92

AML 4/MDS 7,ALL 2, MPD 5, NHL 21, HL 5, MM13 100 std AML 29/MDS 12,, ALL 16, MPD 23, NHL 12, HL 0, MM 8

Martino et al. 215 RIC [25] MDS 215

Shimoni et al. [21]

AML 34, MDS 7 71 std (BuCy&flu/bu4) AML 61, MDS 10

Hari et al. [29]

88 RIC Follicular NHL 88

NMT nonmyeloablative transplantation, RIC reduced-intensity conditioning, std standard, flu/bu fludarabine/busulfan, BuCy busulfan/cyclophosphamide, Cy-TBI cyclophosphamide/total-body irradiation, OS overall survival, PFS progression-free survival, NRRM non-relapse-related mortality, LFS leukemia-free survival, TRM treatment-related mortality

recent report from Maruyama et al. demonstrated no difference in grade II–IV acute GvHD between patients undergoing RIC (flu/bu +/− TBI) and myeloablative conditioning [26]. In addition, recent data defining the critical role of host antigen-presenting cells in the initiation of acute GvHD [34] would suggest that low-intensity regimens, which one could postulate, would have less inhibitory impact on host APC, might even potentiate acute GvHD. Although there have been no large-scale, systematic studies to adequately address this issue, recently published outcomes from single centers comparing standard to

Chapter 25  Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic 

nonmyeloablative conditioning provide some intriguing insights. Despite the potentially confounding impact of traditional definitions and grading systems on more recently recognized syndromes of “late-onset acute GvHD” and mixed manifestations of acute and chronic GvHD in the same patient, several retrospective studies [33, 35–37] allow for some generalizations regarding the timing, severity, and response to therapy of acute GvHD after RIC/NMT: (1) initial observations that RIC/NMT may be associated with less AGvHD have to be tempered based on studies with longer follow-up. Although the incidence of AGvHD may be lower in the early post-transplant period (<100 days), the development of “late-onset AGvHD” (particularly when one takes into account the additional immunologic modifications often associated with NMT such as immunosuppressant withdrawal for relapse or mixed chimerism, and DLI) after NMT brings the overall incidence of AGvHD up to that seen after standard conditioning [28, 38]; (2) although there are inadequate data to confirm whether the incidence of steroid refractoriness differs between standard transplants and NMT, there is no data to suggest that there is a difference in response to salvage therapy once patients are refractory to corticosteroids; (3) although theoretically sound, choosing a specific immunosuppressive strategy based on organ involvement (e.g., infliximab for gut, extracorporeal photopheresis for skin) has not yet been demonstrated to improve overall outcome for GvHD after NMT, highlighting the need for well-designed prospective trials in this area. Two additional factors that impact on the overall incidence of GvHD, distinct from the intensity of the conditioning regimen, are (1) the use of peripheral blood stem cells (as opposed to bone marrow) in the overwhelming majority of patients receiving NMT/RIC conditioning [1] with its associated increase in acute and chronic GvHD, and (2) the use of T cell-depleting antibodies (e.g., alemtuzumab) as part of the conditioning regimen. The decrease in acute GvHD afforded by the use of these agents [39] has not yet been translated into improvement in overall outcomes , due in part, to potential increase in mortality associated with risk of infection and relapse. 3.2. TRM after NMT Despite the high risk nature of the patient cohorts undergoing RIC/NMT based on age and comorbid conditions, there is broad consensus across investigators that the 100-day NRRM after RIC/NMT is clearly lower than that after standard transplantation; in the range of 5% vs. 15–20%. However, the 1-year NRRM (or beyond) after both strategies are remarkably similar due to the the late impact of GvHD and infection. (Tables 25-3–25-5).

4. Exploration of Novel Regimens At present, there is no consensus regarding a single optimal regimen for either reduced-intensity or nonmyeloablative conditioning, nor should there be. A “one regimen fits all” philosophy is unlikely to fulfill the criteria for a successful balance of the critical endpoints required for patients with hematologic malignancies (minimizing NRRM, GvHD, while maximizing donor chimerism and disease control). In addition, although beyond the scope of this chapter, one must recognize that the preparative regimen itself cannot be viewed in

447

No

No

Giralt [76] F 125 M 140–180

Nakamura [77]

24 URD

Mult dx

AML 41

MDS 11

AML 56

MDS 20

40 at 3 years

33 at 1 year

17 at 100 D

19 at 1 year

9 at 100 D

15 at 100D

35 at 2 years

27 at 100D

37 at 100D 45 at 2 years

NRRM (%)

PFS (%)

62 at 1 year

39 at 1 year

28 at 2 years

39 at 2 years 31 at 2 years

49 at 1 year

C n = 8

A n = 28

C 8%

A 21%

C 62%

C 62%

A 53%

C 18%

40% at 2 A 33% years

41 at 3 years 37 at 3 years 27/76

76 at 1 year

39 SIB

7 URD

A 63%

A 49% C 68%

GvHD Relapse (II–IV)

54 at 2 years 51 at 2 years 16.3

28 at 2 years 23 at 2 years

OS (%)/ timepoint

26 m

18 m

11 m

38 m

Comments

No grade III/IV aGvHD

46/47 received BM, low incidence of chronic extensive GvHD

Not FM with higher toxdescribed icity profile than FB

Not 62% with active described AML or MDS >5%, Tumor burden strongly correlated with poor outcome

9

6

Not Lower relapse among described URD

7

Median f/u DLI

F fludarabine, M melphalan, sib sibling, URD unrelated donor, NRRM non-relapse-related mortality, OS overall survival, PFS progression-free survival, GvHD graft-versus-host disease, DLI donor lymphocyte infusion

55 sib

M100–140

79

Shimoni [16]

F150

25 URD

M 140

NO

27 sib

52

VanBesien [17]

F 150

41 URD

M 140

YES

35 sib

76

F 150

Tauro [79]

M 140

YES

47 URD Mult dx

Chakraverty [78] Yes

F 150

24 URD

AML 15

19 sib

M 140

MDS 28

Mult dx

DX

F 125

43

78

Alemtuzumab N

Author/Dose (total mg/m2)

Table 25-4.  Fludarabine/melphalan +/− alemtuzumab (selected references).

448  S.C. Goldstein and S. Luger

8 mg/kg PO

Bu 4 mg/kg/d × 2d, flu 180 mg/m2, ATG

Blaise [85]

24

25

29

46

49

42

19

27

31

64

33

nr

73

56

33

43

45

79% 2 years

39% 2 years

51% 1 year

40%

41% (2.5 years)

54% (CR1 pts 2 years)

PFS 65%

OS nr

81% DFS

85% OS

66% IV Bu 52% all pts

6

32

42%

33%

53% (not CR)

33% (CR)

5% 1 year

15%

19% IV Bu 39% 1 year all pt

Graft failure (%)

0

0

nr

4

5

5

0

Apr-36

flu fludarabine, Bu busulfan, urd unrelated donor, ATG anti-thymocyte globulin, GvHD graft-versus-host disease, OS overall survival, nr not reported, CR complete remission, NRRM nonrelapse-related mortality

33

6.6 mg/kg IV

8 mg/kg PO

Bu 3.3 mg/kg/d × 2 d IV, flu 150 mg/m2

Bu 4 mg/kg/d × 2d, flu 180 mg/m2

3.2 mg/kg IV

8 mg/kg

6.6 mg/kg IV

8 mg/kg PO

Bornhauser [84] 24 sib

28

Hamaki [83]

71

38 Bu 1 mg/kg × 8, Flu 150/m

Bu 0.8 mg/kg/d × 4d, flu 120 mg/m2

83 total/53 myeloid

Alyea [28]

2

45 Bu 1 mg/kg × 8, Flu 180/m2, ATG

23 other

36 Bu 6.6 mg/kg, flu 120–180

113

Schetelig [82]

54 Bu 8 mg/kg, flu 90–180/m2

Sayer [18]

37(sib)

Martino [75]

10 mg/kg po

Bu 4 mg/kg/d × 2 d/flu 180/m2, ATG

Slavin [81]

Bu 1 mg/kg × 10, flu 150/m2

8 mg/kg po

(11) Bu 0.8 mg/kg × 8 IV [(8)1 mg/kg po × 8], flu 150 mg/m2, ATG

Shimoni [80] 36/19flu-bu (>55 years urd)

25

6.4 mg/kg IV 8 mg/kg po

Dose/schedule

GvHD% OS Total Bu dose/ route Acute Chronic NRRM

N

Reference

Table 25-5.  Fludarabine/busulfan (selected references).

Chapter 25  Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic  449

450 

S.C. Goldstein and S. Luger

isolation from the GvHD prophylaxis regimen, stem cell source, and use of DLI. Indeed, each regimen is likely to require its own counterbalance strategy, which takes into account its potential inherent weaknesses. For example, the excellent early protection from acute GvHD provided by alemtuzumab may have a less desirable impact on maintaining full-donor chimerism and/or minimizing risk of relapse thus warranting a planned strategy such as “prophylactic” DLI in order to maximize immune and hematopoietic reconstitution later in the post-transplant course. 4.1. Disease-Specific Regimens? Published prospective, comparative trials across regimens within specific diseases are lacking. Whereas many clinicians may consider one drug or regimen more “anti-lymphoid” (e.g., fludarabine/melphalan or fludarabine/TBI) or “anti-myeloid” (e.g., fludarabine/busulfan), there is no conclusive evidence at present that customizing the regimen based on disease influences outcome (though disease state, i.e., remission vs. MRD vs. active disease, is likely to correlate with outcome based on the “intensity” of the regimen as described above), highlighting the need for disease-specific multi-institutional prospective trials [40]. 4.2. Fludarabine-Based Conditioning Purine analogs, primarily fludarabine, have become the foundation around which most nonmyeloablative and reduced-intensity regimens have been constructed. Indeed, there is also a growing trend in purine analog-based myeloablative conditioning as well [12–15]. Among NMT and RIC regimens, the most common combinations published to date have been fludarabine (125–180 mg/2) with melphalan, busulfan, or TBI with or without the addition of serotherapy in the form of anti-thymocyte globulin (ATG) or alemtuzumab [Tables 25.4, 25.5]. 4.3. Fludarabine-Melphalan vs. Fludarabine-Busulfan In an important retrospective (nonrandomized) study of 151 patients with both myeloid and lymphoid malignancies, comparing 72 patients conditioned with fludarabine/busulfan (flu/bu) vs. 79 patients conditioned with fludarabine/ melphalan (flu/mel), Shimoni et  al. [16] described a lower cumulative incidence of acute GvHD (33 vs. 53%), death from organ toxicity (10 vs. 23%) and graft-versus-host disease (6 vs. 17%) in the flu/bu cohort as compared to flu/mel group, respectively, but did not identify a significant difference in the overall survival between the two regimens. The higher reported incidence of acute GvHD after flu/mel is intriguing in that the flu/mel cohort had significantly fewer patients with unrelated donors and therefore expected to be at a lower risk for acute GvHD as a group. Confounding factors in this observation include the use of ATG in the conditioning of recipients of unrelated grafts, but not sibling grafts, as well as the higher incidence of mucositis among flu/ mel patients as compared to flu/bu (49 vs. 29%), likely not coincidental to the higher observed incidence of gut GvHD in the flu/mel cohort. Although no difference in the overall survival between the two regimens can be identified when patients with active disease are included, a subset analysis of patients

Chapter 25  Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic 

in remission at the time of transplant suggests a possible overall survival advantage of flu/bu over flu/mel, for patients in remission, likely , due in large part,to a lower rate of NRRM despite a similar rate of relapse. 4.4. Fludarabine/TBI The largest experience in combining low-dose total-body irradiation (200 cGy) with and without fludarabine (90  mg/m2) has been described by the Seattle Consortium [41, 42]. Retrospective cohort analyses of >100 patients with myeloid malignancies (AML, MDS, CML) have demonstrated feasibility with sustained engraftment in recipients of both sibling and unrelated grafts with a 1-year nonrelapse mortality rate of ~15–20% [27, 43]. Of note, though several early patients (with sibling donors) received TBI alone, the relatively high rate of graft failure prompted the addition of fludarabine to the remaining cohort of sibling and all unrelated recipients. This series was one of the first to note the potential for divergent outcome based on donor source, rather than regimen. Specifically, there was a statistically significant decrease in relapse at 2 years among CR1 recipients of unrelated grafts when compared to CR1 recipients of sibling grafts (16 vs. 50%; p = .005) and a trend towards improved 2 years overall survival (63 vs. 44%; p = 0.13), despite a similar incidence of acute and chronic graft-versus-host disease [43]. The potential for an improved GvL effect, putatively attributed to the use of unrelated donors in this cohort, did not translate into a lower relapse rate in patients beyond CR1 however and may have been confounded by differences in the use of fludarabine, prior autologous transplantation, and DLI between the two groups. 4.5. Addition of Alemtuzumab Although the use of serotherapy in the form of pre-transplant conditioning with anti-CD52 monoclonal antibody (alemtuzumab) either in  vivo or ex vivo/“in the bag” has been in clinical practice for more than a decade [44–46], the immunologic mechanism and optimal utilization of this agent remains an area of active investigation. Early reports investigating the addition of alemtuzumab to ablative and nonmyeloablative conditioning regimens have illustrated the double-edged sword of this strategy. While there is a consensus among investigators regarding the reduction of acute graft-versus-host disease among recipients of alemtuzumab, most apparent in the higher-risk unrelated cohort [47], ambiguity persists in the literature in terms of its longerterm impact on donor chimerism, infection and risk of relapse [46, 48–53]. Analyzing these studies in terms of total dose of alemtuzumab, timing of administration, and the specific endpoints being reported provide insights into potential explanations for the disparate conclusions between investigators. For example, depending on the timing and dose of administration of alemtuzumab, the prolonged half-life likely impacts on clinical endpoints via its dual role in both depleting donor T cells, B cells, and APCs in the graft [45] and host lymphoid and antigen-presenting cells in the patient [54, 55]. As the monoclonal antibody may be still in circulation on day 0 at the time of the stem cell infusion, the depleting impact on donor cells may amelioriate GvHD, but may poteniate risk of infecton, relapse, and failure to achieve full-donor chimerism, whereas the impact of alemtuzamab on host lymphoid cells and APCs may facilitate engraftment and decrease GvHD [34]. Although surprisingly little data is

451

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S.C. Goldstein and S. Luger

available in terms of alemtuzumab levels at the time of stem cell infusion and its relevance to in vivo T cell depletion and outcome, recent reports [49, 52, 53, 56] evaluating a range of alemtuzumab dosages provide some important insights. By titrating the alemtuzumab dose to 50 mg from 100 mg given subcutaneously from d-7 to d-3, Khouri et al. [56] may have separated the hostdepletion of lymphoid cells and APCs from the Tcell depletion of the donor graft based on their finding that no detectable alemtuzumab could be identified in circulation via ELISA [57] after only 48 h beyond the last dose. This is in contrast to the findings of Rebello et al. [57] which suggested that the 50-mg dose given over a similar time frame prior to stem cell infusion was associated with peak concentrations of alemtuzumab (2.5 ucg/ml) on day 0 that was still above the level necessary for opsonization of lymphocytes. Indeed, circulating alemtuzumab was still detected up to 11 days after the 50-mg total dose with a terminal half-life of 15–21 days. Other investigators have postulated that “in vitro” alemtuzumab doses, as low as 20 mg, added “to the bag” may provide an alternate strategy, though immune reconstitution remains a concern [49, 53]. While retrospectively reconciling these disparate findings is not possible, one explanation may be that Khouri et al. cohort analysis was limited to patients with B-cell malignancies (i.e., CD-52 positive), thereby serving as a potential “sink” for circulating antibody (as compared to patients undergoing transplantation with nonlymphoid malignancies [57]), and possibly decreasing the levels of detectable antibody [58]. Potentiating functional depletion of host APCs while minimizing T cell depletion of the donor graft may be critical for achieving the long-sought balance between decreasing GvH and maximizing GvT [59]. Optimal titration of both the dose and timing of alemtuzumab [53] (as well as considering the impact of CD-52 positivity of the disease) remains an active area of investigation. 4.6. Extracorporeal Photopheresis-Based Conditioning On the basis of phase I/II results [60, 61] exploring the reduced-intensity combination of 2-deoxycoformycin, TBI (600 rads) and extracorporeal photopheresis (ECP) in patients with hematologic malignancies, a cooperative group Phase II study has been initiated for patients with myelodysplasia. Although the putative immunomodulatory effects of the specific components of the conditioning regimen cannot be distinguished from each other, it is postulated that ECP may (1) attenuate Th1-mediated cytokine secretion by activated T-helper cells, (2) cause a shift in the DC1/DC2 ratio favoring plasmacytoid rather than monocytoid dendritic cell profiles, and (3) decrease antigen responsiveness by dendritic cells [62]. The low rates of GvHD with this regimen appear promising, but the impact on disease control and relapse rates will require longer follow-up. Relating an ECP-mediated increase in Treg’s as a possible mechanism for lowering acute GvHD is supported by the work of Lamioni et  al. who demonstrated an in increase in Treg’s after ECP as therapy for cardiac transplant rejection [63]. 4.7. TLI-Based Conditioning Murine models [64, 65] demonstrating the protective effect of total lymphoid irradiation (TLI) against acute GvHD, (as opposed to total-body irradiation) prompted a phase I trial in humans in which total lymphoid irradiation, rather than TBI was used in conjunction with ATG as protective conditioning against GvHD. This strategy was based on the postulated skewing of the T cell to a

Chapter 25  Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic 

Th2 phenotype by TLI (but not TBI) and enhanced IL-4 (inhibitory) secretion with “downstream” abrogating effects on the inflammatory cytokine cascade. Another proposed mechanism is the selection of regulatory T cells and NK-T cells by low-dose lymphoid radiation, without compromising the effector CD8+ population, thus allowing for the potential separation of the GvL effect mediated by donor CD8+ T cells from the GvHD-abrogating effect of regulatory T cells and NK-T cells. Thirty-seven patients with lymphoid (n = 24) and myeloid (n = 13) disease received 80  cGy TLI over 11 days with ATG prior to receiving PBSC grafts from matched sibling (n = 23) and unrelated (n = 14) donors, followed by cyclosporine and mycophenolate mofetil [66]. Despite the advanced disease status in the majority of patients entering transplant, the response rates and disease-free survival appear very promising when compared to historical controls, though longer-term follow-up in a larger cohort is required. The incidence of grade II–IV acute GvHD was remarkably low for the entire cohort, 1/37 (3%). Of particular interest is the demonstration of a tenfold increase in NK-T cells after TLI as well as an increase in IL-4 production and decrease in proliferative response in CD4+ cells after TLI when compared to CD4+ cells after TBI-containing regimens [66]. 4.8. Future Directions Despite the rising number of patients undergoing allogeneic transplantation with nonmyeloablative or reduced-intensity conditioning, the proportion of patients achieving cure remains disappointing. Improving overall outcomes will require a two-tiered approach. While smaller, single-center phase I/II studies will continue to explore novel strategies to enhance the graft-versusmalignancy effect while minimizing graft-versus-host disease via incorporation of novel methods of immunosuppression (e.g., TLI [66], targeting of APCs [34, 67]), cellular therapy (e.g., NK infusions [68], preemptive DLI [69–72]), vaccines [73], and gene therapy [74], it remains critical that multicenter, prospective, randomized trials comparing disease-specific regimens in the NMT/RIC and myeloablative setting are prioritized to ultimately define the optimal strategy for the individual patient.

References 1. Pasquini MC (2006) Current use and outcome of hematopoietic stem cell transplantation: part I - CIBMTR Summary Slides, 2005. CIBMTR Newslett 12(1):5–8 2. Storb R et al (1997) Stable mixed hematopoietic chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation. Blood 89(8):3048–3054 3. Yu C et  al (1995) DLA-identical bone marrow grafts after low-dose total body irradiation: effects of high-dose corticosteroids and cyclosporine on engraftment. Blood 86(11):4376–4381 4. Giralt S et al (1997) Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: harnessing graft-versus-leukemia without myeloablative therapy. Blood 89(12):4531–4536 5. Hartman AR, Williams S, Dillon JJ (1998) Survival, disease-free survival and adverse effects of conditioning for allogeneic bone marrow transplantation with busulfan/cyclophosphamide vs total body irradiation: a meta-analysis. Bone Marrow Transplant 22:439–443 6. Socie G et al (2001) 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 98(13):3569–3574

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S.C. Goldstein and S. Luger 7. Blaise D, Maraninchi D, Archimbaud E (1992) Allogeneic bone marrow transplantation for acute myeloid leukemia in first remission: a randomized trial of a busulfancytoxan versus cytoxan-total body irradiation as preparative regimen: a report from the Groupe d’Etudes de la Greffe de M. Blood 79:2578–2582 8. Ringden O, Ruutu T, Remberger M (1994) A randomized trial of 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 83:2723–2730 9. Clift RA et  al (1994) Marrow transplantation for chronic myeloid leukemia: a randomized study comparing cyclophosphamide and total body irradiation with busulfan and cyclophosphamide. [comment]. Blood 84(6):2036–2043 10. Devergie A, Blaise D, Attal M (1995) Allogeneic bone marrow transplantation for chronic myeloid leukemia in first chronic phase: a randomized trial of busulfancytoxan versus cytoxan-total body irradiation as preparative regimen: a report from the French Society of Bone Marrow. Blood 85:2263–2268 11. Litzow MR et al (2002) Comparison of outcome following allogeneic bone marrow transplantation with cyclophosphamide-total body irradiation versus busulphancyclophosphamide conditioning regimens for acute myelogenous leukaemia in first remission. Br J Haematol 119(4):1115–1124 12. Russell J et al (2002) Once-daily intravenous busulfan given with fludarabine as conditioning for allogeneic stem cell transplantation: study of pharmacokinetics and early clinical outcomes. Biol Blood Marrow Transplant 8:468–476 13. deLima M, Couriel D, Thall PF (2004) Once-daily intravenous busulfan and fludarabine: clinical and pharmacokinetic results of a myeloablative, reducedtoxicity conditioning regimen for allogeneic stem cell transplantation in AML and MDS. Blood 104:857–864 14. Bornhauser M et al (2003) Conditioning with fludarabine and targeted busulfan for transplantation of allogeneic hematopoietic stem cells. Blood 102:820–826 15. Field T et al (2006) Busulfan area-under-the-curve finding study within a busulfan/ fludarabine (BuFlu) conditioning regimen before allogeneic hematopoietic cell transplantation. Blood 108:832a 16. Shimoni A et  al (2007) Comparison between two fludarabine-based reducedintensity conditioning regimens before allogeneic hematopoietic stem-cell transplantation: fludarabine/melphalan is associated with higher incidence of acute graft-versus-host disease and non-relapse mortality and lower incidence of relapse than fludarabine/busulfan. Leukemia 21(10):2109–2116 17. van Besien K et al (2005) Fludarabine, melphalan, and alemtuzumab conditioning in adults with standard-risk advanced acute myeloid leukemia and myelodysplastic syndrome. J Clin Oncol 23(24):5728–5738 18. Sayer HG et al (2003) 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 31(12):1089–1095 19. de Lima M et  al (2004) Nonablative versus reduced-intensity conditioning regimens in the treatment of acute myeloid leukemia and high-risk myelodysplastic syndrome: dose is relevant for long-term disease control after allogeneic hematopoietic stem cell transplant. Blood 104(3):865–872 20. Kebriaei P et  al (2005) Impact of disease burden at time of allogeneic stem cell transplantation in adults with acute myeloid leukemia and myelodysplastic syndromes. Bone Marrow Transplant 35(10):965–970 21. Shimoni A et al (2006) Allogeneic hematopoietic stem-cell transplantation in AML and MDS using myeloablative versus reduced-intensity conditioning: the role of dose intensity. Leukemia 20(2):322–328 22. Aoudjhane M et al (2005) Comparative outcome of reduced intensity and myeloablative conditioning regimen in HLA identical sibling allogeneic haematopoietic

Chapter 25  Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic  stem cell transplantation for patients older than 50 years of age with acute myeloblastic leukaemia: a retrospective survey from the Acute Leukemia Working Party (ALWP) of the European group for Blood and Marrow Transplantation (EBMT). Leukemia 19(12):2304–2312 23. Valcarcel D et al (2005) Conventional versus reduced-intensity conditioning regimen for allogeneic stem cell transplantation in patients with hematological malignancies. Eur J Haematol 74(2):144–151 24. Massenkeil G et  al (2005) Survival after reduced-intensity conditioning is not inferior to standard high-dose conditioning before allogeneic haematopoietic cell transplantation in acute leukaemias. Bone Marrow Transplant 36(8):683–689 25. Martino R et al (2006) Retrospective comparison of reduced-intensity conditioning and conventional high-dose conditioning for allogeneic hematopoietic stem cell transplantation using HLA-identical sibling donors in myelodysplastic syndromes. Blood 108(3):836–846 26. Maruyama D et  al (2007) Comparable antileukemia/lymphoma effects in nonremission patients undergoing allogeneic hematopoietic cell transplantation with a conventional cytoreductive or reduced-intensity regimen. Biol Blood Marrow Transplant 13(8):932–941 27. Scott BL et al (2006) Myeloablative vs nonmyeloablative allogeneic transplantation for patients with myelodysplastic syndrome or acute myelogenous leukemia with multilineage dysplasia: a retrospective analysis. Leukemia 20(1):128–135 28. Alyea EP et al (2005) Comparative outcome of nonmyeloablative and myeloablative allogeneic hematopoietic cell transplantation for patients older than 50 years of age. Blood 105(4):1810–1814 29. Hari P et al (2008) Allogeneic transplants in follicular lymphoma: higher risk of disease progression after reduced-intensity compared to myeloablative conditioning. Biol Blood Marrow Transplant 14(2):236–245 30. Ferrara J, Deeg H (1991) Graft-versus-host disease. N Engl J Med 324:667–674 31. Mielcarek M et al (2003) Graft-versus-host disease after nonmyeloablative versus conventional hematopoietic stem cell transplantation. Blood 102(2):756–762 32. Sorror M et  al (2005) Lessened severe graft-versus-host after “minitransplantations”. Blood 105(6):2614 33. Couriel DR et al (2004) Acute and chronic graft-versus-host disease after ablative and nonmyeloablative conditioning for allogeneic hematopoietic transplantation. Biol Blood Marrow Transplant 10(3):178–185 34. Shlomchik W et al (1999) Prevention of graft-versus-host disease by inactivation of host antigen-presenting cells. Science 285:412–415 35. Mielcarek M, Storb R (2005) Graft-vs-host disease after non-myeloablative hematopoietic cell transplantation. Leuk Lymphoma 46(9):1251–1260 36. Mielcarek M et al (2005) Prognostic relevance of “early-onset” graft-versus-host disease following non-myeloablative haematopoietic cell transplantation. Br J Haematol 129(3):381–391 37. Levine J et al (2003) Lowered-intensity preparative regimen for allogeneic stem cell transplantation delays acute graft-versus-host disease but does not improve outcome for advanced hematologic malignancy. Biol Blood Marrow Transplant 9:189–197 38. Couriel D, Giralt S (2005) Graft vs Host Disease in Nonmyeloablative Transplant. In: Ferrara JL, Cooke KR, Deeg HJ (eds) Graft vs Host Disease. Marcel Dekker, New York 39. Loren A et  al (2005) Intensive graft-versus-host disease prophylaxis is required after unrelated donor non-myeloablative stem cell transplantation. Bone Marrow Transplant 35:921–926 40. Deeg HJ et al (2006) Optimization of allogeneic transplant conditioning: not the time for dogma. Leukemia 20(10):1701–1705 41. McSweeney PA et  al (2001) Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graftversus-tumor effects. Blood 97(11):3390–3400

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S.C. Goldstein and S. Luger 42. Maris MB et al (2003) HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative conditioning for patients with hematologic malignancies. Blood 102(6):2021–2030 43. Hegenbart U et al (2006) Treatment for acute myelogenous leukemia by low-dose, total-body, irradiation-based conditioning and hematopoietic cell transplantation from related and unrelated donors. J Clin Oncol 24(3):444–453 44. Hale G et  al (1998) Improving the outcome of bone marrow transplantation by using CD52 monoclonal antibodies to prevent graft-versus-host disease and graft rejection. Blood 92(12):4581–4590 45. Hale G, Cobbold S, Waldmann H (1988) T cell depletion with CAMPATH-1 in allogeneic bone marrow transplantation. Transplant 45(4):753–759 46. Kottaridis PD et al (2000) In vivo CAMPATH-1H prevents graft-versus-host disease following nonmyeloablative stem cell transplantation. Blood 96(7):2419–2425 47. Loren AW et al (2005) Intensive graft-versus-host disease prophylaxis is required after unrelated-donor nonmyeloablative stem cell transplantation. Bone Marrow Transplant 35(9):921–926 48. Perez-Simon JA et  al (2002) Nonmyeloablative transplantation with or without alemtuzumab: comparison between 2 prospective studies in patients with lymphoproliferative disorders. Blood 100(9):3121–3127 49. Morris EC et  al (2003) Pharmacokinetics of alemtuzumab used for in  vivo and in vitro T-cell depletion in allogeneic transplantations: relevance for early adoptive immunotherapy and infectious complications. Blood 102(1):404–406 50. Morris E et  al (2004) Outcomes after alemtuzumab-containing reduced-intensity allogeneic transplantation regimen for relapsed and refractory non-Hodgkin lymphoma. Blood 104(13):3865–3871 51. Michaelis L et al (2007) Chimerism does not predict for outcome after alemtuzumab based conditioning. Bone Marrow Transplant 40(2):181 52. Juliusson G et  al (2006) Subcutaneous alemtuzumab vs ATG in adjusted conditioning for allogeneic transplantation: influence of Campath dose on lymphoid recovery, mixed chimerism and survival. Bone Marrow Transplant 37(5):503–510 53. Hale G et  al (2001) CAMPATH-1 antibodies in stem-cell transplantation. Cytotherapy 3(3):145–164 54. Klangsinsirikul P et  al (2002) Campath-1G causes rapid depletion of circulating host dendritic cells (DCs) before allogeneic transplantation but does not delay donor DC reconstitution. Blood 99(7):2586–2591 55. Ratzinger G et  al (2003) Differential CD52 expression by distinct myeloid dendritic cell subsets: implications for alemtuzumab activity at the level of antigen presentation in allogeneic graft-host interactions in transplantation. Blood 101(4):1422–1429 [erratum appears in Blood. 2005 Apr 15;105(8):3018 Note: dosage error in text] 56. Khouri IF et al (2004) Low-dose alemtuzumab (Campath) in myeloablative allogeneic stem cell transplantation for CD52-positive malignancies: decreased incidence of acute graft-versus-host-disease with unique pharmacokinetics. [see comment]. Bone Marrow Transplant 33(8):833–837 57. Rebello P et  al (2001) Pharmacokinetics of CAMPATH-1H in BMT patients. Cytotherapy 3(4):261–267 58. Hale G et  al (2004) Blood concentrations of alemtuzumab and antiglobulin responses in patients with chronic lymphocytic leukemia following intravenous or subcutaneous routes of administration. Blood 104(4):948–955 59. Russell NH, Byrne JL (2004) In vivo Campath for the prevention of GvHD following allogeneic HSCT: effects of dose, schedule and antibody type. [comment]. Bone Marrow Transplant 34(12):1101–1102 60. Chan GW et al (2003) Reduced-intensity transplantation for patients with myelodysplastic syndrome achieves durable remission with less graft-versus-host disease. [see comment]. Biol Blood Marrow Transplant 9(12):753–759

Chapter 25  Concepts and Controversies in the Use of Novel Preparative Regimens for Allogeneic  61. Miller KB et  al (2004) A novel reduced intensity regimen for allogeneic hematopoietic stem cell transplantation associated with a reduced incidence of graft-versus-host disease. Bone Marrow Transplant 33(9):881–889 62. Foss FM, Gorgun G, Miller KB (2002) Extracorporeal photopheresis in chronic graft-versus-host disease. Bone Marrow Transplant 29(9):719–725 63. Lamioni A et al (2005) The immunological effects of extracorporeal photopheresis unraveled: induction of tolerogenic dendritic cells in vitro and regulatory T cells in vivo. Transplantation 79(7):846–850 64. Lan F et al (2003) Host conditioning with total lymphoid irradiation and antithymocyte globulin prevents graft-versus-host disease: the role of CD1-reactive natural killer T cells. Biol Blood Marrow Transplant 9(6):355–363 65. Lan F et  al (2001) Predominance of NK1.1+TCR alpha beta+ or DX5+TCR alpha beta+ T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: “natural suppressor” cells. J Immunol 167(4):2087–2096 66. Lowsky R et al (2005) Protective conditioning for acute graft-versus-host disease. [see comment]. N Engl J Med 353(13):1321–1331 [erratum appears in N Engl J Med. 2006 Feb 23;354(8):884] 67. Reddy P et  al (2005) A crucial role for antigen-presenting cells and alloantigen expression in graft-versus-leukemia responses. Nat Med 11(11):1244–1249 68. Miller JS et al (2005) Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 105(8):3051–3057 69. Baron F, Beguin Y (2002) Preemptive cellular immunotherapy after T-cell-depleted allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 8:351–359 70. Barrett AJ et al (1998) T cell-depleted bone marrow transplantation and delayed T cell add-back to control acute GVHD and conserve a graft-versus-leukemia effect. Bone Marrow Transplant 21(6):543–551 71. Massenkeil G et  al (2003) Reduced intensity conditioning and prophylactic DLI can cure patients with high-risk acute leukaemias if complete donor chimerism can be achieved. Bone Marrow Transplant 31(5):339–345 72. Montero A et al (2006) T-cell depleted peripheral blood stem cell allotransplantation with T-cell add-back for patients with hematological malignancies: effect of chronic GVHD on outcome. Biol Blood Marrow Transplant 12(12):1318–1325 73. Barrett AJ, Rezvani K (2007) Translational mini-review series on vaccines: Peptide vaccines for myeloid leukaemias. Clin Exp Immunol 148(2):189–198 74. Rossig C, Brenner MK (2004) Genetic modification of T lymphocytes for adoptive immunotherapy. Mol Ther 10(1):5–18 75. Martino R et  al (2002) Evidence for a graft-versus-leukemia effect after allogeneic peripheral blood stem cell transplantation with reduced-intensity conditioning in acute myelogenous leukemia and myelodysplastic syndromes. Blood 100(6):2243–2245 76. Giralt S et al (2001) Melphalan and purine analog-containing preparative regimens: reduced-intensity conditioning for patients with hematologic malignancies undergoing allogeneic progenitor cell transplantation. Blood 97(3):631–637 77. Nakamura R et al (2007) Reduced-intensity conditioning for allogeneic hematopoietic stem cell transplantation with fludarabine and melphalan is associated with durable disease control in myelodysplastic syndrome. Bone Marrow Transplant 40(9):843–850 78. Chakraverty R et  al (2002) Limiting transplantation-related mortality following unrelated donor stem cell transplantation by using a nonmyeloablative conditioning regimen. Blood 99(3):1071–1078 79. Tauro S et al (2005) Allogeneic stem-cell transplantation using a reduced-intensity conditioning regimen has the capacity to produce durable remissions and long-term disease-free survival in patients with high-risk acute myeloid leukemia and myelodysplasia. J Clin Oncol 23(36):9387–9393

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S.C. Goldstein and S. Luger 80. Shimoni A et  al (2005) Hematopoietic stem-cell transplantation from unrelated donors in elderly patients (age >55 years) with hematologic malignancies: older age is no longer a contraindication when using reduced intensity conditioning. [see comment]. Leukemia 19(1):7–12 81. Slavin S et al (1998) 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 91:756–763 82. Schetelig J et al (2004) Reduced-intensity conditioning with busulfan and fludarabine with or without antithymocyte globulin in HLA-identical sibling transplantation–a retrospective analysis. Bone Marrow Transplant 33(5):483–490 83. Hamaki T et al (2004) Reduced-intensity stem cell transplantation from an HLAidentical sibling donor in patients with myeloid malignancies. Bone Marrow Transplant 33(9):891–900 84. Bornhauser M et al (2000) Dose-reduced conditioning for allogeneic blood stem cell transplantation: durable engraftment without antithymocyte globulin. Bone Marrow Transplant 26(2):119–125 85. Blaise DP et al (2005) Reduced intensity conditioning prior to allogeneic stem cell transplantation for patients with acute myeloblastic leukemia as a first-line treatment. Cancer 104(9):1931–1938

Chapter 26 Allogeneic Haematopoietic Stem Cell Transplantation and Natural Killer Cell Alloreactivity Franco Aversa and Andrea Velardi

1. Introduction Despite advances in chemotherapy, most adults with acute lymphoblastic leukemia (ALL) or Acute Myeloid Leukemia (AML) relapse and few survive when they have unfavorable cytogenetics at diagnosis, when they do not achieve complete remission (CR) after the first induction cycle and when they are in second or later remission [1–3]. Under these circumstances, an allogeneic hematopoietic stem cell transplant (HSCT) is preferred as post-remission therapy [4–6]. As only 30% of patients have a matched sibling donor, the only option is transplantation from an alternative donor. Phenotypically matched unrelated donors are the most widely sought-after for allogeneic transplant but have two major limitations. Molecular analysis ensures more accurate close matching, which lowers the risk of GvHD, but reduces the chance of finding a suitable matched donor [7–9]. The time-lapse from registration to donor identification can lead to disease progression in patients who urgently need transplantation. Unrelated umbilical cord blood transplantation (UCBT) has emerged as a viable option, at least in children. It offers the advantages of immediate availability of cryopreserved samples, easy procurement with no risk to the donor, and acceptance of minor mismatching (2/6 antigens). For adults UCBT is seldom considered because the divergence between body weight and the number of hematopoietic cells in a cord blood unit, particularly if associated with a two-antigen mismatch, increases the risk of graft failure and delays hematopoietic reconstitution [10–13]. Another source of stem cells is the family donor with whom the patient shares only one HLA haplotype for HLA-A, B, C and DR. These donors offer several advantages: (a) immediate availability for all transplant candidates; (b) selection of the best of many relatives on the basis of age, infectious disease status and natural killer (NK) cell alloreactivity (see below) [14–16]; (c) option to change donor if a poor stem cell mobiliser or if optimal graft

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_26, © Springer Science + Business Media, LLC 2003, 2010

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Table 26-1.  HSC Transplantation from alternative donors. MUD

UCB

HAPLO

Candidate donors with HLA-A + B + DRB1 typing 16–56%

~80%

100%

Median search time

3–6 months

<1 month

1 week

Optimal donor selection criteria

HLA matching

Cell dose

NK-mismatch

Donor availability

5–60%

40–60%

~100%

Donor identified but unavailable

30%

1%

1%

Risk of congential disease

No

Yes

No

Risk to donors

Yes

No

Yes

Graft processing

Usually none

Usually none

Stem cell selection

Conditioning regimen

Myeloablative/RIC Myeloablative/RIC Myeloablative

Post transplant immunosuppression

Yes

Yes

No

Adoptive cell therapy, if required

Possible

Impossible

Possible

Major complications

GvHD

engraftment

infections

MUD Matched unrelated donor; UCB unrelated cord blood; RIC reduced intensity conditioning; NIK natural killer cells

composition is not achieved; and (d) access to donor-derived cellular therapies if required after transplantation. Furthermore, unlike transplants from other alternative stem cell sources, for nearly all patients who reject the graft, the haploidentical transplant offers the advantage of another family member who is immediately available as an alternative donor or even a second graft from the original donor (Table 26.1). 1.1. Natural Killer Cell Alloreactivity Another striking advantage which has emerged from the unique setting of the one haplotype mismatched transplant was the benefits of NK cell alloreactivity [17, 18]. NK cell activation is regulated by a balance between inhibitory and activating receptors. In humans, currently 16 inhibitory killer-cell Ig-like receptors (KIR) genes and pseudo genes are known to codify for inhibitory and activating KIRs. Inhibitory KIRs recognize amino acids in the COOH-terminal portion of the MHC class I a1 helix [reviewed in refs. 17–20]. They possess two (KIR2D) or three (KIR3D) extra-cellular C2-type Ig-like domains and a long cytoplasmic tail (L) containing immunoreceptor tyrosine-based inhibition motifs (ITIM), which recruit and activate SHP-1 and SHP-2 phosphatases for inhibitory signal transduction. KIR2DL1 recognizes HLA-C alleles characterized by a Lys80 residue (HLA-Cw4 and related, “Group 2” alleles). KIR2DL2 and KIR2DL3 (which are allele variants) recognize HLA-C with an Asn80 residue (HLA-Cw3 and related, “Group 1” alleles). KIR3DL1 is the receptor for HLA-B alleles sharing the Bw4 supertypic specificity (Table 26.2) CD94NKG2A, another type of human NK cell inhibitory receptor involved in HLA recognition, binds to the non-conventional class I molecule HLA-E. Several HLA class I alleles provide signal sequence peptides that bind HLA-E and allow its expression at the cell surface. Consequently, it is expressed in every individual.

Chapter 26  Allogeneic Haematopoietic Stem Cell Transplantation and Natural Killer 

Table 26-2.  Human leukocyte antigen (HLA) class I specificity of the main inhibitory killer immunoglobulin-like receptors (KIRs) expressed by human natural killer (NK) cells. KIR genesa

Encoded protein

HLA specificityb

KIR 2DL1

P58.1 receptor

HLA-C group 2 i.e., −Cw2, −Cw4, −Cw5, −Cw6 Sequence: Asn77, Lys80

KIR 2DL2/3

P58.2 receptor

HLA-C group 1  i.e., Cw1, −Cw3, −Cw7, −Cw8  Sequence: Ser77, Asn80

KIR 3DL1

P70/NKB1 receptor

HLA-Bw4-associated  i.e., B27

a

 KIR 2D refer to receptor molecules with two immunoglobulin-like domains whereas KIR 3D to those displaying three immunoglobulin-like domains. Receptors having a long inhibitory cytoplasmic tail are designated as L (long), whereas those having a short activating tail are termed S (short) b  Two groups of HLA-C alleles can be distinguished on the basis of alternative amino acid sequence motif at position 77 and 80 of the a1 helix

Inhibitory KIRs, CD94/NKG2 and HLA-class I genes determine individual NK cell repertoires during development. As they are located on different chromosomes, receptors and ligands segregate independently in human pedigrees. The HLA class I genotype selects a self-tolerant repertoire by dictating which KIR and/or NKG2A receptor combinations are to be used as inhibitory receptors for self HLA class I [21]. Consequently, every functional NK cell in the mature repertoire expresses at least one inhibitory receptor for self HLA; co-expression of two or more receptors is not frequent. Since inhibitory KIRs recognize specific groups of HLA class I molecules, i.e., HLA-C group 1, HLA-C group 2, HLA-Bw4 alleles, NK cells with the potential to exert alloreactions use KIRs as inhibitory receptors for self [17–19, 22]. NK cells which express, as their only inhibitory receptor for self, a KIR for the HLA class I group which is absent on allogeneic targets, sense the missing expression of the self class I KIR ligand and mediate alloreactions (“missing self” recognition). Most individuals possess a full complement of inhibitory KIR genes and can exert NK cell alloreactions [17–19]. Activating KIRs, which regulate NK and T cell functions, are molecular homologues of the inhibitory KIRs with shorter cytoplasmic tails (S) [reviewed in refs. 17, 19, 20] and a charged residue in their transmembrane domain that allows association with ITAM containing signaling polypeptides. Knowledge of their ligand specificity is limited. Studies have reported a weak interaction between KIR2DS1 and Lys80 HLA-C molecules, despite its homology to KIR2DL1, and an even weaker interaction between KIR2DS2 and Asn80 HLA-C molecules, despite its homology to KIR2DL2 and KIR2DL3. Unlike inhibitory KIRs, activating KIRs exhibit extensive variation in gene number and content, which leads to heterogeneity within the general population and diverse ethnic groups [reviewed in ref. 20]. Indeed, activating KIRs may not even be present in approximately 25% of Caucasians who are homozygous for the so-called group A KIR gene haplotypes which contain inhibitory KIR

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genes and the KIR2DS4 activating KIR gene (encoding for a non-functional protein in 2/3 of individuals). On the other hand, 75% of Caucasians are either heterozygous or homozygous for B haplotypes which carry not only inhibitory KIR genes but also various combinations of activating KIR genes (KIR2DS1-2-3-5 and KIR3DS1).

2.  Haploidentical Transplantation: Engraftment and GvHD Until the early 1990s obstacles to haploidentical transplant in leukemia patients were unmanageable T cell alloreactions across the HLA barrier, which caused a high incidence of severe GvHD after unmanipulated HSCT [23]. The benefit of preventing GvHD by T-cell depletion of the graft was largely offset by the high incidence of graft rejection [24] despite proof that a haploidentical graft could be successfully transplanted without causing GvHD in SCID patients receiving a lectin-separated bone marrow transplantation from haploidentical parents [25]. 2.1. Megadose of CD34+ Cells and T-Cell Depletion In mouse models, engraftment was achieved without GVHD by transplanting high doses of T-cell-depleted bone marrow cells [26–29]. In vivo, the megadose of haematopoietic stem cells reduces the frequencies of anti-donor cytotoxic T-lymphocyte precursors (CTL-ps). Cells within the CD34+ cell population exhibit “veto” activity i.e., in bulk mixed lymphocyte reactions they neutralize specific CTL-ps directed against their antigens but not against a third party. Early myeloid CD33+ cells, harvested 7–12  days after ex vivo expansion of CD34+ cells are also endowed with marked veto activity, which is not found in late myeloid cells expressing CD14 or CD11b [26–28]. Therefore, soon after transplantation, infused CD34+ cells and their CD33+ progeny inhibit residual anti-donor CTL-ps in recipients, probably through deletion mediated by tumor necrosis factor-a (TNF-a) [29]. In 1993, the Perugia Bone Marrow Transplant Centre created a megadose of haematopoietic stem cells by adding G-CSF-mobilized peripheral blood progenitor cells to bone marrow cells [30]. Grafts contained a median of 10 × 106 CD34+ cells/kg recipient b.w. After a lectin-based technique which ensured over 3 log T-cell depletion, T cells in the graft were in the range of 1–2 × 105/ kg recipient b.w. Colony-forming granulocyte-macrophage units were sevento tenfold greater than in bone marrow alone. Conditioning included 8  Gy single fraction TBI, thiotepa (10  mg/kg over 2  days), cyclophosphamide (100 mg/kg over 2 days) and rabbit anti-thymocyte globulin (ATG) at 25 mg/ kg over 5 days. About 16/17 patients with end-stage leukemia achieved primary engraftment. Even without post-transplant immune suppressive treatment, acute GvHD grade II–IV occurred in only one patient; there were no cases of chronic GvHD. 2.1.1. Improving the Haploidentical Transplantation Procedure After this successful pilot study, attention focused on optimizing graft processing and conditioning regimen for haploidentical transplantation. The original soybean agglutinin and E-rosetting were replaced by positive immuno-selection of the CD34+ cells, which ensured patients received a median of 2 × 104

Chapter 26  Allogeneic Haematopoietic Stem Cell Transplantation and Natural Killer 

T-cell/kg, one log less than in the previous series. This transplant protocol was used in 43 adult patients with high-risk acute leukemia [31]. Since 1999, CD34+ cells have been positively selected from peripheral blood using the Clinimacs device which is a one-step, fully automated instrument [32]. Besides yielding highly purified CD34+ cells, it ensures a median of 4.5 log T cell depletion. The concomitant 3.2 log B cell depletion helps prevent EBVrelated lymphoproliferative disorders in T-cell-depleted HSCT [33]. Preclinical data [34] indicated fludarabine (40 mg/m2 for 5 days) could be substituted for cyclophosphamide in the TBI-based conditioning regimen to minimize extrahematological toxicity. Post-transplant G-CSF administration was suspended because it induced immunosuppression in haploidentical transplant recipients [35]. Figure 26.1 illustrates the current strategy that is adopted by the Perugia group. The outcomes of the first 104 patients were reported in 2005 [32]. These studies confirmed: (a) a megadose of CD34+ cells is essential to promote engraftment across the HLA barriers in leukemia patients; and (b) the threshold dose of 2 × 104 CD3+ cell/kg prevents severe GvHD as long as it is associated with ATG in the conditioning. ATG, with its plasma half-life of several days, exerts in vivo T cell depletion, thus lowering the incidence and severity of GvHD [36, 37].

sTBI 8 Gy

TT 5 mg/Kg x2

No Post-Transplant GvHD Prophylaxis

rATG -9

-8

-7

-6

-5

-4

Fludarabine 40 mg/kg x 5

-3

-2

-1

0 days

Megadose of CD34+ cells

Fig. 26-1.  Haploidentical transplantation procedure. sTBI 8 Gy: 9 days before transplantation (on day −9), patients receive 8 Gy TBI in a single fraction. Single dose total body irradiation is delivered by two anterior-posterior and posterior-anterior fields with patient in a right lateral decubitus. 18 MV photons from a linear accelerator are used. The homogeneity of the dose is assured by adopting head-neck and leg lead compensators. The dose to the lungs is reduced to 4 Gy by constructing personalized shields from a drawing made on a radiograph taken under the linear accelerator. The thickness of the shield is calculated on the basis of lung thickness and density as determined by CT images done in the therapy position. The instantaneous dose-rate is 12–24 cGy/min/ midplane. The dose administered to the neck, mediastinum, lungs, abdomen and pelvis is monitored directly by semiconductor detectors, which are calibrated before each treatment. The exact position of the lung shields and compensators is checked before initiating therapy by radiographs. TT (Thiotepa) is given i.v. on days −8 and −7 at a dose of 5 mg/kg (4 h for each infusion). Fludarabine is administered each day, from −7 to −3, at a dose of 40 mg/m2 in half an hour i.v. infusion. rATG (Rabbit Anti-thymocyte Globulin) is infused each day, from −6 to −2, at a dose of 1.2  mg/kg (Sangstat) or 5  mg/kg (Fresenius) over an 8  h-period. During the course of ATG, patients will receive methylprednisolone 2  mg/kg/day. On Day 0, the graft is rapidly thawed and infused through a central line following standard procedure and safety measures. No post-transplant immunosuppression is given as extensive T-cell depletion of the graft in combination with ATG in the conditioning, prevents GvHD

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3. Haploidentical Transplantation: Leukemia Relapse Leukemia relapse, a high rate of which was always correlated with extensive T cell depletion, was not an issue after haploidentical transplantation in patients who were not transplanted in end-stage disease. Under the Perugia protocol, the cumulative incidence of leukemia relapse was 18% and 30%, respectively in AML and ALL patients transplanted in first or later complete remission (CR). These figures were lower than what might have been expected in highrisk patients who did not benefit from a GvHD-related GvL effect. Major factors which may have contributed to control post-translant relapse included: (a) the highly myeloablative conditioning regimen, which successfully debulked the leukemia burden; (b) no post-transplant immunosuppression, which may have allowed the few T cells in the graft to exert a sub-clinical GvL/ GvHD effect; and (c) donor-vs-recipient NK-cell-alloreactivity, which exerted a specific Graft-vs-AML effect in susceptible patients. Indeed, transplantation from NK-alloreactive donors was associated with a significantly lower relapse rate in patients transplanted in CR (3% vs. 47%; p < 0.003) [22].

4.  Haploidentical Transplantation: Transplant Related Mortality Transplant-related mortality, a major problem in all transplants from alternative sources, depends mainly on slow immunological recovery which increases susceptibility to life-threatening infections. For up to 1 year after transplant, early immune recovery in adults with their decayed thymic function, stems from expansion of mature T cells in the graft. Naïve T cells are produced months after transplantation because conditioning-induced tissue damage prevents T cell homing to peripheral lymphoid tissues, where T cell memory is generated and maintained. All the studies from the Perugia group reported ~40% non-relapse mortality in haploidentical transplant recipients who tended to remain susceptible to opportunistic infections. Most deaths were caused by infections, the majority of which were CMV and aspergillus [31, 32]. After about 1 year post-transplant life-threatening episodes rarely occurred, confirming that immunological reconstitution was practically complete by this stage. The time-lapse was due to two overlapping factors:(1) the ex vivo extensive T cell depletion of the graft which limited the engrafted T cell repertoire and (2) ATG-related in vivo inhibition of homeostatic expansion of the mature donor T cells that had been transplanted. Unlike recipients of unmanipulated grafts from alternative donors who, for months/years after transplantation, remain at risk of TRM because of GvHD and its immunosuppressive treatments which antagonize T cell expansion and function, almost all recipients of haploidentical transplant had not received immunosuppression and had not developed chronic GvHD. Another aspect of the post-transplant immune deficiency, which has emerged from recent studies, is the impact of G-CSF in transplant recipients. G-CSF promotes Th-2 immune deviation which, unlike Th-1 responses, does not protect against fungi, bacteria and viruses. As G-CSF blocks IL12 production in antigen presenting cells, it decreases pathogen specific responses. Patients who do not receive G-CSF after transplant recover CD4+ cells count faster and most post-transplant CD4+ cell clones exhibit usual Th1-Th0 features [35].

Chapter 26  Allogeneic Haematopoietic Stem Cell Transplantation and Natural Killer 

4.1. Reducing TRM with Adoptive Cell Therapy Some strategies were attempted to hasten immune re-building after haploidentical transplantation but however, alloreactivity is attenuated, T-cell–based adoptive therapy is problematic in the adult haploidentical transplant because of the need to transfer donor T cells across the HLA histoincompatibility barrier soon after transplant when patients are most vulnerable to GvHD and are not receiving GvHD prophylaxis. One strategy was described for transferring safely donor pathogen-specific immune responses to haploidentical recipients [38]. Donor T cell clones raised against Aspergillus fumigatus and CMV antigens were screened for crossreactivity to host alloantigens by MLR. Non-host-reactive clones, presumably devoid of GvHD potential, were pooled and infused into recipients soon after transplant. This study demonstrated the maximum tolerated dose of one million CD3+ cell/kg recipient b.w. was clinically effective as CMV reactivation and Aspergillus galactomannan antigenemia tended to disappear over time. In patients who were infused, the frequencies of anti-aspergillus and anti-CMV specific T cell clones increased rapidly unlike the control group, where they developed in  vitro more than 9  months post-transplant. In other transplant centers, similar results were achieved when ex vivo-expanded EBV-specific allogeneic CTL clones were used to prevent or manage EBV-associated diseases, including post-transplant lymphoproliferative disorders [39, 40].The main drawbacks of both approaches were that they were time-consuming, labor intensive and, at present, unsuitable for routine clinical use as cloning and screening procedures do not always satisfy quality controls. Genetic manipulation of donor lymphocytes with a suicide gene is a promising strategy that was developed in Milan [41–43]. Should GvHD develop, after genetic engineering of donor lymphocytes with the herpes simplex virusthymidine kinase (HSV-TK) suicide gene, transduced cells can be eliminated by ganciclovir treatment. In 17 patients, TK cells provided protection against CMV reactivation and disease. Overall, the cumulative infectious mortality at 6 months’ post-transplant was 12.5% with only 6.0% CMV-related mortality. This strategy may find a place in preventing relapse, selectively controlling GVHD and/or enhancing wide spectrum immunological reconstitution after transplantation.

5.  Haploidentical Transplantation and Event Free Survival Outcomes after haploidentical transplantation compare very favorably with those of other alternative sources of stem cells, such as the matched unrelated donor and cord blood units. Indeed, a metanalysis of alternative HSCT showed that UCBT in adult and pediatric patients had equivalent survival outcomes compared with MUD transplantation [44]. Event Free Survival (EFS) was closely related to disease and disease status at transplant ranging from 30% to over 50% in acute leukemia patients who were transplanted in CR. With no chronic GvHD, all these long-term survivors enjoy an excellent quality of life. Donor-vs-recipient NK-cell alloreactivity impacted so favorably upon survival in AML patients (see below) [22] that transplantation from non-NK alloreactive haploidentical donors appears justified only for AML patients in remission. Lack of an NK alloreactive donors is a counter-indication to transplant for patients in chemo-resistant relapse as very few survived.

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6.  Haploidentical Transplantation and NK Cell Alloreactivity Several observations suggested alloreactive NK cells were responsible for favorable transplantation outcomes. Transfer of human alloreactive NK cells to NOD-SCID mice eradicated previously transplanted human AML cells [15]. KIR ligand mismatches correlated with donor NK cell clone killing of cryopreserved haematopoietic recipient cells, including leukemia cells [14, 15, 22]. As depicted in Fig. 26.2, engrafted stem cells, upon transplantation from NK alloreactive donors, gave rise to an NK cell repertoire which included donor-vs-recipient alloreactive NK clones that were detected in vivo in recipients for up to 1 year after transplant [14, 22]. When exerted in the donor-versus-recipient direction, NK cell alloreactivity emerged as a crucial factor in improving outcomes of haploidentical transplantation [15, 18]. It reduced the risk of leukemia relapse, did not cause graft-versus-host disease (GvHD) and markedly improved event-free survival in a series of haploidentical transplants (57 acute myeloid leukemia (AML) patients, 20 of whom were transplanted from natural killer (NK) alloreactive donors) [14]. In an updated analysis [22], 112 high-risk AML patients received haploidentical transplants from natural killer (NK) alloreactive (n = 51) or nonNK alloreactive donors (n = 61). Transplantation from NK-alloreactive donors was associated with: a significantly lower relapse rate in patients transplanted in any CR (3% vs. 47%) (p < 0.003); better EFS whether patients transplanted in relapse (34% vs. 6%, p = 0.04) or in remission (67% vs. 18%, p = 0.02); overall reduced risk of relapse or death (relative risk vs. non-NK-alloreactive donor: 0.48 [95% CI 0.29–0.78], p < 0.001). 6.1.  NK Cell Alloreactivity and Immunity to Infection After Haploidentical Transplantation The role of donor activating KIR genetics (see introduction) was evaluated in (Mancusi et  al. manuscript submitted for publication) a series of 84 haploidentical transplants for AML. The impact of donor KIR genetics (group A vs. group B KIR gene haplotypes) was assessed separately in NK alloreactive

NK repertoire Host targets Myeloablative TBI based Conditioning + Megadose of CD34 + cells from allo-NK donor + No post-transplant Immune suppression

Haploidentical HSCT strategy

600 cells/cmm

466 

400

KIR2DL1

HLA-C group1 Cw2/Cw4

KIR2DL 2/3

Missing self

KIR3DL1

HLA-Bw4

CD16 CD8

200

CD4 0 0

50 100 150 200 days

Post-transplant Lymphocyte recovery

Donor-derived NK cell repertoire

Fig. 26.2.  Left: Haploidentical stem cell transplant strategy. Middle: Post-transplant lymphocyte recovery. Right: Engrafted stem cells give rise to an NK cell repertoire which includes donor-vs-recipient alloreactive NK clones

Chapter 26  Allogeneic Haematopoietic Stem Cell Transplantation and Natural Killer 

and non-NK alloreactive transplants. Forty-seven recipients were transplanted from NK alloreactive donors (12 with group A KIR gene haplotypes vs. 35 with B haplotypes) and 37 recipients from non-NK alloreactive donors (8 with group A KIR gene haplotypes vs. 29 with B haplotypes). KIR gene haplotypes had no impact in non-NK alloreactive transplants. In transplants from NK alloreactive donors, presence of group B haplotype KIR genes in the donors was associated with reduced incidence of TRM (largely infection-related) (B vs. A haplotypes: 20% vs. 67% TRM, p < 0.005). In multivariate analyses against disease status at transplant, age, patient and donor sex, conditioning regimens, and the number of CD34+ and CD3+ cells in the graft, it was the only significant variable predicting protection from TRM (RR: 0.24; 95% CI:0.14–0.42; p < 0.01) and resulted in a trend towards better EFS (60% vs. 33%, p < 0.1). When the number of activating KIR genes in the donor was taken into account, donors carrying ³3 activating KIR genes provided significant protection from TRM and significantly better EFS compared with A haplotype donors (TRM: 12% vs. 67%, p < 0.003) (EFS: 71% vs. 33%, p = 0.02). In multivariate analysis, transplantation from alloreactive donors carrying ³3 group B haplotype activating KIR genes was the only variable predicting protection from TRM (RR: 0.40; 95% CI: 0.27–0.58; p < 0.02) and significantly improved EFS (RR: 0.56; 95% CI: 0.32–0.98; p < 0.05). Thus, while NK-alloreactive donors protect against leukemia relapse, those who also carry activating KIRs protect against infectious mortality and help improve survival. Protection against infection may be mediated directly by NK cells or indirectly through other mechanisms. Activating KIRs could enhance NK-cell cytokine secretion and cytotoxicity against pathogen infected cells in the context of missing self. A notable example of direct recognition of a pathogen by activating NK receptor was provided by murine CMV protein m157 and the murine Ly49H NK receptor [45]. In humans, progression to AIDS was slower in patients who had KIR3DS1 and the HLA-Bw4 allotype, the putative ligand of KIR3DS1 [46]. More NK cells expressing NKG2C were present in CMVexposed individuals, suggesting this activating NK cell receptor played a role in the immune response to this infection [47]. Therefore, associations between activating NK receptors and enhanced immunity against infections have been documented. Activating KIRs could also help control infections indirectly through the interaction between NK cells and dendritic cells (DCs) [reviewed in ref. 48]. NK cells regulate DC homeostasis and maturation. Mature DCs can, in turn, activate NK cells. In vivo NK/DC interactions in lymphoid organs or non-lymphoid tissues can lead to Th1 polarization. NK cells in lymph nodes provided early IFN-g production, which is essential for Th1 polarization. Consequently, the interaction between NK cells and DCs influences the quality and the strength of adaptive immune response. The clinical data suggested either or both these mechanisms could operate in haploidentical transplants from NK-alloreactive donors who possess activating KIRs. 6.1.1. NK Cell Alloreactivity in Matched Unrelated Donor Transplants Donor-vs-recipient NK cell alloreactivity may also occur in unrelated donor transplants because approximately half are mismatched for one or more HLA class I alleles. Some retrospective studies showed no advantage in transplantation from KIR ligand-mismatched donors [49, 50] probably because of differences in conditioning regimens, patient populations and diseases.

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Most protocols used unmanipulated grafts which contained ~4 log more T cells and up to 1 log fewer stem cells than haploidentical grafts. These factors, combined with post-transplant immune suppression created conditions that were associated with poor reconstitution of potentially alloreactive, KIR-bearing NK cells [51]. Other studies observed an increased GvL effect [52–55]. A marked survival advantage was reported in patients who received ATG pre-transplant and a graft containing two- to threefold more nucleated cells than usual in unrelated-donor transplants [52]. Prospective studies are needed to determine whether high doses of stem cells, T-cell depletion, no post-transplant immune suppression, i.e., strategies which harness donor-vsrecipient NK cell alloreactivity in haploidentical transplantation, can be implemented to improve outcome in unrelated donor transplants. 6.1.1.1. NK Cell Alloreactivity in Cord Blood Transplants The Eurocord-Netcord and Acute Leukemia Working Party of the EBMT assessed outcomes of 218 patients with AML (n = 94) or ALL (n = 124) in CR who received a single unit UCBT from a KIR-ligand compatible or incompatible donor [56]. Grafts were HLA-A,-B,-DRB1-matched (n = 21) or -mismatched (n = 197). Patients and donors were categorized according to their degree of KIR-ligand compatibility by determining whether or not they expressed HLA-C group 1 or 2, HLA-Bw4 or HLA-A3/-A11. Sixty-nine patient-donor pairs were KIR-ligand incompatible and 149 were compatible. KIR-ligand incompatible UCBT showed decreased relapse incidence (Hazard Ratio (HR) = 0.53, p = 0.05), increased Disease Free-Survival (HR = 2.05, p = 0.016) and overall survival (HR = 2.0, p = 0.004). Benefits were significantly more marked in patients with AML. The incidence of relapse and overall survival were 5% and 73% in recipients of NK alloreactive transplants vs. 36% and 38% after non-NK alloreactive transplants. These findings in UCBT concur with reports from the Perugia group. Haploidentical stem cell transplantation and UCBT are both characterized by a rapid post-transplant recovery of NK-cells and severe, long-lasting T-cell immuno-incompetence after transplantation due to extensive T cell depletion for the haploidentical graft and T cell naïvety after UCBT. In this setting UCBT and haploidentical SCT may allow “unmasking” of the effects of NK-cell alloreactivity. 6.1.1.1.1.  The Missing Ligand Model of NK Cell Alloreactivity The original report on NK cell alloreactivity in haploidentical transplantation stated it rested upon KIR ligand mismatching and donor NK cell recognition of “missing self” on recipient targets [15]. The “missing ligand” model was proposed as a powerful algorithm for predicting favorable transplant outcomes not only in haploidentical transplants [57] but also in matched sibling [58] and in unrelated donor transplants [59]. It hypothesized NK alloreactions occur when KIR ligand-matched donors possess an “extra” KIR for which neither donor nor recipient have an HLA ligand. These donors may carry KIR-bearing NK cells in an anergic/regulated state which, upon transfer into the recipient, become activated and exert a GvL effect. No studies have, as yet, determined whether self-tolerant NK cells which do not express inhibitory receptors for self-MHC [60] acquire/resume cytotoxic effector function after transplant. The “missing ligand” algorithm was applied to an adult series of AML patients who received haploidentical transplants. The “missing ligand” transplant recipients had a worse prognosis than patients transplanted from

Chapter 26  Allogeneic Haematopoietic Stem Cell Transplantation and Natural Killer 

NK-alloreactive (KIR ligand mismatched) donors [22] and the analysis showed that donor NK cell recognition of “missing self” on recipient targets was essential for triggering powerful NK cell alloreactions that impacted beneficially on transplantation outcomes.

7.  Selecting an NK Alloreactive Donor One hundred percent of individuals possess the KIR2DL2 and/or KIR2DL3 receptors for HLA-C group 1 alleles. If they have HLA-C group 1 allele(s) in their HLA type, these individuals possess HLA-C1-specific NK cells which are alloreactive against cells from individuals who do not express HLA-C group 1 alleles. Ninety-seven percent of individuals possess the KIR2DL1 receptor for HLA-C group 2. If they possess HLA-C group 2 allele(s) in their HLA type, these individuals have HLA-C2-specific NK cells which mediate alloreactions against cells from individuals who do not express HLA-C group 2 alleles. Finally, ~90% of individuals possess the KIR3DL1 receptor for HLA-Bw4 alleles. When they have HLA-Bw4 allele(s) in their HLA type, these individuals may have HLA-Bw4-specific NK cells that are alloreactive against Bw4-negative cells. These KIR ligand mismatches often occur in haploidentical donor-recipient transplant pairs (Fig. 26.3). Recipients who express alleles belonging to 1 or 2 of the class I allele groups that are recognized by KIRs may find NK alloreactive donors. Donors who are HLA-C group mismatched with their recipients possess high-frequency NK clones which are alloreactive against recipients’ target cells [15, 17, 18]. Thus, high-resolution HLA-C typing is a good predictor of NK cell alloreactivity. Since 3% of individuals do not possess the KIR2DL1 gene, the combination of a KIR2DL1-negative donor and a recipient without HLA-C group 2 alleles

Donors HLA HLA-C group 1

Recipients

NK repertoire KIR2DL 2/3

HLA Missing HLA-C group 1

Target

Lysis!

KIR genes in 100% of individuals allo-NK clones detected in 100% of >50 donors

HLA-C group 2

KIR2DL1

Missing HLA-C group 2

Lysis!

KIR gene in97% of individuals allo-NK clones detected in 100% of >50 donors

HLA-Bw4

KIR3DL1

Missing HLA-Bw4

Lysis!

KIR gene in~90% of individuals, allo-NK clones detected in 2/3 donors

Fig. 26.3.  Frequency of each type of KIR ligand mismatch in haploidentical donorrecipient transplant pairs

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could result in a 1.5% incidence of false positivity. KIR2DL1 gene typing of the donor may be necessary to assess the NK alloreactive potential of this combination. In HLA-Bw4 mismatches, even when the KIR3DL1 gene is present in the donor (~90% of individuals), NK repertoire studies show alloreactive NK clones are non-detectable ~1/3 of individuals [22]. In some, allelic variants in the HLA-Bw4 inhibitory NK receptor gene KIR3DL1 may not allow full receptor expression at the cell membrane and affect NK cell inhibition by HLA-Bw4 ligand [61]; others apparently express alloreactive NK clones in very low frequencies. Thus, for HLA-Bw4 mismatches, functional assessment of the donor NK repertoire appears necessary. Table 26.3 gives a full list of C1,

Table 26-3.  HLA-C group 1, HLA-C group 2 and HLA-Bw4 group alleles. Group 1 HLA-C alleles (Ser 77, Asn 80)

Group 2 HLA-C alleles (Asn 77, Lys 80)

Cw1a

HLA-Bw4 alleles B5 B13

Cw3 (except C*0307, C*0310b and C*0315)

Cw7 (except C*0707 and C*0709)

Cw2

B17

C*0307 and C*0215

B27

Cw4

B37

Cw5

B38

Cw6

B44

C*0707 and C*0709

B47

Cw8

B49 c

Cw12 (except C*1204 and C*1205) C*1204 and C*1205

B51

Cw13

B52 d

Cw14 (except C*1404 )

B53

C*1507

Cw15 (except C*1507)

B57

Cw16 (except C*1602)

C*1602

B58

Cw17

B59

Cw18

B63 B77 B*1513 B*1516 B*1517 B*1523 B*1524

a

 Each serologically-defined group includes all alleles except where noted b  C*0310 (Ser77, Lys80) belongs to both HLA-C groups 1 and 2 [142]. C*0310 blocks NK cells expressing all HLA-C-specific receptors, but it does not block clones expressing the Bw4 receptor c  C*1207 Gly77, Asn80, cannot be assigned to either group based on its aminoacid sequence, and still needs to be tested functionally d  C*1404 (Asn77, Asn80) does not belong to either HLA-C group 1 or 2 and does not block NK cells expressing any HLA-C-specific receptor [142]. Expression of C*1404 in a patient behaves with respect to NK cell recognition, as if the patient does not express that HLA-C allele

Chapter 26  Allogeneic Haematopoietic Stem Cell Transplantation and Natural Killer 

471

Table 26-4.  Donor/recipient HLA class I combinations associated with NK cell alloreactivity in the GvH direction. Recipient HLA type

HLA type of NK alloreactive donora

Group 1 HLA-C, Group 2 HLA-C, HLA-Bw4

No NK alloreactive donor

Group 1 HLA-C, Group 2 HLA-C

HLA-Bw4

Group 1 HLA-C, HLA-Bw4

Group 2 HLA-C

Group 2 HLA-C, HLA-Bw4

Group 1 HLA-C

Group 1 HLA-C

Group 2 HLA-C and/or HLA-Bw4

Group 2 HLA-C

Group 1 HLA-C and/or HLA-Bw4

a

 Recipients who express class I alleles belonging to the 3 major class I groups (HLA-C group 1, HLA-C group 2, and HLA-Bw4 alleles) will block all NK cells from every donor. Donors may exert donor-versus-recipient NK cell alloreactivity when HLA-C and HLA-B typing show KIR ligand mismatches in the GvH direction, i.e., the recipient do not possess one HLA-C allele group (C1 or C2) and/or the HLA-Bw4 group which are present in the donor. In the donor’s HLA typing, the alleles listed are associated with the potential to exert NK cell alloreactions against the specific HLA type of the recipient, whatever other alleles may be present. The HLA-C group 1 receptor genes (KIR2DL2 and/or KIR2DL3) are present in 100% of individuals. High-frequency alloreactive NK clones are detected in these individuals. The HLA-C group 2 receptor gene (KIR2DL1) is present in ~97% of donors. When the gene is present, high-frequency alloreactive NK clones are detected in donors. The KIR3DL1 HLA-Bw4 receptor gene is present in only ~90% of individuals. Even when the gene is present, alloreactive NK clones occur in highly variable frequencies and are detected in 2/3 donors. Thus, functional assessment of the donor NK repertoire appears necessary.

C2 and Bw4 alleles. Table 26.4 shows donor recipient C1, C2 and Bw4 KIR ligand mismatches which trigger NK cell alloreactivity. The chance of finding an NK alloreactive donor is ~50% of haploidentical transplants and the odds of finding an NK alloreactive donor carrying activating KIRs are ~30% of haploidentical transplants.

8.  Conclusions and Future Directions The past few years have seen growing interest in haploidentical transplantation. At present, after T cell depleted haploidentical transplant rejection, GvHD and leukemia relapse are no longer major issues in patients who are not transplanted in end-stage disease. Exploiting donor-vs-recipient NK cell alloreactivity emerged as a crucial factor in improving outcomes of haploidentical transplantation. It reduced the risk of leukemia relapse, did not cause GvHD and markedly improved event-free survival. Survival rates overlap after haploidentical transplants for high risk acute leukemia and transplants from other alternative sources. However, it is worth noting that the probability of EFS after matched unrelated transplants is confounded by the gap between the number of activated searches and de facto transplants. All these advances encourage extending the haploidentical transplant to patients with an indication to transplant. Haploidentical donors are found within the family for almost all patients with no undue delay between decision-making and transplantation, which is a crucial factor in urgent cases. It is to hoped that transplant physicians will be less hesitant to treat patients in better condition and earlier disease stage and that the mismatched transplant will be offered, not as a last resort, but as a routine option to high risk acute

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Table 26-5.  Assessing haploidentical transplantation. Strength

Weakness

~100% donor availability from extended family 3 weeks from decision to transplant:

Need for haematopoietic stem cell selection

  1 week search time,

(skilled staff)

  1 week donor mobiliazation,

Minimum 3 h laboratory time

  1 week recipient conditioning

Cost of each leukapheresis processing (€6–7,000)

Selection of NK cell alloreactive donors Low rejection rates of 7%

Slow immune re-building

Immediate availability of same or other donor

High rate of opportunistic infections

No post-transplant immunosuppressive therapy

High incidence of CMV reactivation

Low incidence of GvHD of <5%

Adoptive immune cell therapy may be required

Excellent quality of life Low AML relapse rate(3% with NK alloreactive donors)

High relapse rate in adult ALL

High AML event free survival(67% with NK alloreactive donors)

leukemia patients who need an allogeneic haematopoietic stem cell transplant (Table 26.5). As post-transplant infections still need to brought under control, re-building immunity after haploidentical transplantation is the one outstanding clinical issue. Preliminary results of innovative strategies such as ex vivo-expanded immunomodulatory cells (Tregs, NK/Tregs, MSCs and donor-derived NK) [62–64], adoptive transfer of allogeneic T cells that are specific for viral [38, 65–69] appear promising. Some groups are incorporating KGF-1 into the preparative regimen/supportive care in an attempt to prevent GvHD, minimize toxicity and improve outcome [70].

References   1. Byrd JC, Mrozek K, Dodge RK et al (2002) Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 100:4325–4336   2. Giles FJ, Keating A, Goldstone AH, Avivi I, Willman CL, Kantarjian HM (2002) Acute myeloid leukemia. Hematology (Am Soc Hematol Educ Program) 73–110   3. Hoelzer D, Gokbuget N, Ottmann O, et al (2002) Acute lymphoblastic leukemia. Hematology (Am Soc Hematol Educ Program) 162–192   4. Burnett AK, Wheatley K, Goldstone AH et al (2002) The value of allogeneic bone marrow transplant in patients with acute myeloid leukaemia at differing risk of relapse: results of the UK MRC AML 10 trial. Br J Haematol 118:385–400   5. Tavernier E, Le QH, Elhamri M, Thomas X (2003) Salvage therapy in refractory acute myeloid leukemia: prediction of outcome based on analysis of prognostic factors. Leuk Res 27:205–214   6. Litzow MR (2007) Progress and strategies for patients with relapsed and refractory acute myeloid leukemia. Curr Opin Hematol 14(2):130–137

Chapter 26  Allogeneic Haematopoietic Stem Cell Transplantation and Natural Killer  7. Lee SJ, Klein J, Haagenson M, Baxter-Lowe LA, Confer DL, Eapen M, FernandezVina M, Flomenberg N, Horowitz M, Hurley CK, Noreen H, Oudshoorn M, Petersdorf E, Setterholm M, Spellman S, Weisdorf D, Williams TM, Anasetti C (2007) High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood 110(13):4576–4583 8. Perz JB, Szydlo R, Sergeant R, Sanz J, O’Shea D, Khan T, Davey N, Loaiza S, Davis J, Apperley JF, Olavarria E (2007) Impact of HLA class I and class II DNA high-resolution HLA typing on clinical outcome in adult unrelated stem cell transplantation after in vivo T-cell depletion with alemtuzumab. Transpl Immunol 18(2):179–185 9. Madrigal A, Shaw BE (2008) Immunogenetic factors in donors and patients that affect the outcome of hematopoietic stem cell transplantation. Blood Cells Mol Dis 40(1):40–43 10. Takahashi S (2007) Leukemia: cord blood for allogeneic stem cell transplantation. Curr Opin Oncol 19(6):667–672 Review 11. Sanz MA, Sanz GF (2002) Unrelated donor umbelical cord blood transplantation in adults. Leukemia 16:1984–1991 12. Rocha V, Labopin M, Sanz G et al (2004) Transplants of umbilical cord blood or bone marrow from unrelated donors in adult with leukemia. N Engl J Med 351:2276–2285 13. Brunstein CG, Setubal DC, Wagner JE (2007) Expanding the role of umbilical cord blood transplantation. Br J Haematol 137(1):20–35 14. Ruggeri L, Capanni M, Casucci M et  al (1999) Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation. Blood 94:333–339 15. Ruggeri L, Capanni M, Urbani E et al (2002) Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 295:2097–2100 16. Velardi A, Ruggeri L, Moretta A, Moretta L (2002) NK cells: a lesson from mismatched hematopoietic transplantation. Trends Immunol 23:438–444 17. Ruggeri L, Aversa F, Martelli MF, Velardi A (2006) Haploidentical transplantation and natural killer cell recognition of missing self. Immunol Rev 214:202–218 18. Ruggeri L, Mancusi A, Burchielli E, Aversa F, Martelli MF, Velardi A (2007) Natural killer cell alloreactivity in allogeneic hematopoietic transplantation. Curr Opin Oncol 19(2):142–147 19. Moretta L, Moretta A (2004) Killer immunoglobulin-like receptors. Curr Opin Immunol 16:626–633 20. Parham P (2005) MHC class I molecules and KIRs in human history, health and survival. Nat Rev Immunol 5:201–214 21. Yawata M, Yawata N, Draghi M, Little AM, Parteniou F, Parham P (2006) Roles for HLA and KIR polymorphisms in natural killer cell repertoire selection and modulation of effector function. J Exp Med 203:633–645 22. Ruggeri L, Mancusi A, Capanni M, Urbani E, Carotti A, Aloisi T et  al (2007) Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value. Blood 110:433–440 23. Mickelson EM, Petersdorf EW, Hansen JA (2002) HLA matching and hematopoietic cell transplant outcome. Clin Transpl 263–271 24. Terenzi A, Aversa F, Albi N, Galandrini R, Dembech C, Velardi A, Martelli MF (1993) Residual clonable host cell detection for predicting engraftment of T cell depleted BMTs. Bone Marrow Transplant 11:357–361 25. Reisner Y, Kapoor N, Kirkpatrick D et al (1983) 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 61:341–348 26. Bachar-Lusting E, Rachamim N, Li HW et al (1995) Megadose of T cell-depleted bone marrow overcomes MHC barriers in sublethally irradiated mice. Nat Med 1:1268–1273

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F. Aversa and A. Velardi 27. Rachamin N, Gan J, Segall R et  al (1998) 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 65:1386–1393 28. Gur H, Krauthgamer R, Berrebi A et al (2002) Tolerance induction by megadose hematopoietic progenitor cells: expansion of veto cells by short-term culture of purified human CD34(+) cells. Blood 99:4174–4181 29. Gur H, Krauthgamer R, Bachar-Lustig E, Katchman H, Arbel-Goren R, Berrebi A et al (2005) Immune regulatory activity of CD34+ progenitor cells: evidence for a deletion-based mechanism mediated by TNF-alpha. Blood 105(6):2585–2593 30. Aversa F, Tabilio A, Terenzi A, Velardi A, Falzetti F, Giannoni C et  al (1994) 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 84:3948–3955 31. Aversa F, Tabilio A, Velardi A, Cunningham I, Terenzi A, Falzetti F et al (1998) 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 339:1186–1193 32. Aversa F, Terenzi A, Tabilio A, Falzetti F, Carotti A, Ballanti S et al (2005) Fullhaplotype mismatched hematopoietic stem cell transplantation: a phase II study in patients with acute leukemia at high risk or relapse. J Clin Oncol 23:3447–3454 33. Liu D, Tammik C, Zou JZ, Ernberg I, Masucci MG, Ringden O, Levitsky V (2004) Effect of combined T- and B-cell depletion of allogeneic HLA-mismatched bone marrow graft on the magnitude and kinetics of Epstein-Barr virus load in the peripheral blood of bone marrow transplant recipients. Clin Transplant 18(5):518–524 34. Terenzi A, Aristei C, Aversa F et  al (1996) Efficacy of fludarabine as an immunosuppressor for bone marrow transplantation conditioning: preliminary results. Transplant Proc 28:3101 35. Volpi I, Perruccio K, Tosti A et al (2001) Post-grafting granulocyte colony-stimulating factor administration impairs functional immune recovery in recipients of HLA haplotype-mismatched hematopoietic transplants. Blood 97:2514–2521 36. Russell JA, Turner AR, Larratt L, Chaudhry A, Morris D, Brown C et al (2007) Adult recipients of matched related donor blood cell transplants given myeloablative regimens including pretransplant antithymocyte globulin have lower mortality related to graft-versus-host disease: a matched pair analysis. Biol Blood Marrow Transplant 13(3):299–306 37. Waller EK, Langston AA, Lonial S, Cherry J, Somani J, Allen AJ et  al (2003) Pharmacokinetics and pharmacodynamics of anti-thymocyte globulin in recipients of partially HLA-matched blood hematopoietic progenitor cell transplantation. Biol Blood Marrow Transplant 9(7):460–471 38. Perruccio K, Tosti A, Burchielli E, Topini F, Ruggeri L, Carotti A et  al (2005) Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation. Blood 106:4397–4406 39. Liu Z, Savoldo B, Huls H et al (2002) Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes for the prevention and treatment of EBV-associated post-transplant lymphomas. Recent Results Cancer Res 159:123–133 40. Comoli P, Basso S, Zecca M, Pagliara D, Baldanti F, Bernardo ME et  al (2007) Preemptive therapy of EBV-related lymphoproliferative disease after pediatric haploidentical stem cell transplantation. Am J Transplant 7(6):1648–1655 41. Marktel S, Magnani Z, Ciceri F, Cazzaniga S, Riddell SR, Traversari C et al (2003) Immunologic potential of donor lymphocytes expressing a suicide gene for early immune reconstitution after hematopoietic T-cell-depleted stem cell transplantation. Blood 101(4):1290–1298 42. Ciceri F, Bonini C, Gallo-Stampino C, Bordignon C (2005) Modulation of GvHD by suicide-gene transduced donor T lymphocytes: clinical applications in mismatched transplantation. Cytotherapy 7(2):144–149

Chapter 26  Allogeneic Haematopoietic Stem Cell Transplantation and Natural Killer  43. Traversari C, Marktel S, Magnani Z, Mangia P, Russo V, Ciceri F et al (2007) The potential immunogenicity of the TK suicide gene does not prevent full clinical benefit associated with the use of TK-transduced donor lymphocytes in HSCT for hematologic malignancies. Blood 109(11):4708–4715 44. Hwang WYK, Samuel M, Tan D et al (2007) A meta-Analysis of unrelated donor umbilical cord blood transplantation versus unrelated donor bone marrow transplantation in adult and pediatric patients. Biol Bone Marrow Transplant 13:444–453 45. Smith HRC, Heusel JW, Mehta IK et al (2002) Recognition of a virus-encoded ligand by a natural killer cell activation receptor. Proc Natl Acad Sci USA 99:8826–8831 46. Martin MP et al (2002) Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nat Genet 31:429–434 47. Guma M et al (2004) Imprint of human cytomegalovirus infection on the NK cell receptor repertoire. Blood 104:3664–3671 48. Degli Esposti MA, Smyth MJ (2005) Close encounters of different kinds: dendritic cells and NK cells take centre stage. Nat Rev Immunol 5:112–124 49. Davies SM, Ruggieri L, DeFor T et al (2002) Evaluation of KIR ligand incompatibility in mismatched unrelated donor hematopoietic transplants. Blood 100:3825– 3827 50. Farag SS, Bacigalupo A, Eapen M et al (2006) The effect of KIR ligand incompatibility on the outcome of unrelated donor transplantation: a report from the center for international blood and marrow transplant research, the European blood and marrow transplant registry, and the Dutch registry. Biol Blood Marrow Transplant 12:876–884 51. Cooley S, McCullar V, Wangen R et al (2005) KIR reconstitution is altered by T cells in the graft and correlates with clinical outcomes after unrelated donor transplantation. Blood 106:4370–4376 52. Giebel S, Locatelli F, Lamparelli T et al (2003) Survival advantage with KIR ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors. Blood 102:814–819 53. Elmaagacli AH, Ottinger H, Koldehoff M et al (2005) Reduced risk for molecular disease in patients with chronic myeloid leukemia after transplantation from a KIRmismatched donor. Transplantation 79:1741–1747 54. Beelen DW, Hottinger HD, Ferencic S et  al (2005) Genotypic inhibitory killer immunoglobulin-like receptor ligand incompatibility enhances the long-term antileukemic effect of unmodified allogeneic hematopoietic stem cell transplantation in patients with myeloid leukemias. Blood 105:2594–2600 55. Dawson MA, Spencer A (2005) Successful use of haploidentical stem-cell transplantation with KIR mismatch as initial therapy for poor-risk myelodysplastic syndrome. J Clin Oncol 23:4473–4474 56. Willemze R, Arrais Rodrigues C, Labopin M, et al (2008) Inhibitory KIR-ligend mismatching is associated with decreased incidence of relapse and improved disease-free survival after unrelated cord blood stem cell transplantation for patients with acute leukaemia. Bone Marrow Transplant 41(suppl 1); Abstr 83 57. Leung W, Iyengar R, Turner V et al (2004) Determinants of antileukemia effects of allogeneic NK cells. J Immunol 172:644–650 58. Hsu KC, Keever-Taylor CA, Wilton A et  al (2005) Improved outcome in HLAidentical sibling hematopoietic stem-cell transplantation for acute myelogenous leukemia predicted by KIR and HLA genotypes. Blood 105:4878–4884 59. Hsu KC, Gooley T, Malkki M et al (2006) KIR ligands and prediction of relapse after unrelated donor hematopoietic cell transplantation for hematologic malignancy. Biol Blood Marrow Transpl 12:828–836 60. Fernandez NC, Treiner E, Vance RE, Jamieson AM, Lemieux S, Raulet DH (2005) A subset of natural killer cells achieves self-tolerance without expressing inhibitory receptors specific for self-MHC molecules. Blood 105:4416–4423 61. Pando MJ, Gardiner CM, Gleimer M, McQueen KL, Parham P (2003) The protein made from a common allele of KIR3DL1 (3DL*004) is poorly expressed at cell

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F. Aversa and A. Velardi surfaces due to substitution at position 86 in Ig domain 0 and 182 in Ig domain 1. J Immunol 171:6640–6647 62. Dey BR, Spitzer TR (2006) Current status of haploidentical stem cell transplantation. Br J Haematol 135(4):423–437 63. Zorn E (2006) CD4 + CD25+ regulatory T cells in human hematopoietic cell transplantation. Semin Cancer Biol 16(2):150–159 64. Le Blanc K, Ringden O (2006) Mesenchymal stem cells: properties and role in clinical bone marrow transplantation. Curr Opin Immunol 18(5):586–591 65. Schattenberg AV, Dolstra H (2005) Cellular adoptive immunotherapy after allogeneic stem cell transplantation. Curr Opin Oncol 17(6):617–621 66. Peggs KS, Verfuerth S, Pizzey A, Khan N, Guiver M, Moss PA, Mackinnon S (2003) Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stemcell transplantation with virus-specific T-cell lines. Lancet 362:1375–1377 67. Einsele H, Hebart H (2004) CMV-specific immunotherapy. Hum Immunol 65(5):558–564 68. Feuchtinger T, Matthes-Martin S, Richard C, Lion T, Fuhrer M, Hamprecht K et al (2006) Safe adoptive transfer of virus-specific T-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation. Br J Haematol 134:64–76 69. Thorne SH, Negrin RS, Contag CH (2006) Synergistic antitumor effects of immune cell-viral biotherapy. Science 311:1780–1784 70. Seggewiss R, Einsele H (2007) Hematopoietic growth factors including keratinocyte growth factor in allogeneic and autologous stem cell transplantation. Semin Hematol 44(3):203–211

Chapter 27 Therapeutic Potential of Mesenchymal Stem Cells in Hematopoietic Stem Cell Transplantation Luis A. Solchaga and Hillard M. Lazarus

1. Introduction Mesenchymal Stem Cells (MSCs) are non-hematopoietic multipotent cells residing in the stroma of the bone marrow that are capable of differentiating into both mesenchymal and non-mesenchymal lineages [1]. In fact, in addition to bone, cartilage, fat, and myoblasts, it has been demonstrated that MSCs are capable of differentiating into neurons and astrocytes in vitro and in vivo [2, 3]. Multipotent Adult Progenitor Cells (MAPCs) are also multipotent cells that can be isolated from the bone marrow. Although these cells share some features with MSCs, they differ from MSCs at several levels; MAPCs are expanded at low densities under low oxygen tension; they are CD90+, CD49C+, CD10+, CD45− and class II HLA−; they have active telomerase, are genetically stable and exhibit greater neurologic and hematopoietic potential than MSCs [4]. MSCs and related cells produce cytokines, chemokines and extracellular matrix proteins that support in vitro hematopoietic stem cell (HSC) survival and proliferation and facilitate in  vivo HSC engraftment [5]. MSCs possess immunomodulatory properties and can inhibit alloreactive T-cell responses. Their capacity for extensive in  vitro expansion and immuno-suppressive capacity have made MSCs very attractive therapeutic agents for allo- and auto-immune disorders.

2. Bone Marrow-Derived Mesenchymal Stem Cells Bone marrow is the most common tissue source for the isolation of adult mesenchymal stem cells (MSCs) [6]. The notion that adult stem cells are present in bone marrow was first suggested by Friedenstein [7]. In the mid to late 1980’s, Owen [8], and Owen and Friedenstein [9] proposed a model of differentiation

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_27, © Springer Science + Business Media, LLC 2003, 2010

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in which marrow stromal stem cells give rise to cells from the fibroblastic, reticular, adipogenic, osteogenic, and possibly other lineages. This model of pathways available to MSCs was later termed mesengenesis [10]. Haynesworth et  al. [1] demonstrated that cultured human bone marrowderived cells generate bone when loaded into porous ceramic cubes and implanted subcutaneously in immunodefficient mice. These bone-forming cells were isolated from the bone marrow, recovered from the top fraction of a density gradient (1.073 g/ml), and seeded into tissue culture dishes in a medium containing selected lots of fetal bovine serum (FBS) pre-screened for their ability to support MSC proliferation [11]. A subset of these cells becomes anchored to the substrate and proliferate forming colonies. This ability to attach to the culture substrate along with the selection of the appropriate lot of FBS [11] are the cornerstones of the technology that allows the isolation of MSCs from the total nucleated cell population and their selective expansion in vitro. After subcultivation, MSCs exhibit a strong, albeit variable proliferative potential [12–14]. Human bone marrow-derived MSCs conform to the criteria defined by the International Society for Cellular Therapy; they are plastic-adherent in culture, CD105+, CD73+, CD90+, CD45−, CD34−, CD14−, CD19− and HLA-SR surface molecule negative and they can differentiate into osteoblasts, chondrocytes and adipocytes in  vitro [15]. However, these preparations are not established cell lines and the preparation-to-preparation (donor-to-donor) variability in features such as proliferation rate, differentiation potential and eventual senescence [13] pose a significant challenge that must be overcome if stem cell technology is to be translated into functional therapies.

3. Multipotentiality of Mesenchymal Stem Cells One of the identifying characteristics of MSCs is their capacity to differentiate into different phenotypes. A number of in vitro assays, specific for different lineages, can be used to assess the multipotentiality of MSC preparations [16]. Osteogenic differentiation of MSCs can be triggered by exposure to specific culture supplements including dexamethasone and ascorbate [12, 17]. Aggregate or pellet cultures can be established in a defined medium containing transforming growth factor-beta (TGF-b) to promote chondrogenic differentiation of MSCs [16, 18–21]. Differentiation of MSCs to adipocytes can be induced through the use of an induction medium supplemented with insulin, indomethacin and 1-methyl13-isobutylxanthine in cultures seeded at high density [16]. The ability of MSCs to differentiate along these various phenotypic lines is strongly suggestive of their stem cell nature. MSCs, although sometimes referred to in different terminology (e.g., marrow stromal fibroblasts [22], marrow stromal cells [23], mesenchymal stem cells [10, 24], mesenchymal progenitor cells [25]), have also been shown to differentiate into other lineages, including skeletal [26] and cardiac muscle [27], and hematopoietic supportive tissue [28]. In addition they can differentiate into non-mesenchymal tissues including neurons [2, 3] and retinal cells [29]. However, MSCs do not maintain their stem cell characteristics indefinitely; with extensive subcultivation, MSCs senesce in vitro and lose their multipotential properties [30, 31].

Chapter 27  Therapeutic Potential of Mesenchymal Stem Cells 

4. Mesenchymal Stem Cell Senescence Human MSC preparations have a significant, although variable and not unlimited, proliferative potential [6]. This variability may be due to factors, such as the method used to obtain the bone marrow [12–14, 32], and the age and condition of the donor [13, 33]. Despite their high proliferative potential, MSCs do not lose their normal karyotype or telomerase activity [16]. However, extensive subcultivation results in loss of differentiation potential and the onset of senescence [13]. Interestingly, the senescence-related loss of potential is not generalized. MSCs maintain their osteogenic potential through extensive subcultivation [12, 13]. Although these cells approach senescence they lose their ability to differentiate into adipocytes [13]. The ability to differentiate into chondrocytes is lost in earlier passages [30].

5. Immunologic Properties of Mesenchymal Stem Cells An emerging body of data suggests that MSCs inhibit T-lymphocyte activation and proliferation induced by mitogens, recall antigens and alloantigens in vitro. This immunosuppressive capacity makes MSCs a very attractive therapeutic agent in allogeneic hematopoietic stem cell transplantation whereby MSCs could be used to reduce the incidence and severity of graft-versus-host disease (GVHD). To date, the mechanism of the MSC-mediated inhibition of T-lymphocyte activation has not been fully understood. Most groups have reported that this effect is mediated by soluble factors, yet others report that cell-to-cell contact is necessary. Several candidate cytokines have been studied with conflicting results [34, 35]. The lack of a clearly defined mechanism despite a number of independent and contradictory reports suggests that the interactions are complex and likely involve multiple pathways. The interest in the immunosuppressive effects of MSCs was generated by observations of McIntosh et al. who reported MSC-mediated suppression of both primary and secondary T-lymphocyte proliferation in response to allogeneic stimuli [36]. Subsequently a number of laboratories reported on the inhibitory effects of MSCs on T-lymphocyte activation and proliferation [37–40]. Di Nicola et al. suggested that the mechanism of this suppression is mediated by soluble factors, including hepatocyte growth factor and transforming growth factor-b (TGF-b) [37], yet other groups have not confirmed these findings. Le Blanc et al. demonstrated dose-dependent inhibition of mixed lymphocyte cultures by addition of autologous, allogeneic or third-party MSCs [38]. T-cell activation and proliferation in response to phytohemagglutinin (PHA), concanavalin A (ConA), and ProteinA were also suppressed by MSCs, indicating a non-specific effect. Tse and coworkers demonstrated suppression of T-cells by MSCs, an effect that did not appear to be mediated by interleukin (IL)-10, TGF-b, or PGE2 [39]. Allogeneic HLA-unmatched MSCs did not activate T-cells in any of the individuals tested. Most importantly, a significant reduction of T-cell activation occurred in mixed lymphocyte reactions (MLR) performed in the presence of MSCs unrelated to either of the lymphocyte donor [40]. These immunosuppressive effects of MSCs were mediated by soluble

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factors as shown by trans-well chambers. The immunosuppressive effects of MSCs were shown to require an activation step that involves interaction with CD14+ monocytes [40, 41]. More recently MSCs were found to have multiple effects on both T-cells and antigen presenting cells (APCs) [35, 42]. MSCs were shown to decrease tumor necrosis factor (TNF) secretion from mature type 1 dendritic cells, IL-10 secretion from mature type 2 dendritic cells, decrease IFN-g secretion of TH1 cells and increase secretion of IL-4 from TH2 cells [35]. In addition it was found that MSCs increased the proportion of CD4+CD25+ regulatory cells in an MLR, although the significance of this proportional increase was not clear. MSCs were also suggested to block APC maturation and activation necessary for allogeneic T-cell activation and proliferation [42].

6.  Mesenchymal Stem Cells Facilitate Crossing the Major Histocompatibility Complex Barrier Experimental evidence indicates that major histocompatibility complex (MHC) mismatch between the donor hematopoietic progenitors and the host bone marrow microenvironment may be disadvantageous to donor cells, particularly in the non-myeloablative setting. Engraftment of hematopoietic cells in a mismatched allogeneic transplant model was shown to be facilitated by MHC-matched bone grafting which is known to contain MSC-like cells [43]. Similarly, MHC matched stromal and CD8+, CD3+, TCRneg “facilitator cell” co-transplantation was shown to improve engraftment with purified allogeneic hematopoietic progenitors [44, 45]. Furthermore, using a baboon skin graft model, Bartholomew and co-workers showed that infusion of ex vivo-expanded donor (baboon) MSCs at a dose of 20 × 106 MSC/kg recipient weight prolonged time to rejection of histoincompatible skin grafts [46]. Even third-party baboon MSCs, derived neither from the donor nor the recipient, appeared to suppress allo-reactivity in vivo. Co-infusion of murine MSCs and murine tumor cell lines have also been shown to lead to tumor formation in immuno-competent mice due to immunosuppressive effects of MSCs whereas the tumor cell line by itself was rejected [47]; a potential drawback for the clinical application of MSCs. Most dramatic in vivo demonstration of MSCs immunosuppressive activity comes from a clinical report describing a case of severe GVHD in a human recipient of matched unrelated donor allogeneic bone marrow cells. This patient had a dramatic improvement in clinical condition after infusion of haplo-identical (parental donor) MSCs and again upon a second infusion following GVHD relapse and the patient survived this otherwise lethal complication [48]. These data indicate that T-cell inhibitory effects of MSCs are not limited to in vitro experimental settings.

7.  Clinical Autologous and Allogeneic Mesenchymal Stem Cell Transplantation Our group established the feasibility and safety of clinical-scale autologous and allogeneic human MSC expansion and intravenous infusion [28, 49–51]. We are responsible for the first trial with clinical ex vivo expansion and infusion of MSCs [49]; in this study, autologous MSCs from patients with

Chapter 27  Therapeutic Potential of Mesenchymal Stem Cells 

hematological cancers in complete remission were isolated and expanded in culture for 4–7  weeks and then were reinfused intravenously. Patients were grouped into three classes and received 1, 5, and 10 × 106 MSCs/kg, respectively. No toxicity or adverse reactions were observed, suggesting that MSCs are safe and can be used in a transplant setting. In a second study, MSCs were culture expanded in the Cell and Gene Therapy facility of the Ireland Cancer Center under an FDA-approved IND. A total of 1–2.2 × 106 autologous MSCs/kg were infused into 28 breast cancer patients to augment hematopoietic engraftment after peripheral blood progenitor cell (PBPC) transplantation [28]. No toxicity related to intravenous MSC infusion was observed. Clonogenic MSCs were detected in venous blood up to 1 h after infusion in 13 out of 21 (62%) patients. Hematopoietic engraftment was prompt in all patients with median neutrophil recovery (>500/ml) in 8 (range: 6–11) days and platelet count recovery >20,000/ml and >50,000 unsupported in 8.5  days (range: 4–19) and 13.5  days (range: 7–44), respectively. As this investigation was not a randomized trial one cannot confirm the role of MSCs in rapid hematopoietic recovery. Nonetheless, these results justify testing whether co-transplantation of MSCs provide an engraftment advantage in difficult-to-engraft hematopoietic grafts such as umbilical cord blood, T-depleted grafts and those grafts given in nonmyeloablative settings. Allogeneic MSC transplantation was investigated in a multi-center clinical trial under the leadership of the Case Comprehensive Cancer Center/ Ireland Cancer Center to test the hypothesis whether infused culture expanded MSCs obtained from the histocompatible sibling donor would promote faster hematopoietic engraftment or limit GVHD after sibling-matched myeloablative allogeneic bone marrow or peripheral blood progenitor cell (PBPC) transplantation in patients with hematological malignancies [52]. Forty-three patients received myeloablative chemo-radiation therapy, HLA-identical sibling marrow (n = 17) or mobilized peripheral blood (n = 26), and escalating doses of culture-expanded MSCs from the histocompatible sibling HSC donor (1–5 × 106/kg). There were no instances of MSC infusion-related toxicities. Median (range) time to neutrophil recovery >500/ml and platelets >20,000/ml (untransfused) for all patients was 14 [11–22] days and 21 (15–78) days, respectively. Grade 0–I acute GVHD occurred in 29 patients and grade II–III in 10 patients; none had grade IV. Of 36 patients at-risk, chronic GVHD did not develop in 17 subjects, was of limited extent in 13 patients, and extensive in 6 subjects. The expected incidence of acute and chronic GVHD in siblingmatched allogeneic stem cell transplant varies and in the range of 40–60% for grade II–IV and 10–25%, grade III–IV acute GVHD [53, 54]. Extensive chronic GVHD rates in published reports range between 30–70%. Our results compare favorably with these data despite the use of less intensive GVHD prophylaxis used. A randomized trial is necessary to determine the activity of MSC co-infusion in limiting incidence and severity of GVHD in allogeneic HSC transplantation. Based on analyses of these early data, co-transplantation of culture-expanded MSCs with an HLA-identical sibling HSC graft appears to be safe and feasible and may reduce the incidence and severity of GVHD. Elucidating the mechanism of this putative MSCs effect on GVHD and vigorous pre-clinical testing is essential for proper assessment and translation of this novel cellular therapy.

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Table 27-1.  Clinical experience with MSCs. Indication

Patients

Results

Source

Cardiac disease

67

Reduction in area of damaged heart tissue

[68]

Cardiac disease

10

Improved heart function in all patients

[67]

Cardiac disease

75

Moderate improvement in heart function Infusion safe and feasible

[66]

Cardiac disease

39

Improved heart function in all patients

[65]

Crohn’s fistula

4

75% of fistulas healed

[70]

Stroke

30

Improved outcome; Barthel Index and Rankin score

[69]

Spinal cord injury

2

Improved motor and sensitive levels

[64]

Multiple sclerosis

10

Improvement in Expanded Disability Status Scale

[73]

Bone graft

32

Improved integration of grafts

[71]

Diabetic foot

1

Decrease of wound size and increase in vascularity and thickness of dermis

[62]

Amyotrophic lateral sclerosis

9

Slower decline of forced vital capacity

[72]

Skin burns

1

Accelerated healing and rehabilitation

[60]

Skin graft

1

Accelerated healing and rehabilitation

[61]

Other groups have also established the feasibility and safety of MSC infusions and documented positive outcomes in acceleration of engraftment [5] and graft enhancement [55, 56]. Additionally, MSC infusions have been successfully used for repair of tissue injury secondary to allogeneic HSC transplantation [57] and treatment of GVHD [58]. MSC-based therapies have been or are being investigated also for applications unrelated to the field of hematopoietic stem cell transplantation. Some of these applications include the treatment of skin burns [59, 60] and grafts [61], diabetic foot [62], joint disease [63], chronic spinal cord injury [64], cardiac disease [65–68], stroke [69], Crohn’s disease [70], bone grafts [71], amyotrophic lateral sclerosis [72], and multiple sclerosis [73] (Table 27.1)

8.  Growth Factor Supplementation for Expansion of Mesenchymal Stem Cells Experimental observations have accumulated evidence of the strong mitogenic effect that fibroblast growth factor 2 (FGF-2) exerts on MSCs [74]. Human MSCs isolated from normal donor bone marrow aspirates were divided in two subsets and cultured either in standard medium; Dulbecco’s Modified Eagle’s Medium; low glucose (DMEM-LG) supplemented with 10% fetal bovine serum (FBS) or in standard medium supplemented with FGF-2. These cell subsets were maintained in these conditions and expanded for up to eight passages (approximately 60  days). MSCs grown in the presence of FGF-2 achieved more population doublings (31 ± 3) than control (23 ± 2) (Fig. 27.1).

Chapter 27  Therapeutic Potential of Mesenchymal Stem Cells 

Fig.  27-1.  MSCs grown in the presence of FGF-2 exhibit shorter population doubling times and achieved a greater number of population doublings than those expanded in control conditions

Fig. 27-2.  The immunosuppressive potential of hMSCs is measured by adding them to the lymphocyte mixture and assessing whether the T-cell response (i.e., the number of interferon-gamma–positive spots) is reduced compared to that observed in the absence of hMSCs. We demonstrate that culture expansion of hMSCs from four donors in the presence of FGF-2 results similar and, in some cases greater, effect on IFN-g production

Cells expanded in the presence of FGF-2 maintained their immunomodulatory potential when compared to matched control cells in IFN-g Elispot assays. Figure 27-2 shows that the T-cell response (i.e., the number of IFN-g-positive spots) is reduced compared to that observed in the absence of MSCs.

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9. Limitations in the Expansion of Mesenchymal Stem Cells A critical technical difficulty, which hinders the clinical application of MSC-based therapies, is the need for substantial culture-expansion. Practical therapeutic applications require cell numbers on the order of 108–109 cells but, with the increasing in  vitro expansion necessary to attain these cell doses, loss of multipotentiality and population senescence rapidly become limiting factors. The current state-of-the-art in MSC technology has not overcome this technical obstacle. The lack of unique markers, the need for long periods of ex vivo expansion, the onset of senescence [6, 13, 25] and loss of potential [6, 13, 25, 30] that occur during mitotic expansion of MSCs hinder the development of MSC-based therapies. For example, a minimum dose of 2 × 106 cells/kg for an 80-kg patient would require 1.6 × 108 cells; in an average MSC preparation, this fold-expansion is not reached until the fourth or the fifth passage; some MSC preparations may not exhibit sufficient proliferative capacity to reach this target and/or may lose their potential before the target dose is reached. Many factors contribute to inadequate expansion; some of these factors are susceptible of technical improvement, such as the aspiration of the bone marrow specimen or the culture conditions (base medium, supplementation, oxygen tension, etc.), while others, such as the titers of MSCs in the donor bone marrow or their intrinsic physiology, are not. In our ongoing study (CWRU 3Y03) involving isolation and expansion of MSCs from matched sibling donors, we have encountered, a number of specimens failed to expand to the desired target cell dose. Twenty culture expansions were attempted from 19 different donors. Eleven donors were expanded to the target infusion dose with a mean of 161 ± 54 × 106 MSCs at harvest and a median cell expansion time of 41  days (range 23–66) (Table  27-2). Nine other cultures failed to reach an infusion cell dose of 0.5 × 106 cells/kg during an 8-week culture period (Fig.  27-3). In preliminary analyses, there was no correlation between donor age, gender, volume of the bone marrow aspirate or the initial number of MNCs in the marrow harvest and the success or failure of the culture.

Table 27-2.  CWRU 3Y03 donor and MSC preparation characteristics. Culture medium

Control

FGF-supplemented

Number of donors

18

4

Cultures meeting minimum infusion dose

9

4

Median (range) age of donors

52 (38–67)

49 (39–58)

Median volume of marrow aspirate

29 ml

a

37 ml 6

1.7–2.4 × 106

0.5–2.4 × 10

Infusion dose range

a

Mean number of MSCs at harvest

161.0 ± 0.5 × 10

158 ± 0.5 × 106

Median (range) days to harvesta

41 (23–66)

24.5 (20–41)

a

Successful cultures only

6

Chapter 27  Therapeutic Potential of Mesenchymal Stem Cells 

Fig. 27-3.  MSCs from siblings of patients enrolled on CWRU 3Y03 were grown in standard culture medium to achieve a minimum target number based on the recipients’ weight. Cultures that did not expand sufficiently (gray lines) were terminated

Fig.  27-4.  MSCs from siblings of patients enrolled on CWRU 3Y03 were grown in culture medium supplemented with FGF-2 to achieve a minimum target number based on the recipients’ weight. All four cultures grown in the presence of FGF-2 met the minimum cell dose

10.  Improved Conditions for Clinical Expansion of Mesenchymal Stem Cells We have obtained FDA approval to modify the conditions for the expansion of MSCs with the addition of FGF-2. All MSC cultures grown in the presence of FGF to date were successful (N = 4) and reached infusion doses of 1.65–2.4 × 106 cells/kg. The mean number of MSCs was 158.0 ± 0.5 × 106 and the median days to harvest was 24.5 (range 20–41) (Table 27-2). Furthermore, a second harvest from one donor that failed to expand in our standard culture media reached 171 ± 27 × 106 MSCs in 27 days when cultured in the presence of FGF, suggesting that FGF may rescue a cell preparation that otherwise would be unable to expand (Fig.  27-4). As an added potentially beneficial

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feature, FGF-treated cells are smaller in size and, upon reinfusion, they may not get trapped in the lungs to the extent that control MSCs do.

11.  Discussion Mesenchymal Stem Cells are isolated from adult tissues and they do not raise the ethical controversy that surrounds embryonic stem cells. MSC-based therapies are based on the principle that, once delivered, the cells will home to the damaged tissues and trigger, enhance or modulate intrinsic repair processes within the host tissue. Unfortunately, MSCs are very difficult to track in vivo and the hypothesis that MSCs home to injured tissues has not yet been unequivocally proven. Factors contributing to this problem are the lack of unique markers for MSCs, the relatively low number of cells infused and the entrapment in the lungs of cells administered intravenously. To exploit the potential of MSC-based therapies, we must be able to isolate and expand MSCs so that they possess the necessary characteristics for successful transplantation, engraftment, and in vivo function but this is only one of the technical hurdles that need to be overcome. The following is a simplified list of goals that need to be accomplished to bring such treatments to the clinic; MSCs should be easy to obtain with minimal morbidity to the donor, innocuous to the host, proliferate extensively without loss of functionality, survive in the host and engraft in the target tissue after implantation and achieve proper in vivo function sustained long term. Unfortunately, not all the items in this list have been achieved and many questions remain unanswered. It is not clear what tissue is the best reservoir of MSCs in humans. The lack of specific markers makes it difficult for this identification. The endpoints of the clinical studies performed with MSCs are clouded because the majority of these studies deal with heterogeneous diseases, patients and treatment regimens. There is also concern about the risk of tumor growth secondary to the immunosuppression induced by the MSCs or the possibility of tumorogenicity due to genetic instability of the MSCs themselves as a consequence of the ex vivo expansion. Last but not least is the high expense associated with the isolation and culture expansion of these cells.

References 1. Haynesworth SE, Goshima J, Goldberg VM, Caplan AI (1992) Characterization of cells with osteogenic potential from human bone marrow. Bone 13:81–88 2. Black IB, Woodbury D (2001) Adult rat and human bone marrow stromal stem cells differentiate into neurons. Blood Cells Mol Dis 27(3):632–636 3. Woodbury D, Schwarz EJ, Prockop DJ, Black IB (2000) Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 61(4):364–370 4. Breyer A, Estharabadi N, Oki M et al (2006) Multipotent adult progenitor cell isolation and culture procedures. Exp Hematol 34(11):1596–1601 5. Ball LM, Bernardo ME, Roelofs H et  al (2007) Co-transplantation of ex vivo expanded mesenchymal stem cells accelerates lymphocyte recovery and may reduce the risk of graft failure in haploidentical hematopoietic stem cell transplantation. Blood 110(7):2764–2767 6. Minguell JJ, Erices A, Conget P (2001) Mesenchymal stem cells. Exp Biol Med (Maywood) 226(6):507–520

Chapter 27  Therapeutic Potential of Mesenchymal Stem Cells  7. Friedenstein AJ (1976) Precursor cells of mechanocytes. Int Rev Cytol 47: 327–359 8. Owen M (1988) Marrow stromal stem cells. J Cell Sci Suppl 10:63–76 9. Owen M, Friedenstein AJ (1988) Stromal stem cells: marrow-derived osteogenic precursors. Ciba Found Symp 136:42–60 10. Caplan AI (1994) The mesengenic process. Clin Plast Surg 21(3):429–435 11. Lennon DP, Haynesworth SE, Bruder SP, Jaiswal N, Caplan AI (1996) Human and animal mesenchymal progenitor cells from bone marrow: identification of serum for optimal selection and proliferation. In Vitro Cell Dev Biol 32(10):602–611 12. Bruder SP, Jaiswal N, Haynesworth SE (1997) Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 64(2): 278–294 13. Digirolamo CM, Stokes D, Colter D, Phinney DG, Class R, Prockop DJ (1999) Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol 107(2):275–281 14. Phinney DG, Kopen G, Righter W, Webster S, Tremain N, Prockop DJ (1999) Donor variation in the growth properties and osteogenic potential of human marrow stromal cells. J Cell Biochem 75(3):424–436 15. Dominici M, Le Blanc K, Mueller I et  al (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8(4):315–317 16. Pittenger MF, Mackay AM, Beck SC et al (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284(5411):143–147 17. Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP (1997) Osteogenic differentiation of purified culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 64:295–312 18. Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU (1998) In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 238(1):265–272 19. Yoo JU, Barthel TS, Nishimura K et  al (1998) The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg Am 80(12):1745–1757 20. Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD (2000) Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 18(6):675–679 21. Mackay AM, Beck SC, Murphy JM, Barry FP, Chichester CO, Pittenger MF (1998) Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng 4(4):415–428 22. Kuznetsov SA, Friedenstein AJ, Robey PG (1997) Factors required for bone marrow stromal fibroblast colony formation in vitro. Br J Haematol 97(3):561–570 23. Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276(5309):71–74 24. Caplan AI (1991) Mesenchymal stem cells. J Orthop Res 9(5):641–650 25. Conget PA, Minguell JJ (1999) Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 181(1):67–73 26. Wakitani S, Saito T, Caplan AI (1995) Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve 18(12):1417–1426 27. Tomita S, Li RK, Weisel RD et al (1999) Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 100(19 Suppl):II247–II256 28. Koc ON, Gerson SL, Cooper BW et  al (2000) Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow

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L.A. Solchaga and H.M. Lazarus mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 18(2):307–316 29. Tomita M, Adachi Y, Yamada H et al (2002) Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina. Stem Cells 20(4):279–283 30. Pittenger MF, Mbalaviele G, Black M, Mosca JD, Marshak DR (2001) Mesenchymal stem cells. In: Koller MR, Palsson BO, Masters JRW (eds) Primary mesenchymal cells. Kluwer, Dordrecht, pp 189–207 31. Banfi A, Bianchi G, Notaro R, Luzzatto L, Cancedda R, Quarto R (2002) Replicative aging and gene expression in long-term cultures of human bone marrow stromal cells. Tissue Eng 8(6):901–910 32. Blazsek I, Delmas Marsalet B, Legras S, Marion S, Machover D, Misset JL (1999) Large scale recovery and characterization of stromal cell-associated primitive haemopoietic progenitor cells from filter-retained human bone marrow. Bone Marrow Transplant 23(7):647–657 33. Galotto M, Berisso G, Delfino L et al (1999) Stromal damage as consequence of high-dose chemo/radiotherapy in bone marrow transplant recipients. Exp Hematol 27(9):1460–1466 34. Meisel R, Zibert A, Taskaya D, Daeubener W, Dilloo D (2003) Bone marrow stromal cells inhibit allogeneic T-cell responses by indolamine 2;3-dioxygenase mediated tryptophan depletion. Blood 102:19a 35. Aggarwal S, Pittenger M (2005) Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105:1815 36. McIntosh K, Klyushnenkova E, Shustova V, Moseley A, Deans R (1999) Suppression of alloreactive T cell response by human mesenchymal stem cells involves CD+ cells. Blood 94:133a 37. DiNicola M, Carlo-Stella C, Magni M et al (2002) Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 99:3838 38. Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O (2003) Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 57(1):11–20 39. Tse W, Pendleton J, Beyer W, Egalka M, Guinan E (2003) Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 75:389 40. Maitra B, Szekely E, Gjini K et al (2004) Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation. Bone Marrow Transplant 33(6):597–604 41. Krampera M, Glennie S, Dyson J et al (2003) Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood 101:3722 42. Beyth S, Borovsky Z, Mevorach D et al (2005) Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness. Blood 105:2214 43. El-Badri N, Wang B, Cherry, Good R (1998) Osteoblasts promote engraftment of allogeneic hematopoietic stem cells. Exp Hematol 29:110 44. Ishida T, Inaba M, Hisha H et  al (1994) Requirement of donor-derived stromal cells in the bone marrow for successful allogeneic bone marrow transplantation: complete prevention of recurrence of autoimmune diseases in MRL/MP-Ipr/Ipr mice by transplantation of bone marrow plus bones (stromal cells) from the same donor. J Immunol 152:3119 45. Kaufman C, Colson Y, Wren S, Watkins S, Simmons R, Ildstad S (1994) Phenotypic characterization of a novel bone marrow-derived cell that facilitates engraftment of allogeneic bone marrow stem cells. Blood 84:2436

Chapter 27  Therapeutic Potential of Mesenchymal Stem Cells  46. Bartholomew A, Sturgeon C, Siatskas M et  al (2002) Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 30(1):42–48 47. Djouad F, Plence P, Bony C et al (2003) Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood 102:3837 48. Le Blanc K, Rasmusson I, Sundberg B et  al (2004) Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 363(9419):1439–1441 49. Lazarus HM, Haynesworth SE, Gerson SL, Rosenthal NS, Caplan AI (1995) Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant 16(4):557–564 50. Koç O, Day J, Nieder M, Gerson S, Lazarus H, Krivit W (2002) Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH). Bone Marrow Transplantation 30:215 51. Lazarus H, Curtin P, Devine S, McCarthy P, Holland K, Moseley A (2000) Role of mesenchymal stem cells in allogeneic transplantation: early phase I clinical results. Blood 96:392a 52. Lazarus HM, Koc ON, Devine SM et al (2005) Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant 11(5):389–398 53. Bensinger W, Martin P, Storer B et al (2001) Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives in patients with hematologic cancers. N Engl J Med 344:175–181 54. Couban S, Simpson D, Barnett M et  al (2002) A randomized multicenter comparison of bone marrow and peripheral blood in recipients of matched sibling allogeneic transplants for myeloid malignancies. Blood 100:1525 55. Le Blanc K, Samuelsson H, Gustafsson B et al (2007) Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells. Leukemia 21(8):1733–1738 56. Fouillard L, Chapel A, Bories D et al (2007) Infusion of allogeneic-related HLA mismatched mesenchymal stem cells for the treatment of incomplete engraftment following autologous haematopoietic stem cell transplantation. Leukemia 21(3):568–570 57. Ringden O, Uzunel M, Sundberg B et al (2007) Tissue repair using allogeneic mesenchymal stem cells for hemorrhagic cystitis, pneumomediastinum and perforated colon. Leukemia 21:2271–2276 58. Le Blanc K, Ringden O (2005) Use of mesenchymal stem cells for the prevention of immune complications of hematopoietic stem cell transplantation. Haematologica 90(4):438 59. Francois S, Mouiseddine M, Mathieu N et al (2007) Human mesenchymal stem cells favour healing of the cutaneous radiation syndrome in a xenogenic transplant model. Ann Hematol 86(1):1–8 60. Rasulov MF, Vasilchenkov AV, Onishchenko NA et al (2005) First experience of the use bone marrow mesenchymal stem cells for the treatment of a patient with deep skin burns. Bull Exp Biol Med 139(1):141–144 61. Bystrov AV, Polyaev YA, Pogodina MA, Rasulov MF, Krasheninnikov ME, Onishchenko NA (2006) Use of autologous bone marrow mesenchymal stem cells for healing of free full-thickness skin graft in a zone with pronounced hypoperfusion of soft tissues caused by arteriovenous shunting. Bull Exp Biol Med 142(1):123–128 62. Vojtassak J, Danisovic L, Kubes M et  al (2006) Autologous biograft and mesenchymal stem cells in treatment of the diabetic foot. Neuro Endocrinol Lett 27(Suppl 2):134–137

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Chapter 28 Hematopoietic Stem Cell Transplantation for Thalassemia Javid Gaziev and Guido Lucarelli

1. Introduction The thalassemias are a group of hemoglobin disorders characterized by a reduced synthesis of one or more of the globin chains (a, b, g, db, gdb, d and egdb) and are the commonest monogenic disorders to cause a major public health problem in the world [1]. It is estimated that there are 270 million carriers of hemoglobin disorders, of whom 80 million are carriers of b thalassemia. The clinical manifestations of b-thalassemia are extremely diverse, ranging from the transfusion–dependent state of thalassemia major to slightly less severe transfusion-dependent state of thalassemia intermedia or to the asymptomatic state of thalassemia trait. The most severe form of this disease is characterized by the complete absence of b-globin production and results from the inheritance of two b0 thalassemia allels, homozygous or compound heterozygous states. These combinations usually result in b-thalassemia major and the patients present themselves within 6  months of life with severe anemia, and if not treated with regular blood transfusions, die within the first 2 years. The anemia is due to a combination of ineffective erythropoiesis, excessive peripheral red blood cell hemolysis, and progressive splenomegaly. The treatment of thalassemia remains a challenge. Conventional regular life-long transfusions and iron chelation have improved survival of these patients especially in the developing world, although patients are not cured with this approach. In contrast, stem cell transplantation has been shown to be curative in 85–91% of patients with thalassemia [2, 3]. Until recently, limited donor availability was the major limiting factor to the widespread using of stem cell transplantation for patients with thalassemia. Recent advances in tissue typing and supportive care have enabled physicians to use stem cell transplantation from alternative donors such as HLA matched unrelated and mismatched related donors with encouraging results.

