Post-Transplant Lymphoproliferative Disorders

Post-Transplant Lymphoproliferative Disorders

Vikas R. Dharnidharka • Michael Green Steven A. Webber (Eds.)

Post-Transplant Lymphoproliferative Disorders

Vikas R. Dharnidharka, MD, MPH Associate Professor and Chief, Fellowship Program Director Division of Pediatric Nephrology University of Florida College of Medicine Medical Director, Pediatric Kidney Transplantation, Shands Hospital at UF Gainesville, Florida, USA [email protected]

Steven A. Webber MBChB, MRCP Professor of Pediatrics, University of Pittsburgh School of Medicine Chief, Division of Cardiology Co-Director, Heart Center Medical Director, Pediatric Heart and Heart-Lung Transplantation Children’s Hospital of Pittsburgh Pittsburgh, Pennsylvania, USA

Michael Green, MD, MPH Professor of Pediatrics and Surgery University of Pittsburgh School of Medicine Division of Infectious Diseases Children’s Hospital of Pittsburgh Pittsburgh, Pennsylvania, USA michael.green@ chp.edu

ISBN: 978-3-642-01652-3

e-ISBN: 978-3-642-01653-0

DOI: 10.1007/978-3-642-01653-0 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2009934493 © Springer-Verlag Berlin Heidelberg 2010 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Cover design: eStudio Calamar, Figueres/Berlin Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Dedication

We dedicate this book to the clinicians and scientists who cared for transplant patients and began the work of understanding the problem of PTLD, to the transplant recipients and their families that we care for who motivate these efforts, and to the colleagues we work with to provide care to our patients. Finally, special thanks to our families who had put up with us and supported our efforts in preparing this book and in all other things: Ramnath, Pushpa, Dimple, Shrey and Ria; Jenny, Dave, Erin, Molly, and Allison; Elizabeth, Hannah, and Katie.

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Foreword

By the mid 1960s, there was evidence that the loss of tumor surveillance in immunosuppressed organ recipients could result in (1) accidental engraftment of donor malignancies, (2) accelerated growth of microscopic metastatic neoplasms of either donor or recipient origin, and (3) an increased incidence of de novo malignancies. Although real, the risks from the first two kinds of complications were promptly minimized by appropriate donor and recipient screening. In contrast, the de novo malignancies, most notably those of lymphoid origin (called posttransplantation lymphoproliferative disorders or PTLDs), have been endemic, or at times epidemic, particularly just after the advent of the T-cell-directed agents, cyclosporine, tacrolimus, and the antilymphoid antibodies. The PTLDs resemble the malignancies found in immune deficiency states, including acquired immunodeficiency syndromes. Originally referred to as reticulum cell sarcomas, they constitute a spectrum of lymphopoietic neoplasms, of which most are B-cell lymphomas that are highly, but not invariably, associated with Epstein–Barr virus infection. Because PTLDs are frequently subject to immune surveillance, the pathogenesis and treatment of these lymphoblastic lesions have been of special interest to tumor biologists as well as to clinicians. Heavy immunosuppression, either applied by protocol or in a management response to rejection, usually precedes the appearance of PTLD. Most of these tumors are of host origin in the organ recipients, whereas almost all are of donor origin after bone marrow transplantation. Although PTLDs occur in all kinds of organ recipients, the highest incidence has been after transplantation of nonrenal organs. Reduction or discontinuance of immunosuppression in organ recipients allows recovery of tumor surveillance and may be followed by tumor regression. The most effective treatment is complete discontinuance of immune suppression. However, this drastic step is usually avoided in kidney recipients and is rarely taken in liver, heart, and lung recipients for whom “treatment rest” and artificial organ support followed by retransplantation are not feasible. Even in such cases, however, stopping immunosuppression as a last resort has been effective in some nonrenal organ recipients without rejection of their allografts. Such examples of donor-specific, but not tumor-specific, immune nonreactivity and other features of PTLD could not be explained until the mechanisms of leukocyte chimerism-dependent alloengraftment and allogeneic tolerance were elucidated with the discovery that organ recipients had leukocyte microchimerism.

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The resulting fresh insight into mechanisms has suggested treatment options for PTLDs beyond stopping immunosuppression or the use of multiple-agent antilymphoma and antiviral therapies. In fact, PTLDs have evolved into a free-standing model in which some mysteries of immunology, virology, and tumor biology have been resolved while others await further discoveries that may be exploited. Meanwhile, many books have been written about transplantation, but none specifically about PTLDs. The three editors of this book are to be commended for their efforts to fill the gap. There is something here for all clinicians, and perhaps for basic scientists who also are looking for new worlds to investigate and conquer. Thomas E. Starzl, MD, PhD Professor of Surgery Thomas Starzl Transplant Institute University of Pittsburgh PA, USA

Contents

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Introduction ........................................................................................................ Vikas R. Dharnidharka, Michael Green, and Steven Webber

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Historical Perspective on the Early Studies of Posttransplant Lymphoproliferative Disorders (PTLD)............................ Douglas W. Hanto

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Epidemiology of PTLD ...................................................................................... Vikas R. Dharnidharka

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The Biology of Epstein–Barr Virus and Posttransplant Lymphoproliferative Disease ........................................... Olivia M. Martinez

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Epstein–Barr Viral Load Testing: Role in the Prevention, Diagnosis and Management of Posttransplant Lymphoproliferative Disorders ........... Jutta K. Preiksaitis

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Clinical Features and Diagnostic Evaluation of Posttransplant Lymphoproliferative Disorder ........................................... Upton D. Allen

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Pathology ............................................................................................................ Steven H. Swerdlow

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Prognostic Factors for PTLD ............................................................................ Tapan Maniar and Donald Tsai

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Treatment of PTLD............................................................................................ Steven A. Webber

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Prevention of Epstein–Barr Virus Infection and Posttransplant Lymphoproliferative Disease Following Transplantation ........................... Michael Green and Marian Michaels

11.a Organ Specific Issues of PTLD – Kidney ..................................................... Sophie Caillard 11.b Posttransplantation Lymphoproliferative Disorder (PTLD) in Liver and Small Bowel Transplant Recipients......................................... Jaime Pineda and George V. Mazariegos 11.c Heart and Lung Transplantation .................................................................. Silke Wiesmayr and Steven A. Webber

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11.d Posttransplant Lymphoproliferative Disease (PTLD) in Hematopoietic Stem Cell Transplantation (HSCT)................................. Thomas G. Gross

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Research Priorities and Future Directions ................................................... Vikas R. Dharnidharka, Michael Green, and Steven A. Webber

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Index ..........................................................................................................................

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Contributors

Upton D. Allen Department of Pediatrics, Division of Infectious Diseases, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada

Douglas W. Hanto Division of Transplantation, The Transplant Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA

Sophie Caillard Nephrology-Transplantation Department, Hopitaux Universitaires de Strasbourg, Strasbourg, France

Tapan Maniar University of Pennsylvania, Philadelphia, PA, USA

Vikas R. Dharnidharka Division of Pediatric Nephrology, University of Florida College of Medicine, Gainesville, FL, USA Pediatric Kidney Transplantation, Shands Hospital at UF, University of Florida College of Medicine, Gainesville, FL, USA Michael Green Departments of Pediatrics and Surgery, Division of Infectious Diseases, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA Thomas G. Gross Department of Pediatrics, The Ohio State University School of Medicine, Columbus, OH, USA Division of Hematology/Oncology/BMT, Nationwide Children’s Hospital, Columbus, OH 43205, USA

Marian Michaels Division of Infectious Diseases, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Olivia M. Martinez Department of Surgery, Division of Transplantation and the Program in Immunology, Stanford University School of Medicine, Palo Alto, CA 94305-5492, USA George V. Mazariegos Hillman Center for Pediatric Transplantation, Children’s Hospital of Pittsburgh, Thomas E. Starzl Transplantation Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA xi

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Jaime Pineda Hillman Center for Pediatric Transplantation, Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA Jutta K. Preiksaitis Division of Infectious Diseases, University of Alberta, Edmonton, AB, Canada Provincial Public Health Laboratory, University of Alberta, Edmonton, AB, Canada T6G 2J2 Steven H. Swerdlow Division of Hematopathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA

Contributors

Donald E. Tsai Division of Hematology/Oncology, University of Pennsylvania, Philadelphia, PA, USA Steven A. Webber Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Division of Cardiology, Children’s Hospital of Pittsburgh, 3705 5th Avenue, Pittsburgh, PA 15213, USA Silke Wiesmayr Division of Cardiology, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Introduction

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Vikas R. Dharnidharka, Michael Green, and Steven Webber

Core Messages

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Posttransplant lymphoproliferative disorders (PTLDs) straddle the disciplines of immunology, infection, and malignancy. The study of PTLD could lead to advances that have ramifications beyond transplantation. Early observations revealed that lightening of immunosuppression could lead to regression in some cases, eliminating the hopelessness of the word “lymphoma.” As newer and more potent immunosuppressive agents were introduced, the frequency of PTLD increased, responses were often suboptimal, and the need for additional strategies for prevention and treatment became apparent. PTLD is now recognized as a major complication of transplantation, such that we now consider its study a discipline by itself.

Posttransplant lymphoproliferative disorder (PTLD) is at once both “bad” and “fascinating” in organ and tissue transplantation. It is “bad” since it represents an unwanted complication of transplantation, with significant morbidities and mortality. It is “fascinating” since this disease, for the most part, results from lost immune control of a long-lived virus that has infected the body’s cells, leading to uncontrolled proliferation of the infected cell. Thus, PTLD straddles the disciplines of immunology, infection, and malignancy. These are three of the basic disease processes involved in a large percentage of all diseases in the general population. The study of PTLD, therefore, could lead to advances that have ramifications, beyond just transplantation, in the very basic processes of how the body’s immune system deals with any long-lived microbe, especially when placed in any nonphysiological situation, such as allotransplantation. In the intial stages of the organ transplant field, the focus was on how to prevent acute rejection of the allograft by the recipient’s immune system. The concepts that (1) a virus

V. Dharnidharka (*) Division of Pediatric Nephrology, University of Florida College of Medicine, Gainesville, Florida, USA e-mail: [email protected]fl.edu V. R. Dharnidharka et al. (eds.), Post-Transplant Lymphoproliferative Disorders, DOI: 10.1007/978-3-642-01653-0_1, © Springer Verlag Berlin Heidelberg 2010

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could be long-lived in a latent form in an organ being transplanted and that (2) this virus would have far-reaching consequences because of therapeutic immune suppression were well beyond people’s imagination at the time. Occasional cases of PTLD were seen in the late 1960s and early 1970s, beginning with the first reports by Dr Thomas Starzl in 1968 in a written discussion of Murray’s transplant results [1], referring to the malignancies as “reticulum cell sarcomas.” The B-cell lymphoid origin and the link to Epstein–Barr virus (EBV) became apparent later. As more cases were reported, this disease presented many more complexities than originally thought. Back then, the disease was considered as a rare nuisance, a necessary but very infrequent evil of the good that all of us were trying to do [2]. Many investigators seized the opportunity provided by this experiment of nature (and man). “Lightening” of immunosuppression, as shown by Starzl in his Lancet series [3], seemed enough in some cases to cause regression, and eliminate the hopelessness of the word lymphoma. What our transplant community did not anticipate then was that as newer and more potent immunosuppressive agents were developed and introduced, the PTLD frequency would increase, responses would be suboptimal, and more strategies would be needed. PTLD, thus, developed into a major complication of transplantation, such that we now consider its study a discipline by itself. In the chapters within this book, the authors tackle each of these fascinating aspects and more. The amount of knowledge and information accumulated over the last four decades no longer fits within the confines of medical journal review articles; hence, we felt the need for this book. Dr. Starzl, the pre-eminent transplantation scientist, discusses his thoughts about PTLD when it first presented, and how those thoughts have evolved over 40 years. Dr. Hanto provides a detailed historical overview of PTLD in the early years, including some of his own seminal work. Dr. Dharnidharka details the epidemiological aspects, including the changing incidence over time and the relationship to immunosuppression. Dr. Martinez discusses the current state of knowledge with respect to EBV biology and the pathogenesis of PTLD, an ever changing field. Dr. Preiksaitis provides an overview of EBV PCR technology and the caveats involved in the assay and its interpretation. Dr. Allen presents the myriad clinical features possible, which force transplant clinicians to keep PTLD in the differential diagnoses in so many situations. Dr. Swerdlow presents the pathological classification currently in use, which incorporates genetic and molecular information that is continually being updated under the auspices of the World Health Organization. Drs. Green and Michaels summarize the very important topic of EBV disease and PTLD prevention. Dr. Webber then discusses the therapeutic options currently available and the evidence on which their usage is based. Drs. Tsai and Maniar explore the prognostic factors reported so far. Several organ subspecialists then summarize the epidemiology, presentation, and treatment of PTLD in recipients of thoracic (Drs.Wiesmayr and Webber), liver (Drs. Pineda and Mazariegos), kidney (Dr. Caillard), or hematopoietic cell transplants (Dr. Gross). Finally, we end by summarizing much that is still unknown and the directions required for future research to solve this perplexing and many-faceted disease. This book should be of value to transplant professionals in all disciplines including transplant surgery, nephrology, immunology, infectious disease, hepatology, cardiology, pulmonology, and pathology. We wish to thank all the authors who have contributed their expert knowledge and hard work to this text, and also wish to offer our thanks to Springer for recognizing the need for such a book and translating our dream to reality.

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References 1. Murray JE, Wilson RE, Tilney NL, Merrill JP, Cooper WC, Birtch AG, et al. Five years’ experience in renal transplantation with immunosuppressive drugs: survival, function, complications, and the role of lymphocyte depletion by thoracic duct fistula. Ann Surg. 1968;168(3):416–35; Discussion by Starzl TE, p. 433–4 2. Nalesnik MA, Makowka L, Starzl TE. The diagnosis and treatment of posttransplant lymphoproliferative disorders. Curr Probl Surg. 1988;25(6):367–472 3. Starzl TE, Nalesnik MA, Porter KA, Ho M, Iwatsuki S, Griffith BP, et al. Reversibility of lymphomas and lymphoproliferative lesions developing under cyclosporin-steroid therapy. Lancet. 1984;1(8377):583–7

Historical Perspective on the Early Studies of Posttransplant Lymphoproliferative Disorders (PTLD)

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Douglas W. Hanto

Core Messages

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Cases of cancer started appearing soon after introduction of chronic immunosuppression in organ transplant recipients Posttransplant lymphoproliferative disorders (PTLD) initially presented with varied features but a pathophysiologic paradigm was developed The linkage of PTLD to EBV was initially made serendipitously but then confirmed through several types of investigations Early treatments published included acyclovir and reducing immunosuppression

2.1 Malignancies in Immunocompromised Hosts Less than a decade after the introduction of immunosuppression, an increased risk of cancer in renal allograft recipients and five cases of malignant lymphoma were reported [1, 2]. In the 1970s, it was reported that the incidence of malignant lymphomas arising in patients with genetically determined immunodeficiency diseases (e.g., congenital immunodeficiency, ataxia telengiectasia, and X-linked lymphoproliferative syndrome (XLP) ) and immunosuppressed renal allograft recipients was increased [3–7].There were many hypotheses about the causes including impaired immune surveillance, chronic antigenic stimulation,

D. W. Hanto Division of Transplantation, The Transplant Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, 110 Francis Street, 7th Floor, Boston, MA 02215, USA e-mail: [email protected]

V. R. Dharnidharka et al. (eds.), Post-Transplant Lymphoproliferative Disorders, DOI: 10.1007/978-3-642-01653-0_2, © Springer Verlag Berlin Heidelberg 2010

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reactivation of latent oncogenic herpesviruses, and direct oncogenic effects of immunosuppressive drugs, but none were proven [8]. By 1980, malignant lymphomas had been described in kidney, liver, heart, bone marrow, and thymic epithelial transplant recipients (reviewed in [9]).

2.2 The Epstein–Barr Virus In 1964, the Epstein–Barr virus (EBV) was identified by electron microscopy in cultured cells from African Burkitt’s lymphoma (BL) biopsies [10]. Over the next two decades, EBV was shown to transform B lymphocytes inducing a polyclonal proliferation in vitro [11] and in vivo [12]; to cause infectious mononucleosis (IM) in humans [13] and a fatal lymphoproliferative disorder in cotton-top marmosets [14]; and was linked to BL [15], nasopharyngeal carcinoma (NPC), and the lymphoproliferative disorders (LPD) occurring in patients with XLP [5, 6]. At that time, it was known that EBV caused two types of cellular infections: a productive replicative or lytic infection in which mature infectious virus particles were assembled and released resulting in cell death; and a nonproductive infection in which the virus is incorporated into and replicates with the host DNA, but remains in the latent state in transformed B-cells [16]. EBV proliferation was controlled by EBV specific cytotoxic T lymphocytes and by specific antibodies directed against virally determined antigens [9, 13, 17]. It was shown that oropharyngeal shedding of EBV occurred in 47–87% of renal transplant recipients [18, 19] compared to 17% in normal seropositive individuals [20], and that rises in EBV antibody titers occurred commonly after transplantation, but clinical illness was rare [21, 22].

2.3 The Serendipitous Intersection of Immunosuppression and EBV In May 1976, a patient at the University of Minnesota underwent a living donor kidney transplant 1 month after his college roommate developed IM. The recipient died 4 weeks later of a rapidly progressive and invasive polyclonal LPD associated with an elevated heterophil antibody titer, a rise in the anti-VCA (viral capsid antigen) IgM and IgG titers, and a polyclonal increase in serum immunoglobulins. This was thought to represent a case of severe fatal IM transmitted from the roommate, but also had features suggestive of an aggressive lymphocytic malignancy and was reported at a pathology meeting because of the unusual morphologic features and polyclonality [23]. Subsequent cases in other renal transplant recipients were soon observed. The clinical and pathologic presentation and subsequent clinical course in these patients were varied, providing early evidence of the range of histology, progression, and outcome of these lesions. The unusual clinical presentations, pathologic findings, and hint of a role for EBV led to a great interest in studying these and additional patients over the next 2 years [24–28].

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2.4 Initial Studies of “Posttransplant Lymphoma” The initial analyses of the patient with possible IM and two new patients with oropharyngeal lesions raised many questions. The former patient had an invasive and fatal lymphoproliferative disorder that had many characteristics of an aggressive lymphoma, but was polyclonal, not monoclonal. Was it an EBV infection (i.e., fatal IM) accelerated by immunosuppression (a form of iatrogenic XLP) or an unusual manifestation of an aggressive malignancy? Two subsequent patients had, on initial biopsy, an invasive polymorphic B-cell hyperplasia that in one patient evolved into a polymorphic B-cell lymphoma with characteristics of a monoclonal malignancy (see below). Again the question was whether these were infectious in origin or an unusual presentation of a malignant lymphoma? Cell marker studies done by immunofluorescence for surface (sIg) and cytoplasmic immunoglobulin (cIg) and immunoperoxidase staining for kappa (k) and lamda (l) light chains demonstrated on the initial biopsies of these two patients that they were both polyclonal proliferations. Clonal cytogenetic abnormalities in one patient, however, suggested the development of a malignant cell clone, not yet identifiable morphologically or by cell marker studies. Tissue from both patients demonstrated EBV specific sequences. With a reduction of immunosuppression, lesions in both of these patients resolved. However, the second patient developed metastatic lymph node disease, which, on biopsy, had changed morphologically and now manifested features more characteristic of a malignant lymphoma and, as in the first patient, now contained a subpopulation of cells with cytogenetic abnormalities. This patient subsequently developed widely metastatic disease and died. Based on our experience with these three patients, we speculated that EBV induced a polyclonal lymphoproliferation that initially proceeded unchecked in the immunosuppressed patients; that a reduction in immunosuppression could restore the immune balance and lead to resolution of the lymphoproliferation; that chronic antigenic stimulation from the allograft may further stimulate continued B-cell proliferation; and that a cytogenetic event might then occur leading to the emergence of a malignant clone represented by the undifferentiated cell population that did not stain for Ig [25]. This hypothesis fits well with the three-step theory proposed by Klein for the development of African BL [15].

2.5 Growing Experience with EBV and Posttransplant Lymphoproliferative Diseases The patients described were immunosuppressed with azathioprine, prednisone, and Minnesota anti-lymphocyte globulin. In early studies of cyclosporine, there were reports of a higher incidence of lymphomas [29]. Two reports, about this same time, provided additional evidence for the relationship between EBV and these diseases using serologic techniques [30] and Epstein– Barr nuclear antigens (EBNA) staining of biopsy tissue [31]. We subsequently presented and published a detailed analysis of six patients with posttransplant lymphoproliferative disorders (PTLD), and began to classify these lymphoproliferations according to their clinical,

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morphologic, immunologic, and virologic characteristics with the hope that this would aid in determining the best therapy [24, 26]. Subsequently, there were two other major series of patients reported from Stanford University and the University of Pittsburgh, and we extended our observations to include 19 patients. These studies provided important observations that have stood the test of time and are important to review in light of current knowledge.

2.6 Clinical Spectrum of Lymphoproliferative Disorders Two groups of patients were initially identified [26, 27]. First, there were tumors arising in younger patients (mean age, 23 years) who presented soon (mean, 9 months) after transplantation or antirejection therapy with fever, pharyngitis, and lymphadenopathy resembling IM. Second, older patients (mean age, 48 years) presented later (mean, 6 years) after transplantation with symptoms and signs related to solid tumor masses. Weintraub and Warnke [32] reported that lymphomas in first heart transplant recipients occurred later (775 days) compared to lymphomas occurring in retransplant patients (365 days), and that the incidence of lymphoma was greater in the retransplant population. All patients presented with localized extranodal lesions and 5/7 had central nervous system (CNS) involvement. Starzl reported 17 cases in kidney, liver, heart, and heart–lung transplant recipients that included six with an IM-like syndrome and nine with gastrointestinal lymphomas manifested by perforation, obstruction, or hemorrhage [33]. Eleven patients had solid tumors in multiple extranodal sites. Most were young and the diseases occurred in the first year posttransplant, and all had received cyclosporine and prednisone.

2.7 Morphology Early efforts were made to histologically characterize these lesions. Frizzera described the unique morphological features of the lymphoid lesions in these patients and interpreted them as being different expressions of a morphological and biological spectrum of abnormal B-cell proliferations [24]. His proposed schema categorized lesions as either polymorphic B-cell hyperplasias (PDBH) or as polymorphic B-cell lymphomas (PBL). PDBH differed from nonspecific lymphoid hyperplasias, including infectious mononucleosis in immunologically normal hosts. Although the obliteration of nodal architecture suggested a malignancy, the reactive cellular component without atypia and the polyclonal nature of the proliferations argued against this interpretation and suggested an unrestrained B-cell hyperplasia in an immunocompromised host. PBL were characterized by nuclear atypia and extensive necrosis and differed from the immunoblastic sarcomas described in the literature that were viewed as morphologically and immunologically pure neoplasias of immunoblasts. PBL arising in transplant recipients and other immunocompromised hosts

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were thought to represent immunoblastic malignancies that still bear morphological and immunological evidence of their origin from a reactive lymphoid process. Early descriptions of PTLD in cardiac transplant recipients at Stanford were all classified as diffuse large cell lymphomas, though, by description, some seem to correspond to Frizzera’s PBL [32]. In the Pittsburgh series, two patients had B-cell hyperplasia, 11 had large noncleaved lymphoma, and three immunoblastic malignant lymphoma [33].

2.8 Clonality We and others carried out studies to define the clonality of these lesions. Our initial studies demonstrated the presence of polyclonal B-cell proliferations indicating a common reactive nonneoplastic origin [24]. However, some biopsies manifest features suggestive of evolution into a malignant lymphoproliferation. In a subsequent larger series, we found that although the majority were polyclonal B-cell proliferations [27], the presence of monoclonal B-cell tumors, a monoclonal immunoblastic lymphoma, and the evolution from polyclonal to monoclonal proliferations were also documented. Using similar techniques, the lymphoproliferations in the Pittsburgh series were found to be polyclonal in six, monoclonal in three, and indeterminate in six [33]. All patients in their series with polyclonal B-cell proliferations were interpreted as hyperplasia. Using immunoglobulin gene rearrangement (IgR) techniques, Weintraub and Warnke found that the lymphoid lesions arising in the Stanford cardiac allograft patients were monoclonal proliferations [34]. On further study, however, an important observation was made that although each individual biopsy had a single clonal rearrangement of immunoglobulin gene DNA, there were different rearrangements in biopsy tissues from separate sites in the same patient [35]. These lymphoproliferations were designated multiclonal or oligoclonal, rather than polyclonal, because no specimen demonstrated more than two rearranged bands per immunoglobulin gene. Our subsequent evaluation of five renal transplant recipients and one cardiac transplant recipient with EBV-related PTLD led to several important observations [36]: 1. Biopsies from two patients with lesions that were hyperplastic, polyclonal by sIg and cIg staining, and had diploid karyotypes, had no detectable IgR and were, therefore, consistent with benign reactive processes. These patients were alive at 37 and 57 months after diagnosis without evidence of disease. 2. In one patient with a lesion that was malignant lymphoma, monoclonal, and had clonal cytogenetic abnormalities, clonal IgR were detected in a majority of cells, confirming their neoplastic nature. 3. In biopsies from an intermediate group of three patients with morphologically malignant proliferations that were composed predominantly of a polyclonal population of B-cells, clonal gene rearrangements were also found, consistent with early malignant transformation in a subpopulation of cells.

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These data affirmed our earlier hypotheses regarding the evolution of these PTLD. Later chapters in this book detail the ongoing evolution of the classifications, which have led to serial WHO classifications including the most recent.

2.9 EBV It has been clearly established that EBV plays a critical role in the development of most PTLD by serologic studies, EBNA staining of tumor tissue, and EBV DNA hybridization studies that demonstrate the presence of EBV-specific sequences in tissue specimens. In our series of IM-like illness, a third had evidence of a primary EBV infection, a third reactivation, and a third long-past infection. Staining of tumor tissue for EBNA has been positive in several patients [26, 31, 33], but EBV DNA hybridization techniques that were available at the time were more sensitive and were positive in 10/12 patients studied in our series [37], and in four Stanford cardiac surgery patients [38]. In the Pittsburgh series, 6/12 patients had EBNA-positive PTLD and hybridization studies were not reported [33].

2.10 Therapy We and others reported on our initial efforts in the treatment of PTLD. Because many patients had EBV-carrying polyclonal B-cell proliferations and serologic evidence of a primary or reactivation EBV infection, we hypothesized that interruption of EBV replication with acyclovir might be an effective therapy. Acyclovir had been shown to inhibit EBV DNA replication only in virus-producing cell lines by blocking the EBV associated DNA polymerase, but did not affect the synthesis of the latent EBV genome in nonproducer cell lines. We reported a kidney transplant recipient with an EBV-associated polyclonal PBL that underwent regression on two occasions during treatment with acyclovir in association with suppression of oropharyngeal shedding of EBV [39]. This was the first clinical report of the use of acyclovir in a clinical EBV infection. We suggested that acyclovir was effective early in the disease by interrupting the lytic cycle of EBV replication that was responsible for the polyclonal B-cell proliferation. Unfortunately, the patient progressed to a monoclonal B-cell proliferation, presumably composed of latently infected (and therefore acyclovir insensitive) B-cells. Therefore, we suggested that acyclovir might need to be combined with other therapies including a reduction in immunosuppression or chemotherapy. The potential therapeutic role of reduction of immunosuppression was illustrated by Starzl et al. [33] who noted regression of lymphoproliferative lesions in several patients with a reduction in immunosuppression. This report also illustrated the value of other therapies including resection of localized gastrointestinal lymphomas. However, even in this series, five of six patients with widespread lymph node disease died in spite of

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a reduction in immunosuppression. During this time, there were also reports suggesting that a-interferon and g-globulin were efficacious in bone marrow [40] and organ allograft recipients [41] who do not respond to antiviral therapy or reduction in immunosuppression, or who have severe and rapidly progressive disease. These initial as well as our ongoing experience led to proposed management recommendations for these patients. Patients with benign polyclonal PDBH appear to respond to acyclovir and that a reduction in immunosuppression might or might not be required depending on the severity of EBV infection and response to acyclovir [42]. If there was evidence of progression from polyclonal to monoclonal (morphologically, cytogenetically, or by cell marker studies), we recommended that immunosuppression should be reduced and rejection be allowed to proceed should it occur to avoid development of a fatal monoclonal lymphoma. For patients with monoclonal PTLD, a reduction in immunosuppression combined with chemotherapy/radiation therapy was suggested. These initial strategies formed the basis of treatment for PTLD for years to come. Current concepts on the role of antiviral therapy and other treatment options for EBV/PTLD will be addressed in Chap. 9.

2.11 Summary Classification of PTLD Based on this work, we argued that PTLD could be divided into four main categories based on clinical presentation, morphology, immunologic cell typing, immunoglobulin gene rearrangements, cytogenetic abnormalities, and virologic characteristics [27] reviewed in [9, 43], and that this classification scheme was important for determining treatment strategies. The first category was uncomplicated IM. We proposed that acyclovir might be beneficial in these patients and suggested it might have a role recognizing that most patients had a self-limited clinical course. The second category was benign polyclonal polymorphic B-cell hyperplasia which is characterized by: an IM-like illness with serologic evidence of a primary or reactivated EBV infection and EBV DNA sequences within the tissue; and tissue biopsies that are morphologically PDBH and are polyclonal by standardized testing. Recommended therapy at the time included anti-viral therapy and a reduction of immunosuppression in severe cases. The third category was an intermediate group that is difficult to characterize because patients exhibit characteristics of both benign and malignant B-cell proliferations. These patients have evidence of early malignant transformation in polyclonal polymorphic B-cell lymphoma characterized by: an IM like illness or in some cases, solid tumor masses containing EBV; and biopsies that are morphologically malignant B-cell proliferations and are polyclonal by light-chain phenotyping. Yet in a small subpopulation of cells, clonal cytogenetic abnormalities and immunoglobulin gene rearrangements can be demonstrated and have the potential to evolve into a monoclonal or oligoclonal lymphoma. Recommended therapy at the time was antiviral therapy for the polyclonal B-cell proliferation presumably dependent on EBV replication and a reduction in immunosuppression. Finally, the fourth category are patients who present or evolve into monoclonal polymorphic B-cell lymphoma. These patients are older, develop disease longer

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posttransplant, and develop solid tumor masses in the organ allograft, soft tissue, brain, gastrointestinal tract, lung, or liver. These PTLD are morphologically malignant and monoclonal by phenotypic and genotypic characterizations. A reduction in immunosuppression, along with chemotherapy, radiation therapy, and surgical resection when appropriate, was recommended.

2.12 Pathogenesis A proposed pathogenesis of these diverse PTLD was developed and is illustrated in Fig. 2.1. In transplant recipients, immunosuppression and chronic antigenic stimulation may explain why these events can occur over a few months rather than years.

Fig. 2.1 Proposed pathogenesis of the spectrum of EBV- associated PTLD

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2.13 Conclusions This early work has been the foundation for subsequent work. It has stood the test of time, and is still relevant in many ways today. Having said this, however, there have been many important and remarkable advances that have improved our ability to prevent, diagnose, and treat patients with a wide spectrum of PTLD, as well as identify patients at risk. The leaders in the field will no doubt highlight this work in the other chapters of this book.

2.14 Take Home Pearls

• •

Early work confirmed, through several means, the critical linkage between EBV and PTLD. The pathophysiology was hypothesized to involve several progressive steps, a paradigm that has stood the test of time.

References 1. McKhann CF. Primary malignancy in patients undergoing immunosuppression for renal transplantation. Transplantation. 1969;8:209–12 2. Penn I, Hammond W, Brettschneider L, Starzl TE. Malignant lymphomas in transplantation patients. Transplant Proc. 1969;1(1):106–12 3. Gatti RA, Good RA. Occurrence of malignancy in immunodeficiency diseases. A literature review. Cancer. 1971;28(1):89–98 4. Penn I. Malignancies associated with immunosuppressive or cytotoxic therapy. Surgery. 1978;83(5):492–502 5. Purtilo DT. Pathogenesis and phenotypes of an X-linked recessive lymphoproliferative syndrome. Lancet. 1976;2(7991):882–5 6. Purtilo DT, DeFlorio D Jr., Hutt LM, et al. Variable phenotypic expression of an X-linked recessive lymphoproliferative syndrome. N Engl J Med. 1977;297(20):1077–80 7. Spector BD, Perry GS, III, Kersey JH. Genetically determined immunodeficiency diseases (GDID) and malignancy: report from the immunodeficiency–cancer registry. Clin Immunol Immunopathol. 1978;11(1):12–29 8. Matas AJ, Simmons RL, Najarian JS. Chronic antigenic stimulation, herpesvirus infection, and cancer in transplant recipients. Lancet. 1975;1(7919):1277–9 9. Hanto DW, Frizzera G, Gajl-Peczalska KJ, Simmons RL. Epstein–Barr virus, immunodeficiency, and B cell lymphoproliferation. Transplantation. 1985;39(5):461–72 10. Epstein MA, Achong B. The Epstein–Barr virus. New York: Springer; 1979 11. Rosen A, Gergely P, Jondal M, Klein G, Britton S. Polyclonal Ig production after Epstein–Barr virus infection of human lymphocytes in vitro. Nature. 1977;267(5606):52–4 12. Robinson JE, Brown N, Andiman W, et al. Diffuse polyclonal B-cell lymphoma during primary infection with Epstein–Barr virus. N Engl J Med. 1980;302(23):1293–7

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13. Henle G, Henle W. The virus as the etiologic agent of infectious mononucleosis. In: Epstein M, Achong B, editors. The Epstein–Barr virus. New York: Springer; 1979. p. 297–320 14. Frank A, Andiman WA, Miller G. Epstein–Barr virus and nonhuman primates: natural and experimental infection. Adv Cancer Res. 1976;23:171–201 15. Klein G. Lymphoma development in mice and humans: diversity of initiation is followed by convergent cytogenetic evolution. Proc Natl Acad Sci USA. 1979;76(5):2442–6 16. Epstein MA, Achong BG. Introduction: discovery and general biology of the virus. In: Epstein MA, Achong BG, editors. The Epstein–Barr virus. New York: Springer; 1979. p. 1–22 17. Klein E, Masucci MG. Cell-mediated immunity against Epstein–Barr virus infected B lymphocytes. Springer Semin Immunopathol. 1982;5(1):63–73 18. Chang RS, Lewis JP, Reynolds RD, Sullivan MJ, Neuman J. Oropharyngeal excretion of Epstein–Barr virus by patients with lymphoproliferative disorders and by recipients of renal homografts. Ann Intern Med. 1978;88(1):34–40 19. Strauch B, Andrews LL, Siegel N, Miller G. Oropharyngeal excretion of Epstein–Barr virus by renal transplant recipients and other patients treated with immunosuppressive drugs. Lancet. 1974;1(7851):234–7 20. Chang RS, Lewis JP, Abildgaard CF. Prevalence of oropharyngeal excreters of leukocytetransforming agents among a human population. N Engl J Med. 1973;289(25):1325–9 21. Cheeseman SH, Henle W, Rubin RH, et al. Epstein–Barr virus infection in renal transplant recipients. Effects of antithymocyte globulin and interferon. Ann Intern Med. 1980;93(1):39–42 22. Marker SC, Ascher NL, Kalis JM, Simmons RL, Najarian JS, Balfour HH Jr. Epstein–Barr virus antibody responses and clinical illness in renal transplant recipients. Surgery. 1979;85(4):433–40 23. Hertel B, Rosai J, Dehner P, Simmons R. Lymphoproliferative disorders in organ transplant recipients. Lab Invest. 1977;36:340 24. Frizzera G, Hanto DW, Gajl-Peczalska KJ, et al. Polymorphic diffuse B-cell hyperplasias and lymphomas in renal transplant recipients. Cancer Res. 1981;41(11 Pt 1):4262–79 25. Hanto DW, Frizzera G, Gajl-Peczalska J, et al. The Epstein–Barr virus (EBV) in the pathogenesis of posttransplant lymphoma. Transplant Proc. 1981;13(1 Pt 2):756–60 26. Hanto DW, Frizzera G, Purtilo DT, et al. Clinical spectrum of lymphoproliferative disorders in renal transplant recipients and evidence for the role of Epstein–Barr virus. Cancer Res. 1981;41 (11 Pt 1):4253–61 27. Hanto DW, Gajl-Peczalska KJ, Frizzera G, et al. Epstein–Barr virus (EBV) induced polyclonal and monoclonal B-cell lymphoproliferative diseases occurring after renal transplantation. Clinical, pathologic, and virologic findings and implications for therapy. Ann Surg. 1983;198(3):356–69 28. Hanto DW, Sakamoto K, Purtilo DT, Simmons RL, Najarian JS. The Epstein–Barr virus in the pathogenesis of posttransplant lymphoproliferative disorders. Clinical, pathologic, and virologic correlation. Surgery. 1981;90(2):204–13 29. Calne RY, Rolles K, White DJ, et al. Cyclosporin A initially as the only immunosuppressant in 34 recipients of cadaveric organs: 32 kidneys, 2 pancreases, and 2 livers. Lancet. 1979;2(8151): 1033–6 30. Nagington J, Gray J. Cyclosporin A immunosuppression, Epstein–Barr antibody, and lymphoma. Lancet. 1980;1(8167):536–7 31. Crawford DH, Thomas JA, Janossy G, et al. Epstein–Barr virus nuclear antigen positive lymphoma after cyclosporin A treatment in patient with renal allograft. Lancet. 1980;1(8182):1355–6 32. Weintraub J, Warnke RA. Lymphoma in cardiac allotransplant recipients. Clinical and histological features and immunological phenotype. Transplantation. 1982;33(4):347–51 33. Starzl TE, Nalesnik MA, Porter KA, et al. Reversibility of lymphomas and lymphoproliferative lesions developing under cyclosporin-steroid therapy. Lancet. 1984;1(8377):583–7 34. Cleary ML, Warnke R, Sklar J. Monoclonality of lymphoproliferative lesions in cardiac-transplant recipients. Clonal analysis based on immunoglobulin-gene rearrangements. N Engl J Med. 1984;310(8):477–82

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35. Cleary ML, Sklar J. Lymphoproliferative disorders in cardiac transplant recipients are multiclonal lymphomas. Lancet. 1984;2(8401):489–93 36. Hanto DW, Birkenbach M, Frizzera G, Gajl-Peczalska KJ, Simmons RL, Schubach WH. Confirmation of the heterogeneity of posttransplant Epstein–Barr virus-associated B cell proliferations by immunoglobulin gene rearrangement analyses. Transplantation. 1989;47(3):458–64 37. Saemundsen AK, Purtilo DT, Sakamoto K, et al. Documentation of Epstein–Barr virus infection in immunodeficient patients with life-threatening lymphoproliferative diseases by Epstein– Barr virus complementary RNA/DNA and viral DNA/DNA hybridization. Cancer Res. 1981;41(11 Pt 1):4237–42 38. Saemundsen AK, Klein G, Cleary M, Warnke R. Epstein–Barr-virus-carrying lymphoma in cardiac transplant recipient. Lancet. 1982;2(8290):158 39. Hanto DW, Frizzera G, Gajl-Peczalska KJ, et al. Epstein–Barr virus-induced B-cell lymphoma after renal transplantation: acyclovir therapy and transition from polyclonal to monoclonal B-cell proliferation. N Engl J Med. 1982;306(15):913–8 40. Shapiro RS, Chauvenet A, McGuire W, et al. Treatment of B-cell lymphoproliferative disorders with interferon alfa and intravenous gamma globulin. N Engl J Med. 1988;318(20):1334 41. Kane RE, Bunchman TE, Vogler C, Brems JJ. Treatment of a B-cell lymphoproliferative disorder in a liver transplant patient with interferon alpha and gamma globulin. A case report. Clin Transplant. 1992;6:154–8 42. Hanto DW, Frizzera G, Gajl-Peczalska J, Balfour HH, Jr., Simmons RL, Najarian JS. Acyclovir therapy of Epstein–Barr virus-induced posttransplant lymphoproliferative diseases. Transplant Proc. 1985;17:89–92 43. Hanto DW. Classification of Epstein–Barr virus-associated posttransplant lymphoproliferative diseases: implications for understanding their pathogenesis and developing rational treatment strategies. Annu Rev Med. 1995;46:381–94

Epidemiology of PTLD

3

Vikas R. Dharnidharka

Core Messages

› › ›

Several different types of risk factors (host, infectious, transplant, immunosuppression) are associated with posttransplant lymphoproliferative disorders (PTLD) development Epstein–Barr virus (EBV) infection is the single most important risk factor PTLD is a significant cause of earlier graft loss and added mortality

3.1 Introduction This chapter is devoted to a detailed discussion of (1) the incidence of posttransplant lymphoproliferative disorders (PTLD) by organ system and over time; (2) the time to PTLD; (3) the different risk factors involved; (4) the risk for graft loss after PTLD; (5) mortality rates after PTLD; and (6) results of re-transplantation if PTLD occurred in a prior transplant. The prognostic factors after PTLD was diagnosed are covered in Chap. 8.

3.2 Incidence of PTLD Large registry data show that lymphoma (a subgroup of PTLD) incidence in transplant recipients, as compared to the general population, is several-fold elevated [1, 2]. The degree of elevation in incidence varies by recipient age. Within the Australia–New Zealand database

V. R. Dharnidharka Division of Pediatric Nephrology, University of Florida College of Medicine, Gainesville, FL, USA e-mail: vikasmd@ufl.edu V. R. Dharnidharka et al. (eds.), Post-Transplant Lymphoproliferative Disorders, DOI: 10.1007/978-3-642-01653-0_3, © Springer Verlag Berlin Heidelberg 2010

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(ANZDATA) registry of kidney transplants, recipients under 35 years of age had a 23–37 fold higher incidence of lymphoma. The risk dropped with each 10-year-older age group, yet the >55 years old recipients still experienced a six fold increase in incidence over the general population [2]. From the Germany-based multinational Collaborative Transplant Study (CTS) registry data across multiple different organ transplants, recipients under 10 years age were found to experience a 200–1,200 fold increased incidence [1]; and recipients above 60 years age experienced 7–16 fold increases in incidence over the general population. Compared to HIV-infected patients, the increase in lymphoma risk in transplant recipients appears to be of the same magnitude [3]. Since PTLD can occur at any time posttransplant and the cumulative incidence goes up over time posttransplant [4], the appropriate unit of measurement should be incidence density, not incidence per patient. Incidence density takes into account those patients whose allografts survive longer, thereby increasing their chance of developing PTLD. This is a simple concept, yet rarely followed in PTLD publications, most of which list PTLD incidence on a per patient percentage. Examples of PTLD incidence density were provided by McDiarmid et al. in the pediatric liver transplant population in the 1980–1990s [5]. This group reported a very high incidence density of 1,800 per 100,000 patient years of followup. Dharnidharka et al. published data from a multicenter pediatric kidney transplant registry. In the period 1987–1992, the incidence density was 320 per 100,000 patient years [6]; this doubled to 603 per 100,000 patient years in the time period 1993–1998 [7]. An alternative method of displaying incidence density is rate per patient per fixed time posttransplant, e.g., rate of PTLD by 1-, 3-, or 5-years posttransplant, as shown in Table 3.1. For example, Kremers et al. documented a cumulative incidence of PTLD post liver transplant of 1.1% at 18 months and 4.7% at 15 years [8]. Cumulative incidence implies a “fixed” cohort with no losses to follow up, a situation that is rare in transplantation, so incidence density, where losses are acceptable, is preferable. When analyzing PTLD incidence by the more common per patient incidence, a clear trend could be seen. Initially PTLD cases were stray and anecdotal through the 1950s–1970s. This was also a time period of two-drug immunosuppression (azathioprine Table 3.1 Reported incidence density or cumulative incidence of PTLD by organ system and recipient age Organ

Recipient age

Kidney Adulta [17, 23] Pediatric Liver Adult [8] Pediatric [70] Heart Adultb (ISHLT 2008 data) Children (ISHLT 2008 data) Lung Adultb (ISHLT 2008 data) Children (ISHLT 2008 data)

1-year (%)

3-years (%)

5-years (%)

0.46 1.73 1.1

0.87 2.45 3

0.67 1.7 1.6 4.8

Not stated Not stated Not stated

1.18 Not stated 4 6 1.3 4.6 2.1 11.1

>5 years (%)

4.7 (15 years) 2.0 (10 years) 7.9 (10 years) 5.6 (10 years) 10.3

a Adult kidney data are from USRDS and French PTLD registry. Pediatric kidney data are from NAPRTCS registry from 1999–2003 b Heart and lung data from International Society of Heart and Lung Transplantation (ISHLT) are based on recipient survival till that time point

3 Epidemiology of PTLD

19

and prednisone) and relatively poor graft survival. After the introduction of cyclosporine A in 1983, graft survival improved dramatically, but the frequency of PTLD reports started rising [9–11]. The pediatric kidney transplant population, heretofore relatively spared from high PTLD rates, showed a steady increase in per patient incidence from <1 to 2.2 to 6% from the early 1990s to the early 2000s [6, 12, 13]. These rises coincided with the advent of more potent immunosuppressive agents and more intense regimens, frequently incorporating an induction antibody with three-drug maintenance immunosuppression. In contrast, in the same time period the pediatric liver transplant population, a group particularly hard hit by PTLD, showed reductions in per patient incidence from 10 to 5% with the use of multiple interventions [14]. It is also possible that some of the increase could be due to better physician awareness and recognition of PTLD and better reporting.

3.3 Time to PTLD The time to PTLD varies greatly by series, era, and organ system. In general, time to PTLD has shortened in more recent eras with more potent immunosuppression [6, 10, 15]. Early PTLDs tend to be B cell proliferations and EBV-positive, while late PTLDs are more likely to be non-B cell proliferations and EBV-negative [8, 16]. Series with predominantly adult populations [17–19] show longer median times to PTLD (25–72 months) vs. series with significant pediatric populations [20–23], where the median times range from 5.5 to 25 months.

3.4 Risk Factors for PTLD There are many different risk factors for PTLD development that have been described in the medical literature. Briefly, these risk factors can be grouped under the following headings: 1. 2. 3. 4. 5.

Infection related: Epstein–Barr virus (EBV) and other infectious agents Host related Primary disease related Graft organ related Immunosuppression related

3.4.1 Infection Related Risk Factors One of the seminal events in PTLD pathogenesis was the epidemiologic linkage of PTLD risk to EBV serostatus in donor and recipient. Focus on EBV had come from the knowledge that some cases of PTLD resembled lymphomas, of which Burkitt’s lymphoma was already linked to EBV. The linkage of EBV to posttransplant lymphoproliferative disease

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was established in the late 1970s [24, 25] and early 1980s [26, 27]. Walker et al. demonstrated that an EBV-seronegative adult recipient of a heart or lung allograft was at 24-fold higher risk for subsequent PTLD development [28]. This finding has since been borne out by multiple other investigators in other organ type transplants and across age groups (Table 3.2). In fact, besides having stood the test of time, the magnitude of risk conveyed (ranging from 3–33-fold) exceeds by far the magnitude of risk conveyed by any immunosuppressive agent risk factors. The reason for this strong epidemiological relationship between EBV and PTLD will become clear in Chap. 4, which is about the biology of EBV and pathogenesis of PTLD. Beyond EBV, other viruses may play a role in PTLD pathogenesis. CMV co-infection certainly has been implicated. Walker’s studies also showed that CMV mismatch, a surrogate for primary CMV infection being transmitted to a seronaive recipient with the allograft, increased PTLD risk by six fold [29]. Manez et al. analyzed 40 adult liver transplant recipients, all of whom were EBV seronegative [30]. Of these, 33% developed PTLD, and a diagnosis of CMV disease posttransplant increased the relative risk to 7.3. CMV infection and EBV infection/PTLD are now clear representations of over-immunosuppression, a malady that afflicted the transplant community in the mid-1990s. A few cases of PTLD are not EBV-tumor positive and CMV co-infection cannot be documented. In these cases, it is possible that a heretofore undetected virus may play a role. Two abstracts presented at American Transplant Congress 2009 provide some more single center information about T cell or EBV-negative PTLD. EBV-negative PTLD comprised 28% of one series of 118 PTLDs at University of Pennsylvania, median time to presentation was 63 vs. 23 months for EBV-positive PTLD, was less likely to involve the allograft, more likely monomorphic, and had worse median survival of 22 vs. 42 months. A University of Minnesota series on T cell PTLDs showed a prevalence of 10% (22/209), median time to presentation 8.2 years (vs. 6.3 years for B cell PTLD), median survival time only 44 days vs. 1.7 years for B cell PTLDs. With the development of peripheral blood PCR assays to measure Epstein–Barr DNAemia, the development of EB DNAemia posttransplant can also be considered a risk factor for PTLD development. This discussion is handled in great depth by Preiksaitis in Chap. 5, and hence, not discussed further here.

3.4.2 Host-Related Risk Factors Dharnidharka et al. reviewed data from several large registries, such as the United Network of Organ Sharing/Organ Procurement and Transplant Network (UNOS/OPTN) and North American Pediatric Renal Transplant Cooperative Studies (NAPRTCS). Younger recipient of age <18 years, male sex, and Caucasian race were all higher risk factors for PTLD development, individually and synergistically [12]. At the other end of the age spectrum, transplant recipients above 50 or 60 years are also at higher risk for PTLD [1, 17, 21]. Since these registries did not, or do not, collect donor/recipient EBV serology data for most of their existence, it is possible that host-related risk factors may be indirect markers of those recipients who are more likely to be EBV seronegative. Such a possibility is

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supported by data from Newell et al. [31], demonstrating highest risk for PTLD in the youngest infant recipients of pediatric liver transplants. This point brings up an important issue when evaluating small single-center vs. large registry data: how to judge the relative impact of other risk factors if EBV serology data are largely missing (as in large registries); or where EBV serology data are present in all but the small sample size (as in single center series). There is no good answer at present; so each reader must try to develop an overall assessment of the big picture. UNOS now collects EBV donor/recipient serology data since 2002 and 2005, respectively; however these fields are often unpopulated by centers, and thus, missing data percentages are high. The same is true for the ANZDATA registry in Australia and New Zealand [15]. Within the most recent UNOS and ANZDATA reports, where some data on EBV recipient serostatus are taken into account, certain immunosuppressive agents were still noted to increase the PTLD risk [15, 32]. Caillard et al., utilizing data from the USA-based United States Renal Data System (USRDS) registry, showed that pretransplant malignancy and fewer HLA matches are also associated with higher PTLD rates [23], though one study of lung transplants showed the opposite [33]. HLA matching and specific alleles have been the subject of several studies; B mismatch, A3 allele or Bw22 allele have all been associated with higher PTLD risk [34–36].

3.4.3 Primary Disease Related Factors Within the liver transplant population, some single center series have documented certain primary liver diseases to be associated with higher risk for subsequent PTLD. These diseases include hepatitis C [37] or associated cirrhosis [21], alcoholic cirrhosis [21], autoimmune hepatitis [38], Langerhans cell histiocytosis [39], or fulminant hepatitis [8]. However, these diseases are also among the most common reasons to receive a liver transplant; hence, they may have been overrepresented in individual series.

3.4.4 Graft Organ Related Risk Factors Data from the multinational CTS and the U.S.-based OPTN/UNOS/USRDS have demonstrated differences in PTLD frequency by organ system. In general, intestinal transplants [40, 41] and thoracic organ transplants are associated with higher frequencies of PTLD, followed by liver transplants, and the lowest frequency has been in kidney transplants. From the most recent International Intestinal Transplant Registry (IITR) report for 2003 (www. intestinaltransplant.org), the incidence of PTLD in adults was 3.3% and in children was 13.3%. Table 3.2 depicts recent incidence density or cumulative incidence data for PTLD by organ system and recipient age at transplant. Figure 3.1 shows (a) cumulative incidence of nonHodgkin lymphoma by organ system from the CTS; or (b) incidence of PTLD by organ system from UNOS/OPTN data.

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Table 3.2 Epstein–Barr virus associated risk ratios for PTLD development Study

Risk ratio

Organ

Age group

Walker et al. [28] Cockfield et al. [67] Mendoza et al. [68] Katz et al. [48] Caillard et al. [17] Faull et al. [15] Kirk et al. [32] Funch et al. [56] Allen et al. [69] McDonald et al. [13]

24 33 12.84 3.94 3.01 3.1 5.28 7.05 4.5 7.7

Heart–Lung Kidney Heart Heart Kidney Kidney Kidney Kidney Several Kidney

Adult Adult Pediatric Pediatric Adult Adult and pediatric Adult and pediatric Adult and pediatric Pediatric Pediatric

Cumulative Incidence (per 100 000)

a

6000 Heart–Lung

n=1222

Lung Heart

n=4414 n=25 485

Liver Pancreas

n=15 631 n=4081

Cadaver Kidney

n=145 104

5000

4000

3000

2000

1000

0 0

1

2

3

4

5

Years

b

PTLD incidence % by organ (UNOS) 6 Intestine = 19/319 Heart-Lung = 29/532

5

Heart = 950/24,500 4

Lung = 228/6207 Liver = 375/39,974

3

K-P = 59/7719 Pancreas = 13/1625

2

Kidney = 692/124,638 1 0 Intestine HeartLung

Heart

Lung

Liver

Kidney- Pancreas Kidney Pancreas

Organ

Fig. 3.1 (a) Cumulative 5 year incidence of nonHodgkin lymphoma by organ system, Collaborative Transplant Study [1], reproduced with permission. (b) Incidence of PTLD by organ system, UNOS/OPTN data [12]

3 Epidemiology of PTLD

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3.4.5 Immunosuppression Related Risk Factors PTLD was very rare in the era of two drug immunosuppression with azathioprine and oral steroids. With the advent of more potent immunosuppression such as cyclosporine A, reports of PTLD started increasing [9] and then exploded in the mid-1990s with the emergence of multiple newer agents (tacrolimus, mycophenolate mofetil, OKT3, anti-thymocyte antibodies, and IL-2R antibodies). Swinnen’s seminal paper brought attention to the increased risk with OKT3 use [42]. Many investigators have, thus, focused attention on the differential risk for PTLD with individual agents. While this type of study is attractive since immunosuppression is a modifiable risk factor, the reader should understand that cumulative totality of immunosuppression is probably the real measure of risk [4, 43]. Yet measuring totality of immunosuppression has been almost impossible so far; hence, the attention to each agent. In this regard, for most of the agents mentioned above, there are several retrospective studies that demonstrate higher PTLD risk with that agent, and at least one study with each agent that does not demonstrate such increase in risk. Almost all prospective studies show no increase in PTLD risk with any agent. These prospective studies tend to have short follow-up time, patient selection biases and more standardized follow-up than general practice. The reader is advised to look at the weight of the published literature, in both quality and quantity, as opposed to any individual study. The magnitude of risk with an immunosuppressive agent, in almost all cases, is much lower than the risk from EBV seromismatch. Thus, some studies show higher risk with (a) cyclosporine A [15, 44, 45] or not [46], (b) with OKT3 [1, 8, 31, 42, 47] or not [17, 48], (c) with equine anti-thymocyte polyclonal antibody [23, 47], (d) with rabbit anti-thymocyte polyclonal antibody [1, 23, 32] or not [23, 49, 50], (e) with Il-2R antibodies [51] or not [1, 23, 52], and (f) with tacrolimus [5, 23, 46, 53] or not [7]. A meta-analysis comparison of cyclosporine to tacrolimus from prospective trials revealed no significant difference in lymphoma/PTLD rates [54]. Mycophenolate mofetil and oral steroids, to date, have not been reported to increase risk for PTLD [52, 55–57], though high dose steroids for acute rejection has been implicated in one study [8]. Azathioprine use was never previously considered as a risk factor for PTLD in the era of two drug immunosuppression, but some recent studies suggest that azathioprine use (vs. mycophenolate) increases PTLD risk [52], not replicated in other studies [23]. Such findings should be interpreted with caution. Sirolimus is unique in that in vitro studies have suggested a protective effect from PTLD [58, 59], which seemed borne out by a clinical retrospective registry study [60]. However, a more recent study by the same group suggested the opposite [32] and sirolimus was part of a protocol that was associated with a high PTLD frequency [13], so the issue may not be as clear-cut. Among the newer biologic agents, alemtuzumab does not seem to be associated with an increased risk for PTLD, based on recent, though early, UNOS registry data [32] in almost 1,700 patients, but only 25 of whom were children and only 12 were EBV-seronegative children. A phase II trial of belatacept in adult kidney transplant recipients showed a few cases of PTLD in the two belatacept arms [61]. But a recent follow-up abstract from the study presented identical incidence density rates of 3 per 100 patient years for the belatacept and cyclosporine arms (Muhlbacher et al., Transplantation Society Meeting August 2008, Sydney, Australia). In a small study of efalizumab, the arm receiving the highest dose of this agent and full dose of CsA experienced three PTLD cases in ten subjects, a concerning number [62].

