Biologic Therapy of Leukemia

CONTEMPORARY HEMATOLOGY

BIOLOGIC THERAPY OF LEUKEMIA Edited by

Matt Kalaycio, MD

BIOLOGIC THERAPY OF LEUKEMIA

CONTEMPORARY HEMATOLOGY Gary J. Schiller, MD, SERIES EDITOR Biologic Therapy of Leukemia, edited by MATT KALAYCIO, 2003 Chronic Lymphocytic Leukemia: Molecular Genetics, Biology, Diagnosis, and Management, by GUY B. FAGUET, 2003 Modern Hematology: Biology and Clinical Management, by REINHOLD MUNKER, ERHARD HILLER, AND RONALD PAQUETTE, 2000 Red Cell Transfusion: A Practical Guide, edited by MARION E. REID AND SANDRA J. NANCE, 1998

BIOLOGIC THERAPY OF LEUKEMIA Edited by

MATT KALAYCIO, MD The Cleveland Clinic Foundation, Cleveland, OH

HUMANA PRESS TOTOWA, NEW JERSEY

© 2003 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 humanapress.com For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel: 973-256-1699; Fax: 973-256-8341; E-mail: [email protected]; website at humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. All articles, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. Due diligence has been taken by the publishers, editors, and authors of this book to ensure the accuracy of the information published and to describe generally accepted practices. The contributors herein have carefully checked to ensure that the drug selections and dosages set forth in this text are accurate in accord with the standards accepted at the time of publication. Notwithstanding, as new research, changes in government regulations, and knowledge from clinical experience relating to drug therapy and drug reactions constantly occurs, the reader is advised to check the product information provided by the manufacturer of each drug for any change in dosages or for additional warnings and contraindications. This is of utmost importance when the recommended drug herein is a new or infrequently used drug. It is the responsibility of the health care provider to ascertain the Food and Drug Administration status of each drug or device used in their clinical practice. The publisher, editors, and authors are not responsible for errors or omissions or for any consequences from the application of the information presented in this book and make no warranty, express or implied, with respect to the contents in this publication. This publication is printed on acid-free paper. ∞ ANSI Z39.48-1984 (American National Standards Institute) Permanence of Paper for Printed Library Materials. Production Editor: Robin B. Weisberg. Cover Illustration: From Fig. 5 in Chapter 4, “Drug Immunoconjugate Therapy of Acute Myeloid Leukemia” by Arthur E, Frankel, Bayard L. Powell, Eli Estey, and Martin S. Tallman. Cover design by Patricia F. Cleary. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $20.00 per copy is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [1-58829-071-9/03 $20.00].

Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging-in-Publication Data Biologic therapy of leukemia / edited by Matt Kalaycio. p. ; cm.-Includes bibliographical references and index. ISBN 1-58829-071-9 (alk. paper) 1-59259-383-6 (e-book) 1. Leukemia—Immunotherapy. 2. Leukemia—Gene therapy. 3. Cytokines—Therapeutic use. 4. Biological products—Therapeutic use. I. Kalaycio, Matt. II. Series. [DNLM: 1. Leukemia‚therapy. 2. Biological Therapy. 3. Cytokines-therapeutic use. 4. Gene Therapy. 5. Immunotherapy. 6. Oligonucleotides, Antisense—therapeutic use. WH 250 B615 2003] RC643.B457 2003 616.99'41906—dc21 2002192220

To Linda, Mollie, Jason, and Rachel

PREFACE In the latter part of the 20th century, hematologists and medical oncologists were trained to treat leukemia with systemic therapy that was cytotoxic to both normal and malignant cells. Some of these therapies, such as methotrexate and L-asparaginase, were developed within the context of known biologic pathophysiology, but most were developed in relative ignorance of biologic mechanisms and cannot therefore be considered “biologic.” The usual goal of treatment was to eliminate rapidly dividing, or malignant, cells with DNA-damaging agents that spared normal tissue only in a relative sense. The paradigm of systemic, nonspecific therapy dominated oncologic thought at the time: Leukemia is by very definition a wide-spread systemic disease at the time of diagnosis. For this reason systemic therapy which reaches simultaneously every cell in the body is the most logical form of treatment and is probably the only type which offers, theoretically, the possibility of complete cure. (1)

To an extent, the systemic, nonspecific treatment approach was successful and certainly resulted in cures when before none were possible. However, this approach failed to cure the majority of patients with leukemia and is usually associated with significant toxicity. No other way was known, and for a time, no other way seemed possible. The frequent failure of nonspecific treatments, remarkable advances in molecular biology, and well-timed serendipity, led to new approaches that are revolutionizing the management of leukemia as we enter the 21st century. In contrast to the treatments of the past, the new approaches can collectively be classified as truly “biologic” therapies because they take advantage of the known biology of leukemia. Thus, treatment can often be directed at the leukemia, sparing normal tissues and causing less tissue damage. These new targeted treatments represent the beginning of a new age in leukemia therapeutics. As exciting as these are, clinicians often find it difficult to access appropriate medical information on these new treatments when faced with a patient who may benefit from them. The advances are coming so often, and so quickly, that treatments are sometimes approved for use before the information that supports their claimed efficacy can be published in peer-reviewed literature. Large textbooks attempting to publish accurate and current information on leukemia are doomed to obsolescence before reaching print. These practical concerns prompted the publication of this book. Biologic Therapy of Leukemia is devoted to these new biologic therapies and provides a vii

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rapidly accessible, authoritative source of practical information for clinicians attempting to use these treatments for their patients. Some of the treatments described in this text, such as interferon and all-trans retinoic acid, have been available for some time and are well-described in the medical literature. However, that information is difficult to access when contrasting their efficacy with newer treatments, such as imatinib mesylate and arsenic trioxide, which are also described in this text. Other treatments, such as P-glycoprotein inhibitors and interleukins, have been dancing on the edges of clinical practice and may yet find their place based on emerging data. The graft vs leukemia effect has been better defined and promises to completely alter the way allogeneic stem cell transplant is employed in the future. Finally, therapeutic approaches that reverse failure of apoptosis, alter genetic codes, and modulate immunologic mechanism are no longer mere theory, but are now being tested in the clinic and warrant close attention by the oncologic community. The authors and I hope that clinicians treating patients will find Biologic Therapy of Leukemia helpful. We all share the goal of eradicating leukemia and I believe the information contained in these pages moves us closer to that goal. I thank the contributors for their expertise and willingness to share it. I stand in awe of their knowledge and dedication. Matt Kalaycio, MD

REFERENCE 1. Burchenal, J.H. Treatment of the leukemias. Semin Hematol 1966;3:122.

CONTENTS Preface ........................................................................................................... vii Contributors .................................................................................................... xi PART I: IMMUNOTHERAPY 1 Human Leukemia-Derived Dendritic Cells as Tools for Therapy .......................................................................................... 3 David Claxton 2 The Graft vs Leukemia Effect ............................................................... 13 Brian J. Bolwell 3 Unconjugated Monoclonal Antibodies ................................................. 29 Matt Kalaycio 4 Drug Immunoconjugate Therapy of Acute Myeloid Leukemia ........................................................................................... 43 Arthur E. Frankel, Bayard L. Powell, Eli Estey, and Martin S. Tallman 5 Radiolabeled Monoclonal Antibodies .................................................. 59 John M. Burke and Joseph G. Jurcic PART II: CYTOKINES 6 Interferons.............................................................................................. 81 Thomas Fischer 7 Interleukin-2 Treatment of Acute Leukemia ........................................ 93 Peter Kabos and Gary J. Schiller PART III: TARGETED THERAPEUTICS 8 Antisense Therapy ............................................................................... 109 Stanley R. Frankel 9 Signal Transduction Inhibitors ............................................................127 Michael E. O’Dwyer and Brian J. Druker 10 P-Glycoprotein Inhibition in Acute Myeloid Leukemia .................... 145 Thomas R. Chauncey

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Targeting the Apoptotic Machinery as a Potential Antileukemic Strategy ....................................................................163 Benjamin M. F. Mow and Scott H. Kaufmann

PART IV: DIFFERENTIATION AGENTS 12 Arsenicals: Past, Present, and Future ................................................189 Chadi Nabhan and Martin S. Tallman 13 All-Trans-Retinoic Acid in the Treatment of Acute Promyelocytic Leukemia ................................................................205 Pierre Fenaux and Laurent Degos PART V: GENE THERAPY 14 Gene Therapy ......................................................................................225 Paul J. Orchard and R. Scott McIvor Index ............................................................................................................. 261

CONTRIBUTORS BRIAN J. BOLWELL, MD • Bone Marrow Transplant Program, Department of Hematology and Medical Oncology, Cleveland Clinic Foundation, Cleveland, OH JOHN M. BURKE, MD • Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY THOMAS R. CHAUNCEY, MD, PhD • University of Washington School of Medicine, VA Pugent Sound Health Care System, Seattle, WA DAVID CLAXTON, MD • Division of Hematology/Oncology, Penn State Hershey Medical Center, Hershey, PA LAURENT DEGOS, MD, PhD • Institut d’Hematologie, Hôpital Saint Louis, Paris, France BRIAN J. DRUKER, MD • Leukemia Center, Oregon Health & Science University Cancer Institute, Portland, OR ELI ESTEY, MD • Section of Acute Leukemia and Myelodysplastic Syndromes, MD Anderson Cancer Center, Houston, TX PIERRE FENAUX, MD • Service des Maladies du Sang, CHU, Lille, France THOMAS FISCHER, MD • Johannes Gutenberg University of Mainz, Mainz, Germany ARTHUR E. FRANKEL, MD • Department of Medicine, Wake Forest University School of Medicine, Winston Salem, NC STANLEY R. FRANKEL, MD, FACP • Medical Operations, Genta Incorporated, Chicago, IL, and Department of Medicine, Greenbaum Cancer Center, University of Maryland, Baltimore, MD JOSEPH G. JURCIC, MD • Leukemia Service, Memorial Sloan-Kettering Cancer Center, New York, NY PETER KABOS, MD • Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, Los Angeles, CA. MATT KALAYCIO, MD • Leukemia Program, Department of Hematology and Medical Oncology, Cleveland Clinic Foundation, Cleveland, OH SCOTT H. KAUFMANN, MD, PhD • Division of Oncology Research, Mayo Clinic, and Department of Molecular Pharmacology, Mayo Graduate School, Rochester, MN R. SCOTT MCIVOR, MD, PhD • Department of Genetics, Cell Biology, and Development, Institute of Human Genetics, University of Minnesota, Minneapolis, MN BENJAMIN M. F. MOW, MD • Division of Hematology, National University Hospital, Singapore xi

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CHADI NABHAN, MD • Division of Hematology/Oncology, Northwestern University Medical School, Chicago, IL MICHAEL E. O’DWYER, MD • Leukemia Center, Oregon Health & Science University Cancer Institute, Portland, OR PAUL J. ORCHARD, MD • Department of Pediatrics, Institute of Human Genetics, University of Minnesota, Minneapolis, MN BAYARD L. POWELL, MD • Section of Hematology/Oncology, Wake Forest University School of Medicine, Winston Salem, NC GARY J. SCHILLER, MD • Division of Hematology-Oncology, UCLA School of Medicine, Los Angeles, CA MARTIN S. TALLMAN, MD • Division of Hematology/Oncology, Northwestern University Medical School, Chicago, IL

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Human Leukemia-Derived Dendritic Cells as Tools for Therapy David Claxton, MD CONTENTS INTRODUCTION: IMMUNOTHERAPY OF LEUKEMIA DENDRITIC CELLS AS TOOLS FOR THERAPY HUMAN DENDRITIC CELLS DERIVED FROM MYELOID LEUKEMIAS CLINICAL STUDIES USING LEUKEMIA-DERIVED DENDRITIC CELLS FUTURE STUDIES AND OPPORTUNITIES REFERENCES

1. INTRODUCTION: IMMUNOTHERAPY OF LEUKEMIA Excluding selected subsets of human leukemia, progress in the therapy of this group of disorders has been relatively modest since the late 1980s. In contrast, the elucidation of the pathogenesis and mechanisms of progression of these diseases has been substantial. The development of specific agents targeted at selected molecular changes specific to leukemic cells offers great hope for the therapy of these disorders. Even these targeted therapies, such as the recently developed tyrosine kinase inhibitor Imatinib Mesylate, may fail to control advanced forms of the target disease (1). Additionally, for many human leukemias, no such well-defined genetic background is available at present. Accordingly, efforts to advance the therapy for these diseases must focus on other approaches. There is substantial experimental and clinical data suggesting the usefulness of T-cell-mediated antigen-specific immunotherapy for leukemia. The central principle common to all these approaches is the development of effective cell-mediated immunity able to selectively or semiselectively target leukemic or malignant cells. Thus, these approaches ultimately yield an active immune process with the potential for ongoing control of residual malignant cellular elements. From: Biologic Therapy of Leukemia Edited by: M. Kalaycio © Humana Press Inc., Totowa, NJ

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Alloimmune antileukemic effects are well established, both experimentally and clinically. Several investigators have described animal models that document the ability of allogeneic immune cells to cure transplantable experimental leukemias. In human clinical studies, this effect is also clearly active. Thus, for both matched sibling and unrelated donor hematopoietic stem cell transplants, the acquisition of an allodisparate T-cell population and the recognition of host allo-antigens expressed on leukemias have been shown to be active in the ultimate control of chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML). Relevant antigens targeted in clinical studies and identified as active in this phenomenon include the minor histocompatiblity antigen HA-1 (2). In certain cases, such antigens have been shown to be hematopoietic-specific polymorphic proteins (3). Thus, the graft vs hematopoietic effects developing after allogeneic stem cell transplantation control the normal hematopoietic and leukemic elements derived from the host simultaneously. Autologous cellular immune activity directed toward leukemias has also been described in experimental and clinical systems. Several murine systems have been described that demonstrate the activity of autologous immunity (4–7) against transplantable congenic leukemia. Several authors have published clinical series suggesting therapeutic effects of systemic IL-2 with (8) or without (9–12) IL-2 activated cells for human leukemias. Antileukemic T-cell clones or lines have been described (13).

2. DENDRITIC CELLS AS TOOLS FOR THERAPY Human dendritic cells represent the so-called “professional antigen-presenting cells” responsible for initiating all antigen-specific cell-mediated immunity from naïve T-cell elements. The development of techniques for the ex vivo differentiation of dendritic cells from peripheral blood-derived monocytes or bone marrow-derived CD34 cells has presented clinical investigators with the opportunity to use these cells for the initiation of antitumor immunity in experimental and clinical studies. Several clinical trials have been reported in which allogeneic or autologous tumor lysates or antigen-specific peptides have been pulsed into dendritic cells derived in one of these two ways (14–17). The development of tumor-specific autologous cellular cytotoxicity has been demonstrated in several human tumor systems, including human AML (18). Common to all of these systems, however, is the use of normal autologous dendritic cells brought to a state of maturation that are suitably primed to acquire fresh antigen from the extracellular environment. These antigens then are provided by the investigator and effectively presented to autologous T-cells through the antigen processing and presenting machinery of the dendritic cells. In the following pages, a similar system for the initiation of antileukemic cell-mediated immunity is described that, however, stems from the ability of the original leukemic cells to differentiate directly into effective antigen-presenting cells and to pre-

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sent the endogenously expressed leukemic antigens. It is our contention that this system may be the ideal one in which to study the therapeutic potency of human dendritic cells for the initiation of antitumor cell-mediated activity.

3. HUMAN DENDRITIC CELLS DERIVED FROM MYELOID LEUKEMIAS In 1994, two groups independently described cellular differentiation of cells with the characteristics of myeloid dendritic cells from AML (19,20). Cells derived from patients and cultured ex vivo in recombinant cytokines, including GM-CSF and tumor necrosis factor-alpha (TNF-α), matured into cells with the morphology and activity of typical mature dendritic cells (19). These cells generated brisk allogeneic mixed lymphocyte reactions, as would be expected of mature dendritic cells. In 1996, Choudhury et al. demonstrated that CML cells could differentiate into cells with the morphology, phenotype, and allostimulatory function of mature dendritic cells (21). Simultaneously, these cells were shown to be derived from the malignant clone by in situ hybridization to nucleic acid probes specific for the Philadelphia chromosome breakpoints of the bcr-abl genes. These leukemia-derived dendritic cells were able to stimulate autologous preactivated T-cells to acquire cytotoxicity specific for the leukemic clone and not for normal human leukocyte antigen (HLA)-matched targets or autologous remission bone marrow cells. The cytotoxic activity demonstrated was found within the CD8 cellular compartment. There was no NK type cytotoxicity given the near absence of cytotoxicity for K562 cells. This was the first report of a leukemia-derived dendritic cell-inducing autologous leukemia-specific cell-mediated cytotoxicity (21). Since this publication, a total of 30 peer-reviewed publications describing human leukemia-derived dendritic cells have been identified by literature search. These publications have examined the differentiation of both CML and AML into active antigen-presenting cells. Several authors have documented the clonal origin of the culture-derived dendritic cells (22–26). Most reports have shown the acquisition of relatively high expression of the critical costimulatory molecules CD80 and 86, together with the mature dendritic cell marker CD83, on the leukemia-derived dendritic cells. Five independent groups have demonstrated the acquisition of leukemia-specific cellular cytotoxicity in autologous T-cells stimulated by leukemia-derived dendritic cells in CML (22,27–30). Five independent investigative groups, including our own group, have also shown that AML may similarly differentiate into clonal dendritic cells capable of stimulating cytotoxic leukemia-specific T-cell activity (23,25,31–33). In the case of both anti-CML and anti-AML activities, these autologous cytotoxic reactions have been shown to be major histocompatibility complex (MHC) restricted as demonstrated by partial blocking of cytotoxicity

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by HLA class 1- or class 2-specific monoclonal blocking antibodies (23,27). Thus, the cytotoxic activities identified have the characteristics expected for dendritic cell-initiated T-cell mediated responses. Several questions arise from this large body of work. Fundamental questions exist about the uncertainty concerning the details of the immune response to leukemia-derived dendritic cells. Specifically, it is unclear what antigens are being responded to and which subsets of effector cells are participating. Currently, there is little data to document which antigens may be responsible for eliciting responses to leukemia-derived dendritic cells; however, our current work has suggested that a limited number of target antigens may be shared between patients. Yasukawa et al. have shown that CML-derived dendritic cells may specifically present a bcr-abl B3A2 junction-specific peptide fragment to CD4-positive T cells via an HLA-DRB1*0901 restricted mechanism (34). On the other hand, Bertazzoli et al. have shown that T-cells derived from CML patients respond less well to a B3A2 bcr-abl-specific peptide in a proliferative assay than those from normal donors (35). Thus, the role of bcr-abl junctionderived antigens and the responses elicited by leukemia-derived dendritic cells remain unresolved but dubious.

4. CLINICAL STUDIES USING LEUKEMIA-DERIVED DENDRITIC CELLS Fujii et al. described a single patient with CML who was treated with autologous peripheral stem cell transplantation followed by a series of infusions of leukemia-derived dendritic cells (22). The leukemia-derived dendritic cells were generated in the cytokine combination GM-CSF, TNF-α, and IL-4. After receiving four infusions of post-transplant-administered culture-derived autologous leukemic dendritic cells, the patient developed a partial cytogenetic response because the total number of Philadelphia (Ph) chromosome-positive metaphases in the bone marrow fell from 20 out of 20 to 7 out of 20 within several months. We carried out a study using CML-derived dendritic cells to stimulate autologous T-cells to develop a therapeutic cell line for adoptive immunotherapy. This study, which enrolled five patients and treated two, has been briefly reported and described elsewhere (36). A schematic diagram of this study is shown in Fig. 1. Patients had CML cells collected by apheresis when they had substantial numbers of circulating dendritic cells or after stimulation with GMCSF. These cells were subsequently used to stimulate autologous T-cells preactivated with OKT3 and IL-2. After coculture, cells were cryopreserved in aliquots and subsequently administered to patients. A schematic view of the cell culture approach is given in Fig. 2. Two days before cell infusion, modest doses of cyclophosphamide were administered to cytoreduce the disease. After receiving the activated lympho-

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Fig. 1. Schematic view of dendritic cell-activated lymphocyte therapy for CML.

Fig. 2. Culture procedures for the generation of autologous T-cells activated by leukemiaderived dendritic cells. * Culture A (DC) must be >20% CD86 + and >40% HLA-DR +. Culture B(AL) must be >60% CD3 +.

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Fig. 3. Time course of patient 2 treated with DC-AL for accelerated-phase CML. The horizontal axis represents months (April through August). Leukocytosis and thrombocytopenia reverted to normal on protocol.

cytes, patients received intermittent subcutaneous low-dose IL-2. This was given 5d on, 2d off and 5d on with each treatment of chemotherapy. The therapy was well tolerated. Mild fever and local skin reactions were seen in association with IL-2 therapy, as might be expected. Disease stabilized in both patients, and the second patient treated, who was in frank accelerated phase of CML before initiation of therapy, reverted into a chronic phase. Marrow metaphases, which had been marked with trisomy 8 in addition to the Ph1 chromosome, transiently reverted toward simple Ph-positive metaphases. The previous thrombocytopenia in this patient was reversed, and the patient was well for the duration of the therapy, as shown in Fig. 3. Thus, this trial showed that it was feasible to generate large numbers of active dendritic cells that had the characteristics of professional antigen-presenting cells. Administration of these cells to patients was not associated with adverse effects. Adverse effects seen with the program were those expected from the concomitant chemotherapy or the IL-2 given.

5. FUTURE STUDIES AND OPPORTUNITIES AMLs and myelodysplastic syndromes are often ultimately untreatable with available conventional therapies. Accordingly, the ability to generate potent dendritic cells from the majority of patients with advanced AML or myelodysplastic syndrome presents great opportunities for study. This group of diseases is attractive for study for several reasons. First, it is straightforward to harvest large numbers of malignant cells from patients by venipuncture or bone marrow collection. Second, subsets of patients who may be successfully treated into remission but who have a poor prognosis with con-

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ventional chemotherapy administered in remission are easily identified. Thus, these diseases offer the opportunity for administration of immunotherapy in the context of minimal residual disease, which is nonetheless known to portend a poor prognosis with conventional therapy. This is perhaps the optimal setting in which to test the activity of novel immunologic treatments. Significant questions remain concerning the optimal mode of delivery of dendritic cell immunotherapy to such patients, however. It is unclear whether vaccination with activated antigen-presenting cells via the subcutaneous, intradermal, or intravenous infusion routes or administration of ex-vivo activated Tcells after dendritic cells stimulation is the optimal approach for initiation of disease-specific immunity. Certainly the latter approach (adoptive transfer of activated T-cells), which was pursued in the CML protocol described, presents considerable technical challenges. The logistic and regulatory challenges of the laboratory processing and culture of cells in a program of ex vivo immune activation are formidable. We are currently pursuing clinical trials of dendritic cells as vaccination-based immunotherapy for the treatment of poor prognosis, newly diagnosed patients with AML, and patients whose diseases relapsed but are believed to have a reasonable chance of successful remission induction. Patients will receive standard remission-induction chemotherapy and will receive dendritic cell vaccination when they are recovering from consolidation therapy. Vaccination therapy will continue for a few months, and patients will ultimately have immune cell populations assayed for antileukemic T-cell responses. Additionally, cutaneous cellmediated T-cell responses to vaccinating dendritic cells will be followed. An additional appealing arena for the study of therapeutic activity of leukemia-derived dendritic cells is that of allogeneic stem cell transplantation. Although this therapy is active, for patients with poor-prognosis AML, disease relapse after transplantation is still frequent. Thus, the treatment of disease relapse after transplantation with chemotherapy followed by vaccination with dendritic cell-activated lymphocytes is of great importance. There are experimental data to suggest that T-cell vaccination after allogeneic transplantation may lead to control of disease. The development of novel therapies is often slower than might be initially expected. At the time when such therapies are identified as potentially active in initial experimental laboratory studies, the natural belief is that clinical proof of principle will follow rapidly. Monoclonal antibodies were identified approx 20 yr ago, but the first monoclonal anticancer therapies to show antitumor activity have only been available recently. Thus, it may be expected that initial frustrations with the development of cellular anticancer therapies may be expected to yield to success in the treatment of many human tumors. There is certainly abundant experimental evidence for the potent cellular immune activities that should be active in the control of human cancer.

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The author believes that human myeloid leukemias and myelodysplastic syndromes may offer a nearly ideal system in which to test the hypothesis that human malignancies may be controlled through dendritic cell or antigen-presenting cell therapy. Trials of several vaccine-based and adoptive immunotherapeutic approaches are urgently needed.

REFERENCES 1. Druker BJ, Sawyers CL, Kantarjian H, et al. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome [see comments] [erratum appears in N Engl J Med 2001;345(3):232]. N Engl J Med 2001;344:1038–1042. 2. Brossart P, Spahlinger B, Grunebach F, et al. Induction of minor histocompatibility antigen HA-1-specific cytotoxic T-cells for the treatment for leukemia after allogeneic stem cell transplantation [letter; comment]. Blood 1999;94:4374–4376. 3. Mutis T, Verdijk R, Schrama E, Esendam B, Brand A, Goulmy E. Feasibility of immunotherapy of relapsed leukemia with ex vivo-generated cytotoxic T lymphocytes specific for hematopoietic system-restricted minor histocompatibility antigens [see comments]. Blood 1999;93:2336–2341. 4. Matulonis U, Dosiou C, Freeman G, et al. B7-1 is superior to B7-2 costimulation in the induction and maintenance of T-cell-mediated antileukemia immunity. Further evidence that B7-1 and B7-2 are functionally distinct. J Immunol 1996;156:1126–1131. 5. Enright H, McGlave PB. Biology and treatment of chronic myelogenous leukemia. Oncology (Huntington) 1997;11:1295–1300. 6. Runyon K, Lee K, Zuberek K, Collins M, Leonard JP, Dunussi-Joannopoulos K. The combination of chemotherapy and systemic immunotherapy with soluble B7-immunoglobulin G leads to cure of murine leukemia and lymphoma and demonstration of tumor-specific memory responses. Blood 2001;97:2420–2426. 7. Dunussi-Joannopoulos K, Runyon K, Erickson J, Schaub RG, Hawley RG, Leonard JP. Vaccines with interleukin-12-transduced acute myeloid leukemia cells elicit very potent therapeutic and long-lasting protective immunity. Blood 1999;94:4263–4273. 8. Benyunes MC, Massumoto C, York A, Higuchi CM, Buckner CD, Thompson JA, et al. Interleukin-2 with or without lymphokine-activated killer cells as consolidative immunotherapy after autologous bone marrow transplantation for acute myelogenous leukemia. Bone Marrow Transplant 1993;12:159–163. 9. Toren A, Ackerstein A, Slavin S, Nagler A. Role of interleukin-2 in human hematological malignancies [review]. Med Oncol 1995;12:177–186. 10. Messina C, Zambello R, Rossetti F, et al. Interleukin-2 before and/or after autologous bone marrow transplantation for pediatric acute leukemia patients. Bone Marrow Transplant 1996;17:729–735. 11. Mandelli F, Vignetti M, Tosti S, Andrizzi C, Foa R, Meloni G. Interleukin-2 treatment in acute myelogenous leukemia. Stem Cells 1993;11:263–268. 12. Fefer A, Benyunes M, Higuchi C, et al. Interleukin-2 +/– lymphocytes as consolidative immunotherapy after autologous bone marrow transplantation for hematologic malignancies. Acta Haematol 1993;89(Suppl 1):2–7. 13. Coleman S, Fisher J, Hoy T, Burnett AK, Lim SH. Autologous MHC-dependent leukemiareactive T lymphocytes in a patient with CML. Leukemia 1996;10:483. 14. Tjoa BA, Erickson SJ, Bowes VA, et al. Follow-up evaluation of prostate cancer patients infused with autologous dendritic cells pulsed with PSMA peptides. Prostate 1997;32:272–278. 15. Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells [see comments]. Nat Med 1998;4:328–332.

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16. Kugler A, Stuhler G, Walden P, et al. Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids [see comments]. Nat Med 2000;6:332–336. 17. Morse MA, Deng Y, Coleman D, et al. A Phase I study of active immunotherapy with carcinoembryonic antigen peptide (CAP-1)-pulsed, autologous human cultured dendritic cells in patients with metastatic malignancies expressing carcinoembryonic antigen. Clin Cancer Res 1999;5:1331–1338. 18. Fujii S, Fujimoto K, Shimizu K, et al. Presentation of tumor antigens by phagocytic dendritic cell clusters generated from human CD34+ hematopoietic progenitor cells: induction of autologous cytotoxic T lymphocytes against leukemic cells in acute myelogenous leukemia patients. Cancer Res 1999;59:2150–2158. 19. Santiago-Schwarz F, Coppock DL, Hindenburg AA, Kern J. Identification of a malignant counterpart of the monocyte-dendritic cell progenitor in an acute myeloid leukemia. Blood 1994;84:3054–3062. 20. Srivastava BL, Srivastava A, Srivastava MD. Phenotype, genotype and cytokine production in acute leukemia involving progenitors of dendritic Langerhans’ cells. Leukemia Res 1994;18:499–411. 21. Choudhury A, Gajewski JL, Liang JC, et al. Use of leukemic dendritic cells for the generation of antileukemic cellular cytotoxicity against Philadelphia chromosome-positive chronic myelogenous leukemia. Blood 1997;89:1133–1142. 22. Fujii S, Shimizu K, Fujimoto K, et al. Analysis of a chronic myelogenous leukemia patient vaccinated with leukemic dendritic cells following autologous peripheral blood stem cell transplantation. Jpn J Cancer Res 1999;90:1117–1129. 23. Woiciechowsky A, Regn S, Kolb HJ, Roskrow M. Leukemic dendritic cells generated in the presence of FLT3 ligand have the capacity to stimulate an autologous leukemia-specific cytotoxic T-cell response from patients with acute myeloid leukemia. Leukemia 2001;15:246–255. 24. Oehler L, Berer A, Kollars M, et al. Culture requirements for induction of dendritic cell differentiation in acute myeloid leukemia. Ann Haematol 2000;79:355–362. 25. Harrison BD, Adams JA, Briggs M, Brereton ML, Yin JA. Stimulation of autologous proliferative and cytotoxic T-cell responses by “leukemic dendritic cells” derived from blast cells in acute myeloid leukemia. Blood 2001;97:2764–2771. 26. Cignetti A, Bryant E, Allione B, Vitale A, Foa R, Cheever MA. CD34(+) acute myeloid and lymphoid leukemic blasts can be induced to differentiate into dendritic cells. Blood 1999;94:2048–2055. 27. Choudhury A, Toubert A, Sutaria S, Charron D, Champlin RE, Claxton DF. Human leukemia-derived dendritic cells—ex-vivo development of specific antileukemic cytotoxicity. Crit Rev Immunol 1998;18:121–131. 28. Nieda M, Nicol A, Kikuchi A, et al. Dendritic cells stimulate the expansion of bcr-abl-specific CD8+ T-cells with cytotoxic activity against leukemic cells from patients with chronic myeloid leukemia. Blood 1998;91:977–983. 29. Eibl B, Ebner S, Duba C, et al. Dendritic cells generated from blood precursors of chronic myelogenous leukemia patients carry the Philadelphia translocation and can induce a CMLspecific primary cytotoxic T-cell response. Genes Chromosomes Cancer 1997;20:215–223. 30. Chen X, Regn S, Raffegerst S, Kolb HJ, Roskrow M. Interferon alpha in combination with GM-CSF induces the differentiation of leukaemic antigen-presenting cells that have the capacity to stimulate a specific anti-leukaemic cytotoxic T-cell response from patients with chronic myeloid leukaemia. Br J Haematol 2000;111:596–607. 31. Choudhury A, Liang JC, Thomas EK, et al. Dendritic cells derived in vitro from acute myelogenous leukemia cells stimulate autologous, antileukemic T-cell responses. Blood 1999;93:780–786.

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32. Charbonnier A, Gaugler B, Sainty D, Lafage-Pochitaloff M, Olive D. Human acute myeloblastic leukemia cells differentiate in vitro into mature dendritic cells and induce the differentiation of cytotoxic T, cells against autologous leukemias. Eur J Immunol 1999;29:2567–2578. 33. Hagihara M, Shimakura Y, Tsuchiya T, et al. The efficient generation of CD83-positive immunocompetent dendritic cells from CD14-positive acute myelomonocytic or monocytic leukemia cells in vitro. Leukemia Res 2001;25:249–258. 34. Yasukawa M, Ohminami H, Kojima K, et al. HLA class II-restricted antigen presentation of endogenous bcr-abl fusion protein by chronic myelogenous leukemia-derived dendritic cells to CD4(+) T lymphocytes. Blood 2001;98:1498–1505. 35. Bertazzoli C, Marchesi E, Passoni L, et al. Differential recognition of a BCR/ABL peptide by lymphocytes from normal donors and chronic myeloid leukemia patients. Clin Cancer Res 2000;6:1931–1935. 36. Choudhury A, Toubert A, Sutaria S, et al. Human leukemia-derived dendritic cells—ex vivo development of specific antileukemic cytotoxicity. Critical Reviews in Immunology 1998;18:121–131.

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The Graft vs Leukemia Effect Brian J. Bolwell, MD CONTENTS INTRODUCTION THE RELATIONSHIP OF GRAFT VS HOST DISEASE WITH THE GRAFT VS LEUKEMIA EFFECT THE RELATIONSHIP OF T-CELL DEPLETION WITH THE GVL VS LEUKEMIA EFFECT DONOR LEUKOCYTE INFUSIONS NONMYELOABLATIVE ALLOGENEIC TRANSPLANTATIONS SUMMARY REFERENCES

1. INTRODUCTION The original rationale of bone marrow transplantation (BMT) was solely based on the concept of dose intensity. The logic was as follows: the ability to deliver anticancer therapy (chemotherapy and/or radiation therapy) is limited by dose toxicities, primarily toxicity to normal bone marrow; tumors not susceptible to repetitive doses of modest amounts of chemotherapy might be completely obliterated with one extremely large dose of chemotherapy and/or radiation therapy; a consequence of one large dose of therapy is destruction of normal hematopoiesis, resulting in permanent aplasia; if normal matched marrow were available for transplantation, then these “lethal” doses of chemotherapy could be administered to a patient, the tumor might be eradicated, and the infusion of donor allogeneic bone marrow would restore normal hematopoiesis and save the patient from iatrogenic death. Clinical success with autologous BMT has shown validity of this theory of dose intensity. However, it has become clear throughout the past 20 yr that powerful immunologic forces contribute to the potential for cure in allogeneic BMT (alloBMT). The immunologic reaction by which donor cells from the graft generate an anticancer effect From: Biologic Therapy of Leukemia Edited by: M. Kalaycio © Humana Press Inc., Totowa, NJ

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is known as the graft vs leukemia (GVL) or graft vs tumor (GVT) effect. This chapter focuses on clinical aspects of the GVL effect: the relationship between graft vs host disease (GVHD) and the GVL effect, T-cell depletion and its relationship to the GVL effect, donor leukocyte infusions (DLI) as a treatment of disease relapse after alloBMT, and current results of nonmyeloablative allogeneic transplantation.

2. THE RELATIONSHIP OF GRAFT VS HOST DISEASE WITH THE GRAFT VS LEUKEMIA EFFECT One of the major complications of alloBMT is GVHD. GVHD is an immunologic phenomenon occurring when immunocompetent donor cells perceive host tissues to be “foreign” and mount an immunologic attack against them. Acute GVHD usually occurs within the first 100 d after transplantation and affects the skin, liver, and gastrointestinal tract. Chronic GVHD occurs 100 or more d after transplantation and is generally belived to be less of a fulminate disorder; clinical manifestations can affect almost any organ but commonly involve the liver, skin, eyes, bone marrow, mouth (sicca syndrome), and lungs. GVHD prophylaxis and treatment employ potent immunosuppressive therapy directed toward reducing lymphocyte number and function. Unfortunately, the immunosuppressive therapy predisposes patients to opportunistic infections. Therefore, both the development of GVHD and its treatment are clinically vexing problems associated with significant morbidity and mortality. Despite these toxicities, the development of GVHD may be beneficial, because GVHD is frequently associated with the GVL effect, resulting in a lower risk of disease relapse after transplantation. Although it was originally postulated more than 40 yr ago that donor hematopoietic cells might generate an anticancer effect (1,2), the clinical relationship of GVHD with leukemic relapse was not documented until the 1970s and early 1980s. Odom et al. described two children with acute lymphoblastic leukemia (ALL) who relapsed after alloBMT. When clinical GVHD developed, the children subsequently developed a remission (3). Weiden et al. compared leukemic patients undergoing a syngeneic BMT with those receiving an alloBMT and observed differences in the relative relapse rates for those with and without clinical GVHD (4). The patients with clinical GVHD had a relapse rate 2.5 times less than patients without GVHD. Additionally, the relapse rate was higher in syngeneic patients than in allogeneic transplantation recipients who did not develop GVHD. This finding suggested that cells in the allogeneic graft produced a GVL effect. Subsequent studies by the Seattle transplantation group further defined the relationship between GVHD and leukemic relapse. One study of patients with leukemia receiving an alloBMT showed that clinical GVHD augmented the

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GVT effect, because patients with clinically significant chronic GVHD had a 27% risk of long-term relapse, compared with a 55% risk of relapse for patients with subclinical GVHD (p = 0.0003) (5). Thus, clinical chronic GVHD was strongly associated with a long-term GVL effect. A second study of more than 1200 recipients of alloBMT reported that patients with acute leukemia transplanted when in relapse had a lower relapse rate if they subsequently developed either acute or chronic GVHD (6). The conclusion was, again, that chronic GVHD leads to a durable antileukemic, or GVL, effect. The International Bone Marrow Transplant Registry reported a landmark analysis of more than 2000 recipients of human leukocyte antigen (HLA)-identical sibling BMTs, examining the relationship between GVHD and disease relapse. Decreased risk of relapse was observed in recipients of non-T-celldepleted allografts with acute (relative risk 0.68, p = 0.03), chronic (relative risk 0.43, p = 0.01), and both acute and chronic GVHD (relative risk 0.33, p = 0.0001) when compared with recipients without GVHD (7). This large multiinstitutional trial confirmed an unequivocal relationship of both acute and chronic GVHD with decreased leukemic relapse. Many trials have substantiated these findings, including specific associations with a GVT effect in leukemia, lymphoma, and myeloma (8–17). In summary, abundant data emerged from 1978 to 1992 describing a strong relationship between the development of clinical GVHD, both acute and chronic, with reduced risk of relapse following alloBMT. The logical conclusion of these observations was that cells in the donor graft, which resulted in GVHD, also led to a profound antitumor effect.

3. THE RELATIONSHIP OF T-CELL DEPLETION WITH THE GVL VS LEUKEMIA EFFECT Although it was evident by 1990 that a relationship existed between GVHD and reduced risk of leukemic relapse, the development of GVHD itself was unfortunately associated with significant morbidity and mortality. Therapeutic options to treat and prevent GVHD were limited. Mortality from GVHD could overshadow the risks of leukemic relapse for some patients. Therefore, many centers began clinical trials of T-cell depletion in alloBMT to reduce the incidence and severity of GVHD. It was believed that the T-cells in the donor graft were, in part, responsible for the development of clinical GVHD. Removing these T-cells might reduce the risk of morbidity and mortality from clinical GVHD. Unfortunately, many studies subsequently showed that T-cell depletion was associated with an increased risk of leukemic relapse. Several small studies of T-cell-depleted alloBMT showed a trend toward increased risk of relapse after transplantation (18,19). Two large reports sub-

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sequently demonstrated an association of an increased risk of disease relapse following alloBMT in patients receiving T-cell-depleted bone marrow. Goldman et al. described 405 patients with chronic myelogenous leukemia (CML) receiving alloBMTs when they were in the chronic phase. The probability of relapse was higher for recipients of T-cell-depleted marrow compared with non-T-cell-depleted marrow (relative risk 5.4, p < 0.0001) (20). The International Bone Marrow Transplant Registry compared more than 731 recipients of T-cell-depleted HLA-identical sibling BMT with 2480 recipients of non-T-cell-depleted marrow. Although T-cell depletion did reduce the risk of acute and chronic GVHD leukemic relapse increased. Leukemic relapse was 2.75 times more likely after T-cell depletion for patients with acute leukemia in first remission or patients with CML in chronic phase (p < 0.0001) (21). Thus, although T-cell depletion did reduce the risk of GVHD, leukemia-free survival was not enhanced because of the loss of the GVL effect. Most of these studies used pan T-cell depletion, or removal of all T-cell subsets from the HLA-matched sibling donor graft. Subsequent reports have suggested that less-than-full T-cell depletion might reduce the risk of GVHD while retaining a GVL effect. In particular, selective CD8+ T-cell depletion has been reported to reduce the risk of GVHD without losing the GVL effect (22,23). Additionally, it has been suggested that T-cell depletion in recipients of unrelated BMTs might reduce the risk of GVHD without losing the GVL effect (24). Strategies in which T-cell depletion is used to reduce the risk of acute GVHD have also been described, but T cells are then subsequently infused (“add back”) to generate a GVL effect (25,26). However, no large multicenter trial has investigated these strategies and such data remains preliminary. In summary, data concerning T-cell depletion demonstrates that manipulating the cellular composition of the allogeneic marrow graft can influence the risk of leukemic relapse. These powerful data confirm that the cells themselves, specifically the donor T-cells, have the capacity to mount an antileukemic effect. Given the relationship of clinical GVHD with the GVL effect and because T-cell depletion reduces the GVL effect, it is clear that T-cells are critical mediators in the GVL effect. The precise cellular mechanism, however, remains unknown. In particular, a fundamental question is whether the GVL effect is independent of the GVHD effect. Thus, is the GVL effect simply immunologic GVHD directed against alloantigens shared by host tissues and leukemia cells, or, alternatively, are there donor cells that specifically recognize tumor antigens and generate the GVL effect? The association of clinical GVHD with the GVL effect would strongly imply that the GVL effect is simply an alloantigen reaction directed against all host tissues, both normal and leukemic. However, abundant data exist suggesting that it is possible to sepa-

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rate the GVL effect from the alloantigen GVHD reaction (27–33). Both natural killer cells and different T-cell subsets, may have a role in the GVL effect (34–35). An ongoing up-to-date elusive goal is to harness the GVL effect and minimize clinical GVHD toxicity. Theoretically, if one accepts the hypothesis that the GVL effect operates through mechanisms other than simple alloantigen recognition, one might decrease the morbidity and mortality of GVHD if a state of immune tolerance could be obtained. Therefore, if both donor and recipient cells are present in a given host, without clinical GVHD, then a state of immune tolerance would theoretically exist and hopefully the donor cells might still be able to generate a GVL effect. This situation has been described clinically and is known as mixed hematopoietic chimerism. It is generally defined as the coexistence of both donor and recipient cells after alloBMT. Mixed hematopoietic chimerism has long been known to exist after transplantation, with conflicting clinical implications. Although some authors have found that the detection of mixed chimerism may be associated with increased risk of relapse in certain disease states (36–38), others have found that mixed hematopoietic chimerism after alloBMT is common and is not necessarily associated with an increased risk of disease relapse (39–44). For example, Huss and Deeg described mixed hematopoietic chimerism in patients with aplastic anemia or CML undergoing alloBMT; the incidence of rejection was higher (but not significantly) in patients with aplastic anemia with mixed chimeras. Intriguingly, among patients with CML, both overall survival and relapse-free survival were superior in mixed as opposed to complete chimeras (45). The development of stable mixed chimerism is theoretically attractive; however, in clinical practice, the majority of patients undergoing either an ablative or a nonmyeloablative allogeneic transplantation clinically either evolve into a fully chimeric state or experience disease relapse (46).

4. DONOR LEUKOCYTE INFUSIONS The use of the GVL effect as adoptive immunotherapy was proven conclusively with results obtained from a treatment known as DLI. The use of DLI was pioneered in patients who relapsed after alloBMT. The theory was straightforward: if a patient relapsed after receiving an ablative alloBMT, and if that patient also did not have overt clinical GVHD, then the infusion of additional donor cells (DLI) might be sufficient to produce a cellular immunotherapeutic effect and result in clinical remission. Initially, small studies investigated the use of donor buffy coat leukocytes for patients with CML who relapsed after alloBMT and found that a combination of α interferon and DLI resulted in both clinical and cytogenetic remissions (47,48).

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The largest series examining the efficacy of DLI was a survey of 25 North American BMT programs regarding their use of DLIs (49). One-hundred forty patients who received DLI relapsed after alloBMT. Diseases included CML (n = 56), acute myeloid leukemia (AML) (n = 46), ALL (n = 15), myelodysplastic syndrome (MDS) (n = 6), non-Hodgkin’s lymphoma (NHL) (n = 6), multiple myeloma (n = 5), Hodgkin’s disease (n = 2), and other (n = 4). Donor leukocytes were obtained by leukopheresis from nonprimed donors in a median of leukopheresis sessions during a median 7-d period. The leukocytes were not manipulated in vitro. The cell yield was variable, but most centers obtained a mean mononuclear cell (MNC) dose of approx 5 × 108 MNCs per kilogram. CML responded best to DLIs. Of the 55 evaluable patients with CMI, 60% achieved a complete response to DLI. Patients who relapsed in chronic phase had a 74% chance of achieving a remission with DLI, patients in accelerated phase had a 33% response rate, and only one of six patients in blast crisis achieved a remission. Responses were more modest in other diseases. Fifteen percent of the patients with AML who relapsed achieved a complete response, 18% of patients with ALL, 40% of MDS, and 50% of myeloma. Median time to remission in patients with CML was 85 d, and 34 d for patients with AML. Sixty percent of evaluable patients developed acute GVHD and 61% developed chronic GVHD. The median time to development of acute GVHD was 32 d. Eighteen percent developed pancytopenia related to DLI at a median of 21 d after infusion. This pancytopenia resolved without treatment in 13 patients, resolved with granulocyte colony-stimulation factor (G-CSF) treatment in 8, resolved after bone marrow boost in 2, and did not resolve in 3. Importantly, there was a clear correlation of disease response with development of clinical GVHD. Of 45 evaluable completely responding patients, 42 developed acute GVHD, and 36 of 41 developed chronic GVHD. The correlation of acute and chronic GVHD with complete remission was statistically significant (p < 0.0001). Of the 23 patients who did not develop either acute or chronic GVHD, only 3 obtained a complete response to DLI. This landmark study by Collins et al. conclusively demonstrated that adoptive immunotherapy with DLIs in a large series of patients has the potential to lead to clinical and cytogenetic remissions in several diseases, with CML appearing to be the most amenable to this therapy. Correlation of clinical response with GVHD was strong. Long-term follow-up of this cohort of patients was recently published (50). Seventy-three patients achieved a complete remission after DLI, and long-term follow-up was available for 66, with a median follow-up of 32 mo. The probability of survival at 1, 2, and 3 yr was 83%, 71%, and 61%, respectively. Patients with CML had 1-, 2-, and 3-yr survival rates of 87%, 76%, and 73%; for other diseases, survival probability at 1 and 2 yr was 77% and 65%. This

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follow-up study concluded that the majority of remissions achieved with DLI persist for years. Additional data have confirmed that DLI-induced remissions are durable (51). Although adoptive immunotherapy with DLI has excellent efficacy in patients with CML, its efficacy is somewhat more modest in patients with lymphoid malignancies. Fewer than 50% of patients with ALL or multiple myeloma have been reported to achieve complete responses with DLI (55,56). The use of G-CSF (filgrastim) for the treatment of relapse after allogeneic BMT has been described as a potential alternative to DLI (57,58). The largest series reported 14 patients relapsing after allogeneic transplantation (n = 5 CML, n = 5 AML, n = 2 MDS, n = 1 Chronic lymphocytic leukemia [CLL], n = 1 ALL). Filgrastim was given at 5µg/kg subcutaneously for 21 d. Of the participant, 43% achieved a complete response. Most patients developed chronic GVHD. In summary, infusions of donor leukocytes induce remission in the absence of any other therapy, proving that donor hematopoietic cells have the capacity to generate a GVL or GVT effect and resulting in meaningful and potentially durable clinical remissions.

5. NONMYELOABLATIVE ALLOGENEIC TRANSPLANTATIONS We now know that a major component of cure in alloBMT is the GVT effect. Indeed, some chemotherapy-resistant malignancies are potentially cured by alloBMT. In this setting, the GVL effect may be the most important contributor to cure. Therefore, if one were to hypothesize that a given group of patients might be cured by the GVL effect but would probably not benefit by high doses of chemotherapy, then why should such patients receive highdose chemotherapy? Instead, it would make more sense to significantly decrease the intensity of the pretransplant conditioning regimen and simply deliver enough immunosuppressive therapy to prohibit graft rejection. One would then infuse donor hematopoietic cells and rely entirely on the GVT effect to generate a tumor response. This is the fundamental concept of nonmyeloablative (“mini”) allogeneic transplantation. To summarize, the rationale is straightforward: 1. Some malignancies will not be cured by high-dose chemotherapy. 2. Some malignancies may be cured by the GVT effect. 3. If so, a minimal BMT preparative regimen would be desirable to prevent graft rejection and minimize toxicity. 4. Once the donor cells engraft, a GVT effect will hopefully result, leading to a clinical remission.

Mini-transplantations are attractive for several reasons. A significant reduction in the ablative preparative regimen will generate less acute toxicity for patients undergoing alloBMT. Regimen-related toxicity of the traditional abla-

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tive BMT preparative regimen can be severe (57), and it has been suggested that gastrointestinal mucosal damage can result in a release in inflammatory cytokines and actually stimulate the production of acute GVHD (58). A minitransplantation could avoid many of these regimen-related toxicities. Reduction in treatment-related morbidity and mortality might also facilitate alloBMT in older patients and patients with concurrent medical illnesses who might be otherwise ineligible for fully ablative transplantation regimens. Additionally, some mini-transplantation regimens allow the procedure to be performed as an outpatient, which is certainly attractive for some patients undergoing transplantation. Early data have suggested that a mini-transplantation is feasible and often effective. Slavin et al. reported data on 26 patients with a variety of disorders who underwent a nonmyeloablative transplant using fludarabine, anti-T-lymphocyte globulin, and moderate dose busulfan (8 mg/kg) (59). The patients then received G-CSF mobilized peripheral blood progenitor cell (PBPC) allogeneic transplantation, with cyclosporine as the sole GVHD prophylactic agent. Of the 26 patients, 17 achieved complete chimerism and the remainder partial chimerism. Fourteen patients did not experience GVHD; severe GVHD was the cause of death for four patients. With a median follow-up of 8 mo, 85% of patients were alive and 81% were disease free. The conclusion was that nonmyeloablative allogeneic transplants were well tolerated and offered exciting promise. Giralt et al. reported on 15 patients undergoing nonmyeloablative stem cell transplantation to treat refractory AML or MDS (60). The nonmyeloablative regimen was not uniform. GVHD prophylaxis consisted of cyclosporine and methylprednisolone. Acute GVHD occurred in only three patients. Bone marrow chimerism (greater than 90% donor cells) occurred in 75 patients by d 30 after infusion. The procedure was well tolerated, and again, the conclusion was that nonmyeloablative transplantation offered exciting promise for a generally elderly (median age 59 yr; range 27–71 yr) patient population. Although reported follow-up for most nonmyeloablative allogeneic transplantations is relatively brief, several series do have somewhat mature followup. The M.D. Anderson experience with mini-transplantations was recently reported (61). Seventy-eight patients received fludarabine and melphalan as a preparative regimen, and eight received cladribine and melphalan. The median patient age was 52 yr (22–70 yr range). Most patients had advanced hematologic malignancies. The median percentage of donor cells at 1 mo in 75 patients was 100%. The probability of grades 2–4 and 3–4 acute GVHD was 0.49 and 0.29, respectively. Disease-free survival at 1 yr was 57% for patients in first remission and 49% for patients with more advanced disease. The conclusion was that disease control can be achieved by nonmyeloablative alloBMT. McSweeney et al. reported on 45 patients with hematologic malignancies in HLA-identical sibling donors receiving low-dose total body irradiation

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(200 cGy) and cyclosporine plus mycophenolate for GVHD prophylaxis (62). Of the eligible patients 53% had the transplantation performed entirely as an outpatient. Nonfatal graft rejection occurred in 20% of patients, and fludarabine was later added to this preparative regimen to control graft rejection. The incidence of grade 2–3 acute GVHD was 47%. With a median follow-up of 417 d, overall survival was 67%, nonrelapse mortality was 7%, and relapse mortality was 27%. This minimally ablative regimen was extremely well tolerated and demonstrated significant potential efficacy for elderly patients in need of alloBMT. Possibly the prototypic experience of mini-allogeneic transplantations was reported by Childs et al. using mini-transplantations for metastatic renal cell carcinoma (63). Renal cell carcinoma is refractory to chemotherapy but occasionally responds to immunologic therapy such as IL-2. Because some patients respond to immunologic therapy and because the GVT effect is potentially powerful immunologic therapy, the goal of this trial was to use the GVT effect to treat metastatic renal cell carcinoma. Nineteen patients with refractory metastatic renal cell carcinoma received a preparative regimen of cyclophosphamide and fludarabine followed by infusion of peripheral blood stem cell allograft from HLA-identical siblings or a sibling with a one HLA antigen mismatch. The median follow-up was 402 d. Of the 19 patients, 9 survived, 2 died of transplant-related causes, and 8 died of progressive disease. Of the 19 patients, 53% showed disease regression. Of these patients, 30% had a complete response and 70% had a partial response. There was a dramatic correlation of development of disease response with the development of clinical GVHD. Prolonged tumor regression occurred in the majority of patients with grade 2–4 acute GVHD (9 of 10 patients) and in a minority of those without acute GVHD (1 of 9, p = .005). The conclusion was that mini-allogeneic stem cell transplantation can lead to sustained tumor regression in patients with refractory metastatic renal cell carcinoma and was strongly associated with the development of clinical GVHD. This group has also emphasized the development of full donor chimerism of T-cells as a requirement for the GVT response (64). Early data concerning the use of nonmyeloablative alloBMT is exciting. The initial toxicity is diminished compared with a traditional ablative transplantation. However, it is not certain whether mini-transplantation will be as effective as fully ablative transplantation in controlling disease relapse. A retrospective study comparing ablative and nonmyeloablative patients with hematologic malignancies showed that survival was actually decreased in nonmyeloablative recipients (52% vs 28%), with the majority of deaths secondary to disease relapse (65). From January 2000 through September 2001, 20 evaluable patients received a nonmyeloablative alloBMT using a uniform preparative regimen at the

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Table 1 Clinical Characteristics of Patients Who Received Nonmyeloablative Transplantations at the Cleveland Clinic Foundation 2000–2001 No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Age at Transplantation, Yr 57 51 62 62 38 62 62 48 44 61 52 52 57 45 48 60 52 45 49 48

Disease Status at Transplantation

Current Disease Status

AML–CR 2 CLL–Refractory NHL–Rel 2 AML–Rel 2 MM–PR 2 CML–Chronic MDS–RA RCC–Progressive MM–PR 3 Waldenstrom’s–Refractory MDS–Unclass MFB–Stable CML–Chronic AML–CR 2 RCC–Progressive MDS–RAEB CML–Chronic MM–PR 3 MM–CR 1 CLL–Rel 2

Dead (acute GVHD) PR CR CR Dead (chronic GVHD) CR Dead (chronic GVHD) Dead (progressive disease) Progressive disease PR Dead (cGVHD) CR Progressive disease Dead (progressive disease) Progressive disease Progressive disease Progressive disease Progressive disease Progressive disease Progressive disease

AML = acute myeloid leukemia; GVHD = graft vs host disease; CLL = chronic lymphocytic leukemia; PR = partial remission; NHL = non-Hodgkin’s lymphoma; Rel = relapse; CR = complete remission; MM = multiple myeloma; MDS = myelodysplastic syndrome; RA = refractory anemia; RCC = renal cell carcinoma; MFB = myelofibrosis; RAEB = refractory anemia with excess blasts.