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_28, © Springer Science + Business Media, LLC 2003, 2010

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2.  Hematopoietic Stem Cell Transplantation: Allogeneic Gene Therapy of Thalassemia At present, allogeneic HSCT is the only cure of b-thalassemia major. It is a form of gene therapy that uses allogeneic stem cells as vectors for genes essential for normal hematopoiesis. Eventually the vector may well be autologous stem cells transformed by the insertion of normal genes but there is no indication that this approach will be a clinical option in the foreseeable future. There are no controlled trials of BMT versus medical treatment for thalassemia major and few studies comparing quality of life. Regular blood transfusion and iron chelation have improved both survival and quality of life of patients with thalassemia and have changed a previously fatal disease with early death to a chronic, although progressive disease compatible with prolonged survival [4, 5]. Despite the prolonged life expectancy, a recent study from U.K. Thalassemia Registry showed a steady decline in survival starting from the second decade, with fewer than 50% of the patients remaining alive beyond 35  years mainly because of poor compliance with chelation therapy [6]. The acute toxicities of transplantation are challenged by the observation that in the developed world patients with thalassemia are well served by medical treatment, and in some countries patients receiving regular blood transfusion and chelation could have a better survival rate [4, 5, 7]. Unfortunately, these same patients with increasing age currently experience poor outcome on conventional treatment, even in well-resourced countries with universal access to good medical treatment [6, 8]. The survival rate of most patients with thalassemia in the developing world, where the disease is more common is less than 20 years because of the unavailability of safe blood products and/or expensive iron chelating drugs as deferoximine. However, recently developed oral iron-chelator deferasirox (Exjade, ICL670) at 20 mg/kg/day given once daily showed similar efficacy to deferoxamine 40/mg/kg/day given by subcutaneous infusion 5 days each week [9], and could allow better compliance with chelation, therefore could have a favorable impact on survival of patients with thalassemia. However, even an ideal iron-chelator with rigorous adherence only substantially reduces, but does not eliminate, the iron overload of patients on lifelong transfusions. The first two transplant procedures for the treatment of thalassemia with marrow from matched related donors were performed in December 1981, in Seattle, WA, and in Pesaro, Italy. The Seattle approach was based on the assumption that the risks associated with BMT would be increased by the iron overload and by sensitization to human leukocyte antigens (HLAs) induced by hypertransfusion. Therefore, it was decided that early clinical studies would be conducted in very young patients who had received very few transfusions. On December 3, 1981 a 14-month-old child with b-thalassemia major untransfused until the time of the transplant, received BMT from his HLA-identical sister in Seattle. The treatment was completely successful. The Pesaro approach was based on an assessment that restricting transplants to untransfused patients was impracticable. On December 17, 1981 the Pesaro team performed a transplant in a 16-year-old thalassemic patient who had received 150 RBC transfusions, using marrow from his HLA identical brother. This patient rejected the graft and was the first of an extensive series of transplants for thalassemia that provides most of the data supporting this chapter.

Chapter 28  Hematopoietic Stem Cell Transplantation for Thalassemia 

2.1. Preparatory Regimens Preparatory regimens for BMT of patients with thalassemia must achieve two objectives: elimination of the disordered marrow and establishment of a tolerant environment that will permit transplanted marrow to survive and thrive. Total body irradiation (TBI) can satisfy both these objectives, but there are many reasons to avoid the use of this marrow-ablative modality. These include the known growth-retarding effects of TBI in young children and the increased risk of secondary malignancies which has been reported in patients treated for leukemia, lymphoma and aplastic anemia [10, 11]. These hazards are particularly objectionable in very young patients with a potential for a long life span. The risk of these toxicities has not yet been fully explored for cytotoxic regimens that do not involve TBI. There is a considerable body of experience with the use of busulfan (BU) and its derivatives in ablating marrow in patients undergoing HCT for the treatment of non-malignant conditions such as the Wiskott–Aldrich syndrome and inborn errors of metabolism [12, 13]. Cyclophosphamide (CY) is an agent that is well established as providing immunosuppression adequate for allogeneic engraftment of patients with aplastic anemia [14, 15]. Santos et  al. reported on its use in high doses (200  mg/kg over 4  days) as the sole antitumor agent in patients receiving allogeneic transplants for leukemia and demonstrated that it was sufficiently immunosuppressive to permit sustained allogeneic engraftment [16]. Experimental data and clinical experience in the use of chemotherapy-only transplant regimens for the treatment of malignant diseases [17, 18] have been pivotal in developing regimens appropriate for the treatment of thalassemia by transplantation, using a combination of BU and CY which can eradicate thalassemia and facilitate sustained allogeneic engraftment. Busulfan pharmacokinetic (PK) variability following oral administration is well documented in both adult and especially in pediatric patients. Therefore, therapeutic drug monitoring of BU with targeted AUCs is recommended because of the relationship between systemic exposure to the drug and both toxicity and success of treatment [19–22]. The recently developed intravenous formulation of BU showed good tolerability, less toxicity profile and high inter-and intrapatient pharmacokinetics consistency, allowing for predictable systemic exposure without PK monitoring [23]. In an attempt to reduce transplant related toxicity reduced-intensity conditioning regimens have been developed. Unfortunately, such regimens in non malignant disorders often resulted in higher rates of graft failure. Recently developed reduced-intensity regimen for non malignant diseases based on using Campath-1H in a novel manner -3 weeks before stem cell transplantation to provide more host immune suppression and less in vivo donor T-cell depletion showed higher engraftment rates [24]. This approach could be useful also for selected patients with thalassemia. 2.2. Disease Eradication The disease is characterized by extreme marrow hyperplasia with aggressive extension of a rapidly proliferating erythron into intra- and extra-medullary areas not usually occupied by marrow. This results in major bone remodeling together with marked hepatomegaly and splenomegaly. By analogy with the behavior of malignant tissue, it might be supposed that this large mass of

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rapidly proliferating hematopoietic tissue would be more difficult to eradicate than normal hematopoietic tissue, and more likely to recur after transplantation. Although post-transplant thalassemic recurrence is a problem, it occurs in circumstances that differ from those usually observed with leukemic relapse. The most common presentation of leukemic relapse is the return of host-type leukemia in the presence of a persisting immune system of donor origin. In contrast, the recurrence of thalassemia usually occurs in the context of a return of host type immune and hematologic reconstitution. Thus, this event has aspects of both relapse and rejection and it is customary to speak of this phenomenon as rejection. 2.3. Engraftment Nearly all thalassemic patients undergoing transplantation have been transfused repeatedly. This lead to sensitization to HLA antigens therefore could increase the risk of rejection as it has been demonstrated in patients with aplsatic anemia [25, 26]. There is a substantial incidence of graft rejection in thalassemia patients even after myeloablative preparatory regimens, and this seems to be related to the stage of disease at the time of transplant. In most cases of graft rejection the thalassemic marrow will regrow and subsequent survival will be long albeit with thalassemia. Occasionally, patients will reject grafts without recurrence of thalassemia. Unless they are rescued by a second transplant such patients will die from the consequences of marrow aplasia. As the preferred graft source nearly all transplant centers worldwide use bone marrow. The higher rate of chronic GvHD observed following peripheral blood stem cells may be offset by lower relapse rates in some hematological malignancies. In contrast, there is no benefit of chronic GvHD for nonmalignant diseases [27]. 2.4. Transplant-Related Morbidity and Mortality Preparatory regimens capable of eradicating a diseased marrow and facilitating persistent engraftment are necessarily toxic, and the consequences of successful allogeneic marrow engraftment include acute and chronic graft-vs.-host disease (GVHD), syndromes associated with severe immune incompetence. Transplant related toxicity may be aggravated by GVHD and by measures aimed at the prevention and treatment of this complication. Such toxicity can be categorized either as regimen-related toxicity (RRT) or as GVHD. Regimen-related toxicity has been well described in patients treated by BMT for hematologic malignancies [28, 29]. The lungs and liver are the organs most at risk for toxicity induced by TBI and BU while the heart is the main site of CY-induced damage. Increasing age of the patients, previous exposure to cytotoxic agents and the presence of latent viruses such as hepatitis C and cytomegalovirus adversely influence these toxicities. Patients transplanted for the treatment of thalassemia derive benefit from the fact they are usually young and without prior exposure to cytotoxic agents.

Chapter 28  Hematopoietic Stem Cell Transplantation for Thalassemia 

2.5. Risk Classes Analysis of the influence of pretransplant characteristics on the outcome of transplantation was conducted in 161 patients aged less than 17 years who were all treated with exactly the same regimen [2, 30]. In multivariate analysis hepatomegaly more than 2  cm, portal fibrosis and irregular chelation history were associated with a significantly reduced probability of survival. The quality of chelation was characterized as regular when deferoxamine therapy was initiated no later than 18 months after the first transfusion and administered subcutaneously for 8–10  h continuously for at least 5  days each week. Any deviation from this regimen was defined as irregular chelation. On the basis of these risk factors patients were categorized into three risk classes. Class 1 patients had none of these adverse risk factors, class 3 patients had all three and class 2 patients had one or two adverse risk factors. 2.6. Outcome of Transplant from HLA Matched Related Donors 2.6.1. Class 1 Patients These are younger patients without risk factors. Between October 1985 and January 2003 145 class 1 patients with median age of 4  years (range 1–16  years) were given bone marrow transplantation following conditioning (Protocol 6) with Bu 3.5 mg/kg/day for four consecutive days and CY 50 mg/ kg/day for the subsequent 4 days. Graft-versus-host disease prophylaxis consisted of cyclosporine (CSA) and low-dose methylprednisolone (MP) for all but the last 37 patients received CSA, MP and a modified “short course” of methotrexate (MTX). The probability of overall survival, thalassemia-free survival and rejection for these patients were 90, 87 and 3% respectively [2, 31]. 2.6.2. Class 2 Patients These patients have one or two risk factors as a consequence of poor transfusion therapy and/or irregular chelation. Three hundred and thirty tree patients with median age of 9  years (range 2–16  years) were given transplant after preparation with the same conditioning regimen used for class 1 patients. They had an overall survival rate, thalassemia-free survival and rejection of 87, 85 and 3% respectively [2, 31]. 2.6.3. Class 3 Patients When class 3 younger patients (age <17  years) were treated with the same conditioning as class 1 and class 2 patients (BU14  mg/kg and CY200  mg/ kg), they showed a low probability of thalassemia-free survival (53%) and a higher probability of non-rejection transplant related mortality (39%) [32]. In an attempt to improve results in class 3 patients, new treatment regimens were devised using BU 14 mg/kg and lower doses of CY (160 or 120 mg/kg). These regimens improved the probability of survival in class 3 younger (age <17 years) patients from 53 to 79%, but were associated with an increase of rejection probability from 7 to 30%, probably due to inadequate immunosuppression and failure to eradicate the massive erythroid hyperplasia characteristic of these patients [3, 31, 32]. Therefore in April 1997 a new preparative regimen (Protocol 26) was adopted for class 3 patients <17  years of age in an attempt to decrease the

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higher rejection rate through reducing erythroid expansion and increasing immunosuppression over a period of time to avoid unacceptable drug toxicity during conditioning regimen [33]. Protocol 26 was devised on the assumption that preparation with BU 14  mg/kg and CY 160  mg/kg was inadequate to eradicate thalassemic hematopoiesis in class 3 patients <17 years of age. This protocol involved an intensified preparation with 3 mg/kg of azathioprine and 30  mg/kg hydroxyurea daily from day -45 from the transplant, fludarabine 20  mg/m2 from day -17 through day -13, followed by the administration of BU 14 mg/kg total dose and CY 160 mg/kg total dose. Graft-versus-host disease prophylaxis consisted of CSA, low-dose methylprednisolone a modified “short course” of MTX. During this time interval patients received a regimen of hypertransfusion to keep the level of hemoglobin between 14 and 15 g/dL, continuous 24-h infusions of 40 mg/kg of deferoxamine via the central venous catheter and growth factors, granulocyte colony-stimulating factor (G-CSF) and erythropoietin twice weekly to maintain stem cell proliferation in the face of hypertransfusion, thereby facilitating the effect of the hydroxyurea. The probabilities of survival, thalassemia-free survival, rejection and non-rejection mortality in 33 such patients treated with Protocol 26 were 93, 85, 8 and 6%, respectively [33]. Interestingly this regimen improved the probability of thalassemia-free survival for patients with class 3 thalassemia aged younger than 17 years at the time of the transplantation from 58 to 85% together with a reduction of the probability of rejection from 30 to 8% as compared with previous preparative regimens. 2.6.4. Adult Patients Adult thalassemia patients (age >17 years) have more advanced disease and treatment related organ complications mainly due to prolonged exposure to iron overload. One hundred and seven patients with thalassemia aged from 17 through to 35 years (median 20 years) were given transplant from HLA identical siblings between November 1988 and September 1996 [34, 35]. Eighteen patients were in class 2 and these received BU 14  mg/kg and CY 200  mg/ kg. The other patients were in class 3 and received BU 14–16  mg/kg and CY 120–160  mg/kg. The probability of survival, thalassemia-free survival, rejection and non-rejection mortality for the entire group of these patients were 66, 62, 4 and 37% respectively. Interestingly, unlike class 3 younger patients, reduced dose intensity of conditioning in adults was not associated with a higher rejection rate (4%) suggesting that donor-host tolerance might be induced even among extensively transfused patients. Therefore, these results show that adult patients could be given less intensive transplant conditioning to overcome excessive transplant related toxicity. From April 1997 all adult patients were prepared for transplantation according to Protocol 26 with the only difference that the dose of Cyclophosphamide was reduced to 90 mg/kg total dose. The probability of survival, thalassemiafree survival, rejection and non-rejection mortality in 15 high risk group patients were 65, 65, 7 and 28% respectively [36]. Table  28.1 reports Pesaro experience of BMT for thalassemia from HLA matched related donors between May 1985 and December 2003. Bone marrow transplant programs for thalassemia have been established in several countries worldwide with results equivalent to those obtained in Pesaro [37–46].

Chapter 28  Hematopoietic Stem Cell Transplantation for Thalassemia 

497

Table 28-1.  Outcome of BMT for thalassemia from HLA matched related donors between May 1985 and December 2003. Treatment Protocols in use. Class 1

Class 2

Class 3 (age <17 years) (since 1997)

Adult (age >17 years) (since 1997)

Patients, n

145

333

33

15

Treatment Protocol

6

6

26

26

Survival %

90a

87a

93b

65b

a

a

b

65b

Thalassemia-free survival %

87

85

85

Death %

10

13

 6

28

Rejection %

3

 3

 8

 7

a

 Survival and thalassemia-free survival at 15 years  Survival and thalassemia-free survival at 5 years

b

2.7. Mixed Chimerism Mixed hematopoietic chimerism (MC) is a common phenomenon after myeloablative transplantation for thalassemia. In fact the incidence of MC at 2 months was 32.2% [3]. While none of the patients with complete chimerism at 2 months rejected their grafts, 35 of 108 patients with MC determined at the same time, lost the graft suggesting that MC after BMT for thalassemia is a risk factor for rejection. The percentages of residual host hematopoietic cells (RHCs) after transplant were predictive for graft rejection with nearly all patients experiencing graft rejection when RHCs exceeded 25%, and only 13% of patients showing rejection when RHCs were less than 10% [47, 48]. Patients with RHCs between 10 and 25% had the risk of rejection of 41%. Stable mixed chimerism is an exciting phenomenon after HSCT. Ten percent of patients receiving BMT for thalassemia following myeloablative conditioning became persistent mixed chimeras and are transfusion-independent, suggesting that few engrafted donor cells might be sufficient for correction disease phenotype in patients with thalassemia major, once donor-host tolerance has been established. 2.8. Graft Failure or Rejection Patients with engraftment failure and without a functioning marrow have a bleak prospect because an early second transplant with a second course of conditioning is usually not a reasonable option. However, occasionally patients have late graft failure without thalassemia recurrence and in this situation second transplant attempts with intensive conditioning may provide the only treatment option to offer a chance of prolonged survival. Patients who reject their grafts and have a return of host hematopoiesis do not have an urgent need for second transplants, and such interventions can be delayed until the toxic effects of the conditioning regimen for the first transplants have ameliorated. At least a year should be allowed to elapse between the first and second transplant. We have performed second transplants in 21 patients who had complete thalassemia recurrence and in 11 patients who developed irreversible aplasia following the first transplant. Patients with thalassemia recurrence received BUCY alone or in association with total lymphoid

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irradiation or anti-lymphocyte globulin, while most patients with aplasia were given CY with or without total lymphoid irradiation [49]. The probability of survival and thalassemia-free survival were 55 and 29% in patients with thalassemia recurrence, and 41 and 41% in patients with aplasia respectively. Survival and thalassemia-free survival were better in patients who had late graft failure (over 60  days) compared with patients developing graft failure within 60 days after the first transplant.

2.9. Graft-Versus-Host Disease The probability of grade II to IV acute GvHD was significantly low (17%) in patients given CSA and a short course of MTX as compared to patients receiving CSA and methylprednisolone as GvHD prophylaxis (32%). The probability of moderate or severe chronic GVHD in thalassemia patients was 8 and 2% respectively [50]. This low incidence of chronic GVHD probably related to normal thymic function in thalassemic patients. 2.10. Stem Cell Transplantation from Alternative Donors 2.10.1. Alternative Related Donors The possibility of having an HLA matched related donor is 25–30%. As HSCT is the only effective cure for thalassemia there is the need to develop alternative stem cells donations. Our past experience with BMT from alternative donors for 29 patients with b-thalassemia major who received phenotypically matched grafts or haploidentical grafts mismatched for one, two, or three antigens was characterized by higher graft failure (55%), and low thalassemia-free survival (21%) [51]. Haploidentical T-cell depleted HCT from mother to child using megadose of CD34 cells is under study at our Center. 2.10.2. Unrelated Bone Marrow Transplantation Results of HSCT from unrelated donors for the treatment of malignant disease have improved steadily, mainly due to the introduction of high-resolution molecular techniques for histocompatibility testing and improvements in the management of post-transplant complications. A recent analysis of the outcome of BMT from matched volunteer unrelated donors prospectively selected using high-resolution molecular typing for HLA class I and class II loci for 32 patients with b-thalassemia major who received BUCY ± tiothepa regimen showed thalassemia-free survival of 66% and mortality of 25%, although patients sharing at least one extended haplotype had a better survival rate [52]. Encouraging results were also obtained in adult thalassemic patients who received bone marrow from unrelated donors with the probability of survival, thalassemia-free survival, transplant-related mortality and rejection of 70, 70, 30 and 4% respectively [53]. Although there has only been a small number of patients transplanted so far, these data show that unrelated marrow transplantation not only in younger patients but also in adult patients from well-selected donors may offer a success rate similar to those obtained from HLA-identical sibling transplantation. The main limitation of the experience with unrelated BMT for thalassemia is that, using such stringent criteria, only about one-third of thalassemia patients who started the search found a suitable donor in a median time of 3–4 months. One possibility to increase the donor

Chapter 28  Hematopoietic Stem Cell Transplantation for Thalassemia 

pool could be adopting less stringent criteria of HLA matching for selection donors with one or two allelic disparity, but the results of such transplants remain to be determined. 2.10.3.  Unrelated Cord Blood Transplantation Advantages such as faster availability, tolerance of 1–2 HLA mismatches, and low incidence of acute GVHD have made UCB transplants attractive for patients with some non malignant diseases [54]. Recently, data on 36 patients with transfusion-dependent thalassemia and 2 with sickle cell disease (SCD) who received unrelated cord blood transplants in 14 transplant centers were reported [55]. The probabilities of survival and thalassemia-free survival are 77 ± 7 and 65 ± 8% respectively. These data are encouraging and show that patients who need transplantation and do not have a suitable either related or unrelated matched donor can benefit from unrelated cord blood transplantation. 2.11. Management of “the Ex-thalassemic” After Transplant After successful transplantation, we call thalassemic patients as “ex-thalassemic” after BMT. Ex-thalassemic patients still carry the clinical complications acquired during years of transfusion and chelation therapy. Among the issues requiring long-term management in such patients are iron overload, chronic hepatitis, liver fibrosis and endocrine dysfunction. Management of these complications in ex-thalassemics is an important issue. Iron stores after transplantation remain elevated in most patients receiving HSCT in advanced stage of disease (class 2 and class 3 patients) [56]. Analysis of the natural history of liver fibrosis following BMT for thalassemia showed that ion overload and hepatitis C virus infection are independent risk factors for liver fibrosis progression and their concomitant presence results in a striking increase in risk [57]. Therefore, the toxic effect of iron overload contributing to progression of already-present organ damage should be avoided as soon as possible using post-transplant iron depletion. Either regular phlebotomy or chelation therapy can successfully remove excess iron from the body by normalizing the iron pool, which results in marked improvement in liver and cardiac function [58–61]. Growth failure and endocrine dysfunction are common in thalassemia major patients treated by conventional treatment. While the role of busulfancontaining regimens on growth velocity after transplant remains controversial, the negative effect of this regimen on gonadal damage is well documented. Children receiving transplant before 8 years of age showed a normal growth rate, while older children, class 3 patients, and patients who developed chronic GVHD had impaired growth [62]. Gonadal damage is a common side effect of BUCY conditioning. Indeed, approximately one third of boys and two thirds of girls failed to spontaneously enter puberty following transplant [63]. Nevertheless some patients can restore their fertility after transplant, which is supported by our observations of five successful pregnancies and four spontaneous paternities in our ex-thalassemics. These data demonstrate that patients exposed to BUCY regimen are not inevitably infertile. Infection with the HCV is common in thalassemic patients; particularly in those transfused before second generation enzyme-linked immunosorbent assay (ELISA) tests became available for detecting HCV in donated blood. In thalassemia, liver damage due to HCV infection is exacerbated by iron

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overload, and liver disease is a recognized cause of mortality and morbidity. Chronic HCV infection and transplant-related complications are probably the only factors that may limit survival in ex-thalassemics. Thus, avoidance of progression of liver damage to cirrhosis must be a primary goal. Present standard therapy is the peginterferon/alfa-ribaverin for adults and children without thalassemia [64]. Concern about ribaverin –induced hemolysis and worsening of the anemia and iron overload limit the use of this therapy in patients with thalassemia. Ex-thalassemics with complete donor chimerism and chronic hepatitis C will experience similar spectrum of side effects compared to those patients without thalassemia. Therefore ex-thalassemics should be offered treatment with the peginterferon/alfa-ribaverin combination after they have completed their iron depletion program. It has been shown that with the increasing number of transplant survivors there is a risk for secondary malignancies. Patients who received BMT for thalassemia major and SCD had a low incidence (0.8%) of malignancies. The type of malignancies observed in our patients consisted of three early and one late non-Hodgkin’s lymphomas and four solid tumors (squamous cancer, Kaposi’s sarcoma, melanoma, and colon cancer, respectively). Four of these patients are alive and well.

3. Summary and Future Considerations HSCT from HLA matched related or unrelated donor offers the only chance of cure and the return of both life expectancy and quality of life to normal. Current results of HSCT clearly demonstrate that 80–87% of patients with thalassemia could be cured if transplants are given at a younger age. Performing transplants at a younger age before the patient has developed disease- and treatment-related complications is essential. Adult patients with thalassemia treated with myeloablative conditioning continue to have inferior results because of their advanced stage of disease. They might be more suitable candidates for current fludarabine-based reduced intensity conditioning regimens. Ex-thalassemic patients still carry the clinical complications acquired during years of transfusion and chelation therapy. Therefore management of these complications is essential. Some late complications of BMT such as growth disturbances, endocrine complications, and infertility are important issues in evaluating the role of HSCT for hemoglobinopathies. Therefore, studies comparing the growth and development of patients treated by medical therapy with those treated with BMT are warranted. Despite substantial progress made in the field of HSCT in thalassemia such complications as increased rates of graft failure and transplant related mortality remain major obstacles to successful outcome. Future research will focus on decreasing the transplantation-related morbidity and mortality and on increasing thalassemia-free survival. This will include designing effective, reduced toxicity conditioning regimens, discovering targeted therapies to prevent and treat GvHD, in vitro expansion and modification of stem cells, and using embryonic stem cells to eliminate the need for HLA typing and toxic conditioning regimens. Use of gene therapy with targeting autologous or allogeneic stem cells also may hold promise for the correction of genetic diseases such as thalassemia and sickle cell anemia.

Chapter 28  Hematopoietic Stem Cell Transplantation for Thalassemia 

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J. Gaziev and G. Lucarelli 60. Giardini C, Galimberti M, Lucarelli G et al (1995) Desferrioxamine therapy accelerates clearance of iron deposits after bone marrow transplantation for thalassemia. Br J Haematol 89:868–873 61. Mariotti E, Angelucci E, Agostini A, Baronciani D, Sgarbi E, Lucarelli G (1998) Evaluation of cardiac status in iron-loaded thalassemia patients following bone marrow transplantation: improvement in cardiac function during reduction in body iron burden. Br J Haematol 103(4):916–921 62. Gaziev D, Galimberti M, Giardini C, Baronciani D, Lucarelli G (1993) Growth in children after bone marrow transplantation for thalassemia. Bone Marrow Transplant 12(Suppl 1):100–101 63. De Sanctis V, Galimberti M, Lucarelli G et al (1997) Growth and development in ex-thalassemic patients. Bone Marrow Transplant 19(Suppl 2):126–127 64. Strader DB, Wright T, Thomas DL, Seef LB (2004) Diagnosis, management, and treatment of hepatitis C. Hepatology 39(4):1147–1171

Chapter 29 Viral Infections in Hematopoietic Stem Cell Transplant Recipients Per Ljungman

1. Introduction Viral infections are important as causes of morbidity and mortality after allogeneic stem cell transplantation (SCT). Severe viral infections are more common after unrelated and mismatched donor SCT and in particular after haploidentical SCT. B-cell function and specific antibodies are the main defense mechanisms against infection with exogenous viruses, thus reducing the risk for reinfection in already seropositive individuals. On the other hand, T-cell function in particular cytotoxic T-cell function is the main mechanism for preventing severe viral disease and also for the control of viruses such as herpesviruses that can cause latency and thus reactivate in an immunocompromised individual. The immune defects in SCT-patients are frequently complex with defects in cytotoxic T-lymphocyte, helper T-lymphocyte, NK-cell, and B-lymphocyte functions. T-cell dysfunction is usually most important early after SCT while deficient B-cell reconstitution can remain for many years after SCT. Furthermore, since loss of specific antibodies occurs frequently over time after allogeneic SCT, this will also increase the risk for reinfections with previously encountered viruses such as measles or varicella-zoster virus (VZV) and allow reactivation of viruses controlled by antibodies such as hepatitis B virus (HBV) [1, 2].

2. Diagnosis of Viral Infections Many different techniques have been developed for diagnosis of viral infections. A summary is shown in Table  29-1. During recent years, important advances have been made through the use of rapid nucleic acid testing improving sensitivity and thereby making specific diagnosis and monitoring of viral infections feasible. The most commonly used technique is polymerase chain reaction (PCR) especially when used for determining viral load. Other techniques such as the hybrid capture assay, NASBA or branched-DNA have been applied and have shown good sensitivity and high specificity. The source of From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_29, © Springer Science + Business Media, LLC 2003, 2010

505

Baseline risk stratification

Baseline risk stratification

Baseline risk stratification

Baseline risk stratification

Not useful

Not useful

Not useful

Not useful

HSV

VZV

CMV

EBV

HHV-6

Respiratory viruses

Adenovirus

BK-virus/JC-virus

Sometimes useful

Diagnosis of disease

Diagnosis of disease

Diagnosis of disease

Diagnosis of disease

Diagnosis of disease

Diagnosis of disease

Diagnosis of disease

Not applicable

Diagnosis of infection

Diagnosis of infection

Rarely useful

Rarely useful

Monitoring (antigenemia)

Diagnosis of acute infection

Diagnosis of acute infection

EM electron microscopy; IF direct immunofluorescence; q quantitative; PCR polymerase chain reaction

Serology

Virus

Histopathology/ immune histochemistry/DNA hybridization IF

Table 29-1.  Main use of diagnostic tests for virus infections in stem cell transplant recipients.

Diagnosis of infection (urine, blood); diagnosis of CNS disease

Monitoring (blood); diagnosis of CNS disease

Diagnosis of infection

Diagnosis of CNS disease

Monitoring (blood)

Monitoring (blood); diagnosis of CNS disease. Other applications need further studies

Diagnosis of disease especially in CNS

Diagnosis of disease especially in CNS

(q) PCR

Rarely useful

Diagnosis of acute infection; typing

Diagnosis of acute infection

Rarely useful

Rarely useful

Diagnosis of disease

Diagnosis of acute infection

Diagnosis of acute infection

Cell culture

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Chapter 29  Viral Infections in Hematopoietic Stem Cell Transplant Recipients 

the specimen, the timing of collection in relation to onset of symptoms, the rapidity and method of delivery to the laboratory, and the clinical and epidemiological data provided to the laboratory are important factors that directly affect the likelihood of successful isolation and/or identification of a viral pathogen.

3. Cytomegalovirus 3.1. Risk Factors Cytomegalovirus (CMV) remains one of the most important complications to allogeneic bone marrow and stem cell transplantation. CMV can cause multi-organ disease after SCT including pneumonia, hepatitis, gastroenteritis, retinitis, and encephalitis. CMV disease can occur both early and late after transplantation [3–6]. Seropositivity of the patients remains a risk factor for transplant related mortality in unrelated transplant patients despite major advances in early diagnosis and management [7–9] Seronegative patients with seropositive stem cell donors develop primary CMV infection in about 30% and have an increased mortality in bacterial and fungal infections [10]. In a study using the EBMT registry database, CMV seropositive patients receiving seropositive unrelated donor grafts had improved survival and reduced TRM compared to those receiving seronegative grafts and a similar result was found in a single center study [11, 12]. The mechanism for this positive effect was hypothesized to be the transfer of CMV-specific donor cells with the grafts. Other studies have, however, failed to find this correlation and therefore, it remains controversial [13]. Other identified risk factors include acute and chronic GVHD and the use of mismatched or unrelated donors. Sirolimus as prophylaxis against acute GVHD has been reported to result in a lower risk for CMV reactivation [14]. The mechanism behind this reduced risk is unknown. CMV might also be one factor in the pathogenesis of chronic GVHD [15, 16]. 3.2. Prophylaxis Against CMV Infection and Disease Since the prognosis of established CMV disease is still poor, preventive measures are very important. The available strategies can be divided into prevention of a primary infection, recurrence of CMV (prophylaxis), and prevention of development of disease when a reactivation has occurred (preemptive therapy). Serology should be performed before SCT in both patients and donors. Patients who are CMV seronegative before SCT should if possible be transplanted from a CMV seronegative donor [17]. To reduce the risk of CMV transmission from blood products, blood products from CMV seronegative donors or leukocyte depleted blood products should be used, as CMV is mainly harbored in the leukocyte fraction [18–20]. Which strategy is preferable is still not definitively settled [21, 22]. In many centers, and even in entire countries, leukocyte filtration is obligatory for all blood products and no study has in a controlled fashion compared the benefit of use of seronegative blood to that of already filtered blood products. IV immune globulin has at best a minor effect and has therefore been replaced by other more effective strategies. High doses of acyclovir and valacyclovir can reduce the risk for CMV

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infection [23–26]. Valacyclovir can reduce the need for preemptive therapy to approximately 50% [26]. I.v. ganciclovir is effective for prevention of CMV disease [27, 28]. However, these studies were performed before the widespread use of growth factors such as G-CSF and ganciclovir induced neutropenia was a problem in both studies. Valganciclovir is the product of ganciclovir giving similar blood levels as i.v ganciclovir but no study has evaluated its efficacy as a prophylactic agent. 3.3. Preemptive Therapy Preemptive therapy based on early detection of CMV has become the most commonly used strategy for prevention of CMV disease after allogeneic SCT [29, 30]. As early identification of patients at risk for developing viral disease reduces virus-related morbidity and mortality, monitoring with sensitive techniques such as antigenemia or quantitative PCR is indicated in all allogeneic SCT patients. Viral load monitoring seems to be of importance for assessing the risk for CMV disease or the efficacy of antiviral therapy [31–35]. Ganciclovir is the most used drug for preemptive therapy. Valganciclovir has been studied in several uncontrolled studies and in a small randomized pharmacokinetic study and gives higher drug exposure compared to i.v. ganciclovir and similar efficacy [36]. Both drugs are associated with bone marrow suppression and renal toxicity. Antiviral resistance can develop on the basis of mutations in the CMV genes, which the drugs inhibit. However, virus mediated antiviral resistance is quite rare in SCT patients while “clinical” resistance based on rapid replicating virus in the severely immunocompromised patients is quite common especially early after initiation of antiviral therapy. Increasing antigenemia or CMV DNA is therefore commonly not a sign of antiviral resistance and does not necessitate change of therapy [32, 37]. Although foscarnet is as effective as ganciclovir [38], it is more commonly used today as a second line drug. Foscarnet is associated with renal toxicity and electrolyte disturbances. The duration of preemptive therapy has varied in the published studies. One strategy is to continue therapy until day 100 after SCT [39] while the other possibility is to treat until the indicator test becomes negative, usually resulting in a shorter duration of therapy [38, 40]. Also the combination of ganciclovir and foscarnet has been used [41, 42]. Cidofovir (3–5 mg/kg per week) has also been used as a second-line agent but is associated with renal toxicity [43–45]. Case reports have been published of treatment with leflunomide or artesunate in patients failing other antiviral therapies [46–48]. 3.4. CMV Disease Appropriate diagnostic procedures should be undertaken in patients suspected to have CMV end-organ disease [49]. The prognosis in patients with established CMV disease is still poor [3, 50]. Standard therapy of CMV pneumonia has been intravenous ganciclovir combined with high dose immune globulin but this standard was questioned by the results of an uncontrolled study suggesting that the advantage of adding immune globulin is limited with no improvement in survival over ganciclovir therapy given alone [50]. For patients with CMV disease other than pneumonia, the addition of immune

Chapter 29  Viral Infections in Hematopoietic Stem Cell Transplant Recipients 

globulin does not seem to be beneficial [51]. A retrospective survey reported that cidofovir could salvage nine of 16 patients with CMV pneumonia failing therapy with ganciclovir, foscarnet, or the combination [43]. 3.5. Immune Monitoring and Immune Therapy The lack of specific immunity to CMV, both regarding cytotoxic T-cell (CTL) response and helper T-cell response to CMV, has been associated with a high risk for CMV disease [3, 5, 52, 53]. Monitoring of CD8 and/or CD4 CMV specific T-cells has therefore been studied and different techniques can be applied including detection by tetramers containing immunodominant peptides from CMV or measurement of peptide-specific intracellular cytokine staining [54–59]. Riddell et al. have shown that specific CTL can be cloned in vitro, safely be given to the patient, and their activity be detectable during follow-up [52, 60, 61]. Techniques for isolation of CTL including the use of peptide pulsed dendritic cells, selection by tetramer technology, or vaccination with peptide pulsed dendritic cells have been developed and several centers are testing these strategies in clinical trials [58, 62–66].

4. Herpes Simplex Virus Herpes simplex virus (HSV) can cause local and rarely disseminated infections after SCT. Serology is useful for determining the risk for reactivated HSV infection and should be performed before transplantation. The manifestations in transplant patients can be atypical causing generalized inflammation and pain from the mucous membranes without classical vesicular or ulcerative lesions. Generalized and invasive disease can occur but encephalitis is not more frequent in immunocompromised compared to immune competent patients. Acyclovir prophylaxis is indicated in all HSV seropositive SCT recipients [67]. The duration of antiviral prophylaxis should be at least during the aplastic phase but a longer duration should be considered in patients with GVHD or a history of frequent reactivations before the transplantation [67]. A recent study has shown a 2-year probability for HSV disease of 32% when acyclovir was given for 30  days compared to 0% if prolonged prophylaxis was given [68]. Acyclovir resistant virus strains are still quite rare but seem to be more common in high risk patients such as unrelated donor transplants or patients with severe GVHD [69–71]. However, the risk was reported to be very low in patients receiving prolonged prophylaxis [68]. The recommended drug for acyclovir-resistant HSV is foscarnet [72–74]. Two reports have described mutants resistant to both acyclovir and foscarnet [69, 70]. Currently, the only available antiviral drug available for treatment of double resistant HSV is cidofovir.

5. Varicella-Zoster Virus A primary VZV infection (varicella) is an uncommon but severe complication in SCT patients [75]. Seronegative patients are at risk for developing varicella and preventive measures are therefore indicated. The risk is highest in children but cases of varicella-like disease in seropositive adults becoming seronegative

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after SCT have been described. Serology is therefore important to identify patients at risk for varicella and should be performed in all patients before and at regular intervals after SCT. Varicella-zoster immune globulin is the recommended prophylactic measure in seronegative patients if it can be given within 4 days after a household or other type of close exposure [67]. Another option is prophylaxis with acyclovir or valacyclovir but there are no published data regarding effectiveness. Reactivated VZV infection – herpes zoster – develops in approximately one third of the SCT recipients in the absence of prophylaxis [78–79]. Severe and fatal cases have also been reported after allogeneic SCT with reduced conditioning. Herpes zoster is usually dermatomal but disseminated and potentially fatal infections with visceral involvement can occur [75]. The clinical picture might be atypical with gastro-intestinal, liver, or CNS disease occurring in the absence of skin lesions. The risk of herpes zoster is highest between 3 and 6 months after transplantation and decreases thereafter steadily over the first 2 years after SCT [80]. Therefore, the duration of antiviral prophylaxis must be long to be effective. A rebound phenomenon occurs when prophylaxis is given for 6 months [81, 82] but does not exist if prophylaxis is given for 12 months [83]. Longer prophylaxis reduces the rates even further especially in patients with chronic GVHD [80]. The recommended therapy for primary varicella, disseminated herpes zoster, or localized zoster developing early after SCT or in patient on treatment for GVHD is intravenous acyclovir 10 mg/kg (or 500 mg/m2) three times daily. For localized dermatomal herpes zoster occurring late after SCT especially on patients of immunosuppression, clinical experience suggests that oral therapy with acyclovir, famciclovir, or valaciclovir is effective in the majority of patients [84, 85].

6. Epstein-Barr Virus Epstein-Barr Virus (EBV) is frequently detected after allogeneic SCT [86–90]. However, only a few case reports have suggested that it directly causes significant disease such as meningo-encephalitis [91]. EBV induced post-transplant lymphoproliferative disease (PTLD) is a serious complication to allogeneic SCT. Although the incidence of EBV-PTLD is generally lower than 2% following allogeneic SCT, it may increase up to 20% in patients with risk factors such as mismatched donor SCT, the use of an EBV positive donor to an EBV negative recipient, T-cell depletion, ATG therapy, and other forms of intensified immunosuppression for prevention and treatment of GVHD [92, 93]. Cord-blood SCT recipients receiving reduced intensity conditioning including ATG were reported to have a high risk for EBV associated complications [94]. EBV-PTLD usually occurs during the first 3 months after SCT although it can present later. PTLD usually presents during the first months after SCT as a polymorphic polyclonal lymphoproliferation that may result in monoclonal malignant lymphoma if left untreated. EBV DNA load monitoring in peripheral blood has been studied as a predictor for EBV-PTLD but the variations in the “in house” developed assays and the use of different sample types such as whole blood, serum/plasma, or PBL make it difficult to draw firm conclusions [88, 95–97]. The usefulness of viral load monitoring depends on the likelihood for a patient developing PTLD. The positive predictive values vary greatly between different studies [98] with the highest for patients having risk factors for EBV-PTLD

Chapter 29  Viral Infections in Hematopoietic Stem Cell Transplant Recipients 

[95, 96, 98]. Despite these uncertainties, monitoring of viral load seems to be a valuable tool especially in high risk patients. As many different techniques using different materials and primers exist, no cut-off viral load for initiating therapy can be recommended. However, rapid increase in viral load has been suggested to be associated with a high risk for EBV-PTLD. The first management option in a patient at high risk for PTLD is, if possible, to reduce the immunosuppression. Antiviral therapy might lower the EBV viral load but whether this influences the risk for PTLD is doubtful. Rituximab has been used as “preemptive therapy” in several patient series with good results but no controlled data exist [88, 94, 98, 99]. Another prophylactic option is to give infusions with EBV specific CTL [100–102]. There is no established therapy for treatment of PTLD. Rituximab has been used after both solid organ and SCT [90, 103–106]. Cloned EBV specific donor T-cells [100, 102], partially HLA-matched allogeneic donor CTL [107], and unspecific donor lymphocyte infusions have also been used as treatment of PTLD [108].

7. Human Herpes Virus Type 6 Human Herpes Virus Type 6 (HHV-6) exists in two subtypes (A and B) that differ from each other in 4–8% of the DNA. Subtype B is the cause of exanthema subitum in childhood. HHV-6 infection is very common early in life; hence the rate of seropositivity in older children and adults is more than 95%. Serology is therefore not helpful in patient management. There is no “gold standard” diagnostic test for HHV-6 infection but quantitative PCR has been used to better define of the contribution of HHV-6 to post transplant complications [109–111]. A possible confounding factor is that the HHV-6 genome can be integrated into cellular DNA resulting in high levels of HHV-6 DNA in blood samples including PBL [112, 113]. The best documented clinical manifestations of HHV-6 are encephalitis and bone marrow suppression. HHV-6 has a propensity for the CNS and although HHV-6 DNA can occasionally be detected in the CSF of asymptomatic SCT recipients [114, 115], the combination of symptoms of encephalitis with detection of HHV-6 DNA is suggestive of HHV-6 disease of the CNS. Approximately 35 case reports have been published [110, 114–128]. A summary of published information around these cases regarding patient characteristics, diagnostic findings, and outcome of HHV-6 CNS disease in SCT patients is shown in Table 29-2. Lethargy, confusion, convulsions, and decreased consciousness are the predominant clinical manifestations of HHV-6 encephalitis. Focal neurological findings have been reported but are less common. Magnetic resonance imaging can show abnormalities but it can also be normal. These changes included multiple, non-enhancing, low attenuation lesions in the gray matter. EEG usually shows diffuse changes. The prognosis is poor unless the encephalitis is treated with antiviral drugs. Both ganciclovir and foscarnet have been reported being effective against HHV-6 meningo-encephalitis after SCT (Table 29.2) [114, 129]. Another possible manifestation of HHV-6 is bone marrow suppression or delayed engraftment as HHV-6 can infect hematological progenitor cells and reduce colony formation [87, 110, 130–132].

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Table 29-2.  Patient characteristics, diagnostic findings, therapy, and outcome of SCT patients with suspected or proven HHV-6 encephalitis. Patient characteristics

N = 37

Median age

41 (12–66)

Unrelated or mismatched

26

Sibling donor

6

Autologous

1

Acute GVHD grade II–IV

16/25

CSF findings   Pleocytosis

15/32

  Increased protein

21/32

  HHV-6 DNA

35/35

Radiographic findings   MRI changes

22/34

  CT changes

4/17

  EEG changes

22/22

Survival of patients receiving therapy

17/28

Ganciclovir/valganciclovir

5/7

Foscarnet

13/15

Acyclovir

1/2

Foscarnet + ganciclovir given in combination or consecutively

3/10

8. Respiratory Viruses Respiratory viruses including respiratory syncytial virus (RSV), parainfluenza viruses, coronaviruses, rhinoviruses, and influenza A and B are widespread in the community with major seasonal variations. Recently several new viruses have been discovered including bocavirus and two papovaviruses that can cause respiratory disease. The epidemiological situation in the local community has been shown to influence the risk for infection in the SCT patients. This at least partly explains the variation in frequencies of diagnosed infections between different studies [133–136]. Respiratory viruses can be spread nosocomially through immune competent staff and patient relatives and outbreaks of both RSV and parainfluenza infections have been documented in transplant units [137–141]. Thus, infection control measures including isolation of symptomatic patients, use of sensitive diagnostic procedures, and as far as possible avoidance of exposure to infected persons including family and staff are important in the management of respiratory infections. The influence of respiratory virus infections on transplant related mortality has been estimated by a study

Chapter 29  Viral Infections in Hematopoietic Stem Cell Transplant Recipients 

by the EBMT. In that study 1.1% of allogeneic patients transplanted at the participating centers died of a respiratory virus infection [133]. Furthermore, RSV [142, 143] and parainfluenza infections [143] have also been implicated in the development of late respiratory dysfunction after SCT.

9. Respiratory Syncytial Virus RSV has been the cause of outbreaks in SCT patients forcing closure of transplant units [136, 144–146]. In a prospective survey, the overall mortality in patients with a RSV lower respiratory tract infection was 30% and the RSV associated mortality 17% [133]. More recently, the impact of RSV seems to have been reduced [134, 147] possibly as a result of identification of patients with lesser degree of symptoms. Several studies have analyzed risk factors for progression to lower respiratory tract disease. The outcome is worse after allogeneic and in patients with lymphopenia [133, 148]. Patients having documented RSV infection pretransplant should have their allogeneic transplant postponed if possible [149], while this does not seem to be as important for patients undergoing autologous SCT for myeloma [150]. There is no established therapy for RSV. In a small randomized trial there was no difference between patients receiving ribavirin or no therapy regarding the risk for progression to pneumonia, but there was a tendency for a greater viral load decrease in ribavirin treated patients (p = 0.07) [151]. In an uncontrolled study, 4 of 14 patients treated with the combination of ribavirin and high dose iv immune globulin developed pneumonia [152]. In the prospective EBMT survey, no regimen was superior to any other [133]. In a small phase I study of the RSV monoclonal antibody palivizumab, three patients were treated for an upper respiratory tract infection and none developed lower respiratory tract disease [153]. Only uncontrolled phase II treatment studies of RSV pneumonia have been reported. There are no proven benefits with any drug or combination, but patients treated when ventilator dependent usually have dismal outcome [136]. Ljungman et al reported similar outcomes with ribavirin given intravenously and as aerosol [133]. On the other hand, McCarthy et al. reported 26 patients with RSV infections and no apparent effect on outcome with ribavirin therapy [154]. DeVincenzo et  al. reported that 10/11 children treated with a high-titer anti-RSV immune globulin in combination with ribavirin survived [155]. In a series of 16 patients with RSV pneumonia treated with ribavirin aerosol and/or RSV Ig, 14 survived [147]. Despite the lack of controlled data, many centers use ribavirin to treat RSV infections especially in allogeneic SCT recipients. 9.1. Parainfluenza Viruses Parainfluenza viruses can give severe and fatal infections after SCT. The subtype most associated with severe infections is type 3 [141, 156–158]. In a retrospective study, unrelated donor transplant was the only identified risk factor for parainfluenza infection [141] and parainfluenza infection was an independent predictor of mortality [141]. The usefulness of antiviral therapy is doubtful. Wendt et  al and Nichols et  al both failed to find any effect of ribavirin therapy [141, 158]. Other studies have shown some indications of effectiveness by ribavirin therapy [142, 157, 159].

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10. Influenza Viruses Influenza is an important problem to consider in SCT recipients. The mortality has been reported to be around 15% in untreated patients [133, 160]. The mortality is highest in patients developing pneumonia [161]. Fatal influenza infections can occur several years after an allogeneic SCT in particular in patients with chronic GVHD [133]. The primary mode for prevention of influenza is vaccination and should be given to all transplant patients from 4  months after transplantation and yearly while the patients are immunosuppressed [67, 162]. The antibody responses have been poor when vaccinations are performed early after SCT [163, 164] but clinical protection might still be achieved [165]. Vaccination of family members and hospital staff to reduce the risk for transmission of influenza is recommended [133]. The possibilities for prevention with antiviral agents include today mainly the neuramidase inhibitors (zanamivir and oseltamivir). No controlled study has been performed in SCT patients, but in an uncontrolled study oseltamivir was given to 41 patients with influenza of whom two developed pneumonia and none died [166]). In another series 6 of 34 untreated patients, one of eight treated with rimantadine, and none of nine patients treated with oseltamivir developed pneumonia [161]. One concern is the reported rapid development of oseltamivir resistant influenza viruses.

11. Other Respiratory Viruses Metapneumovirus is a paramyxovirus causing upper and lower respiratory tract infections in children. Martino et al found in a prospective study an incidence of 5% in allogeneic and 3% in autologous SCT recipients [135]. Forty-four percent of the patients with metapneumovirus infections in allogeneic SCT recipients developed pneumonia. Fatal infections have been reported [167]. The impact of other respiratory viruses including rhinoviruses, coronaviruses, and the recently discovered boca- and respiratory papovaviruses needs further study. No therapy exists for any of these recently discovered respiratory viruses.

12. Adenoviruses Adenoviruses cause a number of clinical syndromes in immune competent individuals that are usually mild and self-limiting, but more severe manifestations have also been reported. Although 51 distinct adenovirus serotypes have been identified, most human diseases are associated with only one-third of these types. Adenovirus infections can result in morbidity and mortality after allogeneic SCT. The frequencies of adenovirus infections vary between studies. Overall, there is a higher frequency in children. Flomenberg et al. reported a frequency of 31% in children compared to 14% in adults [168]. In a study using PCR monitoring of pediatric SCT recipients, Lion et al. reported that 27% of the patients had adenovirus DNA detected [169] while Hoffman et al. in a study of pediatric SCT recipients detected adenovirus in 47% of the patients [170]. Other reports give frequencies of 3–29% [171–176]. The factors influencing the detection frequency seem to be the age of the population studied, whether

Chapter 29  Viral Infections in Hematopoietic Stem Cell Transplant Recipients 

the study was prospective or retrospective, and the diagnostic technique used, but there also seems to be a center effect with some centers experiencing a major adenovirus problem while it is rather rare in other centers. The most serious disease manifestations are pneumonia, encephalitis, and fulminant hepatitis. However, hemorrhagic cystitis and gastroenteritis seem to be more common. The most commonly recognized risk factors for adenovirus disease in allogeneic SCT recipients are younger age, T-cell depletion, GVHD, the use of mismatched and unrelated donors, the use of unrelated cord blood grafts, and adenovirus detected from more than one site [168, 170–174, 177, 178]. Identification of adenovirus in peripheral blood has also been associated with an increased risk for adenovirus disease [169]. There is no established either prophylaxis or therapy for adenovirus infections in SCT recipients. Ribavirin has been used in case reports with varying outcome [173, 179–185]. Morfin et al. reported that the in vitro efficacy varied among different subgenera of adenovirus with group C isolates being more sensitive in vitro to ribavirin compared to other subgenera [186]. This might explain some of the inconsistencies in the treatment results with ribavirin. Cidofovir might have effect against adenovirus infections but no controlled studies have been performed in SCT recipients. Reported results have been varying but it seems probable that cidofovir has an anti-adenoviral effect in many patients but it alone cannot give long-term control as development of a specific T-cell response is necessary [187–193]. Similar to CMV and EBV, CTL based immunotherapy is under development for adenovirus [194, 195].

13. Hepatitis B and C Viruses In patients who are HBsAg positive before transplantation there does not seem to be an obvious increased risk for severe liver complications after transplantation [196, 197] and long-term survival is similar in HBV-positive and negative patients [198]. Patients who are anti-HBs positive at the time of transplant can during long-term follow-up become HBsAg and HBV-DNA positive and also develop a flare of acute hepatitis because of loss of specific antibodies to HBV [2, 199–202]. In a seronegative recipient, the use of an HBV antigen positive marrow donor should if possible be avoided as the risk for transfer of HBV is high and hepatitis is likely to develop [203]. If a seropositive donor must be used, vaccination of the patient before transplant would be logical as patients who are antibody positive to HBV before transplant are less likely to develop severe liver complications [196, 204]. HBV specific immune globulin can be given to the patient before transplantation [196]. Lamivudine has been used in SCT patients to prevent reactivation [205–211]. Patients with hepatitis C virus (HCV) and abnormal liver function tests were reported having an increased risk for hepatic VOD [212, 213]. If the stem cell donor is HCV RNA positive the risk for transmission to the patient is very high [214]. Therefore, the use of an HCV positive donor should be avoided if alternatives exist. HCV-infected long-term survivors of allogeneic SCT have a high risk for development of liver cirrhosis [215, 216]. Therapy with interferon together with ribavirin using similar dose and duration as in non-transplant individuals seems to be safe and effective although no controlled study exists [217–219].

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14. Papovaviruses Papovaviruses are a group of DNA-viruses with two members – JC-virus and BK-virus – that can be pathogenic in SCT patients. JC-virus is the agent causing progressive multifocal leukoencephalopathy (PML) and BK-virus has been implicated in hemorrhagic cystitis and nephropathy in transplant recipients. Both BK- and JC-viruses are excreted in the urine of many patients after transplant. Papovaviruria has been associated with hemorrhagic cystitis although there is no absolute correlation. Higher viral loads in urine, mutations in a viral gene, and BK-viremia have been correlated to hemorrhagic cystitis [220–226]. However, also transplant factors such as allogeneic rather than autologous SCT, myeloablative conditioning, and the use of mismatched or unrelated donors have also been shown to correlate to hemorrhagic cystitis [223, 224, 227, 228]. Thus, the pathogenesis of hemorrhagic cystitis seems to be multifactorial [229]. Cidofovir has in small uncontrolled studies been reported to be effective against polyoma virus-associated HC [230, 231]. There is no established therapy for PML although cidofovir and ara-C have been given with varying results.

15. Other Viruses Measles can be fatal in immunocompromised hosts [232, 233] and severe cases have been reported in SCT recipients [234, 235]. Most patients will lose immunity during extended follow up and are therefore vulnerable to infection [236]. Vaccination against measles has been shown to be safe in patients without GVHD or ongoing immunosuppression. The seroconversion rates varied between 54 and 100% [1, 237, 238]. Parvovirus B19 exhibits a marked tropism for human bone marrow and replicates only in erythroid cells. Occasional case reports of protracted parvovirus infections have been published after stem cell transplantation [239, 240]. Rotavirus infections mostly affect otherwise normal children below 3 years of age. Reinfection in adults can occur. The symptoms are usually diarrhea and vomiting. In BMT recipients, gastroenteritis caused by rotavirus has been described [241]. Electron microscopy and ELISA can confirm the diagnosis. There is no proven effective treatment, although two cases described by Kanfer et al. [242] appeared to respond to oral immunoglobulin (6 g daily for 5 days). Coxsackie A1 virus infection with diarrhea and a significant mortality has been reported in BMT patients [241]. The diagnosis can be obtained with virus isolation from stool, cerebrospinal fluid, secretions from nose and pharynx, tears, and urine and by serology. Prolonged enteroviral infection has been described in a BMT recipient who developed pericarditis and heart failure posttransplant [243]. Although no formal study has been performed, it seems likely that these outbreaks are associated with the epidemiological situation in the community and awareness of the local situation can be of value. West Nile virus can be transmitted by blood products or from the stem cell donor and has been associated with severe diseases including fatal outcome after SCT [244–248].

Chapter 29  Viral Infections in Hematopoietic Stem Cell Transplant Recipients 

16. Summary Viral infections remain important challenges for the physician taking care of SCT patients. This includes “old” pathogens that might change the clinical presentations when new techniques are included in the treatment of SCT patients for example the use of haploidentical donors, cord blood grafts, or new immunosuppressive agents. New viral pathogens might also be introduced into the SCT patient population. On the other hand new management options need to be carefully evaluated both regarding new diagnostic options and antiviral agents.

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P. Ljungman receiving myeloablative chemotherapy and autologous stem cell transplantation. Bone Marrow Transplant 26(11):1243–1245 207. Nakagawa M, Simizu Y, Suemura M, Sato B (2002) Successful long-term control with lamivudine against reactivated hepatitis B infection following intensive chemotherapy and autologous peripheral blood stem cell transplantation in nonHodgkin’s lymphoma: experience of 2 cases. Am J Hematol 70(1):60–63 208. Ohnishi M, Kanda Y, Takeuchi T, Won Kim S, Hori A, Niiya H et  al (2002) Limited efficacy of lamivudine against hepatitis B virus infection in allogeneic hematopoietic stem cell transplant recipients. Transplantation 73(5):812–815 209. Hsiao LT, Chiou TJ, Liu JH, Chu CJ, Lin YC, Chao TC et  al (2006) Extended lamivudine therapy against hepatitis B virus infection in hematopoietic stem cell transplant recipients. Biol Blood Marrow Transplant 12(1):84–94 210. Lin PC, Poh SB, Lee MY, Hsiao LT, Chen PM, Chiou TJ (2005) Fatal fulminant hepatitis B after withdrawal of prophylactic lamivudine in hematopoietic stem cell transplantation patients. Int J Hematol 81(4):349–351 211. Moses SE, Lim ZY, Sudhanva M, Devereux S, Ho AY, Pagliuca A et  al (2006) Lamivudine prophylaxis and treatment of hepatitis B Virus-exposed recipients receiving reduced intensity conditioning hematopoietic stem cell transplants with alemtuzumab. J Med Virol 78(12):1560–1563 212. Locasciulli A, Testa M, Valsecchi MG, Bacigalupo A, Solinas S, Tomas JF et al (1999) The role of hepatitis C and B virus infections as risk factors for severe liver complications following allogeneic BMT: a prospective study by the Infectious Disease Working Party of the European Blood and Marrow Transplantation Group. Transplantation 68(10):1486–1491 213. Strasser SI, Myerson D, Spurgeon CL, Sullivan KM, Storer B, Schoch HG et al (1999) Hepatitis C virus infection and bone marrow transplantation: a cohort study with 10-year follow-up. Hepatology 29(6):1893–1899 214. Shuhart MC, Myerson D, Childs BH, Fingeroth JD, Perry JJ, Snyder DS et  al (1994) Marrow transplantation from hepatitis C virus seropositive donors: transmission rate and clinical course. Blood 84(9):3229–3235 215. Strasser SI, Sullivan KM, Myerson D, Spurgeon CL, Storer B, Schoch HG et al (1999) Cirrhosis of the liver in long-term marrow transplant survivors. Blood 93(10):3259–3266 216. Peffault de Latour R, Levy V, Asselah T, Marcellin P, Scieux C, Ades L et  al (2004) Long-term outcome of hepatitis C infection after bone marrow transplantation. Blood 103(5):1618–1624 217. Ljungman P, Johansson N, Aschan J, Glaumann H, Lönnqvist B, Ringdén O et al (1995) Long-term effects of hepatitis C virus infection in allogeneic bone marrow transplant recipients. Blood 86(4):1614–1618 218. Giardini C, Galimberti M, Lucarelli G, Polchi P, Angelucci E, Baronciani D et  al (1997) Alpha-interferon treatment of chronic hepatitis C after bone marrow transplantation for homozygous beta-thalassemia. Bone Marrow Transplant 20(9):767–772 219. Peffault de Latour R, Asselah T, Levy V, Scieux C, Devergie A, Ribaud P et al (2005) Treatment of chronic hepatitis C virus in allogeneic bone marrow transplant recipients. Bone Marrow Transplant 36(8):709–713 220. Biel SS, Held TK, Landt O, Niedrig M, Gelderblom HR, Siegert W et al (2000) Rapid quantification and differentiation of human polyomavirus DNA in undiluted urine from patients after bone marrow transplantation. J Clin Microbiol 38(10):3689–3695 221. Leung AY, Suen CK, Lie AK, Liang RH, Yuen KY, Kwong YL (2001) Quantification of polyoma BK viruria in hemorrhagic cystitis complicating bone marrow transplantation. Blood 98(6):1971–1978 222. Priftakis P, Bogdanovic G, Kalantari M, Dalianis T (2001) Overrepresentation of point mutations in the Sp1 site of the non-coding control region of BK virus

Chapter 29  Viral Infections in Hematopoietic Stem Cell Transplant Recipients  in bone marrow transplanted patients with haemorrhagic cystitis. J Clin Virol 21(1):1–7 223. Giraud G, Bogdanovic G, Priftakis P, Remberger M, Svahn BM, Barkholt L et al (2006) The incidence of hemorrhagic cystitis and BK-viruria in allogeneic hematopoietic stem cell recipients according to intensity of the conditioning regimen. Haematologica 91(3):401–404 224. Bogdanovic G, Priftakis P, Giraud G, Kuzniar M, Ferraldeschi R, Kokhaei P et al (2004) 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 42(11):5394–5396 225. Erard V, Kim HW, Corey L, Limaye A, Huang ML, Myerson D et al (2005) BK DNA viral load in plasma: evidence for an association with hemorrhagic cystitis in allogeneic hematopoietic cell transplant recipients. Blood 106(3):1130–1132 226. Erard V, Storer B, Corey L, Nollkamper J, Huang ML, Limaye A et al (2004) BK virus infection in hematopoietic stem cell transplant recipients: frequency, risk factors, and association with postengraftment hemorrhagic cystitis. Clin Infect Dis 39(12):1861–1865 227. Gorczynska E, Turkiewicz D, Rybka K, Toporski J, Kalwak K, Dyla A et al (2005) Incidence, clinical outcome, and management of virus-induced hemorrhagic cystitis in children and adolescents after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 11(10):797–804 228. Peinemann F, de Villiers EM, Dorries K, Adams O, Vogeli TA, Burdach S (2000) Clinical course and treatment of haemorrhagic cystitis associated with BK type of human polyomavirus in nine paediatric recipients of allogeneic bone marrow transplants. Eur J Pediatr 159(3):182–188 229. Dropulic LK, Jones RJ (2008) Polyomavirus BK infection in blood and marrow transplant recipients. Bone Marrow Transplant 41(1):11–18 230. Held TK, Biel SS, Nitsche A, Kurth A, Chen S, Gelderblom HR et  al (2000) Treatment of BK virus-associated hemorrhagic cystitis and simultaneous CMV reactivation with cidofovir. Bone Marrow Transplant 26(3):347–350 231. Savona MR, Newton D, Frame D, Levine JE, Mineishi S, Kaul DR (2007) Lowdose cidofovir treatment of BK virus-associated hemorrhagic cystitis in recipients of hematopoietic stem cell transplant. Bone Marrow Transplant 39(12):783–787 232. Breitfeld V, Hashida Y, Sherman FE et al (1973) Fatal measles infection in children with leukemia. Lab Invest 29:279–281 233. Kaplan L, Daum R, Smaron M, McCarthy C (1992) Severe measles in immunocompromised patients. JAMA 267(9):1237–1241 234. Nakano T, Shimono Y, Sugiyama K, Nishihara H, Higashigawa M, Komada Y et al (1996) Clinical features of measles in immunocompromised children. Acta Paediatr Jpn 38(3):212–217 235. Machado CM, Goncalves FB, Pannuti CS, Dulley FL, de Souza VA (2002) Measles in bone marrow transplant recipients during an outbreak in Sao Paulo, Brazil. Blood 99(1):83–87 236. Ljungman P, Lewensohn-Fuchs I, Hammarstrom V, Aschan J, Brandt L, Bolme P et al (1994) Long-term immunity to measles, mumps, and rubella after allogeneic bone marrow transplantation. Blood 84(2):657–663 237. Machado CM, de Souza V, Sumita LM, da Rocha I, Dulley FL, Pannuti CS (2005) Early measles vaccination in bone marrow transplant recipients. Bone Marrow Transplant 35(8):787–791 238. King SM, Saunders EF, Petric M, Gold R (1996) Response to measles, mumps and rubella vaccine in paediatric bone marrow transplant recipients. Bone Marrow Transplant 17(4):633–636 239. Kaptan K, Beyan C, Ural AU, Ustun C, Cetin T, Avcu F et al (2001) Successful treatment of severe aplastic anemia associated with human parvovirus B19 and EpsteinBarr virus in a healthy subject with allo-BMT. Am J Hematol 67(4):252–255

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P. Ljungman 240. Heegaard ED, Laub Petersen B (2000) Parvovirus B19 transmitted by bone marrow. Br J Haematol 111(2):659–661 241. Yolken RH, Bishop CA, Townsend TR, Bolyard EA, Bartlett J, Santos GW et al (1982) Infectious gastroenteritis in bone-marrow-transplant recipients. N Engl J Med 306(17):1010–1012 242. Kanfer EJ, Abrahamson G, Taylor J, Coleman JC, Samson DM (1994) Severe rotavirus-associated diarrhoea following bone marrow transplantation: treatment with oral immunoglobulin. Bone Marrow Transplant 14(4):651–652 243. Galama JM, de Leeuw N, Wittebol S, Peters H, Melchers WJ (1996) Prolonged enteroviral infection in a patient who developed pericarditis and heart failure after bone marrow transplantation. Clin Infect Dis 22(6):1004–1008 244. Brenner W, Storch G, Buller R, Vij R, Devine S, DiPersio J (2005) West Nile Virus encephalopathy in an allogeneic stem cell transplant recipient: use of quantitative PCR for diagnosis and assessment of viral clearance. Bone Marrow Transplant 36(4):369–370 245. Hong DS, Jacobson KL, Raad II, de Lima M, Anderlini P, Fuller GN et al (2003) West Nile encephalitis in 2 hematopoietic stem cell transplant recipients: case series and literature review. Clin Infect Dis. 37(8):1044–1049 246. Martin SE, Grubbs S, Della Valla J, Reinhardt JF, Lilly N, Getchell J et al (2004) Fatal West Nile virus encephalitis following autologous peripheral blood stem cell transplantation. Bone Marrow Transplant 34(11):1007–1008 247. Reddy P, Davenport R, Ratanatharathorn V, Reynolds C, Silver S, Ayash L et al (2004) West Nile virus encephalitis causing fatal CNS toxicity after hematopoietic stem cell transplantation. Bone Marrow Transplant 33(1):109–112 row transplant patients. Bone Marrow Transplant 34(9):823–824

Chapter 30 Fungal Infections John R. Wingard

1. Introduction Invasive fungal infections (IFI) are important causes of infectious morbidity and mortality after allogeneic stem cell transplant (ASCT). Candida, Aspergillus, and Pneumocystis are the chief fungal pathogens and historically have accounted for most of the IFIs. In recent years, Zygomycosis and several other fungal pathogens such as Fusarium and Scedosporium have grown in frequency. A bimodal distribution in the time of occurrence of IFIs has been noted: an early peak prior to engraftment due mostly to Candida and a later peak during the second and third months due mostly to Aspergillus. Risk for Pneumocystis is mostly during the second to the sixth month. In recent years, the second peak has extended to later periods and late infections by IFI have been noted 6 months or later. Over the past two decades, a climb in the rate of Aspergillosis has been noted in many centers.

2. Candida Infections Candida infections historically occur in 15–20% of allogeneic stem cell transplant (ASCT) recipients. They are caused by C. albicans in half of cases. C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and other species account for the remaining half. Candida ordinarily colonizes the mucosal surfaces and skin in healthy persons as well as ASCT patients. Mucosal injury due to the cytotoxic effects of the transplant conditioning regimen or gastrointestinal manifestations of graft versus host disease (GVHD), allows easier entry of organisms through the weakened mucosal barrier into the tissue and bloodstream. Similarly, breaches in the skin due to venous catheters or wounds allow entry of organisms into the bloodstream. Neutrophils are the major second line of defense to eradicate organisms that may enter the tissue and bloodstream. Neutropenia heightens the vulnerability for bloodstream infection Mucosal Candida infections commonly occur and are manifest by oral thrush, vulvovaginal candidiasis, and Candida esophagitis. However, more

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_30, © Springer Science + Business Media, LLC 2003, 2010

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serious is fungemia, the most common form of invasive Candida infection in ASCT patients (Table 30-1). Chronic hematogenous infections may result in granulomas or abscesses in the liver or spleen (so-called hepatosplenic Candidiasis or chronic hematogenous Candidiasis). Fever may be the only symptom of Candida fungemia. Rarely is Candida the cause of a first fever during neutropenia. More commonly, it is a cause of persistent or recurrent fever and should be a major diagnostic consideration during the second or third week of neutropenia. Occasional clinical manifestations of Candida fungemia are polyarthalgias, polymyalgias, cutaneous erythematous maculonodular lesions, and new onset azotemia. Endophthalmitis may also be seen in patients who are not neutropenic. Diagnosis of mucosal infection can be made by microscopic examination of scrapings of the whitish plaque with demonstration of budding yeasts and pseudohyphae. Culture is also useful to determine the Candida species, information that is useful in choosing a therapy, as discussed below. Bloodstream infections are best diagnosed by cultures. Cultural techniques of blood samples have improved yields in recent years, but some patients with systemic infection still may have negative blood cultures and even when positive, cultures may take several days to demonstrate organisms. Species identification may take several additional days. The beta glucan assay is a serologic assay which can detect several fungal species (including Candida, Aspergillus, Fusarium) and may identify infection several days before cultures become positive [1]. This has the potential for earlier diagnosis, but as noted it is not specific for Candida. Once cultures demonstrate yeast, the PNA-FISH assay is useful to determine whether a yeast isolate is Candida albicans. It takes only several hours and species isolation can be determined more quickly than conventional means. This is useful in selecting therapy. Because of the lack of specific symptoms in most cases of Candidemia during neutropenia and the difficulty in establishing a diagnosis early, empirical therapy with antifungal agents to cover Candida pathogens has been a mainstay of the management of persistent neutropenic fever (lasting more than 3–5  days) not responding to antibiotics [2]. Several antifungal agents have been shown to be effective as empirical antifungal therapy, including amphotericin B deoxycholate, the lipid amphotericin B formulations, itraconazole, and caspofungin. Although voriconazole did not meet the non-inferiority criterion in a randomized trial, comparing it to liposomal amphotericin B, many experts believe it to be suitable in view of its demonstrated effectiveness in randomized trials of treatment of documented Candida and Aspergillus infections,– the major fungal pathogens in neutropenic fever. Prophylaxis was studied more than two decades ago in several trials of amphotericin formulations, azoles, and more recently, echinocandins [3]. Fluconazole is a triazole available in oral and intravenous formulations. It is well tolerated and has excellent activity against most Candida species. Notable exceptions in its spectrum of activity are C. krusei (not susceptible) and C. glabrata (many strains are susceptible but only to high concentrations and some strains are resistant). Clinical trials have shown fluconazole to be highly effective when given as prophylaxis from the time of transplant until to either the time of engraftment or for 75  days after ASCT. Consensus panels recommend its routine use as prophylaxis after ASCT [4]. Other agents have also been found to be effective, including itraconazole, amphotericin B deoxycholate,

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amphotericin B lipid formulations, and micafungin. The advantages and disadvantages of the various prophylaxis options are presented in Table 30-2. In patients given non-ablative conditioning regimens, neutropenia is short, risk for Candida infection is lessened, and prophylaxis may not be required. There are a number of treatment options as noted in Tables 30-2 and 30-3 reviewed in consensus guidelines [5]. The same agents useful for prophylaxis are suitable for treatment. Certainly, fluconazole is highly effective but its use as prophylaxis would make it not suitable for treatment of a breakthrough Table 30-1.  Common clinical syndromes of invasive fungal infections after ASCT. Common clinical manifestations

Less frequent clinical manifestations

Comments

Persistent fever (often fever is the sole manifestation)

Polyarthralgias, polymyalgias, azotemia, erythematous macronodular cutaneous lesions

Frequent cause of neutropenic fever persisting despite antibiotics in patients not receiving antifungal prophylaxis with systemically active antifungal agents; infrequent if fluconazole prophylaxis (or other antifungal prophylaxis) is used

Chronic disseminated Fever Candidiasis (hepatosplenic candidiasis)

Abdominal pain

Often becomes apparent at neutrophil recovery; elevated alkaline phosphatase is characteristically seen, target lesions visualized on CT scan in liver and/or spleen; blood cultures frequently negative and organisms often difficult to recover from biopsy; glucan assay may be useful

Pulmonary Aspergillosis

Fever, cough

Hemoptysis, pleuritic Nodules on chest CT, halo sign occasionally present; sometimes localized nonpain, physical findspecific infiltrates; cavitary lesions at ings typical of neutrophil recovery and later in course consolidation, of infection (including the air-crescent disseminated sign) infection

Fungal sinusitis

Nasal or sinus pain, Epistaxis, nasal fever ulceration or eschar

Opacification of sinuses on CT scan is common (but is non-specific), bony erosion highly suggestive of fungal etiology; Zygomycetes and Aspergillus most common fungal etiologies; important to distinguish between Aspergillus and Zygomycetes to decide optimal therapy (voriconazole for Aspergillus and lipid amphotericin formulation for Zygomycetes

Zygomycosis

Sinusitis, pneumonia

Disseminated infection

Syndrome similar to Aspergillosis; sinusitis more common than pneumonia but both occur with regularity

Fusarium

Pneumonia, paronychia

Disseminated infection

Syndrome similar to Aspergillosis; fungemia is frequent and blood cultures useful (in contrast to Aspergillosis and Zygomycosis)

Scedosporium

Pneumonia

Disseminated infection

Syndrome similar to Aspergillosis; fungemia is frequent and blood cultures useful (in contrast to Aspergillosis and Zygomycosis)

Fungal syndrome Candida fungemia

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Candidemia. Agents with a broader spectrum of activity and not cross-resistant would be more suitable, such as the echinocandins, polyenes, or perhaps voriconazole. Duration of the therapy is generally recommended to continue for 2 weeks beyond resolution of signs and symptoms attributable to infection, cultures becoming negative, and improvement of the host deficit responsible for susceptibility. In addition to antifungal therapy, most experts recommend removal of the central venous catheters, since they may be a portal of entry or harbor organisms that may lead to persistence of fungemia. If the catheter is not removed at onset of fungemia, it certainly should be removed if fungemia persists.

3.  Aspergillus Infections Aspergillus infections occur in 10–15% of ASCT patients. Aspergillus fumigatus is the most frequent species; less common species include flavus, terreus, niger, and others. Although Aspergillosis can occur during prolonged neutropenia, much like it occurs in non-transplant treatment of acute leukemia, it more commonly occurs later after transplant. Early Aspergillus infections generally are seen mostly in patients who had prior infection by Aspergillus, those who fail to engraft, or those who have prolonged times to engraftment, such as some cord blood graft recipients. The greatest vulnerability for Aspergillus infections is after engraftment during times of profound T cell immunodeficiency: the early post-engraftment period (second and third months after transplant), during treatment for acute or chronic GVHD, or if high dose steroids are given over several weeks or longer. Infection mostly occurs in patients with low CD4 counts (<50  cells/mL). Aspergillus is an airborne organism and the nasal passages and respiratory tract are the usual portals of entry. Not surprisingly, pneumonia is the most common manifestation of infection. Sinusitis is another occasional manifestation. Occasionally, organisms can enter through breaches in the skin and cause cutaneous or deep facial infections or abscesses. Fever is frequently a symptom of Aspergillosis, as with Candida and other IFIs. Symptoms and signs of lower respiratory tract infection also are typically present: cough, dyspnea, sputum, and rales or rhonchi may be heard on examination. Less frequently, hemoptysis or pleuritic pain may be present, and a pleural friction rub may be heard. For sinus and nasal infections, nasal or sinus pain, epistaxis, or presence of a nasal eschar or ulceration can occur. Radiology has been the mainstay of diagnostic assessment (Table 30-3). CT scans are much more sensitive than radiographs to detect and characterize the pulmonary infiltrates of Aspergillosis. Nodules of at least 1 cm in diameter are found in most patients with pulmonary Aspergillosis [6]. In many instances, a “halo” of ground glass density surrounds one or more nodules early in the course of infection. Wedged-shaped, peripheral nodules, or cavitary infiltrates suggest that a fungal mold etiology is a more likely cause than bacteria. In the sinuses, opacification or air-fluid levels may be observed. Such findings are also seen with bacterial infections. However, bony destruction, if present, strongly suggests a fungal rather than bacterial etiology. Radiology should be followed by additional testing aimed to establish a microbiological diagnosis. This is important since the clinical and radiological findings of pneumonia and sinusitis can be caused by a variety of pathogens.

Chapter 30  Fungal Infections 

For pneumonia, bronchoscopy is a logical next step. Both bronchoalveolar lavage and transbronchial biopsy are desirable, but the latter may not be an option if the patient is deeply thrombocytopenic. For peripherally located lesions, a percutaneous needle aspiration or video-assisted transthoracic surgical biopsy are other options. Microscopy with fungal stains and fungal cultures are important. Unfortunately, the yield for Aspergillus (or other mold pathogens) is not high even in patients subsequently documented to have pulmonary Aspergillosis by other diagnostic means. The yield of the bronchoalveolar lavage can be improved by testing the lavage fluid with the galactomannan assay (see below). Because of the low sensitivity for detecting Aspergillus, a bronchoscopic evaluation that is unrevealing should not be the sole ground for excluding the diagnosis of Aspergillosis. Notwithstanding, the bronchoscopic evaluation is important. Other pathogens are sometimes found on bronchoscopy in patients with suspected Aspergillosis; moreover, cytomegalovirus, respiratory viruses, and bacteria may co-infect patients with pulmonary Aspergillosis. For sinus or nasal infections, endoscopic examination allows for aspiration of material for microscopy and culture and biopsy of mucosal lesions. An ELISA assay for galactomannan (a substance released by Aspergillus in the course of invasive infection) is licensed for Aspergillus testing. Serial sampling of serum conducted twice weekly after ASCT for galactomannan has been found to be useful in detecting Aspergillus [7–9]. A single test result that is negative is less reliable when it is tested only at the time a patient is suspected to be infected. False positives are seen in patients receiving certain beta lactam antibiotics, especially piperacillin-tazobactam and ticarcillinclavulinate. The galactomannan test is fairly specific for Aspergillus. Another serologic assay, the beta glucan assay, can also be used to evaluate patients suspected to have Aspergillosis, but it is less useful since other fungal pathogens will also give a positive test result. Yield of the bronchoalveolar lavage procedure can be improved by testing the lavage specimen for galactomannan. Use of Plasmalyte in the lavage solution can produce false positives. Some investigators advocate the use of serial testing of the galactomannan assay twice weekly, along with intensive monitoring for clinical symptoms and signs for Aspergillosis, and prompt investigation by CT scans, for all ASCT patients. If the CT scan demonstrates an infiltrate, bronchoscopy (or percutaneous needle biopsy) is then performed. Such an aggressive screening approach allows prompt identification of patients who may be infected and permits initiation of presumptive therapy even while the evaluation is proceeding. Prompt initiation of therapy is the key to optimize successful treatment. Thus, it is recommended that treatment should be started while the evaluation proceeds. Voriconazole, a triazole with excellent anti-aspergillus activity is the treatment of choice [10, 11]. For patients receiving a calcineurin inhibitor (e.g., cyclosporine or tacrolimus), reduction of the calcineurin inhibitor by approximately 50% should be done at initiation of voriconazole due to an interaction that raises the blood levels; monitoring of blood levels of the calcineurin inhibitor should be continued to ensure that therapeutic levels are maintained. Voriconazole, if given concomitantly with sirolimus, an immunosuppressive agent increasingly used for GVHD prophylaxis or treatment, results in potentiation of sirolimus. In one study evaluating the coadministration of the two, sirolimus doses had to be reduced by 90% in order

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to achieve similar concentrations as sirolimus given without voriconazole coadministration [12]. For patients who are unable to take voriconazole, a lipid amphotericin B formulation is an alternative choice. A recent trial that tested two initial dose schedules of liposomal amphotericin B (10 mg/kg/d vs. 3 mg/ kg/d) found no therapeutic advantage to the higher dose regimen but greater toxicity [13]. Of note, Aspergillus terreus is intrinsically resistant to polyenes and an azole must be given to provide adequate treatment. For patients who do not respond to the initial therapy, it is important to confirm the correct diagnosis, especially in patients who did not have microbiological documentation at the outset, and to evaluate for the possibility of co-infecting pathogens. Failure of initial therapy should not be judged precipitously. Fever may be slow to resolve in many patients who respond to initial therapy and radiologically the infiltrates characteristically increase during the first week even in the face of clinical response; this is particularly so as neutropenia resolves or immune competence improves. Thus, such issues need to be considered in determination of whether the patient is truly not responding. For progressive or non-responding Aspergillosis, second line therapies include the lipid amphotericin formulations, echinocandins, or other azoles (itraconazole or posaconazole). There is some experience using combination antifungal therapy also (such as voriconazole plus an echinocandin or an amphotericin B product plus an echinocandin) but the role of combination therapy has not been well studied. In addition to antifungal therapy, surgical resection of necrotic material is sometimes performed to obviate secondary infection, hemorrhage from erosion into major pulmonary vasculature, invasion of the pericardium, pleura, or invasion of the chest wall. Such decisions must be individualized. Some clinicians advocate resection of cavitary lesions prior to ASCT, but there are no formal studies of this approach. Granulocyte transfusions have been used in neutropenic patients not responding to antifungal therapy. Currently, no randomized trials have been performed although there is a suggestion of benefit from case series. Currently, there is a national multi-center randomized trial underway to evaluate this strategy. At present, the role of surgery and granulocyte transfusions must be individualized in the absence of definitive studies. Itraconazole, voriconazole, and posaconazole have all been evaluated as prophylaxis [14–18]. Toxicities and tolerance have been issues for itraconazole. Posaconazole has been found to be effective in patients with GVHD at high risk for Aspergillosis. Lipid amphotericin B formulations and echinocandins have anti-aspergillus activity and have been studied in limited trials, but they are less attractive due to the requirement for intravenous administration and the need for prolonged prophylaxis in the outpatient setting.

4. Zygomycetes Infections Infections by Zygomycetes (Rhizopus, Mucor, Rhizomucor, Absidia, Cunninghamella spp.) have similar manifestations as Aspergillosis. There is a greater propensity for sinonasal infections, but pneumonia (with nodular and/ or cavitary infiltrates) and cutaneous infections at sites of skin wounds (or dissemination) also occur. Several centers have noted an increase in infections over the years. There is some suggestion that the wider use of voriconazole

Chapter 30  Fungal Infections 

(an azole without activity against Zygomycetes) may increase the risk for Zygomycosis. Diagnostic assessment is similar to that used for Aspergillosis. Important to note is that micoscopically Zygomycetes have a different morphologic appearance from Aspergillus: broad, waxy, hyaline bands without septa. Tissue necrosis is often extensive. Zygomycetes are more difficult to recover from tissue by culture. As with Aspergillus, even with disseminated infection, organisms are not recovered from blood cultures. Prompt initiation of therapy is important to optimize the success of the treatment. The lipid formulations of amphotericin B are the mainstay of therapy [19]. Voriconazole has no activity but posaconazole is active and has been found to be an effective option as salvage therapy for patients unresponsive to first line therapy. Surgical resection of necrotic tissue is an important adjunct to antifungal therapy. Repeated debridement may be necessary during therapy.

5. Other Mycoses Pneumocystis carinii (now known as Pneumocystis jiroveci) is now considered a fungus. It presents typically after engraftment as one of the several causes of diffuse pneumonitis. Low grade fever, and non-productive cough are frequently the initial manifestations. Bronchoscopy with microscopy using special stains of bronchoalveolar lavage fluid has a high yield and sensitivity and specificity exceeds 90%. Prophylaxis with trimethoprim-sulfamethoxazole is recommended for 6 months and longer if chronic GVHD occurs. It should be continued for as long as active immunosuppression is given. For allergy or intolerance, dapsone, atovaquone, or inhaled pentamidine may be substituted, but these appear to be somewhat less effective. Treatment of cases is with the same agents. Corticosteroids with initial therapy may reduce inflammation and improve treatment outcome. Fusarium is an occasional pathogen in ASCT patients. Its clinical manifestations are similar to Aspergillosis. Its portal of entry is also similar to that of Aspergillus, but also prominent is its entry through breaches in skin. Fusarium should be strongly considered as an important cause of paronychia during neutropenia. Fungal cultures of blood may be positive in disseminated infection in contrast to Aspergillosis. Occasionally the organisms may morphologically resemble yeasts (and thus may be initially confused with Candida until distinguished by culture). Most antifungal agents are poorly active against Fusarium. Amphotericin B formulations have been used widely but outcomes have been poor. Voriconazole and posaconazole demonstrate activity in vitro against some isolates and case series with voriconazole demonstrates efficacy as salvage therapy (Table 30-2). Scedosporium apiospermum and S. prolificans are occasional causes of pneumonia after ASCT. Hematogenous dissemination may occasionally occur. Evaluation is similar to that for Aspergillosis. Fungal blood cultures may be positive in contrast to Aspergillus. Amphotericin B lipid preparations in high doses have been the usual therapy but outcomes have been poor. Voriconazole has better in vitro activity and there are some clinical data supporting its use as salvage therapy.

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Table 30-2.  Antifungal agents useful for prophylaxis or treatment of invasive fungal infections after ASCT. Agent

Candida

Fluconazole

P, T

Aspergillus

Other

Comments Oral and intravenous formulations Excellent safety profile Metabolized by cytochrome p450, but drug interactions usually not problematic No activity against C. krusei Activity against C. glabrata less in many strains and no activity in some strains Although isolated breakthrough infections and several outbreaks by less susceptible species have been reported, emergence of widespread resistance has not been seen after more than 15 years of widespread use after ASCT

Itraconazole

P,T

P,T

Oral and intravenous formulations Bioavailability may be erratic and measurement of blood levels is advisable Oral suspension is preferable to oral capsules due to better absorption Metabolized by cytochrome p450 and drug interactions can be considerable: it should be avoided as concomitant therapy in patients receiving chemotherapy metabolized by the liver (e.g., cyclophosphamide) and doses of the calcineurin inhibitors need to be reduced (and monitored by blood levels) with concomitant use Generally safe but some studies have observed excess renal and hepatic toxicity in patients given concomitant calcineurin inhibitors; cardiac contractility has been impaired in some patients leading to congestive heart failure

Voriconazole

T

T

Fusarium, Scedosporium

Oral and IV formulations Excellent bioavailability but some studies suggest variable absorption in ASCT recipients; measurement of blood concentrations advisable Preferred first line treatment for Aspergillosis No activity against Zygomycetes Cross-resistance noted in many fluconazoleresistant C. glabrata strains Metabolized by cytochrome p450 and drug interactions can be considerable: although not well studied, one would anticipate similar interactions as with itraconazole with concomitant chemotherapy metabolized by the liver (e.g., cyclophosphamide) and doses of the calcineurin inhibitors need to be reduced (and monitored by blood levels) with concomitant use (continued)

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Table 30-2.  (continued) Agent

Candida

Aspergillus

Other

Comments

Posaconazole

P

P

Zygomycosis

Oral formulation only Bioavailability not well studied after ASCT but appears adequate with limited data to date Metabolized by cytochrome p450 and drug interactions can be considerable: although not well studied, one would anticipate similar interactions as with itraconazole with concomitant chemotherapy metabolized by the liver (e.g., cyclophosphamide) and doses of the calcineurin inhibitors need to be reduced (and monitored by blood levels) with concomitant use Limited data for first line therapy of Candida and Aspergillus; Excellent efficacy as salvage therapy for Zygomycosis

Amphotericin P,T B deoxycholate

P,T

Zygomycosis, some activity against most other mycoses

Excellent spectrum of activity Available only in intravenous formulations Toxicity even in low doses makes it undesirable for prophylaxis Inexpensive, but considerable infusional toxicity and nephrotoxicity Difficult for patients receiving calcineurin inhibitors to tolerate (nephrotoxicity)

Lipid ampho- P,T tericin B formulations

P,T

Zygomycosis, some activity against most other mycoses

Excellent spectrum of activity Available only in intravenous formulations Better tolerated than amphotericin B deoxycholate but toxicities may still occur Liposomal amphotericin B formulation most studied and best tolerated May be effective in lower doses in some studies and may be effective in 2–3 doses per week in limited studies

Echinocandins P,T

T

Excellent spectrum of activity Available only in intravenous formulations Micafungin best studied for prophylaxis (with FDA-approved indication); caspofungin evaluated in one trial Caspofungin and anidulafungin approved for Candida treatment Caspofungin approved for salvage therapy for Aspergillus None well studied for first-line therapy for Aspergillosis Poor activity against other mycoses Drug interactions not clinically significant Excellent safety profile

P = prophylaxis, T = therapy

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Table 30-3.  Evaluation and treatment approaches to clinical syndromes to determine fungal etiology. Syndrome

Evaluation

Treatment approach

Neutropenic fever

Fungal cultures Galactomannan and/or glucan assays

Persistent fever for 96 h or longer suggests a fungal etiology and is grounds for consideration of empirical fungal therapy (if patient is not on systemic antifungal prophylaxis)

CT scan of chest (and/or sinuses, abdomen) Pneumonia

CT scan of chest

Nodular infiltrates more likely to be Aspergillus than diffuse infiltrates; presumptive antifungal therapy should be initiGalactomannan and/or ated when fungal etiology is suspected while diagnostic glucan assays assessment is underway; pursuit of a microbiological Bronchoscopy (or needle diagnosis is strongly urged since multiple bacterial and biopsy or VATS biopsy fungal pathogens may be etiologic and many infections as indicated) with funare mixed; since yield of bronchoscopy for Aspergillus is gal smears and cultures low, strong suspicion in the face of a negative bronchosand galactomannan copy warrants continued antifungal therapy and further evaluation; fungal differential includes Aspergillus (most likely), Zygomycetes, Fusarium, Scedosporium

Sinusitis

CT scan of sinuses Nasal endoscopy with fungal smears and cultures

Cutaneous lesions

Biopsy with fungal smears and cultures; blood cultures for fungus

Boney erosion warrants strong suggestion of fungal etiology; presumptive antifungal therapy should be initiated when fungal etiology is suspected while diagnostic assessment is underway; surgical debridement may be necessary while antifungal therapy treatment course proceeds; fungal differential includes Zygomycetes and Aspergillus (the two organisms can be distinguished microscopically in most cases by different morphologies by an experienced mycologist); Nocardia may mimic fungal pneumonia or disseminated infection Infected wounds may be localized but skin lesions may also be manifestation of disseminated infection; fungal differential includes Aspergillus, Zygomycetes, Fusarium, Scedosporium, paronychia is commonly caused by Fusarium

With all of these emerging but still uncommon mycoses, there are scant clinical data to determine the best treatment options. As antifungal activity of current agents is suboptimal, adjunctive measures remain extremely important. Myeloid growth factors or granulocyte transfusions for infected patients who are neutropenic and neutrophil, recovery is not imminent. Surgical resection of necrotic tissue may also be useful, but this must be individualized (Table 30-3).

References 1. Ostrosky-Zeichner L, Alexander BD, Kett DH et  al (2005) Multicenter clinical evaluation of the (1–>3) beta-D-glucan assay as an aid to diagnosis of fungal infections in humans. Clin Infect Dis 41(5):654–659 2. Wingard JR (2004) Empirical antifungal therapy in treating febrile neutropenic patients. Clin Infect Dis 39(Suppl 1):S38–S43 3. Wingard JR (2002) Antifungal chemoprophylaxis after blood and marrow transplantation. Clin Infect Dis 34(10):1386–1390

Chapter 30  Fungal Infections  4. Dykewicz CA, Jaffe HW, Spira TJ, Jarvis WR, Kaplan JE, Edlin BR, Chen RT, Hibbs B, Bowden RA, Sullivan K, Emanuel D, Longworth DL, Rowlings PA, Rubin RH, Sepkowitz KA, Wingard JR, Modlin JF, Ambrosino DM, Baylor NW, Donnenberg AD, Gardner P, Giller RH, Halsey NA, Le CT, Molrine DC, Sullivan KM (2000) Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients. MMWR Recomm Rep 49(RR-10):1–125 5. Pappas PG, Rex JH, Sobel JD, Filler SG, Dismukes WE, Walsh TJ, Edwards JE (2004) Guidelines for treatment of candidiasis. Clin Infect Dis 38(2):161–189 6. Greene RE, Schlamm HT, Oestmann JW, Stark P, Durand C, Lortholary O, Wingard JR, Herbrecht R, Ribaud P, Patterson TF, Troke PF, Denning DW, Bennett JE, de Pauw BE, Rubin RH (2007) Imaging findings in acute invasive pulmonary aspergillosis: clinical significance of the halo sign. Clin Infect Dis 44(3):373–379 7. Maertens J, Van Eldere J, Verhaegen J, Verbeken E, Verschakelen J, Boogaerts M (2002) Use of circulating galactomannan screening for early diagnosis of invasive aspergillosis in allogeneic stem cell transplant recipients. J Infect Dis 186(9):1297–1306 8. Maertens J, Theunissen K, Verhoef G, Verschakelen J, Lagrou K, Verbeken E, Wilmer A, Verhaegen J, Boogaerts M, Van Eldere J (2005) Galactomannan and computed tomography-based preemptive antifungal therapy in neutropenic patients at high risk for invasive fungal infection: a prospective feasibility study. Clin Infect Dis 41(9):1242–1250 9. Pfeiffer CD, Fine JP, Safdar N (2006) Diagnosis of invasive aspergillosis using a galactomannan assay: a meta-analysis. Clin Infect Dis 42(10):1417–1427 10. Walsh TJ, Anaissie EJ, Denning DW et  al (2008) Treatment of Aspergillosis: Clinical Practice Guidelines of the Infectious Diseases Society of America (IDSA). Clin Infect Dis 46(3):327–60 11. Herbrecht R, Denning DW, Patterson TF, Bennett JE, Greene RE, Oestmann JW, Kern WV, Marr KA, Ribaud P, Lortholary O, Sylvester R, Rubin RH, Wingard JR, Stark P, Durand C, Caillot D, Thiel E, Chandrasekar PH, Hodges MR, Schlamm HT, Troke PF, de Pauw B (2002) Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J Med 347(6):408–415 12. Marty FM, Lowry CM, Cutler CS et al (2006) Voriconazole and sirolimus coadministration after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 12(5):552–9 13. Cornely OA, Maertens J, Bresnik M, Ebrahimi R, Ullmann AJ, Bouza E, Heussel CP, Lortholary O, Rieger C, Boehme A, Aoun M, Horst HA, Thiebaut A, Ruhnke M, Reichert D, Vianelli N, Krause SW, Olavarria E, Herbrecht R (2007) Liposomal amphotericin B as initial therapy for invasive mold infection: a randomized trial comparing a high-loading dose regimen with standard dosing (AmBiLoad trial). Clin Infect Dis 44(10):1289–1297 14. Winston DJ, Maziarz RT, Chandrasekar PH, Lazarus HM, Goldman M, Blumer JL, Leitz GJ, Territo MC (2003) Intravenous and oral itraconazole versus intravenous and oral fluconazole for long-term antifungal prophylaxis in allogeneic hematopoietic stem-cell transplant recipients. A multicenter, randomized trial. Ann Intern Med 138(9):705–713 15. Marr KA, Crippa F, Leisenring W, Hoyle M, Boeckh M, Balajee SA, Nichols WG, Musher B, Corey L (2004) Itraconazole versus fluconazole for prevention of fungal infections in patients receiving allogeneic stem cell transplants. Blood 103(4):1527–1533 16. Mattiuzzi GN, Estey EH, Hernandez M, Cabanillas ME, Giles F, Cortes JE, O’Brien S, Verstovsek S, Kantarjian HM (2005) Voriconazole and liposomal amphotericin B (Ambisome) effectively prevent mold infections in patients (pts) with acute myelogenous leukemia (AML) following remission induction chemotherapy. Abstract 2773. Presented at ASH Annual Meeting in Atlanta, GA, 2005 17. Rojas R, Serrano J, Martin C, Tabares S, Capote M, Fernandez M, Arqueros V, Molina JR, Martin V, Torres A (2005) Voriconazole in primary prophylaxis reduces

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J.R. Wingard the incidence of IFI in high risk hematologic patients. Abstract 5349. Presented at ASH Annual Meeting in Atlanta, GA, 2005 18. Ullmann AJ, Lipton JH, Vesole DH, Chandrasekar P, Langston A, Tarantolo SR, Greinix H, Morais de Azevedo W, Reddy V, Boparai N, Pedicone L, Patino H, Durrant S (2007) Posaconazole or fluconazole for prophylaxis in severe graftversus-host disease. N Engl J Med 356(4):335–347 19. Roden MM, Zaoutis TE, Buchanan WL et al (2005) Epidemiology and outcome of zygomycosis: a review of 929 reported cases. Clin Infect Dis 41:634–53

Chapter 31 Immune Reconstitution and Implications for Immunotherapy Following Hematopoeitic Stem Cell Transplantation Kirsten M. Williams and Ronald E. Gress 1.  Innate Immune Reconstitution-Natural Killer, Neutrophil, and Dendritic Cells The innate immune system comprises a collection of cells that recognize and eradicate pathogens or aberrant cells without priming or antigen presentation. Natural killer cells, neutrophils, monocytes, dendritic cells, and macrophages contribute to innate immunity. Natural killer (NK) cells purge tumor or virusinfected cells. By one month post-transplant, natural killer cells circulate at normal levels and confer adept immune protection [1–6] (see Fig. 31-1). These donor derived NK cell clones effectively lyse recipient leukemia in vitro [7]. Studies have also shown that the number of natural killer cells, early posttransplant, correlate with remission rates, implicating the function of these early cells in the clearance of residual tumor [8–10]. Data also suggest that killer immunoglobulin-like receptor (KIR) mismatch may play a critical role in NK cell-mediated tumor eradication [7, 9]. Many, but not all, studies have associated NK cell KIR mismatch with protection from relapse and GVHD in HLA haplotype-mismatched and matched unrelated transplants for myeloid and acute lymphoid malignancies [9, 11, 12]. Recovery of neutrophils and monocytes is rapid post-transplant akin to NK cells; however, the timeline for dendritic cell (DC) reconstitution is delayed and falls between that of innate and adaptive immune recovery [3, 4, 13–20]. Dendritic cells, process and present antigens to the adaptive immune system, while producing inflammatory cytokines important for stimulation of the innate immune system. After SCT, though donor DC can be detected in the peripheral blood within the first few weeks, the total number may not approach normal for more than a year in adults [21, 22] (Fig. 31-1). Furthermore, studies suggest

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_31, © Springer Science + Business Media, LLC 2003, 2010

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Fig. 31-1.  Representative timelines of immune reconstitution in adult patients without extensive GVHD, infections, or relapse [1, 2, 4, 18, 20–22, 31, 103, 105]

that while the peripheral blood DC are largely of donor origin (> 80% by day 14), up to 70% of tissue DC may be of host origin 1 month after allogeneic stem cell transplantation and host DC can persist up to a year after stem cell infusion [23–25]. Furthermore, the functional competency of these newly derived dendritic cells is not yet well understood in the context of stem cell transplantation. Because DC-loaded peptides engender better infectious responses than vaccine alone post-transplant, there is evidence that DC function may be impaired posttransplant, though these studies are limited by differing diseases and transplant conditions [26–28]. Collectively, these data suggest that many of the components of the innate immune system reconstitute relatively quickly and competently in the early post-transplant period. Furthermore, although some studies have noted slight differences between host or transplant characteristics for innate immune reconstitution, these do not appear to be significant for restoration of at least some level of functional immunity. While this quick, functional recovery would lend NK cells to immunotherapy, to date, there is little evidence that the innate immune system can be manipulated to maximize graft-versus-leukemia effects with the one notable exception of acute myeloid leukemia (AML). Studies have suggested that a graft with KIR mismatch may predict a high probability of NK alloreactivity and augment the graft-versus-AML effect [7, 9, 29]. Unfortunately, for most patients and diseases, NK cells offer few opportunities for anti-tumor interventions following stem cell transplantation. Additionally, because little is known about the kinetics and function of tissue DC post-transplant, mechanisms to exploit this biology in vivo have yet to be elucidated. Indeed, the few studies that have investigated anti-tumor vaccination strategies after transplantation have included the administration of DCs loaded with specific peptides to obviate the need for functional donor DCs in vivo (see Sect. 4.3).

Chapter 31  Immune Reconstitution and Implications for Immunotherapy 

2. Adaptive Immune Reconstitution – B and T cells In contrast to innate immune cells, the lymphoid, adaptive compartment reconstitutes later and with persistent recognized deficits in terms of global immunity. In large part, deficiencies in T and B function after transplant are due to impaired thymopoiesis. As a result, T cell reconstitution relies upon an alternate pathway of development, termed homeostatic peripheral expansion (HPE). In HPE, mature donor T cells expand, rendering a limited repertoire of T cells available to recognize and signal B cell counterparts [30]. B and T cells require priming by antigen and engender long-term specific responses using unique receptor sequences. In non-transplant subjects, because B and T cells have the potential for long-term and specific immunity, they are attractive targets for cellular immunotherapy. However, the protracted timeline for lymphoid recovery after transplant remains a challenge to this approach. Circulating B cells may not reach normal numbers for 12 months posttransplant and T cell reconstitution may be delayed beyond 2 years [4, 5, 14, 19, 31–34]. Despite this fact, there are aspects of the immediate post-transplant milieu that may be especially conducive to anti-tumor T cell responses, including high levels of cytokine that can fuel cytotoxic T cell proliferation and activation. Thus, an understanding of the cytokine and antigen-driven responses that are possible post-transplant may permit the genesis of a platform for antitumor treatments, with cautious consideration to the potential for exacerbation of graft-versus-host disease.

3. B Cell Immune Reconstitution 3.1. B Cell Development B cells are lymphocytes generated in the bone marrow from common lymphoid progenitors (CLPs). B cells possess immunoglobulin receptors that are generated by somatic recombination, whereby genes are rearranged to create diverse receptor sequences. B cells undergo selection in the bone marrow prior to release into the peripheral blood. Naïve mature B cells emerge from the marrow with surface IgM and IgD receptors then migrate to secondary lymphoid structures. Once a B cell encounters an antigen, often through interactions with CD4+ T cells or dendritic cells, it becomes activated and releases IgM. The activated B cell then processes the antigen for presentation to T cells. After T cell stimulation, the B cell may isotype switch to express IgG, IgA, or IgE on its surface. The B cell receptor/immunoglobulin molecule may then undergo further receptor modification in the variable regions (VH), through somatic hypermutation, increasing the avidity of the antibody/epitope interaction. 3.2.  B Cell Development Following allogeneic stem cell transplant, B cell development recapitulates that of normal ontogeny in terms of circulating cell types and numbers. Although B cell numbers are very low for the first few months, they may reach levels exceeding normal adults by 1–2 years, approaching the range of normal neonates [32] (Fig. 31-1). The levels then gradually fall. In normal ontogeny, B cell numbers rise until the toddler years, after which there is a gradual descent

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to normal adult levels in the peripheral blood. Post-transplant, donor B cells emerge with a naïve phenotype (IgM+IgD+) initially, with memory B cell development up to 5 years later [35, 36]. Consistent with this, IgM levels recover first, by 2–6 months [6, 37], followed by IgG levels which approach normal between 3 and 18 months post-transplant. Finally, IgA reconstitution may be delayed for up to 3 years [6, 37]. It is important to note that the IgG levels may, in part, reflect residual host plasma cell production and thus may not be entirely suggestive of donor B cell competence because host plasma cells are relatively resistant to current preparative regimens and may persist for up to 2 years [38, 39]. 3.3. B Cell Function Post-Transplant In contrast to the quick quantitative recovery of B cells, function remains compromised for 1–2 years following SCT. In part, this has been attributed to the T cell defects during this time frame, with maturation arrest at the naïve stage due to insufficient CD4+ T cell signaling. After transplant, memory B cells that require CD4+ help for isotype switching show a skewed pattern of complementary determining regions 3 (CD3) of the immunoglobulin heavy chain [40, 41]. In addition to the lack of CD4+ help, there appears to be an environmental defect after transplantation, because the rate of somatic hypermutation is decreased in mature B cells even in the presence of normal donor CD4+ T cells [42]. Thus, post-transplant B cell immunity is impaired due to (1) prolonged low levels of circulating B cell numbers, (2) a relative deficit of mature B cells secondary to decreased isotype switching, and (3) a diminished ability to undergo somatic hypermutation. Importantly, this decreased B cell function post-SCT has resulted in diminished vaccine responses to infectious antigens even after normal B cell numbers have been achieved [26, 43, 44].

4. T Cell Reconstitution 4.1. Thymic Dependent T Cell Reconstitution The thymus is the fundamental site for T cell development. When peripheral T cell populations are severely depleted, renewed thymic activity can contribute to T cell reconstitution, producing naïve CD4 helper, CD8 cytotoxic effector, and CD4+CD25+ regulatory T cells. During thymopoiesis, bone marrow-derived T progenitors migrate to the thymus, expand and mature. Developing thymocytes acquire a T cell receptor (TCR), generated through recombinant rearrangement of Variable (V), Diversity (D), and Joiner (J) genes. The rearrangement of these genes within the thymus ensures T cell receptor diversity. Prior to emigration as naïve T cells, these TCR undergo positive and negative selection, deleting autoreactive clones. Renewed thymopoiesis may be assessed by several techniques including thymic imaging, naïve T cell frequency, T cell receptor excision circle quantity, and spectrotype pattern. With renewal of thymopoiesis, the proportion of cells with a “naïve” phenotype increases in peripheral T cell populations [45–47]. Measurement of T cell receptor rearrangement excision circles (TREC) provides a means of quantifying thymic productivity [48].

Chapter 31  Immune Reconstitution and Implications for Immunotherapy 

TREC are episomal DNA circles that are generated as a by-product from the spliced DNA elements remaining after rearrangement of the VDJ genes encoding the TCR a and b chains. TREC circles are not replicated during cell division but are retained in the thymic emigrant cell, and thus are diluted with cell division. Robust thymopoiesis will be reflected in high frequencies of TREC-bearing cells, termed recent thymic emigrants (RTE). In contrast, with intense peripheral expansion, peripheral TREC frequencies remain low. Finally, thymic function may also be evaluated by spectratyping, a PCR-based analysis of length variation in the CDR3 of the TCR b chain. A productive thymus is reflected in a Gaussian distribution of CDR3 lengths because of the enormous diversity generated by VDJ rearrangement occurring in maturing thymocytes. In contrast, clonal expansion results in an oligoclonal pattern of limited CDR3 lengths. Renewal of thymopoiesis thus re-establishes polyclonal Gaussian patterns in the CDR3 spectratypes, first in naïve cells and subsequently in memory T cells [47, 49]. 4.2. T Cell Reconstitution by Homeostatic Peripheral Expansion An alternate pathway for T cell development is through the rapid cell division of mature T cell clones, termed homeostatic peripheral expansion (HPE) [46, 50]. This was first demonstrated in a murine model in which two very distinct patterns of T cell reconstitution ensued following the adoptive transfer of syngeneic bone marrow (BM) cells and congenic lymph node cells into C57BL/6 irradiated, thymus-intact, or thymectomized recipients. Thymus-bearing hosts largely reconstituted with syngeneic marrow-derived T cells, through a thymus-dependent mechanism. Thymectomized mice derived the majority of the peripheral T cells from the congenic lymph node innocula, via HPE [46]. HPE drives T cell reconstitution in lymphopenic hosts and is associated with a shift from naïve to memory/activated phenotype in the proliferating cells [50]. HPE is dependent upon both the support of homeostatic cytokines and cognate antigen-driven and regulatory cellular interactions. Interleukin 7 (IL-7) and interleukin 15 (IL-15) are the cytokines that drive HPE of naïve and CD8+ memory T cells, respectively. Regulatory cells and TGFb provide cellular constraints on HPE. Finally, antigen presentation can drive HPE in lymphopenic and transplant recipients. IL-7 is a critical, non-redundant cytokine required for stimulating naïve T cell expansion and sustaining naïve T cell survival. Human and animal studies have shown that IL-7 markedly increases naïve CD4+ and CD8+ T cells, diminishing TREC frequency [51, 52]. Conversely, IL-7 depletion restricts T cell expansion through cytokine depletion by a consumption-based mechanism. Current data suggest that lymphocyte depletion following preparative regimens leads to an increase in cytokine availability (presumably due to lack of cells consuming the cytokine). Upon infusion of donor T cells, the surplus IL-7 drives naïve cells into proliferation until T cell numbers return to a level at which IL-7 is once more a limiting factor. Human studies initially revealed this inverse correlation between serum levels of IL-7 and T cell reconstitution after lymphodepletion; high serum levels of IL-7 coincided with severe lymphopenia, which decreased rapidly with lymphocyte recovery post-transplant [53, 54]. Recent evidence suggests that IL-15 as well as IL-7 may act as a homeostatic cytokine, supporting HPE in lymphopenic hosts. IL-15 enhances proliferation of human CD8+ memory populations in  vitro [55, 56]. It is both produced

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constitutively by tissues and antigen presenting cells and is upregulated in the setting of inflammation. When lymphocytes have been severely depleted as in the setting of marrow transplant, plasma IL-15 levels increase dramatically and exceed normal levels concomitant with a disproportionate expansion of CD8+ memory cells [57–60]. Consistent with this finding, IL-15R expression and responsiveness is highest on activated memory CD8+ T cells [61–63]. Since IL-15 and IL-7 are constitutively produced and could lead unchecked T cell expansion, these are balanced by factors that negatively regulate T cell subsets. TGFß antagonizes the effect of IL-15, curtailing the proliferation and persistence of CD8 central memory populations [64, 65]. Administration of TGFb to human and murine CD8+ memory cells in  vitro decreases CD8+ proliferation and attenuates effector function [66, 67]. Similarly, regulatory T cells (Tregs) constrain the HPE of naïve CD4+ and CD8+ T cells, influencing the host-reactivity of T cells during immune reconstitution [68]. Treg are CD127−CD25++ CD4+ T cells that function to control autoimmune responses [69, 70]. Characterized by the expression of the transcription factor FoxP3 and a high level of glucocorticoid-induced TNF receptor, Tregs have been shown to modulate the peripheral expansion of T cells to low and high affinity antigen in lymphopenic hosts [68, 70]. Treg levels appear to be low following transplant or chemotherapy but expand rapidly in the first month [71, 72]. In addition to cytokine and cellular regulation of HPE, antigen presentation can also direct the recovering T cell compartment. First demonstrated in murine models, peripheral expansion of TCR transgenic cells was substantially increased in the presence of cognate antigen [73]. This mechanism was further characterized as a rapid, IL-7 independent, expansion of CD4+ and CD8+ T cells that is dependent on MHC II interactions and CD28 ligation [74], and termed endogenous proliferation to differentiate it from the homeostatic proliferation which is driven by cytokine signals [75]. In irradiated lymphopenic hosts, both processes could be demonstrated for subsets of CD4+ and CD8+ cells during immune reconstitution [75]. In allogeneic transplantation, the influence of endogenous proliferation is best demonstrated by the disproportionate expansion of CMV-reactive CD8+ T cells observed in CMV+ individuals [76, 77]. Thus, although homeostatic proliferation permits the expansion of the T cell compartment as a whole post-SCT, endogenous proliferation directs T cells to proliferate in response to specific antigen stimuli early in T cell recovery. 4.3.  T Cell Reconstitution by Peripheral Expansion in the Initial Posttransplant Period and Implications for Immunotherapy In the early period post-transplant, elevated cytokines of IL-7 and IL-15 drive homeostatic expansion of residual (host) or infused (donor) T cell subsets. Thymopoiesis is significantly impaired due to thymic injury from the preparative regimen, the lack of circulating T cell precursors, and the absence of critical cytokines or growth factors. This early reliance of the T cell compartment on HPE has profound consequences. The relative frequencies of the T subsets shift toward a preponderance of activated and cycling memory T cells [78]. As T cells expand through HPE, TREC frequency declines [78]. T cells, especially CD8+ subsets, expand rapidly in the first weeks, but this expansion is often unstable, with longitudinal studies describing a sharp decline in overall

Chapter 31  Immune Reconstitution and Implications for Immunotherapy 

T cell numbers by 3–6 months [58]. Robust CD8+ proliferation and impaired CD4+ reconstitution lead to an inverted CD4:CD8 ratio in the months to years following SCT [6, 33]. The TCR repertoire resulting from these early expansions is typically skewed and oligoclonal. This oligoclonal peripheral expansion of relatively few donor T cells potentially presents an opportunity for cellular immunotherapy. Murine models have shown that severe lymphodepletion establishes optimal conditions to promote graft-versus-tumor due to the high levels of homeostatic cytokines (IL-7 and IL-15) and the depletion of regulatory T cells that impede immune responses. Higher doses of irradiation yielded superior anti-tumor responses when mice were given either transgenic melanoma-antigen-specific T cells [79–81]. Furthermore, this effect was abrogated in the absence of IL-7 and IL-15 when these knock-out mice were used as recipients [79]. Depletion of regulatory T cells also enhanced the tumoricidal effect [82]. In the setting of vaccination, while Treg can also rapidly proliferate and hamper effective anti-tumor responses in lymphoreplete hosts, following SCT, the Treg compartment is constrained (by IL-2) while anti-tumor effectors expand and outcompete Tregs [83, 84]. Murine SCT models validated that the post-transplant period provides an optimal milieu for cellular immunotherapy, demonstrating specific anti-melanoma immunity and tumoricidal effect when either a vaccine and cytokine adjuvant, a tumor lysate DC-pulsed vaccine, or GM-CSF producing tumor vaccine were administered in post-transplant lymphopenic setting [80, 85, 86]. Human studies have corroborated murine data, showing maximal antitumor T cell expansion in the setting of severe lymphopenia. Studies in the highly immunogenic tumor melanoma have demonstrated the potential for this platform. Ex vivo-expanded melanoma-infiltrating lymphocytes administered with IL-2 after cytoreductive therapy led to clinical responses in up to 50% of patients evaluated [87, 88]. Furthermore, a single study has even demonstrated that T cells can be genetically engineered to target melanoma and affect clinical responses when given during intense lymphopenia [89]. Vaccination strategies using peptide-pulsed dendritic cell vaccines for melanoma have also demonstrated T cell-specific responses with some clinical responses as well [90–92]. It should be noted that these DC-pulsed peptide vaccines did not incorporate lymphodepletion in the regimens. In hematologic malignancies, this framework for vaccines has only recently been incorporated into clinical trials with fewer studies reported to date. In contrast to melanoma, when peptide-pulsed vaccines were administered in post-transplant setting (autologous or allogeneic) for multiple myeloma or acute myeloid leukemia, while some assays could detect an immunologic response, clinical responses were absent [93–95]. Notably, these studies administered the vaccines months posttransplant, after exponential T cell proliferation has likely occurred and IL-7 and IL-15 cytokine levels would be expected to be low. These studies suggest peptide-pulsed vaccines may be used safely after SCT, and that alternative timing strategies may actually lead to a clinical response. Given the successful strategies in melanoma, future trials may seek to incorporate infusion of tumor-targeted T cells and/or the administration of DC vaccines in the earlier post-transplant period to capitalize on the cytokine storm and absent Treg populations to potentiate tumorcidial effects.

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4.4.  T cell Reconstitution by Thymopoiesis Post-transplant and Implications for Cellular Immunotherapy While early post-transplant T cell reconstitution relies upon HPE, an optimal T cell armamentarium with a diverse TCR repertoire is only achieved through the (delayed) recovery of thymopoiesis [47, 48, 96–100] (Fig. 31-2). This may have important implications for cellular immunotherapy. Without renewal of thymopoiesis, naïve T cells and total CD4+ T cell reconstitution is impaired indefinitely [19, 45, 47, 48]. In pediatric patients, CD4+ naïve T cells are delayed for a year or more, until thymic activity has resumed [31]. Even 20 years after transplant, deficits in naïve CD4 population persist in patients with inadequate renewal of thymopoiesis [34]. While the CD8+ rich, memory, oligoclonal population of T cells due to HPE may be beneficial as a platform for vaccination therapy, the delay in thymopoiesis post-transplant may ultimately restrict vaccine efficacy. The lack of diversity of T cell receptors could diminish the likelihood of an appropriate

Fig. 31-2.  Diagrammatic representation of the clinical consequences of the two types of T cell reconstitution evident in the post-transplant period. Early after stem cell infusion, homeostatic proliferation (HPE) drives the T cell repertoire due to the high levels of cytokines and delayed thymic recovery. As a result, T cells in the donor product expand and mature into the memory pool, leading to an expanded memory pool with decreased diversity in both naïve and memory lymphocytes. Given the rapid proliferation of few T cells, this immediate post-transplant period presents an opportunity for immunotherapy, through the administration of tumor-targeted T cells or through vaccine administration. Later, in patients with the potential for thymic recovery (including younger patients without GVHD), renewed thymopoiesis enriches the naïve T cell pool. Due to the T cell receptor rearrangement in the thymus, these recent thymic emigrants enhance the diversity of the naïve, and subsequently, the memory T cell repertoire (diagrammatically shown as multi-patterned cells). This has significant implications for long-term immunity. Not only are these patients less prone to life-threatening infections and GVHD, the enhanced T cell diversity and renewed CD4+ cells likely confer improved tumor immunosurveillance, as shown by decreased rates of tumor relapse

Chapter 31  Immune Reconstitution and Implications for Immunotherapy 

TCR for tumor antigen recognition. Murine models have demonstrated that decreased TCR diversity due to age or thymectomy correlated with diminished response to an infectious antigen [101]. Furthermore, since CD4+ T are necessary for long lasting B and CD8+ T cell immunity, the persistent defects in CD4+ T cells likely contribute to impaired responses to infectious and tumor vaccines post-transplant. Murine data suggest that optimal vaccination responses occur in the setting of active thymopoiesis [19, 102]. In human trials, impaired thymic function has been correlated with increased incidence of relapse, suggesting that renewed thymopoiesis may enhance anti-tumor immunity [103, 104]. In addition to mitigating the effects of cellular immunotherapy, thymic defects are associated with increased risk of acute and chronic GVHD which further impedes de novo immune responses [105–109]. In a pediatric population, GVHD and low TREC levels indicative of poor thymic function even conferred an increase risk of mortality [109]. Post-transplant thymic dysfunction contributes to the onset and persistence of GVHD. While thymic damage permits the escape of alloreactive T cells that may engender GVHD, [46, 110], the thymus is also a target of these alloreactive T cells, limiting thymic renewal [111, 112]. Thus, the regeneration of thymic structures is stunted and de novo diverse T production indefinitely impaired [111, 112]. Of critical importance when considering T cell immunotherapy in the context of SCT is the barrier that factors that support GVL may worsen GVHD. In murine studies, high doses of IL-7 and IL-15, through exogenous administration or transgenic models, increased the severity and frequency of GVHD [113–115]. In human studies, persistence of high IL-15 levels following allogeneic stem cell transplant was correlated with severe acute GVHD as well [116, 117]. In contrast, factors that reduce peripheral expansion can reduce GVHD. Regulatory T cells could control homeostatic expansion in lymphopenic mice and prevent acute GVHD in murine models [68, 118]. Similarly in clinical trials, the level of FoxP3+ T regulatory cells in the donor inoculum was inversely correlated with development of acute GVHD [71].