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3.5 Mortality After PTLD In general, PTLD has been a significant cause of added mortality after transplantation [31], though at least one study suggested no increase in mortality risk [48]. The mortality rates need to be interpreted in light of the varying baseline mortality by organ system (e.g., kidney transplant recipients have lower mortality rates than heart/lung transplant recipients). Similar to incidence rates, mortality rate should ideally be expressed as a cumulative incidence over time as opposed to per PTLD percentage. The 1 year survival rates after PTLD range from 56 to 73% [1, 8, 17], while 5 year survival rates are lower at 40–61% [1, 8, 17]. Older eras and series showed higher mortality rates [15], with improvement in each subsequent decade. Children also did better than elderly adults [15]. A mid-1990s study of PTLD after pediatric kidney transplantation reported a 48% mortality [63], whereas a recent repeat study showed only 13% mortality and 5 year cumulative survival at 87.8% (Dharnidharka et al., unpublished data). Higher grades of PTLD, late-onset PTLD and central nervous system involvement are generally associated with higher mortality [17, 64, 65].

3.6 Risk for Graft Loss After PTLD Does PTLD itself elevate the risk for earlier graft loss, independent of its effect on patient survival? Reduction of immunosuppression is typically the first strategy employed after PTLD diagnosis, and can be associated with a higher risk of rejection episodes. For thoracic organs, rejection can also be graft threatening or life-threatening. In a multicenter pediatric heart transplant study, death from graft loss was as frequent as death from PTLD. In kidney transplants, the availability of dialysis means that most clinicians might be willing to lower immunosuppression considerably and accept a risk of losing the graft. In a NAPRTCS study presented by Dharnidharka et al. at the International Pediatric Transplant Association conference in 2007, PTLD was highly significant as a predictor of worse graft survival (hazard ratio 4.3, 95% CI 3.4–4.5) after adjustment for other factors.

3.7 Re-Transplantation After PTLD in Prior Transplant The only multicenter and relatively large series comes from UNOS/OPTN, where 69 retransplants have been recorded after PTLD [66]. Time from PTLD to re-transplant was <1 year in 24.6%, 1–3 years in 37.7%, 3–5 years in 17.4%, and 5–10 years in 20.3%. A variety of immunosuppression agents have been used in the re-transplant, and no recurrence of PTLD was reported in the re-transplants.

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3.8 Take Home Messages

• • • •

The incidence of PTLD varies with several host, transplant and immunosuppression related risk factors; highest risk is seen in EBV seronegative very young or very old recipients receiving intestinal/thoracic organs and a high overall level of immunosuppression. EBV infection, especially primary infection acquired through the allograft, is the single most important risk factor. PTLD is a significant cause of earlier graft loss and added mortality. Fortunately, recurrence of PTLD in a re-transplant after prior transplant complicated by PTLD has so far not been reported.

References 1. Opelz G, Dohler B. Lymphomas after solid organ transplantation: a collaborative transplant study report. Am J Transplant. 2004;4(2):222 2. Webster AC, Craig JC, Simpson JM, Jones MP, Chapman JR. Identifying high risk groups and quantifying absolute risk of cancer after kidney transplantation: a cohort study of 15,183 recipients. Am J Transplant. 2007;7(9):2140 3. Srisawat N, Avihingsanon A, Praditpornsilpa K, Jiamjarasrangsi W, Eiam-Ong S, Avihingsanon Y. A prevalence of posttransplantation cancers compared with cancers in people with human immunodeficiency virus/acquired immunodeficiency syndrome after highly active antiretroviral therapy. Transplant Proc. 2008;40(8):2677 4. Boubenider S, Hiesse C, Goupy C, Kriaa F, Marchand S, Charpentier B. Incidence and consequences of post-transplantation lymphoproliferative disorders. J Nephrol. 1997;10(3):136 5. Younes BS, McDiarmid SV, Martin MG, et al. The effect of immunosuppression on posttransplant lymphoproliferative disease in pediatric liver transplant patients. Transplantation. 2000;70 (1):94 6. Dharnidharka VR, Sullivan EK, Stablein DM, Tejani AH, Harmon WE. Risk factors for posttransplant lymphoproliferative disorder (PTLD) in pediatric kidney transplantation: a report of the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS). Transplantation. 2001;71(8):1065 7. Dharnidharka VR, Ho PL, Stablein DM, Harmon WE, Tejani AH. Mycophenolate, tacrolimus and post-transplant lymphoproliferative disorder: a report of the North American Pediatric Renal Transplant Cooperative Study. Pediatr Transplant. 2002;6(5):396 8. Kremers WK, Devarbhavi HC, Wiesner RH, Krom RA, Macon WR, Habermann TM. Posttransplant lymphoproliferative disorders following liver transplantation: incidence, risk factors and survival. Am J Transplant. 2006;6(5 Pt 1):1017 9. Harmon WE, Dharnidharka VR. Lymphoproliferative disease in children. Transplant Proc. 1999;31(2B):1268 10. Alfrey EJ, Friedman AL, Grossman RA, et al. A recent decrease in the time to development of monomorphous and polymorphous posttransplant lymphoproliferative disorder. Transplantation. 1992;54(2):250 11. Ciancio G, Siquijor AP, Burke GW, et al. Post-transplant lymphoproliferative disease in kidney transplant patients in the new immunosuppressive era. Clin Transplant. 1997;11(3):243

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12. Dharnidharka VR, Tejani AH, Ho PL, Harmon WE. Post-transplant lymphoproliferative disorder in the United States: young Caucasian males are at highest risk. Am J Transplant. 2002;2(10):993 13. McDonald RA, Smith JM, Ho M, et al. Incidence of PTLD in pediatric renal transplant recipients receiving basiliximab, calcineurin inhibitor, sirolimus and steroids. Am J Transplant. 2008;8 (5):984 14. McDiarmid SV, Jordan S, Lee GS, et al. Prevention and preemptive therapy of postransplant lymphoproliferative disease in pediatric liver recipients. Transplantation. 1998;66(12):1604 15. Faull RJ, Hollett P, McDonald SP. Lymphoproliferative disease after renal transplantation in Australia and New Zealand. Transplantation. 2005;80(2):193 16. Leblond V, Sutton L, Dorent R, et al. Lymphoproliferative disorders after organ transplantation: a report of 24 cases observed in a single center. J Clin Oncol. 1995;13(4):961 17. Caillard S, Lelong C, Pessione F, Moulin B. Post-transplant lymphoproliferative disorders occurring after renal transplantation in adults: report of 230 cases from the French Registry. Am J Transplant. 2006;6(11):2735 18. Patel H, Vogl DT, Aqui N, et al. Posttransplant lymphoproliferative disorder in adult liver transplant recipients: a report of seventeen cases. Leuk Lymphoma. 2007;48(5):885 19. Saadat A, Einollahi B, Ahmadzad-Asl MA, et al. Posttransplantation lymphoproliferative disorders in renal transplant recipients: report of over 20 years of experience. Transplant Proc. 2007;39(4):1071 20. Cacciarelli TV, Green M, Jaffe R, et al. Management of posttransplant lymphoproliferative disease in pediatric liver transplant recipients receiving primary tacrolimus (FK506) therapy. Transplantation. 1998;66(8):1047 21. Duvoux C, Pageaux GP, Vanlemmens C, et al. Risk factors for lymphoproliferative disorders after liver transplantation in adults: an analysis of 480 patients. Transplantation. 2002;74(8):1103 22. Fernandez MC, Bes D, De Davila M, et al. Post-transplant lymphoproliferative disorder after pediatric liver transplantation: characteristics and outcome. Pediatr Transplant. 2009;13(3):307 23. Caillard S, Dharnidharka V, Agodoa L, Bohen E, Abbott K. Posttransplant lymphoproliferative disorders after renal transplantation in the United States in era of modern immunosuppression. Transplantation. 2005;80(9):1233 24. Chang RS, Lewis JP, Reynolds RD, Sullivan MJ, Neuman J. Oropharyngeal excretion of Epstein-Barr virus by patients with lymphoproliferative disorders and by recipients of renal homografts. Ann Intern Med. 1978;88(1):34 25. Marker SC, Ascher NL, Kalis JM, Simmons RL, Najarian JS, Balfour HH Jr. Epstein-Barr virus antibody responses and clinical illness in renal transplant recipients. Surgery. 1979;85(4):433 26. Hanto DW, Frizzera G, Purtilo DT, et al. Clinical spectrum of lymphoproliferative disorders in renal transplant recipients and evidence for the role of Epstein-Barr virus. Cancer Res. 1981;41 (11 Pt 1):4253 27. Hanto DW, Sakamoto K, Purtilo DT, Simmons RL, Najarian JS. The Epstein-Barr virus in the pathogenesis of posttransplant lymphoproliferative disorders. Clinical, pathologic, and virologic correlation. Surgery. 1981;90(2):204 28. Walker RC, Paya CV, Marshall WF, et al. Pretransplantation seronegative Epstein-Barr virus status is the primary risk factor for posttransplantation lymphoproliferative disorder in adult heart, lung, and other solid organ transplantations. J Heart Lung Transplant. 1995;14(2):214 29. Walker RC, Marshall WF, Strickler JG, et al. Pretransplantation assessment of the risk of lymphoproliferative disorder. Clin Infect Dis. 1995;20(5):1346 30. Manez R, Breinig MC, Linden P, et al. Posttransplant lymphoproliferative disease in primary Epstein-Barr virus infection after liver transplantation: the role of cytomegalovirus disease. J Infect Dis. 1997;176(6):1462 31. Newell KA, Alonso EM, Whitington PF, et al. Posttransplant lymphoproliferative disease in pediatric liver transplantation. Interplay between primary Epstein-Barr virus infection and immunosuppression. Transplantation. 1996;62(3):370

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32. Kirk AD, Cherikh WS, Ring M, et al. Dissociation of depletional induction and posttransplant lymphoproliferative disease in kidney recipients treated with alemtuzumab. Am J Transplant. 2007;7(11):2619 33. Sundin M, Le Blanc K, Ringden O, et al. The role of HLA mismatch, splenectomy and recipient Epstein-Barr virus seronegativity as risk factors in post-transplant lymphoproliferative disorder following allogeneic hematopoietic stem cell transplantation. Haematologica. 2006;91(8):1059 34. Bakker NA, van Imhoff GW, Verschuuren EA, et al. HLA antigens and post renal transplant lymphoproliferative disease: HLA-B matching is critical. Transplantation. 2005;80(5):595 35. Pourfarziani V, Einollahi B, Taheri S, Nemati E, Nafar M, Kalantar E. Associations of Human Leukocyte Antigen (HLA) haplotypes with risk of developing lymphoproliferative disorders after renal transplantation. Ann Transplant. 2007;12(4):16 36. Wheless SA, Gulley ML, Raab-Traub N, et al. Post-transplantation lymphoproliferative disease: Epstein Barr virus DNA levels, HLA A3 and survival. Am J Respir Crit Care Med. 2008; 178(10):1060 37. McLaughlin K, Wajstaub S, Marotta P, et al. Increased risk for posttransplant lymphoproliferative disease in recipients of liver transplants with hepatitis C. Liver Transpl. 2000;6(5):570 38. Shpilberg O, Wilson J, Whiteside TL, Herberman RB. Pre-transplant immunological profile and risk factor analysis of post-transplant lymphoproliferative disease development: the results of a nested matched case-control study. The University of Pittsburgh PTLD Study Group. Leuk Lymphoma. 1999;36(1–2):109 39. Newell KA, Alonso EM, Kelly SM, Rubin CM, Thistlethwaite JR Jr., Whitington PF. Association between liver transplantation for Langerhans cell histiocytosis, rejection, and development of posttransplant lymphoproliferative disease in children. J Pediatr. 1997;131(1 Pt 1):98 40. Quintini C, Kato T, Gaynor JJ, et al. Analysis of risk factors for the development of posttransplant lymphoprolipherative disorder among 119 children who received primary intestinal transplants at a single center. Transplant Proc. 2006;38(6):1755 41. Finn L, Reyes J, Bueno J, Yunis E. Epstein-Barr virus infections in children after transplantation of the small intestine. Am J Surg Pathol. 1998;22(3):299 42. Swinnen LJ, Costanzo-Nordin MR, Fisher SG, et al. Increased incidence of lymphoproliferative disorder after immunosuppression with the monoclonal antibody OKT3 in cardiac-transplant recipients. N Engl J Med. 1990;323(25):1723 43. Dharnidharka VR, Harmon WE. Management of pediatric postrenal transplantation infections. Semin Nephrol. 2001;21(5):521 44. Penn I. Cancers following cyclosporine therapy. Transplantation. 1987;43(1):32 45. Penn I. Neoplastic complications of transplantation. Semin Respir Infect. 1993;8(3):233 46. Guthery SL, Heubi JE, Bucuvalas JC, et al. Determination of risk factors for Epstein-Barr virus-associated posttransplant lymphoproliferative disorder in pediatric liver transplant recipients using objective case ascertainment. Transplantation. 2003;75(7):987 47. Opelz G, Naujokat C, Daniel V, Terness P, Dohler B. Disassociation between risk of graft loss and risk of non-Hodgkin lymphoma with induction agents in renal transplant recipients. Transplantation. 2006;81(9):1227 48. Katz BZ, Pahl E, Crawford SE, et al. Case-control study of risk factors for the development of post-transplant lymphoproliferative disease in a pediatric heart transplant cohort. Pediatr Transplant. 2007;11(1):58 49. Dharnidharka VR, Stevens G. Risk for post-transplant lymphoproliferative disorder after polyclonal antibody induction in kidney transplantation. Pediatr Transplant. 2005;9:622 50. Hardinger KL, Rhee S, Buchanan P, et al. A prospective, randomized, double-blinded comparison of thymoglobulin versus Atgam for induction immunosuppressive therapy: 10-year results. Transplantation. 2008;86(7):947 51. Bustami RT, Ojo AO, Wolfe RA, et al. Immunosuppression and the risk of post-transplant malignancy among cadaveric first kidney transplant recipients. Am J Transplant. 2004;4(1):87

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52. Cherikh WS, Kauffman HM, McBride MA, Maghirang J, Swinnen LJ, Hanto DW. Association of the type of induction immunosuppression with posttransplant lymphoproliferative disorder, graft survival, and patient survival after primary kidney transplantation. Transplantation. 2003; 76(9):1289 53. Cox KL, Lawrence-Miyasaki LS, Garcia-Kennedy R, et al. An increased incidence of EpsteinBarr virus infection and lymphoproliferative disorder in young children on FK506 after liver transplantation. Transplantation. 1995;59(4):524 54. Webster AC, Woodroffe RC, Taylor RS, Chapman JR, Craig JC. Tacrolimus versus ciclosporin as primary immunosuppression for kidney transplant recipients: meta-analysis and metaregression of randomised trial data. BMJ. 2005;331(7520):810 55. Birkeland SA, Hamilton-Dutoit S. Is posttransplant lymphoproliferative disorder (PTLD) caused by any specific immunosuppressive drug or by the transplantation per se? Transplantation. 2003;76(6):984 56. Funch DP, Ko HH, Travasso J, et al. Posttransplant lymphoproliferative disorder among renal transplant patients in relation to the use of mycophenolate mofetil. Transplantation. 2005;80(9):1174 57. Robson R, Cecka JM, Opelz G, Budde M, Sacks S. Prospective registry-based observational cohort study of the long-term risk of malignancies in renal transplant patients treated with mycophenolate mofetil. Am J Transplant. 2005;5(12):2954 58. Majewski M, Korecka M, Kossev P, et al. The immunosuppressive macrolide RAD inhibits growth of human Epstein-Barr virus-transformed B lymphocytes in vitro and in vivo: a potential approach to prevention and treatment of posttransplant lymphoproliferative disorders. Proc Natl Acad Sci U S A. 2000;97(8):4285 59. Nepomuceno RR, Balatoni CE, Natkunam Y, Snow AL, Krams SM, Martinez OM. Rapamycin inhibits the interleukin 10 signal transduction pathway and the growth of Epstein Barr virus B-cell lymphomas. Cancer Res. 2003;63(15):4472 60. Kauffman HM, Cherikh WS, Cheng Y, Hanto DW, Kahan BD. Maintenance immunosuppression with target-of-rapamycin inhibitors is associated with a reduced incidence of de novo malignancies. Transplantation. 2005;80(7):883 61. Vincenti F, Larsen C, Durrbach A, et al. Costimulation blockade with belatacept in renal transplantation. N Engl J Med. 2005;353(8):770 62. Vincenti F, Mendez R, Pescovitz M, et al. A phase I/II randomized open-label multicenter trial of efalizumab, a humanized anti-CD11a, anti-LFA-1 in renal transplantation. Am J Transplant. 2007;7(7):1770 63. Hebert D, Sullivan EK. Malignancy and post transplant lymphoproliferative disorder (PTLD) in pediatric renal transplant recipients: a report of the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS). Pediatr Transplant. 1998;2(S 1):107:57A 64. Hayashi RJ, Kraus MD, Patel AL, et al. Posttransplant lymphoproliferative disease in children: correlation of histology to clinical behavior. J Pediatr Hematol Oncol. 2001;23(1):14 65. Buell JF, Gross TG, Hanaway MJ, et al. Posttransplant lymphoproliferative disorder: significance of central nervous system involvement. Transplant Proc. 2005;37(2):954 66. Johnson SR, Cherikh WS, Kauffman HM, Pavlakis M, Hanto DW. Retransplantation after posttransplant lymphoproliferative disorders: an OPTN/UNOS database analysis. Am J Transplant. 2006;6(11):2743 67. Cockfield SM, Preiksaitis JK, Jewell LD, Parfrey NA. Post-transplant lymphoproliferative disorder in renal allograft recipients. Clinical experience and risk factor analysis in a single center. Transplantation. 1993;56(1):88 68. Mendoza F, Kunitake H, Laks H, Odim J. Post-transplant lymphoproliferative disorder following pediatric heart transplantation. Pediatr Transplant. 2006;10(1):60 69. Allen UD, Farkas G, Hebert D, et al. Risk factors for post-transplant lymphoproliferative disorder in pediatric patients: a case-control study. Pediatr Transplant. 2005;9(4):450 70. Ng VL, Fecteau A, Shepherd R, et al. Outcomes of 5-year survivors of pediatric liver transplantation: report on 461 children from a north american multicenter registry. Pediatrics. 2008;122 (6):e1128.

The Biology of Epstein–Barr Virus and Posttransplant Lymphoproliferative Disease

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Olivia M. Martinez

Core Messages

› › › › › ›

Epstein–Barr virus (EBV) hijacks the normal B cell differentiation process to access, and persists in the memory B cell compartment in healthy individuals. Infection of bystander B cells or failure of EBV-infected B cells to successfully transit through the B cell differentiation process could result in EBV+ B cell lymphomas in immunosuppressed individuals. Over 90% of posttransplant lymphoproliferative disorders (PTLD) associated tumors are EBV+ and the vast majority are B cell lymphomas. EBV is present in a latent state in PTLD-associated B cell lymphomas and expresses a limited set of viral genes that drive tumor growth and survival. LMP1 is a key latent cycle gene of EBV that activates a variety of cellular signaling pathways to promote tumor growth and survival. EBV has evolved to actively evade and subvert host immune effector pathways and these maneuvers may impact the development of PTLD.

4.1 Introduction Epstein–Barr virus (EBV) was discovered over 40 years ago in the cells of a patient with Burkitt’s lymphoma. Since that time we have learned that primary infection with EBV can have widely diverse outcomes ranging from asymptomatic infection to self-resolving infectious mononucleosis, to the development of EBV-associated malignancies including Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinomas, and B cell lymphomas

O. M. Martinez Department of Surgery, Division of Transplantation and the Program in Immunology, Stanford University School of Medicine, Palo Alto, CA 94305-5492, USA e-mail: [email protected] V. R. Dharnidharka et al. (eds.), Post-Transplant Lymphoproliferative Disorders, DOI: 10.1007/978-3-642-01653-0_4, © Springer Verlag Berlin Heidelberg 2010

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in transplant recipients and AIDS patients. In this chapter, we will discuss the biology of EBV, its life cycle and interaction with the host immune system – and how these factors impact the pathogenesis of posttransplant lymphoproliferative disorders (PTLD).

4.2 Biology of EBV 4.2.1 Infection EBV is a double stranded DNA virus of the gammaherpes family that was first identified by Epstein, Achong, and Barr in the tissue obtained from a patient with Burkitt’s lymphoma [1]. Serologic evidence indicates that over 90% of the world’s population is infected with EBV. Typically the virus is transmitted through the saliva, though in the setting of organ transplantation, EBV can be acquired through co-transfer with an organ from a seropositive donor to a seronegative donor. The EBV genome is packaged in a nucleocapsid of approximately 100 nm surrounded by a viral tegument and enclosed within a lipid bilayer envelope containing glycoprotein spikes. The major vial envelope glycoprotein is gp350/220, which participates in viral infection by interacting with the CD21 molecule (complement receptor 2) on B cell membranes, thereby mediating the initial attachment of the virion to the cell. The interaction of gp350/220 and CD21 also induces capping of CD21 on the membrane and triggers endocytosis of the virus. Viral entry into the cell requires fusion of the viral envelope with the B cell membrane, a process mediated by the interaction between the viral envelope glycoprotein, gp42, and major histocompatibility complex (MHC) class II proteins (HLA-DR, -DQ, or -DP) expressed on the cell membrane. gH, gL, and gP are other viral envelope glycoproteins required for the fusion event [2]. While B cells are the primary cellular host for EBV, epithelial cells are also susceptible to infection. However, the physiologic role of the gp350/220-CD21 complex in attachment of EBV to epithelial cells remains unclear and other mechanisms of virion-epithelial cell membrane interaction have been proposed, including a role for resting B cells as a transfer vehicle for membrane-bound EBV to epithelial cells [3, 4]. Other cell types that EBV has been reported to infect includes T cells, NK cells, and possibly monocytes, though the mechanisms of viral entry into these cell types are very poorly understood. Nevertheless, rare cases of EBV-associated T cell or NK cell posttransplant lymphomas have been reported [5].

4.2.2 The Viral Life Cycle EBV is generally acquired through close contact with oral secretions from a seropositive person. Whether oropharyngeal epithelial cells or B cells in the submucosal lymphoid layer is the site of initial infection has been a topic of extensive, ongoing discussion. In either case, the virus undergoes productive replication in cells of the oropharynx region and infectious viral particles are shed in the saliva of healthy carriers [6]. Ultimately, EBV persists for the lifetime of the host in a subset of circulating, memory B cells. As with other

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herpes viruses, EBV persistence is linked to viral latency, though a small number of B cells remain permissive for viral replication. Thus, the EBV life cycle takes two forms; the latent phase in which the virus remains dormant within B lymphocytes, and the lytic phase in which the virus is actively replicated and infectious virions are released that can go on to infect bystander cells or be shed to infect naive individuals. Using this life cycle strategy, EBV is highly successful at achieving widespread infection among the human population, while perpetuating viral survival and minimizing the pathologic consequences for the host. An important component of this strategy is that EBV has achieved a state of détente with the host immune system, though this is likely a delicately balanced co-existence [7]. Indeed, disruption of the viral–host equilibrium predisposes individuals to the development of EBV-associated B cell lymphomas as in immunosuppressed transplant recipients with PTLD or immunocompromised people co-infected with HIV. How EBV gains access to the memory B cell compartment is an ongoing question [8]. One prominent model is that the virus initially infects a naïve B cell and then exploits the normal B cell differentiation process [9] (Fig. 4.1). During the early stages of infection of

Epithelial Layer

Lytic Infection

NaÏve B cell

Infected Lymphoblast

CD8+ T cell

EBNA1, EBNA2, EBNA3s, EBNALP,LMP1, LMP2A,LMP2B, EBERs

Secondary Lymphoid Tissue Memory B cell EBNA1, LMP1, LMP2A, EBERs

Germinal center

(EBNA1) (LMP2A) EBERs

Fig. 4.1 EBV infection and the viral life cycle. EBV infects naïve host through transfer in saliva of infected individuals. Viral particles pass through the epithelial layer and infect epithelial cells or naïve B cells. Primary infection can lead to either a productive lytic infection where new virions are produced or a latent infection which confers the ability of autonomous proliferation in infected B lymphoblasts. CD8 T cells of immunocompetant hosts can control expansion of B lymphoblasts. Alterations in viral gene expression programs is likely to promotes the survival of infected cells that migrate to secondary lymphoid organs and transit through germinal center reactions in the absence of encounter with antigen. EBV persists in a subset of memory B cells with minimal viral gene expression.

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naïve B cells, the linear EBV genome circularizes and is subsequently maintained as an extrachromosomal episome. The first of several latent cycle gene programs is triggered within 12–16 h of infection and nine key latent cycle genes are expressed that lead to cellular activation and autonomous proliferation of the infected cell. This program of viral gene expression has been termed latency type III (or the growth program) and is characterized by expression of Epstein Barr nuclear antigens (EBNAs) 1, 2, 3A, 3B, 3C, LP, latent membrane protein (LMP1, LMP2A, LMP2B) in addition to polyadenylated viral RNAs (EBERS 1 and 2), and a group of transcripts from the BamH1A region of the genome whose function is unknown. The resulting infected B cells resemble antigen-activated B lymphoblasts. In immunocompetent individuals, the outgrowth of the EBV-activated lymphoblasts is controlled by a robust, antiviral cytotoxic T lymphocyte (CTL) response. However, disruption of host immunity can lead to development of EBV+ B cell lymphomas, as seen in PTLD, that are also characterized by the latency III program of viral gene expression. Finally, the same viral gene expression program is found in B cells infected with EBV in vitro, resulting in the generation of immortalized lymphoblastoid cell lines (LCL). In the normal course of EBV infection, the activated lymphoblasts can migrate to the B cell follicles of secondary lymphoid tissue where the growth program is silenced and replaced by latency type II (default program) characterized by expression of EBNA1, LMP1, and LMP2A. In conventional T cell-dependent immune responses, the follicles are sites where activated B cell blasts that have encountered antigen undergo isotype switching and somatic mutation of immunoglobulin genes to differentiate into antibody forming cells or memory cells bearing high affinity B cell receptors (BCR). This process of differentiation depends upon encounter with antigen-presenting follicular dendritic cells and T helper cells. Cells expressing BCR that do not bind antigen die via apoptosis. In type II latency, the expression of EBNA2, LMP1, and LMP2A provides key signals that ensure survival of the infected cell and drive it through the B cell differentiation process associated with the GC reaction without requirement for interaction with antigen, follicular dendritic cells or T helper cells. Infected memory cells that emerge from GC then switch to type I, or latency program, where either no viral genes are expressed or only EBNA1 is expressed during cell division to ensure maintenance of the EBV episome. Other latency classification schemes have dubbed the latency pattern of resting memory B cells as latency 0, and may include expression of LMP2A [10]. The virtual absence of viral gene expression when EBV is harbored in resting memory B cells in the periphery promotes viral persistence and escape from host antiviral immune mechanisms. This scenario suggests that EBV has co-evolved with the host immune system and utilizes its ability to induce autonomous proliferation of infected B cells only transiently as a means to exploit the process of B cell development to transit safely through to the memory B cell compartment [11]. Thus, the development of EBV-associated B cell lymphomas, including PTLD, may be an inadvertent and unintended consequence of this process in the context of impaired immunity or when additional mutations arise.

4.2.3 Latent Cycle Genes of EBV How do latent cycle proteins shepherd EBV through the process of B cell differentiation? In this section, the key properties of the eleven gene products expressed during type III latency and in PTLD-associated B cell lymphomas are summarized (Table 4.1).

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Table 4.1 Association of latent cycle viral gene expression with types of EBV+ malignancies Latency

Viral Gene

Malignancy

I II

EBER (EBNA1) EBNA1, LMP1, LMP2A EBER EBNA1, EBNA2, EBNA3s, EBNA-LP, LMP1, LMP2A, l LMP2B, EBER

Burkitt’s Lymphoma Hodgkin’s Disease, NPC PTLD, AIDS-related lymphomas

III

1. EBNA1: a DNA binding protein that binds to the origin of plasmid replication (OriP) of EBV, and is required for episomal replication of the viral genome. Further, EBNA tethers the viral genome to mitotic chromosomes and is sufficient to ensure passage of the viral genome to daughter cells during cell division. 2. EBNA2: a transcriptional activator that regulates the function of several viral genes including LMP1 and LMP2A, as well as numerous cellular genes. EBNA2 does not directly interact with DNA regulatory sequences, but mimics Notch signaling by interacting with the DNA-binding protein, RBP-Jk, to prevent B cell differentiation. EBNA2 is required for transformation of human B cells in vitro. 3. EBNA3A, 3B, 3C: encoded by genes which lie in tandem within the EBV genome. All three proteins interact with cellular DNA binding protein RBP-Jk and modulate transactivation by EBNA2. EBNA3A and 3C are required for immortalization of B cells in vitro, but EBNA3B is dispensable. EBNA3C, through interaction with cyclin proteins, can disrupt cell cycle checkpoints. 4. EBNA-LP: important in transformation of B cells in vitro and enhances the ability of EBNA2 to transactivate cellular and viral genes. 5. LMP1: the major oncogene of EBV since it is sufficient to transform rodent fibroblasts in vitro and is required for generation of LCL from human B cells. LMP1 is an integral membrane protein with a short intracellular N-terminal tail, six membrane-spanning domains and a long cytoplasmic c-terminal tail. LMP1 mimics a constitutively active member of the tumor necrosis factor receptor (TNFR) superfamily and activates multiple cellular signaling pathways including NF-kB, the mitogen activated protein kinases (MAP) p38, Erk, and JNK, and AKT/PI3K through the use of the cellular adaptor proteins TRAF and TRADD. LMP1 signaling induces expression of cell adhesion molecules, anti-apoptotic proteins including bcl-2, cFLIP, A-20, and the production of the B cell lymphoma autocrine growth factor IL-10 [12, 13]. Thus, LMP1 provides critical growth and survival signals to infected B cells. Indeed, the ability of LMP1 to inhibit apoptosis through death receptors [14–16] suggests it may play an important role in survival of B cells through the GC in the absence of encounter with antigen by providing signals normally delivered through T cell help. Mice expressing a transgene for LMP1 under the control of the immunoglobulin promoter develop lymphomas at three times the frequency as LMP1-negative, control mice [17].

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6. LMP2A: expressed in the membrane of infected cells and contains immunoreceptor tyrosine-based activation motifs (ITAM), motifs similar to the BCR. Like LMP1, LMP2A is aggregated in the membrane and constitutively signals. LMP2A sequesters key tyrosine kinases, including syk, from the BCR and thus, inhibits BCR-mediated cell activation, thereby inhibiting entry into the lytic phase of infection. However, LMP2A supplies the tonic signals normally provided by the BCR for cell survival. 7. LMP2B: the second isoform of LMP2 and is controlled by a separate promoter from LMP2A. Neither LMP2A nor LMP2B is essential for B cell transformation in vitro. LMP2B has been one of the most enigmatic of the EBV latent cycle proteins. Recent studies suggest that LMP2B can physically associate with LMP2A [18] and may negatively regulate the ability of LMP2A to inhibit switching from the latent to the lytic cycle [19]. 8. EBER: EBERs 1 and 2 are small polyadenylated, noncoding RNAs expressed in each of the three forms of latency. Though the EBER are abundant in EBV-transformed cells, their function has not been fully elucidated. However, they have been reported to inhibit apoptosis [20] and induce IL-10 production in Burkitt’s lymphoma cells [21].

4.2.4 Characteristics of the T Cell Response to EBV in Healthy Individuals The outgrowth of potentially pathogenic latency III lymphoblasts during primary infection or following intermittent reactivation of the virus in healthy carriers is controlled primarily by EBV-specific CD8+ CTL that recognize viral peptides in the context of MHC class I proteins. Studies using MHC/peptide tetramers to detect antigen-specific T cells revealed that seropositive humans devote a significant proportion of the T cell repertoire to controlling EBV. Early after primary infection a massive expansion of viral-specific CD8 T cells occurs, such that upwards of 40% of CD8+ T cells can be directed toward a single lytic cycle protein epitope in individuals with recent onset infectious mononucleosis [22]. ELIspot assays as well as intracellular staining and flow cytometry have been used to demonstrate that these cells can rapidly produce IFN-g and show cytotoxic potential when restimulated ex vivo [23]. It has been estimated that approximately 1 in 106 B cells carries the virus in a latent state in healthy EBV carriers. EBV-specific CD8+ T cells can constitute about 1–3% of the T cell repertoire and display an activated/memory phenotype. In general, responses to lytic cycle proteins, including BZLF1 and BMLF1, predominate by about tenfold over responses to latent cycle proteins [24]. Of latent cycle proteins, EBNA1, EBNA2, and LMP1 are generally weakly immunogenic for CD8+ T cells, while EBNA3A, 3B, and 3C are the most immunogenic followed by LMP2. However, the frequencies of various antigen-specific populations can vary greatly over time [23]. In contrast to the CD8+ T cell response, relatively little is known about the CD4+ T cell response to EBV [25]. In general, the magnitude of primary CD4+ T cell responses to EBV is much smaller than CD8+ T cell responses. BZLF1, a lytic cycle product, dominates the hierarchy of EBV proteins recognized by CD4+ T cells, followed by BMLF1 and the latent cycle proteins EBNA3A and EBNA1 [25]. The function of EBNA1-specific CD4+ T cells has been studied and shown to be primarily of the Th1 type with IFN-g producing cells most

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prevalent [26, 27]. The CD4+ memory response to EBV has been correspondingly small and difficult to measure. Further hindering the analysis of EBV-specific CD4+ T cells is that the relevant epitopes have not been extensively defined. Nevertheless, CD4+ T cells specific for EBNA- and LMP-derived epitopes have been shown to be present in healthy donors and are capable of producing cytokines in ex vivo assays [28, 29]. As discussed previously, EBNA1, EBNA2, EBNA3A, B, and C, LMP1, and LMP2 proteins are expressed in most EBV+ B cell lymphomas in PTLD. Thus, cells in which peptides derived from these proteins are presented by class I molecules constitute potential targets of host CTL. Efforts to generate autologous or allogeneic EBV-specific effector CTL ex vivo as a form of cellular immunotherapy for infusion into patients with PTLD yielded polyclonal populations of both CD4+ and CD8+ T cells directed at latent and lytic targets [30, 31]. The broad specificity of these populations may be beneficial as a safeguard measure since some PTLD lesions may have incomplete or variable expression of latency antigens.

4.2.5 Characteristics of the T Cell Response to EBV in Transplant Recipients Immunosuppression of host T cell immunity is clearly a principal factor in the development of EBV+ B cell lymphomas in PTLD. Along these lines, it is plausible that immunosuppressive drugs prevent the development and expansion of EBV-specific T cells during primary EBV infection posttransplant as well as the maintenance, activation, and function of EBVspecific memory T cells in recipients with prior exposure to the virus. Nevertheless, the analysis of the T cell response to EBV has unequivocally demonstrated that transplant recipients are fully capable of developing and maintaining a significant EBV-specific CD8+ T cell population. Falco et al. [32] analyzed the frequency of EBV-specific T cells in pediatric liver and kidney recipients posttransplant. These patients had readily detectible CD8+ T cells specific for the immunodominant epitopes of the EBV proteins BZLF1, BMLF1, and EBNA3A. The levels of EBV-specific CD8+ T cells and the dominance of CD8+ T cells specific for lytic epitopes were similar to findings in healthy adults. Further, the majority of antigen-specific cells showed an activated/memory phenotype. Macedo et al. [33] analyzed EBV-specific CD8+ T cells in adult solid organ recipients and found that the frequency of BMLF1 (lytic cycle protein) specific cells was similar in patients and healthy controls, but the proportion of CD8+ T cells specific for latent cycle proteins (LMP2A and EBNA3A) was increased in patients compared to controls. Phenotypic studies indicated that CD8+ T cells in patients specific for latent cycle proteins were predominantly CD45RO+CD62L− indicative of an “effector memory” population. In contrast, the EBV-specific CD8+ T cells in healthy controls were CD45RO+CD62L+, characteristic of a “central memory” population. Moreover, patients cells showed somewhat decreased ability to produce IFN-g when stimulated with EBV-specific peptides compared to cells from healthy controls. Interestingly, Falco et al. [32] detected EBV-specific CD8+ T cells in the blood of seronegative recipients of grafts from seropositive donors within 4 weeks of transplantation, indicating that even in the setting of immunosuppression, EBV-specific CD8+ T cells can develop rapidly and expand in number. In contrast, the development of EBV-specific antibodies, even in the presence of high EBV DNA load, is markedly delayed or even absent

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posttransplant. Davis et al. [34] studied four solid organ transplant recipients, who were seronegative at the time of transplant and developed early onset of PTLD. In each case, the development of PTLD and the generation of EBV-specific CTL preceded production of EBV-specific antibodies. These results suggest that monitoring the T cell response to EBV posttransplant is more informative than monitoring EBV serology. Indeed, the reversal of PTLD when immunosuppression is reduced is generally attributed to the effector function of antiviral CD8+ T cells. However, only a few studies have directly correlated EBVspecific CTL function with disease course and PTLD. Two studies in bone marrow transplant recipients that developed PTLD monitored the CD8+ T cell population over time and showed that reduction of immunosuppression was associated with expansion of oligoclonal CTL populations and resolution of disease [35, 36]. While there is good evidence that host CTL can induce tumor regression, there are many cases where tumor progression proceeds unabated, even when immunosuppression is relaxed. There may be multiple factors at play in this unfortunate outcome. First, conversion of PTLD from the polyclonal to monoclonal form may be associated with more aggressive or advanced growth characteristics that are not amenable to control by immune surveillance mechanisms. Second, chronic exposure to the virus may lead to “exhaustion” of EBV-specific CD8 T cells. Such exhausted T cells express high levels of the negative co-stimulatory molecule programed death (PD)-1, and have been identified in humans with chronic HIV infection or hepatitis C virus (HCV) infection. In experimental animal models of chronic viral infection, both PD-1 expression on T cells and high levels of IL-10 have been linked to viral persistence [37, 38]. It will be of interest to determine if EBV-specific T cells express PD-1 and display an “exhausted” phenotype in transplant recipients with chronically high loads of EBV or in patients who develop PTLD. Finally, emerging studies indicate that EBV itself can contribute to immune evasion through a variety of strategies including induction of human IL-10 by infected cells [13] and active subversion of apoptotic pathways used by effector CTL to eliminate virally infector cells [15, 16].

4.3 PTLD PTLD represents a heterogeneous group of disorders, the majority of which are B cell proliferations associated with EBV. PTLD tumors can arise during primary infection with EBV, or as a result of viral reactivation in individuals who were seropositive for the virus at the time of transplantation. Further, EBV-associated PTLD lymphomas can be polyclonal or monoclonal, with polyclonal tumors arising more often in the early posttransplant period, while tumors that occur more than 1 year posttransplant tend to be of the monoclonal variety, but are more biologically heterogeneous. Thus, the factors contributing to the pathogenesis of PTLD are multiple and complex (Fig. 4.2). They include an immunosuppressed host, a virus with the ability to confer autonomous growth on infected cells and to invoke clever strategies of immune evasion, and the direct effects of immunosuppressive drugs on virally-infected or transformed cells – all in the setting of alloreactivity. Despite these common factors, most transplant recipients do not develop PTLD. How then does PTLD arise?

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EFFECTOR CELL FasL/TRAIL Death receptor DISC

LMP1

Caspase 8 NF-κB PI3K/AKT p38

cFLIP

apoptosis

EBV+ B CELL LYMPHOMA

cFLIP hIL-10 Survival proteins

hiL-10R

hIL-10

Fig. 4.2 Examples of viral strategies to promote cell survival and proliferation. The latent cycle gene LMP1 can activate a variety of cellular signaling pathways that drive expression of a host of survival proteins and cytokines. These molecules include cFLIP that can interfere with propagation of the apoptotic signal from death receptor pathways utilized by cytotoxic T cells and NK cells, as well as cellular IL-10 that is utilized in an autocrine growth pathway by EBV-infected cells.

Transplant recipients generally maintain higher EBV loads than healthy individuals, and have increased numbers of latently infected memory B cells and increased frequency of viral reactivation [39]. The elevated viral loads and lytic replication could lead to more viral infection events in naïve B cells raising the number of cells that initially express the latency III growth program. If these cells cannot exit the cell cycle or fail to successfully progress through the differentiation program, lymphomas could arise. Similarly, infection of bystander GC B cells or memory B cells could lead to aberrant expression of the growth program without the ability to differentiate, and subsequent clonal expansion. Alternately, latently infected GC B cells or memory B cells could inappropriately turn on the growth program, perhaps due to accumulated mutations or as yet unidentified signals [11]. Coupled with the impaired T cell response, the autonomous growth properties of EBV+ lymphoblasts that result in each of these scenarios could culminate in PTLD. In support of this, analysis of immunoglobulin gene sequences shows that PTLD tumors can originate from naïve B cells, GC cells, or memory cells. Extensive molecular and phenotypic studies of EBV+ monoclonal PTLD indicate that the majority appears to be GC-experienced cells that reflect different stages of B cell differentiation [40]. The high rate of proliferation in these cells could lead to additional mutations that further drive oncogenesis, in some cases, perhaps independent of EBV. While sporadic alterations in c-Myc [41] and p53 [42] have been described in PTLD lesions, mutations in the Bcl-6 proto-oncogene were found in ~40% of PTLD cases and correlated with poor outcome [43]. Monoclonal forms of PTLD tend to carry a higher frequency of mutations in tumor-suppressor genes and altered proto-oncogene expression. A variety of other factors could influence the development and progression of PTLDassociated B cell lymphomas. These factors can be classified into three broad categories: (1) viral determinants that drive tumor growth and survival; (2) viral mechanisms of immune

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T Cell Compartment

EBV infected B cell

Graft

? Immunosuppression

Fig. 4.3 Immune and viral factors influence the development of PTLD. The T cell compartment of transplant recipients includes cells that can respond to alloantigens on the graft and cells that are potentially reactive with viral antigens expressed on EBV-infected B cells. Immunosuppressive drugs impair the function of graft- reactive cells but also inhibit function of viral-specific cells. EBV can also impair the anti-viral response by actively subverting or evading T cell function. Finally, the direct effects of immunosuppressive drugs on EBV-infected B cells can also impact on their growth and survival.

evasion or subversion; and (3) direct effects of immunosuppression on tumor cell growth. The following section will highlight some specific examples pertinent to each of these categories.

4.3.1 Viral Determinants that Drive Growth and Survival of PTLD-Associated B Cell Lymphomas EBV has evolved to effectively co-opt several cellular signaling pathways within the host B cell to promote growth and survival of infected cells. The cellular cytokines IL-6 and IL-10 are both well-described autocrine growth factors in EBV+ B cell lymphomas [12, 44]. In addition, elevated levels of IL-6 and IL-10 are found in the circulation of patients with PTLD [45, 46]. In the case of IL-10, it has been definitively shown that the EBV-encoded protein, LMP1, activates the cellular mitogen activated protein kinase p38, and the PI3K/ AKT pathway to induce production of IL-10 [13] (Fig. 4.3). The latent cycle protein, LMP2A, acts as a constitutively active mimic of the BCR to deliver tonic signals to EBVinfected B cells through activation of the syk tyrosine kinase pathway. Furthermore, LMP2A can provide signals for survival and differentiation of B cells in the absence of BCR signaling through constitutive activation of the ERK/MAPK pathway [10, 47].

4.3.2 Viral Mechanisms of Immune Evasion or Subversion EBV-encoded proteins that can counter apoptotic signals are a common theme in viral subversion strategies. LMP1 can actively block apoptotic signals delivered through the

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Fas/Fas ligand and TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) death receptors pathways [16] (Fig. 4.3). This function of LMP1 could help ensure survival of infected cells through the process of B cell differentiation and also prevent elimination of EBV+ lymphoblasts by viral-specific CTL. LMP1 is also able to block apoptotic signals in EBV-infected B cells through upregulation of a variety of survival proteins including bcl-2, A20, mcl-1, and bfl-1. The EBV lytic cycle gene, BHRF1, encodes a viral homolog of bcl-2 that can inhibit apoptosis induced by multiple stimuli including anti-Fas antibodies and TNF-a. EBNA1 can block apoptosis induced by p53 expression, which may be particularly relevant in Burkitt’s lymphoma where EBNA1 is the sole latent cycle protein expressed [48]. A second EBV nuclear antigen, EBNA2, interferes with apoptosis induced by some stimuli through the intrinsic pathway by sequestering Nur77 in the nucleus and preventing its translocation to the cytoplasm where it can induce cytochrome C release from the mitochondria [49]. Immunomodulatory cytokines or their receptors, either encoded by EBV or induced by EBV, are also an important tactic utilized by the virus to evade host immunity. The lytic cycle gene BCRF1 encodes viral IL-10 (vIL-10), a functional homolog of cellular IL-10. vIL-10 is expressed early following infection of B cells by EBV, and because of its immunosuppressive properties, may facilitate transformation by impairing T cell and macrophage responses. In particular, vIL-10 can inhibit production of IFN-g by T cells and production of IL-12 by monocytes. As discussed earlier, LMP1 induces cellular IL-10, which acts as an autocrine growth factor for EBV+ B cell lymphomas. Cellular IL-10 can also have potent inhibitory effects on host T cells and monocytes during viral latency as in PTLD-associated lymphomas. The lytic cycle EBV gene, BARF1, encodes a functional, soluble receptor for colony stimulating factor 1 (CSF-1) that can interfere with the ability of CSF-1 to augment monocyte/macrophage proliferation and produce IL-12. Finally, EBV infection of B cells induces expression of a cellular protein, EB13, that is a functional homolog of the IL-12 p40 subunit. Thus, it has been suggested that EB13 can antagonize IL-12 activity [50]. EB13 can also pair with p28, an IL-12p35-related protein, to form the cytokine IL-27. IL-27 is a complex cytokine with diverse pro and anti-inflammatory properties, but strong evidence exists to indicate that IL-27 can inhibit a variety of effector functions by T cells [51]. Clearly, there are multiple avenues by which EBV can modulate host immunity that could impact the development and progression of PTLD.

4.3.3 Direct Effects of Immunosuppression on Viral Infection and Tumor Cell Growth Finally, it is likely that immunosuppressive medications may also have direct, cellautonomous effects on EBV-infected B cells, apart from effects on host immunity. Indeed, the frequency of outgrowth of immortalized EBV-infected B lymphoblasts is increased in the presence of the calcineurin inhibitor cyclosporine A (CS) [52]. Both CS and tacrolimus enhance the survival of EBV-infected B cell lymphoma lines from patients with PTLD, but do not affect the rate of cell division in vitro [53]. In contrast, the mTOR inhibitor, Rapamycin (Rapa), significantly inhibits proliferation of EBV-infected B lymphoma lines from PTLD patients in vitro. Importantly, Rapamycin is also a potent inhibitor of EBV+ B cell lymphoma growth in vivo in a xenogeneic SCID mouse model of PTLD [54]. At least

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part of the inhibitory growth effect of Rapa can be ascribed to its ability to inhibit production of the autocrine growth factor IL-10 in EBV+ B cell lines. Rapa also inhibits proliferation of EBV+ B cell lines through modulation of cell cycle protein expression [55].

4.4 Conclusion EBV is a highly successful virus that has developed effective strategies to persist in memory B cells of healthy individuals with minimal clinical consequences. However, disruption of the delicate balance between EBV and antiviral immunity, as in transplant recipients, can result in the development of EBV+ B cell lymphomas. In addition to immunosuppression, host–viral interactions play an important role in the development of EBV-associated PTLD. Elucidating the underlying host–viral mechanisms in the pathogenesis of PTLD could identify new therapeutic opportunities for the treatment of EBV-associated PTLD.