Cleveland Clinic Foundation. The patient characteristics and clinical outcomes are shown in Table 1. All patients were treated with a nonmyeloablative preparative regimen consisting of fludarabine (30 mg/m2/d for 3 d), followed by TBI (200 cGy). The patients received donor PBPCs the day after TBI completion. The median patient age was 52 yr (range 28–62, with seven patients older than 60). All patients initially experienced prompt hematopoietic engraftment with neutrophil recovery by day +10 after transplantation, and most patients were treated as outpatients. As shown in Table 1, 6 patients achieved either a complete response or an excellent partial response, 8 patients were alive with progressive disease, and 6 died. The most common cause of death was chronic GVHD. Infection complications have been common, especially cytomegalovirus viremia (66). Lineage-specific chimerism analysis has shown a significant difference in the kinetics of peripheral blood-nucleated cell chimerism and T-cell

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chimerism. The mean peripheral buffy coat donor chimerism, using all nucleated cells, is 95% donor cells by day +21. In contrast, the kinetics of T-cell chimerism are more variable. Most patients ultimately achieve 100% donor Tcell chimerism; however, some patients experience rapid T-cell chimerism by day +49, and others do not experience complete T-cell chimerism until day +200 or longer. Five patients never achieved 100% donor T-cell chimerism, and all five patients relapsed. Fifteen patients achieved 100% T-cell chimerism; nevertheless, 5 of these patients developed progressive disease, including 3 with multiple myeloma. All patients who achieved a complete or an excellent partial response achieved 100% T-cell chimerism. Several conclusions can be drawn from this data. First, the preparative regimen described is associated with limited early toxicity and reduced treatmentrelated mortality compared with an ablative allogeneic transplantation. Peripheral blood–nucleated cell chimerism develops rapidly. Complete T-cell chimerism appears to be a requirement for an ongoing disease response, although the development of complete T-cell chimerism does not guarantee absence of progressive disease. The leading cause of death in our small cohort of patients is chronic GVHD. Our experience demonstrates the feasibility of minitransplantations even for an elderly population. Continued obstacles are disease relapse and clinical GVHD.

6. SUMMARY The author believes that the GVL effect is the most potent immunologic therapy ever described in man. The GVL effect associated with DLI can save patients who are relapsing after alloBMT who would otherwise be incurable. The clinical outcome data of nonmyeloblative allogeneic transplantation, although preliminary, demonstrate the exciting therapeutic promise of the GVL effect. The single biggest clinical problem of the GVL effect is its almost universal association with clinical GVHD. GVHD remains the major cause of morbidity and mortality after alloBMT. Those who perform basic and clinical research involving alloBMT have a simple and straightforward research goal: to separate the GVL effect from GVHD in a clinically meaningful way. To date, we have been unable to achieve this goal. As we become more knowledgeable about the biochemical nature of the GVL effect, graft engineering, and the causes and treatments of clinical GVHD, our ability to maximize the GVL effect and minimize the toxicity of GVHD will result in better and more powerful oncologic immunotherapy.

REFERENCES 1. Barnes D, Corp M, Loutit J, Neal F. Treatment of murine leukaemia with x-rays and homologous bone marrow: II. Br J Haematol 1957;3:241–252. 2. De Vries M, Vos O. Treatment of mouse lymphosarcoma by total-body X irradiation and by injection of bone marrow and lymphnode cells. J Natl Cancer Inst 1958;21:1117–1129.

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3. Odom L, Githens J, Morse H, et al. Remission of relapsed leukaemia during a graft-versushost reaction. Lancet 1978;2:537–540. 4. Weiden P, Flournoy N, Thomas E, et al. Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 1979;300:1068–1073. 5. Sullivan K, Storb R, Buckner C, et al. Graft-versus-host disease as adoptive immunotherapy in patients with advanced hematologic neoplasms. N Engl J Med 1989;320:828–834. 6. Sullivan K, Weiden P, Storb R, et al. Influence of acute and chronic graft-versus-host disease on relapse and survival after bone marrow transplantation from HLA-identical siblings as treatment of acute and chronic leukemia. Blood 1989;73:1720–1728. 7. Horowitz M, Gale R, Sondel P, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990;75:555–562. 8. Jones R, Ambinder R, Piantadosi S, Santos G. Evidence of graft-versus-lymphoma effect associated with allogeneic bone marrow transplantation. Blood 1991;77:649–653. 9. Kersey J, Weisdorf D, Nesbit M, et al. Comparison of autologous and allogeneic bone marrow transplantation for treatment of high-risk refractory acute lymphoblastic leukemia. N Engl J Med 1987;317:461–467. 10. Weisdorf D, Nesbit M, Ramsay N, et al. Allogeneic bone marrow transplantation for acute lymphoblastic leukemia in remission: prolonged survival associated with acute graft-versushost disease. J Clin Oncol 1987;5:1348–1355. 11. Doney K, Fisher L, Appelbaum F, et al. Treatment of adult acute lymphoblastic leukemia with allogeneic bone marrow transplantation. Multivariate analysis of factors affecting acute graftversus-host disease, relapse, and relapse-free survival. Bone Marrow Trans 1991;7:453–459. 12. Passweg J, Tiberghien P, Cahn J, et al. Graft-versus-leukemia effects in T lineage and B lineage acute lymphoblastic leukemia. Bone Marrow Trans 1998;21:153–158. 13. Uzunel M, Mattsson J, Jaksch M, Remberger M, Ringdén O. The significance of graft-versus-host disease and pretransplantation minimal residual disease status to outcome after allogeneic stem cell transplantation in patients with acute lymphoblastic leukemia. Blood 2001;98:1982–1985. 14. Mendoza E, Territo M, Schiller G, Lill M, Kinkel L, Wolin M. Allogeneic bone marrow transplantation for Hodgkin’s and non-Hodgkin’s lymphoma. Bone Marrow Trans 1995;15:199–303. 15. Ratanatharathorn V, Uberti J, Karanes C, et al. Prospective comparative trial of autologous versus allogeneic bone marrow transplantation in patients with non-Hodgkin’s lymphoma. Blood 1994;84:1050–1055. 16. Björkstrand B, Ljungman P, Svensson H, et al. Allogeneic bone marrow transplantation versus autologous stem cell transplantation in multiple myeloma: a retrospective case-matched study from the european group for blood and marrow transplantation. Blood 1996;88:4711–4718. 17. Tricot G, Vesole D, Jagannath S, Holton J, Munshi N, Barlogie B. Graft-versus-myeloma effect: proof of principle. Blood 1996;87:1196–1198. 18. Maraninchi D, Blaise D, Rio B, et al. Impact of T-cell depletion on outcome of allogeneic bone-marrow transplantation for standard-risk leukaemias. Lancet 1987;2:175–178. 19. Mitsuyasu R, Champlin R, Gale R, et al. Treatment of donor bone marrow with monoclonal anti-T-cell antibody and complement for the prevention of graft-versus-host disease. Ann Int Med 1986;105:20–26. 20. Goldman J, Gale R, Horowitz M, et al. Bone marrow transplantation for chronic myelogenous leukemia in chronic phase. Ann Int Med 1988;108:806–814. 21. Marmont A, Horowitz M, Gale R, et al. T-cell depletion of HLA-identical transplants in leukemia. Blood 1991;78:2120–2130. 22. Champlin R, Ho W, Gajewski J, et al. Selective depletion of CD8+ T lymphocytes for prevention of graft-versus-host disease after allogeneic bone marrow transplantation. Blood 1990;76:418–423.

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23. Champlin R, Jansen J, Ho W, et al. Retention of graft-versus-leukemia using selective depletion of CD8-positive T lymphocytes for prevention of graft-versus-host disease following bone marrow transplantation for chronic myelogenous leukemia. Trans Proc 1991;23:1695–1696. 24. Drobyski W, Ash R, Casper J, et al. Effect of T-cell depletion as graft-versus-host disease prophylaxis on engraftment, relapse, and disease-free survival in unrelated marrow transplantation for chronic myelogenous leukemia. Blood 1994;83:1980–1987. 25. Barrett A, Mavroudis D, Tisdale J, et al. T cell-depleted bone marrow transplantation and delayed T cell add-back to control acute GVHD and conserve a graft-versus-leukemia effect. Bone Marrow Trans 1998;21:543–551. 26. Johnson B, Truitt R. Delayed infusion of immunocompetent donor cells after bone marrow transplantation breaks graft-versus-host tolerance and allows for persistent antileukemic reactivity without severe graft-versus-host disease. Blood 1995;85:3302–3312. 27. Nieda M, Nicol A, Kikuchi A, et al. Dendritic cells stimulate the expansion of bcr-abl specific CD8+ T cells with cytotoxic activity against leukemic cells from patients with chronic myeloid leukemia. Blood 1998;91:977–983. 28. Krijanovski O, Hill G, Cooke K, et al. Keratinocyte growth factor separates graft-versus-leukemia effects from graft-versus-host disease. Blood 1999;94:825–831. 29. Jiang Y, Barrett A, Goldman J, Mavroudis D. Association of natural killer cell immune recovery with a graft-versus-leukemia effect independent of graft-versus-host disease following allogeneic bone marrow transplantation. Ann Hematol 1997;74:1–6. 30. Glass B, Uharek L, Zeis M, et al. Graft-versus-leukaemia activity can be predicted by natural cytotoxicity against leukaemia cells. Br J Haematol 1996;93:412–420. 31. Tsukada N, Kobata T, Aizawa Y, Yagita H, Okumura K. Graft-versus-leukemia effect and graftversus-host disease can be differentiated by cytotoxic mechanisms in a murine model of allogeneic bone marrow transplantation. Blood 1999;93:2738–2747. 32. Weiss L, Weigensberg M, Morecki S, et al. Characterization of effector cells of graft vs leukemia following allogeneic bone marrow transplantation in mice inoculated with murine B-cell leukemia. Cancer Immunol Immunother 1990;31:236–242. 33. Pan L, Teshima T, Hill G, et al. Granulocyte colony-stimulating factor-mobilized allogeneic stem cell transplantation maintains graft-versus-leukemia effects through a perforin-dependent pathway while preventing graft-versus-host disease. Blood 1999;93:4071–4078. 34. Hsieh M, Korngold R. Differential use of FasL- and perforin-mediated cytolytic mechanisms by T-cell subsets involved in graft-versus-myeloid leukemia responses. Blood 2000;96:1047–1055. 35. Jiang Y, Kanfer E, Macdonald D, et al. Graft-versus-leukaemia following allogeneic bone marrow transplantation: emergence of cytoxic T lymphocytes reacting to host leukaemia cells. Bone Marrow Trans 1991;8:253–258. 36. Bertheas M, Lafage M, Levy P, et al. Influence of mixed chimerism on the results of allogeneic bone marrow transplantation for leukemia. Blood 1991;78:3103–3106. 37. Mackinnon S, Barnett L, Heller G, O’Reilly R. Minimal residual disease is more common in patients who have mixed T-cell chimerism after bone marrow transplantation for chronic myelogenous leukemia. Blood 1994;83:3409–3416. 38. Delage R, Soiffer R, Dear K, Ritz J. Clinical significance of bcr-abl gene rearrangement detected by polymerase chain reaction after allogeneic bone marrow transplantation in chronic myelogenous leukemia. Blood 1991;78:2759–2767. 39. van Leeuwen J, van Tol M, Joosten A, et al. Persistence of host-type hematopoiesis after allogeneic bone marrow transplantation for leukemia is significantly related to the recipient’s age and/or the conditioning regimen, but it is not associated with an increased risk of relapse. Blood 1994;83:3059–3067. 40. Petz L, Yam P, Wallace R, et al. Mixed hematopoietic chimerism following bone marrow transplantation for hematologic malignancies. Blood 1987;70:1331–1337.

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41. Fishleder A, Bolwell B, Lichtin A. Incidence of mixed chimerism using busulfan/cyclophosphamide containing regimens in allogeneic gone marrow transplantation. Bone Marrow Trans 1992;9:293–297. 42. Ely P, Miller W. bcr/abl mRNA detection following bone marrow transplantation for chronic myelogenous leukemia. Transplantation 1991;52:1023–1028. 43. Miyamura K, Tahara T, Tanimoto M, et al. Long persistent bcr-abl positive transcript detected by polymerase chain reaction after marrow transplant for chronic myelogenous leukemia without clinical relapse: a study of 64 patients. Blood 1993;81:1089–1093. 44. van Leeuwen J, van Tol M, Joosten A, et al. Mixed T-lymphoid chimerism after allogeneic bone marrow transplantation for hematologic malignancies of children is not correlated with relapse. Blood 1993;82:1921–1928. 45. Huss R, Deeg H, Gooley T, et al. Effect of mixed chimerism on graft-versus-host disease, disease recurrence and survival after HLA-identical marrow transplantation for aplastic anemia or chronic myelogenous leukemia. Bone Marrow Trans 1996;18:767–776. 46. Theil K, Warshawsky I, Tubbs R, et al. Dynamics of T-Cell (CD3+) mixed chimerism after non-myeloablative allogeneic peripheral blood stem cell transplant (PBSCT). Proc Am Society Hem 2001;98:189a. 47. Drobyski W, Keever C, Roth M, et al. Salvage immunotherapy using donor leukocyte infusions as treatment for relapsed chronic myelogenous leukemia after allogeneic bone marrow transplantation: efficacy and toxicity of a defined T-cell dose. Blood 1993;82:2310–2318. 48. Kolb H, Mittermuller J, Clemm C, et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 1990;76:2462–2465. 49. Collins R, Shpilberg O, Drobyski R, et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 1997;15:433–444. 50. Porter D, Collins R, Shpilberg O, et al. Long-term follow-up of patients who achieved complete remission after donor leukocyte infusions. Biol Blood Marrow Trans 1999;5:253–261. 51. Dazzi F, Szydlo R, Cross N, et al. Durability of responses following donor lymphocyte infusions for patients who relapse after allogeneic stem cell transplantation for chronic myeloid leukemia. Blood 2000;96:2712–2716. 52. Collins R, Goldstein S, Giralt S, et al. Donor leukocyte infusions in acute lymphocytic leukemia. Bone Marrow Trans 2000;26:511–516. 53. Salama M, Nevill T, Marcellus D, et al. Donor leukocyte infusions for multiple myeloma. Bone Marrow Trans 2000;26:1179–1184. 54. Lokhorst H, Schattenbert A, Cornelissen J, Thomas L, Verdonck L. Donor leukocyte infusions are effective in relapsed multiple myeloma after allogeneic bone marrow transplantation. Blood 1997;90:4206–4211. 55. Giralt S, Escudier S, Kantarjian H, et al. Preliminary results of treatment with filgrastim For relapse of leukemia and myelodysplasia after allogeneic bone marrow transplantation. N Engl J Med 1993;329:757–760. 56. Bishop M, Tarantolo S, Pavletic Z, et al. Filgrastim as an alternative to donor leukocyte infusion for relapse after allogeneic stem-cell transplantation. J Clin Oncol 2000;18:2269–2272. 57. Bearman S, Appelbaum F, Buckner C, et al. Regimen-related toxicity in patients undergoing bone marrow transplantation. J Clin Oncol 1988;6:1562–1568. 58. Hill G, Crawford J, Cooke K, et al. Total body irradiation and acute graft-versus-host disease: the role of gastrointestinal damage and inflammatory cytokines. Blood 1997;90:3204–3213. 59. Slavin S, Nagler A, Naparstek E, et al. Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood 1998;91:756–763.

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60. Giralt S, Estey E, Albitar M, et al. Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: harnessing graft-versus-leukemia without myeloablative therapy. Blood 1997;89:4531–4536. 61. Giralt S, Thall P, Khouri I, et al. Melphalan and purine analog-containing preparative regimens: reduced-intensity conditioning for patients with hematologic malignancies undergoing allogeneic progenitor cell transplantation. Blood 2001;97:631–637. 62. McSweeney P, Niederwieser D, Shizuru J, et al. Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graftversus-tumor effects. Blood 2001;97:3390–3400. 63. Childs R, Chernoff A, Contentin N, et al. Regression of metastatic renal-cell carcinoma after nonmyeloablative allogeneic peripheral-blood stem-cell transplantation. N Engl J Med 2000;343:750–758. 64. Childs R, Clave E, Contentin N, et al. Engraftment kinetics after nonmyeloablative allogeneic peripheral blood stem cell transplantation: full donor T-cell chimerism precedes alloimmune responses. Blood 1999;94:3234–3241. 65. Couriel D, Giralt S, De Lima M, et al. Graft-versus-host disease (GVHD) after non-myeloablative (NMA) versus myeloablative (MA) conditioning regimens in fully matched sibling donor hematopoietic stem cell transplants (HSCT). Proc Am Society Hem 2000;1996:480a. 66. Mossad S, Avery R, Longworth D, et al. Infectious complications within the first year after nonmyeloablative allogeneic peripheral blood stem cell transplantation. Bone Marrow Trans 2001;28:491–495.

3

Unconjugated Monoclonal Antibodies Matt Kalaycio, MD CONTENTS INTRODUCTION HUM195 RITUXIMAB ALEMTUZUMAB (CAMPATH-1H) OTHER ANTIBODIES CONCLUSIONS REFERENCES

1. INTRODUCTION Monoclonal antibodies (MAbs) have been studied as a treatment for leukemia for approximately 20 years but have only recently been used successfully. The history of MAb development has been reviewed extensively (1–4). The purpose of this chapter is to review the clinical data supporting the use of unconjugated MAbs in the treatment of leukemia. Unconjugated MAbs are those that are not conjugated to a toxin or radioisotope. These antibodies typically begin as mouse antibodies to cell surface determinants and are humanized to reduce immunogenicity and avoid human antimouse antibodies (HAMA). By definition, MAbs depend on immunologic mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) to effect cell death. Other mechanisms, such as complement-mediated cellular toxicity and apoptosis, have also been described, but the true nature of their antileukemic effects remain uncertain. However, MAbs are generally well tolerated and easy to administer. Of course, these features mean little if the antibodies are not efficacious in the treatment of leukemia. There are several unconjugated MAbs available for use or in the late stages of clinical development.

From: Biologic Therapy of Leukemia Edited by: M. Kalaycio © Humana Press Inc., Totowa, NJ

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2. HUM195 CD33 is a surface glycoprotein of uncertain function that is expressed on 90% of clonogenic myeloid blasts (5). There is no known expression of CD33 outside hematopoietic tissue and, importantly, it is not expressed on pluripotential stem cells. Unlike some other antigens, however, CD33 is internalized on binding, thus limiting exposure of any antigen-antibody complexes to the reticuloendothelial system. However, complement-mediated cytotoxicity has been demonstrated in leukemia cell lines (6). M195 was the first murine anti-CD33 antibody generated. In a dose-escalating trial of M195 in patients with advanced acute myeloid leukemia (AML), 10 patients were treated with M195 but no clinical responses were documented (7). However, the study demonstrated complete saturation of CD33 binding sites with rapid modulation. The antibody was well tolerated, but HAMA were detected in 67% of patients, which may have contributed to treatment failure. To overcome HAMA, HuM195, a CDR-grafted (humanized) antibody, was constructed. In addition to having similar pharmacokinetics to M195, HuM195 also demonstrated ADCC, probably due to the human IgG1 isotype framework (8). In another clinical trial, 13 patients with advanced AML were treated with a dose-escalating schedule. In this trial, however, no HAHA developed and one patient experienced a reduction in marrow blasts at the highest dose level of 10 mg/m2 (9). A second trial tested HuM195 at supersaturating doses. Ten patients with advanced AML were treated at doses of 12, 24, or 36 mg/m2/d for 4 d as well as a second cycle of treatment 2 wk later. Clinical responses were seen in four patients, and one patient achieved a complete remission (10). A cytokine-release syndrome, characterized by fevers and rigors, was noted at these higher dose levels. The investigators conducting the study theorized that a potential contributor to the low response rate was the lack of effector cells in the setting of advanced leukemia. To test the theory that HuM195 would work better in the setting of a lower leukemic burden, a clinical trial was designed in which patients in remission of acute promyelocytic leukemia (APL) were treated with HuM195 after achieving complete remission with standard chemotherapy. APL is characterized by the reciprocal translocation of the PML oncogene on chromosome 15 with the RARα gene on chromosome 17. The chimeric PML/RARα gene can be monitored by polymerase chain reaction (PCR) techniques even in the setting of histologic remission. Of 22 patients in first remission of APL but who were still PCR positive for PML/RARα, 11 (50%) achieved molecular complete remission after treatment with HuM195 3mg/m2 twice weekly for six doses (11). This study demonstrates proof of principle that HuM195 is capable of eliminating minimal residual disease in patients with APL.

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In a larger study of patients with advanced AML, HuM195 was administered to 49 patients with active leukemia. Patients were randomized to either a 12-mg/m2 infusion or a 36-mg/m2 infusion administered weekly for 4 wk. Although no responses were seen in patients with more than 30% marrow blasts, the percentage of blasts decreased in 12 (60%) of 20 patients with initial blast counts between 5% and 30% and 2 patients (10%) achieved a complete remission (12). HuM195 was well tolerated, and only one patient experienced infusion-related toxicity that required therapy termination. The most common side effects were self-limited fever, chills, and hypotension that usually required no specific therapy. The study demonstrates that HuM195 is a welltolerated treatment with minimal, but observable, activity in patients with advanced AML. Unfortunately, a subsequent prospective randomized trial comparing standard chemotherapy combined with HuM195 versus standard chemotherapy alone in patients with relapsed refractory AML failed to show a significant advantage for the combination treatment arm (13). The ultimate role, if any, for HuM195 remains to be clarified by future clinical trials.

3. RITUXIMAB CD20 is a B-lymphocyte surface antigen that does not internalize on binding antibody (nonmodulating). Most B-cell lymphoproliferative disorders have high-level CD20 expression, but the function of CD20 is unknown. Rituximab is a chimeric anti-CD20 MAb that induces cytotoxicity when bound to surface CD20. Several potential mechanisms of action may explain rituximab’s therapeutic activity, including ADCC, complement-mediated cytotoxicity, and apoptosis induction (14–17) Rituximab administered as single agent at a dose of 375 mg/m2 weekly for 4 wk results in an approx 50% response rate in relapsed low-grade non-Hodgkin’s lymphoma (18). Rituximab is also well tolerated in patients with lymphoma, with most side effects limited to an initial infusion cytokine-release syndrome characterized by fever, chills, and, occasionally, hypotension. The excellent tolerability and remarkable activity of rituximab have prompted testing in nearly all CD20-positive lymphomas either alone or in combination with standard chemotherapy. Rituximab has also been tested against lymphoid leukemias with CD20 expression.

3.1. B-Cell Chronic Lymphocytic Leukemia B-cell chronic lymphocytic leukemia (B-CLL) is a clonal neoplasm of mature lymphocytes expressing a unique immunophenotype characterized by coexpression of CD5, CD19, and CD23. CD20 is also expressed but only at low levels (19). In early trials of rituximab for low-grade lymphomas, patients with small lymphocytic leukemia (SLL), the nodal equivalent of B-CLL, were included. However, in contrast to the high response rates noted in follicular lym-

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phomas with standard doses of rituximab, patients with relapsed SLL achieved remission rates of no more than 15% (18). When studies of patients with relapsed B-CLL treated with standard doses of rituximab also demonstrated low response rates (20,21), enthusiasm for rituximab as a treatment alternative for BCLL/SLL waned further. In a study of 28 patients treated with rituximab 375 mg/m2 weekly for four doses, only 7 patients (25%) achieved a partial remission that lasted a median of only 20 wk (22). In addition to suggesting a limited role in the management of B-CLL/SLL, early studies of rituximab also revealed potentially serious, and previously undescribed, side effects. The early studies of rituximab given to patients with high white blood cell counts revealed a low incidence of a potentially fatal syndrome characterized by rapid multisystem organ failure, severe coagulopathy, and tumor lysis syndrome (23–26). Patients with white blood cell counts higher than 100,000/µl are most at risk, and caution is warranted if rituximab is given to patients with high levels of circulating malignant cells (27). The white blood cell count should be reduced to a level lower than 100,000/µl whenever possible before treatment. Cautious use of rituximab for patients with high white blood cell counts notwithstanding, investigators at M.D. Anderson Cancer Center in Houston, Texas, sought to overcome the limited expression of CD20 on CLL lymphocytes by escalating the dose of rituximab to maximally tolerated levels in patients with advanced diseases (28). The investigators treated 50 patients with previously treated B-CLL (n = 40) or other low-grade lymphoid leukemias (n = 10). Fifty-three percent of patients were refractory to fludarabine, and 43% were refractory to alkylating agents. In addition, 80% of the patients had advanced stage disease. All patients were treated with an initial intravenous dose of rituximab 375 mg/m2, which was followed by three weekly intravenous infusions of a higher dose. The dose levels ranged from 500 to 2250 mg/m2. Most toxicity was limited to the first dose and largely consisted of mild to moderate fever and chills. However, six patients (12%) experienced severe toxicity manifested as fever, chills, hypoxia, and hypotension. Importantly, only one of these patients had B-CLL. The other five patients had other lymphoid leukemias, such as mantle cell leukemia. Thus, severe first-dose toxicity was noted in 2% of patients with B-CLL, regardless of white blood cell count and 50% of patients with other lymphoid leukemias. There was no dose-limiting toxicity to subsequent higher doses of rituximab. Overall, 40% of patients achieved a partial response (28). No patient achieved a complete remission. Importantly, a dose-response effect was noted when the patients at various dose levels were analyzed (Fig. 1). The response rate of 80% at the highest dose levels, 75% if only patients with B-CLL are considered, is far higher than was previously noted at standard doses of rituximab. Dose escalation, then, appears to overcome either the antibody resistance of the leukemic

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Fig. 1. Dose-response effect of rituximab for patients with chronic lymphocytic leukemia. Data from refs. 22, 28, and 29.

clone or the low-level expression of CD20. Unfortunately, the responses were short lived, with a median progression-free survival of 8 mo. The improved efficacy of higher doses of rituximab was further corroborated by a study reported by Byrd and colleagues, who designed a steppeddose schedule of rituximab to reduce the incidence of severe first infusion reactions (29). The first dose of rituximab was 100 mg/m2. Subsequent cohorts of patients were then treated with either 250 or 375 mg/m2 intravenously three times a week, beginning 2 d after the initial 100 mg/m2 dose and continuing for 12 doses or 4 wk. After the initial infusion, rituximab could be safely administered during 1 h. The stepped-dose approach lowered the incidence of infusion-related toxicity, with only two patients experiencing grade III/IV toxicity. Thirty-three patients with either B-CLL or SLL were treated, 28 of whom were previously treated. In addition to one complete response, 14 patients (42%) achieved a partial response (29). Importantly, of the six previously untreated patients receiving rituximab on this protocol, five achieved a partial remission. This study and the one reported by O’Brien et al. (28) clearly demonstrated the clinical efficacy of rituximab in the treatment of B-CLL (Fig. 1). The two trials also demonstrated the difficulty of achieving a complete remission with singleagent rituximab in this often heavily pretreated patient population. Rituximab generally reduces the white blood cell count, reduces splenomegaly, and improves both anemia and thrombocytopenia. However, rituximab is less effective in clearing the bone marrow of malignant cells and in reducing the size of enlarged lymph nodes. As a result, remissions are usually short lived. The relatively modest benefit of rituximab in previously treated B-CLL and the intriguing activity noted in the six untreated patients in Byrd’s study (29)

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suggest a potential role for rituximab earlier in the natural history of the disease. Furthermore, rituximab’s excellent tolerability profile makes it an ideal agent with which to investigate combination therapies. Indeed, preliminary results of two clinical trials that combine fludarabine, cyclophosphamide, and rituximab for patients with B-CLL have recently been reported. In the first study, 43 patients with previously treated B-CLL were treated first with rituximab 375 mg/m2 on day 1, followed by fludarabine 25 mg/m2 and cyclophosphamide 250 mg/m2 on days 2 through 4 (30). The dose of rituximab was increased to 500 mg/m2, and chemotherapy was started on day 1 for the subsequent five cycles of treatment. Although only 21% of these patients were resistant to fludarabine, 70% of patients achieved a remission and 14% achieved a complete remission. The toxicity of the regimen was different from what would normally be expected with the combination of fludarabine and cyclophosphamide alone in this patient population. However, 12% of patients did develop hypotension with the first infusion of rituximab, further suggesting caution on initiation of treatment. The second study used the same chemobiotherapeutic regimen described. For this study, however, the patients had previously untreated B-CLL. Of the 135 evaluable patients, the median age was 57 yr and the median β2-microglobulin level was 4 mg%. However, only 39% of the patients were Rai stage III or IV at enrollment (31). The first cycle of rituximab was generally well tolerated, but 8% of patients experienced grade III–IV fever, chills, or blood pressure changes. An unprecedented 63% of these patients achieved a complete remission with this combination therapy (31). The overall remission was an impressive 95%, and with a median follow-up of approximately 1 yr, the progression-free survival is more than 22 mo and the median survival has not yet been reached. These results compare favorably with results obtained in previously untreated patients with B-CLL treated with fludarabine and cyclophosphamide alone. With the two-drug regimen, 47% of patients achieved a complete remission, with an overall response rate of 53% (32). Rituximab works synergistically with chemotherapy in the clinic, an observation borne out of experimental observations (33). If the promising results from this preliminary analysis are confirmed in a larger patient, population or if the improved remission rates translate into improved survival when compared with standard chemotherapeutic regimens, rituximab may become an important component of initial therapy for patients with B-CLL.

3.2. Hairy Cell Leukemia Rituximab has also been explored as a treatment for hairy cell leukemia, another low-grade B-cell lymphoproliferative disorder characterized by highlevel CD20 expression (34). In a pilot study of patients with previously treated active hairy cell leukemia, nine patients were treated with standard doses of rit-

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uximab 375 mg/m2 weekly for 8 wk. Although no patients with lymphadenopathy responded, four patients (50%) achieved a remission, three of which were complete (35). The patients were too few and follow-up was too short to accurately determine remission duration, but the clinical activity of rituximab was demonstrated. In another study of patients with hairy cell leukemia, who were previously treated 14 evaluable patients were treated with rituximab 375 mg/m2 weekly for 4 wk. A remission was achieved in three patients (21%), and a partial remission was achieved in one patient, for an overall remission rate of 29% (36). No patient relapsed within the 9-mo median follow-up. These small preliminary studies suggest that rituximab has modest activity against previously treated hairy cell leukemia when administered in standard doses. Whether results would improve in less heavily pretreated patients or with higher doses requires additional study. The cumulative experience suggests that rituximab is an important new therapeutic alternative in patients with low-grade B-cell leukemias. Whether administered alone or as part of a combination treatment regimen, rituximab increases remission rates with little toxicity. Patients who are not heavily pretreated and are without bulky lymphadenopathy are those most likely to derive benefit. Whether rituximab can improve survival rates is currently uncertain, but clear palliative benefit can be obtained in properly selected patients for whom other options are limited.

4. ALEMTUZUMAB (CAMPATH-1H) Most normal and malignant T and B lymphocytes express high levels of CD52. CD52 is a small nonmodulating surface antigen that induces ADCC and binds complement when bound with antibody (37). Investigators at Cambridge Pathology developed the first rat anti-CD52 antibody, CAMPATH-1M. This MAb-induced complement-mediated cytotoxicity in vitro but had little clinical activity (38). A subsequent rat MAb, CAMPATH1G, induced ADCC and could deplete lymphocytes from the circulation in vivo (39). Finally, a genetically reshaped human IgG1 CD52 MAb, CAMPATH-1H or alemtuzumab, was developed that mediates ADCC and demonstrates clinical activity. Alemtuzumab is a powerful immunosuppressant and effectively eliminates circulating lymphocytes. These properties led to the use of alemtuzumab as prophylactic treatment for graft vs host disease (GVHD) and to promote stem cell engraftment after allogeneic bone marrow transplantation (41–43). More recently, alemtuzumab has been used to treat patients with lymphoid malignancy (24). Several small studies and case reports suggest clinical activity, but none convincingly demonstrated a role for alemtuzumab in the treatment of B-cell lymphoma (40,44,45). In contrast to rituximab, the lack of a direct apoptotic mechanism of action and the need for effector cells probably explain alemtuzumab’s failure to effectively treat lymphadenopathy (46).

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4.1. T-Cell Prolymphocytic Leukemia Unlike the experience in B-cell lymphoma, alemtuzumab consistently induces high remission rates in patients with T-cell prolymphocytic leukemia (T-PLL). In one study, 15 patients with refractory T-PLL were treated with alemtuzumab 10-mg intravenous (IV) test dose, followed by 30 mg three times a week as tolerated for 6 wk. A complete remission was achieved in nine patients (60%), and two other patients had a partial remission, for an overall remission rate of 73% (47). The median duration of remission was 6 mo. Other than an acute infusion-related syndrome associated with the test dose characterized by fever and rigors, alemtuzumab was well tolerated. However, serious infections complicated two courses of treatment, and three patients developed local Herpes simplex infections. Hematologic toxicity was generally mild, but two patients developed severe bone marrow aplasia (47). A more recent larger study confirms the effectiveness of alemtuzumab in the treatment of T-PLL. Alemtuzumab induced a remission in 75% of 36 evaluable patients with advanced refractory and 2 patients with untreated T-PLL treated at a dose of 30 mg iv three times a week after an initial dose escalation (48). Patients were treated to their maximal responses, and 23 (60%) achieved a complete remission. Importantly, the remission rate was higher for patients with diseases limited to blood, marrow, and spleen, with lower response rates noted in those patients with hepatic and central nervous system involvement. This study also demonstrated that alemtuzumab can induce remissions in patients in relapse after an initial alemtuzumab-induced remission in as many as 42% of patients. Unfortunately, there was median survival of only 10 mo in this population of heavily pretreated patients. In patients with advanced T-PLL, alemtuzumab was reasonably well tolerated. Tumor lysis syndrome was not observed, and toxicity was limited to the first infusion syndrome of fever, rigors, and nausea. Alemtuzumab did result in prolonged and profound lymphopenia that predisposed patients to significant infections, such as cryptococcal meningitis, cytomegalovirus (CMV) infection, Pneumocystis pneumonia, and Legionella pneumonia (48). Therefore, the accumulated evidence suggests that alemtuzumab is an extremely effective treatment for T-PLL. Because T-PLL is relatively resistant to chemotherapy including purine analogs (49), alemtuzumab should be considered as part of the initial therapy for these patients.

4.2. B-CLL Alemtuzumab has been studied in two small studies of patients with B-CLL. In the first, 29 patients with advanced, relapsed, or refractory CLL were treated with alemtuzumab 30 mg iv three times a week for as many as 12 wk. Circulating lymphocytes were eliminated in 28 patients but only 42% of patients were able to achieve a remission by standard criteria. Only two patients experienced

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regression of lymphadenopathy (50). The first infusion of alemtuzumab was typically complicated by fever and rigors, but subsequent infusions were more easily tolerated. Grade IV neutropenia was noted in 20% of patients, but infections secondary to profound lymphocytopenia and immunosuppression were the most serious toxicities encountered. Fifty-two opportunistic infections occurred, 28 of which were serious. In a second small study, seven patients with advanced B-CLL or B-PLL were treated with alemtuzumab administered subcutaneously. After a test dose of 10 mg, patients were treated with 30 mg three times a week. Four patients responded, but serious infections (such as reactivated CMV infections) occurred in five patients (51). The early experience with these two trials suggested the potential for significant clinical benefit from alemtuzumab but also the potential for serious infectious toxicity that prompted antimicrobial prophylaxis in subsequent clinical trials. The results from a large study of alemtuzumab in patients with advanced BCLL have recently been reported. Patients who had been exposed to alkylating agents and were refractory to fludarabine were treated with alemtuzumab beginning with an initial dose of 3 mg, followed in 2 d with a dose of 10 mg, and escalated to a dose of 30 mg administered three times a week for 4 to 12 wk, depending on response. Of the 93 evaluable patients treated in this study, 33% achieved at least a partial remission (52). Another 59% had antitumor responses (reductions in lymphocyte count, improved blood cell counts, resolution of constitutional symptoms, etc.) but failed to meet criteria for partial remission. Similar to the experience with rituximab, patients with bulky lymphadenopathy responded less well, as did older patients and those with β 2 microglobulin levels greater than 5. The median time to progressive disease in the patients in whom a remission was achieved was approx 9 mo. These results are remarkable, given the population of patients treated. In fact, based largely on the data from this study, the Food and Drug Administration (FDA) of the United States approved alemtuzumab in spring 2001 for use in patients with purine analog refractory CLL. In addition to significant clinical activity, alemtuzumab was relatively well tolerated in the aforementioned study. Premedication with acetaminophen and antihistamines were given to lessen the severity of infusion-related side effects, but fever, rigors, nausea and vomiting, and dyspnea were still common manifestations of the first infusion syndrome (Fig. 2) (52). Despite early prophylaxis against infectious diseases with cotrimoxazole and famciclovir, 27% of patients experienced grade 3–5 infections, most of which were pneumonias. Hematologic toxicity was also noted, although difficulties emerged as the investigators tried to distinguish toxicity from the hematopoietic failure induced by the leukemia before treatment. Grade 3–4 anemia occurred in 47%, neutropenia in 70%, and thrombocytopenia in 52% of patients. Within 4 mo, most patients improved their blood cell counts.

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Fig. 2. Incidence of side effects after treatment with alemtuzumab in patients with advanced chronic lymphocytic leukemia (CLL). Data from ref. 52.

Given alemtuzumab’s effectiveness in advanced disease, investigators have begun to explore alemtuzumab’s potential role in patients with less advanced CLL. In a small study, nine patients with previously untreated B-CLL were treated with alemtuzumab 30 mg three times a week after an initial rapid dose escalation. The first five patients received iv infusions, but the next four were treated by subcutaneous injection. Of the nine patients treated, seven (78%) achieved a complete remission in the bone marrow, but only three out of eight patients with enlarged lymph nodes achieved remission (53). Therefore, only three patients achieved complete remission. Of interest, the median response duration had not been reached with short follow-up, even for patients who only achieved a partial remission. Whether administered subcutaneously or intravenously, the first dose of alemtuzumab caused fevers and rigors and one patient experienced a CMV infection. Several other preliminary studies suggest high remission rates in previously untreated patients, but larger randomized trials are needed to confirm any purported survival advantage compared with currently available standard therapies (54,55).

5. OTHER ANTIBODIES Other antibodies are in development or have been evaluated for the treatment of B-CLL and other low-grade lymphoproliferative disorders. Two of these, Hu1D10 and T101, are unconjugated. Hu1D10 is a humanized IgG1κ directed against human leukocytic atigen (HLA)-polymorphism that is usually expressed on both normal and malignant B-lymphocytes. Hu1D10 induces cytotoxicity by inducing apoptosis in the absence of effector cells and complement through transmembrane signaling (56). T101 is an anti-CD5 antibody that demonstrated few antileukemic effects when tested in patients with advanced CLL. Unfortunately, toxic pulmonary

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reactions and limited efficacy inhibited the clinical development of what would be a more targeted approach to B-CLL therapy (57).

CONCLUSIONS After a slow start, monoclonal antibodies are becoming important tools in leukemia treatment. Although many early studies were disappointing regarding remission rates, those studies provided a framework on which subsequent studies capitalized. The available body of literature provides several general concepts with regard to the clinical application of unconjugated MAbs: 1. Aggressive disease, such as acute leukemia or advanced B-CLL, responds poorly to unconjugated antibodies administered alone. However, less advanced disease responds better, especially if antibody is administered with chemotherapy. Even in the absence of remission, these antibodies effectively clear leukemia cells from the blood that may improve blood counts, reduce transfusion requirements, and lessen constitutional symptoms even in the absence of an objective measurable remission. 2. Unconjugated antibodies are generally well tolerated but have the potential for serious side effects when administered in the setting of a large tumor burden. The antilymphocyte antibodies may also induce profound immunosuppression with risk for infections, especially in the case of alemtuzumab. 3. Purely biologic therapies, such as MAbs, are capable of significant antileukemic effects. This observation alone is enough to warrant additional research into the mechanisms by which these antibodies work and into the potential for combination with other biologic therapies that may enhance those mechanisms.

The specific role MAbs will play in the management of leukemia remains to be determined. For now, unconjugated MAbs have been clearly demonstrated to benefit select patients with advanced leukemias if only temporarily. However, the accumulated evidence to date suggests that they will someday be used as part of a treatment strategy, likely in combination, in early stage patients to maximize response rates and improve survival if not cure the disease. Coupled with the promise of other biologic therapies discussed in this book, the hope of cure does not appear as unattainable as it did even in the early 1990s.

REFERENCES 1. Levy R. A perspective on monoclonal antibody therapy: where we have been and where we are going. Sem Hematol 2000;37:43–46. 2. Maloney DG. Advances in immunotherapy of hematologic malignancies. Curr Opin Hematol 1998;5:237–243. 3. Foon KA, Schroff RW, Bunn PA, Jr. Clinical applications of monoclonal antibodies for patients with leukemia and lymphoma. Prog Clin Biol Res 1986;211:265–284. 4. Hainsworth JD. Monoclonal antibody therapy in lymphoid malignancies. Oncologist 2000;5:376–384.

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5. Legrand O, Perrot JY, Baudard M, et al. The immunophenotype of 177 adults with acute myeloid leukemia: proposal of a prognostic score. Blood 2000;96:870–877. 6. McGraw KJ, Rosenblum MG, Cheung L, Scheinberg DA. Characterization of murine and humanized anti-CD33, gelonin immunotoxins reactive against myeloid leukemias. Cancer Immunol Immunother 1994;39:367–374. 7. Scheinberg DA, Lovett D, Divgi CR, et al. A phase I trial of monoclonal antibody M195 in acute myelogenous leukemia: specific bone marrow targeting and internalization of radionuclide. J Clin Oncol 1991;9:478–490. 8. Caron PC, Co MS, Bull MK, Avdalovic NM, Quenn C, Scheinberg DA. Biological and immunological features of humanized M195 (anti-CD33) monoclonal antibodies. Cancer Res 1992;52:6761–6767. 9. Caron PC, Jurcic JG, Scott AM, et al. A phase 1B trial of humanized monoclonal antibody M195 (anti-CD33) in myeloid leukemia: specific targeting without immunogenicity. Blood 1994;83:1760–1768. 10. Caron PC, Dumont L, Scheinberg DA. Supersaturating infusional humanized anti-CD33 monoclonal antibody HuM195 in myelogenous leukemia. Clin Cancer Res 1998;4:1421–1428. 11. Jurcic JG, DeBlasio T, Dumont L, Yao TJ, Scheinberg DA. Molecular remission induction with retinoic acid and anti-CD33 monoclonal antibody HuM195 in acute promyelocytic leukemia. Clin Cancer Res 2000;6:372–380. 12. Feldman E, Kalaycio M, Schulman P, et al. Humanized monoclonal anti-CD33 antibody HuM195 in the treatment of relapsed/refractory acute myelogenous leukemia (AML): preliminary report of a phase II trial. Proc Am Soc Clin Oncol 1999;18:4a. 13. Feldman E, Stone RM, Brandwein J, et al. Phase III randomized trial of an anti-CD33 monoclonal antibody (HUM195) in combination with chemotherapy compared to chemotherapy alone in adults with refractory of first-relapse acute myeloid leukemia. Proc Am Soc Clin Oncol 2002;21:261a. 14. Hofmeister JK, Cooney D, Coggeshall KM. Clustered CD20 induced apoptosis: src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3dependent apoptosis. Blood Cells Molecules, Dis 2000;26:133–143. 15. Golay J, Zaffaroni L, Vaccari T, et al. Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood 2000;95:3900–3908. 16. Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nature Med 2000;6:443–446. 17. Shan D, Ledbetter JA, Press OW. Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells. Cancer Immunol Immunother 2000;48:673–683. 18. McLaughlin P, Hagemeister FB, Grillo-Lopez AJ. Rituximab in indolent lymphoma: the single-agent pivotal trial. Sem Oncol 1999;26:79–87. 19. Caligaris-Cappio F. B-chronic lymphocytic leukemia: a malignancy of anti-self B cells. Blood 1996;87:2615–2620. 20. Ladetto M, Bergui L, Ricca I, Campana S, Pileri A, Tarella C. Rituximab anti-CD20 monoclonal antibody induces marked but transient reductions of peripheral blood lymphocytes in chronic lymphocytic leukaemia patients. Med Oncol 2000;17:203–210. 21. Winkler U, Jensen M, Manzke O, Schulz H, Diehl V, Engert A. Cytokine-release syndrome in patients with B-cell chronic lymphocytic leukemia and high lymphocyte counts after treatment with an anti-CD20 monoclonal antibody (rituximab, IDEC-C2B8). Blood 1999;94:2217–2224. 22. Huhn D, von Schilling C, Wilhelm M, et al. Rituximab therapy of patients with B-cell chronic lymphocytic leukemia. Blood 2001;98:1326–1331. 23. Dillman RO. Infusion reactions associated with the therapeutic use of monoclonal antibodies in the treatment of malignancy. Cancer Metastasis Rev 1999;18:465–471.

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24. Dyer MJ. The role of CAMPATH-1 antibodies in the treatment of lymphoid malignancies. Sem Oncol 1999;26:52–57. 25. Kunkel L, Wong A, Maneatis T, et al. Optimizing the use of rituximab for treatment of B-cell non-Hodgkin’s lymphoma: a benefit-risk update. Sem Oncol 2000;27:53–61. 26. Yang H, Rosove MH, Figlin RA. Tumor lysis syndrome occurring after the administration of rituximab in lymphoproliferative disorders: high-grade non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. Am Hematol 1999;62:247–250. 27. Byrd JC, Waselenko JK, Maneatis TJ, et al. Rituximab therapy in hematologic malignancy patients with circulating blood tumor cells: association with increased infusion-related side effects and rapid blood tumor clearance. J Clin Oncol 1999;17:791–795. 28. O’Brien SM, Kantarjian H, Thomas DA, et al. Rituximab dose-escalation trial in chronic lymphocytic leukemia. J Clin Oncol 2001;19:2165–2170. 29. Byrd JC, Murphy T, Howard RS, et al. Rituximab using a thrice weekly dosing schedule in B-cell chronic lymphocytic leukemia and small lymphocytic lymphoma demonstrates clinical activity and acceptable toxicity. J Clin Oncol 2001;19:2153–2164. 30. Garcia-Manero G, O’Brien S, Cortes J, et al. Combination fludarabine, cyclophosphamide, and rituximab for previously treated patients with chronic lymphocytic leukemia. Blood 2000;96:757a. 31. Wierda W, O’Brien S, Albitar M, et al. Combined fludarabine, cyclophosphamide, and rituximab achieves a high complete remission rate as initial treatment for chronic lymphocytic leukemia. Blood 2001;98:771a. 32. Flinn IW, Byrd JC, Morrison C, et al. Fludarabine and cyclophosphamide with filgrastim support in patients with previously untreated indolent lymphoid malignancies. Blood 2000;96:71–75. 33. Golay J, Xiao YM, Di Gaetano N, Dastoli G, Rambaldi A, Introna M. Fludarabine synergises with anti CD20 monoclonal antibody rituximab in complement mediated cell lysis. Blood 2000;96:339a. 34. Hagberg H. Chimeric monoclonal anti-CD20 antibody (rituximab)—an effective treatment for a patient with relapsing hairy cell leukaemia. Med Oncol 1999;16:221–222. 35. Thomas DA, O’Brien S, Cortes J, et al. Pilot study of rituximab in refractory or relapsed hairy cell leukemia. Blood 1999;94:705a. 36. Nieva J, Bethel K, Baker T, Saven A. Phase II study of rituximab in the treatment of cladribine-failed patients with hairy cell leukemia. Blood 2001;98:364a–365a. 37. Domagala A, Kurpisz M. CD52 antigen—a review. Med Sci Monitor 2001;7:325–331. 38. Hale G, Bright S, Chumbley G, et al. Removal of T-cells from bone marrow for transplantation: a monoclonal antibody fixes human complement. Blood 1983;62:873–882. 39. Dyer MJS, Hale G, Hayhoe FHJ, Waldmann H. Effects of CAMPATH-1 antibodies in vivo in patients with lymphoid malignancies. Blood 1989;73:1431–1439. 40. Hale G, Dyer MJ, Clark MR, et al. Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-1H. Lancet 1988;2:1394–1399. 41. Hale G, Zhang MJ, Bunjes D, et al. Improving the outcome of bone marrow transplantation by using CD52 monoclonal antibodies to prevent graft-versus-host disease and graft rejection. Blood 1998;92:4581–4590. 42. Kottaridis PD, Milligan DW, Chopra R, et al. In vivo CAMPATH-1H prevents graft-versushost disease following nonmyeloablative stem cell transplantation. Blood 2000;96:2419–2425. 43. Cull GM, Haynes AP, Byrne JL, et al. Preliminary experience of allogeneic stem cell transplantation for lymphoproliferative disorders using BEAM-CAMPATH conditioning: an effective regimen with low procedure-related toxicity. Br J Haematol 2000;108:754–760. 44. Pangalis GA, Dimopoulou MN, Angelopoulou MK, Tsekouras CH, Siakantaris MP. Campath-1H in B-chronic lymphocytic leukemia: report on a patient treated thrice in a 3 year period. Med Oncol 2000;17:70–73.

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45. Lundin J, Osterborg A, Brittinger G, et al. CAMPATH-1H monoclonal antibody in therapy for previously treated low-grade non-Hodgkin’s lymphomas: a phase II multicenter study. European Study Group of CAMPATH-1H Treatment in Low-Grade Non-Hodgkin’s Lymphoma. J Clin Oncol 1998;16:3257–3263. 46. Dyer MJS, Osterborg A. The use of therapeutic monoclonal antibodies in chronic lymphocytic leukemia. In: Cheson BD, ed. Chronic Lymphoid Leukemias. New York: Marcel Dekker; 2001:335–352. 47. Pawson R, Dyer MJS, Barge R, et al. Treatment of T-cell prolymphocytic leukemia with human anti-CD52 antibody. J Clin Oncol 1997;15:2667–2672. 48. Dearden CE, Matutes E, Cazin B, et al. High remission rate in T-cell prolymphocytic leukemia with CAMPATH-1H. Blood 2001;98:1721–1726. 49. Mercieca J, Matutes E, Dearden C, MacLennan K, Catovsky D. The role of pentostatin in the treatment of T-cell malignancies: analysis of response rate in 145 patients according to disease subtype. J Clin Oncol 1994;12:2588–2593. 50. Osterborg A, Dyer MJS, Bunjes D, et al. Phase II multicenter study of human CD52 antibody in previously treated chronic lymphocytic leukemia. J Clin Oncol 1997;15:1567–1574. 51. Bowen AL, Zomas A, Emmett E, Matutes E, Dyer MDC. Subcutaneous CAMPATH-1H in fludarabine-resistant/relapsed chronic lymphocytic and B-prolymphocytic leukaemia. Br J Haematol 1997;96:617–619. 52. Keating MJ, Flinn I, Jain V, et al. Therapeutic role of alemtuzumab (CAMPATH-1H) in patients who have failed fludarabine: results of a large international study. Blood 2002;99:3554–3561. 53. Osterborg A, Fassa AS, Anagnostopoulos A, Dyer MJS, Catovsky D, Mellstedt H. Humanized CD52 monoclonal antibody CAMPATH-1H as first-line treatment in chronic lymphocytic leukemia. Br J Haematol 1996;93:151–153. 54. O’Brien SM, Thomas DA, Cortes J, et al. CAMPATH-1H for minimal residual disease in chronic lymphocytic leukemia. J Clin Oncol 2001;21:284a. 55. Lundin J, Kimby E, Bjorkholm M, et al. Phase II trial of subcutaneous anti-CD52 monoclonal antibody alemtuzuzmab (Campath-1H) as first line treatment for patients with B-cell chronic lymphocytic leukemia (B-CLL). Blood 2002;100:768–773. 56. Kostelny SA, Link BK, Tso JY, et al. Humanization and characterization of the anti-HLADR antibody 1D10. Int J Cancer 2001;93:556–565. 57. Foon KA, Schroff RW, Bunn PA, et al. Effects of monoclonal antibody therapy in patients with chronic lymphocytic leukemia. Blood 1984;64:1085–1093.

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Drug Immunoconjugate Therapy of Acute Myeloid Leukemia Arthur E. Frankel, MD, Bayard L. Powell, MD, Eli Estey, MD, and Martin S. Tallman, MD CONTENTS INTRODUCTION ACUTE MYELOID LEUKEMIA GEMTUZUMAB OZOGAMICIN—STRUCTURE AND FUNCTION GEMTUZUMAB OZOGAMICIN CLINICAL TRIALS PHARMACOKINETICS AND IMMUNE RESPONSE TOXICITIES CLINICAL RESPONSES ONGOING AND PLANNED CLINICAL STUDIES REFERENCES

1. INTRODUCTION Drug immunoconjugates are a novel class of cell surface receptor-targeted agents. They consist of monoclonal antibodies (MAbs) covalently linked to cytotoxic drugs. The first application of this class of compounds is in the treatment of patients with acute myeloid leukemia (AML). In this review, we examine the biology of AML and identify the rationale for the use of the anti-CD33 antibody in the selective targeting of this disease. We look at the structure and function of calicheamicin and how it came to be linked successfully to the antiCD33 antibody. The phase I and II clinical data are discussed, with particular attention to toxicities and mechanisms of resistance. We relate the clinical information with the prior observations on AML biology and antibodycalicheamicin conjugate pharmacology. Finally, we suggest strategies to reduce the normal tissue toxicities and broaden the clinical use of this new exciting therapy. Lessons learned with this agent may be useful in the development and application of future drug immunoconjugates. From: Biologic Therapy of Leukemia Edited by: M. Kalaycio © Humana Press Inc., Totowa, NJ

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2. ACUTE MYELOID LEUKEMIA Patients with AML have excess myeloblasts in their marrow. These leukemic blasts frequently have clonal cytogenetic and immunophenotypic abnormalities. The focus of current chemotherapies or immunotherapies has been to deplete these abnormalities. However, recent experimental observations suggest that in most leukemias (other than acute promyelocytic leukemia [APL]), the self-renewing long-term proliferating stem cells have a phenotype and physiology that is distinct from the majority of blasts. Only rare (1/1000 to 1/106) mononuclear cells from these patients survive and proliferate in liquid culture or immunocompromised mice (1). These leukemic stem cells express CD34 and lack myeloid differentiation antigens, including CD33 and CD38 (2,3). They proliferate slowly and overexpress CD123 death-associated protein kinase (DAPK) and interferon regulatory factor 1 (IRF-1) (4,5). In some but not all cases, they lack the Thy1 antigen (CD90) expressed on normal stem cells (6). Different genetic lesions in these bland lineage-negative mononuclear cells can produce different patterns of differentiated offspring (7). Because cytotoxic chemotherapies and many cell surface-targeted compounds selectively kill mature myeloblasts, how are durable remissions obtained? Two hypotheses are presented. There may be some, albeit reduced, chemosensitivity and differentiation antigen density on the primitive leukemic stem cells. Consequently, repeated high doses of drugs or targeted proteins may kill a fraction of the repopulating cells and permit prolonged leukemia remission. Alternatively, cytoreductions of mature myeloblasts may remove a critical feedback loop. Differentiating myeloid cells may produce cytokines that help the leukemic stem cells to survive and grow (8). Therapies for AML also lead to recovery of normal polyclonal hematopoiesis. How is this achieved when the drugs and immunoconjugates injure normal myeloid progenitors as well as leukemic progenitors? Again, we postulate three nonexclusive theories. The leukemic progenitors may be more sensitive than normal progenitors due to altered signal transduction or apoptotic pathways in the malignant cells. Alternatively, there may be many more normal stem cells than leukemic stem cells. If chemotherapy or immunotherapy depletes an equal number (rather than an equal fraction) of normal and neoplastic stem cells, the normal stem cells may have a numeric regrowth advantage. Finally, leukemic blasts may secrete factors that inhibit normal hematopoiesis (9). With depletion of leukemic blasts, normal hematopoiesis may occur. The ongoing clinical studies with molecularly targeted compounds such as gemtuzumab ozogamicin (Mylotarg™, Wyeth, NY) may help to elucidate the mechanism for leukemia control and normal hematopoietic reconstitution. Ancillary studies with the blood or marrow from these patients may be useful in identifying leukemia stimulatory or normal marrow-suppressive molecules.