5.  Strategies to Optimize Cellular Immunotherapy Post-transplant Because T and B cell reconstitution is critical to successful immunotherapy approaches post-transplant, maximal anti-tumor responses are likely to be elicited when lymphoid reconstitution is both rapid and functional. In part, efforts should be directed toward enhancing thymic recovery while potentially directing the immediate post-transplant HPE toward tumor-specific antigens. Thymic function is influenced by age, stem cell source, GVHD (see above), the degree of damage due to the preparative regimen, and possible cytokine, hormonal, and growth factors. Host age significantly affects thymic recovery, with each successive decade reducing the rate and potential for thymic renewal [47, 48, 100, 106, 119]. In many older adults, recovery of naïve cells through thymopoiesis occurs 3–5 years post-transplant; in some, naïve cells remain below normal levels decades after SCT [34, 120]. While host age is an immutable challenge to thymic recovery, other factors may be influenced by transplant design.

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To reduce thymic damage, less intensive preparative regimens have enhanced CD4+ T cell reconstitution and T cell repertoire diversity, though these may also reduce the levels of HPE cytokines available for early T cell expansion [13, 121, 122]. Increasing the availability of functional T progenitors has enhanced thymopoiesis in murine models [123, 124]. In human studies, umbilical cord stem cells resulted in higher TREC frequencies than adult bone marrow stem cells [125], possibly due to a higher frequency of T progenitors in these marrows. Furthermore, manipulation of thymic cytokines, growth factors or hormones has improved thymic function after SCT. Because IL-7 is an important survival factor for developing thymocytes, administration of this cytokine in murine transplant models has enhanced post-BMT thymopoiesis [126, 127]. Similarly, keratinocyte growth factor (KGF) has shown promise to boost thymic productivity in murine hosts as well [128, 129]. Human studies utilizing KGF have not confirmed these data; however, they were not designed to evaluate enhanced thymopoiesis [130]. Finally, androgen withdrawal and growth hormone have been associated with enhanced thymopoiesis in murine and human studies as well [131–136]. In addition to the factors that affect thymic recovery, the transplant design may enhance immunotherapy by targeting the initial expansion of alloreactive T cells in the graft. Stem cell source has separately been studied and shown to influence HPE-derived early T cell reconstitution. Peripheral blood stem cell grafts initially reconstitute peripheral T cell numbers more rapidly [3, 5]. T cell depletion and GVHD have also been shown to delay T cell reconstitution [105]. Degree of mismatch may also influence early T cell recovery; partially matched or unrelated donors demonstrate delayed T reconstitution with skewed repertoires and very low TREC frequency [1, 137].

6. Summary Immune reconstitution following stem cell transplant does not recapitulate that of ontogeny. This has important implications for the development and maintenance of tumor immunotherapy. While most of the innate immune system recovers quickly and competently, lymphoid reconstitution may be quite delayed in terms of cell number and global function. In large part, lymphoid recovery is hindered by the lack of thymopoiesis. As a result, T cell reconstitution relies upon an alternate pathway of development that expands infused or residual cells in response to cytokines and antigens. Furthermore, donor dendritic cell renewal may also occur late after transplant, minimizing the capacity for these cells to effectively present tumor antigens and alert the developing immune system to the presence of minimal residual disease. These delays in lymphoid and dendritic cell reconstitution have significant clinical ramifications, permitting the exploitation of the initial T cell expansion while challenging effective long-term immunity in the absence of thymopoiesis, appropriate B cell signaling (due to the absence of CD4+ T cells), and functional donor dendritic cells. However, these components of immune reconstitution may be manipulated to aid graft-versus-leukemia effects. The optimal SCT involves eradication of residual tumor, long-term anti-tumor immunity, and minimal GVHD.

Chapter 31  Immune Reconstitution and Implications for Immunotherapy 

For rare patient and donor pairs, the early functional recovery of NK cells affords considerable anti-tumor benefit. However, for most graft-host pairs, the optimal transplant entails achieving a balance between the peripheral expansion of anti-tumor T cells and thymic reconstitution, rendering a continuous infusion of de novo T cells for tumor immunosurveillance with a diverse repertoire to minimize the likelihood of severe GVHD. To maximize the SCT for immunotherapy, future studies may alter the donor graft to direct the initial homeostatic T cell expansion toward tumor eradication, either by infusing genetically engineered anti-tumor T cells, expanding naturally occurring GVL T cells prior to infusion, and/or administering DC vaccines loaded with peptides to drive a specific anti-tumor T cell response. For the best effect, current immunologic data suggest that these therapies should be given in the immediate post-transplant period to capitalize on the surfeit of cytokine levels. Alternatively, one could consider administering IL-7 or IL-15 after the first month to raise the levels and drive HPE of anti-tumor T cells after the cytokine surplus is depleted, although this should be considered cautiously in allo-transplantation because of the heightened risk of GVHD. While typically CD8+ T cells are targeted for these therapies, treatments that employ CD4+ T cells may lead to longer lasting immunity and permit a more effective B cell response. In the initial period, administration of CD4+ T cells may help achieve B cell responses, although the greatest immunotherapy benefit is likely to be obtained when thymopoiesis is augmented as well, replenishing naïve CD4+ T cells. Hopefully, future studies will advance thymic renewal while directing the initial homeostatic T cell expansion toward tumor eradication. In this way, both pathways of T cell development can be maximally exploited toward improved GVL immunotherapy without stimulating GVHD, leading to long-term fully functional lymphoid immunity and successful tumor and infection clearance. 6.1. Considerations for Immunotherapy after Stem Cell Transplant Clinical · Innate immunity including neutrophils and natural killer cells recovers quickly and competently. · Donor dendritic cell competence is delayed. · T cell, and to some extent B cell, immune reconstitution is delayed following stem cell transplant. · The initial post-transplant period is dominated by cytokine-driven peripheral expansion of mature donor T cells. · Recovery of thymopoiesis is impaired after stem cell transplant and contributes to T and B cell incompetence. Research · The initial post-transplant period may provide a platform for successful T cell immunotherapy strategies due to the robust peripheral expansion during this time frame. · Strategies to aid in thymic renewal may improve long-term tumor immunosurveillance.

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Chapter 31  Immune Reconstitution and Implications for Immunotherapy  82. Antony PA, Paulos CM, Ahmadzadeh M, Akpinarli A, Palmer DC, Sato N et al (2006) Interleukin-2-dependent mechanisms of tolerance and immunity in vivo. J Immunol 176(9):5255–5266 83. Zhou G, Drake CG, Levitsky HI (2006) Amplification of tumor-specific regulatory T cells following therapeutic cancer vaccines. Blood 107(2):628–636 84. Mirmonsef P, Tan G, Zhou G, Morino T, Noonan K, Borrello I et al (2008) Escape from suppression: tumor-specific effector cells outcompete regulatory T cells following stem-cell transplantation. Blood 111(4):2112–2121 85. Kochenderfer JN, Chien CD, Simpson JL, Gress RE (2006) Synergism between CpG-containing oligodeoxynucleotides and IL-2 causes dramatic enhancement of vaccine-elicited CD8+ T cell responses. J Immunol 177(12):8860–8873 86. Moyer JS, Maine G, Mule JJ (2006) Early vaccination with tumor-lysate-pulsed dendritic cells after allogeneic bone marrow transplantation has antitumor effects. Biol Blood Marrow Transplant 12(10):1010–1019 87. Dudley ME, Wunderlich JR, Robbins PF, Yang JC, Hwu P, Schwartzentruber DJ et al (2002) Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 298(5594):850–854 88. Robbins PF, Dudley ME, Wunderlich J, El-Gamil M, Li YF, Zhou J et  al (2004) Cutting edge: persistence of transferred lymphocyte clonotypes correlates with cancer regression in patients receiving cell transfer therapy. J Immunol 173(12):7125–7130 89. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM et al (2006) Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314(5796):126–129 90. Thurner B, Haendle I, Roder C, Dieckmann D, Keikavoussi P, Jonuleit H et  al (1999) Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J Exp Med 190(11):1669–1678 91. Fay JW, Palucka AK, Paczesny S, Dhodapkar M, Johnston DA, Burkeholder S et al (2006) Long-term outcomes in patients with metastatic melanoma vaccinated with melanoma peptide-pulsed CD34(+) progenitor-derived dendritic cells. Cancer Immunol Immunother 55(10):1209–1218 92. Banchereau J, Ueno H, Dhodapkar M, Connolly J, Finholt JP, Klechevsky E et al (2005) Immune and clinical outcomes in patients with stage IV melanoma vaccinated with peptide-pulsed dendritic cells derived from CD34+ progenitors and activated with type I interferon. J Immunother 28(5):505–516 93. Reichardt VL, Okada CY, Liso A, Benike CJ, Stockerl-Goldstein KE, Engleman EG et al (1999) Idiotype vaccination using dendritic cells after autologous peripheral blood stem cell transplantation for multiple myeloma – a feasibility study. Blood 93(7):2411–2419 94. Bendandi M, Rodriguez-Calvillo M, Inoges S, Lopez-Diaz de Cerio A, PerezSimon JA, Rodriguez-Caballero A, Rodriguez-Caballero A et al (2006) Combined vaccination with idiotype-pulsed allogeneic dendritic cells and soluble protein idiotype for multiple myeloma patients relapsing after reduced-intensity conditioning allogeneic stem cell transplantation. Leuk Lymphoma 47(1):29–3 95. Kitawaki T, Kadowaki N, Kondo T, Ishikawa T, Ichinohe T, Teramukai S et  al (2008) Potential of dendritic cell immunotherapy for relapse after allogeneic hematopoietic stem cell transplantation, shown by WT1 peptide- and keyhole limpet hemocyanin-pulsed, donor-derived dendritic cell vaccine for acute myeloid leukemia. Am J Hematol 83(4):315–317 96. Bellucci R, Alyea EP, Weller E, Chillemi A, Hochberg E, Wu CJ et  al (2002) Immunologic effects of prophylactic donor lymphocyte infusion after allogeneic marrow transplantation for multiple myeloma. Blood 99(12):4610–4617 97. Hochberg EP, Chillemi AC, Wu CJ, Neuberg D, Canning C, Hartman K et  al (2001) Quantitation of T-cell neogenesis in  vivo after allogeneic bone marrow transplantation in adults. Blood 98(4):1116–1121

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K.M. Williams and R.E. Gress 98. Klein AK, Patel DD, Gooding ME, Sempowski GD, Chen BJ, Liu C et al (2001) T-Cell recovery in adults and children following umbilical cord blood transplantation. Biol Blood Marrow Transplant 7(8):454–466 99. Dumont-Girard F, Roux E, van Lier RA, Hale G, Helg C, Chapuis B et al (1998) Reconstitution of the T-cell compartment after bone marrow transplantation: restoration of the repertoire by thymic emigrants. Blood 92(11):4464–4471 100. Sarzotti M, Patel DD, Li X, Ozaki DA, Cao S, Langdon S et al (2003) T cell repertoire development in humans with SCID after nonablative allogeneic marrow transplantation. J Immunol 170(5):2711–2718 101. Yager EJ, Ahmed M, Lanzer K, Randall TD, Woodland DL, Blackman MA (2008) Age-associated decline in T cell repertoire diversity leads to holes in the repertoire and impaired immunity to influenza virus. J Exp Med 205(3):711–723 102. Zoller M, Rajasagi M, Vitacolonna M, Luft T (2007) Thymus repopulation after allogeneic reconstitution in hematological malignancies. Exp Hematol 35(12):1891–1905 103. Parkman R, Cohen G, Carter SL, Weinberg KI, Masinsin B, Guinan E et al (2006) Successful immune reconstitution decreases leukemic relapse and improves survival in recipients of unrelated cord blood transplantation. Biol Blood Marrow Transplant 12(9):919–927 104. Powles R, Singhal S, Treleaven J, Kulkarni S, Horton C, Mehta J (1998) Identification of patients who may benefit from prophylactic immunotherapy after bone marrow transplantation for acute myeloid leukemia on the basis of lymphocyte recovery early after transplantation. Blood 91(9):3481–3486 105. Fallen PR, McGreavey L, Madrigal JA, Potter M, Ethell M, Prentice HG et  al (2003) Factors affecting reconstitution of the T cell compartment in allogeneic haematopoietic cell transplant recipients. Bone Marrow Transplant 32(10):1001–1014 106. Lewin SR, Heller G, Zhang L, Rodrigues E, Skulsky E, van den Brink MR et al (2002) Direct evidence for new T-cell generation by patients after either T-celldepleted or unmodified allogeneic hematopoietic stem cell transplantations. Blood 100(6):2235–2242 107. Jimenez M, Martinez C, Ercilla G, Carreras E, Urbano-Ispizua A, Aymerich M et al (2006) Clinical factors influencing T-cell receptor excision circle (TRECs) counts following allogeneic stem cell transplantation in adults. Transpl Immunol 16(1):52–59 108. Weinberg K, Blazar BR, Wagner JE, Agura E, Hill BJ, Smogorzewska M et al (2001) Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation. Blood 97(5):1458–1466 109. Olkinuora H, Talvensaari K, Kaartinen T, Siitonen S, Saarinen-Pihkala U, Partanen J et al (2007) T cell regeneration in pediatric allogeneic stem cell transplantation. Bone Marrow Transplant 39(3):149–156 110. Desbarats J, Lapp WS (1993) Thymic selection and thymic major histocompatibility complex class II expression are abnormal in mice undergoing graft-versus-host reactions. J Exp Med 178(3):805–814 111. Ghayur T, Seemayer TA, Xenocostas A, Lapp WS (1988) Complete sequential regeneration of graft-vs-host-induced severely dysplastic thymuses. Implications for the pathogenesis of chronic graft-vs-host disease. Am J Pathol 133(1):39–46 112. Hauri-Hohl MM, Keller MP, Gill J, Hafen K, Pachlatko E, Boulay T et al (2007) Donor T-cell alloreactivity against host thymic epithelium limits T-cell development after bone marrow transplantation. Blood 113. Blaser BW, Roychowdhury S, Kim DJ, Schwind NR, Bhatt D, Yuan W et  al (2005) Donor-derived IL-15 is critical for acute allogeneic graft-versus-host disease. Blood 105(2):894–901 114. Alpdogan O, Eng JM, Muriglan SJ, Willis LM, Hubbard VM, Tjoe KH et  al (2005) Interleukin-15 enhances immune reconstitution after allogeneic bone marrow transplantation. Blood 105(2):865–873

Chapter 31  Immune Reconstitution and Implications for Immunotherapy  115. Chung B, Dudl E, Toyama A, Barsky L, Weinberg KI (2008) Importance of interleukin-7 in the development of experimental graft-versus-host disease. Biol Blood Marrow Transplant 14(1):16–27 116. Kumaki S, Minegishi M, Fujie H, Sasahara Y, Ohashi Y, Tsuchiya S et al (1998) Prolonged secretion of IL-15 in patients with severe forms of acute graft-versushost disease after allogeneic bone marrow transplantation in children. Int J Hematol 67(3):307–312 117. Chik KW, Li K, Pong H, Shing MM, Li CK, Yuen PM (2003) Elevated serum interleukin-15 level in acute graft-versus-host disease after hematopoietic cell transplantation. J Pediatr Hematol Oncol 25(12):960–964 118. Taylor PA, Lees CJ, Blazar BR (2002) The infusion of ex vivo activated and expanded CD4(+)CD25(+) immune regulatory cells inhibits graft-versus-host disease lethality. Blood 99(10):3493–3499 119. de Vries E, van Tol MJ, van den Bergh RL, Waaijer JL, ten Dam MM, Hermans J et  al (2000) Reconstitution of lymphocyte subpopulations after paediatric bone marrow transplantation. Bone Marrow Transplant 25(3):267–275 120. Nordoy T, Kolstad A, Endresen P, Holte H, Kvaloy S, Kvalheim G et al (1999) Persistent changes in the immune system 4–10 years after ABMT. Bone Marrow Transplant 24(8):873–878 121. Chao NJ, Liu CX, Rooney B, Chen BJ, Long GD, Vredenburgh JJ et al (2002) Nonmyeloablative regimen preserves “niches” allowing for peripheral expansion of donor T-cells. Biol Blood Marrow Transplant 8(5):249–256 122. Chen X, Hale GA, Barfield R, Benaim E, Leung WH, Knowles J et  al (2006) Rapid immune reconstitution after a reduced-intensity conditioning regimen and a CD3-depleted haploidentical stem cell graft for paediatric refractory haematological malignancies. Br J Haematol 135(4):524–532 123. Chen BJ, Cui X, Sempowski GD, Domen J, Chao NJ (2004) Hematopoietic stem cell dose correlates with the speed of immune reconstitution after stem cell transplantation. Blood 103(11):4344–4352 124. Zakrzewski JL, Kochman AA, Lu SX, Terwey TH, Kim TD, Hubbard VM et al (2006) Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation. Nat Med 12(9):1039–1047 125. Talvensarri K, Clave E, Douay C, Rabian C, Garderet L, Busson M et al (2002) 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 99(4):1458–1464 126. Bolotin E, Smogorzewska M, Smith S, Widmer M, Weinberg K (1996) Enhancement of thymopoiesis after bone marrow transplant by in  vivo interleukin-7. Blood 88(5):1887–1894 127. Mackall CL, Fry TJ, Bare C, Morgan P, Galbraith A, Gress RE (2001) IL-7 increases both thymic-dependent and thymic-independent T-cell regeneration after bone marrow transplantation. Blood 97(5):1491–1497 128. Alpdogan O, Hubbard VM, Smith OM, Patel N, Lu S, Goldberg GL et al (2006) Keratinocyte growth factor (KGF) is required for postnatal thymic regeneration. Blood 107(6):2453–2460 129. Rossi S, Blazar BR, Farrell CL, Danilenko DM, Lacey DL, Weinberg KI et al (2002) Keratinocyte growth factor preserves normal thymopoiesis and thymic microenvironment during experimental graft-versus-host disease. Blood 100(2):682–691 130. Blazar BR, Weisdorf DJ, Defor T, Goldman A, Braun T, Silver S et  al (2006) Phase 1/2 randomized, placebo-control trial of palifermin to prevent graft-versushost disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Blood 108(9):3216–3222 131. Sutherland JS, Goldberg GL, Hammett MV, Uldrich AP, Berzins SP, Heng TS et  al (2005) Activation of thymic regeneration in mice and humans following androgen blockade. J Immunol 175(4):2741–2753

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Chapter 32 Acute Graft Versus Host Disease: Prophylaxis Corey Cutler, Vincent T. Ho, and Joseph H. Antin

Acute GVHD remains one of the most significant barriers to successful allogeneic stem cell transplantation, accounting for a substantial portion of early transplant-related morbidity and mortality. Acute GVHD results from the complex interaction of donor T cells and host tissues that involves recognition of major and minor histocompatibility antigens in an inflammatory milieu. The pathophysiology of acute GVHD involves both the innate and adaptive immune systems and follows the orderly cycle of host tissue damage (from conditioning or other injury), donor T cell activation and clonal expansion, followed by cellular and inflammatory factor-induced tissue injury [1]. While all three components are critical, it is only the cellular attack on host tissues that is currently specifically targeted by GVHD prophylactic mechanisms, either with the use of a variety of pharmacologic agents or graft manipulation techniques. Other strategies to reduce acute GVHD (such as non-myeloablative or reduced intensity transplantation) may independently reduce the risk of acute GVHD via a reduction in host tissue damage from conditioning. It is clear that prevention of acute GVHD is associated with improved outcomes. In addition to the reduction in mortality associated with acute GVHD itself, the intense immunosuppression required to treat acute GVHD is associated with infectious mortality. This chapter focuses on those strategies most commonly employed after myeloablative stem cell transplantation, and discusses newer strategies being tested today.

1. Graft Manipulation to Prevent GVHD Consistent with the notion that GVHD is induced by donor T cells co-infused with the stem cell graft, graft manipulation to remove T cells (T cell depletion, TCD) is the one method available for preventing GVHD. A full review on T-cell depletion as GVHD prophylaxis has previously been published [2], and a detailed discussion is beyond the scope of this article/chapter. In general, T cell depletion strategies include the ex vivo removal of T cells or their From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_32, © Springer Science + Business Media, LLC 2003, 2010

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subsets from the donor stem cell product (by negative selection of T cells or T cell subsets, or by the positive selection of CD34+ stem cells), or through the in vivo reduction of donor and/or host T cell numbers by anti-T cell antibody administration. In vivo negative selection techniques use horse or rabbit derived antithymocyte globulin (ATG), and alemtuzumab (Campath-1H) to reduce T cell numbers. The benefit of in vivo TCD is that it simultaneously eliminates both recipient T cells that could mediate graft rejection, and donor T cells that might induce GVHD [3–5]. However, other cellular components, such as B cells, NK cells and dendritic cells may be affected by these polyspecific antibodies. The specific depletion of these cellular components may or may not influence the risk of GVHD. Ex vivo TCD can be accomplished via physical separation of T cells (using methods such as lectin agglutination and sheep RBC rosetting or counterflow centrifugal elutriation) [6–9] or by immunologic separation using monoclonal T cell antibodies with complement [10–12]. The increased use of peripheral blood stem cells (PBSC) in allogeneic transplantation has spurred the development of simple, automated, and efficient medical devices to separate the cellular components of the PBSC graft, since the mobilized PBSC product typically contains an average of 1 log-fold increase in T cells relative to marrow grafts, and older separation techniques are not feasible for both economical and practical reasons. These new column-based devices use immunoadsorption or magnetic bead separation techniques, and can reduce the lymphocyte content up to 5 log-fold in the eluted adherent fraction of CD34+ cells. It is therefore important to recognize that the resulting CD34+ selected graft that is delivered after such manipulation is not only T cell depleted, but is also devoid of B cells, NK cells, and other cellular components that could potentially be important for supporting engraftment, immune reconstitution, and anti-tumor immunity. Newer separation devices using immune-magnetic beads coated with anti-CD8 or anti-CD3 monoclonal antibodies are now available, and should allow for simple depletion of T cell subsets only from PBSC products. Studies in both, matched, related and unrelated transplantation have demonstrated that T cell depletion can be an effective strategy to prevent acute GVHD, with rates of acute GVHD as low as 10% in matched, related transplantation [13], and 34–38% in matched unrelated donor transplantation [14]. In a recent prospective multi-center randomized phase II–III trial comparing TCD versus conventional GVHD prophylaxis in unrelated donor marrow transplantation, 410 patients with hematologic malignancies were randomized to receive either T cell depleted bone marrow (by the monoclonal antibody T10B9 or counter flow centrifugal elutriation) with post transplant cyclosporine alone, versus T cell replete bone marrow with cyclosporine and methotrexate as GVHD prophylaxis [15]. Conditioning consisted of cyclophosphamide (1,200 mg/kg over 2 days) and total body irradiation (1,320–1,375 cGy over 4  days) in all patients. Antithymocyte globulin (60  mg/kg over 2  days) was added in recipients of TCD by elutriation, and cytosine arabinoside (9  g/m2 over 3 days) was added in the recipients of TCD by T10B9 antibody. The mean T cell dose infused for the TCD and non-TCD arms were 2.8 vs. 30.1 × 106 CD3+ cells/kg, respectively (p < 0.0001). The incidence of acute GVHD was significantly lower in the TCD arm compared to the non-TCD arm (Gr. II–IV 39% vs. 63%, p < 0.0001; Gr. III–IV 18% vs. 37%, p < 0.0001).

Chapter 32  Acute Graft Versus Host Disease: Prophylaxis 

However, overall disease-free survival at 3 years was 30%, with no difference between the TCD and non-TCD arms (27% vs. 34%, p = 0.16) despite a reduction in the incidence of early treatment-related mortality in the TCD group. The use of TCD has been explored more in the alternative donor and highly mismatched donor setting, where the rates of acute GVHD are notably higher than with HLA-matched donors. In these settings, TCD is often combined with conventional immunosuppression to prevent severe GVHD [16, 17]. Despite a seemingly lower rate of acute GVHD, ex vivo T cell depletion has not gained widespread acceptance because TCD is commonly associated with impaired post-transplant immune reconstitution, increased risks of graft failure, post-transplant EBV-associated lymphoproliferative disorders, and a reduction in graft-versus-tumor activity. Of greater importance, most studies comparing TCD to T replete strategies have not demonstrated a survival benefit for T cell depletion over conventional pharmacologic GVHD prophylaxis [15, 18]. This strategy is again being tested in a Phase II multicenter trial conducted by the Bone Marrow Transplant Clinical Trials Network (BMT CTN). In contrast, in vivo TCD strategies are commonly used as primary prophylaxis, despite the lack of contemporary randomized evidence supporting their use. Part of the continued use of these agents relates more to their role in prevention of chronic GVHD, rather than as prophylaxis of acute GVHD, particularly in alternative donor transplantation [18, 19].

2.  Pharmacologic Prevention of Acute GVHD 2.1. Historical Perspective Pioneered in Seattle using a canine model, methotrexate was the first agent used to prevent GVHD after transplantation for aplastic anemia [20]. When used as a single agent, rates of acute GVHD exceeded 50%, even with HLA-identical sibling donors. Subsequently, cyclosporine was developed as an immune suppressant, and was proven to be more effective as a single agent than methotrexate [21]. Studies performed in Seattle [22, 23] and elsewhere [24, 25] then demonstrated that the two drug combination of cyclosporine and methotrexate was more effective than cyclosporine alone, and the current standard was established. Corticosteroids, the mainstay of therapy of established acute GVHD, have not found a prominent role in GVHD prophylaxis. Several trials were performed that incorporated prednisone into the GVHD prophylactic regimen often as a third agent, however, many of these employed a control arm of cyclosporine and prednisone, rather than the standard of cyclosporine and methotrexate. In one large trial, the rate of acute GVHD in the cyclosporine and prednisone control arm was 23% which was inferior to the three drug combination of cyclosporine, methotrexate and prednisone, where the rate was only 9% [26]. Subsequent trials of the three drug combination, however, were unable to demonstrate an improvement in the prevention of acute GVHD, or improved long-term outcomes using this three drug regimen when compared with the two drug standard [27, 28], and therefore prednisone or other corticosteroids are not routinely used in GVHD prophylaxis. Other novel calcineurin inhibitors, such as tacrolimus, have been developed as GVHD prophylactic agents because of their favorable toxicity profiles in

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comparison with cyclosporine [29]. As monotherapy for prevention of GVHD after allogeneic transplantation, tacrolimus has demonstrated safety and efficacy in rodent studies [30] as well as in trials of human subjects [31]. In the first trial of tacrolimus monotherapy, 27 patients received allogeneic bone marrow transplants from matched donors. Ten of 27 patients (37%) developed grade II acute GVHD, and only one patient developed grade III acute GVHD. All patients in whom GVHD developed responded to steroids [31]. Studies evaluating tacrolimus with tacrolimus-based combination therapy have not been repeated because of extrapolation of favorable findings with cyclosporine combinations. However, in the one trial that did compare GVHD outcomes after peripheral blood stem cell transplantation, where GVHD rates may be higher than after bone marrow transplantation [32], no statistically significant increase in GVHD was noted in patients who did not receive methotrexate [33]. Despite this, tacrolimus monotherapy is not routinely used as GVHD prophylaxis. Large phase III studies comparing tacrolimus and methotrexate versus cyclosporine and methotrexate for both, matched, related and unrelated donors have been performed. In the matched, related donor setting, 329 patients were randomized to receive either tacrolimus with methotrexate or cyclosporine and methotrexate. The incidence of Grade II–IV acute GVHD was 31.9% in the tacrolimus arm and 44.4% in the cyclosporine arm [34]. Similarly, in the unrelated donor study, the incidence of Grade II–IV acute GVHD was 56% among 46 patients randomized to tacrolimus and was 74% among 63 patients randomized to receive cyclosporine [35]. Despite the data from the prospective trials and retrospective database studies [36], the combination of cyclosporine and methotrexate is the most commonly employed regimen for GVHD prophylaxis currently prescribed. Table  32-1 demonstrates current GVHD prophylaxis agent use, according to a recent Center for International Blood and Marrow Transplant Research (CIBMTR) analysis. 2.2. Novel Strategies Beyond Methotrexate Despite its 30 year history as a mainstay of GVHD prophylaxis, there are several reasons to move beyond methotrexate prophylaxis in allogeneic transplantation, notwithstanding the fact that this agent is imperfect in preventing GVHD. Table 32-1.  Current use of GVHD regimens, as reported to the IBMTR. Regimen

Matched, related donor (%) Unrelated donor (%)

Methotrexate alone

 4

 1

Cyclosporine alone

 6

 2

Tacrolimus ± other

 5

 6

Cyclosporine ± other

 6

27

Cyclosporine, methotrexate ± other

42

32

Tacrolimus, methotrexate ± other

24

24

T cell depletion ± other

10

 5

Other

 2

 4

Chapter 32  Acute Graft Versus Host Disease: Prophylaxis 

Methotrexate use may increase the risk of other complications of transplantation such as mucositis and hemorrhagic pneumonia/interstitial pneumonitis [37, 38]. As an antiproliferative agent, methotrexate has been associated with slower engraftment of neutrophils after allogeneic transplantation. In a retrospective study of peripheral blood stem cell transplant trials, the use of fulldose methotrexate was associated with a 2.5 day median delay in the time to neutrophil engraftment [39], but the omission of the fourth dose of methotrexate may be associated with an increased risk of acute GVHD [39–41]. Finally, as an agent that can independently induce tissue injury, there is a theoretical concern that methotrexate may contribute to the mechanisms that incite acute GVHD. Strategies that protect the mucosa from tissue injury, while promising in pre-clinical trials, have not demonstrated clinical efficacy [42]. An antiproliferative agent that did not cause mucositis and lung toxicity and that did not delay engraftment would be highly desirable. 2.3. Sirolimus Sirolimus (Rapamune®, Wyeth) is a naturally occurring compound originally isolated from a soil saprophyte (Streptomyces hygroscopicus) found uniquely on Easter Island (Rapa Nui). In addition to its immunosuppressive properties, sirolimus has antifungal, antiviral and antineoplastic properties. Sirolimus binds uniquely to FK binding protein 12 (FKBP12) and forms a complex with mTOR and the raptor/rictor proteins [43, 44]. Although there is theoretical competition for FKBP binding sites between sirolimus and calcineurin inhibitors, these agents appear to work synergistically [45, 46], as sirolimus does not interact with calcineurin or its downstream effectors. Consequently, sirolimus has a different toxicity profile when compared with the calcineurin inhibitors. The sirolimus-FKBP12-mTOR complex inhibits several biochemical pathways, resulting in a reduction in DNA transcription, DNA translation, protein synthesis and cell cycling, ultimately leading to T cell immunosuppression. Upstream pathways that interact with mTOR include the PTEN/ PI3 kinase/Akt pathway and the Janus kinase pathway, which is important in mediating IL-2 driven signaling from the T cell receptor [47]. In contrast to the calcineurin inhibitors, sirolimus may also be immunosuppressive via inhibition of dendritic cell activity through a reduction in antigen uptake [48, 49], cellular maturation [50], intracellular signaling [51], and apoptosis induction [52, 53]. The differential inhibition of certain T cell subsets (such as the sparing of CD4+CD25+ regulatory T cells) may also be responsible for some of sirolimus’ immunosuppressive properties [54–56]. The combination of sirolimus and tacrolimus is more effective than the combination of sirolimus and cyclosporine in reducing memory T cell production, apoptosis induction and cytokine production [57]. In a trial of sirolimus and tacrolimus as GVHD prophylaxis after HLAmatched, related peripheral blood stem cell transplantation, we have demonstrated excellent control of acute GVHD (Gr. II–IV 18.9%) with minimal transplant-related morbidity and mortality (5.7% 100-day non-relapse mortality, 9.4% incidence each of hepatic veno-occlusive disease and thrombotic microangiopathy). In this trial, all patients received conditioning with cyclophosphamide and total body irradiation (14 Gy) and engrafted promptly (median times to neutrophil and platelet engraftment: 14 and 12  days). Similarly, in

569

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the unrelated donor setting, acute GVHD was well controlled (Gr. II–IV 23.3%) with early engraftment in the absence of methotrexate (median time to neutrophil and platelet engraftment: 13.5 and 12 days). Transplant-related morbidity and mortality was low (3.9% 100-day non-relapse mortality) and major complications of transplantation were uncommon (6.7% incidence of hepatic veno-occlusive disease, 3.3% incidence of thrombotic microangiopathy) [58]. The omission of methotrexate in this unrelated donor experience did not adversely influence the rate of acute GVHD, but significantly lowered the rate of transplant-related morbidity and mortality when compared with a cohort of patients receiving transplants for mismatched related, matched unrelated and mismatched unrelated donor bone marrow and peripheral blood stem cell transplants who received sirolimus, tacrolimus and methotrexate as GVHD prophylaxis [59]. Similarly, when used in the reduced intensity setting, sirolimus use has reduced the rates of acute GVHD to approximately 20%, similar to the rates noted in the myeloablative studies, even without methotrexate. Table 32-2 presents a summary of the clinical results with sirolimus in several transplant prophylaxis settings. Sirolimus is currently being tested in a randomized controlled trial in combination with tacrolimus against the control arm of tacrolimus and methotrexate in a large Phase III randomized trial conducted through the BMT CTN.

Table 32-2.  Clinical results of sirolimus and tacrolimus without methotrexate as GVHD prophylaxis [58]. MRD

URD

Sample size

53

30

Median age (range)

42 (18, 59)

44 (22, 54)

Male sex

26 (49%)

13 (43%)

  M/M

11 (21%)

11 (37%)

  F/F

15 (28%)

7 (23%)

  F/M1

15 (28%)

2 (7%)

  M/F

12 (23%)

10 (33%)

CD34+ cell dose (×106/kg)

7.6

10.2a

  Gr. II–IV

18.9%

23.3%

  Gr. III–IV

5.6%

3.3%

  VOD

9.4%

6.7%

  Thrombotic microangiopathy

9.4%

3.3%

Chronic GVHD

58.3%

59.3%

Relapse-free survival (2 year)

66.0%

66.5%

Overall survival (2 year)

69.8%

69.7%

Gender match (D/R)

Acute GVHD

Transplant-related toxicity

Chapter 32  Acute Graft Versus Host Disease: Prophylaxis 

2.4. Mycophenolate Mofetil Mycophenolate mofetil is the 2-morpholinoethyl ester of mycophenolic acid (MPA). MPA is a potent, selective, uncompetitive, and reversible inhibitor of inosine monophosphate dehydrogenase, and inhibits the de novo pathway of guanosine nucleotide synthesis without incorporation into DNA. As T- and B-lymphocytes are critically dependent for their proliferation on de novo synthesis of purines, MPA has potent cytostatic effects on lymphocytes. In combination with cyclosporine [60], cyclosporine with methotrexate [61, 62], and tacrolimus [63] mycophenolate mofetil has been used for GVHD prophylaxis. The Seattle group recently published their Phase I/II experience using mycophenolate mofetil with cyclosporine. Using a daily dose of 45 mg/ kg/day in three divided doses, the incidence of acute GVHD was 62% [64]. Neumann et al. compared their single center experience of cyclosporine with mycophenolate mofetil in comparison with cyclosporine with methotrexate. The incidence rates of grade II–IV acute GVHD were 38 and 61%, and the overall survival rates were 76 and 55%, although neither of these comparisons were statistically significant [65]. In a small, single center randomized study, the combination of cyclosporine and mycophenolate mofetil was associated with faster hematopoietic engraftment, a decreased incidence of mucositis, a similar incidence of acute GVHD, and comparable survival as compared to cyclosporine and methotrexate [66]. Table  32-3 compares the outcomes of mycophenolate mofetil in GVHD prophylaxis when used in lieu of methotrexate. Mycophenolate mofetil is also commonly employed after reduced intensity transplantation as well. Absorbed well after oral administration, the optimal dose for use in transplantation is unclear, with some centers administering it twice and others three times daily. 2.5. Cyclophosphamide Post-transplant cyclophosphamide was used in the 1980s to prevent GVHD via inhibition of rapidly dividing T cells in a manner similar to methotrexate [67]. Since stem cells contain high levels of aldehyde dehydrogenase which converts 4-hydroxycyclophosphamide into a non-alkylating metabolite, they

Table 32-3.  Clinical results of mycophenolate mofetil randomized trials. Bolwell [66]

Neumann [65]

N

40

93

Design

RCT

Retrospective analysis

Stem cell source

Marrow

Marrow/PBSC (unbalanced)

Conditioning

BuCy

BuCy, CyTBI

5 mg/m

15 mg/m2/10 mg/m2

1, 3, 6, 11

1, 3, 6

Neutrophil engraftment

11 vs. 18 days

12 vs. 18 days

Gr. II–IV GVHD

48 vs. 37%

38 vs. 61%

p = NS

p = NS

63 vs. 64%

50 vs. 45%

Mtx

Chronic GVHD

2

571

572 

C. Cutler et al.

are not susceptible to the anti-proliferative activity of this agent, which makes its administration after transplant feasible. In addition, the gastrointestinal epithelium also contains high levels of aldehyde dehydrogenase, thus affording a protective effect against gastrointestinal mucositis when this cytotoxic agent is given shortly after intensive conditioning regimens. When given as a single agent after myeloablative related and unrelated bone marrow transplantation in 46 patients, the rate of Gr. II–IV acute GVHD was 41%, with few late infections, attributed to the brief nature of immune suppression after transplantation [68]. Ongoing studies are examining the role of this agent in alternative donor transplants. 2.6. Additional Agents and Conclusions There are numerous additional agents that are active as a therapy of established acute GVHD, either in the first-line setting in conjunction with corticosteroids or as second-line therapy, in the steroid refractory setting. Examples of these agents include Pentostatin and Etanercept, and there are active efforts to determine if these agents are effective when used as primary GVHD prophylaxis. However, some biological agents that may have activity in established acute GVHD (such as IL-1 antagonists [69, 70], and CD5 immunotoxins [71–73]) may not be effective as primary prophylaxis. Novel approaches to the prevention of acute GVHD include the prevention of migration of lymphocytes to the target organs of GVHD using chemokine blockade [74] and the use of extracorporeal phototherapy, which may alter host antigen presentation to prevent acute GVHD [75]. These and other approaches that target intermediate steps in the 3-stage model of acute GVHD will eventually mandate a change in this model, to include cellular migration pathways, and to emphasize the role of host-derived professional antigen presenting cells in the pathophysiology of acute GVHD.

References 1. Antin JH, Ferrara JL (1992) Cytokine dysregulation and acute graft-versus-host disease. Blood 80:2964–2968 2. Ho VT, Soiffer RJ (2001) The history and future of T-cell depletion as graft-versushost disease prophylaxis for allogeneic hematopoietic stem cell transplantation. Blood 98:3192–3204 3. Hale G, Jacobs P, Wood L et al (2000) CD52 antibodies for prevention of graftversus-host disease and graft rejection following transplantation of allogeneic peripheral blood stem cells. Bone Marrow Transplant 26:69–76 4. Henslee-Downey PJ, Parrish RS, MacDonald JS et al (1996) Combined in vitro and in vivo T lymphocyte depletion for the control of graft-versus-host disease following haploidentical marrow transplant. Transplantation 61:738–745 5. Perez-Simon JA, Kottaridis PD, Martino R et  al (2002) Nonmyeloablative transplantation with or without alemtuzumab: comparison between 2 prospective studies in patients with lymphoproliferative disorders. Blood 100:3121–3127 6. de Witte T, Hoogenhout J, de Pauw B et al (1986) Depletion of donor lymphocytes by counterflow centrifugation successfully prevents acute graft-versus-host disease in matched allogeneic marrow transplantation. Blood 67:1302–1308 7. Noga SJ, Donnenberg AD, Schwartz CL et al (1986) Development of a simplified counterflow centrifugation elutriation procedure for depletion of lymphocytes from human bone marrow. Transplantation 41:220–229

Chapter 32  Acute Graft Versus Host Disease: Prophylaxis  8. Reisner Y, Kapoor N, Kirkpatrick D et  al (1981) Transplantation for acute leukaemia with HLA-A and B nonidentical parental marrow cells fractionated with soybean agglutinin and sheep red blood cells. Lancet 2:327–331 9. Wagner JE, Donnenberg AD, Noga SJ et al (1988) Lymphocyte depletion of donor bone marrow by counterflow centrifugal elutriation: results of a phase I clinical trial. Blood 72:1168–1176 10. Prentice HG, Blacklock HA, Janossy G et al (1982) Use of anti-T-cell monoclonal antibody OKT3 to prevent acute graft-versus-host disease in allogeneic bonemarrow transplantation for acute leukaemia. Lancet 1:700–703 11. Martin PJ, Hansen JA, Thomas ED (1984) Preincubation of donor bone marrow cells with a combination of murine monoclonal anti-T-cell antibodies without complement does not prevent graft-versus-host disease after allogeneic marrow transplantation. J Clin Immunol 4:18–22 12. Filipovich AH, McGlave PB, Ramsay NK et al (1982) Pretreatment of donor bone marrow with monoclonal antibody OKT3 for prevention of acute graft-versushost disease in allogeneic histocompatible bone-marrow transplantation. Lancet 1:1266–1269 13. Urbano-Ispizua A, Solano C, Brunet S et al (1998) Allogeneic transplantation of selected CD34+ cells from peripheral blood: experience of 62 cases using immunoadsorption or immunomagnetic technique. Spanish Group of Allo-PBT. Bone Marrow Transplant 22:519–525 14. Champlin RE, Passweg JR, Zhang MJ et al (2000) T-cell depletion of bone marrow transplants for leukemia from donors other than HLA-identical siblings: advantage of T-cell antibodies with narrow specificities. Blood 95:3996–4003 15. Wagner JE, Thompson JS, Carter SL, Kernan NA (2005) Effect of graft-versus-host disease prophylaxis on 3-year disease-free survival in recipients of unrelated donor bone marrow (T-cell Depletion Trial): a multi-centre, randomised phase II-III trial. Lancet 366:733–741 16. Rizzieri DA, Koh LP, Long GD et al (2007) Partially matched, nonmyeloablative allogeneic transplantation: clinical outcomes and immune reconstitution. J Clin Oncol 25:690–697 17. Mehta J, Singhal S, Gee AP et al (2004) Bone marrow transplantation from partially HLA-mismatched family donors for acute leukemia: single-center experience of 201 patients. Bone Marrow Transplant 33:389–396 18. Pavletic SZ, Carter SL, Kernan NA et al (2005) Influence of T-cell depletion on chronic graft-versus-host disease: results of a multicenter randomized trial in unrelated marrow donor transplantation. Blood 106:3308–3313 19. Bacigalupo A, Lamparelli T, Barisione G et  al (2006) Thymoglobulin prevents chronic graft-versus-host disease, chronic lung dysfunction, and late transplantrelated mortality: long-term follow-up of a randomized trial in patients undergoing unrelated donor transplantation. Biol Blood Marrow Transplant 12:560–565 20. Storb R, Thomas ED, Buckner CD et  al (1974) Allogeneic marrow grafting for treatment of aplastic anemia. Blood 43:157–180 21. Deeg HJ, Storb R, Thomas ED et al (1985) Cyclosporine as prophylaxis for graftversus-host disease: a randomized study in patients undergoing marrow transplantation for acute nonlymphoblastic leukemia. Blood 65:1325–1334 22. Storb R, Deeg HJ, Farewell V et al (1986) Marrow transplantation for severe aplastic anemia: methotrexate alone compared with a combination of methotrexate and cyclosporine for prevention of acute graft-versus-host disease. Blood 68:119–125 23. Storb R, Deeg HJ, Whitehead J et al (1986) Methotrexate and cyclosporine compared with cyclosporine alone for prophylaxis of acute graft versus host disease after marrow transplantation for leukemia. N Engl J Med 314:729–735 24. Mrsic M, Labar B, Bogdanic V et al (1990) Combination of cyclosporin and methotrexate for prophylaxis of acute graft-versus-host disease after allogeneic bone marrow transplantation for leukemia. Bone Marrow Transplant 6:137–141

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C. Cutler et al. 25. Tollemar J, Ringden O, Sundberg B et  al (1988) Decreased incidence of graftversus-host disease in bone marrow transplantation recipients treated with a combination of cyclosporine and methotrexate. Transplant Proc 20:494–498 26. Chao NJ, Schmidt GM, Niland JC et  al (1993) Cyclosporine, methotrexate, and prednisone compared with cyclosporine and prednisone for prophylaxis of acute graft-versus-host disease. N Engl J Med 329:1225–1230 27. Chao NJ, Snyder DS, Jain M et al (2000) Equivalence of 2 effective graft-versushost disease prophylaxis regimens: results of a prospective double-blind randomized trial. Biol Blood Marrow Transplant 6:254–261 28. Storb R, Pepe M, Anasetti C et al (1990) What role for prednisone in prevention of acute graft-versus-host disease in patients undergoing marrow transplants? Blood 76:1037–1045 29. Przepiorka D, Devine S, Fay J, Uberti J, Wingard J (1999) Practical considerations in the use of tacrolimus for allogeneic marrow transplantation. Bone Marrow Transplant 24:1053–1056 30. Markus PM, Cai X, Ming W et  al (1991) Prevention of graft-versus-host disease following allogeneic bone marrow transplantation in rats using FK506. Transplantation 52:590–594 31. Fay JW, Wingard JR, Antin JH et al (1996) FK506 (Tacrolimus) monotherapy for prevention of graft-versus-host disease after histocompatible sibling allogeneic bone marrow transplantation. Blood 87:3514–3519 32. Cutler C, Giri S, Jeyapalan S et al (2001) Acute and chronic graft-versus-host disease after allogeneic peripheral- blood stem-cell and bone marrow transplantation: a meta-analysis. J Clin Oncol 19:3685–3691 33. Reynolds C, Ratanatharathorn V, Adams P et  al (1998) Comparative analysis of tacrolimus/methotrexate versus tacrolimus in allogeneic peripheral blood stem cell transplants: engraftment, GVHD, relapse, and survival outcomes. Blood 92:449a 34. Ratanatharathorn V, Nash RA, Przepiorka D et al (1998) 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 92:2303–2314 35. Nash RA, Antin JH, Karanes C et al (2000) Phase 3 study comparing methotrexate and tacrolimus with methotrexate and cyclosporine for prophylaxis of acute graftversus-host disease after marrow transplantation from unrelated donors. Blood 96:2062–2068 36. Horowitz MM, Przepiorka D, Bartels P et al (1999) Tacrolimus vs. cyclosporine immunosuppression: results in advanced- stage disease compared with historical controls treated exclusively with cyclosporine. Biol Blood Marrow Transplant 5:180–186 37. Cutler C, Li S, Kim HT et al (2005) Mucositis after allogeneic hematopoietic stem cell transplantation: a cohort study of methotrexate- and non-methotrexate-containing graft-versus-host disease prophylaxis regimens. Biol Blood Marrow Transplant 11:383–388 38. Ho VT, Weller E, Lee SJ et al (2001) Prognostic factors for early severe pulmonary complications after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 7:223–229 39. Cutler C, Antin JH (2002) Omission of day +11 methotrexate after matched-related allogeneic peripheral blood stem cell transplantation does not increase the risk of graft-vs.-host disease. Biol Blood Marrow Transplant 8:85 Abstr 40. Kumar S, Wolf RC, Chen MG et al (2002) Omission of day +11 methotrexate after allogeneic bone marrow transplantation is associated with increased risk of severe acute graft-versus-host disease. Bone Marrow Transplant 30:161–165 41. Nash RA, Pepe MS, Storb R et al (1992) Acute graft-versus-host disease: analysis of risk factors after allogeneic marrow transplantation and prophylaxis with cyclosporine and methotrexate. Blood 80:1838–1845

Chapter 32  Acute Graft Versus Host Disease: Prophylaxis  42. Blazar BR, Weisdorf DJ, Defor T et al (2006) Phase 1/2 randomized, placebo-control trial of palifermin to prevent graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Blood 108:3216–3222 43. Ali SM, Sabatini DM (2005) Structure of S6 kinase 1 determines whether raptormTOR or rictor-mTOR phosphorylates its hydrophobic motif site. J Biol Chem 280:19445–19448 44. Sarbassov DD, Ali SM, Kim DH et  al (2004) Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 14:1296–1302 45. Sehgal SN (1998) Rapamune (RAPA, rapamycin, sirolimus): mechanism of action immunosuppressive effect results from blockade of signal transduction and inhibition of cell cycle progression. Clin Biochem 31:335–340 46. Sehgal SN (2003) Sirolimus: its discovery, biological properties, and mechanism of action. Transplant Proc 35(3 Suppl):S7–S14 47. Kirken RA, Wang YL (2003) Molecular actions of sirolimus: sirolimus and mTor. Transplant Proc 35:S227–S230 48. Hackstein H, Taner T, Logar AJ, Thomson AW (2002) Rapamycin inhibits macropinocytosis and mannose receptor-mediated endocytosis by bone marrow-derived dendritic cells. Blood 100:1084–1087 49. Monti P, Mercalli A, Leone BE et al (2003) Rapamycin impairs antigen uptake of human dendritic cells. Transplantation 75:137–145 50. Hackstein H, Taner T, Zahorchak AF et al (2003) Rapamycin inhibits IL-4–induced dendritic cell maturation in  vitro and dendritic cell mobilization and function in vivo. Blood 101:4457–4463 51. Chiang PH, Wang L, Liang Y et al (2002) Inhibition of IL-12 signaling Stat4/IFNgamma pathway by rapamycin is associated with impaired dendritic [correction of dendritc] cell function. Transplant Proc 34:1394–1395 52. Woltman AM, de Fijter JW, Kamerling SW et al (2001) Rapamycin induces apoptosis in monocyte- and CD34-derived dendritic cells but not in monocytes and macrophages. Blood 98:174–180 53. Woltman AM, van der Kooij SW, Coffer PJ et  al (2003) Rapamycin specifically interferes with GM-CSF signaling in human dendritic cells, leading to apoptosis via increased p27KIP1 expression. Blood 101:1439–1445 54. Baan CC, van der Mast BJ, Klepper M et al (2005) Differential effect of calcineurin inhibitors, anti-CD25 antibodies and rapamycin on the induction of FOXP3 in human T cells. Transplantation 80:110–117 55. Battaglia M, Stabilini A, Roncarolo MG (2005) Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood 105:4743–4748 56. Zeiser RS, Nguyen VH, Beilhack A et al (2006) Inhibition of CD4 + CD25+ regulatory T cell function by calcineurin dependent interleukin-2 production. Blood 108(1):390–399 57. Koenen H, Michielsen E, Verstappen J, Fasse E, Joosten I (2003) Superior T-cell suppression by rapamycin and FK506 over rapamycin and cyclosporine A because of abrogated cytotoxic T-lymphocyte induction, impaired memory responses, and persistent apoptosis. Transplantation 75:1581–1590 58. Cutler C, Li S, Ho VT et  al (2007) Extended follow-up of methotrexate-free immunosuppression using sirolimus and tacrolimus in related and unrelated donor peripheral blood stem cell transplantation. Blood 109:3108–3114 59. Antin JH, Kim HT, Cutler C et al (2003) Sirolimus, tacrolimus, and low-dose methotrexate for graft-versus-host disease prophylaxis in mismatched related donor or unrelated donor transplantation. Blood 102:1601–1605 60. Bornhauser M, Schuler U, Porksen G et  al (1999) Mycophenolate mofetil and cyclosporine as graft-versus-host disease prophylaxis after allogeneic blood stem cell transplantation. Transplantation 67:499–504

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C. Cutler et al. 61. Kasper C, Sayer HG, Mugge LO et al (2004) Combined standard graft-versus-host disease (GvHD) prophylaxis with mycophenolate mofetil (MMF) in allogeneic peripheral blood stem cell transplantation from unrelated donors. Bone Marrow Transplant 33:65–69 62. Wang J, Song X, Zhang W et  al (2002) Combination of mycophenolate mofetil with cyclosporine A and methotrexate for the prophylaxes of acute graft versus host disease in allogeneic peripheral stem cell transplantation. Zhonghua Yi Xue Za Zhi 82:507–510 63. Osunkwo I, Bessmertny O, Harrison L et al (2004) A pilot study of tacrolimus and mycophenolate mofetil graft-versus-host disease prophylaxis in childhood and adolescent allogeneic stem cell transplant recipients. Biol Blood Marrow Transplant 10:246–258 64. Nash RA, Johnston L, Parker P et al (2005) A phase I/II study of mycophenolate mofetil in combination with cyclosporine for prophylaxis of acute graft-versus-host disease after myeloablative conditioning and allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 11:495–505 65. Neumann F, Graef T, Tapprich C et al (2005) Cyclosporine A and mycophenolate mofetil vs cyclosporine A and methotrexate for graft-versus-host disease prophylaxis after stem cell transplantation from HLA-identical siblings. Bone Marrow Transplant 35:1089–1093 66. Bolwell B, Sobecks R, Pohlman B et  al (2004) A prospective randomized trial comparing cyclosporine and short course methotrexate with cyclosporine and mycophenolate mofetil for GVHD prophylaxis in myeloablative allogeneic bone marrow transplantation. Bone Marrow Transplant 34:621–625 67. Santos GW, Tutschka PJ, Brookmeyer R et  al (1987) Cyclosporine plus methylprednisolone versus cyclophosphamide plus methylprednisolone as prophylaxis for graft-versus-host disease: a randomized double-blind study in patients undergoing allogeneic marrow transplantation. Clin Transplant 1:21–28 68. Luznik L, Chen A, Kaup M et al (2006) Post-transplantation high-dose cyclophosphamide (Cy) is effective single agent GVHD prophylaxis that permits prompt immune reconstitution after myeloablative HLA matched related and unrelated bone marrow transplantation (BMT). Blood 108:2891s 69. Antin JH, Weinstein HJ, Guinan EC et al (1994) Recombinant human interleukin-1 receptor antagonist in the treatment of steroid-resistant graft-versus-host disease. Blood 84:1342–1348 70. Antin JH, Weisdorf D, Neuberg D et  al (2002) Interleukin-1 blockade does not prevent acute graft-versus-host disease: results of a randomized, double-blind, placebo-controlled trial of interleukin-1 receptor antagonist in allogeneic bone marrow transplantation. Blood 100:3479–3482 71. Koehler M, Hurwitz CA, Krance RA et al (1994) XomaZyme-CD5 immunotoxin in conjunction with partial T cell depletion for prevention of graft rejection and graft-versus-host disease after bone marrow transplantation from matched unrelated donors. Bone Marrow Transplant 13:571–575 72. Martin PJ, Nelson BJ, Appelbaum FR et al (1996) Evaluation of a CD5-specific immunotoxin for treatment of acute graft-versus-host disease after allogeneic marrow transplantation. Blood 88:824–830 73. Weisdorf D, Filipovich A, McGlave P et al (1993) Combination graft-versus-host disease prophylaxis using immunotoxin (anti-CD5-RTA [Xomazyme-CD5]) plus methotrexate and cyclosporine or prednisone after unrelated donor marrow transplantation. Bone Marrow Transplant 12:531–536 74. Beilhack A, Schulz S, Baker J et  al (2008) Prevention of acute graft-versushost disease by blocking T-cell entry to secondary lymphoid organs. Blood 111(5):2919–2928 75. Miller KB, Roberts TF, Chan G et  al (2004) A novel reduced intensity regimen for allogeneic hematopoietic stem cell transplantation associated with a reduced incidence of graft-versus-host disease. Bone Marrow Transplant 33:881–889

Chapter 33 Chronic Graft-Versus-Host Disease Madan Jagasia and Steven Pavletic

1. Introduction Chronic graft-versus-host disease (cGVHD) is a complex, immune phenomenon that occurs after allogeneic stem cell transplant (SCT) and resembles a plethora of autoimmune diseases [1]. It can have minimal features like a dry eye or can be disabling with sclerodermatous fascitis and bronchiolitis obliterans. The incidence and time course is variable. Despite improvement in other areas of SCT, little significant progress has been made in the treatment of cGVHD [2]. One of the major reasons may be that cGVHD is a heterogeneous disease, with various subtypes having differing natural history. The development of cGVHD has been associated with the protective effect of graft-versus-tumor effect. It is likely that various subtypes of chronic GVHD have varying graft-versustumor effect, with non-relapse mortality from chronic GVHD as a constant competing risk. Traditionally, any GVHD after day 100 from SCT has been classified as cGVHD [1]. Recently, there has been a movement to sub-classify GVHD after day 100, based on the morphology and in some cases, histology of the GVHD lesion. This chapter focuses on the recent advances in the classification and grading of cGVHD and highlights the newer developments in therapeutics of cGVHD.

2. Classification of cGVHD The first attempt to classify GVHD after day 100 was published in 1980. Any GVHD after day 100 was termed as cGVHD to contrast with acute GVHD (aGVHD) that typically occurs in the first 3 months after SCT and presents as a constellation of dermatitis, hepatitis and enteritis [1]. It was recognized in the 1970s that cGVHD features resembled an overlap of several collagen vascular diseases with frequent involvement of skin, liver, eyes, mouth, upper respiratory tract, esophagus and less frequent involvement of the serosal surfaces, lower gastrointestinal tract and skeletal muscles [1, 3]. Scleroderma,

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_33, © Springer Science + Business Media, LLC 2003, 2010

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dry eyes, dry mouth, pulmonary insufficiency and wasting accounted for the major causes of morbidity. cGVHD, based on the pattern of onset in relation to aGVHD, was classified as progressive (continuously active aGVHD gradually progressing into cGVHD), quiescent (resolution of aGVHD, then a phase of no clinical GVD activity, followed by development of cGVHD), and de novo onset (onset of cGVHD without clinical or biopsy evidence of prior aGVHD). The concept of limited (isolated liver and/or localized skin involvement) and extensive cGVHD (generalized skin or localized skin plus liver or any other organ involvement) was introduced based on the outcome of 20 patients. The original limited and extensive classification of cGVHD was revised by the Seattle group [4]. The purpose of the revision was to guide implementation and duration of systemic immunosuppressive therapy. Patients with the revised extensive cGVHD would be candidates for prolonged immunosuppressive therapy, in contrast to the revised limited cGVHD. The prognostic impact or incidence of cGVHD using the revised Seattle classification is not known. More recently, as part of the National Institute of Health (NIH) Consensus Criteria, a more biologically and clinically relevant classification system has been proposed [5]. The NIH consensus criteria propose to divide GVHD into two groups (acute and chronic) based on features at presentation rather than time after transplant (Fig. 33-1). aGVHD is further classified as classic acute or persistent, recurrent or delayed aGVHD. cGVHD is sub-classified as classic cGVHD or overlap cGVHD. The diagnosis of cGVHD now requires the following: (1) distinction from aGVHD; (2) presence of at least one diagnostic clinical sign of cGVHD or presence of at least one distinctive manifestation confirmed by pertinent biopsy or other relevant tests; (3) exclusion of other possible diagnoses. The NIH consensus criteria have proposed a scoring system of 0–3 for evaluation of individual organs and sites. A global assessment of severity (mild, moderate, severe) is computed by combining organ and site-specific scores.

3. Clinical Features of cGVHD In view of the recent NIH consensus criteria, clinical features are best considered as either diagnostic (those manifestations that establish the presence or absence of cGVHD), distinctive (manifestations that are not ordinarily found in aGVHD but are not considered sufficient to establish an unequivocal diagnosis of cGVHD without further testing or additional organ involvement), common (manifestation seen in both aGVHD and cGVHD), and other (rare, controversial or nonspecific features that cannot be used to establish the diagnosis of cGVHD) (Table 33-1). 3.1. Skin Skin is commonly affected in cGVHD [6, 7]. Diagnostic manifestations of cGVHD include poikiloderma, lichen planus-like eruption, deep sclerotic features, morphea-like superficial sclerosis or lichensclerosus-like lesions. Distinctive features include depigmentation. Common features include erythema, maculopapular rash, and pruritis. Other rare features include sweat impairment and intolerance to temperature.

Overlap chronic GVHD

Yes

No

Classic acute GVHD (first episode before day 100)

Fig. 33-1.  Various subtypes of GVHD as proposed by the NIH consensus criteria

Classic chronic GVHD

No

Presence of acute GVHD

Fulfills definition of chronicGVHD (1 diagnostic criteria OR 1 distinctive Criteria PLUS biopsy proof in an organ AND exclusion of other probable causes.

Yes

Any features of GVHD

Recurrent acute GVHD Delayed acute GVHD (first episode after day100) Persistent acute GVHD

Acute GVHD

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Table 33-1.  Signs and symptoms of chronic GVHD. Skin

Diagnostic

Distinctive

Poikiloderma

Depigmentation

Lichen planus-like features Sclerotic features Morphea-like feature Lichen sclerosus-like features Nails

None

Dystrophy Longitudinal ridging, splitting, or brittle Features Onycholysis Pterygium unguis Nail loss (usually symmetric; affects most nails)

Scalp and body hair

None

New onset of scarring or nonscarring scalp alopecia (after recovery from chemoradiotherapy) Scaling, papulosquamous lesions

Mouth

Lichen-type features

Xerostomia

Hyperkeratotic plaques

Mucocele

Restrictions of mouth

Mucosal atrophy

opening from sclerosis

Pseudomembranes Ulcers

Eyes

None

New onset dry, gritty, or painful eyes Cicatricial conjunctivitis Keratoconjunctivitis sicca Confluent areas of punctuate keratopathy

Genitalia

Lichen Planus-like features

Erosions

Vaginal scarring or stenosis

Fissures Ulcers

GI tract

Esophageal web

None

Strictures or stenosis in the upper to mid third of the esophagus Liver Lung

Bronchiolitis obliterans

Bronchiolitis obliterans diagnosed with PFT’s and radiology

Diagnosed with lung biopsy Muscles, fascia, Fasciitis joints

Myositis or polymyositis

Joint stiffness or contractures secondary to sclerosis Hematopoietic None and immune

Thrombocytopenia Eosinophilia Lymphopenia Hypo- or hypergammaglobulinemia Autoantibodies (AIHA and ITP) Pericardial or pleural effusions (continued)

Chapter 33  Chronic Graft-Versus-Host Disease 

Table 33-1.  (continued) Other

Diagnostic

Distinctive

None

Ascites Peripheral Neuropathy Nephrotic Syndrome Myasthenia gravis Cardiac Conduction Abnormality or cardiomyopathy

Skin

Other features

Common (seen with both acute and chronic GVHD)

Sweat impairment

Erythema

Ichthyosis

Maculopapular rash

Keratosis pilaris

Pruritus

Hypopigmentation Hyperpigmentation Scalp and body hair

Thinning scalp hair, typically patchy, None coarse, or dull (not explained by endocrine or other causes) Premature gray Hair

Mouth

None

Gingivitis Mucositis Erythema Pain

Eyes

Photophobia

None

Periorbital Hyperpigmentation GI tract

Exocrine pancreatic insufficiency

Anorexia Nausea Vomiting Diarrhea Weight loss Failure to thrive (infants and children)

Liver

None

Total bilirubin, alkaline Phosphatase > 2x Upper limit of Normal ALT or AST > 2x Upper limit of Normal

Lung

None

Muscles, Edema muscle cramps fascia, joints Arthralgia or arthritis

BOOP

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As the disease advances, skin gets involved with more sclerodermatous features with thickening and limitation of joint movement, loss of dermal appendages (hair and sweat glands). Rarely, skin can become blistered from poor lymphatic drainage and can ulcerate after minor trauma. Skin cancers can often complicate areas affected by cGVHD. 3.2. Nails Distinctive features include longitudinal ridging, nail splitting or brittleness, onycholysis, pterygium unguis and nail loss. 33.3.3 Hair Distinctive features include new scarring and non-scarring scalp alopecia (after previous recovery from chemotherapy and radiation), and loss of body hair. Other features include premature graying, thinning, or brittleness. 3.4. Mouth In recent studies, the oral cavity was found to be involved in 70% of peripheral blood stem cell transplant recipients and 53% of marrow transplant recipients [8]. Diagnostic features include lichenplanus-like changes (white lines and lacy-appearing lesions of buccal mucosa, tongue, palate or lips), hyperkeratotic plaques, or decreased range of motion in patients with sclerotic skin features. Distinctive features include dryness, mucoceles, mucosal atrophy, pseudomembranes and ulcers. In case of ulcers, infectious causes like yeast and herpes need to be ruled out. Common features include gingivitis, musocitis, erythema and pain. 3.5. Eyes Distinctive features include new onset of dry, gritty or painful eyes, cicatrical conjunctivitis, kertoconjunctivitis sicca and confluent areas of punctuate keratopathy. Other features include photophobia, periorbital hyeperpigmentation, and blepharitis. New ocular sicca documented by a Schirmer test performed without anesthesia with a mean value of both eyes £5 mm at 5 min or a new onset of kertoconjunctivitis sicca with mean value of 6–10 mm on Schirmer test along with distinctive feature of GVHD in at least one other organ is sufficient for the diagnosis of cGVHD. The most common manifestation of ocular GVHD is dry eyes or KCS resulting from lacrimal gland destruction form the allogeneic T cells. Patients report ocular fatigue, dry eye sensation, a foreign body sensation, ocular pain, photophobia, blurring, red eye, discharge, heavy eye feeling, dull sensation, difficulty in eye opening, epiphora, and itchy sensation or a burning sensation. In most patients, KCS is permanent once it develops. In a study, none of the patients who developed KCS as part of GVHD had a return of tear function to normal despite 4 years of follow up [9]. In another study, only 8% of patients had some improvement after a follow-up of 0.5 to 3 years [10]. Corneal effects attributed to cGVHD are due to a direct effect of the GVHD, to the effect of KCS, or to lid position abnormalities. Corneal epithelial sloughing is a devastating complication of ocular cGVHD characterized by increased pain, decrease in vision and severe photophopia. Other corneal abnormalities

Chapter 33  Chronic Graft-Versus-Host Disease 

identified include peripheral corneal neovascularization, punctuate staining, keratitis, keratinization, corneal perforation [11], infectious keratitis [12], and corneal scarring. Corneal epithelial thinning present in 75% of patients following SCT with no relation to GVHD, epithelial irregularity, and epithelial dysplasia have been attributed to the ablative conditioning regimen [12, 13]. Cataracts occur in approximately 11% of patients following SCT [12]. Their occurrence has been linked to various risk factors including total body irradiation, steroid usage and GVHD [10, 13–15]. 3.6. Genitalia Diagnostic features include lichen planus-like features and vaginal scarring or stenosis. Vaginal cGVHD often correlates with presence of oral GVHD. 3.7. Gastrointestinal Tract Diagnostic features include esophageal web, stricture or concentric rings as documented by endoscopy or other imaging modalities. Common features include anorexia, nausea, vomiting, diarrhea weight loss and failure to thrive. Some patients can present with a wasting syndrome. 3.8. Liver Liver GVD can present as cholestasis with liver function test abnormalities, and not uncommonly present as a hepatitis. Liver biopsy typically needs to be done to rule out other causes of liver function tests abnormality in a transplant patient. Liver GVHD by itself is not diagnostic of cGVHD and requires distinctive manifestation in at least one other organ. The degree of hyperbilrubinemia and clinical outcome is not as linear as in acute GVHD [6]. A hepatitic form of cGVHD occurs and needs to be differentiated from viral hepatitis after SCT [16]. Patients are usually asymptomatic until advanced stages of liver GVHD. Portal hypertension, cirrhosis and hepatic failure are all rare. 3.9. Lungs The only diagnostic feature of cGVHD in the lung is biopsy proven bronchiolitis obliterans (BO). BO that is diagnosed based on pulmonary function testing or imaging requires presence of a distinctive feature in a separate organ system to establish the diagnosis of cGVHD. Patients with BO can be asymptomatic but typically present with dyspnea, cough, wheezing and in advanced cases have recurrent pneumothoraces, pneumomediastinum, and subcutaneous emphysema. Pulmonary function test and imaging criteria to diagnose BO have been proposed by the NIH consensus recommendation. 3.10. Musculoskeletal System Diagnostic features include fascial involvement, joint stiffness or contractures as a result of fascial involvement near joints. Clinical myositis with tender muscles and elevated muscle enzymes is considered a distinctive feature. Patients can present with arthralgias and arthritis, but are uncommon and often associated with autoantibodies. Muscle cramps are often seen in patients with cGVHD, but the exact etiology remains undefined.

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3.11. Hematopoietic and Immune System cGVHD is often associated with cytopenias, which could be autoimmune in origin or related to stromal damage. Lymphopenia, esoinophilia, hypogammaglobulinemia or hypergammaglobulinemia have been seen in the context of cGVHD. Autoimmune hemolytic anemia, or idiopathic thrombocytopenic purpura have been associated with cGVHD. 3.12. Other cGVHD has been linked to other rare manifestation like serositis (pleural or pericardial effusions or ascites), peripheral neuropathy, myasthenia gravis, nephritic syndrome, and cardiac involvement, typically a coronary arteritis. These manifestations are rare and cannot be considered as diagnostic or distinctive. Most of these are based on immune mechanisms of injury at these target organs. The various organ systems are scored by severity and a composite score, which is proposed to be reflective of global functioning is then calculated (Table 33-2) [5]. cGVHD is scored as mild, moderate or severe. Patients with moderate or severe cGVHD in general should be considered for systemic therapy for cGVHD.

4. Risk Factors for Development and Outcome of cGVHD Historically, the incidence of cGVHD (any GVHD after day 100 after SCT) has been in the range of 40–60% [17]. The incidence depends on donor status (HLA identical sibling vs. matched or mismatched unrelated), and source of stem cells (peripheral blood stem cells vs. marrow stem cells). In a review by Przepiorka et al., the incidence of limited and extensive cGVHD was 6% and 71% after HLA identical sibling transplant [18]. Recent data from the National Marrow Donor Program (NMDP) indicate that in patients surviving upto day 100 after SCT, the incidence of cGVHD is 70%. In the light of the recent NIH classification, Jagasia et al. have reported a GVHD incidence of 67% (73 of 110 patients) after day 100 from SCT [19]. Of these 73 patients, 46 patients (62%) fulfilled criteria of cGVHD as defined by the NIH consensus criteria. The remaining 27 patients had aGVHD after day 100. Understanding the risk factors that predict for the development and outcome of cGVHD is important as it can allow for risk-stratified approach for GVHD prophylaxis in the future. It is important to remember that most risk factor analysis continue to use the historical definition of cGVHD (i.e., any GVHD after day 100). Development of aGVHD remains probably the greatest risk factor for cGVHD. In one study, history of aGVHD and continued use of steroids at day 100 was associated with relative risk (RR) of 3.9 for the development of cGVHD [20]. As the HLA disparity increases, the risk of aGVHD increases [21–23]. The impact of HLA disparity directly on cGVHD is not clear and may be mediated through the development of aGVHD. The incidence of cGVHD increases with recipient age [21–23]. The impact of recipient age on cGVHD incidence in an unrelated donor SCT is less clear. Impact of donor age on cGVHD remains controversial [24, 25]. The use of female donors for male recipients has been associated with an increased risk of cGVHD but also

Chapter 33  Chronic Graft-Versus-Host Disease 

585

Table 33-2.  Severity and scoring of chronic GVHD. Performance Score 0

Asymptomatic and fully active (ECOG 0; KPS or LPS 100%)

Score 1

Symptomatic, fully ambulatory, restricted only in physically strenuous activity (ECOG 1, KPS or LPS 80–90%)

Score 2

Symptomatic, ambulatory, capable of self-care, >50% of waking hours out of bed (ECOG 2, KPS or LPS 60–70%)

Score 3

Symptomatic, limited self-care, >50% of waking hours in bed (ECOG 3–4, KPS or LPS < 60%)

Skin

Clinical features: Maculopapular rash, lichen planus-like features, papulosquamous lesions or ichthyosi, hyperpigmentation, hypopigmentation, keratosis pilaris, erythema, erythrodema, poikiloderma, sclerotic features, Pruritus, hair involvement, nail involvement

Score 0

No symptoms

Score 1

<18% BSA with disease signs but NO sclerotic features

Score 2

19–50% BSA OR involvement with superficial sclerotic features “not hidebound” (able to pinch)

Score 3

>50% BSA OR deep sclerotic features “hidebound” (unable to pinch) OR impaired mobility, ulceration or severe pruitus

Mouth Score 0

No symptoms

Score 1

Mild symptoms with disease signs but not limiting oral intake significantly

Score 2

Moderate symptoms with disease signs with partial limitation of oral intake

Score 3

Severe symptoms with disease signs on examination with major limitation of oral intake

Eyes Score 0

No symptoms

Score 1

Mild dry eye symptoms not affecting ADL (requiring eyedrops £3x per day) OR asymptomatic signs of keratoconjunctivitis sicca

Score 2

Moderated dry eye symptoms partially affecting ADL (requiring drops >3x per day or punctual plugs), WITHOUT vision impairment

Score 3

Severe dry eye symptoms significantly affecting ADL (special eyewear to relieve pain) OR unable to work because of ocular symptoms OR loss of vision caused by keratoconjunctivitis sicca

GI tract Score 0

No symptoms

Score 1

Symptoms such as dysphagia, anorexia, nausea, vomiting, abdominal pain or diarrhea without significant weight loss (<5%)

Score 2

Symptoms associated with mild to moderate weight loss (5–15%)

Score 3

Symptoms associated with significant weight loss >15%, requires nutritional supplement for most calorie needs OR esophageal dilation (continued)

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Table 33-2.  (continued) Liver Score 0

Normal LFT

Score 1

Elevated Bilirubin, AP*, AST or ALT < 2x ULN

Score 2

Bilirubin >3 mg/dl or Bilirubin, enzymes 2–5x ULN

Score 3

Bilirubin or enzymes. 5x ULN

Lungs Score 0

No symptoms

FEV1 > 80% OR LFS = 2

Score 1

Mild symptoms (shortness of breath after climbing one flight of steps)

FEV1 60–79% OR LFS 3–5

Score 2

Moderate symptoms (shortness of breath after walking on flat ground)

FEV1 40–59% OR LFS 6–9

Score 3

Severe symptoms (shortness of breath at rest; require 02)

FEV1 £ 39% OR LFS 10–12

Joints and fascia Score 0

No symptoms

Score 1

Mild tightness of arms or legs, normal or mild decreased range of motion (ROM) AND not affecting ADL

Score 2

Tightness of arms or legs OR joint contractures, erythema thought due to fasciitis, moderate decrease ROM AND mild to moderate limitation of ADL

Score 3

Contractures WITH significant decrease of ROM AND significant limitation of ADL (unable to tie shoes, button shirts, dress self etc.)

Genital tract Score 0

No symptoms

Score 1

Symptomatic with mild signs on exam AND no effect on coitus and minimal discomfort with gynecologic exam

Score 2

Symptomatic with moderate signs on exam AND with mild dyspareunia or discomfort with gynecologic exam

Score 3

Symptomatic WITH advanced signs (stricture, labial agglutination or severe ulceration) AND severe pain with coitus or inability to insert vaginal speculum

a lower relapse rate. The use of peripheral blood stem cell transplantation (PBSCT) has had a significant impact on development of cGVHD. Although initial reports did not support this [26, 27], subsequent reports including a meta-analysis clearly show that PBSCT is associated with a higher incidence of cGVHD [28]. Patients developing cGVHD after PBSCT require longer duration of immunosuppressive therapy compared to patients developing cGVHD after marrow SCT [29]. In HLA-identical peripheral blood SCT, higher cell dose (>8 × 106 CD34+ cells/kg) has been associated with increased extensive cGVHD (RR 2.3) [30, 31]. In contrast higher CD34 cell dose after marrow SCT is associated with less cGVHD [32].

Chapter 33  Chronic Graft-Versus-Host Disease 

Skin biopsies in absence of overt GVHD are done at many transplant centers as part of the day 100 evaluation. Histologic presence of GVHD in absence of clinical features at day 100 is not predictive of clinical cGVHD, although some might argue to monitor these patients more closely for development of cGVHD. Biomarkers have been studied in new onset extensive cGVHD and compared to time-controlled controls without cGVHD. Soluble BAFF (sBAFF), anti-dsDNA antibody soluble IL-2 receptor alpha (sIL-2Ra), and soluble CD13 (sCD13) were elevated in early GVHD compared to controls. sBAFF and anti-dsDNA were elevated in late onset GVHD suggesting that pathogenesis of early and late cGVHD may be different [33, 34]. Candidate gene polymorphisms have been studied for various cytokine and non-cytokine genes to predict aGVHD [35]. Recently, recipient FCRL3169c/G genotype has been associated with a lower risk of cGVHD compared to recipients without this phenotype [36]. As FCRL3 is expressed on B-cells, this raises the question regarding the role of B-cells in cGVHD. IL-10 promoter polymorphism has been studied as a predictor of cGVHD. Patients with ATA/ATA haplotype had a sevenfold higher risk of developing cGVHD compared with other haplotypes. The duration of immunosuppressive therapy was significantly shorter for the ATA/ATA haplotype compared to others (339 days vs. 1,146 days, P = 0.0091) [37]. Risk factor analyses have been done to predict survival after the development of cGVHD. As part of the original description of the limited/extensive cGVHD classification system, patients were classified into three groups based on morbidity and mortality (group I: moderate or resolved disease, alive; Group II: severe disease, alive; and Group III: severe disease, dead) [1]. The authors did make an observation that among patients with extensive cGVHD, the Karnofsky performance status score was the best prognosticator of outcome. Due to lack of other classification systems, this limited/extensive cGVHD classification has been in use since its development, realizing the limited prognostic ability for both relapse and non-relapse mortality. Subsequent studies identified progressive presentation, bilirubin elevation, lichenoid changes of skin histology [38], platelets less than 100 × 109/L [39], and prior steroid refractory or dependent acute GVHD as markers for poor outcome. The next major attempt to develop a prognostic model was made by Lee et al. [17] using survival data on more than a 1,000 patients above the age of 16 years from the International Bone Marrow Transplant Registry (IBMTR) who underwent non-T cell depleted transplant from a Human Leukocyte Antigen (HLA) identical sibling or a matched unrelated donor (matched at HLA-A, HLA-B and HLA-DR by serologic or molecular testing). All patients in this cohort received cyclosporine and methotrexate for GVHD prophylaxis and were disease free for at least 100 days after SCT. A new risk scale was developed and patients were stratified into different groups based on Karnofsky performance status (KPS), chronic diarrhea, weight loss, skin involvement and oral involvement. Patients were classified as low risk (KPS ³ 80%, no weight loss or chronic diarrhea), intermediate risk (KPS ³ 80% with either weight loss or chronic diarrhea or KPS < 80% with or without oral involvement), high risk (KPS ³ 80% with both chronic diarrhea and weight loss or KPS < 80% with chronic diarrhea, weight loss and skin involvement or KPS < 80% with oral involvement but with one or two other features). All analyses to predict

587

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overall survival (OS) and transplant-related mortality (TRM) were adjusted for disease type, stage, recipient age, sex-matching and prior aGVHD. These risk categories were able to predict OS (low and intermediate risks) and TRM (low and intermediate risks) but were not prognostic for relapse. A risk stratification system based on a retrospective review of 150 patients referred for GVHD to the Johns Hopkins Center was developed by Akpek et al. [40]. The most common sites of GVHD involvement (skin and fascia, mouth and eye involvement, performance status, weight loss and infection within 1 month prior to or after diagnosis of cGVHD or primary treatment failure) were coded on a scale of 1 to 3 indicating the extent and severity of cGVHD. The two major statistical outcomes studied were GVHD specific survival from onset of cGVHD and from time of primary treatment failure. According to multivariate analyses, extensive skin involvement in more than 50% body surface area (HR 7.0), thrombocytopenia (platelets < 100 × 109/L) (HR 3.6) and progressive-type onset (HR 1.7) significantly influenced GVHD specific survival. These three factors and Karnofsky performance status influenced survival form primary treatment failure. Patients were divided into three categories depending on their prognostic factor score. Patients with no adverse prognostic factor (score 0) had a 10-year GVHD specific survival of 82%. Patient with prognostic factor score less than 2 (extended skin involvement only or thrombocytopenia and/or progressive type onset) had a 68% 10-year GVHD specific survival. Patients with prognostic factor score of 2 to 3.5 (extended skin involvement and either thrombocytopenia or progressive type onset) had a 34% 10-year GVHD specific survival. Patients with a prognostic factor of higher than 3.5 (presence of all three factors) had a 3% 10-year GVHD specific survival. Similarly, the 5-year survival from primary treatment failure for prognostic factor scores of 0, less than 2, 2 to 3.5 and higher than 3.5 were 91%, 71%, 22% and 4%, respectively. Jagasia et  al. have shown that patients with any acute feature of GVHD (accounting for subtype changes) after day 100 (including late acute, recurrent acute, persistent acute, overlap GVHD) had an inferior survival when compared to classic chronic GVHD (HR 3.36) [19].

5. Impact of GVHD Prophylaxis on cGVHD As aGVHD is the strongest predictor for cGVHD, it is logical to assume that decreasing aGVHD translates into a lower incidence of cGVHD. Unfortunately, this has not been borne out in clinical practice [41–44]. Investigators have speculated on the duration of post-transplant immunosuppression to decrease cGVHD, but studies randomizing patients to 6 or 24 months of cyclosporine have shown no difference in cGVHD incidence [45]. In a study investigating 6 vs. 12 months of cyclosporine after allogeneic PBSCT, the incidence of extensive cGVHD was decreased in the 12-month group, but this did not translate into superior overall of disease free survival [46]. Surprisingly, ex vivo T cell deletion has not translated into a decrease incidence of cGVHD [32, 47]. Use of anti-thymocyte globulin (ATG) at higher doses for GVHD prophylaxis translated into a lower incidence of cGVHD (62% ATG vs. 39% no ATG; P = 0.04). There were no differences in overall survival, cumulative incidence of transplant-related mortality between the two groups [48].

Chapter 33  Chronic Graft-Versus-Host Disease 

6. Treatment of cGVHD Advances in supportive care have reduced morbidity associated with cGVHD, but however, true advances in treatment of cGVHD have been only minimal since the 1980s [2]. A variety of causes including the heterogeneity of cGVHD with varying natural history, and lack of validated response criteria have hampered the progress of clinical trials for cGVHD. Many aspects of cGVHD are not reversible (e.g., dry eyes, advanced sclerodermatous fascitis) and may lead to misinterpretation of clinical trial results. The end point of a cGVHD clinical trial should encompass not only response, but time to stopping steroid, time to stopping all immunosuppressive therapy and patient experience. Biostatistical analysis of cGVHD clinical trials should incorporate considerations like competing events, concomitant therapy, appropriate power calculations, interim analyses and missing measurements. A comprehensive review of various problems with cGVHD clinical trials has been addressed by the NIH cGVHD Working Group [49, 50]. Patient education and infection prophylaxis are integral part of cGVHD management. Prophylaxis against P carinii should be given to all cGVHD patients for 6 months after stopping immunosuppression. All patients on cGVHD treatment are recommended to be on prophylaxis against encapsulated bacteria. Standard antibiotic prophylaxis for dental invasive procedures is also recommended. Patients on systemic steroids should be monitored for CMV reactivation and appropriately treated. Varicella prophylaxis should be recommended if immunosuppression is still needed to control cGVHD. Many patients with cGVHD have hyopgammaglobulinemia, and often are given intravenous IgG if levels are less than 400 mg/dL. Vaccination should be done in accordance with Centers for Disease Control guidelines [51].

Table 33-3.  Risk factors in chronic GVHD. Risk factors for development of cGVHD Donor status (related vs. unrelated) Recipient age Stem cell source (marrow vs. peripheral blood) Acute GVHD Female donor to male recipient FCRL3 genotype IL10 promoter polymorphism Risk factors predicting outcome of cGVHD Progressive presentation Bilirubin elevation (>3 mg/dL) Thrombocytopenia (<100 × 109/L) Prior steroid refractory or steroid dependent acute GVHD Risk status by Lee scale Akpek score NIH classification of cGVHD (classic GVHD associated with better outcome)

589

Calcineurin inhibitor

Photoactivation and subsequent apoptosis of blood mononuclear cells, immunomodulation

mTOR inhibitor, antifibrotic

Nucleoside analog

Monclonal chimeric anti CD20 antibody

Anti-malarial agent

Tacrolimus [57, 58]

Extracorporeal Photophersis (ECP) [59]

Sirolimus [60]

Pentostatin [61]

Rituximab [62–64]

Hydroxychloroquine [65]

Photoactivation of mononuclear cells infiltrating the skin

Polyclonal antibody

Humanized anti IL2 receptor monoclonal antibody

Chimeric TNF alpha monocolonal antibody

Recombinant human soluble TNF receptor fusion protein

Psoralen and UVA [67]

Anti-thymocyte globulin [68]

Daclizumab

Infliximab

Etanercept

Less commonly used

High dose pulse steroids [66] Coticosteroid, lympholytic

De novo pathway purine synthesis inhibitor

Class/mechanism

Mycophenolate mofetil [55, 56]

Commonly used

Agent (reference)

Table 33-4.  Salvage therapy for chronic GVHD.

Thrombotic microangiopathy, central nervous system

Gastrointestinal bleeding, leucopenia, infection

Serious side effects

Infections

Hypersensitivity

Gastrointestinal, dizziness, headache, insomnia, fatigue

Infections

Nausea and phototoxicity

Numerous

Gastrointestinal symptoms

Infusional reaction

Nausea, vomiting

Hyperlipidemia, renal insufficiency, cytopenia, infection

Serious interaction with posaconazole, higher incidence of thrombotic angiopathy when combined with other calineurin inhibitors

High rate of response in salvage trials. Different subtypes of GVHD may have different responses

Use often instead of cyclosporine, but limited success when changing from cyclosporine to tacrolimus

Common first agent. Efficacy results vary. Randomized study underway

Efficacy/comments

Infections

Infections

Infections

Anaphylaxis, serum sickness

Non-melanoma skin cancers

Myopathy, infections

Retinal damage, myositis, psychosis

Infections

Few case reports to show efficacy. May be more effective in non-infectious lung injury

Efficacy in GI GVHD, unusual infections with mycobacteria

Few case reports

Not much data for efficacy in chronic GVHD

Best for skin GVHD, no systemic immunosuppressive effect

Used when rapid response needed

Need annual eye exam. Mostly used as adjuvant.

Best response seen in skin and musculoskeletal systems

Infections, renal insuf- Requires IV hydration pre- and post-adminficiency, central nerv- istration ous system

Thrombotic microangiopathy

Logistics of frequent Line infection, clots pheresis, requirement and malfunction of venous access

Renal insufficiency, nausea

Gastrointestinal

Common side effects

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Chapter 33  Chronic Graft-Versus-Host Disease 

The cornerstone of cGVHD therapy in patients who are deemed to be candidates for systemic immunosuppression is prednisone along with either cyclosporine (CSA) or tacrolimus. A randomized study showed that single agent prednisone is superior to prednisone plus azathioprine [39]. Patients with high risk cGVHD (platelets count < 100 × 109/L) had a 26% 5-year survival [2]. A subsequent study treating high risk group of cGVHD showed that alternate day CSA and prednisone increased the 5-year survival to greater than 50%. Most centers currently start prednisone at 1  mg/kg/day for 1–2 weeks and after initial evidence of response, start a weekly alternate day taper until patients are on 1  mg/kg/every other day. CSA or tacrolimus are continued daily and tapered after steroids are discontinued or decreased to the physiologic replacement dose [52]. If a patient has complete resolution of symptoms, a subsequent taper is started 9 months later with dose reductions every 2 weeks. If a patient has not responded by month 3 or has progression at any time, salvage therapies are needed [53]. Long-term outcome of this treatment strategy suggests that immunosuppression can be discontinued in 60% of standard risk patients and 40% of high-risk patients [29]. A recent study looked at outcome of standard risk (no thrombocytopenia) cGVHD patients when randomized between prednisone or prednisone plus CSA. The cumulative incidences of transplant related mortality, survival, and relapse and need for salvage therapy showed no significant difference between two arms [54]. Thus, continued clinical trials are needed. A large study organized by the Fred Hutchinson Cancer Research Center is studying the role of mycophenolate moefitil when added to prednisone plus CSA in a randomized double blind study. If a patient has no response to initial prednisone-based therapy or has progression of cGVHD or has recurrent flare of systemic cGVHD, salvage therapies are warranted. The first option should be a clinical trial as there is no accepted standard salvage therapy. Table 33-3 outlines the various strategies that have been tried for salvage therapy of cGVHD (Table 33-4). Organ-specific multidisciplinary team approach is ideal in taking care of the complex cGVHD patient. Access to dermatologists, pulmonary specialists, infectious disease consultants, ophthalmologists (specializing in dry eyes/cornea), gynecologists, internists, physical therapists, nutrionists and social workers are essential in treating cGVHD and maintaining the patient’s quality of life.

7. Summary cGVHD continues to be a major obstacle to the successful outcome of allogeneic SCT, with an impact not only on survival but quality of life. Progress in clinical classification, staging and grading has established a common platform. Further progress in identification of accurate biologic and molecular predictors, which are modifiable, will be essential to impact on patient outcome. Advances in non-steroid immunosuppressive therapy with better understanding of mechanism of action are urgently needed. A focused effort on collaborative clinical trials for high risk cGVHD with appropriate surrogate end-points should be undertaken to move therapeutics in cGVHD forward and in the right direction.

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References 1. Shulman HM, Sullivan KM, Weiden PL et  al (1980) Chronic graft-versus-host syndrome in man. A long-term clinicopathologic study of 20 Seattle patients. Am J Med 69:204–217 2. Sullivan KM, Witherspoon RP, Storb R et al (1988) Alternating-day cyclosporine and prednisone for treatment of high-risk chronic graft-v-host disease. Blood 72:555–561 3. Graze PR, Gale RP (1979) Chronic graft versus host disease: a syndrome of disordered immunity. Am J Med 66:611–620 4. Lee SJ, Vogelsang G, Flowers ME (2003) Chronic graft-versus-host disease. Biol Blood Marrow Transplant 9:215–233 5. Filipovich AH, Weisdorf D, Pavletic S et  al (2005) National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versushost disease: I. Diagnosis and staging working group report. Biol Blood Marrow Transplant 11:945–956 6. Sullivan KM, Shulman HM, Storb R et al (1981) Chronic graft-versus-host disease in 52 patients: adverse natural course and successful treatment with combination immunosuppression. Blood 57:267–276 7. Shulman HM, Sale GE, Lerner KG et  al (1978) Chronic cutaneous graft-versushost disease in man. Am J Pathol 91:545–570 8. Imanguli MM, Pavletic SZ, Guadagnini JP, Brahim JS, Atkinson JC (2006) Chronic graft versus host disease of oral mucosa: review of available therapies. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 101:175–183 9. Ogawa Y, Yamazaki K, Kuwana M et  al (2001) A significant role of stromal fibroblasts in rapidly progressive dry eye in patients with chronic GVHD. Invest Ophthalmol Vis Sci 42:111–119 10. Bray LC, Carey PJ, Proctor SJ, Evans RG, Hamilton PJ (1991) Ocular complications of bone marrow transplantation. Br J Ophthalmol 75:611–614 11. Hirst LW, Jabs DA, Tutschka PJ, Green WR, Santos GW (1983) The eye in bone marrow transplantation. I. Clinical study. Arch Ophthalmol 101:580–584 12. Jabs DA, Hirst LW, Green WR et al (1983) The eye in bone marrow transplantation. II. Histopathology. Arch Ophthalmol 101:585–590 13. Jabs DA (1996) Ocular complications of bone marrow transplantation. In: Pepose JS, Holland GN, Wilhelmus KR (eds) Ocular infection and immunity. Mosby, St. Louis, pp 426–434 14. Franklin RM, Kenyon KR, Tutschka PJ et al (1983) Ocular manifestations of graftvs-host disease. Ophthalmology 90:4–13 15. Tichelli A, Gratwohl A, Egger T et al (1993) Cataract formation after bone marrow transplantation. Ann Intern Med 119:1175–1180 16. Strasser SI, Shulman HM, Flowers ME et al (2000) Chronic graft-versus-host disease of the liver: presentation as an acute hepatitis. Hepatology 32:1265–1271 17. Lee SJ, Klein JP, Barrett AJ et  al (2002) Severity of chronic graft-versushost disease: association with treatment-related mortality and relapse. Blood 100:406–414 18. Przepiorka D, Anderlini P, Saliba R et al (2001) Chronic graft-versus-host disease after allogeneic blood stem cell transplantation. Blood 98:1695–1700 19. Jagasia M, Giglia J, Chinratanalab W et  al (2007) Incidence and Outcome of Chronic Graft-versus-Host Disease Using National Institutes of Health Consensus Criteria. Biol Blood Marrow Transplant 13(10):1207–1215 20. Wagner JL, Flowers ME, Longton G et al (1998) The development of chronic graftversus-host disease: an analysis of screening studies and the impact of corticosteroid use at 100 days after transplantation. Bone Marrow Transplant 22:139–146

Chapter 33  Chronic Graft-Versus-Host Disease  21. Storb R, Prentice RL, Sullivan KM et  al (1983) Predictive factors in chronic graft-versus-host disease in patients with aplastic anemia treated by marrow transplantation from HLA-identical siblings. Ann Intern Med 98:461–466 22. Atkinson K, Horowitz MM, Gale RP et  al (1990) Risk factors for chronic graftversus-host disease after HLA-identical sibling bone marrow transplantation. Blood 75:2459–2464 23. Ochs LA, Miller WJ, Filipovich AH et  al (1994) Predictive factors for chronic graft-versus-host disease after histocompatible sibling donor bone marrow transplantation. Bone Marrow Transplant 13:455–460 24. Kollman C, Howe CW, Anasetti C et al (2001) Donor characteristics as risk factors in recipients after transplantation of bone marrow from unrelated donors: the effect of donor age. Blood 98:2043–2051 25. Ratanatharathorn V, Ayash L, Lazarus HM, Fu J, Uberti JP (2001) Chronic graftversus-host disease: clinical manifestation and therapy. Bone Marrow Transplant 28:121–129 26. Bensinger WI, Clift R, Martin P et al (1996) Allogeneic peripheral blood stem cell transplantation in patients with advanced hematologic malignancies: a retrospective comparison with marrow transplantation. Blood 88:2794–2800 27. Schmitz N, Bacigalupo A, Labopin M et  al (1996) Transplantation of peripheral blood progenitor cells from HLA-identical sibling donors. European Group for Blood and Marrow Transplantation (EBMT). Br J Haematol 95:715–723 28. Cutler C, Giri S, Jeyapalan S et al (2001) Acute and chronic graft-versus-host disease after allogeneic peripheral-blood stem-cell and bone marrow transplantation: a meta-analysis. J Clin Oncol 19:3685–3691 29. Flowers ME, Parker PM, Johnston LJ et  al (2002) Comparison of chronic graftversus-host disease after transplantation of peripheral blood stem cells versus bone marrow in allogeneic recipients: long-term follow-up of a randomized trial. Blood 100:415–419 30. Perez-Simon JA, ez-Campelo M, Martino R et al (2003) Impact of CD34+ cell dose on the outcome of patients undergoing reduced-intensity-conditioning allogeneic peripheral blood stem cell transplantation. Blood 102:1108–1113 31. Zaucha JM, Gooley T, Bensinger WI et al (2001) CD34 cell dose in granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cell grafts affects engraftment kinetics and development of extensive chronic graft-versushost disease after human leukocyte antigen-identical sibling transplantation. Blood 98:3221–3227 32. Pavletic SZ, Carter SL, Kernan NA et  al (2005) Influence of T-cell depletion on chronic graft-versus-host disease: results of a multicenter randomized trial in unrelated marrow donor transplantation. Blood 106:3308–3313 33. Fujii H, Cuvelier G, She K et al (2008) Biomarkers in newly diagnosed pediatric extensive chronic graft-versus-host disease: a report from the Children's Oncology Group. Blood 111(6):3276–85 34. Sarantopoulos S, Stevenson KE, Kim HT et  al (2007) High Levels of B-Cell Activating Factor in Patients with Active Chronic Graft-Versus-Host Disease. Clin Cancer Res 13:6107–6114 35. Dickinson AM, Middleton PG, Rocha V, Gluckman E, Holler E (2004) Genetic polymorphisms predicting the outcome of bone marrow transplants. Br J Haematol 127:479–490 36. Shimada M, Onizuka M, Machida S et  al (2007) Association of autoimmune disease-related gene polymorphisms with chronic graft-versus-host disease. Br J Haematol 139:458–463 37. Kim DH, Lee NY, Sohn SK et al (2005) IL-10 promoter gene polymorphism associated with the occurrence of chronic GVHD and its clinical course during systemic

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M. Jagasia and S. Pavletic immunosuppressive treatment for chronic GVHD after allogeneic peripheral blood stem cell transplantation. Transplantation 79:1615–1622 38. Wingard JR, Piantadosi S, Vogelsang GB et  al (1989) Predictors of death from chronic graft-versus-host disease after bone marrow transplantation. Blood 74:1428–1435 39. Sullivan KM, Witherspoon RP, Storb R et al (1988) 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 72:546–554 40. Akpek G, Zahurak ML, Piantadosi S et  al (2001) Development of a prognostic model for grading chronic graft-versus-host disease. Blood 97:1219–1226 41. Storb R, Deeg HJ, Pepe M et  al (1989) Methotrexate and cyclosporine versus cyclosporine alone for prophylaxis of graft-versus-host disease in patients given HLA-identical marrow grafts for leukemia: long-term follow-up of a controlled trial. Blood 73:1729–1734 42. Storb R, Deeg HJ, Farewell V et al (1986) Marrow transplantation for severe aplastic anemia: methotrexate alone compared with a combination of methotrexate and cyclosporine for prevention of acute graft-versus-host disease. Blood 68:119–125 43. Chao NJ, Snyder DS, Jain M et al (2000) Equivalence of 2 effective graft-versushost disease prophylaxis regimens: results of a prospective double-blind randomized trial. Biol Blood Marrow Transplant 6:254–261 44. Cutler C, Antin JH (2004) Sirolimus for GVHD prophylaxis in allogeneic stem cell transplantation. Bone Marrow Transplant 34:471–476 45. Kansu E, Gooley T, Flowers ME et al (2001) Administration of cyclosporine for 24 months compared with 6 months for prevention of chronic graft-versus-host disease: a prospective randomized clinical trial. Blood 98:3868–3870 46. Mengarelli A, Iori AP, Romano A et al (2003) One-year cyclosporine prophylaxis reduces the risk of developing extensive chronic graft-versus-host disease after allogeneic peripheral blood stem cell transplantation. Haematologica 88:315–323 47. Wagner JE, Thompson JS, Carter SL, Kernan NA (2005) Effect of graft-versus-host disease prophylaxis on 3-year disease-free survival in recipients of unrelated donor bone marrow (T-cell Depletion Trial): a multi-centre, randomised phase II–III trial. Lancet 366:733–741 48. Bacigalupo A, Lamparelli T, Barisione G et  al (2006) Thymoglobulin prevents chronic graft-versus-host disease, chronic lung dysfunction, and late transplantrelated mortality: long-term follow-up of a randomized trial in patients undergoing unrelated donor transplantation. Biol Blood Marrow Transplant 12:560–565 49. Martin PJ, Gooley T, Anasetti C, Petersdorf EW, Hansen JA (1998) HLAs and risk of acute graft-vs.-host disease after marrow transplantation from an HLA-identical sibling. Biol Blood Marrow Transplant 4:128–133 50. Martin PJ, Nash RA (2006) Pitfalls in the design of clinical trials for prevention or treatment of acute graft-versus-host disease. Biol Blood Marrow Transplant 12:31–36 51. Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients. (2000) Biol Blood Marrow Transplant 6:659–713 52. Flowers ME, Lee S, Vogelsang G (2003) An update on how to treat chronic GVHD. Blood 102:2312 53. Vogelsang GB (2001) How I treat chronic graft-versus-host disease. Blood 97:1196–1201 54. Koc S, Leisenring W, Flowers ME et al (2002) Therapy for chronic graft-versushost disease: a randomized trial comparing cyclosporine plus prednisone versus prednisone alone. Blood 100:48–51 55. Mookerjee B, Altomonte V, Vogelsang G (1999) Salvage therapy for refractory chronic graft-versus-host disease with mycophenolate mofetil and tacrolimus. Bone Marrow Transplant 24:517–520

Chapter 33  Chronic Graft-Versus-Host Disease  56. Busca A, Saroglia EM, Lanino E et al (2000) Mycophenolate mofetil (MMF) as therapy for refractory chronic GVHD (cGVHD) in children receiving bone marrow transplantation. Bone Marrow Transplant 25:1067–1071 57. Tzakis AG, bu-Elmagd K, Fung JJ et  al (1991) FK 506 rescue in chronic graftversus-host-disease after bone marrow transplantation. Transplant Proc 23: 3225–3227 58. Carnevale-Schianca F, Martin P, Sullivan K et al (2000) Changing from cyclosporine to tacrolimus as salvage therapy for chronic graft-versus-host disease. Biol Blood Marrow Transplant 6:613–620 59. Couriel DR, Hosing C, Saliba R et al (2006) Extracorporeal photochemotherapy for the treatment of steroid-resistant chronic GVHD. Blood 107:3074–3080 60. Couriel DR, Saliba R, Escalon MP et al (2005) Sirolimus in combination with tacrolimus and corticosteroids for the treatment of resistant chronic graft-versus-host disease. Br J Haematol 130:409–417 61. Jacobsohn DA, Chen AR, Zahurak M et  al (2007) Phase II study of pentostatin in patients with corticosteroid-refractory chronic graft-versus-host disease. J Clin Oncol 25:4255–4261 62. Zaja F, Bacigalupo A, Patriarca F et  al (2007) Treatment of refractory chronic GVHD with rituximab: a GITMO study. Bone Marrow Transplant 40:273–277 63. Cutler C, Miklos D, Kim HT et al (2006) Rituximab for steroid-refractory chronic graft-versus-host disease. Blood 108:756–762 64. Ratanatharathorn V, Ayash L, Reynolds C et al (2003) Treatment of chronic graftversus-host disease with anti-CD20 chimeric monoclonal antibody. Biol Blood Marrow Transplant 9:505–511 65. Gilman AL, Chan KW, Mogul A et al (2000) Hydroxychloroquine for the treatment of chronic graft-versus-host disease. Biol Blood Marrow Transplant 6:327–334 66. Akpek G, Lee SM, Anders V, Vogelsang GB (2001) A high-dose pulse steroid regimen for controlling active chronic graft-versus-host disease. Biol Blood Marrow Transplant 7:495–502 67. Vogelsang GB, Wolff D, Altomonte V et  al (1996) Treatment of chronic graftversus-host disease with ultraviolet irradiation and psoralen (PUVA). Bone Marrow Transplant 17:1061–1067 68. Arai S, Margolis J, Zahurak M, Anders V, Vogelsang GB (2002) Poor outcome in steroid-refractory graft-versus-host disease with antithymocyte globulin treatment. Biol Blood Marrow Transplant 8:155–160

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Chapter 34 Post-transplant Lymphoproliferative Disorder Ran Reshef, Alicia K. Morgans, and Donald E. Tsai

1. Introduction The increased risk of malignancy, especially of lymphoid tumors, in solid-organ and hematopoietic stem cell transplant (HST) recipients has been recognized for many years [1–3]. Post-transplant lymphoproliferative disorder (PTLD) represents a heterogeneous group of abnormal lymphoid proliferations, generally of B-cell origin, that occur in the setting of ineffective T-cell function due to pharmacologic immunosuppression after organ transplantation. Unlike most other forms of non-Hodgkin’s lymphoma, nearly all PTLD is associated with Epstein–Barr virus (EBV) infection, as manifested by the presence of EBV within the malignant tissue.

2. Definition and Classification The term PTLD refers to a spectrum of B-cell hyperproliferation ranging from benign conditions similar to infectious mononucleosis and polyclonal lymphoid hyperplasia to monoclonal neoplasms, such as B-cell and occasionally T-cell lymphomas [4]. These conditions differ clinically and morphologically and carry different prognoses. Therefore, a number of attempts have been made to classify this disorder based on morphology, clonality and disruption of nodal architecture [5, 6]. A formal classification was established by two international consensus groups in 1999 [7], determining that the term PTLD may encompass the full range of EBV-related lymphoproliferative states, although the benign forms should be segregated and subclassification should be attempted in each case (see Table 34-1). Further classification of monomorphic PTLD is currently done according to the WHO or REAL classification system for lymphomas [8–10].

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_34, © Springer Science + Business Media, LLC 2003, 2010

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Table 34-1.  WHO Classification of PTLD. Subtype

Classification

Characteristic findings

1

Early/benign PTLD

Plasmacytic or atypical lymphoid hyperplasiaInfectious mononucleosis-like syndrome • Nodal disease with preservation of lymph node architecture • <3 months post-transplantation • Polyclonal

2

Polymorphic PTLD

• Nodal disease with effacement of lymph node architecture or extranodal disease • Full range of B-call maturation • Usually monoclonal • Normal cytogenetics • No oncogenic mutations

3

Monomorphic PTLD (Non-Hodgkin’s B- or T-cell lymphoma)

• Nodal disease with effacement of lymph node architecture or invasive extranodal disease • Monomorphic sheets of transformed B-cells • Monoclonal • Some with abnormal cytogenetics or mutations in ras or p53

4

T-cell-rich large B-cell (Hodgkin’s-like) lymphoma

• Nodal disease • Background of small T-cells with superimposed Reed-Sternberg-like cells • Monoclonal

5

Plasmacytoma-like lesions

• Nodal disease with effacement of lymph node architecture by mature plasma cells • Monoclonal

3. Pathophysiology and Risk Factors EBV was first implicated as a cause for malignancy in 1964, when viral particles were discovered in a patient with Burkitt’s lymphoma [11]. Since then, EBV has also been implicated as a causative factor in nasal NK-T-cell lymphoma, nasopharyngeal carcinoma, Hodgkin’s disease and other malignancies. The association of EBV with PTLD has been well established and it is present in approximately 90% of cases. In the pediatric population, it usually arises as a result of primary EBV infection, whereas in adults it is typically reactivation of a latent infection. The abnormal lymphoid proliferation in PTLD is the acquired and most often iatrogenic equivalent of the rare disorder X-linked lymphoproliferative disease (XLP). XLP is a congenital disorder that presents in childhood as a fulminant form of primary EBV infection. The mortality rate approaches 50% and some of the survivors develop B-cell lymphomas. The genetic defect underlying XLP involves SAP, a gene, involved in modulation of the immune response against EBV [12]. It is believed that the inability to

Chapter 34  Post-transplant Lymphoproliferative Disorder 

mount an appropriate T-cell response to EBV infection is the culprit in creating the abnormal lymphoid proliferation in XLP, similar to PTLD. EBV is a herpes virus, which infects 90% of the world’s adult population. The virus infects B-cells and establishes a compartment of latently-infected memory cells, forming a reservoir for future reactivation. The virus was shown to induce uncontrolled proliferation of infected B-lymphocytes in vitro. Maintaining it in latent phase throughout life is dependent on an intact host immune system. More specifically, CD3+ CD8+ cytotoxic T-lymphocytes (CTLs), commonly seen as “atypical lymphocytes” on the peripheral smear of patients with acute EBV infection, are critical for control of the EBV-infected B-cells [13]. When T-cell immunity is impaired, abnormal lymphoproliferation can occur in the form of PTLD. The origin of EBV in PTLD is varied. In solid organ transplant recipients, PTLD cells are typically of recipient origin, suggesting reactivation of a latent infection [14]. Most cases of PTLD in allogeneic HST involve seropositive donors and recipients. The lymphoproliferation is believed to be donor-derived because the host lymphoid system has been eradicated by the conditioning regimen. Even in cases where PTLD appears with a seronegative donor, the neoplastic cells are of donor origin, suggesting that donor cells were infected after the transplant [14–16]. The theory that an inadequate T-cell response results in EBV-related B-cell proliferation is supported by the observed risk factors for the development of PTLD in transplantation. For solid organ transplant patients, these include: an EBV-seronegative recipient (especially with an EBV-seropositive donor), EBV seroconversion after transplant, high degree of immunosuppression (especially use of antithymocyte globulin or antilymphocyte antibodies such as OKT3), CMV-seronegative recipient, presence of cytomegalovirus disease, and possibly younger age (independent of EBV status) [4, 17–19]. These factors manifest themselves as a higher risk of PTLD in patients with the most suppressed T-cell function. Differences in the incidence of PTLD among solid organ transplants may result from the different type and intensity of immunosuppression used. It may also reflect the amount of lymphoid tissue transplanted within the organ, explaining the high incidence in intestinal transplants (19–30%), followed by combined heart–lung or lung only (8–10%), and heart or liver (3% each). Kidney transplant recipients have the lowest risk for PTLD (1–2%) [18, 20, 21]. The use of different types of immunosuppression may influence the incidence of PTLD not only through the degree of T-cell inhibition but also through other promalignant effects. It is postulated that calcineurin inhibitors may promote malignant transformation through production of cytokines and their effect on cell cycle control mechanisms. OKT3, a monoclonal anti-CD3 antibody was associated with a nine-fold increase in risk for PTLD [22], while the association of some novel agents such as the anti-IL-2 monoclonal antibodies daclizumab and baziliximab with PTLD is controversial [18, 23]. Major risk factors for developing PTLD in solid-organ transplants and HST are summarized in Table  34-2. Risk factors after HST include T-cell depletion, HLA mismatch, specific anti-lymphocyte anti-GVHD therapies, reduced intensity conditioning and HST for primary immunodeficiency disorders [14, 15, 24–27]. The highest-risk group in large scale studies consists of patients who receive T-cell-depleted HLA mismatched bone marrow, where patients

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are faced with near-complete deletion of both donor and host T-cells and high levels of pharmacologic immunosuppression. The incidence in this group has been reported as high as 24% [15]. In most organ transplant recipients who experience lower levels of immunosuppression, such as renal or liver transplants and recipients of standard T-cell replete allogeneic HST, the risk of PTLD is 1–3% [3, 14, 15, 28]. Umbilical cord blood transplants should potentially pose a high risk for PTLD since the conditioning often includes ATG, the infused dose of T-cells is low and there is often HLA-mismatch. Cohorts of umbilical cord blood transplant recipients did not show increased risk for PTLD, but experience in this regard is still limited [29]. Rare EBVrelated lymphoid tumors have been described in recipients of autologous HST [30–33]. These cases may be related to profound chemotherapy-induced immunosuppression rather than T-cell-specific immunosuppression, and are a very rare complication of autologous HST. In order to assess the role of T-cell immunity in the development of PTLD in allogeneic HST recipients, several investigators looked at immune reconstitution of EBV-specific cytotoxic T-cells post-transplant. Reports have been conflicting in recipients of HLA-matched siblings, some of them showing absence of CTLs against EBV-infected B cells for 3–6 months after transplant [34, 35] and others showing rapid redevelopment of immunity with the frequency of EBV-specific T-cells correlating with the level of EBV genome expression in the peripheral blood [36]. Adult recipients of T-cell-depleted HST unequivocally lack EBV-specific T-cells for several months after transplant and in fact, experience significant deficiencies of all CD3+ cells [25, 26, 37] as compared to adult recipients of related HST and pediatric recipients of all types of HST. Rapid immune reconstitution has been observed after low-dose donor leukocyte infusions [37]. While the majority of cases of PTLD are associated with EBV infection, there are cases with no evidence of EBV involvement. EBV- negative PTLD may comprise a distinct subgroup of PTLD. These cases tend to occur later after transplant (median of 50 months post-transplant), and behave more aggressively, with reported median survival of 1 month. Similar to EBVrelated PTLD, EBV-negative cases may still respond to reduction in immunosuppression and conventional chemotherapy, although they probably represent a different pathogenesis related to the increased rate of malignancies in immunosuppressed individuals [38, 39]. Table 34-2.  Risk factors for developing PTLD. Solid organ transplantation

Allogeneic stem cell transplantation

Intestinal, lung or heart-lung transplant

T-cell depletion

EBV-seronegative recipient with EBV-seropositive donor

HLA-mismatching (related or unrelated; synergistic with T-cell depletion

Specific anti-lymphocyte therapy

Specific anti-lymphocyte therapy

High levels of immunosuppression

Primary immunodeficiency as indication for transplant Nonmyeloablative conditioning Chronic GVHD

Chapter 34  Post-transplant Lymphoproliferative Disorder 

It was suggested that hepatitis C virus plays a role in the pathogenesis of PTLD. Hepatitis C virus is known to be associated with B-cell clonal expansions, benign and malignant, arising in immunocompetent individuals. Early reports from small studies showed a higher incidence of PTLD in liver- and heart-transplant recipients who were HCV-positive [40–42] but this association is not supported by a recent retrospective study of 210,763 solid organ transplant recipients [43].

4. Clinical Features The incidence of PTLD after solid organ transplantation is in the range of 1–20% [18, 44–47]. The rate of occurrence following HST is largely unknown. The median onset of disease in the solid-organ transplant population is 6 months and in HST recipients, 70–90 days [4, 48]. However, cases have been reported as early as 1 week post-transplant and as late as 9 years [48]. Symptoms are diverse and may be related to infection, mass effect, organ dysfunction or B symptoms. Most cases of PTLD involve lymph nodes, but extranodal involvement is often seen, most commonly in the liver, lungs and the central nervous system. Not infrequently, the transplanted organ is involved [20]. Clinical features of PTLD in allogeneic HST recipients are similar to those of solid-organ transplant recipients, but there appears to be a greater incidence of fulminant, disseminated disease, perhaps accounting for the increased mortality seen in this population. Opportunistic infections commonly complicate these cases. In general, any transplant patient who experiences adenopathy, mass lesions, unexplained fever, weight loss, or dysfunction of the transplanted organ should be evaluated for PTLD. PTLD should also be considered when empiric increases in immunosuppression result in worsening organ dysfunction [4]. Given the myriad other diagnoses that PTLD may mimic, including infection and rejection, tissue biopsy confirmation of PTLD is always required. Although morphology is often typical, special studies can be used to confirm the diagnosis (Fig.  34-1). Immunophenotyping by flow cytometry or immunohistochemistry confirms B-cell origin and clonality, and when compared to de-novo B-cell lymphomas, it more often reveals lack or significant loss of surface immunoglobulin (36% of cases) and lack of CD20 (16% of cases), the latter having therapeutic implications [49]. Cytogenetic studies are seldom useful in PTLD since some of the cases do not demonstrate any chromosomal aberrations and the ones that are revealed by conventional cytogenetics or comparative genomic hybridization (CGH), are not specific and do not correlate with the phenotype or clinical outcome [50–52]. a

b

c

Fig.  34-1.  Photomicrographs of monomorphic PTLD. Magnification  × 40. (a) H&E stain (b) CD20 immunohistochemical stain. (c) EBER in-situ hybridization

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Molecular studies such as fluorescent in-situ hybridization for EBV early RNA (EBER) and polymerase chain reaction for the EBV genome are used to confirm the viral etiology. The presence of monoclonal gammopathy in a transplant patient with PTLD is common and does not correlate with clinical features or prognosis [53]. Staging of PTLD has not been formally defined. It is generally recommended to follow conventional staging for non-Hodgkin’s lymphoma by obtaining computed tomography of the chest, abdomen, and pelvis as well as a serum LDH for prognostic purposes. PCR studies for EBV in the peripheral blood may be useful at the time of diagnosis and during follow-up as methods of monitoring patient response to treatment. It is unclear whether results of a bone marrow biopsy alter outcome or management. Overall mortality of PTLD is difficult to establish, given the heterogeneity of presentation, underlying conditions, and therapies; however, estimates of 40–70% have been reported after solid organ transplantation [18, 54]. After allogeneic HST, early mortality rates for PTLD approach 90%. Risk factors, which adversely affect survival are stem-cell rather than solid-organ transplant, HST for a hematological malignancy rather than an immunodeficiency disorder, and four or more sites of disease [55]. Survival has improved significantly with the advent of donor leukocyte infusion (DLI) therapy and other forms of therapy.

5. Surveillance Early detection of PTLD may allow for prompt therapy and potentially decrease mortality. Experience with monitoring for PTLD is limited and there are no prospective randomized clinical trials of early interventions. However, several compelling reports do suggest that the surveillance for the presence of primary or reactivated EBV infection may indeed prove useful, and there are small series suggesting that some therapies may thwart the development of PTLD. Several groups have examined both solid-organ transplant and HST recipients for evidence of active EBV infection. EBV activity can be assessed in a number of ways, including evaluation of EBV viral load in the peripheral blood (as measured by polymerase chain reaction amplification), measurement of the number of EBV-infected peripheral blood mononuclear cells, and ex vivo spontaneous growth of EBV-transformed B-cells [56–61]. Serologic testing of antibodies to EBV viral capsid antigen or nuclear antigen is less sensitive than these methods, but is more specific for EBV disease. By all of these measures, it has been demonstrated that EBV activity is greater in transplant recipients with PTLD than in transplant recipients without PTLD, healthy EBV-seropositive adults, or healthy adults with infectious mononucleosis. Furthermore, these EBV-related markers tend to rise in the weeks prior to development of clinical PTLD and to peak at the time of diagnosis of PTLD, implying that their measurement may be used as predictors of the development of PTLD [57, 59, 60, 62]. Although the studies were small, negative predictive values (94–100%) and sensitivities (100%) were high [60, 61, 63]. It is interesting to note that EBV DNA levels decrease with therapy [59, 61, 64], although this may not always correlate with PTLD tumor response [65]. Several protocols employing EBV viral load monitoring in the first few months following HST were tested in small studies showing some success

Chapter 34  Post-transplant Lymphoproliferative Disorder 

in predicting PTLD, but none of them became part of the standard of care for these patients [66]. The cutoff used in most studies was 1,000 genome equivalents/milliliter. Patients with a higher viral load were considered at high risk to develop EBV-related complications and using preemptive therapy with rituximab for these high risk patients showed promising results in reduction of the risk for PTLD. However, randomized studies of preemptive therapy were not published and this approach may not be specific enough due to the relatively low positive predictive value of a high viral load [67, 68]. To summarize, measurement of EBV viral load is biologically appealing as a predictive test, and preemptive therapy may offer significant benefit to high risk patients, but the clinical utility remains to be determined. Following diagnosis of PTLD, weekly monitoring of EBV viral load is useful in monitoring response to therapy, especially indicating lack of response. Following successful treatment for PTLD, changes in EBV viral load are less predictive for recurrent disease. Guidelines for trials in solid organ transplants recommend following EBV viral load in high risk patients, especially pediatric patients and seronegative recipients who have a seropositive organ donor [69]. No clinical guidelines support use of these methods to date.