4.5 Take Home Pearls

• •

The development of EBV+ B cell lymphomas in PTLD is a complex and multifactorial process Understanding the host–viral interactions at play in transplant recipients with EBV infections could reveal new opportunities for therapies in the treatment of PTLD

References 1. Epstein MA, Achong BG, Barr YM. Virus particles in cultured lymphoblasts from Burkitt’s lymphoma. Lancet. 1964;1(7335):702–3. 2. Spear PG, Longnecker R. Herpesvirus entry: an update. J Virol. 2003;77(19):10179–85. 3. Hutt-Fletcher LM. Epstein-Barr virus entry. J Virol. 2007;81(15):7825–32 4. Shannon-Lowe CD, Neuhierl B, Baldwin G, Rickinson AB, Delecluse HJ. Resting B cells as a transfer vehicle for Epstein-Barr virus infection of epithelial cells. Proc Natl Acad Sci U S A. 2006;103(18):7065–70 5. Swerdlow SH. T-cell and NK-cell posttransplantation lymphoproliferative disorders. Am J Clin Pathol. 2007;127(6):887–95 6. Cohen JI. Epstein Barr virus infection. N Engl J Med. 2000;343:481–92 7. Snow AL, Martinez OM. Epstein-Barr virus: evasive maneuvers in the development of PTLD. Am J Transplant. 2007;7(2):271–7 8. Young LS, Rickinson AB. Epstein-Barr virus: 40 years on. Nat Rev Cancer. 2004;4(10):757–68 9. Thorley-Lawson DA. Epstein-Barr virus: exploiting the immune system. Nat Rev Immunol. 2001;1(1):75–82

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10. Caldwell RG, Wilson JB, Anderson SJ, Longnecker R. Epstein-Barr virus LMP2A drives B cell development and survival in the absence of normal B cell receptor signals. Immunity. 1998; 9(3):405–11 11. Thorley-Lawson DA. EBV the prototypical human tumor virus – just how bad is it? J Allergy Clin Immunol. 2005;116(2):251–61; quiz 62 12. Beatty PR, Krams SM, Martinez OM. Involvement of IL-10 in the autonomous growth of EBV-transformed B cell lines. J Immunol. 1997;158(9):4045–51 13. Lambert SL, Martinez OM. Latent membrane protein 1 of EBV activates phosphatidylinositol 3-kinase to induce production of IL-10. J Immunol. 2007;179(12):8225–34 14. Snow AL, Vaysberg M, Krams SM, Martinez OM. EBV B lymphoma cell lines from patients with post-transplant lymphoproliferative disease are resistant to TRAIL-induced apoptosis. Am J Transplant. 2006;6(5 Pt 1):976–85 15. Snow AL, Chen AL, Nepomuceno RR, Krams SM, Esquivel CO, Martinez OM. Resistance to Fas-mediated apoptosis in EBV-infected B cell lymphomas is due to defects in the proximal Fas signaling pathway. J Immunol. 2001;167:5404–11 16. Snow AL, Lambert SL, Natkunam Y, Esquivel CO, Krams SM, Martinez OM. EBV can protect latently infected B cell lymphomas from death receptor-induced apoptosis. J Immunol. 2006;177(5):3283–93 17. Thornburg NJ, Kulwichit W, Edwards RH, Shair KH, Bendt KM, Raab-Traub N. LMP1 signaling and activation of NF-kappaB in LMP1 transgenic mice. Oncogene. 2006;25(2):288–97 18. Rovedo M, Longnecker R. Epstein-Barr virus latent membrane protein 2B (LMP2B) modulates LMP2A activity. J Virol. 2007;81(1):84–94 19. Rechsteiner MP, Berger C, Zauner L, Sigrist JA, Weber M, Longnecker R, et al. Latent membrane protein 2B regulates susceptibility to induction of lytic Epstein-Barr virus infection. J Virol. 2008;82(4):1739–47 20. Ruf IK, Lackey KA, Warudkar S, Sample JT. Protection from interferon-induced apoptosis by Epstein-Barr virus small RNAs is not mediated by inhibition of PKR. J Virol. 2005;79(23): 14562–9 21. Samanta M, Iwakiri D, Takada K. Epstein-Barr virus-encoded small RNA induces IL-10 through RIG-I-mediated IRF-3 signaling. Oncogene. 2008;27:4150–60 22. Callan MF, Tan L, Annels N, Ogg GS, Wilson JD, O’Callaghan CA, et al. Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus In vivo. J Exp Med. 1998;187(9):1395–402 23. Catalina MD, Sullivan JL, Bak KR, Luzuriaga K. Differential evolution and stability of epitope-specific CD8(+) T cell responses in EBV infection. J Immunol. 2001;167(8):4450–7 24. Tan LC, Gudgeon N, Annels NE, Hansasuta P, O’Callaghan CA, Rowland-Jones S, et al. A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers. J Immunol. 1999;162(3):1827–35 25. Hislop AD, Taylor GS, Sauce D, Rickinson AB. Cellular responses to viral infection in humans: lessons from Epstein-Barr virus. Annu Rev Immunol. 2007;25:587–617 26. Bickham K, Munz C, Tsang ML, Larsson M, Fonteneau JF, Bhardwaj N, et al. EBNA1-specific CD4+ T cells in healthy carriers of Epstein-Barr virus are primarily Th1 in function. J Clin Invest. 2001;107(1):121–30 27. Heller KN, Upshaw J, Seyoum B, Zebroski H, Munz C. Distinct memory CD4+ T-cell subsets mediate immune recognition of Epstein Barr virus nuclear antigen 1 in healthy virus carriers. Blood. 2007;109(3):1138–46 28. Leen A, Meij P, Redchenko I, Middeldorp J, Bloemena E, Rickinson A, et al. Differential immunogenicity of Epstein-Barr virus latent-cycle proteins for human CD4(+) T-helper 1 responses. J Virol. 2001;75(18):8649–59 29. Long HM, Haigh TA, Gudgeon NH, Leen AM, Tsang CW, Brooks J, et al. CD4+ T-cell responses to Epstein-Barr virus (EBV) latent-cycle antigens and the recognition of EBVtransformed lymphoblastoid cell lines. J Virol. 2005;79(8):4896–907

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30. Haque T, Wilkie GM, Jones MM, Higgins CD, Urquhart G, Wingate P, et al. Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial. Blood. 2007;110(4):1123–31 31. Leen AM, Rooney CM, Foster AE. Improving T cell therapy for cancer. Annu Rev Immunol. 2007;25:243–65 32. Falco DA, Nepomuceno RR, Krams SM, Lee PP, Davis MM, Salvatierra O, et al. Identification of Epstein-Barr virus-specific CD8+ T lymphocytes in the circulation of pediatric transplant recipients. Transplantation. 2002;74(4):501–10 33. Macedo C, Donnenberg A, Popescu I, Reyes J, Abu-Elmagd K, Shapiro R, et al. EBV-specific memory CD8+ T cell phenotype and function in stable solid organ transplant patients. Transpl Immunol. 2005;14(2):109–16 34. Davis JE, Sherritt MA, Bharadwaj M, Morrison LE, Elliott SL, Kear LM, et al. Determining virological, serological and immunological parameters of EBV infection in the development of PTLD. Int Immunol. 2004;16(7):983–9 35. Khatri VP, Baiocchi RA, Peng R, Oberkircher AR, Dolce JM, Ward PM, et al. Endogenous CD8+ T cell expansion during regression of monoclonal EBV-associated posttransplant lymphoproliferative disorder. J Immunol. 1999;163(1):500–6 36. Kuzushima K, Kimura H, Hoshino Y, Yoshimi A, Tsuge I, Horibe K, et al. Longitudinal dynamics of Epstein-Barr virus-specific cytotoxic T lymphocytes during posttransplant lymphoproliferative disorder. J Infect Dis. 2000;182(3):937–40 37. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, et al. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature. 2006;439(7077):682–7 38. Brooks DG, Trifilo MJ, Edelmann KH, Teyton L, McGavern DB, Oldstone MB. Interleukin-10 determines viral clearance or persistence in vivo. Nat Med. 2006;12(11):1301–9 39. Babcock GJ, Decker LL, Freeman RB, Thorley-Lawson DA. Epstein-Barr virus-infected resting memory B cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients. J Exp Med. 1999;190(4):567–76 40. Capello D, Cerri M, Muti G, Berra E, Oreste P, Deambrogi C, et al. Molecular histogenesis of posttransplantation lymphoproliferative disorders. Blood. 2003;102(10):3775–85 41. Polack A, Hortnagel K, Pajic A, Christoph B, Baier B, Falk M, et al. c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1. Proc Natl Acad Sci U S A. 1996;93(19):10411–6 42. Knowles DM, Cesarman E, Chadburn A, Frizzera G, Chen J, Rose EA, et al. Correlative morphologic and molecular genetic analysis demonstrates three distinct categories of posttransplantation lymphoproliferative disorders. Blood. 1995;85(2):552–65 43. Cesarman E, Chadburn A, Liu YF, Migliazza A, Dalla-Favera R, Knowles DM. BCL-6 gene mutations in posttransplantation lymphoproliferative disorders predict response to therapy and clinical outcome. Blood. 1998;92(7):2294–302 44. Tosato G, Tanner J, Jones KD, Revel M, Pike SE. Identification of interleukin-6 as an autocrine growth factor for Epstein-Barr virus-immortalized B cells. J Virol. 1990;64:3033–41 45. Martinez OM, Villanueva JC, Lawrence-Miyasaki L, Quinn MB, Cox K, Krams SM. Viral and immunologic aspects of Epstein-Barr virus infection in pediatric liver transplant recipients. Transplantation. 1995;59(4):519–24 46. Tosato G, Jones K, Breinig MK, McWilliams HP, McKnight JL. Interleukin-6 production in posttransplant lymphoproliferative disease. J Clin Invest. 1993;91(6):2806–14 47. Anderson LJ, Longnecker R. EBV LMP2A provides a surrogate pre-B cell receptor signal through constitutive activation of the ERK/MAPK pathway. J Gen Virol. 2008;89(Pt 7):1563–8 48. Kelly GL, Milner AE, Baldwin GS, Bell AI, Rickinson AB. Three restricted forms of EpsteinBarr virus latency counteracting apoptosis in c-myc-expressing Burkitt lymphoma cells. Proc Natl Acad Sci U S A. 2006;103(40):14935–40

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49. Lee JM, Lee KH, Weidner M, Osborne BA, Hayward SD. Epstein-Barr virus EBNA2 blocks Nur77- mediated apoptosis. Proc Natl Acad Sci U S A. 2002;99(18):11878–83 50. Cohen JI. The biology of Epstein-Barr virus: lessons learned from the virus and the host. Curr Opin Immunol. 1999;11(4):365–70 51. Kastelein RA, Hunter CA, Cua DJ. Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation. Annu Rev Immunol. 2007;25:221–42 52. Chen C, Johnston TD, Reddy KS, Merrick JC, Mastrangelo M, Ranjan D. Cyclosporine directly causes oxidative stress and promotes Epstein-Barr virus transformation of human B cells. J Surg Res. 2001;100(2):166–70 53. Beatty PR, Krams SM, Esquivel CO, Martinez OM. Effect of cyclosporine and tacrolimus on the growth of Epstein-Barr virus-transformed B-cell lines. Transplantation. 1998;65(9):1248–55 54. Nepomuceno RR, Balatoni CE, Natkunam Y, Snow AL, Krams SM, Martinez OM. Rapamycin inhibits the interleukin 10 signal transduction pathway and the growth of Epstein Barr virus B-cell lymphomas. Cancer Res. 2003;63(15):4472–80 55. Vaysberg M, Balatoni CE, Nepomuceno RR, Krams SM, Martinez OM. Rapamycin inhibits proliferation of Epstein-Barr virus-positive B-cell lymphomas through modulation of cellcycle protein expression. Transplantation. 2007;83(8):1114–21

Epstein–Barr Viral Load Testing: Role in the Prevention, Diagnosis and Management of Posttransplant Lymphoproliferative Disorders

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Jutta K. Preiksaitis

Core Messages

›

›

›

›

Epstein–Barr virus (EBV) viral load (VL) assessment in peripheral blood represents a potentially powerful tool for surveillance as part of pre-emptive programs for posttransplant lymphoproliferative disorders (PTLD) prevention, for PTLD and EBV disease diagnosis in symptomatic patients, to monitor response to PTLD therapy and predict relapse, for safety monitoring in clinical trials of new immunosuppressive agents and for tailoring immunosuppression in individual patients. However good quality evidence to guide the application EBV VL testing and interpretation resulting data in these clinical settings is limited. Lack of an international reference standard for EBV VL has resulted in significant variability in both qualitative and quantitative EBV VL results reporting among centers, limiting the validity of inter-institutional result comparison. In populations at high risk of early EBV-positive PTLD development, a high EBV VL observed during serial monitoring has high sensitivity but poor specificity for determining PTLD risk; adjunctive assays specifically monitoring of EBV-specific T cells responses in this setting may improve the positive predictive value of EBV VL assays. The preferred sample type- whole blood (WB), peripheral blood mononuclear cells (PBMC)/lymphocytes (PBL), or plasma/serum, reporting units, specific quantitative levels for use trigger points for intervention or PTLD diagnosis, optimal monitoring algorithms and the cost-effectiveness of EBV VL testing in populations at high and low risk of PTLD remain unresolved issues.

J. K. Preiksaitis Division of Infectious Diseases, Provincial Public Health Laboratory, 1B1.17 WMC, 8440-112 Street, Edmonton, AB, Canada T6G 2J2 e-mail: [email protected] V. R. Dharnidharka et al. (eds.), Post-Transplant Lymphoproliferative Disorders, DOI: 10.1007/978-3-642-01653-0_5, © Springer Verlag Berlin Heidelberg 2010

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5.1 Introduction In the early 1990s, several investigators studying solid organ transplant (SOT) and hemopoietic stem cell transplant (HSCT) recipients observed that peak Epstein–Barr virus (EBV) viral load (VL) measured in peripheral blood lymphocytes (PBL) or oropharyngeal excretions was higher in primary infection than in reactivation infection and in patients who developed posttransplant lymphoproliferative disorders (PTLD) vs. those who did not [1–3]. These peak VLs were generally observed prior to the onset of clinical signs and symptoms. Since then, technologic advancements have made EBV VL assays more sensitive and precise. EBV VL assessments are now extensively used for laboratory surveillance as part of preemptive programs for PTLD prevention, for PTLD, and EBV disease diagnosis in symptomatic patients, and to monitor response to PTLD therapy [4]. Their use has also been recommended for safety monitoring in clinical trials of new immunosuppressive agents [5] and tailoring immunosuppression in individual patients. This chapter summarizes the quality of evidence supporting the use of EBV VL assessments in the settings noted above, examines what is being measured when EBV VL assays are performed, and outlines some current limitations and unknowns associated with the use and interpretation of this laboratory tool.

5.2 EBV VL Assessments-What Is Being Measured? The form in which EBV DNA exists in a variety of biologic compartments in transplant recipients is summarized in Table 5.1. Similar to immunocompetent EBV seropositive patients, most of the EBV DNA in the peripheral blood of immunosuppressed patients is found in latently-infected transcriptional silent antigen-selected memory B cells containing 1–2 genome copies/nucleus in episomal form [6, 7, 13, 8]. In pediatric SOT patients with high VL, Schauer et al. [9] also found that up to 30% of EBV infected cells were aberrant “crippled” or “forbidden” Ig-null cells containing 30–60 genomes/nucleus. Cells in the peripheral blood are rarely believed to exist as EBV “blasts” expressing “growth pattern” transcripts or demonstrate evidence of lytic infection [6]. However, in a study of EBV seropositive adult cardiothoracic transplant recipients, both “growth” and lytic gene expression were found surprisingly frequently in non-PTLD with high VLs, though this expression was transient [14]. Although HIV-infected children with chronic high EBV VL have evidence of EBV-infected T cells as well as B cells [15], it is not known whether nonB cell EBV infection also occurs in transplant recipients. The status of EBV DNA detected in cell-free serum or plasma is uncertain. Encapsidated virions may be present, but EBV DNA can also exist as free DNA released from latently infected cells, most notably tumor cells undergoing apoptosis. In patients with infectious mononucleosis (IM), EBV DNA is detectable in serum for only approximately 7 days after symptom onset, even though VL remains detectable significantly longer in the cellular

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Table 5.1 Epstein-Barr virus (EBV) viral load in biologic compartments Compartment

EBV DNA state

Peripheral blood Transcriptionally silent latently infected lymphocytes (PBL) (low genome copy number) resting or mononuclear memory B cells (CD19+, IgD negative cells (PBMC) Ki67−, CD23−, CD80−, IgM+) Highly atypical B cells (high genome copy number) having an aberrant Ig-null cellular phenotype Serum/plasma Encapsidated virus and free DNA in acute infection Free DNA only in EBV-associated malignancies Whole blood (WB) PBL/PBMC plus serum/plasma components Oropharyngeal Lytic virus produced in plasma cells secretions (CD38hi, CD10−, CD19+, CD201o) as they differentiate Role for epithelial cells

Reference Babcock et al. [6], Rose et al. [7]

Rose et al. [8], Schauer et al. [9] Ryan et al. [10]

Laichuk and Thorley-Lawson [11] Borza and Hutt-Fletcher [12]

fraction [16]. Limited studies of EBV DNA in the serum from immunocompetent IM patients suggest that EBV DNA exists in both an encapsidated form and as free DNA [10]. In contrast, EBV DNA existed as only free DNA in specimens from transplant patients and patients with AIDS-related lymphomas [10]. However, the number of transplant patients in this study was small and details regarding serostatus, the presence of primary infection, and timing after transplant are lacking [10]. A recent model of EBV dynamics in IM stresses the importance of lytic viral infection in driving the one-way self-amplifying circuit of new B cell infection, latency, and reactivation that continues until the cellular immune response is activated. The virion peak and most lytic viral production occur before the onset of symptoms. Delay in development of the immune response in transplant patients with primary infection could prolong the one-way self-amplifying circuit, increase the virion production peak, and result in virions present in serum/plasma, even though the circulation is not the major site of virion production. Observations regarding EBV VL distribution in plasma vs. whole blood (WB) and comparison with the cytotoxic T cell response in a renal transplant patient experiencing primary infection are consistent with this model [17]. Although the impact of antiviral therapy on EBV VL measured in the plasma compartment is uncertain, this therapy is unlikely to have an immediate and direct effect on EBV VL measured in the latently-infected cells of peripheral blood [18]. WB VL assays detect EBV DNA present in both the lymphocyte and plasma/serum compartments. The major site of lytic virus production is the oropharynx where infectious virus with a linear genome is produced by memory B cells as they differentiate into plasma cells [11]. The role of transient epithelial cell infection in sustaining orophayngeal lytic viral infection remains controversial [12]. Antiviral drugs significantly reduce EBV VL detected at this site [19, 20]. Both the immunocompetent and immunocompromised host are simultaneously infected with multiple EBV strains. Clear differences in the relative abundance of strains in

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distinct compartments, the oral cavity, PBLs, and plasma exist. The impact of these differences on quantitative VL assessments in these compartments is uncertain [21, 22].

5.3 Limitations and Unresolved Issues Related to EBV VL Assays With advances in molecular technology, EBV VL assays have evolved rapidly. Historical techniques, which involved counting EBV transformed B cell clones resulting from the spontaneous outgrowth of B cells cultured under predetermined conditions, were replaced by semi-quantitative methods that detected EBV DNA directly by comparing Southern blot or agarose gel signals to calibrated controls or used end point dilutions. Assays moved from the research to the diagnostic laboratory when quantitative competitive assays that compared EBV DNA amplified from a clinical sample with an internal calibration standard were introduced. These assays have been almost entirely replaced in hospital and commercial laboratories by more rapid, quantitative, and precise assays that use real time nucleic acid testing (NAT). Review of data from studies carried out over the last 20 years should take these technologic advances into perspective. Despite these advances, significant limitations and unresolved issues persist preventing the optimal use of this laboratory tool as discussed below (reviewed by De Paoli et al. [23]).

5.4 Lack of Standardization and Cross-Referencing of Assays Although commercial assays and analyte-specific reagents using quantitative NAT (QNAT) technology for EBV VL testing have recently become available, many laboratories in North America continue to use laboratory developed and validated assays. Both commercial and laboratory developed assays employ a variety of extraction methods, gene targets, and instruments. Assays are calibrated using standard curves created using EBV-infected cell lines with known genome copy numbers or plasmids. Recent studies suggest that significant and extreme interlaboratory variability exists in both qualitative and quantitative VL assessments in clinical laboratories, raising questions regarding the validity of interinstitutional result comparison [24, 25]. In most laboratories, intralaboratory result reproducibility and result linearity over the dynamic range of the assay was reasonable, suggesting that result trending in patients over time within individual institutions using a single assay is valid. The use of a common calibrator among laboratories reduces quantitative EBV VL variability highlighting the urgent need for the development of a common internationally accepted reference standard, a process that is currently underway. Although interlaboratory quantitative result variability remained high even when extraction method was removed as a variable [24], extraction methodology is often identified as a major source of result variability [23]. Nucleic acid extraction from cellular matrices,

5 Epstein–Barr Viral Load Testing

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such as whole blood or lymphocytes, may also be less efficient than extraction from cell free plasma samples [25]. A variety of genes are the targets of current QNAT assays. Some assays have targeted the Bam HI-W EBV region, but this region exists in a variable number of repeats in individual subjects that may affect accurate quantitative result generation. Single copy genes may be more appropriate targets, though the analytic sensitivity of individual assays targeting specific genes is highly dependent on the primer design used [26]. It is uncertain whether variability in EBV gene expression in PTLD is important in target choice. It has also been suggested that the primer design and product size should consider that free DNA in serum may be highly fragmented [23], so primers that result in small product sizes may be preferable for accurate EBV VL assessment in serum or plasma. The availability of an international reference standard for calibration would allow further standardization, optimization of extraction methodology, gene targets, and primer design, and minimization of other technical sources of result variability This, combined with rising standards for quality systems and laboratory participation in proficiency programs, should result in significant improvements in the inter and intralaboratory reproducibility of quantitative and qualitative EBV VL results.

5.5 Specimen Type and Reporting Units Although there is general consensus that peripheral blood is the preferred sampling site for EBV VL testing in transplant patients, the optimal sample type- WB, peripheral blood mononuclear cells (PBMC)/lymphocytes (PBL), or plasma/serum remains a hotly contested issue. In immunocompetent patients with EBV – associated malignances (undifferentiated carcinoma of nasopharyngeal type, Hodgkin’s disease, and NK/T cell lymphomas), EBV VL acts as a true tumor biomarker with plasma, rather than PBMC, being the preferred specimen type [23, 27]. In the transplant setting, EBV VL result interpretation is more complex and the optimal specimen type is less clear. Continuously changing levels of immunosuppression that strongly influence EBV reactivation and control of infection, the high incidence of primary EBV infection because of donor-recipient EBV mismatch particularly in pediatric populations, HLA-mismatch, organ-specific factors, and other systemic events that might directly stimulate B cells and increase EBV VL, independent of immunosuppression, all contribute to the complexity of EBV VL result interpretation. From a laboratory perspective, the use of either plasma/serum or WB is preferable to peripheral PBMC, all other issues being equal, because of the additional processing steps, increased expense and increased volume of blood required with the latter sample type. Several recent studies have shown comparable sensitivity, a close quantitative correlation of EBV VL results, and similar dynamic trending in individual patients in the PBMC and WB fractions of samples obtained from both pediatric HSCT and adult and pediatric SOT, suggesting there is little added benefit derived from the additional processing steps required for PBMC isolation. [28–30]. Although it has been suggested that DNA amplification

50

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inhibitors were more common in WB compared to PBMC or B cell samples [31], recent investigators have not observed this effect, possibly because of improved specimen preparation methodologies [28, 29]. There is general agreement that EBV VL measured in plasma is less sensitive than similar assessments in PBMC or WB for the detection of early infection and reactivation events in adult and pediatric SOT and HSCT recipients [26, 28–30, 32–35]. Stevens et al. [36] suggested WB to be the preferred specimen type after observing that WB was more sensitive than serum /plasma samples for EBV VL detection in PTLD patients using a quantitative competitive assay. In contrast, using a QNAT assay and primer set with greatest analytical sensitivity, Tsai et al. [26] found EBV VL measurement in plasma had comparable sensitivity and improved specificity to WB for diagnosing EBV-positive PTLD cases in adult EBV seropositive SOT recipients presenting with signs and symptoms suggestive of PTLD. Although plasma samples are more likely to be positive in patients with high EBV VLs in matched WB or PBMC fractions, the quantitative correlations between EBV VL detected in the cellular and acellular compartments of specimens are poor [28–30]. These observations are not really surprising when one considers that the cellular and acellular peripheral blood compartments reflect aspects of viral dynamics that may differ significantly in the setting of either acute primary or reactivation infection, time after transplantation, and the presence or absence of antiviral therapy [18]. It is likely that the optimal specimen type may be dependent on the purpose of the EBV VL testing (patient surveillance, diagnosis of disease, monitoring response to therapy) and the population being examined (adult vs. pediatric, EBV seropositive vs. seronegative, and transplant type). Ideally direct comparisons of EBV VL in different peripheral compartments in these populations and clinical settings using standardized assays are required to definitively determine optimal specimen type. A one-sizefits-all solution may not be possible. Currently, EBV VL results reporting formats are variable and include reporting per unit volume (usually copies/ml) in copies/mg DNA (when testing cellular compartments such as WB or PBMC) or in copies or number of positive cells /number of isolated cells (when PBMC, PBL, or leukocytes are isolated). In a study of pediatric HSCT recipients, a close correlation between copies/ml and copies/mg DNA was observed with similar dynamic trending in patients using both reporting formats, suggesting normalization to cell number or genomic DNA in cellular specimens may not be necessary [28]. Reporting in copies/ml has the advantage of reduced costs and processing time in the laboratory. Testing for EBV VL in the CSF of transplant recipients is often used to assist in the diagnosis of CNS PTLD alone or in the setting of multiorgan involvement to avoid the need for more invasive biopsy procedures. However, result interpretation is extremely difficult as specific studies of the sensitivity, specificity, and positive and negative predictive values for both qualitative and quantitative EBV VL assessments in the CSF of SOT and HSCT recipients are not available. Data are often extrapolated from the HIV-infected population where a recent study suggested that qualitative EBV DNA detection in CSF has a poor positive, but a good negative predictive value for primary CNS lymphoma diagnosis [37]. VL quantitation in CSF may improve the specificity and the positive predictive value [37]. Concomitant review of CSF leukocyte counts might aid VL result interpretation [38].

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Whether simultaneously measuring VL in CSF and peripheral blood adds value is uncertain and has not been studied.

5.6 The Use of EBV VL Assessments for Specific Clinical Purposes 5.6.1 EBV VL Assessments for Preemptive Programs Targeting PTLD Prevention EBV VL assessments are critical for prevention strategies using “pre emptive” approaches. Although controversy exists regarding whether pre-emptive approaches result in better patient outcomes than prompt treatment of patients with early symptoms and signs of EBV disease, or clinically apparent PTLD [39, 40], single center studies of both high risk HSCT recipients [41] and pediatric SOT recipients [42] have demonstrated that rates of PTLD or PTLD associated mortality declined significantly after the implementation of routine EBV VL testing and pre emptive programs compared to similar historical populations. Effective pre emptive programs have specific laboratory requirements. The laboratory marker must have a high positive predictive value for identifying patients at risk (or interventions must result in minimal harm to patients falsely identified as being at risk). There must be compliance with a laboratory monitoring algorithm that captures the highest risk period and is frequent enough to allow both, identification of the high risk patient and intervention prior to onset of symptoms or signs. Intervention trigger points that maximize the sensitivity and specificity of the laboratory marker in predicting PTLD risk must be well-defined, ideally in natural history studies. Unfortunately, EBV VL assays used in both the HSCT and SOT setting fall short of this ideal. Much of the data on EBV VL use in pre emptive strategies are retrospective analyses of single center studies (reviewed in [40, 43] and summarized in Tables 5.2 and 5.3 [33, 34, 39, 44–55, 57–64]). Some studies include both high risk and low risk patients, some fail to differentiate patients experiencing primary infection from those reactivating EBV particularly in the SOT setting, and a wide variety of monitoring algorithms and trigger points for intervention are used. The number of cases of PTLD is often small and early PTLD is sometimes not differentiated from late PTLD, nor is EBV-positive PTLD from EBVnegative PTLD.Despite these limitations, some general observations regarding EBV VL have been observed in serially monitored patients. In the SOT setting, peak EBV VLs are higher in primary infection than in reactivation infection and in children compared to adults. In both SOT and HSCT recipients, EBV reactivation is more frequent and peak VLs are higher in patients who have risk factors for early EBV-positive PTLD. Reactivation/infection events are initially most often observed in the 2–3 month period after transplantation in both the HSCT and SOT settings. As a group, patients with symptomatic EBV disease (but not PTLD) have higher peak VLs than asymptomatic transplant patients. However, significant overlap in peak VL is observed in

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Table 5.2 Studies of hematopoietic stem cell or bone marrow transplant recipients suggesting quantitative “threshold values” for pre emptive intervention (P) or diagnosis (D) of posttransplant lymphoproliferartive disorders (PTLD) Author

Assay type

Specimen type

Monitoring protocol (mean time of follow-up)

Study patients Number of Patients

Patient population

41

Adult and pediatric

12

N/A

q 2 weeks × 3 months, weekly if symptoms or high viral load (N/A) q 2 weeks to day 180 N/A q 2 weeks in hospital, q 4 weeks outpatient (NA)

38

Adult and pediatrics

85

Adult

59

Adult and pediatric

Lucas et al. [44]

SQ

PBMC

Ohga et al. [45]

RT

Hoshino et al. [46]

RT

Plasma or WB PBMC

a

Van Esser et al. [47]

RT

Plasma

Gartner et al. [48]

QC

WB

a

RT

Serum or plasma PBMC

q 2 week (88 days)

26

Pediatric

days 3, 30, 60, 90 (314 days)

85

Adults and pediatric

PBMC

q 2 weeks (N/A)

85

Adult and pediatric

Greenfield et al. [51] RT

WB

28

Pediatric

Kinch et al. [33]

RT

WB and plasma

38

Adult and pediatric

a

Aalto et al. [52]

RT

Serum

406

Adult

Meerbach et al. [34]

SQ

PBMC and plasma

q weekly in hospital, all outpatient visits (148 days) q 1–2 weeks × 3 months then at clinical discretion (N/A) no specified protocol (N/A) weekly to 100 days, monthly to 1 year (N/A)

123

Pediatric

Lankester et al. [49]

Sirvent-von SQ Bueltzingsloewen et al. [50] Wagner et al. [39] RT

b

6–8 Weeks post Tx, then q 2–4 weeks (147 days) N/A

PTLD posttransplant lymphoproliferative disorder; SQ semi-quantitative PCR; RT real time PCR; QC quantitative competitive PCR; PBMC peripheral blood mononuclear cells; WB whole blood; Tx transplant; N/A not available; P threshold for pre-emptive therapy; D threshold for PTLD diagnosis; Sen sensitivity; Spec specificity; PPV positive predictive value; NPV negative predictive value

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Table 5.2 (continued)

PTLD cases

Threshold proposal

Number of cases

Viral load range

Value

Purpose

Sen (%)

Spec (%)

PPV (%)

NPV (%)

7

(40–400,000 gEq/ mg DNA)

300 gEq/mg DNA

P

80

94

71

94

200 gEq/ml

P

100

90

67

100

316 gEq/mg DNA

P

100

80

22

100

D

100

97

83

100

P

100

50

39

100

100,000 gEq/mg D DNA

87

91

f

f

100 gEq/ml

P

100

95

86

100

300 gEq/mg DNA

D

100

81

25

100

4,000 gEq/mg P DNA on two occasions e 31, 622 gEq/ml P

100

79

50

100

100

73

22

100

2 c

2+3

(2.3 × 104 − 8.0 × 105 gEq/mg DNA)

10,000/mg DNA 10 9

6 5

1,000 gEq/ml

74

(N/A)

2

(1.6 × 106 − 4.0 × 106 gEq/ml)

3

(1.6 × 104 − 7.6 × 106 gEq/ml) [plasma value] (N/A)

1,000 gEq/ml [plasma value] 50,000 gEq/ml

P

100

89

43

100

D

75

98

68

99

3 × 105 − 1.3 × 107 gEq/105 PBMC

e

P

100

100

100

100

P

48

100

100

100

2 × 103 − 5 × 105 gEq/ml plasma

e

5

3

1,000 gEq/105 PBMC 500 gEq/ml plasma

Natural history study, retrospective testing, physicians unaware of results Preemptive interventions including Rituximab therapy applied to study population c Nonprospectively followed additional PTLD patients added to analysis d Clinical rather than pathologically confirmed PTLD diagnosis e Authors suggest increase preemptive thresholds to 1,000,000 gEq/ml f Calculated for subset with ³3 risk factors for PTLD b

96

8

d

a

(1.8 × 103 − 7.9 × 105 gEq/ml) (1.4 × 103 − 1 × 108 gEq/mg DNA) [median not peak] (>105 gEq/ml in 5/6) (all >300 gEq/mg DNA)

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J. K. Preiksaitis

Table 5.3 Studies of solid organ transplant recipients suggesting quantitative “threshold values” for preemptive (P) intervention or diagnosis (D) of posttransplant lymphoproliferative sorders (PTLD) Author

Assay type

Specimen type

Monitoring protocol (mean time of follow-up) Study patients Number of patients

Rowe et al. [53]

QC

PBMC

Cross-sectional study (N/A) 26

Lucas et al. [54]

SQ

PBMC

a

McDiarmid et al. [55] a Green et al. [56]

SQ

WB

QC

PBMC

a

Gridelli et al. [57]

SQ

PBMC

Vajro et al. [58]

SQ

PBMC

Baldanti et al. [59]

QC

PBMC

a

Allen et al. [60]

SQ

PBMC

Matsukura et al. [61]

RT

PBMC

Cross-sectional study at mean 33 months post Tx follow-up only if viral load positive q 1 – 2 months (2 groups 243, 275 day) q 2 weeks to 3 – 4 months then q 1 – 3 months (2 groups median 16, 31.5 months) q 3 months, and when symptomatic (2 groups median 12, 38 months) q 3 – 6 months and at physician’s discretion (N/A) q 3 – 6 months, q 1 – 3 months If positive (N/A) q 1 – 2 weeks × 6 weeks, q 4 weeks to 6 months, q 1 – 3 months (N/A) At Tx at symptom onset, at convalescence (N/A)

a

Orentas et al. [62]

RT

PBMC

At physician’s discretion (N/A)

56

Yancoski et al. [63]

RT

WB

Cross-sectional study (median 8.3 months

110

Schubert et al. [64]

RT

WB

2 weeks, 13, 6, 12, 18, 24 months, then yearly (N/A)

41

165

Patient population

Pediatric kidney, heart, liver, small bowel, multivisceral Adult and pediatric kidney, pancreas, liver, heart

40

Pediatric liver

30

Pediatric intestinal

31

Pediatric liver

N/A

Pediatric liver

105

Adult and pediatric heart, lung, liver

135

Pediatric liver, heart, kidney

15

Adult and pediatric symptomatic living related liver Pediatric kidney, liver, heart, bone marrow Pediatric kidney, liver and bone marrow Pediatric heart

QC quantitative competitive PCR; SQ semi-quantitative PCR; RT real time PCR; PBMC peripheral blood mononuclear cells; WB whole blood; N/A: not available; Tx transplant; P for preemptive therapy; D for PTLD diagnosis; Sen Sensitivity; Spec Specificity; PPV Positive predictive value; NPV Negative predictive value a Pre emptive interventions including Rituximab therapy in some studies applied to study population b Threshold differentiates asymptomatic from symptomatic patients with EBV disease including PTLD, but not PTLD specifically

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Table 5.3 (continued)

PTLD cases

Threshold proposal

Number of cases

Viral load range

Value

Purpose

Sen (%)

Spec (%)

PPV (%)

NPV (%)

14

20 to >2.5 × 104 gEq/105 PBMC All >4 × 104 Eq/ mg DNA

500 gEq/105 PBMC

D

92

100

100

99

40,000 gEq/mg DNA

D

100

97

38.5

100

2

N/A

1,000 gEq/mg DNA

P

100

55

17

100

5

N/A

200 gEq/105 PBMC

P

100

48

28

100

1

N/A

500 gEq/105 PBMC

P

100

82

14

100

4

All >4× 104 gEq/105 PBMC N/A

40,000 gEq/PBMC

P, D

N/A

N/A

N/A

N/A

b

D

73

97

64

98

3

N/A

1,000 gEq/0.5 mg DNA

16

10 − 104 cells/ 106 1,000 cells/106 PBMC PBMC

D

69

76

28

95

2

4.0 × 104 − 8.1 × 104 gEq/mg DNA

100 gEq/mg DNA

P

N/A

N/A

N/A

N/A

9

N/A

10,000 gEq/mg DNA

P

N/A

N/A

N/A

N/A

9

N/A

66,800 gEq/ml

P

63

90

36

97

6

[Median] 2.11 × 103 − 5.06 × 105

3,000 gEq/mg DNA

P

100

90

63

100

56

J. K. Preiksaitis

patients experiencing EBV primary infection with and without PTLD and in patients with symptomatic non-PTLD EBV infection/disease and PTLD cases. How sensitive and specific is a high EBV VL determined by serial monitoring for identifying patients at risk for PTLD development prior to the onset of symptoms and signs of PTLD? The positive predictive value of a high VL is dependent on the prevalence of PTLD in the population being monitored, and is significantly better in high risk than low risk SOT and HSCT populations [48]. As expected, studies of EBV monitoring in low risk predominantly seropositive adult organ transplant and low risk HSCT found lower frequencies of EBV reactivation and/or that high EBV VL has a very poor positive predictive value for PTLD risk [46–48, 65–67]. Evidence to support patient monitoring in these settings is lacking, though no formal cost-effectiveness analysis has been performed. In high risk SOT and HSCT populations, high VLs have high sensitivity (100% in some studies), but poorer specificity for predicting EBV-positive early PTLD. As a result, even when the prevalence of PTLD risk is high, the negative predictive value is excellent, but the positive predictive value is poor; a circumstance that would result in significant intervention in patients who are actually not at risk. The sensitivity of high VL for PTLD prediction, however, is not perfect. This laboratory marker will not identify patients at risk for EBVnegative PTLD [26], and serial monitoring fails to detect some cases, particularly localized disease including CNS disease [26, 48, 68] and donor-derived localized hepatobiliary disease in liver transplant recipients [69].

5.6.2 Laboratory Tests as Adjuncts to EBV VL Assessments In an attempt to improve the specificity of high VL as a predictor of PTLD, a number of approaches that include adjunctive laboratory testing have been suggested. The best studied and most promising are assays measuring T cell restoration or EBV-specific T cell responses [32, 70–73]. Although data suggest that the specificity and positive predictive value of EBV VL can be significantly improved by using concomitant EBV-specific T cell elispot and tetramer assays, these assays are complex, costly and difficult to implement in a routine diagnostic laboratory. Preliminary data suggest that simpler approaches measuring T cell reconstitution that involve the direct counting of CD4 cells in SOT recipients [72] or CD3 cells in high risk HSCT recipients may be of value [70]. A number of investigators have also examined EBV latent and lytic gene expression in patients with low and high VL to determine whether specific gene expression patterns can identify a subgroup of patients at higher risk for PTLD development [14, 58, 74, 75]. These data suggest that there is no distinctive pattern in PBL that is indicative of PTLD or PTLD risk. However, Hopwood et al. [14] observed that PTLD cases are highly unlikely to have the restricted gene expression pattern found in healthy seropositive adults. All the PTLD cases that they studied had evidence of “growth” latency III or lytic gene expression. Additional markers that have been proposed as potentially useful for improving the specificity of high VL assessments for PTLD risk prediction include marked hypergammagobulinemia [58], serologic measurement of responses to EBV early antigen [76],

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measurement of the cytokines IL-10 and IL-6 in plasma [77, 78], and transplant recipient IFN gamma, SlcIIaI and HLA class 1 gene polymorphisms [79–81], but further validation of these adjunct markers is required.

5.6.3 Laboratory Monitoring Algorithms Studies reporting experiences with serial EBV VL monitoring of transplant recipients have used mean, median, and peak VL in comparing subpopulations and assessing the value of VL in predicting PTLD risk (Tables 5.2 and 5.3). However, these values are highly dependent on monitoring frequency. EBV VL can change dramatically and quickly in individual patients, and limited EBV viral kinetic data in SOT [82] and HSCT recipients [83] suggest that doubling time for EBV DNA in WB or PMBC can be very short, 49–56 h. Monitoring weekly over the highest risk period has been recommended. No published study examining pre emptive therapy and the utility of EBV VL in predicting risk has employed and had patients be compliant with such a frequent monitoring algorithm over the entire high risk period. This may reflect the pragmatic reality of obtaining samples from patients, particularly after they leave hospital. Although many investigators have suggested that trends in EBV VL in individual patients are more useful than single absolute values, there are no detailed studies of VL kinetics in association with risk. Jabs et al. [84] have suggested that normalization of quantitative values of EBV VL to coamplified genomic DNA allows for more accurate quantification and trending of results in individual patients over time. Although the focus has been on peak VL, persistent lower VLs or high total VL exposure over time may also confer risk, perhaps explaining cases of EBV-positive PTLD occurring after the first transplant year. The rate of rise of VL rather than peak VL may also be important, but has not been studied. All patients, particularly in the SOT setting, may not have an unrelenting increase in EBV VL until the time of EBV –positive PTLD diagnosis. Some patients who developed PTLD may have had very high VLs that subsequently decrease or become negative. However, these transient high VLs may have predisposed them to secondary genetic or epigenetic events that ultimately result in PTLD. Infrequent monitoring could miss such patients. How long should EBV VL monitoring be performed? In high risk HSCT recipient, PTLD risk appears greatest during the first 6 months, the time required for T cell reconstitution in most patients, with most cases developing during the first year [40]. In contrast, although the highest rate of PTLD in the SOT setting is also seen in the first year posttransplant, cases occurring in this timeframe represent only one-fifth of the total cumulative 10-year PTLD burden [85]. However, a significant proportion of late PTLD (21–38%) may be EBV-negative and not preventable by EBV VL monitoring [26]. There is no evidence that ongoing VL monitoring after the first 6 months in HSCT recipients or beyond the first transplant year in SOT recipients, who have undetectable VLs at this time and in whom no increases in immunosuppression are planned, is either useful or cost effective. However, no published study performed frequent patient monitoring during these late periods.

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5.6.4 Defining Trigger Points for Preemptive Interventions Trigger points suggested for preemptive interventions are most often based on single center studies and institution-specific assays, sample types and reporting units [Tables 5.2 and 5.3]. Ideally, trigger points should be set based on natural history studies employing the frequency of monitoring prescribed above by determining the quantitative VLs in asymptomatic patients present prior to disease that is most sensitive and specific for determining future PTLD development. It is also important that a significant number of endpoints (i.e., EBVpositive PTLD) cases be achieved. This is almost never possible in a single center study. A natural history study also requires that the clinicians managing the patients be unaware of the VL results. Given this criteria of clinician blinding, only a few natural history studies of EBV VL exist. These retrospective studies used stored specimens and are limited to the HSCT setting [47, 49, 52]. Even these studies are limited by suboptimal monitoring frequency; one involved primarily a low risk population [52]. Most studies that have suggested trigger points for pre emptive interventions have made these suggestions based on peak or median VLs in serially monitored patients that best differentiate symptomatic patients already diagnosed with PTLD from asymptomatic patients without PTLD in settings where clinicians have been aware of serial EBV VL results. In some studies, pre emptive interventions, including rituximab therapy, were aggressively applied [Tables 5.2 and 5.3]. Unfortunately, the extensive use of EBV VL monitoring in transplant settings would make it ethically very difficult to now perform natural history studies to better define trigger points using a multicenter approach and standardized assays.

5.7 EBV VL Assessment for PTLD Diagnosis Pathologic examination of tissue samples currently remains the gold standard for PTLD diagnosis and EBV VL assessments alone are insufficient to confirm the diagnosis of EBVpositive PTLD and direct therapy. However, in clinical practice, EBV VL is being used extensively as an adjunct diagnostic tool, even though result interpretation is often problematic. In asymptomatic prospectively monitored patients, quantitative VLs that should trigger an investigation for PTLD have been suggested (Tables 5.2 and 5.3). In order to determine the sensitivity and specificity of specific trigger points in this setting, all asymptomatic patients who reach a threshold value should be investigated using a standard approach and the resulting yield of PTLD and EBV-positive PTLD should be determined. Data of this kind are not available. Instead, estimates of sensitivity and specificity have been derived by examining the subset of patients who achieve the quantitative threshold, and determining the proportion of patients both symptomatic and asymptomatic who have been investigated and have a diagnosis of PTLD relative to the proportion of asymptomatic patients who have not been investigated for PTLD, but are presumably PTLD-free. This approach is based on the assumption that all cases of PTLD eventually will become clinically obvious during the period of observation and be diagnosed and that spontaneous remission of PTLD will not occur. It is not clear that this is the case, particularly in the SOT setting where normal

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immunosuppression management usually involves the tapering of immunosuppression over time. In high risk asymptomatic SOT and HSCT recipients being serially monitored, the use of EBV VL as a diagnostic test (i.e., levels above a specific quantitative threshold being diagnostic of PTLD) has the same limitations as its use as a preemptive tool. That is, it has good sensitivity for detecting EBV-positive PTLD, but misses EBV negative and some cases of localized PTLD, has poor specificity resulting in good negative but poor positive predictive value in these populations. When used in the diagnostic context, this would result in significant unnecessary investigation of patients for PTLD. Formal evaluation of EBV VL assessments as a diagnostic tool using a single evaluation in patients presenting with symptoms and/or signs (usually mass lesions) with no history of recent or previous monitoring has not been carried out in populations at high risk for PTLD. In a retrospective study, Tsai et al. [86] examined the utility of a single EBV VL assessment determined using a quantitative competitive assay in the peripheral blood leukocytes of seropositive adult transplant recipients presented for investigation with signs and symptoms compatible with PTLD. They observed, that at a threshold level of >1,000 copies/2 × 106 leukocytes, the assay lacked sensitivity, missing all cases of EBV-negative PTLD and approximately half of the cases of EBV-positive PTLD, but was highly specific (100%) with all patients having a load greater than this value ultimately diagnosed with EBVpositive PTLD. A followup prospective study was carried out by the same group of investigators using plasma samples and more sensitive real-time PCR assays. They found qualitative detection of EBV DNA in plasma was associated with good sensitivity (77%), specificity (72%), positive predictive value (100%), and negative predictive value (93%), for diagnosing EBV-positive PTLD. EBV-negative PTLD cases and a case of EBV-positive PTLD localized to the CSF were missed. These investigators corroborated the findings of others [33, 35] who found that EBV VL measured in plasma significantly improves the specificity of the test as a diagnostic tool for EBV-positive PTLD, while not significantly lowering its sensitivity relative to assessments in cellular blood compartments. These data suggest plasma might be the preferred sample type when EBV VL is used for PTLD diagnosis. Although Tsai et al.’s data suggest that qualitative EBV DNA detection in plasma is sufficient for PTLD diagnosis and quantitative assessment might not be necessary, EBV DNA has often been found in the plasma of patients experiencing primary EBV infection [16] and in symptomatic HSCT and SOT recipients with EBV disease that is not PTLD [29, 33, 86]. Tsai et al.’s [26] study because of its size, oncology referral bias, and restriction to adults may not have included patients of this type. Quantitative rather than qualitative assessments alone may be required to retain good specificity for the EBV VL in the plasma fraction when used to assist in EBV-PTLD positive diagnosis in high risk populations presenting with symptoms or signs of disease.

5.8 EBV VL Assessments for Monitoring Response to PTLD Therapy and Predicting Relapse Clinicians observed that elevated VLs, measured in the PBLs of PTLD patients and patients being monitored serially after transplant, fell rapidly in response to pre emptive interventions or PTLD treatment. These observations were initially made in pediatric

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SOT populations where the incidence of primary EBV infection was high and both pre emptive therapy and PTLD treatment most often consisted of reduction of immunosuppression with or without antiviral drug therapy or immunoglobulin [55, 56]. Green et al. [87] found that VL was “cleared” in the PBL of seven pediatric liver transplant patients with PTLD (six managed as above, one received chemotherapy) at a mean of 18.8 days, and clearance coincided with clinical and histologic regression. In contrast, two groups of investigators found that in adult SOT recipients with EBV-positive PTLD receiving rituximab therapy, EBV VL measured in PBLs fell rapidly and dramatically and could remain low even in the face of progressive disease and disease relapse [88, 89]. However, van Esser et al. [90] who measured EBV VL in plasma rather than the cellular compartment after PTLD treatment of HSCT recipients found monitoring at this site rapidly and accurately reflected response to therapy and distinguished responders (>50% reduction in VL) from nonresponders (increases in VL) as soon as 72 h after initiation of therapy. Seven of the 14 patients studied had received rituximab therapy. Tsai et al. [26] directly compared VL measured in WB and plasma for monitoring PTLD treatment response and observed that while plasma VL correlated closely with treatment response and radiographic evidence of complete remission, WB VL did not (four of the 13 patients had received rituximab). Although the preliminary data from these studies suggest EBV VL monitoring in plasma correlates better with disease response than monitoring in the cellular compartment, the total number of PTLD patients studied receiving specific types of treatment is small. Green et al. [87] observed that all pediatric liver transplant patients with PTLD experienced intermittent or persistent VL rebound after initially achieving VL “clearance” in PBL often to pretreatment levels. This short-term VL rebound was not associated with evidence of clinical or histological PTLD relapse and appeared to occur in patients experiencing primary infection at a median time of 4 months after transplant [91]. PTLD relapse has also been observed in adult PTLD patients in the presence of a persistently low VL measured in WB [88]. These data suggest that in the short term, monitoring of EBV VL in cellular blood compartments cannot predict disease relapse. Although preliminary data are promising, it is uncertain whether monitoring in plasma might be more predictive. Investigators have described a subpopulation of asymptomatic pediatric SOT recipients that they have termed “chronic high load carriers”, defined as patients who have EBV VL >16,000copies/ml of WB or >200copies/105 PMBC over a minimum period of 6 months after asymptomatic infection or after complete resolution of symptomatic EBV disease including PTLD [91, 92]. Although some patients appear to have spontaneous regression of VL with no obvious sequelae, a retrospective analysis of cardiothoracic transplant recipients found that 45% of 20 high load carriers developed late-onset EBVpositive PTLD 2.5–8.4 years later [92]. This risk appears to be organ-specific and was not observed in pediatric liver transplant patients, though follow-up in this latter group is shorter [91]. Further, long-term EBV VL monitoring studies of transplant recipients, particularly those experiencing primary EBV infection, are required to determine whether a “chronic high load carrier state” identifies a patient subpopulation at increased risk for late onset PTLD.

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5.9 EBV VL Assessments for Determining the Impact of New Immunosuppressive Regimens and Tailoring Individual Immunosuppression Because EBV infection occurs almost universally in man, is persistent and highly dependent on T cell responses for ongoing control, EBV VL has been proposed as a biomarker of global immunosuppression in individual patients [64, 66]. Because PTLD rates are low in most transplant populations, routine EBV VL monitoring, particularly in patients experiencing primary infection after transplant, has been recommended as a surrogate marker of PTLD risk and to more accurately determine the impact of new immunosuppressive regimens on the control of EBV infection in clinical trials [5]. Because current assays are not standardized, it is critical that EBV VL testing in clinical trials occurs in a centralized reference laboratory or in participating laboratories that cross-reference the assays being used. Preliminary reports suggesting or examining tailoring of individual immunosuppression based on EBV VL in pediatric kidney and adult lung transplant recipients with a goal of improving graft outcomes have recently been published [92–94]. Although an interesting idea, immunosuppression tailoring based on EBV VL has not been validated and must be approached with caution, particularly when applied to populations at low risk of PTLD. Because “acceptable” EBV VLs that might translate into “just right” individual immunosuppression in specific transplant settings have not been defined, tailoring immunsuppression is associated with the risk of acute rejection and impaired long-term graft function. “Expected” VLs in primary infection differ from those “expected” in reactivation infection independent of global T cell competence. EBV proteins produced during its lytic cycle are themselves immunomodulatory resulting in a relationship between global immunosuppression and EBV VL that is unlikely to be linear. In HIV-infected patients, high EBV VL is believed to be the result of chronic B cell stimulation and chronic hyper immune activation, rather than T cell deficiency alone [95]. Clearly, better understanding of the pathogenesis and nature of EBV VL in the peripheral blood compartment of transplant patients is required before implementing changes in patient management based on its measurement.

5.10 Take Home Pearls

• • •

EBV VL assessment represents a potentially powerful tool for the prevention and management of PTLD and for improving outcomes in transplant patients. A number of obstacles currently limit the optimal use of EBV VL including assay standardization, which will require the development of international reference standards for assay calibration and cross-referencing. Carefully controlled multicenter trials are required to improve our understanding of the biology of EBV infection in the transplant setting, and to definitively answer many

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remaining questions regarding optimal specimen type and the utility and design of programs using VL assessments for pre emptive PTLD treatment, PTLD diagnosis, PTLD treatment monitoring, and tailoring of immunosuppression in all transplant recipients.