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3. GEMTUZUMAB OZOGAMICIN—STRUCTURE AND FUNCTION The mouse hybridoma producing the IgG1 MAb p67 was prepared by immunizing a mouse with AML blasts and screening hybridoma supernatants for reactivity to AML cells but not B cells (10). The p67 antibody reacted with myeloid, differentiation antigen CD33. This antigen was also recognized by anti-My9 and M195 (11,12). CD33 is absent from platelets, T-cells, erythrocytes, B-cells, and early normal stem cells (LTC-IC) (13). The antigen is present on peripheral blood monocytes and marrow myeloblasts, promyelocytes, and weakly on metamyelocytes and granulocytes. CD33 is also present on the myeloid progenitors (CFUGM and CFU-GEMM) and 80% to 90% of AML and blast crisis chronic myelogenous leukemia (CML) blasts. Thus, the p67 anti-CD33 antibody selectively targets AML blasts but not early normal stem cells. In APL, the antibody binds the entire leukemic stem cell pool (14). For other AMLs, as noted, leukemic stem cells may still be affected if they have some CD33 expression or if they are dependent on cytokines produced by CD33-positive leukemic blasts. The p67 antibody was radiolabeled with 131I and used for imaging and therapy of patients with AML (15). The radioimmunoconjugate was selective to marrow and leukemic compartments in patients but was rapidly dehalogenated. As a result, there was poor tumor dosimetry and significant deposition of free radioiodine in the thyroid, stomach, and kidneys. Preclinical work showed that the radioiodinated antibody-retained antigen affinity (Ka = 4 × 1010 M–1) but that rapid internalization of the antibody occurred with 38% removal of surface-bound antibody in 4 h (16). The antibody–antigen endocytosis properties of the p67 antibody make it attractive for construction of a drug immunoconjugate. Before further study, the complementarity-determining regions of p67 were grafted onto a human IgG4 framework (Fig. 1) (17) to reduce the risk of immunogenicity in patients. The human IgG4 isotype was chosen because it has fewer Fc-dependent effector functions and is thus considered less likely to cause unwanted clinical reactions. The humanized p67 retained the affinity of the murine antibody for p67. It was weakly immunogenic in monkeys. Antibodies formed were against antihuman Ig framework epitopes. The selection of the proper drug for drug immunoconjugates is critical. The drug must be potent—able to kill a cell with fewer than 1000 molecules in the cell. It must be linkable to antibody so that it retains bioactivity when released. A class of drugs that meets these requirements is the calicheamicins isolated from the Kerrville, Texas, soil organism, Micromonospora echinospora ssp. Calichensis (18). These small molecular weight compounds are sequence specific, minor groove binding, deoxyribonucleic acid (DNA) damaging antibiotics. Calicheamicin γ1I, the parent compound, is shown in Fig. 2. It consists of

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Fig. 1. Illustration of humanized antibody displaying heavy and light chains of human immunoglobulin IgG4 (gray) and mouse anti-CD33 CDR grafts (white). Disulfide bonds are shown in black.

a methyl trisulfide portion (the trigger), a enediyne moiety that aromatizes to a 1,4-dehydrobenzene-diradical (the warhead), and a sugar aromatic ring backbone—4,6-didoxy-4[hydroxyamino]-β-D-glucopyranoside A ring, N–O glycosidic linkage to the thio sugar B ring, 4-ethylamino sugar E-ring attached to the A ring, a persubstituted dimethoxy aromatic ring attached to the B ring, and a 3-O-methyl-α-L-rhamnopyranoside D-ring (the targeting region). Bioreductive cleavage by reaction with cell glutathione leads to β-addition of the resulting thiol to the enone form to form dihydrothiophene. This compound undergoes cyclization to the 1,4-diyl6, also called the 1,4-dehydrobenzenediradical (Fig. 3) (19). This reactive species abstracts hydrogen from the deoxyribose backbone when DNA is present. Interestingly, the H abstraction on the deoxyribose ring and subsequent oxidative strand scission, as shown in Fig. 4, only occurs at sequence-specific cleavage sites on DNA. In particular, the sequence TCCT is required. Figure 5 shows the cleavage that generates double-strand breaks (20). The drug is active at subpicogram/mL concentrations and is approx 1000-fold more active than doxorubicin against murine tumors (21). The DNA damage leads to cell death. The unique chemical prop-

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Fig. 2. Chemical structure of calicheamicin γ11. The important aspects of its structure are the methyltrisulfide (trigger), the diyne-ene moiety that aromatizes to a 1,4-dehydrobenediradical (the warhead), and a sugar-aromatic ring backbone (the 4,6-didoxy-4 [hydroxyamino]-β-D-glucopyranoside A ring, N-O glycosidic linkage to the thio sugar B ring, and attached to the A ring—the 4-ethylamino sugar E-ring and attached to the B ring, through a methyl,diacetyl iodinated aromatic ring, the 3-O-methyl-α-L-rhamnopyranoside D-ring).

erties of the molecule made it an excellent candidate for linkage to antibodies and selective release inside cells. Conjugation of calicheamicin to humanized p67 antibody so that the drug would be released only intracellularly was accomplished by taking advantage of the methyltrisulfide trigger. Because the thiol-leaving group is not required for drug action, the methyltrisulfide trigger was derivatized with a mercaptohydrazide to yield the hydrazide analogue. A dimethyl group was employed near the disulfide to reduce spontaneous and premature disulfide reduction in the bloodstream (22). An N-acetyl group was attached to the amino sugar at the ethyl nitrogen, based on empirical experiments that measured activity versus toxicity of conjugates (22). The antibody was attached using the bifunctional linker AcBut. This linker attaches to lysines on the antibody and forms a hydrazone with the hydrazide of the NAc-γ-calicheamicin DMH (Fig. 6). This hydrazone is hydrolyzed in the acidic environment of the intracellular endosome/lysosomes through which the antibody is routed after internalization (Fig. 7). The anti-CD33 calicheamicin (previously called CMA-676; now with the generic name gemtuzumab ozogamicin and tradename Mylotarg) is selectively toxic to CD33-bearing normal and malignant myeloid cells.

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Fig. 3. Bioreductive cleavage by reaction with cell glutathione leads to β-addition of the resulting thiol to the enone form to form dihydrothiophene. This compound undergoes cyclization to form the 1,4-diyl6 also called the 1,4-dehydrobenzene-diradical. This is the bioactive intermediate.

4. GEMTUZUMAB OZOGAMICIN CLINICAL TRIALS Gemtuzumab ozogamicin has been studied for treatment of relapsed AML in one phase I clinical trial and three phase II clinical trials. The phase I study was done at the Fred Hutchinson Cancer Research Center and the City of Hope National Medical Center (23). Forty patients were treated with one to three doses of 0.25 mg/m2 to 9 mg/m2 gemtuzumab ozogamicin 2-h intravenous (iv) infusions with 2 or more weeks between doses. Premedications included acetaminophen and diphenhydramine. The median patient age was 54 yr (range 24 to 73); the group included 21 men and 19 women. Half of the patients were in first relapse, and the remainder in second or greater relapse. Fourteen patients had previously received allogeneic bone marrow transplantations (BMT) and 4 had received autologous BMTs. Complex cytogenetic abnormalities were present in the blasts of 22 patients, and single abnormalities were present in 7 patients (2 each with inv[16] and t[9;11] and 1 each with t[8;21], t[4;11], and t[6;11]). Nine patients had previous myelodysplasia syndrome (MDS). The phase II studies were multi-institutional, and the data from the three studies have been pooled because treatment with gemtuzumab ozogamicin was the same in each study (24–26). The 201 study in the United States and Canada

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Fig. 4. Cleavage mechanism of the TCCT site at the 5′C. The carbon-centered radical abstracts a 5′H from the deoxyribose sugar. The sugar is then attacked by dioxygen to form a peroxyl radical that, in the presence of thiol, forms an aldehyde and causes strand scission.

included 13 centers, the 202 study in Europe included 19 centers, and the 203 study in both the United States and Europe included 21 centers. Seventy-two patients were evaluable from the 201 study, 62 patients from the 202 study, and 54 patients from the 203 study. All patients had to be in first relapse AML and older than 18 years. In the 203 study, the patients had to be older than 60 years. In the 201 and 202 studies, the duration of first remission (CR1) had to be 6 mo or more. In the 203 study, the CR1 had to be 3 mo or more. All patients had to have a white blood cell count (WBC) less than 30,000/µL and no prior MDS. Prior BMT was only permitted in the 202 study. All patients had to have CD33 expression on leukemic blasts (staining intensity > 4 × unstained cells on ≥ 80% of cells). Patients were scheduled to receive two infusions of gemtuzumab ozogamicin at 9 mg/m2 given 14 d apart. To date, 188 patients are evaluable. The median age was 60 yr (range 22 to 87), with 56% (105) men and 44% (83) women. Most of the patients were white (93%). The mean duration of CR1 was 11.1 mo. Most had undergone postremission consolidation therapy (94%), which frequently included high-dose cytarabine (67%). Favorable cyto-

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Fig. 5. The calicheamicin double-stranded DNA cleavage site. The cleavage is at the 3′-T and 5′-A of TCCT/AGGA.

genetics at relapse were rare (4% in the patients who had cytogenetic studies performed at relapse).

5. PHARMACOKINETICS AND IMMUNE RESPONSE Serum concentrations of the hP67.6 and calicheamicin component of gemtuzumab ozogamicin and peripheral blast saturation were measured in the phase I and II studies (23). The half-life of hP67.6 was approx 67 h, and the peak concentration was proportional to the dose. The free calicheamicin area

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Fig. 6. Illustration showing the molecular structure of gemtuzumab ozogamicin. The hP67.6 is the humanized anti-CD33 antibody shown as a circle, which is attached through an ε amino group of lysine to an Ac-But acid labile linker and, finally, to calicheamicin 1γ1.

Fig. 7. Cell intoxication by gemtuzumab ozogamicin: (a) binding to cell surface CD33; (b) internalization to endosomes; (c) cleavage of acid labile bond and release of calicheamicin; (d) diffusion through endosome or lysosome membrane bilayer to cytosol; (e) reduction of disulfide followed by diradical formation; (f) insertion into DNA and double-strand DNA cleavage; and (g) programmed cell death.

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under the concentration curve was 0.3% of the hP67.6 area under the concentration curve. Saturation of circulating blasts was observed at 4 mg/m2 or greater. Antibodies to gemtuzumab ozogamicin were measured in all of the clinical trials. In the phase I study, one patient developed antibody to the calicheamicin-linker complex after the third dose and a second patient developed antibodies to the calicheamicin-linker during the second dose of a second course of gemtuzumab ozogamicin. In the phase II studies, no humoral immune response to gemtuzumab ozogamicin occurred in the 188 evaluable patients.

6. TOXICITIES Three classes of side effects were observed in the clinical trials. An acute infusion reaction occurred in most patients with fever and chills, and less commonly, hypotension, hypertension, diarrhea, abdominal pain, headaches, dyspnea, nausea, vomiting, and asthenia. Thirty-eight percent of patients had one or more of these as grade 3 or 4 events. Symptoms occurred usually within 8 to 12 h. The symptoms resembled those seen with other antibody- and recombinant protein-based therapeutics (27). Myelosuppression occurred in all patients, and 97% and 99% of patients showed grade 3 to 4 neutropenia and thrombopenia, respectively. Secondary complications of severe (grades 3 and 4) infections (27%) and bleeding (14%) were seen. The median time to recovery of neutrophils to more than 500/µL was 42 d from the first dose of gemtuzumab ozogamicin for remission patients. The median time to recovery of platelets to 25,000/µL was 36 d and 75 d from the first dose of gemtuzumab ozogamicin for patients achieving a complete remission (CR) or complete remission, except for incomplete platelet recovery (CRp), respectively. Transient myelosuppression was expected, because normal committed myeloid precursors express CD33 (28). The duration of myelosuppression was not significantly different from that seen with high-dose cytarabine salvage regimens (29). Liver injury is the unique and most clinically significant drug-related toxicity observed in the preapproval clinical trials and in postapproval studies (24,30,31). Grade 3 or 4 elevations in liver transaminases (AST and ALT) or in total bilirubin occurred in 16% and 26% of patients, respectively. These signs of liver injury were delayed, with maximal abnormalities 1 to 2 wk after therapy. A fraction of patients (2% in the phase II trials and up to 11% of some combination therapy studies) also developed a veno-occlusive disease (VOD)-like syndrome with liver tenderness and enlargement, jaundice, and fluid retention. The incidence of liver damage was increased by previous bone marrow transplantation (both autologous and allogeneic). Co-administration of other cytotoxic drugs and cytokines (such as interleukin-11) may also predispose patients to the liver lesion (31). Ultrasound, computed tomography scan, and magnetic resonance imaging may show reversal of portal flow consistent with portal hypertension. Rarely have patients

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had transjugular liver biopsies that show sinusoidal cell apoptosis, increased extracellular matrix, and centrilobular necrosis and congestion. Because CD33 is not expressed by hepatocytes or sinusoidal endothelium, the hypothesis has been advanced that gemtuzumab ozogamicin reacts with the fixed macrophages in the liver Kupffer cells. The dying or activated Kupffer cells may release cytokines that activate stellate cells (32). Activated stellate cells secrete collagen and other extracellular matrix material, which can lead to constriction of sinusoids causing portal hypertension and centrilobular ischemia. In mild cases, only transaminasemia is seen, but in severe cases, a VOD-like syndrome ensues. No approaches to date have been effective as prophylaxis or treatment. Despite these various side effects, most patients tolerate gemtuzumab ozogamicin well. Mucositis was minimal, and no evidence of cardiac or central nervous system (CNS) toxicity was seen.

7. CLINICAL RESPONSES Among the 40 patients treated in the phase I trial, there were three CRs and five CRps (23). Remissions occurred only in patients receiving at least two doses and in patients showing at least 80% saturation of circulating blasts. Based on these encouraging results, the phase II trials were undertaken (24,33). The overall remission rate was 31% (n = 188), with 15% CRs and 16% CRps. In the 201 study, the remission rate was 35%, with 18% CRs and 17% CRps (n = 72). In the 202 study, the remission rate was 33%, with 15% CRs and 18% CRps (n = 62). In the 203 study, the remission rate was 24%, with 9% CRs and 15% CRps (n = 54). The lower remission rate in the 203 study may have been due to the older age and shorter CR1 in these patients. When analyzed for prognostic factors, age and duration of CR1, but not cytogenetics, predicted response rate. Patients who were younger than 60 yr had a remission rate (CR + CRp) of 31% compared with 28% for patients equal to or older than 60 yr. Similarly, patients with CR1 ≥ 12 mo had a remission rate of 39% (32 of 82) versus 25% (26 of 106) for those with CR1 < 12 mo. The combination of older age and short CR1 was as expected. The remission rate for patients younger than 60 yr with CR1 ≥ 12 months was 39% (16 of 41). The remission rate for patients older than 60 yr with CR1 for less than 6 mo was 10% (2 of 21). Most patients who achieved remission went on to other therapies. Thus, analysis of overall and disease-free survival is complicated. Nevertheless, a fraction of patients in remission were alive (30%) and disease-free (25%) at 30 mo (24). The type of remission—CR versus CRp—did not influence relapse-free or overall survival. The total (remission plus nonresponding) patient median overall survival was 5.5 mo, which compares favorably with other salvage regimens (29). One of the patients in the 202 study had a t(9;22)-positive FAB M5 AML and responded to gemtuzumab ozogamicin (33). A three-log cytoreduction was documented by quantitative polymerase chain reaction (PCR).

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Since Food and Drug Administration (FDA) approval, gemtuzumab ozogamicin has been used for a several patients with relapsed or refractory AML. Forty-three patients treated at Wake Forest, Cornell, and New York Medical Colleges who had early relapse of AML (< 6 mo), refractory AML, AML in second or greater relapse, CML blast crisis, or AML after MDS received two doses of gemtuzumab ozogamicin 9 mg/m2 on days 1 and 15 (34). Side effects were transient and similar to those observed in the phase I/II trials and included infusion reactions (2%), myelosuppression (95%), and hepatic injury (21%). The overall remission rate was 14% in all patients, with 9% CR and 5% CRp. In patients with relapsed or refractory AML (excluding the CML patients), the remission rate was 17%. This is similar to the 17% remission rate found in the phase II studies for elderly patients with CR1 duration of 3 to 12 mo. The mean duration of gemtuzumab ozogamicin remissions was 4 mo. Only one patient did not have normal cytogenetics—a CRp patient who had t(5;6). Clinical resistance to gemtuzumab ozogamicin may occur by several pharmacologic barriers. The protein conjugate may fail to reach sanctuary sites such as the CNS, testes, or other extramedullary sites. This is not truly cellular resistance but an explanation for the refractory disease in these patients. In the phase I clinical trial, relapses after gemtuzumab ozogamicin remissions were observed in these sites (23). Systemic gemtuzumab ozogamicin treatment of a patient with CNS disease failed to clear blasts from the cerebrospinal fluid (CSF), and drug levels were unmeasurable in the CSF (35). Alternatively, the leukemic blasts may be resistant to gemtuzumab ozogamicin because of rapid efflux by a drug transporter or failure of CD33 internalization. Multidrug-resistant (MDR-1+) AML cell lines were not sensitive to gemtuzumab ozogamicin, but the addition of multidrug-resistant modifiers restored sensitivity (36). Bernstein and colleagues have recently shown a correlation between CD33 internalization and sensitivity to gemtuzumab ozogamicin (unpublished data). The importance of any of these hypothetical resistance mechanisms on gemtuzumab ozogamicin clinical activity is presently unknown.

8. ONGOING AND PLANNED CLINICAL STUDIES Based on the results from the phase II studies, gemtuzumab ozogamicin was approved by the FDA in May 2000 for the treatment of CD33-positive relapsed AML in patients equal to or older than 60 yr (37). Since approval, additional studies with Mylotarg have been initiated to evaluate different schedules, different side-effect prophylaxis regimens, combinations with cytotoxic chemotherapy, and new indications. An alternative schedule with day 1 and 8 treatments will be tested. Because of the several-day half-life of gemtuzumab ozogamicin, there is significant circulating drug remaining at day 8. To avoid higher than necessary levels, the

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second dose will be reduced to 6 mg/m2. This new schedule is more consistent with the current AML treatment regimens and should shorten the duration of myelosuppression. Anecdotal reports suggest that steroids block the acute infusion reactions. Corticosteroid prophylaxis (2 mg dexamethasone the day before and the morning of therapy) will be tested at Wake Forest and several other sites. Combination therapy with cytarabine, cytarabine/daunorubicin, mitoxantrone, idarubicin/cytarabine, daunorubicin/cytarabine/6TG, or cytarabine/mitoxantrone/amifostine will be tested in phase I/II trials. A Wyeth-Ayerst–sponsored phase I/II multicenter trial, protocol 205, examines gemtuzumab ozogamicin on days 1 and 8 with cytarabine continuous infusion on days 1 to 7. Older patients (> 60 years) with both de novo and relapsed/refractory disease will be treated. Gemtuzumab ozogamicin doses will be 6 mg/m2 and 4 mg/m2 initially. The initial cytarabine dose will be 100 mg/m2. Dose escalation will proceed to gemtuzumab ozogamicin 9 mg/m2 and 6 mg/m2, along with cytarabine 200 mg/m2. Another WyethAyerst–sponsored phase I/II trial, protocol 206, treats relapsed/refractory patients and those younger than 60 yr with de novo AML with gemtuzumab ozogamicin on day 4, daunorubicin on days 1 to 3, and cytarabine on days 1 to 7. There is dose escalation. Twelve patients have been treated to date. Other studies will explore different combination regimens including (a) gemtuzumab ozogamicin on days 1 and 8, with five daily doses of cytarabine 1 gm/m2; (b) gemtuzumab ozogamicin on days 7 and 14, with cytarabine 3 gm/m2 on days 1 to 5; (c) gemtuzumab ozogamicin on days 1 and 14, with mitoxantrone; (d) gemtuzumab ozogamicin on day 3 with cytarabine 2 gm/m2, mitoxantrone, and amifostine; and (e) gemtuzumab ozogamicin plus daunorubicin, cytarabine, and 6TG. Thus, soon we should have a wealth of information on the safety and efficacy of combining gemtuzumab ozogamicin with other cytotoxic drugs. Gemtuzumab ozogamicin is also being tested as a single agent in several new disease states, including pediatric-relapsed AML, MDS, acute lymphocytic leukemia (ALL), and elderly de novo AML. The Wyeth-Ayerst pediatric AML protocol 102 has accrued 28 patients, including 9 evaluable at 9 mg/m2 for two doses. Because of a transient grade 4 transaminase elevation, additional patients are being accrued at 7.5-mg/m2 doses. The trial is ongoing, but the response rate resembles that seen in adults. In the Wyeth-Ayerst multicenter protocol 207, intermediate and high-risk MDS patients receive a single dose or two doses of gemtuzumab ozogamicin followed by up to three additional doses at monthly or greater intervals. Nine patients have been enrolled, and marrow blast cytoreductions have been seen. It is too early to assess response. Elderly patients with de novo AML will be treated as noted in protocols 205 and 206 and the ECOG study. In addition, these elderly patients with de novo AML are being treated at M.D. Anderson. Patients with normal cytogenetics have had a 50% CR rate (Estey, unpublished data). However, at M.D.

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Anderson, unlike the phase II trials in which older patients with poor-risk cytogenetics had an overall remission rate of 30%, patients with complex cytogenetic abnormalities or chromosome 5 or 7 changes have had a CR rate of 10%. Additional sites performing studies of single-agent gemtuzumab ozogamicin in elderly patients with AML include EORTC, Northwestern, Rush, and the MRC. Patients with CD33-positive ALL are being treated with single-agent Mylotarg salvage protocols, and remissions have been observed. Patients with AML in remission are being randomized to receive Mylotarg as part of consolidation therapy and as part of the pretransplantation or posttransplantation conditioning regimens. These trials should define new disease indications for gemtuzumab ozogamicin. The next decade should see a better understanding of mechanisms of resistance and toxicity to this novel agent, as well as provide evidence for the optimal disease setting and combination regimens for patients with CD33-positive hematologic malignancies.

REFERENCES 1. Lapidot T, Fajerman Y, Kollet O. Immune-deficient SCID and NOD/SCID mice models as functional assays for studying normal and malignant human hematopoiesis. J Mol Med 1997;75:664–673. 2. Brendel C, Neubauer A. Characteristics and analysis of normal and leukemic stem cells: current concepts and future directions. Leukemia 2000;14:1711–1717. 3. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997;3:730–737. 4. Jordan CT, Upchurch D, Szilvassy SJ, et al. The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells. Leukemia 2000;14:1777–1784. 5. Guzman ML, Upchurch D, Grimes B, et al. Expression of tumor-suppressor genes interferon regulatory factor 1 and death-associated protein kinase in primitive acute myelogenous leukemia cells. Blood 2001;97:2177–2179. 6. Blair A, Hogge DE, Ailles LE, Lansdorp PM, Sutherland HJ. Lack of expression of Thy-1 (CD90) on acute myeloid leukemia cells with long-term proliferative ability in vitro and in vivo. Blood 1997;89:3104–3112. 7. Pereira DS, Dorrell C, Ito CY, et al. Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells. Proc Natl Acad Sci U S A 1998;95:8239–8244. 8. Demetri GD, Griffin JD. Hemopoietins and leukemia. Hematol Oncol Clin North Am 1989;3:535–553. 9. Lichtman MA. Interrupting the inhibition of normal hematopoiesis in myelogenous leukemia: a hypothetical approach to therapy. Stem Cells 2000;18:304–306. 10. Andrews RG, Torok-Storb B, Bernstein ID. Myeloid associated differentiation antigens on stem cells and their progeny identified by monoclonal antibodies. Blood 1983;62:124–131. 11. Griffin JD, Linch D, Sabbath K, Larcom P, Schlossman SF. A monoclonal antibody reactive with normal and leukemic human myeloid progenitor cells. Leuk Res 1984;8:521–534. 12. Tanimoto M, Scheinberg DA, Cordon-Cardo C, Huie D, Clarkson BD, Old LJ. Restricted expression of an early myeloid and monocytic cell surface antigen defined by monoclonal antibody M195. Leukemia 1989;3:339–348.

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13. Andrews RG, Takahashi M, Segal G, Powell JS, Bernstein ID, Singer JW. The L4F3 antigen is expressed by unipotent and multipotent colony-forming cells but not by their precursors. Blood 1986;68:1030–1035. 14. Turhan AG, Lemoine FM, Debert C, et al. Highly purified primitive hematopoietic stem cells are PML-RARA negative and generate nonclonal progenitors in acute promyelocytic leukemia. Blood 1995;85:2154–2161. 15. Appelbaum FR, Matthews DC, Eary JF, et al. The use of radiolabeled anti-CD33 antibody to augment marrow irradiation prior to marrow transplantation for acute myelogenous leukemia. Transplantation 1992;54:829–833. 16. van der Jagt RH, Badger CC, Appelbaum FR, et al. Localization of radiolabeled antimyeloid antibodies in a human acute leukemia xenograft tumor model. Cancer Res 1992;52:89–94. 17. Parren PW. Preparation of genetically engineered monoclonal antibodies for human immunotherapy. Hum Antibodies Hybridomas 1992;3:137–145. 18. Lee MD, Dunne TS, Chang CC, et al. Calicheamicins, a novel family of antitumor antibiotics. 4. Structure elucidation of calicheamicins β1Br, γ1Br, α2I, α3I, β1I, γ1I, and δ1I. J Am Chem Soc 1992;114:985–997. 19. De Voss JJ, Hangeland JJ, Townsend CA. Characterization of the in vitro cyclization chemistry of calicheamicin and its relation to DNA cleavage. J Am Chem Soc 1990;112:4554–4556. 20. Zein H, Poncin M, Nilakantan R, Ellestad GA. Calicheamicin γ1I and DNA: molecular recognition process responsible for site-specificity. Science 1989;244:697–699. 21. Zein N, Sinha A, McGahren WJ, Ellestad GA. Calicheamicin γ1I: an antitumor antibiotic that cleaves double-stranded DNA site specifically. Science 1988;240:1198–1201. 22. Hinman LM, Hamann PR, Wallace R, Menendez AT, Durr FE, Upeslacis J. Preparation and characterization of monoclonal antibody conjugates of the calicheamicins: a novel and potent family of antitumor antibiotics. Cancer Res 1993;53:3336–3342. 23. Sievers EL, Appelbaum FR, Spielberger RT, et al. Selective ablation of acute myeloid leukemia using antibody-targeted chemotherapy: a phase I study of an anti-CD33 calicheamicin immunoconjugate. Blood 1999;93:3678–3684. 24. Sievers EL, Larson RA, Stadtmauer EA, et al. Efficacy and safety of Mylotarg (gemtuzumab ozogamicin, CMA-676) in patients with CD33-positive acute myeloid leukemia in first relapse. J Clin Oncol, in press. 25. Stadtmauer EA, Larson RA, Sievers EL, et al. An updated report of the efficacy and safety of gemtuzumab ozogamicin in 188 patients with acute myeloid leukemia in first relapse. Proc Am Soc Clin Oncol 2001;20:301a. 26. Sievers EA, Larson RA, Estey E, Lowenberg B, Berger MS, Appelbaum FR. Update of prolonged disease-free survival in patients with acute myeloid leukemia in first relapse treated with gemtuzumab ozogamicin followed by hematopoietic stem cell transplantation. Proc Am Soc Clin Oncol 2001;20:302a. 27. Byrd JC, Murphy T, Howard RS, et al. Rituximab using a thrice weekly dosing schedule in B-cell chronic lymphocytic leukemia and small lymphocytic lymphoma demonstrates clinical activity and acceptable toxicity. J Clin Oncol 2001;19:2153–2164. 28. Bernstein ID, Singer JW, Andrews RG, et al. Treatment of acute myeloid leukemia cells in vitro with a monoclonal antibody recognizing a myeloid differentiation antigen allows normal progenitor cells to be expressed. J Clin Invest 1987;79:1153–1159. 29. Karanes C, Kopecky KJ, Head DR, et al. A phase III comparison of high dose ARA-C (HIDAC) versus HIDAC plus mitoxantrone in the treatment of first relapsed or refractory acute myeloid leukemia. Southwest Oncology Group Study. Leuk Res 1999;23:787–794. 30. Neumeister P, Eibl M, Zinke-Cerwenka W, Scarpatetti M, Sill H, Linkesch W. Hepatic venoocclusive disease in two patients with relapsed acute myeloid leukemia treated with antiCD33 calicheamicin (CMA-676) immunoconjugate. Ann Hematol 2001;80:119–120.

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31. Giles F, Kantarjian H, Kornblau S, et al. Mylotarg (gemtuzumab ozogamicin) therapy is associated with hepatic venoocclusive disease in patients who have not received stem cell transplantation. Cancer, in press. 32. DeLeve LD, McCuskey RS, Wang X, et al. Characterization of a reproducible rat model of hepatic veno-occlusive disease. Hepatology 1999;29:1779–1791. 33. de Vetten MP, Jansen JH, Van der Reijden BA, Berger MS, Zijlmans JM, Lowenberg B. Molecular remission of Philadelphia/bcr-abl-positive acute myeloid leukaemia after treatment with anti-CD33 calicheamicin conjugate (gemtuzumab ozogamicin, CMA-676). Br J Haematol 2000;111:277–279. 34. Roboz GJ, Knovich MA, Schuster MW, et al. Efficacy and safety of gemtuzumab ozogamicin in patients with poor-prognosis acute myeloid leukemia, submitted. 35. Weinthal JA, Lenarsky C, Goldman S, et al. Central nervous system and plasma gemtuzumab ozogamicin (Mylotarg, CMA-676) levels in a patient with medullary and extramedullary relapsed acute myeloid leukemia: a case report. Blood 2000;96:219b. 36. Naito K, Takeshita A, Shigeno K, et al. Calicheamicin-conjugated humanized anti-CD33 monoclonal antibody (gemtuzumab ozogamicin, CMA-676) shows cytocidal effect on CD33-positive leukemia cell lines, but is inactive on P-glycoprotein-expressing sublines. Leukemia 2000;14:1436–1443. 37. Niculescu-Duvaz I. Technology evaluation: gemtuzumab ozogamicin, Celltech group. Curr Opin Mol Ther 2000;2:691–696.

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Radiolabeled Monoclonal Antibodies John M. Burke, MD and Joseph G. Jurcic, MD CONTENTS INTRODUCTION ANTIGENIC TARGETS RADIOISOTOPE SELECTION CONJUGATION OF RADIOISOTOPES TO ANTIBODIES PHARMACOKINETICS DOSIMETRY RADIOIMMUNOTHERAPY FOR AML WITH β-PARTICLE EMITTERS ALPHA-PARTICLE IMMUNOTHERAPY FOR AML RADIOIMMUNOTHERAPY FOR ADULT T-CELL LEUKEMIA/LYMPHOMA: 90Y-ANTI-TAC CONCLUSIONS REFERENCES

1. INTRODUCTION By targeting therapy to specific cell types and disease sites, monoclonal antibodies (MAbs) offer the possibility of improved efficacy and decreased toxicity compared with conventional chemotherapy. Nonetheless, the optimistic view of the early 1980s that MAbs were “magic bullets” has now been replaced by a more realistic understanding of their therapeutic potential. Since the 1980s various strategies employing MAbs for the treatment of cancer have evolved. Native MAbs can be used to focus an inflammatory response against a tumor cell. The binding of a MAb to a target cell can result in complement activation, thereby initiating several biologically important effects, including This work was supported by the Lauri Strauss Leukemia Foundation. From: Biologic Therapy of Leukemia Edited by: M. Kalaycio © Humana Press Inc., Totowa, NJ

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the induction of chemotaxis for phagocytic cells and the production of the membrane attack complex that disrupts cell membrane integrity. Another important mechanism for tumor cell killing is antibody-dependent cell-mediated cytotoxicity (ADCC), in which an effector cell expressing an Fc receptor binds to a cell-bound MAb and is triggered to kill the target cell. Examples of antibodies with intrinsic immunologically mediated antitumor activity include the chimeric anti-CD20 antibody rituximab (1) and the humanized anti-CD52 antibody CAMPATH-1H for chronic lymphocytic leukemia (CLL) (2). Because of the lack of potency of many unconjugated MAbs, investigators have studied another approach in which MAbs are used as delivery vehicles for cytotoxic agents. Chemotherapeutic agents such as doxorubicin, calicheamicin, methotrexate, and vinca alkaloids have been conjugated to various MAbs. Toxins used clinically have been either bacterial products, such as diphtheria toxin and Pseudomonas exotoxin A, or plant products, such as ricin, gelonin, pokeweed antiviral protein, and saponin. Significant antileukemic effects have been observed with the anti-CD33-calicheamicin conjugate gemtuzumab ozogamicin for acute myeloid leukemia (AML) (3) and the anti-CD22pseudomonas exotoxin construct BL22 for hairy cell leukemia (4). In an alternative strategy, antibodies can be used to target radioisotopes directly to sites of disease to increase the antitumor effects of native MAbs. Because radioisotopes emit particles capable of inducing lethal deoxyribonucleic acid (DNA) damage to cells lying within a fixed range, radioimmunoconjugates may allow the killing of antigen-negative tumor variants or tumor cells not reached by MAbs. This approach has produced promising results in B-cell lymphomas (5,6). Leukemias are well suited to treatment with radioimmunotherapy for several reasons. First, because of their location in the blood, bone marrow, spleen, and lymph nodes, malignant cells are readily accessible to circulating MAbs. Second, target antigens on leukemic blasts are well known and can be easily characterized in individual patients by flow cytometry. Finally, leukemias are radiosensitive tumors. This chapter focuses on issues affecting MAb pharmacokinetics and the physical properties of various radionuclides used clinically. We also review the results of recent clinical trials in the radioimmunotherapy of leukemia.

2. ANTIGENIC TARGETS Immunophenotypic characterization of the various stages and lineages found during hematopoietic differentiation provides the rationale for selection of MAbs that bind selectively to neoplastic cells while sparing normal tissues (Table 1). Leukemia-associated antigens, however, are not tumor specific, nor are they always stage or lineage specific. For example, CD10, found on B-cell acute lymphoblastic leukemia (ALL), is also expressed by mature B-cell lymphomas and T-cell ALL. CD33, found on most AML cells, is expressed by normal myeloid precursors.

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Table 1 Selected Target Antigens for Immunotherapy of Leukemia Antigen CD5 CD7 CD14 CD15 CD19 CD20 CD22 CD25 CD33 CD45 CD52 HLA-DR

Disease ALL, CLL ALL AML AML ALL, CLL CLL HCL, CLL ATL AML, CML AML, MDS, ALL CLL CLL

Antibody T101, Tp67 Tp41 AML2-23 PM81 Anti-B4 Tositumomab, rituximab, 2B8, 1F5 LL2, huLL2, RFB4 anti-Tac MY9, p67, M195, HuM195 BC8 CAMPATH-1H Lym-1, Hu1D10

ALL = acute lymphocytic leukemia; CLL = chronic lymphocytic leukemia; AML = acute myeloid leukemia; HCL = hairy cell leukemia; ATL = adult T-cell leukemia; CML = chronic myelogenous leukemia; MDS = myelodysplastic syndrome.

The earliest myeloid progenitors express CD34; more committed progenitors acquire CD33 and HLA-DR. Most AML cells express CD33, CD13, and CD15. HLA-DR is typically found on all subtypes of AML except acute promyelocytic leukemia (APL). Monocytic leukemias express antigens associated with more mature granulocytes and monocytes, including CD11a/18, CD11c, CD14, and CD15. Cell-surface antigen expression in early phase chronic myelogenous leukemia (CML) resembles that of mature granulocytes but is heterogeneous in blast crisis. Nearly all neoplasms of B-cell origin express CD19 and HLA-DR. ALLs of B-cell lineage are derived from the earliest stages of B-cell differentiation. Most express CD10 and CD34, and a small proportion express CD20. CD5 and CD19 are found on small lymphocytic lymphoma and CLL. Additionally, they weakly express CD20. Most T-cell ALL and lymphoblastic lymphoma are the malignant counterpart of the earliest T-cells that express CD2, CD5, and CD7.

3. RADIOISOTOPE SELECTION Radioisotopes have unstable nuclei and decay by emitting charged particles (either α particles or β particles) with or without photons (γ rays). Properties of several radioisotopes that have been used in the treatment of leukemia are listed in Table 2.

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Isotope Iodine-131 Rhenium-188 Yttrium-90 Bismuth-213 Actinium-225

Particle(s) Emitted

Half-Life

Particulate Energy (keV)

β, γ β, γ β α, γ α, γ

8.0 d 17 h 64 h 46 min 10.0 d

970 2120 2280 5982 5935

Mean Range of α- or β-Particle Emission (mm) 0.8 2.4 2.7 0.05–0.08 0.05–0.08

To date, most clinical radioimmunotherapy trials have used β particle-emitting radionuclides. β particles are electrons that, compared with α particles, have a relatively long range (0.8–5 mm) and low linear energy transfer (~ 0.2 keV/µm). Because of the distances they travel, β particles create a “field effect” that can destroy tumor cells to which the radioimmunoconjugate is not directly bound, including nearby antigen-negative tumor cells. This property makes radioimmunotherapy with β particle-emitters theoretically more useful for the treatment of bulky disease and for myeloablation before bone marrow transplantation (BMT). On the other hand, β particle-emitting radioimmunoconjugates can damage normal “bystander” cells, resulting in nonspecific tissue toxicity. β particle emitters that have been used in the treatment of leukemias include iodine-131 (131I), yttrium-90 (90Y), and rhenium-188 (188Re). α particles are helium nuclei with a range of only a few cell diameters (0.05–0.08 mm) and a linear energy transfer of approx 100 keV/µm. Because of the high linear energy transfer, only one or two α particles traversing a nucleus can destroy a cell, making α particles among the most potent cytotoxic agents (7). The short range of α particles potentially results in decreased irradiation of normal bystander cells and reduced toxicity. These properties make α particles ideally suited for treating minimal residual disease, in which the goal of therapy is to kill individual tumor cells selectively, not bulky tumor masses. α particle emitters that have been investigated in the treatment of leukemias include bismuth-212 (212Bi), bismuth-213 (213Bi), and actinium225 (225Ac) (8). γ rays are photons that can travel several centimeters in human tissue. They are often emitted together with α and β particles. γ emissions facilitate biodistribution and dosimetry studies because quantitative imaging is possible using a γ camera. Treatment with large doses of isotopes such as 131I, associated with high-energy photon emissions, however, requires patient isolation, can result in significant exposure to hospital staff, and poses a waste hazard. Although the use of a pure β-emitting isotope such as 90Y can overcome the radiation safety issues associated with γ irradiation, biodistribution studies require the adminis-

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tration of MAb trace-labeled with a second isotope, typically indium-111 (111In), whose biodistribution is not identical to 90Y. Positron emission tomography (PET) imaging of MAbs trace-labeled with 86Y is one strategy that may improve radiation dosimetry estimates for 90Y-labeled antibodies.

4. CONJUGATION OF RADIOISOTOPES TO ANTIBODIES Various methods can be used to conjugate radioisotopes to MAbs. Because binds to tyrosine residues, it can be conjugated directly to antibodies using the chloramine-T method. Tumor resistance due to internalization of the antigen-antibody complex, followed by rapid degradation of the radioconjugate and expulsion of isotope metabolites, represents a significant disadvantage to therapy with some 131I-labeled MAb constructs. This problem could potentially be overcome by the use of radiometals, which are retained by cells after catabolism (9) or by novel iodination methods, such as tyramine cellobiose, resulting in more stable radioimmunoconjugates (10). The identification of suitable chelating agents for radiometals has been challenging. Studies have shown that the use of the macrocyclic ligand 1,4,7,10tetraazacyclododecane tetraacetic acid (DOTA) can result in stable 90Y immunoconjugates and significantly reduce bone uptake of radioyttrium (11). Because DOTA has been shown to be immunogenic (12), several diethylenetriamine pentaacetic acid (DTPA)-derived chelates have been evaluated. Although none have held yttrium as well as DOTA, cyclohexyl-A (CHX-A)-DTPA was found to be a suitable chelate for 90Y (13). This chelate has also been used to generate bismuth-containing radioimmunoconjugates for clinical use (14). Another radiometal, 188Re, has been directly labeled to the anti-CD66c antibody BW250/183 using tris-(2-carboxyethyl) phosphine as a reducing agent (15). Radiolabeling of MAbs can cause loss of biologic function, especially when they are labeled with 131I at high specific activities (16). This decrease in immunoreactivity is related directly to the number of tyrosine residues in the hypervariable region of the MAb to which radioiodine attaches. Immunoreactivity of MAb fragments is lost at even lower specific activities because there are fewer tyrosine residues in the constant region (17). Although complementarity-determining regions account for less than one tenth of the entire MAb sequence, they typically contain 20% to 30% of the tyrosine residues in the MAb. In contrast, lysine residues, which bind ligands used for radiometal chelation, are more uniformly distributed. Therefore, radiometal constructs may be more suitable for high specific activity labeling. 131I

5. PHARMACOKINETICS Factors such as variability in tumor burden and number of binding sites per cell among patients, MAb specificity and binding avidity, immunoreactivity,

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MAb internalization after binding, and immunogenicity contribute to the difficult and poorly understood pharmacokinetics of radiolabeled MAbs. The number of available antigen sites will alter antibody pharmacokinetics and biodistribution. For example, in a dose-escalation trial of trace-labeled 131Ianti-CD33 MAb M195 for myeloid leukemias, superior targeting to sites of disease as determined by γ camera imaging was seen with a comparatively small dose (18). This may be explained in part by the relatively low number of binding sites (~ 10,000–20,000) on each leukemia cell. Heterogenous antigen expression can provide a mechanism of resistance to the cytotoxic effects of MAbs and account for toxicities associated with therapy. If the targeted antigen is lacking from a subset of tumor cells, residual tumor will remain after antibody therapy. Radioimmunotherapy offers a potential solution to this problem because radiation kills cells within a given range regardless of whether they express the target antigen. On the other hand, target antigen expression on normal tissues, including hematopoietic cells, can result in myelosuppression and other significant side effects. The influence of antigenic modulation on MAb-based treatments relates to specific therapeutic applications. Tumors in which antigen-antibody complexes remain on the cell surface may be better suited to treatments dependent upon immune-mediated cytotoxicity or delivery of radioisotopes with long-ranged emissions, such as 131I. Internalization of the antigen-antibody complex after binding can optimize delivery of some radioisotopes, such as short-ranged α particle emitters. Antigen modulation can shorten the retention time of some 131I-labeled MAbs due to catabolism of the radioimmunoconjugate. Because most MAbs used clinically are derived from mice, they can generate a human antimouse antibody (HAMA) response. HAMA has been implicated in poor therapeutic results by neutralizing MAb on repeated doses and MAb enhancing clearance. Usually no additional toxicities are seen; however, with large MAb doses, circulating immune complexes can lead to serum sickness. The use of chimeric and humanized MAbs remains the most promising strategy to avoid HAMA responses. For some humanized MAbs, however, a prolonged biologic half-life may result in nonspecific dose deposition and toxicity when used to deliver radioisotopes or chemotherapeutic agents.

6. DOSIMETRY In most radioimmunotherapy trials, biodistribution and dosimetry studies are performed routinely. Serial γ camera imaging and measurements of plasma, urine, bone marrow, and tissue biopsy radioactivity are used to estimate absorbed radiation doses to different organs and tumor sites. These techniques are based on the Medical Internal Radiation Dose model (19). The validity of these predictions, however, is limited by the accuracy in measuring activity using γ camera imaging and by the inability to visualize all sites of disease in

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patients. Single-photon emission-computed tomography (SPECT) may increase the accuracy of planar scintigraphy, especially when used in conjunction with computed tomography (20,21). Nevertheless, the quantitative value of SPECT remains unknown. Based on dosimetric data, models, such as the one developed to simulate the distribution of the anti-CD33 MAb M195 (22), may provide information about radiation doses delivered to tissues not directly sampled and may also be used to estimate total tumor burden and tumor burden in individual organs.

7. RADIOIMMUNOTHERAPY FOR AML WITH β-PARTICLE EMITTERS Most clinical trials of radioimmunotherapy in leukemia have focused on the use of β-emitting radionuclides. The anti-CD33 MAbs M195 and HuM195 have been studied extensively at Memorial Sloan-Kettering Cancer Center. At the Fred Hutchinson Cancer Research Center, the anti-CD33 MAb p67 and the antiCD45 MAb BC8 have been investigated. Recently, use of the anti-CD66 MAb BW 250/183 has been reported from the Ulm University Hospital. Results of recent trials of radioimmunotherapy in leukemia are summarized in Table 3.

7.1. 131I-M195 and 131I-HuM195 M195 is a monoclonal IgG2a antibody directed at CD33, a cell-surface glycoprotein expressed on most myeloid leukemia cells (23,24). CD33 is also expressed on normal myelomonocytic and erythroid progenitor cells but not on pluripotent stem cells, mature granulocytes, lymphoid cells, or nonhematopoietic cells (25,26). An early phase I trial showed that trace-labeled 131I-M195 could rapidly and specifically target known sites of leukemia in patients (18). Subsequently, elimination of large leukemic burdens occurred in patients treated with therapeutically labeled 131I-M195. In an initial phase I trial, escalating doses of 131IM195 (50 to 210 mCi/m2) were used to treat 24 patients with relapsed or refractory myeloid leukemias. Whole-body γ camera imaging demonstrated rapid uptake of the radiolabeled antibody in the bone marrow, as well as uptake in the liver and spleen. The isotope remained at these sites for at least 3 d. The maximum tolerated dose of 131I-M195 was not reached, although one patient had grade 4 hepatic toxicity. At doses of 135 mCi/m2 or greater, profound myelosuppression occurred, allowing eight patients to proceed to either allogeneic (n = 5) or autologous (n = 3) bone marrow transplantation (BMT). Of 24 patients, 22 had decreases in bone marrow blasts; only two patients treated at the two lowest dose levels did not respond. Three patients had complete remissions. HAMA developed in 37% of patients. Two patients who developed HAMA were retreated; their plasma 131I-M195 levels could not be maintained, and no therapeutic benefit was seen (27).

Table 3 Recent Clinical Trials of Radiolabeled Antibodies for Leukemia Radiolabeled Antibody

Disease

Isotope Dose

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Results

Comments

Reference

CR in 3 of 8 patients receiving BMT

5 patients received autologous BMT; 3 received allogeneic BMT

27

7

2 of 7 patients with minimal disease transiently became PCR negative

Used after retinoic acid in patients with relapsed disease

34

122–437 mCi

31

CR in 28 patients; however, long-term DFS in 3 patients

Used with Bu/Cy before allogeneic BMT

28

Advanced AML

0.1–0.3 mCi/kg

19

13 patients had reductions in marrow blasts; 1 CR

Higher doses result in prolonged myelosuppression

35

Advanced AML, CMML

0.28–1 mCi/kg

18

14 patients had reductions in marrow blasts; no CRs

First demonstration of safety of α-particle therapy

47

131I-M195

Advanced AML, MDS, blastic CML

50–210

131I-M195

Relapsed APL

50 or 70 mCi/m2

131I-M195,

Advanced AML, MDS, blastic CML

90Y-HuM195

213Bi-HuM195

131I-HuM195

No. of Patients

mCi/m2

24

131I-p67

AML

131I-BC8

110–330 mCi

9

3 of 4 patients treated with therapeutic doses relapsed

Given with Cy/TBI before BMT. Many patients had unfavorable biodistribution

36

Advanced AML, ALL 76–612 mCi

44

7 of 25 patients with AML or MDS and 3 of 9 patients with ALL had long-term DFS

Given with Cy/TBI before BMT

40

131I-BC8

AML in first remission

24

18 patients with longterm DFS

Given with Bu/Cy before allogeneic BMT

41

188Re-BW

High-risk AML, MDS 11.1 GBq (mean)

36

45% DFS at median 18 months

Given as part of preparative regimen before BMT

42

ATL

18

7 patients had PRs; 2 had CRs

6 patients developed HAMA

50

101–263 mCi

250/183 90Y-anti-Tac

5–15 mCi

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AML = acute myeloid leukemia; MDS = myelodysplastic syndrome; APL = acute promyelocytic leukemia; CML = chronic myelogenous leukemia; CMML = chronic myelomonocytic leukemia; ALL = acute lymphoblastic leukemia; ATL = adult T-cell leukemia; CR = complete remission; BMT = bone marrow transplantation; PCR = polymerase chain reaction; DFS = disease-free survival; PR = partial remission; Bu = busulfan; Cy = cyclophosphamide; TBI = total body irradiation; HAMA = human antimouse antibodies.

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Although encouraging, these trials served to illustrate several limitations of M195 as a therapeutic agent for leukemia. Murine M195 was unable to mediate leukemia cell killing by ADCC in vitro, nor did it lead to the death of leukemic cells in the presence of human complement (24). In addition, many patients treated with M195 developed HAMA, which adversely affected the pharmacokinetics of M195 and precluded repeated treatments with it (18,27,28). Humanized M195 (HuM195) was engineered in an attempt to overcome these limitations. HuM195 was constructed by grafting complementarity-determining regions of M195 into a human IgG1 framework and backbone (29). HuM195 maintained the binding specificity of M195 and had increased binding avidity. Unlike M195, HuM195 induced rabbit complement-mediated cytotoxicity against both HL60 cells and fibroblasts transfected with CD33 genes, and it induced ADCC using human peripheral blood mononuclear cells as effectors (30). A phase I trial conducted in patients with advanced myeloid leukemias showed that trace-labeled 131I-HuM195 had similar biodistribution and pharmacology to murine M195, without significant immunogenicity. Moreover, HuM195 proved to be a suitable carrier for radionuclides (31). Treatment with native HuM195 demonstrated activity against minimal residual disease in patients with APL (32) and produced occasional complete remissions in patients with relapsed or refractory myeloid leukemias with low-burden disease (33). For these reasons, HuM195 was used in place of M195 in more recent clinical trials. 131I-M195 and 131I-HuM195 were both studied as part of a preparative regimen before allogeneic BMT, consisting of 131I-labeled antibody (122–437 mCi in 2–4 divided doses) followed by busulfan (16 mg/kg in divided doses over 4 d) and cyclophosphamide (90–120 mg/kg total dose). Sixteen patients with refractory or relapsed AML, 14 patients with CML in accelerated or blastic phases and 1 patient with myelodysplastic syndrome underwent transplantation with this regimen. Nineteen patients received 131I-M195; 12 received 131IHuM195. No significant toxicities were attributable to the addition of the radiolabeled antibody to the preparative regimen. No delays in engraftment were seen. Of the 31 patients, 28 achieved complete remission, and three patients with refractory AML remained in remission for 4.5–8 yr after transplant (28). This study showed that radioimmunotherapy could potentially be used to intensify antileukemic therapy before stem cell transplantation. The role of nonmyeloablative doses of 131I-M195 given in the minimal residual disease setting was investigated in patients with APL. Seven patients with relapsed disease were treated with all-trans retinoic acid until they attained clinical complete remission. Effects of therapy on residual disease were monitored by reverse transcription-polymerase chain reaction (RT-PCR) amplification of PML/RAR-α mRNA. Of the seven patients, six had minimal residual disease detectable by RT-PCR after retinoic acid induction. Patients

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then received either 50 or 70 mCi/m2 of 131I-M195. Toxicity was limited to myelosuppression, but no episodes of febrile neutropenia occurred. Two patients transiently became RT-PCR negative. The median disease-free survival was 8 mo, which compares favorably with the 3-mo median disease-free survival seen in patients with relapsed APL treated with retinoic acid alone. HAMA developed in five patients. Thus, 131I-M195 had activity against APL, but therapy was limited by significant myelosuppression and by the formation of HAMA (34).

7.2. 90Y-HuM195 Early trials showed that myeloablative therapy with 131I-anti-CD33 constructs has several disadvantages. First, because of decreased immunoreactivity when labeled at high specific activities, multiple infusions of 131I-M195 and 131I-HuM195 are needed to deliver adequate radiation doses to the marrow for ablation. Second, the 8-d half-life of 131I delays the time from treatment to stem cell or marrow infusion in patients undergoing transplantation. Additionally, patients receiving high doses of 131I-antibody constructs must be hospitalized and isolated because of high-energy γ emissions. 90Y offers several advantages over 131I for myeloablation. The higher energy longer ranged β emissions of 90Y permit a lower effective dose than 131I. The absence of γ emissions eliminates the need for radiation isolation and allows large doses to be given safely in the outpatient setting. In addition, radiometals such as 90Y are retained within cells better than 131I after internalization of antigen–antibody complexes (9). For these reasons, 90Y-labeled HuM195 was studied in a recent phase I trial. Nineteen patients with relapsed or refractory AML were treated with escalating doses (0.1–0.3 mCi/kg) of 90Y-HuM195, given as a single infusion without marrow support. Biodistribution and dosimetry studies, performed by co-administering trace-labeled 111In-HuM195, demonstrated that up to 560 cGy and 750 cGy were delivered to the marrow and spleen, respectively. Transient low-grade liver function abnormalities occurred in 11 patients. Myelosuppression, lasting 9–62 d, was dose limiting, and the maximum tolerated dose was 0.275 mCi/kg. Thirteen of the 19 patients had reductions in bone marrow blasts. One patient who received 0.275 mCi/kg achieved a complete remission lasting 5 mo. All patients treated at the highest dose level (0.3 mCi/kg) had hypocellular bone marrows without evidence of leukemia on biopsies performed 2 and 4 wk after treatment (35). These results suggest that 90Y-HuM195 may be useful to reduce leukemic burdens and ablate the marrow as part of a preparative regimen for BMT. Clinical trials investigating this strategy in the setting of autologous peripheral blood progenitor transplantation and nonmyeloablative allogeneic transplantation are currently underway.

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7.3. 131I-p67 The murine IgG1 anti-CD33 antibody p67 labeled with 131I was studied as part of a preparative regimen before BMT (36). In a phase I trial, nine patients with advanced AML received p67 labeled with trace doses of 131I. γ camera imaging demonstrated initial specific uptake of 131I-p67 in the marrow in most patients. However, the half-life of the radiolabeled antibody was only 9–41 h; this relatively brief half-life contrasts with that of 131I-M195, in which the radiolabel remained in the marrow for at least 3 d (18). Four of the nine patients had “favorable biodistribution,” defined as greater uptake of 131I in the marrow and spleen greater than in nonhematopoietic organs. These four patients then received therapeutic doses of 131I-p67 (110–230 mCi) followed by cyclophosphamide (120 mg/kg) and total body irradiation (TBI) (12 Gy) followed by allogeneic BMT. The therapy was well tolerated, but three of the four patients eventually relapsed (37). Because of the unfavorable biodistribution in many patients and the short residence time of 131I-p67 in the marrow, the investigators have since focused on the 131I-labeled anti-CD45 antibody BC8 discussed in the following section.

7.4. 131I-BC8 The murine IgG1 MAb BC8 is directed at CD45, a tyrosine phosphatase expressed on virtually all leukocytes, including myeloid and lymphoid precursors in the bone marrow, mature lymphocytes in lymph nodes, and most myeloid and lymphoid leukemia cells. Unlike the anti-CD33 MAbs, BC8 does not internalize after binding to its antigen (38,39). In a phase I trial, 44 patients with advanced acute leukemia or myelodysplasia received BC8 labeled with trace doses of 131I (5-10 mCi). Thirty-seven patients (84%) had favorable biodistribution of the radiolabeled antibody, with higher radiation-absorbed doses to the marrow and spleen than to normal organs. Thirty-four of these patients then received therapeutic doses of 131IBC8 (76 to 612 mCi) in a dose-escalation trial. Patients then received cyclophosphamide (120 mg/kg) and TBI (12 Gy) followed by either allogeneic or autologous stem cell transplantation. The maximum tolerated dose was an estimated radiation-absorbed dose of 10.5 Gy to the liver. One of six patients treated at the maximum tolerated dose had grade 3 hepatic toxicity. Based on average estimates of radiation-absorbed dose, the investigators calculated that the 131I-BC8 delivered an additional 24 Gy to the marrow and 50 Gy to the spleen in patients treated at the maximum tolerated dose. Of the 25 patients with AML or myelodysplastic syndrome (MDS), 7 were alive and free of disease at a median of 65 mo after transplantation. Of the nine patients with ALL, three were alive and free of disease at 19, 54, and 66 mo (40). Based on these promising results, a phase I/II trial of 131I-BC8, together with busulfan and cyclophosphamide, was begun in patients with AML in first

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remission. After trace-labeled 131I-BC8, γ camera imaging showed that 90% of patients had favorable biodistribution. These patients were then treated with therapeutic doses of 131I-BC8, delivering 3.5 Gy (four patients) or 5.25 Gy (all subsequent patients) to the liver, half the maximum tolerated dose defined in the phase I study. Toxicities attributable to the 131I-BC8 were minimal. In an encouraging preliminary report, 18 of 24 patients treated with therapeutic doses were alive and disease free at a median of 42 mo after transplant (41).