6. Prophylaxis and Treatment 6.1. Antiviral Therapy Initial attempts to prevent PTLD in the solid-organ transplant population were focused primarily on using antiviral therapies, such as the nucleoside analogues ganciclovir or acyclovir, to eradicate or control EBV for high-risk patients (Table  34-3A). These drugs inhibit the replication of other herpes viruses, such as herpes simplex or cytomegalovirus. Although it seems intuitive that these medications would be a mainstay in the treatment of PTLD, the antiviral therapies currently available have proven ineffective in vivo. Nucleoside analogues impair EBV viral replication by inhibiting the virus’s DNA polymerase. However, the activity of these medications requires intracellular phosphorylation by a viral-encoded thymidine kinase, which is not expressed by latentlyinfected B-cells and cells of EBV-related lymphomas [70, 71]. One development in this area of PTLD therapy involves the use of the antiviral nucleoside analogues in conjunction with arginine butyrate. Arginine butyrate acts to induce lytic phase gene expression, causing latent EBV in lymphoma cells to express thymidine kinase and allowing phosphorylation of ganciclovir to its active form. Several studies pairing ganciclovir with arginine butyrate in the treatment of EBV positive lymphoma demonstrated response [72, 73]. This therapeutic approach continues to be an area of active investigation. Prophylaxis against PTLD, especially in patients designated to be at high risk for disease development, is another attractive treatment approach. Primary EBV infections and reactivation of EBV in immune compromised patients are targets for antiviral therapies. Several retrospective studies used institutional and historical controls to demonstrate a trend toward reduction in PTLD incidence in solid-organ recipients receiving prophylactic ganciclovir or acyclovir. These results, however, should be interpreted with caution (Table 34-3A) [61, 74, 75]. HST recipients were not included in these trials. European guidelines for renal-transplant recipients suggest use of prophylactic antiviral therapy

603

Pediatric intes- Prospective nonrandtine omized trial

Pediatric liver

Adults solid organ and HST

Green et al. [63]

McDiarmid et al. [61]

Perrine et al. [73]

Adult solid organ

Adult solid organ

Davis et al. [77]

Tsai et al. [48]

Retrospective study

Prospective nonrandomized trial

(B) Local therapy and reduction of immunosuppression

Phase 1/2 trial

Prospective riskstratified trial

Retrospective study with historical controls

Adult solid organ

Davis et al. [75]

Prospective trial with historical controls

Study design

Adult solid organ

Transplant type

Darenkov et al. [74]

(A) Antiviral therapy

Reference

Table 34-3.  Therapeutic options for PTLD – major trials.

2/14 treated patients developed PTLD, compared with 3/4 untreated patients

3/206 Patients developed PTLD, compared with 8% of historical controls

1/198 (0.5%) treated patients developed PTLD, compared with 7/179 (3.9%) historical controls

Response

Complete surgical resection

Complete surgical resection or radiation alone

Continuous IV arginine butyrate (thymidine kinase inducer) in escalating doses with standard dose ganciclovir BID

12/12 patients achieved CR; no relapses were noted in follow-up

3/3 patients with long-term disease-free survival

PR in 6 patients

15 patients with previously treated refractory PTLD; CR in 4 patients

100 days of IV ganciclovir 0/18 PTLD cases in treated patients compared with prophylaxis in “high risk” 10% of historical controls (donor-seropositive, recipient seronegative) cases

Peripheral blood EBV PCR monitoring with preemptive IV ganciclovir and IVIG for patients with high viral loads

Prophylactic IV ganciclovir followed by high-dose oral acyclovir

Prophylactic ganciclovir (if donor or recipient was CMV+) or acyclovir (if both were CMV−) during antilymphocyte therapy

Therapy

604  R. Reshef et al.

Adult solid organ

Tsai et al. [48]

Adult and pediatric solid organ

Haddad et al. [98]

(F) Cytotoxic therapy

Adult and pediatric bone marrow

Gross et al. [24]

(E) Cytokine therapy

Sharman et al. [92] Adult and pediatric solid-organ

Phase 1/2 trial

Retrospective study

Prospective trial

Retrospective study

Adult solid-organ

Prospective trial

Retrospective study

Elstrom et al. [81]

Adult bone marrow and solidorgan

Milpied et al. [90]

Prospective nonrandomized trial

Retrospective study

Retrospective study

Choquet et al. [89] Adult and pediatric solid-organ

Adult bone marrow and solidorgan

Benkerrou et al. [55]

(D) Anti B-cell antibodies

Adult solid organ

Starzl et al. [83]

(C) Reduction of immunosuppression

Anti-IL-6 monoclonal antibody

IFN-a

Anti-CD-20 antibody (rituximab)

Anti-CD-20 antibody (rituximab) vs. chemotherapy

Anti-CD-20 antibody (rituximab)

Anti-CD-20 antibody (rituximab)

Anti-CD-21 and anti-CD-24 murine monoclonal antibodies

Reduction of all immunosuppression to minimum tolerated doses

Variable decreases in cyclosporine and/or prednisone. Some patients received XRT, chemotherapy and/or acyclovir

5/12 CR; 3/12 PR

3/7 CR; 1/7 PR

(continued)

5 year cause-specific survival 69%

n = 24; CR 46%; PR 17%

Overall response 68%; CR 59%

Rituximab (n = 22):

Chemotherapy (n = 23):Overall response 74%; CR 57%

Overall response 44%; CR rate 28%

HST response 83% (5 CR)

Overall response rate 69%; Solid organ response 65% (15 CR; 2 PR)

36/58 patients achieved CR. 1 year survival for HST patients was 0%

19/30 patients (63%) overall response; 14/30 patients (47%) alive in CR

11/17 patients alive and tumor-free

Chapter 34  Post-transplant Lymphoproliferative Disorder  605

Response

Prospective trial

Adult and pediatric bone marrow

Adult bone marrow and solidorgan

Gustafsson et al. [112]

Haque et al. [118]

EBV-specific donor T-cells

EBV-specific donor T-cells to patients with rising EBV DNA titers

EBV-specific donor T-cells as prophylaxis

CHOP cyclophosphamide, doxorubicin, vincristine, prednisone, ProMACE prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, CytaBOM cytarabine, bleomycin, vincristine, methotrexate

Phase 2 multicenter trial

Prospective trial with historical controls

Prospective trial

Rooney et al. [113] Pediatric T-cell-depleted bone marrow

Pediatric T-cell-depleted bone marrow

EBV-specific donor T-cells to 3 patients with evidence of EBV disease (2 PTLD, 1 high EBV viral load)Seven other patients treated prophylactically

Overall response 68%; CR 59%

Other combined regimens (n = 4)

Rooney et al. [58]

Rituximab (n = 22):

R-CHOP (n = 9)

Unselected donor mononuclear cells

Chemotherapy (n = 23):Overall response 74%; CR 57%

Overall survival rates were higher with multidrug regimens (24– 32%) than single-agent chemotherapy (5%)

6/8 patients with late-onset PTLD survived

n = 110/3 patients with early-onset PTLD survived

Anti-CD-20 antibody (rituximab) vs. Chemotherapy:CHOP (n = 10)

Other combined regimens (n = 65) Singleagent chemotherapy (n = 23)

CHOP (n = 90)ProMACE (n = 12)

Aggressive non-Hodgkin’s lymphoma chemotherapy (predominantly ProMACECytaBOM)

Therapy

Papadopoulos et al. Adult T-cell-depleted bone mar- Prospective trial [110] row

(G) Cellular immunotherapy

Adult solid-organ

Elstrom et al. [81]

Retrospective study

Retrospective study

Adult solid organ

Buell et al. [101]

Study design Retrospective study

Transplant type

Swinnen et al. [99] Adult heart

Reference

Table 34-3.  (continued)

606  R. Reshef et al.

Chapter 34  Post-transplant Lymphoproliferative Disorder 

for EBV-seronegative recipients. Therapeutic use of these agents for at least 1 month is encouraged following diagnosis of PTLD [76]. 6.2. Local Therapy For patients with localized disease, surgical resection or involved field radiation therapy is an excellent treatment option. When coupled with reduction of immunosuppression (RI), PTLD-related mortality rates have been reported as low as 0–26% (Table 34-3B) [48, 54, 77–80]. Surgical resection or radiation therapy has also been used with success in conjunction with rituximab [81]. Patients requiring emergent treatment for advanced disease or palliative treatment to a specific area can also be treated successfully with localized radiation therapy [82]. 6.3. Reduction of Immunosuppression The mainstay of therapy for PTLD remains RI. The effectiveness of this intervention was described initially by Starzl in 1984 [83] (Table 34-3C) and has been substantiated since then [48]. RI allows T-cells to target EBV-harboring B-cells and restore EBV-specific cellular immunity. Ultimately, the goal is to create a lymphoma-targeted immune response without causing acute graft rejection. While reduction in immunosuppression is the most established therapy for PTLD, there are significant risks with this treatment. In solid-organ transplant patients, graft rejection occurs as a potential complication in 39% of both responders and nonresponders [48]. The risk of rejection associated with this approach to treatment varies substantially based on the type of transplant. Heart and lung transplant recipients have the highest risk of acute organ rejection. Rejection of these transplants is not always easily diagnosed or tolerated as well by patients as rejection from kidney or liver transplants. For patients with kidney or liver transplants, RI can be more aggressive due to the ease of following lab values for rejection, patients’ ability to tolerate rejection, and the comparatively low risk of allograft rejection [46, 84]. In patients with life-sustaining organ transplants, such as heart, liver and lungs, RI should be moderate and closely monitored as allograft rejection may be swift and fatal. In allogeneic HST recipients complications from decreased immunosuppression typically manifest themselves as increased risk for GVHD with significant risk for morbidity and mortality. Proper RI represents a challenge to the physician and should be individualized [48, 54]. The method varies by institution, and often by individual physician. Patient characteristics that are considered include allograft type, relative risk of allograft rejection, extent and severity of PTLD, and the particular immunosuppressive drugs being used [85]. Most institutions favor discontinuing mycophenolate mofetil and azathioprine and reducing calcinuerin inhibitor and steroid doses [85]. Close monitoring for acute rejection is imperative while this is being done. There is very little information supporting a specific method of RI in HST recipients. Several factors have been shown to predict outcomes in patients undergoing RI. Predictors of lack of response to RI include LDH >2.5 times the upper limit of normal, organ dysfunction, and multiple visceral sites of disease. Patients who lacked all of these risk factors had an 89% response rate, whereas

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the presence of two or more risk factors predicted a very poor prognosis [48]. EBV status does not predict response to RI, and a similar approach should be attempted in patients with EBV-negative tumors [38, 39]. 6.4. Targeted Therapy: Anti-B-cell Antibodies Rituximab, a chimeric IgG monoclonal antibody targeting the CD20 protein on mature B-cells, is a therapy that has had moderate success in treating PTLD. It has been theorized that rituximab causes B-cell death through multiple mechanisms, including direct stimulation of apoptosis, recruitment of cytotoxic effector cells, and complement fixation with subsequent cell destruction [86–88]. Possibly due to cellular dysfunction following EBV infection, the CD20 antigen is not expressed on all PTLD B-cells. Despite this, it has success and low toxicity when used alone or in conjunction with cytotoxic chemotherapy. Evidence supporting the use of rituximab for the treatment of PTLD is fairly abundant (Table 34-3D). Multiple phase II clinical trials and case series demonstrate overall response rates of 44–75% and complete response rates of 35–69% in patients being treated with rituximab alone after failure of RI [81, 89–92]. An early retrospective study identified 32 patients (26 solid organ transplants, 6 HST) treated initially with RI and subsequently with rituximab. Overall response rate in this study was 69%. Among HST patients, 5/6 (83%) achieved CR. Ten patients progressed despite rituximab, but 50% were salvaged with systemic chemotherapy [90]. A more recent phase II multicenter trial included 43 adult and pediatric patients with PTLD who had failed RI. Patients were treated for 4 weeks with rituximab and results were analyzed after 80 days. Response rate overall was 44% with a 35% complete response rate. The 1-year-survival rate was 67% [89]. In these studies, clinical response to therapy was measured as decrease in tumor size, and it was often observed within several days of initiation of therapy. Delayed responses to therapy also occurred. In addition to treatment for existing PTLD, rituximab has been used preemptively to prevent development of PTLD [67, 93]. In one study of patients who had undergone HST, 17 patients deemed to be at high risk for the development of PTLD due to high EBV viral load were given one dose of rituximab preemptively [93]. Of these 17 patients, only two (18%) developed PTLD after receiving rituximab, whereas 48% of high risk patients developed PTLD in historical controls. Notably, in those patients who did not develop PTLD, EBV viral load was undetectable [67]. Rituximab has been used successfully to treat patients who failed initial systemic chemotherapy [90]. It has also been used in conjunction with systemic chemotherapy in high risk patients with good results [94]. A retrospective analysis comparing standard chemotherapy to rituximab showed overall similar complete response rate, but a much lower mortality rate for rituximab (0% as opposed to 26%), making it the treatment of choice in most instances. In this study, patients who failed either treatment were salvageable by the other therapeutic approach [81]. Predictors for poor response to anti-B cell therapy include multivisceral disease, late-onset PTLD (>1 year post-transplant), and CNS involvement [55]. One study of five patients with PTLD treated with rituximab demonstrated a decrease in EBV viral load in all patients, but progression of the PTLD tumor in three patients, questioning the accuracy of following EBV DNA levels in patients receiving rituximab [65].

Chapter 34  Post-transplant Lymphoproliferative Disorder 

In addition to CD20, the surface proteins CD21 and CD24 have been targeted. A prospective multicenter trial using anti-CD21 and anti-CD24 murine monoclonal antibodies included 58 patients (27 HST recipients) and 31 solid organ transplant recipients [55]. There was a 61% complete response rate and an 8% relapse rate, with an overall survival of 46% (35% for bone marrow transplant patients and 55% for solid- organ transplant recipients) after 61 months median follow up. In addition to “naked” monoclonal antibodies, anti-B cell antibodies linked to radionuclides may play a role in PTLD treatment. Drugs such as tositumomab (anti-CD20 linked to iodine-131) and ibritumomab tiuxetan (anti-CD20 linked to yttrium-90) have been used in nonHodgkin’s lymphoma and may warrant additional studies in PTLD. 6.5. Cytokine Therapy Immune modulators such as cytokines with or without immunoglobulins have been used to treat PTLD by attempting to establish a competent immune response against EBV-related lymphoproliferations (Table  34-3E). Although there has been some success with this approach, it is also associated with a high degree of graft rejection due to the nonspecific T-cell stimulation [77, 95]. Several case series and case reports have described responses to interferonalpha and interferon-alpha combined with intravenous immunoglobulins [15, 24]. Interleukin-6, a cytokine that promotes the growth and differentiation of B cells, has been found in higher levels in patients with PTLD and is a potential target [96, 97]. Targeting IL-6 resulted in partial success in a small prospective study of 12 patients with PTLD who failed to respond to RI [98]. Overall response rate was 66%. It is expected that future study will continue in this area. 6.6. Cytotoxic Chemotherapy Chemotherapy has also been used to treat PTLD, generally after patients failed to respond to surgical excision with or without RI (Table 34-3F). It can be used with or without RI when a rapid response is necessary or following failure of rituximab therapy. Cytotoxic chemotherapy can produce a swift response, but it is associated with significant morbidity and mortality [99, 100]. Regimens are similar to those used for non-Hodgkin’s lymphoma, such as CHOP, R-CHOP, and ProMACE-CytaBOM. In one study, 18 patients with late onset PTLD were treated with systemic chemotherapy with RI or after RI had failed. Complete response was achieved in 33% of patients, but there was a 50% mortality rate related to complications of chemotherapy (infection or end organ toxicity) [78]. Additional data were gathered by a retrospective review of the Israel Penn International Transplant Tumor Registry that examined 193 heterogeneous patients with PTLD treated with various chemotherapeutic regimens. High toxicity rates resulted in high mortality rates, including 25% mortality in patients receiving R-CHOP, which was similar to mortality for the other regimens used [101]. Patients that received only single-agent chemotherapy had a significantly decreased 5-year survival rate (5%) as compared to patients receiving regimens with multiple chemotherapeutic medications. However, no adjustments were made for prechemotherapy functional status that may have been worse in patients given single-agent chemotherapy.

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Another approach to treatment with chemotherapy has been reduced intensity chemotherapy [94]. One prospective multi-center study of pediatric patients with PTLD who failed initial therapies (RI, rituximab, local therapy, or interferon-alpha) demonstrated overall response to cyclophosphamide and prednisone of 83%. Patients with widespread, aggressive disease typically did not respond [102]. For the most part, systemic chemotherapy is reserved for patients with aggressive disease that does not respond to RI and requires more immediate therapy such as Burkitt’s lymphoma-type PTLD. Hodgkin’s disease should also be initially treated with chemotherapy due to its high likelihood of cure with relatively low intensity chemotherapy. Delayed onset, advanced stage, monomorphic EBV–PTLD has demonstrated poor response to RI and has an overall poor prognosis, making some suggest up-front chemotherapy for it, as well. In general, systemic chemotherapy is an approach that is rarely used for initial therapy and one that should probably be reserved for patients that do not respond to RI and other low toxicity approaches, such as treatment with rituximab. Additionally, patients with disease that is refractory to induction chemotherapy have been cured with salvage chemotherapy followed by an allogeneic or autologous stem cell transplant [103–105]. 6.7. Cellular Immunotherapy In allogeneic HST patients, there is another option for both prevention and treatment of PTLD: cellular or adoptive immunotherapy. The lymphocyte populations in recipients of allogeneic HST are abnormal, particularly after T-cell depleted or HLA-mismatched transplants. It is clear that the latter group of patients has markedly decreased T and B cell number and function for at least 6 months, and possibly several years, after transplant [34, 36, 37, 106]. PTLD risk increases in the setting of T cell regulatory dysfunction. As the frequency of EBV-specific CD8+ T cells rises after HST, both a decrease in viral load and regression of PTLD have been observed [35, 106–108]. In addition, the efficacy of unselected donor leukocytes in reconstituting the recipient’s immune system has been well described [37]. These two observations have led to attempts to both prevent and treat PTLD with DLI. Early work with DLI focused on unselected donor mononuclear cells and resulted in high response rates, but carried a significant risk of GVHD [109, 110]. However, over 90% of patients may respond to DLI. In donors previously exposed to EBV, the frequency of viral specific cytotoxic T cells (CTL) will be higher than in the initially infected host, and it is possible that low dose DLI may be given to eradicate PTLD, minimizing the risk of GVHD. In an early series, 18 patients with PTLD after HST received DLI from their stem cell donors [111]. Of these patients, 54% were able to achieve CR, but 62% experienced acute or chronic GVHD. Rather than using unselected DLI, utilizing CTLs targeted against EBV hold the promise of more active anti-viral/tumor effect without the risk of GVHD [112]. Rooney showed that EBV specific CTLs could be used prophylactically and as a primary therapy for PTLD after allogeneic bone marrow transplantation [58, 113]. A compelling argument has been made that CTLs may be most effective as prophylaxis in patients with high EBV viral loads because at that point in the disease course there is minimal tumor burden and consequently, a lower chance of selecting escape mutants [114]. In a report in 2001,

Chapter 34  Post-transplant Lymphoproliferative Disorder 

a patient died of progressive PTLD despite treatment with EBV-specific CTLs. Post-mortem examination of this patient’s tumor revealed a deletion in the tumor virus antigen EBNA-3B. This altered the tumor’s HLA-restricted epitopes and rendered it unresponsive to the donor CTL infusion, which was directed against the wild-type EBNA-3B epitope [115]. The mutated virus appeared to have originated in the recipient after transplant. This demonstrates the possibility of EBV “escape mutants” and a potential limitation of this otherwise promising therapy. The reports of successful CTL therapy in the allogeneic HST population prompted a study of autologous lymphokine-activated killer (LAK) cells, given to solid-organ transplant recipients with PTLD [116]. In this seven-patient series, peripheral blood mononuclear cells were collected from patients with PTLD and were reinfused after in vitro expansion using recombinant human IL-2. Four patients with EBV-positive tumors all sustained involution of their tumors; two patients suffered organ rejection as a complication of this therapy. Subsequent studies included patients that donated T-cells prior to solid-organ transplantation [117]. EBV-specific cell lines were cultured and reinfused into the patients after transplant as PTLD prophylaxis. Their circulating EBV DNA levels were suppressed to below pre-transplant levels and EBV-specific CTLs were measurable in the patients’ blood for 3 months after transplant. Another novel approach was recently described, using a bank of EBVspecific T-cells created from EBV-positive blood donors of common Scottish HLA types [118]. Thirty-three patients who had failed prior therapy were included in this trial. Overall response to treatment with EBV-specific CTLs was 52% at 6 months, with complete response in 42%, partial response in 9%, and no response in 48% (5 patients died prior to completing treatment). Patients with closer HLA matches had better responses, and there was no evidence of graft versus host disease or any allograft rejection. Allogeneic unmatched EBV-specific CTLs were successfully used in three patients after solid- organ transplantation with refractory PTLD [119]. Allogeneic CTLs are particularly attractive for patients who were previously treated with rituximab, in whom autologous CTLs are difficult to obtain. In this study, patients were treated with allo-CTLs, with two patients achieving complete remission. Further work in this area is needed.

7. Conclusion PTLD is an often-fatal complication of both solid-organ and stem cell transplantation. Early diagnosis of PTLD is important and requires high level of clinical vigilance. Surveillance for PTLD by monthly PCR for circulating EBV DNA may be appropriate, particularly in high-risk settings as EBVseromismatched (donor-positive, recipient-negative) solid-organ transplants and T-cell-depleted, HLA-mismatched stem-cell transplants. Optimal therapy for PTLD remains to be determined and should be individualized by taking into account factors such as graft type, clinical and pathological prognostic factors and the rate of progression (Fig. 34-2).When and where possible, surgical excision of the tumor should be pursued. Reduction in immunosuppression remains the primary therapy for PTLD and can often result in permanent disease eradication. Additional modalities include anti-B-cell antibodies, antiviral drugs and conventional chemotherapy. Adoptive immunotherapy

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Clinical Suspicion 1. Constitutional symptoms – weight loss, anorexia, malaise, fever, fatigue, pain 2. Palpable or radiographic lymphadenopathy or mass

Diagnosis

1. LDH levels 2. Peripheral blood EBV PCR 3. CT / PET 4. Biopsy from involved site with EBV staining

High Risk

Low Risk

1. High risk of allograft rejection

1. Low risk for allograft rejection

2. Rapidly progressive disease

2. 0-1 poor prognostic features: a. High LDH b. Multi-organ involvement c. End-organ damage

3. 2-3 poor prognostic features

Reduction of immunosuppression (RI)

No response

Response

Reassessment in 2-3 weeks

One of the followings: Follow-up

T-cell or CD20(-) disease Rapidly progressive disease NO

Curable disease with conventional treatment (Hodgkin-like)

Rituxan single agent +/– RI Reassessment in 4 weeks

No response

YES

Cytotoxic chemotherapy +/– RI +/– Rituxan Clinical trial

Response

612 

Follow-up

Local Disease

CNS Disease

RI + surgery/XRT

consider adding XRT to any regimen

Fig. 34-2.  Proposed algorithm for the diagnosis and treatment of PTLD

with EBV-specific donor T-cells is highly effective and appears to be less associated with graft-versus-host disease than unselected allogeneic donor leukocyte infusions. Use of cellular therapy as a prophylaxis against PTLD with pre-emptive infusion of EBV-specific CTLs either in all high-risk HST recipients or in patients with increasing EBV viral loads may be practical for widespread use in the future.

Chapter 34  Post-transplant Lymphoproliferative Disorder 

PTLD is a biologically fascinating disorder that greatly illuminates the roles of the immune system and viral infection in the development of malignancy. Therapy of PTLD provides a prime and exciting example of successful immunotherapy of cancer that may have implications for other malignancies and viral infections.

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R. Reshef et al. 19. Katz BZ et  al (2007) Case-control study of risk factors for the development of post-transplant lymphoproliferative disease in a pediatric heart transplant cohort. Pediatr Transplant 11(1):58–65 20. Penn I (2000) Post-transplant malignancy: The role of immunosuppression. Drug Saf 23(2):101–113 21. Nalesnik MA (1997) Clinicopathologic features of posttransplant lymphoproliferative disorders. Ann Transplant 2(4):33–40 22. Swinnen LJ et al (1990) Increased incidence of lymphoproliferative disorder after immunosuppression with the monoclonal antibody OKT3 in cardiac-transplant recipients. N Engl J Med 323(25):1723–1728 23. Bustami RT et  al (2004) Immunosuppression and the risk of post-transplant malignancy among cadaveric first kidney transplant recipients. Am J Transplant 4(1):87–93 24. Gross TG et al (1999) B cell lymphoproliferative disorders following hematopoietic stem cell transplantation: Risk factors, treatment and outcome. Bone Marrow Transplant 23(3):251–258 25. Micallef IN et al (1998) Lymphoproliferative disorders following allogeneic bone marrow transplantation: The Vancouver experience. Bone Marrow Transplant 22(10):981–987 26. Curtis RE et  al (1999) Risk of lymphoproliferative disorders after bone marrow transplantation: A multi-institutional study. Blood 94(7):2208–2216 27. Cohen JM et  al (2007) EBV-related disease following haematopoietic stem cell transplantation with reduced intensity conditioning. Leuk Lymphoma 48(2): 256–269 28. Herzig KA et al (2003) A single-centre experience of post-renal transplant lymphoproliferative disorder. Transpl Int 16(7):529–536 29. Barker JN et al (2001) Low incidence of Epstein–Barr virus-associated posttransplantation lymphoproliferative disorders in 272 unrelated-donor umbilical cord blood transplant recipients. Biol Blood Marrow Transplant 7(7):395–399 30. Hauke RJ et al (1998) Epstein–Barr virus-associated lymphoproliferative disorder after autologous bone marrow transplantation: Report of two cases. Bone Marrow Transplant 21(12):1271–1274 31. Shepherd JD et al (1995) Polyclonal Epstein–Barr virus-associated lymphoproliferative disorder following autografting for chronic myeloid leukemia. Bone Marrow Transplant 15(4):639–641 32. Lones MA et al (2000) Post-transplant lymphoproliferative disorder after autologous peripheral stem cell transplantation in a pediatric patient. Bone Marrow Transplant 26(9):1021–1024 33. Nash RA et  al (2003) Epstein–Barr virus-associated posttransplantation lymphoproliferative disorder after high-dose immunosuppressive therapy and autologous CD34-selected hematopoietic stem cell transplantation for severe autoimmune diseases. Biol Blood Marrow Transplant 9(9):583–591 34. Crawford DH et  al (1986) Epstein–Barr virus infection and immunity in bone marrow transplant recipients. Transplantation 42(1):50–54 35. Lucas KG et al (1996) The development of cellular immunity to Epstein–Barr virus after allogeneic bone marrow transplantation. Blood 87(6):2594–2603 36. Marshall NA et  al (2000) Rapid reconstitution of Epstein–Barr virus-specific T lymphocytes following allogeneic stem cell transplantation. Blood 96(8): 2814–2821 37. Small TN et al (1999) Comparison of immune reconstitution after unrelated and related T-cell-depleted bone marrow transplantation: Effect of patient age and donor leukocyte infusions. Blood 93(2):467–480 38. Leblond V et al (1998) Posttransplant lymphoproliferative disorders not associated with Epstein–Barr virus: A distinct entity? J Clin Oncol 16(6):2052–2059

Chapter 34  Post-transplant Lymphoproliferative Disorder  39. Nelson BP et al (2000) Epstein–Barr virus-negative post-transplant lymphoproliferative disorders: A distinct entity? Am J Surg Pathol 24(3):375–385 40. Hezode C et al (1999) Role of hepatitis C virus in lymphoproliferative disorders after liver transplantation. Hepatology 30(3):775–778 41. McLaughlin K et  al (2000) Increased risk for posttransplant lymphoproliferative disease in recipients of liver transplants with hepatitis C. Liver Transpl 6(5): 570–574 42. Buda A et al (2000) Lymphoproliferative disorders in heart transplant recipients: Role of hepatitis C virus (HCV) and Epstein–Barr virus (EBV) infection. Transpl Int 13(Suppl 1):S402–S405 43. Morton LM et  al (2007) Hepatitis C virus infection and risk of posttransplantation lymphoproliferative disorder among solid organ transplant recipients. Blood 110(13):4599–4605 44. Cockfield SM (2001) Identifying the patient at risk for post-transplant lymphoproliferative disorder. Transpl Infect Dis 3(2):70–78 45. Bakker NA et al (2007) Presentation and early detection of post-transplant lymphoproliferative disorder after solid organ transplantation. Transpl Int 20(3):207–218 46. Taylor AL, Marcus R, Bradley JA (2005) Post-transplant lymphoproliferative disorders (PTLD) after solid organ transplantation. Crit Rev Oncol Hematol 56(1):155–167 47. Gottschalk S, Rooney CM, Heslop HE (2005) Post-transplant lymphoproliferative disorders. Annu Rev Med 56:29–44 48. Tsai DE et al (2001) Reduction in immunosuppression as initial therapy for posttransplant lymphoproliferative disorder: Analysis of prognostic variables and longterm follow-up of 42 adult patients. Transplantation 71(8):1076–1088 49. Kaleem Z et al (2004) Flow cytometric evaluation of posttransplant B-cell lymphoproliferative disorders. Arch Pathol Lab Med 128(2):181–186 50. Djokic M et al (2006) Post-transplant lymphoproliferative disorder subtypes correlate with different recurring chromosomal abnormalities. Genes Chromosomes Cancer 45(3):313–318 51. Poirel HA et al (2005) Characteristic pattern of chromosomal imbalances in posttransplantation lymphoproliferative disorders: Correlation with histopathological subcategories and EBV status. Transplantation 80(2):176–184 52. Vakiani E et  al (2007) Cytogenetic analysis of B-cell posttransplant lymphoproliferations validates the World Health Organization classification and suggests inclusion of florid follicular hyperplasia as a precursor lesion. Hum Pathol 38(2):315–325 53. Tsai DE et  al (2005) Serum protein electrophoresis abnormalities in adult solid organ transplant patients with post-transplant lymphoproliferative disorder. Clin Transplant 19(5):644–652 54. Benkerrou M, Durandy A, Fischer A (1993) Therapy for transplant-related lymphoproliferative diseases. Hematol Oncol Clin North Am 7(2):467–475 55. Benkerrou M et  al (1998) Anti-B-cell monoclonal antibody treatment of severe posttransplant B-lymphoproliferative disorder: Prognostic factors and long-term outcome. Blood 92(9):3137–3147 56. Riddler SA, Breinig MC, McKnight JL (1994) Increased levels of circulating Epstein–Barr virus (EBV)-infected lymphocytes and decreased EBV nuclear antigen antibody responses are associated with the development of posttransplant lymphoproliferative disease in solid-organ transplant recipients. Blood 84(3):972–984 57. Savoie A et al (1994) Direct correlation between the load of Epstein–Barr virusinfected lymphocytes in the peripheral blood of pediatric transplant patients and risk of lymphoproliferative disease. Blood 83(9):2715–2722 58. Rooney CM et  al (1995) Use of gene-modified virus-specific T lymphocytes to control Epstein–Barr-virus-related lymphoproliferation. Lancet 345(8941):9–13

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Chapter 34  Post-transplant Lymphoproliferative Disorder    79. Koffman BH et al (2000) Use of radiation therapy in posttransplant lymphoproliferative disorder (PTLD) after liver transplantation. Int J Cancer 90(2):104–109   80. Hauke R et  al (2001) Clinical and pathological features of posttransplant lymphoproliferative disorders: Influence on survival and response to treatment. Ann Oncol 12(6):831–834   81. Elstrom RL et al (2006) Treatment of PTLD with rituximab or chemotherapy. Am J Transplant 6(3):569–576   82. Kang SK, Kirkpatrick JP, Halperin EC (2003) Low-dose radiation for posttransplant lymphoproliferative disorder. Am J Clin Oncol 26(2):210–214   83. Starzl TE et al (1984) Reversibility of lymphomas and lymphoproliferative lesions developing under cyclosporin-steroid therapy. Lancet 1(8377):583–587   84. Loren AW, Tsai DE (2005) Post-transplant lymphoproliferative disorder. Clin Chest Med 26(4):631–645 vii   85. Frey NV, Tsai DE (2007) The management of posttransplant lymphoproliferative disorder. Med Oncol 24(2):125–136   86. Shan D, Ledbetter JA, Press OW (1998) Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood 91(5):1644–1652   87. Di Gaetano N et  al (2003) Complement activation determines the therapeutic activity of rituximab in vivo. J Immunol 171(3):1581–1587   88. Svoboda J, Kotloff R, Tsai DE (2006) Management of patients with post-transplant lymphoproliferative disorder: The role of rituximab. Transpl Int 19(4):259–269   89. Choquet S et al (2006) Efficacy and safety of rituximab in B-cell post-transplantation lymphoproliferative disorders: Results of a prospective multicenter phase 2 study. Blood 107(8):3053–3057   90. Milpied N et al (2000) Humanized anti-CD20 monoclonal antibody (Rituximab) in post transplant B-lymphoproliferative disorder: A retrospective analysis on 32 patients. Ann Oncol 11(Suppl 1):113–116   91. Oertel SH et al (2005) Effect of anti-CD 20 antibody rituximab in patients with posttransplant lymphoproliferative disorder (PTLD). Am J Transplant 5(12):2901–2906   92. Sharman JP et al (2006) Mature results of a prospective trial of rituximab in patients (Pt) with post transplant lymphoproliferative disorder (PTLD) unresponsive or ineligible for reduced immunosuppression (RIS). ASH Annu Meet Abstr 108(11):2755   93. Faye A et  al (2001) Chimaeric anti-CD20 monoclonal antibody (rituximab) in post-transplant B-lymphoproliferative disorder following stem cell transplantation in children. Br J Haematol 115(1):112–118   94. Orjuela M et al (2003) A pilot study of chemoimmunotherapy (cyclophosphamide, prednisone, and rituximab) in patients with post-transplant lymphoproliferative disorder following solid organ transplantation. Clin Cancer Res 9(10 Pt 2):3945S–3952S   95. Faro A et al (1996) Interferon-alpha affects the immune response in post-transplant lymphoproliferative disorder. Am J Respir Crit Care Med 153(4 Pt 1):1442–1447   96. Tosato G et al (1993) Interleukin-6 production in posttransplant lymphoproliferative disease. J Clin Invest 91(6):2806–2814   97. Tosato G et al (1990) Identification of interleukin-6 as an autocrine growth factor for Epstein–Barr virus-immortalized B cells. J Virol 64(6):3033–3041   98. Haddad E et al (2001) Treatment of B-lymphoproliferative disorder with a monoclonal anti-interleukin-6 antibody in 12 patients: A multicenter phase 1–2 clinical trial. Blood 97(6):1590–1597   99. Swinnen LJ et al (1995) Aggressive treatment for postcardiac transplant lymphoproliferation. Blood 86(9):3333–3340 100. Mamzer-Bruneel MF et al (2000) Durable remission after aggressive chemotherapy for very late post-kidney transplant lymphoproliferation: A report of 16 cases observed in a single center. J Clin Oncol 18(21):3622–3632 101. Buell JF et  al (2005) Chemotherapy for posttransplant lymphoproliferative disorder: The Israel Penn International Transplant Tumor Registry experience. Transplant Proc 37(2):956–957

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R. Reshef et al. 102. Gross TG et  al (2005) Low-dose chemotherapy for Epstein–Barr virus-positive post-transplantation lymphoproliferative disease in children after solid organ transplantation. J Clin Oncol 23(27):6481–6488 103. Oertel SH et al (2003) Salvage chemotherapy for refractory or relapsed post-transplant lymphoproliferative disorder in patients after solid organ transplantation with a combination of carboplatin and etoposide. Br J Haematol 123(5):830–835 104. Komrokji RS et al (2005) Mini-BEAM and autologous hematopoietic stem-cell transplant for treatment of post-transplant lymphoproliferative disorders. Am J Hematol 79(3):211–215 105. Bobey NA, Stewart DA, Woodman RC (2002) Successful treatment of posttransplant lymphoproliferative disorder in a renal transplant patient by autologous peripheral blood stem cell transplantation. Leuk Lymphoma 43(12):2421–2423 106. Kook H et  al (1996) 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 88(3):1089–1097 107. Kuzushima K et al (2000) Longitudinal dynamics of Epstein–Barr virus-specific cytotoxic T lymphocytes during posttransplant lymphoproliferative disorder. J Infect Dis 182(3):937–940 108. Khatri VP et  al (1999) Endogenous CD8+ T cell expansion during regression of monoclonal EBV-associated posttransplant lymphoproliferative disorder. J Immunol 163(1):500–506 109. Porter DL, Orloff GJ, Antin JH (1994) Donor mononuclear cell infusions as therapy for B-cell lymphoproliferative disorder following allogeneic bone marrow transplant. Transplant Sci 4(1):12–14 discussion 14–6 110. Papadopoulos EB et al (1994) Infusions of donor leukocytes to treat Epstein–Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation. N Engl J Med 330(17):1185–1191 111. O'Reilly RJ et  al (1997) Biology and adoptive cell therapy of Epstein–Barr virus-associated lymphoproliferative disorders in recipients of marrow allografts. Immunol Rev 157:195–216 112. Gustafsson A et al (2000) Epstein–Barr virus (EBV) load in bone marrow transplant recipients at risk to develop posttransplant lymphoproliferative disease: Prophylactic infusion of EBV-specific cytotoxic T cells. Blood 95(3):807–814 113. Rooney CM et al (1998) Infusion of cytotoxic T cells for the prevention and treatment of Epstein–Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 92(5):1549–1555 114. Wagner HJ, Rooney CM, Heslop HE (2002) Diagnosis and treatment of posttransplantation lymphoproliferative disease after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 8(1):1–8 115. Gottschalk S et al (2001) An Epstein–Barr virus deletion mutant associated with fatal lymphoproliferative disease unresponsive to therapy with virus-specific CTLs. Blood 97(4):835–843 116. Nalesnik MA et  al (1997) Autologous lymphokine-activated killer cell therapy of lymphoproliferative disorders arising in organ transplant recipients. Transplant Proc 29(3):1905–1906 117. Haque T et  al (1998) Reconstitution of EBV-specific T cell immunity in solid organ transplant recipients. J Immunol 160(12):6204–6209 118. Haque T et  al (2007) Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: Results of a phase 2 multicenter clinical trial. Blood 110(4):1123–1131 119. Gandhi MK et  al (2007) Immunity, homing and efficacy of allogeneic adoptive immunotherapy for posttransplant lymphoproliferative disorders. Am J Transplant 7(5):1293–1299

Chapter 35 Psychological Care of Adult Allogeneic Transplant Patients Flora Hoodin, Felicity W.K. Harper, and Donna M. Posluszny

1. Introduction Allogeneic Hematopoietic Stem Cell Transplant (HCT) is a complex and intense physical treatment, accompanied by intense psychological reactions, which can be further complicated by pre-morbid psychopathology or lingering and latent long-term psychological difficulties. The challenge in the clinical management of HCT patients is to accurately recognize psychological presentations that exceed normative responses to HCT and to draw on the strengths and expertise of psychosocial members of the multidisciplinary symptom management team for assessment and treatment. This chapter is a guide through the literature about psychological responses to and effects of HCT (anxiety, depression, cognitive difficulties) and the literature on evidence-based practice about psychological treatment. The unique contributions mental health professionals can make to care of HCT patients is also detailed. Clinical practice guidelines for psychological care are suggested, ranging from standard pre-transplant work-up of candidates to long-term follow-up of survivors. Possible adjustments in organizational systems and health care policies are outlined to assist transplant teams in providing more pro-active psychological care as called for by the 2006 Joint EBMT/CIBMTR/ ASMBT Recommendations. The gold standard of psychological patient care is to express appropriate concern and intervention at the appropriate point in recovery with the ultimate goals of ameliorating current distress in a timely way, preventing future distress, and maximizing patient physical and psychological well-being after allogeneic HCT.

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_35, © Springer Science + Business Media, LLC 2003, 2010

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1.1. Overview of Psychological Impact of HCT Studies of post-transplant adjustment indicate a number of those surviving beyond 1 year fare relatively well. Despite often significant treatment-related sequelae, patients who remain in remission report being grateful for a “second birthday” and a renewed chance to live [5]. They report good physical health and little psychological distress, they display good performance status and psychosocial functioning, and they have often resumed many of their previous activities ([6–10]; for reviews of QOL outcomes across allogeneic and autologous patients, see [11–13]). Despite these signs of positive recovery, there is clear evidence of variability in functioning and recovery among survivors of allogeneic HCT. A significant percentage suffers long-term negative physical and psychological effects and poor quality of life [8, 14–22]. Moreover, patients often have impairments across multiple domains of functioning including physical, emotional, role, vocational, social, sexual, and cognitive domains [13, 23, 24]. Some patients even report feeling psychologically worse despite having improved physical health [25]. The variability in physical and psychological functioning appears largely related to the numerous challenges patients face over time from pre- to posttransplant. Regardless of previous treatment history, past treatment failure, familiarity with the medical system, prognosis, or risk of relapse, all transplant patients face the strain of medical decision-making, lengthy pre-transplant work-ups, conditioning regimen and side effects, and a long, tedious, and uncertain path to recovery. The acute phase just before and after transplant is a period with perhaps the greatest challenges and demands, and therefore, not surprisingly, tends to show the greatest variability in patient functioning [24, 26–28]. Patients are challenged by acute and chronic treatment toxicities (e.g., hepatic or pulmonary complications), treatment side effects (e.g., fatigue, nausea, chronic immunosuppression, infertility and other sexual dysfunction, changes in body image) [13, 29, 30], and practical demands (e.g., numerous medical visits, physical isolation, complicated infection control procedures, lengthy hospital stays, intense outpatient regimens, and even relocation to be in close proximity to the hospital). Further, once the acute physical and psychological impact of treatment passes, longer-term recovery brings a new set of challenges for patients including fears about recurrent disease; difficulty with social reintegration; financial concerns; psychological disturbance including anxiety and depression [19, 20, 31–34], chronic physical complications (e.g., chronic graft versus host disease), and “chemo-brain” or cognitive dysfunction [35–37]. Given the intensity and prolonged nature of these treatment challenges, it is not surprising that many, if not the majority of patients, show transitory and temporary difficulties in psychological functioning across the transplant period. Psychologically, the period from pre-transplant admission to immediate post-transplant recovery (e.g., within 7–14 days post) seems particularly difficult for patients, who are both anticipating the transplant and subsequently adjusting to periods of isolation and infection control [38]. Many studies report the highest levels of patient depressive and anxiety disorders during this time [39–42] with pre-transplant rates of depression as high as 30% [8, 43, 44] and rates of anxiety as high as 40% [8, 25, 38, 44, 45] before admission to the hospital.

Chapter 35  Psychological Care of Adult Allogeneic Transplant Patients 

The good news is that most studies find emotional (anxiety and depression) and cognitive symptoms slowly decrease post-transplant with many patients reaching “recovery” within the first year post-transplant [15, 25, 36, 38, 39, 46]. The bad news is that a maximal point of “recovery” for some patients might be less than optimal. Thus, while many patients may improve during the first year after transplant, some may continue to experience psychological impairment beyond the initial transplant period [47, 48], reach only a suboptimal level of recovery, and have no further gains in functioning beyond that point [8, 10, 14, 16, 17, 23, 49, 50]. As many as 40% of a sample of autologous and allogeneic patients who had returned home (average 25 months post-HCT) were currently depressed [41]. In fact, only a minority describe themselves as “returned to normal” in periods up to 3 years post-HCT; even 6-years post-HCT patients report adjustment difficulties and feeling “stuck” in their recovery [50]. A number of studies have also found pre-transplant psychological functioning to be a robust predictor of post-transplant functioning [8, 22, 26, 40, 48, 51]. There is even some suggestion that self-reported pre-transplant psychological as well as physical functioning might be more important predictors of posttransplant self-reported recovery than clinical or demographic factors such as disease stage and type or patient age [26]. Studies have also shown a relationship between psychological impairment, particularly depression, and post-transplant mortality [47, 52–57]. Although evidence for a causal relationship between depression and mortality has been mixed, owing in large part to differences in assessment methods and patient groups [47, 51, 53, 54, 57–64], this line of research strongly suggests a need to consider the influence of patient psychological functioning on long-term recovery from HCT [65] and underscores the need for careful and close surveillance of patients’ psychological well-being. 1.2.  Clinically Significant Psychological Problems and When to Refer The psychological impact of HCT can manifest in two major domains: emotional disorders or cognitive dysfunction. 1.2.1.  Emotional Disorders Given that HCT involves the threat of serious medical complications and even potential mortality, some amount of psychological distress is expected in most patients. HCT teams should therefore be particularly alert in recognizing when transitory (even normative) changes transition to severe distress and reach a clinically significant (and therefore, diagnostic) level of psychological impairment. As shown in Table 35-1, clinically impaired patients may develop psychiatric diagnoses related to mood, substance abuse or dependency difficulties, or body image issues. Anxiety and depressive disorders tend to be the most common diagnoses. Patients may display symptoms of either both anxiety and depressive disorders, or equally possible, high levels of one but not the other [18, 25]. Symptoms of anxiety may include excessive worry, difficulty controlling, ignoring, or redirecting upsetting thoughts, ideas, or visual images; poor concentration; restlessness; irritation; and emotional numbness [66]. Anxiety disorders may also prompt a number of distressing patient behaviors. For example, anxious patients may exhibit poor medical adherence, refuse

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Table 35-1.  Psychological symptoms requiring clinical attention. Emotional issues • Anxiety (e.g., post-traumatic stress disorder, panic disorder, generalized anxiety disorder, obsessive-compulsive disorder) • Depression (e.g., major depressive disorder, bipolar disorder, adjustment disorder with anxiety and depression) Substance Abuse – Dependence or excessive use of substances that may be a contraindication for treatment and/or interfere with medical treatment • Nicotine • Prescription pain (e.g., oxycodone, morphine) or psychiatric (e.g., Xanax) medications • Alcohol • Illegal drugs Miscellaneous issues • Excessive preoccupation, concern, or focus on physical symptoms • Body image issues (e.g., secondary to development of fibrosis, Hickman catheter) • Anticipatory nausea • Poor adherence to medical regimen (e.g., missed appointments, refusal to adhere to diet restrictions, or to engage in physical therapy)

to take medications as prescribed, fail to follow treatment recommendations, delay making appointments or initiating treatment, or frequently cancel or miss scheduled appointments. Anxious patients may also develop nausea prior to treatment (in contrast to more normative nausea during treatment), reflecting a learned response called anticipatory nausea [67]. Anxiety can manifest as worrisome, intrusive, or ruminative thoughts about the future including discharge from the hospital (e.g., “Who will take care of me? What if my caregivers or I don’t know what to do?”), success of the treatment, recurrence, relapse, treatment complications, and even death (e.g., “What if I develop GvHD? What if the disease comes back? What if I die?”). Patients may also display an excessive, almost obsessive, focus on medical symptoms or concerns (e.g., frequently consulting the medical team about minor or multiple symptoms); express excessive fears about developing infection through exposure to certain environments, situations, or people; or engage in compulsive behaviors (e.g., excessive hand-washing) because of fears of developing an infection. A common side effect of anxiety often noticed by medical professionals is insomnia (i.e., patients are unable to fall asleep or frequently awaken secondary to worry thoughts). Patients can develop depressive symptoms from coping with the many disease- and treatment-related challenges of HCT: loss of personal independence, loss of identity as capable or strong, diminished social roles at work or home, impaired memory, compromised health, loss of fertility, and decreased physical attractiveness. These concerns can engender feelings of hopelessness or helplessness, which are hallmark emotions underlying clinical depression. As with anxiety, the challenge in recognizing clinical depression is that many of its symptoms, such as tearfulness, pessimism, feeling down or “blue,” or feeling guilty (e.g., for becoming ill and causing stress to the family) can be considered normal reactions to diagnosis, diminished health status, or poor

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prognosis. Transitory feelings of hopelessness about the future, having little interest in spending time with people or doing previously enjoyed activities may simply reflect feeling physically ill; chronic feelings of hopelessness are more worrisome. The clinical presentation of depression is further complicated because somatic symptoms of the disorder (i.e., fatigue, difficulty concentrating, weight loss or gain, and insomnia or hypersomnia) may mimic transplant side effects [66]. Thus, distinguishing treatment-related symptoms (e.g., depressive or agitated mood due to corticosteroids) from symptoms of clinical depression, and even simple boredom [68] resulting from long hospital stays and physical isolation, can also present a challenge to health care providers. Given the often fine nuances in symptom presentation, accurately distinguishing between normative reactions and clinical anxiety or depression requires special training and underscores the need for early referrals to qualified mental health professionals for assessment and intervention 1.2.2. Cognitive Dysfunction In addition to the emotional impact of HCT, a significant proportion of patients (8–63%) display post-transplant impairment in cognitive functioning. Appearing either during or in the few months after transplant, adverse effects often involve deficits in memory, executive functioning (abstract reasoning, planning, decision-making), and psychomotor skills (motor speed and dexterity) (see Table 35-2). As with emotional disorders, some studies show recovery to pre-transplant levels of cognitive functioning within 12 months of transplant [36, 69] except for grip strength and motor dexterity [36], suggesting that acute neurocognitive side effects may not result in permanent or long-term impairment. However, continuing impairment in at least one domain has been documented around the 1-year mark for 51% [35] to 74% [36] of patients: specifically 11–22% in executive functioning, 33% in verbal fluency and memory [36], and 11–46% in psychomotor functioning [36, 37]. A further subset of patients (11%) displayed impairment in verbal memory at 12 months that had not otherwise been present before [37], suggesting a latent effect of treatment. Neuropsychological side effects may result from multiple potential biomedical sources. Treatment regimens expose patients to a variety of potential CNS toxicities (e.g., conditioning agents, immunomodulatory agents, infection, and metabolic effects of treatments) [77, 78], and neurological changes such as cortical atrophy or white matter lesions have been shown in imaging studies of HCT patients after conditioning chemotherapy, total body irradiation, and receipt of cyclosporine or tacrolimus treatments for GvHD [35, 74, 79, 80]. Of particular relevance for allogeneic patients is that neurological complications occur more frequently in these grafts than autologous grafts [81]. Specific risk factors for cognitive impairment in allogeneic patients include previous chemotherapy prior to transplant [36]; total body irradiation [82]; cyclosporine [36, 74], tacrolimus, and mycophenolate mofetil [36]; CNS changes [35, 74] or delirium during HCT [83] (see Table  35-3). The mechanisms underlying cognitive declines are not yet well understood [84, 85], but are hypothesized to involve direct neurotoxicity leading to demyelination, secondary inflammatory response, or microvascular injury, modulated by individual genotypes such as the e4 allele of Apolipoprotein E [86, 87]. It is noteworthy that prior to HCT conditioning, up to half of patients (4–53%) demonstrate cognitive deficits in attention, memory, executive functioning, and psychomotor functioning (see Table 35-2). Even in one study in

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2–11

40

Executive Memory Motor Total function

23–32

Executive function

15–20

2

37

Memory Motor Total

Short-term post-HCT (<365 days) Long-term post-HCT (≥365 days)

0–29

25

5–26

35

Memory Motor Totald

Notes: (1) percentages are rounded to nearest full digit; (2) studies which do not provide percentages are not reported in this table; (3) samples that contain autologous in addition to allogeneic patients are noted in column 1. MRD matched related donor, MUD matched unrelated donor, BMT bone marrow transplantation a Executive Function: Abstract reasoning including planning, organization, problem-solving; also may reflect processing speed and verbal fluency b Memory: Verbal or visual immediate and/or delayed recall c Motor: Psychomotor strength, speed, dexterity d Total: Index comprised of average z score for each test in battery e Impairment on at least one test

Harder et al. [76]; 66 allogeneic: 42 MRD, 24 MUD 35 autologous, 82 hematology controls

Harder et al. [75]; 35 allogeneic: 26 MRD, 9 MUD 5 autologous; 22–82 months post BMT

Padovan et al. [74]; 59 allogeneic, 7 autologous; 34 ± 26 months post BMT

Booth-Jones et al. [73]; 12 allogeneic, 53 autologous; 6 months post BMT

34–43

8–16

Executive Memory Motor Total function

Source; Sample size; Timing of assessments

Andrykowski et al. [72]; 55 BMT candidates; pre-BMT 4–33

Pre-HCT

20

Cross-sectional studies

11

0–41

Meyers et al. [71]; 42 allogeneic: 25 MRD, 17 MUD 19 autologous: pre-BMT, 2 weeks post BMT, on discharge from hospital, 8 months post BMT

8–12

58e 8–24

0

5–26

Harder et al. [37]; 16 allogeneic, 5 autologous pre-HCT; 6 months (n = 12); 12 months (n = 9)

Sostak et al. [35]; 71 allogeneic; pre-HCT, 14 ± 3 months

Schulz-Kinderman et al. [70]; 39 allogeneic pre-HCT; 16–21 100 days (n = 19)

27

Source; Sample size; Timing of assessments 32

Pre-HCT Executive Executive Executive function Memory Motor Totald function Memory Motor Totald function

Prospective studies

21

Table  35-2.  Cognitive impairmente of allogeneic HCT recipients: percentage of sample impaired in executive functiona, memoryb, and psychomotor functionc.

Syrjala et al. [36]; 161 allogeneic; pre-HCT, 80, 365 days

F. Hoodin et al.

Short-term post-HCT (<365 days) Long-term post-HCT (³365 days)

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Chapter 35  Psychological Care of Adult Allogeneic Transplant Patients 

625

which verbal memory recovered to baseline after transplant [36], the baseline level of memory was itself below expected norms for 32% of patients, suggesting that HCT treatment may not be solely responsible for compromising cognitive function. Other contributing factors may be prior chemotherapy (see Table 35-3) or the disease process itself. Emotional distress has also been implicated as a cause of cognitive impairment. Studies show an inverse relationship between distress and cognitive functioning across all transplant phases including pre-transplant [71], immediately post-transplant [37, 71, 73], and long-term recovery [37, 75]. However, multivariate analyses, which take into consideration disease and

Table  35-3.  Risk factors for cognitive impairment in patients undergoing HCT (measured using objective neuropsychological tests). Cognitive functions affected Risk factors

Executive functiona

Psychomotor Memoryb functionc Totald Source

Chemotherapy X

Pre-transplant systemic chemotherapy, but not hydroxyurea alonee Pre-transplant high -dose ara-C Pre-transplant CNS disease plus X intrathecal chemotherapy

X

Syrjala et al. [36]

X

Andrykowski et al. [72]

X

Andrykowski et al. [72]

Radiation Cranial radiation Total body irradiation (TBI)

X

X

Xf

Andrykowski et al. [72] Andrykowski et al. [82]

GvHD Chronic GvHD developing from acute GvHD

X

GvHD (unspecified whether acute or chronic)

X

Padovan et al. [74] X

X

Jacobs et al. [88]

Immunosuppressants Cyclosporine (> 1 year)

X

Padovan et al. [74]

CyclosporineTacrolimus

X

Mycophenolate mofetil

X

Syrjala et al. [36]

X Delirium during HCT X

Fann et al. [83]

Older age: >40 years X a

Padovan et al. [74]

Executive function: Abstract reasoning including planning, organization, problem-solving; also may reflect processing speed and verbal fluency b Memory: Verbal or visual immediate and/or delayed recall c Psychomotor function: Motor strength, speed, dexterity d Total: Index comprised of average z score for each test in battery e Almost entire sample received busulfan and cyclophosphamide as conditioning chemotherapy f Self-reported, not measured by objective neuropsychological tests

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treatment history, show the relative contribution of emotional distress fades over time [72], suggesting that disease and treatment (not emotional factors) are likely the primary causes of neuropsychological impairments. Transplant-related cognitive deficits can have a significant negative effect on overall quality of life, and social, role, and occupational functioning. More critically, difficulties with short-term memory, even mild ones, may affect a person’s ability to recall medically- relevant names, dates, and instructions or complete tasks requiring sustained or coordinated attention, thus potentially compromising the patient’s ability to adequately participate in their medical care. Cognitively impaired patients may also experience difficulties keeping follow-up appointments, adhering to treatment and medication regimens, or engaging in appropriate self-care, which may contribute to increased post-transplant morbidity, and possibly, even mortality: Syrjala et  al. [36] documented a mortality hazard ratio of 2.84 for patients with at least one neurocognitive impairment at 80 days post-HCT compared to patients with no cognitive impairments. As with emotional disorders, detecting and quantifying the extent of the cognitive impairments in HCT patients requires special training, hence the need for referrals to qualified mental health professionals for appropriate assessment and follow-up care. 1.3.  Issues in Psychological Assessment A number of different factors have been shown to place patients at higherrisk for poor psychological outcomes. Although findings have been mixed for some factors (e.g., gender, marital status, time since transplant, chronic GvHD), all studies (see Table  35-4) agree on the impact of factors from three patient domains: demographic, biomedical or treatment-related, and psychosocial. 1.3.1.  Clinical Interview Given the difficulty in distinguishing clinical impairment from normal reactions to transplant, good clinical practice dictates that HCT candidates be continuously monitored for psychological problems including emotional and neuro-cognitive impairments. This assessment should begin prior to transplant with a full psychological evaluation including clinical interview, and, ideally, a brief neuropsychological battery to establish a baseline for each patient. The psychological evaluation would assess, within the context of patient values and goals, functioning in three psychosocial sub-domains that influence the course of coping with and recovery from transplant: psychological stability, history of healthy lifestyle and self-care behaviors, and transplant-specific coping and resources (see Table  35-5). Quantifying these facets as specified by the Transplant Evaluation Rating Scale (TERS; [97]) has been shown to have clinical utility in effectively flagging HCT candidates at risk for poorer posttransplant adjustment [98]. If a neuropsychology service is available to conduct a brief neuropsychological assessment, the assessment battery should include evaluation of memory, executive functioning, and psycho-motor skills. 1.3.2. Assessment Tools Assessments of psychological and cognitive (if warranted by concerns of the treatment team, patient, or family) functioning should ideally recur at frequent intervals throughout the transplant period. Post-transplant assessments at 6- and 12- months and annually thereafter are also advocated by the

Chapter 35  Psychological Care of Adult Allogeneic Transplant Patients 

Table 35-4.  Risk factors of poor psychological adjustment and quality of life after HCT. Demographic Domain Agea [9, 14, 16, 20, 21, 44, 89–92] Gendera [9, 14, 15, 20, 21, 26, 44, 47, 92, 93] Marital statusa [8, 26, 93] Lower education [9, 16, 20, 90] Biomedical/Treatment Domain Pre- and peri-transplant   History of previous medical treatment [15] (pre)   Advanced disease at time of transplant [16] (pre)   Pain [44] (pre)   Poorer phFysical functioning [8, 26, 44, 47] (pre); [22, 42] (peri)   Higher dosage total-body irradiation during pre-BMT conditioninga [16, 89, 90] (peri) Post-transplant   More physical symptoms [18, 20, 22] (post)   Greater treatment-related toxicity [44] (post)   More transplant-related medications [47] (post)   Shorter time since transplanta [14, 20, 39, 89–91] (post)   Disease relapse [26, 91] (post)   Impotence [20] (post)   History of more major infections [20] (post)   Chronic GVHDa [7, 8, 14–16, 26, 89, 94] (post)   Poorer physical functioning [9, 93] (post) Psychosocial Domain General psychological stability, pre-transplant   Past psychiatric history [44] (pre) Transplant-related coping and resources, pre-transplant   Low emotional functioning (e.g., distress, poor QOL) at transplant [22, 26, 40, 47, 48] (pre)   Poor perceived personal control [40] (pre, peri, post)   Discordance between pre- and post-transplant expectations [95] (pre)   Low vitality and energy [47] (pre)   More optimistic expectations [96] (pre)   History of smoking [44] (pre)   Family conflict [8] (pre)   Poor social support [15] (pre) Transplant-related coping and resources, post-transplant   Return to work [18, 47] (post)   Poor perceived health [47] (post)   Current psychiatric history (e.g., depression, anxiety) [18, 41] (post)   Poor social support [9] (post) a

Indicates findings have been mixed across studies

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Table 35-5.  Recommendations for psychosocial sub-domains to be assessed in pre-transplant clinical evaluations. Clinical interview of pre-transplant candidates: sub-domains to be evaluated General psychological stabilitya Past and present psychiatric history   Mood, anxiety, and thought disorders   Past and present psychological treatment   Past and present psychiatric medications   Family history of psychological or psychiatric difficulties Past and present substance use History of coping with life stress and crises Healthy lifestyle and self-care behaviorsa History of healthy lifestyle behaviors   Exercise   Diet   Non-smoking History of coping with prior disease and treatment   Adjustment to and experiences during prior long hospitalization  Relationship with and ability to communicate with treatment team History of adherence with previous treatment Transplant-specific coping and resourcesa Current emotional stability, volatility, distress Anticipatory anxiety Current mental status   Ability to comprehend diagnosis, treatment plan, prognosis Family/social support   Caregiver availability  Caregiver emotional stability Brief Neuropsychological Batteryb Executive function   Abstract reasoning   Problem-solving   Verbal fluency   Processing speed Memory   Verbal immediate and delayed recall   Visual immediate and delayed recall Psychomotor function   Motor speed   Motor dexterity a

Based on Transplant Evaluation Rating Scale [97] If neuropsychology service is available to conduct the evaluation

b

2006 Joint Recommendations of the European Group for Blood and Marrow Transplantation, Center for International Blood and Marrow Research, and the American Society for Blood and Marrow Transplantation (EMBT/ CIBMTR/ ASBMT) [34]. The use of evidence-based, psychological instruments is highly

Chapter 35  Psychological Care of Adult Allogeneic Transplant Patients 

recommended to establish accurate levels of pre-transplant psychological functioning in patients and allow monitoring of changes throughout transplant and recovery. Many assessment instruments have been previously used in clinical and research contexts. However, use of any one specific instrument is less important than ensuring that instruments have sound psychometric features (e.g., reliability and validity), validation in the HCT population, and relatively low patient burden. One screening instrument with extremely low patient burden is the National Comprehensive Cancer Network’s (NCCN) Distress Thermometer (DT) [99, 100]. In preliminary validation, the DT demonstrated a low false negative rate for HCT patients, suggesting its success in avoiding the problem of under-recognition and under-treatment of patient distress. A relatively high false-positive rate (23%), however, could also unnecessarily burden providers [101] and drain typically limited psychosocial resources. Several alternate instruments may serve as brief supplemental screening tools for HCT patients at any phase of treatment (see Table 35-6 for a list of suggested and alternate measures). All these instruments, however, assess only for symptoms and should not be used for clinical diagnosis. Patients who trigger psychological concerns in either their screening or presentation to staff should be clinically evaluated by a licensed clinical psychologist who has specialized training in diagnosing psychological impairment. Given the negative effects of psychological distress on medical adherence [115] and satisfaction with medical care [116], the use of brief psychological assessments maximizes the ability of transplant teams to detect emerging psychological issues, seek appropriate referrals, and manage or ameliorate patient distress or impairment. Such a comprehensive and programmatic approach to assessment undoubtedly has beneficial effects on ongoing patient observation, consultation within the transplant team, psychological intervention, and clinical management.

2.  To Whom to Refer and Why: The Role of the Clinical Psychologist Multidisciplinary treatment teams, which draw on the strengths and expertise of individual disciplines, are the gold standard of patient care [117]. In addition to physicians and physician extenders (certified registered nurse practitioners, physician assistants), these teams can include a range of other medical professionals including pharmacists, occupational therapists, nutritionists, radiologists, pain specialists, hospice workers, and patient educators. Critical psychosocial expertise and support can be provided to these teams by social workers, clinical psychologists, and psychiatrists, each of whom has distinct skill-sets with which to address HCT patients’ psychosocial needs. 2.1.  Social Workers: Practicalities, Resources, and Patient/Family Support Groups Social workers in transplant centers typically handle resource-assessment and management for patients both in planning for transplant and in discharge from the hospital. They address practical issues such as facilitating referrals for childcare, transportation to treatment, financial assistance through federal, state, and charitable programs (e.g., My Friends Care), insurance concerns, and legal assistance (e.g., in family medical leave rights for caregivers).

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Table  35-6.  Suggested instruments for psychosocial assessment battery for baseline and repeated follow-up (based on ease of administration, low patient burden, good psychometrics, and clinical and research utility). Suggested basic assessment battery Domains assessed

Instrument

Patient burden:   Number of items   Time to completea

DSM-IV Axis I Psychiatric conditions

Mood disordersAnxiety dis- Patient Health Questionnaire 15 questions with skip-outs orders (PHQ) [102, 103] of additional sub-questions when appropriate Eating disorders criteria are not met Alcohol abuse disorders < 3 min Somatization

Quality of life

Physical well-being Social/family well-being Emotional well-being

Functional Assessment of 47 items Cancer Therapy – Bone 5–10 min Marrow Transplant (FACT – BMTv4) [104]

Functional well-being Quality of patient’s relationship with MD BMT/HCT-specific concerns Cognitive functioning

Mental acuityConcentration Functional Assessment of 50 items Cancer Therapy Cognitive 5–10 min* (may take Verbal memory Scale (FACT-Cog) [105] patients who are physiNon-verbal memory cally compromised longer) Verbal fluency

Impact of cognitive functioning

Functional interference Deficits noted by others Change from previous functioning

Distress in past week Rated 0–10 Patient current prob- Practical lem list Family

Distress Thermometer (DT) [99–101]

1 item plus checklist 5 min

Emotional Spiritual/religious Physical Alternate assessment instruments (non-HCT specific; can be added for in-depth assessment in select areas) Domains assessed

Instrument

Patient burden:   Number of items   Time to complete

Depressed mood

Beck Depression Inventory-II 21 items (BDI-II) [106] 5 min Center for Epidemiological Studies-Depression Scale(CES-D) [107]

20 items 10 min

Brief Symptom Inventory (BSI-18) [108]

6/18 items 4 min

Hospital Anxiety and 7/14 depressive items Depression Scale (HADS) 5 min [109] (continued)

Chapter 35  Psychological Care of Adult Allogeneic Transplant Patients 

Table 35-6.  (continued) Anxiety

Brief Symptom Inventory (BSI-18) [108]

5/18 items 4 min

State-Trait Anxiety Inventory 40 items (STAI) [110] 10 min Hospital Anxiety and 7/14 anxiety items Depression Scale (HADS) 5 min [109] Posttraumatic stress

Quality of life

PTSD Checklist – Civilian Version (PCL-C) [111, 112]

17 items 5–7 min

Impact of Events Scale – Revised (IES-R) [113]

22 items <20 min

Functional Living IndexCancer (FLIC) [114]

22 items <5 min

a

Patients who are physically compromised will likely need longer than the listed completion times

Social workers or nurses may also lead weekly or monthly support groups for patients and families during and after hospitalization. These groups typically provide emotional support and information to educate patients about the “normal trajectory” of recovery [118]. In some transplant centers, these professionals also provide individual supportive counseling with a focus on managing the stress of transplant and its impact on the patient and family. Depending on the setting, social workers are often among the first to identify those patients in need of referral to psychology and/or psychiatry. 2.2.  Clinical Psychologists: Review of Evidence-Based HCT Psychological Treatment Clinical psychologists in transplant centers can make a vital contribution to HCT teams. Not only do these professionals have doctoral-level training in the assessment and treatment of psychiatric disorders, but they also frequently have additional training in behavioral medicine, health psychology, and/ or psycho-oncology. Trained at the interface of psychology and medicine, clinical psychologists can quickly identify cognitive, emotional, or behavior changes in patients across the transplant process – from pre- to post-transplant – as well as provide consultation to the treatment team for problematic patients or patients being discharged to problematic home environments. Psychologists can also facilitate communication between patients and medical or palliative care professionals. Moreover, once clinically significant distress or dysfunction has been identified, psychologists are trained to provide clinical interventions and psychotherapy to patients and their families. The review of psychological interventions (below) summarizes the results of randomized controlled trials that have targeted somatic and emotional symptoms in both allogeneic and autologous HCT patients (see Table 35-7). Given the limited study in this area, the review is supplemented by findings from the general psycho-oncology intervention literature and an overview of its implications for HCT patients.

631

Fatigue

Nausea

Pain (mucositis)

Somatic side effects

Outcome variables

RCT, n = 110

+

Effective postdischarge to 6 months, not 6–12 months post HCTb

7 days post HCT

7 days post HCT

Autologous Autologous

RCT, n = 60

Autologous RCT, n = 110

RCT, n = 110

Unrandomized Allogeneic n = 23, matched controls n = 19

Relaxation with music therapy

+

Unspecified

RCT, n = 94

Unspecified

Autologous

0

+

0

+

Unspecified

Graft type

Unrandomized Allogeneic n = 23, matched controls n = 19

RCT, n = 94

Study design, sample sizea

Relaxation with RCT, n = 45 guided imagery

7 days post HCT

0

Comments

+

+

Supportive/ expressive

Relaxation with music therapy

+

Relaxation +  mindfulness

Psychodynamic intervention

+

+

Relaxation

Relaxation +  cognitive restructuring

Cognitive-behavioral intervention

Table 35-7.  Impact of psychological interventions with HCT patients on somatic and emotional symptoms.

Frick et al. [123]

Gaston-Johnson et al. [121]

Gaston-Johnson et al. [121]

Sahler et al. [120]

Syrjala et al. [119]

Syrjala et al. [122]

Gaston-Johnson et al. [121]

Sahler et al. [120]

Syrjala et al. [119]

Source

632  F. Hoodin et al.

+

0 +

+

RCT, n = 42

Autologous

Effective postdischarge to 6 months, not 6–12 months post HCTa

RCT, n = 60

Autologous and allogeneic

Autologous

Allogeneic

Allogeneic

Autologous

Uncontrolled pilot, n = 24

RCT, n = 110

RCT, n = 42

Single case

Not reported

Autologous

Allogeneic

Minimal contact RCT, n = 26 workbook, 2–6 months postHCT

During hospitalization

7 days post HCT

Relaxation with “exercise”

Pre-HCT

3 years post BMT Single case

7 days post HC T RCT, n = 110

Relaxation with “exercise”

Frick et al. [123]

Trask et al. [128]

Horton-Deutsch et al. [127]

Gaston-Johnson et al. [121]

Kim and Kim [124]

Kopp et al. [126]

DuHamel et al. [125]

Gaston-Johnson et al. [121]

Kim and Kim [124]

“+” = statistically significant positive response of intervention; “0” = no statistically significant response due to intervention; blank cells indicate the effect of intervention was not assessed a RCT denotes Randomized Controlled Trial b During hospitalization all patients received psychotherapeutic support consisting mainly of CBT components of counseling, relaxation, guided imagery, biofeedback

Quality of life

Negative mood

+

+

Depression

Claustrophobia

0

+

+

PTSD

Anxiety

Emotional symptoms

Chapter 35  Psychological Care of Adult Allogeneic Transplant Patients  633

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2.2.1.  Psychological Intervention Literature for HCT Patients Somatic side-effects and complications of treatment, including pain, nausea, and fatigue, have been targeted behaviorally with moderate success in well-controlled trials that utilized relaxation training alone [119, 120] or in combination with cognitive restructuring (e.g., modifying unrealistic perceptions or beliefs) [119, 121]. Both these treatment strategies belong to the larger category of interventions known as Cognitive Behavioral Therapy (CBT). Emotional effects of treatment (e.g., distress, depression, and anxiety) have responded positively to CBT techniques of relaxation alone [124] or in combination with focused awareness or “mindfulness” [127]. Two case studies also suggest anxiety disorders specific to HCT may remit with CBT intervention [125, 126]. Further, quality of life at 2- and 6-months post-transplant has been shown to improve with a “guided imagery” intervention [123] and a “minimal contact” CBT intervention (educational workbook supplemented by an orientation and instructions) [128]. The effects of individual supportive counseling and/or weekly or monthly support groups run by paraprofessionals, social workers, or advanced nurse practitioners have not been empirically examined in controlled trials, so their effects are unknown. 2.2.2.  General Psycho-Oncology Intervention Literature Although the modest HCT literature points to a critical shortage of sound empirical intervention studies in HCT patients, the findings are generally consistent with the larger evidence-base of psychological intervention for oncology patients. As in the HCT literature, meta-analyses in oncology patients indicate CBT yielded moderate mean effect sizes1 when provided preventatively to all patients, regardless of whether they had clinical or subclinical levels of somatic symptoms such as pain [129–131] and nausea [129, 130] or emotional symptoms of anxiety [129, 130], depression [129, 130], and illness-related distress [130–132]. Moderate effect sizes are considered to be indicative of medium-sized clinically meaningful effects [133]. However, large effect sizes which are sufficient to shift a patient from the diagnostic or clinical range into the subclinical range [134] have been achieved in cancer patients with pain, or patients pre-selected for significant nausea and vomiting [129], or clinical (as opposed to sub-clinical) levels of anxiety or depression [134]. Thus, in general, psychological interventions show moderate benefit for patients with symptoms at sub-clinical levels, and large benefit for patients with clinical or diagnostic-level symptoms. The more extensive psycho-oncology literature has important implications for designing, testing, and implementing HCT psychological interventions, since HCT and cancer patients experience similar psychological concerns and

1 Effect size is an index of the size of a treatment effect. It is independent of sample size, but takes into consideration the variation of scores around the mean (standard deviation) of the measure being used. Said another way, effect size is a measure of change in standard deviation units, where change might be the difference between two groups or the difference between the mean pre- and post-scores of a single group. For example, one formula for calculating effect size is Cohen’s d. It is derived by dividing the difference between the mean pre- and post-scores on a measure by the standard deviation of that measure, or dividing the difference between the mean post-scores of the experimental group and control group by the pooled standard deviation of that measure.

Chapter 35  Psychological Care of Adult Allogeneic Transplant Patients 

reactions when facing their diagnosis, treatment, and prognosis. First, psychological interventions with more CBT/Social Cognitive Theory components2 have produced significantly larger effect sizes than those with fewer or no such components [135] in outcomes including global mood, depression, and objective measures of physical quality of life. Second, different modalities of treatment show effectiveness at different time points: psycho-education during diagnosis and pre-treatment when patients and families have high information needs [136]; CBT during extended treatments when stress-management, problem-solving, and emotional self-regulation skills are more critical [137]; and CBT and supportive groups for end-of-life patients when existential issues and life-priorities are especially salient [138]. Third, although current evidence indicates psychological intervention is unlikely to lengthen cancer patients’ survival [139–143], such intervention can significantly improve patients’ somatic and psychological functioning. Fourth, peer-led discussion/support groups are not adequate substitutes for intervention by skilled professionals [144], thus underscoring the need to include mental health professionals as part of treatment teams. So strong is the accumulated evidence for the efficacy of psychological intervention in cancer patients, that some advocate offering such services to cancer patients is not only ethically justified [136], but should be considered on the same footing as adjunctive medical therapies such as chemotherapy [145]. 2.3. Psychiatrists: Psychopharmacological Intervention Psychiatrists, who have expert knowledge in the assessment and psychotropic medication management of patients with severe medical illness, can be an invaluable resource to the HCT team. Psychiatric consultations for psychotropic medication should be considered for patients with impaired psychological functioning, particularly those not responsive to psychotherapy who are experiencing significant and persistent distress, or for those with a longstanding history of mental impairment illness. Psychiatrists can be especially beneficial if HCT physicians are uncertain about prescribing psychotropic medications, if the initial course of psychotropic medication prescribed was not effective, and/or if patients have organic psychological symptoms such as psychoses. Due to the dearth of controlled clinical trials of psychiatric medications with HCT patients and the relatively limited number with cancer patients [146], caution must be used when evaluating HCT patients for psychotropic medication. In patients with cancer, drug pharmacokinetics and pharmacodynamics can be significantly altered by psychotropic medications [147, 148]. Similar effects have been found in HCT patients [47], and therefore, any potential risk for negatively impacting their medical situation (e.g., organ toxicity) should be carefully balanced against any expected benefit of psychotropic medication. When psychotropic medications are determined to be appropriate, ­several factors need to be considered. First, some commonly used psychiatric

2

Social Cognitive components: Relaxation training, Coping (physical, spiritual, affect), Cognitive restructuring, Goal-setting, Problem-solving, Role-playing, Observational learning (modeling), Skill rehearsal, Self-monitoring thoughts, Goal-setting skills, etc.

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­ edications have potential immunological effects. For example, prolactinm elevating medications like risperidone may facilitate engraftment, but promote GvHD [149]. In addition, potential pharmacokinetic drug interactions between cyclosporine and some psychotropic medications should be monitored [150, 151]. Second, some psychotropic medications may be contra-indicated given the drug–drug interaction with any number of medications in the patients’ medical regimen, and the interaction between the psychotropic drug mechanism and the patients’ current medical status [152]. Drugs with longer halflives and higher potential to accumulate in the liver, for example, would be contraindicated for patients with hepatic failure, whereas shorter-acting SSRIs (sertraline, paroxetine) would be less problematic [153]. Third, the severity of patient psychological distress should be weighed against the 2- to 4-week therapeutic latency of many antidepressants [148]. Patients with more severe distress or depression may receive more immediate benefit from shorter acting drugs like psycho-stimulants [154]. Finally, the complexities of using psychopharmacological agents are multiplied when patients have a history of poor medical adherence, substance abuse, or over-use of prescription medications such as hydromorphone or benzodiazepines. A more positive benefit of psychotropic medications is that some have side effect profiles that provide relief for other common treatment-related symptoms. For example, the side effects of the antidepressant mirtazapine (Remeron) include increased appetite, weight gain, and sedation, which may be helpful for patients with cachexia and/or agitation [155]. Some anti-depressants have analgesic (e.g., amitriptyline) or soporific (e.g., trazodone) effects to treat pain and insomnia, respectively [153, 156]. Other medications (e.g., olanzapine) have similar beneficial effects on patient symptoms (e.g., nausea and emesis; [157, 158]). In general, psychotropic medications can provide powerful clinical benefit and serve as an effective complement to HCT treatment. However, they should be considered as only one facet of treatment for psychological concerns. In non-medical populations, psychotropic medications can lead to rapid symptom relief of psychiatric symptoms but their effects often do not last beyond the end of treatment (i.e., when patients stop taking them) [159, 160]. In contrast, CBT has lasting effects relative to medications, particularly in the treatment of anxiety and depression [161]. Similarly in HCT patients, as reviewed above, CBT interventions also provide much benefit for somatic and emotional symptoms. Therefore, treatment with a combination of medication and psychotherapy (CBT) for HCT patients who need psychotropic medication may yield maximum gains [47]. 2.4. Pathways for Psychosocial Care Optimal care for transplant patients includes consideration of how, when, and under what conditions to provide psychosocial services. Two sets of pathways for care can facilitate a systematic and programmatic approach to match the type or intensity of psychosocial screening or intervention to patients’ psychosocial needs. One set of psychosocial care pathways would focus on psychological needs during hospitalization period and preparation for discharge from the hospital. The second set would focus on psychological care during the extended recovery period thereafter.

Chapter 35  Psychological Care of Adult Allogeneic Transplant Patients 

2.4.1.  Pre- and Peri-hospitalization: Primary and Secondary Prevention, Intervention The model provided by the Transplant Evaluation Rating Scale (TERS; [97, 98, 162]) is useful for categorizing patients pre-transplant according to levels of psychosocial risk: none–-low, moderate, or high in each of several subdomains assessed in the psychological evaluation. For example, patients in the high risk category are likely those with tenuous psychological resources (e.g., poor coping strategies), difficulty maintaining self-control (e.g., problems with anger management or substance abuse), and a tendency to disregard social norms of behavior. These characteristics may predispose patients to nonadherence, poor adjustment to HCT, and/or poor post-transplant quality of life [98], thus reinforcing the need for closer screening. Further, this pre-transplant categorization of risk can be used to guide the level of care provided to patients during hospitalization. Patients at all levels of risk will likely benefit from basic training in coping with somatic side effects (e.g., pain, nausea) and the emotional stress of HCT. A stepped-care paradigm of care, however, which matches level of intervention to level of risk, will maximize resources and optimize patient care. Thus, low or no risk patients likely require only routine periodic monitoring (e.g., weekly). Moderate risk patients might require more frequent monitoring (e.g., twice weekly) during hospitalization and individual intervention as indicated. In contrast, patients in the high risk group would not only receive more frequent monitoring (e.g., three times per week) but also receive more frequent and intensive psychological intervention as needed. For example, these patients would likely benefit from brief bedside interventions (e.g., 15-min coping skill-oriented sessions), more in-depth outpatient sessions (depending on medical status), and/or psychiatric consultations for psychotropic medications. Preparation for discharge from the hospital is another important opportunity for strategic prevention and intervention. Patients and caregivers often feel overwhelmed with the practical and emotional aspects of discharge. Compliance with a multi-faceted self-care and daily medication regimen can be daunting, particularly in contrast to the intense vigilance and support of the medical and allied health care staff during hospitalization. Reintegration into social roles might also be demanding for patients who are still experiencing physical sequelae (e.g., fatigue) and feeling taxed by the demands of frequent follow-up appointments. A slower return to “normal” than expected can jeopardize patient well-being and create depression, anxiety, or anger, which might adversely affect capacity for self-care. Again, a stepped-care paradigm of care can guide the allocation of psychosocial resources during this critical transition. Patients at all levels of risk (and their caregivers) likely would benefit from basic psycho-education and instruction in coping skills to enhance adherence and self-care, while promoting gradual reintegration into personal and social roles. Social workers can support these efforts through resource planning and management for services patients might need once they return home (e.g., home health care). In contrast, moderate and high risk patients might need more proactive intervention (e.g., “relapse prevention”), which can be offered on an individual basis. Relapse prevention helps patients identify personal “triggers” (e.g., events or situations) that heighten stress, trigger negative emotions, or decrease motivation for

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appropriate medical adherence. Once triggers are identified, a collaborative problem-solving process can be used to develop a tailored coping plan for each patient. Including caregivers of these higher risk patients in relapse prevention interventions could have the added benefit of enhancing caregiver coping and/or decreasing strain or burden, thus providing further, if indirect, benefit to patients. 2.4.2. Post-hospitalization: Surveillance, Screening, Intervention A final cornerstone of proactive planning for psychological well-being is arranging for appropriate outpatient psychological or psychiatric follow-up care for patients who need psychological support and intervention after discharge. The 2006 Joint EBMT/CIBMTR/ASBMT Recommendations, which call for regular and scheduled monitoring of post-hospitalization psychosocial adjustment [34], can be categorized into three pathways for psychosocial care. The model described below follows the recommendations of Holland and Reznik [163], who, as an extension of the NCCN clinical guidelines for distress management, advocate matching level of psychosocial care to need in cancer survivors. Again, low or no risk patients (i.e., those with only sub-clinical symptoms and/or existential concerns) might benefit from more traditional psychotherapy sessions to enhance adjustment. Although Holland and Reznik [163] advocate for referrals to community mental health professionals, it could be argued that HCT-related issues are so complex that community psychologists do not have the specialized understanding of issues required for this patient group, and therefore, HCT patients might be better served in HCT clinics. Low-to-no risk patients should also receive brief screenings at 6 and 12 months post-HCT, and annually thereafter, to assess any long-term or latent psychological issues. Moderate risk patients (i.e., those who are physically healthy but have psychological sequelae) should receive regular (e.g., monthly) brief screenings (such as the assessment battery suggested above). Further, a HCT team psychologist should be available for brief sessions on an as-needed basis if concerns are raised by these brief screens. These patients may also benefit from brief 15- or 30-min therapy sessions in the HCT outpatient clinic to provide rapid responsive attention to their psychological problems. Finally, as with during hospitalization and discharge planning, high risk patients (i.e., those with physical and psychological sequelae including cognitive or neuropsychological problems) likely need the most attention. Routine assessment for psychological problems should be provided at medical follow-up appointments for this group. Further, medical follow-up should routinely include appointments with the HCT team psychologist at intervals dictated by patient need and stamina. Psychiatric consultation and surveillance should also be considered, ideally with appointments also through the HCT outpatient clinic to minimize patient burden and maximize patient adherence to appointments. 2.5. Summary The matching of treatment to psychological need begins with the pre-transplant psychological evaluation and is followed by a screening protocol designed to identify at-risk or symptomatic patients throughout the transplant process and during the extended recovery period. Early identification permits timely inter-

Chapter 35  Psychological Care of Adult Allogeneic Transplant Patients 

vention, thereby reducing acute distress and likely preventing future distress. Especially but not only for transplant teams with limited resources to provide psychological services, the tailoring of psychological interventions to patient needs can provide both powerful clinical benefit and cost-effective care.

3. Special Issues: Treatment Adherence and Caregiver Role Newer HCT procedures and disease management guidelines have resulted in an increasingly greater proportion of treatment being administered on an outpatient, rather than inpatient, basis. In addition, the advent of nonmyeloablative transplantation, in which the toxicity of the pre-HCT conditioning regimen is reduced, has allowed patients who are not candidates for traditional HCT (due to advanced age or presence of medical co-morbidities) to undergo this milder form of HCT. Although these medical advances have increased treatment options and prolonged life, they have also exponentially increased the importance of patients’ adherence to stringent post-HCT care regimens at home, and in parallel, increased responsibilities for caregivers. In fact, some patients may not be able to take advantage of newer procedures because they do not have a caregiver at home [164]. Despite the centrality of treatment adherence and caregiving assistance to positive patient outcomes, the impact of treatment advances on these areas in HCT patients and caregivers has received little attention. 3.1.  Adherence to Treatment Regimen Medical adherence (i.e., the extent to which patient behavior coincides with medical recommendations; [165]) is crucial for patients undergoing allogeneic HCT. The risky and potentially life-threatening nature of an allogeneic HCT often requires an intense and lengthy outpatient regimen, which is even lengthier for nonablative HCT, or mini-transplants, that may be accomplished with a much shorter inpatient stay. Difficulties in treatment adherence for HCT patients are partly explained by general adherence theory: adherence tends to decrease as specific attributes (e.g., complexity, intrusiveness, side-effects and duration) of the regimen increase [166]. These attributes well describe the medical regimen required of HCT patients after hospital discharge. In addition to numerous, scheduled daily medications and multiple outpatient clinic appointments in the first 3 months post-transplant, patients must also closely manage their immuno-compromised state to prevent complications and opportunistic infections [167]. The National Marrow Donor Program’s [168] recommended restrictions for the first 100 days after HCT include wearing a mask in public places, and avoiding crowds, small children, and pets. Further, to prevent bacterial, viral, and fungal infections, proper care of catheters and frequent and thorough hand-washing is essential [169]. Patients are usually required to monitor their daily temperature in order to quickly recognize signs of infection. Further, patients who are neutropenic must follow a diet that avoids bacteria found in foods; most fresh fruits and vegetables, salad bars, raw and rare-cooked meat, fish, eggs, yogurt, and fresh deli meats are all off-limits. Although restrictions lessen as time from transplant increases, patients remain at risk for infection and should continue to exercise some caution for 2 or more years beyond HCT [34].

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Non-adherence to infection precautions may translate into readmission to the hospital. In one study, over 80% of allogeneic HCT patients had an unscheduled readmission within 6 months of initial discharge, with the majority due to bacterial infection or infection at catheter site [170]. Although adherence rates have not been specifically investigated in HCT patients, adherence to anti-neoplastic agents may be as low as 27% in general oncology patients (see [171] for review). Even adherence rates up to 75–81% with immunosuppressant medication regimens in solid organ transplant patients translate into one in four patients not taking medication as prescribed [172], which unfortunately can lead to losing grafts or death [173]. HCT professionals often consider a history of medical regimen non-adherence as a “red flag” for transplant [174], and it is strongly recommended that patients’ prior adherence to medical regimens be assessed during a pre-transplant evaluation [175]. In general, interventions conducted to improve patient adherence to medical regimens are effective. Providing patients with both education and behavioral skills training demonstrate the best overall efficacy for various outcomes (e.g., appointment making and keeping, medication adherence) [176]. For patients with a history of non-adherence, further interventions may be needed. For example, renal transplant patients who were non-adherent with their immunosuppressive medication improved their adherence after a home visit by a nurse and three follow-up telephone calls designed to increase patient self-efficacy in adhering to and problem-solving difficulties with the regimen [177]. To decrease non-adherence in HCT patients, specific recommendations offered by Bishop et al. [178] include reducing the number of medications or doses to simplify regimens, prioritizing key health behavior recommendations (such as increased emphasis on hand-washing), and tailoring regimens to fit patients’ daily routine. For all patients, education about the importance of the outpatient treatment regimen, particularly of immunosuppressant medication [179], needs to be part of discharge planning instructions to both patients and family caregivers [170, 180]. Moreover, all of these recommendations should take into consideration the patient’s level of cognitive functioning, particularly given the potential for treatment-related decrements in cognitive functioning (described earlier). Helping HCT patients to decrease psychological distress may also indirectly improve adherence and ultimately health outcomes. In general, depressed patients are much more likely than nondepressed patients to be non-adherent with medical regimens [181]. In HCT patients, distress has been associated with self-reports of medication non-adherence [48], and the relationship between distress and adherence may be one pathway to explain findings that depressive symptoms at pre-transplant [56] and post-transplant [182] are negatively related to survival. Additionally, psychological distress can be ameliorated by social support, which has also its own positive effects on health behaviors. For example, cancer patients who reported more social support also reported more positive health behavior change after cancer diagnosis and treatment [183]. Practical support may also be particularly helpful for treatment adherence [184]. 3.2. Caregiver Issues Through both practical and social support, caregivers can play a vital role in patient care and transplant success. Not only can caregivers provide tangible

Chapter 35  Psychological Care of Adult Allogeneic Transplant Patients 

help (e.g., picking up prescriptions, preparing meals), but their social and emotional support (e.g., companionship, emotional reassurance), has been shown to be positively related to patient emotional and physical well-being 1 year after allogeneic HCT [185]. Given the importance of caregivers to positive patient outcomes, it is surprising that caregivers are often overlooked by medical professionals, who tend to focus attention on patient well-being. Not surprisingly, caregiver burden is an often unrecognized consequence of HCT. The emotional impact of care-giving, like the extent of caregiving required, can vary across the time-course of transplant (see Table 35-8). Making the decision to undergo HCT can have an almost immediate effect on caregiver distress. The hospitalization period can be especially difficult for caregivers who experience financial strain and social isolation due to time spent at the hospital with patients. Caregiver burden also tends to increase shortly after transplant [186], perhaps because patients begin to experience difficult medical symptoms and/or because caregivers must begin preparing the home environment to accommodate patients’ immuno-compromised status after discharge (e.g., thoroughly cleaning the home to minimize infection risk). Although caregivers Table 35-8.  Effects of HCT on caregivers through the course of treatment. Psychological impact on caregivers of HCT patients Pre-transplant Fatiguea [188] Anxietya [188] Burden of carea [188] Decreased quality of lifea [188] Distress levels similar to patients [45] or higher [189] Stressed by feeling obligated to be a caregiver, regardless of knowledge or skill [190] Hospitalization Strained by financial loss due to time off work, commuting, or hotel expenses [191] Social isolation from own network of friends and family [192] Stressed by feeling obligated to provide physical and emotional support [175] After hospitalization Burden of care due to practical and physical assistance to patient with   Activities of daily living [192, 193]   Travel to and from medical appointments [192, 193]   Taking medications [192, 193]   Monitoring vital signs (e.g., daily temperature) [192, 193]   Charting adverse medical symptoms [192, 193] Long-term Fear [194] Depression and anxiety, higher than that of patients or normal controls [189] Impaired quality of life [195] Loneliness [196] Less social support, relationship satisfaction than patients or normal controls [196] Less spiritual well-being than patients or normal controls [196] a

Denotes caregivers of autologous HCT patients

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do not experience the physical demands of the transplant, research has also shown a physical impact of caregiving: partners of HCT patients experienced decrements in the immune system functioning immediately pre- and postHCT [187]. After hospital discharge, the majority of care for many types of patients shifts to the family caregiver [197], and this is particularly true for HCT patients. Due to often intense and complex treatment protocols and the need for continuous monitoring, caregivers for allogeneic HCT patients must be available 24 hours a day, from the day of hospital discharge through at least Day 100 post-transplant. For patients with an uncomplicated recovery, the burden of caregiving may gradually decrease over time [198], but many other HCT patients continue to experience difficulties, which require assistance for extended periods of time (e.g., [199]). Not surprisingly, even after the initial transplant recovery period, caregivers continue to report adverse emotional effects. In a study of partners of HCT patients (on average 7 years post-transplant), 25% of both partners and patients reported elevated distress levels. In contrast to patients, partners reported little posttraumatic growth (i.e., personal growth or benefit after a traumatic event; [200]), again suggesting that caregivers are at risk for the same negative outcomes as patients but do not have the benefit of positive growth seen in some survivors of HCT [196]. Clearly, HCT caregivers experience burden and emotional distress, and in light of this, interventions to minimize caregiver burden have been recommended (e.g., [201, 202]). One recurring theme is the need for information [192], particularly regarding homecare after discharge [194]. Caregivers of autologous transplant patients who described themselves as being prepared for caregiving also reported experiencing more positive aspects of caregiving [198]. However, they also described the provision of emotional support to patients to be the most difficult aspect of caregiving [198]. Given the difficulties experienced by caregivers, and the benefits of psychosocial interventions for cancer patients described earlier in this chapter, it is likely that providing psychosocial interventions or assistance to caregivers, whether alone or in conjunction with patients, would provide benefit not only to caregivers but also to the patients who depend on them [203].

4.  Systems Issues: Multidisciplinary/Collaborative/ Integrated Care 4.1. The Need for HCT Psychological Services in Integrated Teams The 2006 Joint EBMT/CIBMTR/ASBMT Recommendations that call for “a high level of vigilance for psychological symptoms” ([34]; p. 259) in longterm HCT survivors are significant in recognizing that, although many cope well with HCT, a considerable proportion may experience transitory, latent, or long-term psychological and/or cognitive problems. Increased screening alone may be insufficient, however, without timely follow-up of patients whose screens raise concerns and without access to psychological treatment for patients in need. Clinical psychologists can play a vital role in bridging this gap; unfortunately, their inclusion in routine care procedures for HCT patients is not yet standard practice.

Chapter 35  Psychological Care of Adult Allogeneic Transplant Patients 

Implementing the EBMT/CIBMTR/ASBMT recommendations may thus necessitate a restructuring of systems of providing psychological services. Although systems using consultation–-liaison models or referring patients to community psychologists are steps in the right direction, an integrated structure would significantly increase the likelihood of truly collaborative care. A fully integrated system would include a dedicated staff psychologist on the transplant service, who would conduct psychological assessments as part of standard pre-transplant evaluations of all candidates, integrate psychosocial concerns into treatment planning and medical rounds, monitor patient psychological well-being and intervene as necessary, and facilitate the integration of psychosocial services (social work, psychology, and psychiatry) for patients during hospitalization and post-discharge long-term follow-up. 4.2.  Funding of Psychological Services in HCT: Implications for Mental Health Parity Policy 4.2.1. The Health and Behavior Codes Historically, a major barrier to integrating psychological services into HCT treatment teams has been financial: no mechanism previously existed for the mental health third party payment system to adequately reimburse for psychological services to medical patients without a mental health diagnoses. That situation changed in 2002 with the advent of the Health and Behavior Current Procedural Terminology (H & B CPT) codes, which allow psychologists and other health care providers to bill for psychological services to patients with medical but not mental health diagnoses. Now being underwritten by all Medicaid/Medicare carriers and many, but not all, private health plans [204], the H & B CPT codes reflect a biopsychosocial model of care [205] that can be used to reimburse HCT team psychologists for services such as smoking cessation, medical adherence enhancement, or relaxation training for nausea and pain. These codes (96150–96154 series) provide for reimbursement of 15 min increments (e.g., 15, 30, 45 min) of psychological assessment, reassessment, and individual and family intervention. Further, as primarily medical and not mental health codes, these services are paid from medical benefits and usually require no pre-authorization. The H & B CPT codes have moved closer towards meeting the NCCN mandate that “medical care contracts should include reimbursement for … evaluating and treating distress” ([117], p. MS-3). However, more progress has yet to be made. Many private insurance carriers do not yet know how to apply these reimbursement codes, or selectively pay for some services and not others. Further, the H & B CPT codes reimburse for medical patients who also have psychiatric diagnoses if and only if the focus of the psychological services is the behavioral, social, or psychophysiological conditions related to the treatment of their medical condition. For medically ill patients who also require psychiatric care, integrated medical and mental health care is often overlooked by mental health plans not covering these services. At the time of going to press, mental health parity bills are under consideration in the Senate and House of Representatives; however, the proposed parity would not be universal due to exclusions of certain segments of the insurance market. Consequently, it is essential that providers, cancer centers, and professional

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organizations (e.g., American Psychosocial Oncology Society; American Society of Blood and Marrow Transplantation) continue to advocate for reimbursement of H & B or mental health CPT codes not only with medical directors or advisory committees of individual carriers [204], but also at a policy-making level. 4.2.2. Resources to Support Psychological Services An integrated model of care – in which clinical psychologists operate as fully integrated members of the HCT team – is a paradigm shift, and its implementation and success require the contribution of clinical psychologists to be assessed within a broader health care economics framework than simply third party reimbursement. From the perspective of hospitals as well as medical and mental health care insurance plans, interventions that promote positive patient and family adaptation to illness, cooperation with care, and adherence to treatment have the potential to offset medical costs (e.g., reduce length of inpatient stays and number of unplanned outpatient clinic visits and hospital readmissions). Although yet to be empirically evaluated in the HCT population, studies in oncology patients indicate a 23.5% savings in direct health care billing for breast cancer survivors who participated in six weekly CBT group meetings [206]. If similar cost-savings could be achieved directly through the provision of psychological services to HCT patients and indirectly through consultation with HCT treatment teams, and an additional proportion is recouped by reimbursable CPT code, it might be feasible to support the remainder of HCT psychologists’ salary with capital and/or operating funds as a component of the administrative overhead. At present, however, philanthropy is not uncommonly a significant source of support for psychosocial services, even at major cancer centers, and although not financially ideal, does provide much needed benefit to patients. 4.3. Vision for Research in the Area The need to determine “predictors and most importantly preventive and coping strategies” ([207], p. 261) to reduce psychological morbidity has been emphasized by Blood and Marrow Clinical Trials Network (BMT CTN) as critical for the management of HCT survivors. At present, the BMT CTN plans to include extended follow-up of patient QOL outcomes in select medical trials. This very important initiative will likely yield vital information to guide the next generation of trials for which the HCT field has a dire need: randomized controlled clinical trials (RCTs) evaluating psychological interventions to prevent and minimize psychosocial difficulties specific to HCT patients. Simply adapting interventions validated in oncology patients, although an important first step, is not sufficient. HCT brings unique acute and chronic physical and emotional challenges for patients that need further study in this population. In particular, RCTs are needed to address several key areas that directly impact patient coping and medical adherence: (a) extend known effective methods of behavioral self-management of physical discomfort (such as pain and nausea) to symptoms engendered by GvHD; (b) reduce emotional distress, such as anxiety and depression, related to the unique effects of HCT; (c) teach patients adaptive compensatory strategies to cope with treatmentrelated cognitive impairment; and (d) increase the quality of caregiver support and decrease caregiver burden. Ideally, these RCTs would be multi-center

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clinical trials with ample sample size and grant funding to ensure sufficient therapist training, treatment integrity (maintenance of accurate treatment methodology across time, therapists, and transplant centers), and evaluation of treatment efficacy. Such clinical trials would also serve to increase the presence of expertly trained psychologists in transplant centers, thus improving the clinical care of all patients. 4.4. Chapter Summary Although HCT is a complex and challenging procedure, post-transplant outcomes for survivors are often positive, and many surviving patients successfully adjust to the physical and psychological effects of transplant. However, the empirical evidence clearly illustrates that a significant percentage of patients do not recover on their own and reach clinically significant levels of psychological impairment. For medical professionals, providing optimal patient care should include attending to patients’ emotional, neurocognitive, and psychosocial needs with equal attention as their physical needs. Not only has poor psychological functioning been linked to poor quality of life but also a lower likelihood of post-transplant survival [36, 47, 53–55, 57]. Thus, it is critical for medical professionals to recognize when psychological symptoms reach clinical (e.g., diagnostic) levels, and patients exceed their ability to cope with the physical, emotional, and cognitive effects of transplant without intervention. Attending to these symptoms and referring patients to a clinical psychologist for routine assessment and therapeutic intervention is essential to both improve current functioning and prevent future longer-term negative outcomes. Of particular importance is that the medical team be aware of differences in patient needs over time. Not all patients need help and not all patients need help at the same time during their transplant process. As noted by Andrykowski et al. [92], the gold standard of patient care should be expressing appropriate concern and intervention at the appropriate course in recovery. Centers seeking to integrate psychologists into their multidisciplinary HCT team can optimize their collaborative care system by building on several key elements. Firstly, the clinical psychologists should have training and expertise in behavioral medicine and oncology to equip them for the complexities of psychological distress in HCT patients. Secondly, any psychological assessment and treatment methods should be empirically validated. Thirdly, the pathways and protocols for implementing care should be clearly delineated to maximize pro-active matching of treatment to patient need. Finally, strategic shifts in resource allocation and third party reimbursement policies can facilitate the emergence and continued development of this collaborative care model. The optimal outcome would be transplant centers that tailor care to individual physical, psychological, and social needs, thereby maximizing the long-term recovery and physical and psychological well-being of patients after allogeneic HCT. Acknowledgments.  The authors acknowledge David Saunders-Scott and Jessica Manculich for their assistance with preparation of various aspects of this chapter.

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F. Hoodin et al. 150. Lewis BR, Aoun SL, Bernstein GA, Crow SJ (2001) Pharmacokinetic interactions between cyclosporine and bupropion or methylphenidate. J Child Adolesc Psychopharmacol 11:193–198 151. Strouse TB, Fairbanks LA, Skotzko CE, Fawzy FI (1996) Fluoxetine and cyclosporine in organ transplantation: failure to detect significant drug interactions or adverse clinical effects in depressed organ recipients. Psychosomatics 37:23–29 152. Kalash G (1998) Psychotropic drug metabolism in the cancer patient: clinical aspects of management of potential drug interactions. Psychooncology 7:307–320 153. National Cancer Institute. Supportive Care: Depression. US National Institutes of Health (2007) (Accessed October 7, 2007 at http://www.cancer.gov/cancertopics/ pdq/supportivecare/depression/HealthProfessional/page3#Section_305) 154. Homsi J, Nelson KA, Sarhill N et al (2001) A phase II study of methylphenidate for depression in advanced cancer. Am J Hosp Palliat Care 18:403–407 155. Joshi N, Breitbart W (2003) Psychopharmacologic management during cancer treatment. Semin Clin Neuropsychiatry 8:241–252 156. Breitbart W, Payne DK (1998) Pain. In: Holland JC (ed) Psycho-oncology. Oxford University Press, New York, NY, pp 450–467 157. Passik SD, Navari R, Jung S, Nagy C, Vinson J, Kirsh KL, Loehrer P (2004) A phase I trial of olanzapine (Zyprexa) for the prevention of delayed emesis in cancer patients: A Hoosier Oncology Group Study. Cancer Invest 22(3):383–388 158. Passik SD, Lundberg J, Kirsh KL, Theobald DE, Donaghy KD, Holtsclaw E, Cooper M, Dugan W (2002) A pilot exploration of the antiemetic activity of olanzapine for the relief of nausea in patients with advanced cancer and pain. J Pain Symptom Manage 23:526–532 159. Hollon SD, DeRubeis RJ, Shelton RC et al (2005) Prevention of relapse following cognitive therapy vs medications in moderate to severe depression. Arch Gen Psychiatry 62:417–422 160. Hollon SD, Jarrett RB, Nierenberg AA, Thase ME, Trivedi M, Rush AJ (2005) Psychotherapy and medication in the treatment of adult and geriatric depression: which monotherapy or combined treatment? J Clin Psychiatry 66:455–468 161. Hollon SD, Stewart MO, Strunk D (2006) Enduring effects for cognitive behavioral therapy in the treatment of depression and anxiety. Annu Rev Psychol 57:285–315 162. Hoodin F, Kalbfleisch KR (2001) How psychometrically sound is the Transplant Evaluation Rating Scale for bone marrow transplant recipients? Psychosomatics 42:490–496 163. Holland JC, Reznik I (2005) Pathways for psychosocial care of cancer survivors. Cancer 104:2624–2637 164. Frey P, Siston A, Knight S et al (2002) Lack of caregivers limit use of outpatient hematopoietic stem cell transplant program. Bone Marrow Transplant 30:741–748 165. Sabate E ed (2003) Adherence to long-term therapies: evidence for action. Geneva: World Health Organization, 13. (Accessed October 9, 2007 at http:// www.who.int/chp/knowledge/publications/adherence_full_report.pdf) 166. Meichenbaum D, Turk DC (1987) Facilitating treatment adherence. Plenum, New York, NY 167. Copelan E (2006) Hematopoietic stem-cell transplantation. N Engl J Med 354:1813–1826 168. National Marrow Donor Program (2007) Patient care: post-transplant guidelines. National Marrow Donor Program. (Accessed October 17, 2007 at http://www. marrow.org/PHYSICIAN/Patient_Care_Post_Tx/index.html) 169. National Comprehensive Cancer Network (2007) NCCN clinical practice guidelines in oncology: prevention and treatment of cancer-related infections.

Chapter 35  Psychological Care of Adult Allogeneic Transplant Patients  National Comprehensive Cancer Network (Accessed 6/20/07 at http://www.nccn. org/professionals/physician_gls/PDF/infections.pdf) 170. Grant M, Cooke L, Bhatia S, Forman S (2005) Discharge and unscheduled readmissions of adult patients undergoing hematopoietic stem cell transplantation: implications for developing nursing interventions. Oncol Nurs Forum 32:E1–E8 171. Partridge AH, Avorn J, Wang P, Winer E (2002) Adherence to therapy with oral angioplastic agents. J Natl Cancer Inst 94:652–661 172. Dew M, DiMartini A, Dabbs A et al (2007) Rates and risk factors for nonadherence to the medical regimen after adult solid organ transplantation. Transplantation 83:858–873 173. Morrissey P, Flynn M, Lin S (2007) Medication noncompliance and its implications in transplant recipients. Drugs 67:1463–1481 174. Foster LW, McLellan LJ, Rybicki LA, Dabney J, Welsh E, Bolwell BJ (2006) Allogeneic BMT and patient eligibility based on psychosocial criteria: a survey of BMT professionals. Bone Marrow Transplant 37:223–228 175. Andrykowski MA, McQuellon RP (2004) Psychosocial issues in hematopoietic cell transplantation. In: Blume KG, Forman SJ, Appelbaum FR (eds) Thomas’ hematopoeitic cell transplantation, 3rd edn. Blackwell, Cambridge, MA, pp 497–506 176. Roter D, Hall J, Rolande M, Nordstom B, Dretin D, Svarstad B (1998) Effectiveness of interventions to improve patient compliance: a meta-analysis. Med Care 36:1138–1161 177. De Geest S, Schafer-Keller P, Denhaerynck K et al (2006) Supporting medication adherence in renal transplantation (SMART): a pilot RCT to improve adherence to immunosuppressive regimens. Clin Transplant 20:359–368 178. Bishop MM, Rodrigue JR, Wingard JR (2002) Mismanaging the gift of life: noncompliance in the context of adult stem cell transplantation. Bone Marrow Transplant 29:875–880 179. Butler J, Peveler R, Roderick P, Smith P, Horne R, Mason J (2004) Modifiable risk factors for non-adherence to immunosuppressants in renal transplant patients: a cross-sectional study. Nephrol Dial Transplant 19:3144–3149 180. McDonald J, Stetz K, Compton K (1996) Education interventions for family caregivers during marrow transplantation. Oncol Nurs Forum 23:1432–1439 181. DiMatteo M, Lepper H, Croghan T (2000) Depression is a risk factor for noncomplicance with medical treatment: a meta-analysis of the effects of anxiety and depression on patient adherence. Arch Intern Med 160:2101–2107 182. Chang G, Orav E, McNamara T, Tong M, Antin J (2004) Depression, cigarette smoking, and hematopoietic stem cell transplantation outcome. Cancer 101:782–789 183. Harper F, Schmidt J, Beacham A et al (2007) The role of social cognitive processing theory and optimism in positive psychosocial and physical behavior change after cancer diagnosis and treatment. Psychooncology 16:79–91 184. DiMatteo M (2004) Social support and patient adherence to medical treatment: a meta-analysis. Health Psychol 23:207–218 185. Hochhausen N, Altmaier E, McQuellon R et al (2007) Social support, optimism, and self-efficacy predict physical and emotional well-being after bone marrow transplantation. J Psychosoc Oncol 25:87–101 186. Foxall MJ, Gaston-Johansson F (1996) Burden and health outcomes of family caregivers of hospitalized bone marrow transplant patients. J Adv Nurs 24: 915–923 187. Futterman A, Wellisch D, Zighelboim J, Luna-Raines M, Weiner H (1996) Psychological and immunological reactions of family members to patients undergoing bone marrow transplantation. Psychosom Med 58:472–480 188. Gaston-Johansson F, Lachica EM, Fall-Dickson JM, Kennedy MJ (2004) Psychological distress, fatigue, burden of care, and quality of life in primary

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F. Hoodin et al. caregivers of patients with breast cancer undergoing autologous bone marrow transplantation. Oncol Nurs Forum 31:1161–1169 189. Langer S, Abrams J, Syrjala K (2003) Caregiver and patient marital satisfaction and affect following hematopoietic stem cell transplantation: a prospective, longitudinal investigation. Psychooncology 12:239–253 190. Burridge L, Winch S, Clavarino A (2007) Reluctance to care: a systematic review and development of a conceptual framework. Cancer Nurs 30:E9–E19 191. Meehan K, Fitzmaurice T, Root L, Kimitis E, Patchett L, Hill J (2006) The financial requirements and time commitments of caregivers for autologous stem cell transplant recipients. J Support Oncol 4:187–190 192. Grimm P, Zawacki K, Mock V, Krumm S, Frink B (2000) Caregiver responses and needs: an ambulatory bone marrow transplant model. Cancer Pract 8:120–128 193. Stetz KM, McDonald JC, Compton K (1996) Needs and experiences of family caregivers during marrow transplantation. Oncol Nurs Forum 23:1422–1427 194. Aslan O, Kav S, Meral C et  al (2006) Needs of lay caregivers of bone marrow transplant patients in Turkey. Cancer Nurs 29:E1–E7 195. Boyle D, Blodgett L, Gnesdiloff S et  al (2000) Caregiver quality of life after autologous bone marrow transplantation. Cancer Nurs 23:193–203 196. Bishop M, Beaumont J, Hahn E et al (2007) Late effects of cancer and hematopoietic stem-cell transplantation on spouses or partners compared with survivors or survivor-matched controls. J Clin Oncol 25:1403–1411 197. Nijboer C, Tempelaar R, Sanderman R, Triemstra M, Spruijt RJ, van den Bos GA (1998) Cancer and caregiving: the impact on the caregiver’s health. Psychooncology 7:3–13 198. Eldredge D, Nail L, Maziarz R, Hansen L, Ewing D, Archbold P (2006) Explaining family caregiver role strain following autologous blood and marrow transplantation. J Psychosoc Oncol 24:53–74 199. Bhatia S, Francisco L, Carter A et al (2007) Late mortality after allogeneic hematopoietic cell transplantation and functional status of long-term survivors: report from the BMT Survivor Study. Blood 110(10):3784–3792. doi:10.1182/blood2007-03-082933 200. Tedeschi RG, Calhoun LG (1996) The Post-Traumatic Growth Inventory: measuring the positive legacy of trauma. J Trauma Stress 9:455–471 201. Wochna V (1997) Anxiety, needs, and coping in family members of the bone marrow transplant patient. Cancer Nurs 20:244–250 202. Williams L (2003) Informal caregiving dynamics with a case study in blood and marrow transplantion. Oncol Nurs Forum 30:679–686 203. Martire L (2005) The “relative” efficacy of involving family in psychosocial interventions for chronic illness: are there added benefits to patients and family members? Fam Syst Health 23:312–328 204. Meyers L (2006) New codes give psychologists more treatment flexibility. Monit Psychol 37(5):51 (Accessed September 21, 2007, at http://www.apa.org/monitor/ may06/codes.html) 205. Engel GL (1977) The need for a new medical model: a challenge for biomedicine. Science 196:129–136 206. Simpson JSA, Carlson LE, Trew ME (2001) Effect of group therapy for breast cancer on healthcare utilization. Cancer Pract 9:19–26 207. Weisdorf D, Carter S, Confer D, Ferrara J, Horowitz M (2007) Blood and Marrow Transplant Clinical Trials Network (BMT CTN): Addressing unanswered questions. Biol Blood Marrow Transplant 13:257–262

Chapter 36 Second Allogeneic Transplantation: Outcomes and Indications Koen van Besien, Dan Pollyea, and Andrew Artz

1. Introduction Allogeneic transplantation is usually thought of as “curative” or “definitive” therapy. The single use of this treatment modality is meant to eradicate illness and/or restore hematopoiesis. Unfortunately, disease recurrence commonly occurs after allogeneic transplantation for hematologic malignancies as does graft failure or graft rejection. In such instances, a second attempt at allogeneic stem cell rescue may become necessary that we will briefly discuss. 1.1. Second Transplantation for Relapsed Acute Leukemia Relapse after transplantation portends a poor prognosis, with an overall survival (OS) of less than 6  months for those receiving no further therapy [1–4]. Salvage therapies include withdrawal of immunosuppression, colonystimulating factors, immunotherapy with alpha-interferon (SS-reference), donor lymphocyte infusion (DLI), and chemotherapy with or without second transplantation. A DLI is the treatment of choice for patients with CML or a smoldering relapse, with CR rates ranging from 60% to 86% [3, 5–9]. However, DLI is less effective for patients with relapsed acute leukemia [8, 10–13]. Chemotherapy alone can provide a short remission, but the results are usually transient, and most patients succumb to relapse or infection [3, 14]. Second transplantation as post-relapse salvage therapy can result in prolonged disease-free survival and has an acceptable cost per year of life saved [15], but has been utilized in only a select group of patients who are sufficiently healthy and have access to donor stem cells [16–18]. Procuring donor cells is usually straightforward in presence of an HLA-identical sibling donor, but repeat collection of an unrelated donor is subject to strict regulation and may not always be possible within a reasonable time frame. For this reason, many centers cryopreserve stem cells collected in excess (usually >10 × 106 CD34 cells/kg) during the first harvest. Graft failure is extremely unusual when using the cells from the same donor as the initial transplant because in most cases lymphopoiesis of donor origin persists even after relapse [19]. In case no back up stem cells are available, another donor may be sought. From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_36, © Springer Science + Business Media, LLC 2003, 2010

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Few if any prospective studies on second transplantation have been performed and practically all information derives from case series or registry analysis. A summary of the largest series, mostly from the European and the International Bone Marrow Transplant Registry, is provided in Table 36-1 [2, 3, 12, 14, 16, 20, 21]. These studies, reported over a period of approximately 20  years and pertaining to patients transplanted over a 20-year period, have remarkable similarities. The patients to whom repeat transplants were offered were usually young. Their median age was in the mid-twenties and very few patients over the age of 50 have been reported. The large majority of cases are recipients of HLA-identical sibling transplantation and the same donor was used for first and second transplant in more than 90% of cases. The use of an alternative donor has not been well studied. Across studies, a long-term disease-free survival rate of approximately 25% is obtained, but second relapse as well as treatment-related mortality constitute formidable problems. An early series from Seattle described a very high incidence of VOD/SOS probably related to the use of high-dose busulfan [14]. But other toxicities including interstitial pneumonitis, multi-organ failure, infections, and deaths related to GVHD are common as well. The important question of whether a second transplant enhances survival compared to other strategies persists. In several small studies that appropriately adjusted for the time elapsed prior to transplant, no survival benefit was identified. But these studies were underpowered to detect clinically significant differences. Most series of second transplant for relapsed acute leukemia report approximately 20–25% long-term disease-free survival, which would be unlikely from chemotherapy alone. A number of prognostic factors for outcome after second transplantation have been identified. The duration of remission after initial transplantation has been the most consistent predictor of outcome. A remission duration of less than 6 months after initial transplantation is associated with a very poor outcome after a second transplant. Such patients are prone to subsequent relapse but are also at higher risk for treatment-related mortality. In most series, transplant in remission results in better outcomes than transplant for patients with overt relapse [20, 22]. Younger patients (i.e., those younger than 20!) tend to do better than older ones. Age, however, is often a surrogate for comorbidities and for performance status. Most studies do not provide information on these health-related parameters, but in the most recent study from CIBMTR performance status was a predictor of poor outcome [12]. The optimal preparative regimen for second transplantation is unknown [23]. The type of regimen selected depends upon the indications for transplantation, the co-morbidities of patients, and the regimen used for first transplantation, making comparisons across studies difficult. Patients treated with more intensive conditioning regimens predictably have a decreased relapse rate [3, 12], and toxicity can be decreased if TBI therapies are alternated with non-TBI between the first and second transplantations [24]. The best GVHD prophylaxis also remains unclear. Table 36-1 shows that the majority of patients have received some form of GVHD prophylaxis, but the duration and intensity of prophylaxis are usually not detailed. Because mixed donor chimerism usually persists after an initial transplant, it has been hypothesized that the risk for GVHD is reduced after second transplantation. In an effort to optimize graft-versus-leukemia effects,

1978– 22 AML (87) 1997 (1–46) ALL (83)

Bosi 170 et al., EBMTR

AML (125) 48 ALL (72) CML(82)

1984– 23 AML (29) 48 1993 (2–42) ALL (27) CML (6), MDS (4)

1990– 25 2000

66

Eapen 279 et al., CIBMTR

Kishi et al., Japan

Reference

Nr Year of Median pts TX age Diagnosis

89

100

87

89

85

83

23

33

77

48

Various, not specified

17

27

14

14

25

38

28

26

41

46%

26%

59%

36%

20/66 52%

Transplant in relapse

HSCT 1 to HSCT 1 to relapse relapse <6 months <6 months

Increase TRM

HSCT 1 to relapse <10 mo,

HSCT to relapse >10 months

TBI at second HSCT

No aGVHD at second transplant

second HSCT,

(continued)

HSCT 1 to Transplant in aGVHD at relapse > relapse, first trans10 months, ALL vs plant, CR at second AML, no TBI at secHSCT, ond HSCT PBSCT for

No aGVHD with HSCT 1,

Non myeloablative transplant,

HSCT 1to HSCT 1 to relapse relapse HSCT1 to <6 months <6months Relapse >6 months Transplant in Age >20, relapse,

Age <20

HSCT 1 to relapse >6 months

Increased recurrence

Prognostic factors

Not in remission or HLA-id Same NonNo not in sib donor TBI NonGVHD chronic for TX1 ablaablaprophphase TX2 and 2 TBI tive tive ylaxis PFS OS Improved (%) (%) (%) (%) (%) (%) (%) (%) (%) TRM Relapse survival

Conditioning

Table 36-1.  Summary of large studies evaluating second allogeneic hematopoietic stem cell transplant after a prior transplant.