References 1. Preiksaitis JK, Diaz-Mitoma F, Mirzayans F, et al. Quantitative oropharyngeal Epstein-Barr virus shedding in renal and cardiac transplant recipients: relationship to immunosuppressive therapy, serologic responses and the risk of post-transplant lymphoproliferative disorder. J Infect Dis. 1992;166:986–94 2. Riddler SA, Breinig MC, McKnight JLC. 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. 1994;84:972–84 3. Savoie A, Perpete C, Carpentier L, et al. Direct correlation between the load of Epstein-Barr virus-infected lymphocytes in the peripheral blood of pediatric transplant patients and risk of lymphoproliferative disease. Blood. 1994;83:2715–22 4. Green M, Avery RK, Preiksaitis JK, editors. Guidelines for the prevention and management of infections complications of solid organ transplantation. Am J Transplant. 2004;4(Suppl 10):59–65 5. Humar A, Michaels M; on behalf of the AST ID Working Group on Infectious Diseases Monitoring. American Society of Transplantation recommendations for screening, monitoring and reporting of infectious complications in immunosuppression trials in recipients of organ transplantation. Am J Transplant. 2006;6:262–74 6. Babcock GJ, Decker LL, Freeman RB, et al. Epstein-Barr virus-infected resting memory B cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients. J Exp Med. 1999;190:567–76 7. Rose C, Green M, Webber S, et al. Pediatric solid-organ transplant recipients carry chronic loads of Epstein-Barr virus exclusively in the immunoglobulin D-negative B-Cell compartment. J Clin Microbiol. 2001;39:1407–15 8. Rose C, Green M, Webber S, et al. Detection of Epstein-Barr virus genomes in peripheral blood B cells from solid-organ transplant recipients by fluorescence in situ hybridization. J Clin Microbiol. 2002;40:2533–44 9. Schauer E, Webber S, Green M, et al. Surface immunoglobulin-deficient Epstein-Barr virusinfected B cells in the peripheral blood of pediatric solid-organ transplant recipients. J Clin Microbiol. 2004;42:5802–10 10. Ryan JL, Fan H, Swinnen LJ, et al. Epstein-Barr virus (EBV) DNA in plasma is not encapsidated in patients with EBV-related malignancies. Diagn Mol Pathol. 2004;13:61–8 11. Laichalk LL, Thorley-Lawson DA. Terminal differentiation into plasma cells initiates the replicative cycle of Epstein-Barr virus in vivo. J Virol. 2005;79:1296–307 12. Borza CM, Hutt-Fletcher L. Alternate replication in B cells and epithelial cells switches tropism of Epstein-Barr virus. Nat Med. 2002;8:594–9 13. Souza TA, Stollar BD, Sullivan JL, et al. Peripheral B cells latently infected with Epstein-Barr virus display molecular hallmarks of classical antigen-selected memory B cells. Proc Natl Acad Sci U S A. 2005;102:18093–8 14. Hopwood PA, Brooks L, Parratt R, et al. Persistent Epstein-Barr virus infection: unrestricted latent and lytic viral gene expression in healthy immunosuppressed transplant recipients. Transplantation. 2002;74:194–202 15. Bekker V, Scherpbier H, Beld M, et al. Epstein-Barr virus infects B and non-B lymphocytes in HIV-1-infected children and adolescents. J Infect Dis. 2006;194(9):1323–30

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16. Fafi-Kremer S, Morand P, Brion JP, et al. Long-term shedding of infectious Epstein-Barr virus after infectious mononucleosis. J Infect Dis. 2005;191:985–9 17. Piriou ER, van Dort K, Weel JFL, et al. Detailed kinetics of EBV-specific CD4+ and CD8+ T cells during primary EBV infection in a kidney transplant patient. Clin Immunol. 2006;119:16–20 18. Hadinoto V, Shapiro M, Greenough TC, et al. On the dynamics of acute EBV infection and the pathogenesis of infectious mononucleosis. Blood. 2008;111:1420–7 19. Balfour HH Jr., Hokanson KM, Schacherer RM, et al. A virologic pilot study of valacyclovir in infectious mononucleosis. J Clin Virol. 2007;39:16–21 20. Cockfield SM, Preiksaitis JK, Jewell LD, Parfrey NA. Post-transplant lymphoproliferative disorder in renal allograft recipients: clinical experience and risk factor analysis in a single center. Transplantation. 1993;56:88–96 21. Fafi-Kremer S, Morand P, Germi R, et al. A prospective follow-up of Epstein-Barr virus LMP1 genotypes in saliva and blood during infectious mononucleosis. J Infect Dis. 2005;192: 2108–11 22. Sitki-Green DL, Edwards RH, Covington MM, et al. Biology of Epstein-Barr Virus during infectious mononucleosis. J. Infect Dis. 2004 Feb 1;189:483–92 23. De Paoli P, Pratesi C, Bortolin MT. The Epstein Barr virus DNA levels as a tumor marker in EBV-associated cancers. J Cancer Res Clin Oncol. 2007;133:809–15 24. Hayden RT, Hokanson KM, Pounds SB, et al. Multicenter comparison of different real-time PCR assays for quantitative detection of Epstein-Barr virus. J Clin Microbiol. 2008;46:157–63 25. Preiksaitis JK, Pang XL, Fox JD, et al. Inter-laboratory comparison of Epstein-Barr virus (EBV) viral load assays. Am J Transplant. 2009;9:269–79 26. Tsai DE, Douglas L, Andreadis C, et al. EBV PCR in the diagnosis and monitoring of posttransplant lymphoproliferative disorder: results of a two-arm prospective trial. Am J Transplant. 2008;8:1016–24 27. Ambinder RF, Lin L. Mononucleosis in the laboratory. J Infect Dis. 2005;192:1503–4 28. Hakim H, Gibson C, Pan J, et al. Comparison of various blood compartments and reporting units for the detection and quantification of Epstein-Barr virus (EBV) in peripheral blood. J Clin Microbiol. 2007;45:2151–5 29. Pang XL, Lee B, Preiksaitis JK. Comparison of various blood compartments for the detection and quantification of Epstein-Barr Virus (EBV) in the peripheral blood of solid organ transplant recipients. J Clin Microbiol (Submitted) 30. Wadowsky RM, Laus S, Green M, et al. Measurement of Epstein-Barr virus DNA loads in whole blood and plasma by Taqman PCR and in peripheral blood lymphocytes by competitive PCR. J Clin Microbiol. 2003;41:5245–9 31. Wagner HJ, Jabs W, Smets F, et al. Real-time polymerase chain reaction (RQ-PCR) for the monitoring of Epstein-Barr virus (EBV) load in peripheral blood mononuclear cells. Klin Padiatr. 2000;212:206–10 32. Clave E, Agbalika F, Bajzik V, et al. Epstein-Barr virus (EBV) reactivation in allogeneic stemcell transplantation: relationship between viral load, EBV-specific T-cell reconstruction and Rituximab therapy. Transplantation. 2004;77:76–84 33. Kinch A, Oberg G, Arvidson J, et al. Post-transplant lymphoproliferative disease and other Epstein-Barr virus diseases in allogeneic haematopoietic stem cell transplantation after introduction of monitoring of viral load by polymerase chain reaction. Scand J Infect Dis. 2007;39: 235–44 34. Meerbach A, Wutzler P, Hafer R, et al. Monitoring of Epstein-Barr virus load after hematopoietic stem cell transplantation for early intervention in post-transplant lymphoproliferative disease. J Med Virol. 2008;80:441–54 35. Wagner HJ, Wessel M, Jabs W, et al. Patients at risk for development of posttransplant lymphoproliferative disorder: plasma versus peripheral blood mononuclear cells as material for quantification of Epstein-Barr viral load by using real-time quantitative polymerase chain reaction. Transplantation. 2001;72:1012–9

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36. Stevens SJC, Pronk I, Middeldorp JM. Toward standardization of Epstein-Barr virus DNA load monitoring: unfractionated whole blood as preferred clinical specimen. J Clin Microbiol. 2001; 39:1211–6 37. Corcoran C, Rebe K, van der Plas H, et al. The predictive value of cerebrospinal fluid EpsteinBarr viral load as a marker of primarcy central nervous system lymphoma in HIV-infected persons. J Clin Virol. 2008;42:433–6 38. Weinberg A, Li S, Palmer M, et al. Quantitative CSF PCR in Epstein-Barr virus infections of the central nervous system. Ann Neurol. 2002;52:543–8 39. Wagner HJ, Cheng YC, Huls MH, et al. Prompt versus preemptive intervention for EBV lymphoproliferative disease. Blood. 2004;103:3979–3881 40. Weinstock DM, Ambrossi GG, Brennan C, et al. Preemptive diagnosis and treatment of Epstein-Barr virus-associated post transplant lymphoproliferative disorder after hematopoietic stem cell transplant: an approach in development. Bone Marrow Transplant. 2006;37:539–46 41. van Esser JWJ, Niesters HGM, van der Holt B, et al. Prevention of Epstein-Barr viruslymphoproliferative disease by molecular monitoring and preemptive rituximab in high-risk patients after allogeneic stem cell transplantation. Blood. 2002;99:4364–9 42. Lee TC, Savoldo B, Rooney CM, et al. Quantitative EBV viral loads and immunosuppression alterations can decrease PTLD incidence in pediatric liver transplant recipients. Am J Transplant. 2005;5:2222–8 43. Stevens SJC, Verschuuren EAM, Verkuulen AWM, et al. Role of Epstein-Barr virus DNA load monitoring in prevention and early detection of post-transplant lymphoproliferative disease. Leuk Lymphoma. 2002;43:831–40 44. Lucas KG, Burton RL, Zimmerman SE, et al. Semiquantitative Epstein-Barr virus (EBV) polymerase chain reaction for the determination of patients at risk for EBV-induced lymphoproliferative disease after stem cell transplantation. Blood. 1998;92:3977–8 45. Ohga S, Kubo E, Nomura A, et al. Quantitative monitoring of circulating Epstein-Barr virus DNA for predicting the development of posttransplantation lymphoproliferative disease. Int J Hematol. 2001;73:323–6 46. Hoshino Y, Kimura H, Tanaka N, et al. Prospective monitoring of the Epstein-Barr virus DNA by a real-time quantitative polymerase chain reaction after allogenic stem cell transplantation. Br J Haematol. 2001;115:105–11 47. van Esser JWJ, van der Holt B, Meijer E, et al. Epstein-Barr virus (EBV) reactivation is a frequent event after allogeneic stem cell transplantation (SCT) and quantitatively predicts EBVlymphoproliferative disease following T-cell-depleted SCT. Blood. 2001;98:972–8 48. Gartner BC, Schafer H, Marggraff K, et al. Evaluation of use of Epstein-Barr viral load in patients after allogeneic stem cell transplantation to diagnose and monitor posttransplant lymphoproliferative disease. J Clin Microbiol. 2002;40:351–8 49. Lankester AC, van Tol MJD, Vossen JM, et al. Epstein-Barr virus (EBV)-DNA quantification in pediatric allogeneic stem cell recipients: prediction of EBV-associated lymphoproliferative disease. Blood. 2002;99:2630–1 50. Sirvent-von Bueltzingsloewen A, Morand P, Buisson M, et al. A prospective study of EpsteinBarr virus load in 85 hematopietic stem cell transplants. Bone Marrow Transplant. 2002;29: 21–8 51. Greenfield HM, Gharib MI, Turner AJL, et al. The impact of monitoring Epstein-Barr virus PCR in paediatric bone marrow transplant patients: can it successfully predict outcome and guide intervention? Pediatr Blood Cancer. 2006;47:200–5 52. Aalto SM, Juvonen E, Tarkkanen J, et al. Epstein-Barr viral load and disease prediction in a large cohort of allogeneic stem cell transplant recipients. Clin Infect Dis. 2007;45:1305–9 53. Rowe DT, QU L, Reyes J, et al. Use of quantitative competitive PCR to measure Epstein-Barr virus genome load in the peripheral blood of pediatric transplant patients with lymphoproliferative disorders. J Clin Microbiol. 1997;35:1612–5

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54. Lucas KG, Filo R, Heilman DK, et al. Semiquantitative Epstein-Barr virus polymerase chain reaction analysis of peripheral blood from organ transplant patients and risk for the development of lymphoproliferative disease. Blood. 1998;92:3977–9 55. McDiarmid SV, Jordan S, Lee G, et al. Prevention and preemptive therapy of posttransplant lymphoproliferative disease in pediatric liver recipients. Transplantation. 1998;66:1604–11 56. Green M, Bueno J, Rowe D, et al. Predictive negative value of persistent low Epstein-Barr virus viral load after intestinal transplantation in children. Transplantation. 2000;70:593–6 57. Gridelli B, Spada M, Riva S, et al. Circulating Epstein-Barr virus DNA to monitor lymphoproliferative disease following pediatric liver transplantation. Transpl Int. 2000;13(Suppl1): S399–401 58. Vajro P, Lucariello S, Migliaro F, et al. Predictive value of Epstein-Barr virus genome copy number and BZLF1 expression in blood lymphocytes of transplant recipients at risk for lymphoproliferative disease. J Infect Dis. 2000;181:2050–4 59. Baldanti F, Grossi P, Furione M, et al. High levels of Epstein-Barr virus DNA in blood of solidorgan transplant recipients and their value in predicting posttransplant lymphoproliferative disorders. J Clin Microbiol. 2000;38:613–9 60. Allen U, Hebert D, Petric M, et al. Utility of semiquantitative polymerase chain reaction for Epstein-Barr virus to measure virus load in pediatric organ transplant recipients with and without posttransplant lymphoproliferative disease. Clin Infect Dis. 2001;33:145–50 61. Matsukura T, Yokoi A, Egawa H, et al. Significance of serial real-time PCR monitoring of EBV genome load in living donor liver transplantation. Clin Transplant. 2002;16:107–12 62. Orentas RJ, Schauer DW Jr., Ellis FW, et al. Monitoring and modulation of Epstein-Barr virus loads in pediatric transplant patients. Pediatr Transplant. 2003;7:305–14 63. Yancoski J, Danielian S, Ibanez J, et al. Quantification of Epstein-Barr virus load in Argentinean transplant recipients using real-time PCR. J Clin Virol. 2004;31:58–65 64. Schubert S, Renner C, Hammer M, et al. Relationship of immunosuppression to Epstein-Barr viral load and lymphoproliferative disease in pediatric heart transplant patients. J Heart Lung Transplant. 2008;27:100–5 65. Benden C, Aurora P, Burch M, et al. Monitoring of Epstein-Barr viral load in pediatric heart and lung transplant recipients by real-time polymerase chain reaction. J Heart Lung Transplant. 2005;24:2103–8 66. Doesch AO, Konstandin M, Celik S, et al. Epstein-Barr virus load in whole blood is associated with immunosuppression, but not with post-transplant lymphoproliferative disease in stable adult heart transplant patients. Transpl Int. 2008;21:963–71 67. Loginov R, Aalto S, Piiparinen H, et al. Monitoring of EBV-DNA Aemia by quantitative realtime PCR after adult liver transplantation. J Clin Virol. 2006;37:104–8 68. Axelrod DA, Holmes R, Thomas SE, et al. Limitations of EBV-PCR monitoring to detect EBV associated post-transplant lymphoproliferative disorder. Pediatr Transplant. 2003;7:223–7 69. Mutimer D, Kaur N, Tang H, et al. Quantitation of Epstein-Barr virus DNA in the blood of adult liver transplant recipients. Transplantation. 2000;69:954–9 70. Annels NE, Kalpoe JS, Bredius RG, et al. Management of Epstein-Barr virus (EBV) reactivation after allogeneic stem cell transplantation by simultaneous analysis of EBV DNA load and EBV-specific T cell reconstitution. Clin Infect Dis. 2006;42:1743–8 71. Meij P, van Esser JWJ, Niesters HGM, et al. Impaired recovery of Epstein-Barr virus (EBV)specific CD8+ T lymphocytes after partially T-depleted allogenic stem cell transplantation may identify patients at very high risk for progressive EBV reactivation and lymphoproliferative disease. Blood. 2003;101:4290–7 72. Sebelin-Wulf K, Nguyen TD, Oertel S, et al. Quantitative analysis of EBV-specific CD4/CD8 T cell numbers, absolute CD4/CD8 T cell numbers and EBV load in solid organ transplant recipients with PLTD. Transpl Immunol. 2007;17:203–10

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73. Smets F, Latinne D, Bazin H, et al. Ratio between Epstein-Barr viral load and anti-Epstein-Barr virus specific T-cell response as a predictive marker of posttransplant lymphoproliferative disease. Transplantation. 2002;73:1603–10 74. Germi R, Morand P, Brengel-Pesce, et al. Quantification of gp350/220 Epstein-Barr virus (EBV) mRNA by real-time reverse transcriptions-PCR in EBV-associated diseases. Clin Chem. 2004;50:1814–7 75. Qu L, Green M, Webber S, et al. Epstein-Barr virus gene expression in the peripheral blood of transplant recipients with persistent circulating virus loads. J Infect Dis. 2000;182:1013–21 76. Carpentier L, Tapiero B, Alvarez F, et al. Epstein-Barr virus (EBV) early-antigen serologic testing in conjunction with peripheral blood EBV DNA load as a marker for risk of posttransplantation lymphoprilferative disease. J Infect Dis. 2003;188:1853–64 77. Jones RJ, Seaman WT, Feng WH, et al. Roles of lytic viral infection and IL-6 in early versus late passage lymphoblastoid cell lines and EBV-associated lymphoproliferative disease. Int J Cancer. 2007;121:1274–81 78. Muti G, Mancini V, Ravelli E, Morra E. Significance of Epstein-Barr virus (EBV) load and Interleukin-10 in post-transplant lymphoproliferative disorders. Leuk Lymphoma. 2005;46: 1397–407 79. Barshes NR, Lee TR, Goss JA, et al. Sic11a1 (formerly Nramp1) polymorphisms and susceptibility to post-transplant lymphoproliferative disease following pediatric liver transplantation. Transpl Infect Dis. 2006;8:108–12 80. Lee TC, Savoldo B, Barshes NR, et al. Use of cytokine polymorphisms and Epstein-Barr virus viral load to predict development of post-transplant lymphoproliferative disorder in paediatric liver transplant recipients. Clin Transplant. 2006;20:389–93 81. McAulay KA, Higgins CD, Macsween KF, et al. HLA class I polymorphisms are associated with development of infectious mononucleosis upon primary EBV infection. J Clin Invest. 2007; 117:3042–8 82. Stevens SJC, Verschuuren EAM, Pronk I, et al. Frequent monitoring of Epstein-Barr virus DNA load in unfractionated whole blood is essential for early detection of posttransplant lymphoproliferative disease in high-risk patients. Blood. 2001;97:1165–71 83. Biasolo MA, Calistri A, Cesaro S, et al. Case report: kinetics of Epstein-Barr virus load in a bone marrow transplant patient with no sign of lymphoproliferative disease. J Med Virol. 2003; 69:220–4 84. Jabs WJ, Hennig H, Kittel M, et al. Normalized quantification by real-time PCR of EpsteinBarr virus load in patients at risk for posttransplant lymphoproliferative disorders. J Clin Microbiol. 2001;39:564–9 85. Opelz G, Dohler B. Lymphomas after solid organ transplantation: a collaborative transplant study report. Am J Transplant. 2004;4:222–30 86. Tsai DE, Nearey M, Hardy CL, et al. Use of EBV PCR for the diagnosis and monitoring of post-transplant lymphoproliferative disorder in adult solid organ transplant patients. Am J Transplant. 2002;2:946–54 87. Green M, Cacciarelli TV, Mazariegos GV, et al. Serial measurement of Epstein-Barr viral load in peripheral blood in pediatric liver transplant recipients during treatment for Posttransplant Lymphoproliferative disease. Transplantation. 1998;66:1641–4 88. Oertel S, Trappe RU, Zeidler K, et al. Epstein-Barr viral load in whole blood of adults with posttransplant lymphoproliferative disorder after solid organ transplantation does not correlate with clinical course. Ann Hematol. 2006;85:478–84 89. Yang J, Tao Q, Flinn IW, et al. Characterization of Epstein-Barr virus-infected B cells in patients with posttransplantation lymphoproliferative disease: disappearance after rituximab therapy does not predict clinical response. Blood. 2000;96:4055–63 90. van Esser JWJ, Niesters HGM, Thijsen SFT, et al. Molecular quantification of viral load in plasma allows for fast and accurate prediction of response to therapy of Epstein-Barr

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virus-associated lymphoproliferative disease after allogeneic stem cell transplantation. Br J Haematol. 2001;113:814–21 91. Green M, Soltys K, Rowe DT, et al. Chronic high Epstein-Barr viral load carriage in pediatric liver transplant recipients. Pediatr Transplant. 2009;13:319–23 92. Bingler MA, Feingold B, Miller SA, et al. Chronic high Epstein-Barr viral load state and risk for late-onset posttransplant lymphoproliferative disease/lymphoma in children. Am J Transplant. 2008;8:442–5 93. Ahya VN, Douglas LP, Andreadis C, et al. Association between elevated whole blood EpsteinBarr virus (EBV) – encoded RNA EBV polymerase chain reaction and reduced incidence of acute lung allograft rejection. J Heart Lung Transplant. 2007;26:839–44 94. Bakker NA, Verschuuren EAM, Erasmus ME, et al. Epstein-Barr virus-DNA load monitoring late after lung transplantation: a surrogate marker of the degree of immunosuppression and a safe guide to reduce immunosuppression. Transplantation. 2007;83:433–8 95. Pietersma F, Piriou E, van Baarle D. Immune surveillance of EBV-infected B cells and the development of non-Hodgkin lymphomas in immunocompromised patients. Leuk Lymphoma. 2008;49:1028–41

Clinical Features and Diagnostic Evaluation of Posttransplant Lymphoproliferative Disorder

6

Upton D. Allen

Core Messages

› › › › ›

The major risk factor for posttransplant lymphoproliferative disorder (PTLD) is primary infection after transplantation The presenting signs and symptoms of PTLD are non-specific Epstein–Barr virus (EBV) serology is of limited value in the diagnosis of PTLD EBV load by itself has poor specificity for the diagnosis of PTLD Histopathologic examination of tissue is the gold standard for the diagnosis of PTLD

6.1 Introduction It is well recognized that the Epstein–Barr virus (EBV) is associated with the majority of cases of PTLD occurring after transplantation. EBV-associated posttransplant lymphoproliferative disorder (PTLD) encompasses a spectrum of clinical entities in the posttransplant period. These syndromes range from uncomplicated infectious mononucleosis to true malignancies [1–3]. While these manifestations of PTLD are often conveniently classified into discreet entities, in reality they often represent a spectrum of illnesses where more benign entities may be followed by more serious syndromes. The heterogenous nature of PTLD makes generalization problematic. This notwithstanding, one can recognize two primary modes of presentation of PTLD in the solid organ transplant recipient, namely, early-onset and late-onset PTLD.

U. D. Allen Department of Pediatrics, Division of Infectious Diseases, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8 e-mail: [email protected] V. R. Dharnidharka et al. (eds.), Post-Transplant Lymphoproliferative Disorders, DOI: 10.1007/978-3-642-01653-0_6, © Springer Verlag Berlin Heidelberg 2010

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Although the time demarcation between these entities is somewhat arbitrary, the former occurs within the first 1–2 years, while the latter occurs after the first 1–2 years. Among hematopoietic stem cell transplant recipients, there are some notable characteristics of PTLD that deserve special mention as discussed below.

6.1.1 Severe Infectious Mononucleosis, Clinical Categories, and Sites of PTLD Severe Infectious Mononucleosis. Infectious mononucleosis is the prototype of primary EBV infection [4–6]. The clinical spectrum of this entity ranges from asymptomatic infection to severe, sometimes fatal disease in immunodeficient patients. Infectious mononucleosis is typically characterized by fever, exudative pharyngitis, lymphadenopathy, hepatosplenomegaly and atypical lymphocytosis. In symptomatic individuals, adenotonsillar disease is often a prominent feature (Fig. 6.1). The features of severe infectious mononucleosis may be seen in some cases of acutely symptomatic PTLD. In complicated cases or the more severe cases in the immunocompromised host, patients may develop hepatitis, upper airway obstruction due to enlarged adenotonsillar tissue, pneumonitis, encephalitis, aseptic meningitis, splenic rupture, decreased blood cellular elements, disseminated intravascular coagulation and hemophagocytic syndrome, bacterial superinfection, renal, cardiac, and other complications [4–6]. PTLD Presenting early after Organ Transplantation. It has been observed that PTLD presenting within the first 1–2 years after transplantation may be characterized by marked constitutional symptoms and rapid enlargement of lymphoreticular tissue. Patients with this entity may have rapidly progressive disease of a disseminated nature and a systemic sepsislike syndrome as a result of a cytokine storm. The clinical picture includes some features that are consistent with severe EBV disease [7, 8], as outlined above (e.g., hemophagocytosis and disseminated intravascular coagulation). In some patients, the diagnosis of PTLD is unfortunately made at autopsy due to difficulty in diagnosis [9, 10]. Mass lesions may not

Fig. 6.1 Exudative tonsillopharyngitis in infectious mononucleosis. Reproduced with permission, Slide Library, Hospital for Sick Children, Toronto.

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be present; pyrexia is present and the disease may be extranodal. It can be difficult to separate this entity from patients who have overwhelming sepsis and multiorgan failure. The above notwithstanding, some cases of early PTLD may be present in a less aggressive form with nodal involvement and less constitutional symptomatology. PTLD Presenting late after Organ Transplantation. PTLD that presents after the first 1–2 years after transplantation is likely to be more anatomically defined, have few systemic symptoms and is less rapidly progressive. This form of PTLD is now the form that is frequently seen in most centers, as the early-onset, rapidly progressive form is less frequently seen in recent years [11, 12]. One possible explanation is that in recent years, the enhanced surveillance for EBV after transplantation has enabled the early recognition of upregulation of EBV activity prior to the development of PTLD, allowing for early intervention, including reduction in immunosuppression. PTLD Occurring after Haematopoietic Stem Cell Transplantation. In the HSCT patients, PTLD usually affects recipients of allogeneic grafts and very rarely affects autologous recipients. Among affected patients, very few cases of PTLD occur after the first year in the absence of chronic graft versus host disease. This is due to the fact that immune restoration occurs as engraftment takes place with advancing time after HSCT. This is in contrast to the solid organ transplant recipient who requires ongoing immunosuppression to prevent organ rejection. The occurrence of PTLD at a relatively early stage after HSCT poses a challenge, with a tendency for fulminant multisystem disease in some patients. While HSCT patients, may experience the full spectrum of PTLD seen in solid organ transplantations, it occurs significantly less frequently after hematopoietic stem cell transplantation (HSCT) compared with solid organ transplantation. Among HSCT recipients, PTLD lesions are usually of donor origin in contrast to recipient origin in solid organ transplant recipients [13–16]. Sites of PTLD Lesions. The dominant presenting signs and symptoms of PTLD are related to the organs affected and the sites of PTLD lesions. Virtually no site is exempt from PTLD involvement and a high index of suspicion is required when assessing lesions in any location in the body of patients after transplantation. This is illustrated by the fact that at the author’s center, for example, PTLD has been documented at the following sites: bone, bone marrow, small bowel, large bowel, stomach, central nervous system, diaphragm, kidneys, liver, lung, lymph nodes, orbits, ovary, paraspinal tissues, salivary glands, paranasal sinuses, skin, soft palate, spleen, stomach, testes, tonsils, and uterus. Table 6.1 shows the relative frequency of the sites of PTLD involvement in children and adults based on a survey of transplant centers in Canada [17]. It shows that in the vast majority of cases, the organs of the reticuloendothelial systems were affected and the transplanted organs were often the sites of involvement. With respect to the transplanted organs, the heart is the only organ that is not usually the primary site of PTLD. Data from a recent review of PTLD cases in children over a 10-year period at The Hospital for Sick Children in Toronto revealed that single site involvement occurred in 40% of cases, while involvement of multiple sites occurred in approximately 60% of cases. Lymph node involvement occurred in 65% [18]. The gastrointestinal (GI) tract is often affected, with up to 43% of cases having such involvement in the above series [18]. Proportionately high rates of GI involvement also occur in adult patients. In a recent series involving adult renal patients, the frequency of GI involvement was comparable to graft and lymph node involvement (22, 20, and 23%,

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Table 6.1 Presenting symptoms and signs in patients with lymphoproliferative disorder Symptoms/complaints

Signs

Swollen lymph glands Weight loss Fever or night sweats Sore throat Malaise and lethargy Chronic sinus congestion and discomfort Anorexia, nausea and vomiting Abdominal pain Gastrointestinal bleeding Symptoms of bowel perforation Cough and shortness of breath Headache Focal neurologic deficits

Lymphadenopathy Hepatosplenomegaly Subcutaneous nodules Tonsillar enlargement Tonsillar inflammation Signs of bowel perforation Focal neurologic signs

respectively) [19]. The nature of GI involvement may include isolated solitary or multisite lesions or disease that is part of a more disseminated process. Easily resectable intestinal lesions that are solitary are associated with better outcomes compared with disease that is either multisite or part of a more generalized PTLD process. Patients with GI PTLD may present with a variety of gastrointestinal manifestations, including vomiting, diarrhea, evidence of protein-losing enteropathy, intussusception, bleeding, and in some cases, evidence of bowel perforation. The latter is also a known complication during the treatment phase of intestinal PTLD, during which necrosis of transmural lesions can occur. Patients with head and neck PTLD disease may present with a spectrum of findings including asymptomatic adenotonsillar hypertrophy, tonsillitis, palatal ulcerative lesions, cervical lymphadenopathy, and disease of the paranasal sinuses [20–25]. The latter has been documented to be one of the manifestations of PTLD in patients who have undergone lung transplantation [25]. Among these findings, enlarged adenoids and tonsils represent the most frequent presentation of head and neck PTLD (Fig. 6.2). In a recent series, adenotonsillar biopsies yielded PTLD in approximately 40% of children who were referred to the Otolaryngology service for assessment to rule out PTLD following initial screening by clinicians [20]. Pulmonary involvement is most frequently seen in heart and lung transplant patients. In most cases, it is characterized by solitary or multiple pulmonary nodules or an infiltrative process [10, 19, 26, 27]. In addition, there may be pulmonary dysfunction in the lung allograft. In the latter situation, clearly discernible lesions might not be apparent in the setting of diffuse consolidation on chest X-rays. Liver involvement usually occurs in liver transplant recipients where there may be evidence of diffuse hepatitis or nodular disease. Nonliver transplant recipients may also have liver involvement as a component of multisystem disease. Among renal transplant recipients, PTLD may involve the allograft or distant sites. This influences the nature of the presenting signs and symptoms. When PTLD affects the renal graft, a significant proportion of patients may present with renal dysfunction [19]. However,

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Fig. 6.2 Older child after liver transplantation. CT reveals left tonsillar nodal mass and mediastinal adenopathy. Courtesy of Dr. David Manson, Hospital for Sick Children, Toronto

when alterations in renal function occur presumably due to PTLD affecting the kidneys, other cause of renal dysfunction after transplant should be considered in the differential diagnosis. These conditions include rejection and BK virus nephropathy. Patients may also present with skin nodules. These should be differentiated from nonPTLD malignancies, including donor-derived malignancies in adult patients. Rarely, EBVassociated smooth muscle tumors have been described [28]. Central nervous system (CNS) disease is usually seen in the setting of extensive multisystem disease. However, solitary CNS disease may occur. Patients may present with evidence of intracranial pathology with headaches, seizures, and focal neurologic deficits. Generally, patients presenting with CNS PTLD tend to have poorer prognoses [11, 19, 26]. As indicated above, several other sites may be affected by PTLD. Their clinical importance may relate to the fact that their involvement may be indicative of disseminated disease, and/or may be suggestive of poorer outcomes. For example, as is the case with CNS PTLD, bone marrow is regarded as a poor prognostic indicator.

6.1.2 Clinicopathologic Correlates The histopathologic examination of suspected PTLD lesions is crucial for the diagnosis of PTLD [29–31]. A detailed description is provided in Chap. 7. The PTLD lesions that present early after transplantation are generally EBV-associated and usually fall into the categories of “early lesions” (plasmacytic hyperplasias and infectious mononucleosis-like PTLD) or polymorphic PTLD [31]. These lesions are most commonly associated with primary EBV infection posttransplantation. The so called “early lesions” are generally polyclonal, while the destructive polymorphic PTLD are frequently clonal or oligoclonal in

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nature. The spectrum of genotypic abnormalities in these lesions is limited and structural chromosomal abnormalities are rarely seen [31, 32]. The histology of PTLD lesions presenting late after transplantation is highly variable. In children and adults undergoing late-onset primary EBV infection, “early lesions” and polymorphic disease may still be observed. However, with increasing time from transplantation, a greater proportion of lesions are monomorphic and many are EBV-negative, especially in adults. These lesions may resemble non-Hodgkin lymphomas, Hodgkin lymphoma, or malignancies with plasma cell predominance. Their clinical behaviors are variable and may be different from the histologically equivalent lesions in nontransplant recipients. Monomorphic lesions are clonal proliferations, and genetic abnormalities and structural chromosomal changes are much more prevalent than in polymorphic lesions.

6.1.3 Diagnostic Evaluation Early diagnosis of PTLD is essential in order to maximize favorable outcomes. The initial diagnostic evaluation of patients with suspected PTLD is influenced by the appropriate historical information, as this relates to symptoms as well as background patient information and the physical examination findings. The diagnostic work up is guided by the presenting symptoms and signs as outlined above and in Table 6.1, taking into account the locations of suspected lessions (Table 6.2) and the differential diagnosis. Therefore, clinicians need to be aware of the conditions that must be differentiated from PTLD in order that these alternative diagnoses are not missed and are managed appropriately.

6.1.3.1 Background Information on Patients Clinical information that should be recorded includes the patient’s age, the underlying disease resulting in transplantation, the date(s) and type(s) of transplant received, and the date of onset of symptoms. It is also necessary to obtain other information that will assist in determining the risk of PTLD or guiding the subsequent management of the patient [33, 34]. This is covered in detail in Chap. 3. The donor and recipient EBV serostatus are important given the fact that the primary risk factor for PTLD is primary EBV infection [33, 34]. Pediatric patients are more likely to have primary EBV infection after transplantation, due to the fact that the majority is EBV-seronegative at transplantation compared with their adult counterparts. Additional data include the types of organ transplanted and the dose and types of immunosuppression used. Thus, as indicated in Chap. 11, the risk of PTLD varies, depending on the types of organ transplanted. In addition, patients who have received specific anti-T cell therapies are known to be at an increased risk of PTLD [34]. The types and doses of antiviral agents used and the CMV donor and recipient serostatus are relevant, given the fact that CMV infection/disease is a known risk factor [33, 34].

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Table 6.2 Sites of PTLD lesions in disease presenting with solitary site involvement and multiple site involvement – Canada Sites affected

Frequency

Percentage of patients

++++++++++++ +++++++++ +++++++++ ++++++ ++++++ +++++ ++++ ++++ +++ ++

20 15 15 10 10 8.3 6.6 6.6 5 3.3

a

Solitary site involvement Lymph node Kidney Bowel Liver Mediastinum Skin Spleen Lung Tonsil Central nervous system

Multiple site involvementb Lymph node Liver Kidney Bowel Tonsil Bone marrow Lung Mediastinum Central nervous system Skin Spleen Bone

++++++++++++++++ +++++++ +++++++ ++++++ +++++ ++++ +++ +++ +++ +++ ++ +

a The above is based on 60 cases where the sites of PTLD were reported in a Canadian study. Each + represents one patient b Multiple site involvement was defined as two or more sites. Each + is represents two patients or is rounded up to represent two patients

6.1.3.2 Initial Clinical Examination In keeping with regular clinical practice, a thorough physical examination is required to detect the manifestations of PTLD, which may be quite nonspecific (Table 6.1). The general physical examination might elicit evidence of pallor or signs referable to the site(s) of organs affected by PTLD. Given the predilection for the reticuloendothelial system to be involved, this clinical examination should include a meticulous assessment for lymphadenopathy. In selected cases, clinicians may choose to supplement clinical examinations with chest radiographs and abdominal ultrasounds as they screen for lymphadenopathy. In some major transplant centers, the clinical examination includes periodic assessment by an otolaryngologist in high risk cases, given the frequency with which the adenotonsillar tissues are involved.

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Table 6.3 Diagnostic evaluation of patients with symptoms or signs consistent with PTLD General investigations

Selective diagnostic tests

EBV-specific

CBC, WBC differential

EBV serologies (anti-EA, EBNA and VCA)

Liver functions tests

Evaluation for specific infectious agents based on clinical presentation Lumbar puncture

Renal function tests

Bone scan

Serum electrolytes, calcium

Bone marrow biopsy

Lactate dehydrogenase Uric acid

Brain CT/MRI Gastrointestinal endoscopy PET scan

Serum immunoglobulins Stools for occult blood Chest radiographs CT scan of chest/ abdomen/pelvis

EBV viral load in peripheral blood–blood compartments EBV status of lesions (PCR, in-situ hybridization) Excision or core needle biopsy of lesions for histopathology

6.1.3.3 Diagnostic and Screening Tests The diagnostic tests (Table 6.3) that are performed for PTLD can be group into four main categories. These are (1). General test; (2). Non-EBV specific tests; (3). EBV specific tests; and (4). Histopathlogy. Given the importance of early diagnosis, the development of screening tests has been the subject of research for many years. Such screening is aimed at detecting subclinical PTLD or more overt PTLD in its earliest stages. There are data to suggest that in some patients, a definite subclinical phase of PTLD exists [35]. This is based on examination of liver biopsy samples obtained prior to the diagnosis of PTLD. Examination of such samples has indicated the presence of EBV by PCR or EBV-encoded small nuclear RNA (EBER) staining in 70% of patients who went on to develop PTLD compared with 10% of those who did not develop PTLD [35]. In addition, the histopathological examination of enlarged adenoidal tissue may indicate evidence of occult PTLD in asymptomatic individuals. In order to assist in the early diagnosis of PTLD, viral load surveillance is employed in most centers. The utility of viral load testing is discussed below and further elucidated in Chap. 5. Tests are performed to rule out other diagnoses, as appropriate. This takes into account the likely differential diagnosis (Sect. 6.1.5). Specific tests are performed to establish the histologic diagnosis of PTLD and to characterize PTLD lesions, including the presence or absence of EBV in biopsy tissue. General tests are performed to determine the presence or absence of complications of PTLD or related conditions. Depending on the nature of the tests, these are performed concurrently or sequentially.

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General Tests and Non-EBV Specific Tests Blood Tests: Initial tests include a complete blood count with white blood cell differential. In some patients with PTLD, there may be evidence of anemia, which is usually normochromic, normocytic. In patients with gastrointestinal tract PTLD and occult bleeding over a prolonged period of time, there may be evidence of iron-deficiency anemia with hypochromia and microcytosis. The source of bleeding can be determined by performing additional testing, namely, examination of the stools for occult blood. The blood elements may be depressed with evidence of leucopenia, atypical lymphocytosis, and thrombocytopenia. Thrombocytopenia and neutropenia have been shown to be associated with poorer outcomes, though the precise mechanism underlying this association is unclear [11, 12]. Depending on the location of PTLD lesions, there may be evidence of disturbance in serum electrolytes, liver and renal function tests. Elevations in serum uric acid and lactate dehydrogenase may occur. Serum immunoglobulin levels may be elevated as part of an acute phase reaction. However, serum IgE levels have been observed to be elevated in some cases of PTLD [36]. Serum IgE levels may be elevated in the setting of a TH2 response profile, and such levels may function as a proxy assay for TH2 activity. PTLD is believed to occur in the setting of a TH2 response profile. However, the relationship between PTLD and serum IgE levels has been found to be inconsistent. The presence of monoclonocal or oligoclonal gammopathy has been shown to precede the detection of overt PTLD, but the specificity of this maker is poor [37]. IL6 is a B-cell growth factor that promotes the expansion of B cell populations [38]. Thus, in the setting of PTLD, it could potentially expand populations of EBV-infected B cells. There is some suggestion that the measurement of serum IL6 may be of value in PTLD [39], notably when this is combined with some measure of EBV viral burden. The relationship between serum IL6 levels and PTLD is sufficiently inconsistent to limit its utility in the evaluation of PTLD. IL10 is a major cytokine in the pathogenesis of EBV infection and disease as indicated in Chap. 4. The role of this cytokine in the pathogenesis of lymphoproliferative disorders has been studied [40–44]. It has been suggested that the usefulness of IL-10 assay for early diagnosis of PTLD is similar to that of EBV load quantification [45]. However, as in the case with IL6, the relationship between IL 10 levels and PTLD is inconsistent and neither is routinely tested for in the use evaluation of PTLD in the clinical setting. Evaluation for the presence of cytomegalovirus is usually performed in patients with suspected PTLD. Cytomegalovirus may contribute to the net state of immunosuppression, and is known to be a risk factor for PTLD. Diagnostic tests would include CMV quantitative PCR on blood as well as the examination of biopsy tissue for viral inclusions, PCR testing, and immunohistochemistry for CMV. HHV6 may also be an indirect co-factor for PTLD due to the potential for interaction with CMV [46]. Radiographic Imaging: Imaging is essential in the evaluation of PTLD. Most centers employ a total body CT scan (head to pelvis) as part of the initial assessment. Beyond this, the choice of tests depends largely on the location of suspected lesions and the historical sequence of prior recent radiographic testing. Many experts recommend that a head CT or MRI be included as part of the initial work-up. This is due to the fact that the presence of central nervous system lesions will influence treatment and such lesions may be solitary and

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a2

b

Fig. 6.3 CT (a) reveals multiple pulmonary parenchymal nodules and small mediastinal lymph nodes (b). Biopsy of the parenchymal nodules confirmed PTLD. Courtesy of Dr. David Manson, Hospital for Sick Children, Toronto

may not be associated with disease in extracranial locations. CNS lesions often tend to fail therapy and are associated with high relapse rates, based on the fact that the CNS is a site that is relatively immunologically privileged. Given the frequency of adenotonsillar involvement in PTLD, CT scanning of the neck may help to define the extent of involvement or detect subtle early changes that necessitate biopsy to rule out PTLD. Figure 6.2 shows the CT findings in a patient who was subsequently shown to have PTLD involving the adenoids. In some patients, adenotonsillar involvement is the only site of PTLD. It is likely that at least a proportion of these asymptomatic cases with adenotonsillar involvement resolve spontaneously as immunosuppression is minimized and stabilized beyond the early months after organ transplantation. Pulmonary lesions that are visible on chest radiographs may require high-resolution CT scanning for better delineation prior to biopsy (Fig. 6.3). Furthermore, CT of the chest may reveal mediastinal adenopathy and small pulmonary nodules that are not visible on the plain chest radiograph. Suspected intra-abdominal lesions may be evaluated with ultrasonography and CT scanning. This is in addition to other modalities of assessment, including, for example,

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Fig. 6.4 Abdominal US of PTLD lesions: Older child after HSCT and liver transplantation with elevated EBV titers. Multiple images show peri-pancreatic and retroperitoneal lymphadenopathy. Courtesy of Dr. David Manson, Hospital for Sick Children, Toronto

GI endoscopy in the case of intestinal hemorrhage. Figure 6.4 shows peripancreatic and retroperitoneal node involvement in a patient with PTLD. Such findings are not specific for PTLD and other causes of lymphadenopathy should be considered in the differential diagnosis. Positron emission tomography–computerized tomography (PET–CT) is emerging to be a useful test in the evaluation of PTLD [47, 48], though additional data are needed on its utility across the known heterogenous spectrum of PTLD lesions. PET is a diagnostic scanning method that directly measures metabolic, physiological, and biochemical functions of the human body. A PET scan uses a small dose of a radionuclide combined with glucose (fluoro2-deoxy-D-glucose – FDG) [49]. The radionucleotide enables glucose metabolism to be traced and it emits positrons, which are then detected by a scanner. Since certain tumors or lesions are known to grow at a faster rate compared to healthy tissue, the former cells will use up more of the glucose that is coupled with the radionuclide attached. The PET scan computer uses the measurements of glucose consumed to produce a color-coded picture. PET-CT utilizes a PET scanner with a computed tomography scanner in an integrated system, such that the CT provides accurate localization of lesions and the PET scan assists in interpretation of the suspected PTLD lesions. It has also proved to be of value in assessing the extent of remission after treatment (Figs. 6.5a, b).

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a1

a2

a3

a4

b1

b2

b3

b4

Fig. 6.5 Pretreatment PET/CT (a) reveals FDG-avid right paratracheal lymph node in a teenager after lung transplantation. Posttreatment study (b) reveals resolution of the FDG-avidity and diminution of the node. Courtesy of Dr. David Manson, Hospital for Sick Children, Toronto

Once the diagnosis of PTLD has been determined, or is highly suspected, additional diagnostic tests may be performed to assist in defining the extent of disease. These investigations include a bone scan, a bone marrow biopsy, and a lumbar puncture to assist in ruling out bone, bone marrow, and CNS disease, respectively.

EBV Specific Tests EBV serology: In immunocompetent patients, primary EBV infection can be determined by measuring IgM and IgG antibodies to EBV viral capsid antigen, antibodies to early antigen (EA), and antibodies to Epstein–Barr nuclear antigen. Persistence of anti-EA antibodies has been shown to be more likely in PTLD patients [50] and patients who are known to be

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seropositive before transplantation may have falling anti-EBNA-1 titers in the setting of elevated EBV loads and the presence of PTLD [51]. However, experience has shown that serology is unreliable as a diagnostic tool for either PTLD or primary EBV infection in immunocompromised patients. These patients show a marked delay in their humoral response to EBV antigens, and many fail to develop immunoglobulin (Ig) M antibodies altogether. Another important drawback is that these patients often receive blood or blood products with the passive transfer of antibodies that render EBV IgG antibody assays difficult to interpret. In the above context, the most important role of EBV serology in the setting of transplantation is the categorization of serostatus of donors and recipients in order to determine the likely risk of PTLD. Detection of EBV nucleic acids or protein in tissue: It has been determined that 85–90% of PTLD lesions are EBV-positive. In situ analysis of biopsy specimens by polymerase chain reaction, viral antigen [52] or (EBER) [52, 53] are of value in the diagnosis of EBVassociated PTLD. These modalities establish the presence or absence of EBV in the PTLD lesions. Polymerase chain reaction detection of EBV DNA in tissue is more useful in ruling out the presence of EBV in lesions than in indicating its presence, as it is difficult to determine if the DNA is originating in the specific tissue as opposed to being deposited in the tissue by passenger lymphocytes. Immunohistochemistry staining may indicate the presence of viral genes, such as LMP-1. In-situ hybridization for EBER labels EBV-encoded early RNA transcripts in infected cells. This is a rapid and reliable approach that is performed in most transplant centers. Viral load determination in the peripheral blood: The measurement of EBV load is addressed in detail in Chap. 5. This test was first shown to be of value in the surveillance for PTLD as a result of the work by Rocchi et al. [54], who indicated a relationship between PTLD and the number of EBV-infected cells in peripheral blood. In 1994, Riddler et al. [55] and Savoie et al. [56] independently reported that an abnormally elevated EBV viremia correlated with PTLD development. Data from the Riddler et al. study indicated that using semiquantitative polymerase chain reaction (PCR), patients with PTLD had a viral load greater than 5,000 EBV genome copies/106 PBMC [55]. Other studies confirmed this relationship between viral loads and PTLD [57–62]. Recent data highlight the need for the creation of an international reference standard for EBV vital load assay calibration as an initial important step in quality improvement [63]. These studies have advocated for the establishment of a threshold value for EBV viremia to distinguish high risk PTLD patients from those at low risk. The characteristics of this test as a diagnostic indicator of the presence of PTLD indicate that it is more useful in ruling out PTLD than in indicating its presence, in keeping with a poor positive predictive value and a high negative predictive value. Serial measurements of EBV load are more useful than single values. The addition of complementary tests might increase the overall utility of viral load in the diagnostic evaluation of PTLD. In the future, these tests might include EBV specific cytotoxic T-lymphocyte measurements with or without the integration of cytokine/chemokine or viral gene expression profiling, using quantitative realtime reverse-transcription PCR and/or microarray technology. Patients with asymptomatic sustained elevation of such loads (chronic carriers) require monitoring, as a proportion of these patients’ clinical course evolves into PTLD. It has been suggested that pediatric heart transplant recipients are more likely to develop PTLD in the setting of chronic viral load carriage than their liver counterparts [64, 65]. However, additional data from prospective studies are needed to confirm these observations. In the author’s

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experience, in HSCT patients, chronic high viral load carriage is not a frequent occurrence in the absence of chronic graft versus host disease with the resulting need for ongoing immunosuppression.

Histopathology The pathologic examination of biopsy material is the gold standard for the diagnosis of PTLD. This is discussed in detail in Chap. 7. The presence of certain features in the lesions might assist in indicating malignant transformation and prognosis. Such criteria include monoclonality, oncogene rearrangements, and presence of specific mutations. Depending on the location of lesions, particular procedures may be needed to obtain tissue for histopathologic examination to rule out non-PTLD diagnoses, establish the diagnosis of PTLD, and characterize PTLD lesions. These procedures may include transbronchial biopsies, surgical biopsies of internal organs, skin lesions, tissues or lymph nodes, CT-guided needle biopsies, and endoscopic gastrointestinal biopsies, as indicated.

6.1.4 Clinical Staging of PTLD Once the diagnosis of PTLD has been established, the location of all proven and suspected lesions should be documented. The nature of involvement of the transplanted organ should be documented as reflected in the results of the various investigations cited above. No staging system currently exists for PTLD and no single system totally captures the full spectrum of what is classified as PTLD. At the lymphoma end of the spectrum, approaches that have been used to stage nonHodgkin lymphomas have been modified and used [66]. While the Ann Arbor staging has been used with the Cotswold’s modifications, other staging approaches, such as the Murphy system, have been used in children [67]. The core components of the Ann Arbor staging are as follows:

• • • •

Stage I indicates that the lesion is located in a single region, usually one lymph node and the surrounding area. Stage I will often not have overt symptoms. Stage II indicates that the lesion is located in two separate regions, an affected lymph node or organ within the lymphatic system and a second affected area, and that both affected areas are confined to one side of the diaphragm – that is, both are above the diaphragm, or both are below the diaphragm. Stage III indicates that the lesion has spread to both sides of the diaphragm, including one organ or area near the lymph nodes or the spleen. Stage IV indicates diffuse or disseminated involvement of one or more extralymphatic organs, including any involvement of the liver, bone marrow, or nodular involvement of the lungs.

The Murphy system as used in a relatively large pediatric series from Toronto is as follows [11, 67]:

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Stage I: A single tumor (extranodal) or single anatomic area (nodal) with the exclusion of mediastinum or abdomen. Stage II: A single tumor (extranodal) with regional node involvement; or two or more nodal areas on the same side of the diaphragm; or two single (extranodal) tumors with or without regional node involvement on the same side of the diaphragm; or a primary GI tract tumor, usually in the ileocecal area, with or without involvement of associated mesenteric nodes. Stage III: Two single tumors (extranodal) on opposite sides of the diaphragm; or two or more nodal areas above and below the diaphragm; or all the primary intrathoracic tumors (mediastinal, pleural, thymic); or all extensive primary intra-abdominal disease; or all paraspinal or epidural tumors regardless of other tumors site(s). Stage IV: Any of the above with initial CNS or bone marrow involvement.

•

• •

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At the very minimum, staging should document the presence or absence of symptoms, the precise location of lesions, the involvement of the allograft, and the presence of CNS involvement. In EBV-positive PTLD, the virologic status should be categorized as reflected by the level of viral load. An increase in load from “remission levels” after therapy may be an indicator of relapse following successful initial treatment of PTLD. This may warrant serial monitoring in the follow-up to this elevation.

6.1.5 Differential Diagnoses A wide spectrum of conditions may mimick PTLD, depending on the nature of the presenting symptoms and the location of lesions. Rejection may be confused with PTLD affecting the transplanted organs [68]. This is an important consideration, given that the former requires augmentation of immunosuppression, while reduction in immunosuppression is required in the management of PTLD. The presence of nonspecific constitutional symptoms might suggest the presence of an infectious etiology. Critically ill patients with an acute fulminant presentation may be confused with those with sepsis. Such patients may need to be treated empirically for infections other than EBV, while the diagnosis of PTLD is being established. Patients presenting with pulmonary nodules might have a variety of conditions that can cause pulmonary nodules, including infections due to Mycobacteria tuberculosis, atypical mycobacteria, nocardia, actinomyces, and fungal species, among other infections. In lung transplant patients, the differential diagnosis of pulmonary lesions includes aspergillus. This deserves special mention, as in cases of pulmonary aspergillosis, careful consideration has to be given to the safety of using CT-guided needle biopsies to obtain tissue. These procedures are generally safe to do if the lesions are PTLD, but may result in life-threatening pulmonary hemorrhage if the lesions are due to aspergillus [69]. In hematopoietic stem cell transplant recipients, the differential diagnosis includes graft versus host disease, particularly when the lesions are less well circumscribed with more diffuse involvement of lung parenchyma.

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The differential diagnosis of lymphadenopathy includes the above entities as well as other conditions causing localized or generalized lymphadenopathy. Examples include, but are not limited to, infections caused by Bartonella species and Toxoplasma gondii [70–72]. Patients with gastrointestinal symptomatology, such as diarrhea, may have a variety of other diagnoses other than PTLD. This can be particularly problematic when these symptoms occur in the setting of elevated EBV viral loads. In some patients with EBV enteritis, the boundaries of separation of this entity from PTLD can be blurred. Conditions to rule out besides PTLD or EBV disease include de novo bowel lymphomas, adenoviral disease, rejection in intestinal transplant patients, graft versus host disease in HSCT patients, cytomegalovirus disease, Clostridium difficile infection, intestinal mycobacterial infection, and other infectious etiologies. Clinicians should always be reminded that non-EBV related malignancies may arise in the posttransplant period and enter into the differential diagnosis of PTLD [73, 74]. These malignancies may be classified into three categories: pre existing recipient malignancies, de novo malignancies originating in the recipient, and donor-transmitted malignancies. These entities are generally more frequently seen in adult patients compared with children. The skin represents the most frequently documented site of involvement by these non-PTLD malignancies. A detailed discussion of these is beyond the scope of this chapter. In disseminated PTLD, the extent of hemophagocytosis can be significant enough to create a syndrome that mimicks hemophagocytic lymphohistiocytosis (HLH) [8, 75, 76]. The latter is characterized by fever, splenomegaly, jaundice, and the pathologic finding of hemophagocytosis (phagocytosis by macrophages of erythrocytes, leukocytes, platelets, and their precursors) in bone marrow and other tissues. EBV infection is one of the etiologic agents that has been linked with HLH, even if the patient does not have PTLD. This gives rise to diagnostic confusion between PTLD with some elements of hemophagocytosis and HLH that is driven by EBV in the absence of PTLD. Treatment of the latter includes, but is not limited to, chemotherapy with etoposide and dexamethasone, while the former requires reduction in immunosuppression as discussed in Chap. 9.

6.1.6 Ten Take Home Pearls

• • • • •

Early detection of PTLD is important in maximizing the chances of a successful outcome. EBV load is more useful in ruling out PTLD than in indicating its presence. EBV serology is unreliable as a diagnostic tool for PTLD and primary EBV infection in immunocompromised patients. Clinicians should have a high index of suspicion for PTLD in at-risk patients including, but not limited to, those who have no pretransplant EBV immunity or have received therapies that significantly impair CTL, such as specific T-cell immune globulin. PTLD often affects the transplanted organ with the exception of the heart.

6

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Lymphoid tissue, including nodes, adenoids and tonsils, are frequently the primary sites affected by PTLD. PTLD affecting the central nervous system may present as a solitary lesion. Knowledge of the differential diagnosis is important in preventing missed diagnoses of non-PTLD diseases. Positron emission tomography – computerized tomography is emerging to be a useful test in the evaluation of PTLD. Histopathologic examination is the gold standard for the diagnosis of PTLD.