7.5. 188Re-Anti-CD66 The glycoprotein CD66c, also known as nonspecific cross-reacting antigen (NCA), is expressed on myeloid cells (200,000 molecules per cell) but not on leukemia cells. BW 250/183 is a murine monoclonal IgG1 antibody directed at CD66 (15,42). The radiometal 188Re has a 17-h half-life and emits both β and γ particles. The β emissions of 188Re are well suited for therapy, and the γ emissions facilitate biodistribution and dosimetry studies. In a pilot dosimetry trial, 12 patients undergoing BMT were treated with 188Re-BW 250/183 followed by a standard preparative regimen. Most patients had a favorable biodistribution. After administration of 9.7 GBq (260 mCi) of 188Re-BW 250/183, the median bone marrow dose was 14 Gy (15,43). Subsequently, 36 patients with high-risk AML or MDS were treated with 188Re-BW 250/183. Patients then received one of three preparative regimens: TBI (12 Gy) plus cyclophosphamide (120 mg/kg), busulfan (12.8 mg/kg) plus cyclophosphamide (120 mg/kg), or TBI (12 Gy) plus thiotepa (10 mg/kg) and cyclophosphamide (120 mg/kg). Patients receiving grafts from unrelated donors or from mismatched related donors also received antithymocyte globulin to prevent graft rejection. Thirty-one patients received allogeneic grafts (mostly T-cell depleted), 1 received a syngeneic graft, and 4 received autologous grafts. All patients had a favorable biodistribution of 188Re-BW 250/183. The mean therapeutic dose administered was 11.1 GBq (300 mCi) injected in 1 or 2 fractions, and the median dose delivered to the bone marrow was 14.9 Gy (range, 8.1–28 Gy). The administration of radiolabeled antibody did not result in any significant additional toxicity beyond that associated with the conventional preparative regimen. Six patients (17%), however, developed renal toxicity between 6 and 12 mo after the transplant, possibly due to radiation. Engraftment occurred in all patients and was not delayed. At a median follow-up of 18 mos, 9 of 35 evaluable patients had relapsed, 8 of whom subsequently died. Eight patients (22%) died from transplant-related toxicity. Disease-free survival at a median of 18 mo was 45% and was significantly higher for those undergoing transplantation in remission (67%) than for those transplanted with overt leukemia (31%) (42). This study suggests that 188Re-BW 250/183, like 90Y-HuM195 and 131I-BC8, may deliver significant doses of radiation to the marrow.

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8. ALPHA-PARTICLE IMMUNOTHERAPY FOR AML 8.1. Preclinical Studies To increase the antileukemic activity of native MAbs but avoid the nonspecific cytotoxic effects of β-emitting radionuclides, targeted α particle therapy has been investigated in several experimental systems. In one of the first reports suggesting the feasibility of this approach, 212Bi conjugated to a tumorspecific MAb 103A was used against murine erythroleukemia. Targeting of the construct to neoplastic cells with the spleen was seen within 1 h after injection. When 212Bi-103A was injected 13 d after inoculation with leukemia, reductions in splenomegaly and the absence of liver metastases were noted. When given on day 8, no histologic evidence of leukemia developed (44). Subsequently, administration of the α-emitter 212Bi conjugated to the anti-CD25 MAb anti-Tac after inoculation of nude mice with a CD25-expressing lymphoma cell line led to prolonged tumor-free survival and prevented the development of leukemia in some animals. Treatment of established tumor with 212Bi-anti-Tac, however, failed to produce responses (45). 213Bi is an α-particle emitter with a 46-min half-life. It was prepared from a 225Ac/213Bi generator and conjugated to HuM195 using the bifunctional chelating agent 2-(4-isothiocyanatobenzyl (SCN)-CHX-A-DTPA (14,46). Intraperitoneal injections of up to 20 mCi/kg and intravenous (iv) injections of 10 mCi/kg 213Bi-HuM195 could safely be given to mice. In vitro the application of bismuth-labeled HuM195 resulted in dose-dependent and specific activity-dependent killing of CD33-positive HL60 cells. Approximately 50% of target cells were killed when only 2 bismuth atoms were bound to the cell surface (14).

8.2. Phase I Trial of 213Bi-HuM195 Based on these preclinical data, 18 patients with relapsed or refractory AML or CML were treated with 213Bi-HuM195 (0.28-1 mCi/kg) in a phase I trial (47). Treatment with 213Bi-HuM195 was tolerated well, and dose-limiting toxicity was not observed. Further dose escalation beyond 1 mCi/kg was limited by the availability of 225Ac. Six patients had transient grade 1 or 2 abnormalities in liver function tests. Myelosuppression occurred in all patients and lasted a median of 14 d (range, 8–34 d). γ camera imaging showed that the majority of the administered activity localized to the bone marrow, liver, and spleen within 5 to 10 min after injection. The absorbed dose ratios between these sites and the whole body were 1000-fold greater than those seen with β-emitting constructs in this antigen system (48). Thirteen of 15 evaluable patients (87%) had reductions in circulating blasts after therapy, and 14 of the 18 patients (78%) had reductions in the percentage of bone marrow blasts. However, no complete remissions were observed. Because of the nature of α particle radia-

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tion, complete remission at 30 d after treatment would have required the individual targeting and killing of 99.9% of the leukemia cells. Because the patients treated on this study had tumor burdens of up to 1012 cells, each with an average CD33 density of 10,000 per cell, roughly 1016 leukemic binding sites were available to HuM195. Because approx 1 in 2700 molecules of HuM195 carried the radiolabel at the specific activities injected, it was difficult to deliver one to two 213Bi atoms to every leukemia cell, even if optimal antibody targeting were assumed (47). Therefore, treatment of overt leukemia with 213Bi-HuM195 as a single agent would require extraordinarily high injected activities. Nevertheless, the short range and high linear energy of α particles make 213Bi-HuM195 ideally suited for the treatment of residual disease. Currently, the use of 213Bi-HuM195 for the elimination of minimal disease after partial cytoreduction with cytarabine in patients with myeloid leukemias is under investigation. This trial provides the rationale for continued investigation of α particle immunotherapy in a variety of cancers where small volume, minimal residual, or micrometastatic disease is present.

8.3. Targeted α Particle Nano-Generators More recently, 225Ac has been conjugated to a variety of MAbs using the bifunctional chelate SCN-DOTA. 225Ac has a 10-d half-life and decays by α emission through three atoms, each of which also emits an α particle. In vitro, 225Ac coupled to internalizing MAbs specifically killed leukemia, lymphoma, breast, ovarian, neuroblastoma, and prostate cancer cells at doses 1000 times less than 213Bi-containing radioimmunoconjugates. In xenograft models of disseminated human lymphoma and solid prostate carcinoma, single doses at nanocurie levels of tumor-specific constructs prolonged survival and cured a substantial fraction of animals without toxicity (49). Therefore, in this strategy, 225Ac-SCN-DOTA serves as an atomic nano-generator that delivers a cascade of four α particles to the inside of a cancer cell by an internalizing antibody. A phase I trial of 225Ac-HuM195 in advanced myeloid leukemias is planned.

9. RADIOIMMUNOTHERAPY FOR ADULT T-CELL LEUKEMIA/LYMPHOMA: 90Y-ANTI-TAC Adult T-cell leukemia/lymphoma (ATL) is a malignancy of lymphocytes caused by infection with the human T-lymphotrophic virus type I. The disease is characterized by circulating malignant cells, diffuse lymphadenopathy, and hypercalcemia. In patients with ATL, each leukemic cell expresses 10,000–35,000 molecules of the α subunit of the interleukin (IL)-2 receptor (also called CD25 or Tac). In contrast, normal resting lymphocytes do not express CD25. Anti-Tac is a murine monoclonal antibody that prevents the interaction of IL-2 with CD25 (50).

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In a phase I/II trial, 18 patients with ATL were treated with anti-Tac labeled with 90Y. The first 9 patients were treated in a phase I dose-escalation trial with 5-15 mCi of 90Y-anti-Tac, and the next 9 patients were treated in a phase II trial with a uniform 10-mCi dose. Observed toxicities included grade 3 or 4 hematologic toxicity (12 patients), transient grade 3 hepatic toxicity (3 patients), and transient proteinuria (1 patient). In addition, 1 patient with hypertension and diabetes died of unexplained cardiac asystole 23 d after the administration of 90Yanti-Tac. Six patients developed HAMA. Of 16 evaluable patients, 7 had partial remissions (mean duration, 9.2 mo) and 2 had complete remissions. One patient who attained a complete remission developed MDS and died of secondary AML 3 yr after receiving 90Y-anti-Tac. At autopsy, the patient had persistent ATL in the skin. The other patient with a complete remission remained without evidence of disease for more than 3 yr after the initiation of therapy (50).

10. CONCLUSIONS Radiolabeled antibodies have the potential to improve the outcome of patients with leukemias. Early clinical studies have demonstrated that radiolabeled antibodies have activity in leukemias and can be given safely to patients with advanced disease. Two applications of radioimmunotherapy appear most promising: (1) cytoreduction before BMT and (2) elimination of minimal residual disease. Myeloablative doses of β-emitting radioimmunoconjugates have already demonstrated significant activity when used in several transplantation preparative regimens (28,36,39,40). Subsequent studies have suggested that the use of radiometals, such as 90Y and 188Re, may overcome many of the difficulties associated with 131I (35,42). Whether the use of radioimmunotherapy as part of BMT preparative regimens improves patient outcomes compared with standard preparative treatments remains unknown. Comparative trials are needed to answer this question definitively. The choice of an optimal radioimmunoconjugate can be dictated by specific clinical situations. Therapy with long-ranged β-emitters may be better for killing bulky disease, but the use of shorter ranged α particle-emitting isotopes could potentially result in more efficient single cell killing with less nonspecific toxicity (47). These physical properties make targeted α particle immunotherapy ideal for the treatment of minimal residual disease. Treatment with 225Ac constructs, which can deliver an in vivo generator of four α particles directly to a tumor cell, could further increase the antitumor activity previously seen with 213Bi-containing constructs (49).

REFERENCES 1. O’Brien SM, Kantarjian H, Thomas DA, et al. Rituximab dose-escalation trial in chronic lymphocytic leukemia. J Clin Oncol 2001;19:2165–2170.

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2. Osterborg A, Dyer MJ, Bunjes D, et al. Phase II multicenter study of human CD52 antibody in previously treated chronic lymphocytic leukemia. European Study Group of CAMPATH1H Treatment in Chronic Lymphocytic Leukemia. J Clin Oncol 1997;15:1567–1574. 3. Sievers EL, Larson RA, Stadtmauer EA, et al. Efficacy and safety of gemtuzumab ozogamicin in patients with CD33-positive acute myeloid leukemia in first relapse. J Clin Oncol 2001;19:3244–3254. 4. Kreitman RJ, Wilson WH, Bergeron K, et al. Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia. N Engl J Med 2001;345:241–247. 5. Kaminski MS, Zelenetz AD, Press OW, et al. Pivotal study of iodine I 131 toxitumomab for chemotherapy-refractory low-grade or transformed low-grade B-cell non-Hodgkin’s lymphomas. J Clin Oncol 2001;19:3918–3928. 6. Witzig TE, White CA, Wiseman GA, et al. Phase I/II trial of IDEC-Y2B8 radioimmunotherapy for treatment of relapsed or refractory CD20+ B-cell non-Hodgkin’s lymphoma. J Clin Oncol 1999;17:3793–3803. 7. Macklis RM, Lin JY, Beresford B, Atcher RW, Hines JJ, Humm JL. Cellular kinetics, dosimetry, and radiobiology of alpha-particle radioimmunotherapy: induction of apoptosis. Radiat Res 1992;130:220–226. 8. McDevitt MR, Sgouros G, Finn RD, et al. Radioimmunotherapy with alpha-emitting nuclides. Eur J Nucl Med 1998;25:1341–1351. 9. Scheinberg DA, Strand M. Kinetic and catabolic considerations of monoclonal antibody targeting in erythroleukemic mice. Cancer Res 1983;43:265–272. 10. Ali SA, Warren SD, Richter KY, et al. Improving tumor retention of radioiodinated antibody: aryl carbohydrate adducts. Cancer Res 1990;50(suppl):783s–788s. 11. Deshpande SV, DeNardo SJ, Kukis DL, et al. Yttrium-90-labeled monoclonal antibody for therapy: labeling by a new macrocyclic bifunctional chelating agent. J Nucl Med 1990;31:473–479. 12. Kosmas C, Snook D, Gooden CS, et al. Development of humoral immune responses against a macrocyclic chelating agent (DOTA) in cancer patients receiving radioimmunoconjugates for imaging and therapy. Cancer Res 1992;52:904–911. 13. Camera L, Kinuya S, Garmestani K, et al. Evaluation of the serum stability and in vivo biodistribution of CHX-DTPA and other ligands for yttrium labeling of monoclonal antibodies. J Nucl Med 1994;35:882–889. 14. Nikula TK, McDevitt MR, Finn RD, et al. Alpha-emitting bismuth cyclohexylbenzyl DTPA constructs of recombinant humanized anti-CD33 antibodies: pharmacokinetics, bioactivity, toxicity and chemistry. J Nucl Med 1999;40:166–176. 15. Seitz U, Neumaier B, Glatting G, Kotzerke J, Bunjes D, Reske SN. Preparation and evaluation of the rhenium-188-labelled anti-NCA antigen monoclonal antibody BW 250/183 for radioimmunotherapy of leukaemia. Eur J Nucl Med 1999;26:1265–1273. 16. Larson SM. A tentative biological model for the localization of radiolabelled antibody in tumor: the improtance of immunoreactivity. Nucl Med Biol 1986;13:393–399. 17. Nikula TK, Bocchia M, Curcio MJ, et al. Impact of the high tyrosine fraction in complementarity-determining regions: measured and predicted effects of radioiodination on IgG immunoreactivity. Molec Immunol 1995;32:865–872. 18. Scheinberg DA, Lovett D, Divgi DR, et al. A phase I trial of monoclonal antibody M195 in acute myelogenous leukemia: specific bone marrow targeting and internalization of radionuclide. J Clin Oncol 1991;9:478–490. 19. Society of Nuclear Medicine. MIRD Primer for Absorbed Dose Calculations. Washington, DC: Society of Nuclear Medicine; 1988. 20. Koral KF, Zasadny KR, Kessler ML, et al. CT-SPECT fusion plus conjugate views for determining dosimetry in iodine-131-monoclonal antibody of lymphoma. J Nucl Med 1994;35:1714–1720.

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21. Sgouros G, Chiu S, Pentlow KS, et al. Three-dimensional dosimetry for radioimmunotherapy treatment planning. J Nucl Med 1993;34:1595–1601. 22. Sgouros G, Graham MC, Divgi CR, Larson SM, Scheinberg DA. Modeling and dosimetry of monoclonal antibody M195 (anti-CD33) in acute myelogenous leukemia. J Nucl Med 1993;34:422–430. 23. Scheinberg DA, Tanimoto M, McKenzie S, Strife A, Old LJ, Clarkson BD. Monoclonal antibody M195: a diagnostic marker for acute myelogenous leukemia. Leukemia 1989;3:440–445. 24. Tanimoto M, Scheinberg DA, Cordon-Cardo C, Huie D, Clarkson BD, Old-LJ. Restricted expression of an early myeloid and monocytic cell surface antigen defined by monoclonal antibody M195. Leukemia 1989;3:339–348. 25. Andrews RG, Torok-Storb B, Bernstein ID. Myeloid-associated differentiation antigens on stem cells and their progeny identified by monoclonal antibodies. Blood 1983;62:124–132. 26. Griffin JD, Linch D, Sabbath K, Larcom P, Schlossman SF. A monoclonal antibody reactive with normal and leukemic human myeloid progenitor cells. Leuk Res 1984;8:521–534. 27. Schwartz MA, Lovett DR, Redner A, et al. Dose-escalation trial of M195 labeled with iodine 131 for cytoreduction and marrow ablation in relapsed or refractory myeloid leukemias. J Clin Oncol 1993;11:294–303. 28. Jurcic JG, Caron PC, Nikula TK, et al. Radiolabeled anti-CD33 monoclonal antibody M195 for myeloid leukemias. Cancer Res 1995;55:5908s–5910s. 29. Co MS, Avdalovic NM, Caron PC, Avdalovic MV, Scheinberg DA, Queen C. Chimeric and humanized antibodies with specificity for the CD33 antigen. J Immunol 1992;148:1149–1154. 30. Caron PC, Co MS, Bull MK, Avdalovic NM, Queen C, Scheinberg DA. Biological and immunological features of humanized M195 (anti-CD33) monoclonal antibodies. Cancer Res 1992;52:6761–6767. 31. Caron PC, Jurcic JG, Scott AM, et al. A phase 1B trial of humanized monoclonal antibody M195 (anti-CD33) in myeloid leukemia: specific targeting without immunogenicity. Blood 1994;83:1760–1768. 32. Jurcic JG, DeBlasio T, Dumont L, Yao TJ, Scheinberg DA. Molecular remission induction with retinoic acid and anti-CD33 monoclonal antibody HuM195 in acute promyelocytic leukemia. Clin Cancer Res 2000;6:372–380. 33. Caron PC, Dumont L, Scheinberg DA. Supersaturating infusional humanized anti-CD33 monoclonal antibody HuM195 in myelogenous leukemia. Clin Cancer Res 1998;4:1421–1428. 34. Jurcic JG, Caron PC, Miller WH Jr, et al. Sequential targeted therapy for relapsed acute promyelocytic leukemia with all-trans retinoic acid and anti-CD33 monoclonal antibody M195. Leukemia 1995;9:244–248. 35. Jurcic JG, Divgi CR, McDevitt MR, et al. Potential for myeloablation with yttrium-90HuM195 (anti-CD33) in myeloid leukemia [Abstract]. Proc Am Soc Clin Oncol 2000;19:8a. 36. Appelbaum FR, Matthews DC, Eary JF, et al. The use of radiolabeled anti-CD33 antibody to augment marrow irradiation prior to marrow transplantation for acute myelogenous leukemia. Transplantation 1992;54:829–833. 37. Ruffner KL, Matthews DC. Current uses of monoclonal antibodies in the treatment of acute leukemia. Semin Oncol 2000;27:531–539. 38. Matthews DC, Appelbaum FR, Eary JF, et al. Radiolabeled anti-CD45 monoclonal antibodies target lymphohematopoietic tissue in the macaque. Blood 1991;78:1864–1874. 39. Matthews DC, Appelbaum FR, Eary JF, et al. Development of a marrow transplant regimen for acute leukemia using targeted hematopoietic irradiation delivered by 131I-labeled antiCD45 antibody combined with cyclophosphamide and total body irradiation. Blood 1995;85:1122–1131. 40. Matthews DC, Appelbaum FR, Eary JF, et al. Phase I study of (131)I-anti-CD45 antibody plus cyclophosphamide and total body irradiation for advanced acute leukemia and myelodysplastic syndrome. Blood 1999;94:1237–1247.

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41. Matthews DC, Appelbaum FR, Eary JF, Mitchell D, Press OW, Bernstein ID. 131I-antiCD45 antibody plus busulfan/cyclophosphamide in matched related transplants for AML in first remission [abstract]. Blood 1996;88:142a. 42. Bunjes D, Buchmann I, Duncker C, et al. Rhenium 188-labeled anti-CD66 (a, b, c, e) monoclonal antibody to intensify the conditioning regimen prior to stem cell transplantation for patients with high-risk acute myeloid leukemia or myelodysplastic syndrome: results of a phase I-II study. Blood 2001;98:565–572. 43. Kotzerke J, Glatting G, Seitz U, et al. Radioimmunotherapy for the intensification of conditioning before stem cell transplantation: differences in dosimetry and biokinetics of 188Reand 99mTc-labeled anti-NCA-95 MAbs. J Nucl Med 2000;41:531–537. 44. Hueneke RB, Pippin CG, Sguire RA, Brechbiel MW, Gansow OA, Strand M. Effective alphaparticle-mediated radioimmunotherapy of murine leukemia. Cancer Res 1992;52:6095–6100. 45. Hartmann F, Horak EM, Garmestani K, et al. Radioimmunotherapy of nude mice bearing a human interleukin 2 receptor α-expressing lymphoma utilizing the α-emitting radionuclideconjugated monoclonal antibody 212Bi-anti-Tac. Cancer Res 1994;54:4362–4370. 46. McDevitt MR, Finn RD, Ma D, Larson SM, Scheinberg DA. Preparation of alpha-emitting 213Bi-labeled antibody constructs for clinical use. J Nucl Med 1999;40:1722–1727. 47. Jurcic JG, Larson SM, Sqouros G, et al. Targeted α particle immunotherapy for myeloid leukemia. Blood 2002;100:1233–1239. 48. Sgouros G, Ballangrud AM, Jurcic JG, et al. Pharmacokinetics and dosimetry of an alphaparticle emitter labeled antibody: 213Bi-HuM195 (anti-CD33) in patients with leukemia. J Nucl Med 1999;40:1935–1946. 49. McDevitt MR, Ma D, Lai LT, et al. Tumor therapy with targeted atomic nanogenerators. Science 2001;294:1537–1540. 50. Waldmann TA, White JD, Carrasquillo JA, et al. Radioimmunotherapy of interleukin-2R alphaexpressing adult T-cell leukemia with Yttrium-90-labeled anti-Tac. Blood 1995;86:4063–4075.

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Interferons Thomas Fischer, MD CONTENTS INTRODUCTION IFN SUBTYPES AND BIOSYNTHESIS IFN RECEPTOR AND SIGNAL TRANSDUCTION BIOLOGIC ACTIVITY PHARMACOKINETICS AND TOXICITY IFN-α THERAPY OF CML REFERENCES

1. INTRODUCTION Interferons (IFNs) represent a family of pleiotropic proteins. They were first described as antiviral agents in 1957 by Issacs and Lindenmann (1). It was later shown that they play an important role not only in antiviral control but also in cellular proliferation control and in immune system modulation. According to the cellular origin, IFNs can be classified as leukocyte, fibroblast, or immune IFN (2). Leukocyte and fibroblast IFNs are also called type I IFNs, and immune IFN is often called type II IFN (2). IFNs were the first cytokines used in clinical trials of patients with cancer. Results of pivotal clinical studies using IFN-α for treatment of chronic myelogenous leukemia (CML) were already published in 1983 by Talpaz et al. (3). Further development of IFNs as antitumor agents developed rapidly, and today recombinant IFN-α is approved worldwide in more than 40 countries for treatment of various malignancies and viral diseases (4).

2. IFN SUBTYPES AND BIOSYNTHESIS IFN subtypes are summarized in Table 1. IFN-α is coded by more than 20 closely related genes, which show an 80% to 85% homology in amino acid sequence (2). Biologic stimuli of IFN-α biosynthesis include viruses, bacteria, From: Biologic Therapy of Leukemia Edited by: M. Kalaycio © Humana Press Inc., Totowa, NJ

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Fischer Table 1 Interferon Subtypes IFN Type I

IFN Type II

Subtypes

IFN-α 1–22 IFN-β IFN-ω

IFN-γ

Genes

27

1

No. of amino acids

165–166

143

pH stability

Stabile

Labile

Cellular origin

Leukocytes Fibroblasts

T lymphocytes NK cells

Receptor

IFNα-R1 IFNα-R2c

IFNγ-R1 IFNγ-R2

microplasma, and protozoa (5). In addition to these organisms, some cytokines and growth factors, such as CSF-1, interleukin (IL)-1, IL-2, or tumor necrosis factor (TNF), can also induce IFN-α synthesis. IFN-β is coded by a single gene on chromosome 9 (2). At the deoxyribonucleic acid (DNA) level, there is only a 30% homology with IFN-α subtypes. Similar to IFN-α, biosynthesis of IFN-β is also induced by most microorganisms. IFN-γ is coded also by a single gene located on chromosome 12 (2). IFN-γ does not show homology with IFN-α or IFN-β. IFN-γ is synthesized by stimulated lymphocytes. Classical stimuli are alloantigens or mitogenes. In addition, NK cells are also capable of producing IFN-γ (5).

3. IFN RECEPTOR AND SIGNAL TRANSDUCTION Because IFN-β and IFN-γ play no role in the biologic therapy of leukemia, this chapter focuses on only IFN-α receptors and signal transduction. Two subunits of the human IFN-α receptor have been identified: IFN-αR1 and IFN-αR2 (4,6). The major ligand-binding chain of the IFN-α receptor is IFN-αR2. This compound can be expressed as a short form (IFN-αR2a) or as a soluble (IFN-αR2b) or large variant (IFN-αR2c). The large-form IFN-αR2c is composed of 550 amino acids and is the functional receptor in IFN-α signaling (4,6). IFN-αR1 is composed of 557 amino acids and is critical in IFN-α signaling (7,8,9). Binding of IFN-α to the IFN-αR2 receptor chain results in oligomerization of receptor components. The next step in molecular events is activation of intracellular kinases, which are closely associated with receptor chains (4,10–14). These kinases have been identified as JAK1 and TYK2 kinases. Activation of JAK1 and TYK2 then results in phosphorylation of tyrosine residues on the cytoplasmic domains of the IFN-α

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Fig. 1. IFN-α signaling. Upon binding IFN-α to its receptor, the JAK/STAT signal transduction pathway is activated, which leads to enhanced transcription of IFN-induced genes.

receptor chains. This event then results in activation of specific docking sites for cytoplasmic signal transducer and activator of transcription (STAT) proteins. STAT proteins then dock to these specific IFN-α receptor chain sites. This enables JAK kinases to also phosphorylate STAT proteins at specific tyrosine residues. As a consequence of these events, homodimers and heterodimers of STAT proteins are formed and translocate to the nucleus. The major STAT heterodimer in IFN-α signaling is called IFN-stimulated gene factor 3 (ISGF3) and is composed of STAT1, STAT2, and the so called P48 protein, a member of the interferon regulatory factor (IRF) family. ISGF3 binds of specific recognition sites within the promoter of IFN-stimulated genes (Fig. 1). These recognition sites for ISGF3 are termed IFN-stimulated response elements (ISRE), which are necessary and sufficient to mediate IFN-α activation of transcription (15,16). In addition to ISGF3, STAT1 homodimers are also involved in signal transduction of IFN-α. STAT1 homodimers do not bind to the ISRE sequence but to the palindromic γ-IFN activatine site GAS sequence. The important role of JAK and STAT proteins in intracellular signaling has first been elucidated for IFN-α signal transduction. However, recently it has been shown that this is a common theme involved in signaling of many cytokines and growth factors.

4. BIOLOGIC ACTIVITY IFNs play an important role in the defense of viral, bacterial, and parasitic infections. In addition, they have antitumor activities. Recently, significant progress has been made in the identification and characterization of a multi-

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Fischer Table 2 Potential Mechanisms of Direct Antitumor IFN Activities

Protein PKR (= P68 Kinase) IRF-1 (IFN regulatory factor-1) RNase L RB (retinoblastoma gene) Cyclins

Function

Modification by IFN

Translation control, potential tumor suppressor gene Transcription factor, potential tumor suppressor gene Endoribonuclease, potential tumor suppressor gene Cell cycle regulation, tumor suppressor gene Cell cycle regulation

Induction by IFN-α, -β, -γ

Cyclin dependant kinases (Cdks)

Cell cycle regulation

c-myc

Transcription factor, oncogene

Induction by IFN-α, -β, -γ Induction by IFN-α, -β Modification of phosphorylation by IFN-α Downregulation of expression by IFN-α Downregulation of expression and inhibition of kinase activity by IFN-α Downregulation of c-myc mRNA by IFN-α

IFN = interferon.

tude of IFN-inducible proteins (5). The molecular targets for antiviral activities of IFN are viral replication, viral transcription, and viral translation. The IFNinducible 2–5 oligoadenylate synthetase/RNase L system and the Mx-protein are particularly important for these functions (2,5,17,18). Although the antiviral activity mechanisms of IFNs are understood, little is known about their antitumor activity mechanisms. Basically, a direct antitumor effect can be separated from more indirect activities. A selection of IFN-inducible and IFNmodified proteins that are believed to play an important role in direct IFN antitumor activity are summarized in Table 2 (19–23). IFN-α exerts direct antiproliferative effects on several cell types. The following mechanisms are believed to play an important role in this function: inhibition of cell-cycle transition, modulation of apoptosis, and induction of IFN-inducible genes directly involved in growth control. In addition, IFN-α is profoundly involved in the regulation of adhesion molecules and thereby mediated signals of hematopoietic progenitor cells. The effects of IFN-α on cell-cycle transition involve modulation of the following cell-cycle regulatory elements: IFN-α upregulates cyclin-dependent kinase (Cdk) inhibitors and inhibits expression of cell cyclins (21,22,23). As a consequence of these events, cyclin-/Cdk-associated kinase activities are suppressed by IFN-α in the G1 phase of the cell cycle. This results in increased expression of the underphosphorylated retinoblastoma gene that ultimately leads to reversible arrest in the G1 phase of the cell cycle.

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IFN-α also has modulatory functions in apoptosis. Depending on the cellular context, IFN may either stimulate or inhibit apoptotic processes. Stimulation of apoptosis is involved in the growth inhibitory effects of IFN-α. One of the molecular mechanisms is enhancement of CD95L-induced apoptosis via activation of caspase 3 (24). Recently, it has also been found that IFN-α upregulates phospholipid-scramblase I (PLSCRI) and thus may have a modulatory function in apoptosis via regulation of this protein (25). IFN-α is significantly involved in regulation of cell adhesion between hematopoietic progenitor cells and bone marrow stromal cells or extracellular matrix components. Extracellular matrix is produced by stromal cells, including endothelial cells, fibroblasts, osteoblasts, and macrophages and is composed of fibronectine, hyaluronic acid, integrins, and other components (26,27). IFN-α is able to modulate fibronectine- and integrin-dependent adhesion function in normal and malignant hematopoietic progenitor cells (28). Indirect mechanisms involved in growth control of malignant hematopoiesis by IFN-α include activation of immune effector mechanisms and modulation of cytokine synthesis at the level of bone marrow stroma. For hematopoiesis, a finely tuned balance of stem cell renewal and regulation of differentiation is a prerequisite (29). This involves the action of positive and negative regulatory cytokines at the level of hematopoietic cells and bone marrow stromal cells. As an example, hematopoietic growth factors, such as IL-7, IL-11, stem cell, and granulocyte-macrophase colony-stimulating factor (GM-CSF), are constantly produced by bone marrow stromal cells and have a stimulatory effect on hematopoiesis (30–33). IFN-α significantly inhibits the production of stimulatory effectors in the bone marrow microenvironment, such as GM-CSF or CSF (29). IFN-α exerts pleiotropic functions on the immune system. It modulates the growth, differentiation, and function of various immunologic effector cells, and it regulates the ability of these effector cells to interact with infected or malignant cells. However, the main mechanism that enhances the interaction between immune effector cells and their target cells is IFN-induced upregulation of major histocompatibility complex (MHC) class I antigen expression and adhesion molecule expression. In a recent report, upregulation of MHC expression was suggested to be involved in clearing malignant cells by T-cell immunity (34). This study demonstrated that a strong correlation between the presence of T cells specifically recognizing a peptide (PR1) from proteinase 3 and clinical responses after IFN-α therapy could be observed in patients with CML. Proteinase 3 is a myeloid tissue–restricted antigen that is overexpressed in CML cells. It has been proposed that IFN-α may have induced clinical remissions by facilitating expansion of autologous leukemia-reactive cytotoxic T-lymphocytes (CTLs). The molecular mechanism involved may be upregulation of MHC class I antigen expression or upregulation of tumor antigens in leukemic cells, thereby precipitating an immune response.

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5. PHARMACOKINETICS AND TOXICITY Upon subcutaneous injection of recombinant IFN-α, C-max in plasma is reached within 4 to 8 h (35). There are no significant differences in intravenous (iv), subcutaneous, or intramuscular (im) routes with regard to bioavailability. The T1/2 is approx 4 h. Elimination of IFN-α mainly occurs by proteolytic degradation in the kidneys. Penetration into the central nervous system (CNS) is usually not observed when applying doses below 50 × 106 IU (35). IFN-α side effects can be separated into acute and chronic toxicity (35). Acute toxicity is represented by a flu-like syndrome, characterized by fever, chills, bone pain, and headache. These side effects usually appear 2 to 4 h application IFN-α after. Acute side effects can be attenuated significantly by prophylactic application of acetaminophen at a dose of 500 to 1000 mg orally (po) taken 2 h before and 2 h after subcutaneous injection of IFN. Side effects are generally dose related but tend to disappear within days due to tachyphylaxis. A less common side effect is gastrointestinal toxicity, such as nausea, vomiting, and diarrhea. Cardiovascular toxicity, such as hypotension, angina pectoris, or tachyarrhythmia, is also rarely observed. The chronic toxicity of IFN generally manifests as fatigue, depression, polyneuropathy, and asthenia. Autoimmune phenomena, such as autoimmune hepatitis or hypothyroidism, may also be observed during prolonged IFN-α therapy. Occlusion of retinal veins or arteries and cotton wool spots are relatively rare events. Side effects can be managed by interruption of IFN-α therapy and subsequent dose reduction by 25% to 50%. However, several patients do not tolerate IFN-α in the long run.

6. IFN-α THERAPY OF CML The pivotal study by Talpaz et al. showed that IFN-α induces complete hematologic and cytogenetic remissions in CML (36). Subsequently, several clinical trials demonstrated that IFN-α is an active drug in the treatment of CML (37). IFN-α induces complete hematologic responses in 70% to 80% of patients in the chronic phase (CP) within a few weeks to months of therapy (37). IFN-α has also been shown to induce durable cytogenetic remissions. The rate of cytogenetic remissions reported varies widely and ranges from 0% to 44% (37). Today, most centers use the well-established criteria of the Houston group (37,38) to define hematologic and cytogenetic remissions (Tables 3 and 4). Based on the results of four prospective randomized studies and a meta-analysis, IFN-α is generally accepted as prolonging survival in patients with CML (39–43). Recently, a European meta-analysis (44) showed that patients achieving a complete cytogenetic response with IFN have an excellent survival rate: the 10yr survival rate from first CCR is 72% (Table 5) and is related to the risk profile.

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Table 3 Definitions of Hematologic Remissions for Chronic Phase with CML Patients (37,38) Complete hematologic response (CHR) Normal white blood cell count (WBC) (< 10.000/µl) Normal platelet count (< 450,000/µl) Normal differential (no immature cells) Disappearance of all symptoms and signs of CML Partial hematologic response (PHR) Decrease in WBC to < 50% of the pretreatment level and < 20.000/µl or Normalization of the WBC but persistent splenomegaly or immature cells in the differential Table 4 Definitions of Cytogenetic Remissions (37,38)* Remission Complete cytogenetic remission (CCR) Partial cytogenetic remission (PCR) Minor cytogenetic remission (mCR) No cytogenetic response (NCR)

Definition Absence of metaphases harboring the Ph+ chromosome 1% to 34% Ph+ metaphases 35% to 90% Ph+ metaphases 91% to 100% Ph+ metaphases

* At least 20 metaphases should be evaluated. Complete and partial cytogenetic responses are referred to as major cytogenetic responses (Ph+ < 35%).

Patients with low-risk Sokal and Euro scores have a 10-yr survival probability of 89% and 81%, respectively (44). The median time to first CCR was 19 mo (44). Recent evidence suggests that survival on IFN-α is better if treatment is initiated within the first 6 months of diagnosis (45) and in patients with fewer than 10% blasts in the peripheral blood (45). The Sokal score and the recently improved Hasford score (http://www.Pharmacoepi.de) have recently been shown to be strong predictors of survival on IFN-α (46). Therapy of chronic phase CML can be initiated with hydroxyurea at a dose of 1 to 3 g daily. This results in a faster tumor debulking than using IFN-α. In addition, allopurinol is given at a dose of 300 to 600 mg per day. Once the WBC drops below 20,000/µl, IFN-α may be started and hydroxyurea plus allopurinol gradually tapered (38). The optimal dose of IFN-α is still a matter of dabate. Usually, a dose of 5 × 6 10 IU/m2 is recommended (38). However, in the long run, the IFN-α dose must be adjusted once the WBC decreases to below 2000/µl or the platelet count to below 50,000/µl. The goal should be to decrease the WBC to a level of 2000 to 4000/µl and to achieve a complete hematologic remission. For this

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Fischer Table 5 Survival Estimates (Kaplan-Meier) for Patients in the Chronic Phase of CML Therapy

Probability of Survival

Busulfan Hydroxyurea IFN-α CCR on IFN-α Unrelated allogeneic SCT Related allogeneic SCT Autologous PBSCT

32% at 5 yr 44% at 5 yr 59% at 5 yr 72% at 10 yr from CCR 57% at 5 yr 58% at 7 yr 66% at 4 yr

goal, a maximal dose of IFN-α that does not induce grade III/IV toxicity is recommended. Initially, IFN-α therapy may be started at a lower dose and then dose escalated. A possible schedule would be 3 × 106 IU/d for 1 wk. Subsequently, the dose could be increased to 5 × 106 IU/d for 1 wk and then 5 × 106 IU/m2/d. This schedule has the advantage that tachyphylaxis is induced early and higher doses may be tolerated more easily. Initial side effects may be suppressed by giving 500 to 1000 mg acetaminophen po 2 hr before and 2 hr after administration of IFN-α at bedtime. Patients achieving a cytogenetic response should continue IFN-α therapy and minimal residual disease (MRD) should be monitored by quantitative PCR regularly. Patients should be followed by bone marrow cytogenetic analysis every 3 to 6 mo. There is a significant difference in the risk of relapse in patients with relatively high bcr-abl transcript levels as compared to patients with low levels (47). Particularly, relapse was rare in patients with a bcr-abl/abl ratio below 0.045%. This suggests that it is advisable to continue IFN-α therapy until low MRD levels are observed (47). Once desired MRD level is achieved and IFN-α has been given at least for 3 yr after documentation of a CCR, IFN-α may be tapered gradually (38,47). Further treatment decisions may then be based on the dynamics of MRD levels. IFN-α may have deleterious effects on the outcome of subsequent bone marrow transplantation (48). However, recent studies demonstrate that prior treatment with IFN-α does not adversely influence allogeneic BMT, providing IFN is discontinued at least 90 d before bone marrow transplantation or if only a short course of IFN-α has been given (45,49).

6.1. IFN-α Treatment of Hairy Cell Leukemia The standard of care for hairy cell leukemia is induction therapy with cladribine (2 CDA) or pentostatin. Alternatively, IFN-α may be used as induction therapy if there is a history of infection, which puts the patients at high risk for

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myelosuppression (50). Nucleoside analogs show a more rapid therapeutic onset in comparison to IFN-α but are significantly more myelosuppressive. Therefore, IFN-α may be useful as a prephase therapy. In addition, IFN-α may be effective inˇ20 the event of relapse after cladribine or if there is primary resistance to cladribine. As for CML, the mode of action in the treatment of hairy cell leukemia is currently unknown. IFN-α has been reported to induce partial or complete remission in more than 80% of patients with hairy cell leukemia (50). The dose recommended for induction therapy is 2 to 3 × 106 IU IFN-α twice a week. However, doses as low as 0.2 to 0.5 × 106 IU may be effective. The first response is a decrease in spleen size, followed by an increase in platelets and hemoglobin. Later, there is normalization of granulocytes and monocytes. Maintenance therapy is 2 to 3 × 106 IU IFN-α once a week (50).

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18. Staeheli P. Interferon-induced proteins and the antiviral state. Adv Virus Res 1990;38: 147–200. 19. Ben Dori R, Resnitzki D, Kimchi A. Reduction in p53 synthesis during differentiation of Friend-erythroleukemia cells. Correlation with the commitment to terminal cell division. FEBS Lett 1983;162:384–389. 20. Lengyel P. Tumor-suppressor genes: news about the interferon connection. Proc Natl Acad Sci USA 1992;90:5893–5895. 21. Tiefenbrun N, Melamed D, Levy N, et al. Alpha interferon suppresses the cyclin D3 and cdc25A genes, leading to a reversible G0-like arrest. Mol Cell Biol 1996;16:3934–3944. 22. Yamada H, Ochi K, Nakada S, et al. Interferon modulates the messenger RNA of G1-controlling genes to suppress the G1-to-S transition in Daudi cells. Mol Cell Biochem 1995;152:149–158. 23. Zhang K, Kumar R. Interferon-alpha inhibits cyclin E- and cyclin D1-dependent CDK-2 kinase activity associated with RB protein and E2F in Daudi cells. Biochem Biophys Res Commun 1994;200:522–528. 24. Roth W, Wagenknecht B, Dichgans J, Weller M. Interferon-alpha enhances CD95L-induced apoptosis of human malignant glioma cells. J Neuroimmunol 1998;87(1–2):121–129. 25. Zhou Q, Zhao J, Al-Zoghaibi F, et al. Transcription control of the human plasma membrane phospholipid scramblase 1 gene is mediated by interferon-alpha. Blood 2000;95(8):2593–9. 26. Verfaillie CM, McCarthy JB, McGlave PB. Mechanisms underlying abnormal trafficking of malignant progenitors in chronic myelogenous leukemia. Decreased adhesion to stroma and fibronectin but increased adhesion to the basement membrane components laminin and collagen type IV. J Clin Invest 1992;90:232. 27. Simons PJ, Masinovsky B, Longenecker BM, et al. Vascular cell adhesion molecule-1 expressed by bone marrow stromal cells mediates the binding of hematopoietic progenitor cells. Blood 1992;80:388. 28. Bhatia R, McCarthy JB, Verfaillie CM. Interferon-α restores normal β1-integrin mediated negative regulation of chronic myelogenous leukemia progenitor proliferation. Blood 1996;87:3883. 29. Aman J, Keller U, Derigs G, Mansour M, Huber C, Peschel C. Regulation of cytokine expression by Interferon-α in human bone marrow stromal cells: inhibition of hematopoietic growth factors and induction of Interleukin-1 receptor antagonist. Blood 1994;84:4142–4150. 30. Namen AE, Schmierer AE, March CJ, et al. B cell precusor growth-promoting activity. Purification and characterization of a growth factor active an lymphocyte precursors. J Exp Med 1988;167:988. 31. Paul SR, Bennett F, Calvette JA, et al. Molecular cloning or a cDNA encoding interleukin 11, a stromal cell-derived lymphopoietic and hematopoietic cytokine. Proc Natl Acad Sci USA 1990;87:512. 32. Heinrich MC, Dooley DC, Freed AC, et al. Constitutive expression of steel factor gene by human stromal cells. Blood 1993;82:771. 33. Charbord P, Tamayo E, Sealand S, et al. Granulocyte-macrophage colony-stimulating factor (GM-CSF) in human long-term bone marrow cultures: endogenous production in the adherent layer and effect of exogenous GM-CSF in granulomonopoiesis. Blood 1991;78:1230. 34. Molldrem J, Lee P, Wang C, et al. Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia Nature Med 2000;6:1018–1023. 35. Dorr RT. Interferon-alpha in malignant and viral diseases. A review. Drugs 1993;45:177–211. 36. Talpaz M, Kantarjian HM, McCredie K, Trujillo JM, Keating MJ, Gutterman JU. Hematologic remission and cytogenetic improvement by recombinant human interferon alpha A in CML. N Engl J Med 1986;314:1065–1069. 37. Silver RT, Woolf SH, Hehlmann R, et al. An evidence-based analysis of the effect of busulfan, hydroyurea, interferon, and allogneic bone marrow transplantation in treating the

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7

Interleukin-2 Treatment of Acute Leukemia Peter Kabos, MD and Gary J. Schiller, MD CONTENTS INTRODUCTION ADOPTIVE IMMUNE THERAPY DONOR LEUKOCYTE INFUSION AUTOLOGOUS IMMUNE ACTIVATION BIOLOGY OF INTERLEUKIN-2 AND PRECLINICAL STUDIES IN ACUTE LEUKEMIA TOXICITY AND BIOLOGIC EFFECT OF IL-2 USE OF INTERLEUKIN-2 IN OTHER MALIGNANCIES CLINICAL USE OF INTERLEUKIN-2 IN ACUTE LEUKEMIA CONCLUSIONS REFERENCES

1. INTRODUCTION Significant progress has been made in chemotherapy treatment of patients with acute leukemia, including effective strategies for both remission induction and dose-intensive consolidation. Long-term survival, however, remains elusive. Despite a better understanding of disease biology, including pretreatment characteristics and unique cytogenetic features, the prognosis for adult patients with acute leukemia remains poor. In acute myeloid leukemia (AML), 60–80% of patients will initially achieve complete remission (CR) but the majority of them will relapse (1,2). Only 10%–30% of patients achieve prolonged leukemiafree survival (3,4,5). In the case of acute lymphocytic leukemia (ALL), 60–65% of children can achieve long-term survival but only 10–30% of adults survive 5 yr free of recurrence (6,7). Although induction chemotherapy for acute leukemia involves a cytoreductive strategy, the main clinical investigation effort involves preventing recurrence. From: Biologic Therapy of Leukemia Edited by: M. Kalaycio © Humana Press Inc., Totowa, NJ

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Despite an improvement in remission duration, relapse remains the major cause of death for more than 50% of adult patients (5,6). Therefore, leukemia patients typically receive regimens of dose-intensive chemotherapy with or without stem cell support to consolidate CR (6,8). Relapse of disease is presumably due to proliferation of leukemia cells that survive induction, consolidation, and even preparative conditioning. Alternative approaches to cytotoxic chemotherapy for maintenance of CR include nonspecific immune stimulation and adoptive immunotherapy through allogeneic transplantation (see the following section). For decades there have been attempts to stimulate the host immune system to control the population of leukemia cells. Initial approaches involved immunization with Corynebacterium parvum, bacille Calmette-Guerin, and transfusion of nonirradiated blood. These modalities were unsuccessful. On the other hand, allogeneic transplantation offers a potentially curative mechanism of adoptive immune therapy, leading to a re-examination of the role of immunotherapy in the management of acute leukemia (9). Currently, allogeneic bone marrow transplantation (alloBMT) represents the best method for an immune-mediated eradication of minimal residual disease. Initially, alloBMT was viewed as supportive care used for restoring hematopoesis after supralethal doses of chemoradiotherapy (10,11). It has been recognized, however, that high-dose chemoradiotherapy often does not eradicate leukemia and that the allogeneic graft itself provides an additive antileukemia effect (12,13). There are several lines of evidence supporting the concept of graft vs leukemia (GVL) after alloBMT. First, patients with AML with acute and chronic graft vs host disease (GVHD) have a reduced risk of leukemia relapse (14–17). Second, the risk of relapse is higher after syngeneic BMT (18–20). Direct comparison of allogeneic and autologous transplantation for acute leukemia suggests that the antileukemia effect of alloBMT relies on the immunoreactive action of the graft as much as the myeloablative preparative therapy itself (5). In addition, T-cell depletion of allotransplantations also significantly increases the risk of relapse (14,21). Allotransplantation, however, is associated with major therapeutic limitations. A histocompatible sibling donor is available to only a limited number of younger patients. Allogeneic transplantation also carries a higher potential for complications mostly from regimen-related toxicity, infection, and especially GVHD, accounting for high treatment-related morbidity and mortality (22).

2. ADOPTIVE IMMUNE THERAPY The mechanism of GVL in alloBMT has been the focus of research and is believed to be due to adoptive immunotherapy. The importance of adoptive immunotherapy was first demonstrated with the use of lymphokine-activated killer (LAK) cells in combination with interleukin (IL)-2 in patients with metastatic cancer. This combination produced objective tumor regression in patients with renal and colon cancers and CR in a patient with metastatic

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melanoma (23,24). In these early studies, LAK cells were derived from peripheral blood mononuclear cells and activated ex vivo with high-dose IL-2. Ex vivo activated LAK cells were capable of lysing tumor cells in a process unrelated to major histocompatibility proteins (25). After initial preclinical success, clinical efforts focused on the use of LAK cells and IL-2 in the treatment of solid tumors such as renal cell carcinoma and melanoma. Unfortunately, no advantage has been clearly demonstrated for the administration of LAK cells and IL-2 over administration of IL-2 alone in metastatic cancer (26). Recent studies have offered an explanation for the limited therapeutic efficacy of LAK cells, given their inability to home exclusively to the tumor (27,28). A search for more potent killer cells has led to the discovery of tumor-infiltrating lymphocytes (TILs) (29). With the phenotype of cytotoxic lymphocytes, these cells kill in a major histocompatability complex (MHC)-restricted manner and also appear to be more potent than LAK cells. More importantly, they are more efficient in homing to the tumor (30,31). Immunologic treatment with TILs can be highly successful, as demonstrated in patients with cytomegalovirus (CMV) infections after alloBMT (32,33). TIL therapy of malignancies has great potential but depends on identifying a tumor-specific antigen. Currently, due to the lack of leukemia-specific antigens, nonspecifically acting donor leukocytes are being used in the setting of alloBMT as means of adoptive immunotherapy.

3. DONOR LEUKOCYTE INFUSION Initially, donor leukocytes were used in combination with interferon-α to successfully re-induce remission in patients with relapsed chronic myelogenous leukemia (CML) after BMT (34). This success led to studies of donor-leukocyte infusion in multicenter trials for patients with CML, as well as acute leukemia relapsed after alloBMT (35,36). Based on these studies, donor leukocyte infusion (DLI) is most effective in patients with relapsed CML, in which 80% of patients achieve CR, whereas patients with more advanced acute leukemia have a much lower likelihood of response. In patients with AML who relapsed after alloBMT, response rates range from 15% to 20%; few patients with ALL respond (37–39). DLI is associated with major complications, including GVHD and pancytopenia. Predictors for developing GVHD include previous T-lymphocyte–depleted transplantation and the concomitant use of interferon-α. However imperfect, the fact that some patients with acute leukemia may be re-induced to enter remission after DLI strongly supports the concept of an immune-mediated antileukemia effect (38,40–43).

4. AUTOLOGOUS IMMUNE ACTIVATION A major limitation of autologous stem cell transplantation in the management of acute leukemia is the absence of an immune effect. Autologous transplantations, however, offer many advantages when compared with allogeneic transplan-

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tations, allowing for the use of myeloablative preparative conditioning in a larger unselected population of patients without the risk of treatment-related morbidity and mortality. Autologous transplantation is also not limited by age or prognostic features. Therefore, a combination of GVL effect with the advantages of autologous transplantation would be desirable (44). Several drugs, including cyclosporine (17,45), IL-1-α (46), and linomide (47) have been used in patients with acute leukemia to boost the GVL effect after autologous transplantation. More recently, IL-2 has been identified as a candidate for immunotherapy with the potential to mimic the immune effect seen in allogeneic transplantation (48–50). IL-2 has been the focus of extensive clinical investigations in acute leukemia and has shown promise as a potential immunologic activator.

5. BIOLOGY OF INTERLEUKIN-2 AND PRECLINICAL STUDIES IN ACUTE LEUKEMIA The IL-2 gene is located on chromosome 4 and encodes a 15-17 kDa glycoprotein that is secreted in response to antigen activation primarily by CD4-positive T-cells, playing a critical role in the immune response. IL-2 induces proliferation and activation of T-cells, B-cells, and natural killer (NK) cells, and also leads to the generation of LAK cells. Furthermore, it stimulates the production of secondary cytokines by IL-2-responsive cells, potentiating its biologic effect. Preclinical leukemia studies should sensitivity to cytolysis and/or growth inhibition mediated by IL-2-activated effector cells in vitro (52–55). These experiments also demonstrated that human primary leukemia blasts resistant to NK cells were sensitive to normal cytotoxic effectors activated by IL-2 (56). In animal models, IL-2 with or without LAK cells was capable of eradicating murine leukemias (57,58). LAK cells, normally decreased in the majority of newly diagnosed patients with acute leukemia, were also shown to stop the in vivo growth of human leukemia cell lines (51). Few leukemia blasts express a functional IL-2 receptor. Therefore, the possibility of stimulating leukemia clones is limited, and, in most instances, IL-2 does not induce proliferation of blasts in vitro (59,60). Furthermore, experiments on nude mice demonstrated that IL-2 inhibits the growth of leukemia cells (59). These encouraging preclinical results, together with the inverse correlation of the LAK cell activity and disease activity in acute leukemia, as well as the limited clinical success of IL-2 in other metastatic cancers, formed the basis for testing IL-2 in acute leukemia.

6. TOXICITY AND BIOLOGIC EFFECT OF IL-2 IL-2 is administered by continuous infusion, by bolus intravenous (iv) injection, or subcutaneously. Its use is associated with dose-dependent toxicity. Most patients experience side effects that may require dose reduction or discontinua-

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tion of treatment and are mainly related to the capillary-leak syndrome (61–63). More common side effects may include a fever, nausea, diarrhea, cutaneous rashes, pruritus, increases in liver and renal parameters, fluid retention, weight gain, hypotension, confusion, and cardiac arrhythmias (62). All patients show some degree of hepatomegaly and/or splenomegaly and variable leukocytosis. The major side effects, although potentially lethal, usually disappear after discontinuation of IL-2 treatment and their duration can be shortened by corticosteroids. High-dose IL-2 (> 8 × 106/m2/d) treatment is usually administered under close supervision in the hospital for no more than 4–5 d due to its toxicity. The leukocyte differential count is affected by IL-2 and varies throughout the regimen. IL-2 infusion is accompanied by neutrophilia and increased circulating eosinophils. After discontinuation of IL-2, a rebound lymphocytosis, with an increase in large granular lymphocytes, can be noted. These changes are most likely in response to the secondary release of cytokines triggered by IL-2. Changes in peripheral blood are accompanied by an increase in lymphocytes and eosinophils in the bone marrow (62). One of the major complications of long-term high-dose IL-2 treatment is marked thrombopenia, primarily due to sequestration (64) and the antimegakaryocytic effects of LAK cells (65). Lowdose IL-2 treatment (0.5×106/m2/d), on the other hand, may be safely administered for weeks (66,68). As expected, the side effects are much less pronounced, but this regimen can also effectively increase the number of circulating cytotoxic cells (69). Patients with IL-2 maintenance therapy develop a persistent lymphocytosis with an increase in large granular lymphocytes. The infusion of high-dose IL-2 induces marked phenotypic and functional modifications of the immune markers in patients with acute leukemia (70–71), leading to an increase in the number of circulating and bone marrow CD3-positive cells and an amplification of cytotoxic cells (CD16 positive, CD56 positive), especially in the CD3-negative NK population. Expression of activation markers HLA-Dr and TAC on circulating and bone marrow lymphocytes can also be observed. There is an increase in NK cell activity and IL-2-induced LAK activity. LAK cells are activated not only in the peripheral blood but also in the bone marrow, with the potential to attack the site of minimal residual leukemia. The peak of biologic effects coincides with maximum lymphocytosis that occurs 2 to 3 d after IL-2 discontinuation. As mentioned, a secondary effect of IL-2 administration is the increase of circulating cytokines and growth factors (TNF, IFN, IL-3, IL-5, and GM-CSF). This increase in cytokines can explain transient leukocytosis and eosinophilia, whereas the increase in growth factors may potentially increase the susceptibility to cytotoxic agents, as documented in some patients receiving IL-2 (72).

7. USE OF INTERLEUKIN-2 IN OTHER MALIGNANCIES Before its use in hematologic malignancies, IL-2 was tested and shown to be beneficial in other malignancies. IL-2 administered to patients with metastatic

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melanoma and renal cell cancer induced CR in 1% to 5% and partial remission with shrinkage of tumor metastases in 5% to 15% of patients (73). Even more encouraging were the initial reports of IL-2 use in combination with LAK cells in renal cell carcinoma, with CR rates in up to 30% of patients (74–76). Larger multicenter studies show, however, a more modest response independent of the use of LAK cells (77).

8. CLINICAL USE OF INTERLEUKIN-2 IN ACUTE LEUKEMIA 8.1. Interleukin-2 as a Single Agent The first IL-2 clinical trials in patients with acute leukemia focused on demonstrating an antileukemia effect and included patients with advanced disease refractory to chemotherapy (63,69,78). These initial European studies also evaluated the toxicity of high-dose IL-2 regimens and documented the feasibility of administration to patients with acute leukemia (57). An objective response was seen in 20% of patients (79). Complete remission with IL-2 as a single agent was achieved only in 17% of patients with AML. In patients with ALL, IL-2 alone failed to induce CR but significantly reduced the peripheral blast counts. An antitumor effect was also documented with lower doses of IL-2, and these regimens produced significant immune stimulation as well (79). For some patients who did not respond to single-agent treatment with IL-2, there was evidence of new sensitivity to previously ineffective chemotherapy (80).