114

Barrett 90 et al., EBMTR

Mrsic et al., IBMTR

Reference

NHL (2)

MDS (1),

CML (27),

1977– 23 AML(47), 1988 (1–53) ALL(19),

1978– 26 ALL (29), 1989 (1–52) AML (46) CML (37)

Nr Year of Median pts TX age Diagnosis

Table 36-1.  (continued) Conditioning

60

87

100

95

90

10

7

90

54

20

10

11

21

12

65%

45/90 25/90

41%

Increase TRM

female donors

KPS <80%

No or mild Chronic GVHD after HSCT 2 Long interval between HSCT 1 and HSCT 2

No chronic GVHD after HSCT 1,

male recipients from female donors

KPS <80%,

remission,

HSCT 1to Acute leuke- HSCT 1 to relapse mia in relapse >6 months, relapse <6 months, vs remisAcute Age >26 sion or vs leukemia CML, not in

Increased recurrence

Prognostic factors

Not in remission or HLA-id Same NonNo not in sib donor TBI NonGVHD chronic for TX1 ablaablaprophphase TX2 and 2 TBI tive tive ylaxis PFS OS Improved (%) (%) (%) (%) (%) (%) (%) (%) (%) TRM Relapse survival

HSCT hematopoietic stem cell transplant PFS progression-free survival OS overall survival TRM transplant-related mortality

CML (28)

1983– 25 AML (32), 1991 (2–51) ALL(15),

77

Radich et al., Seattle

79

AML,MDS, 95 CML MBC

1989– 42 2003 (14– 75)

Hosing 72 et al., MDACC

Reference

Nr Year of Median pts TX age Diagnosis

92

67

100

79

51

100

47

11

NS

14

15

18

45%

70%

28/72 32/72

Refractory relapse

HSCT 1 to relapse >1 year

Increased recurrence

Prognostic factors

Not in remission or HLA-id Same NonNo not in sib donor TBI NonGVHD chronic for TX1 ablaablaprophphase TX2 and 2 TBI tive tive ylaxis PFS OS Improved (%) (%) (%) (%) (%) (%) (%) (%) (%) TRM Relapse survival

Conditioning

Age >10

Increase TRM

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some cases have been treated without GVHD prophylaxis. In one series, it was found that this approach could safely be done in those who did not have GVHD after their first transplant [2], but others found a very high incidence of severe and sometimes fatal GVHD [25]. Interestingly, one series found an overall better outcome for those without acute GVHD after first transplant [16] and others found improved outcomes for those without chronic GVHD after first transplant [2]. One might interpret this as indicating that those without GVHD after first transplant are also at low risk of GVHD with the second transplant. But it may also simply mean that they are on average in better condition at the time of second transplantation. Finally, consistent with numerous other studies, developing chronic GVHD is protective of relapse [2]. It is however not associated with an improved overall survival. Second allogeneic transplantation continues to be utilized because of the lack of other treatment alternatives for disease relapse. The increased availability of active drugs for leukemia may paradoxically increase the use of second transplant by offering therapies that can achieve temporary disease control after relapse from the first transplant. There is also a recent trend toward using non-myeloablative or reduced intensity conditioning regimens for second transplant [12, 21]. Predictably this is associated with an increased rate of disease recurrence. Such transplants constitute a larger fraction of patients in the recent MD Anderson series [21]. The median age in this series is considerably higher than historical registry studies, as is the percentage of unrelated donor recipients. It is therefore probably more representative of current practice than many of the older reports. Reduced intensity conditioning regimens also frequently are used as the initial transplant in patients thought to be poor candidates for myeloablative conditioning. Predictably, patients who receive reduced intensity conditioning regimens for the first transplant have higher relapse rates [12], but they have less TRM and a reasonable CR rate [26]. Recent single institution series demonstrate the potential for successful salvage with myeloablative transplant in a fraction of such patients, but long-term follow up or registry data are still lacking [26]. 1.2. Second Allogeneic Transplantation After Graft Failure Sustained engraftment is routinely achieved after allogeneic stem cell transplantation from an HLA-identical-related donor. But when unrelated or mismatched donors are utilized, graft failure or graft rejection occur in 5–10% of recipients. Patients with certain diseases such as aplastic anemia or sickle cell disease are particularly prone to graft rejection [27–29]. The risk for graft failure is also increased with the use of T-cell depleted transplants [30–32]. Two syndromes are recognized. In primary graft rejection, no recovery of hematopoiesis occurs and the graft is immediately rejected. In such cases, there is usually absence of donor engraftment in all lineages including myeloid, erythroid, and lymphoid lineages. Such graft rejection is usually mediated by immunologic rejection of the donor cells by residual host effector cells, although viral infections such as CMV and defects in the host microenvironment also may play a role. Secondary graft rejection or failure occurs after an initial period of engraftment. While immunologically mediated graft rejection also plays a role in a proportion of cases, in many series cases are included

Chapter 36  Second Allogeneic Transplantation: Outcomes and Indications 

where despite evidence of persistent donor chimerism there is very limited effective hematopoiesis. A better term for such syndromes may be poor graft function. The pathophysiology of these cases remains very poorly understood. Both primary and secondary graft failure have a high mortality and their management represents a considerable challenge. The risk for graft rejection upon second transplant can in theory be mitigated by increasing donor cell doses, by additional immunosuppression of the recipient, or by infusion of cells from a donor that the patient has not previously been exposed to. The use of an alternative donor is limited by practical issues but has been utilized in a fairly percentage of patients with graft failure. Controversy continues to exist on the optimal timing and preparation of a patient for second transplant. Early studies showed that reinfusion of cells without conditioning was associated with a high incidence of second rejection. A variety of conditioning regimens have been used with the consistent observation that durable engraftment can be achieved in a fraction of patients, but toxicity, opportunistic infections, and graft resistance are frequent [33–35]. A retrospective study from France described 82 patients with primary and secondary graft failure. Fifty-one of the patients engrafted after second transplants (although five subsequently suffered secondary graft failure). The use of peripheral blood stem cells, and a long interval (<80 days) between first and second transplant was associated with a higher likelihood to engraft. In other words, those with secondary graft failure had a better chance for engraftment than those with primary graft failure. Positive CMV serology was also associated with a better chance to engraft, an observation that was difficult to explain. The use of another donor for second transplant did not improve engraftment, perhaps because other donors were not as well matched as the initial donors. Various conditioning regimens were used for preparation for second transplant, and no clear association was found between conditioning and engraftment or survival. Younger age (<34), the use of Cyclosporin for GVHD prophylaxis, a long interval (>80  days) between first and second transplant, and positive CMV serology were all associated with less transplant-related mortality and improved overall survival. The advantage to using CSA-based GVHD prophylaxis was consistent with previous data from Seattle [36] and suggests that stringent GVHD prophylaxis is necessary to prevent fatal GVHD even after second transplant. In addition, cyclosporin A may have graft enhancing properties [37]. Infections and particularly fungal infections constituted the major cause of failure. This may explain why they observed an excessive risk of treatment-related mortality when a combination of cyclosporine and steroids was used for GVHD prophylaxis. Non-myeloablative transplantation is often used in the elderly and those with impaired health. When such patients experience graft rejection, the lack of conditioning tends to be blamed, but on the other hand they are poor candidates for more intensive conditioning. Byrne et al. recently reported on 11 such patients who were retransplanted using a nearly identical non-myeloablative conditioning [38]. Engraftment occurred in 9 of the 11 patients, perhaps suggesting that there is cumulative immunosuppression from two consecutive non-myeloablative regimens. The Sloan–Kettering group also used non-myeloablative conditioning after graft rejection mostly after rejection of an initial T-cell depleted transplant. All patients engrafted and 6 of 16 remained alive with prolonged follow up [39].

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For patients with poor graft function, but persistent chimerism, growth factor support [40] or boosts of donor stem cells have been utilized with variable success [41, 42]. This can sometimes improve graft function, but can also induce severe graft versus host disease. A recent retrospective analysis from the Genoa group suggests that the risk of life-threatening GVHD may be minimized by use of CD34 selected stem cells [43]. In case of primary graft rejection one can also consider infusion of previously collected (the so-called “back-up”) autologous stem cells [44, 45]. This represents a reasonable safe salvage modality that usually results in hematopoietic recovery within 10–14  days after infusion. Unfortunately back-up stem cells are available in only a minority of cases and even then, there is a considerable risk of recurrence of the underlying hematologic disorder. In cases of HLA-mismatched transplantation (cord or adult), the risk of graft rejection is increased if anti HLA antibodies directed against the donor graft are present in the recipient. When retransplanting such patients, an attempt should be made to identify donors against whose HLA no antibodies can be detected [46,47].

References 1. Mortimer J, Blinder MA, Schulman S et al (1989) Relapse of acute leukemia after marrow transplantation: natural history and results of subsequent therapy. J Clin Oncol 7:50–57 2. Barrett AJ, Locatelli F, Treleaven JG et al (1991) Second transplants for leukaemic relapse after bone marrow transplantation: high early mortality but favourable effect of chronic GVHD on continued remission. A report by the EBMT Leukaemia Working Party. Br J Haematol 79:567–574 3. Kishi K, Takahashi S, Gondo H et al (1997) Second allogeneic bone marrow transplantation for post-transplant leukemia relapse: results of a survey of 66 cases in 24 Japanese institutes. Bone Marrow Transplant 19:461–466 4. Mielcarek M, Storer BE, Flower MED et al (2007) Outcomes among patients with recurrent high-risk hematologic malignancies after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 13:1160–1168 5. Nagler A, OR R, Naparstek E, Varadi G, Slavin S (2000) Second allogeneic stem cell transplantation using nonmyeloablative conditioning for patients who relapsed or developed secondary malignancies following autologous transplantation. Exp Hematol 28:1096–1104 6. Gilleece MH, Dazzi F (2003) Donor lymphocyte infusions for patients who relapse after allogeneic stem cell transplantation for chronic myeloid leukaemia. Leuk Lymphoma 44:23–28 7. Mackinnon S, Papadopoulos EB, Carabasi MH et  al (1995) Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versusleukemia responses from graft-versus-host disease. Blood 86:1261–1268 8. Kolb HJ, Schattenberg A, Goldman JM et al (1995) Graft-versus leukemia effect of donor lymphocyte transfusion in marrow grafted patients. Blood 86:2041–2050 9. Porter DL, Roth MS, McGarigle C, Ferrara JLM, Antin JH (1994) Induction of graft-versus-host disease as immunotherapy for relapsed chronic myeloid leukemia. N Engl J Med 330:100–106 10. Ferrara F, Palmieri S, Mele G (2004) Prognostic factors and therapeutic options for relapsed or refractory acute myeloid leukemia. Haematol 89:998–1008 11. Verdonck LF, Petersen EJ, Lokhorst HM et al (1998) Donor leukocyte infusions for recurrent hematologic malignancies after allogeneic bone marrow transplantation: impact of infused and residual donor T cells. Bone Marrow Transplant 22:1057–1063

Chapter 36  Second Allogeneic Transplantation: Outcomes and Indications  12. Eapen M, Giralt SA, Horowitz MM et al (2004) Second transplant for acute and chronic leukemia relapsing after first HLA-identical sibling transplant. Bone Marrow Transplant 34:721–727 13. Schmid C, Labopin M, Nagler A et  al (2007) Donor lymphocyte infusion in the treatment of first hematological relapse after allogeneic stem-cell transplantation in adults with acute myeloid leukemia: a retrospective risk factors analysis and comparison with other strategies by the EBMT Acute Leukemia Working Party. J Clin Oncol 25:4938–4945 14. Radich JP, Sanders JE, Buckner CD et al (1993) Second allogeneic marrow transplantation for patients with recurrent leukemia after initial transplant with totalbody irradiation-containing regimens. J Clin Oncol 11:304–313 15. Messori A, Bosi A, Bacci S et al (1999) Retrospective survival analysis and costeffectiveness evaluation of second allogeneic bone marrow transplantation in patients with acute leukemia. Gruppo Italiano Trapianto di Midollo Osseo. Bone Marrow Transplant 23:489–495 16. Bosi A, Laszlo D, Labopin M et al (2001) Second allogeneic bone marrow transplantation in acute leukemia: results of a survey by the European Cooperative Group for Blood and Marrow Transplantation. J Clin Oncol 19:3675–3684 17. Mehta J, Powles R, Kulkarni S, Treleaven J, Singhal S (1997) Induction of graft-versushost disease as immunotherapy of leukemia relapsing after allogeneic transplantation: single-center experience of 32 adult patients. Bone Marrow Transplant 20:129–135 18. Wagner JE, Vogelsang GB, Zehnbauer BA et  al (1992) Relapse of leukemia after bone marrow transplantation: effect of second myeloablative therapy. Bone Marrow Transplant 9:205–209 19. Sanders JE, Buckner CD, Clift RA et  al (1989) Second marrow transplants in patients with leukemia who relapse after allogeneic marrow transplants. Bone Marrow Transplant 3:11 20. Mrsic M, Horowitz MM, Atkinson K et  al (1992) Second HLA-identical sibling transplants for leukemia recurrence. Bone Marrow Transplant 9:269–275 21. Hosing C, Saliba R, Shahjahan M et al (2005) Disease burden may identify patients more likely to benefit from second allogeneic hematopoietic stem cell transplantation to treat relapsed acute myelogenous leukemia. Bone Marrow Transplant 36:157–162 22. Sierra J (1997) 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 89:4226–4235 23. Giralt SA, Champlin RE (1994) Leukemia relapse after allogeneic bone marrow transplantation: a review. Blood 84:3603–3612 24. Wagner JE, Santos GW, Burns WH, Saral R (1989) Second bone marrow transplantation after leukemia relapse in 11 patients. Bone Marrow Transplant 4:115–118 25. Al-Qurashi F, Ayas M, Al SF et  al (2004) Second allogeneic bone marrow transplantation after myeloablative conditioning analysis of 43 cases from single institution. Hematology 9:123–129 26. Pollyea DA, Artz AS, Stock W et al (2007) Outcomes of patients with AML and MDS who relapse or progress after reduced intensity allogeneic hematopoietic cell transplantation. Bone Marrow Transplant 40:1027–1032 27. Storb R, Champlin RE (1991) Bone marrow transplantation for severe aplastic anemia. Bone Marrow Transplant 8:69–72 28. Vermylen C, Cornu G, Ferster A et al (1998) Haematopoietic stem cell transplantation for sickle cell anaemia: the first 50 patients transplanted in Belgium. Bone Marrow Transplant 22:1–6 29. Martin PJ (1992) Determinants of engraftment after allogeneic marrow transplantation. Blood 79:1647–1650 30. Martin PJ, Hansen JA, Torok-Storb B et al (1989) Graft failure in patients receiving T cell-depleted HLA-identical allogeneic marrow transplants. Bone Marrow Transplant 3:445

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K. van Besien et al. 31. Champlin RE, Passweg JR, Zhang MJ et al (2000) T-cell depletion of bone marrow transplants for leukemia from donors other than HLA-identical siblings: advantage of T-cell antibodies with narrow specificities. Blood 95:3996–4003 32. O’Reilly RJ (1992) T-cell depletion and allogeneic bone marrow transplantation. Semin Hematol 29(Suppl. 1):20–26 33. Kernan NA, Bordignon C, Heller G et al (1989) 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 74:2227–2236 34. Davies SM, Weisdorf DJ, Haake RJ et al (1994) Second infusion of bone marrow for treatment of graft failure after allogeneic bone marrow transplantation. Bone Marrow Transplant 14:73–77 35. Grandage VL, Cornish JM, Pamphilon DH et  al (1998) Second allogeneic bone marrow transplants from unrelated donors for graft failure following initial unrelated donor bone marrow transplantation. Bone Marrow Transplant 21:687–690 36. Stucki A, Leisenring W, Sandmaier BM et  al (1998) Decreased rejection and improved survival of first and second marrow transplants for severe aplastic anemia (a 26-year retrospective analysis). Blood 92:2742–2749 37. Zaucha JM, Yu C, Zellmer E et  al (2001) Effects of extending the duration of postgrafting immunosuppression and substituting granulocyte-colony-stimulating factor-mobilized peripheral blood mononuclear cells for marrow in allogeneic engraftment in a nonmyeloablative canine transplantation model. Biol Blood Marrow Transplant 7:513–516 38. Byrne BJ, Horwitz M, Long GD et al (2008) Outcomes of a second non-myeloablative allogeneic stem cell transplantation following graft rejection. Bone Marrow Transplant 41(1):39–43 39. Chewning JH, Castro-Malaspina H, Jakubowski A et al (2007) Fludarabine-based conditioning secures engraftment of second hematopoietic stem cell allografts (HSCT) in the treatment of initial graft failure. Biol Blood Marrow Transplant 13:1313–1323 40. Nemunaitis J, Singer JW, Buckner CD et  al (1990) Use of recombinant human granulocyte-macrophage colony-stimulating factor in graft failure after bone marrow transplantation. Blood 76:245–253 41. Remberger M, Ringden O, Ljungman P et  al (1998) Booster marrow or blood cells for graft failure after allogeneic bone marrow transplantation. Bone Marrow Transplant 22:73–78 42. Bolger GB, Sullivan KM, Storb R et al (1986) Second marrow infusion for poor graft function after allogeneic marrow transplantation. Bone Marrow Transplant 21:30 43. Larocca A, Piaggio G, Podestá M et al (2006) A boost of CD34+-selected peripheral blood cells without further conditioning in patients with poor graft function following allogeneic stem cell transplantation. Haematologica 91:935–940 44. Fouillard L, Deconinck E, Tiberghien P et  al (1998) Prolonged remission and autologous recovery in two patients with chronic myelogenous leukemia after graft failure of allogeneic bone marrow transplantation. Bone Marrow Transplant 21:943–946 45. Mehta J, Powles R, Singhal S, Horton C, Treleaven J (1996) Outcome of autologous rescue after failed engraftment of allogeneic marrow. Bone Marrow Transplant 17:213–217 46. Anasetti C, Amos D, Beatty P et al (1989) Effect of HLA compatibility on engraftment of bone marrow transplants in patients with leukemia or lymphoma. N. Engl. J. Med. 320:197–204. 47. Gutman JA, McKinney SK, Pereira S et al (2009) Prospective monitoring for alloimmunization in cord blood transplantation: "virtual crossmatch" can be used to demonstrate donor-directed antibodies. Transplantation 87:415–418

Chapter 37 Minimal Residual Disease Mehmet Uzunel

1. Introduction During the past 30 years, survival rates after allogeneic stem cell transplantation (SCT) have been improved substantially. These improvements have mostly been due to better graft matching using genomic HLA typing, GVHD prophylaxis, infection management, and supportive care [1–3]. The incidence of leukemia relapse and death rates after relapse, however, have not been significantly decreased [4]. This makes relapse still a major obstacle to successful SCT. A patient with leukemia is considered to be in complete remission (CR) when <5% blast cells are detected by light microscopic examination of the bone marrow (BM). At the time of diagnosis, the number of leukemic cells is approximately 1012, which means that a patient in CR can still harbor as many as 1010 leukemic cells, cells which are responsible for relapse if they are not eradicated by chemotherapy or SCT. Minimal residual disease (MRD) refers to the presence of leukemic cells in the BM of patients in CR. A number of techniques have been developed that are substantially more sensitive than morphology for detecting MRD and assessing response to treatment. In the first section of this chapter, the most commonly used MRD methods after SCT are described and the specific advantages and disadvantages of each method are discussed. The clinical significance of MRD detection, using different techniques, will also be discussed in the second half of the chapter.

2. Methods The MRD methods are summarized in Table 37-1. 2.1. Immunophenotype Analysis Immunological detection of MRD is on the basis of identifying combinations of leukocyte antigens found on leukemic cells, but not on normal cells. These phenotypes can be determined by double or triple color staining with From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_37, © Springer Science + Business Media, LLC 2003, 2010

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Table 37-1.  Characteristics of the techniques currently used for MRD detection after SCT in patients with ALL, AML, and CML. Flow cytometric immunophenotyping

PCR analysis of fusion PCR analysis of Ig/ gene transcripts TcR rearrangements

Chimerism analysis STR/SNP

60–90% B-ALL

40–45% B-ALL

90–95%

>95%

90–95% T-ALL

15–35% T-ALL

80%

20–40%

10%

>95%

Applicability   ALL   AML   CML

~95%

>95%

Sensitivity

10−4

10−4–10−6

10−4–10−5

10−2 (10−4 with Realtime PCR)

Advantages

– Applicable for most patients

– Easy

– Sensitive and patient specific

– Applicable for most patients

– Applicable in most ALL patients

– Useful for engraftment analysis

– Material from diagnosis or relapse needed

– Limited sensitivity

– Quantification simple Disadvantages

– Limited sensitivity – Immunophenotypic shifts can occur between diagnosis and relapse

– Sensitive and leukemia specific – Stable target – RNA degradation – Limited applicability in AML and ALL – High risk of cross-contamination

– Time-consuming at diagnosis

– Not leukemia specific

– Risk of false negative results due to clonal exchange

Ig Immunoglobulin; TcR T-cell receptor; STR short tandem repeats; SNP single nucleotide polymorphism

antibodies conjugated to different fluorochromes and the labeled cells can be analyzed with flow cytometry. With the new cytometers, four and five color analysis is possible, increasing the specificity and informativity of the MRD analysis. Applicability. Immunophenotype analysis can be performed in 60–90% of ALL and AML patients [5–7]. Sensitivity. The sensitivity of MRD detection with flow cytometry depends mainly on two variables: (1) the degree of morphological and phenotypic difference between the target cells and normal cells and (2) the number of cells that can be analyzed. The maximum sensitivity that can be achieved is 10−5. However, most studies report a sensitivity of 10−4. Advantages and disadvantages. Because both leukemic and normal cells are counted directly in the flow cytometry, MRD quantification is more simple and accurate as compared with molecular methods [8]. The method is difficult to perform and therefore restricted to specialized laboratories. Another limitation of the method is that the immunophenotype of leukemic cells may change during the course of treatment and disease progression, leading to false negative results. This problem can be overcome with the use of several immunophenotypes per patient [9]. 2.2. Antigen Receptor Rearrangement Analysis The junctional regions of rearranged immunoglobulin (Ig) and T-cell receptor (TcR) genes, also called the third complementarity determining

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regions (CDR3), are unique sequences that are assumed to be different in each lymphoid precursor. Because ALL cells are clonal proliferations of one precursor cell, analysis of Ig and TcR gene rearrangements can be used as “DNA-fingerprints” for each particular ALL [10]. Applicability. This method is restricted to lymphoid malignancies although Ig and TcR rearrangements have been reported in ~10% of the AML cases [11]. IgH and TcR rearrangements can be detected in >90 of the ALL cases. In ALL, IgH and TcR rearrangements are not lineage-restricted and this is referred to as lineage infidelity or cross lineage rearrangements. Thus, clonal rearrangements of TcR genes are seen in a large proportion of B-ALL and a smaller proportion of IgH rearrangements are found in T-ALL [12]. Sensitivity. The detection limit of PCR analysis of junctional regions generally varies between 10−4 and 10−6. Advantages and disadvantages. The main advantages of this method are its high sensitivity and applicability in virtually all ALL patients. The need to sequence junctional regions and to develop probes and primers for each ALL case is time-consuming and a limiting factor of the method. The main disadvantage of using Ig and TcR rearrangements as MRD targets is that continuing rearrangements can occur during the disease course [13–15]. Such changes in rearrangement patterns will lead to false negative PCR results. It is now generally accepted that at least two Ig/TcR gene targets should be used for reliable and sensitive MRD detection in ALL patients. Some studies have made methodological comparisons between flow cytometry and rearrangement analysis for MRD detection [16–18]. High concordance was found between both methods. Discrepant results were usually due to low sample cellularity or the presence of PCR inhibitors [16]. 2.3. Fusion Gene Transcript Analysis Chromosomal abnormalities are present in 70–80% of the patients with AML and ALL and in >95% of the patients with CML [19]. Some of these abnormalities, especially the chromosome translocations, are recurrent and have been associated with leukemogenesis [20]. Chromosome abnormalities can be used as leukemia specific targets for MRD analysis and with the polymerase chain reaction (PCR) technique, the RNA transcripts of fusion genes can be detected with high sensitivity. Applicability. The Philadelphia (Ph) - chromosome was the first specific chromosome abnormality described in leukemia [21]. The product of this translocation, the fusion gene BCR-ABL, can be found in almost all CML patients and also in a fraction of patients with ALL (10–30%) and AML (1%) [22–26]. In AML and ALL, there is no specific translocation associated with disease. There are several numbers of translocations, which occur in 1–30% of the cases, with larger frequencies in specific leukemia subtypes. Sensitivity. Because chromosome abnormalities are highly disease specific, PCR amplification of the fusion gene transcripts can usually detect one leukemic cell among 104–106 normal cells. Advantages and disadvantages. One of the major advantages of the PCR technique is the high sensitivity. In addition, these translocations are leukemia-specific and stable during the disease course. The high sensitivity can be a problem if cross-contamination of PCR products occurs, leading to

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false-positive results. RNA degradation and variations in efficiency of cDNA synthesis may also affect sensitivity of the method. One important methodological aspect regarding the analysis of fusion gene transcripts is the use of an internal control gene [27]. Internal control genes (housekeeping genes, reference genes) are constitutively expressed genes, which are used for quality control of the patient samples. Usually, the number of fusion gene transcripts (BCR-ABL) is normalized to the number of transcripts of a control gene in order to compensate for variations that can occur between samples. The ABL gene is considered to be a suitable control gene in different diseases [28, 29]. 2.4. Chimerism Analysis After SCT, a state of chimerism develops when donor cells in the graft reconstitute the hematological and immunological system [30]. However, in some cases, host cells of hematopoietic origin survive the conditioning treatment and co-exist with donor cells. This state, which is termed mixed chimerism, may be stable or transient. There are some terms describing the chimeric status after SCT [31]. – Donor chimerism (full chimerism, complete chimerism) means that all the circulating hematopoietic cell populations are of donor origin. – Mixed chimerism means that there is a mixture of donor and host cells in peripheral blood (PB) or BM. The percentage of host cells varies between 5 and 95. – Split chimerism describes the situation when one cell lineage is of host origin and another cell lineage is of donor origin – e.g., B-cells are host and T-cells are donor. Mixed chimerism and split chimerism can be difficult to distinguish if chimerism analysis is performed using whole blood cells without prior cell separation. – Microchimerism is a term usually used after organ transplantation where <1% cells in the circulation can originate from the transplanted organ. This term should perhaps also be used after SCT as more sensitive methods based on realtime quantitative PCR (RQ-PCR) are in use nowadays and host cells <1% are observed at high frequency [32]. The most widely used method for chimerism analysis is PCR amplification of short tandem repeats (STR, microsatellites) and variable number of tandem repeats (VNTR, minisatellites) [33–36]. New approaches based on RQ-PCR have been developed for chimerism analysis. Using this method, single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms are used as PCR targets. This new approach seems to be more sensitive than VNTR/STR analysis and appears promising for chimerism analysis [32, 37, 38]. Applicability. Using a panel of 5–10 VNTRs/STRs, an informative marker can be detected in >95% of the SCT cases [39]. Because of differences in primer sensitivity and other methodological considerations, the frequency of patients analyzed under optimal conditions will be decreased [35, 40]. Sensitivity. With VNTR/STR markers the sensitivity for detecting the minor population ranges from 1 to 5% but can be increased if cell separation is performed before the PCR analysis. Using this approach, the sensitivity can be increased by more than one log [34]. High sensitivity (10−4–10−5) can also be

Chapter 37  Minimal Residual Disease 

achieved if Y-chromosome specific sequences are used as PCR targets [41]. However, this is only applicable in sex-mismatched transplants, male patients with female donors. Initial studies with RQ-PCR of SNPs report a sensitivity of 10−3–10−4 [38]. Advantages and disadvantages. The main advantage of the chimerism analysis is the high applicability, regardless of the underlying disease. Analysis in different cell population allows the investigation of the engraftment process, which is especially important after nonmyeloablative transplants. Limitations of the method are the low sensitivity and that it is not leukemia specific. These problems can be overcome partly by cell separation and the use of RQ-PCR.

3. Clinical Significance of MRD Detection 3.1. Acute Lymphoblastic Leukemia MRD analysis before SCT. Many ALL patients transplanted in CR still relapse, which indicates the presence of leukemic cells before SCT, not detected by standard morphological analysis. Therefore, MRD studies before SCT have been performed in order to identify patients with persistent disease and to see whether the MRD levels are correlated to patient outcome after SCT. Most of these studies show that patients with persistent MRD before SCT are at higher risk of relapse as compared to MRD negative patients [42–47]. Furthermore, among MRD positive patients, relapse free survival is lowest in the patient group with high MRD levels (³10−3). Interestingly, a GVL effect is usually observed in MRD positive patients who remained in CR. GVHD protects against relapse [43, 44], while T-cell depletion is associated with an increased risk of relapse in patients with high MRD levels [42]. Given the fact that MRD levels prior to SCT represent an important prognostic factor in ALL patients, the question is what to do with this information. There are different possible approaches that can be used to in order to improve transplant outcome. Additional cytoreductive therapy to convert MRD positive patients to MRD negative before transplant is one possibility. Other approaches can be modification of transplant protocols in order to induce stronger alloreactivity and/or the use of prophylactic DLI in high risk patients. These strategies should off course be used cautiously as there is an increased risk of transplant related complications such as GVHD and infections. Another important question is what to do with the MRD negative group which shows very low risk of relapse in most studies. Should they be transplanted or are there other less toxic treatment options? On the basis of these encouraging results, a multicenter study has been initiated to evaluate the role of pre-SCT MRD in prospective studies by adopting a common protocol for MRD assessment [48, 49]. It will be interesting to see whether overall outcome in ALL patients can be improved using risk adapted stratification treatment based on MRD levels before SCT. MRD analysis after SCT. All studies of MRD after SCT clearly show that MRD negativity is a good predictor of remission in patients with ALL [50–54]. However, the clinical significance of MRD positive samples is less clear. While most studies have found a strong correlation between MRD positivity and relapse [46, 50–52, 54, 55], regardless of the MRD quantity, some studies report a high frequency of MRD positive patients who do not relapse [15, 53, 56].

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The median time interval between a positive MRD signal and relapse has varied between 1 and 5.5 months in different studies [50, 52, 54]. Prospective studies using adoptive immunotherapy based on MRD results are rare. In a study by Sramkova et al. the use of adoptive immunotherapy in MRD positive patients was of limited success despite a time frame of >300 days between the first MRD positive sample and hematological relapse in some patients [47]. These results need of course to be supported by larger studies but it is possible that more effort should be aimed to control the pre-transplant MRD levels. This is especially important in patients who relapse early after transplant, <6 months, where there may not be enough time for immunotherapy to have an antileukemic effect. 3.2. Acute Myeloid Leukemia The lack of widely expressed molecular markers in AML limits the systematic study of MRD by PCR. Therefore, correlative studies between MRD and treatment outcome have been performed only in selected groups of patients. After SCT for AML, very few MRD studies have been reported. In addition, most of these studies have usually included a small number of patients. PCR analysis of fusion gene transcripts have been used in some studies, but the clinical significance of MRD detection is still not clear [57–59]. Using WT1 as a MRD marker, Ogawa et al. reported that quantitative analysis of the WT1 gene transcript could be useful for predicting relapse in ALL and AML patients after SCT [60]. However, this has not been confirmed by others [15, 61, 62]. 3.3. Chronic Myeloid Leukemia The initial MRD studies after SCT were performed using qualitative PCR analysis. It was found that BCR-ABL transcripts could be detected in most patients for some months after SCT. Patients who were persistently MRD negative, especially for more than 6 months after SCT, had a very low risk of relapse [63]. Long persistent MRD could be detected in some patients with increased risk of relapse [64, 65]. A GVL effect in CML was evident by the fact that MRD detection was more common in patients with less severe GVHD and that T-cell depletion was associated with higher incidence of MRD and relapse [66–68]. Using qualitative MRD analysis it was also shown that MRD could be detected several months before relapse although this approach could not predict relapse for individual patients [69]. With the introduction of quantitative PCR methods, the kinetics of BCRABL transcripts could be followed in more detail [70–72]. Serial quantitative RT-PCR analysis can distinguish patients who will most probably relapse (high or increasing BCR-ABL levels) from those who will remain in clinical remission (low or decreasing BCR-ABL levels) [73, 74]. Using ABL as the internal control gene, molecular relapse has in some laboratories been defined as a BCR-ABL/ABL ratio of >0.02% in three consecutive samples [28]. DLI treatment at the time of molecular relapse is associated with higher response rates as compared to DLI given at the time of hematological relapse [75–77]. The definition of molecular relapse is also important to avoid unnecessary treatment of patients who show very low MRD levels after SCT without having an increased risk of relapse [78].

Chapter 37  Minimal Residual Disease 

In addition to the importance of serial quantitative analysis, some studies have also shown that BCR-ABL quantification at single time points early after SCT can have a predictive value for relapse. Patients with high BCRABL transcript levels at 3–5 months post SCT have much higher probability of relapse, ~80%, as compared to low level/ MRD negative patients, <30% [28, 79]. 3.4. Chimerism Results Chimerism testing is used for routine analysis of engraftment after SCT and has been of great value for this purpose. Successful engraftment is associated with stable complete donor chimerism (DC). Whether chimerism analysis can be a useful tool for predicting relapse has been a matter of debate [80–85]. Although some studies have shown an association between detection of mixed chimerism (MC) and relapse [33, 34, 86–90], others have failed to find such a correlation [91–93]. These conflicting results in the literature may be explained by a number of factors. The time and frequency of sampling are important factors that influence the detection of MC. During the early posttransplant period, most patients will show some degree of MC. Investigating the kinetics of engraftment, Dubovsky et al. showed that DC was usually achieved by day 28 after SCT [94]. Although, frequent sampling during this early time period may lead to a high incidence of MC without an association with relapse, it may be more valuable at later time points. Serial and quantitative chimerism analysis of samples taken at short intervals after SCT has been useful for prediction of relapse [86, 87]. Longer time intervals between samples, especially during the first year when the majority of the relapses occur, can make it difficult to identify patients at high risk of relapse and the time window between MC detection and relapse will be too short. PCR analysis of VNTRs and STRs yield similar sensitivities, 1–5%. In some studies, PCR analysis of Y-chromosome specific sequences has been performed in sex-mismatched transplants (female to male). This approach increases the sensitivity of the chimerism method by at least two logs, to 10−4–10−5 [41, 95, 96]. Using this methodology, MC can be detected at low levels (10−4), several years after SCT [97]. Whether these recipient cells are long-lived normal hematopoietic cells, malignant cells, or contaminating non-hematopoietic cells in the samples is not known. Fehse et  al. showed that the level of MC was higher in BM compared to PB, which may indicate the presence of host-derived cells – e.g., stroma cells, collected during BM sampling [98]. They also showed that complete DC could be achieved after cell sorting. Preliminary results using RQ-PCR with SNP markers also show that MC is more frequently detected as compared to the less sensitive method based on STR markers. Whether this increased sensitivity will improve the predictive value for relapse remains to be seen although the few studies published so far indicate that so may be the case [32, 99]. Most chimerism studies have been performed using DNA samples obtained from whole PB or BM without prior cell separation. This approach has the disadvantage that sensitivity is limited to 1% if VNTRs/STRs are used. In addition, if MC is detected, the identity of the recipient cells will not be

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known – i.e., whether detected recipient cells are potential malignant cells or not. Therefore, in recent years, immunophenotypes of the original leukemic clone have been used for FACS or immunomagnetic separation of specific cell populations expected to harbor tumor cells. After cell separation, the sensitivity and specificity of the chimerism analysis for detecting MRD are increased by reducing the irrelevant background of other cell types. This approach has been successfully applied in ALL, AML, and CML patients [34, 88, 100, 101]. In addition, lineage-specific chimerism analysis may be useful in predicting response to DLI, as shown in some studies [102–104]. Because GVHD and GVL are closely related, T-cell chimerism analysis may be of special interest. Indeed, it has been found that T-cell MC is significantly correlated to a decreased risk of acute GVHD [105]. While no clear association between T-cell MC and relapse has been found in acute leukemia patients, higher incidence of MRD positivity and relapse has been found in CML patients with T-cell MC [106–108]. This is probably due to the GVL effect of T-cells, which is stronger in CML than in acute leukemia. In a study by Zeiser et  al., the predictive value of conventional (no cell separation) chimerism analysis for relapse was compared with lineage-specific chimerism analysis of CD3+ and CD34+ cells [109]. Including only AML and MDS patients and monthly chimerism testing, the conventional and lineagespecific chimerism analyses were comparatively sensitive and specific for predicting relapse. Studies reporting the use of pre-emptive immunotherapy based on chimerism results are increasing in number [110–112]. In one study, Bader et  al. reported 31 ALL patients with increasing MC who received further immunotherapy consisting of withdrawal of immunosuppression and/or DLI [113]. The event free survival (EFS) in this patient group was significantly higher (37%) than that of the group of 15 patients with increasing chimerism who did not receive immunotherapy (EFS 0%). Similar results were also found in patients with MDS and AML [114, 115]. These studies also show that low DLI doses can be effective without causing fatal GVHD and that acute GVHD is not necessary for response to immunotherapy. 3.5. MRD and Chimerism After Reduced Intensity Conditioning In leukemia patients, the conditioning treatment given before SCT is meant to eradicate recipient hematopoietic cells, normal and malignant cells. Therefore, less intensive nonmyeloablative conditioning regimens are expected to give higher incidence of MC and MRD after SCT. Indeed, a higher incidence of MC has been reported after reduced intensity conditioning (RIC) [31, 116–120]. In most cases DC is obtained after a transient MC while in other cases further immunotherapy is needed for conversion from MC to DC [117, 121]. The incidence and level of chimerism after RIC seem to be affected by factors like conditioning regimen [122], previous chemotherapy [119, 123], underlying disease, stem cell source [124], graft composition [125, 126], and immunosuppression [127]. Similar to myeloablative SCT, chimerism analysis after RIC can be useful to predict graft rejection [128], GVHD [129], and relapse [130, 131]. While MRD data are rare in AML and ALL patients [132], some studies have focused on chimerism and MRD monitoring in CML

Chapter 37  Minimal Residual Disease 

patients [133–137]. In one study, the kinetics of MRD and chimerism was compared between patients receiving RIC and those receiving a conventional myeloablative conditioning regimen [134]. In the early posttransplant period (<3 months), a higher incidence of MC and MRD was found in RIC patients. However, during the first year, most RIC patients achieved DC and molecular remission.

4. Conclusions During the last decade, MRD analysis has become an important tool in the management of leukemia patients. In many studies, MRD detection has been shown to be an independent prognostic factor for patient outcome. However, there is still some controversy regarding whether MRD results should be used in clinical decision-making. Different factors may contribute to the conflicting results found in different studies. 1. Patient population. The patient group under study may have an effect on the clinical outcome in relation to MRD results. For instance, adult patients with ALL respond to treatment more slowly than children and therefore, the MRD statuses at later time-points have shown to be more predictive for relapse in adult ALL. 2. Transplant regimens. The type of conditioning regimen and the type of graft given may affect the incidence of MRD and chimerism after SCT. The use of PB as stem cell source has been associated with lower incidence of MC and MRD as compared to BM. 3. Sensitivity of the method used may have a major impact on the predictive value of MRD detection. A sensitivity of at least 10−4 is usually recommended for MRD analysis. 4. Qualitative versus Quantitative MRD analysis. In CML patients, a qualitative MRD analysis is of limited value and does not allow identification of individual patients as quantitative analysis does. In addition, quantitative analysis provides the possibility to find threshold values, which may differentiate between patients at high risk of relapse and those who will most probably remain in CR. 5. Time and frequency of sampling. MRD analysis at a single time point is usually not sufficient to identify patients with poor prognosis. MRD information from different time points after treatment allows kinetic studies of tumor load and appears to be highly informative. The currently used MRD assays are heterogeneous as regards to the markers and techniques used in the analysis. In a routine laboratory, a combination of these methods is needed in order to make MRD analysis available for most patients. This requires skilled personnel and different equipments and materials. Despite this, a sensitive MRD target will not be found in all patients. The search for new MRD markers may allow identification of markers that can be used across different leukemia types. Finally, standardization of MRD protocols is necessary in order to come to a consensus on the significance of MRD detection for each type of disease and treatment. With the introduction of RQ-PCR, this may be easier to achieve than before. In Europe, several protocols have been established to develop

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common guidelines for MRD analysis. Some of these networks include the Europe Against Cancer Program (RQ-PCR analysis of fusion gene transcripts), the International Study Group on Standardization of Residual Disease Detection in BCR-ABL positive leukemias, and the European Study Group for MRD analysis in SCT for ALL [48]. Publications addressing these issues are increasing in number and include methods for BCR-ABL analysis [138, 139], fusion gene transcript analysis [29, 140], IgH and TcR rearrangement analysis [10, 141, 142], and chimerism analysis [31, 35, 143].

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Chapter 37  Minimal Residual Disease  109. Zeiser R, Spyridonidis A, Wasch R et  al (2005) Evaluation of immunomodulatory treatment based on conventional and lineage-specific chimerism analysis in patients with myeloid malignancies after myeloablative allogeneic hematopoietic cell transplantation. Leukemia 19(5):814–821 110. Formankova R, Sedlacek P, Krskova L, Rihova H, Sramkova L, Star J (2003) Chimerism-directed adoptive immunotherapy in prevention and treatment of posttransplant relapse of leukemia in childhood. Haematologica 88(1):117–118 111. Bader P, Klingebiel T, Schaudt A et al (1999) Prevention of relapse in pediatric patients with acute leukemias and MDS after allogeneic SCT by early immunotherapy initiated on the basis of increasing mixed chimerism: a single center experience of 12 children. Leukemia 13(12):2079–2086 112. Shaw BE, Byrne JL, Das-Gupta E, Carter GI, Russell NH (2007) The impact of chimerism patterns and predonor leukocyte infusion lymphopenia on survival following T cell-depleted reduced intensity conditioned transplants. Biol Blood Marrow Transplant 13(5):550–559 113. Bader P, Kreyenberg H, Hoelle W et  al (2004) 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 22(9):1696–1705 114. Bader P, Kreyenberg H, Hoelle W et  al (2004) Increasing mixed chimerism defines a high-risk group of childhood acute myelogenous leukemia patients after allogeneic stem cell transplantation where pre-emptive immunotherapy may be effective. Bone Marrow Transplant 33(8):815–821 115. Bader P, Niemeyer C, Willasch A et  al (2005) Children with myelodysplastic syndrome (MDS) and increasing mixed chimaerism after allogeneic stem cell transplantation have a poor outcome which can be improved by pre-emptive immunotherapy. Br J Haematol 128(5):649–658 116. Spitzer TR (2000) Nonmyeloablative allogeneic stem cell transplant strategies and the role of mixed chimerism. Oncologist 5(3):215–223 117. Mattsson J, Uzunel M, Brune M et al (2001) Mixed chimaerism is common at the time of acute graft-versus-host disease and disease response in patients receiving non-myeloablative conditioning and allogeneic stem cell transplantation. Br J Haematol 115(4):935–944 118. Childs R, Clave E, Contentin N et al (1999) Engraftment kinetics after nonmyeloablative allogeneic peripheral blood stem cell transplantation: full donor T-cell chimerism precedes alloimmune responses. Blood 94(9):3234–3241 119. Valcarcel D, Martino R, Caballero D et al (2003) Chimerism analysis following allogeneic peripheral blood stem cell transplantation with reduced-intensity conditioning. Bone Marrow Transplant 31(5):387–392 120. Mohr B, Koch R, Thiede C, Kroschinsky F, Ehninger G, Bornhauser M (2004) CD34+ cell dose, conditioning regimen and prior chemotherapy: factors with significant impact on the early kinetics of donor chimerism after allogeneic hematopoietic cell transplantation. Bone Marrow Transplant 34(11):949–954 121. Massenkeil G, Nagy M, Lawang M et al (2003) Reduced intensity conditioning and prophylactic DLI can cure patients with high-risk acute leukaemias if complete donor chimerism can be achieved. Bone Marrow Transplant 31(5):339–345 122. Carvallo C, Geller N, Kurlander R et al (2004) Prior chemotherapy and allograft CD34+ dose impact donor engraftment following nonmyeloablative allogeneic stem cell transplantation in patients with solid tumors. Blood 103(4):1560–1563 123. Baron F, Baker JE, Storb R et al (2004) Kinetics of engraftment in patients with hematologic malignancies given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning. Blood 104(8):2254–2262 124. Maris MB, Niederwieser D, Sandmaier BM et al (2003) HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative conditioning for patients with hematologic malignancies. Blood 102(6):2021–2030

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M. Uzunel 125. Baron F, Maris MB, Storer BE et  al (2005) High doses of transplanted CD34+ cells are associated with rapid T-cell engraftment and lessened risk of graft rejection, but not more graft-versus-host disease after nonmyeloablative conditioning and unrelated hematopoietic cell transplantation. Leukemia 19(5):822–828 126. Panse JP, Heimfeld S, Guthrie KA et al (2005) Allogeneic peripheral blood stem cell graft composition affects early T-cell chimaerism and later clinical outcomes after non-myeloablative conditioning. Br J Haematol 128(5):659–667 127. Maris MB, Sandmaier BM, Storer BE et al (2006) Unrelated donor granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cell transplantation after nonmyeloablative conditioning: the effect of postgrafting mycophenolate mofetil dosing. Biol Blood Marrow Transplant 12(4):454–465 128. Matthes-Martin S, Lion T, Haas OA et  al (2003) Lineage-specific chimaerism after stem cell transplantation in children following reduced intensity conditioning: potential predictive value of NK cell chimaerism for late graft rejection. Leukemia 17(10):1934–1942 129. McSweeney PA, Niederwieser D, Shizuru JA et  al (2001) Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graft-versus-tumor effects. Blood 97(11): 3390–3400 130. Baron F, Maris MB, Sandmaier BM et al (2005) Graft-versus-tumor effects after allogeneic hematopoietic cell transplantation with nonmyeloablative conditioning. J Clin Oncol 23(9):1993–2003 131. Keil F, Prinz E, Moser K et al (2003) Rapid establishment of long-term cultureinitiating cells of donor origin after nonmyeloablative allogeneic hematopoietic stem-cell transplantation, and significant prognostic impact of donor T-cell chimerism on stable engraftment and progression-free survival. Transplantation 76(1):230–236 132. Perez-Simon JA, Caballero D, Diez-Campelo M et  al (2002) Chimerism and minimal residual disease monitoring after reduced intensity conditioning (RIC) allogeneic transplantation. Leukemia 16(8):1423–1431 133. Childs R, Epperson D, Bahceci E, Clave E, Barrett J (1999) Molecular remission of chronic myeloid leukaemia following a non- myeloablative allogeneic peripheral blood stem cell transplant: in vivo and in vitro evidence for a graft-versusleukaemia effect. Br J Haematol 107(2):396–400 134. Uzunel M, Mattsson J, Brune M, Johansson JE, Aschan J, Ringden O (2003) Kinetics of minimal residual disease and chimerism in patients with chronic myeloid leukemia after nonmyeloablative conditioning and allogeneic stem cell transplantation. Blood 101(2):469–472 135. Kreuzer KA, Schmidt CA, Schetelig J et al (2002) Kinetics of stem cell engraftment and clearance of leukaemia cells after allogeneic stem cell transplantation with reduced intensity conditioning in chronic myeloid leukaemia. Eur J Haematol 69(1):7–10 136. Or R, Shapira MY, Resnick I et al (2003) Nonmyeloablative allogeneic stem cell transplantation for the treatment of chronic myeloid leukemia in first chronic phase. Blood 101(2):441–445 137. Kerbauy FR, Storb R, Hegenbart U et al (2005) Hematopoietic cell transplantation from HLA-identical sibling donors after low-dose radiation-based conditioning for treatment of CML. Leukemia 19(6):990–997 138. Branford S, Cross NC, Hochhaus A et al (2006) Rationale for the recommendations for harmonizing current methodology for detecting BCR-ABL transcripts in patients with chronic myeloid leukaemia. Leukemia 20(11):1925–1930 139. Hughes T, Deininger M, Hochhaus A et  al (2006) Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood 108(1):28–37

Chapter 37  Minimal Residual Disease  140. Gabert J, Beillard E, van der Velden VH et al (2003) Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – a Europe Against Cancer program. Leukemia 17(12):2318–2357 141. van der Velden VH, Cazzaniga G, Schrauder A et al (2007) Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data. Leukemia 21(4):604–611 142. van der Velden VH, Panzer-Grumayer ER, Cazzaniga G et al (2007) Optimization of PCR-based minimal residual disease diagnostics for childhood acute lymphoblastic leukemia in a multi-center setting. Leukemia 21(4):706–713 143. Thiede C, Bornhauser M, Ehninger G (2004) Evaluation of STR informativity for chimerism testing – comparative analysis of 27 STR systems in 203 matched related donor recipient pairs. Leukemia 18(2):248–254

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Chapter 38 Functional Assessment Tools and Co-morbidity Scoring in Hematopoietic Progenitor Cell Transplantation Sergio Giralt and Uday Popat

1. Introduction Predicting risk of treatment-related toxicities in patients undergoing high dose chemotherapy with progenitor cell support (HDT) has always been an integral part of the decision process for physicians and patients involved in this procedure [1]. Initially when the procedure was used only as salvage therapy for patients with advanced disease performance status and age were the most common factors taken into consideration when deciding whether or not to proceed with high dose chemotherapy [2]. HDT has now become a standard therapy for a variety of malignant and non- malignant disorders, improvements in supportive care as well as novel reduced intensity conditioning regimens have now made HDT a commonly used therapeutic strategy even in patients in the sixth and seventh decades of their life. The increased use of HDT in a more vulnerable patient population has made the transplant community focus more attention on pre-transplant factors that can predict transplant outcomes. In this chapter, we will summarize the value of current pre-transplant assessment in predicting transplant outcomes, as well as the value of comorbidity scores, and novel ways of measuring transplant toxicities and outcomes.

2.  Predicting Transplant Outcomes-Current Trends and Historical Perspectives Single-organ function has been the most frequently studied pre-transplant variable used to predict outcome. Pre-transplant cardio-pulmonary testing and other visceral organ function assessment do not reliably predict for adverse

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_38, © Springer Science + Business Media, LLC 2003, 2010

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outcomes with the exception of pre-transplant abnormal serum liver functions tests which predict for hepatic veno-occlusive disease [3–11]. Performance status (PS) as defined by either the Karnofsky or Zubrod scales, represents a simplified tool of functional assessment and does not account for the presence or absence of specific co-morbidities [12, 13]. PS has been extensively explored as a prognostic factor for outcomes after progenitor cell transplantation, with the largest series of both allogeneic and autologous transplant demonstrating that non-relapse mortality and survival are related to PS [14–16]. However, since very few patients with PS>2 or Karnofsky<70 are transplanted, the utility of PS as a discriminator is limited. Co-morbidity indices have been developed and applied to a wide variety of medical conditions [17–20]. These tools appear to be useful for intervention studies and as a prognostic aid for clinicians and are important factors to consider when comparing or performing clinical trials. The use of standard co-morbidity indices as predictors of transplant outcomes or as instruments to allocate patients to specific transplant therapies has been studied, only recently, in certain detail. Shahjahan et  al. [21] at MD Anderson retrospectively analyzed a cohort of acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS) patients in order to determine the impact of co-morbidities on the outcome of allogeneic HSCT. They used an adaptation of the Charlson Comorbidity Index (CCI) to obtain a weighted composite score for all pretransplant comorbid conditions. In their analysis of 78 AML first remission patients, the authors reported that higher CCI scores (CCI>2 vs. CCI 0–2) correlated with higher non-relapse mortality at 100 days (14 vs. 3%, p = 0.08) and at 1 year (26 vs. 4%, p = 0.03) after transplant. Sorror et al. reported their results of a retrospective analysis of hematologic malignancy patients, who received matched unrelated HSCT after a myeloablative (N = 74) or nonmyeloablative (N = 60) conditioning regimen. Pre-transplant co-morbidities also were scored per CCI in that study. The authors concluded from their analysis that higher pre-transplant CCI score was an independent risk factor for higher transplant-related toxicity and non-relapse mortality [22, 23]. Arzt et  al. [24] compared the performance of the CCI and the Kaplan– Feinstein scale in predicting outcomes after reduced-intensity conditioning with fludarabine and melphalan transplant in 81 patients with a variety of hematologic malignancies. Median age was 51 (range 17–68) years and 55% of the patients received cells from an HLA-identical sibling donor. In total, 53% of patients scored at least one point for a co-morbid condition as assessed by KF, as opposed to 25% by CCI (p < 0.001) and 28% scored at least two points by KF vs. 8% by CCI (p < 0.001). Co-morbidity, PS and age greater than 50 years predicted increased non-relapse mortality. Sorror et al. [25] recently reported a refinement of the CCI to account for some transplant-specific factors such as prior infection. This modified CCI segregated patients into three risk categories for transplant-related mortality. Patients with scores of 0, 1–2, and greater than 2 had transplant-related mortality rates of 13, 24, and 40%, respectively. When the score was applied to patients receiving either non-ablative or ablative conditioning regimens, the transplant-related mortality rate was significantly different only in patients with scores of less than 3 (17 vs. 27% p = 0.002) for scores 1–2, and 39 vs. 42% (p = 0.18) for scores greater than 2 [24]. Recently, this scoring system has been validated with data from patients with AML or MDS in remission from M.D.

Chapter 38  Functional Assessment Tools and Co-morbidity Scoring 

Table 38-1.  Elements of the seattle comorbidity scoring system. Comorbidity

Weighted score

Bleeding Headache Osteoarthritis Gastrointestinal disease Coagulopathy Osteoporosis Renal, mild Endocrine disease Asthma Pulmonary, mild Hypertension Arrhythmia

1

Cardiac

1

Inflammatory bowel disease

1

Diabetes

1

Cerebrovascular disease

1

Psychiatric disturbance

1

Hepatic, mild

1

Obesity

1

Infection

1

Rheumatologic

2

Peptic ulcer

2

Renal, moderate/severe

2

Pulmonary, moderate

2

Prior solid tumor

3

Heart valve disease

3

Pulmonary, severe

3

Hepatic, moderate/severe

3

Anderson Cancer Center and the Fred Hutchinson Cancer Research Center. Elements of the Seattle Co-morbidity Score are summarized in Table 38-1. Traditionally, transplant outcomes have been measured primarily as survival, event-free survival, or degree of organ toxicities, with most quality of life studies limited to long- term follow- up studies. Recently, comprehensive prospective longitudinal studies of symptom burden post transplant have opened the door for a potential new measurement of transplant outcome [26].

3. Functional Assessment Tools HDT represents the treatment of choice for many patients with hematological cancers as well as some solid tumors. Despite the benefits associated with BMT, e.g., improved relapse-free survival, there are also numerous associated

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sequelae, such as pancytopenia, fatigue, and organ toxicities that may limit patients’ function and activities. These health problems compromise physical function thereby adding to distress and disease burden for individuals and their families. Given that long-term survival is now a reality, it is important to focus on the quality of that survival i.e., enhancing quality of life and of living with a chronic and potentially fatal disease. Average life expectancy in the United States is approximately 85 years for females and 80 years for males. Thus a 65-year-old patient with acute myeloid leukemia who has a median survival of less than a year could potentially benefit from aggressive therapy with curative intent. How to balance the risk/ benefit ratio in older patients has become an important area of research in the recent years. Through its focus on symptom management (pain and fatigue) and improvement in physical function, rehabilitation plays an important role in the management of patients with BMT. Optimal management of function is predicated on a sound, comprehensive assessment using psychometrically sound (valid and reliable) assessment tests. In recent years, standardized performance-based tests have been developed and used in patients with a variety of chronic health conditions including those with cancers. Standardized tests of physical performance quantify task performance and complement patient self-reports of function. A variety of performance test batteries have been developed for and tested in different patient groups. Based on simple measures of time taken or distance covered, these task batteries have clinical utility and excellent psychometric properties. They are used to assess the magnitude and burden of physical dysfunction, predict outcome – including survival, identify the factors that limit physical performance (e.g., pain, fatigue, fear, muscle weakness), guide and determine the effectiveness of treatment protocols. Performance tests are sensitive to improvement following rehabilitation, are good predictors of long-term (5-year) survival and may be sensitive to change in disease status. Five functional tests have been validated in cancer and normal patients are relatively easy to perform in a short period of time and are easily reproducible, these are summarized in Table 38-2 [27–29]. These tests have not been prospectively validated as prognostic factors for outcomes in SCT, but prospective studies are currently underway.

Table 38.2.  Measures of functional status.

TEST

30–59 years (Cancer patient/ control)

>59 years (Cancer patient/control)

Comments

Coin test

6.7/3.9

8.5/5.5

Time to pick up four coins

Sock test

9.0/5.1

10.3/7.0

Time to put on a loose fitting sock

50 foot walk

15.9/9.2

30.3/11.6

Time to walk 50 ft at a comfortable pace

Sit to stand

8.0/2.8

9.0/4,0

Time to stand up and sit down again from a chair

6 min walk in meters

320/602

240/465

Distance walked during 6 min at comfortable pace

Chapter 38  Functional Assessment Tools and Co-morbidity Scoring 

4.  Other Potential Prognostic Factors of Transplant Outcomes Atrial natriuretic peptide (ANP) is a hormone that is released from myocardial cells in response to volume expansion. Brain natriuretic peptide (BNP) is a natriuretic hormone that is similar to ANP. It was initially identified in the brain but is also present in the heart, particularly the ventricles. ANP and BNP are increased in heart failure [30]. The plasma concentrations of both hormones are increased in patients with asymptomatic and symptomatic left ventricular dysfunction. Both ANP and BNP have diuretic, natriuretic, and hypotensive effects. They also inhibit the renin-angiotensin system, endothelin secretion, and systemic and renal sympathetic activity [31]. Recently, plasma BNP and troponin levels have been shown to predict transplant outcomes in patients with amyloidosis [32, 33]. Based on these results we have been checking plasma BNP levels on all patients prior to beginning high dose chemotherapy. Although no patients have had their chemotherapy canceled due to an extremely high plasma BNP level, the measurement has provided a useful baseline to assist in the differential diagnosis of dypsnea during the peritransplant period. C Reactive Protein (CRP) is an acute phase protein that is produced predominantly by hepatocytes in response to cytokines such as interleukin (IL)-6 and tumor necrosis factor-alpha [34]. Despite a lack of specificity for the cause of inflammation, data from more than 30 epidemiologic studies have shown a significant association between elevated serum or plasma concentrations of CRP and the prevalence of underlying atherosclerosis, the risk of recurrent cardiovascular events among patients with established disease, and the incidence of first cardiovascular events among individuals at risk for atherosclerosis [35]. CRP has been shown to be predictive of transplant outcomes in a small number of HDT and SCT studies [36–38]. Iron overload has been associated with increases in treatment-related mortality (TRM) for patients with hematologic malignancies undergoing hematopoietic stem cell transplantation (HSCT). Armand et al. recently studied 590 patients who underwent myeloablative allogeneic HSCT. An elevated pretransplantation serum ferritin level was strongly associated with lower overall and disease-free survival. Subgroup multivariable analyzes demonstrated that this association was restricted to patients with acute leukemia or myelodysplastic syndrome (MDS); in the latter group, the inferior survival was attributable to a significant increase in TRM. There was also a trend toward an increased risk of veno-occlusive disease in patients with high ferritin [39–42]. These studies have the limitation that hyperferritenemia is not always correlated with iron overload, although at ferritin levels of greater than 1,000 ng/ ml liver iron concentration as measured by MRI did have a modest correlation with serum ferritin levels [43].

5.  Summary As high dose therapy is performed in more elderly and debilitated patients, the potential deterioration in quality of life and functional independence after therapy may be higher. These outcomes are not routinely measured and are

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now becoming important aspects of progenitor cell transplantation research. Efforts in determining the prognostic value of co-morbidities, functional assessment tools will be an important first step in designing intervention and prevention trials with anabolic agents or anti-inflammatory cytokines. This area is a field in which transplant specialists can benefit from close collaborations with geriatricians, rehabilitation medicine specialists, endocrinologists and physical therapists. Finally, the potential role of genetic polymorphisms has not been reviewed in this chapter, but has been shown to be an important determinant of transplant outcomes [44]. The challenge will be to incorporate this information with that derived from the tests explored in this chapter to provide the optimal therapy for each individual patient.

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S. Giralt and U. Popat patients with primary systemic amyloidosis undergoing peripheral blood stem cell transplantation. Blood 94(6):1881–1887 34. Kushner I (1982) The phenomenon of the acute phase response. Ann NY Acad Sci 389:39 35. Pearson TA, Mensah GA, Alexander RW et  al (2003) Markers of inflammation and cardiovascular disease: Application to clinical and public health practice: A statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association. Circulation 107:499 36. Fuji S, Kim SW, Fukuda T, Mori S et al (2008) C Reactive protein (CRP) value may predict acute graft-versus-host disease and nonrelapse mortality after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 14:510–517 37. Pihusch M, Pihusch R, Fraunberger P et al (2006) Evaluation of C-reactive protein, interleukin-6, and procalcitonin levels in allogeneic hematopoietic stem cell recipients. Eur J Haematol 76:93–101 38. Hinguranage A, Goldschmidt H, Cremer FW et al (2006) Preoperative elevation of serum C-reactive protein is predictive for prognosis in myeloma bone disease after surgery. Br J Cancer 95(7):782–787 39. Armand P, Kim HT, Cutler CS (2007) Prognostic impact of elevated pretransplantation serum ferritin in patients undergoing myeloablative stem cell transplantation. Blood 109:4586–4588 40. Ho GT, Parker A, MacKenzie JF, Morris AJ, Stanley AJ (2004) Abnormal liver function tests following bone marrow transplantation: Aetiology and role of liver biopsy. Eur J Gastroenterol Hepatol 16:157–162 41. Miceli MH, Dong L, Grazziutti ML et al (2006) Iron overload is a major risk factor for severe infection after autologous stem cell transplantation: A study of 367 myeloma patients. Bone Marrow Transplant 37:857–864 42. Altes A, Remacha AF, Sureda A et al (2002) Iron overload might increase transplant-related mortality in haematopoietic stem cell transplantation. Bone Marrow Transplant 29:987–989 43. Majhail N, DeFor T, Lazarus H, Burns L (2008) High prevalence of iron overload in adult allogeneic hematopoietic cell transplant survivors. Biol Blood Marrow Transplant 14:790–794 44. Cullup H, Dickinson AM, Cavet J, Jackson GH, Middleton PG (2003) Polymorphisms of IL-1alpha constitute independent risk factors for chronic graft versus host disease following allogeneic bone marrow transplantation. Br J Haematol 122:778–787

Chapter 39 Unique Thrombotic and Hemostatic Complications Associated with Allogeneic Hematopoietic Stem Cell Transplantation Amber A. Petrolla, Hillard M. Lazarus, and Alvin H. Schmaier

1. Introduction Hemorrhage and thrombosis are two potential sources of morbidity and mortality of hematopoietic stem cell transplantation (HSCT). In this chapter, we will examine the incidence, pathophysiology, and treatment of bleeding and nonphysiologic clot formation in the setting of HSCT. The focus of this work will be to emphasize the unique hemorrhagic and thrombotic complications associated with HSCT, and not hemorrhage and thrombosis due to intrinsic disorders. The patient, in a constitutive anticoagulated intravascular state, responds appropriately to blood vessel damage and hemorrhage with clot formation and hemostasis, blood vessel repair and subsequent thrombolysis to return to equilibrium. Endothelial cells line and maintain the subendothelial matrix critical for blood vessel integrity. Endothelial cells and their matrix secrete shed factors, substances and proteins that are involved in regulating hemostasis and thrombosis and in maintaining the constitutive intravascular anticoagulated state (Table 39-1). Damage to endothelial cells with exposure of the subendothelium increases the risk for initial hemorrhage and subsequently, non-physiologic thrombosis as part of the repair system. One can view the hemostatic and thrombotic system that addresses the integrity of blood flow as part of the inflammatory system. This chapter will be divided into three sections. As graft-vs-host disease (GVHD) is a major complication of HSCT, it will be briefly reviewed because its presence often suffuses the underlying clinical states where the hemorrhagic and thrombotic complications associated with HSCT are seen. Next, this chapter will review the unique hemorrhagic and thrombotic complications and syndromes associated with HSCT. It is not the intent of this chapter to outline

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_39 © Springer Science + Business Media, LLC 2003, 2010

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Table 39-1.  Vessel wall products and their influence on hemostatic and platelet reactions. Platelet activators

Anti-platelet agents

Tissue-plasminogen activator

vWF

Nitric oxide

Single chain urokinase

Platelet activating factor

Prostacyclin

Endothelial cell protein C receptor

Collagen

Ecto-ADPase

Antithrombin

E-Selectin

Procoagulants

Anticoagulants

Profibrinolytics

Tissue factor

Tissue factor pathway inhibitor

Factor V

Thrombomodulin

Protease-activated receptor-1 Plasminogen activator inhibitor-1

a clinical approach to the diagnosis of hemostatic and thrombotic disorders; this information has been recently reviewed [1, 2]. It is important to remember than when approaching a HSCT patient with a thrombotic or hemostatic disorder, the same rigorous approach for diagnosis should be given to any patient with a similar presentation. However, the complications need to be viewed within the context of the unique hemostatic and thrombotic disorders associated with HSCT, to be discussed below.

2. Graft-vs-Host Disease GVHD is one of the most significant complications reportedly affecting more than half of allogeneic HSCT patients, and is the most common cause of treatment-related mortality [3–6]. Acute GVHD (aGVHD) usually manifests within the first few days to as many as 100 days after HSCT. The mortality rate associated with grade IV aGVHD can exceed 90% [4]. The pathophysiology and susceptibility of HSCT patients to develop aGVHD and chronic GVHD (cGVHD) are indeed complex and are described more fully in Chapters XXXX (D. Porter, et al. and other). Briefly, however, aGVHD has been described in murine models as occurring in several steps, commencing with damage to host tissues by the conditioning regimen, infection, and underlying disease and the release of cytokines. Major histocompatibility complex (MHC) antigen expression is subsequently increased and antigens are presented to donor T-lymphocytes by antigen presenting cells (APCs), most notably by dendritic cells. In this first stage, the gastrointestinal (GI) tract barrier is also damaged, especially when total body irradiation (TBI) is employed. Therefore, microbial products in the GI tract, such as lipopolysaccharides (LPS), further stimulate the immune system, and gain access to the circulation. Next, in the second stage, donor T-lymphocytes are activated and react to the recipient’s “foreign” antigens, presented by recipient APCs. A multitude of cytokines are also expressed in this stage, further amplifying this process. Finally, the microbial products introduced during stage 1 activate monocytes [4]. Ultimately in aGVHD, donor T-lymphocytes in the graft damage recipient tissues, including epithelial cells in the GI mucosa, skin, and liver. The incidence of aGVHD and thrombocytopenia is higher in mismatched and unrelated donor (MUD) HSCT than in matched related donor transplantations (MRD). At least one-third of patients undergoing MRD transplantations suffer from aGVHD [7]. In one study of 807 patients who underwent allogeneic

Chapter 39  Unique Thrombotic and Hemostatic Complications Associated with Allogeneic 

HSCT, 45% of mismatched HSCT patients and 23% of MRD HSCT patients suffered a hemorrhagic complication. Hemorrhagic cystitis (see below) followed by bleeding from the GI tract (see below) were the most common sources [5]. In a study of 364 patients receiving allogeneic HSCT, in which 250 had aGVHD, the GI tract was a significant source for bleeding, affecting 37.6%. The GI tract followed behind the skin, nose and mouth as most common site of bleeding. In those with aGVHD grade II or higher, 89.2% had a hemostatic event and 87.6% had a bleeding complication. Only 72.6 and 70.8% of those HSCT patients without aGVHD suffered a hemostatic event and a bleeding complication, respectively [8]. In a study of 807 allogeneic HSCT patients, those with grade III or IV aGVHD had a higher tendency to bleed (55.0%) as compared to patients with grade 0 to II aGVHD (42.0%) [5]. Chronic GVHD arises after donor stem cells ultimately yield progeny lymphocytes but in the setting of immune dysregulation with a thymus that is not fully functional. cGVHD, occurring between 80 and 200 days after HSCT, is due to autoantibodies/self-reactive lymphocytes which the recipient fails to regulate. Multiple recipient tissues are affected, including the skin, eyes, lungs, oral cavity, GI tract, and lungs. Patients with cGVHD also have a higher risk of bleeding. Histologically, cGVHD is characterized by marked sclerosis in many tissues, including the dermis of the skin, the lamina propria of the GI tract, the biliary triads, and the salivary and lacrimal glands. The vascular endothelium and mucosal epithelium are subjected to destructive insults, leading to a local proliferation of blood vessels, which contributes to the increased bleeding risk in affected patients (Table  39-2) [8, 9]. Patients with cGVHD Table 39-2.  Mechanisms of bleeding and thrombosis due to aGVHD and cGVHD. Bleeding   cGVHD    Destruction of epithelium and endothelium    Hyperperfusion of affected tissues    Proliferation of vessels    Mucosal scaring and possible trigger of superinfections   aGVHD    Destruction of epithelium and endothelium    Marrow involvement and subsequent severe thrombocytopenia    Decreased Factor XIII Thrombosis   aGVHD    Endothelial damage    TA-TMA   cGVHD    Acquired lupus anticoagulant    Circulating thrombogenic platelet microparticles    Increased PAI-1    Endothelial damage

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are also at higher risk for venous thrombosis and pulmonary embolism, likely mediated through damage to endothelial cells secondary to cGVHD, TNFalpha, and circulating thrombogenic platelet microparticles [10]. We also can postulate that with the inflammation associated with cGVHD, there should be increased C4b binding protein with reduced free protein S with secondary reduced activated protein C function and increased risk for clot formation. The inflammatory state associated with cGVHD probably creates the conditions increasing thrombosis risk.

3. Bleeding Bleeding is especially problematic in patients who receive HSCT. These patients are often thrombocytopenic, a problem further complicated by the fact that platelet production is usually the last cell line to recover after engraftment. Bleeding does not occur in a vacuum and typically needs a precipitating event to become a problem – such as epithelial tissue injury, endothelial cell disruption, damage to blood vessel integrity, or combinations of both. In one retrospective study of 364 patients who underwent allogeneic HSCT, 85.4% had either a hemorrhagic or thrombotic event, or both, and 84.3% experienced a bleeding complication [8]. Hemorrhagic events occurred primarily within the first 4 weeks after transplant and the majority were mild, mostly involving the skin, nose and mouth. In this study, allogeneic HSCT patients had a higher risk of bleeding complications than autologous HSCT patients, thus hemorrhagic risk is great in HSCT. These data indicate that hemostatic complications (hemorrhage and thrombosis) occur in a high percentage of HSCT patients and bleeding is a far more frequent complication than thrombosis (Table  39-3). The following section will discuss the range of common and rare etiologies of bleeding in HSCT. 3.1. Hemorrhagic Cystitis Hemorrhagic cystitis is a well-recognized and common complication after allogeneic HSCT occurring in up to 60% of patients [8, 9, 11, 12], usually within the first few months, but a range up to 166 days after transplantation has been reported [11, 12]. In a study of 807 allogeneic HSCT reported by Bacigalupo, hemorrhagic cystitis was the most frequently encountered hemorrhagic complication [5]. Patients can present with gross or microscopic hematuria, dysuria, frequency, and even overt renal failure; often, they do not have an identifiable urinary tract infection. Initially, this complication was attributable to the direct toxic effect of cyclophosphamide metabolites on the bladder mucosa, first described in 1959. Hemorrhagic cystitis also has been associated with aGVHD and cGVHD and re-activation of several viruses, including Polyomavirus hominis 1 (the BK virus) and adenovirus. With the effective use of prophylactic agents such as mesna, which reacts with toxic metabolites of cyclophophamide to form nontoxic compounds, hemorrhagic cystitis after HSCT is due almost entirely to BK virus. It is interesting that patients with aGVHD who have been treated with cyclophosphamide have been shown to develop hemorrhagic cystitis 100 days or longer after transplantation. This event reflects an unrecognized long-term damage conferred by aGVHD, cyclophosphamide or a combination of the two [8, 9]. Late onset hemorrhagic

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Table 39-3.  Bleeding complications associated with allogeneic HSCT. Complication

Incidence in allogeneic Time of onset after HSCT patients allogeneic HSCT Predisposing factors

Hemorrhagic cystitis

Up to 25% [8, 9]

166 days [11]

BK virus infection Cyclophophamide Ifosfamide aGVHD

Gastrointestinal hemorrhage

5–35% [3, 8, 15]

33–159 days [3, 8]

aGVHD TBI Factor XIII deficiency

Diffuse alveolar hemorrhage (DAH)

Subdural hematoma

1–21% [21–24]

0–11.9%, autopsy studies

0–30 days, range to 26 months reported [21]

Severe aGVHD

2–60 days [29]

Intrathecal chemotherapy

2–5%, antemortema [29, 31]

Increased age Myeloablative conditioning [21–23, 27]

Radiation Intracranial metastases Trauma Thrombocytopenia [29, 32]

Intracerebral hemorrhage (ICH) (a) Intraparenchymal hemorrhage (IPH)

2–11%a [33]

0–100 daysa [31]

(b) Subarachnoid hemorrhage (SAH)

4.4% [8]

Leukemic relapse

(c) Subdural hemorrhage (SH)

32.3%, post mortem studya [33]

Intracranial hygromas

Infection

FK506 (rare) Ocular posterior segment complications (a) Microvascular retinopathy/ transplant retinopathy

a–d: 12.8%a [35]

a: 0–6 months, range to 62 monthsa [35]

a: cyclosporine

(b) Vitreous hemorrhage

b–d: 3.5%a

b–d: 0–6 months, median 52 days, range to 360 daysa [36]

  TBI

(c) Intraretinal hemorrhage

b: 1%a [36]

  Multifactorial with conditioning

(d) “Other hemorrhage”

c: 2.5%a [36]

  Regimens contributing a [35, 36] b–d: Thrombocytopeniaa [35]

a

Study does not differentiate complications occurring in allogeneic vs. autologous HSCT (all one group)

cystitis is associated with BK virus infection. Primary treatment for BK virus-associated cystitis consists of maintaining adequate hydration, pain control, and continuous bladder irrigation. Secondary treatments for persistent BK virus-associated cystitis include: systemic antimicrobial agents – most notably

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cidofovir, intravesical cidofovir, formalin, alum, estrogen, and vesical artery embolization, all with varying degrees of success [11, 13, 14]. 3.2. Gastrointestinal Bleeding GI bleeding a very common hemorrhagic complication from allogeneic HSCT, with the incidence ranging from 5 to 35% and onset ranging from 33 to 159 days after HSCT [3, 8, 15]. The most common site of bleeding within the GI tract is the distal small bowel or cecum; however, cases of gastric bleeding due to aGVHD have been reported [3]. Most bleeding events are mild, but the occurrence of any GI bleeding event carries a higher overall mortality. ErtaultDaneshpouy assessed the duodenal biopsies of 68 allogeneic HSCT patients with GI symptoms occurring less than 100 days after HSCT using endoscopy. In 13 patients, all of whom had grade III or IV GI aGVHD, capillary damage, including pericapillary hemorrhage was found. Both ultrastructural and immunohistochemical studies demonstrated capillary basal membrane rupture and endothelial cell alterations [16]. The GI epithelial cells are especially susceptible to damage from chemotherapy, radiation and GVHD. Endothelial cells in the vasculature also sustain injury from these same insults leading to compromise of blood vessel integrity with resulting endothelial cell proliferation [16]. In addition to the direct damage to the endothelium and epithelium, patients with GI aGVHD are at increased risk for bleeding also because they have been shown to have a relative Factor XIII deficiency, which is responsible for the final cross linking of the fibrin clot [8, 17]. The mechanism of Factor XIII reduction in patients with GVHD, like patients with chronic inflammatory bowel disease, is not completely known, but it is believed to be acquired from increased consumption of Factor XIII in the inflammatory process. In a study of 27 allogeneic HSCT patients with severe aGVHD of the GI tract who were treated with Factor XIII, those who responded to treatment were shown to have low initial values of Factor XIII [18]. Causes of GI bleeding other than those due to GVHD in HSCT patients have been reported, including gastrointestinal vascular ectasia (GAVE) [19]. Ulcers and infections, both preexisting and new (notably due to CMV), can also be responsible for GI bleeding in the allogeneic HSCT setting. Treatment of other disorders, such as the use of defibrotide, an adenosine receptor antagonist, for veno-occlusive disease (VOD) of the liver after allogeneic HSCT, has been associated with GI bleeding due to increased anticoagulation [20]. 3.3. Diffuse Alveolar Hemorrhage Diffuse Alveolar Hemorrhage (DAH) is a pulmonary complication seen after both autologous and allogeneic HSCT, with a reported incidence of 1–21% in the allogeneic setting [21–24]. DAH most commonly occurs within the first 30 days post transplant, with a reported range of 3 days to 26 months [21]. DAH is diagnosed by the acute onset of alveolar infiltrates, hypoxemia, and worsening bloody alveolar lavage, in the absence of infection [21, 22]. This complication must be differentiated from infection-associated alveolar hemorrhage (IAH). This is difficult to distinguish clinically from DAH as the infectious etiology can be missed. One hundred day DAH mortality rates range from 60 to 76% [21–23]. At this time, the exact mechanism and pathology underlying

Chapter 39  Unique Thrombotic and Hemostatic Complications Associated with Allogeneic 

DAH is not completely understood, but it is most likely multi-factorial. Vascular damage, inflammation, and alveolar hemorrhage secondary to radiation and chemotherapeutic agents, activation of the immune system, and aGVHD have all been implicated as possible etiologies of this disease. Afessa looked at 1,919 HSCT patients, of which 116 cases of alveolar hemorrhage were diagnosed; 89% of whom received an allogeneic HSCT. Forty-five patients were diagnosed with DAH, and 71 with IAH. There was no difference in time of onset of hemorrhage between these two groups of patients; however, the patients who received an unrelated umbilical cord blood transplant and TBIconditioning regimen were more likely to be diagnosed with IAH rather than DAH. In the patients with confirmed IAH, Staphylococcus spp. was the most common organism identified [22]. DAH is not associated with thrombocytopenia and is not corrected with platelet transfusion [22]. Standard treatment for DAH is high dose corticosteroids with a gradual taper, thought to counteract inflammation, which is one of the main contributors to the underlying pathogenesis of DAH. There have not been clinical trials, however, to investigate this treatment. Reports of treatment of DAH with recombinant Factor VIIa showed conflicting results, since some patients received therapy for other reasons than DAH, such as for gastrointestinal bleeding and hemorrhagic cystitis, and had a variety of hematologic malignancies [24–26]. Amicar plus standard corticosteroids led to an improved 100-day mortality rate in one study; however, many serious side effects have been observed, including thrombosis, seizure, stroke, and dysrhthmias [23]. Decreased GVHD from T-cell depletion prior to allogeneic HSCT led to a lower incidence of GVHD and a lower incidence of pulmonary complications [27]. Reported risk factors for developing DAH include older age, myeloablative conditioning and aGVHD [21–23, 27]. A recent study showed the same incidence of DAH in both myeloablative and reducedintensity conditioning (RIC) regimens, thus demonstrating the need for further study in this area [28]. 3.4. Central Nervous System Bleeding The Central nervous system (CNS) can also be involved with hemorrhagic complications after HSCT. Although this site is not commonly involved, it is nonetheless important to recognize these potentially fatal complications. Subdural hematoma (SDH) represents one rare but important complication of HSCT and is frequently bilateral [29, 30]. Autopsy studies have reported and incidence between 0 and 11.9%; however, reportedly only 2–5% are clinically recognized ante mortem, although these studies grouped both autologous and allogenetic HSCT patients [29–31]. The reported time to diagnosis ranges between 2 and 60 days post-transplantation. There can be a range of symptoms, but the most common is headache. Risk factors indicated include intrathecal chemotherapy, radiation, intracranial metastases, trauma and thrombocytopenia, with platelet counts usually below 20,000/ml [29, 30, 32]. Computer tomography (CT) without contrast is the usual mode of diagnosis, although MRI studies have been utilized as well. These patients are often treated conservatively with platelet transfusion and coagulation protein replacement as needed. Intracranial hemorrhage (ICH), including intraparenchymal hemorrhage (IPH), subarachnoid hemorrhage (SAH), and subdural hemorrhage (SH)

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have also been reported. In a study of 364 allogenetic HSCT patients, 4.4% suffered an intracranial hemorrhage [8]. Other studies cite and incidence of 2–11% after HSCT, but do not differentiate between autologous and allogeneic patients [33]. An autopsy study of 180 patients who died after HSCT, 32.2% experienced ICH; however, this study did not indicate which patients had allogeneic HSCT and autologous HSCT. In this study, following IPH in decreasing order of frequency were SAH and SH, and many patients had more than one type of hemorrhage [33]. Tacrolimus (FK 506) has been infrequently implicated in leukoencephalopathy and hemorrhage, possibly due to endothelial cell damage and subsequent capillary leak. In a study of 642 allogeneic HSCT patients, 1.6% developed FK 506 leukencephalopathy and cerebral hemorrhage [34]. 3.5. Ocular Bleeding Ocular complications can manifest in the post-HSCT patient as well. Etiologies include the underlying disease, the conditioning regimen used, cyclosporine, TBI, immunosuppression or a combination of these entities. The posterior segment bleeding complications, with an overall incidence of 12.8%, include microvascular retinopathy/BMT retinopathy, vitreous hemorrhage, intraretinal hemorrhage, and those grouped under “other hemorrhage” [35]. The incidence of the latter three complications taken together has been reported to be 3.5%. These studies, however, did not differentiate between autologous and allogeneic HSCT patients [35]. Microvascular retinopathy/BMT retinopathy usually occurs within the first 6 months after HSCT. Patients have a variety of visual disturbances including decreased bilateral acuity or deficit. Retinal exam shows multiple cotton wool spots, telangiectasias, microaneurysms, macular edema, and retinal hemorrhages. Cyclosporine is thought to contribute to the etiology of this disorder, but has not been shown to cause microvascular retinopathy in autologous HSCT patients [35]. This disorder usually does not require aggressive treatment. Vitreous and intraretinal hemorrhages can occur as well, with reported incidences of 1% and 2.5%, respectively, although there are very few series which do not separate autologous and allogeneic HSCT patients documenting these findings [36]. Thrombocytopenia appears to be the most important factor rendering HSCT patients to the hemorrhagic complications [35, 36]. Retrohyaloid vitreous hemorrhage with retinal neovascularization has been reported in a patient after allogeneic HSCT that did not receive radiation treatment [37]. The neovascularization seen was thought to be secondary to hypoxic insults and endothelial progenitors in the transplanted cells [37].

4.  Thrombosis Non-physiologic thrombosis occurs when an accumulation of risk factors overcome the mechanisms that maintain the constitutive anticoagulated state. Risk factors for thrombosis include external compression, damage to endothelial cells, excess concentration of intravascular procoagulants, turbulent blood flow, general medical conditions leading to inflammation, and familial and acquired hypercoaguable disorders. HSCT recipients manifest many of these risk factors including prolonged immobility, endothelial cell damage from

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chemotherapy and radiation, presence of indwelling catheters, acquired lupus anticoagulant and an inflammatory state from infections and GVHD. The etiologies of thrombosis in patients with HSCT are shown in Table 39-4. Table 39-4.  Mechanisms of thrombosis risk associated with allogeneic HSCT. Complication Catheter-related thrombosis

Incidence in allogeneic HSCT patients

Time of onset after allogeneic HSCT

Predisposing factors

4.1%: catheter thrombosis [8]

0–7 weeks [8]

aGVHD [8]

5.8%: extremity or PE [8, 39]

a,b

PAI-1 4G/4G polymorphism [10]

12%: any thrombotic event [39]a,b

Catheter size/material-larger, polytheylene, triple lumen [38, 40] Placement of catheter-high in SVC [38, 40] cGVHD [10]

Hepatic venoocclusive disease (VOD)

5–60% [20, 42–44, 46]

0–30 days

TBI Busulfan Cycophosphamide GVHD prophylaxsis Increased age Female gender HLA mismatch Increase PAI-1 Gemtuzumab ozogamicin (Mylotarg) [45, 46, 53, 56]

Transplantationassociated thrombotic microangiopathy (TA-TMA)

0.5–63.6% [61, 62]

1 day pre-HSCT to 400 days after HSCT [8, 62, 63]

Complex   aGVHD   Conditioning regimen   Increased age   Infection   Cyclosporine   Siroliums   FK506 [59, 62, 63]

Venous sinus thrombosis

Very rare

Pulmonary veno-occlusive disease

Rare

Range 5–298 days [73, 74]

Infection Prothrombotic state Cyclosporine [73]

Acquired hypercoagulable states

6–8 weeks [67, 69]

BCNU Bleomycin [69] a

3% (75 )

Pulmonary cytolytic Very rare thrombi (PCT) a

Infections

34 days [76]

aGVHD

Median 72 days [70]

aGVHD [70, 71]

Study does not differentiate complications occurring in allogeneic vs. autologous HSCT (all one group) Events not reported exclusively during the post-transplant time period

b

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4.1. Catheter-Related Thrombosis In a retrospective study of 364 patients undergoing allogeneic transplants, 4.1% experienced a catheter-associated thrombosis and 5.8% experienced an extremity thrombosis or pulmonary embolus (PE) [8]. Catheter thrombosis occurred very early, within several weeks after transplant. Recipients of an allogeneic transplant had a higher risk of an extremity thrombosis or PE than those who underwent an autologous transplant; the risk was higher if the allogeneic transplant patients experienced GVHD. One of the explanations revolves around the endothelial damage and increased PAI-1 levels from mechanisms due to GVHD [8]. Currently, the American College of Chest Physicians notes that there is no evidence to support routine prophylaxis for prevention of catheter-related thrombosis in patients with both solid tumors and hematologic malignancies. However, clinical discretion should be used with the subset of patients undergoing HSCT. Several studies have demonstrated they are at a greater risk for catheter-related thrombosis, although the events were not reported exclusively in the post-transplant period [38, 39]. The mechanism behind catheter-related thrombosis is multifactorial including the prothrombotic state inherent in malignancy. It is unknown whether inherited or acquired risk factors for thrombosis render a patient more likely to suffer a catheter-related thrombosis [40]. One study showed a 5.7-fold increase risk for catheter-related thrombosis in patients homozygous for the -675 4G/4G polymorphism of the plasminogen activator inhibitor-1 (PAI-1) gene [10]. The PAI-1 4G/4G polymorphism is associated with higher levels of PAI-1. Placement of the catheter, especially when located high in the superior vena cava, is associated with a higher risk of thrombosis. The material, size, and the number of lumens of the catheter are of importance as well, with larger, polyethylene and triple lumen catheters being associated with a higher thrombosis risk [38, 40]. Lastly, patients with cGVHD have been shown to be at higher risk for venous thrombosis and PE likely mediated through damage to endothelial cells secondary to GVHD, TNF-alpha, and circulating thrombogenic platelet microparticles [10]. It is essential for clinicians to realize that the presence of an upper extremity thrombosis, PE, or the combination in the transplant patient requires standard levels of anticoagulant for the same duration of time as for any other patient with a deep venous thrombosis. 4.2. Hepatic Veno-Occlusive Disease (VOD) VOD is a serious, life-threatening complication of allogeneic HSCT, with reported incidence in the literature ranging from 5% to as high at 60% [20, 41–44]. Hepatic VOD occurs most commonly, but not exclusively, within the first 30 days post transplant. The conditioning regimen used greatly influences the reported incidence, with busulfan, cyclophosphamide, and TBI being most commonly associated with the development of VOD [45]. “Sinusoidal obstruction syndrome” (SOS) has recently been suggested as an alternative nomenclature to VOD. Although sinusoidal obstruction has found as a contributing factor in the pathogenesis of this type of liver injury in both humans and rats, it is not the only essential element. This term may be more applicable to VOD secondary to gemtuzumab ozogamicin [46]. The diagnosis of VOD is based on clinical criteria and presentation ­including: hyperbilirubinemia and jaundice, hepatomegaly, right upper quadrant

Chapter 39  Unique Thrombotic and Hemostatic Complications Associated with Allogeneic 

pain, ascites, edema and weight gain. There are several clinical criteria used including the Jones Criteria, which consists of: hyperbilirubinemia greater than or equal to 2  mg/dL before day 21 post transplantation and two of the following: jaundice, hepatomegaly and right upper quadrant pain, and ascites and/or unexplained weight gain [47]. Fluid retention and hepatomegaly are typically the first signs of developing VOD and can be apparent on the first day of HSCT [47]. The clinical severity of this syndrome ranges from mild to severe disease. In mild VOD, the liver dysfunction can resolve spontaneously, whereas with moderate VOD, treatment is required for complete resolution. In severe VOD, defined as moderate to severe liver dysfunction combined with organ failure in at least one other organ system, survival is poor, with a mortality rate near 100% by day 100 post-transplant [44, 46]. Few noninvasive studies are available to help differentiate hepatic VOD from other hepatic disease. A recent retrospective study of 18 allogeneic HSCT patients reported abdominal CT scanning to be helpful to differentiate hepatic VOD from hepatic aGVHD, because they can have similar non-specific clinical symptoms and often patients do not get liver biopsies to confirm diagnoses. In this study, a small right hepatic vein diameter, periportal edema and ascites were more common in patients with hepatic VOD, and small-bowel wall thickening was much more common in aGVHD [48]. The pathogenesis of hepatic VOD is directly and indirectly related to many factors associated with HSCT. Pre-disposing factors identified include GVHD prophylaxis, the type and intensity of the transplant conditioning, advanced age, female gender and HLA mismatch, among others [46]. TBI dose, radiation dose rate and busulfan route and dose have the greatest impact the development of severe VOD. The pathogenesis, in part, stems from direct injury to the sinusoidal endothelium by the chemotherapeutic agents used in the conditioning regime, notably cycophosphamide and the TBI dose. Also contributing to VOD diasthesis are the multitude of cytokines elaborated after both tissue injury and HSCT as well as the development of microthrombi in the vasculature. The diagnosis of VOD is confirmed histologically after liver biopsy. The initial microscopic observation is widening of the subendothelial space between the basement membrane and the lumen of central venules, followed by edema and increased expression of von Willebrand factor. Sinusoidal endothelium is damaged without inflammatory cell involvement; fibrin deposition and collagenous obstruction of the venules and necrosis of hepatocytes ensue [46, 49]. Other studies suggest that injury to the sinusoidal endothelial cells is more significant than the damage to the venules [49, 50]. Contributing to the pathogenesis is a decrease in the amount of major anticoagulants (antithrombin III, protein C) in the plasma of patients after receiving high-dose cytoreductive treatment [46, 49]. Several studies have focused on the increased levels of PAI-1 found in the plasma of patients with VOD [50–53]. When wild type and PAI-1 deficient mice are exposed to nitrous oxide synthetase inhibition, the wild type mice develop extensive hepatic dysfunction and fibrinoid hepatic venous thrombi whereas the PAI-1 deficient group do not [53]. Wildtype mice treated with tiplaxtinin, a PAI-1 antagonist, are protected from the fibrosis development and hepatic vein thrombosis [53]. One important agent whose exposure can result in VOD is gemtuzumab ozogamicin (Mylotarg). Gemtuzumab ozogamicin is a monoclonal antibody directed against CD33, present on progenitor cells of myeloid origin, and is

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used in the treatment of acute myelogenous leukemia (AML). Unfortunately, endothelial cells and stellate cells in the liver also express CD33 and therefore, may be damaged by this agent. A number of studies have shown that patients previously treated with gemtuzumab ozogamicin have an increased propensity to develop hepatic VOD after HSCT. Most of these studies, however, investigated patients that were treated with more than one agent in addition to gemtuzumab ozogamicin [41, 54]. In a retrospective study of 62 patients who underwent allogeneic HSCT, it was shown that exposure to gemtuzumab ozogamicin prior to and within three- and- a- half months of HSCT were at increased risk of developing hepatic VOD [55]. A retrospective study of 47 patients who received gemtuzumab ozogamicin as single agent showed that if given alone, it does not increase risk of developing VOD [56]. Given the high morbidity and mortality associated with hepatic VOD, its prevention and treatment is of paramount importance. A reduced-intensity pre-transplant conditioning regimen has been shown to have a lower incidence of hepatic VOD; however, this approach is not appropriate for all patients. A number of agents used for prophylaxis have been (prescribed?) including glutathione, prostaglandin E and antithrombin III, tissue plasminogen activator (tPA), heparin, ursodeoxycholic acid and steroids. Higher serum busulfan concentrations are associated with the development of hepatic VOD and “targeted dose” busulfan dosing during the preparative regimen or use of parenteral rather than oral busulfan dosing reduce the likelihood of developing hepatic VOD [55, 57]. Defibrotide is an agent with anti-thrombotic, anti-ischemic, anti-inflammatory and thrombolytic properties. Defibrotide is an adenosine receptor agonist and acts on endothelial cells, increasing thrombomodulin and tissue plasminogen activator (t-PA), and decreasing the activity of PAI-1 [51]. It has been shown to be an effective treatment in several studies and its role as a prophylactic agent is promising [42, 50, 51]. Prophylaxis using urosodeoxycholic acid, a hydrophilic bile acid that stabilizes the hepatocyte membrane and may reduce the release of inflammatory cytokines, has shown conflicting results in several studies [46, 58]. Tissue plasminogen activator, no longer, is used as treatment, and the use of antithrombin III has only been studied in a small number of patients [47]. 4.3. Transplantation-Associated Thrombotic Microangiopathy Transplantation-associated thrombotic microangiopathy (TA-TMA) remains a serious thrombotic complication in the post-HSCT setting. The term “posttransplantation TMA” was recently suggested by the Toxicity Committee of the BMT CTN [59, 60]. The incidence of TA-TMA reported varies greatly from 0.5 to 63.6% [61, 62] and can occur anywhere from 1 day prior to transplantation to 400 days post-transplantation [8, 62, 63]. This much-debated disorder, also referred to as (but now thought to be distinct from) thrombotic thrombocytopenia purpura/hemolytic uremic syndrome (TTP/HUS), is characterized by thrombocytopenia, evidence of microangiopathic hemolytic anemia, and the presence of schistocytes on peripheral blood smear, although exact criteria will differ depending on the source. Presentation can vary greatly, and other disorders including disseminated intravascular coagulation (DIC) and infection must be excluded [59, 64]. Presentation always includes schistocytes on perhipheral blood smear, usually accompanied by thrombocytopenia. Serum lactate dehydrogenase (LDH) usually is increased, serum

Chapter 39  Unique Thrombotic and Hemostatic Complications Associated with Allogeneic 

haptoglobin is decreased, and indirect hyperbilirubinemia is present, usually with associated hematuria [65]. Endothelial injury due to a multitude of factors including conditioning regimen, underlying disease and infection all appear to play a central role in the development of TA-TMA. Microscopically, nonspecific lesions of arteriolar thrombi and injury are seen [59, 61]. Platelets are activated leading to platelet-rich thrombi, which enter the circulation and cause multiorgan dysfunction. TA-TMA is a complication solely of allogeneic HSCT and often occurs in the setting of aGVHD. Traditional TTP (sometimes referred to as “primary” TTP/HUS) has been linked to either a severe deficiency (defined as <5% acitivity) of or an antibody to ADAMTS13, which cleaves ultra large vonWillebrand (vWF) multimers [65]. Changes in ADAMTS13 do not appear to be a main mechanism of pathogenesis in TA-TMA (categorized under “secondary TTP/HUS”), as several studies, both retrospective and prospective, did not demonstrate a severe ADAMTS13 deficiency in the vast majority of patients [61, 65–67]. The development of TA-TMA is complex and associated with many factors such as aGVHD, conditioning treatments, age, diagnosis, and infection, among others [62]. Drugs such as cyclosporine (CsA), sirolimus, and FK506 have all been implicated as inciting agents [59, 63]. Benefit from plasma exchange, a standard treatment for conventional TTP, has proven disappointing [66]. Also, plasma exchange has many complications inherent in the procedure. Calcineurin inhibitors should be withdrawn when the diagnosis of TA-TMA is made [60]. Supportive care for this highly morbid and potentially fatal disorder remains a mainstay of treatment, but other treatment modalities are evolving. A number of agents including defibrotide, aclizumab, rituximab, and eicosapentaenoic acid (EPA) have all been suggested as possible treatments for this disorder, although further studies are needed [59, 65]. Mortality in TA-TMA is high, reported in the literature exceeding 60%, and often is a result of myocardial and cerebral ischemia [61, 65]. 4.4. Pulmonary Veno-Occlusive Disease Another rare complication of HCST is pulmonary veno-occlusive disease (VOD). Pulmonary VOD usually occurs 6–8 weeks after transplantation [68, 69]. These patients develop worsening dyspnea and can have signs of pulmonary hypertension. Open lung biopsy demonstrates intimal proliferation of fibrous tissue seen histologically in the pulmonary venules which is distinct from pulmonary cytolytic thrombi (PCT) (see below). Pulmonary VOD has occurred in non-transplant patients as well as in those who have undergone chemotherapy and radiation and in patients with viral infections. These findings suggest that HSCT-associated pulmonary VOD is a result of endothelial injury from infections, bis-chlororethyl nitrosurea (BCNU) or bleomycin [69]. Treatment for pulmonary VOD with high-dose corticosteroids has been reported anecdotally [68, 69]. 4.5. Pulmonary Cytolytic Thrombi PCT, which typically occurs on the background of GVHD, has been observed almost exclusively in a minority of pediatric allogeneic HSCT patients without any reported mortalities. The median onset is approximately 72 days after transplantation, with cough and fever as presenting symptoms [70]. Chest

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CT, while not helpful in the diagnosis of pulmonary hemorrhage, may show multiple peripheral nodules in PCT. PCT can be definitively diagnosed only through lung biopsy, which demonstrates occlusive vascular lesions that can be associated with hemorrhagic infarcts. The thrombi are composed of an amorphous, basophilic substance admixed with lymphocytes [22, 70–72]. In one case study, monocytes were shown by immunohistochemical stains to be the predominate cells in the thrombi, and were shown by fluorescent in situ hybridization (FISH) to be derived from both the donor and recipient [72]. This disorder may be a manifestation of aGVHD, with monocytes being recruited to an area of the lung that has been damaged by mechanisms such as GVHD or infection [70–72]. In a study of 12 allogeneic HSCT patients with PCT, the pulmonary symptoms and lesions resolved over a period of months [71]. Single-agent corticosteroids, or amphotericin or both were used as treatment [70]. 4.6. Dural (Venous) Sinus Thrombosis Venous sinus thrombosis is a very rare complication of allogeneic HSCT, with very few cases reported in the literature. Three patients reported had non-specific neurological symptoms (grand mal seizures and headache), and presented between 25 and 43 days after HSCT [73]. Another report of three patients had a range of presentation of 5–298 days after HSCT [74]. Patients with sagittal sinus thrombosis have a worse course than those with transverse sinus thrombosis. Magnetic resonance imaging with angiography (MRV) is the preferred imaging study over CT scan, since the latter can be normal or show non-specific abnormalities. Treatment consists of intravenous heparin, local thrombolytic therapy, oral anticoagulants, or combinations [73]. The cause of the venous sinus thrombosis is unknown, but the authors postulate infections, prothrombotic states, subclavian line insertion, L-asparaginase, and cyclosporine as possible contributing factors [73]. 4.7. Acquired Hypercoagulable Disorders Acquired antiphospholipid antibodies with antiphospholipid antibody syndrome after both autologous and allogeneic HSCT have been described. In a retrospective study of 1,292 patients who underwent HSCT (465 autologous and 827 allogeneic), 3% developed a de novo lupus anticoagulant [75]. In one report, a patient who underwent allogeneic HSCT who also had aGVHD and cGVHD subsequently developed a lupus anticoagulant (LA) 34 days after transplant, but did not experience a thrombotic event [76]. The development of the LA in this case has been postulated to be attributed to antibodies developed due to the exposure of antigens such as the hexagonal phase lipids, which are not present on intact cell membranes. These lipids may be exposed after inflammatory damage to cell membranes secondary to aGVHD [76]. 4.8. Reduced Intensity and Non-Myeloablative Conditioning Regimens Non-myeloablative (NM) and RIC regimens are being used more frequently as this approach relies upon on the mechanisms of the graft-versus malignancy effect for their success [77]. Relapse, however, is a major problem in this modality. In a study of patients ineligible for myeloablative conditioning regimen, sustained engraftment rate was 93% and 3-year non-relapse mortality and

73 MA GI mucositis = 70%

Hepatic VOD = 18%

Hemorrhage = 8%

HUS 2nd to CSP = 1 pt.

CNS bleeds = 2 pts.

GI mucositis = 0

Hepatic VOD = 0

16

19

36 Hemorrhage = 1%

74 MA

73 NM

12

12

34 RIC

64 NM

59

91

77

43

56

40

14

61

41

No difference

No difference

aGVHD (%) cGVHD (%)

24 NM

Hemorrhage = 34%

Hepatic VOD = 11%

Hemorrhage = 13%

Hepatic VOD = 0%

Toxicity

30

16

2 year: 19%

2 year: 15

32

20

Non-relapse mortalitya (%)

15

42

Transplant-related mortality (%)

Non-relapse mortality at 1 year unless otherwise noted MA Myeloablative, RIC Reduced intensity, NM Non-myeloablative, CSP cyclosporine, HUS hemolytic uremic syndrome, pt(s). patient(s), GI gastrointestinal Grade III and above hemorrhage and aGVHD were recorded in results

a

Diaconescu [81]

Couriel [80]

LeBlanc [79]

60 NM

Sorror [78]

74 MA

# of patients and regimen used

Study

0

7

3

25

Graft failure (%)

Table  39-5.  Comparison of transfusion-related mortality, GVHD, Bleeding and Thrombosis in selected nonmyeloablative and reduced-intensity regimens to myeloablative regimens. Chapter 39  Unique Thrombotic and Hemostatic Complications Associated with Allogeneic  709

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progression-free survival were 23% and 36%, respectively [77]. Sorror et al. retrospectively compared NM and myeloablative HSCT patients; despite the increased age, more advanced disease, and failed prior high dose myeloablative HSCT in those given a NM conditioning, there was less toxicity, less aGVHD, less than 1- year non-relapse mortality, and fewer hemorrhagic events than in those that received myeloablative regimens [78]. In another retrospective study, Le Blanc et al. compared NM HSCT patients to RIC HSCT patients. In this study, the NM group had less leukopenia and less transfusion requirements, but higher incidences of graft failure, more aGVHD, and more TRM than the RIC group. However, both the NM and RIC groups had more graft failure than myeloablative patients [79]. In a study of MRD sibling HSCT patients, the myeloablative group had a higher incidence of both aGVHD (grades II–IV) and cGVHD than the NM [80]. In another retrospective study by Diaconescu et  al., myeloablative and NM patients who underwent MRD HSCT had less regimen-related toxicities (RRTs) and a lower non-relapse mortality than the myeloablative patients. RRTs included GI and other hemorrhage, VOD, and neurologic toxicities, among others [81]. In one study of 193 allogeneic HSCT patients, no cases of hepatic VOD were noted [44]. Although RIC and NM conditioning regimens for allogeneic HSCT are promising, more prospective studies with larger numbers of patients need to be done to fully address this complex clinical issue (Table 39-5).

5. Conclusions HSCT is associated with a number of acute hemorrhagic and thrombotic complications unique to this treatment modality. The clinician needs to be aware of these conditions and prospectively anticipate the possibility of managing these complications, as all adversely impact on outcome. Management and amelioration of these complications should improve long-term efficacy of this therapeutic modality. The increasing use of RIC and NM regimens should show a decrease in the incidence in a number of these complications, which would translate to better outcomes.

Note Added in Proof Since this chapter was submitted, it has become recognized that atypical HUS, a microangiopathic hemolytic anemia picture associated with renal injury, due to a large number of defects or deficiencies in complement regulatory proteins may arise as result of infectious or inflammatory disorders. The complement proteins associated with atypical HUS include factor H, factor I, membrane cofactor protein, C3, or thrombomodulin. Patients with TM-TMA need to be evaluated for defects or acquired deficiencies in these proteins if another etiology is not evident

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Chapter 39  Unique Thrombotic and Hemostatic Complications Associated with Allogeneic  73. Harvey CJ, Peniket AJ, Miszkiel K et al (2000) MR angiographic diagnosis of cerebral venous sinus thrombosis following allogeneic bone marrow transplantation. Bone Marrow Transplant 25:791–795 74. Bertz H, Laubenberger J, Steinfurth G et al (1998) Sinus venous thrombosis: An unusual cause for neurologic symptoms after bone marrow transplantation under immunosuppression. Transplantation 66:241–244 75. Greeno EW, Haake R, McGlave P, Weisdorf D, Verfaillie C (1995) Lupus inhibitors following bone marrow transplant. Bone Marrow Transplant 15(2):287–291 76. Karfan-Dabaja MA, Morgensztern D, Santos E, Goodman M, Fernandez HF (2003) Acute graft-versus-host disease (aGVHD) presenting with an acquired lupus anticoagulant. Bone Marrow Transplant 31(2):129–131 77. Devetten M, Armitage JO (2007) Hematopoietic cell transplantation: Progress and obstacles. Ann Oncol 18:1450–1456 78. Sorror ML, Maris MB, Storer B et al (2004) Comparing morbidity and mortality of HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative and myeloablative conditioning: Influence of pretransplant comorbidities. Blood 104(4):961–968 79. Le Blanc K, Remberger M, Uxunel M, Mattsson J, Barkholt L, Ringden O (2004) A comparison of nonmyeloablative and reduced-intensity conditioning for allogeneic stem-cell transplantation. Transplantation 78(7):1014–1020 80. Couriel DR, Saliba RM, Giralt S et al (2004) Acute and chronic graft-versus-host disease after ablative and nonmyeloablative conditioning for allogeneic hematopoietic transplantation. Biol Blood Marrow Transplant 10:178–185 81. Diaconescu R, Flowers CR, Storer B et  al (2004) Morbidity and mortality with nonmyeloablative compared with myeloablative conditioning before hematopoietic cell transplantation from HLA-match related donors. Blood 104(5):1550–1558

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Chapter 40 How Much Isolation Is Enough for Allografts? Brandon Hayes-Lattin

Since the beginning of allogeneic transplant procedures, isolation precautions have been used in an effort to prevent infectious complications. However, the medical benefit has been increasingly questioned and, given the substantial costs associated with each measure, practices have been subject to institutional variability. Recent examples from our own institution range from determining the needs for and planning the construction of a new clinical facility, to halting the spread of vancomycin-resistant Enterococcus (VRE), understanding the source of a parainfluenza outbreak, and analyzing the potential for delays in response time to acute emergencies while putting on contact isolation materials (gowns, masks, gloves, etc.). Standards for isolation may be subject to change when managing infection outbreaks or with new construction, and may be influenced by the changing financial pressures of the institution. In an era of ongoing cost-containment, the clinical value of each isolation intervention and the associated expenditures are due to be examined. With changes in the number of patients undergoing allogeneic transplantation, the financial constraints of care, and advances in supportive care, there has been a trend to relax the degree of patient isolation in the absence of definitive data to support its use (Fig. 40-1). Even the use of protected hospital environments is being questioned, with recent trials assessing the safety and efficacy of outpatient care during the pre-engraftment phase after allogeneic transplants. Guidelines for infection control were published in 2000 by the Centers for Disease Control (CDC) with the Infectious Disease Society of America (IDSA) and the American Society of Blood and Marrow Transplantation (ASBMT), but most recommendations were based on uncontrolled studies or expert opinion rather than randomized controlled trials [1]. The Foundation for the Accreditation of Cellular Therapy (FACT) calls for a designated inpatient unit that minimizes airborne microbial contamination and a designated area for outpatient care that reasonably protects the patient from transmission of infectious agents and can provide, as necessary, appropriate patient isolation. The Joint Commission on Accreditation of Healthcare Organizations (JCAHO) standards call for incorporation of an infection control program with ongoing

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_40, © Springer Science + Business Media, LLC 2003, 2010

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HEPA →

Filtration

Standard Room, →

Neutropenic Precautions

Home →

1970’s ————————————————————————————→ 2000’s Influences: a) Increased number of patients undergoing transplantation b) Increased financial constraints: patient, payer, and institution c) Advances in supportive care: hematopoieitic growth factors and antibiotics d) Lack of evidence supporting aggressive isolation

Fig.  40-1.  Trends in the intensity of isolation practices for patients after allogeneic transplantation. Influences: (a) Increased number of patients undergoing transplantation. (b) Increased financial constraints: patient, payer, and institution. (c) Advances in supportive care: hematopoieitic growth factors and antibiotics. (d) Lack of evidence supporting aggressive isolation

assessments to identify risks for the acquisition and transmission of infectious agents using an epidemiological approach of surveillance, data collection, and trend identification. In this chapter, we will consider various interventions used to control patient environments, examine the evidence behind many common interventions, and discuss some of the associated costs.

1. Reasons for Patient Isolation Caring for patients after an allogeneic stem cell transplant within a protective hospital environment has been considered standard of care based upon the spectrum and severity of infections to which this immune-compromised population is susceptible. The risks of infection after allogeneic transplant are highlighted by reports of infection outbreaks from conventional and opportunistic pathogens. Early studies of patients receiving chemotherapy for acute leukemia or undergoing bone marrow transplantation for aplastic anemia suggested they may be getting clinical benefit due to aggressive infection control measures. 1.1. Scope of Infectious Complications Opportunistic infections from bacterial, viral, and fungal organisms are common after allogeneic hematopoietic stem cell transplantation. Infections of varying severity occur in >90% of patients after allogeneic transplantation, and are considered the most common single cause of mortality. The scope and timing of infectious complications after transplant have been reviewed [1]. Periods of varied risk after myeloablative allogeneic transplant have been defined as pre-engraftment, post-engraftment (through day +100), and late phase (Table 40-1).

Chapter 40  How Much Isolation Is Enough for Allografts? 

Table  40.1.  High incident (>10%) opportunistic infections by phases after myeloablative allogeneic hematopoietic stem cell transplantation (adapted from CDC guidelines). Pre-engraftment (<30 days) Herpes simplex virus

Post-engraftment (30–100 days) a

a

Cytomegalovirus

Late phase (>100 days) Cytomegalovirusa

Gram-negative bacilli

Staphylococcus epidermidis

Varicella-zoster virus

Staphylococcus epidermidis

Candida species

Encapsulated bacteria (e.g., pneumococcus)

Streptococci species

Aspergillus species

Aspergillus species

Candida species

Pneumocystis pneumonia (PCP) Pneumocystis pneumonia (PCP)

Aspergillus species a

Without standard prophylaxis

The epidemiology of these infections continues to evolve based on the degree, type, and duration of immune suppression, the use of prophylactic antibiotics, surveillance for organisms associated with nosocomial infections, the emergence of drug-resistant organisms, and the use of isolation precautions. The advent of reduced-intensity allogeneic transplants has also significantly changed the timing and duration of the phases of infection risk. Even with reduced-intensity conditioning, substantial risks for infectious complications have been reported [2]. Despite a shorter duration of neutropenia and less mucosal damage, quantitative and qualitative T-cell defects over the first 12 months are similar to those after myeloablative transplants [3]. In matched controlled studies of patients after nonmyeloablative and myeloablative transplants in Seattle, there were fewer episodes of bacteremia during the first 30 days after reduced-intensity conditioning (9 vs. 27%, p = 0.01), but the difference was less pronounced by day +100 (27 vs. 41%, p = 0.07) [4]. There was no decrease observed in the rate of invasive aspergillosis during the first year (15 vs. 9%). The overall risk of CMV disease was also similar between these groups, but was delayed in onset among recipients of reduced-intensity transplants (median day +130 vs. day +52) [5]. The epidemiology of invasive fungal infections has also changed, reflecting the use of antifungal prophylaxis as well as changes in conditioning regimens. There has been a reduction in the incidence of Candida infections, but a relative increase in non-albicans species, often resistant to fluconazole [6–8]. There has also been an increased incidence of Aspergillus infections, including non-fumigatus species [9]. Temporally related to the increased use of Aspergillus-active antifungal drugs for prophylaxis, there have been recent reports of an increasing incidence of non-Aspergillus molds [9]. 1.2. Infection Outbreaks In addition to understanding the spectrum of routinely acquired pathogens among immuno-compromised hosts, infectious outbreaks in stem cell transplant units have highlighted the potential importance and need for controlled environments.

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Infection outbreaks occurring on stem cell transplant units include reports involving bacterial, viral, and fungal pathogens. A recent survey from the EBMT reported 23 outbreaks among 13 centers involving 231 patients, including 56 attributable deaths [10]. In this series of 10 bacterial, 8 viral, and 5 fungal outbreaks, all were reported to be hospital acquired and 12 centers reported a partial or total unit closure prior to resolution. Notably, all viral, 4 of 10 bacterial, and 3 of 5 fungal outbreaks occurred in high-efficiency particulate air (HEPA) filtered rooms. Aspergillus is a common airborne infection and outbreaks have been widely reported in association with construction activity but also with contamination of water supplies [11–14]. Outbreaks of infection with other fungi have also been reported, including a series of infections with Pseudomonas associated with contaminated skin ointment [15]. Bacterial outbreaks have included water-borne pathogens such Legionella and Pseudomonas species [16, 17]. The risk of Clostridia difficile-associated diarrhea is substantial in a population heavily exposed to antibiotics, and recommendations highlight outbreaks reported in association with non-disposable rectal thermometers or infected gastrointestinal endoscopes [1]. Respiratory viral outbreaks, including seasonal pathogens such as respiratory syncytial virus (RSV) and influenza, are not prevented by HEPA filtration and require increased attention to recognizing individuals with symptoms and removing them from the vicinity of vulnerable patients [18–20]. Outbreaks of RSV in transplantation units illustrate the potential for the rapid spread of lethal pathogens. One such outbreak occurred among 30 patients in a 13-week period and included an additional 35 family members and healthcare workers [21]. Of the 18 patients with lower respiratory infections from RSV, the mortality was 78%. Another center reported an outbreak in which 45% of hospitalized BMT patients with acute respiratory illness in a 2-month period had RSV disease, and two-thirds were considered hospitalacquired [22]. Mortality for those untreated or those treated requiring mechanical ventilation was 100%. In another outbreak, genotyping showed eight of nine isolates from a BMT unit were the same RSV strain [23]. Management of RSV outbreaks in transplant units has included a range of responses, including patient isolation in single rooms with contact and droplet precautions, cohorting of nursing care, screening of patients with respiratory symptoms using naso­ pharyngeal swabs, limiting contact with any staff or visitor who endorsed upper respiratory tract infection symptoms, and implementing educational campaigns for ward staff. 1.3. Vancomycin-Resistant Enterococcus (VRE) VRE is an emerging bacterial pathogen reported in transplant unit outbreaks that is associated with the widespread use of vancomycin [24–26]. The link between VRE colonization and subsequent bloodstream infection and mortality risk has been reported among hematopoietic stem cell transplant patients [27]. A recent description of an outbreak of VRE illustrates the importance of hospital infection surveillance and control programs [10]. After noting a marked increase in colonization with VRE as well as the emergence of VRE infections, initial control measures included strict hand-washing protocols, isolation of patients, attention to ward cleaning and the restriction of vancomycin use.

Chapter 40  How Much Isolation Is Enough for Allografts? 

However, after detecting continued VRE infections with a molecular typing confirming a shared predominant strain of Entercoccus faecalis, the unit was closed for 6 months. Patients colonized or infected with VRE were cohorted in units where all staff were required to wear gowns and gloves. A computerflagging system was implemented to alert for VRE-carriers on re-admissions. Since re-opening the transplant unit, only one new case of VRE was observed in 3 years. In our own institution, VRE has also become problematic. The organism was not detected at OHSU until 1996, but by the year 2000, 30 new cases were identified among immune compromised adults. In 2002, a cluster of 16 patients in the adult oncology unit was found to have VRE infection (14) and colonization (2). This led to the creation of a VRE Management Team and the implementation of enhanced contact precautions, environmental cultures, compliance monitoring, and educational programs for patients and families, nursing, housekeeping, transportation, imaging, and associated medical staff. Specific interventions included the regular use of gloves for each patient encounter and contact precautions with gloves and gown for patients with VRE. Patients were screened for VRE with rectal swabs upon hospital admission, and colonized patients were flagged in our hospital computer system for care in dedicated VRE outpatient clinic rooms. By February 2003, after a period of identifying no new cases in 4 weeks, enhanced isolation was relaxed, but active surveillance in oncology units continued. Sporadic cases were reported until 1 week in October 2004 when 9 new cases of VRE colonization were found among 80 patients screened. Isolates including those 9 and an additional 4 from preceding weeks were sent for DNA analysis, which revealed that 6 isolates were genetically identical and an additional 3 were closely related, suggesting a shared source. Since that time, enhanced isolation was re-instituted with ongoing monitoring for the transplant ward as well as the intensive care unit. 1.4. Benefits of Isolated Environments: Early Studies Early studies among patients undergoing chemotherapy for leukemia suggested that environmental infection control measures might have a positive impact on patient outcomes. These included the use of laminar air flow (LAF) units, patient isolation units, prophylactic antibiotics, sterile and low-microbial diets, and antimicrobial decontamination [28–37]. A randomized trial among patients with acute leukemia performed in 1978 showed a decrease in fatal infections, an increase in complete remission rates, and an improved survival among patients treated in a protected environment with prophylactic antibiotics [38]. However, even in these early studies, there was recognition of emerging antibiotic-resistant strains of bacteria when using antibiotics for patient protection [32]. Early prospective, randomized studies of protective environments among bone marrow transplant recipients suggested that laminar air flow isolation and decontamination procedures resulted in a significant reduction in infections, but no difference in survival, with most deaths due to interstitial pneumonia or recurrent disease [39]. Five years later, the same group published retrospective data suggesting that these protective environments reduced mortality associated with a reduction in and delayed onset of acute GVHD

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after allogeneic transplantation for the treatment of aplastic anemia [40]. Further studies of intestinal bacterial decontamination, including suppression of anaerobic bacteria, decreased the severity of acute GVHD [41]. Finally, retrospective studies of the use of HEPA filtration units showed a reduction in the number of Aspergillus organisms in the air and a decrease in the risk of nosocomial Aspergillus infections [11, 13, 42]. However, no overall survival benefit was demonstrated.

2. Specific Interventions The Centers for Disease Control and Prevention have published strategies to prevent healthcare-associated infections in acute care hospitals [43]. Recommendations for infection control among hematopoietic stem cell transplantation recipients have focused on issues of ventilation, construction, room cleaning, isolation and barrier precautions, interactions with healthcare workers and visitors, skin and oral care, infection surveillance, and prevention of specific nosocomial and seasonal infections [1]. The strength of the recommendation and the quality of supporting evidence is graded (Table 40-2). 2.1. CDC Level I Evidence While the CDC/IDSA/ASBMT guidelines contain over 200 recommendations for infection control among hematopoietic stem cell recipients, only seven are supported by level I (randomized trial) evidence (Table 40-3). Six recommendations hold clinical benefit and one (use of humidifiers) is associated with harm. These level I recommendations , however, do not include the use of patient isolation units, ventilation systems, construction or cleaning guidelines, skin or oral care, or the prevention of catheter-associated infections.

Table  40-2.  Evidence-based rating system (A) strength of recommendation (B) quality of evidence. (A) A  Strongly recommended. Strong evidence for efficacy and substantial clinical benefit B Generally recommended. Moderate evidence for efficacy or only limited clinical benefit C Optional. Insufficient evidence for efficacy or benefit may not outweigh the risk or cost D Generally not recommended. Moderate evidence against efficacy or of adverse outcome E Never recommended. Strong evidence against efficacy or of adverse outcome (B) I

Evidence from at least one well-executed randomized controlled trial

II Evidence from at least one well-designed nonrandomized trial, cohort or case-control studies, multiple time-series studies, or dramatic results from uncontrolled experiments III Evidence from expert opinions based on experience, descriptive studies, or committees

Chapter 40  How Much Isolation Is Enough for Allografts? 