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References 1. Hanto DW, Frizzer G, Gajl-Peczalska KJ, et al. Epstein–Barr virus immunedeficiency, and B-cell proliferation. Transplantation. 1985;39:461–72 2. Nalesnik MA, Jaffe R, Starzl, et al. The pathology of posttransplant lymphoproliferative disorders occurring in the setting of cyclosporine A-prednisone immunosuppression. Am J Pathol. 1988; 133:173–92 3. Preiksaitis JK, Keay S. Diagnosis and management of posttransplant lymphoproliferative disorder in solid-organ transplant recipients. Clin Infect Dis. 2001;33(Suppl):S38–46 4. Auwaerter, PG. Infectious mononucleosis in middle age. JAMA. 1999;281:454–9 5. Grotto I, Mimouni D, Huerta M, et al. Clinical and laboratory presentation of EBV positive infectious mononucleosis in young adults. Epidemiol Infect. 2003;131:683–9 6. Tattevin P, Le Tulzo Y, Minjolle S, et al. Increasing incidence of severe Epstein–Barr virusrelated infectious mononucleosis: surveillance study. J Clin Microbiol. 2006;44:1873–4 7. Imashuku S. Systemic type Epstein–Barr virus-related lymphoproliferative diseases in children and young adults: challenges for pediatric hemato-oncologists and infectious disease specialists. Pediatr Hematol Oncol. 2007;24:563–8 8. Rouphael NG, Talati NJ, Vaughan C, et al. Infections associated with haemophagocytic syndrome. Lancet Infect Dis. 2007;7:814–22 9. Nalesnik MA, Makowa L, Starzl TE. The diagnosis and treatment of posttransplant lymphoproliferative disorders. Curr Probl Surg. 1988;25:367–472 10. Swinnen LJ, Mullen GM, Carr TJ, et al. Aggressive treatment for postcardiac transplant lymphoproliferation. Blood. 1995;86:3333–40 11. Dror Y, Greenberg M, Taylor G, et al. Lymphoproliferative disorders after organ transplantation in children. Transplantation. 1999;67:990–8 12. Green M, Webber S. Posttransplantation lymphoproliferative disorders. Pediatr Clin North Am. 2003;50:1471–91 13. Cen H, Breinig MC, Atchison RW, et al. Epstein–Barr virus transmission via donor organ in solid organ transplantation: polymerase chain reaction and restriction fragment length polymorphism analysis of IR2, IR3 and IR4. J Virol. 1991;65:976–80 14. Larson RS, Scott MA, McCurley TL, et al. Microsatellite analysis of posttransplant lymphoproliferative disorders: determination of donor/recipient origin and identification of putative lymphomagnetic mechanism. Cancer Res. 1996;56:4378–81 15. Shapiro RS, McClain K, Frizzera G, et al. Epstein–Barr virus associated B-cell lymphoprolifetaive disorders following bone marrow transplantation. Blood. 1988;71:1234–43 16. Weissman DJ, Ferry JA, Harris NL, et al. Posttransplant lymphoproliferative disorders in solid organ recipients are predominantly aggressive tumors of host origin. Am J Clin Pathol. 1995; 103:748–55

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17. Allen UD, Hébert D, Moore D, Wasfy S; The Canadian PTLD Survey Group. Survey of posttransplant lymphoproliferative disease in Canada 1988–1997. Pediatr Transplant. 2001;32:1235–7 18. Barton M, Melbourne T, Dipchand A, et al. Evolving patterns of post-transplant lymphoproliferative disorder (PTLD) in solid organ transplant recipients at a major pediatric transplant centre. Joint IDSA/ICAAC Conference; Washingston, DC. 2008. Abstract V-3562 19. Caillard S, Lelong C, Pessione F, et al. post-transplant lymphoproliferative disorders occurring after renal transplantation in adults: report of 230 cases from the French registry. Am J Transplant. 2006;6:2735–42 20. Campisi P, Allen UD, Ngan BY, et al. Utility of head and neck biopsies in the evaluation of posttransplant lymphoproliferative disorder. Otolaryngol Head Neck Surg. 2007;137:296–300 21. Herrmann BW, Sweet SC, Hayashi RJ, et al. Otolaryngological manifestations of posttransplant lymphoproliferative disorder in pediatric thoracic transplant patients. Int J Pediatr Otorhinolaryngol. 2006;70:303–10 22. Herrmann BW, Sweet SC, Molter DW. Sinonasal posttransplant lymphoproliferative disorder in pediatric lung transplant patients.Otolaryngol Head Neck Surg. 2005;133:38–41 23. Roy S, Vivero RJ, Smith LP. Adenotonsillar pathology in post-transplant patients. Int J Pediatr Otorhinolaryngol. 2008;72:865–8 24. Shapiro NL, Strocker AM. Adenotonsillar hypertrophy and Epstein–Barr virus in pediatric organ transplant recipients. Laryngoscope. 2001;111:997–1001 25. Williamson RA, Huang RY, Shapiro NL. Adenotonsillar histopathology after organ transplantation. Otolaryngol Head Neck Surg. 2001;125:231–40 26. Leblond V, Sutton L, Dorent R, et al. Lymphoproliferative disorders after organ transplantation : a report of 24 cases observed in a single institution. J Clin Oncol. 1995;13:961 27. Starzl TE, Nalesnik MA, Porter KA, et al. Reversibility of lymphomas and lymphoproliferative lesions developing under cyclosporin-steroid therapy. Lancet. 1984;1:583–7 28. Lee ES, Locker J, Naslesnickk M et al. The association of Epstein–Barr virus with smooth muscle tumors occurring after organ transplantation. N Engl J Med. 1995;332:19–25 29. Knowles DM, Cesarman E, Chadburn A, et al. Correlative morphologic and molecular genetic analysis demonstrates three distinct categories of posttransplant Iymphoproliferative disorders. Blood. 1995;85:552–65 30. Ranganathan S, Webber SA, Ahuja S, et al. Hodgkin’s-like posttransplant lymphoproliferative disorder in children: does it differ from posttransplant Hodgkin’s lymphoma? Pediatr Dev PathoI. 2004;7:348–60. 31.Swerdlow SH, Webber SA, Ferry JA, Chadburn A. post-transplant lymphoproliferative disorders. In: WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues, 4th ed. Lyon: IARC/WHO; 2008 32. Kaplan MA, Ferry JA, Harris NL, et al. Clonal analysis of posttransplant lymphoproliferative disorders, using both episomal Epstein–Barr virus and immunoglobulin genes as markers. Am J Clin Path. 1994;101:590–6. 33. Allen UD, Farkas G, Hébert D, et al. Risk factors for post-transplant lymphoproliferative disorder in pediatric patients: a case-control study. Pediatr Transplant. 2005;9:450–5 34. Walker RC, Marshall WF, Strickler JG, et al. Pretransplantation assessment of the risk of lymphoproliferative disorder. Clin Infect Dis. 1995;1346–53 35. Randhawa PS, Jaffe R, Demetris AJ, et al. Expression of Epstein–Barr virus-encoded small RNA (by the EBER-1 gene) in liver specimens from transplant recipients with post-transplantation lymphoproliferative disease. N Engl J Med. 1992;327:1710–4 36. Mathur A, Kamat DM, Filipovich AH, et al. Immunoregulatory abnormalities in patients with Epstein–Barr virus-associated B cell lymphoproliferative disorders. Transplantation. 1994;57: 1042–5 37. Badley AD, Portela DF, Patel R, et al. Development of monoclonal gammopathy precedes the development of Epstein–Barr virus-induced posttransplant lymphoproliferative disorder. Liver Transpl Surg. 1996;2(5):375–82

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38. Cohen JI. Epstein–Barr virus infection. N Engl J Med. 2000;343:481–92 39. Tosato G, Jones K, Breinig MK, et al. Interleukin-6 production in posttransplant lymphoproliferative disease. J Clin Invest. 1993;91:2806–14 40. Benjamin D, Knobloch TJ, Dayton MA. Human B-cell interleukin-10: B-cell lines derived from patients with acquired immunodeficiency syndrome and Burkitts lymphoma constitutively secrete large quantities of interleukin-10. Blood. 1992;80:1289–98 41. Birkeland SA, Hamilton-Dutoit SH, Sandvej K, et al. EBV-induced posttransplant lymphoproliferative disorder (PTLD). Transplant Proc. 1995;27:3467–72 42. Birkeland SA, Bentzen K, Moller B, et al. Interleulin-10 and posttranplant lymphoproliferative disorder after transplantation. Transplantation. 1999;67:876–81 43. Randhawa PS, Demetris AJ, Nalesnik MA. The potential role of cytokines in the pathogenesis of Epstein–Barr virus associated post-transplant lymphoproliferative disease. Leuk Lymphoma. 1994;15:383–7 44. Swinnen LJ, Fisher RI. OKT3 monoclonal antibodies induce interleukin-6 and interleukin-10: a possible cause of lymphoproliferative disorders associated with transplantation. Curr Opin Nephrol Hypertens. 1993;2:670–8 45. Muti G, Klersy C, Baldanti F, et al. Epstein-Barr virus (EBV) load and interleukin-10 in EBV-positive and EBV-negative post-transplant lymphoproliferative disorders. Br J Haematol. 2003;122:927–33 46. Humar A, Malkan G, Moussa G, et al. Human herpesvirus-6 is associated with cytomegalovirus reactivation in liver transplant recipients. J Infect Dis. 2000;181:1450–3 47. Bianchi E, Pascual M, Nicod M, et al. Clinical usefulness of FDG-PET/CT scan imaging in the management of posttransplant lymphoproliferative disease. Transplantation. 2008;85:707–12 48. McCormack L, Hany TI, Hübner M, et al. How useful is PET/CT imaging in the management of post-transplant lymphoproliferative disease after liver transplantation? Am J Transplant. 2006;6:1731–6 49. von Falck C, Maecker B, Schirg E, et al. Post transplant lymphoproliferative disease in pediatric solid organ transplant patients: a possible role for [18F]-FDG-PET(/CT) in initial staging and therapy monitoring. Eur J Radiol. 2007;63(3):427–35 50. Carpentier L, Tapiero B, Alvarez F, et al. Epstein–Barr virus (EBV) early-antigen serologic testing in conjunction with peripheral blood EBV DNA load as a marker for risk of posttransplantation lymphoproliferativ disease. J Infect Dis. 2003;188:1853–64 51. Preiksaitis JK, Diaz-Mitoma F, Mirzayans F, et al. Quantitative oropharyngeal Epstein–Barr virus shedding in renal and cardiac transplant recipients: relationship to immunosuppressive therapy, serologic responses, and the risk of posttransplant lymphoproliferative disorder. J Infect Dis. 1992;166:986–94 52. Young L, Alfieri C, Hennessy K, et al. Expression of Epstein–Barr virus transformation-associated genes in tissues of patients with EBV lymphoproliferative disease. N EngI J Med. 1989; 321: 1080–5 53. Fanaian N, Cohen C, Waldrop S, et al.; EBER. Automated in situ hybridization (ISH) vs. manual ISH and immunohistochemistry (IHC) for detection of EBV in pediatric lymphoproliferative disorders. Pediatr Dev Pathol. 2008;1 [Epub ahead of print] 54. Rocchi G, de Felici A, Ragona G, et al. Quantitative evaluation of Epstein–Barr virus-infected mononuclear peripheral blood leukocytes in infectious mononucleosis. N Engl J Med. 1977; 296:132–4 55. Riddler SA, Breinig MC, McKnight JLC. 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. 1994;84:972–8451 56. Savoie A, Perpête C, Carpentier L, et al. Direct correlation between the load of Epstein–Barr virusinfected lymphocytes in the peripheral blood of pediatric transplant patients and risk of lymphoproliferative disease. Blood. 1994;83:2715–22

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57. Allen UD, Hébert D, Tran D, et al. Utility of semiquantitative polymerase chain reaction for Epstein–Barr virus among pediatric solid organ transplant recipients with and without transplant lymphoproliferative disease. Clin Infect. 2001;33:145–50 58. Bai X, Hosler G, Rogers BB, et al. Quantitative polymerase chain reaction for human herpesvirus diagnosis and measurement of Epstein–Barr virus burden in posttransplant lymphoproliferative disorder. Clin Chem. 1997;43:1843–9 59. Kenagy DN, Schlesinger Y, Wesk K, et al. Epstein–Barr virus DNA in peripheral blood leukocytes of patients with posttransplant lymphoproliferative disease. Transplantation. 1995;60:547–54 60. Lucas KG, Burton RL, Zimmerman SE, et al. Semiquantitative, Epstein–Barr virus (EBV) polymerase chain reaction for the determination of patients at risk for EBV-induced lymphoproliferative disease after stem cell transplantation. Blood. 1998;91:3654–61 61. Nakazawa Y, Chisuwa H, Ikegami T, et al. Efficacy of quantitative analysis of Epstein–Barr virus-infected peripheral blood lymphocytes by in situ hybridization of EBER-1 after livingrelated liver transplantation: a case report. Transplantation. 1997;63:1363–6 62. Rowe DT, Qu L, Reyes J. Use of quantitative competitive PCR to measure Epstein–Barr virus genome load in the peripheral blood of pediatric transplant patients with lymphoproliferative disorders. J Clin Microbiol. 1997;35:1612–5 63. Preiksaitis JK, Pang XL, Fox JD, Fenton JM, Caliendo AM, Miller GG; American society of Transplantation Infectious Diseases Community of Practice. Interlaboratory comparison of Epstein-Barr virus load assays. AM J Transplant 2009;9:269-79 63. Kimura H, Morita M, Yabuta Y. Quantitative analysis of Epstein–Barr virus load by using a real-time PCR assay. J Clin Microbiol. 1999;37:132–6 64. Green M, Soltys K, Rowe DT, et al. Chronic high Epstein–Barr viral load carriage in pediatric liver transplant recipients. Pediatr Transplant. 2008;13:319–23 65. Bingler MA, Feingold B, Miller SA, et al. Chronic high Epstein–Barr viral load state and risk for late-onset posttransplant lymphoproliferative disease/lymphoma in children. Am J Transplant. 2008;8:442–5 66. Lister TA, Crowther D, Sutcliffe SB, et al. Report of a committee convened to discuss the evaluation and staging of patients with Hodgkin’s disease: Cotswolds meeting. J Clin Oncol. 1989;7:1630–6. 67. Murphy SB. Classification, staging and end results of treatment of childhood non-Hodgkins lymphoma: dissimilarities with lymphoma in adults. Semin Oncol. 1980;7:332–9 68. Howard TK, Klintmalm GB, Stone MJ. Lymphoproliferative disorder masquerading as rejection in liver transplant recipients – an early aggressive tumor with atypical presentation. Transplantation. 1992;53:1145–7 69. Slatore CG, Yank V, Jewell KD, et al. Bronchial-pulmonary artery fistula with fatal massive hemoptysis caused by anastomotic bronchial aspergillus infection in a lung transplant recipient. Respir Care. 2007;52:1542–5 70. American Academy of Pediatrics. Toxoplasma gondii infections. In: Pickering LK, Baker CJ, Lomg SS, McMillan Ja, editors. Red book: 2006 report of the committee on infectious diseases. 27th ed. Elk Grove Village, IL: American Academy Pediatrics; 2006. p.666–71 71. Dharnidharka VR, Richard GA, Neiberger RE, et al. Cat scratch disease and acute rejection after pediatric renal transplantation. Pediatr Transplant. 2002;6:327–31 72. Friedman AM. Evaluation and management of lymphadenopathy in children. Pediatr Rev. 2008;29:53–60 73. Penn I. De novo malignancies in pediatric organ transplant recipients. Pediatr Transplant. 1998;2:56–63 74. Penn I. Neoplastic complications of organ transplantation. In: Ginns LC, Cosimi AB, Morris PJ, editors. Transplantation. Malden, United Kingdom: Blackwell Science; 1999. p.770–86 75. Fisman DN. Hemophagocytic syndromes and infection. Emerg Infect Dis. 2000;6:601–8. 76. Imashuku S. Clinical features and treatment strategies of Epstein–Barr virus-associated hemophagocytic lymphohistiocytosis. Crit Rev Oncol Hematol. 2002;44:259–72

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Core messages

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The posttransplant lymphoproliferative disorders (PTLD) are a spectrum of lymphoid and plasmacytic proliferations occurring in the posttransplant setting that are frequently, but not always, associated with the Epstein–Barr virus (EBV). Classification of PTLD into four major categories is performed using the 2008 WHO classification. – Early type PTLD are non-destructive polymorphic lymphoplasmacytic proliferations, that are further subdivided into plasmacytic hyperplasia (PH) and infectious mononucleosis (IM) -like PTLD with some cases of florid follicular hyperplasia not easily included in either of these subcategories. – The polymorphic PTLD are the most unique type of destructive PTLD but may be difficult to distinguish in some cases from IM-like PTLD or monomorphic PTLD. They include lymphocytes of varied types, sizes and shapes as well as plasma cells, do not have a predominance of transformed cells, and do not clearly fulfill the criteria for one of the classic malignant lymphomas. – Monomorphic PTLD, originally defined as having numerous large transformed cells, resemble one of the B-cell lymphomas (mostly diffuse large B-cell lymphoma [DLBCL], one of the variants/subtypes of DLBCL or sometimes Burkitt lymphoma), a plasma cell neoplasm or one of the T/NK-cell lymphomas recognized in immunocompetent hosts. They need to be further categorized based on the type of lymphoma they most closely resemble. Small B-cell lymphomas are not considered PTLD. – Rarely posttransplant cases of classical Hodgkin lymphoma type are also recognized.

S. H. Swerdlow Division of Hematopathology, University of Pittsburgh School of Medicine, UPMC-Presbyterian, 200 Lothrop Street, Pittsburgh, PA 15213, USA e-mail: [email protected] V. R. Dharnidharka et al. (eds.), Post-Transplant Lymphoproliferative Disorders, DOI: 10.1007/978-3-642-01653-0_7, © Springer Verlag Berlin Heidelberg 2010

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7.1 Introduction 7.1.1 General Pathologic Features and Classification The posttransplant lymphoproliferative disorders (PTLD) demonstrate a spectrum of pathologic appearances that vary in terms of their degree of resemblance to other reactive and neoplastic lymphoid and plasmacytic proliferations, in terms of their cytologic composition and cell(s) of origin, and whether or not they are associated with the Epstein–Barr virus (EBV). Specifically, it should be noted that EBV positivity is not required for the diagnosis of a PTLD, that at least about 20–30% of cases are EBV negative, which is higher than that seen in the past, and that the EBV negative cases include more monomorphic PTLD and more PTLD of T cell origin [1–3]. Rare cases have been associated with HHV-8 [4]. Most, but not all, PTLD in solid organ transplant patients are of host origin, but they are of donor origin in bone marrow/stem cell transplant patients. It is important to exclude other specific (or nonspecific) lymphoid or plasmacytic proliferations prior to diagnosing PTLD, since transplant patients are also at risk for other infectious or inflammatory processes. In addition, infiltrates associated with rejection in the allograft should not be confused with PTLD. In order to deal with this wide spectrum of PTLD, a consensus classification has evolved from those originally suggested by Frizzera et al., Nalesnik et al., Knowles et al., a Society for Hematopathology slide workshop, and others [5–12 ]. The current classification of the PTLD is part of the 2008 WHO classification of hematopoietic and lymphoid tumors (Table 7.1), and was only minimally revised from the prior 2001 version [13, 14]. In brief, the early lesions show findings that could be seen in reactive proliferations in immunocompetent hosts; the polymorphic PTLD are the most unique and demonstrate heterogeneous populations of lymphocytes and plasma cells with architectural destruction of the underlying tissues, and are not easily categorized as one of the standard lymphomas that occur in immunocompetent hosts; the monomorphic PTLD resemble one of the transformed B cell lymphomas, a plasma cell neoplasm or T/NK (natural killer) cell lymphoma, and classical Hodgkin-type PTLD fulfill the same criteria as those for classical Hodgkin lymphoma in immunocompetent hosts. At the current time, the small B cell lymphomas are not considered PTLD even if occurring in transplant patients. Table 7.1 WHO classification of the posttransplant lymphoproliferative disorders (PTLD) Early lesions Plasmacytic hyperplasia Infectious mononucleosis-like PTLD Polymorphic PTLD Monomorphic PTLD (B- and T/NK-cell types)a Classical Hodgkin lymphoma type PTLD a

Further classify according to the lymphoma they resemble

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While one should try to categorize the PTLD as precisely as possible, and note if they are EBV positive or negative, there is a spectrum of changes ranging from the early lesions to polymorphic to B cell monomorphic lesions making precise classification sometimes impossible and reproducibility questionable. Furthermore, individual patients may have different types of PTLD, sometimes clonally unrelated or even with a different cell of origin, either simultaneously at the same or different sites, or subsequently. In some cases, recurrences may show evidence of progression from a less destructive or more polymorphic PTLD to one that is more lymphoma-like, including B cell or T cell monomorphic PTLD or even Hodgkin lymphoma [3, 15].

7.1.2 Differential Diagnosis Before diagnosing a PTLD, it is important to exclude the possibility of some other type of lymphoid or plasmacytic proliferation that simply happens to be occurring in a patient posttransplantation or, if the biopsy is from the allograft, exclude the possibility of rejection. Features that would favor the diagnosis of a PTLD over rejection (and over many, but not all, other types of inflammatory infiltrates) and that may be useful in difficult situations include the presence of expansile nodules or a mass lesion, numerous transformed cells, lymphoid atypia, a very B cell rich infiltrate, extensive serpiginous necrosis in the infiltrate, a high proportion of plasma cells, and finding numerous EBV+ cells. Not all of these features, however, will be present in a PTLD, and one must also be aware of cases with both rejection and a PTLD. Diagnosis, thus, of the PTLD requires a multiparameter approach that also may take into account the clinical setting. It should be noted that finding a small proportion of EBV positive cells in a lymphoid proliferation is not pathognomonic of a PTLD. Likewise, although the subject is too extensive to review here, there are transplant patients with chronically elevated peripheral blood EBV loads who never develop a PTLD, in spite of the fact that in some settings it may put them at higher risk for one [16, 17]. Following peripheral blood EBV loads has been useful in trying to recognize the earliest signs of a PTLD, so that they can be treated more successfully; however, the findings are not absolute and the best way to monitor EBV loads remains to be determined [16, 18, 19].

7.1.3 Multiparameter Approach to the Diagnosis of PTLD The diagnosis and classification of PTLD requires handling nodal or extranodal biopsies of potential cases using a standard protocol that provides for histologic sections, fresh material for flow cytometric immunophenotypic studies (if possible), and fresh/frozen material for potential genotypic studies. In some instances, sending tissue for classical cytogenetic studies may also be of interest [20, 21]. Adequate evaluation requires morphologic review, at least limited immunophenotypic studies and an EBER in situ hybridization stain to assess EBV status (immunostain for

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EBV-LMP1 is satisfactory if positive). Depending on these results and those of classical cytogenetic studies (if performed), genotypic studies (usually looking for a demonstrably clonal B cell or T cell population) and/or cytogenetic fluorescence in situ hybridization (FISH) studies (looking for one of the lymphoma associated translocations or numerical abnormalities) may be helpful in arriving at a precise diagnosis. Gene profiling studies have provided interesting new information about PTLD, but are not of diagnostic utility at the current time [22, 23].

7.2 Early Lesions The so-called early lesions are defined as lymphoid/plasmacytic proliferations in the posttransplant setting that do not efface the underlying architecture, usually do produce mass lesions, and do not have another explanation, such as a specific non-EBV-associated infectious disorder. There are two relatively well-defined types of early lesion and one more recently proposed variant that must be diagnosed cautiously given the nonspecificity of the histologic/immunophenotypic findings. Some have specifically stressed that transplant patients can have enlarged tonsils with marked follicular hyperplasia and EBV positive cells, who do not have a PTLD and never develop one [24]. In contrast to many PTLD that occur in extranodal sites, the classic early lesions are found most commonly in lymph nodes and tonsils.

7.2.1 Plasmacytic Hyperplasia (PCH) 7.2.1.1 Histopathology Lymph nodes demonstrate intact sinuses with a proliferation of predominantly small lymphocytes and plasma cells with few transformed cells1 (Fig. 7.1a, b). Caution is advised as, especially if EBV cannot be documented, the changes are totally nonspecific.

7.2.1.2 Ancillary Studies PCH are usually either nonclonal (Fig. 7.1c, d) or at best only have a very small clonal population, sometimes only documentable with EBV terminal repeat Southern blot analysis. Cytogenetic or oncogene abnormalities are not expected. 1

Transformed lymphoid cells are relatively large, usually with a round to oval nucleus with one or more nucleoli and basophilic cytoplasm as seen on a Wright-Giemsa type stain. They are like lymphocytes that have been exposed to a mitogen.

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a

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Fig. 7.1 Plasmacytic hyperplasia (PH), lymph node (a) Note there is architectural preservation with many open sinuses. (b) There are many plasma cells and some small lymphocytes, but few transformed cells. Note the open sinus at the lower right. The (c) kappa and (d) lambda immunohistochemical stains demonstrate polyclonal plasma cells. The scattered follicles are negative. (Unless otherwise noted all figures are hematoxylin and eosin stained sections)

7.2.2 Infectious Mononucleosis (IM)-Like PTLD 7.2.2.1 Histopathology Although underlying architectural features are retained, there is a polymorphous lymphoplasmacytic proliferation often in lymph nodes or tonsils/adenoids with more numerous transformed cells/immunoblasts than seen in PCH (Fig. 7.2a, b). Distinction from IM in the normal host is impossible.

7.2.2.2 Ancillary Studies Infectious mononucleosis (IM)-like PTLD are typically EBV positive and do not demonstrate phenotypic aberrancies. Some cases have small clonal or oligoclonal lymphoid populations and occasional cases are reported with simple clonal cytogenetic abnormalities [15, 21].

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Fig. 7.2 PTLD, infectious mononucleosis (IM)-type, lymph node (a) There is a more florid mostly diffuse proliferation in this lymph node that still demonstrates intact paler sinuses (arrows). (b) The proliferation is polymorphic and includes more transformed cells/immunoblasts (arrows) than in PH. The plasma cells were polyclonal

7.2.3 Florid Follicular Hyperplasia 7.2.3.1 Histopathology These cases are indistinguishable from nonspecific florid follicular hyperplasias in the normal host, but should be mass forming lesions. They are reported not to have significant expansion of interfollicular areas and few interfollicular immunoblasts/transformed cells. Distinction from totally nonspecific FH may be impossible, especially in the absence of significant numbers of EBV positive cells or other abnormalities, and there will be a gray zone between these cases and PTLD of IM type.

7.2.3.2 Ancillary Studies Immunophenotypic studies are nondiagnostic and most cases fail to demonstrate a clonal lymphoid population by any method. Occasional cases are reported to demonstrate simple clonal cytogenetic abnormalities [21].

7.3 Polymorphic PTLD Polymorphic (P) PTLD traditionally are destructive polymorphic lymphoplasmacytic proliferations that do not fulfill the criteria for a typical lymphoma as seen in immunocompetent hosts. When originally defined, ancillary studies were very limited, so P-PTLD were

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defined in terms of their polymorphism (lymphoid cells of varied size and shape and at different maturational stages) in contrast to the more uniform transformed cell proliferations that defined the monomorphic (M) PTLD. With the increasing use of more ancillary studies, many classic P-PTLD may have at least some features that do suggest a traditional lymphoma, such as easily identified clonal plasma cells in addition to B cell clones identified by molecular studies. In addition, some M-PTLD share features commonly associated with P-PTLD, such as pleomorphism or more numerous smaller lymphocytes (see below), so that there is now more of a gray zone between these two categories of PTLD.

7.3.1 Histopathology There is destruction of the underlying lymph node or other parenchymal tissue architecture by a diffuse polymorphic proliferation of lymphocytes of varying size, shape and degree of transformation plus plasma cells (Fig. 7.3a, b). The transformed cells/immunoblasts may be “atypical” and resemble classical Reed–Sternberg cells (Fig. 7.3c). The infiltrates may be angiocentric and angiodestructive and, in the lung, may resemble lymphomatoid granulomatosis. Geographic (serpiginous) areas of necrosis are seen in about one third of cases.

7.3.2 Ancillary Studies Typically, immunophenotypic studies demonstrate an admix of variably sized B cells and heterogeneous T cells (Fig. 7.3d, e). Major light chain restricted B cell populations are not expected; however, light chain restricted plasma cell populations are found in some cases that most people would still include in this category and genotypic studies will demonstrate variably sized B cell clones in virtually all cases. Bcl-6 mutations, that in part are physiologic, are reported in some polymorphic PTLD; however, abnormalities in tumor suppressor genes and oncogenes are generally not expected [7, 23, 25]. Some cases do have cytogenetic abnormalities [20, 21]. Many, but not all, cases are EBV positive (Fig. 7.3f).

7.4 Monomorphic PTLD The monomorphic PTLD are a heterogeneous group of lymphoid/plasmacytic proliferations that fulfill the criteria for one of the lymphomas or plasma cell neoplasms that are recognized in the immunocompetent host. The only exception to this definition is that, currently, cases that resemble one of the small B cell lymphomas are not included even though the occurrence of MALT lymphomas in transplant patients has been noted [26]. While classically defined as being composed predominantly of numerous transformed lymphoid cells at one maturational stage (hence monomorphic), they may show pleomorphism, have plasmacytic differentiation, be composed of sheets of light chain restricted

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a

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Fig. 7.3 PTLD, polymorphic, lymph node (a) There is diffuse architectural effacement and extensive areas of eosinophilic geographic necrosis (Nec). (b) The lymphoid cells vary in size and shape and degree of transformation. There are also admixed histiocytes and eosinophils. (c) Atypical immunoblasts are seen especially around the necrotic areas (arrows). (d) There are many CD20+ B cells including the atypical/Reed–Sternberg-like cells (arrows). (e) There are also many admixed CD3+ T cells. (f) Many of the cells are EBV+ as seen in this EBER in situ hybridization stain

plasma cells, or, in the case of monomorphic T cell PTLD, be composed of quite heterogeneous T cell populations as long as they would fulfill the criteria for a lymphoid neoplasm. They are all expected to be monoclonal and it is among this group of cases that the greatest frequency of EBV negative cases will be found.

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Fig. 7.4 PTLD, monomorphic, diffuse large B cell lymphoma type, small intestine (a) Note the angioinvasion (arrow) and foci of eosinophilic necrosis (Nec). (b) There are numerous transformed cells that marked as B cells in the vessel wall. Even here the cells are not completely monotonous

7.4.1 Monomorphic B-Cell PTLD The majority of the monomorphic PTLD resemble one of the types of diffuse large B cell lymphoma with smaller numbers resembling Burkitt lymphoma, plasma cell myeloma, or another type of plasma cell neoplasm.

7.4.1.1 Histopathology The most common monomorphic B cell PTLD are of the diffuse large B cell lymphoma (DLBCL), not otherwise specified type, and are usually composed of sheets of large transformed cells growing with an infiltrative and/or destructive pattern and sometimes showing angiocentricity (Fig. 7.4a, b). There may be pleomorphism and plasmacytic differentiation may be present in some cases. These cases do not have uniform morphologic features and some may fulfill the criteria for one of the subtypes or variants of DLBCL, such as intravascular large B cell lymphoma or plasmablastic lymphoma (Fig. 7.5) [27]. Less frequently, they are of Burkitt type and composed of sheets of intermediate sized transformed cells with amphophilic cytoplasm and a starry sky appearance due to the scattered tingible body macrophages that contain phagocytized apoptotic debris (Fig. 7.6). Other cases are composed of a sheet of plasma cells as seen in plasma cell myeloma or a plasmacytoma (Fig. 7.7a, b).

7.4.1.2 Ancillary Studies Most cases are CD20 positive. Except in cases that lack immunoglobulin expression, immunophenotypic studies should show light chain class restriction and a more detailed phenotype like that seen in the neoplasms they resemble. The DLBCL may have either a

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Fig. 7.5 PTLD, monomorphic, plasmablastic lymphoma type, sinus contents. Note the anaplastic-appearing plasmacytic cells that were CD20−, CD138+, kappa+, and EBV+

Fig. 7.6 PTLD, monomorphic, Burkitt lymphoma type, duodenum. There is a diffuse proliferation of transformed lymphoid cells and a starry sky appearance from the scattered tingible body macrophages (arrows). The cells had a typical Burkitt lymphoma phenotype (CD10+, bcl-6+, bcl-2−, Ki-67 numerous positive cells) and a MYC translocation

a

b

Fig. 7.7 PTLD, monomorphic, plasmacytoma type, small intestine. (a) Note the large mass lesion. (b) In most areas, the mass was composed of a sheet of monoclonal plasma cells

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germinal center type (CD10±, bcl-6+,IRF4/MUM1−) or nongerminal center (CD10−, bcl-6±, IRF4/MUM1+, CD138±) phenotype with the former phenotype more common among the EBV negative cases [28]. Burkitt lymphomas usually have a CD20+, CD10+, bcl-6+, bcl-2− phenotype. The plasma cell neoplasms usually lack B cell associated markers and are CD138+ with cytoplasmic light chain class restriction. EBV is present in a majority of cases, but a significant minority is negative. Genotypic studies can be used to confirm the monoclonality of these PTLD; however, they are usually unnecessary for diagnostic purposes. Among the common types of PTLD, the M-B cell PTLD are the ones most likely to demonstrate abnormalities of tumor suppressor genes (e.g., TP53), oncogenes (N-RAS), perhaps BCL-6 mutations, aberrant somatic hypermutation (e.g., of MYC or Rho/TTF), and translocations such as of MYC (a feature of Burkitt lymphomas) [7, 23, 25]. Cytogenetic abnormalities, including some (not universally agreed upon) recurrent abnormalities, are more common than in the other types of B cell rich PTLD [20, 21]. In addition to finding some recurrent abnormalities associated with conventional lymphomas, such as involving MYC, trisomy 9 and 11 have been found by some with trisomy 11 also associated with other EBV-associated neoplasms [20, 29].

7.4.2 Monomorphic T-Cell PTLD Monomorphic T cell PTLD account for only about 7–15% of PTLD in Western countries and appear to be more common in Japan [3]. They are defined as posttransplant lymphoid proliferations that fulfill the criteria for one of the T cell neoplasms recognized in the WHO classification, and hence, are often not composed of monomorphic large transformed cells [30]. The T cell PTLD most commonly resemble peripheral T cell lymphoma, not otherwise specified, with cases of hepatosplenic lymphoma another of the more common types seen. Many of the recognized peripheral T cell lymphomas have been described in the posttransplant setting including among others, T-lymphoblastic leukemia/lymphoma, T cell large granular lymphocyte leukemia, adult T cell leukemia/lymphoma, mycosis fungoides/Sézary syndrome, and cutaneous and other anaplastic large cell lymphomas (ALK+ and ALK−) [3]. Occasional lymphomas of NK cells, including extranodal NK/T cell lymphoma, nasal type, are also reported. Only about one third are EBV positive.

7.4.2.1 Histopathology The histopathology for the T cell PTLD is very variable but should be the same as for the varied T cell lymphomas seen in immunocompetent hosts. These cases may be confused with polymorphic PTLD or other reactive proliferations because many T cell lymphomas can appear heterogeneous and can include many admixed reactive elements. In general, some features to look for, in addition to a destructive growth pattern, would include prominent cytologically atypical lymphoid cells, or in some cases, very numerous transformed lymphoid cells (like in other monomorphic PTLD) (Fig. 7.8a). A discussion of the histopathology of T cell lymphomas is beyond the scope of this chapter [30].

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Fig. 7.8 PTLD, monomorphic, peripheral T cell, not otherwise specified type (cytotoxic), bone marrow. (a) There is a patchy interstitial infiltrate composed of very large cells with irregular nuclear contours (arrows) in the marrow biopsy. (b) The very abnormal cells are more easily seen in this CD3 immunohistochemical stain that identifies T cells (and natural killer cells). A clonal T cell receptor beta chain rearrangement was documented by Southern blot analysis. EBV was not detected

7.4.2.2 Ancillary Studies Ancillary studies are critical in the diagnosis of the T cell PTLD. Immunophenotypic studies are useful to exclude findings that would suggest one of the other types of PTLD (e.g., abnormal B cells), to document that the cells of concern mark as T cells (pan-T cell and T cell subset marker expression) or NK cells (surface CD3−, CD5−, CD56+), and, in some cases, to demonstrate a population with an aberrant T cell phenotype (e.g., “loss” of one or more pan-T cell markers) or expansion of a T cell subset that is either usually not present in large numbers or is present on cells that clearly appear neoplastic based on their cytologic features and the way they are growing. For example, finding that an intrasinusoidal neoplastic T cell infiltrate in the liver lacks T cell receptor beta chain expression and is positive with TIA-1, but not granzyme B helps make the diagnosis of a hepatosplenic T cell lymphoma type T-PTLD (Fig. 7.8b). Molecular studies (PCR or Southern blot analyses looking for clonal T cell receptor gene rearrangements) are also very important to help identify the presence of a clonal T cell population remembering that clonal populations can be seen in the setting of IM [31] and that NK neoplasms will be negative.

7.5 Classical Hodgkin Lymphoma Type PTLD Classical Hodgkin lymphoma type PTLD are strictly defined and must fulfil the criteria for classical Hodgkin lymphoma. Caution is advised as cells resembling Reed–Sternberg cells are seen in many different types of PTLD, most of which are not of Hodgkin type. The so

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Fig. 7.9 PTLD, classical mixed cellularity Hodgkin lymphoma type, lymph node. There are Reed– Sternberg cells (arrow) in a sea of small lymphocytes and some plasma cells. The Reed–Sternberg cells were CD20−, CD15+ and CD30+

called “Hodgkin-like” PTLD are no longer grouped with the classical Hodgkin type PTLD and are to be categorized in whatever other PTLD category they best fit in [32].

7.5.1 Histopathology Posttransplant classical Hodgkin lymphoma, as in immunocompetent hosts, most typically shows at least partial architectural effacement by a proliferation of variable numbers of small lymphocytes, plasma cells, eosinophils, and histiocytes, together with diagnostic Reed–Sternberg cells and Reed–Sternberg variants (Fig. 7.9). Most cases in the transplant setting fulfill the criteria for the mixed cellularity type. Whether posttransplant Hodgkin lymphoma has any unique features is difficult to assess given the difficulty sometimes in distinguishing it from other PTLD with Hodgkin-like features.

7.5.2 Ancillary Studies Immunohistochemical studies are critical, particularly given the resemblance of many other types of PTLD to classical Hodgkin lymphoma. In the most definite cases, the Reed– Sternberg cells are CD15+, CD30+, CD20−, and CD45− with the majority of the surrounding small lymphocytes of T cell type. It is expected that, as in immunocompetent hosts, the Reed–Sternberg cells are also either Oct-2 (POU2F2) and/or BOB.1 (Pou2a1) negative, a feature that may be helpful in the presence of some CD20 staining or an absence of CD15 expression. The neoplastic cells should also be weakly positive for PAX5 in most cases and positive for IRF4/MUM1. Almost all reported cases have been EBV+. Genotypic studies may demonstrate clonal B cells in some cases.

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7.6 Take Home Pearls

• • • • •

The PTLD include many different types of lymphoid and plasmacytic proliferations that are often, but not always, associated with the EBV. Some cases may be driven by other forms of chronic antigenic stimulation. Classification of the PTLD is important and is best accomplished by working up potential cases as one would work up a potential lymphoma. Categorization requires knowledge both about the WHO criteria for the PTLD and for lymphomas in general. In spite of one’s best efforts, some cases may be difficult to categorize because there are gray zones between the different types of PTLD, particularly those of early, polymorphic, and monomorphic B cell type. There is no sharp border between a PTLD and an overt lymphoma in the posttransplant setting. It is always important to exclude the possibility of some other type of lymphoplasmacytic proliferation including rejection prior to making the diagnosis of a PTLD. In rare cases, there may be both rejection and a PTLD.

References 1. Leblond V, Davi F, Charlotte F, Dorent R, Bitker MO, Sutton L, Gandjbakhch I, Binet JL, Raphael M. Posttransplant lymphoproliferative disorders not associated with Epstein–Barr virus: a distinct entity? J Clin Oncol. 1998;16:2052–9 2. Nelson BP, Nalesnik MA, Bahler DW, Locker J, Fung JJ, Swerdlow SH. Epstein–Barr virusnegative post-transplant lymphoproliferative disorders – a distinct entity? Am J Surg Pathol. 2000;24:375–385 3. Swerdlow SH. T-cell and NK-cell posttransplantation lymphoproliferative disorders. Am J Clin Pathol. 2007;127:887–95 4. Kapelushnik J, Ariad S, Benharroch D, Landau D, Moser A, Delsol G, Brousset P. Post renal transplantation human herpesvirus 8-associated lymphoproliferative disorder and Kaposi’s sarcoma. Br J Haematol. 2001;113:425–8 5. Frizzera G, Hanto DW, Gajl-Peczalska KJ, Rosai J, McKenna RW, Sibley RK, Holahan KP, Lindquist LL. Polymorphic diffuse B-cell hyperplasias and lymphomas in renal transplant recipients. Cancer Res. 1981;41:4262–79 6. Harris NL, Ferry JA, Swerdlow SH. Posttransplant lymphoproliferative disorders: summary of society for hematopathology workshop. Semin Diagn Pathol. 1997;14:8–14 7. Knowles DM, Cesarman E, Chadburn A, Frizzera G, Chen J, Rose EA, Michler RE. Correlative morphologic and molecular genetic analysis demonstrates three distinct categories of posttransplantation lymphoproliferative disorders. Blood. 1995;85:552–65 8. Locker J, Nalesnik M. Molecular genetic analysis of lymphoid tumors arising after organ transplantation. Am J Pathol. 1989;135:977–87 9. Nalesnik MA, Jaffe R, Starzl TE, Demetris AJ, Porter K, Burnham JA, Makowka L, Ho M, Locker J. The pathology of posttransplant lymphoproliferative disorders occurring in the setting of cyclosporine A-prednisone immunosuppression. Am J Pathol. 1988;133:173–92 10. Shapiro RS, McClain K, Frizzera G, Gajl-Peczalska KJ, Kersey JH, Blazar BR, Arthur DC, Patton DF, Greenberg JS, Burke B, et al. Epstein–Barr virus associated B cell lymphoproliferative disorders following bone marrow transplantation. Blood. 1988;71:1234–43

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11. Swerdlow SH. Classification of the posttransplant lymphoproliferative disorders: from the past to the present. Semin Diagn Pathol. 1997;14:2–7 12. Swerdlow SH. Posttransplant lymphoproliferative disorders: a working classification. Curr Diagn Pathol. 1997;4:29–36 13. Harris NL, Swerdlow SH, Frizzera G, Knowles DM. Post-transplant lymphoproliferative disorders. In: Jaffe ES, Harris NL, Stein H Vardiman JW, editors. Pathology and genetics of tumours of haematopoietic and lymphoid tissues. Lyon: IARC ; 2001. p. 264–9 14. Swerdlow SH, Webber SA, Chadburn A, Ferry JA. Post-transplant lymphoproliferative disorders. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J and Vardiman JW, editors. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC; 2008 15. Wu TT, Swerdlow SH, Locker J, Bahler D, Randhawa P, Yunis EJ, Dickman PS, Nalesnik MA. Recurrent Epstein–Barr virus-associated lesions in organ transplant recipients. Hum Pathol. 1996;27:157–64 16. Bingler MA, Feingold B, Miller SA, Quivers E, Michaels MG, Green M, Wadowsky RM, Rowe DT, Webber SA. Chronic high Epstein–Barr viral load state and risk for late-onset posttransplant lymphoproliferative disease/lymphoma in children. Am J Transplant. 2008;8:442–5 17. Green M, Soltys K, Rowe DT, Webber SA, Mazareigos G. Chronic high Epstein–Barr viral load carriage in pediatric liver transplant recipients. Pediatr Transplant. 2009;13:319–23 18. Rowe DT, Webber S, Schauer EM, Reyes J, Green M. Epstein–Barr virus load monitoring: its role in the prevention and management of post-transplant lymphoproliferative disease. Transpl Infect Dis. 2001;3:79–87 19. Tsai DE, Douglas L, Andreadis C, Vogl DT, Arnoldi S, Kotloff R, Svoboda J, Bloom RD, Olthoff KM, Brozena SC, Schuster SJ, Stadtmauer EA, Robertson ES, Wasik MA, Ahya VN. EBV PCR in the diagnosis and monitoring of posttransplant lymphoproliferative disorder: results of a two-arm prospective trial. Am J Transplant. 2008;8:1016–24 20. Djokic M, Le Beau MM, Swinnen LJ, Smith SM, Rubin CM, Anastasi J, Carlson KM. Posttransplant lymphoproliferative disorder subtypes correlate with different recurring chromosomal abnormalities. Genes Chromosomes Cancer. 2006;45:313–8 21. Vakiani E, Nandula SV, Subramaniyam S, Keller CE, Alobeid B, Murty VV, Bhagat G. 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. 2007;38:315–25 22. Craig FE, Johnson LR, Harvey SA, Nalesnik MA, Luo JH, Bhattacharya SD, Swerdlow SH. Gene expression profiling of Epstein–Barr virus-positive and -negative monomorphic B-cell posttransplant lymphoproliferative disorders. Diagn Mol Pathol. 2007;16:158–168 23. Vakiani E, Basso K, Klein U, Mansukhani MM, Narayan G, Smith PM, Murty VV, Dalla-Favera R, Pasqualucci L, Bhagat G. Genetic and phenotypic analysis of B-cell post-transplant lymphoproliferative disorders provides insights into disease biology. Hematol Oncol. 2008; 26:199–211 24. Meru N, Davison S, Whitehead L, Jung A, Mutimer D, Rooney N, Kelly D, Niedobitek G. Epstein–Barr virus infection in paediatric liver transplant recipients: detection of the virus in post-transplant tonsillectomy specimens. Mol Pathol. 2001;54:264–9 25. Cesarman E, Chadburn A, Liu YF, Migliazza A, Dalla-Favera R, Knowles DM. BCL-6 gene mutations in posttransplantation lymphoproliferative disorders predict response to therapy and clinical outcome. Blood. 1998;92:2294–302 26. Hsi ED, Singleton TP, Swinnen L, Dunphy CH, Alkan S. Mucosa-associated lymphoid tissuetype lymphomas occurring in post-transplantation patients. Am J Surg Pathol. 2000;24:100–6 27. Borenstein J, Pezzella F, Gatter KC. Plasmablastic lymphomas may occur as post-transplant lymphoproliferative disorders. Histopathology. 2007;51:774–7 28. Johnson LR, Nalesnik MA, Swerdlow SH. Impact of Epstein–Barr virus in monomorphic B-cell posttransplant lymphoproliferative disorders: a histogenetic study. Am J Surg Pathol. 2006;30:1604–12

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29. Chan WY, Chan AB, Liu AY, Chow JH, Ng EK, Chung SS. Chromosome 11 copy number gains and Epstein–Barr virus-associated malignancies. Diagn Mol Pathol. 2001;10:223–7 30. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: IARC; 2008 31. Callan MF, Steven N, Krausa P, Wilson JD, Moss PA, Gillespie GM, Bell JI, Rickinson AB, McMichael AJ. Large clonal expansions of CD8 + T cells in acute infectious mononucleosis. Nat Med. 1996;2:906–11 32. Ranganathan S, Webber S, Ahuja S, Jaffe R. Hodgkin-like posttransplant lymphoproliferative disorder in children: does it differ from posttransplant Hodgkin lymphoma? Pediatr Dev Pathol. 2004;7:348–60.

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Core Messages

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No therapies are based on data from randomized clinical trials Current outcomes are suboptimal, both in adults and children Death is most common in the first year after diagnosis Success of therapy must include evaluation of allograft outcomes Several new therapies have emerged over the last decade including monoclonal antibody therapies against B cell surface antigens and cellular immunotherapy

9.1 Introduction Despite a growing understanding of the pathophysiology of posttransplant lymphoproliferative disorders (PTLD), its optimal management remains controversial. There is an increasing armamentarium of treatments available to the clinician, but with little evidence base to define how and when to use these treatments, or how best to combine therapies. Of note, no randomized trial of any form of therapy for PTLD has been performed. In the first part of this review, we summarize the rationale and evidence to support individual treatments. Following this, we discuss combinations of therapies and how they might be best applied as first- or second-line therapies. It should be noted that the choice of therapy is often arbitrary (e.g., institutional preference), but may also be driven by predictive factors (real or perceived), such as pathological findings, age at onset, organ transplanted, disease stage, presence or absence of EBV, comorbid conditions, and prior rejection history. This chapter focuses on

S. A. Webber Department of Pediatrics, Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA e-mail: [email protected] V. R. Dharnidharka et al. (eds.), Post-Transplant Lymphoproliferative Disorders, DOI: 10.1007/978-3-642-01653-0_9, © Springer Verlag Berlin Heidelberg 2010

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treatment of PTLD after solid organ transplantation. Treatment of PTLD within the context of hematopoietic stem cell transplantation is dealt with in detail in Chap. 11d.

9.2 Optimal Therapy for PTLD The optimal treatment regimen for PTLD is one that rapidly eradicates the disease, does not increase the risk of allograft rejection (acute or chronic), and which is simple to give, cost-effective, and is associated with minimal adverse events. It is apparent that no single treatment fulfills all these criteria. Furthermore, the treatment must be geared to the individual patient, since the appropriate treatment, for example, for severe PTLD in a lung recipient early after transplant with adverse rejection profile is likely to be very different from that of a renal recipient with benign rejection history late out from transplant. These considerations underscore the enormous challenges involved in designing clinical trials for a rare disease with such clinical heterogeneity. Clinical response is also likely to depend on intrinsic characteristics of the tumor, such as rate of mitosis, presence of oncogenes, presence or absence of EBV, and the ability to be controlled by reconstitution of T cell immune surveillance. Unfortunately, it is currently very hard to predict tumor behavior, even after extensive pathological evaluation is completed. In the nontransplant setting, behavior of similar diffuse large B cell lymphomas (DLBCL) can be partly predicted based on analysis of gene expression profiles of the tumor [1]. Similar studies are underway in PTLD, and might, in time, provide important prognostic information for the clinician.

9.3 Reduction of Immune Suppression In 1984, Starzl et al. reported the reversibility of PTLD by reduction in immunosuppression in cyclosporine treated patients [2]. This strategy remains the initial mainstay of therapy for most patients with polymorphic disease across all age groups. It is also used as first-line therapy in monomorphic disease by some groups (especially in children), though more often nowadays, adjunctive therapies, such as anti-B cell monoclonal antibodies, are being used from the time of diagnosis for this histology. The goal of reduction (or cessation) of immunosuppression is to allow the host to recover natural immune surveillance and subsequently gain control over the proliferation of EBV-infected B cells. Restitution of immune surveillance may also be associated with resolution of EBV negative PTLD, though this seems to occur with lower frequency [3]. The majority of PTLD in children (especially polymorphic lesions) will respond to reduction in immunosuppression, though with significant rates of rebound, acute cellular rejection which may vary by organ [4, 5]. The reported response rates of PTLD to reduction of immunosuppression among adults are highly variable, with excellent results reported by some groups [6] and very poor results by others [7, 8]. These diverse outcomes may reflect differing referral patterns and patient

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characteristics, differences in range of pathology (e.g., proportion with polymorphic vs. monomorphic disease), and perhaps differences in use of adjunctive therapies (such as antiviral agents) that are not always well described. As for children, rebound rejection rates in adults are significant and are an important cause of death after treatment of PTLD by reduced immunosuppression, especially after heart transplantation [7]. In general, most patients show evidence of clinical response within 2–4 weeks of reduction of immune suppression, though a delayed response has been observed as long as several months in some patients. Complete responses to reduced immunosuppression in DLBCL [3, 8], posttransplant Hodgkin disease and other rarer PTLDs (e.g., T cell lymphoma) are less likely, and if they do occur, are often incomplete or not durable. There is, therefore, increased reluctance to use reduction in immunosuppression as sole initial therapy for these histologies. The approach to reduction in immunosuppression varies widely. Most authorities initially hold calcineurin inhibitors (CNI’s) and adjunctive antiproliferative agents, and maintain any corticosteroids that the patient might be receiving. Tacrolimus and cyclosporine levels may initially be high due to impaired hepatic metabolism. Subsequent practice varies widely. Among liver transplant programs, there is a tendency to withhold CNI’s long-term or even indefinitely [4]. This is unlikely to be possible in thoracic and intestinal transplantation. There has been considerable interest in replacing CNI’s with inhibitors of the mammalian target of rapamycin (mTOR) once immunosuppression is reintroduced. Sirolimus has frequently been used in this setting. Interest in the use of this group of agents in PTLD, in part, reflects the anti-neoplastic properties of this class of agents, including inhibition of EBVdriven B cell lymphomas [9, 10]. However, mTOR inhibitors are also potent T cell inhibitors, so will impair T cell immune surveillance. In the clinical setting, it is hard to know whether the use of mTOR inhibitors is beneficial because they may suffice to prevent graft rejection (without the need for reintroduction of CNI’s), or whether these agents are actually exhibiting anti-PTLD properties in vivo. Of note, patients managed with CNI free immunosuppression, using mTOR inhibitors, may still develop PTLD [11].

9.4 Surgery and Radiation Therapy Excisional biopsy, generally performed for diagnostic purposes, may be curative for solitary PTLD lesions, but is usually combined with some reduction in immunosuppression. Thus, almost all patients do receive a systemic approach to treatment and PTLD is probably best thought of as a systemic process. Surgery may also be indicated for management of local complications, such as gastrointestinal hemorrhage, obstruction, or perforation. Radiation therapy has a limited role in the management of PTLD, though many lesions will be responsive. It has been used when rapid local responses are required, for example, when there is acute airway compression from tumor mass, or compression of other critical structures. It may also have a role in the treatment of some cases of central nervous system (CNS) PTLD (see below).

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9.5 Antiviral Therapy Initial interest in the role of antiviral chemotherapy for treatment of PTLD arose in 1982 when Hanto described a patient whose EBV-associated PTLD lesion appeared to wax and wane in association with starting and stopping acyclovir [12]. Both acyclovir and ganciclovir inhibit lytic EBV DNA replication in vitro and may be of value in treating the lytic phase of EBV infection (see Chap. 10). Ganciclovir is approximately 6–10 fold more potent than acyclovir at inhibiting lytic EBV replication in vitro, and has the additional advantage of inhibiting CMV that may be present as a co-pathogen in some cases of PTLD. Use of acyclovir or ganciclovir (or valganciclovir) for the treatment of PTLD has become routine in many centers. However, their efficacy has not been established in prospective, comparative clinical trials, and many investigators have questioned their role in the treatment of PTLD. The vast majority of EBV-infected cells within PTLD lesions have been shown to be transformed B cells that are not undergoing lytic infection. Neither acyclovir nor ganciclovir suppress EBV-driven proliferation of B cells in vitro, nor are they active against B cells that are latently infected with EBV. Furthermore, EBV viral loads in the peripheral blood can climb to very high levels, and PTLD may develop while patients are receiving acyclovir or ganciclovir as CMV or EBV prophylaxis. Recently, there has been progress in the development of other types of antiviral agents for the treatment of severe human herpes virus infections, including those due to EBV [13, 14]. Drugs that act independently of the viral enzyme target, thymidine kinase, may be particularly suitable candidates for investigation for the treatment of PTLD. Cidofovir has potential in this regard, but has important toxicities. Recent work suggests that lipid ester analogues of cidofovir and cyclic cidofovir may have much greater activity against EBV than the parent drug and may be suitable drugs for phase I clinical studies [14]. Another antiviral strategy is to induce EBV thymidine kinase in EBV infected tumors, thereby making the tumors sensitive to nucleoside-type antiviral agents, such as ganciclovir. One agent that may achieve this goal is arginine butyrate. In a phase I/II clinical trial in 15 adults with refractory EBV positive lymphoid tumors, continuous infusion of arginine butyrate along with standard doses of ganciclovir led to significant antitumor responses in two-thirds of patients [15]. More recently, it has been shown that short, discontinuous exposure to arginine butyrate (in contrast to continuous infusion) may suffice to initiate lytic phase EBV gene expression and thymidine kinase induction [16]. This therapy holds promise for patients with EBV-driven PTLD.

9.6 Interferon and Other Cytokines The use of interferon has been described in anecdotal reports as a therapeutic option in the management of PTLD [17, 18]. Interferon is both a proinflammatory cytokine and a natural antiviral agent, and appears capable of controlling proliferation of EBV-infected B

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cells. Since it is a nonspecific immune stimulant, antidonor responses are often seen and severe rejection can develop during therapy. In a recent series of adult PTLD patients unresponsive to reduction in immunosuppression, only 1 of 13 (7%) achieved durable complete remission with interferon-a 2b [8]. At the present time, most centers are not routinely using interferon in the management of PTLD. Interleukin 6 (IL6) has been described as a growth factor for EBV-infected B cells. For this reason, an anti-IL6 monoclonal antibody had been tested in a phase I/II clinical trial [19]. This was well tolerated and complete response was observed in approximately 40% of patients who had not responded to a brief period of reduction in immunosuppression. It is not currently used in routine clinical care. As the biology of EBV-PTLD is further unraveled (see Chap. 4), more targets for biologic intervention are likely to be identified.

9.7 Intravenous Immune Globulin A potential role for the use of intravenous immune globulin (IVIG) for the treatment of PTLD has also been suggested. Several reports have documented an association between loss, or absence of antibody against at least one of the Epstein–Barr nuclear antigens (EBNA) in EBV infected organ recipients and the subsequent development of PTLD [20]. In addition, a correlation between an increasing level of anti-EBNA antibodies (including those introduced through transfusions) with a decrease in EBV viral load has been demonstrated. Taken together, these reports may provide a rationale for considering the use of antibodies in the prevention and/or treatment of EBV disease and PTLD, even though the primary mechanism for controlling EBV infection appears to be cytotoxic T cell mediated immunity. IVIG has been used alone and in combination with interferon-a as treatment for PTLD [18]. Both IVIG and CMV-IVIG have been used in the treatment of some patients with PTLD. As with the use of antiviral agents and interferon, there are no comparative trials evaluating the role of IVIG in general, or CMV-IVIG in particular, in the treatment of PTLD.

9.8 Anti-B Cell Antibodies Most PTLD are of B cell origin. The use of anti-CD21 and anti-CD24 monoclonal antibodies has been reported for the treatment of PTLD in recipients of solid-organ and bone marrow transplantation (BMT) [21]. Therapy was most promising for oligoclonal, but not monoclonal disease. These two products are no longer available. However, an anti-CD20 human/mouse chimeric monoclonal antibody (rituximab; Genentech Inc. & IDEC pharmaceuticals) is currently commercially available for treatment of certain CD20-positive B cell non-Hodgkin lymphomas in adult nontransplant recipients. Most PTLD do express CD20, so trials of rituximab in PTLD were logical. In 2000, clinical investigators in France

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published a retrospective analysis of the use of rituximab in 32 patients with PTLD [22]. The overall response rate was 65% in solid organ transplant recipients, most of whom experienced long-term remission. However, relapse of PTLD developed in approximately 20% of responders a median of 7 months after completing their therapeutic course of rituximab. Several subsequent reports have emerged from single centers [3, 23–25] and from multicenter phase II trials [26, 27]. Overall response rates for adults vary significantly, with complete response rates ranging from 28 to 59% when used as second-line therapy after failed reduction of immunosuppression [23, 26, 27]. However, the recent late followup of the largest series to date (60 patients) has revealed that 57% had progressive disease 1 year after completing treatment with a median progression free survival of only 6 months [28]. Despite the suboptimal results of rituximab in adults with PTLD, it has been pointed out that the drug is well tolerated, that a significant number will achieve durable complete responses without the need for chemotherapy, and those that do demonstrate progressive disease or relapse can still undergo chemotherapy [3]. Interestingly, rituximab may also be effective in some patients who have refractory or relapsed disease and previously been treated with chemotherapy[29]. The results of rituximab may be superior in children, though less data are available. In a recently completed small prospective multicenter phase II trial, 80% of children with refractory PTLD achieved complete response after 4 or 8 doses, but with relapse in 25% [30] (Fig. 9.1). There are several important questions regarding the use of rituximab in the transplant setting. Will the prolonged elimination of B cells result in additional opportunistic infections or other sequelae? Should rituximab be used in all patients at diagnosis, or only those who fail an initial period of reduced immunosuppression? If relapse occurs after the use of rituximab, how should management proceed? Is maintenance therapy every 6 months justified? Is the high relapse rate with rituximab due to its use primarily in high risk patients (i.e., those with refractory disease), or could therapy per se somehow be associated with subsequence risk of relapse? Are clinicians less aggressive in immunosuppression reduction when they use rituximab from the time of diagnosis? It seems likely that durable remission cannot be achieved unless restitution of EBV-CTL responses is achieved [31]. Finally, it should be noted that there has been very little experience with newer radioconjugates of anti-B cell antibodies (so-called radioimmunotherapy). Do these agents offer

Fig. 9.1 Refractory polymorphic PTLD early after bilateral lung transplantation. Disease progression occurred during reduction of immunosuppression (a–b). A complete response was achieved with two courses (8 doses) of rituximab (c)

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any advantage over rituximab and are they safe? Hopefully, ongoing and future studies will answer some of these important questions.