8.2. Use of IL-2 with Other Therapeutic Modalities Although IL-2 had only modest efficacy as a single agent, results were sufficiently encouraging to warrant further investigation. A setting for further IL-2 clinical testing seemed to be in minimal residual leukemia present at the time of first or second remission (81). A variety of postremission treatment strategies, such as myelosuppressive maintenance therapy, multiple and prolonged cycles of consolidation therapy, autologous BMT, and alloBMT achieved leukemia-free survival for 25–45% patients in first CR (82–84). The activity of low-dose maintenance chemotherapy is debatable in adult ALL and is of no benefit in AML. Therefore, clinical trials examined the use of IL-2 in remission and in stem cell transplantation (Table 1). In a phase I trial, Robinson et al. tested the use of IL-2 after alloBMT in children with acute leukemia beyond first remission (85). These patients, in CR without active GVHD after unmodified allogeneic matched-sibling BMT, received escalating doses of IL-2. IL-2 was administered as a continuous infusion for 5 d (0.9, 2.0, 6.0 × 106 IU/m2/d). After 6 d of rest, the patients received IL-2 maintenance therapy (0.9 × 106 IU/m2/d) for 10 d by continuous infusion. At the time of publication, 10 of 17 patients were in sustained CR 5 to 67 mo after transplantation. In a pilot trial by Margolin et al., patients with poor prognosis leukemia and lymphoma received IL2-activated autologous bone marrow and peripheral

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Table 1 Interleukin-2 in the Treatment of Acute Leukemia Author

Year

Subjects

Disease

Robinson et al. (85)

1996

17 children

ALL in CR1

Margolin et al. (86)

1999

12 adults

AML and ALL in CR1

Schiller et al. (87)

2001

49 adults

AML in CR1

Attal et al. (46)

1995

60 adults

ALL in CR1

Blaise et al. (88)

2000

130 adults

AML and ALL in CR1

Therapy

Results

alloBMT fol10 of 17 lowed by patients in IL-2 infusion CR Ex vivo IL-2 CR in 2 of 3 treated PBSC, patients with IL-2 infusion ALL; 3 of 9 patients in AML IL-2 mobilized CR in 49% autologous with no PBPC, SQ clear benefit IL-2 from IL-2 treatment aBMT followed No survival by IL-2 benefit from infusion IL-2 treatment aBMT followed No survival by IL-2 benefit from infusion IL-2 treatment

ALL = acute lymphocytic leukemia; alloBMT = allogenous bone marrow transplantation; BMT = autologous bone marrow transplantation; IL = interleukin; CR = complete remission; AML = acute myeloid leukemia; CR1 = first complete remission, PBSC = peripheral blood stem cells, PBPC = peripheral blood progenitor cells, SQ = subcutaneously.

blood stem cell transplantation followed by IL-2 treatment (86). Patients first received bone marrow or G-CSF-mobilized autologous peripheral blood stem cells that were exposed to IL-2 for 24 h ex vivo. This treatment was followed by low-dose IL-2 infusion until hematologic reconstitution and later by intermediate-dose continuous infusion for six 2-wk maintenance cycles at 1-mo intervals. Of the 12 patients with acute leukemia treated, 2 with ALL were in CR at 38 and 43 mo, 1 with ALL died, and 3 of 9 patients with AML were in CR after 21, 46, and 53 mo. In vivo IL-2 mobilization for the purpose of autologous peripheral blood progenitor cell (PBPC) transplantation was studied by Schiller et al. (87). IL-2 was administered after recovery from high-dose cytarabine-based consolidation chemotherapy with G-CSF as mobilization of peripheral blood progenitor cells for 49 patients with AML in first remission. Forty-one patients received myeloablative chemoradiotherapy followed by the infusion of these IL-2mobilized autologous PBPC. IL-2 was administered after transplantation at the

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same dose and schedule as given after consolidation/mobilization (3 × 106 units subcutaneously twice daily for 10 d). After a median follow-up from remission of 15 mo, actuarial leukemia-free survival for all patients from time of CR was 49%. The authors demonstrated the feasibility of autologous transplantation of IL-2-mobilized PBSC for an unselected population of adult patients with AML in first CR, but there was no clear evidence of survival benefit in this phase II study. In an attempt to simulate a GVL effect, Attal et al. studied the effect of IL-2 after autologous BMT in patients with ALL in first CR (46). In their randomized multicenter prospective study of 60 patients with ALL in first remission undergoing autologous BMT, 30 patients were assigned to receive continuous IL-2 infusion for a total of five cycles every other week. The first 5-d cycle was followed by four 2-d cycles. IL-2 was administered at a dose of 12 × 106 U/m2/d. The probability of continued remission 3 yr after autologous BMT was similar for both groups, 29% for those treated with IL-2, and 27% for those not receiving IL-2. IL-2 did not decrease the high relapse rate after autologous BMT. Unfortunately, the authors chose to examine a disease state in which little GVL can be demonstrated and in which DLIs after unsuccessful alloBMT are ineffective. Blaise et al. tried to clarify the effect of IL-2 immunotherapy in acute leukemia during first CR (88). They conducted a prospective multicenter randomized trial that included patients with both AML and ALL in first CR after autologous BMT. Seventy-eight patients with AML and 52 patients with ALL were randomized to receive five IL-2 cycles (12 × 106 IU/m2/d) after autologous BMT. Of 65 patients randomized into the study group, 38 (59%) started the IL-2 treatment. A total of 13 patients started each of the five cycles, 7 of these patients received more than 95% of the total scheduled dose. The data analysis was based on an intent to treat, with a median follow-up of 7 yr. No difference in outcome was observed between patients in the study and control groups. In the study group, relapse occurred in 66% of patients, compared with 55% of patients in the control group. Survival was 33% vs 43%, and leukemia-free survival was 29% vs 36% in the study and control groups, respectively. Although this study failed to show any benefit of IL-2 treatment in acute leukemia, the low rate of compliance, partly due to toxicity, might have obscured any difference in outcomes. Further trials must address both the timing of IL-2 administration after autologous BMT as well as the toxicity of the regimen.

9. CONCLUSIONS After more than 10 yr of clinical experience with IL-2 in acute leukemia, its place in treatment has not been established. The biologic effects of IL-2, as well as results of some preliminary clinical studies in the relapsed setting, point to the potential benefit of immunotherapy in the treatment of acute

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leukemia. Unfortunately, no single dose or delivery system of IL-2 has been shown to be more effective in improving survival or in alternating surrogate biologic endpoints. Randomized clinical trials of IL-2 as a single agent, in conjunction with autologous or allogeneic transplantation of stem cells, have been disappointing. None of the randomized trials showed benefit in long-term survival when compared with standard regimens, although the Attal study was not optimal. The Blaise study attempted to address a population best suited for an immune-mediated antineoplastic effect; however, small numbers would have made detecting a small benefit impossible. IL-2 has, however, helped to bring attention to an immunologic approach in the treatment of acute leukemia. A better understanding of the biology of the disease and a combination of multiple immunotherapeutic agents may be needed to induce an immunologically active effect and potentially improve survival in patients with acute leukemia.

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17. Yeager A, Vogelsang G, Jones R, et al. Induction of cutaneous graft-versus-host disease by administration of cyclosporine to patients undergoing autologous bone marrow transplantation for acute myeloid leukemia. Blood 1992;79:3031–3035. 18. Fefer A, Cheever MA, Greeberg PD. Identical-twin (syngeneic) marrow transplantation for hematologic cancers. J Natl Cancer Inst 1986;76:1269–1273. 19. Gale RP, Champlin RE. How does bone marrow transplantation cure leukemia? Lancet 1984;2:28–30. 20. Gale RP, Horowitz MM, Ash RC, et al. Identical-twin bone marrow transplants for leukemia. Ann Intern Med 1994;120:646–652. 21. Marmont AM, Horowitz MM, Gale RP, et al. T-cell depletion of HLA-identical transplants in leukemia. Blood 1991;78:2120–2130. 22. Berman E, Little C, Gee T, et al. Reasons that patients with acute myelogenous leukemia do not undergo allogeneic bone marrow transplantation. N Engl J Med 1992;326:156–160. 23. Rosenberg SA, Lotze JT, Muul LM, et al. Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl J Med 1985;313:1485–1492. 24. Rosenberg SA, Packard BS, Aebersold PM, et al. Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma: a special report. N Engl J Med 1988;319:1676–1680. 25. Rosenstein M, Yron I, Kaufman Y, et al. Lymphokine activated killer cells: lysis of fresh syngeneic NK-resistant murine tumor cells by lymphocytes cultured in interleukin-2. Cancer Res 1984;44:1946–1953. 26. Rosenberg SA, Lotze MT, Muul LM, et al. A progress report on the treatment of 157 patients with advanced cancer using lymphokine activated killer cells and interleukin-2 or high dose interleukin-2 alone. N Engl J Med 1987;316:889–897. 27. Foa R, Fierro MT. Cesano, A. et al. Defective lymphokine-activated killer cell generation and activity in acute leukemia patients with active disease. Blood 1991;78:1041–1046. 28. Carson WE, Caligiuri MA. Cellular adoptive immunotherapy with natural killer cells. In: Morstyn G and Sheridan W, eds. Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy. Cambridge: Cambridge University Press; 1996:451–474. 29. Rosenberg SA, Spiess P, Lafreniere R. A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science 1986;223:1318–1321. 30. Fisher B, Packard BS, Read EJ, et al. Tumor localization of adoptively transferred indium111 labeled tumor infiltrating lymphocytes in patients with metastatic melanoma. J Clin Oncol 1989;7:250–261. 31. Griffith KD, Read EJ, Carrasquillo CS, et al. In vivo distribution of adoptively transferred indium-111 labeled tumor infiltrating lymphocytes and peripheral blood lymphocytes in patients with metastatic melanoma. J Natl Cancer Inst 1989;81:1709–1717. 32. Riddell SR, Greenberg PD. Principles for adoptive T cell therapy of human viral disease. Ann Rev Immunol 1995;13:545–586. 33. Riddell SR, Watanabe KS, Goodrich JM, et al. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science 1992;257:238–241. 34. Kolb HJ, Mittermuller J, Clemm CH, et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 1990;76:2462–2465. 35. Collins RH, Shpilberg O, Drobyski WR, et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 1997;15:433–444. 36. Soiffer RJ. Donor lymphocyte infusions: the door is open. J Clin Oncol 1997;15:416–417. 37. Giralt SA, Kolb HJ. Donor lymphocyte infusion. Curr Opin Oncol 1996;8:96–102. 38. Kolb HJ, Schattenberg A, Goldman JM, et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood 1995;86:2041–2050.

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39. van Rhee F, Lin F, Cullis JO, et al. Relapse of chronic myeloid leukemia after allogeneic bone marrow transplant: the case for giving donor leukocyte transfusions before the onset of hematologic relapse. Blood 1994;83:3377–3383. 40. Antin JH. Graft-versus-leukemia: no longer an epiphenomenon. Blood 1993;82:2273–2277. 41. Cullis JO, Jiang YZ, Schwarer AP, et al. Donor leukocyte infusions for chronic myeloid leukemia in relapse after allogeneic bone marrow transplantation. Blood 1992;79:1379–1381. 42. Drobyski WR, Keever CA, Roth MS, et al. Salvage immunotherapy using donor leukocyte infusions as treatment for relapsed chronic myelogenous leukemia after allogeneic bone marrow transplantation: efficacy and toxicity of a defined T-cell dose. Blood 1993;82:2310–2318. 43. Mackinnon S, Papadopoulos EB, Carabasi MH, et al. Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia responses from graft-versushost disease. Blood 1995;86:1261–1268. 44. Klingemann HG. Introducing graft-versus-leukemia into autologous stem cell transplantation. J Hematother 1995;4:261–267. 45. Jones R, Vogelsang G, Hess A, et al. Induction of graft-versus-host disease after autologous transplantation. Lancet 1991;9:754–757. 46. Klingemann H, Grigg A, Wilkie-boyd K, et al. Treatment with recombinant interferon a-2b early after bone marrow transplantation in patients at high risk for relapse. Blood 1991;78:3306–3311. 47. Uckun FM, Kersey JH, Haake R, et al. Pretransplantation burden of leukemic progenitor cells as a predictor of relapse after bone marrow transplantation for acute lymphoblastic leukemia. N Engl J Med 1993;329:1296–1301. 48. Attal M, Blaise D, Marit G, et al. Consolidation treatment of adult acute lymphoblastic leukemia: a prospective, randomized trial comparing allogeneic versus autologous bone marrow transplantation and testing the impact of recombinant interleukin-2 after autologous bone marrow transplantation. Blood 1995;86:1619–1628. 49. Fefer A. Graft-versus-tumor responses: adoptive cellular therapy in bone marrow transplantation. In: Forman SJ, Blume KG, Thomas ED, eds. Bone Marrow Transplantation. Boston; Blackwell; 1994:231–241. 50. Fefer A, Robinson N, Benyunes MC. Interleukin-2 therapy after autologous stem cell transplant for acute myelogenous leukemia in first remission. In: DickeK, Keating A, eds. Autologous Blood and Marrow Transplantation: Proceedings of the Ninth International Symposium. Arlington, TX; Carden Jennings, 1999:46–53. 51. Margolin KA, Wright C, Forman SJ. Autologous bone marrow purging by in situ IL-2 activation of endogenous killer cells. Leukemia 1997;9:723–728. 52. Dawson MM, Johnston D, Taylor GM, et al. Lymphokine activated killing of fresh human leukemias. Leuk Res 1986;10:683–688. 53. Lista P, Fierro MT, Liao X-S, et al. Lymphokine-activated killer (LAK) cells inhibit the clonogenic growth of human laukemic stem cells. Eur J Haematol 1989;42:425–430. 54. Lotzova E, Savary CA, Herberman RB. Inhibition of clonogenic growth of fresh leukemia cells by unstimulated and IL-2 stimulated NK cells of normal donors. Leuk Res 1987;11:1059–1066. 55. Oshimi K, Oshimi Y, Akutsu M, et al. Cytotoxicity of interleukin-2-activated lymphocytes for leukemia and lymphoma cells. Blood 1986;68:938–948. 56. Fierro MT, Biao XS, Lusso P, et al. In vitro and in vivo susceptibility of human leukemic cells to lymphokine activated killer activity. Leukemia 1988;2:50–54. 57. Lim SH, Newland AC, Kelsey S, et al. Continuous intravenous infusion of high-dose recombinant interleukin-2 for acute myeloid leukaemia: a phase II study. Cancer Immunol Immunother 1992;34:337–342. 58. Toze CL, Barnett MJ, Klingemann HG. Response of therapy-related myelodysplasia to lowdose interleukin-2. Leukemia 1993;7:463–465.

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59. Foa R, Caretto P, Fierro MT, et al. Interleukin 2 does not promote the in vitro and in vivo proliferation and growth of human acute leukaemia cells of myeloid and lymphoid origin. Br J Haematol 1990;75:34–40. 60. Touw I, Groot-Loonen J, Broeders L, et al. Recombinant hematopoietic growth factors fail to induce a proliferative response in precursor B acute lymphoblastic leukemia. Leukemia 1989;3:356–362. 61. Blaise D, Viens P, Olive D, et al. Hematologic and immunologic effects of the systemic administration of recombinant Interleukin-2 after autologous bone marrow transplantation. Blood 1990;76:1092–1097. 62. Foa R. Interleukin 2 in the management of acute leukemia. Br J Haematol 1996;92:1–8. 63. Maranichi D, Blaise D, Viens P, et al. High-dose recombinant interleukin 2 and acute myeloid leukemias in relapse. Blood 1991;78:2182–2187. 64. Paciucci PA, Mandeli J, Oleksowiz L, et al. Thrombocytopenia during immunotherapy with interleukin-2 by constant infusion. Am J Med 1990;89:308–312. 65. Guarini A, Sanavio F, Novarino A, et al. Thrombocytopenia in acute leukaemia patients treated with IL2: cytolytic effect of LAK cells on megakaryocytic progenitors. Br J Haematol 1991;79:451–456. 66. Blaise D, Olive D, Stoppa AM, et al. Hematologic and immunologic effects of the systemic administration of recombinant Interleukin-2 after autologous bone marrow transplantation. Blood 1990;76:1092–1097. 67. Meloni G, Foa R, Vignetti M, et al. Interleukin-2 may induce prolonged remissions in advanced acute myelogenous leukemia. Blood 1994;84:2158–2163. 68. Soiffer RJ, Murray C, Gonin R, et al. Effect of low dose interleukin-2 on disease relapse after T-cell depleted allogeneic bone marrow transplantation. Blood 1994;84:964–968. 69. Soiffer RJ, Murray C, Cochran K, et al. Clinical and immunologic effects of prolonged infusion of low-dose recombinant interleukin-2 after autologous and T-cell-depleted allogeneic bone marrow transplantation. Blood 1992;79:517–526. 70. Foa R, Guarini A, Gillio Tos A, et al. Peripheral blood and bone marrow immunophenotypic and functional modifications induced in acute leukemia patients treated with interleukin 2: evidence of in vivo lymphokine activated killer cell generation. Cancer Res 1991;51:964–968. 71. Gottlieb DJ, Prentice HG, Heslop HE, et al. Effects of recombinant interleukin-2 administration on cytotoxic function following high-dose chemo-radiotherapy for hematological malignancy. Blood 1989;74:2335–2342. 72. Foa R, Meloni G, Tosti S, et al. Treatment of acute myeloid leukaemia patients with recombinant interleukin 2: a pilot study. Br J Haematol 1991;77:491–496. 73. Rosenberg SA. Perspectives on the use of IL2 in cancer treatment. Cancer J 1997;3:52–56. 74. Belldegrun A, Figlin R, Haas GP, deKernion JB. Immunotherapy for metastatic renal cell carcinoma. World J Urol 1991;9:157–165. 75. Ilson DH, Motzer RJ, Kradin RL, et al. A phase II trial of IL-2 and interferon alpha-2a in patients with advanced renal cell carcinoma. J Clin Oncol 1992;10:1124–1130. 76. Sleifter DT, Janssen RA, Butler J, et al. Phase II study of subcutaneous IL-2 in unselected patients with advanced renal cell cancer in an outpatient basis. J Clin Oncol 1992;10:1119–1123. 77. McCabe MS, Stablein D, Hawkins MJ. The modified group C experience-phase III randomized trials of IL-2 vs IL-2/LAK in advanced renal cell carcinoma and advanced melanoma. Proc Am Soc Clin Oncol 1991;10:213. 78. Adler A, Chervenick PA, Whiteside TL, et al. Interleukin 2 induction of lymphokine-activated killer (LAK) activity in the peripheral blood and bone marrow of acute leukemia patients. I. Feasibility of LAK generation in adult patients with active disease and in remission. Blood 1988;71:709–716. 79. Blaise D, Maranichi D. Interleukin 2 in the treatment of acute leukemia. Leukemia Res 1998;22:1165–1170.

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80. Foa R. Does interleukin-2 have a role in the management of acute leukemia? J Clin Oncol 1993;11:1817–1825. 81. Macdonald D, Jiang Y, Gordon A, et al. Recombinant interleukin-2 for acute myeloid leukemia in first complete remission: a pilot study. Leuk Res 1990;1:967–973. 82. Applebaum FR, Dahlberg S, Thomas ED, et al. Bone marrow transplantation or chemotherapy after remission induction for adults with acute nonlymphoblastic leukemia. 1984;101:581–588. 83. Mayer RJ, Davis RB, Schiffer CA, et al. Intensive postremission chemotherapy in adults with acute myeloid leukemia. New Engl J Med 1994;331:896–903. 84. Schiller GJ, Nimer SD, Territo MC, et al. Bone marrow transplantation versus high-dose cytarabine-based consolidation chemotherapy for acute myelogenous leukemia in first remission. J Clin Oncol 1992;10:41–46. 85. Robinson N, Sanders JE, Benyunes MC, et al. Phase I trial of interleukin-2 after unmodified HLA-matched sibling bone marrow transplantation for children with acute leukemia. Blood 1996;87:1249–1254. 86. Margolin KA, Van Besien K, Wright C, et al. Interleukin-2-activated autologous bone marrow and peripheral blood stem cells in the treatment of acute leukemia and lymphoma. Biol Blood Marrow Transplant 1999;5:36–45. 87. Schiller G, Wong S, Lowe T, et al. Transplantation of IL-2 mobilized autologous peripheral blood progenitor cells for adults with acute myelogenous leukemia in first remission. Leukemia 2001;15:757–763. 88. Blaise D, Attal M, Reiffers J, et al. Randomized study of recombinant interleukin-2 after autologous bone marrow transplantation for acute leukemia in first complete remission. Eur Cytokine Netw 2000;11:91–98.

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Antisense Therapy Stanley R. Frankel, MD, FACP CONTENTS INTRODUCTION FUNDAMENTAL COMPONENTS OF ANTISENSE OLIGONUCLEOTIDES TARGETS FOR ANTISENSE THERAPY CONCLUSIONS REFERENCES

1. INTRODUCTION The goal of 21st-century leukemia treatment is to discover and validate targets for new active and specific therapies. Improved understanding of the molecular biology of leukemia has identified a series of target genes whose protein products are integral to the pathogenesis of leukemia. Much of this insight has initially been garnered from cytogenetic observations and clinical correlations. These sentinel discoveries have been bolstered by the availability of genomic and proteomic screening. Most of the conventional antileukemic agents have nonspecific cytotoxic effects on deoxyribonucleic acid (DNA) polymerase or protein synthesis. Nucleic acids can be designed to target specific genes. Antisense oligonucleotides are 15–25 bases (mers) of DNA designed to target by Watson-Crick base pairing a unique sequence of messenger ribonucleic acid (mRNA) and lead to degradation of that specific message to eradicate the production of the coded protein. Although other strategies using nucleic acids are being tested (1), the use of antisense oligonucleotides has progressed to the clinic and is the focus of this chapter. Although preclinical studies have identified multiple potential targets, bcl-2 has emerged as the predominant target for advanced-stage large clinical leukemia studies. This chapter briefly reviews the basic components of antiFrom: Biologic Therapy of Leukemia Edited by: M. Kalaycio © Humana Press Inc., Totowa, NJ

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sense oligonucleotide therapy and then focuses on preclinical and clinical leukemia studies.

2. FUNDAMENTAL COMPONENTS OF ANTISENSE OLIGONUCLEOTIDES During the last two decades, advances in chemical formulation and gene sequencing have made available antisense reagents for clinical use. The efficacy of such compounds depends on their ability to enter leukemic cells and to effectively target the mRNA of interest and on the clinical effect of reduction of the target protein. Antisense therapy involves the administration of synthetic oligonucleotides that are complementary to specific mRNA transcripts. Antisense targets the mRNA of interest by Watson-Crick base pairing, thereby providing specificity and avidity. Antisense oligonucleotides must be incorporated into cells to be effective. Although they may be active at nanomolar to micromolar concentrations, uptake varies with the cell type. Intracellular uptake occurs by pinocytosis. Inside the cell, the antisense oligodeoxynucleotide hybridizes to the specific molecule target mRNA to form a DNA/RNA duplex. Ribonuclease H (RNase H) recognizes the DNA/RNA duplex, cleaves the mRNA strand, and renders the message nontranslatable. The specific targeted mRNA fragments are subsequently destroyed by ribonucleases. As a consequence, levels of the target protein are diminished, dependent on the effectiveness of the antisense and the half-life of the mRNA and protein.

2.1. Antisense Classes Antisense compounds may differ in their size, ionic charge, and structural relationship to the natural nucleic acid target. Antisense molecules are generally ionically charged and relatively large. First-generation modifications of the oligo to stabilize them against nuclease digestion of the phosphodiester linkage included phosphoramidates, methyl phosphonates, phosphorothioates, phosphorodiamidate morpholinos, and α-oligonucleotides. The most common backbone replaces one of the oxygen atoms in the phosphodiester linkage with a sulfur atom to form a phosphorothioate. (1,2) Phosphorothioate (PS) oligonucleotides (oligos) contain at least one of the nonbridging oxygens of the internucleotide phosphodiester linkages replaced with sulfur. PS oligos inhibit gene expression by hybridization arrest (i.e., interference with the processing of mRNA by hybridization), followed by cleavage of the mRNA by RNase-H. These oligos are polyanionic and, as a result, can bind to several factors that may produce nonspecific effects. Critical to their function is resistance to nuclease digestion.

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2.2. Mechanism of Antisense Action Antisense oligos must reach the target cell and remain in the cell at a concentration sufficient to trigger their biologic effects. Intracellular uptake of oligos is dependent on physical variable, time, and concentration. At concentrations below 1µ mol/L, uptake of PS oligodeoxynucleotides (ODN) is predominantly via a receptor-like mechanism, while at higher concentrations a fluid-phase endocytosis mechanism predominates (3). ODN have been reported to be endocytosed via clathrin-coated pits on the cytoplasmic membrane. Several classes of receptor-like binding proteins have also been described (3). The intracellular oligos may escape the endosome/lysosome compartment and then migrate rapidly into the nucleus. The mechanism is one of diffusion, with subsequent trapping of the oligo (4). At the intracellular target, the oligo specifically binds to the sequence of interest by Watson-Crick base pairing. Affinity must be sufficient so that the target mRNA is inactivated. This inactivation can be due to either sequestration of the message or, more likely, by cleavage of the mRNA when in the heteroduplex configuration with the antisense oligo. A ubiquitous enzyme, RNase H, cleaves only the RNA strand and leaves the antisense oligo intact to catalytically bind to another strand of specific mRNA. Other mechanisms may also play a role in the antisense effect (5).

3. TARGETS FOR ANTISENSE THERAPY Antisense design requires a known sequence of a target mRNA. Initial cytogenetic observations of translocations in leukemia directed early efforts at gene mapping and sequence identification. Genes located at translocation breakpoints were believed to play a role in the pathogenesis of the disease and became reasonable therapeutic targets. As signaling pathways are unraveled in leukemia, additional targets for therapy with antisense approaches emerge. If the protein encoded by the target gene (such as Bcl-2) is important in tumor cell survival, progression, or resistance to treatment, then antisense administration may be beneficial.

3.1. BCR-ABL The seminal discovery of the Philadelphia (Ph) chromosome by Nowell and Hungerford offered the first rational target for antisense therapy. The subsequent discovery, cloning, and sequencing of BCR-ABL that is the sine qua non for chronic myelogenous leukemia (CML) was an obviously attractive target for antisense development. Antisense therapy directed against the bcr-abl fusion product or the individual components has been investigated both ex vivo and in vivo (5–8). There have been conflicting reports regarding the efficacy and specificity of these

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approaches, and none has led to broad clinical investigation (1). The in vitro results have been provocative, with early exploration of ex vivo purging strategies for autologous transplantation (9–11). There is some modulation of adherence and signaling in response to antisense treatment, which may be modulated by the use of α interferon (12,13). However, the effects of the oligos may not be sequence specific (14). The design of antisense directed against bcr-abl has been considerably more problematic than was initially assumed (8). In addition to drug delivery and targeting, the relatively long half-life of the fusion protein may limit the potential benefit of this approach (15).

3.2. C-myb Despite the initial attraction of a translocation-specific sequence, proteins that played a more central role in neoplastic cell survival and growth based on their overexpression were also rational targets for antisense therapy. MYB protein is a nuclear-binding protein that controls the G1/S transition. C-myb also functions as a transactivator of other cellular control genes (16,17). There may be relatively increased sensitivity to downregulation of myb in leukemic cells. Myb’s ability to transactivate genes required for cell proliferation and growth underlies its importance for normal hematopoietic cell development and suggests that perturbation of its function may play a role in leukemogenesis (18,19). A modest survival benefit was shown when c-myb PS antisense ODN were tested in a human leukemia (K562) SCID mouse model (20). These chimeric mice were treated with PS-modified antisense ODNs for c-myb. Animals treated with antisense c-myb oligos survived 3.5-fold longer than control animals. There was a decreased incidence of central nervous system (CNS) and ovarian infiltration in the antisense treated animals. Although it may be difficult to deliver antisense therapy in vivo, this approach may be feasible for ex vivo purging of bone marrow. Constant infusion of even low doses of antisense ODNs was useful. Suppression of the target gene was clearly demonstrated in a melanoma model (21). A phase I trial of the c-myb PS at doses up to 2 mg/kg/d was tolerable, with some stabilization of a patient with blast-crisis CML. This trial was terminated prematurely because the drug was not available (22). Because of the difficulty and expense of synthesizing large quantities of clinical material, an initial approach was to use the agents in vitro to purge autologous marrow of tumor cells in CML (23). Allograft-ineligible patients with CML had CD34+ marrow cells purged with ODN for either 24 (n = 19) or 72 (n = 5) h. After purging, Myb mRNA levels declined substantially in approximately 50% of patients. Analysis of bcr/abl expression in long-term culture-initiating cells suggested that purging had been accomplished at a primitive cell level in more than 50% of patients and was ODN dependent. Day-100 cytogenetics were evaluated in 13 surviving patients who engrafted and had evaluable metaphases. There were two complete cytogenetic remis-

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sions and three major cytogenetic responses, and eight patients remained 100% Ph-positive.

3.3. c-myc Functional c-myc may be required for the development of the CML phenotype after bcr-abl activation. Antisense to c-myc had an antiproliferative effect against K562 and primary CML-blast crisis cells in a SCID mouse model, although there was a marked synergistic antiproliferative effect when antisense to bcr-abl was also administered (24). A PS antisense to c-myc suppressed growth of HL-60 cells, likely by an apoptotic response. However, c-myc expression rebounded at the end of exposure (25). Although phosphoroamidate constructs were more effective than a PS oligos in vitro, there was therapeutic equivalency in vitro (26). A 15 mer targeted against the translation initiation codon site of c-myc was not active in vitro unless placed in a fusogenic lysosome (26). This compound inhibited 70% of HL-60 growth at a concentration of 2.5 µm. A triple helix-forming oligo with a PS backbone targeting c-myc P2 promoter has also been shown to have sequence specific activity. CEM leukemia cells accumulated in S-phase, and apoptosis was induced (27).

3.4. p53 Another target for modulation was p53. A 20-mer PS was used. Sixteen patients with either refractory acute myeloid leukemia (AML) (n = 6) or advanced myelodysplastic syndrome (MDS) (n = 10) were treated with 1.2–6 mg/kg/day for 10 d by continuous intravenous (iv) infusion. Although leukemic cell growth in vitro was inhibited when compared with pretreatment samples, there were no clinical complete responses (28). A purging strategy was also initiated. Bone marrow cells were incubated with 10 µm OL(1)p53 for 36 h before transplantation. Although safety was established based on reasonable time to platelet and neutrophil engraftment, no efficacy correlates were reported (29).

3.5. bcl-2 3.5.1. BCL-2: ANTI-APOPTOTIC MECHANISMS Much of the understanding concerning the molecular regulation of programmed cell death originates with the B-cell lymphoma/leukemia associated gene 2 (bcl-2) gene family. The best characterized members of this family, Bcl2 and Bax, act by differential homodimerization and heterodimerization. Bcl-2 homodimers act as repressors of apoptosis, whereas Bcl-2/Bax heterodimers act as promoters (30). These effects are more dependent on the balance between Bcl-2 and Bax than on Bcl-2 quantity alone (31). During the last 5 yr, many new members of the Bcl-2 family have been characterized, including the death promoters BCL-xS (34), BAD (35), BIK (34), BAK (35), and the apoptosis suppressors BCL-xL (32) and BFL-1 (36).

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BCL-2 was originally identified because of its association with the t(13,17) chromosomal translocation present in most follicular B-cell non-Hodgkin’s lymphomas (37,38). With this translocation, the Bcl-2 gene is moved from its normal chromosomal location at 18q21 into juxtaposition with the immunoglobulin heavy chain (IgH) locus at 14q32, (resulting in deregulation of the Bcl-2/IgH fusion gene. As a result, Bcl-2 protein is overexpressed. Bcl-2 protein either forms selective membrane pores or more likely controls pre-existing permeability transition pores (37). Bcl-2 is able to stabilize the mitochondrial transmembrane potential (39) and antagonize Bax-induced release of cytochrome c into the cytoplasm (40). Immunoelectron microscopy has shown the association of Bcl-2 with the mitochondrial membrane (41). Functional analysis has shown that Bcl-2 acts upstream of caspases, preventing their activation (42). Moreover, earlier studies on mitochondria showed higher transmembrane potential in tumor cells relative to normal cells (43). Bcl-2 can inhibit the nuclear import of wt p53 after DNA damage (44). Cancers are characterized by dysregulation of proliferation, differentiation, and balance between cell survival and cell death, with the balance typically shifted toward prolonged survival. Such survival plays a major role in the resistance to cytotoxic agents exhibited by these refractory leukemias and is related to abrogation of apoptosis pathways that would normally be activated by key antileukemic drugs, including ara-C and anthracyclines (45,46). One approach to augment net drug cytotoxicity relates to modulation of the pathways, culminating in apoptosis. Apoptosis is a normal physiologic process by which cellular life span is controlled and specific cells are programmed for elimination. Activation of apoptosis pathways may serve as a common final pathway for many cytotoxic agents, irrespective of the primary mechanism of action. In fact, induction of apoptosis in response to DNA strand breakage may at least in part explain the cytotoxic activity of cell cycle-active antileukemic nucleoside drugs (ara-C) against noncycling cells, in addition to the cycling cell population (47). The induction of DNA strand breaks and thus the activation of apoptosis pathways is likely to be a common mechanistic motif for drugs that interact with topoisomerases, including anthracyclines and epipodophyllotoxins. Taking these distinctive findings together, it is apparent that BCL-2 and its family members are convergence points for diverse determinants of normal and malignant hematopoietic cell survival. As such, Bcl-2 presents a pivotal molecular target for therapy of the acute leukemias. The ability to downregulate net Bcl-2 activity could translate into an increase in the antileukemic activity of agents that exert at least some of their cytotoxic effects by inducing apoptosis. 3.5.2. BCL-2 EXPRESSION IN LEUKEMIA Bcl-2 is upregulated in chronic lymphocytic leukemia (CLL) and other major tumor types and is believed to be responsible for maintaining the viabil-

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ity of cancerous cells and contributing to chemotherapy and radiotherapy resistance (38,49–51). 3.5.2.1. BCL-2 Expression and Dysregulation in CLL. B-cell CLL (BCLL) represents a neoplastic disorder caused primarily by defective programmed cell death (PCD) as opposed to increased cell proliferation (52). Overexpression of Bcl-2 in CLL allows for accumulation of CLL cells in GO (53,54). There is an accumulation of long-lived neoplastic B cells expressing Bcl-2 protein. Defects in the PCD pathway also contribute to chemoresistance (53). Expression of a stable amount of Bcl-2 protein blocks apoptosis and prolongs tumor survival. Bcl-2 is universally expressed in CLL cells (55). Increased Bcl-2 protein expression is observed in CLL cells (56). Relative levels of Bcl-2 oncoprotein represent one of the key determinants of the sensitivity of lymphocytic cells to killing by essentially all drugs currently available for the treatment of cancer (57,58). Studies in CLL cells demonstrate a correlation between drug-induced apoptosis and the ratio of endogenous levels of Bcl2 to Bax proteins (59,60). Similarly, the Bcl-2/Bax ratio has been correlated with disease status and is elevated in patients with progressive disease (61) or resistant disease (62). Higher Bcl-2 expression levels were found more often in patients with progressive vs stable CLL (63). By immunoblot analysis, 80% of patients at stage C (p = 0.019) expressed high levels of Bcl2 (64). The clinical relevance of Bcl-2 as a target for CLL therapy has been validated by the finding that high levels of expression of Bcl-2 is an adverse feature for survival in previously untreated patients with CLL (65). Several lymphokines and growth factors have also been reported to upregulate Bcl-2 expression in CLL, suggesting that this increase in Bcl-2 expression may play a role in the delay of fludarabine-induced apoptosis (66). In a multivariate analysis based on measurement of levels of expression of apoptotic control proteins, Bcl-2 levels emerge as the most important protein in predicting survival (67). Reduction of Bcl-2 expression by antisense therapy sensitized cells to chemotherapy-induced apoptosis (60,68,71) supporting study of this approach in vivo. 3.5.2.2. BCL-2 Expression and Dysregulation in Acute Leukemia. In recent clinical studies, abnormal Bcl-2 expression was proven to be predictive of poor response to treatment and adverse clinical outcome in patients with AML, although the data for acute lymphoblastic leukemia are not as robust (69–81). Bcl-2 levels may be higher in relapsed AML than at initial diagnosis (82). Most investigators have shown that the Bcl-2 expression is higher in the CD34+ population of AML blasts (83,84). However, further subset analysis has attributed this to the CD13+CD33+ subsets of CD34 cells and did not show a correlation between bcl-2 expression and prognosis that was independent of

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cytogenetic group (85). As more complicated analyses have been performed, the significance of bcl-2 levels has been questioned (86). Possibly, the Bcl2/Bax ratio might be more important, or in some studies, the bax level alone (87–91). Small samples of pediatric AML cases have not shown a correlation between Bcl-2 expression and outcome (92). The persistence of bcl-2 bearing blasts after chemotherapy treatment suggests an association of Bcl-2 expression with multidrug resistance (93). Interactions with wt1 have also been reported (79). The expression of Bcl-2 protein by flow cytometry correlated with that of CD34 and P170 and with the percentage of blast cells in patients with MDS. Two-color analyses demonstrated that CD34 and Bcl-2 were usually expressed in the same cells. No significant correlation was found with cytogenetic abnormalities. Expression of anti-apoptotic proteins was associated with decreased survival. Consequently, Bcl-2 proteins expression was well correlated with the International Prognostic Scoring System (IPSS) (94). 3.5.3. G3139 THERAPY IN CANCER G3139 (Genasense®, oblimersen sodium, Genta Inc, Berkeley Heights, NJ) is an 18-mer PS antisense ODN directed against the first six codons of the open reading frame of Bcl-2. G3139 studies in xenograft models have shown marked enhancement of the efficacy of standard cytotoxic chemotherapy and rituximab in several cancers, including non-Hodgkin’s lymphoma, melanoma, breast cancer, gastric cancer, and nonsmall-cell lung cancer (95). G3139 clinical activity has been reported in lymphoma, melanoma, and prostate cancer (96–98). In animals, after iv or subcutaneous (sc) injection, G3139 distributes rapidly to highly perfused organs, especially lung and bone marrow. Oligos are generally excreted unchanged, predominantly by the kidney (99). G3139 biodistribution studies in vivo have demonstrated high tissue/plasma ratios, particularly in the kidney and liver, but also significant distribution to the bone marrow and spleen (99). In addition, in vitro and in vivo studies showed both biologic and antitumor activity with submicromolar concentrations (e.g., ~ 170 mM). The first human study of G3139 was performed in patients with nonHodgkin’s lympohoma. Twenty-one patients received G3139 administered by continuous sc infusion (96,100). Thrombocytopenia, infusion site reactions, and fatigue were dose limiting in 2 patients treated at a level of 5.3 mg/kg/day. However, the tolerance to treatment in this study may have been closely linked to the prolonged (2-wk) infusion schedule given by the sc route, and other studies have easily escalated the Genasense doses to 7 mg/kg/d even when given intravenously in combination with cytotoxic chemotherapy (101). Although the administered drug dose was quite low in most patients, i.e., substantially below doses now known to be both safe and optimally effective with respect to Bcl-2 downregulation, several major responses were observed.

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3.5.1.1. G3139 in CLL. Reduction of Bcl-2 expression by antisense therapy sensitized cells to chemotherapy-induced apoptosis (60,68), supporting study of this approach in vivo. In vitro, CLL cells are triggered to undergo apoptosis more vigorously when treated with Bcl-2 antisense than they are by fludarabine, corticosteroids, or rituximab (102). In addition, fresh CLL cells obtained from patients and studied ex-vivo have shown downregulation of Bcl-2 protein after antisense exposure and increased killing by fludarabine (103). The enhanced antitumor activity of fludarabine in fresh CLL cells is consistent with the major enhancement of cytotoxicity by cytosine arabinoside and other antimetabolites (68). Treatment with antisense oligos with the same sequence as oblimersen sodium also decreased Bcl-2 levels in CLL cell lines and in cells taken from patients. In this study, antisense oligos directed against Bcl-2 protein also significantly increased killing of CLL cells when combined with fludarabine and enhanced the immunologic attack from lymphokine-activated killer (LAK) cells (103a). A phase I trial of G3139 in patients with refractory or relapsed CLL established that the maximum tolerated dose (MTD) for cycle 1 in CLL as monotherapy is 3 mg/kg/d, although patients could be safely escalated to 4 mg/kg/d in subsequent cycles. Tumor lysis and transient clinical benefit, including decreased circulating CLL cells, softening and shrinkage of lymph nodes, and splenomegaly reduction, were reported in some patients (104). Patients treated with either G3139 at 7 or 5 mg/kg/d experienced high fever, hypotension, and hypoglycemia. Back pain requiring narcotics for pain control was also observed. This suggests that patients with lymphoma or CLL are more sensitive to the treatment side effects of oblimersen sodium compared with patients with solid tumors, most likely due to cytokine release from the target tumor cells (108). A 3-mg/kg/d dose appears to be well tolerated either as a single agent or combined with fludarabine and cyclophosphamide. A phase II study of this dose level is ongoing in CLL patients. At least two patients have had partial responses to G3139 monotherapy (104a). A randomized phase III trial of fludarabine/cyclophosphamide plus G3139 is underway in patients with refractory or relapsed CLL who have previously received fludarabine therapy. Patients receive fludarabine 25 mg/m2/d with cyclophosphamide 250 mg/m2 for 3 d. Patients randomized to receive G3139 begin a 7-d infusion of 3 mg/kg/d for 4 d before administration of the cytotoxic agents. Cotrimoxazole, allopurinol, acyclovir, and filgrastim are used prophylactically. This trial tests the hypothesis that downregulation of Bcl-2 by G3139 will sensitize CLL cells to undergo apoptosis when treated with cytotoxic chemotherapy. 3.5.1.2. G3139 in AML. Downregulation of Bcl-2 may lower the apoptotic threshold and restore chemosensitivity in chemoresistant leukemic cells (105).

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In vitro experiments had suggested that antisense to Bcl-2 would be effective in the treatment of AML (106–110). Preclinical analysis using liposomal Bcl-2 antisense oligos in myeloid leukemia HL-60 cells, HL-60-doxorubicin-resistant cells, and CD34+ blast cells from patients with primary AML have established Bcl-2 as a critical target for antisense strategies in AML (110). The combination of G3139 and gemtuzumab ozogamicin has been explored in HL60 AML cells. HL-60 cells were cultured for 72 h with Mylotarg™ (Wyeth Laboratories, Philadelphia, PA) (1.25–10 ng/mL), with 20 µM G3139 using the streptolysin O transfection method, or with both. Mylotarg decreased the cell number in a dose-dependent manner (40%, 60%, and 71% growth inhibition at 1.25, 5, and 10 ng/mL). This growth-inhibitory effect was due to a pronounced G2M cell-cycle block arrest (more than 95% at 5 and 10 ng/mL) and modest induction of apoptosis as determined by Annexin V positivity and DNA fragmentation assay (10.2% and 15.9%, respectively, at 10 ng/mL). G3139 alone did not decrease cell numbers at 72 hrs under these conditions. Bcl-2 protein was downregulated by 50% at 72 h of G3139 treatment. However, in combination with Mylotarg, Genasense-enhanced cell death (Annexin V, 20.3% ([+]); sub-G1, 45.4 ([+]) cells) resulting in further decrease in cell number (69%, 75%, and 84% growth inhibition at 2.5, 5, and 10 ng/mL, respectively). The G2M cellcycle block was not affected by Bcl-2 AS. The number of cells with DNA fragmentation (sub G1) increased two- to threefold. These data demonstrated that G3139 downregulates Bcl-2 in AML cells and enhances Mylotarg-induced cell death by lowering the apoptotic threshold (111). At Ohio State University, a phase I/II study was initiated under sponsorship of the National Cancer Institute to evaluate a constant intravenous infusion dose of G3139 with escalating doses of fludarabine, Ara-C, and G-CSF (FLAG) for refractory or relapsed acute leukemia (112). In this dose-escalation study, G3139 (4 or 7 mg/kg/d in successive cohorts) is administered by continuous iv infusion on days 1–10, with fludarabine (starting at 15 mg/m2) and Ara-C (starting at 1000 mg/m2) given daily on days 6–10 and escalated in successive cohorts. Twenty patients were enrolled on this study (13 women and 7 men). The median age was 56 yr. Seventeen patients had AML, 5 with primary refractory disease, 8 in first relapse, and 4 in subsequent relapses. Three patients had ALL, 2 with refractory Philadelphia chromosome-positive disease, and one with t(5;14)(q31;q32) relapsed disease and hypereosinophilia. Of the 20 patients, 9 received high-dose cytarabine (HiDAC) with previous treatments, 1 had autologous stem cell transplantation, and 1 matched unrelated donor (MUD) stem cell transplantation. The median time to relapse from the initial treatment for relapsed patients was 7 mo (range 3 to 21 mo). The median number of previous treatments was two. Of the 20 patients, 9 (45%) had disease response, 6 (5 with AML, 1 with ALL) with complete remission and 3 (2 with AML and 1 with ALL) with no

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evidence of disease but failure to recover normal neutrophil and/or platelet counts, or to remain in remission for 30 d or more (incomplete remission). Two of the 20 patients were removed from protocol therapy due to rapidly rising blast counts before receiving chemotherapy. Median time for neutrophil recovery from start of chemotherapy (i.e., day 6) was 23 d (range 8 to 38 d); median time for platelet recovery (≥ 50,000) was 39 d (range 21 to 56 d). The pharmacokinetic behavior of G3139 was similar to previous reports in solid tumor patients. The mean Css level for the 4-mg/kg dose was 3.19 ± 1.29 µg/mL (range 1.59–5.69 µg/mL), which is significantly lower than the Css of 5.47 ± 2.16 µg/mL (range 2.67–8.38 µg/mL) for the 7-mg/kg dose (p = 0.023). When normalized to dose, the Css values were 0.78 ± 0.33 and 0.78 ± 0.30 µg/mL for the 4- and 7-mg/kg doses, respectively. These results indicated that the Css levels were proportional, and the pharmacokinetics linear for the two doses (i.e., 4 and 7 mg/kg) administered. The linearity in pharmacokinetics was also reflected by dose-dependent differences in area under the curve (AUC) values (p < 0.05) and similar total clearance (4.35 ± 1.85 L/h at 4 mg/kg and 3.89 ± 1.48 L/h at 7 mg/kg, p > 0.5). Bcl-2 mRNA levels were downregulated in 75% of evaluable patients. No unanticipated adverse events were noted. The safety data, coupled with the initial 50% response rate—including patients with refractory acute leukemia and previous high dose cytrabine (HDAC)—support further development of G3139 in combination regimens for refractory or relapsed leukemia. These results prompted initiation of an ongoing phase II trial of G3139 plus gemtuzumab ozogamicin in elderly patients with relapsed AML. Objectives are to determine the complete and overall response rates, duration of response, and safety in this population. G3139 has also been granted designation as an orphan drug from the Food and Drug Administration for the treatment of patients with AML. A pilot study of G3139 combined with daunorubicin and cytarabine in patients older than 60 yr of age with newly diagnosed AMLis underway with plans to proceed to a phase III trial. 3.5.1.3. G3139 in CML. G3139 has been evaluated in xenograft model for Philadelphia chromosome positive leukemia (113). Imatinib mesylate (STI571, Gleevec™, Novartis Pharma AG, Basel) is an inhibitor of the Abelson kinase constitutively activated in the BCR-ABL fusion gene product. In this study, nude mice were transplanted with imatinib mesylate-resistant BCRABL-transformed TF-1 cells and then treated with placebo (n = 5) or G3139 7 mg/kg/d ip for 14 d (n = 5). All of the untreated mice died within 10 wk, while the majority of mice treated with 3139 survived longer than 6 mo and demonstrated reduced tumor volume), with 3 of 5 G3139-treated mice demonstrating complete tumor regression. In addition, cells harvested from mice treated with

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G3139 (7 mg/kg/d for 7 d) were more sensitive to further treatment with imatinib mesylate, daunorubicin, cytarabine, or etoposide, with each combination showing additive or synergistic activity in the induction of apoptosis. When Bcl-2 was analyzed in the transformed cells, there was overexpression noted in the mitochondrial fraction. This is consistent with previous reports that BCRABL may induce Bcl-2 expression (114). These findings support the ongoing evaluation of G3139, both alone and in combination with other chemotherapeutic agents in the treatment of myeloid leukemias. The CALGB has recently initiated a trial of G3139 in patients with chronic phase CML treated with imatinib mesylate who have not had a complete hematologic response or major cytogenetic response.

4. CONCLUSIONS Antisense oligonucleotides have been used for more than a decade to downregulate gene expression. PS structures, such as G3139, have moved forward into clinical testing. These molecules successfully inhibit gene expression. Their clinical success will depend heavily on the appropriateness of the target. Preclinical activity and early clinical results suggest that antisense therapy using Bcl-2 as a target is highly promising with relatively minimal toxicity. Downregulation of Bcl-2 may greatly enhance the activity of other types of standard therapy: chemotherapy, monoclonal antibodies, or radiation therapy. Other targets will require further study as structure-activity relationships and drug delivery and stability are perfected.

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90. Kohler T, Leiblein S, Borchert S, et al. Absolute levels of MDR-1, MRP, and BCL-2 MRNA and tumor remission in acute leukemia. Adv Exp Med Biol 1999;457(10):177–185. 91. Kohler T, Schill C, Deininger MW, et al. High Bad and Bax mRNA expression correlate with negative outcome in acute myeloid leukemia (AML). Leukemia 2002;16(1):22–29. 92. Naumovski L, Martinovsky G, Wong C, et al. BCL-2 expression does not not correlate with patient outcome in pediatric acute myelogenous leukemia. Leuk Res 1998;22(1):81–87. 93. Ahmed N, Sammons J, Hassan HT. Bcl-2 protein in human myeloid leukaemia cells and its down-regulation during chemotherapy-induced apoptosis. Oncol Rep 1999;6(2):403–407. 94. Boudard D, Vasselon C, Bertheas MF, et al. Expression and prognostic significance of Bcl2 family proteins in myelodysplastic syndromes. Am J Hematol 2002;70(2):115–125. 95. Auer R, Corbo M, Fegan C, Frankel S, Cotter F. Bcl-2 antisense (Genasense™) induces apoptosis and potentiates activity of both cytotoxic chemotherapy and rituximab in primary CLL cells [abstract 3358]. Blood 2001;98(11):808a. 96. Waters JS, Webb A, Cunningham D, et al. Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin’s lymphoma [comment]. J Clin Oncol 2000;18(9):1812–1823. 97. de Bono J, Rowinsky E, Kuhn J, et al. Phase I pharmacokinetic (PK) and pharmacodynamic (PD) trial of bcl-2 antisense (Genasense) and Docetaxel (D) in hormone refractory prostate cancer. Proc Am Soc Clin Oncol 2001;20; Abstract 474. 98. Jansen B, Wacheck V, Heere-Ress E, et al. Chemosensitisation of malignant melanoma by BCL2 antisense therapy. Lancet 2000;356(9243):1728–1733. 99. Raynaud FI, Orr RM, Goddard PM, et al. Pharmacokinetics of G3139, a phosphorothioate oligodeoxynucleotide antisense to bcl-2, after intravenous administration or continuous subcutaneous infusion to mice. J Pharmacol Exp Ther 1997;281(1):420–427. 100. Webb A, Cunningham D, Cotter F, et al. BCL-2 antisense therapy in patients with nonHodgkin lymphoma. Lancet 1997;349(9059):1137–1141. 101. Klasa RJ, Gillum AM, Klem RE, Frankel SR. Oblimersen Bcl-2 antisense: facilitating apoptosis in anticancer treatment. Antisense Nucleic Acid Drug Dev 2002;12(3):193–213. 102. Czuczman MS, Fallon A, Mohr A, et al. Phase II Study of rituximab plus fludarabine in patients (pts) with low-grade lymphoma (LGL): final report [abstract]. Blood 2001;98:601a. 103. Vu UE, Dickinson J, Wang P, et al. Differentially expressed genes in B-chronic lymphocytic leukemia: evidence that manipulation of bcl-2 gene leads to increased killing of CLL cells [abstract]. Blood 2000;96:714a 103a. Vu UE, Pavletic ZS, Wang X, Joshi SS. Increased cytotoxicity against B-chronic lymphocytic leukemia by cellular manipulations: potentials for therapeutic use. Leuk Lymphoma 2000;39(5-6):573–582. 104. O’Brien S, Giles F, Kanti R, et al. Bcl-2 antisense (Genasense) as monotherapy for refractory chronic lymphocytic leukemia. Blood 2001;98(11):772a. 104a. Rai KR, O’Brien S, Cunningham C, et al. Genasense (Bcl-2 Antisense) Montherapy in patients with relapsed or refractory chronic lymphocytic leukemia: Phase 1 and 2 results. Blood 2002;100[11],384a. 105. Cotter FE. Antisense therapy of hematologic malignancies. Semin Hematol 1999;36(4 Suppl 6):9–14. 106. Campos L, Sabido O, Rouault JP, Guyotat D. Effects of BCL-2 antisense oligodeoxynucleotides on in vitro proliferation and survival of normal marrow progenitors and leukemic cells. Blood 1994;84(2):595–600. 107. Lin Y, Lu L, Hu J. [Inhibition of Bcl-2 expression in HL60 cells by incubation with antisense phosphorothioate oligodeoxynucleotides]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 1999;16(1):12–15. 108. Reed JC, Stein C, Subasinghe C, et al. Antisense-mediated inhibition of BCL2 protooncogene expression and leukemic cell growth and survival: comparisons of phosphodiester and phosphorothioate oligodeoxynucleotides. Cancer Res 1990;50(20):6565–6570.

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109. Keith FJ, Bradbury DA, Zhu YM, Russell NH. Inhibition of bcl-2 with antisense oligonucleotides induces apoptosis and increases the sensitivity of AML blasts to Ara-C. Leukemia 1995;9(1):131–138. 110. Konopleva M, Tari AM, Estrov Z, et al. Liposomal Bcl-2 antisense oligonucleotides enhance proliferation, sensitize acute myeloid leukemia to cytosine-arabinoside, and induce apoptosis independent of other antiapoptotic proteins. Blood 2000;95(12):3929–3938. 111. Khodadoust M, Konopleva M, Leysath C, Frankel S, Giles F, Andreeff M. Bcl-2 antisense oligodeoxynucleotide (Genasense) enhances gemtuzumab ozogamicin (Mylotarg)-induced cytotoxicity in acute myeloid leukemia. Blood 2001;98(11):102a [Abstract 429]. 112. Marcucci G, Byrd JC, Dai G, et al. Phase I and pharmacodynamic studies of G3139, a bcl2 antisense oligonucleotide, in combination with chemotherapy in refractory or relapsed acute leukemia. Blood 2002;2002–2006. 113. Tauchi T, Nakajima A, Sumi M, Shimamoto T, Sashida G, Ohyashiki K. G3139 (Bcl-2 antisense oligonucleotide) is active against Gleevec-resistant bcr-abl-positive cells. Proc Am Assoc Cancer Res 2002;43:abstract 4702. 114. Sanchez-Garcia I, Grutz G. Tumorigenic activity of the BCR-ABL oncogene is mediated by BCL2. Proc Natl Acad Sci USA 1995;92(12):5287–5291.

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Signal Transduction Inhibitors Michael E. O’Dwyer, MD and Brian J. Druker, MD CONTENTS INTRODUCTION IDENTIFYING THE TARGETS VALIDATING THE TARGETS HITTING THE TARGET TRANSLATING THE SUCCESS OF STI571 TO OTHER MOLECULAR TARGETS REFERENCES

1. INTRODUCTION The development of the first successful leukemia treatments owed much more to empiric observation than rational drug design in an era when the biology of leukemia was poorly understood. Although cytotoxic chemotherapeutic drugs have played and continue to play an essential role in cancer management, their relative lack of specificity, numerous toxicities, and frequency of resistance have limited this approach. Recent efforts have focused on identifying the biologic basis of leukemia, expecting that agents that more precisely target leukemia can be developed to maximize responses while minimizing toxicity. This approach requires the identification of appropriate targets, and the necessary tools to undertake this process have only recently become available. Although any protein in a leukemic cell could be considered a target, this chapter focuses on molecular pathogenetic targets; that is, oncogene products that are clearly responsible for the molecular pathogenesis of the disease. This includes proteins that are mutated or aberrantly expressed, with that event being critical to the malignant process. Advances in the fields of cytogenetics, molecular genetics, and biochemistry during the past 50 yr have greatly From: Biologic Therapy of Leukemia Edited by: M. Kalaycio © Humana Press Inc., Totowa, NJ

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advanced the understanding of leukemia pathogenesis and helped to identify candidate targets. We now know that oncogenic activation can result from several different mechanisms, including chromosomal translocations, activating mutations, loss of function mutations, and deletions. This chapter shows how the application of this knowledge has aided in the development of rational molecularly based treatment approaches, particularly the BCR-ABL tyrosine kinase inhibitor, STI571 (Gleevec™, imatinib mesylate, Novartis, Basel, Switzerland).