Table 40-3.  Level I infection control recommendations, based on at least one properly randomized trial. AI recommendations (1) All persons should wash their hands before entering and after leaving the rooms of HSCT recipients and candidates undergoing conditioning therapy, or before any direct contact with patients regardless of whether they were soiled from the patient, environment or objects (2) All healthcare workers with diseases transmissible by air, droplet, and direct contact (e.g., varicella zoster virus, infectious gastroenteritis, herpes simplex lesions of lips or fingers and upper respiratory tract infections) should be restricted from patient contact and temporarily reassigned to other duties (3) When a case of laboratory confirmed legionellosis is identified in a person who was in the impatient HSCT center during all or part of the 2–10 days before illness onset, or if two or more cases of laboratory-confirmed Legionnaire’s disease occur among patients who had visited an outpatient HSCT center, hospital personnel in consultation with the hospital infection control team should perform a thorough epidemiologic and environmental investigation or determine the likely environmental source(s) of Legionella species (e.g., showers, tap water faucets, cooling towers and hot water tanks) (4) To control VRE exposure, strict adherence to standard infection control measures is necessary, as outlined in the text (5) All HCWs who anticipate contact with a Clostridium difficile-infected patient or the patient’s environment or possessions should put on gloves before entering the patient’s room and before handling the patient’s secretions and excretions (6) HSCT candidates with a recently positive tuberculin skin test or a history of a positive skin test and no prior preventive therapy should be administered a chest radiograph and evaluated for active TB DI recommendation (1) HSCT centers should not use large-volume room air humidifiers that create aerosols (e.g., by Venturi principle, ultrasound, or spinning disk) and, thus, are actually nebulizers

The first AI recommendation (strongly recommended with randomized trial support) involves the long-held infection control practice of handwashing. Guidelines for hand hygiene have been published, which specifically address issues of the indications for hand washing and antisepsis, the hand-washing technique, surgical hand antisepsis, the selection of hand-hygiene agents, and even healthcare worker educational and motivational programs [44]. However, a cross-sectional survey of university hospital physicians showed a dismal 57% average adherence [45]. 2.2. CDC Level II or III Evidence A recent systematic review of randomized controlled trials on the use of parachutes to prevent major trauma from fall identified a complete absence of such trials in the medical literature [46]. This review serves as a captious reminder that many effective interventions can be proven on the basis of observational study alone.

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The CDC/IDSA/ASBMT guidelines address an additional 12 recommendations graded AII (strongly recommended with well-designed nonrandomized trial support) (Table 40-4). While the utility of specific isolation and barrier precautions have not been studied, a level AIII recommendation (strongly recommended with expert opinion support) is given to follow other published guidelines for hospital isolation and the prevention of nosocomial infections such as pneumonia or surgical site infections [47–49]. Additional infection control recommendations can be considered for epidemiologic factors such as the host, human-to-human interactions, fomites, air, food, water, soil, and construction and cleaning. 2.2.1. Host The use of routine bacterial or fungal surveillance cultures among asymptomatic patients is discouraged (DII, generally not recommended with welldesigned nonrandomized trial support). Recommendations are also aimed at

Table 40-4.  Level AII infection control recommendations.   (1) HSCT centers should prevent birds from gaining access to hospital air-intake ducts   (2) Appropriate gloves should be used by all persons when handling potentially contaminated biological materials   (3) Work exclusion policies should be designed to encourage HCWs to report their illnesses or exposures   (4) Visitors who might have communicable infectious diseases should not be allowed in the HSCT center or have direct contact with HSCT recipients or candidates undergoing conditioning therapy   (5) If Legionella species are detected in the water supplying an HSCT center, the water supply should be decontaminated and eradication of Legionella should be verified   (6) HSCT centers should follow basic infection control practices for control of MRSA infection and colonization, including hand washing between patients and use of barrier precautions, including wearing gloves whenever entering the MRSA-infected or MRSA-colonized patient’s room   (7) HSCT personnel should institute prudent use of all antibiotics, particularly vancomycin, to prevent the emergence of staphylococci with reduced susceptibility to vancomycin   (8) Use of intravenous vancomycin is associated with the emergence of VRE; vancomycin and all other antibiotics, particularly antianaerobic agents, should be used judiciously   (9) All patients with Clostridium difficile disease should be placed under contact precautions for the duration of the illness (10) When caring for an HSCT recipient or candidate undergoing conditioning therapy with upper or lower respiratory tract infection, HCWs and visitors should change gloves and wash hands in circumstances outlined in the text (11) Visitors and HCWs with infectious conjunctivitis should be restricted from direct patient contact until the drainage resolves and the ophthalmology consultant concurs that the infection and inflammation have resolved to avoid possible transmission of adenovirus to HSCT recipients (12) For patients with suspected or proven pulmonary or laryngeal TB, HSCT personnel should follow guidelines regarding the control of TB in healthcare facilities

Chapter 40  How Much Isolation Is Enough for Allografts? 

reducing infections from host-derived organisms that reside on the skin, oral or gastrointestinal surfaces. CDC recommendations for skin care are limited to daily showers or baths using mild soap with attention to the hygiene and skin integrity of the perineal area (BIII, generally recommended with expert opinion support). AIII recommendations are listed for dental care before and after transplant. Oral hygiene is recommended by performing rinses 4–6 times daily using sterile water, normal saline, or sodium bicarbonate solutions (AIII). Recent studies of preventing mucosal injury after transplantation further address the loss of mucosal integrity as a portal of entry for host-derived Gram-negative and anaerobic bacterial organisms. A phase III randomized trial of iseganan, a peptide with broad-spectrum microbicidal activity, did not lead to a reduction in mucositis or its clinical sequelae after autologous or allogeneic transplantation [50]. However, a randomized phase III trial of a recombinant human keratinocyte growth factor, palifermin, showed not only a decrease in the duration of advanced grade mucositis, but also a decrease in the incidence of febrile neutropenia and a trend towards fewer episodes of bloodborne infections after autologous transplantation [51]. Trials of palifermin in the allogeneic setting have recently been completed but it is not yet clear from preliminary reports whether the severity of mucositis or the incidence of infections has significantly improved. 2.2.2. Human to Human While handwashing before entering and after leaving patient rooms and before and after direct patient contact carries an AI recommendation, the use of antimicrobial soap and water versus hygienic hand rubs is not mandated (AIII). Gloves should be changed between patients and prior to touching a clean area when they are soiled (AIII). Items that may serve as a nidus such as rings, artificial nails, or bandages should be avoided (BII, generally recommended with well-designed non-randomized trial support). Visitors should be screened for potentially infectious conditions prior to patient contact (BII). 2.2.3. Fomites Established guidelines exist for the sterilization or disinfection and maintenance of hospital equipment and devices (AIII). Due to reports of Aspergillus isolated from their soil or surfaces, exposure to plants and flowers is discouraged (BIII). Recommendations are given for the use of disposable toys (BIII), and the regular cleaning or hot water washing of toys (BIII), the avoidance of water-retaining toys (DII), and the cleaning and disinfecting of occupational and physical therapy items (BIII). 2.2.4. Air Air flow systems capable of least 12 exchanges per hour and point-of-use HEPA filters (0.3 mm) are recommended based largely on expert opinion (AIII). The use of LAF systems for patients with aplastic anemia remain controversial based on conflicting results from retrospective studies (CII, optional with well-designed non-randomized trial support). The use of fittested N95 respirators near construction can be considered (CIII, optional with expert opinion support), but little protection is offered from standard surgical masks near construction (DIII, generally not recommended with expert opinion support).

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2.2.5. Food and Water A low microbial diet is recommended for recipients of allogeneic stem cell transplantation until all immunosuppressive drugs are discontinued (BIII). Patients are discouraged from drinking well water from private wells or public wells in small communities where bacterial contamination is tested less than twice daily (DIII). Naturopathic medications that may contain molds, legionella, or giardia are discouraged too(DIII). 2.2.6. Soil, Construction and Cleaning During construction activities, several recommendations have been made including intensification of the BIII recommendations for Aspergillus-control (AIII), avoidance of construction areas for recipients, healthcare workers, and visitors (AIII), cleaning newly constructed areas per BIII guidelines prior to their use (AIII), and avoiding carpets or vacuuming (AIII). Cleaning of transplant units should be performed at least once daily, with attention to exhaust vents, window sills and all horizontal surfaces using cloths or mop heads premoistened with a registered hospital disinfectant (BIII). These evidence-based infection control recommendations are aimed at the control of selected pathogens of fungal, bacterial, and viral origin based largely on the epidemiology of infections observed in the hospital setting. The burden and spectrum of exposure and associated risk of infection may differ in alternative settings, including the outpatient environment.

3. Costs of Isolation 3.1. Finances The financial costs of hospital protective environments are substantial, and often difficult to calculate. Direct costs of patient care include isolation supplies (gloves, gowns, hand cleansers, etc.), cleaning and maintaining isolation units, and materials and personnel for infection surveillance. In our institution’s nine-room HEPA-filtered BMT unit, the expenditures for “medical materials” last year alone (including gloves, gowns, soaps, hand rubs, and masks as well as nursing items such as tubing and syringes) was $390,940. Other costs include the need to engineer, construct, and maintain an adequate number of patient units with single rooms and unique air and water supplies, and the need to providing staffing for adequate nurse-to-patient ratios. Seemingly simple interventions such as a 10-s hand sanitization or a 1-min scrub with gown-and-glove can be costly when scaled annually for routine clinical practice (Fig. 40-2). Given the high costs and the lack of formal proof of efficacy, institutions have begun to study alternatives to the use of sterile units with positive air pressure and HEPA filtration during the care of patients undergoing intensive chemotherapy. Several preliminary reports have been presented in abstract form. A study of 59 consecutive and nonselected patients undergoing autologous transplantation found comparable toxicities and treatment-related death rate when treated in conventional hospital rooms [52]. In an abstract reporting a retrospective analysis, one institution compared outcomes after first abandoning LAF units, and then changing from routine to targeted IVIG replacement among patients receiving allogeneic transplantation [53]. There were fewer

Chapter 40  How Much Isolation Is Enough for Allografts?  $70,000

$63,875

$60,000 $50,000 $40,000 $30,000 $20,000 $10,000

$15,968 $5,323

$0 10-second hand sanitizer

30-second hand washing 2-minute hand washing with mask, gown and gloves

Fig. 40-2.  Theoretical annual costs of nursing time in preparation to enter patient rooms in a nine-bed BMT unit, based on assumptions of three nurses earning $35 per hour and entering the room 25 times in a 12-h shift

episodes of septicemia per hospital day but not per day with neutropenia in the combined LAF room and routine IVIG cohort. No other differences in outcome, including 100-day treatment-related mortality were observed. To further reduce the costs of inpatient care, several institutions are reporting outcomes after outpatient care for hematologic malignancies and autologous transplantation. Outpatient high-dose chemotherapy and autologous transplantation has been reported in abstract form among patients with multiple myeloma with a trend towards fewer infections compared to historic controls (25 vs. 35%) [54]. A retrospective analysis presented in abstract of bacteremia incidence after 623 cycles of chemotherapy for acute myeloid leukemia suggested a decrease in gram-negative bacteremia among outpatients, with no infection-related deaths in the outpatient group [55]. 3.2. Patient Interactions Beyond the direct and indirect financial costs of treating patients within a protective environment, additional social costs should be considered. Emotional disturbance is common, with one series reporting DSM-IV psychiatric diagnoses among 41% after transplantation [56]. Physical isolation can lead to emotional isolation after transplantation [57]. Major themes in the experience of transplant patients include maintaining control in a seemingly out-ofcontrol situation, intellectualizing the need for isolation, and staying in contact with family and staff [58]. When abandoning protective isolation for patients with prolonged neutropenia, patient satisfaction improved, costs were reduced, and no increases in infection risk were observed [59].

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4. Outpatient Care for Allogeneic Transplantation Despite rigorous attempts to maintain a safe in-hospital microbiological environment for recipients of allogeneic stem cell transplantation, it should be recognized that hospitals are also sources for exposure to resistant opportunistic organisms. Outpatient care, whether in contiguous hospital-affiliated settings or the patient’s home, has been considered as a possible alternative to avoid many of these nosocomial risks. In 1992, a report was published describing 50 consecutive patients receiving an allogeneic transplant from either a sibling or an unrelated donor who were treated in standard single hospital rooms [60]. Twenty patients who lived locally were allowed to go home on a median of 8 days prior to engraftment. The reported incidence of infection (24%), acute GVHD (34%), and 100-day treatment-related mortality (6%) were favorable. An update was published in 2000, after 288 patients were treated in standard single hospital rooms with the ability to leave the room and the hospital, although remaining hospitalized until at least the second week after transplant [61]. Avoidance of crowds was encouraged but no changes were made to the home environment, except to advise against renovations. Approximate one-quarter of “inpatient” days were spent outside of the hospital including 80% during the neutropenic phase. Fifty-seven percent of patients developed fever, with a positive culture or focal infection in 35%. Four patients (1%) died of Aspergillus infection. The overall 100-day treatment-related mortality was 13%. In another series examining homecare after allogeneic transplantation, 36 patients who chose homecare were compared to 18 who chose hospital care and to 36 matched-controls who were not offered homecare [62]. The homecare cohort was allowed home after the graft had been infused, and was visited once or twice daily by a nurse experienced in stem cell transplantation. In a multivariate analysis, homecare patients were discharged earlier, had fewer days of total parenteral nutrition, fewer episodes of grade II–IV acute GVHD, and lower transplant-related mortality. Costs were also lower, with a median cost from day 0 to day +76 of $25,340 in the homecare group versus $36,437 for those choosing hospital care and $33,620 for those not offered homecare. Recently, a review examined the six published studies, including four comparative but non-randomized analyses, describing the experience of homecare for patients with cytopenias after high-dose therapy and stem cell transplantation [63]. The pooled statistics suggest that protective environments provided no benefit in decreasing mortality for the transplant patient. However, it is noted that such non-randomized analyses may well be subject to selection and reporting bias.

5. Conclusions Infectious complications are a major source of morbidity and mortality after allogeneic stem cell transplantation. Only a few infection control and protective environment interventions have been proven useful in randomized trials. In an era of increasing financial pressures, specific interventions such as the use of special transplant unit rooms are being questioned. It is important to remember that expert experience and observational studies are sufficient proof

Chapter 40  How Much Isolation Is Enough for Allografts? 

for some infection control measures, ranging from formal infection control programs with interdisciplinary communication and surveillance for infection outbreaks to interventions as fundamental as strict and regular hand-washing. Given the lack of proof for the necessity of isolated transplant units and encouraging preliminary studies of the safety of outpatient care, randomized trials would be best to establish the safety of caring for recipients of allogeneic transplants outside of a protected in-hospital unit. However, this goal may be logistically difficult due to multiple factors of scale and finance influencing transplant practices (Fig. 40-1). Continued monitoring for trends in infectious complications and communication with a multidisciplinary infection control committee are recommended in any setting.

References 1. Centers for Disease Control and Prevention; Infectious Disease Society of America; American Society of Blood and Marrow Transplantation (2000) Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients. MMWR Recomm Rep 49:1–125 CE121-127 2. Hagen EA, Stern H, Porter D et al (2003) High rate of invasive fungal infections following nonmyeloablative allogeneic transplantation. Clin Infect Dis 36:9–15 3. Busca A, Lovisone E, Aliberti S et  al (2003) Immune reconstitution and early infectious complications following nonmyeloablative hematopoietic stem cell transplantation. Hematology 8:303–311 4. Junghanss C, Marr KA, Carter RA et al (2002) Incidence and outcome of bacterial and fungal infections following nonmyeloablative compared with myeloablative allogeneic hematopoietic stem cell transplantation: A matched control study. Biol Blood Marrow Transplant 8:512–520 5. Junghanss C, Boeckh M, Carter RA et al (2002) Incidence and outcome of cytomegalovirus infections following nonmyeloablative compared with myeloablative allogeneic stem cell transplantation, a matched control study. Blood 99:1978–1985 6. McNeil MM, Nash SL, Hajjeh RA et al (2001) Trends in mortality due to invasive mycotic diseases in the United States, 1980–1997. Clin Infect Dis 33:641–647 7. van Burik JH, Leisenring W, Myerson D et  al (1998) The effect of prophylactic fluconazole on the clinical spectrum of fungal diseases in bone marrow transplant recipients with special attention to hepatic candidiasis. An autopsy study of 355 patients. Medicine (Baltimore) 77:246–254 8. Marr KA, Seidel K, White TC, Bowden RA (2000) Candidemia in allogeneic blood and marrow transplant recipients: Evolution of risk factors after the adoption of prophylactic fluconazole. J Infect Dis 181:309–316 9. Fukuda T, Boeckh M, Carter RA et al (2003) Risks and outcomes of invasive fungal infections in recipients of allogeneic hematopoietic stem cell transplants after nonmyeloablative conditioning. Blood 102:827–833 10. McCann S, Byrne JL, Rovira M et al (2004) Outbreaks of infectious diseases in stem cell transplant units: A silent cause of death for patients and transplant programmes. Bone Marrow Transplant 33:519–529 11. Sherertz RJ, Belani A, Kramer BS et al (1987) Impact of air filtration on nosocomial Aspergillus infections. Unique risk of bone marrow transplant recipients. Am J Med 83:709–718 12. Anaissie EJ, Costa SF (2001) Nosocomial aspergillosis is waterborne. Clin Infect Dis 33:1546–1548 13. Cornet M, Levy V, Fleury L et al (1999) Efficacy of prevention by high-efficiency particulate air filtration or laminar airflow against Aspergillus airborne contamination during hospital renovation. Infect Control Hosp Epidemiol 20:508–513

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B. Hayes-Lattin 14. Wald A, Leisenring W, van Burik JA, Bowden RA (1997) Epidemiology of Aspergillus infections in a large cohort of patients undergoing bone marrow transplantation. J Infect Dis 175:1459–1466 15. Orth B, Frei R, Itin PH et  al (1996) Outbreak of invasive mycoses caused by Paecilomyces lilacinus from a contaminated skin lotion. Ann Intern Med 125: 799–806 16. Lyytikainen O, Golovanova V, Kolho E et al (2001) Outbreak caused by tobramycinresistant Pseudomonas aeruginosa in a bone marrow transplantation unit. Scand J Infect Dis 33:445–449 17. Oren I, Zuckerman T, Avivi I et al (2002) Nosocomial outbreak of Legionella pneumophila serogroup 3 pneumonia in a new bone marrow transplant unit: Evaluation, treatment and control. Bone Marrow Transplant 30:175–179 18. Ljungman P (2001) Respiratory virus infections in stem cell transplant patients: The European experience. Biol Blood Marrow Transplant Suppl 7:5S–7S 19. Champlin RE, Whimbey E (2001) Community respiratory virus infections in bone marrow transplant recipients: The M.D. Anderson Cancer Center experience. Biol Blood Marrow Transplant Suppl 7:8S–10S 20. Nichols WG, Gooley T, Boeckh M (2001) Community-acquired respiratory syncytial virus and parainfluenza virus infections after hematopoietic stem cell transplantation: The Fred Hutchinson Cancer Research Center experience. Biol Blood Marrow Transplant Suppl 7:11S–15S 21. Harrington RD, Hooton TM, Hackman RC et al (1992) An outbreak of respiratory syncytial virus in a bone marrow transplant center. J Infect Dis 165:987–993 22. Whimbey E, Champlin RE, Englund JA et  al (1995) Combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients. Bone Marrow Transplant 16:393–399 23. Taylor GS, Vipond IB, Caul EO (2001) Molecular epidemiology of outbreak of respiratory syncytial virus within bone marrow transplantation unit. J Clin Microbiol 39:801–803 24. Recommendations for preventing the spread of vancomycin resistance (1995) Recommendations of the hospital infection control practices advisory committee (HICPAC). MMWR Recomm Rep 44:1–13 25. Uttley AH, Collins CH, Naidoo J, George RC (1988) Vancomycin-resistant enterococci. Lancet 1:57–58 26. Kirkpatrick BD, Harrington SM, Smith D et al (1999) An outbreak of vancomycindependent Enterococcus faecium in a bone marrow transplant unit. Clin Infect Dis 29:1268–1273 27. Weinstock DM, Conlon M, Iovino C et al (2007) Colonization, bloodstream infection, and mortality caused by vancomycin-resistant enterococcus early after allogeneic hematopoietic stem cell transplant. Biol Blood Marrow Transplant 13:615–621 28. Aker SN, Cheney CL (1983) The use of sterile and low microbial diets in ultraisolation environments. JPEN J Parenter Enteral Nutr 7:390–397 29. Bodey GP, Freireich EJ, Frei E III (1969) Studies of patients in a laminar air flow unit. Cancer 24:972–980 30. Bodey GP, Gehan EA, Freireich EJ, Frei E III (1971) Protected environmentprophylactic antibiotic program in the chemotherapy of acute leukemia. Am J Med Sci 262:138–151 31. Dietrich M, Gaus W, Vossen J et al (1977) Protective isolation and antimicrobial decontamination in patients with high susceptibility to infection. A prospective cooperative study of gnotobiotic care in acute leukemia patients. I: Clinical results. Infection 5:107–114 32. Klastersky J, Debusscher L, Weerts D, Daneau D (1974) Use of oral antibiotics in protected units environment: Clinical effectiveness and role in the emergence of antibiotic-resistant strains. Pathol Biol (Paris) 22:5–12

Chapter 40  How Much Isolation Is Enough for Allografts?  33. Levine AS, Siegel SE, Schreiber AD et  al (1973) Protected environments and prophylactic antibiotics. A prospective controlled study of their utility in the therapy of acute leukemia. N Engl J Med 288:477–483 34. Ribas-Mundo M, Granena A, Rozman C (1981) Evaluation of a protective environment in the management of granulocytopenic patients: A comparative study. Cancer 48:419–424 35. Schimpff SC, Greene WH, Young VM et al (1975) Infection prevention in acute nonlymphocytic leukemia. Laminar air flow room reverse isolation with oral, nonabsorbable antibiotic prophylaxis. Ann Intern Med 82:351–358 36. Schwartz SA, Perry S (1966) Patient protection in cancer chemotherapy. JAMA 197:623–627 37. Yates JW, Holland JF (1973) A controlled study of isolation and endogenous microbial suppression in acute myelocytic leukemia patients. Cancer 32:1490–1498 38. Rodriguez V, Bodey GP, Freireich EJ et al (1978) Randomized trial of protected environment–prophylactic antibiotics in 145 adults with acute leukemia. Medicine (Baltimore) 57:253–266 39. Buckner CD, Clift RA, Sanders JE et al (1978) Protective environment for marrow transplant recipients: A prospective study. Ann Intern Med 89:893–901 40. Storb R, Prentice RL, Buckner CD et  al (1983) Graft-versus-host disease and survival in patients with aplastic anemia treated by marrow grafts from HLAidentical siblings. Beneficial effect of a protective environment. N Engl J Med 308:302–307 41. Beelen DW, Elmaagacli A, Muller KD et al (1999) Influence of intestinal bacterial decontamination using metronidazole and ciprofloxacin or ciprofloxacin alone on the development of acute graft-versus-host disease after marrow transplantation in patients with hematologic malignancies: Final results and long-term follow-up of an open-label prospective randomized trial. Blood 93:3267–3275 42. Passweg JR, Rowlings PA, Atkinson KA et al (1998) Influence of protective isolation on outcome of allogeneic bone marrow transplantation for leukemia. Bone Marrow Transplant 21:1231–1238 43. Yokoe DS, Mermel LA, Anderson DJ et  al (2008) A compendium of strategies to prevent healthcare-associated infections in acute care hospitals. Infect Control Hosp Epidemiol 29:S12–21 44. Boyce JM, Pittet D (2002) Guideline for Hand Hygiene in Health-Care Settings. Recommendations of the Healthcare Infection Control Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force. Society for Healthcare Epidemiology of America/Association for Professionals in Infection Control/Infectious Diseases Society of America. MMWR Recomm Rep 51:1–45 quiz CE41-44 45. Pittet D, Simon A, Hugonnet S et  al (2004) Hand hygiene among physicians: Performance, beliefs, and perceptions. Ann Intern Med 141:1–8 46. Smith GC, Pell JP (2003) Parachute use to prevent death and major trauma related to gravitational challenge: Systematic review of randomised controlled trials. BMJ 327:1459–1461 47. (1994) Guideline for prevention of nosocomial pneumonia. Centers for Disease Control and Prevention. Respir Care 39:1191–1236 48. Garner JS (1996) Guideline for isolation precautions in hospitals. The hospital infection control practices advisory committee. Infect Control Hosp Epidemiol 17:53–80 49. Larson EL (1995) APIC guideline for handwashing and hand antisepsis in healthcare settings. Am J Infect Control 23:251–269 50. Giles FJ, Rodriguez R, Weisdorf D et al (2004) A phase III, randomized, doubleblind, placebo-controlled, study of iseganan for the reduction of stomatitis in patients receiving stomatotoxic chemotherapy. Leuk Res 28:559–565

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B. Hayes-Lattin 51. Spielberger R, Stiff P, Bensinger W et al (2004) Palifermin for oral mucositis after intensive therapy for hematologic cancers. N Engl J Med 351:2590–2598 52. Ketterer N, Zaman K, Luthi F et al (2004) Conventional hospital room is as safe as sterile unit for high-dose chemotherapy and peripheral blood stem cells (PBSC) transplantation. Blood 104:390b abstract 5222 53. Cantoni R, Passweg J, Weisser M et al (2004) Abandoning care in laminar air flow (LAF) units and routine high dose intravenous immunoglobulines (IVIG) in allogeneic hematopoietic stem cell transplantation (HSCT) does not increase mortality and rate of infections. Blood 104:351b abstract 5072 54. Agha ME, Yeager AM, Evans C et al (2004) Outpatient high-dose chemotherapy and autologous hematopoietic stem cell transplantation for multiple myeloma is associated with a low infection safety profile. Blood 104:351b abstract 5070 55. Halim TY, Lavoie JC, Barnett MJ et al (2004) High risk AML outpatient management: A retrospective analysis of bacteremia incidence following chemotherapy. Blood 104:252a abstract 884 56. Sasaki T, Akaho R, Sakamaki H et  al (2000) Mental disturbances during isolation in bone marrow transplant patients with leukemia. Bone Marrow Transplant 25:315–318 57. Cohen MZ, Ley C, Tarzian AJ (2001) Isolation in blood and marrow transplantation. West J Nurs Res 23:592–609 58. Gaskill D, Henderson A, Fraser M (1997) Exploring the everyday world of the patient in isolation. Oncol Nurs Forum 24:695–700 59. Mank A, van der Lelie H (2003) Is there still an indication for nursing patients with prolonged neutropenia in protective isolation? An evidence-based nursing and medical study of 4 years experience for nursing patients with neutropenia without isolation. Eur J Oncol Nurs 7:17–23 60. Russell JA, Poon MC, Jones AR et al (1992) Allogeneic bone-marrow transplantation without protective isolation in adults with malignant disease. Lancet 339: 38–40 61. Russell JA, Chaudhry A, Booth K et al (2000) Early outcomes after allogeneic stem cell transplantation for leukemia and myelodysplasia without protective isolation: A 10-year experience. Biol Blood Marrow Transplant 6:109–114 62. Svahn BM, Remberger M, Myrback KE et al (2002) Homecare during the pancytopenic phase after allogeneic hematopoietic stem cell transplantation is advantageous compared with hospital care. Blood 100:4317–4324 63. van Tiel FH, Harbers MM, Kessels AG, Schouten HC (2005) Homecare versus hospital care of patients with hematological malignancies and chemotherapy-induced cytopenia. Ann Oncol 16:195–205

Chapter 41 Monoclonal Antibodies in Allogeneic Hematopoietic Stem Cell Transplantation for Hematologic Malignancies Maria Corinna Palanca-Wessels and Oliver Press

1. Introduction Monoclonal antibodies have revolutionized the treatment of hematologic malignancies. The monoclonal antibody rituximab plays an integral part in the upfront treatment of both aggressive and indolent CD20-expressing non-Hodgkin’s lymphoma (NHL). Although allogeneic hematopoietic stem cell transplantation (HSCT) can be a curative therapy for many hematologic malignancies, much improvement remains to be made to reduce rates of regimen related-transplant toxicity, disease relapse and graft versus host disease (GVHD). The role of monoclonal antibodies in allogeneic HSCT is still being investigated. We have reviewed recent evidence supporting the use of monoclonal antibodies in allogeneic transplantation and have given our recommendations as to the best way to incorporate them in the transplant process.

2. Description of Immunotherapy The primary advantage of immunotherapy lies in its ability to precisely target tumors on the basis of cell surface antigens. Antibodies complement chemotherapy by eliciting tumor death via alternative pathways. Antibodycoated malignant cells are targets for antibody-dependent cellular toxicity [1, 2], complement-dependent cytoxocity [3] or apoptosis [4, 5]. Antibodies conjugated to toxins or radioisotopes further enhance lethality. Radiolabeled antibodies possess the added benefit of irradiating non-antigen-expressing malignant cells in the local vicinity via a “crossfire” effect. This feature is particularly advantageous in tumors with heterogeneous receptor expression or poor vascularization.

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_41, © Springer Science + Business Media, LLC 2003, 2010

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The anti-CD20 monoclonal antibody rituximab remains the paradigm for monoclonal antibody therapy in hematologic malignancies. Several recent randomized clinical trials have shown its efficacy in the treatment of both aggressive and indolent NHL [6–10]. CD20 is a tetraspan membrane protein expressed on the surface of mature B-lymphocytes and a majority of B-cell lymphomas, but is not expressed on plasma cells or B-lymphoid stem cells. The therapeutic potential of a murine derived anti-CD20 monoclonal antibody for the treatment of NHL was first demonstrated in a small clinical trial nearly two decades ago [11]. Several groups have sought to extend the use of anti-CD20 antibodies to transplantation for NHL but its role is still being established. Antibodies are continually being developed to target other tumor-associated antigens for direct tumor killing. Obvious uses for tumor-directed antibodies in transplant include reduction of toxicity during conditioning regimens or eradication of minimal residual disease after transplantation. Another potential role for antibodies is the prevention or treatment of GVHD through the targeting of donor T-lymphocytes responsible for this toxicity.

3. Monoclonal Antibodies in Conditioning Regimens In allogeneic HSCT, conditioning serves the dual purpose of (1) prevention of graft rejection through ablation of host immunologic barriers prior to infusion of donor cells and (2) eradication of remaining cancer cells. An effective transplant protocol suppresses host immunity yet minimizes GVHD without endangering engraftment or graft versus tumor effect. Ideally, conditioning should eliminate remaining malignant cells and be minimally toxic to normal tissues. High dose myeloablative conditioning effectively reduces immunocompetent cells but is associated with substantial toxicity. Total body irradiation (TBI) is both myeloablative and immunosuppressive. TBI combined with cyclophosphamide is a standard preparative regimen for myeloablative transplantation. Radiation is not associated with crossresistance to chemotherapy. Higher radiation doses are associated with a lower rate of relapse but an increased incidence of death from transplant-related causes [12, 13]. Attempts have been made to avoid the toxicity stemming from TBI by use of regimens containing busulfan (BU) and cyclophosphamide (CY). Use of radioisotype-conjugated antibodies permits targeted delivery of high dose radiation to sites of tumor while sparing normal vital organs from toxicities resulting from TBI. Radioimmunotherapy may be able to substitute for TBI in the preparative regimens for allogeneic transplant. The high risk of regimen-related morbidity generally precludes older individuals from undergoing traditional myeloablative allotransplantation. Reduced intensity conditioning employs lower doses of cytoreductive agents or radiation and incorporates other drugs such as fludarabine, total lymphoid irradiation and/or T-cell depleting strategies to permit engraftment and prevent GVHD. Reduced intensity regimens depend on graft versus tumor effect, rather than high dose therapy to destroy residual cancer cells and may be most efficacious in treating slow-growing malignancies such as indolent NHL. Elderly patients tolerate reduced intensity conditioning better than traditional myeloablative regimens. Use of monoclonal antibodies in conditioning regimens may further reduce transplant-related mortality in older patients who comprise the majority of patients who develop hematologic malignancies.

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3.1. Unlabeled Antibody The anti-CD20 monoclonal antibody rituximab is used extensively in conjunction with chemotherapy for the initial treatment of CD20+ lymphoid malignancies. It has also been used with hematopoietic stem cell harvest for autologous transplantation in NHL. In vitro and in vivo “purging” with rituximab prior to autologous harvest depletes circulating lymphoma cells and reduces the risk of contamination of graft with malignant cells [14, 15]. Use of rituximab in allogeneic transplantation has not been well-explored but given the favorable results in the autologous setting, it will likely show utility in the future. Table 41-1 lists the most recent studies utilizing rituximab in myeloablative and nonmyeloablative conditioning regimens. Notably, these were small phase I or II studies designed to show the feasibility of rituximab addition to transplant regimens. The studies enrolled patients with chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), and NHL (follicular, mantle cell and diffuse large B cell lymphoma). Only one of the studies compared patients receiving rituximab with patients who did not [16]. The analysis of this study compiled data from two sequential trials enrolling CLL patients, the second of which added high dose rituximab to a nonmyeloablative conditioning regimen consisting of fludarabine and CY. The overall survival (OS) of the patients receiving chemotherapy plus rituximab was 100% versus 14% with chemotherapy alone (p = 0.03) at a median follow-up of 21 months. While these results are impressive, we must await randomized trials to determine whether rituximab during conditioning prior to HSCT in lymphoid malignancies improves clinical outcomes. Table 41-1.  Studies utilizing rituximab for conditioning in allogeneic transplantation. Study

Patient

Transplant regimen

Results 2

Khouri [64]

39 consecutive patients, CLL

Fludarabine 30 mg/m + Cyclophosphamide 750 mg/m2 + high dose rituximab→nonmyeloablative

Estimated 4 year OS 48%; median FU 27 mos

Kebriae [65]

35 patients

Cyclophosphamide 120 g/kg

CD20+ ALL patients

TBI 12 Gy

2 year PFS 30%; treatment related mortality 24%, OS 47%

Rituximab 375 mg/m2→matched sib or unrelated donor Khouri [16]

17 patients CLL; previous fludarabine failure

Fludarabine 30 mg/ m2 + Cyclophosphamide 750 mg/m2 ± rituximab→nonmyeloablative

OS 100% for 10 patients receiving chemo+R vs 14% chemo alone (p = 0.03)

Escalon [66]

Continuous CR 18–45 mos, 15 patients (DLBCL, Fludarabine 30 mg/m2 + Cyclophosphamide 750 mg/m2 + median FU 25 months, OS FL, MCL) relapsed, rituximab→Nonmyeloablative 100% failing autologous transplant

Ho [67]

5 patients

Rituximab-BEAM-CAMPATH

Follicular NHL

*R given weekly × 4 weeks, 12 weeks prior to conditioning

13 patients advanced/ relapsed MCL

Fludarabine 30 mg/m2 + Median FU 21 months; OS 77% Cyclophosphamide 750 mg/m2 + rituximab→Nonmyeloablative

Khouri [68]

Median Fu 1.4 year; OS 80%, molecular CR 60%

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3.2. Radiolabeled Antibody The two most extensively studied radiolabeled antibodies are directed against the B-lymphocyte surface antigen CD20. These are 131I-labeled tositumomab and 90Y-labeled ibritumomab tiuxetan. The most compelling data using radiolabeled antibodies was derived from studies of autologous transplantation but the encouraging results in this setting provide the basis for their possible use in allogeneic transplants. Table 41-2 summarizes the most recently published studies using radioimmunoconjugates prior to allogeneic transplantation. The initial study by Press et al. [17] established the use of radiolabeled antibody, prior to autologous bone marrow transplantation. Relapsed or refractory NHL patients who received a myeloablative dose of 131I-labeled antibodies (anti-CD20 and anti-CD37) followed by autologous bone marrow transplant achieved major responses with little toxicity. The follow-up phase II study confirmed the initial favorable results [18]. Analysis of the phase I and II data showed a progression-free survival (PFS) of 62% and OS of 93% after a median follow-up of 2 years. A subsequent study utilizing a combination of anti-CD20 radioimmunotherapy (RIT) with chemotherapy prior to autologous

Table 41-2.  Recent studies using RIT as conditioning prior to allogeneic transplant. Study

Patients

Transplant regimen

Results

Gopal [25] Phase II

14 patients with CD20+ lymphoma

90Y-labeled Ibritumomab tiuxetan anti-CD20→fludarabine 30 mg/ m2, 2 Gy TBI→Allogeneic transplant

Median FU 6 mos for surviving patients, 64% alive, 50% alive and progression free

Pagel [28]

46 patients treated

Phase I/II

Acute myeloid leukemia

131I-labeled murine anti-CD45 (BC8)

Estimated 3 year disease free survival 61%

Zenze [29]

20 patients Philadelphia chromosome+ ALL or advanced CML

188 Rhenium-labeled antiCD66→TBI + Cy ± Thiotepa→Allogeneic transplant

2 patients relapsed CD20+ lymphoma

Ibritumomab tiuxetan→fludarabine 30 mg/m2, 2 Gy TBI→Allogeneic transplant

Phase I/II Fietze [24] Case report Burke [32]

Targeted busulfan (AUC 600–900 ng/ml), cyclophosphamide 120 mg/kg→Allogeneic transplant 4 year OS 29%, diseasefree survival 25%

Phase I

31 patients with relapsed/ 131I-labeled Anti CD33 (M195 or refractory AML, HuM195), busulfan 16 mg/kg, accelerated/blastic cyclophosphamide 90–120 mg/ CML or advanced MDS kg→allogeneic transplant

Median survival 4.9 mos (range 0.2–90+ mos). 3 relapsed AML patients in CR 59, 87,90+ mos

Ringhoffer [31]

20 patients

Phase I/II

AML/MDS

Estimated 2 year survival 52%

Bunjes [30]

57 patients high risk AML/ Rhenium 188-labeled AntiMedian FU 24 mos; 64% MDS CD66→TBI 12 Gy or disease free survival in busulfan+high dose cyclophospatients in CR or good phamide ± thiotepa→Allogeneic/ PR; 8% disease free autologous transplant survival if not in remission

Phase I/II

Rhenium 188-labeled or Yttrium 90-labeled antiCD66→fludarabine ± melphalan

Chapter 41  Monoclonal Antibodies in Allogeneic Hematopoietic Stem Cell  

transplantation demonstrated a 2-year OS of 83% [19]. This compared favorably with historical controls treated with a standard TBI/CY/etoposide regimen and established the feasibility of a combination therapy. A subsequent multivariable comparison of high dose RIT with 131I-tositumomab or conventional high-dose therapy as a conditioning regimen prior to autologous HSCT in patients with relapsed follicular lymphoma (FL) showed that 131I-tositumomab improved clinical outcomes [20]. The estimated 5-year OS and PFS was 67 and 48% respectively with high-dose RIT compared to 53 and 29% for highdose chemotherapy. Early trials using 90Y-ibritumomab tiuxetan have also shown favorable results [21, 22]. The unacceptably high risk of regimen-related toxicity associated with preparative regimens used for standard myeloablative allogeneic transplants generally limits this approach for older individuals. Reduced intensity conditioning or non-myeloablative regimens have been developed to permit individuals who are not candidates for standard ablative allogeneic HSCT to undergo potentially curative therapy. Augmentation with RIT can further reduce toxicity by substituting for TBI. RIT conditioning with 131I-tositumomab prior to autologous transplant was shown to be well-tolerated by elderly patients with relapsed NHL [23]. Anti-CD20 RIT has been investigated as a conditioning regimen prior to allogeneic hematopoietic cell transplantation for lymphoid malignancies. A recent case report described the use of 90Y-labeled ibritumomab tiuxetan as a conditioning regimen with fludarabine 30  mg/m2 and CY 500  mg/m2 in two relapsed lymphoma patients prior to transplantation with grafts from HLA-matched donors [24]. This pilot study showed no delay of engraftment. A Phase II clinical trial by the Seattle group reported at the 2006 ASH annual meeting enrolled 14 patients with CD20+ NHL for treatment with 90 Y-ibritumomab tiuxetan at 0.4  mCi/kg followed by fludarabine 30  mg/m2 and 2 Gy TBI prior to transplantation with either HLA-matched related or unrelated peripheral blood stem cell transplant [25]. With a median followup of 6 months for surviving patients, 64% are alive and 50% are alive and progression-free. These results show that standard dose 90Y-ibritumomab tiuxetan can be delivered with minimal toxicity as part of a reduced intensity conditioning regimen for allogeneic HSCT. RIT prior to allogeneic HSCT for the treatment of leukemia has been studied using monoclonal antibodies directed at a variety of cell surface antigens including CD45, CD66 and CD33. A series of Phase I and II clinical trials employing 131I anti-CD45 (BC8) antibody in conjunction with high dose chemotherapy and HSCT for acute myeloid leukemia (AML), ALL, and myelodysplasia (MDS) have been conducted [26–28]. CD45 is a tyrosine phosphatase that is broadly expressed on the cell surface of normal leukocytes and a high proportion of leukemic cells. In an initial Phase I clinical trial, 34 high risk patients with advanced AML, MDS, or ALL received infusions of 131IBC8 anti-CD45 antibody followed by 120 mg/kg CY, 12 Gy TBI and allogeneic HSCT [26]. The mean radiation absorbed doses delivered to target organs (bone marrow and spleen) from 131I-BC8 antibody were significantly greater than doses delivered to non-target organs (liver, lungs, kidneys, total body). At the maximum tolerated dose (MTD) of 10.5 Gy, it was determined that 131 I-BC8 could deliver 24 Gy to bone marrow and 50 Gy to spleen in addition to CY/TBI. The dose-limiting toxicity was grade 3 mucositis. Nine patients were

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alive and 8 were disease-free 8.9–15 years (median 13.2 years) after SCT. Dose to BM > 7 Gy/mCi. A Phase II study using the same regimen was given to 18 patients with advanced AML or high risk MDS followed by allogeneic HSCT from matched related or unrelated donors [27]. Seven of 18 treated patients (39%) were alive and disease-free after periods up to 3.5 years. A recently published Phase I/II trial utilized 131I-BC8 antibody combined with targeted BU (area-under-curve, 600–900  ng/ml) and CY (120  mg/kg) as a preparative regimen for acute myeloid leukemia patients prior to allogeneic HSCT [28]. The estimated 3-year non-relapse mortality of 21% and disease-free survival of 61% compared favorably to matched historical control patients from the Bone Marrow Transplant Registry who underwent busulfan/ cyclophosphamide alone. The mortality risk was reduced by 35% in antibodytreated patients compared to the registry patients with a p-value approaching significance (p = 0.09). Based on the encouraging outcomes in young patients, 45 elderly patients (median age 62) with advanced AML or high-risk MDS received 131I-BC8 followed by fludarabine, 2 Gy TBI and matched related or unrelated donor allografting. Of the 45 patients treated on this protocol, 13 are alive and 11 remain disease free up to 33 months after HSCT (J. Pagel et al. unpublished data). These favorable results support the use of RIT with antiCD45 antibody in future randomized control trials. Radiolabeled anti-CD66 antibody has been used during conditioning for Philadelphia chromosome positive ALL, advanced CML, and high risk AML/MDS [29, 30]. CD66 is highly expressed on maturing hematopoietic cells but not on leukemic blasts; therefore crossfire effect is responsible for killing neighboring leukemic blasts. The update of a trial utilizing Rhenium-188 labeled antiCD66 antibody for the treatment of Philadelphia chromosome positive ALL or advanced CML showed a 4-year survival of 29% [29]. 188Re-labeled anti-CD66 antibody as part of a conditioning regimen for high-risk AML/MDS showed a 64% disease free survival at a median follow-up of 24 months [30]. A more recent study also treating AML/MDS patients with either 188Re- or 90Y-labeled anti-CD66 antibody showed an estimated 2-year survival of 52% [31]. Burke and others reported the use of 131I anti-CD33 antibody with Bu/Cy conditioning to reduce the burden of the disease prior to allogeneic transplant in leukemic patients [32]. CD33 is a sialic acid adhesion protein highly expressed on myeloid leukemia blasts but not on hematopoietic stem cells. In summary, RIT shows great promise in reducing the toxicities associated with TBI; however, further randomized trials are required to establish its role in allogeneic transplantation.

4. Post-transplant Consolidation Persistence of minimal residual disease after transplant is associated with a higher risk of relapse [33, 34]. Rituximab therapy, after engraftment in patients treated for CD20+ lymphoid malignanies, may eradicate minimal residual disease post-transplant and augment graft versus lymphoma effect by stabilizing the remaining disease. Horwitz et al. reported in a phase II trial the feasibility of rituximab as a consolidation treatment after autologous transplantation for B-cell lymphoma

Chapter 41  Monoclonal Antibodies in Allogeneic Hematopoietic Stem Cell  

with stem cell grafts purged in  vitro with rituximab [35]. All patients were treated with weekly rituximab for 4 weeks beginning day 42 after transplant. A further 14 patients received an additional 4 week course at 6 months post transplant. Immune reconstitution with B-cell recovery was delayed; however, no serious infections were reported. Two-year OS was 80%. Other trials involving autologous transplantation have also shown elimination of minimal residual disease [36–38]. Few published studies describe monoclonal antibody therapy as consolidation after allogeneic transplantation. Shimoni et  al. studied the safety and efficacy of rituximab treatment after autologous (16 patients) or allogeneic (12 patients) transplantation [39]. Nine patients not achieving complete response (CR) converted to CR after treatment with rituximab. The estimated 2-year OS and disease-free survival of patients treated with rituximab were 95% and 64% respectively. Seven patients had recurrent neutropenia and severe hypogammaglobulinemia treated with intravenous immunoglobulin. None of the ten allogeneic recipients treated had severe GVHD. A pilot study investigated the feasibility of treatment with gemtuzumab ozogomicin (anti-CD33 monoclonal antibody) after reduced-intensity allogeneic HSCT in eight children with CD33+ AML [40]. Results showed that two doses of gemtuzumab ozogomicin (first dose between days +60 and +180) were well-tolerated with myeloid suppression being the most commonly observed adverse effect. No episodes of sinusoidal obstructive syndrome occurred. In summary, although there is good data to support the use of rituximab after autologous transplant, there is currently no convincing data for its routine use as maintenance or consolidation therapy after allogeneic transplantation. Use of other monoclonal antibodies for consolidation after transplant similarly remains investigational.

5. Antibody Prophylaxis for Graft Versus Host Disease GVHD remains a serious complication of allogeneic HSCT and accounts for about 15% of deaths after allogeneic transplants [41]. GVHD is a T-lymphocyte mediated inflammatory disease in which donor T-cells attack host cells. Tissue injury during conditioning may contribute partly to development of GVHD. GVHD is associated with reduced risk of relapse presumably due to donor lymphocyte recognition and eradication of recipient tumor cells surviving the preparative regimen. Acute GVHD occurs in 38–60% of transplant recipients while chronic GVHD occurs in 46–67% [42]. Peripheral blood stem cells have increasingly become the source for allogeneic transplantation due to rapid hematopoietic reconstitution after transplant and the relative ease of procurement when compared to bone marrow sources. However, peripheral blood stem cell harvests contain more donor T-cells than marrow harvests. This leads to an increased incidence of acute and chronic GVHD with peripheral blood stem cell transplantation compared with bone marrow transplantation [43]. Depletion of donor T-cells reduces the frequency and/or severity of GVHD; however, this carries an increased risk of graft rejection, viral infection and relapse compared with T-cell replete grafts [44]. In vivo depletion of T-cells with antithymocyte globulin (ATG) has been used in the past. Monoclonal antibodies directed against T-cells are increasingly being used.

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The Campath family of antibodies directed against the human CD52 antigen was first used in the mid-1980s for the purpose of reducing the incidence of acute GVHD [45]. CD52 is highly expressed on both normal and malignant B- and T-lymphocytes. Anti-CD52 antibodies have been used both in vitro and in vivo for purging. Several early studies supported the use of anti-CD52 antibodies in the allogeneic setting to reduce acute GVHD [46, 47]. The most widely used anti-CD52 monoclonal antibody is alemtuzumab (CAMPATH-1H) which is a humanized monoclonal antibody derived from an IgG rat antibody. Several groups have used alemtuzumab as part of conditioning regimens with fludarabine and melphalan prior to allogeneic HSCT. Alemtuzumab appears to permit a reduced intensity of conditioning with no adverse effect on survival in older patients. A recent study showed that older patients tolerated reduced intensity myeloablative conditioning and T cell depleted grafts as well as younger patients with a similar rate of hematopoietic recovery, treatment related mortality and survival [48]. GVHD occurred in only 13% of recipients. However, T-cell depletion of allogeneic grafts has not reliably translated into improved disease-free survival because the rates of graft rejection and relapse are often increased as a consequence. In one randomized phase II–III trial, the risk of CML relapse was significantly increased in patients receiving T-cell depleted grafts [49]. Other studies have also shown that while T-cell depletion reduces GVHD incidence, the risks of relapse, rejection and infection are also significantly increased [44]. In at least one study, reduced GVHD rate as a result of T-cell depletion came at the expense of reduced graft versus myeloma effect [50]. Reduction of the total dose of Campath-1H in one trial was necessary due to loss of graft in five of the six patients [51]. A study comparing the efficacy of alemtuzumab versus methotrexate for GVHD prophylaxis illustrated the pros and cons of anti-CD52 mediated lymphocyte depletion [52]. One hundred twenty-nine patients undergoing nonmyeloablative HLA-matched sibling HSCT for chronic lymphoproliferative disorders enrolled in two prospective studies were treated with melphalan and fludarabine as a conditioning regimen. GVHD prophylaxis consisted of cyclosporin A combined with either alemtuzumab or methotrexate. The alemtuzumab group had a significantly lower rate of both acute (21.7% versus 41.5%, P = 0.006) and chronic GVHD (5% versus 66.7%, P < 0.001) compared to the ­methotrexate group. Eighteen of 78 patients in the alemtuzumab group required donor lymphocyte infusions (DLI) to achieve disease control due to relapse/persistent disease or mixed chimerism compared to four of 51 patients in the methotrexate group. The incidence of GVHD did not increase after DLI. There was no significant difference observed in rates of engraftment, overall survival or disease status. Alemtuzumab treatment delayed platelet recovery by about 1 week (P < 0.001) although granulocyte recovery was faster by about 4 days compared to methotrexate treatment. A greater percentage of deaths in the methotrexate group was attributed to GVHD (33.4% versus 5%) while deaths due to disease progression were more common in the alemtuzumab group (60% versus 20%). The reported studies suggest that use of anti-CD52 antibodies for GVHD prophylaxis does reduce rates of acute and chronic GVHD in allogeneic HSCT. However, this does not necessarily translate into improved OS. This is likely due to reduced graft versus tumor effect requiring DLI rescue and infectious complications.

Chapter 41  Monoclonal Antibodies in Allogeneic Hematopoietic Stem Cell  

6.  Infectious Complications of Antibody Therapy for GVHD Prophylaxis A legitimate concern over the use of lymphocyte depleting antibodies is the lasting effect on immune reconstitution. Several observational studies have shown that the CD4+ T-cell population can remain depressed up to 72 weeks post transplant in patients treated with anti-CD52 antibodies [53–56]. Another group showed that alemtuzumab levels in reduced-intensity conditioning allogeneic HSCT (100  mg administered in  vivo over 5 days) remained at potentially lympholytic levels for approximately 8 weeks after transplant, 26 days longer than in the group that received allografts treated in vitro with alemtuzumab [55]. Total lymphocyte counts were significantly lower in the group treated in vivo and persisted beyond 6 months after transplantation. CD4 reconstitution was delayed until 9 months after transplantation (median time to achieve a CD4 count >200 × 106/L). Since CD52 antigen is expressed on both T- and B-lymphocytes, B-cell recovery can similarly be affected. Recovery of CD19+ B-cells was delayed up to 19 months in a study involving patients undergoing reduced intensity conditioning containing alemtuzumab for multiple myeloma [50]. In contrast, some studies show improved lymphocyte recovery after in vitro treatment with alemtuzumab “in the bag” as opposed to infusion [57]. The spectrum of infections observed after alemtuzumab administration reflects an ongoing suppression of cellular-mediated immunity. Increased rates of viral reactivation or infection including CMV [49, 52], adenovirus [58] and herpes virus [59] have been reported. Reduced reconstitution of EBV T-lymphocyte specific response was described in one study [60]. In a retrospective review at a single institution, cases of infection with Pneumocystis carinii and herpes virus occurred despite prophylaxis [59]. Sequelae of viral infection have included neurological complications. Avivi et  al. examined the incidence of neurological complications after reduced intensity conditioning using fludarabine, melphalan and alemtuzumab [61]. Five of 85 patients developed peripheral radiculo-neuropathy or myelitis, felt to be secondary to viral infection. Close surveillance for CMV reactivation appears to be sufficient to prevent complications from reactivation. In a retrospective study of 173 patients receiving grafts depleted in vitro with Campath, CMV reactivation and viremia occurred in 53% of patients [62]. Pre-emptive treatment with valganciclovir prevented overt CMV disease in all patients, however. Since delayed immune reconstitution exposes patients to the risk of developing infectious complications, prophylactic administration of CD8-depleted DLI was investigated as a strategy to improve reconstitution and reduce incidence of viral complications in a pilot study of unrelated donor PBSCT [63]. Patients received a reduced intensity conditioning regimen of fludarabine, melphalan and alemtuzumab. The leukapheresis product for DLI was depleted of CD8 T-cells through a CliniMACS Plus instrument. Eleven patients without active or previous GVHD received a total of 21 CD8-depleted DLI infusions between +60 and +120 days after transplant. Five of the 11 patients receiving DLI developed transient Grade I acute GVHD after DLI. Two patients with HLA-C mismatched donors developed grade II and III acute GVHD with subsequent limited chronic GVHD. A 2.1-fold median increase in circulating CD4

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T cells was observed within 2 weeks of infusion whereas non-DLI patients did not show comparable rise in counts. Four patients showed enhanced frequency of CD4 and CD8 T cells specific for CMV. This phase I study suggests that infusion of CD8-depleted DLI in lymphopenic transplant recipients previously treated with alemtuzumab may enhance immune reconstitution. While it appears that infectious complications are more prevalent when using anti-CD52 antibodies, close surveillance for CMV reactivation and prompt institution of antiviral therapy prevents overt CMV disease. Recipients of T-cell depleted grafts should also be monitored for other opportunistic infections secondary to prolonged lymphopenia.

7. Conclusions Researchers have investigated the utility of monoclonal antibodies to improve the outcomes of transplant. Antibody-targeted therapy may reduce toxicities that currently limit the utility of HSCT in elderly patients. Conditioning regimens incorporating radiolabeled antibodies prior to myeloablative transplant have been shown to deliver radiation specifically to tumors while sparing normal organs resulting in lower toxicity compared to TBI. Based on the promising preliminary studies, additional randomized trials comparing RIT to TBI containing regimens in the treatment of both NHL and AML are warranted. Donor T-cell depletion of allogeneic grafts utilizing anti-T cell antibodies in vitro and in vivo reduces the incidence of GVHD but carries risk of graft failure, opportunistic infections and disease relapse. Future studies should focus on defining the optimal dosing and administration of antibody therapy to enhance immune reconstitution. A role for antibodies in maintenance therapy after transplant to eradicate minimal residual disease or augment graft versus tumor effect may also exist. Questions regarding the optimal use of antibodies in allogeneic HSCT can only be answered by carefully designed studies. Acknowledgments.  This research was supported by NIH P01 Grant CA44991, SCOR grant #7040-05 from the Leukemia and Lymphoma Society, and gifts from David and Patricia Giuliani, Mary and Geary Britton-Simmons, and the Hext Family Foundation.

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Chapter 41  Monoclonal Antibodies in Allogeneic Hematopoietic Stem Cell   6. Herold M, Haas A, Srock S et al (2007) Rituximab added to first-line mitoxantrone, chlorambucil, and prednisolone chemotherapy followed by interferon maintenance prolongs survival in patients with advanced follicular lymphoma: an East German Study Group Hematology and Oncology Study. J Clin Oncol 25:1986–1992 7. Forstpointner R, Unterhalt M, Dreyling M et al (2006) Maintenance therapy with rituximab leads to a significant prolongation of response duration after salvage therapy with a combination of rituximab, fludarabine, cyclophosphamide, and mitoxantrone (R-FCM) in patients with recurring and refractory follicular and mantle cell lymphomas: results of a prospective randomized study of the German Low Grade Lymphoma Study Group (GLSG). Blood 108:4003–4008 8. van Oers MH, Klasa R, Marcus RE et al (2006) Rituximab maintenance improves clinical outcome of relapsed/resistant follicular non-Hodgkin lymphoma in patients both with and without rituximab during induction: results of a prospective randomized phase 3 intergroup trial. Blood 108:3295–3301 9. Habermann TM, Weller EA, Morrison VA et  al (2006) Rituximab-CHOP versus CHOP alone or with maintenance rituximab in older patients with diffuse large B-cell lymphoma. J Clin Oncol 24:3121–3127 10. Feugier P, Van Hoof A, Sebban C et al (2005) Long-term results of the R-CHOP study in the treatment of elderly patients with diffuse large B-cell lymphoma: a study by the Groupe d’Etude des Lymphomes de l’Adulte. J Clin Oncol 23: 4117–4126 11. Press OW, Appelbaum F, Ledbetter JA et al (1987) Monoclonal antibody 1F5 (antiCD20) serotherapy of human B cell lymphomas. Blood 69:584–591 12. Clift RA, Buckner CD, Appelbaum FR et al (1990) Allogeneic marrow transplantation in patients with acute myeloid leukemia in first remission: a randomized trial of two irradiation regimens. Blood 76:1867–1871 13. Clift RA, Buckner CD, Appelbaum FR et al (1991) Allogeneic marrow transplantation in patients with chronic myeloid leukemia in the chronic phase: a randomized trial of two irradiation regimens. Blood 77:1660–1665 14. Alvarnas JC, Forman SJ (2004) Graft purging in autologous bone marrow transplantation: a promise not quite fulfilled. Oncology (Williston Park) 18:867–876 discussion 876–878, 881, 884 15. Jacobsen E, Freedman A (2004) B-cell purging in autologous stem-cell transplantation for non-Hodgkin lymphoma. Lancet Oncol 5:711–717 16. Khouri IF, Lee MS, Saliba RM et  al (2004) Nonablative allogeneic stem cell transplantation for chronic lymphocytic leukemia: impact of rituximab on immunomodulation and survival. Exp Hematol 32:28–35 17. Press OW, Eary JF, Appelbaum FR et  al (1993) Radiolabeled-antibody therapy of B-cell lymphoma with autologous bone marrow support. N Engl J Med 329: 1219–1224 18. Press OW, Eary JF, Appelbaum FR et  al (1995) Phase II trial of 131I-B1 (antiCD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphomas. Lancet 346:336–340 19. Press OW, Eary JF, Gooley T et  al (2000) A phase I/II trial of iodine-131tositumomab (anti-CD20), etoposide, cyclophosphamide, and autologous stem cell transplantation for relapsed B-cell lymphomas. Blood 96:2934–2942 20. Gopal AK, Gooley TA, Maloney DG et al (2003) High-dose radioimmunotherapy versus conventional high-dose therapy and autologous hematopoietic stem cell transplantation for relapsed follicular non-Hodgkin lymphoma: a multivariable cohort analysis. Blood 102:2351–2357 21. Nademanee A, Forman S, Molina A et  al (2005) A phase 1/2 trial of high-dose yttrium-90-ibritumomab tiuxetan in combination with high-dose etoposide and cyclophosphamide followed by autologous stem cell transplantation in patients with poor-risk or relapsed non-Hodgkin lymphoma. Blood 106:2896–2902

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M.C. Palanca-Wessels and O. Press 22. Shimoni A, Zwas ST, Oksman Y et  al (2007) Yttrium-90-ibritumomab tiuxetan (Zevalin) combined with high-dose BEAM chemotherapy and autologous stem cell transplantation for chemo-refractory aggressive non-Hodgkin’s lymphoma. Exp Hematol 35:534–540 23. Gopal AK, Rajendran JG, Gooley TA et al (2007) High-dose [131I]tositumomab (anti-CD20) radioimmunotherapy and autologous hematopoietic stem-cell transplantation for adults > or = 60 years old with relapsed or refractory B-cell lymphoma. J Clin Oncol 25:1396–1402 24. Fietz T, Uharek L, Gentilini C et al (2006) Allogeneic hematopoietic cell transplantation following conditioning with 90Y-ibritumomab-tiuxetan. Leuk Lymphoma 47:59–63 25. Gopal AK, Rajendran JG, Pagel JM et al (2006) A phase II trial of 90Y-ibritumomab tiuxetan-based reduced intensity allogeneic peripheral blood stem cell (PBSC) transplantation for relapsed CD20+ B-cell non-Hodgkins lymphoma (NHL). Blood 108:316a 26. Matthews DC, Appelbaum FR, Eary JF et  al (1995) 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 85:1122–1131 27. Matthews DC, Appelbaum FR, Eary JF et  al (1999) Phase I study of (131) I-anti-CD45 antibody plus cyclophosphamide and total body irradiation for advanced acute leukemia and myelodysplastic syndrome. Blood 94:1237–1247 28. Pagel JM, Appelbaum FR, Eary JF et  al (2006) 131I-anti-CD45 antibody plus busulfan and cyclophosphamide before allogeneic hematopoietic cell transplantation for treatment of acute myeloid leukemia in first remission. Blood 107: 2184–2191 29. Zenz T, Glatting G, Schlenk RF et  al (2006) Targeted marrow irradiation with radioactively labeled anti-CD66 monoclonal antibody prior to allogeneic stem cell transplantation for patients with leukemia: results of a phase I-II study. Haematologica 91:285–286 30. Bunjes D (2002) 188Re-labeled anti-CD66 monoclonal antibody in stem cell transplantation for patients with high-risk acute myeloid leukemia. Leuk Lymphoma 43:2125–2131 31. Ringhoffer M, Blumstein N, Neumaier B et al (2005) 188Re or 90Y-labelled antiCD66 antibody as part of a dose-reduced conditioning regimen for patients with acute leukaemia or myelodysplastic syndrome over the age of 55: results of a phase I-II study. Br J Haematol 130:604–613 32. Burke JM, Caron PC, Papadopoulos EB et  al (2003) Cytoreduction with iodine131-anti-CD33 antibodies before bone marrow transplantation for advanced myeloid leukemias. Bone Marrow Transplant 32:549–556 33. Gribben JG, Neuberg D, Freedman AS et al (1993) 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 81:3449–3457 34. Zwicky CS, Maddocks AB, Andersen N, Gribben JG (1996) Eradication of polymerase chain reaction detectable immunoglobulin gene rearrangement in nonHodgkin’s lymphoma is associated with decreased relapse after autologous bone marrow transplantation. Blood 88:3314–3322 35. Horwitz SM, Negrin RS, Blume KG et al (2004) Rituximab as adjuvant to highdose therapy and autologous hematopoietic cell transplantation for aggressive nonHodgkin lymphoma. Blood 103:777–783 36. Brugger W, Hirsch J, Grunebach F et al (2004) Rituximab consolidation after highdose chemotherapy and autologous blood stem cell transplantation in follicular and mantle cell lymphoma: a prospective, multicenter phase II study. Ann Oncol 15:1691–1698

Chapter 41  Monoclonal Antibodies in Allogeneic Hematopoietic Stem Cell   37. Neumann F, Harmsen S, Martin S et al (2006) Rituximab long-term maintenance therapy after autologous stem cell transplantation in patients with B-cell nonHodgkin’s lymphoma. Ann Hematol 85:530–534 38. Mangel J, Leitch HA, Connors JM et al (2004) Intensive chemotherapy and autologous stem-cell transplantation plus rituximab is superior to conventional chemotherapy for newly diagnosed advanced stage mantle-cell lymphoma: a matched pair analysis. Ann Oncol 15:283–290 39. Shimoni A, Hardan I, Avigdor A et al (2003) Rituximab reduces relapse risk after allogeneic and autologous stem cell transplantation in patients with high-risk aggressive non-Hodgkin’s lymphoma. Br J Haematol 122:457–464 40. Roman E, Cooney E, Harrison L et al (2005) Preliminary results of the safety of immunotherapy with gemtuzumab ozogamicin following reduced intensity allogeneic stem cell transplant in children with CD33+ acute myeloid leukemia. Clin Cancer Res 11:7164s–7170s 41. Welniak LA, Blazar BR, Murphy WJ (2007) Immunobiology of allogeneic hematopoietic stem cell transplantation. Annu Rev Immunol 25:139–170 42. Giralt S (2006) The role of alemtuzumab in nonmyeloablative hematopoietic transplantation. Semin Oncol 33:S36–S43 43. Cutler C, Giri S, Jeyapalan S, Paniagua D, Viswanathan A, Antin JH (2001) Acute and chronic graft-versus-host disease after allogeneic peripheral-blood stem-cell and bone marrow transplantation: a meta-analysis. J Clin Oncol 19:3685–3691 44. Marmont AM, Horowitz MM, Gale RP et  al (1991) T-cell depletion of HLAidentical transplants in leukemia. Blood 78:2120–2130 45. Wasil T, Rai KR, Mehrotra B (2004) The role of monoclonal antibodies in stem cell transplantation. Semin Oncol 31:83–89 46. Cull GM, Haynes AP, Byrne JL et  al (2000) Preliminary experience of allogeneic stem cell transplantation for lymphoproliferative disorders using BEAMCAMPATH conditioning: an effective regimen with low procedure-related toxicity. Br J Haematol 108:754–760 47. Lush RJ, Haynes AP, Byrne J et al (2001) Allogeneic stem-cell transplantation for lymphoproliferative disorders using BEAM-CAMPATH (+/- fludarabine) conditioning combined with post-transplant donor-lymphocyte infusion. Cytotherapy 3:203–210 48. Novitzky N, Thomas V, Hale G, Waldmann H (2005) Myeloablative conditioning is well tolerated by older patients receiving T-cell-depleted grafts. Bone Marrow Transplant 36:675–682 49. Wagner JE, Thompson JS, Carter SL, Kernan NA (2005) Effect of graft-versus-host disease prophylaxis on 3-year disease-free survival in recipients of unrelated donor bone marrow (T-cell Depletion Trial): a multi-centre, randomised phase II-III trial. Lancet 366:733–741 50. D’Sa S, Peggs K, Pizzey A et al (2003) T- and B-cell immune reconstitution and clinical outcome in patients with multiple myeloma receiving T-cell-depleted, reduced-intensity allogeneic stem cell transplantation with an alemtuzumab-containing conditioning regimen followed by escalated donor lymphocyte infusions. Br J Haematol 123:309–322 51. Shore T, Harpel J, Schuster MW et al (2006) A study of a reduced-intensity conditioning regimen followed by allogeneic stem cell transplantation for patients with hematologic malignancies using Campath-1H as part of a graft-versus-host disease strategy. Biol Blood Marrow Transplant 12:868–875 52. Perez-Simon JA, Kottaridis PD, Martino R et  al (2002) Nonmyeloablative transplantation with or without alemtuzumab: comparison between 2 prospective studies in patients with lymphoproliferative disorders. Blood 100:3121–3127 53. Novitzky N, Davison GM, Hale G, Waldmann H (2002) Immune reconstitution at 6 months following T-cell depleted hematopoietic stem cell transplantation is predictive for treatment outcome. Transplantation 74:1551–1559

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Chapter 42 Treatment of Acute Graft-vs-Host Disease Steven C. Goldstein, Sophie D. Stein, and David L. Porter

1. Introduction Over the last two decades, advances in our understanding of the pathophysiology of acute GVHD (aGVHD) [1, 2] have not yet translated into significant changes in upfront management. Corticosteroids have remained the cornerstone of aGVHD therapy for the last several decades, and still, failure to respond to steroids remains the primary predictor of poor overall outcome. Several issues limit the usefulness of most retrospective reports of therapeutic intervention for aGVHD. First, we must acknowledge that the majority of series using the “temporal onset” definition of acute and chronic GvHD actually included a mix of patients with either late onset acute or early chronic GvHD, confounding their interpretation and extrapolation to recent efforts to define these syndromes on the basis of clinical features regardless of timing of onset. Second is the lack of prognostic tools to identify cohorts who may need more (vs less) aggressive induction. Third, we also must acknowledge the significant variability in clinical practice between transplant physicians, often well-founded in their efforts to individualize therapy on the basis of specific organ involvement, severity of symptoms, etc. Rather than surrendering to the potentially insurmountable task of establishing a single and perhaps suboptimal standard of care for the upfront treatment of aGVHD, advances in the field and improved patient outcomes will require modern and well-designed clinical trials supported throughout the transplant community. 1.1. Indications for Intervention for AGVHD Although the definitions of acute and chronic GvHD have been recently revised to characterize the respective syndromes on the basis of their clinical manifestations rather than timing of onset [3], the indication for intervention among most transplant physicians has remained rather constant, taking into account the level of immunosuppression and severity of symptoms.

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_42, © Springer Science + Business Media, LLC 2003, 2010

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Grade A (aka grade I aGVHD) is typically observed without intervention, though there is the occasional patient in whom a rash may be extremely symptomatic on topical steroids despite its limited body surface area involvement of less than 25%. It may be relevant for the clinician to consider the recent evolution in staging systems for aGVHD when deciding to start steroid therapy as the literature for intervention is primarily on the basis of the traditional grade I–IV staging system [4], typically defining grade II or greater as an indication for the initiation of steroid therapy (see Tables 42-1–42-3). The recent integration of the IBMTR severity index [5] was designed to enhance the predictive value of patterns of organ involvement on outcome on the basis of more objective organ-specific measurements, but may obfuscate traditional criteria for initiation of therapy in certain circumstances. For example, while Index grade A aGVHD may overlap entirely with grade I aGVHD (both typically requiring no intervention), index grade B represents a spectrum of patients with both traditional Glucksberg grade I disease (skin only, less than 50% BSA or S = 2, G = 0, L = 0) and Glucksberg grade II disease (S < 2, G and/or L = 1); the former typically a circumstance where therapy is held, and the latter a circumstance where most clinicians will intervene. The improved likelihood of responding to steroids reported for patients with Index Grade B disease as compared to patients categorized as grade II by Glucksburg likely reflects the inclusion of grade I patients in that cohort [6]. Table 42-1.  Acute graft-vs-host disease: staging table. Stage

Skin

Liver (mg/dL)

Intestine

1

Maculopapular rash <25% of body surface area

Bilirubin 2–3

500–1000 cc diarrhea/day; or nausea, anorexia, or vomiting with biopsy (EGD) confirmation of upper GI GVHD

2

Maculopapular rash 25–50% Bilirubin 3–6 of body surface

>1000–1500 cc diarrhea/ day

3

Maculopapular rash >50% of body surface area of generalized erythroderma

Bilirubin >6–15

>1500 cc diarrhea/day

4

Generalized erythroderma with bullous formation and desquamation

Bilirubin >15

>1500 cc diarrhea/day plus severe abdominal pain with or without ileus

Source: Przepiorka et al. [95]

Table 42-2.  Acute graft-vs-host disease: IBMTR severity index. Index

Skin (max), or

Liver (max), or

Intestine (max)

A

1 (<25%)

0 (<2)

0 (<500 cc)

B

2 (25–50%)

1–2 (2–6)

1–2 (500–1500 cc)

C

3 (>50%)

3 (>6–15)

3 (>1500 cc)

D

4 (bullae)

4 (>15)

4 (severe pain and ileus)

Assign index based on maximum involvement in an individual organ system Source: Rowlings et al. [5]

Chapter 42  Treatment of Acute Graft-vs-Host Disease 

Table 42-3.  Glucksberg (modified) criteria. Grade

Skin

Liver

Intestine

I

1–2 (<25–50%)

0

0

IIa

0

0–1

1

0

1

0–1

1–3

0–1

1

1–3

1

0–1

3

0

0

0–3

2–3

0–2

0–3

0–3

2–3

b

III

IV

0–3

4

0–3

0–3

0–4

4

4

0–4

0–4

a

Grade II–IV aGVHD with only single organ involvement should be biopsy confirmed b If Karnofsky performance status is £30% then grade IV Source: Przepiorka et al. [95]

1.2. Current “Standards of Care” for Acute GVHD Although corticosteroids have remained the primary intervention for aGVHD, there remains a broad range of opinion regarding the optimal timing, dose, duration, and definitions of response to steroid therapy. Traditional distinctions between primary (initial therapy for patients on minimal or no immunosuppression) and secondary treatment (therapy for those failing initial steroids) for aGVHD have evolved into more useful categorizations of “steroid-responsive” vs. “steroid-refractory” aGVHD (SR GvHD), now that initial prophylaxis with prednisone is no longer common among most transplant centers. 1.3. Corticosteroid Dosing A standardized starting dose of corticosteroids in the primary management of aGVHD has not been defined. If the patient has already begun the taper of post-transplant immunosuppressive medications, resumption or increase of tacrolimus/cyclosporine to therapeutic levels in combination with corticosteroids is appropriate. The most common starting dose in the literature is ~2  mg/ kg methylprednisolone (vs oral prednisone) split over two doses daily [6, 7]. A range of 0.5 to >20  mg/kg/d has been reported [8, 9] with few inroads toward improved efficacy, but clearly with higher toxicity at higher doses, predominantly from fungal infection [8, 9] (it is important to note that poor outcomes with high dose steroids were published in an era without effective anti-fungal prophylaxis.). Although the goal of achieving clarity in this area is clouded by decades of empiricism and a lack of outcome data controlled for by starting dose [9], 2 mg/kg of solumedrol is reasonable for the majority of patients. Oral beclomethasone has been reported to benefit a significant number of patients with primarily GI symptoms [10].

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1.4. Duration of Therapy In contrast to the therapy for chronic GvHD in which steroid therapy is prolonged over many months, (reviewed in a later chapter), the goals of therapy for aGVHD focus on rapid suppression of the effector phase of organ damage with rapid taper to minimize the sequelae of both short and long term steroid exposure. The duration of “full dose” therapy and length of taper remain highly individualized between patients and physicians, but some general trends in practice have been established. A typical patient with ³ grade II aGVHD [4] may be treated 14 days at their starting dose before initiating a taper schedule every 4–7 days over approximately 2–3 months with close monitoring for flare. Studies comparing the length of taper demonstrate no advantage of a prolonged taper beyond 2–3 months in duration [11]. In patients on cyclosporine or tacrolimus, tapering of calcineurin inhibitors is usually deferred until after successful steroid taper. Critical early milestones of response and possible triggers for additional salvage therapy often include: progression by day 3, no change by day 5–7, and/or incomplete resolution by day 14. Unfortunately, objective early response data are confounded by the extremely subjective nature of assessing many patients with aGVHD. 1.5. Outcome The response rates to primary corticosteroid therapy for aGVHD have been unfortunately quite consistent over the last three decades. Typically 25–35% of patients achieve complete resolution with an additional 15–20% achieving partial responses [6, 7, 12]; effective for some, yet inadequate for many. The need for improved first line therapy is evident in light of the dismal prognosis for patients failing to respond to prednisone: only 5–30% long term survival was observed among those with steroid resistant GvHD as compared to the 50–60% of patients with stable responses to first line treatment who achieve long term survival. 1.6. Combination Therapy as First-line Treatment These sobering results have prompted efforts to both combine corticosteroids with newer agents in first-line treatment strategies (vs attempts to add second/ third line agents in patients deemed steroid-refractory, see below) and to target more organ-specific modalities. Depending on the timing of onset of aGVHD post-transplant, most physicians will increase cyclosporine/tacrolimus to therapeutic levels if the syndrome develops during the taper phase, and empirically resume a calcineurin inhibitor if they have completed their taper. Although a common practice, no randomized trials assessing corticosteroids ± CSA or tacrolimus as primary treatment for aGVHD have been performed. Rapid progress in the evolution of cell-targeted monoclonal antibody therapy and cytokine inhibition/blockade has coincided with attempts at achieving synergy in this area, with mixed results. Up-front combination strategies of targeting T-cells [ATG [13, 14], anti-CD5 mAb [15], anti-IL2R mAb [16, 17]], cytokines [TNF [18], IL1RA [19]], and T-cell signaling [MMF [20], beclomethasone [10]] have been reported in small Phase II studies with similar themes; initial enthusiasm for improved efficacy, but little long term improvement

Chapter 42  Treatment of Acute Graft-vs-Host Disease 

in overall outcomes when compared to historical controls. Unfortunately, many of these studies lack adequate statistical power and include heterogeneous and small populations making interpretation difficult. However, the case of daclizumab, in particular, speaks to the critical importance of well-designed clinical trials before presuming efficacy of more aggressive therapy; a prospective, randomized, multi-center study of methylprednisone ± Daclizumab as up-front treatment of aGVHD in 102 patients demonstrated statistically significant inferior 100 day survival in the combination arm, in large part due to death from disease and GVHD, prompting early closure of the trial [17]. Although daunting, meeting the challenge of improving outcomes for the primary treatment of aGVHD (as well as improving prophylaxis) is essential for the field to advance. One must account for (1) the limitations of small phase II studies, (2) the poor outcome of patients once they are deemed steroid refractory, (3) the increasing numbers of high-risk patients undergoing allogeneic transplantation, and (4) the plethora of novel immunomodulatory agents now available. Toward this end, well designed studies of newer agents are desperately needed. 1.7. Does Treatment of aGVHD Impact on GvL? A continued concern is whether inhibition of T-cell function by corticosteroids for GvHD also inhibits the graft-vs-leukemia potential. Data are limited and only indirect inferences can be drawn; a retrospective analysis of 197 patients treated for  ³ grade II aGVHD at a single center found no correlation between risk of relapse and prior immunosuppressive therapy for GvHD [12]. In contrast, Kataoka et al. [21] drew a different conclusion from their observation in AML and CML patients that grade I aGVHD (i.e., untreated) was associated with lower relapse rates and improved disease-free survival than grade II or greater GvHD, implying that the primary factor was suppression of moderate/ severe aGVHD rather than potential differences in underlying path physiologic mechanisms and effector pathways between mild and moderate aGVHD and their relevance to the GvL response. Additional indirect evidence for the suppression of GvL via aggressive immunosuppression of GvHD may be inferred from the higher relapse rate and death from disease found in the daclizumab arm of the study of Lee et al. [17]. 1.8. Supportive Care As patients who develop aGVHD may experience a generalized decline secon­ dary to the disease itself as well as from the effects of steroids, it is important to anticipate potential complications and optimize supportive care early in the course of disease. For patients with gastrointestinal symptoms of nausea, vomiting, and diarrhea, bowel rest, hyperalimentation, and pain control are critical ancillary interventions. A trial of octreotide may be of benefit with significant secretory diarrhea [22]. Aggressive prophylaxis and early intervention for opportunistic infections such as HSV, VZV, PCP, and fungus are essential. Many patients will develop hyperglycemia secondary to corticosteroids that requires intervention. Attention to bone mineral retention and ongoing physical therapy is important to minimize the debilitation associated with steroidinduced osteoporosis and myopathy.

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2.  Second Line Therapy Only approximately half of patients treated with corticosteroids for new onset aGVHD will obtain a significant and durable response. Tacrolimus or cyclosporine initiated at the time of transplant is usually continued when steroids are added. The significant morbidity and mortality associated with steroidrefractory aGVHD have led to intense interest in alternative treatments. 2.1. Broad Anti-T Cell Agents/Antibodies 2.1.1. Anti-Thymocyte Globulin Until recently, anti-thymocyte globulin (equine ATG, ATGAM) has been the most common therapy for steroid-refractory GvHD. Unfortunately, outcomes after ATG have been disappointing. Khoury et  al. [23] used three different schedules of equine ATG in 58 steroid-resistant aGVHD patients. Twenty-one days after ATG treatment, only 8% (4/52 subjects) had achieved a complete response. Furthermore, 90% (52/58 subjects) had died at a median of 40 days from the initiation of ATG (see Fig. 42-1). Macmillan et al. [24] and Arai et al. [25] reported similar dismal outcomes with a complete response rate of 20% (N = 79; evaluation at day 28) and 14% (N = 69), respectively. Patients with GVHD involving the skin showed the most significant responses in all studies [23–26] (Table 42-4). Although the more recent introduction of ATG prepared from rabbits (thymoglobulin) has been associated with less infusion related toxicity, there have been no published studies evaluating whether it has any advantages over the equine preparation in the treatment of steroid refractory GvHD [27]. Published studies of thymoglobulin have focused primarily on its potential benefits in the prevention, rather than treatment of GvHD [28–30].

Fig.  42-1.  Kaplan-Meier survival estimate for 58 patients with SR aGVHD treated with ATG. Day 0 represents the first day ATG was initiated. Censored observations are indicated with a plus. Khoury et al. [23]

Chapter 42  Treatment of Acute Graft-vs-Host Disease 

753

Table 42-4.  ATG/thymoglobulin for GvHD therapy-selected trials. Reference

Formulation

N

Response rate

Comments

Khoury et al. [23]

Equine

N = 58

8% CR79% PR/CR in patients with skin gvh

52 of 58 pts expired by d40 ATG

Macmillan et al. [24]

Equine

N = 79

20% CR61% PR/CR in patients with skin gvh

Arai et al. [25]

Equine

N = 69

14% CR

Multiorgan involvement and age >35 predicted no response to ATG

Graziani et al [96]

Rabbit

N = 28

38–74% PR/CR

Steroid refractory not clearly defined

McCaul et al [27]

Rabbit

N = 36

38% CR21% PR

High incidence (25%) of PTLD

2.1.2. Alemtuzumab (Campath; Genzyme) Alemtuzumab is a humanized monoclonal antibody directed against CD52, an antigen known to be expressed on T cells. Unlike the T-cell specificity of ATG, alemtuzumab targets a broader population of cells expressing CD52, such as T-cells, B-cells, and some APCs. A potential advantage of Campath may therefore be a lower incidence of post-transplant lymphoproliferative disease than seen after ATG therapy; a disadvantage would likely be the higher incidence of opportunistic infection as a complication of such broad immunodepletion. There are several studies supporting the use of alemtuzumab to prevent GVHD [31–38] but its role in treating aGVHD has been limited to isolated case reports [39–42], each suggesting possible efficacy in the refractory patient. Although several of the published reports involve patients with hepatic involvement, the numbers are too small to confirm organ specificity. 2.1.3. Visilizumab Another humanized monoclonal antibody, HuM291 (visilizumab, PDL) [43], directed against the invariant CD3 epsilon chain of the T-cell receptor remains under investigation. In a multicenter Phase II study, Carpenter et al. [44] reported an overall response of 32% (14% complete) in a cohort of 44 high-risk patients with predominantly grade III–IV steroid refractory aGVHD. Although response rates may appear to be lower than reported for other agents, this more likely reflects the advanced disease of participants in this study (86% with grades III–IV aGVHD) as compared to other studies enrolling patients with grade I–II aGVHD, rather than lack of efficacy. The relatively high incidence of patients (19/44) with elevation of EBV titers [no patient developed post-transplant lymphoproliferative disease (PTLD), 17 of 19 pre-emptively treated with Rituxan] highlights the importance of following EBV titers in future studies as well as the role for pre-emptive Rituxan in this setting to minimize the risk of PTLD. 2.2. Broad Anti-T Cell Agents/Immunomodulatory Agents 2.2.1. Mycophenolate Mofetil/MMF (CellCept; Roche) In addition to anti-lymphocyte monoclonal antibodies, immunomodulatory drugs targeting signal transduction pathways may have significant activity in

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GVHD [45]. The ability of MMF to selectively inhibit lymphocyte proliferation is on the basis of the blockade of de  novo purine synthesis by MPA, the active metabolite of MMF. Unlike other cell types, lymphocytes do not have a salvage pathway, allowing for a rather selective inhibition of B and T-cell proliferation [45]. Although the availability of MMF in both oral and IV formulations provides the clinician more dosing flexibility, the optimal frequency for dosing and relevance of MPA levels in the plasma remain an active area of investigation. In solid organ transplant recipients, recommendations for specific target levels of MPA AUC have been established [46]. Among stem cell recipients, however, a consensus regarding the utility of drug level monitoring has not been reached, although it will likely be a focus of any prospective study with this agent. Although the role of MMF in the prophylaxis of GvHD has increased over the last several years, particularly in non-myeloablative strategies, data regarding its efficacy once GvHD is established remain rather limited [45]. Most series include both acute and chronic GvHD patients and are divided in terms of whether MMF has similar efficacy in both settings. Kim et  al. [47] evaluated 26 patients with GvHD, split evenly between refractory acute and chronic, and concluded that MMF was more efficacious in patients with chronic GvHD than aGVHD (with response rates of 54 vs. 33%, respectively), although it does not appear that the study carried adequate statistical power to compare outcomes. In contrast, in their retrospective analysis of 21 patients with refractory GvHD (10 acute, 11 chronic), Krejci et  al. [48] reported a similar response rate of ~60% in both cohorts. Basara et  al. [49] reported a response rate of 72% (26 of 36) among patients who developed grade I–IV aGVHD on a prednisone-containing prophylaxis regimen. Interestingly, MMF was given on a QID schedule in this study, potentially providing higher plasma levels when enterohepatic circulation and drug interactions are taken into account. A smaller series by Takami et al. [50] prospectively studied 11 patients with refractory GvHD (6 acute, 5 chronic) and again demonstrated a remarkably similar response rate of 67% among patients with aGVHD. A common theme among these reports was the steroid-sparing effect allowed by MMF for the responding patients (Table 42-5). Table 42-5.  MMF for GvHD-selected trials. Reference

N

Response rate

Kim et al. [46]

N = 26N = 13 with aGVHD

30.8% overall response in agvh 30.8% response in skin agvh 44.4% response in liver agvh 22.9% response in gut agvh

Krejci et al. [47]

N = 21N = 10 with aGVHD

6/10 (60%) with AGVHD 7/11 (64%) with Chronic GvHD

Basara et al. [48]

N = 48N = 36 with AGVHD

72% overall response 86% response in skin agvh 75% response in liver agvh 50% response in gut agvh

Takami et al. [49]

N = 11N = 6 with AGVHD

67% response rate in patients with aGVHD

Chapter 42  Treatment of Acute Graft-vs-Host Disease 

2.2.2. Deoxycoformycin (Pentostatin, Nipent) Deoxycoformycin is a nucleoside analog that inhibits adenosine deaminase (ADA). Lymphocytes contain one of the two isoforms of ADA which metabolizes 2¢-deoxyadenosine to 5¢-triphosphate (dATP). Without the production of dATP, lymphocyte growth is inhibited and apoptosis results. A phase I dose-finding study conducted by Bolaños-Meade et  al. [51] investigated the use of pentostatin in steroid refractory or steroid unresponsive GVHD. The MTD was determined to be 1.5 mg/m2/d × 3 days and careful dose adjustment for renal insufficiency is warranted. In 22 assessable patients, the complete and partial response rates were 64 and 14%, respectively and 1 year overall survival was 26%. Dose limiting toxicity was determined to be infections occurring greater than 3 weeks from the time of enrollment. 2.2.3. Sirolimus (Rapamycin; Wyeth–Ayerst) The IL-2 pathway can be targeted using Sirolimus. Sirolimus binds to FK-binding protein (FKBP12) in the cytosol of cells. The complex then inhibits the mammalian target of Rapamycin (m-tor) and blocks the response to IL-2. In the presence of sirolimus, B- and T-cells cannot become activated. In a pilot study of 21 patients conducted by Benito et al. [52], the overall response rate to sirolimus was 57% (24% complete response and 33% partial response). However, myelosuppression and hypertriglyceridemia were significant dose limiting toxicities. Even more concerning were two cases of hemolytic uremic syndrome reported, although the two study participants were also on cyclosporine. 2.2.4. Extracorporeal Photopheresis Although experience with extracorporeal photopheresis (ECP) has been primarily in the treatment of chronic cutaneous GVHD [53], recent studies suggest a potential benefit for patients with SR aGVHD as well [54]. The exact mechanism by which ECP works is not well understood, but proposed hypotheses focus on the apoptosis of 8-methoxypsoralen-exposed leukocytes induced by UVA irradiation. Upon return to the patient, the apoptic cells are taken up by antigen-presenting cells and ultimately lead to inhibition of T-cell function, cytokine release, and induction of regulatory T-cells [55]. Greinix et  al. [56] reported a series of 21 patients with aGVHD treated with ECP. After 3 months of treatment (median response time was 4 months), the complete response rate was 60%. Nine of the 12 patients who achieved a complete response had grade II aGVHD and the best responses were seen in patients with liver and/or skin involvement. 2.3. Narrow Anti-T Cell Agents/Receptor and Cytokine Targets 2.3.1. Daclizumab (Zenepax; Hoffman-La Roche) Daclizumab is a humanized monoclonal antibody directed against the IL-2 receptor, specifically the alpha chain (CD 25), found on activated immune cells, including T-cells. It competitively inhibits IL-2 binding to CD 25, thereby downregulating the T-cell immune response. Response rates to daclizumab have ranged from 20 to 69% (Table 42-6) with the best responses noted for cutaneous GVHD. Interestingly, when daclizumab was added to corticosteroids as first line therapy for aGVHD, outcomes were worse than that of patients receiving steroids alone [17]. The higher mortality in the daclizumab

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Table 42-6.  Trials of daclizumab in GvHD-selected trials. Reference

Patients

Response/survival

Lee et al. [17]

N = 49 steroids +  placeboN = 53 steroids +  daclizumab

OR:53% (steroid + placebo)

Comments

Survival was worse in combination group due to higher rate 51% (steroid and daclizumab) of relapse and GVHD-related P = 0.85 mortality 100 day survival: 94% (steroid + placebo) 77% (steroid + daclizumab) P = 0.02

Anasetti et al. [97]

N = 19 SteroidCR = 20%PR = 20% refractory Skin GVHD = 56% response GVHDN = 1 primary treatment 100 day survival: 40%; median 76 days

Przepioka et al. [98]

N = 39 steroid CR = 37%PR = 14% refractoryN = 4 Skin GVHD = 54% response primary treatment 120 day survival: 40%; median 77 days

Bordigoni et al. [99]

N = 62 steroid refractory

CR = 68.8%PR = 21.3% Skin GVHD

Patients could receive ATG on study as a salvage regimen but were considered nonrespondersAll 4 patients receiving daclizumab for untreated visceral GVHD failed to respond 64% of study participants were under the age of 18

72.7% CR (stage I–II) 33.3% CR (stage III–IV) P = 0.018 Srinivasan et al. [100]

N = 12 steroid refractory

CR = 100%

Willenbacher et al. [101]

N = 16 steroid refractory

CR = 8%PR = 58%

Majority of subjects were transplanted for solid malignancy;Subjects could receive daclizumab alone (N = 6) or w/atg or infliximab

treated patients was due in part to a higher rate of death from disease and GVHD. As noted above, this study highlights the need to balance effective immunosuppression with GVL activity, infection risks, and other morbidities, and emphasizes the continued need for randomized trials for GVHD therapy. 2.3.2. Denileukin Diftitox (Ontak; Ligand Pharmaceutical Inc.) Denileukin diftitox is another therapy directed against CD25. It is a recombinant fusion protein consisting of an active portion of diphtheria toxin bound to human IL-2, and thus it selectively targets the IL-2 receptor. The toxin inhibits protein synthesis and induces T cell apoptosis once it has gained entry into the cell via the IL-2 receptor. Ho et al. [57] found a 33% (8/24 patients) CR rate and 38% (9/24 patients) PR rate at 29 days after the first denileukin diftitox treatment. Four patients who had a PR converted to a CR after day 29. However, survival data were still quite poor with a median survival of only 7.2 months in the 30 original study

Chapter 42  Treatment of Acute Graft-vs-Host Disease 

subjects. Encouragingly, responses were not limited to patients with grade I–II GVHD. Shaughnessey et al. [58] reported their experience with Ontak in 22 subjects and showed a similar 41% complete response rate on day 36 from initiation of Ontak, but again median survival in the cohort was only 121 days. In these two phase II trials, transaminitis was the dose limiting toxicity, as seen in studies of denilukin diftitox in lymphoma. 2.3.3. ABX-CBL/Anti CD147 This antibody was tested in patients with SR aGVHD with reasonable response rates leading to a phase III trial comparing ABX-CBL to ATG. Unfortunately, as seen repeatedly in other trials, there was no advantage in terms of response or survival using this novel antibody [59]. 2.4. TNF Inhibition 2.4.1. Infliximab (Remicade; Centocor) A number of recent trials have focused on targeting the cytokine cascade involved in the initiation and propagation of SR GVHD [60]. Tumor necrosis factor-alpha (TNFa) has been implicated as central to this process [61]. Infliximab is a humanized TNFa receptor that binds solubleTNFa, causing its neutralization, and the membrane-bound precursor of TNFa, causing cytotoxicity via the complement cascade and antibody-mediated cellular apoptosis. Couriel et al. [62] investigated the use of infliximab in the treatment of aGVHD and reported a 62% (13/21 patients) complete response rate on day 7, the most of which was in subjects who had gastrointestinal involvement. Of note, all of the surviving patients who had a complete response developed chronic GVHD. Over half of the patients in the original group of 21 patients had gastrointestinal GVHD and this may reflect the treatment bias of attending physicians, which is inherent in retrospective analysis. Patriarca et al. [63] conducted another retrospective study of infliximab in 32 patients with steroid refractory aGVHD and found only a 19% complete response rate on day 7. The low response rate in this study may have been impacted by the large percentage of patients (60%) in the group with grade IV GVHD (Table 42-7). Table 42-7.  Categories of agents used in GvHD therapy. Broad

Antibody

Signal transduction

Agent

Target

ATG

CD3

Thymogloblulin

CD3

Alemtuzumab

CD52

Mycophenolate mofetil Sirolimus

M-TOR

Deoxycoformycin Narrow

Cytokine inhibition

?

ECP

?Treg’s

Monoclonal antibody

Denileukin diftitox

IL2-R

Daclizumab

IL2-R

Visilizumab

TCR

Infliximab

TNF

Etanercept

TNF

Monoclonal antibody

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2.4.2. Etanercept (Enbrel; Amgen and Wyeth) In contrast to the neutralizing effect of Infliximab, etanercept is a recombinant human soluble tumor necrosis factor receptor fusion protein that inhibits TNF-a. Busca et al. [64] reported the outcomes of 21 patients with refractory GvHD (13 with SR aGVHD, eight with chronic GvHD) treated with etanercept, in addition to other salvage agents. Six of the 13 patients with aGVHD responded (4 CR, 2 PR) and five of the eight patients with chronic GvHD responded for an overall response rate of 52%. Interestingly, 55% of patients with gastrointestinal involvement responded. In a pre-emptive strategy, Uberti et  al. [18] reported a 75% CR rate in patients treated with up-front etanercept and prednisone in patients with new onset aGVHD. Although greater than 50% of patients demonstrated a response prior to the addition of etanercept and patients with grade IV disease were excluded (potentially exaggerating the response rate to etanercept), the results provide enthusiasm for ongoing efforts to modulate TNF function. 2.4.3. IL1-RA IL1 is another inflammatory cytokine implicated in the pathogenesis of GVHD [60]. Antin and colleagues [19] treated 17 patients with SR AGVHD with an IL-1 receptor antagonist (IL-1RA) in a phase I/II manner and found high levels of response; 63% of patients had improvement of GVHD by at least 1 grade, though this therapy has not yet been pursued further.

3. Related Questions and Future Directions 3.1. Non-Myeloablative Transplantation and GvHD The evolution of non-myeloablative transplantation in patients already at higher risk for the development of aGVHD on the basis of the increasing age of recipient and donor [65–67] has allowed a critical test of “proof of principle” for the established paradigm linking tissue damage and cytokine cascade triggered by myeloablative conditioning to the development of aGVHD [68, 69]. In theory, the low-intensity conditioning in NMT would be associated with a lower incidence and potentially different clinical presentation of acute and chronic GvHD than with classic myeloablative conditioning. Although there have been no large-scale, systematic studies to adequately address this issue, recently published outcomes from single centers comparing standard to non-myeloablative conditioning provide some intriguing insights. Despite the potentially confounding impact of traditional definitions and grading systems on more recently recognized syndromes of “late-onset aGVHD” and mixed manifestations of acute and chronic GvHD in the same patient, several retrospective studies [70–73] allow for some generalizations regarding the timing, severity, and response to therapy of aGVHD after NMT: (1) Initial observations that NMT may be associated with less AGvHD have to be tempered on the basis of studies with longer follow-up. Although the incidence of AGvHD may be lower in the early post-transplant period (<100 days), the development of “late onset AGvHD” (particularly when one takes into account the additional immunologic modifications often associated with NMT such as immunosuppressant withdrawal for relapse or mixed chimerism, and DLI) after NMT brings the overall incidence of AGvHD up to that seen

Chapter 42  Treatment of Acute Graft-vs-Host Disease 

after standard conditioning [74, 75]. (2) Although there are inadequate data to confirm whether the incidence of steroid refractoriness differs between standard transplants and NMT, there are no data to suggest that there is a difference in response to salvage therapy once patients are refractory to corticosteroids. (3) Although theoretically sound, organ-specific therapy has not yet been demonstrated to improve overall outcome for GvHD after NMT, highlighting the need for well-designed prospective trials in this area. 3.2. Future Directions As progress in understanding the basic cellular and molecular mechanisms of graft-vs-host and graft-vs-tumor reactions merges with the advances in cellular therapy and the evolution of monoclonal antibody technology, we face the ultimate challenge optimizing prophylaxis of GvHD without increasing relapse or infection, as well as improving induction therapy. Future efforts will certainly focus on the definition and optimization of cellular subsets that may enhance GvL and/or inhibit GvHD, such as regulatory T cells (Tregs) [76, 77] and mesenchymal stromal cells [78–84]. Previous reports evaluating quantitative assessment of Tregs in patients with and without acute and chronic GvHD have been confounded by the somewhat ambiguous definition of the cell subsets themselves. Depending on the flow cytometric and/or molecular assays (e.g., Foxp3) utilized in earlier studies, conventional CD4+ T-cells that become activated may mimic Tregs in cytometric analysis as CD25 expression is common to both phenotypes, thereby leading to variant conclusions regarding the direct [85] vs. inverse [86, 87] correlations of Treg number to GvHD. More recent studies have demonstrated that the absence of IL-7 receptor (ie., CD127-) on CD4+ CD25+ cells provides a better definition of the Treg subset [88], defining the majority of cells in the population which express Foxp3 and have Treg function [89]. Refinement of cell sorting strategies may allow the isolation of this rather rare subset for ex-vivo expansion to clinically relevant numbers in order to test whether the murine model of its immunomodulatory effect in suppressing GvHD without attenuating GvL can be demonstrated in humans. The unique non-immunogenic and immunosuppressive properties of mesenchymal stromal cells (MSCs) have propelled their entry into clinical trials in the prevention and treatment of GvHD, although the mechanism of their immunomodulatory effects remains unclear [90]. The EBMT has reported the largest series to date in which MSCs were infused in 40 patients with severe, refractory aGVHD (grade III–IV) at a median dose of 1.0 × 106/kg. In 65 total infusions, no infusion-related adverse events were noted. Considering the high-risk nature of this cohort of grade III–IV patients, the complete response rate of 48% and partial response rate of 23% appear quite promising and warrant continued study [90]. Greater understanding of pathophysiologic mechanisms of GVHD is also leading directly to new experimental therapies for GVHD. The vast majority of treatments are targeted to limit T cell number, activation, or function, but newer therapies will clearly need to target the non-T cell pathways that lead to GVHD. For instance, in murine models it is clear that residual host antigen presenting cells present after transplant are necessary for development of GVHD [1]. Newer therapies can therefore be designed to block the

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T cell-APC interaction early after SCT which may prevent initiation of GVHD [91]. Another exciting focus will be on the immunologic mechanisms involved in the specific inhibition of effector cell trafficking to target organs [92–94]. Ultimately, a targeted approach that will minimize the risks associated with AGVHD without limiting GVT activity should lead to broader applications and improved outcomes for patients undergoing allogeneic SCT. Acknowledgments.  This work was supported in part by grants from NIH (K24 CA11787901, DLP; R21 CA119538, SCG), and The Leukemia & Lymphoma Society (7000-02, DLP). The authors have no conflicts of interest to declare.

References 1. Shlomchik W, Couzens M, Tang C et  al (1999) Prevention of graft-versus-host disease by inactivation of host antigen-presenting cells. Science 285:412–415 2. Zhang Y, Joe G, Hexner E, Zhu J, Emerson SG (2005) Host-reactive CD8+ memory stem cells in graft-versus-host disease [see comment]. Nat Med 11(12): 1299–1305 3. Filipovich AH, Weisdorf D, Pavletic S et  al (2005) National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versushost disease: I. Diagnosis and staging working group report. Biol Blood Marrow Transplant 11(12):945–956 4. Glucksberg H, Storb R, Fefer A et al (1974) Clinical manifestations of graft-versushost disease in human recipients of HLA-matched sibling donors. Transplantation 18:295–304 5. Rowlings PA, Przepiorka D, Klein JP et al (1997) IBMTR Severity Index for grading acute graft-versus-host disease: Retrospective comparison with Glucksberg grade. Br J Haematol 97(4):855–864 6. MacMillan M, Weisdorf D, Wagner J et al (2002) Response of 443 patients to steroids as primary therapy for acute graft-versus-host disease: Comparison of grading systems. Biol Blood Marrow Transplant 8:387–394 7. Martin PJ, Schoch G, Fisher L et al (1990) A retrospective analysis of therapy for acute graft-versus-host disease: Initial treatment. Blood 76(8):1464–1472 8. Van Lint MT, Uderzo C, Locasciulli A et al (1998) Early treatment of acute graftversus-host disease with high- or low-dose 6-methylprednisolone: A multicenter randomized trial from the Italian Group for Bone Marrow Transplantation. Blood 92(7):2288–2293 9. Ruutu T, Hermans J, van Biezen A, Niederwieser D, Gratwohl A, Apperley J (1998) How should corticosteroids be used in the treatment of aGVHD? EBMT Chronic Leukemia Working Party. European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 22:614–615 10. McDonald GB, Bouvier M, Hockenbery DM et  al (1998) Oral beclomethasone dipropionate for treatment of intestinal graft-versus-host disease: A randomized, controlled trial [see comment]. Gastroenterology 115(1):28–35 11. Hings IM, Filipovich AH, Miller WJ et  al (1993) Prednisone therapy for acute graft-versus-host disease: Short- versus long-term treatment. A prospective randomized trial. Transplantation 56(3):577–580 12. Weisdorf D, Haake R, Blazar B et al (1990) Treatment of moderate/severe acute graft-versus-host disease after allogeneic bone marrow transplantation: An analysis of clinical risk features and outcome. Blood 75(4):1024–1030 13. Doney KC, Weiden PL, Storb R, Thomas ED (1981) Treatment of graft-versus-host disease in human allogeneic marrow graft recipients: A randomized trial comparing antithymocyte globulin and corticosteroids. Am J Hematol 11(1):1–8

Chapter 42  Treatment of Acute Graft-vs-Host Disease  14. Cragg L, Blazar BR, Defor T et al (2000) A randomized trial comparing prednisone with antithymocyte globulin/prednisone as an initial systemic therapy for moderately severe acute graft-versus-host disease. Biol Blood Marrow Transplant 6(4A):441–447 15. Martin PJ, Nelson BJ, Appelbaum FR et al (1996) Evaluation of a CD5-specific immunotoxin for treatment of acute graft-versus-host disease after allogeneic marrow transplantation. Blood 88(3):824–830 16. Cahn JY, Bordigoni P, Tiberghien P et al (1995) Treatment of acute graft-versushost disease with methylprednisolone and cyclosporine with or without an anti-interleukin-2 receptor monoclonal antibody. A multicenter phase III study. Transplantation 60(9):939–942 17. Lee SJ, Zahrieh D, Agura E et al (2004) Effect of up-front daclizumab when combined with steroids for the treatment of acute graft-versus-host disease: Results of a randomized trial. Blood 104(5):1559–1564 18. Uberti JP, Ayash L, Ratanatharathorn V et al (2005) Pilot trial on the use of etanercept and methylprednisolone as primary treatment for acute graft-versus-host disease. Biol Blood Marrow Transplant 11(9):680–687 19. Antin JH, Weinstein HJ, Guinan EC et al (1994) Recombinant human interleukin-1 receptor antagonist in the treatment of steroid-resistant graft-versus-host disease. Blood 84(4):1342–1348 20. Basara N, Blau WI, Romer E et al (1998) Mycophenolate mofetil for the treatment of acute and chronic GVHD in bone marrow transplant patients [see comment]. Bone Marrow Transplant 22(1):61–65 21. Kataoka I, Kami M, Takahashi S et al (2004) Clinical impact of graft-versus-host disease against leukemias not in remission at the time of allogeneic hematopoietic stem cell transplantation from related donors. The Japan Society for Hematopoietic Cell Transplantation Working Party. Bone Marrow Transplant 34(8):711–719 22. Ippoliti C, Champlin R, Bugazia N et al (1997) Use of octreotide in the symptomatic management of diarrhea induced by graft-versus-host disease in patients with hematologic malignancies. J Clin Oncol 15(11):3350–3354 23. Khoury H, Kashyap A, Adkins DR et al (2001) Treatment of steroid-resistant acute graft-versus-host disease with anti-thymocyte globulin. Bone Marrow Transplant 27(10):1059–1064 24. MacMillan ML, Weisdorf DJ, Davies SM et al (2002) Early antithymocyte globulin therapy improves survival in patients with steroid-resistant acute graft-versus-host disease. Biol Blood Marrow Transplant 8(1):40–46 25. Arai S, Margolis J, Zahurak M, Anders V, Vogelsang GB (2002) Poor outcome in steroid-refractory graft-versus-host disease with antithymocyte globulin treatment. Biol Blood Marrow Transplant 8(3):155–160 26. Remberger M, Aschan J, Barkholt L, Tollemar J, Ringden O (2001) Treatment of severe acute graft-versus-host disease with anti-thymocyte globulin. Clin Transplant 15(3):147–153 27. McCaul KG, Nevill TJ, Barnett MJ et al (2000) Treatment of steroid-resistant acute graft-versus-host disease with rabbit antithymocyte globulin. J Hematother Stem Cell Res 9(3):367–374 28. Duggan P, Booth K, Chaudhry A et  al (2002) Unrelated donor BMT recipients given pretransplant low-dose antithymocyte globulin have outcomes equivalent to matched sibling BMT: A matched pair analysis. Bone Marrow Transplant 30(10):681–686 29. Remberger M, Svahn BM, Mattsson J, Ringden O (2004) Dose study of thymoglobulin during conditioning for unrelated donor allogeneic stem-cell transplantation. Transplantation 78(1):122–127 30. Bacigalupo A, Lamparelli T, Barisione G et  al (2006) Thymoglobulin prevents chronic graft-versus-host disease, chronic lung dysfunction, and late transplantrelated mortality: Long-term follow-up of a randomized trial in patients undergoing unrelated donor transplantation. Biol Blood Marrow Transplant 12(5):560–565

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Chapter 42  Treatment of Acute Graft-vs-Host Disease  48. Krejci M, Doubek M, Buchler T, Brychtova Y, Vorlicek J, Mayer J (2005) Mycophenolate mofetil for the treatment of acute and chronic steroid-refractory graft-versus-host disease. Ann Hematol 84(10):681–685 49. Basara N, Kiehl MG, Blau W et al (2001) Mycophenolate Mofetil in the treatment of acute and chronic GVHD in hematopoietic stem cell transplant patients: Four years of experience. Transplant Proc 33(3):2121–2123 50. Takami A, Mochizuki K, Okumura H et al (2006) Mycophenolate mofetil is effective and well tolerated in the treatment of refractory acute and chronic graft-versushost disease. Int J Hematol 83(1):80–85 51. Bolanos-Meade J, Jacobsohn DA, Margolis J et  al (2005) Pentostatin in steroidrefractory acute graft-versus-host disease. J Clin Oncol 23(12):2661–2668 52. Benito AI, Furlong T, Martin PJ et  al (2001) Sirolimus (rapamycin) for the treatment of steroid-refractory acute graft-versus-host disease. Transplantation 72(12):1924–1929 53. Couriel DR, Hosing C, Saliba R et al (2006) Extracorporeal photochemotherapy for the treatment of steroid-resistant chronic GVHD. Blood 107(8):3074–3080 54. Couriel D, Hosing C, Saliba R et  al (2006) Extracorporeal photopheresis for acute and chronic graft-versus-host disease: Does it work? Biol Blood Marrow Transplant 12(1 Suppl 2):37–40 55. Peritt D (2006) Potential mechanisms of photopheresis in hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 12(1 Suppl 2):7–12 56. Greinix HT, Volc-Platzer B, Kalhs P et al (2000) Extracorporeal photochemotherapy in the treatment of severe steroid-refractory acute graft-versus-host disease: A pilot study. Blood 96(7):2426–2431 57. Ho VT, Zahrieh D, Hochberg E et  al (2004) Safety and efficacy of denileukin diftitox in patients with steroid-refractory acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Blood 104(4):1224–1226 58. Shaughnessy P, Bachier C, Grimley M, LeMaistre C (2003) Phase II study of denileukin diftitox (Ontak) in the treatement of steroid resistant acute graft versus host disease (AGVHD). Biol Blood Marrow Transplant 9(2):98 59. Macmillan ML, Couriel D, Weisdorf DJ et al (2007) A phase 2/3 multicenter randomized clinical trial of ABX-CBL versus ATG as secondary therapy for steroidresistant acute graft-versus-host disease. Blood 109(6):2657–2662 60. Ferrara J, Deeg H (1991) Graft-versus-host disease. New Engl J Med 324: 667–674 61. Piguet PF, Grau GE, Allet B, Vassalli P (1987) Tumor necrosis factor/cachectin is an effector of skin and gut lesions of the acute phase of graft-vs.-host disease. J Exp Med 166(5):1280–1289 62. Couriel D, Saliba R, Hicks K et al (2004) Tumor necrosis factor-alpha blockade for the treatment of aGVHD. Blood 104(3):649–654 63. Patriarca F, Sperotto A, Damiani D et al (2004) Infliximab treatment for steroidrefractory acute graft-versus-host disease. Haematologica 89(11):1352–1359 64. Busca A, Locatelli F, Marmont F, Ceretto C, Falda M (2007) Recombinant human soluble tumor necrosis factor receptor fusion protein as treatment for steroid refractory graft-versus-host disease following allogeneic hematopoietic stem cell transplantation. Am J Hematol 82(1):45–52 65. Kollman C, Howe CW, Anasetti C et al (2001) Donor characteristics as risk factors in recipients after transplantation of bone marrow from unrelated donors: The effect of donor age. Blood 98(7):2043–2051 66. Hagglund H, Bostrom L, Remberger M, Ljungman P, Nilsson B, Ringden O (1995) Risk factors for acute graft-versus-host disease in 291 consecutive HLA-identical bone marrow transplant recipients. Bone Marrow Transplant 16(6):747–753 67. Przepiorka D, Smith TL, Folloder J et  al (1999) Risk factors for acute graftversus-host disease after allogeneic blood stem cell transplantation. Blood 94(4): 1465–1470

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Chapter 42  Treatment of Acute Graft-vs-Host Disease  85. Stanzani M, Martins SL, Saliba RM et al (2004) CD25 expression on donor CD4+ or CD8+ T cells is associated with an increased risk for graft-versus-host disease after HLA-identical stem cell transplantation in humans [see comment]. Blood 103(3):1140–1146 86. Zorn E, Kim HT, Lee SJ et  al (2005) Reduced frequency of FOXP3+ CD4+ CD25+ regulatory T cells in patients with chronic graft-versus-host disease. Blood 106(8):2903–2911 87. Rezvani K, Mielke S, Ahmadzadeh M et al (2006) High donor FOXP3-positive regulatory T-cell (Treg) content is associated with a low risk of GVHD following HLA-matched allogeneic SCT. Blood 108(4):1291–1297 88. Seddiki N, Santner-Nanan B, Martinson J et al (2006) Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med 203(7):1693–1700 89. Negrin R, Hou JZ (2007) Promise and challenges of human regulatory T cells in the clinic. Biol Blood Marrow Transplant 13(Suppl 1):12–16 90. Horwitz E, Andreef M, Frassoni F (2007) Mesenchymal stromal cells. Biol Blood Marrow Transplant 13(Suppl 1):53–57 91. Reddy P, Maeda Y, Hotary K et al (2004) Histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect. Proc Natl Acad Sci U S A 101(11):3921–3926 92. Negrin RS, Contag CH (2006) In vivo imaging using bioluminescence: A tool for probing graft-versus-host disease. Nat Rev Immunol 6(6):484–490 93. Nguyen VH, Zeiser R, Dasilva DL et al (2007) In vivo dynamics of regulatory T-cell trafficking and survival predict effective strategies to control graft-versus-host disease following allogeneic transplantation. Blood 109(6): 2649–2656 94. Beilhack A, Schulz S, Baker J et al (2005) In vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of T-cell subsets. Blood 106(3):1113–1122 95. Przepiorka D, Weisdorf D, Martin P et  al (1995) Consensus conference on aGVHD grading. Bone Marrow Transplant 15:825–828 96. Graziani F, Van Lint MT, Dominietto A et al (2002) Treatment of acute graft versus host disease with low dose-alternate day anti-thymocyte globulin. Haematologica 87(9):973–978 97. Anasetti C, Hansen JA, Waldmann TA et  al (1994) Treatment of acute graftversus-host disease with humanized anti-Tac: An antibody that binds to the interleukin-2 receptor. Blood 84(4):1320–1327 98. Przepiorka D, Kernan NA, Ippoliti C et al (2000) Daclizumab, a humanized antiinterleukin-2 receptor alpha chain antibody, for treatment of acute graft-versushost disease. Blood 95(1):83–89 99. Bordigoni P, Dimicoli S, Clement L et al (2006) Daclizumab, an efficient treatment for steroid-refractory acute graft-versus-host disease. Br J Haematol 135(3): 382–385 100. Srinivasan R, Chakrabarti S, Walsh T et al (2004) Improved survival in steroidrefractory acute graft versus host disease after non-myeloablative allogeneic transplantation using a daclizumab-based strategy with comprehensive infection prophylaxis. Br J Haematol 124(6):777–786 101. Willenbacher W, Basara N, Blau IW, Fauser AA, Kiehl MG (2001) Treatment of steroid refractory acute and chronic graft-versus-host disease with daclizumab. Br J Haematol 112(3):820–823

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Chapter 43 The Importance of Non-Human Primate Models for Pre-clinical Studies in Hematopoiesis Erzsebet Szilagyi, Nadim Mahmud, and Amelia Bartholomew

1.  The Need for Non-Human Primate Models of Hematopoiesis Of all living experimental models, murine studies of hematopoiesis represent the greatest number. Such models can be undeniably elegant reflecting the extensive technological tools available. While mice can be used to study specific genetic pedigrees, specific physiologic, and gene pathways using knockouts, and a variety of immune responses using immunologically deficient mice, murine models are limited in their applicability for clinical use. Rodents differ from humans in many aspects including their short lifespan, difference in the rate of doubling of hematopoietic stem/progenitor cells, and the responses of blood cells to the hematological stresses of radiation and cytotoxic agents [1–5]. The efficacy of human specific reagents and their interaction with the human immune system cannot be tested kinetically in the mouse, as size and metabolism play a substantial role in providing substantially varied results. The ability to perform sequential analyses, such as weekly blood analyses for the phenotypic characterization of hematopoietic engraftment, gene marked cell tracking experiments, and sequential tissue biopsies can be limited in the mouse model because of size constraints. In the case of specific immunologic agents with human determinants, such as monoclonal or polyclonal antibodies or small molecules which specifically interact with human ligands or receptors, the specificity, dose, and efficacy may not always be extrapolated from that used in mice as it is highly likely that such agents are not cross-reactive with murine tissue. Immunologically, mice provide a different model of transplantation than humans and nonhuman primates, as mice lack expression of major histocompatibility complex (MHC) class II on their endothelium [6, 7]. This finding alone can explain why so many transplant-based regimens and outcomes that

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_43, © Springer Science + Business Media, LLC 2003, 2010

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are successful in mice are not similarly successful in humans or nonhuman primates. Advancement of such treatments to clinic may require a large animal model for FDA approval. Canines and pigs have been used successfully as large animal models of hematopoiesis; however, each model has its own limitations, particularly with agents which have limited cross-species reactivity. The pre-clinical large animal model which shares the greatest identity in physiological, immunological, biochemical, and genetic composition to humans is the nonhuman primate model (Table  43-1). Nonhuman primates like baboons resemble humans in rate of cell turn over and the development of their immune and hematopoietic system. In the absence of an appropriate human model, the nonhuman primate has proven useful as a tool for the preclinical evaluation of hematopoietic stem cell (HSC) transplantation protocols [8–13]. There are financial, time, and resource-related benefits in adopting a model which can be rapidly translated to clinical practice, through the ability to determine toxicity, efficacy, and feasibility for clinical development. It has been argued that nonhuman primate studies are prohibitively expensive to pursue; however, these authors would argue that results from early promising nonhuman primate studies can speed up development to clinic while early discouraging results in nonhuman primates can avoid the significant costs and time associated with pursuing an unfruitful phase I clinical trial. Once the commitment is made to perform studies in nonhuman primates, the enthusiasm, dedication, and knowledge of the principle investigator can initiate a significant amount of progress. However, the observation that only a handful of primate facilities worldwide sustain programs in HSC transplantation studies is primarily dependent on the support and experience of the veterinary staff. Veterinary staff, experienced in all levels of nonhuman primate care, can serve to facilitate the care of the animals, troubleshoot protocols, and work with both the principle investigator and the animal care committee, to strengthen and validate the program. In contrast, veterinary staff with less experience, i.e., caring for less than a few hundred primates daily, may not be as comfortable around animals that require intensive medical support. As a result, the lack of experience and/or knowledge may delay the successful launch of protocols, impeding the progress of the investigator. Veterinary support can be nurtured with education and experience; however, most principle investigators do not have the resources or the time to provide such training, and the successful launch of a nonhuman primate program in hematopoiesis can end in frustration resulting in a program that is unsustainable.