9.9 Chemotherapy Chemotherapy is usually used as first-line treatment for aggressive posttransplant lymphomas, such as Burkitt disease (Fig. 9.2) or T cell lymphomas, and also for most cases of posttransplant Hodgkin disease. As discussed earlier, it is unclear whether patients with monomorphic disease with histology of DLBCL should receive a trial of reduction in immunosuppression with, or without, rituximab, or whether they should receive chemotherapy as primary treatment. This is one of the most controversial areas in the current management of PTLD. Pediatric patients with refractory polymorphic PTLD have quite high initial response rates to “low-dose” chemotherapy with cyclophosphamide and prednisone, though 2 year event-free survival is suboptimal at around 58% [32]. The addition of rituximab to this regimen is currently under investigation in children [33]. It is unclear whether chemotherapy has a role before a trial of rituximab has been completed, since rituximab alone has given encouraging results in pediatric refractory disease [33]. Low-dose chemotherapy has not been compared directly to rituximab in children failing to respond to reduced immunosuppression. Chemotherapy should be given to children with progressive disease on rituximab, and is also indicated for the rare cases of active PTLD with concomitant allograft rejection. Burkitt disease in children responds well to aggressive multidrug chemotherapy regimens, similar to those used in the nontransplant setting (Fig. 9.2). The indications for chemotherapy in adults are broadly similar to those in children. Centers are divided on whether DLBCL (the most common pathology in adults) should be treated primarily with chemotherapy, or only after failure to respond to reduced

Fig. 9.2 Burkitt lymphoma in a pediatric heart transplant recipient. There was diffuse disease throughout the abdomen (a), as well as disease in the jaw and orbit. There was almost complete resolution of disease after two courses of chemotherapy (b). Sustained complete response was achieved

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immunosuppression and rituximab. Chemotherapy will generally protect the graft against rejection, but is associated with higher infectious morbidity and mortality than comparable regimes used in the nontransplant setting [3]. Multiple different chemotherapeutic regimens have been used including “CHOP,” “ACVBP,” and “ProMACE-CytaBOM” [3, 8, 34–37]. None have been directly compared to each other in controlled trials. Overall response rates are of the order of 65–75%, though late mortality appears significant [38].

9.10 Cellular Immunotherapy Inadequate EBV-specific T cell responses are an important, if not critical, pathologic step in the development of EBV-driven PTLD, though other mechanisms may contribute. Infusion of EBV-specific CTLs has been employed both as treatment and prophylaxis against PTLD in bone marrow/stem-cell transplantation [39]. In this setting, the PTLD are generally of donor origin, and the donor is usually available to provide a source of CTL for the recipient. The success of this adoptive immunotherapy in stem cell recipients has stimulated investigation into applying this approach as a therapy for PTLD in solid organ transplant recipients [40]. This is a logical therapy as it is directed against the PTLD, and should (in theory) cause little antidonor response (i.e., rejection). However, the use of CTL infusions in solid organ recipients is made more difficult by the fact that the EBV-infected cells within PTLD lesions are typically of recipient origin. Therefore, EBV-specific CTLs should ideally be HLA haplo-identical. For this reason, autologous CTLs are the obvious source for development of EBV-specific CTL infusions for solid organ transplant recipients. However, the development of EBV-specific CTLs from organ recipients is challenging since most patients at risk for EBV-PTLD are EBV-naïve at transplant, CTL generation is impaired by the presence of immunosuppression, and T cell responses are further severely suppressed at the time of development of PTLD. Accordingly, techniques for adoptive immunotherapy of EBV-associated PTLD in solid organ transplant recipients have focused on developing strategies to immunize and stimulate the organ recipient’s own T cells against EBV ex vivo and then subsequently infusing these EBV-specific CTLs back into the recipient at a time when the patient develops refractory EBV infection/PTLD [40–42]. Such an approach could also be used for prevention, with infusion performed when EBV viral loads start to climb after primary posttransplant EBV infection [42]. Ideally, the cells for culture should be obtained prior to transplantation and the initiation of immunosuppression in high risk recipients (EBV seronegative at transplantation). Such an approach is expensive and labor intensive as most candidates will never require CTL therapy. Furthermore, at this time, the success of adoptive immunotherapy using autologous CTL after solid organ transplantation has not paralleled that seen after bone marrow/stem cell transplantation. An alternate approach that has met some success is the use of allogeneic T cell infusions from EBV-positive blood donors that are as closely matched as possible to the recipient’s HLA type. Success in about 50% of patients was observed in a phase II clinical trial using this approach [43]. While the use of adoptive immunotherapy represents an exciting advance in the

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management of PTLD after solid organ transplantation, it remains a research tool at this time, limited to a very small number of solid organ transplant centers that offer cell-based therapies. Current research is focusing on the optimal strategy for developing ex vivo cultures with high cytotoxicity against EBV-infected target cells. The nature of the stimulating cells (e.g., lymphoblastoid cell lines or dendritic cells loaded with EBV antigens) and the cytokine milieu of the culture (e.g., addition of IL7 and IL12) may be critical determinants of the success of development of ex vivo generated CTL bulk cultures for clinical infusion [44, 45].

9.11 Role of Combination Therapies Since no therapies have been tested in randomized controlled trials, it is evident that the use of combination therapies is also not evidence-based. Nonetheless, as with other diseases, there may be logic in combining therapies that work by different mechanisms of action. However, multimodality therapies might lead to a belief that individual therapies can be given at lower doses or for reduced lengths of time. This could lead to reduced efficacy. For example, if rituximab is used without significant reduction in immunosuppression, then there may be little or no recovery in CTL responses. Such responses might be critical for long-term disease control and relapse may be common without them [31]. The use of combination therapies also makes it very challenging to identify the efficacious components of a treatment regimen. For example, autologous EBV-specific CTLs have been used immediately following polychemotherapy combined with rituximab with success of the combined regimen [41]. Combination therapies are likely to remain largely empiric given the enormous challenges of performing randomized controlled trials in this disease.

9.12 Central Nervous System Disease CNS disease may be primary [46, 47] or may be an additional site of disease in patients with extracranial disease [46]. In both situations, the prognosis has generally been considered to be very poor [46, 47]. The CNS is generally considered a protected site in which it is harder for normal immune surveillance to gain control of disease. Nonetheless, normal immune surveillance may be sufficient to control disease in rare cases (Fig. 9.3). Unfortunately, this occurs with insufficient frequency to justify reduction in immunosuppression as the only therapy. In renal transplant patients, complete cessation of immunosuppressive therapy seems justified until the disease is eradicated. A similar argument can be given for liver transplantation, at least until such a time as rebound rejection develops. Adjunctive therapies are generally given, though their efficacy has been hard to establish. These have included systemic or intrathecal rituximab, chemotherapy especially with the use of high dose methotrexate and radiation.

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Fig. 9.3 (a) CNS monomorphic PTLD presenting with sixth nerve palsy. (b) There was complete resolution of disease after temporary withholding of immunosuppression, though with rebound graft rejection requiring temporary mechanical circulatory support. He remains disease free 8 years later

9.13 Monitoring Patients During Therapy 9.13.1 Conventional Monitoring of Graft and PTLD Status Monitoring of tumor responses is performed by conventional methods appropriate to the site of disease. Computed tomography and magnetic resonance imaging are the most commonly used imaging techniques, though there is increasing interest in the use of positron emission tomography for evaluation and follow-up of PTLD [48]. Regression of mass lesions often takes at least 2 weeks, and frequently longer. Following reduction in immunosuppression, anti-EBV CTL responses may recover more quickly, thereby suggesting impending response to therapy, or risk of development of rejection. Thus, there is considerable interest in immunological monitoring in combination with monitoring of clinical disease and status of the allograft. The frequency and method of monitoring allograft status will depend on the clinical setting, including allograft type, prior rejection history, and time from transplantation. As discussed previously, rebound rejection is very common after reduction or cessation of immunosuppression for treatment of PTLD, and death due to allograft loss may be as common as death due to disease progression [5]. In contrast to lymphomas in the nontransplant setting, outcome of PTLD must consider the status of the allograft, and successful therapy should be defined in terms of complete response of PTLD without allograft loss and without development of chronic allograft dysfunction.

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9.13.2 EBV Viral Load Monitoring When EBV-PTLD is associated with high EBV viral load at presentation, serial monitoring appears to provide important information about response of disease after reduction in immunosuppression. This is probably just a simple indirect measure of EBV CTL responses. Viral load may fall prior to clinical response. Most data on viral load monitoring in this setting are from pediatric centers, which predominantly see PTLD in the context of primary EBV infection and high viral loads. A decline in viral load suggests that the patient is responding and may identify the time when the patient is at risk for developing rejection. The role of EBV viral load monitoring during therapy is less well established in adults. It is also important to note that rituximab causes complete elimination of peripheral EBV viral load in almost all patients, and therefore, viral load monitoring is not useful for evaluation of effectiveness of treatment after monoclonal anti-B cell therapy. This topic of viral load monitoring after diagnosis of PTLD is discussed in detail in Chap. 5.

9.13.3 Cellular Immune Responses It would be beneficial to be able to monitor and accurately predict responses to therapy, especially if such techniques could allow for successful modification of ineffective treatments at an early stage. To this end, a number of groups have sought to develop laboratory monitoring techniques for EBV-PTLD. Such techniques would supplement, rather than supplant, EBV-viral load monitoring and careful clinical evaluation of the patient and allograft. Immunological techniques that have been employed include assessment of frequency of EBV-specific T cell precursors by ELISPOT analysis [31], enumeration of EBVspecific CD8 T cells using HLA class I tetramers [49], intracellular cytokine staining for interferon-g producing CD8+ T cells [50], and cytotoxicity assays [51]. A comprehensive immunological assessment might help predict response to therapy, define time of greatest risk of rejection, and also assess potential for relapse, for example, by monitoring persistence or loss of EBV-specific T cells following adoptive cellular immunotherapy [42]. Unfortunately, these specialized tests of cellular immunity to EBV cannot be performed in routine clinical laboratories at this time, and remain research tools in most centers.

9.14 Conclusions Treatment of PTLD remains challenging and no controlled trial of therapy has been performed. A number of new therapies have evolved over the last decade, including use of monoclonal antibodies directed against B cell surface antigens, new chemotherapeutic regimens, and adoptive cellular immunotherapy. In addition, new antiviral agents with activity against a broad range of human herpes viruses are under development. Assessment of outcomes must include evaluation of the allograft and not just PTLD. Tumor behavior

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in the individual remains unpredictable, but new immunological monitoring techniques offer promise for serial evaluation of immune reconstitution in EBV-driven disease. Encouragingly, there is an increasing level of interest in PTLD among clinical and basic investigators, as well as recognition of the need for multicenter trials to define optimal treatment strategies. This offers some optimism for the future.

9.15 Take Home Pearls

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Reduction of immunosuppression remains first-line therapy in many centers Rebound rejection is common after reduction in immunosuppression, and death from both graft loss and progressive disease contributes to post-PTLD mortality The use of anti-CD20 monoclonal antibody therapy (rituximab) is rapidly increasing, but the precise indications for its use have not been established Rituximab is most commonly used for refractory disease, but an increasing number of centers are using this agent as firstline therapy, especially for monomorphic disease The results of chemotherapy are variable and infectious morbidity and mortality are high compared to nontransplant populations treated for lymphoma Cellular immunotherapy offers promise for the treatment of refractory EBV-driven PTLD, but remains a research tool at this time CNS disease may be primary or associated with extracranial disease and is associated with very poor prognosis

References 1. Rosenwald A, Wright G, Chan WC, et al. The lymphoma/leukemia molecular profiling project. The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med. 2002;346:1937–47 2. Starzl TE, Nalesnik MA, Porter KA, et al. Reversibility of lymphomas and lymphoproliferative lesions developing under cyclosporin-steroid therapy. Lancet. 1984;1:583–7 3. Elstrom RL, Andreadis C, Aqui NA, et al. Treatment of PTLD with rituximab or chemotherapy. Am J Transplant. 2006;6:569–76 4. Hurwitz M, Desai DM, Cox KL, et al. Complete immunosuppression withdrawal as a uniform approach to post-transplant lymphoproliferative disease in pediatric liver transplantation. Pediatr Transplant. 2004;8:267–72 5. Webber SA, Naftel DC, Fricker FJ, et al. Lymphoproliferative disorders after paediatric heart transplantation: a multi-institutional study. Lancet. 2006;367:233–9 6. Tsai DE, Hardy CL, Tomaszewski JE, et al. Reduction in immunosuppression as initial therapy for posttransplant lymphoproliferative disorder: analysis of prognostic variables and long-term follow-up of 42 adult patients. Transplantation. 2001;71:1076–88 7. Aull MJ, Buell JF, Trofe J, et al. Experience with 274 cardiac transplant recipients with posttransplant lymphoproliferative disorder: a report from the Israel Penn International Transplant Tumor Registry. Transplantation. 2004;78:1676–82

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8. Swinnen LJ, LeBlanc M, Grogan TM, et al. Prospective study of sequential reduction in immunosuppression, interferon alpha-2B and chemotherapy for posttransplant lymphoproliferative disorder. Transplantation. 2008;86:215–22 9. Nepomuceno RR, Balatoni CE, Natkunam Y, et al. Rapamycin inhibits the interleukin 10 signal transduction pathway and the growth of Epstein Barr virus B-cell lymphomas. Cancer Res. 2003;63:4472–80 10. Vaysberg M, Balatoni CE, Nepomuceno RR, et al. Rapamycin inhibits proliferation of EpsteinBarr virus-positive B-cell lymphomas through modulation of cell-cycle protein expression. Transplantation. 2007;83:1114–21 11. Traum AZ, Rodig NM, Pilichowska ME, et al. Central nervous system lymphoproliferative disorder in pediatric kidney transplant recipients. Pediatr Transplant. 2006;10:505–12 12. Hanto DW, Frizzera G, Gajl-Peczalska KJ, et al. Epstein-Barr virus induced B-cell lymphoma after renal transplantation. N Eng J Med. 1982;306:913–18 13. Prichard MN, Hartline CB, Harden EA, et al. Inhibition of herpes virus replication by hexadecyloxypropyl esters of purine- and pyramidine-based phosphonomethoxyethyl nucleoside phosphonates. Antimicrob Agents Chemother. 2008;52:4326–30 14. Williams-Aziz SL, Hartline CB, Harden EA, et al. Comparative activities of lipid esters of cidofovir and cyclic cidofovir against replication of herpesviruses in vitro. Antimicrob Agents Chemother. 2005;49:3724–33 15. Perrine SP, Hermine O, Small T, et al. A phase I/II trial of arginine butyrate and ganciclovir in patients with Epstein-Barr virus associated lymphoid malignancies. Blood. 2007;109:2571–8 16. Ghosh SK, Forman LW, Akinsheye I, et al. Short discontinuous exposure to butyrate effectively sensitizes latently EBV-infected cells to nucleoside analogue antiviral agents. Blood Cells Mol Dis. 2007;38:57–65 17. Davis CL, Wood BL, Sabath DE, et al. Interferon-alpha treatment of posttransplant lymphoproliferative disorder in recipients of solid organ transplants. Transplantation. 1998;66:1770–9 18. Shapiro RS, Chauvenet A, McGuire W, et al. Treatment of B-cell lymphoproliferative disorders with interferon alfa and intravenous gamma globulin [letter]. N Engl J Med. 1988; 318:1334 19. Haddad E, Paczesny S, Leblond V, et al. Treatment of B-lymphoproliferative disorder with a monoclonal anti-interleukin-6 antibody in 12 patients: a multicenter phase 1–2 clinical trial. Blood. 2001;97:1590–7 20. Riddler SA, Breinig MC, McKnight JLC. Increased levels of circulating Epstein-Barr virusinfected lymphocytes and decreased EBV nuclear antigen antibody responses are associated with the development of posttransplant lymphoproliferative disease in solid-organ transplant recipients. Blood. 1994;84:972–84 21. Fisher A, Blanche S, Le Bidois J, et al. Anti-B-cell monoclonal antibodies in the treatment of severe B-cell lymphoproliferative syndrome following bone marrow and organ transplantation. N Engl J Med. 1991;324:1451–6 22. Milpied N, Vasseur B, Parquet N, et al. Humanized anti-CD20 monoclonal antibody (rituximab) in post transplant B-lymphoproliferative disorder: a retrospective analysis on 32 patients. Ann Oncol. 2000;11(Suppl 1):113–6 23. Blaes AH, Peterson BA, Bartlett N, et al. Rituximab therapy is effective for posttransplant lymphoproliferative disorders after solid organ transplantation: results of a phase II trial. Cancer. 2005;104:1661–7 24. Jain AB, Marcos A, Pokharna R, et al. Rituximab (chemeric anti-CD20 antibody) for posttransplant lymphoproliferative disorder after solid organ transplantation in adults: long-term experience from a single center. Transplantation. 2005;80:1692–8 25. Knoop C, Kentos A, Remmelink M, et al. Post-transplant lymphoproliferative disorders after lung transplantation: first-line treatment with rituximab may induce complete remission. Clin Transplant. 2006;20:179–87

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26. Choquet S, Leblond V, Herbrecht R, et al. Efficacy and safety of rituximab in B-cell posttransplantation lymphoproliferative disorders: results of a prospective multicenter phase 2 study. Blood. 2006;107:3053–7 27. Oertel SH, Verschuuren E, Reinke P, et al. Effect of anti-CD20 antibody rituximab in patients with post-transplant lymphoproliferative disorder (PTLD). Am J Transplant. 2005;5:2901–6 28. Choquet S, Oertel S, Leblond V, et al. Rituximab in the management of post-transplantation lymphoproliferative disorder after solid organ transplantation: proceed with caution. Ann Hematol. 2007;86:599–607 29. Trappe RU, Choquet S, Reinke P, et al. Salvage therapy for relapsed posttransplant lymphoproliferative disorders (PTLD) with a second progression of PTLD after upfront chemotherapy: the role of single agent rituximab. Transplantation. 2007;84:1708–12 30. Webber S, Harmon W, Faro A, et al. Anti-CD20 monoclonal antibody (rituximab) for refractory PTLD after pediatric solid organ transplantation: multicenter experience from a registry and from a prospective clinical trial. Blood. 2004;104:213a 31. Salvado B, Rooney CM, Quiros-Tejeira RE, et al. Cellular immunity to Epstein-Barr virus in liver transplant recipients treated with rituximab for post-transplant lymphoproliferative disease. Am J Transplant. 2005;5:566–72 32. Gross TG, Bucuvalas JC, Park JR, et al. Low-dose chemotherapy for Epstein-Barr virus-positive post-transplantation lymphoproliferative disease in children after solid organ transplantation. J Clin Oncol. 2005;23:6481–8 33. Orjuela M, Gross TG, Cheung YK, et al. A pilot study of chemoimmunotherapy (cyclophosphamide, prednisone, and rituximab) in patients with post-transplant lymphoproliferative disorder following solid organ transplantation. Clin Cancer Res. 2003;9(10 Pt 2):3945S–52 34. Choquet S, Trappe R, Leblond V, et al. CHOP-21 for the treatment of post-transplant lymphoproliferative disorders (PTLD) following solid organ transplantation. Haematologica. 2007;92:273–4 35. Fohrer C, Caillard S, Koumarianou A, et al. Long-term survival in post-transplant lymphoproliferative disorders with a dose-adjusted ACVBP regimen. Br J Haematol. 2006;134:602–12 36. Swinnen LJ, Mullen GM, Carr TJ, et al. Aggressive treatment for postcardiac transplant lymphoproliferation. Blood. 1995;86:3333–40 37. Trappe R, Riess H, Babel N, et al. Salvage chemotherapy for refractory and relapsed posttransplant lymphoproliferative disorders (PTLD) after treatment with single-agent rituximab. Transplantation. 2007;83:912–8 38. Buell JF, Gross TG, Hanaway MJ, et al. Chemotherapy for post-transplant lymphoproliferative disorder: the Israel Penn International Transplant Tumor Registry. Transplant Proc. 2005;37: 956–7 39. Rooney CM, Smith CA, Ng CYC, et al. Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr virus related lymphoproliferation. Lancet. 1995;345:9–13 40. Swinnen LJ. Immune-cell treatment of Epstein-Barr-virus-associated lymphoproliferative disorders. Best Pract Res Clin Haematol. 2006;19:839–47 41. Comoli P, Maccario R, Locatelli F, et al. Treatment of EBV-related post-renal transplant lymphoproliferative disease with a tailored regimen including EBV-specific T cells. Am J Transplant. 2005;5:1415–22 42. Savoldo B, Goss JA, Hammer MM, et al. Treatment of solid organ transplant recipients with autologous Epstein Barr virus-specific cytotoxic T lymphocytes (CTLs). Blood. 2006;108:2942–9 43. Haque T, Wilkie GM, Jones MM, et al. Allogeneic cytotoxic T cell therapy for EBV-positive posttransplant lymphoproliferative disease: results of a phase 2 multicenter clinical trial. Blood. 2007;110:1123–31 44. Comoli P, Ginevri F, Maccario R, et al. Successful in vitro priming of EBV-specific CD8+ T cells endowed with strong cytotoxic function from T cells of EBV seronegative children. Am J Transplant. 2006;6:2169–76

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45. Metes D, Storkus W, Zeevi A, et al. Ex vivo generation of effective Epstein-Barr Virus (EBV)specific CD8+ cytotoxic T cells from peripheral blood of immunocompetent EBV-seronegative individuals. Transplantation. 2000;70:1507–15 46. Buell JF, Gross TG, Hanaway MJ, et al. Post-transplant lymphoproliferative disorder: significance of central nervous system involvement. Transplant Proc. 2005;37:954–5 47. Castellano-Sanchez AA,Li S, Qian J, et al. Primary central nervous system posttransplant lymphoproliferative disorders. Am J Clin Pathol. 2004;121:246–53 48. Bianchi E, Pascual M, Nicod M, et al. Clinical usefulness of FDG-PET/CT scan imaging in the management of posttransplant lymphoproliferative disease. Transplantation. 2008;85:707–12 49. Sebelin-Wulf K, Nguyen TD, Oertel S, et al. Quantitative analysis of EBV-specific CD4/CD8 T cell numbers, absolute CD4/CD8 T cell numbers and EBV load in solid organ recipients with PTLD. Transpl Immunol. 2007;17:203–10 50. Guppy AE, Rawlings E, Madrigal JA, et al. A quantitative assay for Epstein-Barr virus-specific immunity shows interferon-gamma producing CD8+ T cells increase during immunosuppression reduction to treat post-transplant lymphoproliferative disease. Transplantation. 2007;84: 1534–9 51. Baudouin V, Dehee A, Pedron-Grossetete B, et al. Relationship between CD8+ T-cell phenotype and function, Epstein-Barr virus load, and clinical outcome in pediatric renal transplant recipients: a prospective study. Transplantation. 2004;77:1706–13

Prevention of Epstein–Barr Virus Infection and Posttransplant Lymphoproliferative Disease Following Transplantation

10

Michael Green and Marian Michaels

Core Messages

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Increasing attention is focused on the prevention of EBV disease and PTLD. A variety of potential approaches to the prevention of EBV disease and PTLD are currently under consideration including chemoprophylaxis using antiviral therapies, immunoprophylaxis (including adoptive immunotherapy), and viral load monitoring to inform pre emptive strategies. Although many centers have endorsed the use of the antiviral agents acyclovir and ganciclovir for the prevention of EBV/PTLD, there is little data to support this practice At present, the use of serial monitoring of the EBV viral load as a stimulus to reduce immunosuppression (for solid organ transplant recipients) or initiate adoptive immunotherapy or treatment with rituximab (for stem cell transplant recipients) appears to be the most promising strategies for the prevention of EBV disease and PTLD in these populations.

10.1 Introduction The recognition of the importance of Epstein–Barr virus (EBV) infection in recipients of solid organ and bone marrow transplantation has grown in parallel with the growth and success of these procedures. Despite an increasing understanding of EBV disease, the

M. Green () Departments of Pediatrics and Surgery, Division of Infectious Diseases, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA e-mail: [email protected] V. R. Dharnidharka et al. (eds.), Post-Transplant Lymphoproliferative Disorders, DOI: 10.1007/978-3-642-01653-0_10, © Springer Verlag Berlin Heidelberg 2010

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optimal management of this important complication remains unclear with ongoing concerns for morbidity and mortality attributable to pathogens [1, 2]. Accordingly, attention has begun to focus on the prevention of EBV/PTLD in transplant recipients. A variety of potential approaches to the prevention of EBV disease and PTLD are currently under consideration including chemoprophylaxis using antiviral therapies, immunoprophylaxis (including adoptive immunotherapy), and viral load monitoring to inform pre emptive strategies. This chapter reviews the scientific rationale behind, and clinical experience with these potential strategies for the prevention of EBV/PTLD.

10.2 Chemoprophylaxis Using Antiviral Therapy 10.2.1 Mechanisms of Action of Acyclovir and Ganciclovir Chemoprophylaxis using antiviral agents, such as acyclovir and ganciclovir, is one possible approach to the prevention of EBV disease and PTLD. Both acyclovir and ganciclovir actively inhibit lytic EBV replication in vitro [3, 4] through inhibition of the late phase lytic replication without affecting the expression of immediate early or early lytic viral genes 15. Ganciclovir is phosphorylated to levels 100 times greater than acyclovir; it is approximately six times more potent against EBV[3], and has a prolonged effect in suppressing EBV genome replication in vitro compared to acyclovir [4]. Accordingly, if these agents are efficacious ganciclovir is likely to be the more effective agent in the treatment of EBV/PTLD. However, while both antiviral agents suppress the lytic phase of EBV replication, neither has any effect on EBV in its latent state or on the proliferation of EBVtransformed B cells [2–4]. Analyses of pathologic specimens have shown that the vast majority of EBV-infected cells within PTLD lesions are transformed B cells that do not undergo lytic replication, and thus, their ongoing proliferation should not be inhibited by exposure to acyclovir or ganciclovir [1, 3, 5, 6]. Similar studies have been attempted evaluating the state of EBV infection in the steps leading to the development of symptomatic EBV disease and PTLD. The correlation between EBV loads in the peripheral blood and the development of EBV disease and PTLD [7–11] suggests characterization of the state of EBV infection in the blood of patients with elevated EBV loads could offer insight into the utility of antiviral therapy as prophylaxis against the EBV disease and PTLD. Babcock et al. characterized the state of EBV-infected B cells from a small number of asymptomatic EBV-seropositive organ transplant recipients with elevated viral loads shortly after transplant [12]. These investigators found that the EBV load in the peripheral blood was maintained within resting memory B cells, and that although some patients only had episomal EBV DNA (characteristic of latently infected or immortalized B cells), others had both episomal and linear EBV DNA (characteristic of active, lytic replication) [12]. Qu investigated the state of EBV gene expression in the peripheral blood of transplant recipients with elevated viral loads using RT-PCR, including some with active PTLD [13]. In this study, mRNA for ZEBRA

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(the immediate early transcriptional activator of EBV and a marker of entrance into the lytic cycle) was only detected in 6/40 specimens from nine children with persistent high EBV load states who had serial samples available for evaluation, and from only 3/8 specimens obtained from children at or near the time of PTLD. Further analyses suggested that even when positive, only a few EBV infected B cells in the peripheral blood expressed ZEBRA RNA at any given time. While both studies identify the presence of some component of lytic gene expression, neither study confirms the presence of lytic replication in these patients. Additional studies are necessary to confirm the state of EBV viral infection in patients at risk for the development of EBV disease and PTLD.

10.2.2 Animal Models of Chemoprophylaxis The potential role of acyclovir and ganciclovir in the prevention of EBV/PTLD has been explored in studies using the SCID mouse model of EBV/PTLD. Boyle demonstrated minimal activity for ganciclovir and none for acyclovir in reducing the frequency of EBVassociated B cell lymphoma in the SCID mouse model of both active and latent infection [14]. Hong further evaluated the impact of acyclovir on the development of EBVlymphoproliferative disease (LPD) in a similar model [15]. In their system, EBV lymphoblastoid cell lines (LCLs) derived from an EBV wildtype strain, as well as two mutant EBV clones in which one or the other of the two immediate early (IE) genes (BZLF1 or BRLF1) had been knocked out were infused into SCID mice. Growth of LPD was impaired in mice that had been infused with the two mutant strains of EBV. However, the use of acyclovir on SCID mice receiving wildtype EBV-derived LCL did not impact the rate of growth of LPD. These results suggest that early lytic gene expression but not the release of infectious particles (which would be blocked by the presence of acyclovir) contributes to enhanced growth of LPD, and raise doubts as to the likely effectiveness of acyclovir and ganciclovir to prevent the development of PTLD.

10.2.3 Clinical Studies of Chemoprophylaxis Limited evidence is available to address the efficacy of antiviral therapy in the prevention of EBV/PTLD in humans. Two retrospective studies evaluated the rate of development of PTLD in adult organ transplant recipients who received acyclovir or ganciclovir as part of CMV prevention strategies [16, 17]. Although both of these studies appeared to demonstrate a beneficial effect of antiviral therapy against the development of EBV/PTLD, both were limited by the use of either historical [16] or, in the case of the latter study, no specific controls [17]. The difficulty in interpreting the results of such retrospective studies lacking concurrent controls is illustrated by a third study by Malouf, which reported a drop in the incidence of PTLD from 4.2 to 1.34% after the introduction of ganciclovir prophylaxis in 1996 in lung transplant recipients [18]. Unfortunately, the introduction of ganciclovir was coincident with the elimination of antilymphocyte globulin as immunosuppression.

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Accordingly, it is impossible to determine if the drop in incidence of EBV/PTLD was attributable to antiviral therapy or other changes in their management. More recently, Funch and colleagues conducted a multicenter case-control study examining the impact of antiviral therapy on the development of PTLD in kidney transplant recipients [19]. Univariate analysis suggested a protective effect of antiviral treatment with ganciclovir or acyclovir. However, the study also showed that although pretransplant EBV seronegativity was associated with developing PTLD (odds ratio 5.39), these patients were statistically less likely to receive antiviral therapy. To control the possibility that the apparent protective effect of antiviral therapy might be a consequence of this confounding, additional analysis eliminating all individuals known to be EBV seronegative prior to transplant were performed, which again demonstrated significant protective effect of ganciclovir and a trend toward protection with the use of acyclovir or both drugs. Unfortunately, a similar analysis was not carried out for those kidney transplant recipients who were EBV seronegative prior to transplant. In contrast to the results reported by Funch, a retrospective registry study of 44,828 kidney transplant recipients carried out by Opelz failed to identify any impact of antiviral prophylaxis with ganciclovir or acyclovir on the development of posttransplant nonHodgkin’s lymphoma [20]. These investigators found that the incidence of development of lymphoma was essentially identical in patients who received CMV prophylaxis with ganciclovir or acyclovir when compared to kidney recipients who did not receive any antiviral prophylaxis. The authors of this studies concluded that the absence of an antilymphoma effect by the use of antiviral drugs was virtually proven. The limits of retrospective analyses to accurately determine the potential efficacy of antiviral therapy in the prevention of EBV/ PTLD are further illustrated by the discrepant results of these two studies. To date, only a single randomized, controlled trial has been completed evaluating the role of antiviral agents in the prevention of EBV/PTLD [21]. This randomized trial compared 2 weeks of intravenous ganciclovir alone to 2 weeks of ganciclovir followed by 50 weeks of high-dose oral acyclovir in pediatric liver transplant recipients. PTLD developed in eight of 24 patients who received ganciclovir followed by acyclovir compared to five cases of PTLD in 24 children who received the short course of ganciclovir alone (P = NS) [21]. This study suggested that the prolonged use of acyclovir did not prevent EBV/PTLD. Although it is possible that prolonged use of the more potent ganciclovir in lieu of acyclovir might have resulted in a different outcome, development of PTLD in patients while receiving prolonged courses of intravenous ganciclovir has been reported [22].

10.3 Immunoprophylaxis 10.3.1 Cellular Therapy Cellular therapy has been considered both as a potential treatment and as a preventive strategy against EBV/PTLD. The rationale behind using cellular therapy is based on the critical role that EBV-specific cytoxic T lymphocytes (CTLs) are known to play in the

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control of EBV infection in immunocompetent children and adults (see Chap. 4). The use of EBV specific CTLs as a treatment for EBV/PTLD was first reported by Papdopoulos et al. in BMT recipients using white blood cells from EBV seropositive blood donors [23]. While successful in treating PTLD, this approach was associated with complications, such as graft vs. host disease and pulmonary embolism, which were attributed to the infusion of mature nonrelated lymphocytes. In an important modification of this approach, Rooney and colleagues used EBV specific CTL derived from the actual bone marrow donors of the affected BMT recipient to both treat PTLD and prevent development of EBV disease in patients with elevated EBV loads in their blood [24]. Because of the requirement for HLAmatching for the effect of CTLs and the established observation that EBV/PTLD in BMT recipients most often involves donor B cells, this work involved the ex-vivo stimulation and growth of preexisting EBV-specific CTLs obtained from the BMT donor. Given their initial successes, these investigators expanded their prophylaxis work to include 39 children who were at high risk for PTLD due to having undergone T cell depleted BMT. None of these children developed PTLD; however, there was no control group [25]. Subsequently, others have also demonstrated the feasibility of this approach in the BMT population. While the use of cellular immunotherapy is clearly feasible for recipients of BMT, implementation of this strategy for patients undergoing SOT has proven problematic. Unlike BMT recipients, PTLD developing in patients undergoing SOT typically involve B cells of recipient origin and most commonly occur in patients who were immunologically naïve to EBV prior to transplantation. Accordingly, patients most likely to benefit from prophylaxis using cellular immunotherapy will not have preexisting EBV-specific CTLs available for ex-vivo stimulation. However, Savoldo and colleagues demonstrated that autologous EBV-specific CTLs could be derived from patients at high risk for PTLD even before PTLD developed and given safely to prevent PTLD [26]. Twenty-three solid organ recipients with persistently high EBV-DNA viral load (but without evidence of PTLD) and four patients with early posttransplant EBV seroconversion were enrolled in an EBV-CTL generation protocol. Kinetics of CTL derivation were similar to healthy donors. Six of these patients had CTLs infused for prevention of PTLD; none had recognized toxicity. The number of EBV responsive cells increased after infusion. No PTLD developed within 1 year of the infusion, though the viral load levels did not fall substantially.

10.3.2 Passive Immunization Although CTL are thought to play the central role in the control of EBV infections, recent studies have raised questions regarding the role of antibodies in controlling the rapid proliferation of EBV-infected cells [27]. Several reports have documented an association between loss or absence of antibody against at least one of the Epstein-Barr nuclear antigens (EBNA) in EBV-seropositive organ transplant recipients and subsequent development of PTLD [8, 28]. It has also been recognized that many patients undergoing primary EBV infection following transplantation fail to develop anti-EBNA antibodies. Thus, the absence of antibodies against EBNA appears to correlate with an increased risk of developing PTLD. Riddler further demonstrated a correlation between increasing levels of

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anti-EBNA antibodies, including those introduced through transfusions, with decreasing EBV load [8]. Taken together, these data suggest a potential role for antibody in controlling EBV infected cells, and therefore, provide a potential rationale for the use of antibodies in the prevention and/or treatment of EBV/PTLD. Several investigators have evaluated the potential of antibody treatment to prevent EBV/PTLD using the SCID mouse model. Abedi demonstrated that weekly infusions of two different commercial gammaglobulin preparations, as well as purified immunoglobulin from EBV-seropositive blood donors prevented development of PTLD in this model [29]. In contrast, these investigators found that infusion of purified immunoglobulin from EBVseronegative blood donors, as well as rabbit anti-gp340 anti-serum (a potentially protective anti-EBV antibody) failed to protect SCID mice from development of PTLD. Nadal also evaluated the ability of human immunoglobulin preparations to suppress the occurrence of EBV-associated lymphoproliferation in this model [30]. These investigators found that the infusion of human immunoglobulin after reconstitution with human tonsillar mononuclear cells followed by infection with supernatant from B95–8 (a lytic replicationpermissive cell line) delayed or prevented the development of EBV-associated lymphoma in their model. The potential role of intravenous immunoglobulin (IVIG) in the prevention of EBV/ PTLD is further supported by the previously mentioned registry study carried out by Opelz [20]. As mentioned earlier, this international registry review of 44,824 kidney transplant recipients evaluated the impact of the use of strategies to prevent CMV infection on the subsequent development of posttransplant lymphoma. In contrast to the absence of any benefit at all for ganciclovir or acyclovir, these investigators found that none of 2,103 kidney recipients who received anti-CMV immunoglobulin developed lymphoma during the first year following kidney transplantation (P = 0.012). Of interest, the demographics of patients receiving CMV-IVIG did not appear to differ from those who had received ganciclovir or acyclovir as a method of preventing CMV. The protective effect of CMV-IVIG did not appear to persist beyond the first year as the rate of lymphoma development in the subsequent 5 years was similar for recipients of CMVIVIG, anti-viral therapy with ganciclovir or acyclovir, and those kidney recipients who received prophylaxis to prevent CMV. The potential prophylactic benefit of IVIG against the development of EBV/PTLD was evaluated in a randomized, multicentered, controlled trial of CMV-IVIG for prevention of EBV/PTLD in pediatric liver transplant recipients [31]. No significant differences were seen in the adjusted 2 year EBV disease free rate (CMV-IVIG 79%, placebo 71%) and PTLD free rate (CMV-IVIG 91%, placebo 84%) between treatment and placebo groups at 2 years (p >0.20). Although statistically significant differences were not observed, rates of EBV disease and PTLD were somewhat lower in recipients of CMV-IVIG than in those who received placebo. This was particularly true for children less than 1 year of age, where 25% of children receiving CMV-IVIG developed EBV disease compared with 38% receiving placebo. While differences in the rates of development of PTLD in the children <1 year of age were less dramatic, the advantage again favored the recipients of CMV-IVIG (12 vs. 19%). Of note, the use of EBV load monitoring to inform reductions in immune suppression occurred with increasing frequency during the latter part of the study and potentially confounded its ability to identify differences between the two treatment groups.

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10.3.3 Active Immunization Active immunization would be another potential immunoprophylactic strategy. At present, there is no commercially available vaccine to prevent EBV infection or disease. A recombinant glycoprotein 350 ((gp350)/AS04) vaccine is currently in clinical trials. Results of phase I and phase II trials using this candidate vaccine in both EBV seropositive and seronegative healthy volunteers have been published [32, 33]. Use of this vaccine resulted in a reduction in symptomatic primary EBV infectionand development of infectious mononucleosis, but had little impact on EBV seroconversion rates. Of note, the vaccine had no reliable effect on the development of cell-mediated immunity. A second vaccine approach is to generate EBV-specific CD8+ T cells that control the expansion of EBV-infected B cells after infection. Results of a small phase I CD8+ T cell epitopebased EBV vaccine trial in 14 previously healthy seronegative volunteers have recently been published [34]. The vaccine comprised the HLAB*0801-restricted CD8+ T cell epitope FLRGRAYGL (FLR) from the latent EBNA3 using tetanus toxoid as a source of CD4+ T cell help. The vaccine was well tolerated with no serious side effects recognized during the course of the study. All but one of eight volunteers receiving vaccine demonstrated production of FLR-specific T cell response postvaccination as measured by ELISPOT. Although results of these studies are encouraging, additional studies are needed to determine if this vaccine will have any impact on the prevention of EBV disease and PTLD in transplant recipients.

10.4 Viral Load Monitoring and Preemptive Strategies of Prevention The observation that EBV load in the peripheral blood rose prior to the development of overt PTLD and likewise fell with the resolution of disease (see Chap. 5) provided a model similar to CMV preemptive therapy for instituting prevention strategies. However, the lack of impact of antiviral agents on EBV loads raised questions as to what is the most appropriate preemptive intervention. Potential strategies have included reduction or cessation of immunosuppression, use of antiviral medications, such as ganciclovir or acyclovir alone or in combination with reduction of immune suppression, as well as the use of monoclonal anti-CD20 (rituximab) therapy. Each of these strategies is reviewed below. McDiarmid and colleagues reported their experience of monitoring EBV loads, to inform the pre emptive use of the combination of decreasing immunosuppression and intravenous ganciclovir (either reinitiation or continuation if patients were on it already) in pediatric liver transplant recipients [35]. EBV seronegative children were classified as high risk and received 100 days of intravenous ganciclovir (followed by oral acyclovir), and were followed with frequent viral load measurement. Children who were EBV seropositive prior to transplantation were considered low-risk; they received a shorter course of IV ganciclovir followed by oral acyclovir and were monitored less frequently. Elevated EBV viral loads were observed in most of the high risk group, while they were

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still on their initial course of ganciclovir prophylaxis. Accordingly, the only change made in their management in response to the elevated loads was a drop in immunosuppression. However, no PTLD occurred in this group. Interestingly, two children both under a year of age had been seropositive pretransplant, and hence, classified as lowrisk developed PTLD. It is likely that EBV seropositivity was present on the basis of passive maternal antibody and that these infants were really at high risk. The overall rate of PTLD of 5% in this experience was lower than their previous rate of 10%. However, the investigators were unable to determine the relative impact of ganciclovir vs. reduction of immunosuppression on the decreased rate of PTLD observed in this experience. Subsequently, Lee et al. evaluated 43 pediatric liver transplant recipients who underwent prospective EBV load monitoring with a rapid tapering of immunosuppression if their load reached a critically high threshold without addition of antiviral therapy [36]. The rates of PTLD and rejection were compared to 30 historical controls that had been consecutively transplanted just prior to the intervention group at their center. The rates of PTLD were 16% in the historical control compared with only 2% once the rapid weaning protocol was established. Only one patient received valganciclovir for concurrent CMV reactivation. Rejection occurred in one patient who required decreased immunosuppression and responded to steroid pulsing with cessation of tacrolimus tapering. These results are provocative, but suffer from having a historic control in which EBV serologic status was not known before transplantation. Accordingly, it is possible that the differences observed in this experience could in part be due to a larger high risk population. In a similar approach, Bakker and colleagues used EBV load monitoring in 75 adult lung transplant recipients to inform reduction in immunosuppression with the hope of preventing PTLD [37]. This population differed somewhat from the experience in pediatric transplant recipients in that most of the patients were EBV seropositive prior to transplant. Thirty-five percent of patients in this study demonstrated reactivation of EBV as evidenced by elevated viral loads. However, immunosuppression was only able to be reduced in 19 of 26 patients with an elevated EBV load. Overall, no patient developed EBV-associated PTLD regardless of the inability to modify immune suppression in seven of the patients, though one of the 75 subjects did develop an EBVnegative PTLD. Importantly, there was no accelerated rejection of the graft or worse survival in the patients who had immunosuppression reduced due to EBV viral load monitoring [37]. Because of concerns for EBV load having low positive predictive value for development of PTLD particularly in a previously immune population [38], some investigators sought to ascertain if viral load monitoring combined with evaluation of cellular immune response to phytohemagluttinin (PHA) would improve the safety of intervening with decreased immunosuppression [39]. Eighteen children undergoing liver transplantation were followed in this fashion; those children with moderate to high levels of EBV viremia were also found to have a decreased response to PHA suggesting a state of overimmunosuppression. Three of the patients had immunosuppression lowered in response to EBV viral load; all had increased PHA responses and no development of PTLD. EBV viral load monitoring failed to predict the development of PTLD in one

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child whose EBV load remained low; however, his PHA response had also been low suggesting he was overimmunosuppressed. His episode of PTLD could have been prevented by reducing immune suppression in response to either an elevated EBV load or a low PHA. A final approach that has been considered is the use of the anti-B cell monoclonal antibody rituximab as a preemptive therapy in response to an elevated EBV load. Rituximab was used as a preemptive therapy with successful outcome in high risk hematopoietic transplant recipients [40, 41]. Seventeen prospectively monitored BMT recipients showed a high level of EBV reactivation; 15 of the 17 were given rituximab preemptively. Only one of the 15 developed PTLD, but ultimately responded to two further doses of rituximab [41]. A similar approach was taken by Gruhn and colleagues in three children at high risk for PTLD after T cell depleted HSCT. The children received rituximab prophylactically when they were found to have critically high viral loads for EBV; all remained PTLD free 7–9 months after HSCT [40]. Meerbach and colleagues took it one step further and used a single dose of rituximab in combination with two doses of intravenous cidofovir a week apart in four hematopoieitc stem cell transplant recipients who had persistently elevated EBV viral loads [42]. The viral load fell in all cases and no PTLD developed. However, while this approach is potentially attractive as rituximab is uniformly available, the efficacy and importantly safety of this strategy have not been established in clinical trials. Further, although limited data have been published in the BMT population, to date, no experience using rituximab for preemptive prevention of EBV disease and PTLD has been reported for SOT recipients.

10.5 Take Home Pearls

• •

• •

Increasing attention regarding EBV disease and PTLD is being focused on prevention strategies prompting some centers to routinely use antiviral and/or immunoglobulin agents as standard prophylaxis against the development of EBV/PTLD despite the absence of strong data in support of these approaches. At present, the use of serial monitoring of the EBV viral load as a stimulus to reduce immunosuppression (for solid organ transplant recipients) or initiate adoptive immunotherapy or treatment with rituximab (for stem cell transplant recipients) appears to be the most promising strategy for the prevention of EBV disease and PTLD in these populations. Well designed clinical trials are necessary to evaluate the potential role of both antiviral and immunoglobulin agents in the prevention of EBV/PTLD in organ transplant recipients. Finally, the development of an effective EBV vaccine to provide to EBV naïve transplant candidates would likely prove to be an extremely effective strategy in the prevention of this complication.

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References 1. Green M, Michaels MG, Webber SA, Rowe D, Reyes J. The management of Epstein-Barr virus associated post-transplant lymphoproliferative disorders in pediatric solid-organ transplant recipients. Pediatr Transplant. 1999;3:271–81 2. Paya CV, Fung JJ, Nalesnik MA, Kieff E, Green M, Gores G, Habermann TH, Wiesner RH, Swinnnen L, Woodle ES, Bromberg JS. Epstein-Barr virus-induced posttransplant lymphoproliferative disorders. Transplantation. 1999;68:1517–25 3. Davis CL. The antiviral prophylaxis of post-transplant lymphoproliferative disorder. Springer Sem Immunopathol. 1998;20:437–53 4. Lin JC, Smith MC, Pagano JS. Prolonged inhibitory effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine against replication of Epstein-Barr virus. J Virol. 1984;50:50–5 5. Haque T, Crawford DH. Role of donor versus recipient Epstein-Barr virus in post-transplant lymphoproliferative disorders. Springer Semin Immunopathol. 1998;20:375–87 6. Knowles DM, Cesarmen E, Chadburn A, et al. Correlative morphologic and molecular genetic analysis demonstrates three distinct categories of posttransplantation lymphoproliferative disorders. Blood. 1995;85:552–65 7. Kenagy DN, Schlesinger Y, Weck K, Ritter JH, Gaudreault-Keener MM, Storch GA. EpsteinBarr virus DNA in peripheral blood leukocytes of patients with posttransplant lymphoproliferative disease. Transplantation. 1995;19:547–54 8. Riddler SA, Breinig MC, McKnight JLC. Increased levels of circulating Epstein-Barr virusinfected lymphocytes and decreased EBV nuclear antigen antibody responses are associated with the development of posttransplant lymphoproliferative disease in solid-organ transplant recipients. Blood. 1994;84:972–84 9. Rooney CM, Loftin SK, Holladay MS, Brenner MK, Krance RA, Heslop HB. Early identification of Epstein-Barr virus-associated post-transplant lymphoproliferative disease. Br J Haematol. 1995;89:98–103 10. Rowe DT, Qu L, Reyes J, Jabbour N, Yunis E, Putnam P, Todo S, Green M. Use of quantitative competitive PCR to measure Epstein-Barr virus genome load in peripheral blood of pediatric transplant recipients with lymphoproliferative disorders. J Clin Microbiol. 1997;35:1612–15 11. Savoie A, Perpete C, Carpentier L, Joncas K, Alfieri C. Direct correlation between the load of Epstein-Barr virus-infected lymphocytes in the peripheral blood of pediatric transplant patients and risk of lymphoproliferative disease. Blood. 1994;83:2715–22 12. Babcock GJ, Decker L, Freeman RB, Thorley-Dawon DA. Epstein-Barr virus-infected resting memory B-cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients. J Exp Med. 1999;190:567–76 13. Qu L, Green M, Webber S, Reyes J, Ellis D, Rowe D. Epstein-Barr virus gene expression in the peripheral blood of transplant recipients with persistent circulating viral loads. J Infect Dis. 2000;182:1013–21 14. Boyle TJ, Tamburini M, Berend KR, Kizilbash AM, Borowitz MJ, Lyerly HK. Human B-cell lymphoma in severe combined immunodeficient mice after active infection with Epstein-Barr virus. Surgery. 1992;112:378–86 15. Hong GK, Gulley ML, Feng WH, Delecluse HJ, Holley-Guthrie E, Kenney SC. Epstein-Barr virus lytic infection contributes to lymphoproliferative disease in a SCID mouse model. J Virol. 2005;79:1393–403 16. Darenkov IA, Marcarelli MA, Basadonna GP, Friedman AL, Lorber KM, Howe JG, Crouch J, Bia MJ, Kliger AS, Lorber MI. Reduced incidence of Epstein-Barr virus-associated posttransplant lymphoproliferative disorder using preemptive antiviral therapy. Transplantation. 1997;64:848–52 17. Davis CL, Harrison KL, McVicar JP, Forg P, Bronner M, Marsh CL. Antiviral prophylaxis and the Epstein Barr virus-related post-transplant lymphoproliferative disorder. Clin Transplant. 1995;9:53–9

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18. Malouf MA, Chhajed PN, Hopkins P, Plit M, Turner J, Glanville AR. Anti-viral prophylaxis reduces the incidence of lymphoproliferative disease in lung transplant recipients. J Heart Lung Transplant. 2002;21:547–54 19. Funch DP, Walker AM, Schneider G, Ziyadeh NJ, Pescovitz MD. Ganciclovir and acyclovir reduce the risk of post-transplant lymphoproliferative disorder in renal transplant recipients. Am J Transplant. 2005;5:2894–900 20. Opelz G Daniel V Naujokat C Fickenscher H Dohler B. Effect of cytomegalovirus prophylaxis with immunoglobulin or with antiviral drugs on post-transplant non-Hodgkin lymphoma: a multicentre retrospective analysis. Lancet Oncol. 2007;8:212–8 21. Green M, Kaufmann M, Wilson J, Reyes J. Comparison of intravenous ganciclovir followed by oral acyclovir with intravenous ganciclovir alone for the prevention of cytomegalovirus and Epstein-Barr virus after liver transplantation in children. Clin Infect Dis. 1997;25:1344–9 22. Kuo PC, Dafoe DC, Alfrey EJ, Sibley RK, Scandling JD. Posttransplant lymphoproliferative disorders and Epstein-Barr virus prophylaxis. Transplantation. 1995;59:135–8 23. Papadopoulos EB, Ladanyi M, Emanuel D, Mackinnon S, Boulad F, Carabasi MH, CastroMalaspina H, Childs BH, Gillio AP, Small TN, Young JW, Kernan NA, O’Reilly RJ. Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation. N Engl J Med. 1994;330:1185–91 24. Rooney CM, Smith CA, Ng CYC, Loftin S, Li C, Krance RA, Brenner MK, Heslop HE. Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr virus-related lymphoproliferation. Lancet. 1995;345:9–13 25. Rooney CM, Smith CA, Ng CYC, Loftin SK, Sixbey JW, Gan Y, Srivastava DK, Bowman LC, Krance RA, Brenner MK, Heslop HE. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood. 1998 92:1549–55 26. Savoldo B, Goss JA, Hammer MM, Zhang L, Lopez T, Gee AP, Lin YF, Quiros-Tejeira RE, Reinke P, Schubert S, Gottschalk S, Finegold MJBrenner MK, Rooney CM, Heslop HE. Treatment of solid organ transplant recipients with autologous Epstein Barr virus–specific cytotoxic T lymphocytes (CTLs). Blood. 2006;108:2942–49 27. McKnight JLC, Cen H, Riddler SA, Breinig MC, Williams PA, Ho M, Joseph PS. EBV gene expression, EBNA antibody responses and EBV+ peripheral blood lymphocytes in post-transplant lymphoproliferative disease. Leuk Lymphoma. 1994;15:9–16 28. Walker RC, Marshall WF, Strickler JG, Wiesner RH, Velosa JA, Habermann TM, McGregor CGA, Paya CV. Pretransplantation assessment of the risk of lymphoproliferative disorder. Clin Infect Dis. 1995;20:1346–53 29. Abedi MR, Linde A, Christensson B, Mackett M, Hammarstrom L, Smith C. Preventive effect of IgG from EBV-seropositive donors on the development of human lymphoproliferative disease in SCID mice. Int J Cancer. 1997;71:624–29 30. Nadal D, Guzman J, Frohlich S, Braun DG. Human immunoglobulin preparations suppress the occurrence of Epstein-Barr virus-associated lymphoproliferation. Exp Hematol. 1997;25: 223–31 31. Green M, Michaels MG, Katz BZ, Burroughs M, Gerber D, Shneider BL, Newell K, Rowe D, Reyes J. CMV-IVIG for prevention of Epstein-Barr virus disease and post transplant lymphoproliferative disease in pediatric liver transplant recipients. Am J Transplant. 2006;6:1906–12 32. Moutschen M, Leonard P, Sokal EM, Smets F, Haumont M, Mazzu P, Bollen A, Denamur F, Peeters P, Dubin G, Denis M. Phase I/II studies to evaluate safety and immunogenicity of a recombinant gp350 Epstein-Barr virus vaccine in healthy adults. Vaccine. 2007;25:4697–705 33. Sokal EM, Hoppenbrouwers K, Vandermeulen C, Moutschen M, Leonard P, Moreels A, Haumont M, Bollen A, Smets F, Denis M. Recombinant gp350 vaccine for infectious mononucleosis: a phase 2, randomized double-blind, placebo-controlled trial to evaluate the safety, immunogenicity and efficacy of an Epstein-Barr Virus vaccine in healthy young adults. J Infect Dis. 2007;196:1749–53

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34. Elliott SL, Suhrbier A, Miles JJ, Lawrence G, Pye SJ, Le TT, Rosenstengel A, Nguyen T, Allworth A, Burrows SR, Cox J, Pye D, Moss DJ, Bharadwaj M. Phase I trial of CD8+ T-cell peptide epitope-based vaccine for infectious mononucleosis. J Virol. 2008;82:1448–57 35. McDiarmid SV, Jordan S, Lee GS, et al. Prevention and preemptive therapy of posttransplant lymphoproliferative disease in pediatric liver recipients. Transplantation. 1998;66:1604–11 36. Lee TC, Savoldo B, Rooney CM, Heslop HE, Gee AP, Caldwell Y, Barshes NR, Scott JD, Bristow LJ, O’Mahony CA, Goss JA. Quantitative EBV viral loads and immunosuppression alterations can decrease PTLD incidence in pediatric liver transplant recipients. Am J Transplant. 2005;5:2222–8 37. Bakker NA, Verschuuren EAM, Erasmus ME, Hepkema BG, Veeger NJGM, Kallenberg CGM, van der Bij W. Epstein-Barr virus DNA load monitoring late after lung transplantation: a surrogate marker of the degree of immunosuppression and a safe guide to reduce immunosuppression. Transplantation. 2007;83:433–8 38. Benden C, Aurora P, Burch M, Cubitt D, Lloyd C, Whitmore P, Neligan S, Elliot MJ. Monitoring of Epstein-Barr viral load in pediatric heart and lung transplant recipients by real-time polymerase chain reaction. J Heart Lung Transplant. 2005;24:2103–8 39. Lee TC, Goss JA, Rooney CM, Heslop HE, Barshes NR, Caldwell YM, Gee AP, Scott JD, Savoldo B. Quantification of a low cellular immune response to aid in identification of pediatric liver transplant recipients at high-risk for EBV infection. Clin Transplant. 2006;20:689–94 40. Gruhn B, Meerbach A, Häfer R, Zell R, Wutzler P, Zintl F. Pre-emptive therapy with rituximab for prevention of Epstein-Barr virus-associated lymphoproliferative disease after hematopoietic stem cell transplantation. Bone Marrow Tranplant. 2003;31:1023–5 41. van Esser JWJ, Niesters HGM, van der Holt B, Meijer E, Osterhaus ADME, Gratama JW, Verdonck LF, Löwenberg B, Cornelissen JJ. Prevention of Epstein-Barr virus-lymphoproliferative disease by molecular monitoring and preemptive rituximab in high-risk patients after allogeneic stem cell transplantation. Blood. 2002 99:4364–9 42. Meerbach A, Wutzler P, Häfer RZintl F, Gruhn B. Monitoring of Epstein-Barr virus load after hematopoietic stem cell transplantation for early intervention in post-transplant lymphoproliferative disease. J Med Virol. 2008;80:441–54

Organ Specific Issues of PTLD – Kidney

11.a

Sophie Caillard

Core Messages

› › › › ›

Incidence of Posttransplant lymphoproliferative disorders (PTLD) in kidney transplant recipients is relatively low: 0.4% at 1 year, 1.2% at 5 years, and 1.6% after 10 years. Risk factors of PTLD in kidney transplant recipients are principally EBV seronegativity and high level of immunosuppression. Clinical presentation is heterogeneous, but PTLD could arise in kidney allograft in about 20% of cases. Treatment begins by immunosuppression tapering, which is facilitated by the possibility of return to dialysis; transplant nephrectomy is another option. Rituximab and chemotherapy are used in second step, but toxicity can be life threatening. Prognosis is better when PTLD developed in graft kidney. For other localizations, survival is between 40 and 60% after 5 years. Kidney retransplantation is possible after a diagnosis of PTLD.