2. IDENTIFYING THE TARGETS 2.1. Chromosomal Translocations The identification of the Philadelphia (Ph) chromosome as the first consistent chromosomal abnormality in a human malignancy and the subsequent discovery that many types of leukemia and lymphoma are associated with recurrent chromosomal translocations suggested that these chromosomal abnormalities had a role in the pathogenesis of these diseases (1). Numerous recurrent chromosomal translocations have since been identified, and the molecular consequences of some of these are now well characterized (2). Chromosomal translocations result in cellular transformation by one of two principal mechanisms. In the first, juxtaposition to a transcriptionally active region of the genome leads to increased expression of the target gene. This is commonly seen in lymphoid malignancies, in which target genes are translocated to the immunoglobulin or T-cell receptor loci in the case of B- and T-cell malignancies, respectively. In follicular lymphomas, the t(14;18) leads to increased expression of the anti-apoptotic protein Bcl-2 as a result of the juxtaposition of the bcl-2 gene on chromosome 18 with the immunoglobulin heavy chain locus on chromosome 14 (3,4). A similar mechanism operates in both Burkitt’s lymphoma as a consequence of t(8;14) and mantle cell lymphoma as a consequence of t(11;14), with overexpression of the c-myc and cyclin D1 genes, respectively (5–7). The second mechanism, which is more common in leukemia, results in the generation of chimeric oncogenes, which alter the phenotype of the affected cell. The production of chimeric oncogenes involving transcription factors can disrupt the normal function of these factors, leading to altered cellular differentiation. In acute promyelocytic leukemia (APL) associated with the t(15;17) translocation, the normal response of the retinoic acid receptor-α (RAR-α) to its ligand, retinoic acid (RA), is disrupted by the fusion protein PML-RAR-α, resulting in a blockade in myeloid differentiation. However, pharmacologic doses of RA (1 M) are capable of relieving this repression, restoring normal cellular differentiation (8–11). RA therapy may also lead to degradation of PMLRAR-α by a proteolytic pathway, contributing to its ability to restore normal

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differentiation (12). The therapeutic efficacy of RA was first demonstrated in a study from China that showed that all-trans retinoic acid (ATRA) produced differentiation and remission in patients with APL with less toxicity than conventional chemotherapy (13). This was the first example of a small molecule successfully targeting the underlying molecular lesion in a form of leukemia. However, this was an empiric discovery, because the molecular basis of APL had not yet been elucidated. Further experience with ATRA in APL showed that the majority of patients relapsed if treated with ATRA alone. Subsequent clinical trials have now refined the treatment of this disease, and using a combination of chemotherapy and ATRA, high rates of durable remission are seen. Disruption of the transcription factor known as core binding factor (CBF) occurs in several different types of leukemia. CBF is a heterodimeric protein consisting of acute myeloid leukemia 1 (AML1; also known as CBFa2), the deoxyribonucleic acid (DNA)-binding component, and CBFβ, which binds to AML1 and stabilizes its binding to DNA (14). Twelve percent of cases of acute myeloid leukemia (AML) have a translocation between chromosomes 8 and 21, t(8;21) (15). This translocation generates a dominant negative AML1/ETO fusion protein, which interferes with the normal trans-activating function of AML1 (16). The AML1 protein regulates the expression of genes crucial in normal hematopoietic development, differentiation, and function, including the genes for myeloperoxidase, neutrophil elastase, interleukin (IL) 3 and GM-CSF. The ETO fusion partner binds to the nuclear receptor corepressor complex (NCoR), which then recruits Sin3 and histone deacetylase. This complex inhibits the expression of normal AML1-responsive genes, disrupting hematopoiesis (17). A related form of AML associated with inv(16) also interferes with normal AML1-dependent transcriptional regulation. In this case, the fusion protein CBFβ-MYH11 functions as a dominant repressor, again recruiting a corepressor with histone deacetylase activity (18). CBFβ-MYH11 transcripts have been detected in up to 10% of patients with newly diagnosed AML (19). Finally, the most frequent chromosomal translocation seen in childhood acute lymphoblastic leukemia (ALL), t(12;21), accounting for approx 25% of cases of common ALL, results in the generation of a TEL-AML1 chimeric protein. TEL-AML1 contributes to leukemogenesis through the recruitment of a nuclear corepressor complex with histone deacetylase activity (20). Clearly, the AML1 transcription factor would be an ideal target for an ATRA-like agent that restored normal transcriptional activity to this protein. In the absence of such a specific agent, a shared theme between t(8;21), inv(16), and t(12;21), as well as t(15;17) in APL, is that of transcriptional repression related to excessive histone deacetylase activity. Thus, histone deacetylase inhibitors might be expected to be a general class of agents that could find use in these leukemias. Clinical trials of such agents, including sodium phenylbutyrate and trichostatin A, among others, are in progress.

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Another common consequence of translocation events is activation of a tyrosine kinase. In translocations involving genes encoding tyrosine kinases, the partner gene usually encodes a dimerization motif, which leads to constitutive activation of the tyrosine kinase. Chronic myelogenous leukemia (CML) is the classic example. The Ph chromosome, a shortened chromosome 22, was the first consistent chromosomal abnormality identified in a human malignancy (1). With the development of improved chromosomal-banding techniques in the early 1970s, it became apparent that the Ph chromosome was the result of a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11) (21). The molecular consequences of this translocation were subsequently shown to be the juxtaposition of the c-Abl oncogene from chromosome 9 with sequences from chromosome 22, the breakpoint cluster region (BCR), giving rise to a fusion BCR-ABL gene (22). The size of the protein generated by the fusion gene varies depending on where the breakpoint occurs in the BCR region. A 210kDa fusion protein (p210) is seen in approx 95% of patients with CML and up to 20% of adult patients with ALL. A 185kDa fusion protein (p185) is seen in 10% of adults with ALL and is the predominant BCRABL fusion protein in Ph-positive pediatric patients with ALL, although only 5% of pediatric patients with ALL are Ph positive. The product of this fusion gene is a constitutively active tyrosine kinase with markedly enhanced enzymatic activity compared with the ABL kinase. This enhanced tyrosine kinase activity is critical for its transforming activity (23). Other examples of translocations involving genes encoding tyrosine kinases include t(5;12), producing a TEL-PDGFR fusion tyrosine kinase associated with some cases of chronic myelomonocytic leukemia (CMML), and t(2;5), producing a NPM-ALK fusion tyrosine kinase associated with T-cell anaplastic large-cell lymphoma (ALCL) (24,25).

2.2. Activating Mutations Activating mutations in cytokine receptors, specifically receptor tyrosine kinases, is being increasingly recognized as a means of cellular transformation in hematopoietic malignancies. Activating mutations of two members of the class III receptor tyrosine kinase family, c-kit and Flt3, have been documented in leukemic cells from patients with AML. An internal tandem duplication (ITD) of the juxtamembrane-coding region of the Flt3 gene on chromosome 13 has been reported in up to 30% of adult patients with AML and up to 16.5% of pediatric patients with AML (26–29). More recently, another 7% of patients with AML and 3% of patients with myelodysplasia had a point mutation in the activation loop of the Flt3 kinase domain (30). Consequently, mutant Flt3 receptors dimerize in the absence of ligand with constitutive tyrosine kinase activation, resulting in growth factor independence. Clinically, the presence of Flt3-ITD is associated with poor prognosis, with affected patients having lower

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rates of complete remission (CR) after induction chemotherapy and higher relapse rates. Activating mutations in c-kit are less common and occur predominantly in patients with CBF-related AML (31,32). These could be important “second-hits” in the development of these subtypes of leukemia (see following section) and are the targets of ongoing clinical trials with specific kinase inhibitors.

3. VALIDATING THE TARGETS Any cell protein that is involved in a growth or survival pathway could be considered a target for a therapeutic agent. Agents that target these proteins and pathways may be of use and would be expected to result in incremental advances in the treatment of cancer. However, to recapitulate the quantum improvements seen with agents such as ATRA and STI571, the target should be an oncogene that has clearly been shown to be responsible for malignant transformation. Rendering cell lines growth factor independent after retroviral expression of an oncogene is supportive evidence of a role for an oncogene in malignant transformation. However, the strongest evidence comes from animal models in which knock-in (translocations or activating mutations) or knockout (deletions or loss of function mutations) strategies can recapitulate the human disease. In the case of CML, experiments in transgenic mice and murine recipients of BCR-ABL transduced hematopoietic stem cells demonstrate that expression of BCR-ABL alone can induce leukemia (33,34). Similarly, retroviral insertion of the NPM-ALK gene into murine hematopoietic cells followed by transplantation into lethally irradiated recipients causes lymphoid malignancy in mice (35). Finally, transfection of the murine IL-3-dependent cell line, 32D, with a mutant Flt3 gene, results in growth factor independence, and transplantation of these cells into syngeneic mice leads to the rapid development of leukemia (36). Whether hematopoietic progenitors expressing a similar Flt3 mutant would rapidly develop leukemia must be determined. The situation may be more complex for other oncogenes. Transgenic mice expressing PML-RAR-α develop APL, although with relatively long latency (37). However, by adding the reciprocal translocation partner, RAR-α-PML, disease latency is significantly shortened. Experiments in mice expressing chimeric CBF proteins also indicate that these abnormalities alone may be insufficient to induce leukemia. When AML1 was replaced by AML1-ETO in mice using a knock-in strategy, a block in hematopoiesis was seen but mouse embryos died in midgestation due to development of severe central nervous system (CNS) hemorrhages (38). To overcome this embryonic lethality, subsequent experiments employed a model in which the AML1-ETO expression was inducible under the control of a tetracycline-responsive element. Despite high expression of AML1-ETO in the bone marrow cells of these mice, no leukemia

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was seen during 24 mo of observation, although a partial block of myeloid differentiation was seen (39). Although mice expressing AML1-ETO do not develop leukemia, they are at a much higher risk of developing leukemia after exposure to alkylating agents as compared with control mice (40). Similarly, mice expressing CBFβ-MYH11 did not develop leukemia but after exposure to low-dose alkylating agents, had a high rate of leukemic transformation, whereas no cases of leukemia were seen in similarly treated control mice (41). This suggests that secondary mutations cooperate with AML1-ETO to induce leukemia and that AML1-ETO–induced leukemia may be a multistep process. In the case of Burkitt’s lymphoma, transgenic mice overexpressing c-myc develop lymphomas (42). However, it is likely that other abnormalities cooperate with c-myc to induce lymphoma. Up to 80% of cases of Burkitt’s lymphoma exhibit mutations in the p53 tumor suppressor gene, and when wild-type p53 is expressed in a Burkitt’s lymphoma cell expressing a mutated form of p53, rapid cell death by apoptosis ensues (43). In fact, enforced expression of c-myc causes apoptosis as well as cellular proliferation (44,45). Therefore, it may only be with the development of secondary abnormalities that circumvent apoptosis, such as Bcl-2 overexpression or p53 mutations, that c-myc is capable of malignant transformation.

4. HITTING THE TARGET For maximal utility, the identification of crucial early events in malignant progression is the first step in the successful development of a targeted therapy. An equally important issue is the selection of patients for clinical trials based on the presence of the appropriate target. Finally, the normal cellular function of the target will determine the toxicity of a targeted agent, and an ideal target would be dispensable for normal cellular function. Currently, kinase inhibitors are easier to develop than agents that target transcription factors. This is in part due to kinases having well-defined hydrophobic ATP-binding pockets to which inhibitors can easily be targeted, as opposed to transcription factors that often have broad flat hydrophilic-binding surfaces that do not present useful structures for disrupting protein–protein interactions.

4.1. Tyrosine Kinase Inhibitors Because deregulated tyrosine kinase activity is involved in the pathogenesis and disease progression of CML, CMML associated with t(5;12), ALCL associated with t(2;5), and AML associated with ITDs of Flt3, these diseases are obvious choices for the development of specific signal transduction inhibitors. The following discussion focuses on the development of STI571 for the treatment of CML, because this serves as a paradigm for the development of similar therapies.

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4.1.1. CHRONIC MYELOGENOUS LEUKEMIA CML accounts for 20% of all cases of leukemia, with an annual incidence of 1 to 1.5 cases per 100,000. The median age of onset is approx 60 yr, but all age groups are affected (46). Three clinical phases are recognized: a chronic phase lasting 4–6 yr, an accelerated phase lasting 6–18 mo, and a blast phase lasting 3–6 mo. The chronic phase is characterized by a massive proliferation of maturing myeloid cells (white cells and frequently platelets), which, as the disease progresses, lose their capacity to differentiate until eventually the disease terminates in an acute leukemia, termed blast crisis. The accelerated phase is an intermediate phase characterized by increasing myeloid immaturity, systemic symptoms, and refractoriness to therapy. The only therapy known to cure CML is allogeneic stem cell transplantation, but because most patients are too old or lack suitable donors for this procedure, less than one third of patients are candidates for this treatment. The 5–10-yr survival after allogeneic stem cell transplantation is 65%, with significant procedural-related morbidity and mortality. Oral chemotherapy agents, such as hydroxyurea or busulfan, can control blood counts in most patients in chronic phase but do not delay the onset of blast crisis (47). Patients treated with interferon-α live an average of 2 yr longer than patients treated with chemotherapy (48). The best survival advantage is seen in those patients who achieve a major cytogenetic response (a reduction in the percentage of marrow metaphases containing the Ph chromosome to less than 35%), although this occurs in less than one third of patients. Moreover, as many as 20% of patients discontinue interferon-α therapy due to intolerable toxicity. The addition of subcutaneous ara-C to interferon-α has increased response rates but at the cost of increased toxicity (49). These shortcomings provided the impetus for the development of a more effective, less toxic therapy for CML. 4.1.2. BCR-ABL: THE IDEAL TARGET BCR-ABL affects numerous downstream signaling pathways, which leads to increased cellular proliferation, decreased adhesion, inhibition of apoptosis, and possibly genetic instability (50). However, because all of these events are dependent on the tyrosine kinase activity of the fusion protein, it is clear that inhibition of the enzymatic activity of BCR-ABL should be an effective treatment for CML because BCR-ABL is present in the majority of patients with CML, is the causative abnormality of the disease, and its kinase activity is essential for transformation. Moreover, because Abl knock-out mice are viable, it is likely that ABL kinase activity would be dispensable for normal cellular function, suggesting that an ABL kinase inhibitor would have a relatively selective effect on BCR-ABL-expressing cells (51). 4.1.3. DEVELOPING AN INHIBITOR OF THE BCR-ABL TYROSINE KINASE Tyrosine kinases, such as BCR-ABL, catalyze the transfer of phosphate from adenosine triphosphate (ATP) to selected tyrosine residues on substrate proteins.

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With their tyrosine residues in the phosphorylated form, substrate proteins assume conformational changes leading to association with other downstream effectors, propagating signal transduction. Tyrosine kinases have a vital role in cell growth, differentiation, and survival. Because all protein kinases use ATP as a phosphate donor and as there is a high degree of conservation among kinase domains, particularly in the ATP-binding sites, it was believed that inhibitors of ATP binding would lack sufficient target specificity to be clinically useful. This was the case with the first tyrosine kinase inhibitors identified, such as herbimycin-A, which were all natural plant derivatives. However, in 1988, Yaish et al. published a series of compounds known as tyrphostins that demonstrated that specific tyrosine kinase inhibitors could be developed (52). Near the same time, scientists at Ciba Geigy (now Novartis) were performing high throughout screens of chemical libraries searching for compounds with kinase inhibitory activity. They eventually identified a lead compound with kinase inhibitory of the 2-phenylaminopyrimidine class. Although of low potency and poor specificity, this lead compound served as a base from which a series of related compounds were synthesized. By analyzing the relationship between structure and activity, this series of compounds was optimized to inhibit several targets (53). One series of compounds, optimized against the platelet-derived growth factor receptor (PDGF-R) proved to be equally active against the ABL tyrosine kinase. STI571 (formerly CGP57148, now Gleevec; imatinib mesylate) emerged as the lead compound for clinical development based on its superior in vitro selectivity against CML cells and its drug-like properties, including pharmacokinetics and formulation properties (53). 4.1.3.1. Preclinical Studies. Experiments in our laboratory showed that STI571 was a potent and selective inhibitor of the ABL tyrosine kinases, including BCR-ABL (54). The concentration (IC50) of STI571 that resulted in a 50% reduction in substrate phosphorylation and cellular tyrosine phosphorylation induced by BCR-ABL was 0.025 µM and 0.25 µM, respectively. The only other tyrosine kinase found to be inhibited by STI571, besides ABL and the PDGF-R, was c-kit. STI571 specifically inhibited the proliferation of myeloid cell lines containing BCR-ABL. In addition, colony-forming assays from patients with CML showed a marked decrease (92–98%) in the number of BCR-ABL colonies formed with no inhibition of normal colony formation when grown in the presence of 1 µM STI571. Similar results were reported elsewhere (55). Long-term marrow culture experiments showed that prolonged exposure to STI571 produced a sustained inhibitory effect on CML progenitors, with little toxicity to normal progenitors (56). Subsequent experiments showed that p185- and p210-expressing cells were equally sensitive to STI571 (57,58). Dose-dependent inhibition of tumor growth was seen in BCR-ABLinoculated mice treated with STI571, but a daily dosing failed to eradicate the tumors (54). Gambacorti and colleagues subsequently showed that three

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times/day oral dosing of STI571 effectively eradicated BCR-ABL-containing tumors in nude mice (59). Because the half-life of STI571 in mice is approx 4 h, it seemed that continuous exposure to STI571 would be required for optimal antileukemic effects. 4.1.3.2. Clinical Trials of STI571 in CML. Based on the efficacy of STI571 in several preclinical models and an acceptable animal toxicology profile, a phase I clinical trial with STI571 started in June 1998. This was a doseescalation study designed to establish the maximum tolerated dose (MTD), with clinical efficacy being a secondary endpoint. Patients were enrolled in 14 successive dose cohorts ranging from 25 to 1000 mg of STI571. Patients were eligible if they were in the chronic phase of CML and had failed therapy with interferon-α. STI571 was administered as a once daily oral therapy, and no other cytoreductive agents were allowed. Once doses of 300 mg or greater were reached, 53 of 54 patients achieved a complete hematologic response (60). Responses were typically seen within the first 3 wk of therapy and have been maintained in 96% of patients, with a median follow-up of 310 d. At this dose level (≥ 300 mg), major cytogenetic responses were seen in 31% of patients, while 13% achieved a complete cytogenetic response. Side effects have been minimal, with no dose-limiting toxicities encountered. Grade 2 and 3 myelosuppression was observed at a dose ≥ 300 mg in 21% and 8% of patients, respectively. Myelosuppression is likely consistent with a therapeutic effect because the Ph-positive clone contributes the majority of hematopoiesis in these patients. Pharmacokinetic studies showed that the half-life of STI571 is 13–16 h, which is sufficient to permit once-daily dosing. Although the follow-up on this group of patients is relatively short (median 1 yr), these data indicate that an ABL-specific tyrosine kinase inhibitor has significant activity in CML, even in interferon refractory patients. Given the effectiveness of STI571 in patients in chronic phase who had failed interferon, the phase I studies were expanded to include patients with CML in myeloid and lymphoid blast crisis and patients with relapsed or refractory Ph chromosome-positive ALL. Patients have been treated with daily doses of 300 to 1000 mg of STI571. Twenty-one of 38 (55%) patients with myeloid blast crisis responded to therapy, defined by a decrease in percentage of marrow blasts to less than 15%. Eight of 38 (21%) patients had marrow blasts cleared to < 5% (61). Seven of 38 (18%) of the patients in myeloid blast crisis have remained in remission on STI571, with follow-up ranging from 101 to 349 d. Fourteen of 20 (70%) patients with lymphoid phenotype disease, CML in lymphoid blast crisis, or Ph-positive ALL responded, with 11 of 20 (55%) clearing their marrows to < 5% blasts. Unfortunately, all but one of the lymphoid phenotype patients relapsed between days 42 and 123. Thus, STI571 has remarkable single-agent activity in CML blast crisis and Ph-positive ALL, but responses tend not to be durable. However, these studies demonstrate that in the majority of cases, the

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leukemic clone in BCR-ABL–positive acute leukemias, including CML blast crisis, remains at least partially dependent on BCR-ABL kinase activity for survival. In late 1999, phase II studies were initiated to evaluate the safety and efficacy of STI571 in larger cohorts of patients. Patients in all phases of the disease (chronic phase [having failed interferon], accelerated, and blast crisis) were enrolled in studies at 27 institutions in six countries. Evaluation of response and pharmacokinetic data from the phase I study indicated that doses of 400 to 600 mg should be optimal for phase II testing (62). Between December 1999 and May 2000, 532 patients in chronic phase who were refractory to or intolerant of interferon-α were started on treatment with STI571 at a 400-mg daily dose. In recently updated results, after a median exposure of 18 mo, 60% and 41% of patients achieved major and complete cytogenetic responses, respectively. Only 8% of patients discontinued treatment due to disease progression, with only 2% of all patients stopping therapy due to adverse events (63). Of 235 treated patients in accelerated phase 51% achieved a complete hematologic response (CHR) with or without peripheral blood recovery (neutrophils > 1.0 × 109/L and platelets > 100 × 109/L). Eighteen percent achieved a complete cytogenetic response (64). Again, these results were achieved without substantial toxicity, though not surprising, up to 30% of patients experienced grade 3/4 myelosuppression in this study. Nevertheless, only 2% of patients developed febrile neutropenia. Of 260 treated patients in myeloid blast crisis 52% had some form of response, with 24% clearing their marrows to less than 5% blasts (65). Major and complete cytogenetic responses were seen in 15% and 7% of patients, respectively. Toxicity was comparable to that seen in the accelerated-phase study. Median survival was 6.9 mo, and 20% of patients are projected to be alive at 18 mo. Historically, patients treated with chemotherapy for myeloid blast crisis have had a median survival of approx 3 mo. 4.1.4. DOSE SELECTION From the dose-finding study, complete hematologic responses occurred in almost all patients in chronic phase treated at doses of 300 mg and higher and cytogenetic responses were seen once this dose level was reached. In addition, pharmacokinetic data showed that this dose level achieved in vivo concentrations approaching the predicted in vitro IC90 for cellular proliferation of 1 µM (62). Finally, an analysis of white blood and platelet count responses over time suggested that doses of 400 to 600 mg were on the plateau of a dose-response curve, indicating that this dose range would be an efficacious dose for phase II testing. However, in the dose-escalation study, an MTD of STI571 was never reached (60). Although traditional drug development uses dose escalation until an MTD is established, with molecularly targeted therapies, this may not be an appropriate endpoint. A more appropriate endpoint may be the dose that achieves molecular target inhibition. Therefore, in the case of STI571 and CML, an optimal dose

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should approximate that which achieves maximal BCR-ABL kinase inhibition. An analysis of BCR-ABL kinase inhibition, assaying for decreases in phosphorylation of the BCR-ABL substrate, Crkl, has suggested that a plateau in inhibition is seen with more than 250 mg (60). Additional experiments are being conducted to determine the percentage of kinase activity that is inhibited at these dose levels (66). CML lends itself to this kind of molecular monitoring because tumor cells are easily accessible and the kinase itself or its substrates can be monitored for inhibition. These types of assays are clearly more problematic for solid tumors, but are necessary to determine the penetration of these types of agents into solid tumors. In the absence of specific assays, even information about intracellular drug levels in tumor samples would be a useful surrogate. This type of data regarding maximal kinase inhibition could be particularly useful in explaining response variability and could also be useful in individualizing therapy. 4.1.5. FUTURE DIRECTIONS IN CML THERAPY The clinical data presented here demonstrate that STI571 is employed to optimum effect in CML when used early, before disease progression. An ongoing phase III randomized study is comparing STI571 with interferon and ara-C in newly diagnosed patients. The results of this study, when combined with more mature data from the phase II studies, will help to determine the place of STI571 in future CML treatment algorithms. It is tempting to speculate that as BCR-ABL may be the sole oncogenic abnormality driving proliferation in early stage disease, STI571 alone may be sufficient therapy in some patients with CML. However, as additional genetic abnormalities accumulate with disease progression, CML cells may no longer be solely dependent on BCR-ABL for survival. Thus, in blast crisis patients, therapy with STI571 alone is clearly insufficient for most patients. Here, the paradigm of APL and ATRA may be particularly instructive. It is unlikely that PML-RAR-α is the sole molecular abnormality that causes APL, but it is clearly one of the critical pathogenetic events. Targeting PML-RAR-α with ATRA yields a high response rate, but most patients relapse on single-agent therapy. However, combinations of ATRA with chemotherapy lead to high rate of cure. Similarly, in blast crisis, it is unlikely that BCR-ABL is the sole oncogenic abnormality, yet it remains critical to the survival of the leukemic clone. Thus, combinations of STI571 with other antileukemic agents for blast crisis patients could lead to a significantly improved prognosis. This paradigm is likely to apply to most leukemias and lymphomas where multistep disease pathogenesis is the rule. 4.1.6. THE PROBLEM OF RESISTANCE AND THE RATIONALE FOR COMBINATION THERAPY Despite the high initial response rates in patients in blast crisis many patients relapse and not all respond to STI571. One of the most useful catego-

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rizations of relapse/resistance mechanisms has been separation of patients with persistent inhibition of the BCR-ABL kinase and those with reactivation of the BCR-ABL kinase. Patients with persistent inhibition of the BCR-ABL kinase are predicted to have additional molecular abnormalities other than BCR-ABL driving the growth and survival of the malignant clone. In contrast, patients with persistent BCR-ABL kinase activity or reactivation of the kinase are believed to have resistance mechanisms that either prevent STI571 from reaching the target or render the target insensitive to BCR-ABL. In the former category are mechanisms such as drug efflux or protein binding of STI571. In the latter category would be mutations of the BCR-ABL kinase that render BCRABL insensitive to STI571 or amplification of the BCR-ABL protein (67–70). In patients who relapse after an initial response to STI571, the majority of these patients have reactivation of the BCR-ABL kinase (71). No changes in drug levels have been observed, and leukemic cells from these patients have decreased cellular sensitivity to STI571. This suggests that resistance is due to intrinsic cellular properties rather than protein binding of drug or drug metabolism. Interestingly, half of these patients have developed point mutations in the ABL kinase that render the kinase variably less sensitive to STI571 (71). At least one of the point mutations is at a site predicted to be a contact site between STI571 and the ABL kinase based on the crystal structure (71,72). Several others are at residues adjacent to contact points, whereas others are in the kinase activation loop (73,74). Finally, approximately one third of patients who relapse after an initial response have BCR-ABL amplification (71). BCR-ABL mutation and amplification have not been commonly seen in patients with de novo STI571 resistance, and ongoing studies are aimed at identifying mechanisms of resistance in these patients. To circumvent resistance, the combination of STI571 with other active antileukemic agents seems desirable. We have shown that inhibition of BCRABL by STI571 can reverse the intrinsic drug resistance seen in CML cells and that combinations with drugs such as daunorubicin, ara-C, and interferon-α are associated with additive or even synergistic effects in vitro, providing a strong rationale for combination studies (75). Similar studies have been performed with etoposide and ara-C (76). Combination studies with low-dose interferon and ara-C are currently underway for patients in chronic phase, while combinations of STI571 with high-dose chemotherapy regimens (vincristine, daunorubicin, and prednisone and high-dose ara-C) are also in progress for patients in lymphoid and myeloid blast crisis, respectively. As mechanisms of CML disease progression and relapse become apparent, it is hoped that agents that target these specific abnormalities could also be developed. 4.1.7. OTHER THERAPEUTIC TARGETS FOR STI571 Although STI571 was tested as a treatment for BCR-ABL-associated leukemias, its original target was the PDGF-R, and it was subsequently shown to

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inhibit the c-kit tyrosine kinase. Thus, STI571 should also have activity in diseases associated with constitutive activation of these kinases. In the case of c-kit, activating mutations are associated with a gastrointestinal stromal tumor (GIST) (77,78), which is highly refractory to chemotherapy. Results from an ongoing phase I study using STI571 to treat patients with GIST have shown response rates close to 60% (79,80). A particularly interesting finding from this study was that activating mutations of c-kit correlated with response, whereas patients expressing wild-type c-kit had a significantly lower response rate (79). This suggests that other tumors where c-kit is expressed but not activated by mutation may also be less likely to respond to STI571. Tumors that express c-kit include germ cell tumors, small-cell lung cancer (SCLC), AML, neuroblastoma, melanoma, ovarian cancer, and myeloma. The exception to this might be the fraction of patients with AML who express c-kit-activating point mutations. In any case, studies are ongoing with STI571 to determine whether tumors that express c-kit will respond to STI571. The majority of cases of systemic mastocytosis have a mutation of aspartic acid 816 to valine (D816V) in the kinase domain of c-kit, resulting in activation of c-kit. Unfortunately, the kinase activity of the D816V mutant isoform was recently shown to be resistant to STI571 (81). Thus, STI571 is unlikely to be useful in this disorder. With respect to PDGF-R as a target, patients with CMML with a (5;12) translocation resulting in expression of the constitutively active Tel-PDGF-R fusion protein tyrosine kinase are an ideal target. STI571 has shown in vitro inhibition of leukemic cell lines expressing Tel-PDGF-R, and this finding has been corroborated in patient studies where remarkable clinical benefits with STI571 have been observed (57,82). Glioblastomas, the most common brain tumor and a highly chemotherapy- and radiation-resistant tumor, are associated with an autocrine growth loop involving PDGF and its receptor. STI571 inhibits the growth of glioblastoma cells injected into the brains of nude mice, suggesting that this agent could have potential as therapy for this currently incurable disease (83). Numerous other malignancies have also been reported to have autocrine activation of PDGF-R, including non-small cell lung, breast, and prostate cancers and several sarcomas; however, the data supporting a role for PDGF-R activation in these diseases are less compelling (84). Nevertheless, clinical trials with STI571 in these diseases could be envisioned to test this hypothesis. Unlike the case with BCR-ABL (and possibly Tel-PDGF-R), however, it is unlikely that a defect in a single protein kinase is responsible for malignant transformation in most of the aforementioned tumors; therefore, it is unreasonable to expect results as dramatic as those seen in the treatment of CML when using STI571 alone for these other indications. Greater efficacy may be expected when the kinase inhibitor is used in combination with chemotherapy or even other molecularly targeted therapies. Finally, PDGF-R

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activation may have a role in several fibrotic disorders, such as myelofibrosis, pulmonary fibrosis, and hepatic fibrosis (85). Given the acceptable toxicity profile, an exploration of the activity of STI571 in these disorders may also be warranted.

5. TRANSLATING THE SUCCESS OF STI571 TO OTHER MOLECULAR TARGETS The clinical trials with STI571 are a dramatic demonstration of the potential of targeting molecular pathogenetic events in a malignancy. In applying this paradigm to other malignancies, it is important to recognize that BCR-ABL and CML have several features that were critical to the success of this agent. As noted, BCR-ABL tyrosine kinase activity has clearly been demonstrated to be critical to the pathogenesis of CML. Thus, not only was the target of STI571 known but it also was directed against the critical event in the development of CML. Another important feature that the results demonstrate, as with most malignancies, is that treatment of early stage disease yields better results. Specifically, the rate and durability of responses have been notably superior in patients in chronic phase as opposed to those in blast phase. Therefore, to reproduce the success of STI571 in other malignancies, it is imperative to identify the critical early events in malignant progression. It is equally important that selection for clinical trials is limited to those patients whose malignancies express the appropriate target. In clinical trials using STI571, this was clearly feasible because patients with activation of BCR-ABL were easily identifiable by the presence of the Ph chromosome. In this regard, as reagents to analyze molecular endpoints are developed, these same reagents should be useful in identifying appropriate candidates for treatment with a specific agent. With the combination of a critical pathogenetic target that is easily identifiable early in the course of the disease and an agent that targets this abnormality, remarkable results can be achieved. The obvious goal is to identify these early pathogenetic events in each malignancy and to develop agents that specifically target these abnormalities.

ACKNOWLEDGMENTS BJD is funded by grants from the NCI, a Specialized Center of Research Award from The Leukemia and Lymphoma Society, a Clinical Scientist Award from the Burroughs Wellcome Fund, and the T. J. Martell Foundation.

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Inhibition 10 P-Glycoprotein in Acute Myeloid Leukemia Thomas R. Chauncey, MD, PhD CONTENTS INTRODUCTION BACKGROUND PROGNOSIS OF DRUG RESISTANCE PROFILES CLINICAL TRIALS OF P-GLYCOPROTEIN REVERSAL CONCLUSION REFERENCES

1. INTRODUCTION The expression of P-glycoprotein (Pgp) in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) is almost invariably associated with a poor prognosis. In multiple clinical series, Pgp has been associated with a poor response to chemotherapy, early relapse, decreased remission duration, and shorter overall survival. Expression of Pgp is typically higher in patients with more advanced disease, such as those who have relapsed or progressed after initial response. Pgp shares significant structural homology with other efflux pumps comprising the large superfamily of adenosine triphosphate (ATP)-binding cassette (ABC) transporters. Immunologic detection of the drug efflux pumps, Pgp, and multidrug resistance-associated protein 1 (MRP1) typically correlate with their respective functional drug resistance assays. Other markers of resistance described in AML include lungresistance protein (LRP), bcl-2, and breast cancer resistance protein (BCRP), although their pathophysiology and clinical relevance is less clear and methodology for their quantification are not as well standardized. Preclinical studies have shown that small molecules capable of reversing efflux can restore drug sensitivity in resistant tumor models. Initial clinical studies were limited by both potency and specificity of the reverser, whereas From: Biologic Therapy of Leukemia Edited by: M. Kalaycio © Humana Press Inc., Totowa, NJ

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later studies with more effective agents have, in many instances, been limited by pharmacokinetic interactions exacerbating the clinical toxicities of chemotherapy. Although one large randomized study has demonstrated a proven survival advantage without increased toxicity using cyclosporine, inconsistent results with other modulators raise doubt about the use and overall strategy of using drug efflux blockers in patients with established Pgp overexpression. Many of these patients have additional resistance mechanisms and achieving meaningful clinical responses will likely require more complex clinical strategies. Preventing or delaying development of drug resistance in chemosensitive patients remains another therapeutic strategy for evaluation.

2. BACKGROUND Primary chemotherapy for patients with AML leads to high complete response rates in younger patients with significantly lower rates in older patients as well as those with leukemia secondary to antecedent myelodysplastic syndromes (MDS) or previous chemoradiotherapy. For patients who achieve complete remission (CR), response durability is also significantly lower in older patients or those with a clinically apparent secondary onset (1–7). AML features in older patients and those with previous MDS include an increased incidence of cytogenetic abnormalities associated with a poor prognosis, surface phenotypes suggestive of a more primitive etiology, and increased incidence of inherent biologic resistance. Of the cellular mechanisms of drug resistance described for patients with acute leukemia, the best characterized resistance profile is the phenotype of multidrug resistance (MDR) mediated by Pgp. Pgp expression consistently emerges as a clinically significant marker of resistance in patients with AML (8–15), although biologic resistance can be attributed to several different mechanisms. Although the role of Pgp as a marker for resistance in adult ALL has not been as rigorously evaluated, there is increasing evidence that Pgp resistance plays an important role in clinical outcome (16–18). Pgp confers cross-resistance to a variety of mechanistically and structurally unrelated cytotoxic drugs, such as anthracyclines, taxanes, Vinca alkaloids, and epipodophyllotoxins (8,9,19). All anthracyclines are subject to Pgp-mediated resistance, despite evidence that idarubicin has greater cellular retention and is less susceptible to Pgp-mediated efflux (20,21). Pgp is a member of the ABC gene superfamily, which is conserved throughout species evolution. Common to ABC transporters is a heterodimeric transmembrane glycoprotein complex, each with six transmembrane domains and an ATP binding site with conserved sequences known as the Walker A and Walker B domains (19,22). ABC transporters are involved in a range of transport functions from the epithelial transporter mutated in cystic fibrosis (CTFR), the canalicular multispecific organic anion (cMOAT) and canalicular bile acid (cBAT) transporters of liver, mono-

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cyte secretion of IL-1β (ABC1), antigen processing in T-cells (TAP), cholesterol and phospholipid efflux (ABCA1), and non-Pgp-mediated anthracycline transport (BCRP), also known as the mitoxantrone resistance gene (MXR), ABCP, and ABCG2. Current ABC nomenclature has been reviewed (19,22,23) and is available on the Web (24). The phylogenetic conservation of ABC transporters suggests a broad role in protection from naturally occurring toxic compounds (xenobiotics). In mammalian species, the systemic elimination of xenobiotics by the liver and kidney, the integrity of the blood–brain barrier, the isolation of germ cells from the systemic circulation, fetal isolation from maternal circulation, and protection of the stem cell compartment are all mediated in part by ABC transporters. To standardize assays, consensus recommendations have outlined criteria for determining Pgp in clinical samples (25), encouraging both immunophenotypic assessment as well as functional assays. Anthracyclines and, alternatively, fluorescent dyes such as rhodamine 123 (Rh123), Di(OC)2, or calcein are used to determine functional efflux (11,12,26), calculated as the decrease in cellular fluorescence compared with baseline assessment. Efflux measurements and phenotypic determinations on different cell populations can be compared using the Kolmogorov-Smirnov (KS) statistic. Expression profiling for resistance genes is an intriguing research tool (27) that may provide future clinical use. Multidrug-resistance associated protein (MRP) is also a member of the ABC superfamily and is capable of efflux and intracellular sequestration in conjunction with glutathione conjugation or cotransport (26,28). MRP describes a group of transporters, of which MRP1 is the only member implicated in drug transport. The substrate specificity of MRP1 is similar but more limited than Pgp, and its normal physiologic role may be detoxification of intracellular oxidants. It has been suggested that the location of MRP1 genes on chromosome 16 may contribute to the favorable prognosis found in patients with AML with inv(16) abnormalities. MRP1 is typically assessed using flow cytometry. Functional assays using fluorescent dyes in the presence or absence of reversers, such as cyclosporine and probenecid (26), or after glutathione depletion (11), can specifically assess MRP-mediated efflux. Lung-resistance protein was initially identified in a lung cancer cell line during in vitro selection for drug resistance (29–32). It has significant homology with rodent vault proteins, which are subcellular organelles likely involved in nuclear-cytoplasmic transport. Enforced expression of LRP in transfection experiments is not sufficient to confer resistance, suggesting that other cofactors or posttranslational assembly is necessary for biologic function (29). Of interest, LRP expression was increased in patients with relapsed AML after response to induction therapy that included cyclosporine to overcome Pgp resistance (32), suggesting that modulating Pgp-mediated resistance may result in selection or upregulation of LRP as a secondary resistance mechanism after

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Pgp reversal. LRP evaluation can be assessed with flow cytometry, immunocytochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR), although given the subcellular cytoplasmic localization, permeabilizing agents must be used with conventional flow cytometric techniques. Furthermore, posttranslational regulation can make immunologic detection inconsistent. These techniques have been compared on clinical samples and cell lines, and the relative advantages and highlights of each have been described (33). The BCRP/MXR/ABCP/ABCG2 transporter was initially described in breast cancer cell lines and represents a “half-transporter,” consisting of six transmembrane domains and an ATP binding site. It is believed that the functional complex involves formation of a homodimer or heterodimeric partnering with another subunit (19,34,35). BCRP is characterized by high affinity for mitoxantrone and topoisomerase I inhibitors, as well as a resistance spectrum that includes other anthracyclines but unlike Pgp does not include Vinca alkaloids or taxanes (19,36). BCRP has been assessed using RT-PCR, monoclonal antibodies, and selective efflux inhibitors, although optimal evaluation of clinical samples has not been determined. With the availability of many small molecules capable of reversing Pgp, several clinical trials have been designed and executed to overcome this specific resistance. Despite initial promise from encouraging preclinical data, many of these trials have been unsuccessful, attributable to toxicities arising from the pharmacokinetic effects of Pgp modulation on the cytotoxic anticancer drugs.

3. PROGNOSIS OF DRUG RESISTANCE PROFILES Studies from different investigators using both flow cytometry and functional efflux assays have shown that clinical specimens expressing Pgp do worse than patients who are Pgp negative (8–15,26), with a progressive increase in Pgp expression with advancing age and significant correlation of Pgp expression with decreasing remission rates and increasing incidence of resistant disease (11,12,14). The increase in Pgp expression at relapse may also account for differences in daunorubicin sensitivity in patients with acute promyelocytic leukemia (APL) at presentation compared with first relapse (37). Most studies confirm strong correlation between phenotypic Pgp expression and functional efflux, although the Southwest Oncology Group (SWOG) has also shown a small but consistent blast population with discordance between functional and structural profiles, with some cells showing efflux resistant to cyclosporine and no phenotypic evidence of Pgp expression (11), a phenomena also suggested by other investigators (38,39). This profile may represent expression of alternate transporters; however, the prognostic impact of this resistance profile is not yet characterized.

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In the same SWOG studies, MRP1 was expressed at relatively low levels and decreased with advancing age, although neither MRP1 nor LRP detection provided any clinically significant prognostic value (11). Although some studies confirm MRP1’s lack of prognostic use (28,40), others have shown that MRP1 detection is predictive of outcome and adds prognostic value to Pgp for both CR rates, relapse-free survival, and overall survival for patients expressing both phenotypes (26). These discrepancies may be attributable to the different size populations tested or to methodologic differences with SWOG assessing MRP-specific efflux after glutathione depletion and others using efflux inhibition by probenecid and alternate fluorescent substrates. In several series, LRP expression in AML has been more predictive than Pgp for remission rates, resistant disease, and overall survival (20,32,40), although this has not been a consistent finding (9,11,41). These discrepant findings may be attributable to variation in technique (33). Studies evaluating BCRP in patients with AML show a range of expression, generally poor correlation with Pgp expression (38,42,43) and inconsistent prognostic value (43–46). Inasmuch as the resistance spectrum of anticancer drugs for BCRP is similar to Pgp, this may represent other clinically significant transporters in patients with acute leukemia as well as other malignancies, and BCRP may account for some of the non-Pgp-cyclosporine-resistant efflux described (11). Many investigations have suggested that coexpression of drug resistance markers is significantly more predictive of clinical outcomes than expression of a single mechanism (9,11,47–49). These discordant findings can again be attributed not only to assay technique but also to selection of populations studied as well as the biologic heterogeneity of acute leukemia. Patients with adult ALL have not been as well characterized but have been shown to have a relatively high incidence of phenotypic Pgp expression (16–18) with variable prognostic implications. A recent study demonstrated high Pgp expression along with strong concordance of phenotypic Pgp with increased functional efflux measured by Di(OC)2 (16). Of interest, this study confirmed the discrepant finding of immunologically undetectable Pgp in cells with cyclosporine-reversible efflux, suggesting alternate membrane transporters as previously described in clinical AML samples.

4. CLINICAL TRIALS OF P-GLYCOPROTEIN REVERSAL Encouraged by substantial preclinical evidence that small molecules can overcome drug resistance in cell culture, rodent xenografts, and transgenic murine models, clinical trials have tested whether Pgp efflux reversers can improve outcome in patients with AML with high Pgp expression. Initial trials in several malignancies, using drugs such as verapamil and quinine showed

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limited efficacy because toxicities from these compounds did not allow adequate serum levels to reverse Pgp. Confirming this unfavorable therapeutic index, a prospective comparative trial of quinine with a mitoxantrone and cytarabine regimen in patients with high-risk AML, ALL, and transformed myelodysplasia and myeloproliferative disorders failed to demonstrate a consistent clinical advantage, although encouraging trends in Pgp-expressing cases were observed (50). Similarly, attempts with quinine and verapamil together have also been unsuccessful in patients with non-Hodgkin’s lymphoma (NHL) (51). Because many Pgp blockers affect the pharmacokinetics of anticancer drug excretion by the kidney and liver and in some instances compete for hepatic metabolic pathways such as the P450 system, it has typically been necessary to reduce the chemotherapy dose during concurrent therapy with Pgp blockers to achieve a comparative regimen of equivalent clinical toxicity. These interactions may be further complicated by evidence that some Pgp substrates can activate the hepatic and intestinal orphan nuclear receptor, SXR, which in turn can induce expression of both P450 enzymes and Pgp, thereby enhancing inactivation and excretion of the cytotoxic compound (52). To accommodate these pharmacokinetic effects, many phase I and phase II trials were designed to establish regimens equitoxic to similar regimens without the Pgp-reverting drug (8,9,53–61) (Table 1). Of the drugs initially tested, cyclosporine and later its analog, PSC833, showed the highest Pgp-reversing activity and the most promising therapeutic indices. Using cyclosporine, a phase I–II study in patients with high-risk AML that included relapsed and refractory disease demonstrated clinical efficacy with a response rate greater than expected based on historical reference, remission “inversions” with durations of clinical remission longer than previous remissions, and absence or decrease of MDR1 expression in patients with leukemia at later relapse (61). Based on the encouraging results from this pilot study, a comparative phase III study in an identical population was developed and recently completed. The SWOG study used cyclosporine at high doses (16 mg/kg/d) concurrent with daunorubicin by continuous infusion (45 mg/m2 × 3) after 5 d of high-dose ara-C (3 gm/M2/d), while the control arm received the same regimen without cyclosporine. This study is significant because it showed advantages for the addition of cyclosporine in complete response rate, decreased incidence of resistant disease, and importantly improvement in both diseasefree and overall survival that are durable beyond 2 yr (62). Although the daunomycin dose was not reduced on the cyclosporine arm, toxicities were equivalent, except for reversible hyperbilirubinemia in those patients receiving cyclosporine. Of note, an identical regimen in patients with myeloid blast crisis of chronic myelogenous leukemia (CML) did not show any benefit for the addition of cyclosporine (63).

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Table 1 Phase I–II Trials of Pgp Modulation in Acute Myeloid Leukemia Institution/Author (Reference) List (61) M.D. Anderson/Kornblau (60) CALGB 9420/Lee (59) Advani (58) POG 9222/Dahl (57) SWOG 9617/Chauncey (56) Visani (55) CALGB 9621/Kolitz (54) Dorr (53) SWOG 9918/Chauncey (79)

Phase I–II I I II II I I I I–II II

Size 42 110 110 37 66 31 23 398 43 16

Indication

Modulator

Regimen

Rel/ref Rel/ref ≥60 Rel/ref Rel/ref (≤20) >55 >60 <60 Rel/ref >55

CSA PSC833 PSC833 PSC833 CSA PSC833 PSC833 PSC833 PSC833 PSC833

HIDAC-D (civi) ME ADE MEC ME ME DA ADE HIDAC-D (civi) D (civi) A

Rel/ref = relapsed/refractory; CSA = cyclosporine; HIDAC = high-dose cytarabine; ME = mitoxantrone, etoposide; ADE = cytarabine, daunorubicin, etoposide; MEC = mitoxantrone, etoposide, cytarabine; DA = daunorubicin, cytarabine; CALGB = Cancer and Leukemia Group B; POG = Pediatric Oncology Group; SWOG = Southwest Oncology Group; civi = continuous intravenous infusion.

Results of the SWOG study contrast similar trials using cyclosporine in high-risk AML conducted by the Medical Research Council (MRC) (64) and Eastern Cooperative Oncology Group (ECOG) (65) treated with daunomycin, etoposide, and cytarabine in conventional pulse doses, which showed no benefit from the addition of cyclosporine in adults (Table 2). Another single-arm study in children with high-risk AML by the Children’s Cooperative Group/Pediatric Oncology Group (CCG/POG) (57) using mitoxantrone, etoposide, and cyclosporine showed no benefit compared with historical controls. Differences in trial design, study population, and dosing could account for these conflicting results. The MRC study used a lower cyclosporine dose that does not consistently lead to serum levels adequate for reversing Pgp, whereas the MRC, ECOG, and CCG/POG studies also gave cytotoxic drugs by conventional pulse administration, leading to greater peak pharmacokinetic effects and likely exacerbating clinical toxicity. The MRC, ECOG, and CCG/POG studies also used etoposide, which is likely a poorer Pgp substrate (62,66) and a less effective antileukemic agent for primary AML therapy (2), yet one that is significantly modulated by the pharmacokinetic effects of cyclosporine and PSC833. For primary AML therapy, etoposide probably contributes relatively more to toxicity than efficacy, and this discrepancy is exacerbated by the pharmacokinetic effects of cyclosporine and PSC833.

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Chauncey Table 2 Comparative Trials of Pgp Modulation in Acute Myeloid Leukemia

Institution/Author (Ref)

Size Indication Modulator

Regimen

Outcome

ECOG/Tallman (65) MRC AML-R/Liu Yin (64) SWOG 9126/List (62)

38 235 226

Rel/ref Rel/ref Rel/ref

CSA CSA CSA

MEC ADE/HIDAC-DE HIDAC-D (civi)

CML-BP ≥60

CSA PSC833

HIDAC-D (civi) DA

ECOG 2995/Greenberg (78) 127

Rel/ref

PSC833

MEC

HOVON, MRC, C302 (9) C301 (9)

>60 Rel/ref

PSC833 PSC833

DA MEC

No effect No effect Improved RFS, OS No effect Early closure (toxicity) Early closure (efficacy) No effect No effect

SWOG 9032/List (63) CALGB 9720/Baer (77)

82 126

428 256

ECOG = Eastern Cooperative Oncology Group; rel/ref = relapsed/refractory; CSA = cyclosporine; MEC = mitoxantrone, etoposide, cytarabine; MRC = Medical Research Council; ADE = cytarabine, daunorubicin, etoposide; HIDAC = high-dose cytarabine; SWOG = Southwest Oncology Group; RFS = relapse-free survival; OS = overall survival; CML-BP = chronic myelogenous leukemia in blast phase; CALGB = Cancer and Leukemia Group B; DA = daunorubicin, cytarabine; HOVON = Stiching Haemato-Oncologie voor Volwassenen Nederland.

Preclinical investigations and small clinical trials offer rationale for daunorubicin administration by continuous infusion. Small studies evaluating intracellular daunorubicin accumulation show greater blast retention when equivalent doses are administered over a 24-h period compared with a 10-min infusion (67). Furthermore, in Pgp-expressing cell lines, in vitro cytotoxicity studies performed with Pgp sensitizers show that lower total anticancer drug exposures are required to induce equivalent cytotoxicity if given as an extended exposure (68–70). A recent ex vivo study showed increased daunorubicin blast retention when administered by continuous infusion in the presence of the Pgp reverser, PSC833 (71). In the SWOG study (62), full-dose daunorubicin without dose attenuation in the cyclosporine arm was presumably achievable, because the excess toxicities seen in other studies were more directly attributable to the peak pharmacodynamic effects of bolus infusions. Peak daunorubicin levels are blunted with the use of continuous infusion (53,67,72,73), leading to lower peak concentrations albeit higher steady-state levels. In this study, higher steady-state levels in the presence of cyclosporine appear critical to the clinical advantages seen, because increasing daunorubicin steady-state levels show strong correlation with improvement in CR, relapse-free survival, and overall survival, only if cyclosporine was administered. These preclinical and clinical studies suggest that protracted exposure of daunorubicin in the presence of Pgp reverters may be

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not only more efficacious but also less toxic. Furthermore, it is clear from other investigations evaluating steady-state daunorubicin levels that not all patients show modulation of daunorubicin pharmacokinetics with cyclosporines, so attenuating daunorubicin doses to accommodate pharmacokinetic effects may result in lower drug exposures for many patients (53,60). Despite the increase in toxicities seen in entire cohorts without dose attenuation when cyclosporine is combined with daunorubicin given by conventional bolus administration, some patients will have lower serum steady-state levels than expected with full daunorubicin doses in the absence of cyclosporines. The results of SWOG 9126 also raise issues about whether previous exposure to high-dose cytarabine before continuous infusion daunorubicin with cyclosporine represents a critical pharmacologic interaction. PSC833 (valspodar) is a cyclosporine analog with significantly more in vitro Pgp-reverting activity than cyclosporine and a comparative lack of immunosuppressive and nephrotoxic effects. PSC833 retains cyclosporine’s pharmacokinetic effects with inhibition of the metabolism and excretion of anticancer drugs, such as anthracyclines and epipodophyllotoxins (74–76). Several phase I–II studies with PSC833 were performed in patients with AML with high Pgp expression (Table 1). For older patients with newly diagnosed AML as well as those with relapsed and refractory disease with concurrent PSC833, dose reduction of anticancer therapy was necessary to obtain regimens of equivalent toxicity (53–56,58–60). Dose reduction ratios were similar from study to study, with the absolute reduction dependent on the population studied and the relative dose intensity of each regimen tested. Unfortunately, prospective phase III comparative studies of these regimens have not shown improvement in clinical outcome. In older patients with newly diagnosed AML, one study was stopped early due to excessive toxicity in the PSC833 group, despite attenuated doses of daunorubicin (77), whereas other studies evaluating patients with relapsed and refractory AML show no convincing efficacy for PSC833 (78). To mitigate the excessive toxicities seen with PSC833 and daunomycin by conventional bolus administration, the strategy of daunorubicin by continuous infusion with PSC833 was investigated in a phase II study in older patients with AML, but this too was limited by significant regimen-related toxicity and closed before completing its targeted accrual (79). Patients in the SWOG study experienced the expected toxicities from anthracycline dose escalation, including severe mucositis, pneumonia, and sepsis. Reviewing all randomized PSC833 AML trials now complete, pharmacokinetic effects have typically been excessive and/or dose attenuation has limited potential efficacy, despite occasional subset analysis showing trends toward improved outcomes with PSC833 in patients with Pgp-expressing blasts (77). The use of Pgp-reversing compounds with significant pharmacokinetic effects on concomitant anticancer therapy makes clinical trials both difficult to

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perform and difficult to analyze. The successful SWOG study with cyclosporine used a schedule and consolidation schema distinct from other regimens tested. Whether continuous infusion daunomycin with cyclosporine was particularly advantageous, perhaps acting in synergy with high-dose cytarabine in the relapsed/refractory population, or whether cyclosporine and not PSC833 affects other transporters or has other biologic effects leading to improved outcome, are critical speculative issues without current resolution. Cyclosporine may have activity in a broader range of ABC transporters than other current Pgp reversers and may also affect angiogenesis or active paracrine growth factors (62). Current SWOG trials are planned to evaluate the safety and efficacy of cyclosporine with continuous infusion daunorubicin in older patients with AML. Alternatively, Pgp reversers without pharmacokinetic effects have been developed and are being tested in clinical trials (80,81). Zosuquidar (LY335979) is an effective and selective Pgp inhibitor that is 500–1500 times more potent than cyclosporine on in vitro testing (80,82,83). In contrast to other Pgp reverters tested, it has negligible in vivo pharmacokinetic interactions with anthracyclines, making it attractive for use in conjunction with daunorubicin in patients with AML. In a phase II trial of patients that included relapsed and refractory AML, newly diagnosed secondary AML and RAEB-t, a daunorubicin and cytarabine regimen, along with zosuquidar showed predominant dose-limiting toxicities of reversible ataxia, confusion, and agitation. Of interest, reversible ataxia was also seen in PSC833 studies associated with high peak concentrations (53,55,84). The response rates, including 38% CR and 23% CR with incomplete platelet recovery, in this high-risk population warrants further investigation. Ex vivo studies confirm that leukemia samples exposed to in vivo zosuquidar show decreased Rh123 efflux activity consistent with the drug’s anti-Pgp efficacy (82). Strategies to evaluate zosuquidar in newly diagnosed AML in older patients are being developed. Biricodar (VX-710) is another compound without apparent pharmacokinetic effects on doxorubicin when tested in solid tumors (81). It is a particularly attractive compound because it has demonstrated reversing activity for both Pgp- and MRP1-mediated transport. Another potential strategy involves the use of gemtuzumab ozogamicin (Mylotarg; Wyeth Pharmaceuticals, Collegeville, PA) in conjunction with Pgp blockade. Gemtuzumab ozogamicin is a monoclonal antibody to the CD33 epitope linked to calicheamicin, which is an extremely potent naturally occurring compound inducing deoxyribonucleic acid (DNA) cleavage. By design, this agent directly targets CD33-expressing cells, with relative specificity for AML and other myeloid progenitors. Response to gemtuzumab ozogamicin has been shown to correlate both with expression and function of Pgp, as well as inducible apoptosis in ex vivo samples (85–87). Other evidence shows that cyclosporine can increase the proportion of leukemic speci-

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mens undergoing apoptosis. Together, these results suggest that use of gemtuzumab ozogamicin in conjunction with Pgp sensitizers will lead to increased leukemia-directed cytotoxicity (86,88). Limitations to gemtuzumab ozogamicin efficacy include an apparent increased hepatotoxicity, especially in patients who have previously undergone dose-intensive therapy with allogeneic or autologous stem cell transplantation (89). Although this toxicity can be significant in a minority of patients treated with gemtuzumab ozogamicin, it remains unclear whether systemic Pgp blockade will exacerbate this phenomena. It is intriguing to speculate that the somewhat limited therapeutic index of single agent gemtuzumab ozogamicin may improve if used with concurrent Pgp blockade and anecdotal reports indicate the potential tolerability and efficacy of this approach (89) with larger comparative trials currently being developed. Alternatively, targeting populations with established Pgp expression may not be the most efficacious approach for any Pgp-reversing agent. Despite evidence that Pgp-mediated resistance emerges through clonal expansion of spontaneous mutations (90), other data show a variety of cellular stressors, including chemotherapy, are capable of inducing MDR1 expression (91–95). Pgp expression may represent a marker for evolution or selection of other mechanisms of drug resistance (96), whether induced or selected by previous chemotherapy (97) or as an evolution of the leukemic process. Studies demonstrating the frequent occurrence and additive prognostic value of multiple resistance markers are in accord with this concept. Moreover, preclinical data suggest that Pgp reversers can suppress emergence of resistance (90,98,99), whereas other studies show that Pgp expression can occur rapidly after exposure to cytotoxic therapy. One study demonstrated increased Pgp expression and function within hours of ex vivo exposure to cytotoxic agents (94) and another in patients with metastatic sarcoma showed evidence that in vivo Pgp gene expression is rapidly inducible within minutes of treatment (95). There are conflicting data on this issue, with a recent small series of patients with AML followed sequentially from diagnosis to relapse showing no Pgp expression increase as assessed by phenotype, functional efflux, and clonal homozygosity for those patients with informative Pgp polymorphisms (100). Hopefully, these disparate results will be further investigated in larger clinical series. Using Pgp-blocking agents in patients without Pgp expression or other resistance mechanisms is an alternate strategy that is currently being pursued in clinical trials such as CALGB 19808 (54). However, because patients without Pgp expression have a better prognosis, determining the efficacy of this approach will require longer follow-up and must account for the distinct prognostic categories of better risk patients. New insight into Pgp function suggests roles in cellular physiology beyond xenobiotic efflux. The Pgp apparatus may serve broad anti-apoptotic functions

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that interfere with caspase-dependent apoptosis, perhaps in concert with membrane signal transduction (101,102). In many tissues, ABC transporters play a central role in lipid transport as well as membrane sphingomyelin polarity and content, the latter known to affect caspase-dependent apoptotic signaling. Evidence from both cell lines and ex vivo leukemia samples (103) suggests that the anti-apoptotic effects of Pgp may be mediated through the sphingomyelinceramide pathway, an effect reversible with exogenous sphingomyelin given in vitro. Pgp can lower membrane sphingomyelin content, making less available for ceramide production, and can also enhance sphingomyelin redistribution, thereby limiting TNF-α-driven apoptosis, an effect antagonized by PSC833 (104). Pgp can inhibit caspase-3-dependent apoptosis initiated by stimuli such as Fas ligation or ultraviolet irradiation, an effect blocked by the Pgp reverser, verapamil (105), whereas PSC833 has been shown to augment the apoptosis induced by serum and growth factor withdrawal (103). Although blocking Pgp with currently available pharmaceuticals may not achieve the clinical responses eagerly anticipated in clinical trials, future trial design using drug combinations effecting caspase-independent pathways may be enhanced by concurrent Pgp blockade. With a better understanding of Pgp’s role in cellular apoptotic processes, therapeutic strategies can be designed to accommodate these complex interrelationships.