2.  Choice of Non-Human Primate Species and Specific Attributes Man, along with chimpanzees, gorilla, orangutan, and gibbons comprise the hominoid primates, (order Primata, infraorder Catarrhini, superfamily Hominoidea) [21–23]. Old World monkeys, the cercopithecoids (order Primata, infraorder Catarrhini, superfamily Cercopithecoidea), are the sister group of the hominoids. The cercopithecid genera are distributed throughout tropical Africa, Asia, as well as the colder climates of the Mediterranean and Japan, making this primate group the most widely geographically distributed

12–45 kg [14] 15–25 years [14]

Pigs (miniature swine)

Susceptibility to human chemotherapy/radiation Available reagents

14–29 kg

250 g

Marmosets

+++

Most distantly related ++ when compared to the common nonhuman primate models

+ modestly homologous, ++ moderately homologous, +++ highly homologous

12 years

25–30 years Nearly identical

+++

Baboons

31 years

Highly similar

2.5–8.3 kg

Cynomolgus macaque

Small size may limit sequential blood/bone marrow/tissue samples; siblings can be natural chimeric twins enabling transplant studies with paired responses

Most human reagents crossreact; necessity for larger cages and separate housing (differentfrom macaques) can reduce the desirability/ feasibility of this model

Most human reagents crossreact; smaller size promotes greater utility in studying preclinical pharmacology

Rhesus genome has been sequenced;Most human reagents crossreact. Larger macaque more physiologically robust following cytotoxic or radiotherapy

+++

25 years

5.3–10 kg

Rhesus macaque

+large amounts of subcutaneous fat can Limited numbers of reagents; Demonstrated confound radiation studies model for xenotransplantation [15]; Inbred swine exist with characterized major histocompatibility antigens (Swine leukocyte antigens, SLA) [16] Common model used for toxicology stud++some chemotherapy agents are not ies; Dog leukocyte antigens characterized crossreactive; size and metabolism (DLA);well-established model of bone marmay constrain predicitive capabilities row transplantation exists [18, 19]; differin human immune system, radiation ences in gut flora may affect post-transplant dose is dissimilar to achieve 30%, course [20]; 50%, or 70% lethal dose irradiation when compared to humans [17] Highly similar

+short lifespan,

Mouse genome has been sequenced, +some chemotherapy agents are not crossreactive; size and metabolism Limited ability to obtain sequential analyses significant difference may constrain predicitive capabilities (i.e., bone marrow, peripheral blood, tissue in doubling rate of in human immune system, radiation biopsies) in the same organism over time; hematopoietic stem dose is dissimilar to achieve 30, 50 transplantation not similar as mice lack cells and progenitors, or 70% lethal dose irradiation when MHC class II on endothelium as present in compared to humans humans and nonhuman primates

Similarity to human hematopoiesis

Canines (beagle)

24 months

20–40 g

Mice

Lifespan

Size

Animal model

Table 43-1.  Attributes of in vivo models of hematopoiesis.

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group resembling humans. Chimpanzees are the most closely related to man by genetic study [22]; however, because of large geographic availability and more frequent breeding habits of the cercopithecoids, nonhuman primate studies of hematopoiesis have generally been undertaken in baboon and macaque primates. 2.1. Baboons Common long tailed baboons (genus Papio, species hamadryas) have significant genetic similarity to humans evident at the level of overall DNA sequence, the sequences of specific genes, and the arrangement of genetic loci on chromosomes [24]. A notable difference is the genome size in humans (3,500  cM) which is significantly longer than in baboons and domesticated animals (3,000  cM) [25]. There are at least five different subspecies which can successfully interbreed: sacred baboons (P.h. hamadyas), yellow baboons (P.h. cynocephalus), chacma baboons (P.h. ursinus), red baboons (P.h. papio), and olive baboons (P.h. anubis) [26]. While many baboons in US primate centers contain P.h. anubis from East Africa [27], there is heterogeneity in the baboon population. Because there are genetic difference between these subspecies, lack of recognized heterogeneity within the baboon population, and differences in mean values between control and experimental groups may be attributed to sub-species variation. Careful documentation of visual assessment, pedigree information and/or capture site, and biochemical marker data can be used to define the sub-species appropriately [28]. Hematologic clinical parameters of baboons are nearly identical to humans, permitting ease in translational research [29, 30]. In terms of baboon size and maturation, mature papio anubis reach 24–29 kg (males), or 14.7–17 kg (females), and have a life span of 25–30 years [31, 32]. Their relatively short gestation period of 6 months can be useful for cord blood stem cell experiments. In comparison to macaques, the larger fetal size lends itself to in utero transplants which have been performed with success [33–37]. Recently, their use has been reduced because of the necessity of larger cage sizes which are more costly to purchase and maintain, the difficulty in transporting the animals to laboratories world-wide, and, to a lesser extent, the necessity for greater amounts of reagent use in adult animals when compared to macaques which are smaller. 2.2. Macaques Baboons and macaques are closely related as indicated by the fossil record, their identical karyotypes and gene linkages, and their ability to produce hybrids. There are at least 20 species of macaques (genus Macaca), but the two most commonly used in hematopoietic research are the long-tailed macaque (macaca fascicularis) and the rhesus macaque (macaca mulatta), although the stump-tailed, (m. arctoides), Japanese (m. fuscata), pigtail (m. nemestrina), and crested black macaques (m. nigra) have also been used but far less frequently. M. fascicularis, also known as M.cynomolgus or the crab-eating macaque, have a life span of approximately 31 years. Adults can weigh 4.7–8.3  kg (males), and 2.5–5.7  kg (females) [38, 39]. Their gestation period of 5.5 months is similar to baboons, but the fetal size is significantly smaller. M.

Chapter 43  The Importance of Non-Human Primate Models for Pre-clinical Studies 

Olive Baboon (Papio anubis) from the U.S. Fish and Wildlife Service’s online digital media library. Check http://images.fws.gov/ for higher quality version. Metadata Title: Olive Baboon Alternative Title: (none) Creator: Stolz, Gary M. Source: WO-5690031 Publisher: U.S. Fish and Wildlife Service Contributor: DIVISION OF PUBLIC AFFAIRS Language: EN – ENGLISH Rights: (public domain) Audience: (general) Subject: Kenya, animal Date Issued: December 03 2001.

mulatta, also referred to as the rhesus monkey, has a slightly shorter life span of 25 years, with the average weights of adult animals being slightly greater than that of their crab-eating cousins, 7.7 and 5.34  kg, respectively [40]. Rhesus macaques can be found in India and China and are the most studied nonhuman primate. The Rh blood factor, found in humans as well as monkeys, is named after the rhesus. Cynomolgus macaques are found in mainland Asia, and islands of Southeast Asia, Malau, Mauritius, Indonesia, and India. Several islands including Malau, Mauritius, and Tinjil, Indonesia have been established as cynomolgus breeding colonies to supply biomedical research. In both rhesus and cynomolgus monkeys, their country of origin is associated with genetic differences [41–43]. Genetic diversity within the species can contribute to variability in immunological responses, a particular concern in transplantation research. It is likely that intraspecies differences are not limited to immunological responses and may affect other experimental parameters. Determining mitochondrial DNA haplotypes is likely to become a useful method for genetically defining the origin of the species, thereby ensuring a greater relative homogeneity within experimental groups. The recent sequencing of the genome of the Indian-origin rhesus monkey led by the Baylor College of Medicine Human Genome Sequencing Center in collaboration with the J. Craig Venter Institute Joint Technology Center and the Genome Sequencing Center at Washington University, St. Louis will prove invaluable in understanding the strengths of this model for human hematopoiesis [44]. 2.3. Marmosets There has also been some interest in using marmosets in the study of hematopoiesis. These New World monkeys are less related to man, diverging at the phylogenetic infraorder, (order Primata, infraorder Platyrrhini, family Cebidae, subfamily Callitrichinae, genus Callithrix). Marmosets are exceptionally small primates with their adult size similar to a newborn baboon, approximately 250 g [45]. Marmosets are endemic to South America and have been observed to have a lifespan in the wild of approximately 12 years [45]. Callitrichid primates typically have multiple births after a 5 month gestation. The births are as a result of separate products of conception, however, unlike

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human dizygotic twins, the separate marmoset zygotes have intermingling placental circulations. A twin from an opposite sex will contain lymphocytes with both XX and XY karyotypes [46, 47]. Studies on peripheral blood lymphocytes revealed that nearly fifty percent of circulating lymphocytes were shared between each twin. No MHC Class II sharing was observed; however MHC class I gene products expressed by one member of a twin pair were observed to be nearly identical to those expressed by its co-twin [48]. The presence of genetic diversity with extensive polymorphism in the DNA of these animals did not support the notion that common MHC class I expression was due to inbreeding, but it is rather through the production of stable chimeras in utero from shared placental circulation. The ability to study chimeric twins can be advantageous in transplant studies [49]. They can be particularly useful in studies involving paired responses, thereby reducing the number of nonhuman primates necessary for an adequate sample size. Bone marrow functional parameters such as hematopoietic colony forming potential (CFU) and response to hematopolic stress (radiation, chemotherapy), as well as to human cytokines, growth factors, and genetic transfer appear to be similar to human hematopoietic cells enabling pre-clinical correlative studies [50–52]. Their small size can be an ideal feature which permits longterm administration of newly discovered agents and their long-term effects on hematopoiesis [53]. Despite these attractive features, marmosets are less studied than other nonhuman primates. The majority of studies undertaken in models of hematopoiesis have been performed in macaques and baboons.

3.  Hematopoietic Stem Cell Grafts and Hematopoietic Growth factors in Transplantation HSC grafts can be obtained by bone marrow harvests from posterior iliac crests and humerii or by aphaeresis of growth factor mobilized peripheral blood stem cell grafts from baboon or macaques. Both baboons and macaques effectively mimic the conditions and toxicities encountered during human stem cell transplantation. Because of the genetic similarities to human, many of the reagents and cytokines developed for use during human stem cell transplantation can be successfully employed for nonhuman primate HSC transplantation [8–13]. Antibodies conjugated with immunomagnetic bead cell selection systems can be used to prepare HSC grafts enriched for CD34+ cells (a surrogate marker for hematopoietic stem/progenitor cells) from bone marrow, growth factor mobilized peripheral blood, or umbilical cord blood. Growth factor mobilized peripheral blood harvests in baboons (>6 kg) can be performed by using apheresis machines used for collection of peripheral blood stem cell grafts in children, by administering granulocyte colony stimulating factor (rhG-CSF,100 mg/kg) [54–57] in combination with stem cell factor (SCF) as a mobilizing agent to stimulate bone marrow cells [6, 13]. For optimal peripheral blood stem cell collection, the animal is first treated with SCF alone at 25 mg/kg/day subcutaneously for 3 days beginning on day 0. On day 3, a dose of 100 mg/kg of G-CSF is combined with the 25 mg/kg/day dose of SCF on a daily basis. SCF alone resulted in minimal CD34+ cell mobilization; the addition of G-CSF to SCF led to a remarkable egress of CD34+ cells into the peripheral blood. On day 7, the mobilized peripheral blood stem cells can be collected by a single leukapheresis [12, 58]. The CD34+ cell contents

Chapter 43  The Importance of Non-Human Primate Models for Pre-clinical Studies 

of grafts isolated from seven baboons mobilized with G-CSF alone or G-CSF and SCF are shown in Table 43-2. These data indicate that there is some animal to animal variation but the combination of SCF and G-CSF is more effective in stem cell mobilization than G-CSF alone. A mobilization strategy using SCF is not useful in humans because of the severe adverse effects associated with its use. Human umbilical cord blood grafts have been established as an important alternative source of transplantable HSC grafts [14, 15]. Baboon cord blood grafts can be isolated following full term delivery by cesarean section. It is important to note that veterinary support is critical in this model for several reasons: Nonhuman primates deliver spontaneously at night and tend to consume their own placenta following delivery, thereby negating the opportunity of obtaining cord blood stem cells. Therefore, it is essential to preemptively schedule a cesarean section to deliver the live neonate and obtain the placenta and cord blood. Hematopoietic progenitor cells from the placenta and umbilical cord blood can be collected by using standard methods following aseptic techniques [16]. Several cord blood units have been collected from pregnant baboons at the University of Illinois using such techniques and have been utilized for experimental transplantation or other preclinical studies. The volume of cord blood collected following such deliveries ranged between 10 and 75 ml and the total nucleated cell number ranged between 1 and 20 × 107 cells. Low density mononuclear cells can be separated from baboon cord blood using similar techniques utilized to separate bone marrow cells from red blood cells, by incorporating density gradient separation methods. CD34+ cells can be enriched by immunomagnetic beads conjugated to monoclonal antibodies specific for CD34. These cells can be cryopreserved and thawed successfully for various experimental purposes including in vitro functional studies, such as transplantation of cryopreserved HSC grafts and reconstitution of all blood cell lineages. One of the major limitations of using such a nonhuman primate model is the retrieval and cost of maintaining the pregnant primate, and, if performing an autologous transplant, the cost of maintaining the offspring until they can undergo conditioning and transplantation (approximately 24 months). Achieving statistical power can be quite challenging, because of the

Table  43-2.  Growth factor mobilized peripheral blood stem cell product in baboon. Animal

Cytokine used

CD34 + Cells/kg 106

1

G-CSF

4.40

2

G-CSF

3.06

3

G-CSF

13.33

4

G-CSF, SCF

9.49

5

G-CSF, SCF

9.86

6

G-CSF, SCF

61.13

7

G-CSF, SCF

24.38

a

The calculations are based on an animal weighing 8 kg as a recipient G-CSF granulocyte colony stimulating factor, SCF stem cell factor CD34 cell number indicates product of single day leukapheresis

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costs involved. This model may be best suited to demonstrate safety of the proposed therapy rather than superiority of treatment.

4. Conditioning Regimens 4.1.  Radiation Based Regimens Radiation studies involving nonhuman primates have provided early and important insights on hematopoiesis, demonstrating hematopoietic deficiencies in response to radiation injury [4, 5]. This model is particularly useful for investigating in vivo kinetics of blood cells following radiation or chemotherapy. For example, exposure to sub-lethal (250  cGy) total body irradiation, in which endogenous recovery occurs, can be used to study the cellular and molecular responses involved in the kinetics of blood cell recovery and immune system in  vivo. A myeloablative conditioning regimen has been utilized by several investigators to perform allogeneic HSC transplantation in baboons using bone marrow or growth factor mobilized peripheral blood stem cells grafts [5, 59]. Successful protocols have been established to support animals during periods of prolonged pancytopenia (~60 days) [10]. Beginning 4 days prior to transplant, the animals are given two daily doses of 125 cGy total body irradiation from a linear accelerator over 4 days for a total dose of 1,000  cGy. The delivery of a fractionated dose of radiation has been shown to be lethal and myeloablative but results in relatively low nonhematologic toxicities [10]. The baboons that have received such myeloablative regimens have had tolerable gastrointestinal toxicities and almost no detectable pulmonary toxicity [8]. Support for these animals following the delivery of the conditioning regimen requires aggressive supportive care including prophylactic antimicrobial drugs, IV fluid and drug administration, blood component transfusions, and nutritional support via gavage. Conservative myeloablative conditioning regimens prior to clinical HSC transplantation promote hematopoietic cell engraftment and permit transplantation across limited histocompatability barriers (Table  43-3). In allogeneic transplants a combination of cyclophosphamide with busulfan or total body irradiation is commonly employed. The fully myeloablative approach is associated with higher risks of infectious complications when compared to novel nonmyeloablative regimens. Nonmyeloablative combined regimens are largely utilized in primate studies using combined approaches with lower doses of chemotherapy and radiation [60]. Major aims of these studies [60–66] are to induce stabile mixed chimerism for transplantation tolerance or gene therapy for hematologic or metabolic genetic disorders. One approach has been to include nonlethal total body irradiation (TBI), local thymic irradiation (TLI), and T-cell depletion with monoclonal antibodies like antithymocyte globulin (ATG) or antiCD2. Nonlethal low dose TBI ranging from 1.5 to 5 Gy is principally used in NHP HSC transplant studies [56, 60] with or without supplemental local thymic radiation with higher 7 Gy doses [67]. ATG 50 mg/kg has been generally employed for T-cell depletion [68], while adjunctive splenectomy has been used to further complete the reduction of donor-reactive lymphocyte population. Such regimens as reported by Kawai and others have led to stabile chimerism for a transient period of time, but this has been sufficient for the induction of tolerance to renal allografts

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775

Table 43-3.  Conditioning regimens in allogeneic transplantation. Conditioning regimen T-cell depletion/ suppression

Drugs

2 × 150 rad 700 rad

ATG

CsA

+ (multiline- 196, 198, 150, >40 Kawai [64, 67] age)

1.5 Gy

7 Gy

ATG, splenectomy,

CsA

+11/13

>3,478, >2,569, 834, 771, 405, 260, 198, 196, 137, 72, 44, 40, 37

Kawai [61]

1.5 Gy

7 Gy

ATG, anti-CD154

CsA

+8/8

>1,710, >1,167, 837i, 755, 401, 373, 206, 58

Kawai [61]

–

–

Thymoglobulin, fludarabine

Melphalan

8/8

180, 110, 100

Bartholomew [71]

–

–

Anti-IL2, betalecept Busulfan, + (ave 119 Not tested (CD28block), sirolimus days, max H106 196 days) (antiCD154)

TBI

TLI

Stabile chimerism

In vivo donor specific tolerance

[63, 69]. This approach has been successfully translated into clinical practice, using HLA mismatched donor and recipient pairs [70]. 4.2. Non-radiation Based Regimens In other studies, radiation can be entirely eliminated for the induction of mixed chimerism [66], with thymoglobulin, fludarabine, melphalan, or busulfan, the anti-IL2-receptor antibody basiliximab, blockade of CD28 signaling with the belatacept fusion protein, CD154 blockade with the H106 monoclonal antibody, and mTOR inhibitor sirolimus.

5. Models of Stem Cell Transplantation 5.1. Autologous Autologous HSC transplants can successfully engraft in rhesus [54, 56, 72], cynomolgus [73] and baboon [57] models and have been employed in the study of gene therapy, gene marking, mobilizing cytokines [74], and hematopoiesis. In addition to the mobilization strategy described above with recombinant human G-CSF [54–57], new strategies using SDF [54], myelopoetin [75], or other chemotactic factors [74] have also been studied. HSCs have been administered either intravenously or, more recently, directly intramarrow [57] HSC to deliver genetically modified grafts for transplant. 5.2. Autotransplantation for Studies in Gene Therapy HSCs are ideal targets for genetic manipulation in the treatment of several congenital and acquired disorders affecting the hematopoietic compartment. Large animals such as rhesus monkeys, baboons, cats, and dogs have similar

Reference

Kean [66]

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stem cell dynamics, cytokine responsiveness, and retroviral receptor properties as humans. Murine retroviruses based on the Moloney murine leukemia virus (Mo-MLV) were the first and still are the most widely used vectors in gene transfer studies [76]. They are one of the vector systems that have been used to transduce human hematopoietic stem or progenitor cells in a clinical trial. They stably integrate into the genome of the marked cell allowing the transduced cell to be tracked for its entire life span and for the lifespan of its progeny cells. Low level expression of the transgene in vivo, the potentially limiting level of specific cell surface receptors on certain target cells, their requirement for cell cycling, and safety issues including insertional mutagenesis from replication-competent retroviruses have resulted in an intense search for alternative viral and nonviral vectors. To overcome low level of cell surface receptors limiting transduction efficiency, pseudotyping with envelope proteins from different viruses like gibbon ape leukaemia virus (GALV) 10A1, or RD114, a feline endogenous retrovirus with higher receptor densities on target cells was developed [77, 78]. In the baboon model, direct comparisons between GALV and amphotropic vectors favor the GALV pseudotype to be studied [79]. Lentiviral vectors, based on disabled HIV-1 or HIV-2 genomes, are promising transducers without prolonged ex vivo stimulation of haematopoietic stem cells [80]. However, there is evidence that cytokine stimulation may be necessary for optimal transduction of haematopoietic cells using lentiviral vectors [81]. In the macaque, early generation vectors demonstrated low-level long-term in  vivo marking, even with cells cultured in vitro for short time periods without multiple stimulatory cytokines [82, 83]. In vivo gene transfer levels of 5–10% or higher have been achieved [84]. The use of G-CSF and stem cell factor (SCF)-mobilized peripheral blood (PB) CD34+ cells provides significantly higher in  vivo marking levels compared with that of G-CSF alone or that of G-CSF + Flt3-L-mobilized cells in the rhesus macaque competitive repopulation model [54]. Using the autologous HSC transplant model, several studies have illustrated the feasibility of tranduced HSCs to successfully and stably engraft [85–87]. To date, the efficiency of transduction may still be insufficient to attain therapeutic expression of gene products; however, the nonhuman primate model remains the best model to further refine this approach prior to moving to clinical trials. Using this preclinical model has additional benefits in testing the safety of this approach. Five years after receiving replication-defective retroviral transfected autograft a rhesus macaque developed a fatal myeloid sarcoma [88]. This observation highlights the significance of the nonhuman primate as a necessary preclinical stepping-stone. 5.3. Allogeneic Allogeneic HSCT in NHP models can reproduce the immunologic sequelae following fully mismatched allogeneic HSC transplant between nonhaploidentical individuals [71]. With access to breeding colonies, haploidentical transplants can also be performed either between parent and offspring or between siblings. HSCs derived from bone marrow and leukopheresis products both were found to be successful in inducing high-level hematopoietic mixed chimerism in a NHP model [66]. Stabile mixed chimerism is in the

Chapter 43  The Importance of Non-Human Primate Models for Pre-clinical Studies 

focus of organ transplant tolerance research as well [61]. Various promising conditioning regimens may provide mixed chimerism [61, 66] but its duration can be transient, and GVHD and infectious or malignancy complications due to immunosuppressive state still pose as fertile areas of study. 5.4. GVHD One of the most threatening complications of allogeneic bone marrow transplant is the graft-versus-host disease (GVHD). Experimental research on GVHD with laboratory animals has been performed with rodents, rhesus monkeys, and dogs. The basic immunological mechanisms operative in GVHD are largely similar in these three species and in human patients, although the patterns of GVHD in the three animal species show differences. The predictive value for clinical GVHD of the results obtained in the different animal species is different in histocompatibility, T cell numbers in the graft, and the intestinal microflora. Rhesus monkeys score highest as regards clinical relevance for the first two variables [89]. 5.5. MHC Typing In humans, apes, and Old World monkeys, the class II loci (DR) are highly conserved; however, there are species specific differences [90–94]. Like humans, chimpanzees, gorillas, and rhesus macaques have variable numbers of MHC-DRB loci per haplotype [95, 96]. Rhesus macaques (macaca mulatta, hence the MHC designation “Mamu” after the first two letters of macacca and mulatta, respectively), consangineously bred to achieve homozygosity at their MHC region and revealed that the number of Mamu-DRB loci per haplotype varies from two to six with up to three -DRB genes expressed [97]. Recently, Prasad et al. [98] identified also marmoset major MHC Class II DRB genes (Caja-DRB*W1201, Caja-DRB1*03, Caja-DRB*W16) using sequence-based typing techniques. They investigated whether matching at MHC Class II DRB loci alone could predict alloreactivity, as assessed in vitro by two-way mixed lymphocyte reactions. Fully mismatched and partially mismatched animal pairs exhibited significant in  vitro T-cell proliferation above single cell controls. Using DRB genotyping, suitable alloreactive donor-recipient pairs may therefore be rapidly and accurately identified for use in NHP studies of cellular and solid organ transplantation. Lymphohematopoietic chimerism in the primates was investigated in MHCmismatched allogeneic bone marrow transplantation (BMT) in the rhesus monkey detecting restriction fragment length polymorphism. A human MHC (HLA) class II DR beta gene cDNA probe was tested on rhesus peripheral blood mononuclear cell DNA digested with any of three restriction enzymes. The human DR beta probe hybridized to as many as 15 restriction fragments per rhesus DNA sample, suggesting cross-hybridization at more than one locus of rhesus MHC class II beta genes; restriction fragment length polymorphisms were common among outbred monkeys as a result of class II beta gene polymorphisms and would be sufficient for chimerism detection in the majority of random pairs of outbred monkeys utilizing only a single restriction enzyme. Sensitivity (5–10% chimerism) was comparable to that reported for restriction fragment length polymorphism assays utilizing nonMHC probes in

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clinical HLA-identical BMT. [99] Efforts are underway to provide MHC typed rhesus for haploidentical transplants using molecular characterization of the MHC (personal communication, Leslie Kean, Emory). These efforts will be used to direct breeding of rhesus monkeys to obtain specific haplo-identical pairs for further study. 5.6. Combined Stem Cell and Whole Organ Transplants Inducing whole organ tolerance through mixed chimerism after HSC transplant is a prominent area of investigation using NHP hematopoietic transplantation models. Kawai et  al. [60] developed a nonmyeloablative preparative regimen that can produce mixed chimerism and renal allograft tolerance between MHC-disparate nonhuman primates. The induction of transient mixed hematopoietic chimerism led also to long-term heart allograft survival in MHC disparate monkeys without chronic immunosuppression. However, unlike kidney allografts, full tolerance to cardiac allografts was not achieved. Moreover, chronic cellular and humoral immune responses were elicited by cardiac allografts [67]. Significantly, modifications of these early studies were used to define a successful clinical regimen which has recently demonstrated renal transplant tolerance [70]. In this regimen, the same investigators have observed human subjects off all immunosuppression for greater than 6 years (personal communication David Sachs, Harvard). 5.7. Fetal and In Utero Transplants BMT is a promising treatment to reconstitute defective hematopoietic cell lines in children with congenital defects but is limited by donor availability, graft rejection, and GVHD. These problems can be limited by transplanting normal preimmune fetal HSCs or adult HSCs into an unrelated preimmune fetal recipient. In clinical cases only fetuses with immunological defects were transplanted with adult HSC with limited outcomes [100]. Fetal transplantation addressing the composition and cell number of graft, and recipient age has been studied systematically in sheep models. The few NHP studies did provide some valuable insights that can be directly transferred to human clinical situations. Initial studies [33, 101] showed no chimerism after adult bone marrow transplant more than 0.44 (baboons) and 0.42 (cynomolgus) gestation time of the recipient. Zanjani et al. in their studies demonstrated first that the injections of allogeneic fetal stem cells into preimmune fetal monkeys result in long-term stable hematopoietic chimerism. HSCs harvested from the livers of preimmune fetal monkeys when injected into the peritoneal cavity of young unrelated fetal monkey recipients lead to stable, long-term postnatal multilineage hematopoietic chimerism. Donor cell engraftment was achieved without the use of cytoablative procedures and without the development of GVHD [102]. Shields et  al. investigated in baboon and macaques models the level of chimerism reached after in utero transplantation of purified, haploidentical CD34+ allogeneic bone marrow cells and the influence of T-cell number on engraftment [36, 103, 104]. Donor HSCs grafts containing different doses of donor T-cells were administered two or three times into the abdominal cavity of the fetuses between 0.38 and 0.42 gestation time. Chimerism was detected in cord blood or bone marrow cells in 87% of the recipients during the first month of life. Successful postnatal engraftment appeared to correlate with

Chapter 43  The Importance of Non-Human Primate Models for Pre-clinical Studies 

the total CD34+ cell dose as well as donor T-cells number. Interestingly, the amount of long term peripheral blood chimerism did not appear to be of sufficient levels to potentially induce therapeutic effect in most diseases; however this requires further study. 5.8. Xenotransplantation The use of xenotransplantation has been used in immunodeficient mouse models to study human hematopoiesis. Nonhuman primate models have also been used to study xenotransplantation, however this has been directed toward the study of organ transplants, typically between pigs and nonhuman primates. This topic has spanned several volumes in its own right and is not covered herein; however, it is important to note that such studies have investigated the ability to induce mixed chimerism through xenogeneic engraftment of porcine HSCs in nonhuman primates. The overarching hypothesis is on the basis of the premise that the induction of stable mixed chimerism will lead to the induction of immunologic tolerance. HSC xenotransplantation is challenged by problems of natural and elicited anti-pig antibodies, recipient platelet adhesion to pig hematopoietic progenitor cells resulting disseminated intravascular coagulation, and the rapid removal of pig HSC by the host macrophage-phagocytic system. Initial bone marrow xenotransplant studies of Sablinski et al. [105] investigated bone marrow transplants in the pig-to-cynomolgus monkey model. Similar to the conditioning regimen described for allotransplants, the recipient underwent pre-transplant splenectomy (day-6), total body irradiation in two fractions of 150  cGy (on days-6 and -5), and thymic irradiation (TI) of 700  cGy (on day-1). Preformed antibodies directed to Gal[alpha]1,3Gal (Gal) determinants were depleted by ex vivo adsorption through a pig liver (on day 0). Cyclosporine (15 mg/kg per day) was administered for 4 weeks, and 15-deoxyspergualin (6  mg/kg per day) for 2 weeks. In one of the two recipients, pig-specific interleukin-3 (IL-3, 10  mg/kg per day) and stem cell factor (SCF, 10 mg/kg per day, which is not pig-specific) were administered for 14 days. BM cells from MHC-defined miniature swine were transplanted into each recipient at doses of 2.3 and 2.5 × 108 cells/kg, respectively, on day 0. This regimen was not sufficient to induce engraftment, with porcine DNA only detectable by PCR on days 180 and 302 after BM Tx in one animal treated with porcine growth factors. In these and subsequent studies, porcine stem cells were rapidly removed from the circulation, suggesting macrophagephagocytic activity [106, 107] with some detectable DNA in animals treated with porcine IL-3 and SCF [108]. These studies highlight the difficulty of hematopoietic engraftment across disparate species and serve as an important base for greater investigation. Platelet Aggregation and anti Gal antibody production remain the major concerns in xenotransplant models. The infusion of pig HSCs alone or following the nonmyeloablative regimen led to a marked and sustained fall in platelet count and a rise in LDH with hemorrhagic events and even fatal outcome [109]. Alwayn et al. [110] demonstrated that porcine PBPC directly mediate aggregation of baboon platelets and that this process likely contributes to the thrombotic microangiopathy observed after porcine HSC transplant in the pig-to-baboon model. A combination of heparin and eptifibatide appeared to be the most beneficial in preventing a thrombotic disorder while maintaining

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adequate hemostatic responses [111]. Other studies showed that combination of eptifibatide-heparin-methylprednisolone could also prevent thrombotic events [109, 112, 113]. A significant biotechnical advance was the use of [alpha]1,3-galactosyltransferase gene-knock out pigs as donors of HSCs. Such grafts were infused (11 × 108 cells/kg) to three baboons [114]. Although the platelet count fell because of the conditioning regimen and following the infusion of the BM cells, the profound loss of platelets seen previously did not occur. Pig cell chimerism was detected by flow cytometry during the second posttransplant week, suggesting transient pig cell engraftment in baboon BM. With the elimination of this significant obstacle, attention is now directed toward engineering the bone marrow microenvironment by grafting pig spleen [115] with the HSC transplant. Such dramatic advances could never be accomplished nor insights gained in smaller animal models. Nonhuman primate models provide a critical stepping stone for sorting viable and nonviable directions for clinical development. 5.9.  The Hematopoietic Bone Marrow Microenvironment and Mesenchymal Stem Cells The bone marrow microenvironment plays a pivotal role in HSC engraftment. Osteoblasts of the endosteal niche and endothelial cells of the vascular niche can regulate stem cell quiescence and proliferation, respectively [116]. Mesenchymal stem cells (MSC), also active within the bone marrow microenvironment, give rise to osteoblasts, have been implicated in control of hematopoiesis, and are now being used therapeutically to improve hematopoietic engraftment [117–120]. MSC serve another function, providing important immunoregulatory signals that can control both adaptive and innate immune responses. The nonhuman primate model was pivotal in illustrating the powerful effect of a single infusion of MSC on the allogeneic immune response, revealing that such a treatment was capable of prolonging skin graft survival in baboons [12]. Extensions of these studies have been undertaken in humans, using MSC for the successful control of GVHD following BMT [121–123]. Additional pre-clinical studies undertaken in the nonhuman primate have included MSC tracking studies to demonstrate their ability to take up residence in a variety of tissues [11, 124] and gene transfection studies illustrating the feasibility of using such cells for gene therapy [125] and as vehicles in regeneration, promoting their administration to facilitate healing following radiation injury in clinical practice [126, 127].

References 1. Mahmud N, Devine SM, Weller KP et al (2001) The relative quiescence of hematopoietic stem cells in nonhuman primates. Blood 97:3061–3068 2. Schmidt M, Zickler P, Hoffmann G et al (2002) Polyclonal long-term repopulating stem cell clones in a primate model. Blood 100:2737–2743 3. Horn PA, Thomasson BM, Wood BL, Andrews RG, Morris JC, Kiem HP (2003) Distinct hematopoietic stem/progenitor cell populations are responsible for repopulating NOD/SCID mice compared with nonhuman primates. Blood 102: 4329–4335 4. Mauch P, Constine L, Greenberger J et al (1995) Hematopoietic stem cell compartment: Acute and late effects of radiation therapy and chemotherapy. Int J Radiat Oncol Biol Phys 31:1319–1339

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Chapter 43  The Importance of Non-Human Primate Models for Pre-clinical Studies  83. An DS, Wersto RP, Agricola BA et al (2000) Marking and gene expression by a lentivirus vector in transplanted human and nonhuman primate CD34(+) cells. J Virol 74:1286–1295 84. Kiem HP, Andrews RG, Morris J et al (1998) Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor. Blood 92:1878–1886 85. Van Beusechem VW, Bart-Baumeister JA, Bakx TA, Kaptein LC, Levinsky RJ, Valerio D (1994) Gene transfer into nonhuman primate CD34 + CD11b-bone marrow progenitor cells capable of repopulating lymphoid and myeloid lineages. Hum Gene Ther 5:295–305 86. Hanawa H, Hematti P, Keyvanfar K et al (2004) Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virusbased lentiviral vector system. Blood 103:4062–4069 87. Kiem HP, Sellers S, Thomasson B et al (2004) 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 9:389–395 88. Seggewiss R, Pittaluga S, Adler RL et al (2006) Acute myeloid leukemia is associated with retroviral gene transfer to hematopoietic progenitor cells in a rhesus macaque. Blood 107:3865–3867 89. van Bekkum DW (1994) Biology of acute and chronic graft-versus-host reactions: Predictive value of studies in experimental animals. Bone Marrow Transplant 14(Suppl 4):S51–S55 90. Gyllensten U, Sundvall M, Ezcurra I, Erlich HA (1991) Genetic diversity at class II DRB loci of the primate MHC. J Immunol 146:4368–4376 91. Otting N, Bontrop RE (1995) Evolution of the major histocompatibility complex DPA1 locus in primates. Hum Immunol 42:184–187 92. Thiel C, Bontrop RE, Lanchbury JS (1995) Structure and diversity of the T-cell receptor alpha chain in rhesus macaque and chimpanzee. Hum Immunol 43: 85–94 93. Heise ER, Cook DJ, Schepart BS et al (1987) The major histocompatibility complex of primates. Genetica 73:53–68 94. Otting N, de Vos-Rouweler AJ, Heijmans CM, de Groot NG, Doxiadis GG, Bontrop RE (2007) MHC class I A region diversity and polymorphism in macaque species. Immunogenetics 59:367–375 95. Kenter M, Otting N, de Weers M et al (1993) Mhc-DRB and -DQA1 nucleotide sequences of three lowland gorillas. Implications for the evolution of primate Mhc class II haplotypes. Hum Immunol 36:205–218 96. Schonbach C, Vincek V, Mayer WE, Golubic M, O’HUigin C, Klein J (1993) Multiplication of Mhc-DRB5 loci in the orangutan: Implications for the evolution of DRB haplotypes. Mamm Genome 4:159–170 97. Balner H (1980) The DR system of rhesus monkeys: A brief review of serology, genetics, and relevance to transplantation. Transplant Proc 12:502–508 98. Prasad S, Humphreys I, Kireta S et al (2007) The common marmoset as a novel preclinical transplant model: Identification of new MHC class II DRB alleles and prediction of in vitro alloreactivity. Tissue Antigens 69(Suppl 1):72–75 99. Moses RD, Beschorner WE, Singer D et  al (1989) Restriction fragment length polymorphism analysis with a cross-reactive HLA class II DR-beta gene probe for the detection of engraftment of MHC-mismatched marrow in the rhesus monkey. Bone Marrow Transplant 4:475–481 100. Flake AW, Roncarolo MG, Puck JM et  al (1996) Treatment of X-linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow. N Engl J Med 335:1806–1810 101. Brent L, Linch DC, Rodeck CH et al (1989) On the feasibility of inducing tolerance in man: A study in the cynomolgus monkey. Immunol Lett 21:55–61

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E. Szilagyi et al. 102. Zanjani ED, Mackintosh FR, Harrison MR (1991) Hematopoietic chimerism in sheep and nonhuman primates by in utero transplantation of fetal hematopoietic stem cells. Blood Cells 17:349–363 discussion 364–346 103. Shields LE, Gaur L, Delio P, Potter J, Sieverkropp A, Andrews RG (2004) Fetal immune suppression as adjunctive therapy for in utero hematopoietic stem cell transplantation in nonhuman primates. Stem Cells 22:759–769 104. Shields LE, Gaur L, Delio P et al (2005) The use of CD 34(+) mobilized peripheral blood as a donor cell source does not improve chimerism after in utero hematopoietic stem cell transplantation in non-human primates. J Med Primatol 34:201–208 105. Sablinski T, Emery DW, Monroy R et al (1999) Long-term discordant xenogeneic (porcine-to-primate) bone marrow engraftment in a monkey treated with porcinespecific growth factors. Transplantation 67:972–977 106. Kozlowski T, Ierino FL, Lambrigts D et al (1998) Depletion of anti-Gal(alpha)13Gal antibody in baboons by specific alpha-Gal immunoaffinity columns. Xenotransplantation 5:122–131 107. Kozlowski T, Monroy R, Xu Y et  al (1998) Anti-Gal(alpha)1-3Gal antibody response to porcine bone marrow in unmodified baboons and baboons conditioned for tolerance induction. Transplantation 66:176–182 108. Kozlowski T, Monroy R, Giovino M et al (1999) Effect of pig-specific cytokines on mobilization of hematopoietic progenitor cells in pigs and on pig bone marrow engraftment in baboons. Xenotransplantation 6:17–27 109. Buhler L, Awwad M, Treter S et al (2002) Pig hematopoietic cell chimerism in baboons conditioned with a nonmyeloablative regimen and CD154 blockade. Transplantation 73:12–22 110. Alwayn IP, Buhler L, Appel JZ III et al (2001) Mechanisms of thrombotic microangiopathy following xenogeneic hematopoietic progenitor cell transplantation. Transplantation 71:1601–1609 111. Alwayn IP, Appel JZ, Goepfert C, Buhler L, Cooper DK, Robson SC (2000) Inhibition of platelet aggregation in baboons: Therapeutic implications for xenotransplantation. Xenotransplantation 7:247–257 112. Appel JZ III, Alwayn IP, Correa LE, Cooper DK, Robson SC (2001) Modulation of platelet aggregation in baboons: Implications for mixed chimerism in xenotransplantation. II. The effects of cyclophosphamide on pig peripheral blood progenitor cell-induced aggregation. Transplantation 72:1306–1310 113. Appel JZ III, Alwayn IP, Buhler L, DeAngelis HA, Robson SC, Cooper DK (2001) Modulation of platelet aggregation in baboons: Implications for mixed chimerism in xenotransplantation. I. The roles of individual components of a transplantation conditioning regimen and of pig peripheral blood progenitor cells. Transplantation 72:1299–1305 114. Tseng YL, Dor FJ, Kuwaki K et al (2004) Bone marrow transplantation from alpha1, 3-galactosyltransferase gene-knockout pigs in baboons. Xenotransplantation 11:361–370 115. Dor FJ, Tseng YL, Kuwaki K, Ko DS, Cooper DK (2004) Pig spleen transplantation induces transient hematopoietic cell chimerism in baboons. Xenotransplantation 11:298–300 116. Shiozawa Y, Havens AM, Pienta KJ, Taichman RS (2008) The bone marrow niche: Habitat to hematopoietic and mesenchymal stem cells, and unwitting host to molecular parasites. Leukemia 22:941–950 117. Koc ON, Gerson SL, Cooper BW et  al (2000) Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 18:307 118. Angelopoulou M, Novelli E, Grove JE et al (2003) Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice. Exp Hematol 31:413–420

Chapter 43  The Importance of Non-Human Primate Models for Pre-clinical Studies  119. Fibbe WE, Noort WA (2003) Mesenchymal stem cells and hematopoietic stem cell transplantation. Ann NY Acad Sci 996:235–244 120. Le Blanc K, Samuelsson H, Gustafsson B et al (2007) Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells. Leukemia 21:1733–1738 121. Le Blanc K, Rasmusson I, Sundberg B et  al (2004) Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 363:1439–1441 122. Lazarus HM, Koc ON, Devine SM et  al (2005) Cotransplantation of HLAidentical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant 11:389–398 123. Le Blanc K, Frassoni F, Ball L et  al (2008) Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: A phase II study. Lancet 371:1579–1586 124. Devine SM, Cobbs C, Jennings M, Bartholomew A, Hoffman R (2003) Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates. Blood 101:2999–3001 125. Bartholomew A, Patil S, Mackay A et al (2001) Baboon mesenchymal stem cells can be genetically modified to secrete human erythropoietin in vivo. Hum Gene Ther 12:1527–1541 126. Chapel A, Bertho JM, Bensidhoum M et al (2003) Mesenchymal stem cells home to injured tissues when co-infused with hematopoietic cells to treat a radiationinduced multi-organ failure syndrome. J Gene Med 5:1028–1038 127. Lataillade JJ, Doucet C, Bey E et al (2007) New approach to radiation burn treatment by dosimetry-guided surgery combined with autologous mesenchymal stem cell therapy. Regen Med 2:785–794

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Chapter 44 In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation Lisbeth A. Welniak and William J. Murphy

1. Introduction Animal models have been vital to the development of allogeneic hematopoietic stem cell transplantation (AlloHSCT) as well as our understanding of its biology. Rodent models led the way in the demonstration of the whole body radiotherapy for effective anti-tumor responses [1] and the ability to rescue mice from high dose radiation with a transfusion of bone marrow cells [2, 3] in the early 1950s. However, the ability to promote prolonged tumor-free survival in mice using allogeneic bone marrow transplantation after myeloablative doses of radiation was offset by the recognition that allogeneic bone marrow transplant (BMT) could result in a lethal “secondary” disease of wasting, diarrhea and skin lesions [4] now known as graft-versus host disease. Interestingly, graft-versus-tumor (GVT) activity was also recognized in studies during this time period. [5, 6].

2. Preclinical Models for the Study of Allogeneic HSCT After the initial studies described above, much of the development of protocols for alloHSCT as well as the prevention and treatment of GVHD were performed in dogs [7]. Donnall Thomas, in collaboration with other investigators at Fred Hutchinson Cancer Center, pioneered the field of alloHSCT with his work in beagles [8]. Other animal models that have been used include the miniature swine model for work in allogeneic and xenogeneic hematopoietic cell and/or organ transplantation [9, 10] and the fetal sheep model for the study of human stem cells in a tolerant xenogeneic transplant [11]. Rodent models and in particular mouse models are the principle animal species used for the study of hematopoiesis and immunology. Mouse models remain an essential tool to understanding the biology of alloHSCT due to the availability of resources, the advantage of inbred strains,

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_44, © Springer Science + Business Media, LLC 2003, 2010

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the availability of genetically engineered strains and the ability of researchers to perform studies with sufficient numbers of animals for statistical power. Genetic and instrumentation technologies have evolved around the mouse model, which take advantage of the small body size of the mouse and aid in our understanding of alloHSCT. One of the most used technologies is in vivo bioluminescent-based imaging (BLI), which is based, on the observation that light can pass through tissues. Cells or mice of interest can be modified to express a bioluminescent marker such as the luciferase gene or green fluorescence protein (GFP). These cells can then be infused and tracked in unlabeled recipient mice using low light sensitive cameras. Trafficking and proliferation of labeled tumor cells, donor-derived bone marrow or specific cell populations such CD4+ and CD8+ T cells in mice have been examined using this technique and provide powerful real-time physical orientation and temporal information in engraftment, GVHD and GVT/GVL studies [12–14]. Other advantages of the technique are that it is non-invasive, provides quantitative information, and requires the use of far fewer numbers of animals than necropsy followed by analysis of multiple tissues per animal. Work in the mouse model is the foundation for our knowledge of the hematologic and the immunological mechanisms related to alloHSCT. However, there are many differences too, between man and mouse that can complicate the interpretation of findings. Some of the differences are obvious such as variation between species but other factors can be less apparent but can exert just equally important influences on the outcome and interpretation of results. The following are some of the important considerations that need to be taken into account when evaluating the results of animal studies. 2.1. Species Differences Evolution has conserved many aspects of the immune system in mammalian development where it has allowed for the development of functional similarities in molecularly disparate systems (i.e., Ly49 and KIR in mouse and human NK cells, respectively). However, other differences can result in discrepancies between pre-clinical mouse studies and clinical findings. The physical structure of lymphoid tissue in mouse and man are very similar but many differences that affect the innate as well as the adaptive immune systems exist between these two species and are detailed in depth in the 2004 review by Mestas and Hughes [15]. In addition, other physiological differences between the two species exist that can alter the action and or toxicity of drugs that are commonly used in alloHSCT. Finally, many biological targets are not sufficiently conserved between mouse and man. The use of species-specific biological reagents such as cytokines or antibodies can have unexpected differences in the specificity or strength of reactions. For example, the small variation between non-human primate and human CD28 may have been responsible for the differences in the disastrous outcome in the preclinical and clinical trial with agonist anti-CD28 [16]. 2.2. Conditioning Regimens Typically in mouse alloHSCT studies, unfractionated or split dose myeloablative irradiation is used to prepare the recipient but treatments can range from no conditioning for the adoptive transfer of allogeneic T cells in GVHD

Chapter 44  In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation 

studies to myeloablative doses of whole body irradiation followed by HSC rescue. Despite the increasing use of reduced intensity conditioning in clinical practice, most mouse studies have failed to reflect this change although some studies have investigated these therapies in mice [17, 18]. Part of the problem arises from the differences between species. Fludarabine is a common component in reduced intensity conditioning regimens. However, the metabolism and pharmokinetics of this drug differs in mice and man, due to differences in the activity of the enzyme that converts the prodrug into its active form [19], thus hindering its use in mouse models. Work in the mouse model has demonstrated the influence of conditioning on various outcome parameters after alloHSCT. Conditioning not only induces hematopoietic suppression, including lympho-depletion to facilitate engraftment of donor hematopoietic stem cells, but can also induce tissue damage and the translocation of lipopolysaccharide (LPS) from the intestine resulting in immune-stimulation and increased acute GVHD mortality and morbidity [20], as well as increased anti-tumor responses [21]. The intensity and composition of the conditioning regimen may also affect not only the incidence and intensity of GVHD but also the physiopathology as demonstrated in studies by Claman et al. [22] and studies by Xun and Widmer [23]. In addition, mouse models have shown that under lower intensity conditioning regimens, residual host T cells can provide a veto effect on the alloreactive donor T cells [24] that can result not only in reduced rates of GVHD [25] but also result in a loss of the beneficial activity of GVL [26]. These studies demonstrate that the use of different conditioning regimens can result in varied outcomes in the induction, intensity and clinical presentation of GVHD, GVL as well as affecting donor chimerism. In addition, species differences between mouse and man may alter the influence of various conditioning regimens on outcomes in alloHSCT. 2.3. Mouse Strains and Immunologic Disparity Several different combinations of major and/or minor histocompatible mismatches between donor and host are commonly transplanted in humans. However, the histocompatibility differences between mouse strains commonly used in experimental models do not always reflect the common clinical situations. This may be influenced in part by the maintenance of animals in specific-pathogen free housing. The availability of inbred strains, MHC congenic strains and semi-allogeneic (parent into F1) strains also allows for experimental designs to address or control for particular histocompatibility disparities between donor and host. Thus, lethal acute GVHD, which is mediated primarily by CD4+ cells, CD8+ cells or both, can be investigated using the appropriate combination of mouse strains [27]. Issues of host T cell mediated rejection of donor T cells can be eliminated by using parent into F1 models of alloHSCT. Selection of donor-host combinations is usually dependent on the amount of MHC and MiHA mismatched desired, the availability of reagents, histocompatible tumor cell lines, and the background genes of specialize mice such as transgenics and mice with targeted mutations. While genetically engineered mice are invaluable to the study of immunology, they do have limitations that are not always apparent, as the genetic manipulation may result in

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unexpected and/or subtle differences that may confound the interpretation of the experiment. Unexpected influences on neighboring genes, antigenicity of the inserted gene product in transplanted cells and developmental changes in these animals are all complications that have arisen in different experimental settings. Additionally, the time and expense of backcrossing genetically modified mice (gene knockout or transgenics) onto different genetic backgrounds can also limit the choice of strain combinations for alloHSCT. 2.4. Tissue Source and Cellular Composition of the Graft While peripheral blood and/or bone marrow or umbilical cord blood are the primary source of T cells in the grafts of human HSCTs, spleen cells and/ or lymph node cells are added to the bone marrow graft to provide sufficient numbers of T cells for the induction of GVHD in mice. The number of T cells required for GVHD is dependent on the frequency of allo-reactive precursors and therefore is dependent on the strain combination. Until very recently it was less clear as to the importance of expression of homing receptors on T cells taken from different tissues. CD62L, CCR7 and MAdCAM (a4b7 integrin) are differentially expressed on cells found in the blood or tissues such as the skin, liver and intestine and in lymphoid tissues. The homing receptors CD62L and CCR7 have been shown to play a critical role in the development of GVHD but expression of these receptors also partially define the maturation status of the T cell. CD62L and CCR7 are expressed on naïve T cells, which comprise the alloreactive T cell population in unsensitized donor mice [28, 29]. Forced expression of CD62L on mouse T memory cells has shown that it is not required for the induction of GVHD [30]. However, expression of the mucosal addressin molecule, MAdCAM, on donor T cells does appear to be important in liver and intestinal GVHD [31, 32]. In support of these findings, a study from Beilhack et al. [33] has shown that while homing of T cells to lymph nodes or spleen is required for the induction of GVHD, organ associated lymphoid tissue is not required. Thus, mesenteric lymph nodes and Peyer’s Patches are not required for the induction of intestinal GVHD. The caveat to these observations are discussed in Sect. 2.2, as it has been shown that in the absence of conditioning, intestine associated lymphoid tissue is critical to the development of GVHD [34, 35]. In addition to T cells, other cell populations in the graft such as NKT, NK and myeloid cells can affect GVHD and GVT. The role of NKT and NK cells in alloHSCT are described in greater detail in Sect. 4. 2.5. Endogenous Microflora and Opportunistic Pathogens Mice are maintained in specific-pathogen-free facilities which can alter the immune responses in marrow rejection [36] and GVHD. This practice can result in the use of larger numbers of T cells or greater amounts of conditioning than would be required if animals were maintained in conventional housing. The use of non-absorbable antibiotics can influence the outcome in both murine studies and clinical studies [37]. Gut flora is an especially important factor for the induction of intestinal lesions of acute GVHD in mice, since bacterial flora may up-regulate minor histocompatibility antigens in the recipient animals and result in the activation of toll-like receptors following translocation of microorganisms resulting in the induction of cytokine production and

Chapter 44  In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation 

increased immune responses including GVHD [20, 38, 39] and anti-tumor responses [21]. 2.6. Age and Sex of the Donors and Recipients The age of donor and recipient can be important variables for the immune reconstitution and for the development and severity of GVHD. However, most animal studies use young adults of a single sex to minimize the variables in the study and improve reproducibility. A few murine studies have examined the influence of age on GVHD development by using middle aged or old mice as either donors or recipients [40–43].

3. Immunobiology of Allogeneic HSCT Animal models have been instrumental in understanding the biology of alloHSCT. The roles of conditioning, immunodepletion, alloreactive and immunoregulatory cells on the various clinically relevant facets of a hematopoietic cell transplants as illustrated in Fig. 44-1 are described in the remainder of this chapter. 3.1. Graft Rejection Successful allogeneic transplants require that the immunocompetent host not reject the graft. Allogeneic HSC graft rejection can be mediated by NK cells [44, 45],

Fig. 44-1.  Sequelae of alloHSCT and factors influencing their development. Donor T cells recognition of host antigens are responsible for both GVHD and GVT while donor NK cells may provide GVT effects. Host NK and/or T cell recognition of the donor graft can result in graft rejection. Other parameters affecting a successful outcome after alloHSCT include immunodepletion or immunosuppression of the donor or host

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NKT cells [46] and/or T cells [47–50] that recognize histocompatibility determinants on the donor cells. The mechanisms of graft rejection have been extensively studied in animal models. In fact, NK cells were originally described based on their ability to reject bone marrow grafts in a MHC independent manner [44, 45]. To this day, the graft rejection model remains one of the few functional assays for NK activity in mice. Mouse models have also been used to demonstrate that increasing the intensity of host conditioning decreases the number of immunocompetent cells in the recipient to overcome rejection of T-cell depleted grafts [49]. It was also through the use of mouse models that the strategy of transplanting “megadoses” of T-depleted or CD34+ selected HSC partially matched (haploidentical) grafts from related donors was developed and shown that these large doses may tolerize the recipient to the engrafting cells [51, 52]. A great deal of our understanding of the effector pathways used by recipient T cells to reject hematopoietic grafts comes from work with mice. It has been shown that in naïve, unsensitized recipient mice, CD8+ T cells can mediate rejection through the use of the effector molecules, perforin, granzyme B and fas/fasL [36, 53, 54]. CD4+ T cells can also mediate MHC mismatched bone marrow destruction [55] and this activity is dependent on donor CD4+ T cell derived interferon-gamma [56]. Still to be determined are all of the effector pathways that can mediate rejection of bone marrow since CD8+ T cells in recipients pre-sensitized to alloantigen can reject the bone marrow by an unknown mechanism [57]. In addition, as in solid organ transplants, antibodies can induce rapid bone marrow graft rejection in presensitized recipients [58]. These animal studies have been used to demonstrate the types of cells and the effector mechanisms that are involved in graft rejection. 3.2. Immune Reconstitution A major hurdle limiting the efficacy of alloHSCT is prolonged immune suppression in patients due to factors including cytoreductive conditioning, the immunosuppressive drugs to prevent GVHD and the small proportion of transplanted T cells compared to size of the T cell compartment in an immunocompetent person. In addition, lymphoid hypoplasia, resulting from suppression of both thymic dependent and independent expansion of lymphocytes, is associated with acute GVHD [59–63]. These factors leave the patient susceptible to a number of opportunistic infections. Unfortunately, very few pre-clinical models that have been developed to study these opportunistic infections in the allogeneic transplant setting and the complicating effects of GVHD. Studies in rodent models have been employed to investigate therapies to enhance immune reconstitution in young and more importantly mature mice, as it has been demonstrated that conditioning protocols for alloHSCT can damage the cells that support lymphohematopoiesis [64–66]. A variety of experimental approaches have been examined in mice as a means to enhance immune reconstitution following alloHSCT. These approaches include the administration of keratinocyte growth factor (KGF) prior to transplant to protect the thymus resulting in increased immune reconstitution post-transplant [67–69], the administration of the T cell growth factor, interleukin-7, to increase thymopoiesis [70–72] although it also enhances peripheral T cell expansion [72–74], and sex steroid hormone blockade to remove negative regulators of thymopoiesis [75–77].

Chapter 44  In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation 

The mouse model has also allowed investigators the opportunity to explore the consequences of improved T cell reconstitution post-transplant. Responses to infectious diseases following alloHSCT and the promotion or aggravation of GVHD can be tested to examine the function of the immune system following these experimental manipulations. However, it has been shown that administration of growth factors after alloHSCT can promote peripheral expansion of T cells that may exacerbate GVHD [78–80]. 3.3. Acute GVHD Graft-versus-host disease (GVHD) is a major complication of alloHSCT. Shortly after the development of experimental bone marrow transplantation as a cure for radiation sickness, it was recognized that the animals developed a wasting syndrome with tissue destruction to the gut, liver and skin. Based on the work performed in animal models, Billingham put for a set of principles necessary for the development of GVHD [81]. These principles are (1) GVHD requires that the host must be incapable of adequately rejecting the graft; (2) the graft must contain immunocompetent cells and (3) there must be incompatibilities in transplantation antigens between the host and donor such that the host tissues express antigens not present on the donor cells [81]. In general these principles have been upheld through vigorous investigation into the biology of GVHD. However, it has been shown that GVHD can develop after syngeneic or autologous transplants due to a loss of tolerance in the reconstituted immune system [82]. Mouse models have been instrumental in understanding the role of effector mechanisms in acute GVHD. Evaluation of clinical trials has provided insight into the roles of cell populations and their products on alloHSCT outcome, however there is a limit to the definitive cause and effect that can be derived. Mouse models provide a means for in-depth evaluation which may lead to better and more specific targeting of therapeutic strategies in the clinic. For example, work in mouse models of GVHD have not only been able demonstrate the critical role of cytokines, Tumor Necrosis Factor-a (TNFa), and interferon-g (IFNg) in acute GVHD and GVT, but have led to the knowledge that only the production of these cytokines by specific T cell subpopulations are responsible for potentiating GVHD and GVT, respectively. Thus this provides a greater understanding of how TNFa blockade modulates GVHD disease [83–86] and why neutralization of IFNg [87, 88] can lead to deleterious effects in both GVHD and GVT. The commonly used mouse models for acute GVHD have been well characterized for the requirement of strain specific doses of irradiation for myeloablation, the extent of major and minor histocompatibility differences, the involvement of CD4+ and/or CD8+ T cells in pathophysiology, severity of disease and prevalence of organ-specific disease. A few of the most frequently utilized strain combinations for GVHD are listed in Table 44-1. 3.4. Chronic GVHD Mouse models of acute GVHD provide a wide range of clinically relevant BMT models that allow for the investigation of either CD4+ and/or CD8+ T cell mediated acute GVHD, as well as the selection of histocompatibility antigen mismatch. In addition, the spectrum of clinical features of acute GVHD

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Table 44-1.  Mouse models of GVHD. Donor

Host

BALB/c (H2d)

C57BL/6 (H2b)

b

d

Histocompatibility differences

Disease manifestations

Primary T cell dependence

MHC I, II, mHAs

Acute GVHD

CD4+

C57BL/6 (H2 )

BALB/c (H2 )

MHC I, II, mHAs

Acute GVHD

CD4+

B10.BR (H2d)

C57BL/6 (H2b)

MHC I, II

Acute GVHD

CD4+

b

b

C3H.SW (H2 )

C57BL/6 (H2 )

mHAs

Acute GVHD

CD8+

Bm1 (H2bm1)

C57BL/6 (H2b)

MHC I

Acute GVHD

CD8+

C57BL/6 (H2 )

MHC II

Acute GVHD

CD4+

BALB/c (H2d)

MHC I, II, mHAs

Bm12 (H2

bm12

FvB (H2q) d

)

b

d

CD4+ or CD8+

B10.D2 (H2 )

BALB/c (H2 )

mHAs

Chronic GVHD-scleroderma

CD4+

LP/J (H2b)

C57BL/6 (H2b)

mHAs

Chronic GVHD-scleroderma

CD4+

Chronic GVHD-SLE

CD4+

d

DBA (H2 )

(DBA x C57BL/6) MHC I, II, mHAs F1 (H2d/b)

MHC Major histocompatibility complex; mHA minor histocompatibility antigen; SLE systemic lupus erythematosus

in mice resembles the spectrum seen in humans. Unfortunately, the situation is not the same for chronic GVHD. The five most common murine models of chronic GVHD are all dependent on CD4+ T cells and only two models use conditioning and bone marrow transplant. Of these two models, the B10. D2 donor cells into BALB/c recipient, after sub lethal or lethal irradiation, is probably the most commonly utilized strain combination. Both the B10.D2 → BALB/c and the second transplant model, LP/J → C56BL/6, model the scleroderma features of chronic GVHD that is observed clinically in patients [89, 90]. 3.5. Graft Versus Tumor Graft versus Leukemia (GVL) or the broader term Graft versus Tumor (GVT) refers to the anti-tumor response that is associated with alloHSCT. Since the earliest rodent studies it has been recognized that GVL/GVT is associated with the occurrence GVHD [5, 6]. While the antigenic targets for GVL/GVT are not always clear in MHC-matched HSCT (it is speculated to be minor HAs and/or tumor associated or tumor-specific antigens), alloantigens in MHC-mismatched transplants may elicit potent anti-tumor responses. Mouse tumor models allow for study of GVL/GVT in both MHC-matched and MHCmismatched models. However, a caveat to all tumor studies in mice is the use of tumor cell lines that are immunogeneic to some degree in syngeneic strains, as many of these tumors were initiated by viruses, and because the hosts are not tolerized/tolerant to the tumor-associated antigens. In addition, many but not all of the tumor cell lines commonly used in GVL/GVT studies do not express MHC II and thus are not recognized by CD4+ T cells and not susceptible to CD4+ T cell mediated killing.

Chapter 44  In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation 

4.  Insights from Mouse Studies into the Role of Non-T Cell Lymphoid Populations on Recovery After Allogeneic HSCT Murine models have been instrumental in delineating the role of non-T cell lymphoid populations, present in both the recipients and in the donor graft, which can affect the outcome of graft rejection, induction of tolerance, GVHD and GVT after alloHSCT. 4.1. T Regulatory Cells CD4+ T regulatory cells are defined by the expression the IL-2 receptor alpha chain (CD25) and the restricted expression of the transcription factor, Foxp3. These cells have a potent immunoregulatory suppressor activity that has been shown to have profound effects in mouse models. Depletion of T regulatory cells in the mouse can be accomplished by the administration of the PC61, a monoclonal antibody that recognizes CD25. Reducing the number of CD25+ cells from the graft or in the recipient immediately after alloHSCT promotes GVHD in several mouse models [91–94]. Conversely, adding more CD4+CD25+ T cells to the graft can reduce GVHD in both acute and chronic murine models [92–94]. Interestingly, GVT does not appear to be diminished by the addition of Treg cells [95]. In addition to reducing GVHD, donor-derived Treg cells have been shown to increase engraftment and tolerance of MHC disparate allografts after sub lethal conditioning [96, 97]. Expression of CD62L is critical for the protective effects of infused donor-derived Treg cells, suggesting that homing of these cells to secondary lymphoid tissues and inhibition of alloreactive T cell priming is the mode of action of this cell population during GVHD [96, 98]. The adoptive cell transfer studies with Treg cells usually used ex vivo activated and expanded populations due to the low frequency of this population in mice, as well as man. This expansion protocol provides evidence of feasibility and potential efficacy in human trials but it is worth noting that freshly isolated Treg cells are also capable of inhibiting GVHD lethality [99] which demonstrates that passenger Tregs in the graft or host-derived Tregs may play an important function in suppressing GVHD, which could be manipulated to improve outcome. Immunosuppressive drugs given to prevent or control GVHD have also been shown to affect Treg cell expansion and function. In animal studies, cyclosporine A can lead to a reduction in donor Treg cell proliferation and function resulting in increased GVHD severity [100], while rapamycin can expand functional murine Treg cells in ex vivo culture [101]. The hematopoietic graft is not the only source of Treg cells. CD4+CD25+ Treg cells are less radiosensitive than conventional T cells and host-derived Treg cells are found at a higher frequency in recipient animals then other T cell populations [92]. The presence of this cell population in conditioned hosts can also be advantageous to the outcome of alloHSCT. Recipient-derived CD4+CD25+ Treg cells also can reduce acute and chronic GVHD in murine models [92, 102], inhibit NK cell-mediated BM graft rejection [103] as well as improve immune reconstitution and GVT [102].

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4.2. Gamma/Delta (gd) TCR T Cells gd TCR T cells comprise a small proportion of the T cell population (0.5–3% in the PB and spleen of mice). In experimental murine models, recipient gd T cells can reject allogeneic hematopoietic cell grafts [104, 105] while donortype gd TCR T cells can promote engraftment in mice [105, 106] and can promote GVHD [107]. Unlike recipient ab TCR T cells that may reduce GVHD by rejecting donor T cells, it has been proposed that recipient gd TCR T cells promote GVHD by activating stimulating host APCs even in heavily irradiated hosts [108] however, another investigator found no role for recipient gd TCR T cells in GVHD [92]. 4.3. Natural Killer Cells Unlike B and T cells, natural killer (NK) cells are primitive immune cells that provide one of the first lines of defense during an immune response. NK cells lack the T-cell markers CD3 and TCR and express NK specific markers (NK1.1 and DX5 in mouse, CD56 in humans). Although early mouse studies suggested host NK cells could reject donor BM in a non-MHC restricted manner [44, 45] it is now recognized that NK cells express inhibitory and activating receptors on their cell surface, that are directed to MHC and other cellular determinants on target cells that are critical for target identification and subsequent NK cell mediated killing (reviewed in [109]). Murine models have also been used to demonstrate that adoptive transfer of activated NK cells early after transplant inhibit GVHD and promote GVT [110] although, administration of activated NK cells later in the course of GVHD could exacerbate the disease [110]. Additional studies in animals are needed to determine how one can best exploit the potential benefit of NK cells in alloHSCT. 4.4. NKT Cells Natural Killer T (NKT) cells are characterized by the expression NK cell markers and a TCR. NKT cells can be stimulated through the TCR by recognition of glycolipid antigens and peptides presented in the non-classical MHC I molecule CD1d. Upon stimulation these cells can produce IFNg or IL-4 and can enhance or suppress responses in a wide range of immunological models. Murine studies have demonstrated that both donor and host NKT cells can attenuate GVHD and that this protection is dependent on the production of IL-4 [46, 111, 112] of invariant TCR type NKT or IFNg [113] by CD8+ NKT cells. While NKT cells suppressed GVHD in these experimental models, NKT cells can also provide anti-tumor activity to the graft [113, 114] and promote graft rejection by the recipient [46].

5. Concluding Remarks Animal models have been instrumental in the development of allogeneic as a medical therapy for the treatment of a number of hematologic, immunologic and oncology related disorders in humans. Animal models remain an important tool in pre-clinical and translational medicine and mouse models, in particular, continue to provide insight into the mechanisms underlying cellular therapy and greater understanding of the basic workings of the immune and hematopoietic systems.

Chapter 44  In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation 

Acknowledgments.  The work from the authors’ laboratories was supported by NIH R01 CA93527, R01 HL089905, R01 CA102282 and R01 AG022661.

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L.A. Welniak and W.J. Murphy 20. Hill GR, Crawford JM, Cooke KR, Brinson YS, Pan L, Ferrara JL (1997) Total body irradiation and acute graft-versus-host disease: the role of gastrointestinal damage and inflammatory cytokines. Blood 90(8):3204–3213 21. Paulos CM, Wrzesinski C, Kaiser A et al (2007) Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4 signaling. J Clin Invest 117(8):2197–2204 22. Claman HN, Jaffee BD, Huff JC, Clark RA (1985) Chronic graft-versus-host disease as a model for scleroderma. II. Mast cell depletion with deposition of immunoglobulins in the skin and fibrosis. Cell Immunol 94(1):73–84 23. Xun C, Thompson J, Jennings C, Brown S, Widmer M (1994) Effect of total body irradiation, busulfan-cyclophosphamide, or cyclophosphamide conditioning on inflammatory cytokine release and development of acute and chronic graftversus-host disease in H-2-incompatible transplanted SCID mice. Blood 83(8): 2360–2367 24. Weiss L, Nusair S, Reich S, Sidi H, Slavin S (1996) Induction of graft versus leukemia effects by cell-mediated lymphokine-activated immunotherapy after syngeneic bone marrow transplantation in murine B cell leukemia. Cancer Immunol Immunother 43(2):103–108 25. Sykes M, Szot GL, Swenson KA, Pearson DA (1997) Induction of high levels of allogeneic hematopoietic reconstitution and donor-specific tolerance without myelosuppressive conditioning. Nat Med 3(7):783–787 26. Truitt RL, Atasoylu AA (1991) Impact of pretransplant conditioning and donor T cells on chimerism, graft-versus-host disease, graft-versus-leukemia reactivity, and tolerance after bone marrow transplantation. Blood 77(11):2515–2523 27. Sprent J, Schaefer M, Gao EK, Korngold R (1988) Role of T cell subsets in lethal graft-versus-host disease (GVHD) directed to class I versus class II H-2 differences. I. L3T4+ cells can either augment or retard GVHD elicited by Lyt-2+ cells in class I different hosts. J Exp Med 167(2):556–569 28. Anderson BE, McNiff J, Yan J et al (2003) Memory CD4+ T cells do not induce graft-versus-host disease. J Clin Invest 112(1):101–108 29. Chen BJ, Cui X, Sempowski GD, Liu C, Chao NJ (2004) Transfer of allogeneic CD62L- memory T cells without graft-versus-host disease. Blood 103(4): 1534–1541 30. Anderson BE, Taylor PA, McNiff JM et al (2008) Effects of donor T cell trafficking and priming site on GVHD induction by naive and memory phenotype CD4 T cells. Blood 111(10):5242–5251 31. Dutt S, Ermann J, Tseng D et al (2005) L-selectin and beta7 integrin on donor CD4 T cells are required for the early migration to host mesenteric lymph nodes and acute colitis of graft-versus-host disease. Blood 106(12):4009–4015 32. Petrovic A, Alpdogan O, Willis LM et al (2004) LPAM (alpha 4 beta 7 integrin) is an important homing integrin on alloreactive T cells in the development of intestinal graft-versus-host disease. Blood 103(4):1542–1547 33. Beilhack A, Schulz S, Baker J et  al (2008) Prevention of acute graft-versushost disease by blocking T-cell entry to secondary lymphoid organs. Blood 111: 2919–2928 34. Murai M, Yoneyama H, Ezaki T et  al (2003) Peyer’s patch is the essential site in initiating murine acute and lethal graft-versus-host reaction. Nat Immunol 4(2):154–160 35. Welniak LA, Kuprash DV, Tumanov AV et al (2006) Peyer patches are not required for acute graft-versus-host disease after myeloablative conditioning and murine allogeneic bone marrow transplantation. Blood 107(1):410–412 36. Bennett M, Taylor PA, Austin M et  al (1998) Cytokine and cytotoxic pathways of NK cell rejection of class I-deficient bone marrow grafts: influence of mouse colony environment. Int Immunol 10(6):785–790

Chapter 44  In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation  37. Holler E, Rogler G, Brenmoehl J et  al (2006) Prognostic significance of NOD2/ CARD15 variants in HLA-identical sibling hematopoietic stem cell transplantation: effect on long-term outcome is confirmed in 2 independent cohorts and may be modulated by the type of gastrointestinal decontamination. Blood 107(10): 4189–4193 38. Hill GR, Teshima T, Gerbitz A et  al (1999) Differential roles of IL-1 and TNFalpha on graft-versus-host disease and graft versus leukemia. J Clin Invest 104(4):459–467 39. Cooke KR, Gerbitz A, Crawford JM et al (2001) LPS antagonism reduces graftversus-host disease and preserves graft-versus-leukemia activity after experimental bone marrow transplantation. J Clin Invest 107(12):1581–1589 40. Fitzgerald PA, Bennett M (1983) Aging of natural and acquired immunity of mice. I. Decreased natural killer cell function and hybrid resistance. Cancer Invest 1(1):15–24 41. Chen MG, Price GB, Makinodan T (1972) Incidence of delayed mortality (secondary disease) in allogeneic radiation chimeras receiving bone marrow from aged mice. J Immunol 108(5):1370–1378 42. Gorczynski RM, Kennedy M, MacRae S (1983) Alteration in lymphocyte recognition repertoire during aging. II. Changes in the expressed T-cell receptor repertoire in aged mice and the persistence of that change after transplantation to a new differentiative environment. Cell Immunol 75(2):226–241 43. Ordemann R, Hutchinson R, Friedman J et  al (2002) Enhanced allostimulatory activity of host antigen-presenting cells in old mice intensifies acute graft-versushost disease. J Clin Invest 109(9):1249–1256 44. Cudkowicz G, Bennett M (1971) Peculiar immunobiology of bone marrow allografts. II. Rejection of parental grafts by resistant F 1 hybrid mice. J Exp Med 134(6):1513–1528 45. Cudkowicz G, Bennett M (1971) Peculiar immunobiology of bone marrow allografts. I. Graft rejection by irradiated responder mice. J Exp Med 134(1):83–102 46. Haraguchi K, Takahashi T, Matsumoto A et al (2005) Host-residual invariant NK T cells attenuate graft-versus-host immunity. J Immunol 175(2):1320–1328 47. Blazar BR, Hirsch R, Gress RE, Carroll SF, Vallera DA (1991) In vivo administration of anti-CD3 monoclonal antibodies or immunotoxins in murine recipients of allogeneic T cell-depleted marrow for the promotion of engraftment. J Immunol 147(5):1492–1503 48. Slavin S, Reitz B, Bieber CP, Kaplan HS, Strober S (1978) Transplantation tolerance in adult rats using total lymphoid irradiation: permanent survival of skin, heart, and marrow allografts. J Exp Med 147(3):700–707 49. Soderling CC, Song CW, Blazar BR, Vallera DA (1985) A correlation between conditioning and engraftment in recipients of MHC-mismatched T cell-depleted murine bone marrow transplants. J Immunol 135(2):941–946 50. Trambley J, Bingaman AW, Lin A et al (1999) Asialo GM1(+) CD8(+) T cells play a critical role in costimulation blockade-resistant allograft rejection. J Clin Invest 104(12):1715–1722 51. Aversa F, Tabilio A, Terenzi A et  al (1994) Successful engraftment of T-celldepleted haploidentical 3-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 84(11): 3948–3955 52. Bachar-Lustig E, Rachamim N, Li HW, Lan F, Reisner Y (1995) Megadose of T cell-depleted bone marrow overcomes MHC barriers in sublethally irradiated mice. Nat Med 1(12):1268–1273 53. Martin PJ, Akatsuka Y, Hahne M, Sale G (1998) Involvement of donor T-cell cytotoxic effector mechanisms in preventing allogeneic marrow graft rejection. Blood 92(6):2177–2181

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L.A. Welniak and W.J. Murphy 54. Graubert TA, Russell JH, Ley TJ (1996) The role of granzyme B in murine models of acute graft-versus-host disease and graft rejection. Blood 87(4):1232–1237 55. Sprent J, Surh CD, Agus D, Hurd M, Sutton S, Heath WR (1994) Profound atrophy of the bone marrow reflecting major histocompatibility complex class II-restricted destruction of stem cells by CD4+ cells. J Exp Med 180(1):307–317 56. Welniak LA, Blazar BR, Anver MR, Wiltrout RH, Murphy WJ (2002) Opposing roles of interferon-gamma on CD4+ T cell-mediated graft-versus-host disease: effects of conditioning. Biol Blood Marrow Transplant 6:605–612 57. Komatsu M, Mammolenti M, Jones M, Jurecic R, Sayers TJ, Levy RB (2003) Antigen-primed CD8+ T cells can mediate resistance, preventing allogeneic marrow engraftment in the simultaneous absence of perforin-, CD95L-, TNFR1-, and TRAIL-dependent killing. Blood 101(10):3991–3999 58. Taylor PA, Ehrhardt MJ, Roforth MM et al (2006) Mechanisms responsible for and strategies to overcome bone marrow (BM) rejection in allosensitized recipients. Submitted 59. Dulude G, Roy DC, Perreault C (1999) The effect of graft-versus-host disease on T cell production and homeostasis. J Exp Med 189(8):1329–1342 60. Weinberg K, Blazar BR, Wagner JE et al (2001) Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation. Blood 97(5):1458–1466 61. van den Brink MR, Moore E, Ferrara JL, Burakoff SJ (2000) Graft-versus-hostdisease-associated thymic damage results in the appearance of T cell clones with anti-host reactivity. Transplantation 69(3):446–449 62. Gendelman M, Yassai M, Tivol E, Krueger A, Gorski J, Drobyski WR (2003) Selective elimination of alloreactive donor T cells attenuates graft-versus-host disease and enhances T-cell reconstitution. Biol Blood Marrow Transplant 9(12):742–752 63. Brochu S, Rioux-Masse B, Roy J, Roy DC, Perreault C (1999) Massive activationinduced cell death of alloreactive T cells with apoptosis of bystander postthymic T cells prevents immune reconstitution in mice with graft-versus-host disease. Blood 94(2):390–400 64. Chung B, Barbara-Burnham L, Barsky L, Weinberg K (2001) Radiosensitivity of thymic interleukin-7 production and thymopoiesis after bone marrow transplantation. Blood 98(5):1601–1606 65. Mackall CL, Fleisher TA, Brown MR et  al (1995) Age, thymopoiesis, and CD4+ T-lymphocyte regeneration after intensive chemotherapy. N Engl J Med 332(3):143–149 66. Mackall CL, Fleisher TA, Brown MR et al (1997) Distinctions between CD8+ and CD4+ T-cell regenerative pathways result in prolonged T-cell subset imbalance after intensive chemotherapy. Blood 89(10):3700–3707 67. Panoskaltsis-Mortari A, Taylor PA, Rubin JS et al (2000) Keratinocyte growth factor facilitates alloengraftment and ameliorates graft-versus-host disease in mice by a mechanism independent of repair of conditioning-induced tissue injury. Blood 96(13):4350–4356 68. Min D, Taylor PA, Panoskaltsis-Mortari A et  al (2002) 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 99(12):4592–4600 69. Rossi S, Blazar BR, Farrell CL et al (2002) Keratinocyte growth factor preserves normal thymopoiesis and thymic microenvironment during experimental graftversus-host disease. Blood 100(2):682–691 70. Alpdogan O, Schmaltz C, Muriglan SJ et al (2001) Administration of interleukin-7 after allogeneic bone marrow transplantation improves immune reconstitution without aggravating graft-versus-host disease. Blood 98(7):2256–2265 71. Bolotin E, Smogorzewska M, Smith S, Widmer M, Weinberg K (1996) Enhancement of thymopoiesis after bone marrow transplant by in  vivo interleukin-7. Blood 88(5):1887–1894

Chapter 44  In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation  72. Mackall CL, Fry TJ, Bare C, Morgan P, Galbraith A, Gress RE (2001) IL-7 increases both thymic-dependent and thymic-independent T-cell regeneration after bone marrow transplantation. Blood 97(5):1491–1497 73. Tan JT, Dudl E, LeRoy E et al (2001) IL-7 is critical for homeostatic proliferation and survival of naive T cells. Proc Natl Acad Sci USA 98(15):8732–8737 74. Alpdogan O, Muriglan SJ, Eng JM et  al (2003) IL-7 enhances peripheral T cell reconstitution after allogeneic hematopoietic stem cell transplantation. J Clin Invest 112(7):1095–1107 75. Greenstein BD, Fitzpatrick FT, Adcock IM, Kendall MD, Wheeler MJ (1986) Reappearance of the thymus in old rats after orchidectomy: inhibition of regeneration by testosterone. J Endocrinol 110(3):417–422 76. Windmill KF, Meade BJ, Lee VW (1993) Effect of prepubertal gonadectomy and sex steroid treatment on the growth and lymphocyte populations of the rat thymus. Reprod Fertil Dev 5(1):73–81 77. Sutherland JS, Goldberg GL, Hammett MV et  al (2005) Activation of thymic regeneration in mice and humans following androgen blockade. J Immunol 175(4):2741–2753 78. Chung B, Dudl E, Toyama A, Barsky L, Weinberg KI (2008) Importance of interleukin-7 in the development of experimental graft-versus-host disease. Biol Blood Marrow Transplant 14(1):16–27 79. Blaser BW, Roychowdhury S, Kim DJ et al (2005) Donor-derived IL-15 is critical for acute allogeneic graft-versus-host disease. Blood 105(2):894–901 80. Blaser BW, Schwind NR, Karol S et al (2006) Trans-presentation of donor-derived interleukin 15 is necessary for the rapid onset of acute graft versus host disease but not for graft versus tumor activity. Blood 108(7):2463–2469 81. Billingham RE (1966) The biology of graft-versus-host reactions. Harvey Lect 62:21–78 82. Hess A, Thoburn C (1997) Immunobiology and immunotherapeutic implications of syngeneic/autologous graft-versus-host disease. Immunol Rev 157:111–123 83. Korngold R, Marini JC, de Baca ME, Murphy GF, Giles-Komar J (2003) Role of tumor necrosis factor-alpha in graft-versus-host disease and graft-versus-leukemia responses. Biol Blood Marrow Transplant 9(5):292–303 84. Cooke KR, Hill GR, Gerbitz A et al (2000) Tumor necrosis factor-alpha neutralization reduces lung injury after experimental allogeneic bone marrow transplantation. Transplantation 70(2):272–279 85. Schmaltz C, Alpdogan O, Muriglan SJ et al (2003) Donor T cell-derived TNF is required for graft-versus-host disease and graft-versus-tumor activity after bone marrow transplantation. Blood 101(6):2440–2445 86. Levine J, Paczesny S, Mineishi S et al (2008) Etanercept plus methylprednisolone as initial therapy for acute graft-versus-host disease. Blood 111(4):2470–2475 87. Murphy WJ, Welniak LA, Taub DD et al (1998) Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice. J Clin Invest 102(9):1742–1748 88. Yang YG, Qi J, Wang MG, Sykes M (2002) Donor-derived interferon gamma separates graft-versus-leukemia effects and graft-versus-host disease induced by donor CD8 T cells. Blood 99(11):4207–4215 89. Jaffee BD, Claman HN (1983) Chronic graft-versus-host disease (GVHD) as a model for scleroderma. I. Description of model systems. Cell Immunol 77(1): 1–12 90. DeClerck Y, Draper V, Parkman R (1986) Clonal analysis of murine graft-vs-host disease. II. Leukokines that stimulate fibroblast proliferation and collagen synthesis in graft-vs. host disease. J Immunol 136(10):3549–3552 91. Martin PJ, Pei J, Gooley T et al (2004) Evaluation of a CD25-specific immunotoxin for prevention of graft-versus-host disease after unrelated marrow transplantation. Biol Blood Marrow Transplant 10(8):552–560

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L.A. Welniak and W.J. Murphy 92. Anderson BE, McNiff JM, Matte C, Athanasiadis I, Shlomchik WD, Shlomchik MJ (2004) Recipient CD4+ T cells that survive irradiation regulate chronic graftversus-host disease. Blood 104(5):1565–1573 93. Cohen JL, Trenado A, Vasey D, Klatzmann D, Salomon BL (2002) CD4(+) CD25(+) immunoregulatory T Cells: new therapeutics for graft-versus-host disease. J Exp Med 196(3):401–406 94. Hoffmann P, Ermann J, Edinger M, Fathman CG, Strober S (2002) Donor-type CD4(+)CD25(+) regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. J Exp Med 196(3):389–399 95. Edinger M, Hoffmann P, Ermann J et al (2003) CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nat Med 9(9):1144–1150 96. Taylor PA, Panoskaltsis-Mortari A, Swedin JM et al (2004) L-Selectin(hi) but not the L-selectin(lo) CD4+25+ T-regulatory cells are potent inhibitors of GVHD and BM graft rejection. Blood 104(12):3804–3812 97. Hanash AM, Levy RB (2005) Donor CD4+CD25+ T cells promote engraftment and tolerance following MHC-mismatched hematopoietic cell transplantation. Blood 105(4):1828–1836 98. Ermann J, Hoffmann P, Edinger M et  al (2005) Only the CD62L+ subpopulation of CD4+CD25+ regulatory T cells protects from lethal acute GVHD. Blood 105(5):2220–2226 99. Taylor PA, Noelle RJ, Blazar BR (2001) CD4(+)CD25(+) immune regulatory cells are required for induction of tolerance to alloantigen via costimulatory blockade. J Exp Med 193(11):1311–1318 100. Zeiser RS, Nguyen VH, Beilhack A et al (2006) Inhibition of CD4+CD25+ regulatory T cell function by calcineurin dependent interleukin-2 production. Blood 108(1):390–399 101. Battaglia M, Stabilini A, Roncarolo MG (2005) Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood 105(12):4743–4748 102. Trenado A, Charlotte F, Fisson S et al (2003) Recipient-type specific CD4+CD25+ regulatory T cells favor immune reconstitution and control graft-versus-host disease while maintaining graft-versus-leukemia. J Clin Invest 112(11):1688–1696 103. Barao I, Hanash AM, Hallett W et  al (2006) Suppression of natural killer cellmediated bone marrow cell rejection by CD4+CD25+ regulatory T cells. Proc Natl Acad Sci USA 103(14):5460–5465 104. Xu H, Exner BG, Cramer DE, Tanner MK, Mueller YM, Ildstad ST (2002) CD8(+), alphabeta-TCR(+), and gammadelta-TCR(+) cells in the recipient hematopoietic environment mediate resistance to engraftment of allogeneic donor bone marrow. J Immunol 168(4):1636–1643 105. Blazar BR, Taylor PA, Bluestone JA, Vallera DA (1996) Murine gamma/deltaexpressing T cells affect alloengraftment via the recognition of nonclassical major histocompatibility complex class Ib antigens. Blood 87(10):4463–4472 106. Drobyski WR, Majewski D (1997) Donor gamma delta T lymphocytes promote allogeneic engraftment across the major histocompatibility barrier in mice. Blood 89(3):1100–1109 107. Blazar BR, Taylor PA, Panoskaltsis-Mortari A, Barrett TA, Bluestone JA, Vallera DA (1996) Lethal murine graft-versus-host disease induced by donor gamma/ delta expressing T cells with specificity for host nonclassical major histocompatibility complex class Ib antigens. Blood 87(2):827–837 108. Maeda Y, Reddy P, Lowler KP, Liu C, Bishop DK, Ferrara JL (2005) Critical role of host gammadelta T cells in experimental acute graft-versus-host disease. Blood 106(2):749–755 109. Barao I, Murphy WJ (2003) The immunobiology of natural killer cells and bone marrow allograft rejection. Biol Blood Marrow Transplant 9(12):727–741

Chapter 44  In Vivo Models of Allogeneic Hematopoietic Stem Cell Transplantation  110. Asai O, Longo DL, Tian ZG et al (1998) Suppression of graft-versus-host disease and amplification of graft-versus-tumor effects by activated natural killer cells after allogeneic bone marrow transplantation. J Clin Invest 101(9):1835–1842 111. Zeng D, Lewis D, Dejbakhsh-Jones S et  al (1999) Bone marrow NK1.1(-) and NK1.1(+) T cells reciprocally regulate acute graft versus host disease. J Exp Med 189(7):1073–1081 112. Lan F, Zeng D, Higuchi M, Huie P, Higgins JP, Strober S (2001) Predominance of NK1.1+TCR alpha beta+ or DX5+TCR alpha beta+ T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: “natural suppressor” cells. J Immunol 167(4):2087–2096 113. Baker J, Verneris MR, Ito M, Shizuru JA, Negrin RS (2001) Expansion of cytolytic CD8(+) natural killer T cells with limited capacity for graft-versus-host disease induction due to interferon gamma production. Blood 97(10):2923–2931 114. Morris ES, MacDonald KP, Rowe V et al (2005) NKT cell-dependent leukemia eradication following stem cell mobilization with potent G-CSF analogs. J Clin Invest 115(11):3093–3103

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Chapter 45 Dendritic Cells Jacalyn Rosenblatt and David Avigan

1. Introduction Dendritic cells (DCs) represent a complex network of antigen-presenting cells that play a crucial role in the initiation of primary immunity, as well as maintaining the balance between immune tolerance and reactivity [1]. The modern field of DC biology was initiated in 1973 by Steinman and Cohn, who identified a subpopulation of murine splenocytes that had distinctive morphologic and phenotypic characteristics and powerfully stimulated T cell responses [2]. DCs have subsequently been described as the most potent antigen-presenting cells, which demonstrate the unique capacity to induce primary immune responses. Stimulation of naive T cells requires antigen presentation in the context of co-stimulatory and adhesion molecules, which serve as secondary signals needed for the activation of primary immunity. Antigen-presenting cells, such as B cells and macrophages, are effective in maintaining immune responses, but are incapable of initiating primary responses to novel antigens. In contrast, DC richly express MHC class I, II, co-stimulatory and adhesion molecules and are uniquely potent in initiating cellular immunity (Fig. 45-1) [3–6]. DCs also mediate humoral responses through the activation of helper T cells and direct effects on B cells [7]. DC activation of innate immunity has been demonstrated through their effects on NK cells and NKT cells [8, 9]. DCs have emerged as an area of intense interest in the fields of tumor immunotherapy and transplant biology. Although DCs represent only a small fraction of circulating mononuclear cells, large number of DCs can be generated from precursor populations derived from blood, marrow, and cord blood, allowing for the potential clinical use for immunotherapy [10–12]. A major focus of tumor immunotherapy has been the use of DCs to reverse tumor-induced anergy by the presentation of antigen in the context of co-stimulatory molecules [13]. Tumor cells evade host immunity through a variety of mechanisms including the presentation antigen in the absence of co-stimulation, secretion of inhibitory cytokines that suppress T cell function and DC maturation, and

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_45, © Springer Science + Business Media, LLC 2003, 2010

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Fig. 45-1.  Antigen presentation

the associated increase in inhibitory cells such as regulatory T cells. In contrast, DCs with a normal phenotype may be generated ex vivo from patients with malignancy. DCs, manipulated to express tumor antigens, have been shown to induce tumor-specific immunity in preclinical animal and human studies, and are now being studied in clinical trials [14]. DCs are also essential for maintaining tolerance toward self antigens and may mediate rejection or tolerance towards allogeneic tissue [15–18]. Distinct DC populations participate in the process of clonal deletion of autoreactive T cell clones and the establishment of peripheral tolerance by preventing expansion and activation of those clones that escaped central deletion in the thymus [19]. As such, DCs are thought to play a crucial role following allogeneic hematopoietic stem cell transplantation with the capacity to promote or inhibit graft versus host disease dependent on the phenotypic characteristics of the DC population [20]. Recent studies have sought to exploit this issue by manipulating DCs to minimize the risk of GVHD while maintaining the potent graft versus disease effect. Understanding the pattern DC reconstitution post-transplant and the balance between donor and recipient cells is crucial for this endeavor [21]. This review will focus on hematopoietic development of DC populations and the intimate link between the circumstances of DC development and the nature of its impact on cellular immunity. The use of ex vivo generated DC populations for tumor immunotherapy will be discussed including attempts to translate these finding into the clinical setting. The role of DCs in eliciting tolerance will be reviewed. Studies examining the role of DCs in allogeneic transplantation and the risk of graft versus host disease will be discussed.

Chapter 45  Dendritic Cells 

2. DC Subsets DC arise from precursor populations that differentiate along distinct pathways of maturation [22, 23]. In mice, CD11c+ DCs are found in the lymph nodes, spleen, and thymus and are subdivided by the expression of CD4 and CD8, the latter of which is expressed by thymic DCs responsible for deletion of autoreactive lymphocytes [24]. Other subsets include skin-derived DCs (Langerhans Cells) and tissue interstitial DCs which manifest an immature phenotype and migrate to draining lymph nodes upon activation. In humans, two primary pathways of DC development include the generation of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) [25]. mDCs are found at sites of antigen capture in the peripheral tissues, in the secondary lymph nodes at sites of T cell interaction, and in the circulation. In the skin, mDCs are differentiated into classic Langerhans cells (LCs) and interstitial DCs found in the dermis [26]. These two subsets express different cytokines and respond to unique chemokine signaling. Dermal DCs promote the differentiation of B cells into plasma cells, stimulate CD4 mediated help of immunoglobulin class switching, and migrate to the area of the LN adjacent to the B cell follicles. In contrast, LCs are far more potent in inducing primary CTL responses and migrate to T cell areas of the draining lymph node. Blood-derived mDCs are characterized by DR+/lineage-/CD11c+ expression. These cells are likely a reservoir for tissuebased DCs and are often referred to as DC1 because they characteristically induce expression of TH1 cytokines by reactive T cell populations. pDCs are characterized by DR+/lineage-/CD123+ expression, further divided into CD2+/ and CD2− subsets and are potent secretors of type I IFN in response to viral pathogens [27, 28]. In contrast to mDCs, pDCs do not prominently express IL-12 and may polarize T cells towards a TH2 phenotype. However, depending on the nature of the milieu present during their development and T cell interaction, these subsets may be associated with the expansion of activated or suppressor T cells [20, 29, 30].

3. Phenotypic Characterization of Immature DCs DCs pass through a complex life cycle, in which their phenotypic characteristics evolve with maturation (Fig. 45-2) [1, 3, 23, 31, 32]. DCs originate from marrow progenitors and subsequently migrate to sites of exposure to foreign antigens [31]. In murine studies, DC’s have been shown to differentiate from myeloid and lymphoid precursor populations [33]. Lymphoid-derived DCs express CD8a and share a common precursor with T cells, B cells and natural killer (NK) cells [34, 35]. Myeloid DC progenitor are defined by the absence of CD8a, expression and exquisite sensitivity to granulocyte–macrophage colony stimulating factor (GM-CSF). In humans, DCs differentiate from marrow-derived CD34+ precursors and migrate to the sites of antigen uptake [10, 20, 36]. DCs are found in the epithelial surface of the skin, gastrointestinal tract, and lung, as well as the interstium of all organs with the exception of the immunoprivileged sites of the brain, testis, and eye [37]. The precise mechanisms that are responsible for the recruitment and localization of DCs in tissue has not been fully elucidated, but appears to be related to intrinsic features of the progenitor cells, as well as the release of cytokine and inflammatory signals [38]. For example, the presence

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Fig. 45-2.  Dendritic cell life cycle

of a “skin-homing receptor” on a subset of CD34+ cells is associated with their subsequent migration to the epidermis and their acquisition of phenotype of a Langerhans cell (LC) [39]. Expression of E-cadherin by LCs facilitates the binding of these cells into the epidermal layer. The migration of DCs into the brochoepithelium is induced by the presence of aerosolized lipopolysaccharide (LPS), soluble protein, bacterial or viral products, or the release of GM-CSF secondary to local inflammation or by pulmonary tumors. In a murine model, administration of FLt3L resulted in the accumulation of immature DCs in the bone marrow, gastrointestinal lymphoid tissue, peripheral blood, peritoneal cavity, liver, lymph nodes, lung, spleen, thymus, and dermis which strongly express class II, CD 11c, DEC205, and CD86 [40–42]. LCs represent a well-characterized immature DC population found in the skin, which express CD1a, Lag antigen, E-cadherin, and contains cytoplasmic inclusion bodies known as Birbeck granules (Fig. 45-3) [3, 31]. DCs residing in other tissues do not share all of these morphologic characteristics, but demonstrate similar properties with regard to antigen processing and presentation. The morphology of immature DCs is characterized by a highly organized cytoskelatin, slow motility, and the absence of prominent dendrites. They express low levels of co-stimulatory molecules and are poor stimulators of allogeneic T cell proliferation. Immature DCs demonstrate potent capacity to internalize exogenous antigens [38, 43]. Studies of freshly isolated LCs and bone marrow-derived immature DCs demonstrate the ability to internalize protein latex microspheres, bacille Calmette-Guerin (BCG), colloidal gold, apoptotic and necrotic cell fragments, heat shock proteins, viral and bacterial products, as well as whole bacteria. Phagocytic properties of DCs are distinct from that seen with macrophages [3, 38, 43, 44]. Macrophages are responsible for the scavenging and clearance of foreign material, which are transferred to cytoplasmic lysosomal compartments for degradation. DCs are more selective, demonstrating the uptake of smaller quantities of antigen, which are incorporated into MHC class Il compartments for subsequent antigen presentation. DC-mediated antigen uptake

Chapter 45  Dendritic Cells 

Fig. 45-3.  Phenotypic properties of immature and mature dendritic cells

Fig. 45-4.  Functional characteristics of immature and mature dendritic cells

occurs via both receptor-mediated endocytosis as well as macropinocytosis. Immature DCs express Fc receptors, complement, and mannose receptors thought to mediate internalization of exogenous proteins [45, 46]. In contrast to macrophages, DCs express the avB5 integrin, which is crucial for the uptake of apoptotic bodies and the subsequent presentation of antigen along the class l pathway [47]. An essential component facilitating endocytosis is the presence of DEC205, a receptor homologous to the macrophage mannose receptor [45, 48]. Antigen-bound DEC 205 is transferred via coated pits into endosomal compartments for subsequent processing and presentation in the context of MHC class II molecules. Endocytosis mediated by this pathway has been demonstrated to be 100-fold more effective than bulk macropinocytosis [49]. Mannosylyation of the ingested antigen markedly increases in immunogenicity with an increase in the levels of T cell responsiveness by 200- to 10,000fold (Fig. 45-4).

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4. Phenotypic Characteristics of Mature DC Upon maturation, DCs migrate as veiled cells via the afferent lymphatics to regional lymph nodes and designated areas of the spleen, which serve as the site of antigen recognition and T cell activation [1, 3, 31]. During this process, DCs undergo fundamental changes in their phenotypic and functional characteristics. DCs do not recirculate in the efferent lymphatics, and it is thought that those cells that do not present antigen undergo T cell-mediated apoptosis. A variety of factors have been demonstrated to induce the maturation and migration of DCs. Receptors for GM-CSF are prominently expressed by immature DCs and GM-CSF has been shown to support the differentiation, viability, and long-term survival of DC [6, 12, 36, 50–53]. Innate immunity against infectious pathogens are mediated through signaling pathways associated with the toll-like receptors (TLRs) and other receptor families, and is intimately involved in the maturation and development of distinct DC subsets [54]. Response to microbial products, such as lipids (TLR 1, 2, 4, and 6) and nucleic acids (TLR 3, 7, 8, and 9), are mediated by individual TLR receptors. For example, exposure to oligodeoxynucleotides containing CpG motifs signals via TLR9 and augments DCs maturation as manifested by a transient increase in antigen processing followed by loss of capacity to internalize and process exogenous protein antigens [55]. These signaling pathways may act synergistically to activate DCs and promote maturation and the stimulation of inflammatory cytokines such as IL-12 [56]. Myeloid DCs express TLR 1-6 and 8 while plasmacytoid DCs express TLR 7 and 9 [54]. Cell populations of the innate system secrete cytokines that dictate the nature of DC polarization and maturation [22]. For example, IFNg expression by NK cells or plasmacytoid DCs support DC activation, IL-12 production, and the stimulation of TH1 responses. Similarly, mature DC activate innate immune cells such as NKT cells to express IFNg. In this manner, the innate and adaptive immune system have developed a complex interaction that modulate host responses [20]. Material from dying cells has also been shown to induce DC maturation either directly or by stimulation of other accessory cells such as macrophages. Heat shock proteins (HSP), high mobility group box 1 (HMGB1), b-defensin, and uric acid derivatives have been shown to activate DCs via TLR mediated signaling and other pathways [57–59]. Maturation signals also impact properties of cell adhesion promoting the migration of maturing DCs. For example, loss of E-cadherin by LCs is associated with their capacity to migrate from skin epithelium and travel toward lymphocyte-rich areas [60, 61]. Maturing DCs downregulate CCR6 and lose sensitivity towardmacrophage-inhibiting protein (MIP)-3 [3, 62, 63]. Conversely, maturing DCs upregulate expression of CCR7 and demonstrate increased sensitivity towards chemokines MIP-3a and 6Ckine [64]. Expression of these chemokines is found in lymphatic vessels and the T cell-rich paracortical areas of the draining lymph nodes and mediates migration of DCs through the afferent lymphatics [53, 65]. Mature DCs release MIP-3a and 6Ckine, further amplifying the effect, as well as attracting naive T cells to the site of antigen presentation [66]. Absence of MIP-3a and 6Ckine expression is associated with deficient homing of DCs and T cells to lymphatic issue [67, 68]. Adenoviral transfection of tumor cells with MIP-3o resulted in the migration of DCs into the tumor bed and inhibition of tumor growth [69].

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With the onset of maturation, there is a transient increase in the production of cytoplasmic class II molecules and antigen loaded during this period is particularly immunogenic [70]. Terminal maturation associated with a decrease in the synthesis of MHC molecules, and MHC class II are thrust onto the cell surface resulting in stable presentation of the incorporated antigen. Localization of co-stimulatory molecules and peptide–MHC complexes in cytoplasmic compartments is subsequently translated to clustering of these molecules on the membrane surface for antigen presentation. In contrast, exposure to IL-10 inhibits the translocation of antigen expressing class II molecules onto the plasma membrane [71]. Mature DCs are distinguished by the prominent expression of MHC class I, II, adhesion and co-stimulatory molecules [4, 5, 7, 72, 73]. Ligation of corresponding molecules on T cells, most notably CD28, provides the essential secondary signals for the initiation of primary immune responses. Interference with this crucial dialog through antibody blockade abrogates DC-mediated T cell stimulation. Although signaling occurs via the entire network of adhesion and co-stimulatory molecules, disruption of CD86 binding appears paramount, which results in the reduction in T cell stimulation. Mature DCs are distinguished morphologically by the presence of prominent dendrites that facilitate motility and provide a large surface area for the simultaneous interaction with multiple T cells [1]. Morphologic changes are mediated by the actin-bundling protein p55 fascin. Fascin expression is augmented by cytokines that induce DC maturation and has been associated with increased capacity to stimulate T cell proliferation [74]. Mature DCs are distinguished by low buoyant density, lack of adherent properties, absence of expression of lineage-specific surface markers characteristic of T, B, and NK cells, macrophages, and the presence of CD83 in some populations [3, 75]. Expression of Fc receptors and nonspecific esterase is downregulated and Birbeck granules are no longer detected. Mature DCs lack phagocytic capacity and are incapable of processing and presenting exogenous protein. Mature cells are far more effective than macrophages and B cells in stimulating mitogen or allogeneic T cell proliferation and induce significantly higher levels of IL-2 secretion [67, 68, 76, 77]. Unlike other antigen-presenting cells, DCs are uniquely capable of inducing CD8 proliferative and CTL responses in the absence of CD4 helper cells [78]. Mature DCs potently stimulate T cell responses induce TH1 responses following repetitive stimulation of T cells. In contrast, co-culture of T cells with immature DCs resulted in upregulation of the inhibitory molecule CTLA-4, lack of proliferation, and an inability for the T cells to subsequently respond to stimulation with mature DCs [79].

5. DC:T Cell Interactions Endogenously generated antigens are characteristically presented along the class I pathway to CD8+ T cells while exogenous proteins are processed and presented to helper T cells via the class II pathway. DCs may also perform cross presentation in which exogenous protein antigens are presented along the class I pathway [80–83]. DCs initially aggregate with T cells in an antigen-independent manner in an effort to survey the repertoire for T cells with the capacity to recognize the presented antigen. IL-I5 induces the release of chemokines which mediate the migration of T cells to the site of

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antigen presentation, while IL-14 facilitates T cell clustering around the DC [84]. Adhesion molecules such as CD1a, CD54, and CD58 are responsible for DC–T cell binding, which is strengthened by the presence of antigen recognition [85]. T cell activation is significantly impaired by antibody blockade of these interactions or genetic defects in which animals lack the full complement of adhesion molecules. Danger signals expressed at sites of inflammation induce DC maturation [86, 87]. The activation of T cells is mediated through the ligation of co-stimulatory molecules and the subsequent release of a complex network of cytokines (Fig. 45-5). CD40L and IL-12 play an important role in DC-mediated stimulation of T cells [88, 89]. Upon binding to DCs, T cell expression of CD40L is upregulated. This results in increased expression of MHC class II adhesion and co-stimulatory molecules and prolonged DC survival [6, 90–95]. Ligation of RANK, a member of the TNF receptor family, and release of IFNg in response to DC–T cell binding also results in the secretion of IL-12 [96]. IL-12 release is also stimulated by the antigenspecific activation of T cells and exposure of the antigen-presenting cells to inflammatory factors such as TNF, segmental allergen, challenge (SAC), and LPS [97]. IL-12 augments T and NK cell cytotoxicity and biases T cell development towards the TH1 phenotype that is associated with the release of IFNg [98, 99] IL-12 has been shown to be far more potent than IL-2 in amplifying antigen-specific responses mediated by DCs activation of T cells [73]. Exogenous IL-12 has been demonstrated to replace the need for helper T cells in generating effective immune responses directed against tumor lines of poor immunogenicity [100]. Maturation along the DC1 or DC2 pathways is determined by exposures occurring during differentiation such as presence of bacterial or helminth products, respectively [101]. TLRs are expressed by DCs and bind bacterial and viral products and stimulate DC maturation and expression of stimulatory cytokines such as IL-12. TLRmediated signaling operates in concert with cytokines via the TNF receptor family to stimulate DC activation and proliferation [86]. In several animal models, exposure to necrotic cells stimulates DC maturation and activation [102]. Intracellular uric acid that is released as a part of tissue necrosis has been shown to contribute to the DC response. This response may be a critical step in promoting pathways associated with graft versus host disease and transplant rejection.

Fig. 45-5.  DC-T cell interaction

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IL-12 enhances T cell proliferation following stimulation with DCs pulsed with tumor peptide, and transfection of murine DCs with IL-12 gene markedly upregulates their capacity to induce tumor-specific CTL responses. DC-mediated signaling though OX40 and 4-1BB on T cells provides proliferative stimuli to reactive T cell populations [103]. Another approach to enhance DC mediated stimulation is through the suppression of inhibitory signaling mediated by SOCS1 using siRNA [104]. Stimulatory signals such as CD40 deliver antiapoptotic effects that facilitate DC survival and promote DC capacity to stimulate T cells [105]. In contrast, a variety of factors such as IL-10 inhibit DC maturation resulting in the downregulation of co-stimulatory molecule expression, and the suppression of the release of inflammatory cytokines such as IL-1, IL-6, IL-8, TNF, and GM-CSF [106]. DCs generated in the presence of IL-10 induce anergy in potentially reactive T cell populations that is not reversed upon exposure to IL-10 naive DCs. In contrast, DCs that mature in the absence of IL-10 are subsequently resistant to its inhibitory effects. IL-10 secretion has been demonstrated by a variety of malignancies including melanoma, renal cell, and colon cancer, and may play an important role in the tumor evasion of host immunity. DC subsets also impart unique homing characteristics on the responding T cell population [92].

6. Ex Vivo Generation of DCs DCs have been generated from CD34+ or progenitor populations that undergo differentiation through in  vitro exposure to cytokines [36, 51, 93, 94]. In murine models, exposure of bone marrow mononuclear cells to GM-CSF induces the presence of cells with a DC phenotype as characterized by the strong expression of co-stimulatory molecules with potent immunostimulatory capacity. In humans, CD34+ cells cultured with GM-CSF give rise to mixed myeloid colonies of DCs and macrophages [36]. Stem cell factor (SCF) and FLt3L promotes the recruitment and proliferation of early progenitors resulting in an increase in the number and size of the colonies [95]. TNFa acts in concert with GM-CSF in promoting the differentiation of DCs giving rise to pure DCs as well as mixed myeloid colonies. Large yields of DCs may be generated from CD34+ cells isolated from cord blood, bone marrow, and mobilized peripheral blood stem cells that are cultured in the presence of GMCSF and TNF a [10, 21, 22, 51, 135] IL-4 suppresses monocyte maturation and improves the purity of DCs in the resultant population [107]. SCF and Flt3L promote the expansion of early progenitor populations increasing the overall cell yields in suspension cultures. Exposure to Flt3L also facilitates the generation of CDla+ and CDla-immature DC from CD34+ progenitors [108, 109]. DCs derived from CD34+ progenitors that pass through intermediate stages of differentiation with the capacity to mature into DCs or macrophages dependent on the nature of cytokine exposure [110]. Mature myeloid and plasmacytoid DCs can be directly isolated from peripheral blood using CMRF-44 or other antigens [111, 112]. Myeloid DCs (CD11c+) may also be generated in significant numbers in vitro from partially differentiated monocyte precursors in peripheral blood [113]. Plastic adherent PBMC cultured with GM-CSF, TNFx, and IL-4 generate cell populations that are potent in allogeneic mixed leukocyte reaction (MLR) and express CD83

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and CDla [84]. Alternatively, DC precursors may be isolated by magnetic bead isolation of CD14+ cells that are then differentiated into DCs [114]. Monocyte precursors cultured with GM-CSF and IL-4 yield DCs with an immature phenotype that maintain the capacity to internalize and present exogenous protein [115]. Blood precursor populations may also differentiate into an LC phenotype under the influence of TGFb or IL-15 [99, 116] Plasmacytoid DCs are derived from CD11c− cells and their growth and maturation is supported by GM-CSF, IL-3, and TNFa [117]. Some studies have demonstrated that CPG ODN promote plasmacytoid DCs to adopt an immunosuppressive phenotype characterized by an increased expression of IL-10, TGFb, and IL-6 and their capacity to stimulate the expansion of regulatory T cells [29]. Alternatively, CPG has also been shown to increase expression of IFNa and the capacity to stimulate B cell activation [118]. A variety of agents promote terminal maturation of myeloid DCs in vitro with the concomitant loss of phagocytic capacity, increased expression of the co-stimulatory markers CD80, CD86, and CD40, increase in the maturation marker, CD83, and enhanced ability to stimulate T cell responses. These include TNFa, LPS, IL-3, CD40L, LPS, and PG-E2. Exposure to monocyteconditioned media potently induces terminal DC maturation and activation which persists following withdrawal of cytokines. Its effects may be recapitulated by the addition of combination of IL-1b, IL-6, TNFa, and PG-E2 [119, 120]. The effects of PG-E2 on DC development are complex with some studies demonstrating its ability to promote polarization of DCs towards an inhibitory phenotype characterized by IL-10 expression, stimulation of TH2 responses and the expansion of regulatory T cells [121]. Activation of signaling pathways associated with innate immunity also induce DC maturation. Ligation of the TLRs by LPS (TLR4), polyI:C (TLR3), Imiquomod (TLR 7/8), and CpG ODN (TLR9) promote the activation of immature DCs and has been shown to be more potent than stimulation with cytokines [14, 56, 122]. Another strategy for DC maturation involves the combined use of TNF, IL-1, POLY I:C, IFNa and IFNg which results in cells that potently express IL-12 in response to CD40-mediated stimulation [123]. DC maturation may also be induced by relatively brief exposure of monocytes (2 days) to IFNg and LPS or GM-CSF and type I IFN which generates a population of antigen presenting cells that secrete high levels of IL-12 [124]. Vaccination with these rapidly activated DCs pulsed her2neu peptide resulted in anti-tumor immune and clinical responses in patients with breast cancer [125]. Maturation of immature DCs be induced in vivo by exposure to adjuvants such as imiquomod that also facilitate migration to draining lymph nodes [126]. The ideal strategy for DC generation is dependent on the nature of the vaccine design and the method of antigen loading. Immature Dcs have been associated with a tolerogenic phenotype. In one study, vaccination with immature DCs with influenza peptide resulted in flu specific anergy [127]. In contrast, peptide-loaded mature DCs effectively stimulated anti-influenza responses [128]. Several studies have compared the immunologic efficacy of mature vs. immature DCs loaded with tumor antigens [129]. In one study, vaccination of melanoma patients with peptide-pulsed mature DCs resulted in DTH and clinical responses while none were seen following vaccination with immature DCs [130]. Similar findings were observed in comparing the efficacy immature and mature DCs in patients with glioma [131]. Another strategy involves the use

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of immature DCs that undergo antigen-loading techniques that directly induce maturation. Loading of DCs with necrotic tumor cells or DC–tumor fusion has been shown to activate and mature immature DCs [132]. Similarly, vaccination with immature DCs in conjunction with adjuvant has been shown to induce maturation in  vivo [126]. Use of TH1-polarized DCs for immunization was associated with increased IL-12 expression and enhanced responses [133]. In one study, vaccination with Langerhans cells was more potent than monocytederived DCs [134]. Despite these findings, there is considerable complexity regarding the balance of DC-mediated stimulation and inhibition of T cells and the precise strategy for DC generation for vaccine therapy remains to be elucidated. Mature DCs may also be tolerogenic and promote the expansion of regulatory T cell populations. Mature DCs have been shown to express IDO or inhibitory cytokines resulting in an inhibitory phenotype [135]. Therefore, the phenotypic characteristics of DC populations are the product of multiple factors that include maturation state and the nature of the culture stimuli [108]. Another concern regarding optimizing DC vaccination involves identifying the population with the capacity to migrate to sites of T cell traffic. In a study of patients with melanoma, mature DCs were far more capable to reach the draining lymph nodes as compared to immature DCs [109]. A correlated question is “what is the optimal mode of administration to facilitate migration of vaccine cells and immunologic potency?”. In a study of patients with prostate cancer undergoing vaccination with DCs pulsed with tumor antigen, T cell expression of IFNg was most pronounced following intradermal or intralymphatic injection while antibody responses occurred most commonly following intravenous administration [110]. In a study of patients with melanoma, intranodal as compared to intravenous or intradermal administration was associated with greatest DTH responses and T cell cytokine expression to antigen-bearing targets [136]. However, the superiority of the intranodal route has not been uniformly observed. In another study, subcutaneous as compared to intravenous administration was associated with increase in tumor-reactive memory cells in the lymph nodes with protective effects against skin based disease [113].

7. The Role of DCs in Establishing Tolerance The T cell repertoire is generated by random DNA rearrangements of the T cell receptor genes resulting in a diverse population of T cells, many of which with the capacity to recognize self antigens. Tolerance towards self antigens and protection from autoimmunity is provided by thymic deletion of autoreactive T cell clonal populations and peripheral mechanisms by which potentially noxious T cells are anergized [137]. DCs play a crucial role in the maintenance of both central peripheral tolerance and establishing the balance between immune activation of suppression [17, 138]. Autoreactive T cells are eliminated in the thymic medulla via interactions mediated by thymic epithelial cells and mature DCs. Tolerogenic DCs are thought to be essential for suppressing potentially autoreactive T cells that escape thymic deletion. This DC population is most commonly characterized by an immature phenotype, relatively low expression of co-stimulatory molecules, impaired ability to secrete stimulatory cytokines such as IL-12, and strong expression of ­inhibitory

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cytokines [15]. A greater understanding of DC-mediated immune tolerance is crucial for establishing strategies to immunize against shared self and tumor-associated antigens. In addition, the balance of DC-mediated immune activation and tolerance is thought to play a critical role in the development of alloreactive rejection and GVHD and is a potential focus for therapeutic manipulation. The nature of DC development determines the nature of the signaling pattern to reactive T cells and subsequent polarization towards an activated or inhibitory phenotype [1, 3, 16]. Immature DCs promote immune tolerance by processing and presenting self antigens at tissue sites [115]. Presentation of antigen in the absence of co-stimulatory molecules by immature DCs and their expression of cytokines such as IL-10 results in the delivery of an inhibitory signal to reactive T cell populations. Immature DCs also express receptors for apoptotic cells promoting tolerance toward these antigens [116]. Immature DCs may express high level of Indoleamine 2,3-dioxygenase (IDO), an enzyme responsible for tryptophan degradation [139]. Tryptophan is thought to be an essential amino acid for T cell survival and its absence may induce T cell death. Animal models suggest that DC capture and presentation of apoptotic bodies polarizes DCs towards a tolerogenic phenotype which is thought to be responsible, in part, for the therapeutic efficacy of photopheresis in the treatment of graft versus host disease. In contrast, defects in apoptotic mechanisms are thought to contribute in the disruption of immune tolerance in patients with systemic lupus erythematosus [140]. Presence of cytokines such as IL-10, TGFb, and VEGF expressed in the tumor bed prevents DC differentiation and polarizes DCs towards an inhibitory phenotype. Other inhibitory signaling pathways thought to be important in DC–T interactions include the expression of the programmed death ligand-1 (PDL1/B7-H1) by DCs that binds PD-1 on T cells and provides an inhibitory signal to T cell development [141]. Of note, B7-H1 expression is also found on tumor cells and abrogation of expression is associated with T cell activation and autoimmunity. A variety of agents have been identified that inhibit DC maturation and function in  vitro resulting in an inhibitory phenotype. These include IL-10, TGFb, inducers of cyclic AMP such as prostaglandin E2, and immunosuppressive drugs such as rapamycin and corticosteroids [15]. For example, rapamycin-treated DCs produce low levels of IL-12p70, do not respond to TLR-mediated signaling and markedly expand regulatory T cell populations with the capacity to enhance graft survival in an allogeneic transplant model [142]. The activated form of vitamin D3, in concert with other immunosuppressive agents, has also been shown to polarize DCs towards a tolerogenic phenotype. Other agents associated with the development of tolerogenic DCs include G-CSF, M-CSF and IL-4, thrombopoietin, and IFNl [143–147]. DC mediate signaling via CTLA-4. and PD-1 pathways induces tolerance in CD8+ cells [148]. Regulatory T cells represent a thymic-derived population of immunosuppressive T cells and play a vital role in maintaining peripheral tolerance [149–151]. Regulatory T cells co-express CD4 and high levels of CD25, minimally proliferate in response to mitogenic stimuli, and inhibit T cell responsiveness through the expression of inhibitory ligands and cytokines such as TGFb. Distinctive phenotypic characteristics include co-expression of the glucocorticoid-induced tumor-necrosis factor receptor (GITR),

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cytotoxic T lymphocyte antigen-4 (CTLA-4), and, most importantly, the forkhead–winged helix transcription factor, Foxp3 [152]. Mature DCs facilitate the expansion of regulatory T cells in the thymus. Regulatory T cells consist of 5–10% of circulating lymphocytes and their adoptive transfer has been associated with mitigation of autoimmunity and GVHD [153–155]. Regulatory T cells express inhibitory cytokines such as IL-10 and TGFb but also demonstrate the capacity to suppress T cell activation by direct cell contact via the CD95 signaling pathway. T regulatory 1 cells (Tr1) are expanded from peripheral CD4 T cells and suppress T cell activation by IL-10 and TGFb expression in a nonantigen-specific manner, similar to thymic derived regulatory cells [156]. DCs play a crucial role in the maintenance and expansion of regulatory T cell populations in vivo and in vitro [15, 19, 138, 155]. In an animal model, immature DCs loaded with ovalbumin were shown to induce the expansion of T cells which demonstrated specific inhibition of ovalbumin-directed immunity [157]. In a murine model, DCs that have been modified to express co-stimulatory molecules with low levels of co-stimulatory molecules were highly effective in stimulating regulatory T cell responses [158]. Expansion of regulatory T cells may also be facilitated by TGFb expression by DCs (72). However, mature DCs have been shown to the most effective antigen presenting cell with regard to the expansion of regulatory T cells in an antigen-dependent fashion [159, [160]. Of note, the suppressive function of DC-expanded regulatory cells is greater than cells directly isolated from the circulation. Once activated in the presence of a specific antigen, regulatory T cells have the capacity to inhibit T cell activation generally towards other antigens. Mature DCs induce the expansion of both activated effector and regulatory T cell populations [161]. Co-stimulatory molecule-mediated signaling appears to potently stimulate both populations suggesting that regulatory T cells may represent a homeostatic mechanism that ultimately blunts response to DC-mediated stimulation. In concert with these findings is the observation that response to DC-based vaccination is augmented by regulatory T cell depletion [162]. The interaction between DCs and regulatory T cells is bidirectional. Regulatory T cells may interfere with DC maturation and activation further augmenting their inhibitory potential [163]. Regulatory T cells inhibit the upregulation of co-stimulatory molecules on DCs and the interaction of DCs with effector cells [159]. One potential mechanism is CTLA-4-mediated signaling resulting in increased IDO expression in DCs which in turn result in suppressive effects on reactive T cell populations. Tolerogenic DCs may also inhibit DC populations with an immunostimulatory phenotype. Plasmacytoid DCs may direct T cells towards a TH2 phenotype, upregulate IDO expression and induce the differentiation of regulatory T cells [164]. Allograft tolerance may be induced by inhibitory donor-derived DCs or recipient DCs that have been loaded with donor allopeptides [165]. The effect of plasmacytoid DCs in vivo may be determined by their site of tissue migration. In a cardiac allograft model, plasmacytoid DCs homing to the lymph nodes were found to expand regulatory T cells and promote allograft tolerance while those migrating to the spleen-induced rejection [18]. Plasmacytoid DC precursors have been shown to promote hematopoietic stem cell engraftment in the absence of GVHD and are an important subpopulation of transplantfacilitating cells [166]. Regulatory T cells expanded by allogeneic DCs dem-

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onstrate the capacity to suppress rejection specific to the allogeneic stimulus [159]. Similar findings were observed in an in vivo GVHD model in which mice that received donor regulatory T cells expanded by recipient allogeneic DCs were better protected than those receiving regulatory cells expanded by third party DCs. Tumor cells utilize a variety of mechanisms to inhibit DC maturation, activation, and antigen presentation resulting in the polarization of T cell responses towards tolerance [167]. Tumor cells constitutively express STAT3 which inhibits the production of inflammatory cytokines and promotes the release of factors that inhibit DC function. Tumor cells secrete VEGF, IL-10, and IL-6 which have been shown to prevent DC maturation towards an activated phenotype. This results in the localization of tolerizing immature DCs in the tumor bed with mature cell largely confined to the periphery [168]. In contrast, inhibitors of VEGF have restored normal DC differentiation but not necessarily their stimulatory capacity [169]. Tumor-associated glycoproteins such as MUC1 have been shown to disrupt antigen processing, inhibit DC expression of IL-12, and promote the development of TH2 responses [170]. Of note, in a breast cancer model, tumor growth is supported by IL-13, a TH2 associated cytokine [171]. In a human myeloma model, DCs have been shown to support the growth of myeloma cells [172].