Posttransplant lymphoproliferative disorders (PTLD) are a rare but serious complication after renal transplantation. The risk of developing PTLD is approximately 20-fold greater than in the general population. Several characteristics of PTLD occurring after kidney transplantation are common with other organs, but some are specific to kidney recipients in terms of incidence, clinical features, treatment, and outcome.

S. Caillard Nephrology-Transplantation Department, Hopitaux Universitaires de Strasbourg, 1 place de l’Hopital, 67091 Strasbourg, France e-mail: [email protected] V. R. Dharnidharka et al. (eds.), Post-Transplant Lymphoproliferative Disorders, DOI: 10.1007/978-3-642-01653-0_11.a, © Springer Verlag Berlin Heidelberg 2010

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11.a.1 Epidemiology Incidence of PTLD in kidney recipients is relatively low comparing to heart, heart–lung, lung, or intestine recipients [1, 2]. The lower incidence observed in kidney transplantation might be explained by two points: i) the need of fewer immunosuppression in kidney than lung or heart transplant recipients; ii) the presence of less “donor passenger lymphocytes” in renal tissue than in lung or intestine organs. In kidney recipients, PTLD incidence ranges from 0.3 to 0.46% at 1 year posttransplantation, from 0.9 to 1.4% at 3 years posttransplantation, around 1.2% after 5 years, and 1.6% after 10 years [2–5]. The risk of lymphoma is higher during the first year posttransplant, after which the cumulative incidence increases steadily over the 10 years period. There is no significant difference between recipients of first or subsequent transplant [2]. Incidence is greater in pediatric population and may reach 5–10%, especially in kidney recipients treated with tacrolimus [6–8]. This higher frequency in children is explained by a high rate of EBV seronegativity, leading to EBV primo-infection and secondary uncontrolled lymphoid proliferation. Risk factors for lymphoma in kidney transplant recipients are usual: younger and older age (<10 and >60 years-old), Caucasian race, EBV seronegativity, CMV co infection, and higher amount of immunosuppression, especially the T cell depleting agents thymoglobulin and OKT3 [2, 4, 9]. The local immune response against the transplanted organ plays an important role in the cellular dysregulation process that results in lymphomas, and HLA mismatches could be involved in the development of PTLD [10].

11.a.2 Clinical Presentation The clinical presentation of PTLD is heterogeneous and sometimes nonspecific: it ranges from asymptomatic disease, discovered on a graft biopsy, to a fulminant disorder. Nevertheless, clinical presentation can take particular patterns in kidney transplant recipients, particularly if lymphoma occurs in the allograft. A hilar tumor can be responsible for vascular compression or ureteric obstruction revealed by hydronephrosis and acute renal failure [11, 12]. Infiltration of kidney by the lymphoma can induce graft enlargement, creatinine elevation, and mimic rejection (Fig. 11.a.1). Sometimes, PTLD is fortuitously discovered on a systematic sonography. The differential diagnosis of a mass adjacent to a renal allograft includes hematoma or lymphocele. CT scan or MRI could be helpful showing a mild contrast enhancement of a solid mass. The pathological differential diagnosis of renal PTLD is acute rejection. Features that distinguish PTLD include a monomorphic infiltrate of lymphoblasts, patchy area of necrosis cells, and nodular aggregates of immature lymphoid cells with nuclear atypia. Immunostaining can be helpful showing B cells proliferation, whereas the presence of T cells suggests an acute rejection. In the French Registry, 366 PTLD occurring after kidney transplantation in adults were recorded during 10 years. Sixty one occurred in the grafted kidney (17%). The PTLD confined

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Fig. 11.a.1 Lymphoma localized in the hilum of the allograft and infiltrating the kidney parenchyma (CT scan)

to the graft was more likely early-onset lymphomas with a median of diagnosis of 13 months posttransplantation vs. 91 months for lymphomas localized outside the graft (personal datas). More than 2/3 of cases of PTLD developed in the allograft kidney were not disseminated. Acute renal failure was frequent concerning 67% of patients (mean creatininemia = 204 ± 125 mmol/l vs. 157 ± 86 mmol/l for other localizations). It has been shown that early-onset PTLD, especially if localized near the graft kidney, are most often developed from donor passenger lymphocytes [13], conversely to the other PTLD that are most likely of recipient origin. In a French series [14], 12 tumors were analyzed and showed that lymphoma developed from donor cells in four cases. All of these four cases occurred in the kidney allograft during the first year posttransplantation. Among the eight recipient-derived PTLD, tumors were disseminated with only two cases presenting an allograft involvement. Mortality was higher in the latter group. Another study showed that EBV can infect kidney allograft before PTLD diagnosis and established a relationship between this infection and the presence of PTLD near or within the graft [15]. Finally, it seems that central nervous system lymphomas are most common after kidney transplantation than in general population and other organs transplant patients (11% in kidney recipients vs. 3–4% in heart, heart–lung and liver recipients in CTS report, 13% in French Registry) [2, 5].

11.a.3 Therapeutic Aspects Management of lymphoma in kidney transplant recipients is easier than in other organ transplant patients because, unlike in vital organ recipients, the option of return to dialysis exits. In vital organ transplant patients, graft lost following treatment of PTLD leads to death because urgent retransplantation is rarely possible. After lymphoma diagnosis, the first step is to reduce immunosuppression in order to reconstitute the immune system.

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Immunosuppression tapering is more comfortable in kidney recipients with PTLD, especially in children and in early subsets of lymphoma (lymphoid hyperplasia or polymorphic lymphoma) [8]. For example, calcineurin inhibitors may be stopped, MMF dose be decreased, and corticosteroids increased or added. For early or limited forms of PTLD, immunosuppression tapering could be progressive with careful monitoring of tumor size. Switching calcineurin inhibitors to m-tor inhibitors has been proposed because sirolimus and everolimus inhibit the in vitro proliferation of lymphoblastoid cells [16] and the growth of lymphoma in a mouse model [17]. Moreover, activation of m-tor pathway was demonstrated in cells of PTLD regardless of their EBV status [18]. Nevertheless, only few published cases showed regression of PTLD after switching from CNI to m-tor inhibitors as the single treatment [19–21]. Furthermore, a recent study from the United Network for Organ Sharing database observed unexpectedly that therapy using m-tor inhibitors was associated with a higher incidence of PTLD (RR × 2) [22]. Prophylaxis with antiviral therapy, especially ganciclovir and its prodrug valganciclovir, seems helpful to reduce the risk of early-onset PTLD in kidney transplant recipients. The relative risk of developing a PTLD in patients treated with ganciclovir for CMV prophylaxis was 0.32 in an US case control study [23]. When reduction in immunosuppression fails, B cell monoclonal therapy represents an attractive second-line therapeutic option because of its low toxicity. AntiCD20 Rituximab* was proposed after immunosuppression tapering, alone or in association with chemotherapy. Nevertheless, the results of a French multicenter trial assessing the efficacy of Rituximab* in PTLD were disappointing with only 44% responses in 55 SOT recipients and a 2-year actuarial survival lower than 30% [24, 25]. In the French Registry, rituximab was used in 39% of cases, alone or in association with chemotherapy. The combination of chemotherapy and immunotherapy with rituximab is a promising approach; a prospective study is ongoing in France. Chemotherapy is considered in monomorphic PTLD occurring late in the posttransplant course, in EBV-negative, in T cell PTLD, as well as in refractory patients to the first management approaches. Management of chemotherapy in kidney transplant recipients should be done with caution because of kidney dysfunction. Drugs dosages must be adapted to creatinine clearance to avoid cumulative toxicity. In kidney transplant recipients treated by chemotherapy, toxic deaths are frequent and multifactorial: enhanced hematologic toxicity of drugs because of added myelotoxicity of immunosuppression, increased frequency and severity of infections, and accumulation of cytotoxic drugs in case of renal failure. In the French Registry, one third of the deaths were of toxic origin. Low-dose chemotherapy has, therefore, been advocated in kidney transplant recipients with encouraging results [26]. CHOP or adapted ACVBP regimen seems well tolerated and associated with a good clinical outcome [27, 28]. Advices to limit chemotherapy toxicity are the following: drastically reduce immunosuppression during chemotherapy, adapt cyclophosphamide dose to kidney function, systematic prophylactic use of G-CSF, and clotrimazole. In kidney transplant recipients, graft removal is another therapeutic option. Graft nephrectomy can be proposed in case of severe graft dysfunction, or for PTLD occurring in the graft and not responding to other therapies. In the French Registry, graft was removed in

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eighteen patients (30%) when lymphoma was localized in the graft, and in four patients with other localizations (1.3%).

11.a.4 Outcome and Prognostic Factors In Opelz report, the prognosis of kidney transplant patients with lymphoma was poor with a 5 year survival of 40% in the period 1995–2001. This 5 year survival dropped to 38% in patients with central nervous system involvement, and increased to 65% in patients with graft involvement [2]. In the French Registry, overall survival after diagnosis was 73% at 1 year and 61% at 5 years [5]. Patients’ 5 year survival improved to 81% in case of graft PTLD, and decreased to 53% for patients with CNS lymphomas. Patients’ survival was better in patients with early than late PTLD, in patients with involvement of a single site vs. multiple sites, and in patients with graft PTLD as compared with all other localizations. In a report from the Israel Penn International Transplant Tumor Registry [29], factors negatively influencing kidney recipients survival after PTLD were multiple sites and increasing age. Patients with graft involvement alone had better survival, especially if treated by transplant nephrectomy. In case of kidney graft lost, retransplantation after a PTLD seems possible [30–32]. Some criteria have to be fulfilled to minimize recurrence: a 1–2 year observation period between PTLD and retransplantation depending on the widespread of the haematological disease, disappearance of monoclonal immunoglobulin, undetectable or low EBV viral load, and apparition of antiEBNA IgG as this marker is linked to an effective cytotoxic response against EBV [31]. After retransplantation, patients should receive prolonged antiviral prophylaxis, serial measurements of EBV load, and appropriate adjustments of immunosuppression in order to avoid overimmunosuppression. Between 1987 and 2004, 69 patients underwent retransplantation in United States including 27 kidney transplant recipients. No patient has developed recurrent PTLD [32].

11.a.5 Take Home Pearls

• • • • • •

Incidence of PTLD in kidney transplant recipients is relatively low (1–1.5% at 5 years). PTLD can be revealed by a graft dysfunction in kidney recipients. PTLD is often localized within or near the graft. Graft PTLD is more often of donor origin, developed during the first posttransplant year, and localized in a single site. Its prognosis is better. Management of kidney transplant recipients with PTLD is easier because kidney is not a vital organ and management of immunosuppression tapering is facilitated. Prognosis of kidney transplant recipient is poor with a 40–60% survival after 5 years.

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References 1. Dharnidharka VR, Tejani AH, Ho PL, et al. Post-transplant lymphoproliferative disorder in the United States: young Caucasian males are at highest risk. Am J Transplant. 2002;2:993–8 2. Opelz G, Dohler B. Lymphomas after solid organ transplantation: a collaborative transplant study report. Am J Transplant. 2004;4:222–30 3. Kasiske BL, Snyder JJ, Gilbertson DT, et al. Cancer after kidney transplantation in the United States. Am J Transplant. 2004;4:905–13 4. Caillard S, Dharnidharka V, Agodoa L, et al. Posttransplant lymphoproliferative disorders after renal transplantation in the United States in era of modern immunosuppression. Transplantation. 2005;80:1233–43 5. Caillard S, Lelong C, Pessione F, et al. Post-transplant lymphoproliferative disorders occurring after renal transplantation in adults: report of 230 cases from the French registry. Am J Transplant. 2006;6:2735–42 6. Shapiro R, Nalesnik M, McCauley J, et al. Posttransplant lymphoproliferative disorders in adult and pediatric renal transplant patients receiving tacrolimus-based immunosuppression. Transplantation. 1999;68:1851–4 7. Dharnidharka VR, Sullivan EK, Stablein DM, et al. Risk factors for posttransplant lymphoproliferative disorder (PTLD) in pediatric kidney transplantation: a report of the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS). Transplantation. 2001;71:1065–8 8. McDonald RA, Smith JM, Ho M, et al. Incidence of PTLD in pediatric renal transplant recipients receiving basiliximab, calcineurin inhibitor, sirolimus and steroids. Am J Transplant. 2008;8:984–9 9. Opelz G, Naujokat C, Daniel V, et al. Disassociation between risk of graft loss and risk of nonHodgkin lymphoma with induction agents in renal transplant recipients. Transplantation. 2006; 81:1227–33 10. Bakker NA, Van Imhoff GW, Verschuuren EA, et al. Early onset post-transplant lymphoproliferative disease is associated with allograft localization. Clin Transplant. 2005;19:327–34 11. Hestin D, Claudon M, Champigneulles J, et al. Epstein–Barr-virus-associated post-transplant B-cell lymphoma presenting as allograft artery stenosis. Nephrol Dial Transplant. 1996;11: 1164–7 12. Kew CE II, Lopez-Ben R, Smith JK, et al. Postransplant lymphoproliferative disorder localized near the allograft in renal transplantation. Transplantation. 2000;69:809–14 13. Caillard S, Pencreach S, Braun L, et al. Simultaneous development of lymphoma in recipients of renal transplants from a single donor: donor origin confirmed by human leukocytes antigen staining and microsatellite analysis. Transplantation. 2005;79:79–84 14. Petit B, Le Meur Y, Jaccard A, et al. Influence of host-recipient origin on clinical aspects of posttransplantation lymphoproliferative disorders in kidney transplantation. Transplantation. 2002;73:265–71 15. Cosio FG, Nuovo M, Delgado L, et al. EBV kidney allograft infection: possible relationship with a peri-graft localization of PTLD. Am J Transplant. 2004;4:116–23 16. Vaysberg M, Balatoni CE, Nepomuceno RR, et al. Rapamycin inhibits proliferation of Epstein– Barr virus-positive B-cell lymphomas through modulation of cell-cycle protein expression. Transplantation. 2007;83:1114–21 17. Majewski M, Korecka M, Joergensen J, et al. Immunosuppressive TOR kinase inhibitor everolimus (RAD) suppresses growth of cells derived from posttransplant lymphoproliferative disorder at allograft-protecting doses. Transplantation. 2003;75:1710–7 18. El-Salem M, Raghunath PN, Marzec M, et al. Constitutive activation of mTOR signaling pathway in post-transplant lymphoproliferative disorders. Lab Invest. 2007;87:29–39

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19. Zaltzman JS, Prasad R, Chun K, et al. Resolution of renal allograft-associated post-transplant lymphoproliferative disorder with the introduction of sirolimus. Nephrol Dial Transplant. 2005;20:1748–51 20. Cullis B, D’Souza R, McCullagh P, et al. Sirolimus-induced remission of posttransplantation lymphoproliferative disorder. Am J Kidney Dis. 2006;47:e67–72 21. Pascual J. Post-transplant lymphoproliferative disorder–the potential of proliferation signal inhibitors. Nephrol Dial Transplant. 2007;22(Suppl 1):i27–35 22. Kirk AD, Cherikh WS, Ring M, et al. Dissociation of depletional induction and posttransplant lymphoproliferative disease in kidney recipients treated with alemtuzumab. Am J Transplant. 2007;7:2619–25 23. Funch DP, Walker AM, Schneider G, et al. Ganciclovir and acyclovir reduce the risk of posttransplant lymphoproliferative disorder in renal transplant recipients. Am J Transplant. 2005;5: 2894–900 24. Choquet S, Leblond V, Herbrecht R, et al. Efficacy and safety of rituximab in B-cell posttransplantation lymphoproliferative disorders: results of a prospective multicenter phase 2 study. Blood. 2006;107:3053–7 25. Choquet S, Oertel S, Leblond V, et al. Rituximab in the management of post-transplantation lymphoproliferative disorder after solid organ transplantation: proceed with caution. Ann Hematol. 2007;86:599–607 26. Gross TG, Hinrichs SH, Winner J, et al. Treatment of post-transplant lymphoproliferative disease (PTLD) following solid organ transplantation with low-dose chemotherapy. Ann Oncol. 1998;9:339–40 27. Fohrer C, Caillard S, Koumarianou A, et al. Long-term survival in post-transplant lymphoproliferative disorders with a dose-adjusted ACVBP regimen. Br J Haematol. 2006;134:602–12 28. Choquet S, Trappe R, Leblond V, et al. CHOP-21 for the treatment of post-transplant lymphoproliferative disorders (PTLD) following solid organ transplantation. Haematologica. 2007;92: 273–4 29. Trofe J, Buell JF, Beebe TM, et al. Analysis of factors that influence survival in post-transplant lymphoproliferative disorder in renal transplant recipients: the Israel Penn Transplant Tumor Registry experience. Am J Transplant. 2005;5:775–80 30. Birkeland SA, Hamilton-Dutoit S, Bendtzen K. Long-term follow-up of kidney transplant patients with posttransplant lymphoproliferative disorder: duration of posttransplant lymphoproliferative disorder-induced operational graft tolerance, interleukin-18 course, and results of retransplantation. Transplantation. 2003;76:153–8 31. Karras A, Thervet E, Meur YL, et al. Successful renal retransplantation after post-transplant lymphoproliferative disease. Am J Transplant. 2004;4:1904–9 32. Johnson SR, Cherikh WS, Kauffman HM, et al. Retransplantation after post-transplant lymphoproliferative disorders: an OPTN/UNOS database analysis. Am J Transplant. 2006;6:2743–9

Posttransplantation Lymphoproliferative Disorder (PTLD) in Liver and Small Bowel Transplant Recipients

11.b

Jaime Pineda and George V. Mazariegos

Core Messages

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The incidence, morbidity, and mortality of PTLD in liver and intestine transplantations have significantly decreased over time. Incidence of PTLD in pediatric liver transplantation is currently 2–3%, and is approaching the 1–2% rate of PTLD seen after adult liver transplantation. Improvements in immunosuppression, EBV monitoring, pre emptive therapy, and treatment have led to this reduction in incidence as well as to a concomitant reduction in mortality. The incidence of PTLD in pediatric intestine transplantation has been dramatically reduced from historical rates of 30–40% to approximately 8–10%. PTLD occurs in approximately 13% of children as compared to 5% in adults. Therapy for PTLD differs based on the transplanted organ. In liver transplantation, cessation of immunosuppression is recommended as the mainstay of therapy for PTLD. In intestinal transplantation, the extent of the lowering of immunosuppression is limited by the immunogenicity of the transplanted bowel. Antiviral therapies with ganciclovir and intravenous immune globulin (IVIG) are used in both instances. Additional therapies such as rituximab (anti CD20 antibody) and low dose chemotherapy options have significantly contributed to improved outcome after PTLD in intestine transplantation.

G. V. Mazariegos (*) Hillman Center for Pediatric Transplantation, Children’s Hospital of Pittsburgh (CHP), Thomas E. Starzl Transplantation Institute, University of Pittsburgh School of Medicine, 3705 Fifth Avenue, Suite 7950, Pittsburgh, PA 15213, USA e-mail: [email protected] V. R. Dharnidharka et al. (eds.), Post-Transplant Lymphoproliferative Disorders, DOI: 10.1007/978-3-642-01653-0_11.b, © Springer Verlag Berlin Heidelberg 2010

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11.b.1 Introduction Posttransplant lymphoproliferative disease (PTLD) has been an important cause for morbidity, mortality, and one of the most serious complications after adult and pediatric transplant for more than 40 years. As defined earlier in this textbook, this polyclonal or monoclonal lymphoid proliferation of B cells seen after transplant is related to the immunosuppression burden and is often associated with Epstein–Barr virus (EBV) [1]. The rapid development of liver and intestine transplantation has been due, in large part, to the development of immunosuppression regimens to control the recipient’s immune system from rejecting the allograft. In particular, the development of calcineurin inhibitors such as cyclosporine and tacrolimus rapidly advanced early patient and graft survival. As expected, immunosuppression has been associated with multiple medical, infectious, and malignant complications including PTLD. It was noted early on that regression of PTLD after withdrawal or tapering of immunosuppression in solid organ transplantation was possible in recipients of heart, liver, and kidney grafts [2]. The American Society of Transplant Surgeons (ASTS) and the American Society of Transplant Physicians (ASTP) developed a classification system for PTLD to avoid inconsistencies in reports, definition, and management [3]. Classification System: 0. EBV lymphadenitis, hepatitis, not classified as PTLD. 1. Early lesion, low-grade mononucleosis, plasma cell hyperplasia. 2. Polymorphic, diffuse B cell hyperplasia (PDBH) and polymorphic B cell lymphoma (PBC). 3. Monomorphic or Lymphomatous PTLD or lymphoma, Immunoblastic Lymphoma (IBL), diffuse large B cell or diffuse small cell noncleaved (Burkitt-like). 4. Other Hodgkin’s-like PTLD, plasma cell lesions, plasmacytoma, T-cell PTLD. In this chapter, we will focus on describing the current incidence as well as risk factors, characteristics, management, morbidity, prognosis, and mortality of PTLD following liver and small bowel transplant. We will also highlight important differences between the therapeutic approaches to both scenarios and the differences between children and adults.

11.b.2 PTLD Following Liver Transplantation 11.b.2.1 Incidence Historically, the incidence of PTLD following liver transplantation has been reported to be up to 20% in children and 2–4% in adults [1, 4, 5]. One of the largest series was reported

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by Jain et al. who studied 4,000 liver transplant patients [6] and found a global incidence of 4.3% (170 patients). They reported PTLD in 9.7% of the children studied and in 2.9% of the adult population included. Most of the cases presented 2 months after transplantation and almost all the cases occurred within the first 2 years. As expected due to a higher percentage of EBV seronegative patients in the pediatric population at the time of transplantation, a higher incidence of PTLD was found in children when compared to adults. Another large single center experience focusing on adult transplantation [7] also showed an incidence of PTLD of 3%. The Scientific Registry of Transplant Recipients (SRTR) analyzed the number of first-time liver-only transplant recipients diagnosed with PTLD between 1998–2007 and found 144 cases of PTLD in 4,429 (3.25%) pediatric (age 0–17) recipients, and 314 cases in 43,960 (0.7%) adult recipients aged 18 and older (Source: Analysis prepared by the SRTR, November, 2008). With the advent of preemptive modulation of immunosuppression and the possible role of antiviral therapy [8] and EBV monitoring using PCR [9], the incidence of PTLD in children undergoing liver transplant has decreased to 2–3%. Ganshow et al. [10] have also recently reported incidences of PTLD between 1–2%, similar to the authors’ current experience. Multicenter pediatric data are limited to reports of the Studies in Pediatric Liver Transplantation (SPLIT) registry. The SPLIT database has prospectively recorded transplant outcomes in children transplanted at over 40 participating centers in the US and Canada. Through 1 June 2008, 2,997 recipients of primary liver transplants have been enrolled (SPLIT 2008 Annual Report, personal communication). The prevalence of PTLD has declined from 3.7% in children transplanted in years up to 1999 to 3% in those who were transplanted between 2000 and 2003. Overall, 78/2,997 (2.6%) developed PTLD.

11.b.2.2 Risk Factors Several risk factors render the pediatric population at a higher risk for the development of PTLD. Age, recipient EBV seronegativity, long-term immunosuppression, type of allograft, and primary EBV infection [11] make this specific population more susceptible. PTLD is the most common posttransplantation tumor in children. The highest incidence is seen in children younger than 5 years of age, and progressively declines thereafter. Age is not the only factor affecting the development of this condition. Indeed, the type of allograft also plays a role and it has been shown that liver transplants have lower risk of developing PTLD compared to heart, lung, or small bowel transplantation. This may be explained by the fact that liver transplantation typically requires less maintenance immunosuppression than the other types of transplant. Furthermore, liver transplant recipients typically have a wider therapeutic window to lower immunosuppression without rejection risk in cases of infection, drug related morbidities, or malignancy. The most important risk factors are immunosuppression and EBV status. The cumulative use of calcineurin inhibitors, or the additional use of OKT3 and antithymocyte globulin to treat refractory rejection, though rare, increase PTLD risk. Use of mycophenolate mofetil, sirolimus, and anti-IL2 receptor monoclonal antibodies has not been clearly documented to increase the incidence of PTLD [12]. As mentioned earlier, EBV plays a central

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role in the pathogenesis of PTLD. In liver and intestine transplant, EBV mismatch between the donor and recipient status greatly increases PTLD risk. Interestingly, in the report by Allen looking at PTLD among heart, lung, liver, and kidney recipients, 27% of the cases of PTLD occurred in EBV-positive recipients [13]. The impact of underlying disease on incidence of PTLD is controversial. For example, some single center data have reported an increased incidence of PTLD in hepatitis C patients [14], while another large series have not reported any difference in incidence among this population [6].

11.b.2.3 Presentation PTLD in liver transplant recipients most frequently affects lymphatic nodes. Almost 50% of the cases involve the lymph nodes; the graft and the gastrointestinal tract are the next most common sites. Other affected locations include the tonsils, adenoids, spleen, lungs, kidneys, bone marrow, or the central nervous system (CNS). Most patients have a singlesite involvement at the time of presentation. In an SRTR analysis of 220 patients diagnosed with extranodal PTLD from 1998–2007, the most common extra nodal sites were small intestine 15.9%, lung 15.5%, colon 14.5%, liver 14.1%, stomach 10%, and allograft 5.9%. Bone marrow and CNS involvement were noted in 5.7 and 3.2% of patients, respectively. (Source: Analysis prepared by the SRTR, November, 2008). Symptoms are very unspecific, ranging from cases presenting with mononucleosis-like syndromes (including fever, night sweats, weight loss, and fatigue) to other presentations featuring lymphadenitis, otitis media, and tonsillitis. Cases involving the gastrointestinal tract typically present with nausea, abdominal pain, hemorrhage, perforation, or symptoms of an acute abdomen, while CNS presentations may be with seizures and signs of mass effect. Most cases of PTLD in the current era present with slowly developing symptoms; fulminant presentation of PTLD in a compliant patient undergoing routine follow-up is rare. In the SPLIT registry, 81% of the EBV-associated PTLD occurred in children between the ages of 6 months to 5 years (SPLIT 2008 Annual Report) and the majority of the cases had presented within 12 months posttransplant. The median time to presentation was 11.2 ± 1.2 months. The rate of PTLD was 3.9% in children aged 6 months to 5 years as compared to 0.87% in patients who are of 5 years or older at transplant. Only one of the children reported in the SPLIT database developed non-EBV-associated PTLD. Presentation differs between children and adults in that adults tend to present later posttransplant and are more likely to present with monoclonal or malignant disease. In the series reported by Kremers, for example, there was a cumulative incidence of PTLD after adult liver transplant of 1.1% at 18 months and 4.7% at 15 years [7]. In this series, PTLD within 4 years of transplant was more likely to be EBV-related, whereas PTLD occurring thereafter was most likely EBV-negative. Diagnostic strategies include appropriate imaging of the brain, thorax, abdomen, and pelvis, bone marrow aspirates if indicated, EBV PCR, lymphocyte subpopulation studies, and most importantly, the biopsy of the nodules or of the mass [15, 16] if possible.

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11.b.2.4 Treatment Treatment strategies have been changing for the last 30 years. However, reduction or discontinuation of immunosuppression continues to be the mainstay of therapy. It is imperative to begin therapy as soon as possible and maintain close observation and follow-up to assure adequacy of the treatment.

11b.2.4.1 Reduction or Discontinuation of Immunosuppression and Antiviral Therapies Hurwitz et al. discontinued immunosuppression in all of patients with PTLD and in most of their pediatric liver recipients with EBV infection and reported an overall survival of 84% [17]. Our current practice is to withdraw immunosuppression in cases of confirmed PTLD following liver transplant. Antiviral therapies with intravenous ganciclovir and intravenous immune globulin (IVIG) are begun, while disease size and symptoms are assessed with physical examination or serial imaging. Frequent assessment of liver injury tests (bilirubin, AST, ALT, and GGT) is followed and percutaneous liver biopsy is initiated for any abnormal results. One caveat of our practice is to not resume immunosuppression until there is evidence of biopsy confirmed rejection. In this manner, Hurwitz, as well as our group and others, has shown that a significant number of patients have been able to remain off immunosuppression for extended periods of time, without graft loss [18, 19]. Although several reports show that polymorphic PTLDs, rather than monomorphic PTLDs, and early cases of PTLD (< than 2 years after transplantation) respond better to the reduction or discontinuation of immunosuppression [20], most would advocate cessation of immunosuppression as appropriate initial therapy in all cases.

11.b.2.4.2 Surgery and Radiotherapy Surgery is generally indicated to obtain histologic sampling of pathologic tissue. Hemorrhage, perforation, or bowel obstruction are rarely the indications for emergent surgery. Radiotherapy is considered a complement to systemic treatment, but generally is limited to cases of PTLD involving the CNS.

11.b.2.4.3 Monoclonal Antibodies This therapeutic approach is focused on targeting B cell derived PTLD that express CD20 surface antigen. Rituximab (Rituxan; Genetech Inc., San Francisco and IDEL Pharmaceutical Corp., San Diego, CA) is a chimeric monoclonal antibody against CD20 that can be used in patients with CD20 positive PTLD who have failed to respond to conventional therapy

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[21, 22]. In these series, rituximab has primarily been used as rescue therapy when primary withdrawal of immunosuppression has failed [22] and less commonly in conjunction with immunosuppression withdrawal or for prophylaxis [21, 22].

11.b.2.4.5 Conventional Chemotherapy Chemotherapy can be used when the initial treatment has failed or in patients with late-onset monoclonal PTLD. Medications used to provide this alternative are cyclosphosphamide, doxorubicin, etoposide, cytarabine, bleomycin, vincristine, methotrexate, and corticosteroids. [23, 24]. One major limitation of traditional intensive multidrug chemotherapy regimens has been toxicity and resultant infection. Therefore, a low dose regimen consisting of cyclophosphamide (600 mg/m2) and prednisone (2 mg/kg × 5 days) given every 3 weeks was developed by Gross and colleagues [25]. Three patients with disseminated, resistant PTLD treated under this regimen showed remission at 26, 22, and 15 months of follow-up, indicating this approach may have merit for the patients with refractory disease.

11.b.2.5 Prognosis Historically, PTLD was associated with a high mortality approaching 30–40% [17]. Other reports have documented improved long-term mortality from PTLD at 12–14% [4, 11]. In the SPLIT registry, only 2 (2.6%) of 78 children with PTLD had died and 74.4% had complete resolution with 23% having unresolved disease at the time of the annual report (10 with less than 12 month follow-up and 8 with greater than 12 month follow-up) (SPLIT 2008 Annual Report). Adult presentation may also carry a worse prognosis than PTLD in children because of the frequency of nonEBV-associated PTLD. In the series reported by Jain and Nalesnik et al., 15-year long-term survival was 60% for children with PTLD compared to 39% for adults (P = 0.06) [6]. Survival is highly dependant on the type of PTLD (monomorphic vs. polymorphic) and on the number of involved organs. Worst prognosis has been reported with monomorphic PTLD vs. polymorphic PTLD and when multiple organs are affected. EBV-negative PTLD also responds less consistently to withdrawal of immunosuppression and requires more rapid assessment for chemotherapeutic options.

11.b.3 PTLD Following Small Bowel Transplantation Intestine transplantation has been performed as a surgical strategy for treatment of adults and children with surgical loss of intestine (from inflammatory bowel disease, volvulus, gastroschisis, necrotizing enterocolitis, intestinal atresia, etc.) or dysfunctional bowel disorders

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(pseudo-obstruction, microvillus inclusion disease, tufting enteropathy, etc.)[26]. The increased immune requirements of the intestinal transplant recipient have correlated with the potential for important complications such as graft rejection, infections, or posttransplant lymphoproliferative disorder (PTLD).

11.b.3.1 Incidence The incidence of PTLD after intestinal transplantation has been reported to be between 15–20% [27]. More recently, rates of PTLD have fallen to 8–10% [28–31], reflecting advances in prophylaxis, prevention, and management of EBV disease. The Intestinal Transplant Registry (data provided on request at www.intestinetransplant.org) documents intestine transplant activity on a worldwide basis. In analysis from 1985 to 5 September 2007, 1,720 intestine transplants had been performed in 1,608 patients. 168 patients (9.8%) developed PTLD. 50% of the cases developed in the era 1991–2000, 30.4% developing in the era 2001–2004, and 17.9% of the reported cases during the recent era of 2005 to present. The incidence in patients under the age of 18 was 13.2% compared to 5.1% in the adult population.

11.b.3.2 Risk Factors EBV seropositive status is not protective for EBV infection or PTLD after intestine transplantation. For this reason, monitoring of viral loads is recommended for both seronegative and seropositive patients. Monitoring of EBV loads in peripheral blood has been effective in preventing PTLD because low viral loads have been associated with very low risk of developing PTLD. Cumulative immunosuppression and the use of OKT3 in the treatment of refractory rejection have been associated with increased PTLD risk after intestine transplantation [32, 33].

11.b.3.3 Presentation As in liver transplantation, the presentation of PTLD is very variable and nonspecific. Affected sites most often include the transplanted bowel, lymphatic tissue, as well as the liver. PTLD may present as gastrointestinal tract ulcers [34] at endoscopy and lead to bacteremia because of disruption of the mucosal barrier [35]. Of the 168 patients reported in the Intestine Transplant Registry, PTLD was more common in the isolated small bowel (35.7% of reported cases) and the liver small bowel (41.7%) than the multivisceral (22.6%) recipient.

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11.b.3.4 Treatment Mainstay of therapy includes reduction of immunosuppression dose by 25–50%. In contrast to liver transplantation, complete withdrawal of immunosuppression is not possible because rejection of the small bowel is much more likely; therefore, our usual practice is to maintain steroids at lower baseline and lower tacrolimus therapy to levels of 5 mcg/dl, while maintaining antiviral therapy with ganciclovir and IVIG. Frequent endoscopy is performed every 1–2 weeks to monitor antigraft activity, and EBV PCR weekly measurements are used to monitor viral activity. As opposed to experience in other organ transplant recipients, it is not infrequent that rejection may occur while PTLD is resolving or still active. For this reason, rituximab (anti-CD 20 monoclonal antibody) and low dose chemotherapy regimens have been more commonly used, often with successful outcomes [36, 37]. Under current treatment algorithms, mortality after PTLD in intestine transplant has decreased to 10–14% [27] in the era of current monitoring and availability of therapies such as rituximab. In the Intestine Transplant Registry, 54.2% of the patients were reported to have resolved their PTLD, while 6.5% had ongoing PTLD. 39.2% of patients with PTLD subsequently died, but 10-year survival was not different between those with PTLD and those without PTLD (data obtained on request from www.intestinetransplant.org).

11.b.4 Take Home Pearls

• • • • •

The incidence, morbidity, and mortality of PTLD in liver and intestine transplantation have significantly decreased over time. Incidence of PTLD in pediatric liver transplantation is currently 2–3%, and is approaching the 1–2% rate of PTLD seen after adult liver transplantation. Improvements in immunosuppression, EBV monitoring, pre emptive therapy, and treatment have led to this reduction in incidence, as well as to a concomitant reduction in mortality. The incidence of PTLD in pediatric and adult intestine transplantation has been dramatically reduced from historical rates of 30–40% to approximately 13 and 5%, respectively. Therapy for PTLD differs based on the transplanted organ. In liver transplantation, cessation of immunosuppression is recommended as the mainstay of therapy for PTLD. In intestinal transplantation, lowering of immunosuppression is more limited by the immunogenicity of the transplanted bowel. Antiviral therapies with gancyclovir and IVIG are used in both instances. Additional therapies such as rituximab (anti CD20 antibody) or low-dose chemotherapy options have significantly contributed to improved outcome after PTLD in intestine transplantation.

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References 1. Avolio A, Agnes S, Barbarino R, et al. Post transplant lymphoproliferative disorder after liver transplantation: analysis of early and late cases in a 255 patient series. Transplant Proc. 2007;39:1956 2. Starzl TE, Nalesnik MA, Porter KA, et al. Reversibility of lymphomas and lymphoproliferative lesions developing under cyclosporin-steroid therapy. Lancet. 1984;1(8377):583–7 3. Paya C, Fung J, Nalesnik M, et al. Epstein–Barr virus-induced post transplant lymphoproliferative disorder. ASTS/ASTP EBV-PTLD task force and the mayo clinic organized international consensus development meeting. Transplantation. 1999;68:1517 4. D’Alessandro A, Knechtle S, Thomas Chin L, et al. Liver transplantation in pediatric patients: twenty years of experience at the University of Wisconsin. Pediatr Transplant. 2007;11:661 5. Jain A, Mazariegos G, Kashyap R, et al. Pediatric liver transplantation. Transplantation. 2002;73:941–7 6. Jain A, Nalesnik M, Reyes J, et al. Post transplant lymphoproliferative disorder in liver transplantation a 20-years experience. Ann Surg. 2002;236(4):429–37 7. Kremers WK, Devarbhavi HC, Wiesner RH, et al. Post-transplant lymphoproliferative disorders following liver transplantation: incidence, risk factors and survival. Am J Transplant. 2006;6(5 Pt 1):1017–24 8. McDiarmid SV, Jordan S, Lee G, et al. Prevention and preemptive therapy of post transplant lymphoproliferative disease in pediatric liver recipients. Transplantation. 1991;66(12):1604–11 9. Lee T, Savoldo B, Rooney C, et al. Quantitative EBV viral loads and immunosuppression alterations can decrease PTLD incidence in pediatric liver transplant recipients. Am J Transplant. 2005;5:2222–8 10. Ganschow R, Grabhorn E, Schultz A, et al. Long-term results of basiliximab induction immunosuppression in pediatric liver transplant recipients. Pediatr Transplant. 2005;9(6):741–5 11. Cacciarelli TV, Reyes J, Jaffe R, et al. Primary tacrolimus (FK506) therapy and the long-term risk of post-transplant lymphoproliferative disease in pediatric liver transplant recipients. Pediatr Transplant. 2001;5:359–64 12. Guthery S, Heubi J, Bucuvalas J, et al. Determination of risk factors for Epstein–Barr virus associated posttransplant lymphoproliferative disorder in pediatric liver transplant recipients using objective case ascertainment. Transplantation. 2003;75:987–93 13. Allen U, Farkas G, Hebert D, et al. Risk factors for post-transplant lymphoproliferative disorder in pediatric patients: a case-control study. Pediatr Transplant. 2005;9:450–5 14. McLaughlin K, Wajstaub S, Marotta P, et al. Increased risk for posttransplant lymphoproliferative disease in recipients of liver transplants with hepatitis C. Liver Transplant. 2000;6:570–4 15. Fyle A, Vilmer E. Post-transplant lymphoproliferative disorder in children. Incidence, prognosis and treatment options. Pediatr Drug. 2005;7:55–65 16. Patel H, Vogl D, Aqui N, et al. Post transplant lymphoproliferative disorder in adult liver transplant recipients: a report of seventeen cases. Leuk Lymphoma. 2007;48:885–91 17. Hurwitz M, Desai DM, Cox KL, et al. Complete immunosuppressive withdrawal as a uniform approach to post-transplant lymphoproliferative disease in pediatric liver transplantation. Pediatr Transplant. 2004;8:267–72 18. Mazariegos G. Withdrawal of immunosuppression in liver transplantation: lessons learned from PTLD. Pediatr Transplant. 2004;8:210–3 19. Mazariegos G, Sindhi R, Angus T, et al. Clinical tolerance following liver transplantation: long term results and future prospects. Transpl Immunol. 2007;17:114–9 20. Aucejo F, Profile G, Miller C. Who is at risk for post-transplant lymphoproliferative disorders (PTLD) after liver transplantation? J Hepatol. 2006;44:19–23

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21. Hayashida M, Ogita K, Matsuura T, et al. Successful prolonged rituximab treatment for posttransplant lymphoproliferative disorder following living donor liver transplantation in a child. Pediatr Transplant. 2007;11:671–5 22. Jain A, Marcos A, Pokharma R, et al. Rituximab (Chimeric Anti-CD20 Antibody) for post transplant lymphoproliferative disorder after solid organ transplantation in adults: long-term experience from a single center. Transplantation. 2005;80:1692–8 23. Dufour J, Fey M. What is the current treatment of PTLD after liver transplantation? J Hepatol. 2006;44:23–6 24. Smets F, Vajro P, Cornu G, et al. Indications and results of chemotherapy in children with post transplant lymphoproliferative disease after liver transplantation. Transplantation. 2000;69 (5):982–5 25. Gross TG, Hinrichs SH, Winner J, et al. Treatment of post-transplant lymphoproliferative disease (PTLD) following solid organ transplantation with low-dose chemotherapy. Ann Oncol. 1998;9:339–40 26. American Gastroenterological Association (AGA). American Gastroenterological Association medical position statement: short bowel syndrome and intestinal transplantation. Gastroenteroly. 2003;124:1105–10 27. Quintini C, Kato T, Gaynor JJ et al. Analysis of Risk factors for the development of posttransplant lymphoproliferative disorder among 119 children who received primary intestinal transplant at a single center. Transplant Proc. 2006;38:1755–8 28. Abu-Elmagd K, Reyes J, Bond G, et al. Clinical intestinal transplantation: a decade of experience at single center. Ann Surg. 2001;234(3):404–17 29. Bond G, Felmet K, Jafee R et al. Intestinal and multiviceral transplantation. In: Rogers ND, editor. Textbook of pediatric care. Philadelphia, PA: Lippincott Williams & Wilkins; 2008. p.1550–61 30. Bond G, Mazariegos G, Sindhi R, et al. Evolutionary experience with inmunosuppression in pediatric intestinal transplantation. J Pediatr Surg. 2005;40:274–80 31. Pascher A, Kohler S, Neuhaus P et al. Present status and future perspectives of intestinal transplantation. Transpl Int. 2008;21:401–14 32. Green M, Bueno J, Rowe D, et al. Predictive negative value of persistent low Epstein–Barr virus load after intestinal transplantation in children. Transplantation. 2000;70:593–6 33. Green M, Soltys K, Rowe DT, et al. Chronic high load Epstein–Barr viral load carriage in pediatric liver transplant recipients. Pediatr Transplant. 2008;13:319–23 34. Sarkar S, Selvaggi G, Mittal N, et al. Gastrointestinal tract ulcers in pediatric intestinal transplantation patients: etiology and management. Pediatr Transplant. 2006;10:162–16 35. Sigurdsson L, Reyes J, Kocoshis SA, et al. Bacteremia after intestinal transplantation in children correlates temporally with rejection or gastrointestinal lymphoproliferative disease. Transplantation. 2000;70(2):302–35 36. Berney T, Delis S, Kato T, et al. Successful treatment of poattransplant lymphoproliferative disease with prolonged rituximab treatment in intestinal transplant recipients. Transplantation. 2002;74:1000–6 37. Codeluppi M, Cocchi S, Guaraldi G, et al. Rituximab as treatment of posttransplant lymphoproliferative disorder in patients who underwent small bowel/multivisceral transplantation: report of three cases. Transplant Proc. 2005;37:2634–5

Heart and Lung Transplantation

11.c

Silke Wiesmayr and Steven A. Webber

Core Messages

› › › ›

The incidence of PTLD after heart and lung transplantation is higher than for all other types of solid organ transplantation other than intestinal The heart is almost never an involved organ; by contrast, lung involvement by PTLD is very common in heart, lung, and heart–lung recipients Clinical presentation of PTLD is highly variable and may mimic many other disease processes including rejection and (non EBV) infection No therapies are based on data from randomized clinical trials and current outcomes are suboptimal, both in adults and children

11.c.1 Introduction PTLD is a relatively common complication of thoracic transplantation in children. In adults, the incidence is much lower, but the absolute number of cases observed is higher due to the much larger number of transplant procedures performed annually in the adult population. Incidence, patterns of clinical presentation, and outcomes differ between heart and lung recipients, and therefore, each population is dealt with separately in this brief review. PTLD in combined heart–lung recipients tends to behave like PTLD after lung transplantation. This small group is, therefore, considered along with lung recipients. We also emphasize known differences between children and adults.

S. A.Webber (*) Division of Cardiology, Children’s Hospital of Pittsburgh, 45th St. at Penn Avenue Pittsburgh, PA 15201, USA e-mail: [email protected] V. R. Dharnidharka et al. (eds.), Post-Transplant Lymphoproliferative Disorders, DOI: 10.1007/978-3-642-01653-0_11.c, © Springer Verlag Berlin Heidelberg 2010

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11.c.2 PTLD After Heart Transplantation 11.c.2.1 Epidemiology The “incidence” of PTLD is approximately 3–4% after heart transplantation (all age groups combined) [1], but as discussed in Chap. 3, reporting of cases as a percentage of all transplants within a given time period can be misleading due to variable length of follow-up and the reduced risk for patients with short survival time. In adults, “incidence” has been reported within the range of 0.7–7% [1–7 ], but is probably nearer to 1–2% at present. The incidence is undoubtedly higher in children and is frequently quoted as 4–10% [1, 8–13 ]. This probably reflects an increased rate of posttransplant primary EBV infection in the pediatric cohort [9, 10, 13 ]. Finally, it should be noted that the inclusion of “early lesions” (plasmacytic hyperplasias and infectious mononucleosis-like lesions, see Chap. 7) in any study will significantly increase the incidence of PTLD and will improve survival since these lesions generally carry a good prognosis. Therefore, the definition of PTLD must be clear in all reports; unfortunately, this is rarely the case, especially for registry data. The most detailed information in children comes from the prospective database of the Pediatric Heart Transplant Study [12]. Among 1,184 primary transplants, 56 (5%) cases of PTLD were identified with a probability of freedom from PTLD of 98, 94, and 92% at 1, 3, and 5 years, respectively, after transplantation (Fig. 11.c.1a). The major risk factors for the development of PTLD are comparable to other organs, and it is prudent to note that the rare adult who is EBV seronegative at transplant will likely carry a risk comparable to the pediatric population. Such recipients almost invariably receive organs from a seropositive donor putting them at high risk for early posttransplant primary EBV infection. In both children and adults, the risk of developing PTLD after heart transplantation is highest in the first year after transplantation, but a continuous risk persists throughout posttransplant life.

11.c.2.2 Clinical Presentation Presentation of PTLD after heart transplantation is highly variable and clinical features of PTLD are described in detail in Chap. 6. The most specific observation in heart transplantation is that the graft is almost never involved, in stark contrast to all other solid organ transplants where the transplanted organ is a frequent site of disease involvement. In a large series from the Israel Penn Transplant Registry, the majority of patients (51%) had diffuse disease (two or more sites involved) at the time of diagnosis of PTLD and the most commonly affected sites were lymph nodes and lungs (34 and 32%), followed by gastrointestinal tract (24%), liver (23%), and central nervous system (13%) [3]. In the series from the Pediatric Heart Transplant Study, there were multiple sites of disease at presentation in 52% of cases, and the most commonly involved site was the gastrointestinal tract (39%), followed by respiratory system (25%) [12].

11.c Heart and Lung Transplantation

Fig. 11.c.1 (a) Probability of freedom from PTLD (upper curve) and associated hazard function (lower curve) among 1184 pediatric heart transplant recipients within the Pediatric Heart Transplant Study (PHTS). Dotted lines are 70% confidence intervals. (b) Survival after diagnosis of PTLD (N = 56) with hazard function in the same cohort

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Early onset disease, especially in children and following primary EBV infection, typically demonstrates polymorphic histology and is almost invariably EBV positive [12]. Monomorphic histology is seen more often in adults and with late-onset disease, and is more likely to be EBV negative than early-onset PTLD. Both histologic forms (and other rare forms of PTLD) have highly protean clinical presentations, with no specific presentation associated with histology, recipient age, EBV status, or time of onset after transplantation. Burkitt lymphoma may occur at any age, but accounted for almost 40% of all monomorphic disease in the Pediatric Heart Transplant Study (PHTS) study, a markedly higher proportion than in adult studies [12].

11.c.2.3 Treatment of PTLD The principles of treatment of PTLD are discussed in detail in Chap. 9. This section will only deal with particular issues relevant to the heart transplant population. As with other organs, lowering of immunosuppression has been the mainstay of treatment of PTLD after heart transplantation. This is particularly true in pediatric patients with polymorphic disease (which accounts for two thirds of cases) where resolution of disease is common with reduction of immunosuppression [12]. Antiviral agents and immunoglobulin preparations are

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also commonly used with polymorphic disease, though their efficacy is not proven. In adults, while data are limited, reduction in immunosuppression alone is generally considered to be less effective [14], possibly related to the higher incidence of monomorphic disease (most commonly with diffuse large B cell lymphoma [DLBCL] histology). Refractory polymorphic disease unresponsive to reduced immunosuppression can often be successfully treated with rituximab without the need for the addition of chemotherapy [15–19 ]. However, recurrence rates are approximately 20%. Management of monomorphic disease after heart transplantation is more challenging. Some cases will respond to reduced immunosuppression, especially early-onset EBV positive disease. However, because of poor response rates [14, 17 ], it has been suggested that rituximab should be used as first-line therapy in conjunction with reduced immunosuppression for CD20 positive monomorphic disease [17]. This has become common practice, though late outcomes appear less promising than originally hoped for [16]. Burkitt lymphoma after heart transplantation is an aggressive form of PTLD most commonly seen in children who are treated with chemotherapy with generally good results [11, 12 ]. EBV-negative monomorphic PTLDs have been considered to be less responsive to reductions in immunosuppression and have been associated with a worse prognosis [17]. This creates a rationale for not relying on reduced immunosuppression alone as primary therapy, though no specific treatment algorithms for this population have been developed. A key consideration in the treatment of all types of PTLD after heart transplantation is the risk of rebound acute rejection [12, 14 ]. In contrast to liver and kidney transplantation, there are no blood markers for acute rejection, and the first presentation of acute rejection after therapeutic reduction in immunosuppression for treatment of PTLD may be acute graft failure and sudden death. In the PHTS study, over 60% of children developed rebound acute rejection (within 6 months) when reduced immunosuppression was the initial treatment for PTLD, and a high rebound rejection rate has also been observed in adults [3, 14 ]. This concern for graft loss with reduced immunosuppression has led some authorities to recommend early use of chemotherapy irrespective of PTLD histology. The rationale being that chemotherapy will treat the PTLD while protecting against acute rejection. In general, however, chemotherapy is given for refractory disease, or for cases of overt malignancy such as Burkitt lymphoma and T cell lymphomas. Initial therapy for the DLBCL form of monomorphic PLTD has included reduction of immunosuppression, rituximab, or chemotherapy [17]. A large variety of chemotherapeutic regimens have been used including reduced intensity protocols (used in children [20]), or full intensity multi-drug regimens comparable to those used for treatment of acute lymphoma in the nontransplant setting [14, 21, 22 ]. No chemotherapeutic regimens have been compared to reduced immunosuppression or monoclonal antibodies in prospective clinical trials. In children, low dose regimens (with or without rituximab) have been applied to cases of refractory disease (mostly polymorphic) with good success, though with relapse rates similar to those when rituximab is used alone [19, 20 ]. While chemotherapy may protect the allograft from rejection, it has been noted that the complication rates of chemotherapy in transplant recipients are relatively high, especially infectious morbidity and mortality [17, 20 ], thereby supporting a trial of rituximab prior to chemotherapy. It is clear that there is no consensus at present and treatments

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tend to be based on institutional preference rather than knowledge of individual tumor behavior in response to different treatment regimens.