5. CONCLUSION Of the better characterized markers of drug resistance in AML, phenotypic and functional Pgp expression predicts outcome more consistently than LRP and MRP1, although the prognostic utility of these and other resistance markers will improve and change with larger studies and more consistent methodology. Although preclinical experiments have shown that some resistant phenotypes can be overcome with reversing agents, biologic resistance is a complex interaction of multiple cellular alterations, with mutations leading to drug resistance also conferring other biologic advantages. As such, overcoming clinical resistance in leukemia therapy may require not only targeted resistance modifiers but also a more complete biologic and clinical understanding of the leukemic process.

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23. Muller M, Jansen PLM. Molecular aspects of hepatobiliary transport. Am J Physiol 1997;272:G1285–G1303. 24. Available at: http://www.med.rug.nl/mdl/humanabc.htm. 25. Beck WT, Grogan TM, Willman CL, et al. Methods to detect P-glycoprotein-associated multidrug resistance in patients’ tumors: consensus recommendations. Cancer Res 1996;56:3010–3020. 26. Legrand O, Simonin G, Beauchamp-Nicqud A, Zittoun R, Marie J. Simultaneous activity of MRP1 and Pgp is correlated with in vitro resistance to daunorubicin and with in vivo resistance in adult acute myeloid leukemia. Blood 1999;94:1046–1056. 27. Kudoh K, Ramanna M, Ravatn R, et al. Monitoring the expression profiles of doxorubicininduced and doxorubicin-resistant cancer cells by cDNA microarray. Cancer Res 2000;60:4161–4166. 28. Filipits M, Suchomel RW, Zöchbauer S. Multidrug resistance-associated protein in acute myeloid leukemia: no impact on treatment outcome. Clin Cancer Res 1997;3:1419–1425. 29. Dalton WS, Scheper RJ. Lung resistance-related protein: determining its role in multidrug resistance [editorial]. J Natl Cancer Inst 1999;91:1604–1605. 30. Filipits M, Pohl G, Stranzl T, et al. Expression of the lung resistance protein predicts poor outcome in de novo acute myeloid leukemia. Blood 1998;5:1508–1513. 31. Kickhoefer VA, Rajavel KS, Scheffer GL, Dalton WS, Scheper RJ, Rome LH. Vaults are up-regulated in multidrug-resistant cancer cell lines. J Biol Chem 1998;273:8971–8974. 32. List AF, Spier CS, Grogan TM, et al. Overexpression of the major vault transporter protein lung-resistance protein predicts treatment outcome in acute myeloid leukemia. Blood 1996;87:2464–2469. 33. Legrand O, Simonin G, Zittoun R, Marie J-P. Lung resistance protein (LRP) gene expression in adult acute myeloid leukemia: a critical evaluation by three techniques. Leukemia 1998;12:1367–1374. 34. Ross DD, Yang W, Abruzzo LV, et al. Atypical multidrug resistance: breast cancer resistance protein messenger RNA expression in mitoxantrone-selected cell lines. J Natl Cancer Inst 1999;91:429–433. 35. Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci USA 1998;95:15,665–15,670. 36. Honjo Y, Hryeyna CA, Yan Q-W, et al. Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP-overexpressing cells. Cancer Res 2001;61:6635–6639. 37. Michieli M, Damiani D, Ermacora A, Geromin A, Michelutti A, Masolini P. P-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP) expression in acute promyelocytic leukaemia. Br J Haematol 2000;108:703–709. 38. Ross DD. Novel mechanisms of drug resistance in leukemia. Leukemia 2000;14:467–473. 39. Merlin JL, Guerci A-P, Marchal S, et al. Influence of SDZ-PSC833 on daunorubicin intracellular accumulation in bone marrow specimens from patients with acute myeloid leukemia. Br J Haematol 1998;103:480–487. 40. Filipits M, Stranzl T, Pohl G, Heinzl H, Jager U, Geissler K. Drug resistance factors in acute myeloid leukemia: a comparative analysis. Leukemia 2000;14:68–76. 41. Pallis M, Turzanski J, Harrison G, et al. Use of standardized flow cytometric determinants of multidrug resistance to analyse response to remission induction chemotherapy in patients with acute myeloblastic leukaemia. Br J Haematol 1999;104:307–312. 42. Ross DD, Karp JE, Chen TT, Doyle LA. Expression of breast cancer resistance protein in blast cells from patients with acute leukemia. Blood 2000;96:365–368. 43. van den Heuvel-Eibrink MM, Wiemer EA, Prins A, et al. Increased expression of the breast cancer resistance protein (BCRP) in relapsed or refractory acute myeloid leukemia (AML). Leukemia 2002;16:833–839.

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44. Abbott BL, Colapietro AM, Barnes Y, et al. Low levels of ABCG2 expression in adult AML blast samples. Blood 2002;15(100):4594–4601. 45. Suvannasankha A, Minderman H, O’Loughlin KL, et al. Breast cancer resistance protein (BCRP) expression in acute myeloid leukemia (AML) [abstract]. Blood 2001;98(suppl 1):307a. 46. van der Kolk DM, Vellenga E, Scheffer GL, et al. Expression and activity of breast cancer resistance protein (BCRP) in de novo and relapsed acute myeloid leukemia. Blood 2002;99:3763–3770. 47. Borg AG, Burgess R, Green LM, Scheper RJ, Liu Yin JA. P-glycoprotein and multidrug resistance-associated protein, but not lung resistance protein, lower the intracellular daunorubicin accumulation in acute myeloid leukaemic cells. Br J Haematol 2000;108:48–54. 48. Kasimir-Bauer S, Ottinger H, Meusers P, et al. In acute myeloid leukemia, coexpression of at least two proteins including P-glycoprotein, the multidrug resistance-related protein bcl2, mutant p53, and heat-shock protein 27, is predictive of the response to induction chemotherapy. Exp Hematol 1998;26:1111–1117. 49. Goasguen JE, Lamy T, Bergeron C, et al. Multifactorial drug-resistance phenomenon in acute leukemias: impact of P170-MDR1, LRP56 protein, glutathione-transferases and metallothionein systems on clinical outcome. Leukemia Lymphoma 1996;23:567–576. 50. Solary E, Witz B, Caillot D, et al. Combination of quinine as a potential reversing agent with mitoxantrone and cytarabine for the treatment of acute leukemias: a randomized multicenter study. Blood 1996;88:1198–1205. 51. Gaynor ER, Unger JM, Miller TP, et al. Infusional CHOP chemotherapy (CVAD) with or without chemosensitizers offers no advantage over standard CHOP therapy in the treatment of lymphoma: a Southwest Oncology Group study. J Clin Oncol 2001;19:750–755. 52. Synold TW, Dussault I, Forman BM. The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux. Nat Med 2001;7:584–590. 53. Dorr R, Karanes C, Spier C, et al. Phase I/II study of the P-glycoprotein modulator PSC 833 in patients with acute myeloid leukemia. J Clin Oncol 2001;19:1589–1599. 54. Kolitz JE, George SL, Dodge RK, et al. Dose escalation studies of ara-C (A), daunorubicin (D) and etoposide (E) with and without multidrug resistance (MDR) modulation with PSC833 (P) in untreated adult patients with acute myeloid leukemia (AML) < 60 years: final induction results of CALGB 9621 [abstract]. Blood 2001;98(suppl 1):461a. 55. Visani G, Milligan D, Leoni F, et al. Combined action of PSC 833 (Valspodar), a novel MDR reversing agent, with mitoxantrone, etoposide and cytarabine in poor-prognosis acute myeloid leukemia. Leukemia 2001;15:764–771. 56. Chauncey TR, Rankin C, Anderson JE, et al. A phase I study of induction chemotherapy for older patients with newly diagnosed acute myeloid leukemia (AML) using mitoxantrone, etoposide, and the MDR modulator PSC 833: a Southwest Oncology Group Study (9617). Leukemia Res 2000;24:567–574. 57. Dahl GV, Lacayo NJ, Brophy N, et al. Mitoxantrone, etoposide, and cyclosporine therapy in pediatric patients with recurrent or refractory acute myeloid leukemia. J Clin Oncol 2000;18:1867–1875. 58. Advani R, Saba HI, Tallman MS, et al. Treatment of refractory and relapsed acute myelogenous leukemia with combination chemotherapy plus the multidrug resistance modulator PSC833 (valspodar). Blood 1999;93:787–795. 59. Lee EJ, George SL, Caligiuri M, et al. Parallel phase I studies of daunomycin given with cytarabine and etoposide with or without the multidrug resistance modulator PSC-833 in previously untreated patients 60 years of age or older with acute myelogenous leukemia: results of Cancer and Leukemia Group B Study 9420. J Clin Oncol 1999;17:2831–2839. 60. Kornblau SM, Estey E, Madden T, et al. Phase I study of mitoxantrone plus etoposide with multidrug blockade by SDZ PSC-833 in relapsed or refractory acute myelogenous leukemia. J Clin Oncol 1997;15:1796–1802.

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61. List AF, Spier CM, Greer J, et al. Phase I/II trial of cyclosporine as a chemotherapy-resistance modifier in acute leukemia. J Clin Oncol 1993;11:1652–1660. 62. List AF, Kopecky KJ, Willman CL, et al. Benefit of cyclosporine modulation of drug resistance in patients with poor-risk acute myeloid leukemia: a Southwest Oncology Group study. Blood 2001;98:3212–3220. 63. List AF, Kopecky KJ, Willman CL, et al. Cyclosporine inhibition of P-glycoprotein in chronic myeloid leukemia blast phase. Blood 2002;100:1910–1912. 64. Liu Yin JA, Wheatley K, Rees JKH, et al. Comparison of “sequential” versus “standard” chemotherapy as re-induction treatment, with or without cyclosporine, in refractory/relapsed acute myeloid leukaemia (AML): results of the UK Medical Research Council AML-R trial. Br J Haematol 2001;113:713–726. 65. Tallman MS, Lee E, Sikic BI, et al. Mitoxantrone, etoposide and cytarabine plus cyclosporine in patients with relapsed or refractory acute myeloid leukemia: an Eastern Cooperative Oncology Group pilot study. Cancer 1999;85:358–367. 66. Politi PM, Arnold ST, Felsted RL, Sinha BK. P-glycoprotein-independent mechanism of resistance to VP-16 in multidrug-resistance tumor cell lines: pharmacokinetic and photoaffinity labeling studies. Mol Pharmacol 1990;37:790–796. 67. Paul C, Tidefelt U, Liliemark J, Peterson C. Increasing the accumulation of daunorubicin in human leukemic cells by prolonging the infusion time. Leukemia Res 1989;13:191–196. 68. Toffoli G, Turniotto L, Gigante M, et al. Doxorubicin, vincristine, and actinomycin-D, but not teniposide, require long-lasting uninterrupted verapamil pressure to overcome drug resistance in multidrug-resistant cells. Cancer Detect Prev 1993;17:425–432. 69. Slate D, Michelson S. Drug resistance-reversal strategies: comparison of experimental data with model predictions. J Natl Cancer Inst 1991;83:1574–1580. 70. Cass CE, Janowska-Wieczorek A, Lynch MA, et al. Effect of duration of exposure to verapamil on vincristine activity against multidrug-resistant human leukemic cell line. Cancer Res 1989;49:5798–5804. 71. Tidefelt U, Liliemark J, Gruber A, et al. P-Glycoprotein inhibitor valspodar (PSC 833) increases the intracellular concentrations of daunorubicin in vivo in patients with P-glycoprotein-positive acute myeloid leukemia. J Clin Oncol 2000;18:1837–1844. 72. Lewis JP, Meyers FJ, Tanaka L. Daunomycin administered by continuous intravenous infusion is effective in the treatment of acute nonlymphocytic leukaemia. Br J Haematol 1985;61:261–265. 73. DeGregorio MW, Carrera CJ, Klock JC, et al. Cellular and plasma kinetics of daunorubicin given by two methods of administration in a patient with acute leukemia. Cancer Treat Rep 1982;66:2085–2088. 74. Friche E, Jensen PB, Nissen NI. Comparison of cyclosporine A and SDZ PSC833 as multidrug-resistance modulators in a daunorubicin-resistant Ehrlich ascites tumor. Cancer Chemother Pharmacol 1992;30:235–237. 75. Boesch D, Gaveriaux C, Jachez B, et al. In vivo circumvention of P-glycoprotein-mediated multidrug resistance of tumor cells with SDZ PSC 833. Cancer Res 1991;51:4226–4233. 76. Twentyman PR, Bleehen NM. Resistance modification by PSC-833, a novel non-immunosuppressive cyclosporin A. Eur J Cancer 1991;27:1639–1642. 77. Baer MR, George SL, Dodge RK, et al. Phase 3 study of the multidrug resistance modulator PSC-833 in previously untreated patients 60 years of age and older with acute myeloid leukemia: Cancer and Leukemia Group B Study 9720. Blood 2002;100:1224–1232. 78. Greenberg P, Advani R, Tallman M, et al. Treatment of refractory/relapsed AML with PSC833 plus mitoxantrone, etoposide, cytarabine (PSC-MEC) vs MEC: randomized phase III trial (E2995) [abstract]. Blood 1999;94(suppl 1):383a. 79. Chauncey TR, Gundacker H, List AF, et al. personal communication

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80. Dantzig AH, Shepard RL, Cao J, et al. Reversal of P-glycoprotein-mediated multidrug resistance by a potent cyclopropyldibenzosuberane modulator, LY335979. Cancer Res 1996;56:4171–4179. 81. Peck RA, Hewett J, Harding MW, et al. Phase I and pharmacokinetic study of the novel MDR1 and MRP1 inhibitor Biricodar administered alone and in combination with doxorubicin. J Clin Oncol 2001;19:3130–3141. 82. Gerrard G, Ganeshaguru K, Baker R, et al. A phase I study of a Pgp inhibitor Zosuquidar (LY 335979), given by short intravenous infusion in combination with daunorubicin and cytarabine in AML/MDS patients [abstract]. Blood 2001;98(suppl 1):177b. 83. Cripe LD, Tallman M, Karanes C, et al. A phase II trial of Zosuquidar (LY335979), a modulator of P-glycoprotein (Pgp) activity, plus daunorubicin and high-dose cytarabine in patients with newly diagnosed secondary acute myeloid leukemia (AML), a refractory anemia with excess blasts in transformation (RAEB-t) or relapsed/refractory AML [abstract]. Blood 2001;98(suppl 1):595a. 84. Patnaik A, Warner E, Michael M, et al. Phase I dose-finding and pharmacokinetic study of paclitaxel and carboplatin with oral valspodar in patients with advanced solid tumors. J Clin Oncol 2000;18:3677–3689. 85. Bross PF, Beitz J, Chen G, et al. Approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia. Clin Cancer Res 2001;7:1490–1496. 86. Naito K, Takeshita A, Shigeno K, et al. Calicheamicin-conjugated humanized anti-CD33 monoclonal antibody (gemtuzumab ozogamicin, CMA-676) shows cytocidal effect on CD33-positive leukemia cell lines, but is inactive on P-glycoprotein-expressing sublines. Leukemia 2000;14:1436–1443. 87. Sievers EL, Appelbaum FR, Spielberger RT, et al. Selective ablation of acute myeloid leukemia using antibody-targeted chemotherapy: a phase I study of an anti-CD33 calicheamicin immunoconjugate. Blood 1999;93:3678–3684. 88. Linenberger ML, Hong T, Flowers D, et al. Multidrug-resistance phenotype and clinical responses to gemtuzumab ozogamicin. Blood 2001;98:988–994. 89. Giles FJ, Kantarjian HM, Kornblau SM, et al. Mylotarg (gemtuzumab ozogamicin) therapy is associated with hepatic venoocclusive disease in patients who have not received stem cell transplantation. Cancer 2001;92:406–413. 90. Beketic-Oreskovic L, Durán G, Chen G, Dumontet C, Sikic B. Decreased mutation rate for cellular resistance to doxorubicin and suppression of mdr1 gene activation by the cyclosporin PSC 833. J Natl Cancer Inst 1995;87:1593–1600. 91. Chaudhary PM, Roninson IB. Induction of multidrug resistance in human cells by transient exposure to different chemotherapeutic drugs. J Natl Cancer Inst 1993;85:632–639. 92. Chin KV, Tanaka S, Darlington G, Pastan I, Gottesman MM. Heat shock and arsenite increase expression of the multidrug resistance (MDR1) gene in human renal carcinoma cells. J Biol Chem 1990;265:221–226. 93. Mickley LA, Bates SE, Richert ND, et al. Modulation of the expression of a multidrug resistance gene (mdr-1/P-glycoprotein) by differentiating agents. J Biol Chem 1989;264:18,031–18,040. 94. Hu XF, Slater A, Kantharidis P, et al. Altered multidrug resistance phenotype caused by anthracycline analogues and cytosine arabinoside in myeloid leukemia. Blood 1999;93:4086–4095. 95. Abolhoda A, Wilson AE, Ross H, Danenberg PV, Burt M, Scotto KW. Rapid activation of MDR1 gene expression in human metastatic sarcoma after in vivo exposure to doxorubicin. Clin Cancer Res 1999;5:3352–3356. 96. Broxterman HJ, Sonneveld P, Pieters R, et al. Do P-glycoprotein and major vault protein (MVP/LRP) expression correlate with in vitro daunorubicin resistance in acute myeloid leukemia? Leukemia 1999;13:258–265.

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the Apoptotic Machinery 11 Targeting as a Potential Antileukemic Strategy Benjamin M. F. Mow, MD and Scott H. Kaufmann, MD, PHD CONTENTS INTRODUCTION MULTIPLE ROLES FOR CASPASES DURING APOPTOSIS REGULATION OF APOPTOTIC PATHWAYS ALTERED APOPTOTIC PATHWAYS IN LEUKEMIA INDUCTION OF APOPTOSIS BY ANTILEUKEMIC THERAPY NEW AGENTS THAT DIRECTLY TARGET APOPTOTIC PATHWAYS OR THEIR REGULATION SUMMARY REFERENCES

1. INTRODUCTION Apoptosis is a morphologically and biochemically distinctive cell death process that occurs under several physiologic and pathologic conditions (1,2). Morphologically, apoptosis is characterized by plasma membrane blebbing followed by chromatin condensation and disassembly of the cell into multiple membrane-enclosed fragments, which are then engulfed by neighboring cells or professional phagocytes (2). These biochemical changes are believed to reflect, at least in part, the action of caspases, unique intracellular proteases that digest critical polypeptides required for cellular integrity and survival (3,4). Studies performed since the late 1980s, have demonstrated that virtually all of the agents currently used to treat acute leukemia induce apoptosis in susceptible cells (5,6). Conversely, there has also been a growing recognition that the process of leukemogenesis might involve changes that inhibit apoptosis (6,7). From: Biologic Therapy of Leukemia Edited by: M. Kalaycio © Humana Press Inc., Totowa, NJ

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In the following sections, the current understanding of apoptotic pathways is outlined, some of the ways in which these pathways are altered during the process of leukemogenesis are described, and the current status of agents designed to reverse some of these alterations is reviewed.

2. MULTIPLE ROLES FOR CASPASES DURING APOPTOSIS As mentioned above, many of the biochemical and morphological features of apoptotic cells reflect the selective proteolytic cleavage of a subset of cellular polypeptides (3,8). For example, cleavage of the inhibitor of caspase-activated deoxyribonuclease (ICAD) liberates CAD (9), an endonuclease that contributes to the internucleosomal deoxyribonucleic acid (DNA) degradation and chromatin condensation observed in apoptotic cells (10). Likewise, cleavage of lamins, filamentous polypeptides that form a structural meshwork inside the inner nuclear membrane, facilitates apoptotic nuclear fragmentation (11). These cleavages result from the action of caspases, intracellular cysteine proteases that cleave next to aspartate residues (3,4). Of the 11 known human caspases, 5 (caspases 3, 6, 8, 9, and 10) have increasingly well-defined roles in apoptosis. Each is synthesized as a relatively inactive precursor containing a prodomain, a large subunit, and a small subunit. Activation of these zymogens commonly involves proteolytic cleavage at multiple aspartate residues, including one between the large and small subunits and another between the prodomain and the large subunit. After a series of conformational changes, the activated enzymes ultimately consist of two large and two small subunits (12). The fact that activation usually involves digestion at potential caspase cleavage sites opens the possibility that caspases participate in intracellular proteolytic cascades (4). Like other enzyme cascades, the caspase proteolytic cascades consist of upstream and downstream participants with discrete roles. Caspases 3 and 6, the major downstream or “effector” caspases, are responsible for most of the cleavages that disassemble the cell. Caspases 8, 9, and 10, the major upstream or “initiator” caspases, are responsible for transducing various signals into proteolytic activity. These initiator caspases are activated as a consequence of signaling through at least two distinct pathways (Fig. 1).

2.1. The Death Receptor Pathway Signaling through the death receptor (DR) pathway (also called the extrinsic pathway) begins when certain death-inducing cytokines bind to their specialized cell surface receptors (13,14). For example, Fas ligand (FasL) expressed on the surface of cytotoxic lymphocytes binds to Fas (also known as CD95 or Apol-1), a receptor on the surface of target cells. As a consequence of this ligand-receptor interaction, the cytoplasmic domain of Fas undergoes an apparent conformational change and then binds the adaptor molecule FADD (Fas-associated protein with

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death domain). After interacting with Fas, FADD not only oligomerizes (15) but also binds procaspases 8 and 10, which have low but detectable enzymatic activity (16–18). These interactions cause juxtaposition of multiple zymogens and result in apparent autocatalytic liberation of active caspases 8 and 10 (16–18). Other DRs signal similarly, although the adaptor molecules differ in some cases (13). In some lymphoid cells (so-called “type I cells”), the caspase 8 and/or 10 generated by DR ligation directly activates procaspase 3 (19). In other cells (“type II cells”), however, caspase 8 or 10 is insufficient to activate procaspase3. Instead, the cytoplasmic protein Bid is cleaved to produce a fragment that activates the mitochondrial pathway (4).

2.2. The Mitochondrial Pathway The mitochondrial pathway (also called the intrinsic pathway) is activated by the selective release of a subset of mitochondrial polypeptides into the cytoplasm (4). Several models have been advanced to explain the altered localization of these polypeptides (20,21). Some research stresses the importance of a phenomenon known as mitochondrial permeability transition, which involves the opening of a pore in the mitochondrial membranes (22), whereas other research focuses on the insertion of two polypeptides, Bax and Bak, in the outer mitochondrial membrane, where they oligomerize to form pores that mediate cytochrome c release (23–26). Although Bax and Bak are constitutively expressed in many cells, these polypeptides are ordinarily found in the cytoplasm or loosely associated with mitochondria. Recent evidence (27) suggests that their insertion into the mitochondrial outer membrane is induced by “BH3-only” polypeptides, a group of small proapoptotic polypeptides that includes the Bid fragment generated by caspase-8 during DR signaling in type II cells (25,26); Bad, which is dephosphorylated and activated upon withdrawal of interleukin-induced survival signals (28); Bim, which is displaced from microtubules in response to microtubule-directed drugs (29); and PUMA, which is synthesized in a p53-dependent manner in response to DNA damage (30). As a result of Bax and Bak insertion in the outer mitochondrial membrane, several mitochondrial polypeptides are released (31). The most widely studied is the electron transporter cytochrome c, which binds to a cytoplasmic scaffolding protein Apaf-1 (apoptotic protease-activating factor-1), causing an adenosine triphosphate (ATP)- or dATP-dependent conformational change that allows Apaf-1 to bind and activate procaspase 9 (32). Activated caspase 9 in turn cleaves procaspase 3 to yield active caspase 3. Other mitochondrial polypeptides that are released include second mitochondrial activator of caspases (SMAC) and HtRA2, which facilitate caspase activation by binding to caspase inhibitors (see Subheading 3.2.2), as well as endonuclease G, which also enters the nucleus and participates in chromatin digestion in some apoptotic cells (31).

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Fig. 1. (Facing page.) Two major pathways of caspase activation. In the death-receptor pathway (left), binding of specialized cell surface receptors such as Fas to its extracellular ligand FasL induces recruitment of the adaptor molecule FADD followed by procaspase 8 to cytoplasmic “death domains” of the receptors. Assembly of this macromolecular complex results in activation of caspase 8, which can then activate procaspase 3 directly or cleave the proapoptotic Bcl-2 family member Bid to activate the mitochondrial pathway (box). In the mitochondrial pathway (right), multiple polypeptides are released from mitochondria. Once in the cytoplasm, cytochrome c acts as a cofactor for assembly of a macromolecule complex (“apoptosome”) containing Apaf-1 and procaspase 9, which exhibits caspase 9 activity. Although procaspase-9 can be activated without cleavage, some or all of the caspase 9 is typically cleaved upon activation. This cleaved caspase 9 reacts with XIAP to form an enzymatically inactive complex. A similar reaction (not shown) inhibits active caspase 3. SMAC/Diablo, which is also released from mitochondria, reacts with XIAP, liberating the active caspases and facilitating downstream cleavages. Other mitochondrial polypeptides, including endonuclease G and apoptosis inducing factor (AIF) also participate in apoptotic events in some cell types (31). Events leading to cytochrome c release are depicted in greater detail in the box. Some BH3 only Bcl-2 family members, e.g., the fragment of Bid resulting from caspase-8-mediated cleavage (tBid), facilitate insertion of Bax and Bak in the mitochondrial outer membrane, where they participate in cytochrome c release (39), possibly by forming pores. Antiapoptotic Bcl-2 family members, including Bcl-2, Bcl-xL and the adenovirus 19 kilodalton E1B protein, inhibit Bax/Bak mitochondrial membrane insertion and cytochrome c release, most likely by sequestering BH3 only polypeptides (27).

3. REGULATION OF APOPTOTIC PATHWAYS Because of the lethal consequences of inadvertent caspase activation, it is not surprising that the caspase activation pathways are highly regulated. Several factors that affect the activation of one or both pathways have been identified.

3.1. Death Receptor Pathway Regulation Some DR pathway regulation occurs at the DR level. First, DR expression varies according to cell lineage and stage of development (14). DR4 and DR5, for example, are expressed by leukemia cells (33) and other neoplastic cells but not by many normal cells, except for thymocytes (14). These observations provide at least a partial explanation for the selectivity of TNFα-related apoptosisinducing ligand (TRAIL) for neoplastic cells (34,35). Moreover, DR expression can be induced by certain treatments, particularly DNA damaging agents that induce DR5 synthesis (36). Second, DR signaling is regulated by phosphorylation. Protein kinase C activation, for example, inhibits DR recruitment of FADD after treatment with FasL or TRAIL (37). Although the mechanistic basis for these observations is incompletely understood, it has been suggested that protein kinase Cα is the responsible isoform (38). Third, DR signaling can be modulated by the expression of decoy receptors, cell surface, or secreted proteins that bind ligand but do not signal (13).

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Interactions between FADD and initiator caspases are also regulated. Many cells express Flice-like inhibitory protein (FLIP) (3) which contains a caspase8-like prodomain but lacks an active site cysteine. Because FLIP competitively inhibits procaspase 8 binding or activation, FLIP overexpression causes resistance to various DR ligands. Finally, expression of procaspases 8 and 10 can vary widely. In particular, procaspase 10 is expressed at much higher levels in primary lymphocytes than in lymphoid leukemia cell lines (17). Although complete absence of both of these procaspases results in inability to activate DR pathways (18), it is presently unclear how diminished expression of one or the other of these zymogens affects sensitivity to DR ligands.

3.2. Regulation of the Mitochondrial Pathway The mitochondrial pathway is regulated both upstream of cytochrome c release and downstream of caspase 9 activation. Two important polypeptide families, the Bcl-2 family and the inhibitor of apoptosis protein (IAP) family, participate in these events. 3.2.1. REGULATION OF CYTOCHROME C RELEASE BY BCL-2 FAMILY MEMBERS As indicated, insertion of Bax and Bak into the mitochondrial outer membrane is postulated to play a critical role in cytochrome c release (39). The effects of these polypeptides are antagonized by antiapoptotic Bcl-2 family members (40,41). The founding member of this family, Bcl-2, was originally identified as a polypeptide that is expressed at high levels when the corresponding gene is juxtaposed to the immunoglobulin heavy chain promoter as a consequence of the t(14;18) translocation found in indolent B-cell lymphomas. Subsequent analysis demonstrated that Bcl-2 diminishes the induction of apoptosis by several different stimuli (40,41). Several structural homologs, including Bcl-xL and Mcl-1, have similar effects. These effects were traced, at least in part, to the ability of these polypeptides to inhibit mitochondrial cytochrome c release (42). Subsequent studies have demonstrated that these antiapoptotic Bcl-2 family members inhibit Bax and Bak insertion into the outer mitochondrial membrane (23,43), perhaps by binding and sequestering BH3 only family members such as Bid, Bad, Bim, and PUMA (27). The balance between proapoptotic and antiapoptotic Bcl-2 family members is regulated by transcriptional mechanisms as well as posttranslational modifications. The proapoptotic Bcl-2 family members Bax, Noxa, and PUMA are all synthesized in a p53-dependent manner after DNA damage or other cellular stresses (44). Conversely, expression of the antiapoptotic family members Bcl-xL (45) and Mcl-1 (46) is regulated by Stat3 (signal transducer and activator of transcription 3), a transcription factor that is activated by antiapoptotic cytokines (47).

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Activities of certain Bcl-2 family members are also modulated by phosphorylation (42). For example, the antiapoptotic function of Bcl-2 is enhanced by protein kinase Cα- (PKCα) or ERK (extracellular signal-regulated kinase)mediated phosphorylation on 70Ser (48,49). Mcl-1 is also phosphorylated in an ERK-dependent manner (50). Other kinases affect other Bcl-2 family members. Protein kinase A-mediated phosphorylation of BAD on 155Ser inactivates its proapoptotic BH3 domain, whereas cytokine-stimulated Akt-mediated phosphorylation on 112Ser and 136Ser contributes to cytoplasmic sequestration of BAD by 14-3-3 proteins (42). 3.2.2. REGULATION OF CASPASE ACTIVATION BY INHIBITOR PROTEIN FAMILY MEMBERS The presence of IAPs within cells conveys a second level of regulation (51,52). Multiple members of this gene family, including X-linked IAP (XIAP), cellular IAP1 (cIAP1), cIAP2, and neuronal apoptosis inhibitory protein (NAIP), are expressed in various tissues. These polypeptides all contain multiple finger-like baculovirus inhibitor repeat (BIR) domains that bind to the surfaces of certain caspases and allow sequences between the BIRs to block the active sites of target enzymes (53). Consistent with these observations, XIAP, cIAP1, and cIAP2 directly inhibit the activities of active caspases 3, 7, and 9 (51,52). In addition, these IAPs contain domains that transfer ubiquitin to target caspases, thereby marking them for proteasome-mediated degradation (54,55). It is currently believed that IAPs act as a cellular buffer for small amounts of caspases that are inadvertently activated (56). The capacity of this buffer is regulated on at least two levels. First, IAP expression is regulated. Transcription of the genes encoding XIAP, cIAP1, and cIAP2, for example, is stimulated by nuclear factor-κB (NFκB) (3), a transcription factor implicated in antiapoptotic signaling (57). Second, the ability of XIAP, cIAP1, and cIAP2 to bind to active caspases is regulated. In particular, two small polypeptides that are released from mitochondria concomitant with cytochrome c, SMAC and HtRA2, bind to the BIR regions of IAPs and inhibit their ability to interact with caspases (58,59). OF APOPTOSIS

4. ALTERED APOPTOTIC PATHWAYS IN LEUKEMIA 4.1. Chronic Lymphocytic Leukemia The potential role of altered apoptosis in the pathogenesis of leukemia was first demonstrated for chronic lymphocytic leukemia (CLL), a disorder characterized by gradual accumulation of morphologically mature lymphocytes. It was recognized decades ago that CLL cells had a low proliferative index compared with acute leukemias. The demonstration that 70% of CLL specimens contain elevated levels of Bcl-2 (60), a polypeptide now known to inhibit mitochondrial release of cytochrome c (42) and SMAC (61), provided an explana-

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tion for these observations. Although chromosomal rearrangements that juxtapose the Bcl-2 gene to the immunoglobulin light or heavy chain promoters have been described in rare cases of CLL, most CLL specimens lack these rearrangements. Instead, Bcl-2 overexpression reflects signaling initiated by interleukin (IL)-4 (62) as well as Bcl-2 gene methylation (60). In addition to Bcl-2, Mcl-1 and the antiapoptotic Bcl-2 binding protein Bag1 are also overexpressed in a substantial portion of CLL cases (63). Moreover, p53 is mutated in a subset of CLL cases (64), providing another mechanism for the inhibition of apoptotic pathways that could otherwise be activated by DNA damage or cellular stress. Collectively, these alterations not only contribute to the accumulation of CLL, cells but also render CLL cells somewhat more resistant to chemotherapy than they might be otherwise.

4.2. Large Granular Lymphocyte Leukemia Large granular lymphocyte (LGL) leukemia is a rare chronic lymphoproliferative disorder characterized by accumulation of large granular lymphocytes, typically of T-cell or natural killer (NK) cell origin (65). Symptoms are often related to neutropenia and anemia. The LGL leukemia cells typically contain constitutively activated STAT3 and elevated levels of the antiapoptotic Bcl-2 family member Mcl-1 (46). These cells are resistant to Fas-induced apoptosis (66). In addition, they synthesize and release FasL, which has been implicated in arrest of erythroid maturation (67) and induction of granulocyte apoptosis. Thus, resistance of the LGL leukemia cells to apoptosis plays an important role in not only their expansion, but also their ability to suppress normal hematopoiesis.

4.3. Chronic Myeloid Disorders Based in part on analogy to CLL, several groups have tested the hypothesis that apoptosis might be altered in chronic myelogenous leukemia (CML). In 95% of CML cases, a characteristic t(9;22) chromosomal translocation juxtaposes the 5′ end of the BCR gene with the 3′ end of the ABL gene, resulting in expression of a unique constitutively active 210 kDa kinase, p210BCR/ABL (68). Transfection of complementary DNA (cDNA) encoding this kinase into cytokine-dependent murine 32D myeloid cells inhibits drug-induced apoptosis (69). Conversely, downregulation of BCR/ABL using antisense oligonucleotides enhances the rate of spontaneous or drug-induced apoptosis in CML cell lines (70) and clinical samples (71). Subsequent studies have demonstrated that p210BCR/ABL activates the phosphatidylinositol-3 kinase/Akt pathway as well as transcription mediated by STAT5 and NFκB (68,72,73). Collectively, these signals cause phosphorylation (inhibition) of the proapoptotic protein Bad as well as enhanced expression of the antiapoptotic proteins Bcl-xL (74) and XAIP (75). These events are believed to inhibit cytochrome c release from the mitochondria and activation of the caspase cascade.

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Emerging evidence suggests that apoptosis might also be deranged in other chronic myeloproliferative syndromes. Neutrophils from patients with chronic neutrophilic leukemia, a rare BCR/ABL-negative chronic myeloproliferative syndrome characterized by accumulation of mature neutrophils (76), also exhibit diminished apoptosis (77). Moreover, erythroid progenitors from patients with polycythemia vera, a stem cell disorder characterized by expansion of the red cell compartment, contain elevated levels of Bcl-xL (78). The mechanistic bases for both of these observations remain to be established.

4.4. Acute Myeloid Leukemia Current understanding suggests that development of the neoplastic phenotype in solid tumors requires at least two changes, one that enhances proliferation and another that inhibits apoptosis (7). Whether this is also true in acute leukemias is less clear. Perhaps the best studied example involves acute promyelocytic leukemia (APL), a disorder in which an almost universal t(15;17) chromosomal translocation brings together the promyelocytic leukemia gene PML, which encodes a nuclear ubiquitin ligase, and the retinoic acid receptor gene RARα (79). Soon after its discovery, the PML-RARα fusion protein was shown to render leukemia cell lines resistant to the induction of apoptosis (80). Subsequent analysis (reviewed in [81]) has shown that PML-RARα acts as a dominant negative inhibitor of both RARα and PML. It is believed to competitively inhibit binding of RARα to cognate DNA sequences, thereby inhibiting RA-induced expression of genes involved in terminal differentiation of myeloid precursors. In addition, PML-RARα heterodimerizes with and inhibits wildtype PML, a polypeptide that appears to be somehow required for induction of apoptosis by several agents (82). It has been suggested that sensitivity to spontaneous or drug-induced apoptosis is also altered in poor risk acute myeloid leukemia (AML) (83,84). These claims are based on experiments in which AML samples are incubated under serum-free conditions or with drug in vitro. It is important to recognize that the behavior of AML cells might be modified by the artificial conditions employed in these experiments. In particular, contact of blasts with stroma-derived cells (85) or a stromal cell line (86) inhibits spontaneous and drug-induced apoptosis. These observations raise the possibility that resistance to the induction of apoptosis might be underestimated in studies that expose cells to cytokine-free medium or drugs in the absence of stroma. Nonetheless, the available data suggest that sensitivity of AML samples to induction of apoptosis varies (83,84). The biochemical basis for this variation remains to be determined. Several studies have suggested that Bcl-2 overexpression is associated with a poor prognosis in AML (87–89). Another study has reported that Mcl-1 is selectively elevated in approx 50% of AML cases at the time of relapse compared with initial diagnosis (90). Elevated XIAP expression has also been identified

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as a negative prognostic factor in AML (91). Finally, expression of messenger ribonucleic acid (mRNA) encoding several apoptotic regulators is reportedly altered in AML samples with isolated trisomy 8, a harbinger of poor outcome, compared with other leukemias (92). Further studies examining additional alterations in apoptotic pathways in AML are warranted.

4.5. Acute Lymphocytic Leukemia There are also several examples of altered apoptotic pathways in acute lymphocytic leukemia (ALL). First, approx 30% of adult patients with ALL express p190BCR/ABL, a chimeric BCR/ABL kinase with higher constitutive enzymatic activity and stronger transforming ability than p210BCR/ABL. Transfection of cytokine-dependent murine hematopoietic cells with cDNA encoding p190BCR/ABL results in cytokine-independent proliferation and inhibition of apoptosis (93). Interestingly, a mutant p190BCR/ABL kinase that suppresses apoptosis but does not stimulate proliferation has been identified. The poor transforming ability of this kinase suggests that suppression of apoptosis and stimulation of proliferation are both required for transformation by p190BCR/ABL (93). Apoptosis is also inhibited in at least one other ALL variant (reviewed in [94] ). The t(17;19) translocation observed in pro-B lymphocyte ALL results in the formation of a chimeric protein containing domains from E2A, a bHLH (basic helix-loop-helix) transcription factor, and HLF (hepatocyte leukemia factor), a basic leucine zipper (bZIP) transcription factor (95). Expression of this chimeric polypeptide inhibits p53-induced apoptosis and abrogates the interleukin-3 dependence of normal pro-B lymphocytes. Conversely, dominant-negative inhibition of E2A-HLF induces apoptosis, suggesting that the chimeric protein increases the number of immature lymphoid cells by preventing or delaying their death. It has been proposed that E2A-HLF does this by competing with the human homolog of the Caenorhabditis elegans transcription factor CES-2 and activating antiapoptotic target genes that are normally repressed by CES-2-like proteins (96). The E2A-HLF-induced apoptotic delay is believed to allow accumulation of additional mutations that contribute to leukemic transformation (94).

5. INDUCTION OF APOPTOSIS BY ANTILEUKEMIC THERAPY In view of the emerging data suggesting that regulation of apoptotic pathways might be altered in many leukemias, it is not surprising that virtually all antileukemic agents currently in use induce apoptosis in susceptible cells in vitro (5). Apoptosis has also been detected in circulating blasts after initiation of induction chemotherapy (97,97a), providing support for the potential importance of apoptosis in the clinical setting. That antineoplastic agents kill cells by two different mechanisms, that is, their primary effects on cellular metabolism and their ability to activate intrinsic cell death pathways, has led to extensive discussion of two different questions.

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5.1. Which Apoptotic Pathway(s) Are Triggered by Antileukemic Agents? Because the answer to this question has implications for potential mechanisms of resistance, multiple studies have examined the pathways activated by various agents. Studies in fibroblasts or thymocytes from mice with targeted deletions of various DR components have suggested that many of the antileukemic agents activate the mitochondrial pathway (5,98). Whether this activation is a primary event or occurs downstream of caspase 8 activation in type II cells has been more difficult to establish. Because caspase 8 can be activated downstream of caspase 6 when the mitochondrial pathway is activated (99), caspase 8 activation by itself does not establish that caspase 8 is the initiating caspase after a particular treatment. Instead, further studies using dominant negative mutants of caspase 8, caspase 8-deficient cells, or inhibitors of DR signaling are required to establish caspase 8 as an initiator caspase. Results of these types of studies have provided evidence that etoposide and topotecan are among the agents that can initiate apoptosis by inducing caspase 8 activation in at least some cell types (100,101).

5.2. Does Resistance to Apoptosis Translate Into Drug Resistance? Much of the current interest in apoptotic pathways in the chemotherapy community stems from the hope that better understanding of these pathways will provide new insight into drug resistance. Whether this will prove to be the case, however, remains to be established. Some investigators argue that even if apoptotic pathways are inhibited, cells treated with various chemotherapeutic agents will still die as a result of the effects of the primary lesions (e.g., DNA strand breaks). Others argue that the primary lesions are compatible with prolonged survival and possible repair in the absence of caspase activation. There are relatively few studies that address this issue satisfactorily. Studies from Nunez and colleagues revealed that expression of Bcl-xL in FL5.12 cells not only delayed apoptosis but also allowed an increased number of cells to survive treatment with etoposide or methotrexate and ultimately repopulate a culture in vitro (102). Forced overexpression of Bcl-2 has likewise been reported to enhance colony formation after leukemia cell lines are treated with various agents (103), although this has not been a universal finding (104). The long-term protective effects of antiapoptotic Bcl-2 family members presumably reflect the ability of these polypeptides to inhibit mitochondrial release of several proapoptotic factors in addition to cytochrome c. In contrast, none of the papers describing antiapoptotic effects of IAPs (105–107) has demonstrated any long-term protective effect using repopulation or colony-forming assays. Moreover, current understanding (outlined above) suggests that IAPs allow cytochrome c release and caspase 9 cleavage before they act (108). Because other proapoptotic polypeptides, including AIF

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and endonuclease G (31), are released along with cytochrome c, it is more difficult to envision how IAP overexpression might influence long-term survival of cells exposed to proapoptotic stimuli.

6. NEW AGENTS THAT DIRECTLY TARGET APOPTOTIC PATHWAYS OR THEIR REGULATION The improved understanding of apoptotic pathways has led to studies that attempt to alter apoptotic pathways for therapeutic benefit. In the case of leukemias, the intent would be to selectively activate the apoptotic machinery in neoplastic cells or to reverse the antiapoptotic changes that occur during the process of leukemogenesis. These therapies are reviewed in the context of the apoptotic pathways and antiapoptotic changes described. Because of space limitations, the following examples are illustrative rather than comprehensive.

6.1. Activation of Death Receptor Pathways Although TNF-α, the founding member of the DR ligand family, kills a number of cancer and leukemia cell lines, the septic shock-like syndrome caused by this agent limits its ability to be administered systemically (109). Accordingly, TNF-α is reserved for local infusions into isolated perfused limbs. Several observations have given rise to optimism that the TNF-α homolog TRAIL might be a more suitable death ligand for systemic treatment of cancer and leukemia. First, TRAIL is used by interferon (IFN) γ-stimulated lymphocytes and NK cells to kill their targets, which include transformed cells (110). Second, the limited expression of DR4 and DR5 in normal cells (14) raises the possibility that the TRAIL might exhibit some selectivity for solid tumors and leukemia. Consistent with this hypothesis, two recombinant versions of TRAIL have been shown to kill neoplastic cell lines, including leukemia lines, in vitro, and shrink tumors in animals (34,35). Although some investigators have reported that TRAIL is toxic to human hepatocytes in primary culture, these results are not been reproducible (111), and early human clinical trials of TRAIL are anticipated to begin soon. Because hepatocytes do not express DR5, an alternative approach that would avoid the potential hepatotoxicity of TRAIL (if it exists) would be selective crosslinking of DR5 with a suitable antibody. Toward this end, a monoclonal antibody that binds DR5 but not DR4, TRAIL decoy receptors, Fas, or TNF-α receptors has been developed (112). Like recombinant human TRAIL, this antibody kills malignant hepatocytes and certain leukemia cell lines but spares normal human hepatocytes. The effects of all-trans-retinoic acid (ATRA) on APL cells provide an example of the potential importance of TRAIL in antileukemic therapy. Although ATRA has long been viewed as an agent that restores RARα-mediated differentiation in

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APL, recent studies indicate that ATRA also upregulates TRAIL expression in an APL cell line and clinical APL samples in vitro (113). Importantly, soluble TRAIL receptors abolish ATRA toxicity in culture, suggesting that the toxicity reflects paracrine effects of the ATRA-induced TRAIL. The observation that DNA damaging agents upregulate DR5 (36), coupled with these new results, provides a convenient explanation for the efficacy of ATRA in combination with an anthracycline or other DNA-damaging agent to bring about durable remissions in APL (114). DNA damaging agents, including etoposide and cytarabine, also upregulate DR5 and potentiate the effects of TRAIL in other AML lines (115). If TRAIL exhibits antileukemic activity, the effects of these combinations certainly need be tested.

6.2. Reversing Inhibition of the Mitochondrial Pathway 6.2.1. INHIBITING BCL-2 OVEREXPRESSION WITH ANTISENSE OLIGONUCLEOTIDES The observation that elevated Bcl-2 expression is associated with a poor prognosis in AML and ALL (87–89) has led to studies examining the effects of Bcl-2 downregulation. Several studies have reported that cytarabine-induced apoptosis can be enhanced in vitro by treating leukemia cells with Bcl-2 antisense oligonucleotides (116,117). Antisense oligonucleotides are short synthetic single-stranded DNA molecules (15–25 bases) that enter cells, bind to complementary sequences within target mRNA, and either prevent translation of the target message or induce its RNase H-mediated degradation (118,119). Because of bound proteins and conformational factors, not all portions of the mRNA sequence are equally effective targets of antisense oligonucleotides. A series of studies (reviewed in [119] ) has demonstrated that an 18-base phosphorothioate oligonucleotide complementary to the first 18 nucleotides of the Bcl-2 coding sequence (now called G3139 or Genasense™, Genta Corp., Berkeley Heights, NJ) is particularly effective at downregulating Bcl-2 in lymphoma cells containing the t(14;18) chromosomal translocation and inhibiting engraftment of these cells when administered to nude mice for 14 d after cell inoculation. Based on these results, a phase I study of G3139 was completed in patients with relapsed non-Hodgkin’s lymphoma (NHL) (119,120). Twenty-one patients were treated with escalating oligonucleotide doses as a 14-d continuous infusion. Inflammatory reactions at the injection site were noted in all patients, and dose-limiting toxicities were thrombocytopenia, hypotension, fever, and asthenia. Examination of paired lymph node, peripheral blood, or bone marrow samples revealed that G3139 treatment resulted in decreased Bcl2 levels in 58% of cases. Objective responses were noted in 14% of patients, with one complete response that was sustained for ≥ 36 months. It should be noted, however, that this trial examined G3139 alone.

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As indicated in Section 3.2.1., Bcl-2 inhibits the ability of proapoptotic stimuli to induce mitochondrial cytochrome c release. Accordingly, even in neoplasms in which Bcl-2 overexpression is not a primary oncogenic event, Bcl-2 downregulation would be predicted to enhance the induction of apoptosis by other agents. Based on this rationale, G3139 is currently undergoing extensive clinical testing in combination with other antineoplastic agents (121). One current strategy involves combining G3139 with FLAG fludarabine, ara-C, and G-CSF), a popular regimen for the treatment of relapsed and refractory AML (122). For CLL, G3139 is being combined with fludarabine plus cyclophosphamide. Data supporting the safety and improved efficacy of this approach are awaited with interest. 6.2.2. BISPECIFIC ANTISENSE OLIGONUCLEOTIDES Antisense oligonucleotides provide the potential to selectively downregulate a single mRNA. This selectivity is both a strength and a potential weakness. Although some studies analyzing Bcl-2 family members in intermediate grade NHL have suggested a strong correlation between Bcl-2 overexpression and prognosis, others have suggested that Bcl-xL might also contribute (123). BclxL would not be downregulated by G3139 because of differences in mRNA sequence. To circumvent this problem, a Bcl-xL/Bcl-2-bispecific antisense molecule that targets a region of sequence identity between the respective mRNAs has been created (124). The resulting downregulation of both Bcl-2 and Bcl-xL is associated with induction of apoptosis in vitro and in vivo as well as inhibition of variety of tumors grown as xenografts in nude mice (125). Further preclinical and clinical studies of this unique molecule are awaited with interest. 6.2.3. SMALL MOLECULE INHIBITORS OF BCL-2 HOMOLOG SYNTHESIS Current understanding suggests that expression of antiapoptotic Bcl-2 family members is, at least in part, a regulated process. As indicated in Section 3.2.1., expression of Mcl-1 and Bcl-xL is stimulated by activated STAT3 and STAT5 transcription factors (45,46,126). Additional studies have shown that expression of Bcl-2, Mcl-1, and Bcl-xL is also regulated by signaling through the raf/Mek1/ERK pathway (127–129). These observations suggest several strategies for downregulating expression of antiapoptotic Bcl-2 family members using small molecule inhibitors. 6.2.3.1. Inhibition of Signal Transducer and Activator of Transcription Activation. During the course of normal cytokine-mediated signaling, Janus kinases (Jaks) bound to the cytoplasmic domains of cytokine-bound receptors are activated. The Jaks in turn phosphorylate cytosolic STAT proteins, which then dimerize, enter nuclei, and activate transcription of target genes (47). In addition, STAT5 is directly phosphorylated and activated by BCR/ABL

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kinases (reviewed in [73,126]). These observations suggest at least two strategies for downregulating antiapoptotic Bcl-2 family members. AG490, a small-molecule Jak2 inhibitor, downregulates expression of BclxL in multiple myeloma cells (45) and Mcl-1 in LGL leukemia cells in vitro (46). In addition, AG490 has demonstrated antileukemic activity in severe combined immunodeficient (SCID) mice bearing human leukemia xenografts (130). Unfortunately, low potency and poor solubility limit the efficacy of AG490 in vivo. Nonetheless, these observations provide a starting point for the development of more potent and useful Jak inhibitors. STI571 is a potent and somewhat selective BCR/ABL kinase inhibitor. As might be expected, this drug inhibits STAT5 phosphorylation and Bcl-xL expression in CML cells (126). In addition, STI571 inhibits other antiapoptotic signaling pathways downstream of BCR/ABL. Collectively, these effects contribute to the induction of apoptosis by STI571 as well as its ability to sensitize cells to other apoptosis inducing agents (see Chapter 9). 6.2.3.2. Inhibition of the Ras/Mek1/ERK Pathway. Constitutive ERK activation has been observed in approx 50% of human specimens with acute leukemia (131). Recent studies have shown that treatment of AML cell lines with one of two Mek1 inhibitors, PD98059 or PD184352 (now known as CI-1040), results in decreased levels of the antiapoptotic Bcl-2 family members Bcl-xL and Mcl-1 followed by spontaneous induction of apoptosis in vitro (132). In addition, PD98059 inhibits proliferation of leukemic progenitors (but not normal myeloid progenitors) in colony-forming assays (132). Because CI-1040 is well tolerated in phase I trials in patients with solid tumors, there is ample rationale for further preclinical and possible clinical studies of this agent in acute leukemia. 6.2.3.3. Flavopiridol. Although the mechanism of its proapoptotic effects remains to be more fully elucidated, the kinase inhibitor flavopiridol represents another example of a small molecule that downregulates antiapoptotic Bcl-2 family members. Flavopiridol is a semisynthetic flavonoid that was originally identified as an inhibitor of cyclin-dependent kinases (133), the enzymes that enable passage of cells from one cell cycle stage to another (134). Although flavopiridol was believed to be cytostatic, the initial report that it induced apoptosis in HL-60 human leukemia cells (135) was quickly followed by the demonstration that this agent kills several leukemia and lymphoma-derived cell lines as well as clinical CLL isolates (133). In a CLL cell line and in CLL samples, flavopiridol decreases expression of the antiapoptotic proteins Bcl-2, Mcl-1, and/or XIAP (136,137). One potential explanation for these findings is the flavopiridol-induced inhibition of cyclin-dependent kinase 9 and the consequent inhibition of RNA polymerase II, which depends on cyclin-dependent kinase 9-mediated phosphorylation for activation (138). According to this view, polypeptides such as Bcl-2 and Mcl-1 that are synthesized in a regulated

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fashion have short-lived messages. When RNA polymerase II cannot function, mRNA synthesis stops, and the messages for several of the antiapoptotic proteins turnover, quickly leading to decreased polypeptide levels (138). Based on these antiproliferative and proapoptotic effects in preclinical models, flavopiridol is currently undergoing clinical testing. Phase I clinical trials have examined three schedules: a 72-h infusion, a 24-h infusion, and daily 1-h infusions for 5 consecutive d (133,139). The daily schedule is particularly germane to the treatment of hematologic malignancies because it generates peak concentrations in the range previously observed to induce apoptosis in human leukemia cell lines and CLL cells in vitro. In a phase I study performed at the National Cancer Institute, dose-limiting toxicities of neutropenia, diarrhea, and fatigue were observed on this schedule. As might be expected of an agent that inhibits cell cycle progression, flavopiridol has important effects on the action of other anticancer drugs (140). In particular, when flavopiridol is administered before or concomitant with proliferation-dependent agents such as cytarabine, it inhibits their activity. On other hand, if cytarabine is delayed until cells that survive flavopiridol treatment have resumed cycling, the cytotoxic effects of flavopiridol and cytarabine can be synergistic (140). Efforts to harness this sequence-dependent synergy in a clinical trial for patients with relapsed/refractory acute leukemia are currently under way. 6.2.4. INHIBITION OF BCL-2 FUNCTION Rather than inhibiting the synthesis of Bcl-2 family members, an alternative approach would be the inhibition of Bcl-2 function. Two different strategies have been explored. The first strategy involves inhibition of Bcl-2 phosphorylation. As indicated in Section 3.2.1., current understanding suggests that Bcl-2 is a more potent inhibitor of apoptosis when phosphorylated on Ser70. Because PKCα has been implicated in this phosphorylation (48), PKCα inhibition would be expected to facilitate activation of the mitochondrial pathway. Although several small-molecule PKC inhibitors have been identified (141), these agents often lack specificity because of homologies between the various PKC isoforms as well as similarities between PKC and other kinases. Of interest, therefore, is ISIS3521. This antisense oligonucleotide directed against PKCα mRNA is currently being evaluated for potential anticancer activity as a single agent and in combination with various drugs (142). Although PKCα undoubtedly has many targets in cancer cells, it will be interesting to see whether Bcl-2 phosphorylation is inhibited at therapeutic concentrations of this agent. Wang et al. took a different approach in targeting Bcl-2 function (143). Current understanding suggests that Bcl-2 inhibits apoptosis by binding to proapoptotic family members (27). These protein-protein interactions involve a hydrophobic pocket on the Bcl-2 surface. Starting with the predicted structure

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of Bcl-2, Wang et al. simulated the potential binding of a library of compounds to the critical protein-protein interaction domain. Of 28 compounds identified as potential ligands for this pocket, 1 (HA14-1) demonstrated binding to Bcl-2 in vitro and the ability to induce apoptosis in HL-60 human acute leukemia cells, which intrinsically express high Bcl-2 levels (143). This is undoubtedly one of many small molecules that will be identified or designed to inhibit various antiapoptotic Bcl-2 family members. 6.2.5. INHIBITION OF INHIBITOR OF APOPTOSIS PROTEIN SYNTHESIS OR FUNCTION

As indicated in Section 4.4., elevated XIAP expression has been associated with a poor prognosis in AML (91). In light of these results, approaches for inhibiting XIAP synthesis or function are potentially germane to antileukemic therapy. Conceptually, the most straightforward way to inhibit XIAP synthesis would be the use of a suitable antisense oligonucleotide. This approach sensitizes non-small cell lung cancer cells to radiation (144) and ovarian cancer cells to cisplatin-induced apoptosis (145). It remains to be determined whether similar molecules will sensitize AML cells to antileukemic agents. Other approaches for downregulating XIAP expression also are feasible. As indicated in Section 3.2.2., XIAP expression is enhanced by NFκB signaling. Additional studies have demonstrated that NFκB is constitutively activated in a large percentage of primary AML samples (146). Moreover, this NFκB signaling can be inhibited in vitro by NFκB decoy oligonucleotides, i.e., doublestranded oligonucleotides that contain the NFκB binding site and compete activated NFκB away from genomic sequences (147). Although these decoy oligonucleotides sensitize leukemic blasts to cytarabine in vitro (146), further preclinical study is required to determine whether similar effects will be observed in vivo and whether XIAP downregulation plays a role in this process. An alternative approach involves the inhibition of XIAP function by synthetic peptides and peptidomimetics. The proapoptotic mitochondrial protein SMAC binds XIAP and inhibits its interaction with cleaved caspase-9 (108). The N-terminal tetrapeptide on SMAC, ala-val-pro-ile (AVPI), is believed to play a critical role in this process (148). Several investigators are currently introducing this peptide sequence into cells and examining its effect on druginduced apoptosis. To the extent that XIAP contributes to drug resistance in leukemia, it is predicted that AVPI peptides will increase drug-induced apoptosis. If these predictions are confirmed, there will be considerable interest in the design and synthesis of peptide-like molecules that might have similar effects.