8.  The Role of DCs in Immune Reconstitution, Graft Versus Disease and Graft Versus Host Disease Following Hematopoietic Stem Cell Transplantation Mobilization of DC precursors has been studied as a part of stem cell collections for autologous and allogeneic transplantation. Use of GM-CSF as a part of the cytokine regimen has been associated with increased presence of DCs in the mobilized product [173]. Another study demonstrated that mobilization with GM-CSF and G-CSF as compared to G-CSF alone resulted in a decrease in the DC2 subset in the graft suggesting that these cells may be less tolerogenic [174]. Following allogeneic transplantation, DCs are thought to play an important role in the pathogenesis of GVHD [175]. DCs may be activated in the setting of cytokine storm that prevails following transplant conditioning resulting in the presentation of alloantigens to donor T cell populations [176]. DC-mediated signaling of CD4 T cells via OX40 has been shown to be an important pathway for the induction of GVHD in animal models [177]. DCs were found to be predominantly of donor origin in the early post-transplant period [178]. However, DCs are nonproliferating cells and may transiently survive ablative conditioning regimens [20]. Host-derived DCs may persist for a long-time post-transplant particularly after reduced intensity conditioning regimens [179]. In one animal model, as compared to activated B cells, recipient DCs were shown to be uniquely capable of stimulating alloreactive CD4 and CD8 T cells resulting in GVHD [180]. In contrast, in reduced intensity murine transplant model, high autoantibody levels predictive of cGVHD were seen in the setting of mixed chimerism and associated with the persistence of host B cells rather than DCs [181].

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The importance of host-derived DCs was emphasized in a study in which host skin Langerhans cells persist following T cell-depleted transplantation despite the conversion to full donor chimerism in other tissue sites [182]. GVHD responses in the skin following infusion of donor lymphocytes were seen exclusively in animals with residual host Langerhans cells. Donor T cells were shown to be crucial for the eradication of recipient Langerhans cells and the recruitment of donor Langerhans cells to the site. While host DCs are sufficient to initiate GVHD, it is further intensified by the presence of donor antigen-presenting cells which cross prime alloreactive CD8 cells [183]. Of note, donor-derived antigen-presenting cells were not thought to be responsible for stimulating graft versus leukemia responses. A major research focus has been the separation of GVHD from graft versus disease effects. In a murine transplant model, host DCs stimulated T cell subsets differentiated by CD44 expression in which the CD44low/CD8+ fraction was responsible for GVHD and depletion of these cells was protective without impacting graft versus leukemia effects of the graft [184]. The nature of DCs reconstitution following allogeneic transplantation is likely to have profound implications for the development of donor/host tolerance with clinical implications for the incidence of rejection, graft-vs-host disease (GVHD), infection, and disease relapse [185]. The nature of the recovering DCs subpopulations and their functional characteristics is strongly associated with levels of alloreactivity and tolerance. Rapid establishment of donor chimerism for DC populations has been noted in the early posttransplant period [179]. In animal models of solid-organ transplants, treatment with immature DCs, lymphoid-derived DCs, or DCs following blockade of the CD40 pathway resulted in prolonged survival of the allograft tissue [186, 187]. In an effort to manipulate the kinetics of DC reconstitution, investigators have examined the in vivo effect of CAMPATH-1G, an antibody directed against CD52 expressed on T cells and certain DC subsets [188, 189]. In one study, the use of CAMPATH-1G as a part of transplant conditioning resulted in the depletion of host DCs but did not impact recovery of donor DC subsets post-transplant. Following transplantation with G-CSF-mobilized allogeneic stem cells, increased numbers of circulating DC2 cells are found that may mediate tolerance [147]. This finding was thought to potentially explain the lack of increase in acute GVHD associated with allogeneic peripheral blood stem cell grafts, despite the increased numbers of T cells as compared to bone marrow. In patients undergoing allogeneic peripheral blood stem cell transplantation, increased number of DC2 in the stem cell graft was associated with decreased incidence of GVHD and increased risk of relapse [190]. In a pediatric study, decreased circulating levels of monocytoid and plasmacytoid DCs early post-transplant was associated with the subsequent development of acute but not chronic GVHD [191]. In a study of 31 adult patients undergoing allogeneic peripheral blood stem cell transplantation, both myeloid and plasmacytoid DC subsets were suppressed in patients with grade II–IV aGVHD [192]. Similar to these findings, a study of 50 patients undergoing transplant demonstrated that lower levels of circulating DCs was associated with a higher risk of relapse, death, and aGVHD [185]. Analysis of DC1 and DC2 subsets demonstrated a trend towards similar effect as that seen with the total DC population. In another study, an increase in myeloid

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and ­plasmacytoid DCs was observed at the onset of GVHD, the latter of which was promptly suppressed with steroid therapy [193]. Administration of G-CSF was associated with higher initial levels of myeloid DCs but ­subsequently resulted in lower levels of myeloid and plasmacytoid DCs as well as IL-12-producing cells. Persistence of host-derived DCs was associated with graft versus leukemia effects following donor lymphocyte infusions in patients with CML [194]. Umbilical cord blood is an effective source of hematopoietic stem cells used to support allogeneic transplantation. Cord blood transplantation is associated with a greater degree of tolerance as compared to adult bone marrow such that that a greater degree of HLA mismatch results in similar levels of GVHD. Of note, DCs isolated from cord blood demonstrated a more immature phenotype than those from peripheral blood [195]. Cord blood-derived DCs were associated with lower levels of TNFa and IFNg secretion and potently induced the expansion of regulatory T cells. There has been strong interest in developing strategies to expand tolerogenic DC as a means of inhibiting the GVHD response following allogeneic transplantation. In animal models, infusion of tolerogenic DCs was found to inhibit GHVD while preserving graft versus tumor responses [196]. Similarly, DCs differentiated in the presence of vasoactive intestinal peptide induce the expansion of regulatory T cells and blunt GVHD following allogeneic transplantation [197]. In contrast, these cells do not inhibit the capacity of alloreactive CD8+ T cells to lyse leukemia targets. Co-transplantation of TGFb treated DCs resulted in the prolongation of the survival of animals undergoing MHC-disparate allogeneic transplantation [198]. DCs generated in  vitro with GM-CSF, IL-10 and TGFb prevent the development of lethal GVHD but not anti-leukemia responses in a murine allogeneic transplant model [196]. In contrast, transplantation of mature DCs intensified the resulting course of lethal GVHD. Extracorporeal photochemotherapy has become established as a therapeutic strategy for patients with chronic GVHD. A proposed mechanism based on animal models is that apoptotic cells resulting from this procedure are ingested by native DC populations polarizing them towards a tolerogenic phenotype [199, 200]. DCs generated from patients treated with FK506 following allogeneic transplantation demonstrated decreased functional capacity. Exposure to rapamycin or corticosteroids results in inhibited maturation and expression of co-stimulatory molecules [142, 165, 201].

9. DC-Based Immunotherapy for Cancer Tumor cells express unique antigens that serve as potential targets for cancer immunotherapy. Tumor-associated antigens have been identified that are aberrantly expressed by malignant cells allowing for their differentiation from normal tissue [202–204]. T cells with the capacity to recognize tumor antigens have been found in the immune repertoire of patients with malignancy. However, tumor cells evade recognition leading to immunologic tolerance that supports the growth and dissemination of malignant disease [205]. Tumor cells present antigens in the absence of co=stimulatory molecules necessary for the initiation of primary immune responses [206]. As outlined above, tumor cells secrete factors that disrupt function and maturation of native antigen-

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p­ resenting cells [207, 208]. In addition, tumor cells blunt the functional potency of effector cell populations by a variety of mechanisms including the increased presence of regulatory T cells that suppress T cell-mediated response. Tumor cells may exist in an equilibrium with host immunity until progressive tumor-mediated immune suppression and emergence of poorly immunogenic clones result in the promotion of tumor growth [205]. In contrast, DCs potently richly express co-stimulatory molecules and cytokines necessary for immune activation. As such, there has been strong interest in the manipulation of DCs to process and present tumor-associated antigens to generate productive anti-tumor immunity. Strategies to introduce tumor antigens into DCs have been pursued in an effort to induce tumor-specific CTL responses. One approach has been the in vivo loading of tumor antigens by DCs recruited to the site of malignancy. Introduction of DCs into the tumor bed has been shown to directly inhibit tumor growth [209, 210]. Tumor cells genetically engineered to express GM-CSF or co-administered with GM-CSF-secreting cells induce tumorspecific immunity through the recruitment of DCs to the site of inoculation with subsequent internalization and presentation of tumor antigens [211, 212]. Administration of immature DCs in conjunction with the TLR agonist, imiquimod, enhanced the capacity of the DC vaccine to the draining lymph node and the induction of anti-tumor immunity [126]. Systemic administration of Flt3L results in the tissue accumulation of DCs and the potential internalization, processing, and presentation of tumor antigens at the site of malignant disease [41]. Therapy with Flt3L has been shown to induce tumor regression in animal models [213]. Tumor-specific immunity was transferred by CD8+ splenocytes isolated from mFlt3L-treated animals. In another murine model, administration of Flt3L was protective against an otherwise lethal challenge of myeloid leukemia cells and induced anti-leukemia CTL responses but did not generate long-term memory responses and was ineffective for treating established disease [214]. In another study, animals treated with a combination of radiation and FLT3L experienced a decrease in pulmonary metastases and improved survival as compared to those treated with FLI3L alone [215]. The investigators postulated that radiation facilitated the loading of tumor antigens onto infiltrating DCs. DCs isolated from patients with malignancy demonstrate functional deficiencies [207, 216, 217]. As such, the use of native DC populations for cancer immunotherapy is potentially problematic. Alternatively, DCs generated ex vivo from progenitor populations have been shown to be functionally competent [173, 218–220]. As such, manipulation of these population have been pursued to design tumor vaccines. A variety of in vitro strategies to introduce tumor antigens into DCs have also been examined in animal models. (Fig. 45-6) Exogenous loading of DCs with tumor peptides allows for the use of DCs with a mature phenotype. Response to individual peptides is governed by their affinity to MHC binding and their capacity to induce responses against various epitopes [221]. In murine models and pre-clinical human studies, DC pulsed with tumor-associated peptides effectively induce antigen-specific CTL responses resulting in protection from tumor challenge [155–160, 204, 222–224]. In one study, vaccination with DCs pulsed with Her2neu peptide, which was altered to augment binding to the MHC complex resulted in higher levels of CTL activity [225]. Of note, weekly immunization resulted in decreased

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Fig. 45-6.  Strategies to load tumor antigens onto dendritic cells

levels of response, while animals vaccinated every 3 weeks did not experience diminution in CTL immunity. In the hematological malignancy setting, peptides derived from the BCR/ABL fusion region have also been shown to be immunogenic when presented by antigen-loaded DCs [226]. CD4-mediated responses were generated that lysed CML cells containing the associated breakpoint region. T cells stimulated with DCs pulsed with a bcr–abl peptide lyse patient-derived chronic myeloid leukemia (CML) cells containing the same breakpoint, but not autologous monocytes [227]. DCs loaded with peptides eluted from AML cells have also be used to stimulate tumor-specific responses [228]. Peptide-pulsed DC-derived exosomes have also been shown to be potent anti-tumor immunogens resulting in eradication of established disease in murine models [178]. Although effective in animal models, the efficacy of peptide-pulsed DCs in generating tumor-specific immunity is limited. The immunogenicity of identified antigens is variable, the stability of antigen presentation following exogenous pulsing is uncertain, and the clinical efficacy of an immune response directed against a single epitope may be muted. Of note, patientderived CTL induced by DCs pulsed with p53-derived peptides were unable to lyse autologous squamous cell carcinoma cells due to the downregulation of expression of this epitope [229]. In addition, there is a lack of defined tumorspecific peptides in many malignancies, and treatment is limited to patients of a particular HLA genotype. Another approach to designing DC-based tumor vaccines is through the exogenous loading of whole proteins [230]. In this way, multiple epitopes may be presented in the appropriate HLA context. As an example, vaccination with DCs pulsed with lymphoma-derived idiotype protein stimulates antigen-specific T cell response and protection from challenge with idiotypeexpressing tumor cells [231]. The efficacy of protein loading may also be limited in that it is dependent on the use of DCs with the capacity to internalize

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and process antigen, and the ability of T cell repertoire to recognize presented epitopes is less easily defined. The processing of exogenous proteins also results in their presentation along a class II pathway, producing a primary helper as compared to cytotoxic T cell response. However, cross-presentation of internalized antigens to cytotoxic T cells has been documented as an important mechanism of DC-mediated immune responses. One strategy that has been used to use whole proteins as a source of peptide epitopes is the generation of overlapping long peptides that cover the coding sequence of the identified tumor antigen [232]. Another approach to introducing tumor antigens into DCs has been the use of viral vectors to insert genes encoding for tumor-specific proteins. ln this way, tumor antigens are processed through endogenous mechanisms and presented along a class I pathway to reactive CD8+ T cells. Viral transduction may induce DC activation enhancing immune responses. Another potential advantage is that insertion of tumor-associated genes potentially provides an ongoing source of antigens for presentation. In contrast, peptide or protein antigens may be cycled off the cell surface by the time that DCs arrive at sites of T cell interaction [14]. Transduction of DCs with recombinant pox viruses expressing the co-timulatory and adhesion molecules (TriCOM complex) markedly augments their capacity to stimulate antigen-specific responses [233]. DCs infected with vaccinia virus bearing melanoma-derived gp 100 stimulated CTL responses that lysed HLA-matched targets that had been pulsed with a variety of gp100-derived peptides [234]. Transduction of DCs with retroviral or lentiviral vectors has also been investigated [235, 236]. Investigators have explored the feasibility of the retroviral insertion of tumor genes into CD34+ cells that are subsequently differentiated into DCs in the presence of cytokines. Using this approach, stable expression of the MUC-1 tumor antigen was generated in DCs derived from retrovirally transfected precursor cells [237]. Similarly, the MART1 gene was expressed in approx 25% of DC following its retroviral insertion into CD34+ cells that were then cultured with SCF, TNFcr, and GM-CSF [238]. Vaccination with DCs transduced with an adenoviral vector bearing the MAGE-1 gene resulted in suppression of tumor growth in a subcutaneous melanoma model, with 10% of animals experiencing long-term survival [239]. In contrast, vaccination with tumor cells expressing IL-12, GM-CSF, or CD40L were unable to contain tumor progression. Paradoxically, levels of MAGE-I expression were significantly higher in tumor cells as compared to DCs. A potential concern regarding the use of viral vectors is their generation of potent immunologic responses directed against viral proteins. These antigens are potentially far more immunogenic and may overwhelm the response against the designated tumor antigens and prevent repetitive dosing from being effective. Another potential limitation of this approach is the demonstration that viral infection may be associated with decreased DC function [240]. Transfer of tumor-specific genes into DCs has also been accomplished through the use of tumor-derived RNA [241]. This strategy is facilitated by established methods to isolate and amplify RNA from biopsy specimens, allowing for its potential general applicability in the clinical setting. As an example, DCs pulsed with CEA-mRNA stimulate tumor-specific CD8+ CTL [242]. Similarly, DCs pulsed with RNA encoding for prostate-specific antigen (PSA) induced CTL responses against cells expressing PSA but not kallikrein,

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a self antigen that shares homology with PSA [243]. DCs transfected with RNA encoding for the MUC-I tumor antigen induced tumor-specific responses in immunized animals resulting in protection from tumor challenge and regression of established disease [244]. Vaccination with DCs cotransfected with MUC-I RNA and IL-12 resulted in MUC-1-specific responses in a transgenic murine model. Similarly, DCs pulsed with mRNA encoding for human telomerase reverse transcriptase are highly effective in stimulating responses against diverse malignancies for this effective target antigen [245]. One potential concern is that mRNA-based strategies will induce a cytotoxic T cell response in the absence of a helper response. Approaches to concomitantly stimulate CD4 T cells include the insertion of RNA encoding for a lysosomal targeting fusion signal [246]. All of the above mentioned-strategies involve the targeting of known tumor-associated antigens. The use of single gene products for DC-based immune strategies limits one to a small group of potential antigens of uncertain immunogenicity. Immunotherapeutic approaches that rely on induction of immunity against a particular antigen are also potentially subject to tumor cell resistance mediated by the downregulation of expression of that single gene product. One approach to circumvent this limitation is the pulsing of DCs with antigens extracted from whole tumor cells or whole tumor-derived RNA [241, 247]. DCs generated from cord blood CD34+ cells have been successfully transduced with RNA derived from a leukemia cell line [248]. DCs loaded with lysate generated by freeze–thawing of an Epstein–Barr virus (EBV) transformed lymphoblastoid cell lines (LCL) line stimulated tumorspecific CD4+ and CD8+ responses with TH1 phenotype [249]. Loading of DCs with lysate prior to terminal maturation with TNFa, IL-1b, and PG-E2 was the most effective approach in generating tumor immunity. Another study examined the capacity of monocyte-derived immature DCs to process and present tumor antigens from tumor cells that had undergone lethal l irradiation or exposure to anti-Fas antibody [250]. DCs loaded with leukemia lysates generate tumor-specific CTL responses [251]. As a measure of their capacity to stimulate class I responses via cross-priming, DCs pulsed with breast cancer lysate-induced CD8 mediated MUC1 specific responses associated with the production of TH1 cytokines [252]. Another strategy for generating tumor immunity involves the use of apoptotic bodies as a means of introducing tumor antigens into DCs with subsequent cross-presentation along the class I pathway [81, 253]. DCs were found to express a unique receptor which facilitates phagocytosis of apoptotic bodies and is downregulated upon maturation [47]. Mactophages ingest apoptotic bodies more readily, but lack the capacity to cross-present antigenic material from the apoptotic bodies. In one study, DCs demonstrated the capacity to internalize necrotic as well as apoptotic tumor bodies, but only the latter induced DCs maturation and resulted in potent CD8+ tumor-specific responses [81]. ln another study, DCs pulsed with melanoma cells that underwent apoptosis were more effective in generating tumor responses than those loaded with live or necrotic cells [254]. One strategy that is currently being explored for the generation of DC-based immunotherapy for leukemia has been the differentiation in vitro of leukemic clones into DCs. In this manner, tumor antigens retained from the malignant clone can be endogenously processed and presented by a functionally active

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antigen-presenting cell. CML cells cultured with GM-CSF andlL-4 developed phenotypic characteristics of DCs that contained the bcr–abl translocation [255]. Stimulation of autologous T cells resulted in cytotoxic activity against CML cells and bcr–abl-expressing targets as well as the inhibition of growth in CML clonogenic precursors in colony-forming assays in vitro. Retroviral transduction with the gene encoding for IL-7 further increased the potency of bcr–abl-expressing DCs generated from CML patients [256]. Investigators have also demonstrated that functionally active DCs can be generated from acute myeloid and leukemia cells [257, 258]. Leukemic blasts were cultured in cytokines and subsequently found to express co-stimulatory molecules such as CD80, CD86, and CD40, DC-specific markers such as CD83, and retained the chromosomal abnormalities of the original leukemic clone. DCs stimulated autologous T cell-lysed leukemic targets. Of note, immature CD34+/CD38− leukemia progenitors are resistant to differentiating toward DCs [259]. A potent strategy for designing DC-based tumor vaccines involves the fusion of DCs with tumor cells. In this approach, multiple tumor antigens, including those yet unidentified, are presented in the context of DC-mediated co-stimulation. DC/tumor fusions stimulate CD4- and CD8-mediated immunity resulting in greater potential durability of the anti-tumor response. In diverse animal models, vaccination with DC/tumor fusions has been shown to potently induce tumor-specific CTL responses, is protective from an otherwise lethal challenge of tumor cells and may eradicate established metastatic disease [260–263]. DC/tumor fusions were also found to break immunologic tolerance toward the MUCl tumor antigen in transgenic mouse models [264]. In another study, vaccination with fusion cells resulted in protection from tumor challenge as well as efficacy as therapy for metastatic disease in melanoma and lung carcinoma models [265]. Subsequent studies have demonstrated that DC/tumor fusions are potent stimulators of tumor-specific immunity in preclinical human studies in multiple myeloma, breast and ovarian cancer [266–269]. Fusion cells were generated from patient-derived tumor cells and autologous DCs, and were found to co-express tumor antigens and DC-derived co-stimulatory molecules. Fusion cells induced prominent tumor-specific CTL responses in vitro following a single stimulation. CTLs did not lyse autologous monocytes and were inhibited by incubation with anti-class I antibody. Similar findings were demonstrated with DCs fused with AML cells in which fusion cells potently stimulated CTL responses directed against leukemia cells including those with core binding factor mutations [270–272]. Fusion cells were also shown to prevent the spontaneous development of mammary tumors [273].

10. DC Immunotherapy for Cancer: Clinical Studies DC-based tumor immunotherapy is now being pursued in the clinical setting. One strategy involves the attempt to stimulate in  vivo generation and mobilization of native DC populations that then undergo antigen uptake and presentation at the tumor site [274]. In one study, patients with colon cancer were treated with Flt3L prior to resection of metastatic lesions in the lung or liver [275]. Increased number of CD11c/CD14− DC were noted in the peripheral blood-as well as at the tumor margins. Patients receiving Flt3L following autologous stem cell transplant experienced an increase in monocyte and plasmacytoid DCs that could be further matured ex vivo with CPG ODN [276].

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Clinical responses have also been demonstrated in patients with melanoma and breast cancer undergoing injection with autologous DCs directly into the tumor bed [205]. This was further facilitated by radiation therapy into the tumor bed in patients with hepatoma [277]. Another approach is the use of IFNa as an adjuvant to melanoma peptides vaccine to enhance native DC processing and presentation [278]. Exposure of DCs to alpha-GalCer enhances their capacity to stimulate NK T cells as anti-tumor effector cells in lung cancer [279]. Another approach is the use of the leukemia clone to generate DCs expressing leukemia-associated antigens. In patients with CML – vaccination in patients with incomplete response to imatinib or interferon therapy – 4/10 patients had potential evidence of further response to vaccination which was associated with the emergence of tumor-reactive T cells in three patients [280]. In a study of 22 patients with AML, patients underwent vaccination with leukemia derived DCs following completion of chemotherapy. Although T cell responses were documented in a subset of patients, only two patients remained in remission for more than 12 months [281]. In a small study of patients with AML undergoing vaccination with DCs generated from leukemia cells, immune responses were noted against the leukemia-associated antigen, PRAME [282]. Vaccination of patients with DCs pulsed with tumor peptides has been studied in clinical trials particularly in patients with melanoma. In an early trial, 16 patients with melanoma were treated with DCs pulsed with melanoma peptides or lysate as well as KLH to induce helper responses [283]. Following vaccination, 11 out of 16 patients developed DTH responses at the vaccine site and associated tumor-specific CTL responses were noted. Six out of 16 patients showed evidence of clinical response. In a similar study of patients undergoing vaccination with peptide-pulsed DCs activated with IFNa, significant clinical or immunologic responses were not observed [284]. Vaccination with DCs pulsed with MAGE peptide demonstrated that clinical responses were associated with a 20- to 400-fold increase in antigen-specific CTL which demonstrated a polyclonal profile [285]. In another study, 18 patients with metastatic melanoma underwent vaccination with CD34+-derived DCs pulsed with several melanoma peptides [286]. Sixteen out of 18 patients demonstrated evidence of T cell response to the control antigen, influenza, and KLH, and at least one of the melanoma peptides. Of note, clinical response was associated with immunologic response to at least two melanoma peptides as manifested by an increased percentage of T cells expressing IFNg in response to ex vivo exposure to the peptide. In a follow-up report, four patients remained alive 5 years after completion of the study, with survival correlating with tumor peptide-specific immunity [287]. Based on the encouraging pre-clinical data, a study was performed in which 15 patients with melanoma were treated with DC-derived exosomes pulsed with MAGE peptides [288]. Only one patient demonstrated evidence of a partial response and no evidence of circulating antigen-reactive T cells was observed. Several studies have examined the efficacy of vaccination with DCs pulsed with peptides in other malignancies. In one study, 17 previously untreated patients with prostate cancer underwent monthly intravenous infusions with DCs pulsed with prostate-specific membrane-antigen (PSMA) peptides [207, 289] Three partial responders and one complete response were noted. No significant treatment related toxicity was reported. Immunological assessment

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revealed that clinical response was associated with skin test response to recall antigens, T cell responsiveness to cytokines, and, in some patients, cytotoxicity against the immunizing peptides [290]. In other studies of patients with prostate cancer, vaccination with DCs pulsed with tumor-associated peptides has resulted in antigen-specific T cell responses and disease stabilization in a subset of patients [212, 291, 292]. A phase I trial was conducted in which ten patients with breast and ovarian cancer underwent vaccination with DCs pulsed with Her2neu- or MUC1-derived peptides [293]. In five out of ten patients, peptide-specific CTL responses were noted with immunodominance of two particular epitopes. DCs pulsed with Her2neu peptide and activated with IFNg and LPS were administered intranodally to patients with DCIS results in T cell responses, decreased hers2neu expression in the resected tumor tissue and regression in areas of disease [125]. Patients undergoing vaccination with DC pulsed with peptides eluted from CNS tumors demonstrated cellular immune response and intratumoral T cell infiltration [294]. Response to vaccination of patients with glioblastoma with DCs pulsed with tumoreluted peptides was inversely related to expression of TGFb in the tumor bed [295]. Vaccination of patients with hepatocellular carcinoma with DC pulsed with alpha fetoprotein resulted in the development of T cell immunity against this tumor-associated antigen [296]. In a study of 13 patients with colon cancer undergoing vaccination with DCs matured with IFNg and Klebsiella wall fraction, CEA-specific responses were noted in a minority of patients but no clinical responses were noted [297]. In another study, vaccination of patients with colon cancer resulted in immunologic responses against CEA and disease stabilization in a subset of patients [298]. DC-based vaccines using telomerase peptide has also been successfully employed for generating tumor-specific immunity [299, 300]. Pre-clinical models have demonstrated that peptides altered to enhance binding to the MHC complex may be associated with enhanced immunogenicity. Vaccination with DCs pulsed with a mutant CEA or p53 peptide was associated with responses against the wild-type antigen and disease response [301, 302]. Another strategy that is examined in the clinical setting has been the vaccination of patients with DCs pulsed with tumor-associated proteins. In one study, patients with low-grade lymphoma underwent vaccination with DCs pulsed with idiotype protein. In an initial report, all the patients showed evidence of idiotype-specific cellular immunity, while humoral responses were absent [303]. Eight out of ten patients demonstrated idiotype-specific cellular immunity. Disease response was seen in four patients, including two patients who experienced a complete response. In a subsequent report, of patients undergoing vaccination following completion of chemotherapy, 70% remained without evidence of progression with a median follow up of 43 months [304]. In a study of 11 patients with multiple myeloma, who were treated with CD34derived DCs pulsed with idiotype protein, four out of ten patients developed evidence of increased idiotype-specific cellular immunity as determined by ELIspot analysis, and three patients showed evidence of anti-idiotype tumoral response [305]. One patient demonstrated evidence of marrow regression of plasma cells. Another study examined the impact of the infusion of DCs pulsed with idiotype protein following high-dose chemotherapy with stem cell rescue in patients with multiple myeloma [306]. Four out of 26 patients demonstrated evidence of idiotype-specific T cell proliferative responses in the

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post-transplant period. In another study of patients with myeloma, subcutaneous as compared to intravenous vaccination with DCs pulsed with the idiotype protein resulted in T cell anti-tumor immunity [307]. DCs loaded with tumor-associated RNA has been shown to stimulate antitumor immune responses in patients with prostate cancer [308]. Vaccination was associated with reduction of the log slope in PSA and molecular evidence of circulating tumor cells in a subset of patients. Vaccination with DCs pulsed with renal carcinoma-derived RNA-induced immune responses against a broad array of tumor-specific antigens including G250 and oncofetal antigen [309]. In a similar study, immunologic responses were not improved following intranodal as compared to intradermal injection [310]. In another study, vaccination with DCs pulsed with telomerase reverse transcriptase RNA and lysosomal-associated membrane protein 1 (LAMP1) induced antigen-specific CD4 and CD8 T cell responses and was associated with a decrease in levels of disease as measured by surrogate markers [246]. Insertion of tumor-associated antigens into DCs for vaccination has also been examined using viral vectors. Vaccination with DCs transduced ex vivo with fowl pox virus engineered to express CEA resulted in a minor response or transient disease stabilization in 14 patients with advanced colon or lung cancer. T cell responses to CEA were higher in those patients with evidence of clinical benefit [311]. Another strategy that has been explored in clinical trials involves the use of whole tumor cells as a source of antigen for DC-based vaccines. Vaccination with DC pulsed with autologous tumor lysate has been pursued by multiple investigators [312, 313]. In a study of patients with melanoma, DCs pulsed with autologous lysate appeared more immunologically potent than those pulsed with peptides in stimulating T cell-mediated IFNg responses [314]. In a trial of patients with glioma, vaccination resulted in disease response and stabilization in a subset of patients with longer survival seen in patients with undergoing both intratumoral and intradermal vaccination [131]. In a study of nine patients with renal carcinoma undergoing vaccination with DCs pulsed with renal carcinoma lysate, five patients experienced stable disease and one achieved a partial response and these patients demonstrated treater antigenspecific proliferative responses ex vivo than those with progressive disease [315]. Vaccination with DCs pulsed with killed allogeneic melanoma cells induced disease regression and tumor antigen-specific immune responses in a subset of patients with advanced disease [316]. Of note, some patients experienced extended overall survival raising the possibility that anti-tumor immunity may demonstrate clinical benefit even in those patients without overt disease regression. Similarly, vaccination of patients with non-small lung cancer with DCs pulsed with necrotic malignant cells obtained from pleural effusions resulted in the induction of tumor-specific T cells in a minority of patients who experience disease stabilization [317]. Alternatively, patients have undergone vaccination with apoptotic cells derived from a non-small cell carcinoma cell line that overexpressed the tumor antigens, survivin, Her2/neu, CEA, MUC1, and WT1 [318]. Another promising strategy for DC-based immunization involves the use of DCs fused with patient-derived tumor cells. In one study, 23 patients with advanced breast and renal carcinoma, who underwent vaccination with autologous DC/tumor fusions, were considered [319]. Vaccination was well tolerated and not associated with the development of autoimmunity. 10/18 evaluable

Chapter 45  Dendritic Cells 

patients demonstrated evidence of anti-tumor immunity as manifested by an increase in the percent of circulating T cells that expressed IFNg following ex vivo exposure to autologous tumor lysate. Two patients demonstrated evidence of disease regression and an additional six patients had stabilization of the disease. In a study of 15 patients with glioma, vaccination with autologous DC/ tumor fusions in conjunction with IL-12 resulted in >50% regression in four patients [320]. Vaccination of 17 patients with melanoma with patient-derived tumor cells fused with allogeneic DCs resulted in one complete remission, one partial response and six disease stabilization. Fourteen patients demonstrated evidence of immunologic response as manifested by T cell response to tumor antigens. Progression was associated with loss of antigen expression and presentation [321]. In another study, 24 patients with metastatic renal carcinoma underwent vaccination with autologous tumor cells fused with DCs generated from normal donors. Ten patients demonstrated evidence of immunologic response against antigens in autologous tumor lysate. Ten out of 20 evaluable patients experienced either disease regression or stabilization. A statistically significant association between immunologic and clinical response was noted [322].

11. DC Immunotherapy: Potential Limiting Factors While DC-based immunotherapy has emerged as a promising therapeutic strategy, its clinical role has not been defined. In a randomized study of patients with melanoma, DC vaccines were not shown to improve outcomes as compared to standard DTIC therapy [323]. The nature of antigen loading, DC generation, vaccine administration, and the schedule of priming and boosting are all likely to be essential in determining the effectiveness of vaccination [14, 324]. Tumor cells generate an immunosuppressive environment that disrupts the function of host antigen presenting and effector cells and allows for their escape from host immunosurveillance. Tumor-mediated immunosuppression may prevent response to DC-based vaccination. Tumor cells secrete factors such as VEGF, TGFb, IL-6, IL-10, and M-CSF which inhibit DC maturation [207, 325]. Tumor cells secrete high levels of MIP-3a which fosters the migration of immature DCs into the tumor bed and exert a tolerizing influence on host immunity. In contrast, the more functionally active mature DCs are characteristically found in the peritumoral areas [168]. The tumor-associated antigens, MUC-1 and HER-2/neu, are internalized by DCs, but are not consistently transported to late MHC class II endosomes, thus abrogating their ability to undergo appropriate processing and presentation [326]. DCs isolated from peripheral blood and tumor-draining lymph nodes were studied in 93 patients with breast, head and neck, and lung cancer [327]. Decreased numbers of circulating mature DCs was noted and impaired function was seen in DCs derived from both lymph nodes and blood, suggesting a systemic effect of the tumor. Partial reversal of these findings was noted after tumor resection. In contrast, functionally active DCs derived from patients with malignancy can be generated from precursor populations cultured in vitro with cytokines. DCs generated from patients with multiple myeloma, breast cancer, lymphoma, and renal cancer have been shown to prominently express co-stimulatory molecules and stimulate autologous and allogeneic T cell responses [218, 223–225, 328, 329]

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Tumor cells suppress T cell function through a variety of mechanism that limit the capacity of patients with malignant disease to respond to vaccination. T cell dysfunction has correlated with disease bulk. Activation of inhibitory pathways such as those mediated by PDL1 inhibits cytotoxic responses. Migration of tumor-reactive T cells to the tumor bed may also be a target of tumor-mediated immune suppression. In one study, functionally potent Melan-A-specific T cells were detected in the circulation but T cells isolated from the tumor bed demonstrated a suppressive phenotype [330]. Regulatory T cells play a central role in tumor-mediated tolerance, predict for clinical outcomes and may inhibit responses to DC based tumor vaccines [331, 332]. Regulatory T cells are increased in the tumor bed, draining lymph nodes and circulation of patients with malignancy [333]. Circulating levels of regulatory cells may be paradoxically increased following DC-based vaccination. In animal models, depletion of regulatory cells enhances vaccine efficacy and the capacity to induce tumor rejection [103, 162]. In a clinical study, vaccination in conjunction with anti-CD25 linked to diphtheria toxin (ONTAK) resulted in the transient depletion of regulatory cells and a corresponding increase in tumor-reactive T cell responses [103]. The use of chemotherapy prior to vaccination has also been shown to deplete circulating regulatory T cells that may potentially provide an improved platform for DC immunization. Of note, animal models have demonstrated that a transient increase capacity to respond to tumor vaccines is noted following high-dose chemotherapy with stem cell rescue. It is thought that tumor-mediated tolerance is disrupted during the early lymphopoietic reconstitution period post-transplant, potentially due to the elimination of regulatory T cell populations [334]. In an animal model, vaccination with lysate-pulsed DCs following autologous transplantation was associated with heightened and more durable responses [335].

12. Conclusion DCs are potent antigen-presenting cells that play a crucial role in the initiation of cellular immunity and maintenance of the delicate balance between tolerance and immune recognition. DC biology plays an important role in posttransplant immune reconstitution and the development of GVHD. The use of DC-based immunotherapy has emerged as a major field of investigation and has yielded promising preliminary findings. Its integration into hematopoietic stem cell transplantation offers a potential avenue to modulate tumor-specific immunity and improve outcomes: Ongoing efforts in this arena will hopefully bear fruit in the struggle to generate clinically meaningful immunotherapeutic strategies.

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Chapter 45  Dendritic Cells  4. Young JW, Koulova L, Soergel SA, Clark EA, Steinman RM, Dupont B (1992) The B7/BB1 antigen provides one of several costimulatory signals for the activation of CD4+ T lymphocytes by human blood dendritic cells in vitro. J Clin Invest 90:229–237 5. Inaba K, Witmer-Pack M, Inaba M, Hathcock KS, Sakuta H, Azuma M, Yagita H, Okumura K, Linsley PS, Ikehara S et al (1994) The tissue distribution of the B7–2 costimulator in mice: abundant expression on dendritic cells in situ and during maturation in vitro. J Exp Med 180:1849–1860 6. Kelsall BL, Stuber E, Neurath M, Strober W (1996) Interleukin-12 production by dendritic cells. The role of CD40-CD40L interactions in Th1 T-cell responses. Ann N Y Acad Sci 795:116–126 7. Qi H, Egen JG, Huang AY, Germain RN (2006) Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science 312:1672–1676 8. Lucas M, Schachterle W, Oberle K, Aichele P, Diefenbach A (2007) Dendritic cells prime natural killer cells by trans-presenting interleukin 15. Immunity 26:503–517 9. Fujii S, Shimizu K, Kronenberg M, Steinman RM (2002) Prolonged IFN-gammaproducing NKT response induced with alpha-galactosylceramide-loaded DCs. Nat Immunol 3:867–874 10. Bernhard H, Disis ML, Heimfeld S, Hand S, Gralow JR, Cheever MA (1995) Generation of immunostimulatory dendritic cells from human CD34+ hematopoietic progenitor cells of the bone marrow and peripheral blood. Cancer Res 55:1099–1104 11. Caux C, Vanbervliet B, Massacrier C, Dezutter-Dambuyant C, de Saint-Vis B, Jacquet C, Yoneda K, Imamura S, Schmitt D, Banchereau J (1996) CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNF alpha. J Exp Med 184: 695–706 12. Romani N, Gruner S, Brang D, Kampgen E, Lenz A, Trockenbacher B, Konwalinka G, Fritsch PO, Steinman RM, Schuler G (1994) Proliferating dendritic cell progenitors in human blood. J Exp Med 180:83–93 13. Baskar S, Ostrand-Rosenberg S, Nabavi N, Nadler LM, Freeman GJ, Glimcher LH (1993) Constitutive expression of B7 restores immunogenicity of tumor cells expressing truncated major histocompatibility complex class II molecules. Proc Natl Acad Sci USA 90:5687–5690 14. Gilboa E (2007) DC-based cancer vaccines. J Clin Invest 117:1195–1203 15. Morelli AE, Thomson AW (2007) Tolerogenic dendritic cells and the quest for transplant tolerance. Nat Rev Immunol 7:610–621 16. Rutella S, Danese S, Leone G (2006) Tolerogenic dendritic cells: cytokine modulation comes of age. Blood 108:1435–1440 17. Steinman RM, Hawiger D, Nussenzweig MC (2003) Tolerogenic dendritic cells. Annu Rev Immunol 21:685–711 18. Ochando JC, Homma C, Yang Y, Hidalgo A, Garin A, Tacke F, Angeli V, Li Y, Boros P, Ding Y, Jessberger R, Trinchieri G, Lira SA, Randolph GJ, Bromberg JS (2006) Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts. Nat Immunol 7:652–662 19. Steinman RM, Nussenzweig MC (2002) Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc Natl Acad Sci USA 99:351–358 20. Young JW, Merad M, Hart DN (2007) Dendritic cells in transplantation and immune-based therapies. Biol Blood Marrow Transplant 13:23–32 21. Auletta JJ, Lazarus HM (2005) Immune restoration following hematopoietic stem cell transplantation: an evolving target. Bone Marrow Transplant 35:835–857 22. Ueno H, Klechevsky E, Morita R, Aspord C, Cao T, Matsui T, Di Pucchio T, Connolly J, Fay JW, Pascual V, Palucka AK, Banchereau J (2007) Dendritic cell subsets in health and disease. Immunol Rev 219:118–142

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Chapter 45  Dendritic Cells  cells can induce objective clinical responses and MART-1 specific CD8+ T-cell immunity. J Immunother 29:545–557 317. Chang GC, Lan HC, Juang SH, Wu YC, Lee HC, Hung YM, Yang HY, WhangPeng J, Liu KJ (2005) A pilot clinical trial of vaccination with dendritic cells pulsed with autologous tumor cells derived from malignant pleural effusion in patients with late-stage lung carcinoma. Cancer 103:763–771 318. Hirschowitz EA, Foody T, Hidalgo GE, Yannelli JR (2007) Immunization of NSCLC patients with antigen-pulsed immature autologous dendritic cells. Lung Cancer 57:365–372 319. Avigan D, Vasir B, Gong J, Borges V, Wu Z, Uhl L, Atkins M, Mier J, McDermott D, Smith T, Giallambardo N, Stone C, Schadt K, Dolgoff J, Tetreault JC, Villarroel M, Kufe D (2004) Fusion cell vaccination of patients with metastatic breast and renal cancer induces immunological and clinical responses. Clin Cancer Res 10:4699–4708 320. Kikuchi T, Akasaki Y, Abe T, Fukuda T, Saotome H, Ryan JL, Kufe DW, Ohno T (2004) Vaccination of glioma patients with fusions of dendritic and glioma cells and recombinant human interleukin 12. J Immunother 27:452–459 321. Trefzer U, Herberth G, Wohlan K, Milling A, Thiemann M, Sherev T, Sparbier K, Sterry W, Walden P (2004) Vaccination with hybrids of tumor and dendritic cells induces tumor-specific T-cell and clinical responses in melanoma stage III and IV patients. Int J Cancer 110:730–740 322. Avigan DE, Vasir B, George DJ, Oh WK, Atkins MB, McDermott DF, Kantoff PW, Figlin RA, Vasconcelles MJ, Xu Y, Kufe D, Bukowski RM (2007) Phase I/ II study of vaccination with electrofused allogeneic dendritic cells/autologous tumor-derived cells in patients with stage IV renal cell carcinoma. J Immunother 30:749–761 323. Schadendorf D, Ugurel S, Schuler-Thurner B, Nestle FO, Enk A, Brocker EB, Grabbe S, Rittgen W, Edler L, Sucker A, Zimpfer-Rechner C, Berger T, Kamarashev J, Burg G, Jonuleit H, Tuttenberg A, Becker JC, Keikavoussi P, Kampgen E, Schuler G (2006) Dacarbazine (DTIC) versus vaccination with autologous peptide-pulsed dendritic cells (DC) in first-line treatment of patients with metastatic melanoma: a randomized phase III trial of the DC study group of the DeCOG. Ann Oncol 17:563–570 324. Masopust D, Ha SJ, Vezys V, Ahmed R (2006) Stimulation history dictates memory CD8 T cell phenotype: implications for prime-boost vaccination. J Immunol 177:831–839 325. Perrot I, Blanchard D, Freymond N, Isaac S, Guibert B, Pacheco Y, Lebecque S (2007) Dendritic cells infiltrating human non-small cell lung cancer are blocked at immature stage. J Immunol 178:2763–2769 326. Hiltbold EM, Vlad AM, Ciborowski P, Watkins SC, Finn OJ (2000) The mechanism of unresponsiveness to circulating tumor antigen MUC1 is a block in intracellular sorting and processing by dendritic cells. J Immunol 165: 3730–3741 327. Almand B, Resser JR, Lindman B, Nadaf S, Clark JI, Kwon ED, Carbone DP, Gabrilovich DI (2000) Clinical significance of defective dendritic cell differentiation in cancer. Clin Cancer Res 6:1755–1766 328. Avigan D, Wu Z, Joyce R, Elias A, Richardson P, McDermott D, Levine J, Kennedy L, Giallombardo N, Hurley D, Gong J, Kufe D (2000) Immune reconstitution following high-dose chemotherapy with stem cell rescue in patients with advanced breast cancer. Bone Marrow Transplant 26:169–176 329. Chaperot L, Chokri M, Jacob MC, Drillat P, Garban F, Egelhofer H, Molens JP, Sotto JJ, Bensa JC, Plumas J (2000) Differentiation of antigen-presenting cells (dendritic cells and macrophages) for therapeutic application in patients with lymphoma. Leukemia 14:1667–1677

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Chapter 46 Augmentation of Hematopoietic Stem Cell Transplantation with Anti-cancer Vaccines Edward D. Ball and Peter R. Holman

1. Introduction Hematopoietic stem cell transplantation (HSCT) is a curative therapy for a variety of hematological malignancies including acute and chronic leukemia, non-Hodgkin lymphoma, and Hodgkin lymphoma [1]. In addition, better disease control and improved survival can be achieved in patients with multiple myeloma (MM), though it is presently unclear whether patients can be cured with HSCT [2]. However, relapse of the underlying malignant disease is still a significant clinical problem. After autologous HSCT, the relapse rate is as high as 60% while after allogeneic HSCT up to 30% of patients relapse [3]. If relapse occurs, the prognosis is generally poor. Thus, new and more effective treatments of relapse and/or means of preventing relapse are urgently needed. It is widely believed that much of the success of allogeneic HSCT for patients with leukemia and lymphoma is due to the graft-versus-leukemia (GVL) effect [4]. This belief is based on the observations that GVHD correlates with superior disease control [4] and that clinical responses result from maneuvers such as infusions of donor lymphocytes and withdrawing immunosuppression [5]. The target antigens recognized by the donor immune system are not wellcharacterized and certainly are not identified in routine clinical practice [6]. Many tumor-associated antigens (TAA) have been described (see Table 461). For example, leukemia cells from patients with acute myeloid leukemia (AML) express antigens such as WT-1, PR1, and others [7]. The most tumor-specific antigen known is the idiotype of the surface immunoglobulin, expressed by non-Hodgkin lymphoma cells [8]. This unique region of the immunoglobulin molecule allows selective targeting of essentially each malignant lymphoma cell without cross-reaction with normal cells. The general purpose of vaccines in the context of stem cell transplantation is to amplify the immune response to tumor antigens at a time of tumor

From: Allogeneic Stem Cell Transplantation, Contemporary Hematology, Edited by: H.M. Lazarus and M.J. Laughlin, DOI 10.1007/978-1-59745-478-0_46, © Springer Science + Business Media, LLC 2003, 2010

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Table 46-1.  Potential leukemia-associated antigenic targets. Antigen

Disease

Reference

WT-1

AML

[6]

Proteinase 3

AML, CML

[7]

HAGE

CML

[6]

Survivan

CLL, NHL, CML, MM

[6]

RHAMM

AML, MDS, MM, CML, CLL

[6]

PRAME

CML, CLL, ALL

[6]

HTERT

CML, CLL

[6]

MPP11

CML, CLL

[6]

Ig Idiotype

NHL, MM

[8]

vulnerability. Success is dependent on choosing the right antigen, engaging the immune system effectively, and choosing the optimal clinical setting in which to observe definitive results. This chapter will review this promising new approach to improving outcomes through reduction of relapse following HSCT. There are few published clinical trial results but many interesting trials are in progress or in the planning stage. This chapter will focus on the underlying principles of “vaccine”-based therapeutic approaches in hematological malignancy, a review of clinical trials underway, and of vaccine approaches in the developmental phase of clinical application.

2. Types of Vaccines The term vaccine in the setting of cancer vaccines is somewhat misleading. Traditionally, vaccines have been defined as materials used to prevent disease, such as the polio vaccine, an attenuated strain of virus, that induces an immune response in an unaffected individual that protects if challenged with live wildtype virus. In cancer therapy, “vaccine” has evolved to engender the use of both molecular and cellular products in affected individuals to induce primary immune responses to cancer cells. In a sense, they are attempts to break tolerance to cancer-associated antigens. Vaccines may, therefore, consist of a peptide or whole protein with sequences known to be expressed more or less exclusively on cells of the cancer, or, at least, its specific cellular lineage. Alternatively, cytotoxic T lymphocytes may be generated that recognize relatively tumor-specific antigens and that can be directly infused into the circulation of afflicted patients. Further, such TAA-directed T cells may be targeted further through genetic manipulation (transduction of membrane-bound antibodies: e.g., anti-CD19 or 20). Some of the advantages and disadvantages of these approaches are summarized in Table 46-2.

Chapter 46  Augmentation of Hematopoietic Stem Cell Transplantation 

Table 46-2.  Advantages and disadvantages of various vaccine approaches. Advantages

Disadvantages

Protein-based vaccine (e.g., ID, PR-1)

Ease of administration, inexpensive

All malignant cells may not express target antigen. CTL precursors may be limited

Cell-based vaccine

Generation of polyclonal responses

Cumbersome to generate cells. Requires GMP facility. Potential autoimmunity

Immunomodulation e.g., Ease of administration, Potential autoimmune events. anti-CTLA-4 monogeneration of polyclonal No FDA-approved reagents clonal antibody response at present

3. Clinical Settings 3.1. NHL and Multiple Myeloma 3.1.1. Vaccinations in Transplantation for Lymphoma and Myeloma Non-Hodgkin’s lymphoma (NHL) and Multiple Myeloma (MM) are both attractive disorders to target for the development of new immunotherapeutic approaches. Both these groups of diseases are amenable to immunotherapeutic approaches which have already been successful to varying degrees. Supporting the application of immune therapies to lymphoma is the observation of spontaneous remissions seen in a small number of patients with follicular lymphoma [9], the identification of an immune signature within follicular lymphoma as a good prognostic marker [10], the success of the monoclonal antibody rituximab [11] and the efficacy of allogeneic transplantation [12]. Bone marrow- or blood-derived stem cell transplantation is frequently offered to patients with NHL and MM and this setting offers both challenges and opportunities for the development of further innovative immunotherapeutic strategies. The recognition that a graft versus disease effect is primarily responsible for the curative potential of allogeneic transplantation has resulted in the emergence and widespread application of nonmyeloablative allogeneic transplantation protocols. However, owing to the lack of an available matched donor, this form of active immunotherapy is not applicable to the vast majority of patients. The success of monoclonal antibody therapy, a form of passive immunotherapy, and allogeneic transplantation, a form of active immunotherapy, clearly illustrate the susceptibility of these disorders to immunotherapeutic approaches. In NHL and MM, both clonal B cell disorders, the malignant clone as with normal B lymphocytes undergo rearrangement of the V, D and J genes during development. As a result, all cells of the malignant clone express immunoglobulins with the same unique variable sequence termed the idiotype. The idiotype can function as a tumor-specific antigen and as a result, these B cell-derived malignancies should be particularly well suited to a vaccinebased immunotherapeutic approach. Additional methods of inducing an active immune response are also being explored in these diseases.

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3.1.2. Autologous Transplantation High-dose chemotherapy with autologous stem cell transplant (HDT/ASCT) is widely applied in the management of both lymphoma and myeloma. When used to treat patients with indolent lymphoma, progression-free survival is improved but relapse occurs in virtually all patients [13]. Relapse is most likely due to disease surviving the high-dose therapy and/or lymphoma cells surviving in the graft. Similarly, for patients with relapsed aggressive lymphoma an improved OS for patients receiving high-dose therapy and autologous bone marrow transplantation has been established by Philip et al. [14]. Patients with mantle cell lymphoma also frequently undergo HDT/ASCT with an improvement in progression-free survival (PFS) [15, 16]. In MM, even though the introduction of newer targeted therapies is challenging this approach, HDT/ASCT (single or tandem) is widely accepted as initial standard consolidation therapy for younger patients with stage 2 and 3 disease [17, 18]. In all of these situations, however, with the exception of the aggressive lymphomas, HDT/ASCT is not curative. For this reason, many patients will be offered an allogeneic approach. In the case of the aggressive lymphomas, the relapse rate post-autologous transplant is still significant and a further improvement in outcome is required. 3.1.3. Post-autologous Transplant Immunization Idiotype vaccination has been evaluated in the autologous transplant setting in both MM and NHL. A report from the Stanford group described 26 MM patients who received idiotype vaccination following HDT/ASCT. Initially, in the setting of minimal residual disease post-transplant, patients received two intravenous infusions of dendritic cells pulsed with Id or Id-KLH. Subsequently, they received subcutaneous injections of Id-KLH. 24/26 patients developed a KLH-specific immune proliferative response but only 4 (3 in CR) developed an Id-specific immune response [19]. In another report from Stanford, 12 patients with relapsed or refractory B cell lymphomas receiving idiotype vaccination following HDT/ASCT were evaluated for the development of an immune response. Two different vaccination approaches were utilized, one with Id-KLH + GM-CSF and the other also incorporated the use of dendritic cells. Patients received vaccinations starting at 2–12 months post-transplant and all developed KLH-specific immune responses, supporting the use of active immunotherapy in the post-transplant time period. Ten patients developed an Id-specific humoral or cellular immune response [20]. At UCSD, we have conducted a pilot study of idiotype vaccination following HDC/ASCT. Fifteen patients with mantle cell lymphoma, follicular lymphoma grade 1 or 2 or transformed lymphoma received Id-KLH + GM-CSF vaccination. Starting at 3 months following HDT/ASC,T five vaccinations were administered over a 6-month period. Ten patients developed an antiKLH humoral or cellular immune response after one to four immunizations, and seven developed an anti-Id humoral or cellular response after one to five immunizations. Improvements in the clinical response from the early posttransplant status to the end of vaccination were seen in a number of patients and surprisingly, long-lasting remissions were seen in this heavily pre-treated group of patients [21]. At the time this manuscript was prepared, ten of the 15 patients are surviving at a median follow-up time of 55 (range 44–81) months and 7/10 are in complete remission. Further modifications to this approach

Chapter 46  Augmentation of Hematopoietic Stem Cell Transplantation 

could include pre-transplant immunization and continuing vaccinations through the period of immune recovery post-transplant which may allow the response of all arms of the immune system to the vaccination. 3.1.4. Allogeneic Transplantation Allogeneic transplantation is being increasingly offered to carefully selected patients. The development of the reduced intensity and nonmyeloablative conditioning approaches, based on the therapeutic graft-versus-malignancy effect has been increasingly applied and has resulted in a decrease in nonrelapse peri-transplant mortality. A number of trials, mostly small and single center have reported low transplant-related mortality and a low relapse rate in responding patients. There is, however, a significant risk of chronic graft-versus-host disease and other transplant-associated complications which continue to limit the general applicability of this approach. The incidence of graftversus-host disease has tended to correlate with the graft-versus-lymphoma effect but they may occur independently as has been seen in clinical trials [22]. Attempts to separate these effects are ongoing and progress is being made in the identification of lymphocyte subsets that are involved in each of these processes; reviewed in [23]. An improvement in the outcome following allogeneic transplants would occur if the incidence of graft-versus-host disease could be decreased while specific methods to augment graft-versus-malignancy could be enhanced. Disease relapse remains a considerable problem following allogeneic transplantation and molecular remission status is an important predictor of freedom from relapse. Inducing molecular remission following an allogeneic transplant may be achieved with donor lymphocyte infusions; however, this carries a significant risk of graft-versus-host disease. Vaccine approaches may eventually be a safer means of accomplishing this. Meanwhile, these improvements have extended the opportunity to undergo this form of immunotherapy to older patients and those with comorbidities who would have otherwise been excluded from an ablative allogeneic transplant. 3.1.5. Immunotherapy Including Idiotype Directed Therapy Before applying the concept of active immunotherapy with tumor vaccines to the transplant setting for lymphoma and myeloma, a little background information may be helpful. Passive immunotherapy with antibodies such as rituximab has proven to be a very successful therapy [24]. The mechanism of action of this antibody continues to be debated but is thought to function primarily through antibody-dependant cellular cytotoxicity (ADCC). Additionally, the direct induction of apoptosis and complement-mediated cytotoxicity are felt to be important. Recently, it has been postulated that an additional mechanism for rituximab is a vaccinal effect, whereby the dying cell releases components that are taken up by antigen-presenting cells from where an immune response could be initiated [25]. Whether rituximab does have active immunotherapeutic properties remains to be proven. Unlike passive immunotherapy, active immunotherapy with a vaccine has the potential to induce a polyclonal cellular and humoral immune response with a memory component that may prove to be more durable. The potential of idiotype-directed immunotherapy was first demonstrated in the early 1970s. Lynch et al. demonstrated the immunogenicity of myelomarelated proteins and the ability of the resulting antibodies to suppress growth of the corresponding tumor cells [26]. In 1987, it was shown that mice with

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lower, rather than greater, tumor burdens had a greater ability to develop an idiotype-specific immune response. As is the case with most tumor associated antigens, the idiotype protein is a relatively weak immunogen and subsequent studies demonstrated the utility of adding a more immunogenic carrier protein such as KLH to augment the immunogenicity of the administered proteins [27]. A further advance was the administration of an adjuvant in the form of GM-CSF which acts by recruiting antigen-presenting cells to the region of protein administration [8]. Idiotype vaccination has been evaluated in phase 1 and 2 clinical trials. In a pilot study from Stanford, nine patients received idiotype vaccination in the setting of minimal residual disease or complete response following chemotherapy. Two patients developed an idiotype-specific humoral response, four developed an idiotype-specific cellular immune response and one developed a humoral and cellular immune response [28]. Subsequently, the result of a larger cohort of patients was reported. Id-specific immune responses were demonstrated amongst a cohort of 41 patients with follicular lymphoma, also with either a CR or with minimal residual disease following chemotherapy. Forty-one percent of patients developed an anti-idiotype antibody response and 17% developed an anti-idiotype cellular response. Two patients with residual disease achieved a complete response along with the development of an idiotype-specific immune response [29]. Bendandi et al. reported the results of a further clinical trial of idiotype vaccination in 20 patients with follicular lymphoma. All were in the first CR after chemotherapy and were vaccinated starting 6 months after completing chemotherapy. This delay was included to allow recovery of the immune response prior to idiotype vaccination. All were in a CR at the time of vaccination. All patients developed anti-KLH cellular and humoral responses. Anti-idiotype humoral responses developed in 15 of the 20 patients and anti-idiotype cellular responses were noted in 19 patients. Importantly for establishing clinical efficacy, minimal residual disease as demonstrated by a positive test for the t(14;18) was present in 11 patients following chemotherapy, prior to idiotype vaccination. Eight of these patients became negative following vaccination, suggesting an idiotype-specific clinically relevant benefit. In 19 of the 20 patients, an HLA class 1-restricted idiotype-specific cellular proliferation was identified when post-vaccine peripheral blood mononuclear cells were co-cultured with autologous follicular lymphoma cells. This suggested the importance of a cytotoxic cellular response to the anti-lymphoma activity [30]. A long-term follow-up report of this study has been published and with a median follow-up of 9.2 years, the median disease free survival was 8 years and the overall survival rate was 95% [31]. Other studies have suggested the importance of a humoral cellular response for therapeutic benefit. It is likely that components of both may be important; however, following rituximab administration, cellular immune responses can be identified in the absence of humoral responses. The clinical utility of idiotype vaccination is currently being evaluated in three randomized clinical trials. The method of idiotype preparation varies but in all the three, the final product is idiotype protein complexed to the immunogenic carrier protein KLH and administered along with GM-CSF as an adjuvant. In one trial, hybridoma methodology is used for vaccine preparation. In the other two, recombinant DNA techniques are used. The latter

Chapter 46  Augmentation of Hematopoietic Stem Cell Transplantation 

techniques allow for a shorter preparation time, allowing for a more rapid time to administration. The trial designs also differ. In two of the trials, patients receive chemotherapy prior to vaccination. They are required to have at least a partial response in order to receive vaccination. In the third trial, patients are vaccinated following rituximab given alone. Patients who have a response to rituximab, or who have stable disease can be vaccinated. As the role of the humoral response is unclear, maintenance vaccinations are continued as B cell recovery occurs following rituximab and also beyond until there is evidence of disease progression. Individual patient-specific protein vaccines are cumbersome, being laborintensive and time-consuming. Alternative formulations to induce idiotypespecific immunity are under development. These include DNA vaccines [32]. Additionally, insight from studies evaluating the mechanism of action of rituximab and other antibodies is spurring on the development of more effective passive immunotherapies which may render the more cumbersome patient-specific therapies less useful. The peritransplant period has not been extensively evaluated as an opportunity for therapeutic vaccination. In both autologous and allogeneic transplantation, there is a long-lasting immune deficit that was felt to render attempts at augmenting tumor-specific immunotherapy approaches futile. However, a number of different strategies to augment disease control without increasing graft-versus-host disease have been developed in both myeloma and lymphoma. 3.1.6. Donor Immunization One strategy involves immunizing allogeneic stem cell donors. Kwak et al. and Neelapu et  al. have reported the use of myeloma-associated idiotype conjugated to an immunogenic carrier protein and emulsified in an adjuvant to immunize healthy sibling donors prior to allogeneic bone marrow transplant. They demonstrated the induction of Id- and carrier-specific T cell responses in three of the five evaluable recipients who survived more than 30 days post-transplantation. Two of the three patients remained disease free 7 and 8 years post-transplant. The third patient died from renal failure 5.5 years post-transplant while in complete remission [33, 34]. An alternative strategy involves the ex vivo priming of allogeneic donor T cells with recipient Id-KLH-pulsed dendritic cells [35]. Minor Histocompatibility antigendirected donor T cells are felt to mediate graft-versus-malignancy effects. Augmenting the presentation of such antigens is another strategy to augment graft-versus-malignancy following allogeneic transplantation. HA-1 and HA-2 are two such minor histocompatibility antigens that are restricted to hematopoietic lineages. HA-1- and HA-2-positive patients with leukemia and myeloma have been treated with DLI from HA-1- or HA-2-negative donors with the appearance of HA-1- and HA-2-specific CD8+ T cells 5–7 weeks following DLI, coinciding with the induction of a complete remission [36]. Recently, an additional minor histoincompatibility antigen (LB-ADIR-1F) relevant to a myeloma was reported following the study of a tumor-reactive clone that resulted in a complete remission following donor lymphocyte infusion in a patient with relapsed multiple myeloma [37]. These antigens and others may be useful in the development of immunotherapeutic strategies following allogeneic transplantation.

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3.2. Leukemia 3.2.1. Peptide Immunization Leukemia cells are subject to immune attack through their expression of cell surface antigens that are either unique to the particular leukemia or that are lineage-associated. Several leukemia-associated antigens (LAA) expressed on AML cells have been described [6]. These include a peptide-derived from proteinase 3, known as PR1 [6], the Wilm’s tumor antigen WT-1 [6], and many others [6]. Vaccination strategies using these peptide vaccines are underway [7, 38–40]. The group from M.D. Anderson reported that PR1 vaccination elicited immunological responses after hematopoietic stem cell transplantation in 55% of 20 patients [38]. Immune response was defined as a >twofold increase in PR1-CTL by tetramer assay. There was good correlation between immune and clinical responses with 9/11 immunoresponsive patients having a clinical disease response. 3.2.2. Cell-Based Therapies Another therapeutic approach is the generation of cytotoxic T cells reactive with known or unknown antigens expressed on AML cells. At UCSD, we have generated polyclonal T cell cultures, as well as T cell lines that are cytotoxic to autologous AML cells and that could be used in adoptive immunotherapy after autologous stem cell transplantation. This was accomplished by co-culturing AML and normal T cells from the peripheral blood of patients with active disease in the presence of GM-CSF and IL-4 [41, 42]. In these cultures, AML blasts differentiated into dendritic cells that presented antigen(s) to the autologous T cells. The T cells are then expanded through the addition of IL-2 and OKT3 to the cultures. Large quantities of T cells (1010) that are cytotoxic to autologous AML cells can be generated in a closed bag system. We are currently preparing to initiate a clinical trial of autologous stem cell transplantation for patients with AML in remission followed by infusion of the CTL. Blood will be obtained from the patients at diagnosis and the mononuclear cells cryopreserved. The patients will then undergo chemotherapy according to standard treatment protocols. If a patient is considered to be eligible for autologous stem cell transplantation (no HLA-match sibling donor, intermediate- to good-risk cytogenetics) their cryopreserved cells will be thawed and placed into the culture system designed to generate dendritic cell differentiation followed by expansion of autoreactive T cells. The patient will then undergo an autologous stem cell transplant using Bu/ Cy conditioning. After engraftment, the patients will then receive an infusion of CTLs generated from the cultures that were initiated prior to PBSCT. The study will use graded doses of T cells starting with 0.5 × 108 cells/kg body weight, then 108, and then 2 × 108 cells/kg. End points of the study include safety (absence of significant auto-immune events) and efficacy (with comparisons to our historical control database). Given the known correlation of graft-versus-host disease and control of leukemia after allogeneic stem cell transplantation [4], it is attractive to consider specific methods of amplifying anti-leukemia responses after allogeneic transplantation. Toward this end, we have studied the use of a cytotoxic T cell antigen (CTLA)-4 blocking human monoclonal antibody (mAb) in patients relapsing after allogeneic stem cell transplantation [43, 44]. This phase I/II study treated patients relapsing after allogeneic HSCT who relapsed and who

Chapter 46  Augmentation of Hematopoietic Stem Cell Transplantation 

did not have GVHD with safety as the primary end point. Patients received a single dose of ipilimumab, an IgG1 human monoclonal antibody that blocks the binding of CD80/86 to CTLA-4 expressed on activated T cells. Ipilimumab, therefore, blocks the down-regulatory effect of CTLA-4 ligation and allows T cells stimulated by antigen-presenting cells to continue proliferating and potentially mediate anti-tumor activities. Fortunately, we have not seen graft-versus-host disease in any of the 29 patients, though we did observe a few breakthrough autoimmune events (Immune Adverse Events). We have seen several intriguing clinical responses including partial and complete remissions of nodal masses in patients with non-Hodgkin Lymphoma and Hodgkin Disease, and a molecular remission in a patient with chronic myeloid leukemia. Examination of lymphocyte subsets following ipilimumab infusion revealed that T regulatory (Treg) cells were not affected, while there were increased numbers of activated T cells in many patients [44]. Rousseau et  al. studied eight patients with high-risk acute leukemia with a cellular vaccine of autologous leukemia cells mixed with fibroblasts transduced with CD40 ligand and IL-2 [45]. CD40L generates immune responses in leukemia-bearing mice, an effect that is potentiated by IL-2. They studied the feasibility, safety, and immunologic efficacy of an IL-2- and CD40Lexpressing recipient-derived tumor vaccine consisting of leukemic blasts admixed with skin fibroblasts transduced with adenoviral vectors encoding human IL-2 (hIL-2) and hCD40L. Ten patients (including seven children) with high-risk acute myeloid (n = 4) or lymphoblastic (n = 6) leukemia in cytologic remission (after allogeneic stem cell transplantation [n = 9] or chemotherapy alone [n = 1]) received up to six subcutaneous injections of the IL-2/CD40L vaccine. No severe adverse reactions were noted. Immunization produced a 10- to 890-fold increase in the frequencies of major histocompatibility complex (MHC)-restricted T cells reactive against recipient-derived blasts. These leukemia-reactive T cells included both T-cytotoxic/T-helper 1 (Th1) and Th2 subclasses, as determined from their production of granzyme B, interferongamma, and interleukin-5. Two patients produced systemic IgG antibodies that bound to their blasts. Eight patients remained disease free for 27–62 months after treatment (5-year overall survival, 90%). Thus, even in heavily treated patients, including recipients of allogeneic stem cell transplants, recipient-derived anti-leukemia vaccines can induce immune responses reactive against leukemic blasts.

4. Future Directions Augmentation of anti-tumor immune recognition and control is an attractive and important goal, given that relapse after both autologous and allogeneic HSCT continues to be one of the obstacles to cure using this therapy. Tantalizing hints of efficacy of various means of boosting immunity are reviewed above. Continued research into the nature of tumor-associated antigenic targets, means of enhancing antigen presentation, and methods of immune cell activation, proliferation and long-term survival will hopefully result in more specific and rational immunotherapy in the future.

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E.D. Ball and P.R. Holman 31. Santos C, Stern L, Katz L, Watson T, Barry G (2005) BiovaxIDTM vaccine therapy of follicular lymphoma in first remission: long-term follow-up of a phase II trial and status of a controlled, randomized phase III trial. ASH Annu Meet Abstr 106:2441 32. Zhu D, Rice J, Savelyeva N, Stevenson FK (2001) DNA fusion vaccines against B-cell tumors. Trends Mol Med 7:566–572 33. Neelapu SS, Munshi NC, Jagannath S, Watson TM, Pennington R, Reynolds C, Barlogie B, Kwak LW (2005) Tumor antigen immunization of sibling stem cell transplant donors in multiple myeloma. Bone Marrow Transplant 36:315–323 34. Kwak LW, Taub DD, Duffey PL, Bensinger WI, Bryant EM, Reynolds CW, Longo DL (1995) Transfer of myeloma idiotype-specific immunity from an actively immunised marrow donor. Lancet 345:1016–1020 35. Kim SB, Baskar S, Kwak LW (2003) In vitro priming of myeloma antigen-specific allogeneic donor T cells with idiotype pulsed dendritic cells. Leuk Lymphoma 44:1201–1208 36. Marijt WAE, Heemskerk MHM, Kloosterboer FM, Goulmy E, Kester MGD, van der Hoorn MAWG, van Luxemburg-Heys SAP, Hoogeboom M, Mutis T, Drijfhout JW et al (2003) Hematopoiesis-restricted minor histocompatibility antigens HA-1or HA-2-specific T cells can induce complete remissions of relapsed leukemia. Proc Natl Acad Sci USA 100:2742–2747 37. van Bergen CAM, Kester MGD, Jedema I, Heemskerk MHM, van LuxemburgHeijs SAP, Kloosterboer FM, Marijt WAE, de Ru AH, Schaafsma MR, Willemze R et al (2007) Multiple myeloma-reactive T cells recognize an activation-induced minor histocompatibility antigen encoded by the ATP-dependent interferonresponsive (ADIR) gene. Blood 109:4089–4096 38. Qazibash MH, Wieder ED, Thall PF, Wang X, Rios RL, Lu S, Kant S, Giralt S, Estey EH, Cortes J, Komanduri K, Champlin RE, Molldrem JJ (2007) PR1 peptide vaccine-induced immune response is associated with better event-free survival in patients with myeloid leukemia. Blood 110:90a 39. Qazibash MH, Wieder ED, Thall PF, Wang X, Rios RL, Lu S, Kant S, Giralt SA, Estey EH, Cortes J, Komanduri K, Champlin RE, Molldrem JJ (2007) PR1 Vaccine-elicited immunological response after hematopoietic stem cell transplantation is associated with better clinical response and event-free survival. Blood 110:178a 40. Rezvani K, Yong ASM, Mielke S, Savani BN, Musse L, Superata J, Jafarpour B, Boss C, Barrett AJ (2007) Leukemia-associated antigen specific t-cell responses following combined PR1and WT1 peptide vaccination in patients with myeloid malignancies. Blood 110:91a 41. Zhong R-K, Rassenti LZ, Kipps TJ, Chen J, Law P, Yu J-F, Ball ED (2002) Sequential modulation of growth factors – a novel strategy for adoptive immunotherapy of acute myeloid leukemia. Biol Blood Marrow Transplant 8:557–568 42. Zhong R-K, Loken M, Lane TA, Ball ED (2006) CTLA-4 blockade by a human monoclonal antibody enhances the capacity of AML-derived dendritic cells to induce T cell responses against AML cells in an autologous culture system. Cytotherapy 8:3–12 43. Bashey A, Medina B, Corringham S, Pasek M, Carrier E, Vrooman L, Streicher H, Lowy I, Solomon SR, Morris LE, Holland K, Mason JR, Soiffer RJ, Ball ED (2007) Clinical trial of therapeutic blockade of CTLA4 with ipilimumab in pPatients with relapse of malignancy following allogeneic hematopoietic transplantation. Blood 113: 1581–8, 2009 44. Zhou JH, Zhong RK, Corringham S, Sapp T, Soiffer R, Mitrovich RC, Lowy I, Bashey A, Ball ED (2007) Flow cytometry analysis of peripheral blood CD4+/ CD25+/FOXP3+ t regulator cells from 11 patients treated with ipilimumab following

Chapter 46  Augmentation of Hematopoietic Stem Cell Transplantation  relapse of malignancy after allogeneic hematopoietic stem, cell transplantation. Blood 110:952a 45. Rousseau RF, Biagi E, Dutour A, Yvon ES, Brown MP, Lin T, Mei Z, Grilley B, Popek E, Heslop HE, Gee AP, Krance RA, Popat U, Carrum G, Margolin JF, Brenner MK (2006) 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 107:1332–1341

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ERRATUM TO:

Allogeneic Stem Cell Transplantation Second Edition Edited by Hillard M. Lazarus University Hospitals Case Medical Center, Cleveland, OH, USA Mary J. Laughlin Case Western Reserve University, Cleveland, OH, USA

Chapter 35  Psychological Care of Adult Allogeneic Transplant Patients Flora Hoodin1, Felicity W.K. Harper 2, and Donna M. Posluszny 3 1

 Department of Psychology, Eastern Michigan University, Ypsilanti, MI, USA [email protected]

2

 Communication and Behavioral Oncology Program, Barbara Ann Karmanos Cancer Institute and Department of Family Medicine and Public Health Sciences, Wayne State University School of Medicine, Detroit, MI, USA

3

 Department of Medicine, University of Pittsburgh School of Medicine and Behavioral Medicine Clinical Service, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

H.M. Lazarus and M.J. Laughlin (Eds.), pp. 619–656, © Springer Science+Business Media, LLC 2003, 2010

DOI 10.1007/978-1-59745-478-0_47 The abstract for Chapter 35 Psychological Care of Adult Allogeneic Transplant Patients was erroneously substituted for the first three paragraphs of the text. The original paragraphs follow. References are cited at the end of the chapter. This chapter is devoted to the recognition, detection, and management of psychological concerns of adult patients who undergo allogeneic hematopoietic cell transplantation (HCT). As HCT is typically indicated for life threatening conditions, or when other treatment avenues are no longer curative, the resulting psychological distress cuts across disease types, sources of stem cells, and types of transplant. We attempt to focus exclusively on outcomes for allogeneic patients; however, given that the psychosocial literature for this subgroup of patients is limited, we also draw on empirical studies of

DOI 10.1007/978-1-59745-478-0_47 The abstract for Chapter 35 Psychological Care of Adult Allogeneic Transplant Patients was erroneously substituted for the first three paragraphs of the text. The original paragraphs follow. References are cited at the end of the chapter. autologous transplant patients. Thus, in this chapter, we use the term “HCT” to encompass both types of transplant, and we note explicitly when mixed patient samples are referenced. The intensity of psychological distress is likely amplified for allogeneic as opposed to autologous patients, for whom undergoing HCT is a tightrope-walk between the life-extending, sometimes curative effects of transplant and the potential of the treatment to hasten their death. For example, the treatment related mortality for allogeneic patients at 1 year is 16–29% in contrast to 2% for autologous HCT patients [1]. Further, psychological distress persists for the approximately 50% [2] to 77% [3] of allogeneic patients who do survive 2 or more years and face lingering or latent long-term effects [3], negatively impacting the quality of their lives. In the words of Macklin Smith, poet and long-term survivor of a matched unrelated allograft, a HCT brings both long term positive aspects such as “being alive, amazement at being alive, enjoying life, appreciation for the small things in life, living a productive life,” and negative aspects “fatigue, forgetfulness, chronic sorrow, general bewilderment, fear of death, indifference to death, confusion about death” [4]. The challenge, therefore, in effectively caring for the psychological needs of allogeneic HCT patients is to recognize when psychological symptoms and distress exceed normative responses to HCT with its risks and long-term effects and to provide appropriate psychological intervention and support as needed.

The online version of the original chapter can be found at http://dx.doi.org/10.1007/978-1-59745-478-0_35

sdfsdf

Index

A Acquired hypercoagulable disorders, 708 Acute graft versus host disease (aGVHD) graft manipulation ex vivo TCD, 567 T cells removal, 565 in vivo negative selection techniques, 566 PBSCT, 286 pharmacologic prevention cyclophosphamide, 571–572 cyclosporine, 567 IBMTR, regimens, 568 methotrexate, 567 mycophenolic acid (MPA), 571 novel calcineurin inhibitors, 567 sirolimus, 569–570 treatment cellular subsets optimization, 759 corticosteroid dose, 749 definition, 747 first-line treatment, combination therapy, 750–751 Glucksberg criteria, 749 IBMTR severity index, 748 impact, GvL, 751 mesenchymal stromal cells (MSCs), 759 non-myeloablative transplantation, 758–759 pathophysiologic mechanisms, 759–760 second line therapy, 752–758 standards of care, 749 steroid therapy, 748 supportive care, 751 therapy duration, 750 Acute leuekemia, 356 Acute lymphoblastic leukaemia (ALL) biological randomization, 30 CNS disease, 198–199 conditioning regimens, 194 CR1/CR2, 32 definitions, 31 donor lymphocyte infusions, 198–199 first remission, sibling allograft, 194–195 haploidentical donors, 36 palifermin, 199 Philadelphia chromosome positive ALL, 37–38

prognostic factors, 29–30 rationale and GvL effect, 193–194 refractory disease, 199 relapsed disease allograft, 198 remission, 37 RIC regime, 36–37 role, RIC, 196–197 sibling allo HSCT, 34–35 sibling donor, 32 T-cell depletion, 36 UD-SCT, 33 UKALL XII/ECOG 2993 study, 32 umbilical cord blood HSCT, 33 Acute myeloid leukemia (AML), 11 myeloablative conditioning regimen Bu-based regimens, 16 CR1, 13 donor lymphocyte infusions (DLI), 20 donor vs. no-donor analyses, 14 optimal conditioning regimen, 16 optimal stem cell source, 17 outcome, disease relapse, 19 reduced intensity allografts, 18 RIC regimen, 18 salvage chemotherapy, 15 T-cell depletion strategies, 16–17 Acute nonlymphoblastic leukemia, 3 Adenoviruses, 514–515 Adoptive cell therapy, TRM reduction, 465 Adult cord blood transplantation double cord blood, 368–369 refractory lymphoma, single cord blood, 368 Aggressive B-cell lymphomas chemosensitivity, 118 DLBCL, 98, 117 RIT, 119 single arm cohort studies, 99 Alemtuzumab, 451–452, 753 ALL. See Acute lymphoblastic leukaemia Allogeneic gene therapy, thalassemia alternative donors transplantation, 498–499 BMT vs. medical treatment, 492 disease eradication, 493–494 engraftment, 494

871

872 

Index

Allogeneic gene therapy, thalassemia (cont.) ex-thalassemic management, 499–500 graft failure/rejection, 497–498 GVHD, 498 mixed chimerism, 497 morbidity and mortality, 494 preparatory regimens, 493 risk classes, 495 transplant outcome, HLA matched related donors, 495–497 adult patients, 496–497 class 1and 2 patients, 495 class 3 patients, 495–496 transplant procedures, 492 AlloSCT favorable-risk AML HOVON/SAKK study, 186 LFS, 184 myeloablative conditioning therapy, 183 OS, 185 Alternative donors, SCT alternative related donors, 498 unrelated bone marrow transplantation, 498–499 unrelated cord blood transplantation, 499 AMD3100, hematopoietic cells mobilization mononuclear cell (MNC) fraction, 394–395 PBSC, 395 phase 3 clinical trials, 395 SCID-repopulating capacity (SRC), 394 AML. See Acute myeloid leukemia Antibody-dependant cellular cytotoxicity (ADCC), 859 Anti-cancer vaccines, HSCT augmentation leukemia cell-based therapies, 862–863 peptide immunization, 862 NHL and MM allogeneic transplantation, 859 autologous transplantation, 858 donor immunization, 861 idiotype directed therapy, 859–861 post-autologous transplantation immunization, 858–859 potential leukemia-associated antigenic targets, 856 vaccines types, 856–857 Anti-thymocyte globulin (ATG), 588, 752–753 Aplastic anemia, allograft, 3 Aspergillus infections Aspergillosis, 536 ELISA assay, 537 galactomannan, 537 symptoms, 536 voriconazole, 538 B B cell immune reconstitution, immunotherapy development, B cell, 547–548 naïve phenotype, 548 post-transplant, 548 BCR. See Breakpoint cluster region

Bioluminescent-based imaging (BLI), 790 Birbeck granules, 810 Blood and marrow transplant clinical trials network (BMT CTN) GVHD, 570 psychological morbidity, 644 tandem autologous transplantation, 148 TA-TMA, 706 TCD, 567 unrelated donor CB, 381 Breakpoint cluster region (BCR), 54 Bronchiolitis obliterans (BO), cGVHD, 583 Burkitt's lymphoma (BL), 102, 225 C CALGB. See Cancer and leukemia group B Cancer and leukemia group B (CALGB), 178 Candida infections beta glucan assay, 534 common clinical syndromes, 535 mucosal injury, 533 symptoms, 534 Catheter thrombosis, 704 Cell infusion, NKcell CD 56+, 420 clinical application, 419 CliniMACS ®, 421 cytokine activation, 421 NK-DLI, 418–420 UCB, 420 Center for International Blood Marrow Transplant Research (CIBMTR) allograft recipients age, 130 CML, 57 follicular non-Hodgkin's lymphoma (FL), 112 peri-TKI era, 62 RIC transplantation, 134 RIT regimens, 114 Centers for Disease Control (CDC) evidence-based rating system, 722 guidelines, infection control, 723, 724 level I evidence, 722–723 level II/III evidence air, 725 fomites, 725 food and water, 726 host, 724–725 human to human, 725 soil, construction and cleaning, 726 Central nervous system (CNS) ALL, 198–199 bleeding intracranial hemorrhage (ICH), 701 subdural hematoma (SDH), 701 tacrolimus (FK 506), 702 Childhood ALL. See Pediatric ALL Chromosomal abnormalities, 669 Chronic graft-versus-host disease (cGVHD) diagnosis of, 578

Index  eyes, 582–583 gastrointestinal tract, 583 genitalia, 583 grades II–IV, probabilities, 286 GVHD prophylaxis, 591 hairs, 582 hematopoietic and immune system, 584 liver, 583 lungs, 583 mouth, 582 musculoskeletal system, 583 nails, 582 NIH consensus criteria, 579 risk factors development of, 589 HLA, 587 KPC, 587 peripheral blood stem cell transplantation (PBSCT), 586 sBAFF and anti-dsDNA, 587 severity and score, 585–586 signs and symptoms, 580 skin, 578, 582 treatment cyclosporine (CSA)/tacrolimus, 591 salvage therapy, 590 Chronic lymphocytic leukemia (CLL) treatment. See Hematopoietic progenitor cell transplantation Chronic myeloid leukemia (CML), 4 CIBMTR. See Center for International Blood Marrow Transplant Research Clinical psychologist role evidence-based HCT psychological treatment psychological intervention literature, 634 psycho-oncology, 634–635 somatic and emotional symptoms, 632–633 pathways, psychosocial care post-hospitalization, 638–639 pre-and peri-hospitalization, 637–638 psychopharmacological intervention antidepressant mirtazapine, 636 CBT, 636 psychotropic medications, 635 social workers normal trajectory, 631 practical issues, 629 suggested instruments, 630 Clonal hematopoietic cell disorder. See Chronic myeloid leukemia Cognitive impairment, 624 Co-morbidity index, 688–689 Complete response (CR) rates, 153, 180, 657 Conditioning regimens, hematopoiesis non-radiation based regimens, 775 radiation based regimens, 774–775 Cord blood banking, 364 Cord blood units selection vs. peripheral blood progenitor cells, 375 principles, 376 unrelated donor CB

cell dose, 377–378 diagnosis effect, 381 double cord blood units, 381–382 factors, 377–378 product, 376–377 Texas Transplant Institute Perspective, 382–383 untreated donor CB HLA matching, 378–379 non-inherited maternal allele matching, 379–380 Core-binding factor (CBF) abnormality, 179 Corticosteroid dose, 749 CR1. See First complete remission Cryopreservation advantages and disadvantages, 436 bacterial contamination, 430 clinical outcomes BM allograft comparison, 432–433 engraftment and outcome data, 431 GVHD incidence, 433 multivariate analyses, 432 PB allograft comparison, 432 unrelated allogeneic transplantation, 433–434 donor lymphocyte infusion, 434 ethical concerns, 435 graft content donor graft alteration, 428 impact, 428–430 MNC subtypes, 428 logistics, 434–435 methodology, 428 transfusion reactions, 430 Cytokine-primed marrow transplantation, 290 Cytomegalovirus (CMV) immune therapy and monitoring, 509 preemptive therapy, 508 prophylaxis, 507–508 risk factors, 507 Cytotoxic T-lymphocyte precursors (CTL-ps), 462 D Daclizumab, 755–756 Dendritic cells (DC) ex vivo generation IL, 816 SCF, 815 T cell population, 817 immune reconstitution, GVHD vs. HSCT CAMPATH, 821 co-transplantation, 822 cytokine regimen, 820 donor T cells, 821 pathogenesis, 820 umbilical cord blood, 822 immunotherapy cancer, 822–827 clinical studies, cancer, 827–831 potential limiting factors, 831–832

873

874 

Index

Dendritic cells (DC) (cont.) phenotypic characterization Birbeck granules, 810 GM-CSF, 809 immature DC, 810 mature DC, 812–813 skin-homing receptor, 810 TLRs, 812 subsets, 809 T cell interactions adhesion molecules, 814 danger signals, 814 IL-12, 814, 815 stimulatory signals, 815 tolerance establishment IDO, 818 programmed death ligand-1, 818 T cells, 817 thymic-derived population, 818 Tr1, 819 in vivo GVHD model, 820 Denileukin diftitox, 756–757 Deoxycoformycin, 755 Diffuse alveolar hemorrhage (DAH), 700–701 Diffuse large B cell lymphoma (DLBCL), 98, 117 DLBCL. See Diffuse large B cell lymphoma Donor leukocyte infusion (DLI), myeloablative and non-myeloablative SCT, 434 therapy, 602 Donor lymphocyte infusions (DLI) GVL response, 20 reduced intensity regimens, 81 salvage therapies, 657 survival rate, 272 Donor selection. See also Alternative donors, SCT CLL, 50 hematopoietic progenitor cell transplantation, 50 HLA-Bw4 mismatches, 470–471 HLA-C group 1 alleles, 469–470 principles allele level typing, 313–315 first-degree relatives, 315 KIR ligand groups, 315 molecular methods, 313 Dose intensity, novel regimens definition, 441–442 GvHD, impact acute GvHD, 445–447 TRM, 447 myeloablative conditioning vs. NMT/RIC, 442–443 RIC/NMT, 443 standard transplantation, 442–443 Double cord blood transplantation, 368–369 Down syndrome (DS), 224 Dural (venous) sinus thrombosis, 708 E EBMT transplantation risk score, 57 Emotional disorders

anticipatory nausea, 622 effects, 634 insomnia, 622 psychological distress, 621 symptoms, 622 Epratuzumab, 122 Epstein-Barr Virus (EBV), 510–511, 598 Etanercept, 758 European group for Blood and Marrow Transplantation (EBMT), 45, 49, 346 Event free survival (EFS), 161, 465 F Favorable-risk AML alloSCT HOVON/SAKK study, 186 LFS, 184 myeloablative conditioning therapy, 183 OS, 185 ASCT, 181–183 CR1, 181 GVHD, 181 ITT analysis, 181 LFS, 182 overall survival, 182 CALGB, 178 cytarabine-/anthracycline based induction chemotherapy, 178 definition, 178 molecular pathogenesis, 179 post-remission therapy, 179–180 refractory AML, 186 relapsed AML, 186–187 RIC SCT, 187 SCT, 180–181 First complete remission (CR1) LFS vs. ASCT, 182 OS vs. ASCT, 182 pediatric ALL chemotherapy vs. transplantation, 231 disease-free survival, 234 eligibility criteria, 228–230 HSCT outcome, 232 transplantation, 230, 234–235 umbilical cord outcomes, 236 very high risk ALL frontline treatment, 227 FLIPI. See Follicular lymphoma international prognostic index Fludarabine, 450–451 Follicular center cell NHL. See also Non-Hodgkin's lymphoma CD20 vs. chemotherapy, 161 chemoimmunotherapy (R-CHOP), 163 CR/PR, 161 EFS, 161 FLIPI, 160 myeloablative therapy allogeneic HSCT, 163–165

Index  relapsed/refractory FL, 163 vs. RIC, 166–167 nonmyeloablative therapy allogeneic HSCT, 165 graft versus tumor/GvL effect, 166 RIC regime, 165 relapsed follicular lymphoma treatment, 162 transformed follicular NHL, 167–168 Follicular lymphoma international prognostic index (FLIPI), 160 Follicular non-Hodgkin's lymphoma (FL), 110 Foundation for the accreditation of cellular therapy (FACT), 349, 717 Full and reduced intensity, AML GVL effects, 12 leukemic stem cells (LSC), 11 myeloablative conditioning regimen Bu-based regimens, 16 CR1, 13 DLI, 20 donor vs. no-donor analyses, 14 optimal conditioning regimen, 16 optimal stem cell source, 17 outcome, disease relapse, 19 reduced intensity allografts, 18 RIC regimen, 18 salvage chemotherapy, 15 T-cell depletion strategies, 16–17 post-transplant cyclosporine (CyA) impact, 12 Fungal infections antifungal agents, 540–541 Aspergillus infections airborne infection, 720 Aspergillosis, 536 Clostridia difficile-associated diarrhea, 720 ELISA assay, 537 galactomannan, 537 symptoms, 536 voriconazole, 538 Candida infections beta glucan assay, 534 common clinical syndromes, 535 mucosal injury, 533 symptoms, 534 evaluation and treatment approaches, 542 Fusarium, 539 Pneumocystis carinii, 539 Zygomycetes infections, 538–539 G Gamma/delta ( g d ) TCR T cells, 798 Gastrointestinal bleeding, 700 Grade 4 mucositis, 199 Graft failure/rejection, 497–498 Graft versus host disease (GVHD) aGVHD cellular subsets optimization, 759 corticosteroid dose, 749 definition, 747

875

first-line treatment, combination therapy, 750–751 Glucksberg criteria, 749 graft manipulation, 565–567 IBMTR severity index, 748 impact, GvL, 751 mesenchymal stromal cells (MSCs), 759 non-myeloablative transplantation, 758–759 pathophysiologic mechanisms, 759–760 PBSCT, 286 pharmacologic prevention, 569–572 second line therapy, 752–758 standards of care, 749 steroid therapy, 748 supportive care, 751 therapy duration, 750 antibody prophylaxis alemtuzumab vs. methotrexate, 740 peripheral blood stem cells, 739 T-cell depletion, 740 T-lymphocyte, 739 cGVHD cyclosporine (CSA)/tacrolimus, 591 diagnosis of, 578 eyes, 582–583 gastrointestinal tract, 583 genitalia, 583 GVHD prophylaxis, impact, 591 hairs, 582 hematopoietic and immune system, 584 HLA, 587 KPC, 587 liver, 583 lungs, 583 mouth, 582 musculoskeletal system, 583 nails, 582 NIH consensus criteria, 579 peripheral blood stem cell transplantation (PBSCT), 586 salvage therapy, 590 sBAFF and anti-dsDNA, 587 severity and score, 585–586 signs and symptoms, 580 skin, 578, 582 treatment HLA mismatch adverse effect, 311 infectious complications CD8-depleted DLI, 741–742 immunity suppression, 741 lymphocyte count, 741 mitigate graft allodepletion, 325 non-inherited maternal antigens, 324–325 replete graft, T Cell, 326–327 T cell depletion and infusion lymphocytes, 325–326 prophylaxis, 352 Graft vs. leukemia (GVL), 796, Graft vs. lymphoma effect (GVLy), 91 Graft vs. tumor (GVT), 796

876 

Index

Granulocyte–macrophage colony stimulating factor (GM-CSF), 809 GROb, 393–394 GVL/GVT, 796 GVLy. See Graft vs. lymphoma effect H Haploidentical transplantation engraftment and GvHD CD34+ and T-cells, 462 transplantation procedure, 462–463 event free survival (EFS), 465 leukemia relapse, 464 NK cell alloreactivity cytokine secretion and cytotoxicity, 467 KIR genetics activation, 466–467 matched unrelated donor transplants, 467–469 transplant related mortality (TRM) adoptive cell therapy, 465 G-CSF impact, 464 immunological recovery, 464 overlapping factors, 464 procedure, 463 HCT specific comorbidity index (HCT–CI), 136 Health and behavior current procedural terminology (H & B CPT) codes, 643 Hematopoiesis bone marrow microenvironment, 780 conditioning regimens non-radiation based regimens, 775 radiation based regimens, 774–775 HSC grafts and growth factors, 772–774 MHC, 767 non-human primate models baboons, 770 macaques, 770–801 marmosets, 771–772 in vivo models, attributes, 769 SCT models allogeneic, 776–777 autologous, 775 gene therapy, autotransplantation, 775–776 GVHD, 777 MHC typing, 777–778 whole organ tolerance induction, 778 xenotransplantation, 779–780 Hematopoietic bone marrow microenvironment, 780 Hematopoietic cells mobilization cytokines, mobilizing donor cells, 402–403 G-CSF effect, normal donor PBSC mobilization, 400 short-term stimulation effect, 402 splenomegaly, 401 toxicity, 401 mechanisms adhesion molecules, 388 CD26, 389 chemokines, 388 cytokines, 388–389 IL-8 activity, 389

mobilizing agents, 403 normal donors cell doses, 398 factors, 399–400 G-CSF, 399 GM-CSF, 399 immunomodulatory effect, 400 novel agents AMD3100, 394–396 CXCR4 peptide, 393 GRO[$$], 393–394 parathyroid hormone (hrPTH), 393 pegfilgrastim, 391–392 recombinant human growth hormone (rhGH), 391 stem cell factor, 391 thrombopoietin, 392 regimen selection aldehyde dehydrogenase (ALDH), 397 AMD3100, 398 CD34+ cells, 397 chemotherapy/HGF, 396–397 mobilization capacity, 396 stem cell mobilization regulation neural signals, 389–390 osteolineage derived cells Hematopoietic progenitor cell transplantation autologous transplantation, 44 genomic high risk CLL, 48 myeloablative conditioning regime, 45–47 non-myeloablative conditioning regime, 47–48 treatment, 44–45 chronic immunosuppression, 49 co-morbidity index, 688–689 donor selection, 50 functional assessment tools, 689–690 high dose therapy (HDT), 687 indications, 49 performance status (PS), 688 prognostic factors, 691 Hematopoietic stem cell, thalassemia allogeneic gene therapy alternative donors transplantation, 498–499 BMT vs. medical treatment, 492 disease eradication, 493–494 engraftment, 494 ex-thalassemic management, 499–500 graft failurerejection, 497–498 graft-versus-host disease, 498 mixed chimerism, 497 morbidity and mortality, 494 preparatory regimens, 493 risk classes, 495 transplant outcome, HLA matched related donors, 495–497 transplant procedures, 492 clinical manifestations, 491 hemoglobin disorder, 491 treatment, 491 Hemorrhagic cystitis, 698–700 Hepatic veno-occlusive disease (VOD) diagnosis, 704–705

Index  gemtuzumab ozogamicin, 705–706 morbidity and mortality, 706 pathogenesis, 705 sinusoidal obstruction syndrome (SOS), 704 Hepatitis B and C viruses, 515 Herpes simplex virus (HSV), 509 High dose therapy (HDT), 687 Histocompatibility, 5 HLA. See Human leukocyte antigens HLA-haploidentical related donors clinical outcomes allodepletion, 323–324 G-CSF primed donors, 319–321 megadose stem cell transplantation, 318–319 nonmyeloablative HLA-haploidentical SCT, 321–323 replete grafts, T cell, 317 T cell depletion effect, 317–318 complications aGvHD, 311–312 graft failure, 309–311 GVHD, 311–312 impaired immune reconstitution and infection, 312–313 complications reducing strategies mitigate GVHD, 324–327 prevent/treat relapse, 327–329 feature, 299 HLA typing and donor selection principles allele level typing, 313–315 first-degree relatives, 315 KIR ligand groups, 315 molecular methods, 313 iKIRs and HLA ligands interactions, 303 immunobiology human T cell responses, 300–301 limitations, 301–302 natural killer cell alloreactions, 302–304 reactivity models, 304–307 T and NK cells interactions, 308 inhibitory KIR types, 304 natural killer cell alloreactivity models, 305 unrelated donor umbilical cord blood cell dose, 330 double unit UBCT, 330–331 grafts typing, 329 leukemia-free survival, 329 Hodgkin’s lymphoma allogeneic bone marrow transplantation fully ablative regimens, 80 high risk patients, 85 intensity preparative regimens, 82 ASCT, 78 autologous bone marrow transplant, 77 FFS/OS rates, 78 high-dose therapy, 77 reduced intensity regimens chemoresistant disease, 83 donor lymphocyte infusions (DLI), 81 fludarabine-melphalan, 83 GVL effect, 84

877

nonrelapse mortality, 84 peripheral blood stem cells (PBSC), 81 risk factors, 78 standard therapy, 76 Homeostatic peripheral expansion (HPE), 549–550 Human Herpes Virus Type 6 (HHV-6), 511–512 Human leukocyte antigens (HLA), 5, 587 Hyperleukocytosis, 223–224 I IBMTR. See International Blood and Marrow Transplant Research Idiotype directed therapy ADCC, 859 anti-idiotype humoral responses, 860 trial designs, 861 vaccination, 858, 860 Immunobiology, HLA-haploidentical related donors human T cell responses, 300–301 allogeneic response strength, 300 cross-reactivity phenomenon, 301 determinant density, 300–301 determinant frequency, 301 minor histocompatibility Ags (minor H Ags), 300 limitations, 301–302 natural killer cell alloreactions DNA damage, 304 inhibitory KIRs (iKIRs), 302–304 licensing, 304 missing self hypothesis, 302 molecular basis, 302 reactivity models gene–gene model, KIR, 307 KIR ligand incompatibility, 305 missing ligand model, 307 receptor-ligand model, 306–307 T and NK cells interactions, 308 Immunotherapy B and T cells, 547 B cell immune reconstitution B cell development, 547–548 post-transplant, 548 cancer fusion cells, 827 immature DC administration, 823 melanoma cells, 826 mRNA, 826 tumor-associated antigens, 823, 826 vaccination, 825 viral transduction, 825 cellular immunotherapy post-transplant, 553–554 clinical studies, cancer CD11c/CD14, 827 CPG ODN, 827 ELIspot analysis, 829 MAGE peptides, 828 prostate-specific membrane-antigen (PSMA) peptides, 828 vaccination, 831 considerations, 555

878 

Index

Immunotherapy (cont.) immune reconstitution, 546 killer immunoglobulin-like receptor (KIR), 545 potential limiting factors chemotherapy, 832 ONTAK, 832 tumor cells secrete factors, 831 T cell reconstitution cellular immunotherapy, thymopoiesis, 552–553 HPE, 549–550 initial post-transplant period and implications, 550–551 thymic dependent, 548–549 Indoleamine 2,3-dioxygenase (IDO), 818 Indolent lymphoma, NHL dose intensive chemotherapy, 91 EBMT/IBMTR review, 93 European CUP trial, 91 GVLy effect, 92 low treatment-related mortality (TRM), 92 single arm cohort study, 93 transplantation outcome, 94 Infliximab, 757 Influenza viruses, 514 Intent-to-treat (ITT) analysis, 181 Interleukin (IL), 549 International Blood and Marrow Transplant Research (IBMTR), 46, 81, 93 International Prognostic Scoring System (IPSS), 206 Invasive fungal infections (IFI). See Fungal infections In vivo models, AlloHSCT immunobiology aGVHD, 795 cGVHD, 795–796 graft rejection, 793 graft vs. tumor (GVT), 796 immune reconstitution, 794–795 non-T cell lymphoid populations gamma/delta ( g d ) TCR T cells, 798 natural killer cells, 798 NKT cells, 798 preclinical models age and sex, 793 BLI, 790 endogenous microflora, 792–793 mouse strains and immunologic disparity, 791–792 species differences, 790 IPSS. See International Prognostic Scoring System Isolation methodology, allografts CDC level I evidence, 722–723 level II/III evidence, 723–726 guidelines, infection control, 723, 724 isolation costs finances, 726–727 patient interactions, 727 necessity benefits, 721–722 infection outbreaks, 719–720

infectious complications, 718–719 vancomycin-Resistant Enterococcus (VRE), 720–721 outpatient care, 728 K Karnofsky performance status (KPS), 587 Killer-cell immunoglobulin-like receptors (KIR) genes, 460, 545 gene–gene model, 307 ligand incompatibility model, 305–306 L Lentiviral vectors, 776 Leukemia-associated antigens (LAA), 862 Leukemia-free survival (LFS), 180, 182 Leukemic cell characteristics pediatric ALL Burkitt-leukemia, 225 cytogenetics, 225 morphology, 224 precursor B-cell, 224 Lymphoblastic lymphoma (LBL), 103 Lymphohematopoietic chimerism, 777 M Major histocompatibility complex (MHC), 5, 767 Mantle cell lymphoma (MCL) nonmyeloablative/reduced intensity transplantation, 116 nonmyeloablative/RIT ergimes, 117 Matched unrelated donor (MUD), 95. See also MUD BMT MDS. See Myelodysplastic syndromes Megadose stem cell transplantation, 318–319 Mesenchymal stem cells bone marrow-derived MSCs, 477–478 clinical autologous and allogeneic MSCs transplantation, 480–482 clinical expansion, 485–486 growth factor supplementation, 482–483 immunologic properties, 479–480 limitations, 482–483 MHC barrier, 480 multipotent adult progenitor cells (MAPCs), 477 multipotentiality, 478 senescence, 479 Mesenchymal stem cells (MSC), 776 Mesenchymal stromal cells (MSC), 759 MHC. See Major histocompatibility complex Minimal residual disease (MRD), 226, 237 antigen receptor rearrangement analysis, 668–669 chimerism analysis, 670–671 clinical significance acute lymphoblastic leukemia, 671–672 acute myeloid leukemia, 672 chimerism results, 673–674 chronic myeloid leukemia, 672–673 reduced intensity conditioning regimen, 674–675

Index  fusion gene transcript analysis, 669–670 immunophenotype analysis, 667–668 methods, 668 Missing ligand model, 307 Mixed hematopoietic chimerism (MC), 497 Monoclonal antibodies, hematologic malignancies advantage, 733 CD20, 734 conditioning regimens radiolabeled antibody, 736–738 unlabeled antibody, 735 GVHD prophylaxis alemtuzumab vs. methotrexate, 740 infectious complications, 741–742 peripheral blood stem cells, 739 T-cell depletion, 740 T-lymphocyte, 739 morbidity and mortality, 734 post-transplant consolidation, 738–739 total body irradiation (TBI), 734 MSC. See Mesenchymal stem cells Mucosal Candida infections, 533 MUD BMT comparative analyses, 101 GVLy effect, MCL, 96 mantle cell lymphoma (MCL), 95 national marrow donor program (NMDP), 95 RICSCT techniques, 95 Multiple myeloma age, 131, 132 applications, 137–138 donor availability, 133 GVHD, 133 RIC regimens CIBMTR data, 135 multiple retrospective studies, 134 SEER incidence rates, 129 single vs. tandem autologous transplant, HSCT maintenance therapy, 151 novel pre-transplant regimens, 153 optimal time, transplant, 148 salvage therapy, 151 specific comorbidity index, 136 treatment allogeneic stem cell source and alternative donors, 267 vs. autologous transplantation, 265–267 DLI and post-transplant management, 273 phase II studies, 268–269 prognostic factors, 268–270 rationale, 263–264 syngeneic transplantation, 267 tandem autologous, 270–273 T-cell depletion, 267–268 TRM and OS, 264–265 trends in, 130–131 Multipotent adult progenitor cells (MAPCs), 477 Murine retroviruses, 776 Mycophenolate mofetil, 571 Mycophenolic acid (MPA), 571

879

Myeloablative allogeneic transplantation allogeneic stem cell source and alternative donors, 267 vs. autologous transplantation, 265–267 prognostic factors, 268 syngeneic transplantation, 267 T-cell depletion, 267–268 TRM and OS, 264–265 Myeloablative conditioning regime, AML Bu-based regimens, 16 donor lymphocyte infusions (DLI), 20 donor vs. no-donor analyses, 14 first complete remission (CR1), 13 optimal conditioning regimen, 16 optimal stem cell source, 17 outcome, disease relapse, 19 reduced intensity allografts, 18 RIC regimen, 18 salvage chemotherapy, 15 T-cell depletion strategies, 16–17 Myelodysplastic syndromes (MDS) age and disease state, impact, 207 bone marrow transplantation, 206 chemotherapy induction, 209 classification, 205 conditioning regimens, 210–211 donor and stem cell source, 209–210 JMML transplantation, 213 optimal timing, transplantation HLA identical sibling donors, 208 IPSS scores, 207 prognostic factors, 204 transplantation, CMML, 211–212 UD HSCT, 354–356 Myeloid DCs (mDCs), 809 N National marrow donor program (NMDP) CML, 57 cord blood banks, 364 CR2 ALL, 243 cryopreserved unrelated grafts, 427 MUD BMT, 95 Natural killer (NK) cell activation process, 460 donor selection HLA-Bw4 mismatches, 470–471 HLA-C group 1 alleles, 469–470 haploidentical transplantation cytokine secretion and cytotoxicity, 467 engraftment and GvHD, 462 event free survival (EFS), 465 KIR genetics activation, 466–467 leukemia relapse, 464 matched unrelated donor transplants, 467–469 transplant related mortality (TRM), 464–465 HLA haplotype, 459–460 HSC transplantation, alternative donor, 460 immune cells, 798 matched unrelated donors, 459

880 

Index

Natural killer (NK) cell (cont.) post-transplant infection, 492 strength and weakness, 472 treatment adoptive immunotherapy/DLI, 416 cell expansion, 418 cell infusion, 418–421 definition, alloreactivity, 415 donor lymphocyte infusion (DLI), 416–417 extracellular domain, 414 graft vs. host direction, 415–416 haploidentical donors transplantation, 415 haploidentical HSCT, 416 harvesting, 417 inhibitory receptor, 413–414 localization, 413 product, 417 production issues, 422 purification, 417–418 umbilical cord blood transplantation (UCBT), 459 NMDP. See National marrow donor program Non-Hodgkin's lymphoma (NHL) advantages and disadvantages, 91 aggressive B-cell lymphomas chemosensitivity, 118 diffuse large B cell lymphoma (DLBCL), 98, 117 single arm cohort studies, 99 allogeneic reduced intensity conditioning regime follicular (FL) NHL, 112 nonmyeloablative/reduced intensity transplantation, 113 treatment-related mortality (TRM), 111 Burkitt's lymphoma (BL), 102 chemosensitivity importance, 100 indolent lymphoma dose intensive chemotherapy, 91 EBMT/IBMTR review, 93 European CUP trial, 91 GVLy effect, 92 low treatment-related mortality (TRM), 92 single arm cohort study, 93 transplantation outcome, 94 lymphoblastic lymphoma (LBL), 103 mantle cell lymphoma (MCL) nonmyeloablative/reduced intensity transplantation, 116 nonmyeloablative/RIT ergimes, 117 MUD BMT comparative analyses, 101 GVLy effect, MCL, 96 mantle cell lymphoma (MCL), 95 national marrow donor program (NMDP), 95 RICSCT techniques, 95 NK and T-cell lymphomas, 102 T-cell lymphomas allogeneic nonmyeloablative/RIT, 120 novel/emerging therapies, 121–122 Nonmyeloablative allogeneic transplantation phase II studies EBMT data, 269 TRM, 268

prognostic factors, 269–270 tandem autologous, 270–273 O Ocular bleeding, 702 Overall survival (OS), 264–265 P Palifermin, 199 Papovaviruses, 516 Partial remission (PR), 161 PBSC. See Peripheral blood stem cells Pediatric ALL conditioning regimens, 248–249 donor choice, 246–247 first complete remission (CR1) chemotherapy vs. transplantation, 231 eligibility criteria, 228–230 HSCT outcome, 232 transplantation, 230, 234–235 umbilical cord outcomes, 236 very high risk ALL frontline treatment, 227 MRD and allogeneic transplantation, 249–250 NMDP retrospective study, 243 oncology, 220 polychemotherapy, 221 prognostic factors age, 222–223 cytogenetics, 225–226 down syndrome (DS), 224 early multidrug response, 226 extramedullary involvement, 224 hyperleukocytosis, 223–224 immunophenotype, 224–225 morpholog, leukemic cell characteristics, 224 MRD after induction therapy, 226 prednisone poor response (PPR), 226 second complete remission (CR2) ALL in advanced phase, 244 eligibility criteria, 236–238 relapsed ALL, 236 transplantation, 238, 240–244 umbilical cord outcomes, 244 stem cell source, 245–246 toxicity and mortality, 249 UCBT vs. BMT, unrelated donors, 247 UCBT vs. haploidentical transplantation, 247–248 very high risk (VHR)/ultra high risk (UHR), 220 Pediatric cord blood transplantation, 365–366 Pegfilgrastim, 391–392 Peripheral blood progenitor cells (PBPC) AMD3100, 282–283 vs. BMT, 285 CD34, 281–282 CD133+ graft, 282 clinical aspects cytokine-primed marrow transplantation, 290 engraftment, 284 graft characteristics, 283–284

Index  GvHD, 284–287 infections, 288 quality of life, 288–289 survival, 288 unrelated peripheral blood transplants, 289 cost, 291 cytokines, 282 donor considerations severe adverse reactions, 291 short-term adverse effect, 290–291 Peripheral blood stem cells (PBSC), 17, 81 PFS. See Progression-free survival rates Philadelphia chromosome positive ALL, 37–38 Plasmacytoid DCs (pDCs), 809 Polyomavirus hominis 1, 698 Post-transplant lymphoproliferative disorder (PTLD) definition, 597 pathophysiology EBV, 599 hepatitis C virus, 601 OKT3, 599 risk factors, 600 umbilical cord blood transplants, 600 photomicrographs, 601 prophylaxis and treatment anti-B-cell antibodies, 608–609 antiviral therapy, 603–607 cellular immunotherapy, 610–611 cytokine therapy, 609 cytotoxic chemotherapy, 609–610 immunosuppression reduction, 607–608 local therapy, 607 surveillance, 602–603 WHO classification, 598 Potential leukemia-associated antigenic targets, 856 Prednisone poor response (PPR), 226 Preparative regimens dose intensity definition, 441–442 GvHD, impact on, 445–447 myeloablative conditioning vs. NMT/RIC, 442–443 NMT/RIC vs. myeloablative, 446 novel myeloablative regimen, 444 RIC/NMT, 443 standard transplantation, 442–443 regimens exploration alemtuzumab, 451–452 extracorporeal photopheresis, 452 fludarabine-melphalan vs. fludarabine-busulfan, 450–451 fludarabine/TBI, 450–451 total lymphoid irradiation (TLI), 452–453 Prognostic factors pediatric ALL age, 222–223 cytogenetics, 225–226 down syndrome (DS), 224 early multidrug response, 226 extramedullary involvement, 224 hyperleukocytosis, 223–224 immunophenotype, 224–225

881

morpholog, leukemic cell characteristics, 224 MRD after induction therapy, 226 prednisone poor response (PPR), 226 Progression-free survival (PFS) rates, 75 Prophylaxis and treatment, PTLD anti-B-cell antibodies, 608–609 antiviral therapy EBV infections, 603 therapeutic options, 604–607 cellular immunotherapy, 610–611 cytokine therapy, 609 cytotoxic chemotherapy, 609–610 immunosuppression reduction, 607–608 local therapy, 607 proposed algorithm, 612 Psychological care assessment issues clinical interview, 626 tools, 626–629 clinically significant psychological problems cognitive dysfunction, 623–626 emotional disorders, 621–623 clinical psychologist role evidence-based HCT psychological treatment, 631–635 pathways, psychosocial care, 636–638 psychopharmacological intervention, 635–636 social workers, 629–631 HCT psychological services, 642–643 mental health parity policy implications health and behavior codes, 643–644 resources, 644 treatment adherence and caregiver role caregiver issues, 640–642 treatment regimen, 639–640 Pulmonary cytolytic thrombi (PCT), 707–708 R Radiation chimaera, 1 Radiolabeled monoclonal antibodies anti-CD66 antibody, 738 leukemia treatment, 737–738 radioimmunoconjugate, 736 90Y-labeled ibritumomab tiuxetan, 737 Receptor-ligand model, 306–307 Recombinant human growth hormone (rhGH), 391 Reduced intensity conditioning (RIC) regime, 36–37, 187 Respiratory syncytial virus (RSV), 513, 720 Respiratory viruses, 512–513 Rituximab, 122, 735 S SCT models, hematopoiesis allogeneic, 776–777 autologous, 775 gene therapy, autotransplantation, 775–776 GVHD, 777 MHC typing, 777–778 whole organ tolerance induction, 778 xenotransplantation, 779–780

882 

Index

Second allogeneic transplantation graft failure back-up autologous stem cells, 664 cyclosporine, 663 immunologic rejection, 662 positive CMV serology, 663 relapsed acute leukemia DLI, 657 GVHD risk, 659–661 prognostic factors, 658 reduced intensity conditioning regimens, 662 TBI vs. non-TBI therapies, 658 Second complete remission (CR2), pediatric ALL ALL in advanced phase, 244 eligibility criteria, 236–238 relapsed ALL, 236 transplantation, 238, 240–244 umbilical cord outcomes, 244 Second line therapy, aGVHD treatment broad anti-T cell agents/antibodies alemtuzumab, 753 anti-thymocyte globulin, 752 visilizumab, 753 broad anti-T cell agents/immunomodulatory agents deoxycoformycin, 755 extracorporeal photopheresis (ECP), 755 mycophenolate mofetil/MMF, 753–754 sirolimus, 755 narrow anti-T cell agents/receptor and cytokine targets ABX-CBL/anti CD147, 757 daclizumab, 755–756 denileukin diftitox, 756–757 TNF inhibition etanercept, 758 IL-1 receptor antagonist (IL1-RA), 758 infliximab, 757 SEER. See Surveillance epidemiology and end results Single vs. tandem autologous transplant, HSCT complete response (CR) rates, 153 HDT/transplant vs. SDT OS benefit, 147, 150 PFS benefit, 146 maintenance therapy, 151 cancer and leukemia group B (CALGB), 152 thalidomide, 151 novel pre-transplant regimens, 153 optimal time, transplant blood and marrow transplant clinical trials network (BMT CTN), 149 bortezomib, 148 lenalidomide maintenance therapy, 150 single vs. double autologous ASCT, 149 PFS benefit, 146 salvage therapy, 151 United States Intergroup Study, S9321, 146 Sirolimus FK binding protein 12 (FKBP12), 569 Streptomyces hygroscopicus, 569 tacrolimus, 570

Soluble BAFF (sBAFF), cGVHD, 587 Somatic side-effects, 632–634 Stem cell factor (SCF), 815 Surveillance epidemiology and end results (SEER), 129 T T cel depletion (TCD), 565 T cell receptor rearrangement excision circles (TREC), 548 T cell reconstitution, immunotherapy cellular immunotherapy, thymopoiesis anti-tumor immunity, 553 diagrammatic representation of, 552 HPE endogenous proliferation, 550 homeostatic proliferation, 550 IL-7/15, 549 TGFß, 550 initial post-transplant period and implications oligoclonal peripheral expansion, 551 thymopoiesis, 550 thymic dependent, 548–549 naïve phenotype, 548 recent thymic emigrants (RTE), 549 TREC bearing cells, 549 Texas transplant institute perspective, 382–383 Thalassemia, allogeneic gene therapy alternative donors transplantation, 498–499 BMT vs. medical treatment, 492 disease eradication, 493–494 engraftment, 494 ex-thalassemic management, 499–500 graft failure/rejection, 497–498 GVHD, 498 mixed chimerism, 497 morbidity and mortality, 494 preparatory regimens, 493 risk classes, 495 transplant outcome, HLA matched related donors, 495–497 adult patients, 496–497 class 1and 2 patients, 495 class 3 patients, 495–496 transplant procedures, 492 Thrombopoietin, 392 Thrombotic and hemostatic complications acquired hypercoagulable disorders, 708 bleeding central nervous system (CNS), 701–702 complications, 699 diffuse alveolar hemorrhage (DAH), 700–701 events, 698 gastrointestinal, 700 hemorrhagic cystitis, 698–700 ocular, 702 catheter-related thrombosis, 704 dural (venous) sinus thrombosis, 708 endothelial cell, 695 etiology, 703

Index  GVHD acute GVHD, 696–697 chronic GVHD, 697–698 mechanisms, 697 hepatic veno-occlusive disease (VOD), 704–706 non-myeloablative (NM) and RIC regimens, 708–710 pulmonary cytolytic thrombi (PCT), 707–708 pulmonary veno-occlusive disease (VOD), 707 thrombosis risk factors, 702–703 transplantation-associated thrombotic microangiopathy (TA-TMA), 706–707 Toll-like receptors (TLRs), 812 Total lymphoid irradiation (TLI), 452–453 Total nucleated cells (TNC), 376–377 Transplantation-associated thrombotic microangiopathy (TA-TMA), 706–707 Transplant related mortality (TRM) adoptive cell therapy, 465 G-CSF impact, 464 immunological recovery, 464 myeloablative transplantation, 263 overlapping factors, 464 procedure, 463 Treatment related morbidity and mortality (TRM), 57 Treatment-related mortality (TRM), 187 T regulatory 1 cells (Tr1), 819 Tumor-associated antigens (TAA), 855 Tyrosine kinase inhibitors (TKI), CML patients BCR-ABL gene, 54 breakpoint cluster region (BCR), 54 EBMT transplantation risk score, 57 imatinib use, 65 novel kinase inhibitors, 67 Philadelphia (PH) chromosome, 54 survival percentage, 57 treatment related morbidity and mortality (TRM), 57 U UD-SCT. See Unrelated donor stem cell transplantation Umbilical cord blood (UCB) transplantation adult cord blood double cord blood, 368–369 reduced intensity regimen, 367 refractory lymphoma, single cord blood, 368 single unit ablative regimen, 367 unrelated cord blood vs.bone marrow/peripheral blood stem cell, 366–367 challenges, 370 cord blood banking, 364 ethical issues, 365 pediatric cord blood, 365–366 pre-clinical characteristics, 363 strategies, 369–370 Unrelated donor cord blood cell dose, 377–378

883

diagnosis effect, 381 double cord blood units, 381–382 factors, 377–378 product, 376–377 Texas Transplant Institute Perspective, 382–383 Unrelated donor stem cell transplantation (UD-SCT), 33 Unrelated donor transplants actuarial disease free survival (DFS), 357 acute leuekemia, 356 aplastic anemia, 357–358 bone marrow donors, 347 clinical outcome, 348–349 conditioning regimens, 352–353 HLA and matching criteria antigenic matching, 347–348 haplotypes matching, 348–349 HLA mismatch effect, 349 HSC donation collection quality, 350–351 safety issue, 349 human leukocyte antigen (HLA) typing technology, 346 myelodysplastic syndromes, 356–357 patient selection and indications, 350–351 prophylaxis acute GvHD, 352 chronic GvHD, 352 stem cell source, 353–354 V Vancomycin-resistant enterococcus (VRE), 720–721 Varicella-Zoster virus (VZV), 509–510 Veno-occlusive disease (VOD), 707 Veto activity, 462 Viral infections adenoviruses, 514–515 cytomegalovirus (CMV) immune monitoring and immune therapy, 509 preemptive therapy, 508 prophylaxis, 507–508 risk factors, 507 diagnostic tests, 506 Epstein-Barr Virus (EBV), 510–511 hepatitis B and C viruses, 515 herpes simplex virus (HSV), 509 human herpes virus type 6 (HHV-6), 511–512 influenza viruses, 514 papovaviruses, 516 respiratory syncytial virus (RSV), 513 respiratory viruses, 512–513 Varicella-Zoster virus (VZV), 509–510 Y Yakoub-Agha, MDS, 209