11.c.2.4 Outcomes Survival after PTLD diagnosis has been described in many studies. However, following heart transplantation, there is ongoing graft loss and mortality with an almost constant hazard (instantaneous risk) beyond the first year after transplantation, mostly due to chronic rejection. The true impact of PTLD on this “natural history” of the transplanted heart is not well defined in any study. In the PHTS cohort, probability of survival was 75% at 1 year, 68% at 3 years, and 67% at 5 years after diagnosis (Fig.11.c.1b). The highest risk of death being in the first 12–18 months after diagnosis. This far exceeded the expected death rate for a pediatric heart transplant cohort. Importantly, death from graft loss (including acute and chronic rejection) was as frequent as death from progressive PTLD [12]. In adults, similar outcomes as in children have been reported after diagnosis of PTLD [2–6 ]. Again, deaths are due to both failure to eliminate disease and from complications of therapy, including sepsis and graft loss. It is clear that advances in management of PTLD after heart transplantation must focus on strategies to protect the allograft as well as improved therapies for eradicating tumor.

11.c.3 PTLD After Lung and Heart–Lung Transplantation 11.c.3.1 Epidemiology The reported “incidence” of PTLD in lung and heart–lung transplant recipients varies between 1.8 and 20% [6, 23–27 ]. As would be expected, higher incidences have been reported in children [8, 24 ], likely reflecting the higher risk for primary posttransplant EBV infection. In both the Collaborative Transplant Study [28] and the UNOS/OPTN database [1], heart–lung recipients have a higher incidence of PTLD than either heart or lung recipients. Only intestinal transplant recipients appear to have a higher incidence of PTLD (see Chap. 3). Some reports suggest that PTLD in lung/heart–lung recipients occurs earlier than after other types of solid organ transplantation [26], and this may reflect the relatively greater amounts of immunosuppression used in this population of patients. It has also been speculated that a relatively high lymphoid load is transmitted with the graft, and this might increase the risk of donor transmission of EBV to an EBV naïve recipient. Among adults, other risk factors have been identified. In a large study of 400 lung transplant recipients, older age above 55 years was identified as a risk factor for PTLD, as well as chronic obstructive pulmonary disease as the underlying diagnosis leading to transplantation [27]. An equal distribution between polymorphic and monomorphic disease has been reported in adults [27], though children most often present with the so called “early lesions” or polymorphic disease.

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11.c.3.2 Clinical Presentation In contrast to heart transplant recipients, PTLD in lung and heart–lung recipients usually involves the allograft (64–80% of cases) [23–25, 27 ]. Early onset disease is more likely to be confined to the allograft [25]. Late PTLD tends to involve sites outside the lung allograft, especially the gastrointestinal tract (56% of cases in one series) [25]. As with other transplanted organs, the symptoms and signs of PTLD are protean. Even when disease is limited to the allograft, patterns of presentation are diverse. Identification of one of more pulmonary nodules on a routine surveillance chest radiograph in an asymptomatic recipient is not rare. In others, there may be more extensive and confluent infiltrates, often associated with dyspneoa, cough, hypoxia, and fever. The differential diagnosis is then broad and includes allograft rejection, as well as infection with viruses, bacteria, or fungi. Since the treatment of these entities is very different, the need for precise diagnosis is critical and urgent. Bronchoalveolar lavage, transbronchial or percutaneous biopsy, or even open lung biopsy is often indicated. For patients with advanced disease, these diagnostic procedures may lead to the need for mechanical ventilation. High EBV viral load in the peripheral blood is relatively sensitive for the diagnosis of PTLD, but is not specific. Unfortunately, some cases of PTLD are associated with low viral load, even when the disease is EBV-driven. A recent pilot study suggests that measurement of EBV load in bronchoalveolar lavage fluid may be even more sensitive than viral load measurements in peripheral blood [29]. As with all forms of PTLD, a tissue diagnosis is warranted.

11.c.3.3 Treatment There are no special therapies specifically indicated for lung transplant recipients with PTLD. The principles of management are similar to those of other organs (Chap. 9). However, the lung allograft is very immunogenic, and prolonged reductions in immunosuppression are generally poorly tolerated. Some cases of polymorphic disease, especially early onset disease in children, have been successfully treated with reduced immunosuppression alone [8]. However, in one large series of 400 adult lung or heart– lung transplant recipients, only one patient out of 10 responded to reduced immunosuppression [27]. Although there are no prospective treatment trials in this patient population, it seems reasonable to add rituximab to the therapeutic regimen for CD20 positive B cell PTLD when disease is bulky or when there is no rapid response to decreased immunosuppression [27, 30 ]. Others have suggested that rituximab be used as first-line therapy in the treatment of PTLD in lung recipients regardless of clinical, radiographic, or histologic findings at presentation [31]. It should be noted, however, that first dose infusion reactions, including an ARDS-type picture, are more common with rituximab in the setting of pre existing lung injury and hypoxemia. Therefore, careful monitoring, including continuous pulse oximetry, should be employed when rituximab is given to this population. Experience with chemotherapy in PTLD in lung recipients is very limited. Where there is

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severe lung injury and need for mechanical ventilation, chemotherapy is likely to be poorly tolerated. Comorbid infections should be sought in all cases of PTLD as these are common and may complicate recovery if not promptly treated. In particular, CMV infection is observed with increased frequency in patients with PTLD, and must be recognized and treated early.

11.c.3.4 Outcome Mortality is relatively high at all time points after lung/heart–lung transplantation. Therefore, it is exceedingly hard to quantify the excess mortality related to PTLD. It is even harder to quantify associated morbidity and impact on quality of life. In general, prognosis for PTLD is considered poor compared to other organs, and it is frequently stated that treatment (in the form of reduced immunosuppression) leads to acute rejection and obliterative bronchiolitis. Unfortunately, the latter is such a common complication of lung transplantation beyond the first year that it may be very hard to attribute the cause to PTLD. In general, most clinicians would agree that this is one of the most challenging groups of solid organ recipients to treat for PTLD, and results are suboptimal. Mortality rates have been reported from 20 to 50% after PTLD diagnosis, though none of these estimates take into account the “normal” expected mortality in this population [25, 27].

11.c.4 Take Home Pearls

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PTLD should be considered as part of the differential diagnosis in any unexplained illness that is not rapidly self-limiting in a heart or lung transplant recipient. Tissue (excision or core biopsy, not needle aspiration) is required for a complete diagnosis. PTLD in the lung may mimic rejection and infection; or both may occur concurrently. Therefore, treatment must be based on accurate diagnosis of all pathologies. Rebound rejection rates are high following reduction in immunosuppression, and deaths due to acute and chronic rejection are as common as death due to progressive PTLD. No therapies have been tested in randomized controlled trials. Rituximab appears to have a role in treating patients with refractory PTLD who have failed reduction in immunosuppression. Results appear best for polymorphic EBV positive disease. Monomorphic PTLD is less likely to respond to reduction in immunosuppression or rituximab, but treatment with chemotherapy carries significant morbidity. Long-term survival after diagnosis of PTLD remains relatively poor in both adults and children following thoracic transplantation.

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References 1. Dharnidharka VR, Tejani AH, Ho PL, Harmon WE. Post-transplant lymphoproliferative disorder in the United States: young Caucasian males are at highest risk. Am J Transplant. 2002; 2:993–8 2. Armitage JM, Kormos RL, Stuart RS, Fricker FJ, Griffith BP, Nalesnik M, Hardesty RL, Dummer JS. Posstransplant lymphoproliferative disease in thoracic organ transplant patients: ten years of cyclosporine-based immunosuppression. J Heart Lung Transplant. 1991;10: 877–86 3. Aull MJ, Buell JF, Trofe J, First MR, Alloway RR, Hanaway MJ, Wagoner L, Gross TG, Beebe T, Woodle ES. Experience with 274 cardiac transplant recipients with posttransplant lymphoproliferative disorder: a report from the Israel Penn International Transplant Tumor Registry. Transplantation. 2004;78:1676–82 4. Chen JM, Barr ML, Chadburn A, Frizzera G, Schenkel FA, Sciacca RR, Reison DS, Addonizio LJ, Rose EA, Knowles DM. Management of lymphoproliferative disorders after cardiac transplantation. Ann Thorac Surg. 1993;56:527–38 5. Crespo-Leiro MG, Alonso-Pulpón L, Vázquez de Prada JA, Almenar L, Arizón JM, Brossa V, Delgado JF, Fernandez-Yañez J, Manito N, Rábago G, Lage E, Roig E, Diaz-Molina B, Pascual D, Muñiz J. Malignancy after heart transplantation: incidence, prognosis and risk factors. Am J Transplant. 2008;8:1031–9 6. Gao SZ, Chaparro SV,Perlroth M, Montoya JG, Miller JL, DiMiceli S, Hastie T, Oyer PE, Schroeder J. Post-transplantation lymphoproliferative disease in heart and heart–lung transplant recipients: 30-year experience at Stanford University. J Heart Lung Transplant. 2003; 22: 505–14 7. Olivari MT, Diekman RA, Kabo SH, Braunlin E, Jamieson SW, Ring WS. Low incidence of neoplasia in heart and heart–lung transplant recipients receiving triple-drug immunosuppression. J Heart Transplant. 1990;9:618–21 8. Boyle GJ, Michaels MG, Webber SA, Knisely AS, Kurland G, Cipriani LA, Griffith BP, Fricker FJ. Posttransplantation lymphoproliferative disorders in pediatric thoracic organ recipients. J Pediatr. 1997;131:309–13 9. Harwood JS, Gould FK, McMaster A, Hamilton JR, Corris PA, Hasan A, Gennery AR, Dark JH. Significance of EBV status and post-transplant lymphoproliferative disease in pediatric thoracic transplantation. Pediatr Transplant. 1999;3:100–103 10. Katz BZ, Pahl E, Crawford SE, Kostyk MC, Rodgers S, Seshadri R, Proytcheva M, Pophal S. Case-control study of risk factors for the development of post-transplant lymphoproliferative disease in a pediatric heart transplant cohort. Pediatr Transplant. 2007;11:58–65 11. Schubert S, Abdul-Khaliq H, Lehmkuhl HB, Yegitbasi M, Reinke P, Kebelmann-Betzig C, Hauptmann K, Gross-Wieltsch U, Hetzer R, Berger F. Diagnosis and treatment of post-transplantation lymphoproliferative disorder in pediatric heart transplant patients. Pediatr Transplant. 2009;13:54–62 12. Webber SA, Naftel DC, Fricker FJ, Olesnevich P, Blume ED, Addonizio L, Kirklin JK, Canter CE. Lymphoproliferative disorders after paediatric heart transplantation: a multi-institutional study. Lancet. 2006;367:233–9 13. Zangwill SD, Hsu DT, Kichuk MR, Garvin JH, Stolar CJ, Haddad J Jr., Stylianos S, Michler RE, Chadburn A, Knowles DM, Addonizio LJ. Incidence and outcome of primary Epstein–Barr virus infection and lymphoproliferative disease in pediatric heart transplant recipients. J Heart Lung Transplant. 1998;17:1161–6 14. Swinnen LJ, LeBlanc M, Grogan TM, Gordon LI, Stiff PJ, Miller AM, Kasamon Y, Miller TP, Fisher RI. Prospective study of sequential reduction in immunosuppression, interferon alpha2B and chemotherapy for posttransplant lymphoproliferative disorder. Transplantation. 2008; 86:215–22

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15. Choquet S, Leblond V, Herbrecht R, Socié G, Stoppa AM, Vandenberghe P, Fischer A, Morschhauser F, Salles G, Feremans W, Vilmer E, Peraldi MN, Lang P, Lebranchu Y, Oksenhendler E, Garnier JL, Lamy T, Jaccard A, Ferrant A, Offner F, Hermine O, Moreau A, Fafi-Kremer S, Morand P, Chatenoud L, Berriot-Varoqueaux N, Bergougnoux L, Milpied N. Efficacy and safety of rituximab in B-cell post-transplantation lymphoproliferative disorders: results of a prospective multicenter phase 2 study. Blood. 2006;107:3053–7 16. Choquet S, Oertel S, Leblond V, Riess H, Varoqueaux N, Dorken B, et al. Rituximab in the management of post-transplantation lymphoproliferative disorder after solid organ transplantation: proceed with caution. Ann Hematol. 2007;86:599–607 17. Elstrom RL, Andreadis C, Aqui NA, Ahya VN, Bloom RD, Brozena SC, Olthoff KM, Schuster SJ, Nasta SD, Stadtmauer EA, Tsai DE. Treatment of PTLD with rituximab or chemotherapy. Am J Transplant. 2006;6:569–76 18. Verschuuren EA, Stevens SJ, van Imhoff GW, Middeldorp JM, de Boer C, Koëter G, The TH, van Der Bij W. Treatment of posttransplant lymphoproliferative disease with rituximab: the remission, the relapse, and the complication. Transplantation. 2002;73:100–4 19. Webber S, Harmon W, Faro A, Green M, Sarwal M, Hayashi R, Canter C, Thomas D, Jaffe R, Fine R. Anti-CD20 monoclonal antibody (Rituximab) for refractory PTLD after pediatric solid organ transplantation: multicenter experience from a registry and from a prospective clinical trial. Blood. 2004;104–1:213a 20. Gross TG, Bucuvalas JC, Park JR, Greiner TC, Hinrich SH, Kaufman SS, Langnas AN, McDonald RA, Ryckman FC, Shaw BW, Sudan DL, Lynch JC. Low-dose chemotherapy for Epstein–Barr virus-positive post-transplantation lymphoproliferative disease in children after solid organ transplantation. J Clin Oncol. 2005;23:6481–8 21. Choquet S, Trappe R, Leblond V, Jager U, Davi F, Oertel S. CHOP-21 for the treatment of post-transplant lymphoproliferative disorders (PTLD) following solid organ transplantation. Haematologica. 2007;92:273–4 22. Hayashi RJ, Kraus MD, Patel AL, Canter C, Cohen AH, Hmiel P, Howard T, Huddleston C, Lowell JA, Mallory G Jr., Mendeloff E, Molleston J, Sweet S, DeBaun MR. Posttransplant lymphoproliferative disease in children: correlation of histology to clinical behavior. J Pediatr Hematol Oncol. 2001;23:14–18 23. Aris RM, Maia DM, Neuringer IP, Gott K, Kiley S, Gertis K, Handy J. Post-transplant lymphoproliferative disorder in the Epstein–Barr virus naive lung transplant recipient. Am J Respir Crit Care Med. 1996;154:1712–7 24. Cohen AH, Sweet SC, Mendeloff E, Mallory GB Jr., Huddleston CB, Kraus M, Kelly M, Hayashi R, DeBaun MR. High incidence of posttransplant lymphoproliferative disease in pediatric patients with cystic fibrosis. Am J Respir Crit Care Med. 2000;161:1252–5 25. Paranjothi S, Yusen RD, Kraus MD, Lynch JP, Patterson GA, Trulock EP. Lymphoproliferative disease after lung transplantation: comparison of presentation and outcome of early and late cases. J Heart Lung Transplant. 2002;20:1054–63 26. Ramalingam P, Rybicki L, Smith MD, Abrahams NA, Tubbs RR, Pettay J, Farver CF, Hsi ED. Posttransplant lymphoproliferative disorders in lung transplant patients: the Cleveland clinic experience. Mod Pathol. 2002;15:647–56 27. Reams BD, McAdams HP, Howell DN, Steel MP, Davis RD, Palmer SM. Posttransplant lymphoproliferative disorder: incidence, presentation, and response to treatment in lung transplant recipients. Chest. 2003;124:1242–9 28. Opelz G, Dohler B. Lymphomas after solid organ transplantation. A collaborative study report. Am J Transpl. 2003;4:222–30 29. Michelson PH, Watkins B, Webber SA, Wadowsky RM, Michaels MG. Screening for PTLD in lung and heart–lung transplant recipients by measuring EBV DNA load in bronchoalveolar lavage fluid using real time PCR. Pediatr Transplant. 2008;12:464–8

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30. Cook RC, Connors JM, Gascoyne RD, Fradet G, Levy RD. Treatment of post-transplant lymphoproliferative disease with rituximab monoclonal antibody after lung transplantation. Lancet. 1999;354:1698–9 31. Knoop C, Kentos A, Remmelink M, Garbar C, Goldman S, Feremans W, Estenne M. Posttransplant lymphoproliferative disorders after lung transplantation: first-line treatment with rituximab may induce complete remission. Clin Transplant. 2006;20:179–87

Posttransplant Lymphoproliferative Disease (PTLD) in Hematopoietic Stem Cell Transplantation (HSCT)

11.d

Thomas G. Gross

Core Messages

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Posttransplant lymphoproliferative disorders (PTLD) following hematopoietic stem cell transplantation (HSCT) is nearly 100% associated with EBV and occurs 3–6 months posttransplant. T cell depletion (TCD) of the hematopoietic stem cell graft (ex vivo or in vivo) is the strongest risk factor for developing PTLD. Anything that stimulates B cell proliferation and/or delays EBV cytotoxic T cell (EBV-CTL) recovery will increase the risk for PTLD. Comparisons of results in the literature for incidence of PTLD and outcomes of therapies are difficult due to differing definitions, i.e., EBV infection vs. EBV disease vs. PTLD. Preemptive therapy with rituximab for high-risk patients has reduced incidence and mortality of PTLD following HSCT. Adoptive EBV-specific cytotoxic T cell therapy is the most effective therapy in the prevention and treatment of PTLD post-HSCT, but it is not widely available.

11.d.1 Introduction The pathogenesis to posttransplant lymphoproliferative disorders (PTLD) following hematopoietic stem cell transplantation (HSCT) is similar to that of PTLD observed following solid organ transplantation (SOT). As in SOT, PTLD following HSCT can be present in a very heterogeneous fashion, making the definitions of disease confusing and

T. G. Gross Division of Hematology/Oncology/BMT, Nationwide Children’s Hospital, Columbus, OH 43205, USA e-mail: [email protected] V. R. Dharnidharka et al. (eds.), Post-Transplant Lymphoproliferative Disorders, DOI: 10.1007/978-3-642-01653-0_11.d, © Springer Verlag Berlin Heidelberg 2010

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consensus difficult to reach. As opposed to PTLD following SOT, PTLD following HSCT is more predictably associated with Epstein–Barr virus (EBV), i.e., nearly 100%, and rarely occurs later than 6 months posttransplant [10, 13]. The general treatment strategies for PTLD used in SOT apply to PTLD following HSCT, though some differences do exist that are important. In this chapter, definitions of EBV infection/reactivation, EBV disease, and PTLD, as well as known risk factors, focusing on how these relate to therapeutic strategies, and past and current therapeutic approaches and outcomes for PTLD following HSCT will be reviewed.

11.d.2 Pathogenesis In a healthy individual, there exists a very tight balance between EBV-infected B cells and anti-EBV immunity, primarily mediated through CD8+ cytotoxic T cells (EBV-CTL). To illustrate the great proliferative potential of EBV-infected B cells, in an immunocompetent host, only 10−5–10−6 B cells are latently infected with EBV, but approximately 1–5% of all circulating CD8+ T cells are capable of reacting against EBV [19, 31, 36, 37]. In SOT, the recipient B cells are infected with EBV. The source of EBV is most often the donor, though it can be from recipient or third party via the natural oral transmission. In contrast, both the EBV and the infected B cells in PTLD following HSCT are of donor origin in the vast majority of cases. This is explained in part by the fact that the intense conditioning regimens used in HSCT usually eradicates host latent EBV infection [11]. PTLD rarely occurs within 30 days of HSCT and the peak incidence is in the third month posttransplant [8, 10, 13].

11.d.3 Definitions and Diagnosis of EBV Related Disease Post-HSCT EBV infection: Detecting EBV infection in the posttransplant patient is not trivial. The “monospot” test is not specific for EBV infection and antiviral serologic studies have limited value, as many patients will not have the ability to respond normally and/or will have obtained antibodies passively from gamma globulin or blood products. The detection of EBV DNA by polymerase chain reaction (PCR) provides sufficient evidence that the patient has been infected by EBV. EBV disease: As in SOT, EBV infection following HSCT may be asymptomatic or symptomatic with clinical manifestations in any number of organs (Table 11.d.1). PTLD: The World Health Organization (WHO) classification of early lesions, polymorphous PTLD, and monomorphic PTLD applies to PTLD following HSCT [16]. The designation of PTLD is usually limited to lymphoid masses that are often extranodal. A particularly difficult presentation of PTLD to diagnosis is the very rapidly progressive, disseminated disease that clinically resembles septic shock or graft-vs.-host disease (GVHD), which almost always results in death and the diagnosis is often made at autopsy [13]. This very fulminant

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Table 11.d.1 Manifestation of EBV disease post-HSCT Asymptomatic infection Infectious mononucleosis (IM) Typical Fulminant, severe EBV hepatitis Lymphocytic interstitial pneumonitis (LIP) Meningo-encephalitis Posttransplant lymphoproliferative disease (PTLD)

disease appears to be more common following HSCT than SOT, with as many as 35% of all PTLD cases in one series presenting in this fashion [13]. As opposed to SOT, EBV-negative and non-B cell PTLD is exceedingly rare. Clonality and morphology have been poor predictors of outcome in PTLD, especially following HSCT [13]. The diagnosis of PTLD usually requires a tissue biopsy, though fluid examination for abnormal cells may suffice in some cases. The presumptive diagnosis of PTLD is often made in symptomatic patients with clinical or radiographic evidence of “lymphoid masses” in the setting of elevated EBV DNA levels in the peripheral blood. The incidence of PTLD varies depending on the definition ranging from patients with increased EBV viral loads with or without symptoms to patients with only biopsy proven polymorphic or monomorphic PTLD.

11.d.4 Risk Factors The incidence of PTLD following HSCT ranges from <1% to approximately 25% in highest risk population [1, 10, 13]. The risk factors for developing PTLD following HSCT have been well characterized and are shown in Table 11.d.2. In general, any factor that stimulates B cell proliferation and/or decreases or delays T cell immunity will increase the risk of PTLD. In HSCT, unlike SOT, age and EBV status of recipient have not been demonstrated to be risk factors for PTLD [13, 17]. Interestingly it has been demonstrated that the risk of PTLD increases with donor age, and it has been speculated that this is due to fewer memory EBV-CTL with increasing age [13]. PTLD is rare following autologous BMT [13]. The incidence of PTLD is low (about 1%) following HSCT with matched related donors [10, 13]. The use of unrelated donors has been shown to be a risk factor for PTLD [10, 13]. The reason for the difference may be due to earlier EBV-CTL recovery in matched related donors compared to unrelated donors [21]. However, HLA disparity may also provide chronic B cell stimulation and proliferation, which could predispose to PTLD [3, 10, 13]. Since cord blood does not contain EBVinfected B cells, it was felt that PTLD would not occur. However, the incidence of PTLD following cord blood transplant is similar to matched related donors [1]. It appears that the time to PTLD following cord blood transplant is longer than the time when stem cells from

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Table 11.d.2 Risk factors for developing PTLD following HSCT Risk factor

Relative risk of PTLD

Donor sources Allogeneic £1 HLA – Ag mismatched related donor >2 HLA-Ag mismatched related Unrelated marrow donor Unrelated cord blood Recipient age Donor age

1.0 1.5–3.7 3.5 1.0 1.0 1.04a

Preparative regimen TBI, vs. no TBI

2.9

Graft manipulation T cell depletion (TCD), vs. no TCD

5.4–9.1

TCD methods CAMPATH-1 Elutriation Lectins Anti-T cell MoAb SRBC rosetting

2.0 2.6 4.1 12.3 15.6

Immunosuppression ATG/ALG for GVHD prophylaxis or treatment Anti-CD3 MoAb for treatment of GVHD

5.5 35.9

Effect of multiple risk factors HLA mismatched and TCD marrow

15.7

Factors T cell specific TCD (anti-T cell MoAb or SRBC rosetting) HLA-mismatched (³ 2 Ag) Use of ATG/ALG Use of anti-T cell MoAb 2 factors 3–4 factors

8.0 22.3

Ag antigen; TBI total body irradiation; MoAb monoclonal antibodies; SRBC sheep red blood cell; ATG anti-T cell globulin; ALG anti-lymphocyte globulin; GVHD graft-vs.-host disease a Continuous variable

a sibling or unrelated adult donor are used [1, 5]. This is not surprising since the source of EBV infection must be from natural transmission, and immune reconstitution, including EBV-CTL activity, and is slower using cord blood grafts [21]. T cell depletion (TCD), especially in combination with HLA mismatching, has long been known to significantly increase the risk of PTLD [3, 10, 13]. However, methodology of TCD affects PTLD risk. Methods that specifically remove T cells confer a higher risk of PTLD than “pan-lymphocyte” depletion methods [10]. “Pan-lymphocyte” depletion methods decrease the number of EBV-infected B cells as well as T cells, which may delay B cell proliferation from achieving a “critical mass” until EBV-CTL function recovers [10, 12].

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GVHD has not consistently been demonstrated to be a risk factor for PTLD, though the agents used to prevent and/or treat GVHD can increase the risk of PTLD [3, 6 10, 13, 35, 38]. Several analyses have shown that anti-T cell antibodies, especially monoclonal as opposed to polyclonal preparations, used in the prophylaxis or treatment of GVHD greatly increases the risk of PTLD [3, 10, 13, 38]. It is difficult to speculate how the intensity of immunosuppression in the preparatory regimen would affect immune recovery following infusion of the stem cell graft. Nonmyeloablative preparatory regimens are increasingly being used, as the toxic death rate attributed to the preparatory regimen is substantially less. The rationale is to obtain sufficient immunosuppression or immunoablation of the recipient to allow donor lymphoid engraftment, which will then reject recipient myelopoiesis and/or residual cancer. Many of these nonmyeloablative regimens appear to delay EBV-CTL recovery [8]. One report demonstrated in children that the risk of PTLD was increased in recipients of nonmyeloablative vs. myeloablative regimens [8]. Of interest, this study demonstrated that PTLD can be of recipient origin following nonmyeloablative regimens, suggesting these regimens are potent at eradicating recipient EBV-CTL activity, but not recipient EBV-infected B cells [8].

11.d.5 Prevention and Treatment The prognosis of PTLD post-HSCT has been dismal until recently (>90%) [13, 38]. Successful treatment of PTLD necessitates controlling the B cell proliferation while awaiting or ideally facilitating the development of an appropriate memory cytotoxic T cell (EBVCTL) response to maintain a homeostasis. Additional factors that contribute to the difficulty of treating these patients include increased toxicity from therapy and/or secondary infections and potential enhancement of alloreactive T cell immunity, which increases the patient’s risk of developing life-threatening GVHD. Interpretation of results is dependent on the definition of disease being treated, i.e., preemptive therapy for EBV infection/reactivation vs. EBV disease vs. PTLD. Treatment approaches and results are summarized in Table 11.d.3.

11.d.5.1 Prophylactic Therapy There is no study to support the use of antiviral prophylaxis to prevent PTLD following HSCT. In fact, many patients are on an antiviral when PTLD develops [13]. As discussed previously, using “pan-lymphocyte” depletion methods may help reduce the incidence of PTLD by reducing number of EBV-infected B cells infused with the stem cell graft [10, 12]. As opposed to SOT, PTLD in HSCT is nearly always a proliferation of donor B cells and acquisition of donor lymphocytes is often feasible. One caveat for donor leukocyte infusions (DLI) to be effective is that the donor must be EBV-positive [20]. DLI have been demonstrated to be successful in the treatment of PTLD post-HSCT; however, severe

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Table 11.d.3 Treatment for PTLD post-HSCT Prophylactic therapies Antivirals – not been shown to be effective Adoptive cellular therapy Donor leukocyte infusion (DLI) – mixed results EBV-specific CTL – very effective, unavailable for most centers B cell depletion of stem cell graft – partially effective Preemptive therapies Monoclonal antibodies (anit-CD20) – effective Treatment therapies Reduction of immunosuppression – rarely effective Surgical resection/radiation – rarely applicable Adoptive cellular therapy DLI – mixed results EBV-specific CTL – very effective, unavailable for most centers Monoclonal antibodies (anti-CD20) – mixed results Chemotherapy – effective, but toxic Enhancement of viral replication – early results promising

GVHD has also been associated with DLI [20, 25]. To circumvent the GVHD problem, investigators have inserted a suicide gene, i.e., herpes thymidine kinase, into donor lymphocytes, so that if GVHD occurs, it could be treated with ganciclovir [4, 32]. In one study, PTLD still developed in 3/12 patients receiving DLI containing suicide genes as PTLD prophylaxis [32]. Ex vivo EBV-specific CTL has been shown to be very effective as prophylaxis, preemptive therapy, and treatment for PTLD post-HSCT [15, 27]. These studies have provided proof of principle that if adequate EBV-CTL activity can be achieved, PTLD can be prevented and/or cured. EBV-specific CTL therapy is not without problems. This approach is not feasible for most centers due to the cost and regulatory oversight necessary to generate and administer the ex vivo generated EBV-CTL. And finally, even at centers where this technology is available, it has not been found to be cost effective except in perhaps the highest risk population, i.e., >20% risk of PTLD.

11.d.5.2 Preemptive Therapy There are many reports that correlate increased viral load with PTLD [20, 27, 28]. However, for preemptive therapy to be successful, one must have a method of reliably identifying patients who are at high-risk before they develop disease. There are no blinded, prospective studies to determine the predictive value of quantitative PCR for the development of PTLD. Unfortunately, patients can develop PTLD so rapidly that weekly screening has not been helpful [34]. Several studies have demonstrated that quantitative EBV PCR is predictive in patients receiving TCD grafts, but the predictive value for T cell replete grafts is less clear [8, 33]. Others have improved the predictive value of EBV viremia by including

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measures of T cell immunity. One study demonstrated only patients with absolute lymphocyte counts <300/mm3 and EBV viremia were predicted with risk of PTLD [7], and another study demonstrated that only patients with very low EBV-specific T cells and EBV viremia developed PTLD [22]. Investigators are now using anti-CD20 antibody (rituximab) as preemptive therapy [7, 8, 18, 24]. The rationale is to reduce B cell proliferation while EBV-CTL activity recovers. One study demonstrated that in cohort of patients receiving TCD HSCT, using serially EBV monitoring and rituximab preemptively, the incidence of PTLD and mortality due to PTLD was significantly decreased compared to a historical cohort [34]. Though no randomized trials have been published to date, this approach of serially monitoring high-risk patients, e.g., patients who receive ex vivo or in vivo TCD, by weekly EBV blood PCR and giving rituximab for patients with elevated EBV DNA levels or persistently increasing levels has become standard of care at many institutions.

11.d.6 Treatment of PTLD Historically, mortality has been 90% once PTLD has developed [13, 38]. Some recent reports from single centers have mortality rates ranging from 0 to 50% [5, 8, 15, 18]. Despite the use of rituximab and/adoptive cellular therapy, the overall survival for patients who develop PTLD patients remains poor, with death due to sepsis, GVHD, and underlying cancer recurrence being common [24]. As opposed to PTLD following SOT, reduction of immunosuppression is rarely successful as the major defect following HSCT is delayed EBV-CTL recovery, not suppression of EBVCTL function [13, 29]. Antiviral therapy has been successful in some cases of mononucleosislike disease or meningo-encephalitis. However, efficacy in treating PTLD is controversial [13]. A novel approach has been used where viral replication is promoted with the use of arginine butyrate, which upregulates expression of EBV thymidine kinase. Ganciclovir is also given that causes an abortive replicative cycle and no virions are produced, but cell death occurs [26]. Of the 15 patients with EBV-positive tumors treated, three were PTLD following HSCT. All three received at least one regimen of chemotherapy prior to treatment. One patient achieved a complete response and the other two had >50% reduction in tumor mass [26]. These results are very interesting and this is a very novel approach to EBV-related tumors. Anti-B cell antibodies have been used successfully to treat PTLD post-HSCT [2, 5, 8, 9, 18, 23]. With anti-B cell antibodies, EBV-CTL development is neither enhanced nor inhibited. The first report using anti-B cell therapy used anti-CD21 and anti-CD23 antibodies and demonstrated they were well tolerated, though only 35% of patients achieved long-term survival and were the only ones to survive with monoclonal disease [2]. More recently, antiCD20 or rituximab has been used. All reports to date have had small numbers and results vary. But it appears that about 50% of patients will achieve complete remission with rituximab [5, 8, 18, 23]. One must keep in mind the significant effect of rituximab on normal B cells and monitor patients’ immunoglobulin levels closely. As discussed previously, increasing EBV-CTL by adoptive cellular infusions has been demonstrated to be very effective in preventing and/or treating PTLD following HSCT, and

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to date is the most effective and safest therapy for PTLD following HSCT [15, 22, 25, 27]. However, due to the obstacles mentioned previously, this therapy is only available at a few centers. Chemotherapy has been used to treat resistant PTLD following SOT [14, 30]. However, chemotherapy used to treat lymphoma may be too toxic for posttransplant patients, especially following HSCT. Additionally, there is substantial risk of losing the recovery marrow graft. There are anecdotal reports of using standard and low-dose chemotherapy for refractory PTLD following HSCT; however, results have usually been very poor.

11.d.7 Take Home Pearls

• • •

• •

PTLD following HSCT overall is rare (1–2%). TCD of the stem cell graft, either ex vivo or in vivo, is the strongest risk factor. Other factors that either stimulate B cell proliferation and/or delayed T cell recovery will increase the risk of PTLD. It appears that weekly monitoring high-risk patients for 4–6 months post-HSCT by EBV PCR of the blood and preemptive treatment of patients with rituximab can reduce the incidence and mortality of PTLD. This approach is not completely effective and there are no large, prospective, controlled trials to delineate the best time to begin preemptive therapy. Adoptive therapy with EBV-specific CTL is the most effective therapy for prevention and treatment of PTLD, but is not widely available. Though outcome has improved for PTLD when it does develop, data suggest even with rituximab, immunosuppression reduction and/or chemotherapy long-term survival remains no better than 50%. Novel approaches that are easily transferable to all centers are needed.

References 1. Barker JN, Martin PL, Coad JE, et al. Low incidence of Epstein-Barr virus-associated posttransplantation lymphoproliferative disorders in 272 unrelated-donor umbilical cord blood transplant recipients. Biol Blood Marrow Transplant. 2001;7:395–9 2. Benkerrou M, Jais J-P, Leblond V, et al. Anti-B-cell monoclonal antibody treatment of severe post-transplant B-lymphocyte disorder: prognostic factors and long-term outcome. Blood. 1998; 92:3137–47 3. Bhatia S, Ramsay NKC, Steinbuch M, et al. Malignant neoplasms following bone marrow transplantation. Blood. 1996;87:3633–9 4. Bonini C, Ferrari G, Verzeletti S, et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science. 1997;276:1719–24 5. Brunstein CG, Weisdorf DJ, Defor T, et al. Marked increased risk of Epstein-Barr virus-related complications with the addition of anti-thymocyte globulin to a non-myeloablative conditioning prior to unrelated umbilical cord blood transplantation. Blood. 2006;108:2874–89

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6. Buell JF, Gross TG, Woodle ES. Malignancy after transplantation. Transplantation. 2005;80: S254–64 7. Cohen J, Gandhi M, Naik P, et al. Increased incidence of EBV-related disease following paediatric stem cell transplantation with reduced-intensity conditioning. Br J Haemtaol. 2005;129: 229–39 8. Cohen JM, Cooper N, Chakrabarti S, et al. EBV-related disease following hematopoietic stem cell transplantation with reduced intensity conditioning. Leuk Lymph. 2007;48:256–69 9. Comoli P, Basso S, Zecca M, et al. Preemptive therapy of EBV-related lymphoproliferative disease after pediatric haploidentical stem cell transplantation. Am J Transplant. 2007;7:1648–55 10. Curtis RE, Travis LB, Rowlings PA, et al. Risk of lymphoproliferative disorders after bone marrow transplantation: a multi-institutional study. Blood. 1999;94:2208–16 11. Gratama JW, Oosterveer MA, Zwaan FE, et al. Eradication of Epstein-Barr virus by allogeneic bone marrow transplantation: implication for sites of viral latency. Proc Natl Acad Sci USA. 1988;85:8693–6 12. Gross TG, Hinrichs SH, Davis JR, et al. Effect of counterflow elutriation (CE) on Epstein-Barr virus (EBV) infected cells in donor bone marrow. Exp Hematol. 1998;26:395–9 13. Gross TG, Steinbuch M, DeFor T, et al. B cell lymphoproliferative disorder following hematopoietic stem cell transplantation: Risk factors, treatment and outcome. Bone Marrow Transplant. 1999;23:251–8 14. Gross TG, Bucuvalas J, Park J, et al. Low dose chemotherapy for the treatment of refractory post-transplant lymphoproliferative disease in children. J Clin Oncol. 2005;23:6481–8 15. Gustafsson A, Levitsky V, Zou JZ, et al. Epstein-Barr virus (EBV) load in bone marrow transplant lymphoproliferative disease: prophylactic infusion of EBV-specific cytotoxic T cells. Blood. 2000;95:807–14 16. Harris NL, Ferry JA, Swerdlow SH. Posttransplant lymphoproliferative disorders: summary of Society for Hematopathology Workshop. Semin Diagn Pathol. 1997;14:8–14 17. Ho M, Jaffe R, Miller G, et al. The frequency of Epstein-Barr virus infection and associated lymphoproliferative syndrome after transplantation and its manifestations in children. Transplantation. 1998;45:719–27 18. Kuehnle I, Huls MH, Liu Z, et al. CD20 monoclonal antibody (rituximab) for therapy of Epstein-Barr virus lymphoma after hemopoietic stem-cell transplantation. Blood. 2000;95:1502–5 19. Kuzushima K, Hoshino Y, Fujii K, et al. Rapid determination of Epstein-Barr virus-specific CD8+ T-cell frequencies by flow cytometry. Blood. 1999;94:3094–100 20. Lucas KG, Burton RL, Zimmerman SE, et al. Semiquantitative Epstein-Barr virus (EBV) polymerase chain reaction for determination of patients at risk for EBV-induced lymphoproliferative disease after stem cell transplantation. Blood. 1998;91:3654–61 21. Marshall NA, Howe JG, Formica R, et al. Rapid reconstitution of Epstein-Barr virus-specific T lymphocytes following allogeneic stem cell transplantation. Blood. 2000;96:2814–21 22. Meij P, van Esser JWJ, niesters HGM, et al. Impaired recovery of Epstein-Barr virus (EBV)specific CD8+ T lymphocytes after partially T-depleted allogeneic stem cell transplantation may identify patients at very high risk for progressive EBV reactivation and lymphoproliferative disease. Blood. 2003;101:4290–7 23. Milpied N, Vasseur B, Parquet N, et al. Humanized anti-CD20 monoclonal antibody (Rituximab) in post transplant B-lymphoproliferative disorder: a retrospective analysis on 32 patients. Ann Oncol. 2000;11:113–6 24. Ocheni S, Kroeger N, Zabelina T, et al. EBV reactivation and post transplant lymphoproliferative disorders following allogeneic SCT. Bone Marrow Transplant. 2008;42:181–6 25. Papadopoulos EB, Ladanyi M, Emanuel D, et al. Infusions of donor leukocytes to treat EpsteinBarr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation. N Engl J Med. 1994;330:1185–91

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26. Perrine SP, Hermine O, Small T, et al. A phase ½ trial of arginine butyrate and ganciclovir on patients with Epstein-Barr virus-associated lymphoid malignancies. Blood. 2007;109:2571–8 27. Rooney CM, Smith CA, Ng CYC, et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood. 1998;92:1549–55 28. Rowe DT, Webber S, Shcauer EM, et al. Epstein-Barr virus load monitoring: its role in the prevention and management of post-transplant lymphoproliferative disease. Transplant Infect Dis. 2001;3:79–87 29. Shapiro R, Nalesnik M, McCauley J, et al. Posttransplant lymphoproliferative disorders in adult and pediatric renal transplant patients receiving tacrolimus-based immunosuppression. Transplantation. 1999;68:1851–4 30. Swinnen LJ, Mullen GM, Carr TJ, Costanzo MR, Fisher RI. Aggressive treatment for postcardiac lymphoproliferation. Blood. 1995;86:3333–40 31. Tan LC, Gudgeon N, Annels NE, et al. A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy carriers. J Immunol. 1999;162:1827–35 32. Tiberghien P, Ferrand C, Lioure B, et al. Administration of herpes simplex-thymidine kinaseexpressing donor T-cells with a T-cell-depleted allogeneic marrow graft. Blood. 2001;97:63–72 33. van Esser JWJ, van der Holt B, Meijer E, et al. Epstein-Barr virus (EBV) reactivation is a frequent event after allogeneic stem cell transplantation (SCT) and quantitatively predicts EBVlymphoproliferative disease following T-cell-depleted SCT. Blood. 2001;98:972–8 34. van Esser JWJ, Niesters HGM, van der Holt B, et al. Prevention of Epstein-Barr virus-lymphoproliferative disease by molecular monitoring and preemptive rituximab in high-risk patients after allogeneic stem cell transplantation. Blood. 2002;99:4364–9 35. Witherspoon RP, Fisher LD, Schoch G, et al. Secondary cancers after bone marrow transplantation for leukemia or aplastic anemia. N Engl J Med. 1989;321:784–9 36. Yang J, Lemas VM, Flinn IW, et al. Application of the ELISPOT assay to the characterization of CD8 + responses to Epstein-Barr virus antigens. Blood. 2000;95:241–8 37. Yang J, Tao Q, Flinn IW, et al. Characterization of Epstein-Barr virus-infected B cells in patients with posttransplantation lymphoproliferative disease: disappearance after rituximab therapy does not predict clinical response. Blood. 2000;96:4055–63 38. Zutter MM, Martin PJ, Sale GE, et al. Epstein-Barr virus lymphoproliferation after bone marrow transplantation. Blood. 1998;72:520–9

Research Priorities and Future Directions

12

Vikas R. Dharnidharka, Michael Green, and Steven A. Webber

12.1 Introduction The preceding chapters of this book emphasize how much has been learnt about PTLD in the past few decades. Yet, so much still remains to be learned. Listed below are some of the unanswered questions regarding PTLD. Each of these areas is ripe for future research and gaining new knowledge.

12.2 Etiology/Pathogenesis of PTLD What is the point at which proliferation of EBV-infected B cells becomes uncontrolled? We know that EBV penetrates into the B cell genome and drives cellular proliferation. Under normal circumstances, this proliferation is controlled by cytotoxic CD8+ T cells, which are impaired by extrinsic immunosuppressive agents. Yet, <10% of transplant recipients develop PTLD. Is this just an issue of degree of cumulative immunosuppression or is it related to the organs themselves? If so, why are intestinal and lung transplants associated with the highest rates of PTLD? Is cumulative immunosuppression over time most important, or exceeding some threshold of total immunosuppression at any one point in time? How do the different immunosuppressive agents combine to suppress the immune system? Is the combination synergistic in a linear fashion, exponential fashion, or otherwise? Is the use of certain immunosuppressive agents more likely to lead to PTLD, and are any such agents (e.g., mTOR inhibitors) actually protective?

V. R. Dharnidharka (*) Division of Pediatric Nephrology, University of Florida College of Medicine, Gainesville, FL, USA e-mail: vikasmd@ufl.edu V. R. Dharnidharka et al. (eds.), Post-Transplant Lymphoproliferative Disorders, DOI: 10.1007/978-3-642-01653-0_12, © Springer Verlag Berlin Heidelberg 2010

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Why is primary EBV infection so much more likely to lead to PTLD? Multiple studies have shown that the EBV seronegative recipient is much more likely to develop PTLD. What are the unique features of primary infection under an immunosuppressed state that make it different from the reactivation of primary infection that developed in an immunocompetent host? What are the etiologic agent(s) or factors involved in the genesis of EBV-negative PTLD? While we have strong evidence for the role of EBV in the pathogenesis of PTLD, we have minimal knowledge about the triggers for EBV-negative PTLD. Is this entity also induced by a viral infection? If so, which virus or viruses are causal? If this is not attributable to a virus, then what else could drive uncontrolled immune-cell proliferation? What are the differences in pathophysiology for late PTLD or non-B cell PTLD vs. typical early EBV-driven B cell PTLD? Why do they behave so differently? Compared to B cell PTLD, other types, such as T cell PTLD, tend to occur later, are less likely to be EBV-associated, and have a poor response to therapy and higher mortality. What makes the presentation, response, and outcomes so different?

12.3 Surveillance and Monitoring How intense should the PTLD monitoring be? What is both scientifically valid and cost effective? The role and optimal methods for viral infection surveillance, whether for EBV, CMV or BK virus, are currently being intensively studied by transplant professionals. Serial measurements of viral load by PCR can be quite expensive and few studies have addressed cost effectiveness. For CMV and BK virus, viremia is due to viral replication and there is a clear progression of viremia to virus-induced organ involvement that can be prevented in many cases. However, for EBV, viremia may reflect B cell proliferation with or without viral replication. The determinants of progression to EBV disease/PTLD are less clear and many cases of chronic high load carrier do not progress to clinical disease. So what does a single high viral load mean? What do repeated high viral loads mean? Why is it different for different organ systems, such as heart vs. liver, and between adults and children? Is viral load monitoring indicated in patients who are EBV seropositive prior to transplantation? Finally, should optimal surveillance combine viral load monitoring with some assessment of the patients’ immunological status (e.g., cytotoxic T cell frequency and function)?. The evidence is incomplete at this point and more research is needed in order to define optimal and cost-effective surveillance programs.

12.4 Treatment of PTLD What are the optimal treatment strategies for PTLD and how should these be individualized for factors such as patient age, EBV status, histopathology, time of onset post transplant, and prior rejection history and disease stage?

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Research Priorities and Future Directions

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Currently, the accepted initial approach is to reduce immunosuppression in all cases. However, it is unclear when this should be the only initial therapy or when other treatments are indicated at presentation. Should rituximab be tried as a first-line therapy in all CD20 positive tumors? Are antiviral agents and intravenous immunoglobulin therapies effective? What is an adequate trial of reduction in immunosuppression? How should the allograft be best protected from acute and chronic rejection? When should second-line therapies be introduced, and what should they be? Should rituximab be used before consideration of chemotherapy for refractory disease? Toxicities are less, but chemotherapy may help prevent allograft rejection. Are we losing out by not subjecting more patients to chemotherapy early? How should central nervous system disease be treated? Will cellular (adoptive) immunotherapy ever become a practical treatment option in most centers? We hope to find answers to these and other questions in the years to come.

Index

A Active immunization, 139 Acyclovir, 10, 134–135 Allotransplantation, 1 American Society of Transplant Physicians (ASTP), 154 American Society of Transplant Surgeons (ASTS), 154 Anti-B cell antibodies, 121–123 Antiviral therapy, 120 Azathioprine, 23 B B cell lymphomas, 32, 33 B-cell proliferation, 36 Burkitt’s lymphoma (BL), 6 C Calcineurin inhibitors (CNI’s), 119 CD4 cells, , 34, 35 Cellular immunotherapy, 124–125 Central nervous system disease, 125–126 Chemoprophylaxis, EBV infection acyclovir and ganciclovir, 134–135 animal models, 135 clinical studies, 135–136 Chemotherapy, 123–124 Classical Hodgkin lymphoma type PTLD, 100–101 Clonality, 9–10 Combination therapies, 125 Cyclosporine A, 23 Cytomegalovirus (CMV), 20 Cytotoxic T lymphocyte (CTL), 6, 32, 34, 36

E Early lesions florid follicular hyperplasia, 94 infectious mononucleosis (IM)-type, 93–94 plasmacytic hyperplasia (PH), 92–93 EBV-associated posttransplant lymphoproliferative disorder blood tests, 77 clinical staging, 82–83 clinicopathologic correlates, 73–74 diagnostic evaluation, 74–76 differential diagnoses, 83–84 early after organ transplantation, 70–71 EVB nucleic acid detection, 81 EVB serology, 80–81 haematopoietic stem cell transplantation, 71 infectious mononucleosis, 70 initial clinical examination, 75 late after organ transplantation, 71 lesion sites, 71–73, 75 patients, background information, 74 radiographic imaging, 77–80 viral load determination, 81–82 Epidemiology cytomegalovirus (CMV), 20 density report, 18 graft loss, risk, 24 graft organ related risk factor, 21–22 host-related risk factor, 20–21 immunosuppression, 19, 23 infection related, 19–20 lymphoma, 17, 18 mortality, 24 posttransplant, 18 primary disease, 21 re-transplantation, 24 time series, 19 187

188

Epstein–Barr virus (EBV), 2, 6. See also Heterogeneous group disorders B cell lymphomas, 32, 33 Burkitt’s lymphoma (BL), 6 cell marker studies, 7 chemoprophylaxis, 134–136 (see also Chemoprophylaxis, EBV infection) classification of, 11–12 cytotoxic T lymphocytes, 6 DNA hybridization techniques, 10 growing experience, 7 healthy individuals, T cell response, 34–35 immunoprophylaxis, 136–141 (see also Immunoprophylaxis, EBV infection) infection, 30 infectious mononucleosis (IM), 6 latent cycle genes, 32–34 morphology, 8–9 oropharyngeal shedding, 6, 10 pathogenesis, 12 theraphy, 10–11 transplant recipients, T cell response, 35–36 viral life cycle, 30–32 Epstein–Barr virus (EBV) viral load (VL) assessment assay limitations, 48 biologic compartments, 47 immunosuppressive regimens determination, 61 infectious mononucleosis (IM), 46 lack of standardization, 48–49 monitoring response, 59–60 plasma vs. whole blood (WB), 47 PTLD diagnosis, 58–59 specific clinical purposes, 51–58 specimen type and reporting units, 49–51 Epstein–Barr nuclear antigens (EBNA) intravenous immune globulin, 121 passive immunization, 137–138 F Florid follicular hyperplasia, 94 G Ganciclovir, EBV infection, 134–135 Ghobrial index, 114 H Heart transplantation. See also Lung and heart-lung transplantation

Index

clinical presentation, 164–165 epidemiology, 164 outcomes, 167 treatment, 165–167 Hematopoietic stem cell transplantation (HSCT), 50 EBV infection, 174–175 pathogenesis, 174 preemptive therapy, 178–179 prophylactic therapy, 177–178 risk factors, 175–177 treatment, 179–180 Heterogeneous group disorders B-cell proliferation, 36 immune and viral factors, 38 polyclonal tumors, 36 PTLD-associated B cell lymphomas, 38 subversion/immune evasion, 38–39 viral infection and tumor cell growth, immunosuppression, 39–40 viral strategy, 37 I Immune suppression reduction calcineurin inhibitors (CNI’s), 119 clinical response, 119 immune surveillance, 118 response rate, 118–119 target of rapamycin (mTOR) inhibitor, 119 Immunoprophylaxis, EBV infection active immunization, 139 cellular therapy, 136–137 EBV loads, 139–141 passive immunization, 137–138 preemptive therapy, 141 Infectious mononucleosis (IM) active immunization, 139 ancillary studies, 93 clinical features and symptoms, 70 EBV VL assessments, 46 Epstein–Barr virus (EBV), 6 heart transplantation, 164 histopathology, 93, 94 morphological features, 8 oropharyngeal lesions, 7 tonsillopharyngitis, 70 Interferon, 120–121 Interleukin 6 (IL6), 121 International prognostic index vs. PTLDspecific index BCL-2, 114–115

Index

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Ghobrial index, 114 immunosuppression, 113 overall survival, 114 PS and site involved, 113 single-agent rituximab, 113 variable model, 113 Intravenous immune globulin (IVIG), 121

monomorphic B-Cell PTLD, 97–99 monomorphic T-Cell PTLD, 99–100 Mononucleosis, 70

K Kidney transplantation clinical presentation, 146–147 epidemiology, 146 outcomes, 149 prognostic factors, 149 therapeutic aspects, 147–149

P Passive immunization, 137–138 Pathology classical Hodgkin lymphoma type PTLD, 100–101 diagnosis, 91–92 early lesions, 92–94 general features, 90 monomorphic PTLD, 95–100 polymorphic PTLD, 94–96 WHO classification, 90–91 Patients monitoring cellular immune responses, 127 EBV viral load, 127 graft and PTLD status, 126 Peripheral blood mononuclear cells (PBMC)/ lymphocytes (PBL), 49 Phytohemagluttinin (PHA), 140 Plasmacytic hyperplasia (PH) ancillary studies, 92 histopathology, 92, 93 Polymorphic PTLD ancillary studies, 95 histopathology, 95, 96 Preemptive therapy immunoprophylaxis, 141 hematopoietic stem cell transplantation (HSCT), 178–179 Prognostic factors. See also International prognostic index vs. PTLD-specific index adults, 112 allogeneic bone marrow transplant, 109 CD20 negativity, 109 children, 112 CNS involvement, 109 early vs. late onset, 111–112 EBV-positive vs. negative PLTD, 110 monomorphic disease, 110 multifocal disease, 108–109 multivariable PTLD prognostic index, 111 patient age, 107–108 performance status, 107

L Lesion sites CNS disease, 73 GI tract, 71–72 head and neck, 72 liver involvement, 72 pulmonary involvement, 72 renal transplant, 72–73 Liver transplantation antiviral therapies, 157 clinical presentation, 156 conventional chemotherapy, 158 discontinued immunosuppression, 157 incidence, 154–155 monoclonal antibody, 157–158 prognosis, 158 risk factors, 155–156 surgery and radiotherapy, 157 Lung and heart-lung transplantation clinical presentation, 168 epidemiology, 167 outcomes, 169 treatment, 168–169 Lymphoma, kidney transplant clinical presentation, 146–147 outcomes, 149 prognostic factors, 149 risk factors, 146 therapeutic aspects, 147–149 Lymphoproliferative disorders (LPD), 6 M Major histocompatibility complex (MHC), 30 Monomorphic PTLD, 95, 96

O Optimal therapy, 118 Oropharyngeal shedding, 6

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solid organ transplant, 110 T cell PTLD, 110 Prophylactic therapy, 177–178 R Radiation therapy, 119 Research priorities and future directions etiology/pathogenesis, 183–184 surveillance and monitoring, 184 treatment, 184–185 Risk factors graft organ related, 21–22 host-related, 20–21 immunosuppression, 23 infection related, 19–20 primary disease, 21

Index

S Small bowel transplantation clinical presentation, 159 incidence and risk factors, 159 treatment, 160 Solid organ transplant (SOT), 50 Surgery therapy, 119 T Target of rapamycin (mTOR) inhibitor, 119 T cell response, Epstein–Barr virus healthy individuals, 34, 35 transplant recipients, 35–36 Tonsillopharyngitis, 70 W WHO classification, 90–91