7. SUMMARY Biochemical analyses have identified several polypeptides that participate in or regulate the apoptotic process. There is emerging evidence that apoptotic

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pathways are inhibited, at least to some extent, in leukemia cells. On the other hand, as indicated by examples in the preceding section, several experimental treatments can alter the apoptotic machinery and enhance the induction of apoptosis by antileukemic drugs in vitro. Current efforts are directed toward performing suitable preclinical studies and clinical trials to determine whether any of these approaches can improve therapeutic outcomes in vivo. Results of these studies will determine whether the current interest in dysregulation of apoptotic pathways as a potential mechanism of clinical drug resistance is well founded.

8. ACKNOWLEDGMENTS The authors thank Deb Strauss for secretarial assistance and Joya Chandra, Wei Meng, David Steensma, Ruben Mesa, Michael Heldebrant, and Christina Arnt for helpful conversations. Work in the Kaufmann laboratory is support by R01 CA69008.

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IV

DIFFERENTIATION AGENTS

12 Arsenicals Past, Present, and Future Chadi Nabhan, MD and Martin S. Tallman, MD CONTENTS INTRODUCTION MECHANISMS OF ACTION APOPTOSIS AND MITOCHONDRIAL DAMAGE EFFECT ON PML/RAR-α EFFECT ON ANGIOGENESIS ARSENIC TRIOXIDE IN ACUTE PROMYELOCYTIC LEUKEMIA ARSENIC IN OTHER HEMATOLOGIC MALIGNANCIES ARSENIC TRIOXIDE IN MULTIPLE MYELOMA ARSENIC TRIOXIDE IN CHRONIC MYELOGENOUS LEUKEMIA ARSENIC TRIOXIDE IN OTHER HEMATOLOGIC MALIGNANCIES ARSENIC IN SOLID TUMORS ARSENIC TRIOXIDE-ASSOCIATED TOXICITIES CONCLUSION REFERENCES

1. INTRODUCTION Despite its historical reputation as a toxin and a poison, arsenic trioxide has been used therapeutically in a variety of diseases as early as the 18th century. The compound known as Fowler’s solution contained a potassium bicarbonatebased arsenic trioxide (ATO) that was in use until the early 20th century (1,2). The fascination with arsenic was enhanced when Paul Ehrlich developed salvarsan, a formulation that contained organic arsenic used to treat syphilis (3). However, clinicians’ interests in arsenic were abandoned as data accumulated on its toxic effects (1,4). The ATO activity in chronic myelogenous leukemia (CML) was reported in 1938 by Forkner and Scott (5). However, arsenic lost favor as a therapy for From: Biologic Therapy of Leukemia Edited by: M. Kalaycio © Humana Press Inc., Totowa, NJ

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Nabhan and Tallman Table 1 Potential Mechanisms of Action of Arsenic Trioxide Apoptosis induction Caspase cascade activation Mitochondrial injury Relocalization of PML/RARα transcript product Antiangiogenesis effect

CML when radiation was introduced for the treatment of leukemia. Clinical use of arsenic as anticancer therapy ceased in the United States during the 1970s, but recent reports of remarkable activity in acute promyelocytic leukemia (APL) and other in vitro data in many different cell lines, redirected the attention toward arsenic and has rekindled its therapeutic potential (6,7). This chapter focuses the proposed mechanisms of action of ATO, its role in APL, and its potential future directions in other disorders.

2. MECHANISMS OF ACTION Understanding how certain agents work permits one to exploit potential synergism when combined with other therapies. Limited information is available about the specific mechanism(s) of action of ATO. Most in vitro studies were performed in the NB4 (APL) cell line. Several mechanisms have been proposed, including apoptosis induction in several cell lines, partial cellular differentiation, inhibition of angiogenesis, degradation of specific APL fusion transcripts, antiproliferation, and mitochondrial injury (Table 1).

3. APOPTOSIS AND MITOCHONDRIAL DAMAGE Neoplasia generally occurs when the balance between cell proliferation and cell death is disrupted. In most malignancies, apoptotic pathways are perturbed. Among these, upregulation of antiapoptotic genes, such as bcl-2, plays an important role. Caspase cascade activation and deactivation frequently determine the final outcome of cells (8,9). Other contributing factors include p53 mutations, deletion of retinoblastoma (RB) gene, and cyclin D2 overexpression. It has been proposed that ATO exerts its activity through affecting one or more of these pathways (10,11). Several studies have been conducted in an APL cell line (NB4) showing that ATO enhances apoptosis by downregulating bcl-2 protein expression (12,13). This activity is independent of promyelocytic leukemia/retinoic acid receptor-alpha (PML/RAR-α) expression. The apoptotic activity of ATO occurs at a concentration of 1–2 µmol/L. ATO also activates the caspase cascade in APL cells, which promotes proteolysis of intracellular proteins, including poly-ADP-ribose-polymerase (PARP), completing the apoptotic process (10,14).

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Mitochondria are an integral part of the cell machinery and play an important role in apoptosis. This is, in part related to the function of mitochondria as the major site of activity of Bax (proapoptotic) and bcl-2 (antiapoptotic) genes, with the balance between both genes contributing to the fate of the cell (15). In pathologic conditions, apoptotic signals trigger progressive permeabilization of the mitochondrial membrane through the permeability transition pore complex (PTPC). ATO induces such a process, allowing the release of intramitochondrial compounds such as cytochrome C and other apoptosis-inducing factors (16,17). Another mechanism by which ATO induces mitochondrial injury is the release of reactive oxygen species (ROS) that generally causes loss of mitochondrial membrane potential (18). Cells that contain higher levels of glutathione (GSH) are resistant to ATO-induced apoptosis, and manipulation of GSH levels has an effect on ATO activity (18,19). This might explain why NB4-APL cell lines with relatively low levels of reduced GSH, GSH-peroxidase, and catalase are sensitive to ATO. Furthermore, ATO inhibits GSH-peroxidase and increases cellular hydrogen peroxide content (18–20). Additive effects and potential synergism have been explored by combining ATO with agents that reduce GSH levels and allows for more ROS generation. Dai and colleagues showed that by increasing the intracellular levels of hydrogen peroxide with ascorbic acid, the ATO-induced apoptotic activity in lymphoma cell lines increases (19). Gartenhaus and Evens tested a similar concept in a multiple myeloma in vitro model to enhance ATO activity (21,22). In addition, recent data suggest that glutathione depletion could render cells that are resistant to ATO to become sensitive to that agent (23). These data encourage future combination regimens with ATO and other pharmacologic compounds that directly affect the mitochondria or can perturb the redox system.

4. EFFECT ON PML/RAR-α Early studies demonstrated that the characteristic chromosomal translocation t(15;17) in the leukemic cells of patients with APL results in the creation of fusion gene with a protein product that arrests myeloid cell maturation and inhibits differentiation (24). The remarkable activity of all-trans-retinoic acid (ATRA) in APL is attributed to induction of differentiation through interference with the transcript gene (25). ATO also has activity in cellular differentiation in the bone marrow and peripheral blood of patients with APL (26). This was confirmed by demonstrating that leukemic cells treated with ATO lose their primitive markers that are present before therapy and that after therapy, these cells express markers of mature myeloid cells. On the molecular level, Andre and colleagues demonstrated that ATO induces the relocalization of PML and PML/RAR-α onto the nuclear body from the cytoplasm in NB4 cell lines and

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also induces the degradation of these proteins (27). This observation was also seen in non-APL myeloid cell lines (28). This relocalization inhibits the activity of PML and promotes maturation. However, many studies revealed that some ATO effects are independent of both PML and RAR-α. Shao et al. studied ATO activity on cell lines that did not express an intact PML/RAR-α fusion protein and were resistant to ATRA (29). In that study, ATO inhibited cell growth and induced apoptosis despite absence of the chimeric gene product.

5. EFFECT ON ANGIOGENESIS Interrupting the blood supply to tumors can inhibit their growth. The formation of new vessels supplying tumor cells has been proposed as a potential mechanism by which malignant cells grow. Lew et al. studied the effect of ATO on experimental solid tumor system, showing that it produced preferential vascular shutdown and eventually necrosis (30). Proposed mechanisms by which ATO exerts this antiangiogenic effect include activation of endothelial cells, upregulation of endothelial cell adhesion molecules, apoptosis of endothelial cells, and inhibition of vascular endothelial growth factor (VEGF) production (31). Although the proposal that ATO interrupts the vicious circle created by leukemic cells producing VEGF and stimulating endothelial cell production of many cytokines is attractive, the exact mechanisms of ATOrelated antiangiogenesis properties remain under investigation (10).

6. ARSENIC TRIOXIDE IN ACUTE PROMYELOCYTIC LEUKEMIA Initial reports describing the ATO activity in APL (Table 2) first came from China when investigators from the Harbin Medical University observed that 21 of 32 patients treated with an ATO-containing compound achieved complete remission (CR) and a 5-yr overall survival of 50% (32). An updated report from the same group showed a CR rate in 42 previously treated patients of 52% and a CR rate in 30 untreated patients of 73% (33). An initial dose-escalating study conducted at Memorial Sloan-Kettering Cancer Center showed remarkable results (26). In that study, 12 patients with relapsed APL were treated with ATO at doses ranging from 0.06 to 0.2 mg/kg/d until leukemic cells were eliminated from the bone marrow. Of the 12 patients, 11 achieved CR. Furthermore, 8 of these 11 patients who were initially positive for the PML/RAR fusion transcript by reverse transcription-polymerase chain reaction (RT-PCR) tested negative after two courses of therapy. The median duration of treatment in the 11 patients achieving CR was 33 d at a median daily dose of 0.16 mg/kg/d. These results were validated in a multicenter trial conducted in the United States and included 40 patients with previously treated APL, all of whom had been previously exposed to ATRA (34). Thirty-four patients (85%) achieved CR. Of the 40

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Table 2 Patients With Relapsed and Refractory Acute Promyelocytic Leukemia Achieving Complete Remission After One Course of Arsenic Trioxide Therapy Reference

N

Complete Remission

% Complete Remission

Zhang, 1996 (33) Niu, 1999 (65)

42 47 25 12 40

22 40 24 11 35

52 85 96 92 85

Soignet, 1998 (26) Soignet, 2000 (77)

Fig. 1. Relapse-free survival in patients with relapsed APL treated with ATO in the US multicenter trial. Reproduced with permission from ref. 34.

patients, 2 had resistant disease and 3 died early. Analysis of both the pilot study and the multicenter trial revealed that the overall survival for all patients was 66% at 18 months, with a relapse-free survival of 50% (Fig. 1). These data clearly demonstrate that ATO is highly effective in patients with relapsed APL. The role of ATO in patients with previously untreated APL is currently being addressed in a North American Intergroup Trial in which previously untreated patients will receive induction with ATRA and chemotherapy (daunorubicin and cytarabine). Subsequently, all patients in CR are randomized to either two courses of ATO or not before receiving two courses of consolidation with daunourubicin and 1 wk of ATRA.

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Fig. 2. Survival curve of leukemic mice left untreated (▲ ▲) or treated with retinoic acid for 28 d (●), arsenic (■ ■), or both (bold line). Reproduced with permission from ref. 36.

There are data to suggest that ATO and ATRA may be synergistic (35,36). ATO decreases ATRA-induced differentiation in APL cell lines but is synergistic with ATRA in inducing differentiation in resistant cells. Conversely, ATO-induced apoptosis is less with ATRA pretreatment in NB4 cells (35,37). The synergistic activity was shown in a transgenic mouse model when Lallemand-Breitenback demonstrated that mice treated with combined ATRA/ATO have longer CR than these treated with either agent alone (Fig. 2) (36). These observations prompted studies to exploit potential synergism in humans. In a preliminary report, Dombret and colleagues treated six patients who relapsed after combination ATRA/chemotherapy with combined ATO/ATRA, whereas four patients with similar characteristics were treated with ATO alone (38). No differences were observed in toxicity or response. Despite the enhanced activity of ATRA and ATO combined, it remains to be determined if this approach improves survival in patients who are in relapse or are untreated. Some investigators explored administering low-dose ATO to reduce toxicity with maintaining efficacy. Shen and colleagues reported on 20 cases of relapsed APL treated with ATO at 0.08 mg/kg/d intravenously for 28 d, showing 80% CR rate with overall survival and relapse-free survival at 61.55% ± 15.79% and 49.11% ± 15.09% at 2 yr, respectively (39). Cardiac toxicity and gastrointestinal side effects were observed less often in the lowdose cohort compared with historical controls. The efficacy was comparable, suggesting that this approach should be studied further in a prospective clinical trial.

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7. ARSENIC IN OTHER HEMATOLOGIC MALIGNANCIES Because ATO may induce apoptosis independent of PML/RAR-α, it is plausible that ATO could have a broader role in hematologic malignancies. In vitro data in non-APL cell lines complement the known potential mechanisms proposed in APL cells. Huang and colleagues showed that ATO induces caspase activation and apoptosis in myeloid non-APL cell lines (14). This also occurred in multiple myeloma and in cells from a human megakaryocytic cell line (40–42). As in APL cell lines, angiogenesis is also inhibited by ATO in other non-APL lines, and this is believed to occur through an antiproliferative effect on human vascular endothelial cells by inhibiting the production of VEGF (31). Increased angiogenesis and VEGF production have recently been described in patients with APL (43). In that regard, ATO resembles ATRA because they both inhibit angiogenesis, allowing for exploring synergistic effects of combination regimens. These in vitro data formed the basis for several phase II clinical trials exploring the efficacy of ATO in patients with hematologic malignancies.

8. ARSENIC TRIOXIDE IN MULTIPLE MYELOMA The evidence that ATO induces apoptosis in multiple myeloma (MM) cell lines (44) and that this activity is not inhibited by interleukin (IL)-6, a known growth factor in MM, prompted investigators to explore the activity of this agent in MM (45–47). Munshi and colleagues studied the efficacy of ATO in patients with refractory MM. In that study, eligible patients had relapsed or resistant MM, at least one previous cycle of high-dose therapy with autologous stem cell transplant, and normal renal, liver, and cardiac functions (48,49). Patients in that study received ATO at 0.15 mg/kg/d for 60 d. Treatment was continued for an additional 30 d in responding patients. Of nine evaluable patients, six had chromosome 13 abnormalities, five had advanced disease, extensive bone marrow involvement, and seven had two or more courses of high-dose therapy. Preliminary results showed that of the four patients who completed more than 30-d infusion, two had more than a 50% reduction in their paraprotein, one had stable disease, and one progressed (49). Of the five patients who were treated for less than 30 d, two had stable disease and three progressed. Although the clinical responses in that trial were modest, the patient population was heavily pretreated, with the majority failing more than one autologous stem cell transplantation. Because the toxicities reported in that study were minimal, this agent is likely to be explored in combination regimens in MM and can even be considered for incorporation into front-line therapy in well-designed clinical trials, particularly in high-risk patients (48).

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Fig. 3. The apoptotic effects of ATO on Ph+ cell lines expressing p185 (BCR-Abl) and p210 (BCR-Abl) with respect to Ph- lymphoblastic cell lines. All cells were treated with 2 µM of ATO (+ represents treatment, – represents no treatment). Reproduced with permission from ref. 50.

9. ARSENIC TRIOXIDE IN CHRONIC MYELOGENOUS LEUKEMIA Puccetti and colleagues studied the apoptotic effects of ATO in several leukemic cell lines expressing BCR-ABL fusion protein, a pathognomonic feature of CML (50). This study demonstrated that ATO induces apoptosis in cells transfected with this protein compared with cells that do not and that it inhibits the proliferation of leukemic blasts that are Philadelphia chromosome (Ph) positive without affecting peripheral progenitor cells. ATO had minimal apoptotic activity in cells that did not express Ph (Fig. 3). This observation strongly suggests that clinical applications of ATO in Ph+ diseases are warranted.

10. ARSENIC TRIOXIDE IN OTHER HEMATOLOGIC MALIGNANCIES Other studies are being conducted and are currently under way in many hematologic malignancies. Investigators from China reported potential activity of ATO in 27 patients with malignant lymphoma (14 with Hodgkin’s disease, 9 with histiocytosarcoma, 2 with plasmacytoma, and 2 with lympholeukosarcoma), with a remission rate of 48% (51). Although a decade has passed by since these data were reported, the understanding of how ATO exerts its activities in many different cell lines and the outstanding results in APL renewed the

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interest in this agent. Recently, Japanese study revealed that ATO induces apoptosis in a caspase-dependent manner in human-T-leukemia virus cell lines (52). Many US institutions are evaluating ATO in refractory non-Hodgkin’s lymphoma (NHL), acute myeloid leukemia (AML), MM, myelodysplastic syndromes (MDSs) (53,54), and Hodgkin’s disease. Of significance, a phase II trial combining ATO and α-interferon (IFN) in patients with relapsed and/or refractory adult T-cell leukemia was recently reported (55). In that study, eight patients were treated, three with single-agent ATO at 0.15 mg/kg and five with a combination of ATO at 0.15 mg/kg and IFN at 9 million units/d for a maximum duration of 56 d. Median duration of therapy was 20 d, and none of the patients was able to achieve 56 d of therapy, because of either toxicity (three patients) or progression (four patients). The results of this study showed one CR, three PRs, and four progressions. At the time of this report, one patient was still alive at 20 mo of follow-up. Murgo summarized the current ongoing trails (56,57). The results on these trials are currently not published.

11. ARSENIC IN SOLID TUMORS The interest in ATO as an innovative approach in solid tumors was encouraged by the impressive results in APL and the promising preclinical data. Zhou and colleagues studied the effect of ATO in androgen-dependent and independent prostate cancer cell lines and showed apoptosis induction (58). This observation is important because it shows that ATO may have activity in the early stages of prostate cancer and potentially can be used earlier. Traditionally, most chemotherapeutic agents have been used as salvage regimens in prostate cancer when patients fail androgen blockade therapy and their disease becomes androgen independent. The ATO activity in patients with advanced prostate cancer is currently being studied in the context of a clinical trial, the results of which will be available soon. In that study, patients will receive ATO at 0.2 mg/kg/d on days 1–5 and 8–12 of a 4-wk cycle (56). Because renal cell carcinoma is highly vascular, investigators at Memorial Sloan-Kettering Cancer Center are exploring the activity of ATO in advanced disease. The antiangiogenesis properties of ATO might be effective in that setting. Patients with advanced renal cell carcinoma will receive ATO at 0.3 mg/kg/d on days 1–5 of a 4-wk schedule (56). Other studies are being conducted in transitional cell carcinoma of the bladder based on preclinical data of ATO-induced cytotoxicity in these cell lines (59). Zheng and colleagues showed that ATO induces cell death in human papilloma virus-infected cervical cell lines (60). The efficacy of ATO in advanced cervical cancer is currently being tested (56). Some other reports of activity were published in abstract form on ATO in melanoma cell lines, ovarian cancer, and breast and lung cancer cell lines (61,62).

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12. ARSENIC TRIOXIDE-ASSOCIATED TOXICITIES The most prominent adverse events with ATO in APL have included weight gain and fluid retention, leukocytosis, the APL differentiation syndrome, and prolongation of the QTc interval on the electrocardiogram (Table 3). Peripheral neuropathy, hyperglycemia, and cutaneous reactions have also been described (37,63). Among the 52 patients treated on the combined pilot and US multicenter trials, nausea was observed in 75%, cough in 65%, fatigue in 63%, headache in 60%, emesis in 58%, tachycardia in 55%, diarrhea in 53%, hypokalemia in 50%, and skin rash in 43% (34). The APL differentiation syndrome is reminiscent of the retinoic acid syndrome manifested as fever, rash, peripheral edema, pulmonary infiltrates, and pleural and pericardial effusions (64). In general, similar to the therapy for the retinoic acid syndrome, early administration of dexamethasone is effective when ATO is continued. In addition, hyperleukocytosis developed in 55% of patients in some reports, ranging from 11,900 µL to 167,000 µL (65,66). Prolongation of QTc interval on the electrocardiogram and in some situations a suggestion of sudden cardiac death has been reported (67,68). It is important that potassium and magnesium levels be kept within normal limits and that medications known to prolong QTc interval are avoided if at all possible. Among 19 patients with several hematologic malignancies treated with institutionally prepared ATO, 3 cases of torsade de pointes were reported (69). In this report, the torsade de pointes occurred early in each case and in only one case was the QTc interval reported. In the first case, serum potassium was 3.3 meq/L while the magnesium was 1.7 mg/dL. The second case had even lower potassium value at 2.4 meq/L with a magnesium level at 1.7 mg/dL. The third case was the only one with normal electrolyte levels. The calcium values were not reported. A second observation was recently reported by Westervelt and colleagues providing details of another institutionally prepared ATO in

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patients with relapsed or refractory APL, of whom three individuals experienced sudden cardiac death (68). It is important to note, however, that these three patients were all critically ill and intubated, and one had low electrolyte values, making it difficult to conclude that these fatal events are actually attributable to ATO and not other confounding issues. Prolongation of QTc interval has been studied extensively in patients with APL treated with ATO. One-thousand thirty-nine electrocardiograms from 99 patients receiving between 5 and 60 d of ATO were studied, and prolongation of QTc interval (defined as greater than 415 msec in men and greater than 479 msec in women) was observed in 69% of patients (70). All patients, however, were asymptomatic and the QTc interval returned to baseline between cycles. It has been recommended that ATO be held if the QTc interval is prolonged to 500 msec and restarted when the QTc falls to less than 460 msec off the drug. Because some reports showed less cardiac toxicity with low-dose ATO without compromising efficacy, it is crucial to investigate this approach (39). The mechanism of QTc prolongation induced by ATO is not entirely clear. Some suggest that it could be related to neuropathic injury and an effect on the cardiac sympathetic system (71,72). It needs to be determined if there is a direct effect of ATO on the myocardium. These studies are currently under way. Limited data exist on ATO in previously untreated patients with APL. Niu and colleagues reported a CR rate of 72.7% at a median of 35 d, but severe hepatotoxicity occurred in two patients, resulting in fulminant hepatic failure (65). The specific cause for the severe hepatic toxicity has not been established. In some countries, arsenicals have been considered a serious public health problem because chronic exposure can cause many health hazards (4). Arsenic is present in high concentrations in well water in many parts of the western United States, South America, and Taiwan (1). Widespread use of arsenic-containing herbicides and pesticides, its use to promote growth of poultry, and its industrial use caused this compound to be ubiquitous to a certain degree (73). Consequently, it is estimated that the average daily human intake of arsenic is about 300 µg, almost all of which is ingested with food and water (74). Despite this environmental association, arsenic has never been shown to be a carcinogen (75). The reported cases of possible association with secondary malignancies were anecdotal and were obtained from experiments in rodent models (76).

13. CONCLUSION ATO has emerged as the most active agent in patients with relapsed/refractory APL and is clearly the treatment of choice in patients who are resistant to retinoids. This remarkable activity, together with laboratory data suggesting the possible role in other malignancies, provides the foundation to explore potential activity of this agent. Mechanisms of action are still being studied,

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but abundant evidence exists that arsenic induces apoptosis, causes mitochondrial damage, and inhibits angiogenesis. Understanding how this compound acts will promote the investigation of combination therapies that may enhance the activity. Arsenic is well tolerated, with few serious side effects, allowing for this agent to emerge as a part of future combination therapies in many malignancies. It is possible that combining ATRA with arsenic will increase the likelihood of cure in patients with APL.

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60. Zheng J, Deng YP, Lin C, Fu M, Xiao PG, Wu M. Arsenic trioxide induces apoptosis of HPV16 DNA-immortalized human cervical epithelial cells and selectively inhibits viral gene expression. Int J Cancer 1999;82:286–292. 61. Islam M, Kirkwood J. Arsenic trioxide induces apoptosis of human melanoma cell lines invitro [abstract 1435]. Proc ASCO 2001;360a. 62. Zeng L. Arsenic trioxide-induced apoptosis in breast and lung cancer cell lines [abstract 2350]. Proc AACR 2001;437. 63. Che-Pin L, Huang MJ, Chang IY, Lin WY, Sheu YT. Retinoic acid syndrome induced by arsenic trioxide in treating recurrent all-trans retinoic acid resistant acute promyelocytic leukemia. Leukemia Lymphoma 2000;38:195–198. 64. Tallman MS, Andersen JW, Schiffer CA, et al. Clinical description of 44 patients with acute promyelocytic leukemia who developed the retinoic acid syndrome. Blood 2000;95:90–95. 65. Niu C, Yan H, Yu T, et al. Studies on treatment of acute promyelocytic leukemia with arsenic trioxide: remission induction, follow-up, and molecular monitoring in 11 newly diagnosed and 47 relapsed acute promyelocytic leukemia patients. Blood 1999;94:3315–3324. 66. Camacho LH, Soignet SL, Chanel S, et al. Leukocytosis and the retinoic acid syndrome in patients with acute promyelocytic leukemia treated with arsenic trioxide. J Clin Oncol 2000;18:2620–2625. 67. Ohnishi K, Yoshida H, Shigeno K, et al. Prolongation of the QT interval and ventricular tachycardia in patients treated with arsenic trioxide for acute promyelocytic leukemia. Ann Intern Med 2000;133:881–885. 68. Westervelt P, Brown RA, Adkins DR, et al. Sudden death among patients with acute promyelocytic leukemia treated with arsenic trioxide. Blood 2001;98:266–271. 69. Unnikrishnan D, Dutcher JP, Varshneya N, et al. Torsades de pointes in 3 patients with leukemia treated with arsenic trioxide. Blood 2001;97:1514–1516. 70. Barbey JT, Soignet S. Prolongation of the QT interval and ventricular tachycardia in patients treated with arsenic trioxide for acute promyelocytic leukemia. Ann Intern Med 2001;135:842. 71. Roden DM, Lazzara R, Rosen M, Schwartz PJ, Towbin J, Vincent GM. Multiple mechanisms in the long-QT syndrome. Current knowledge, gaps, and future directions. The SADS Foundation Task Force on LQTS. Circulation 1996;94:1996–2012. 72. Viskin S. Long QT syndromes and torsade de pointes. Lancet 1999;354:1625–1633. 73. Klaassen C. Heavy metals and heavy-metal antagonists. In: Hardman G, Limbrid, eds. Goodman & Gilman’s The Pharmacological Basis of Therapeutics. New York: McGrawHill; 1996:1649–1672. 74. Landrigan PJ. Arsenic—state of the art. Am J Ind Med 1981;2:5–14. 75. Novick SC, Warrell RP Jr. Arsenicals in hematologic cancers. Semin Oncol 2000;27:495–501. 76. Chan P, Huff J. Arsenic carcinogenesis in animals and in humans: mechanistic, experimenta, and epidemiological evidence. J Environ Sci Health 1997;C15:83–122. 77. Soignet S, Frankel S, Tallman M, Dour D, Scheinberg D. Arsenic trioxide (ATO) in relapsed acute promyelocytic leukemia (APL): the combined results and follow-up from the US pilot and multicenter trials [abstract 3575]. Blood 2000;96:827a.

Acid 13 inAll-Trans-Retinoic the Treatment of Acute Promyelocytic Leukemia Pierre Fenaux, MD, PhD and Laurent Degos, MD, PhD CONTENTS INTRODUCTION BACKGROUND: RESULTS OF CHEMOTHERAPY ALONE IN ACUTE PROMYELOCYTIC LEUKEMIA FIRST RESULTS OBTAINED WITH ALL-TRANS-RETINOIC ACID ALONE IN ACUTE PROMYELOCYTIC LEUKEMIA ALL-TRANS-RETINOIC ACID FOLLOWED BY INTENSIVE CHEMOTHERAPY IN NEWLY DIAGNOSED ACUTE PROMYELOCYTIC LEUKEMIA CURRENT ISSUES CONCERNING TREATMENT COMBINING ALL-TRANS-RETINOIC ACID AND CHEMOTHERAPY IN NEWLY DIAGNOSED ACUTE PROMYELOCYTIC LEUKEMIA MAINTENANCE TREATMENT IN ACUTE PROMYELOCYTIC LEUKEMIA PROGNOSTIC FACTORS IN PATIENTS TREATED WITH ALL-TRANS-RETINOIC ACID AND CHEMOTHERAPY TREATMENT OF RELAPSING ACUTE PROMYELOCYTIC LEUKEMIA ALL-TRANS-RETINOIC ACID SYNDROME AND OTHER SIDE EFFECTS REFERENCES

1. INTRODUCTION All-trans-retinoic acid (ATRA) can induce complete remission (CR) in most patients with acute promyelocytic leukemia (APL) through in vivo differentiation of APL blasts. However, it cannot eliminate the leukemic clone and must From: Biologic Therapy of Leukemia Edited by: M. Kalaycio © Humana Press Inc., Totowa, NJ

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be used in combination with anthracycline-based chemotherapy. It has now been demonstrated that the combination of ATRA and chemotherapy gave better survival than chemotherapy alone in newly diagnosed APL, due to fewer relapses and to a slightly higher CR rate. It is also probable that maintenance treatment with ATRA, and possibly with low-dose chemotherapy, can further reduce the incidence of relapse. Overall, more than 90% newly diagnosed patients with APL can achieve CR, and approx 75% can be cured by the combination of ATRA and chemotherapy. ATRA syndrome remains the major combination of ATRA treatment, which should be prevented by addition of chemotherapy and/or dexamethasone in case of raising white blood cell (WBC) counts. Until the late 1980s, intensive cytoreductive chemotherapy, usually combining an anthracycline and cytosine arabinoside (AraC) was the only effective treatment for APL, likewise for other types of acute myeloid leukemia (AML). The demonstration that ATRA could differentiate APL blasts both in vitro and in vivo has greatly improved the therapeutic approach for APL. ATRA and other retinoids, on the other hand, have limited efficacy in other types of leukemias.

2. BACKGROUND: RESULTS OF CHEMOTHERAPY ALONE IN ACUTE PROMYELOCYTIC LEUKEMIA APL is a specific type of AML, characterized by the morphology of blast cells (abnormal promyelocyte) by t(15;17) translocation, which fuses the PML gene on chromosome 15 to the retinoid acid receptor (RAR) α on chromosome 17 and by a coagulopathy combining disseminated intravascular coagulation (DIC) and fibrinolysis (1,2). Using anthracycline AraC regimens, CR rates of only 50% to 60% had generally been reported in the 1970s, but results subsequently improved, and CR rates of 70% to 80% were reported in the 1980s (3–5). Failure to achieve CR in early reports was mainly due to central nervous system (CNS) bleeding during the first days of treatment or to sepsis during the phase of aplasia, whereas resistant leukemia was generally seen in less than 10% of the patients, probably reflecting the high sensitivity of APL cells to anthracyclines. Of note is that it remained unclear if anthracycline-AraC combinations were superior to anthracyclines alone if the latter were given at high dose, e.g., at least 300 mg/m2 during induction for daunorubicin. Other anthracyclines, including zorubicin or idarubicin, were at least as effective as daunorubicin in APL, whereas Amsa was probably less effective than anthracyclines (6). Significant coagulopathy, present at diagnosis in 80% of APL cases, is worsened (or triggered in the remaining patients) by the onset of chemotherapy. Intensive platelet support during chemotherapy is crucial in the management of APL coagulopathy and clearly reduces the incidence of hemorrhagic deaths.

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Intensive platelet support is especially important in patients with hyperleukocytosis, who have an increased risk of early death due to bleeding. By contrast, the role of heparin and antifibrinolytic agents is unproven (5,7). The optimal postinduction chemotherapy remains controversial in APL. In AML as a whole, it is proved that intensive consolidation chemotherapy generally improves outcome, by comparison to milder consolidation courses followed by prolonged maintenance therapy. However, in APL, two studies have suggested that prolonged maintenance chemotherapy with 6 mercaptopurine and methotrexate can prolong remissions when compared with shorter consolidation regimens (8,9). Age older than 50 yr (9), hyperleukocytosis at diagnosis (5), microgranular APL variant, severe bleeding at diagnosis, and or major thrombocytopenia were associated with a higher risk of early death in newly diagnosed patients with APL treated with chemotherapy alone. Shorter remissions were seen in patients with hyperleukocytosis (3) and in patients with microgranular APL variant (9). Thus, anthracycline-AraC regimens with sufficient anthracycline dosage, associated with intensive platelet support during induction, yielded CR in 75% to 80% of newly diagnosed patients with APL, with a risk of early death due to bleeding of approx 10% to 15%. With anthracycline-based consolidation and possibly maintenance chemotherapy, median CR duration ranged from 11 to 25 mo so that only 35% to 45% of the patients could be cured by chemotherapy alone. Patients with high leucocyte counts had a particularly poor prognosis with chemotherapy alone, because their CR rates were only 50% to 60% and their risk of relapse was high.

3. FIRST RESULTS OBTAINED WITH ALL-TRANS-RETINOIC ACID ALONE IN ACUTE PROMYELOCYTIC LEUKEMIA In the first reports of ATRA therapy (10–13), CR rates of approx 90% were reported in newly diagnosed and first relapse APL, generally with a 45 mg/m2/d dose of ATRA. The presence of Auer rods in neutrophils, the absence of aplasia, and the study of X chromosome-linked polymorphisms showed that response was not obtained by cytotoxicity but by differentiation of APL blasts into neutrophils, leading to progressive replacement of leukemic hematopoiesis by normal polyclonal hematopoiesis (10–12,14,15). Recent findings from our group also showed a correlation between in vitro differentiation of APL blasts by ATRA and with clinical results obtained in vivo with this drug (16). Rapid improvement of coagulopathy, instead of the initial worsening observed with conventional chemotherapy, was also seen. These experiences, however, showed two major drawbacks to ATRA treatment. The first, mainly in newly diagnosed APL, was a rapid rise in leukocytes

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in one-third to one-half of the patients, accompanied by clinical signs of “ATRA syndrome,” which proved fatal in some patients (11,17,18). Addition of intensive anthracycline-AraC chemotherapy reduced leukocyte counts and allowed most patients to enter CR (17). High-dose dexamethasone also had a favorable effect on ATRA syndrome. ATRA therapy was also associated with development of resistance to this drug: patients who achieved CR with ATRA and received either ATRA alone or low-dose chemotherapy for maintenance therapy generally relapsed within a few months of CR achievement (11,19). These findings led clinicians to administer a treatment that combined ATRA and intensive chemotherapy in APL.

4. ALL-TRANS-RETINOIC ACID FOLLOWED BY INTENSIVE CHEMOTHERAPY IN NEWLY DIAGNOSED ACUTE PROMYELOCYTIC LEUKEMIA Nonrandomized studies and two randomized trials have demonstrated the superiority of combined treatment with ATRA and intensive chemotherapy over intensive chemotherapy alone in newly diagnosed APL.

4.1. Nonrandomized Studies These studies are summarized in Table 1. In the first study, 26 newly diagnosed cases of APL treated with ATRA until CR, followed by three courses of daunorubicin-AraC were compared with a historical control group treated by chemotherapy alone (17,20). ATRA followed by chemotherapy slightly (but not significantly) improved the CR rate and sharply reduced the number of early relapses occurring within 18 mo of CR achievement, whereas the number of late relapses was similar to that seen after chemotherapy alone (20). Those results have been largely confirmed (Table 1).

3.2. Randomized Studies A European trial (APL 91) comparing chemotherapy alone (three courses of daunorubicin and AraC) and ATRA followed by the same chemotherapy in newly diagnosed APL was started in 1991. In the ATRA group, the first chemotherapy course was rapidly added to ATRA if WBC were greater than 5000 mm3 at diagnosis or increased during treatment. The trial was prematurely stopped after 18 mo, because event-free survival (EFS) was significantly better in the ATRA group (20,21). The last interim analysis, performed 73 mo after closing date of the study, confirmed the significantly higher actuarial EFS, relapse rate, and survival rate in the ATRA group (Table 1) (22). These results confirmed that the combination of ATRA and chemotherapy reduced the incidence of early relapses without increasing the overall incidence of later relapses by comparison with chemotherapy alone. Of note, however, was the

Table 1 Published Experience Comparing All-Trans-Retinoic Acid Followed by Intensive Chemotherapy (and Chemotherapy Alone), in Newly Diagnosed Acute Promyelocytic Leukemia

Authors Fenaux et al. (1992) (17)

Warrell et al. (1994) (25)

Complete No. of Remission Patients Rate, % 26

Comparison with Chemotherapy Alone Follow-up

96

85

Kanamaru et al. (1995) (47)

109

89

Fenaux et al. (1993, 1994, and 2000) (20,21,22) (successive updates) Tallman et al. (1997) (23)

54

91

172

72

209

49

Type of Comparison Historical (29 cases)

EFS: 62% at 4 yr DFI: 70% at 4 yr survival: 77% at 4 yr Median CR duration not reached (3+– 38+mo) median survival not reached

Historical (80 cases)

Historical (64 cases) EFS at 23 mo: 75% DFS at 23 mo: 81% EFS: 63% at 4 yr Relapse: 31% at 4 yr*

Survival: 76% at 4 yr* DFS: 67% at 3 yr Survival: 77% at 3 yr

Randomized (47 cases)

Randomized (174 cases)

EFS=Event-free survival; DFI = disease-free interval; DFS = disease-free survival. * Results updated at the reference date of January 1, 2000.

Results of Chemotherapy

p

Complete remission (CR) rate: 76% EFS: 28% at 4 yr DFI: 42% at 4 yr survival: 40% at 4 yr Median CR duration 14 mo Median survival 17 mo

NS <0.01 <0.05 <0.01 0.008 0.0009

CR rate: 70% EFS at 23 mo: 48% DFS at 23 mo: 65% CR rate: 81% EFS: 17% at 4 yr* Relapse: 78% at 4 yr

0.004 0.0007 0.07 NS 10–4 0.03

Survival: 49% at 4 yr* CR rate: 69% DFS: 32% at 3 yr Survival: 50% at 3 yr

NS <0.001 <0.001

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occurrence of two late relapses (at 58 and 74 mo, respectively) in patients treated with ATRA combined with chemotherapy, whereas no relapse was seen beyond 43 mo after chemotherapy alone. It is therefore important to determine, with longer follow-up of other published APL series, if a few late relapses can indeed occur after ATRA combined with chemotherapy and whether maintenance treatment can reduce or eliminate them. A US intergroup study randomized ATRA followed by chemotherapy and chemotherapy alone in newly diagnosed APL, and patients who achieved CR were further randomized between no maintenance and ATRA maintenance (see following). The CR rate was similar in patients who received and did not receive ATRA for induction, but the incidence of relapse was significantly lower in patients who received ATRA during induction, compared with those who received chemotherapy alone, and this translated into survival differences (23) (Table 1). Recently, it has been proved that the addition of ATRA to chemotherapy can somewhat increase CR rates compared with chemotherapy alone. Indeed, two recently closed European trials of ATRA combined with chemotherapy in newly diagnosed APL, which overall included approx 1000 patients, showed CR rates of 92% and 93%, respectively (20,24). Thus, with better knowledge of the use of ATRA (and especially of the prophylaxis of its major side effect, the ATRA syndrome) high CR rates above 90% can be achieved by combining this drug with chemotherapy in newly diagnosed APL. By contrast, CR rates above 80% have rarely been reported in newly diagnosed APL treated with chemotherapy alone. Finally, it was shown that treatment with ATRA led to significantly fewer platelet and red blood cell (RBC) transfusions, less days with fever and with antibiotics, shorter hospital stay, and overall lower cost than treatment with chemotherapy (25).

5. CURRENT ISSUES CONCERNING TREATMENT COMBINING ALL-TRANS-RETINOIC ACID AND CHEMOTHERAPY IN NEWLY DIAGNOSED ACUTE PROMYELOCYTIC LEUKEMIA 5.1. Duration and Dosing of All-Trans-Retinoic Acid During Induction Treatment Most centers administer ATRA until achievement of hematologic CR, which usually occurs after 40 to 60 d if ATRA is used alone and after fewer than 30 d if chemotherapy is combined with ATRA from the onset. A British Medical Research Council randomized study compared a short course (5 d) of ATRA followed by chemotherapy to a long course of ATRA (until CR achievement) combined with the same chemotherapy in newly diagnosed APL. A better outcome was seen in the latter group, demonstrating that longer ATRA adminis-

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tration during induction is required (26). It is, however, unknown whether discontinuation of ATRA before CR achievement (e.g., after 15 to 20 d of treatment) is sufficient to achieve full activity of the drug, particularly in reduction of the incidence of relapses. Although ATRA is generally used at a dose of 45 mg/m2/d, it has been shown that lower doses, i.e., 25 mg/m2/d, gave similar CR results (27). These lower doses are often applied in children when severe headache due to ATRA develops (28). It is not certain, however, if the additive or synergistic effect obtained with ATRA at 45 mg/m2/d and chemotherapy on reducing the incidence of relapses in APL would be similar with lower doses of ATRA.

5.2. Scheduling of All-Trans-Retinoic Acid and Chemotherapy in Acute Promyelocytic Leukemia The original combinations of ATRA and chemotherapy in APL used ATRA alone until CR achievement, followed by chemotherapy. The European group (APL 93 trial) randomized newly diagnosed APL patients with WBC counts ≤ 5000 mm3 between ATRA followed by chemotherapy (ATRA→chemotherapy) and ATRA plus chemotherapy (ATRA + chemotherapy, where chemotherapy was started on day 3 of ATRA treatment). The CR rate was similar in the two groups, but relapses at 2 yr were significantly less frequent in the ATRA + chemotherapy group (69). This suggested that the “additive” or “synergistic” effect of ATRA and chemotherapy on reducing the incidence of relapse in APL was optimal when the two treatment modalities were administered together. Also of note was that early addition of chemotherapy to ATRA reduced the incidence of ATRA syndrome (see following section).

5.3. Role of AraC in Acute Promyelocytic Leukemia Chemotherapy For treatment with chemotherapy alone, it is unclear whether it is useful to combine AraC to anthracyclines in APL treatment, when chemotherapy is combined to ATRA. Recently, Sanz et al. (29) treated 100 cases of newly diagnosed APL with ATRA and four successive courses of chemotherapy with idarubicin or mitoxantrone alone, followed by maintenance treatment with intermittent ATRA and low-dose continuous chemotherapy. The CR rate was 89%, and the 2-yr EFS was 79%. After the end of chemotherapy, 93% of the patients were polymerase chain reaction (PCR) negative. No mortality and limited morbidity were seen during consolidation chemotherapy, whereas a 5% mortality was observed by the European APL group after consolidation chemotherapy with two daunorubicin-AraC courses (69). Updated results of this study, with longer follow up, did not show a increased incidence of late relapses (30). Estey et al. (31) also obtained favorable results in APL using ATRA followed by idarubicin without AraC in 43 patients. The European APL group is currently randomizing newly diagnosed patients with APL between

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treatment with ATRA + daunorubicin and treatment with ATRA + daunorubicin + AraC.

6. MAINTENANCE TREATMENT IN ACUTE PROMYELOCYTIC LEUKEMIA Two randomized studies strongly support that maintenance treatment has a beneficial role in newly diagnosed APL treated with ATRA and consolidation chemotherapy. The US intergroup randomized patients who received ATRA followed by three daunorubicin-AraC courses to continuous maintenance with ATRA (45 mg/m2/d) during 1 yr or no maintenance (23). The incidence of relapse was significantly lower in patients who received maintenance ATRA (10 of 46 cases) than in patients who received no maintenance (21 of 54 patients). Liver toxicity of the treatment was, however, relatively important. Patients who had received no ATRA during induction therapy also benefited from ATRA maintenance. In addition, patients who received chemotherapy alone followed by ATRA maintenance had a similar outcome to patients who received ATRA followed by chemotherapy but no maintenance ATRA (23). The European APL group (APL 93 trial) randomized patients who had achieved CR with a combination of ATRA and chemotherapy (ATRA→ chemotherapy or ATRA + chemotherapy) between no maintenance, maintenance with ATRA (45 mg/m2/d) 15 d every 3 mo, continuous low-dose chemotherapy with 6 mercaptopurine (6MP) and methotrexate, or both during 2 yr, using a 2-by-2 factorial design (69). The rationale for intermittent rather than continuous ATRA for maintenance was based on pharmakokinetic studies showing a progressive decrease of serum peak levels of ATRA after a few weeks of treatment, due to hypercatabolism of the drug (32–34). This hypercatabolism was reversible a few weeks after drug discontinuation (34). The rationale for low-dose maintenance chemotherapy was based on two nonrandomized studies. The incidence of relapse after 2 yr was 25% in patients who received no maintenance ATRA vs 13% in those who received maintenance ATRA (p = 0.02) and 27% in patients who received no maintenance chemotherapy vs 11% in those who received maintenance chemotherapy (p = 0.0003). Furthermore, an additive effect of intermittent ATRA and low-dose chemotherapy in reducing the risk of relapse was seen. Regarding survival, the effect was significant for maintenance with chemotherapy but was only borderline for ATRA, at least with the current follow up (20). In addition, patients with WBC counts higher than 5000/mm3 who remain at higher risk of relapse after ATRA and intensive chemotherapy benefitted particularly from maintenance with both chemotherapy and ATRA. Finally, liver toxicity (and other toxicities) were moderate with intermittent ATRA.

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7. PROGNOSTIC FACTORS IN PATIENTS TREATED WITH ALL-TRANS-RETINOIC ACID AND CHEMOTHERAPY Although a large improvement in outcome has been observed with the combination of ATRA and chemotherapy, not all patients with newly diagnosed APL achieve CR and some patients still relapse.

7.1. Prognostic Complete Remission Achievement Factors Five percent to 10% of patients with APL fail to achieve CR with ATRA and chemotherapy, almost exclusively due to early death, because resistance to ATRA is exceptional (probably less than 1/500) in cytogenetically (t[15;17]) or molecularly (PML-RARα rearrangement) confirmed APL (20,24). Major causes of early death include CNS bleeding, ATRA syndrome (see following section), and sepsis, the latter usually occurring during the chemotherapyinduced aplasia phase. High WBC counts and older age remain the major risk factors for early death in APL (69).

7.2. Prognostic Relapse Factors Using a combination of ATRA and chemotherapy followed by maintenance treatment, approx 10% to 15% of patients with APL still relapse. Both pretreatment factors and evaluation of minimal residual disease (MRD) by reverse transcription (RT)-PCR analysis of PML-RARα rearrangement contribute to evaluation of the relapse risk. 7.2.1. PRETREATMENT FACTORS Pretreatment factors include high WBC counts in all reports (Table 1) (5), even if, as seen in previous section, results of the European APL group suggest that maintenance treatment may particularly reduce the risk of relapse in this population (20). Most of the other risk factors described, including morphologic M3 variant, low platelet count, CD34 or CD2 expression on blast surface, presence of the short isoform (S isoform or bcr3) and possibly the rare bcr2 (or V) breakpoint in PML-RARα, as compared to bcr1 (or L breakpoint), are generally correlated to high WBC counts (35,36). On the other hand, a metaanalysis of Gimema and Pethema protocols in APL, which both included maintenance with ATRA and low-dose chemotherapy, showed that WBC and platelet counts had independent prognostic value for relapse. Disease-free survival (DFS) at 4 yr was 98% in patients with platelets > 50,000/mm3, 90% in patients with platelets < 50,000/mm3 but WBC < 10,000/mm3, and only 68% in patients with platelets < 50,000/mm3 and WBC > 10,000/mm3 (4). Prognostic factors independent of the WBC count include CD13 and CD56 (37) expression, associated with an increased risk of relapse in some studies and importance of in vitro differentiation of APL blasts with ATRA (16),

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whereas cytogenetic abnormalities in addition to t(15;17) do not confer a higher relapse risk (3,30,38). 7.2.2. MONITORING MINIMAL RESIDUAL DISEASE A good correlation has been found in APL between RT-PCR findings and the risk of relapse. Patients with detectable PML-RARα fusion mRNA by RTPCR at the end of consolidation and perhaps, more importantly, patients with positive findings after a phase of negative results are at high risk of relapse (16,24,39,40). RT-PCR results should however always be interpreted carefully. First, for unknown reasons, PML-RARα fusion transcript appears to be easily degraded in bone marrow samples, often making results uninterpretable. In addition, despite consensus meetings (41), there may be a certain interlaboratory variation in the sensitivity of RT-PCR, especially because some laboratories perform one-round PCR and others two-round PCR. Thus, comparison between successive samples rather than interpretation of one given sample is advised. Quantitative PCR techniques, especially with Taqman probes, allow better analysis of an increase or decrease of fusion PML-RARα mRNA in successive samples and should improve clinical interpretation of results and therapeutic decisions (42).

7.3. Extramedullary Relapses A relatively large number of cases of extramedullary relapses have been reported in APL treated with ATRA and chemotherapy: 13 of the 97 relapses in a Gimema group study (43), 3 of the 75 relapses in the European APL 93 trial (69), and 3 of the 28 relapses in an MRC study (43,44). Extramedullary relapse in these studies mainly included the CNS (in 14 of the 21 cases) and less often the skin or other organs. CNS relapse in 10 of the 14 cases was associated with morphologic or cytogenetic evidence of marrow relapse. It is uncertain, however, if there is a real increase in the incidence of extramedullary relapses during the last few years, which could be attributable in some way to the advent of ATRA.

8. TREATMENT OF RELAPSING ACUTE PROMYELOCYTIC LEUKEMIA 8.1. Treatment or Retreatment with All-Trans-Retinoic Acid Most (85% to 90%) of the patients previously treated with chemotherapy alone who are in first relapse can achieve a second CR with ATRA (19,25,45). However, because ATRA is now administered during front-line therapy, most patients in relapse have already received ATRA. Relapses occurring shortly after ATRA discontinuation are usually resistant to ATRA, even at higher doses (25,46). Pharmacokinetic studies have indeed shown that prolonged use of

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ATRA is associated with hypercatabolism of the drug through cytochrome p450 mechanisms, with dramatic reduction of the serum peak levels of ATRA (33). Later relapses, however, may respond to ATRA. Indeed, in the APL 91 European trial, all 10 patients initially treated with ATRA who relapsed and were retreated with ATRA achieved a second CR (38). All those relapses occurred more than 6 mo after CR achievement (i.e., more than 6 mo after ATRA discontinuation), and pharmokinetic studies indeed suggest that the hypercatabolism of ATRA induced by treatment with this drug is reversible after a few weeks of ATRA discontinuation (34,46). Another reason for those good responses could be that in some of those patients, chemotherapy was rapidly added to ATRA, generally due to increasing WBC counts, and this possibly overcame partial resistance to ATRA. Recently, the European APL group completed a study of ATRA followed by intensive chemotherapy with etoposide, mitoxantrone, and AraC (EMA) in 50 patients with APL in first relapse, 39 of whom had received ATRA during their first-line treatments Median duration of first CR was 17 mo (minimum duration 6 mo). Of the 39 patients previously treated with ATRA, 13 received ATRA alone for salvage and they all achieved CR. Twenty-six also received early chemotherapy, due to high WBC counts at relapse or increasing WBC counts with ATRA, and 24 achieved CR (47). The long-term outcome of patients initially treated with ATRA who relapse and achieve a second CR after retreatment with ATRA, with or without chemotherapy, is not known precisely due to the small number of published cases with sufficient follow-up. Long-term results of the APL 91 trial suggest that allogeneic and possibly autologous stem cell transplantation can durably salvage a proportion of those cases (22). In our recent study in relapsing APL, however, allogeneic bone marrow transplantation (BMT) was associated with a high rate of toxic mortality, probably due to the intensity of consolidation chemotherapy (EMA protocol) before the procedure (47). By contrast, 17 of the 22 patients who were autografted were still in second CR, with a 3-yr DFS of 77%. Nine of them had already reached a second CR longer that their first CR. PML-RARα transcript was no longer detectable in eight of the nine patients tested (73). Finally, in a few anecdotal cases, we observed a second CR that was longer than the first CR, using maintenance treatment combining intermittent ATRA and continuous 6MP and MTX.

8.2. Other Retinoids As maintenance treatment with ATRA is used by more and more groups, a large proportion of first relapses now concern patients who are receiving or have recently received ATRA and will probably show resistance to this drug. In addition, in some patients, resistance to ATRA is not due to hypercatabolism of the drug but is irreversible and due to the occurrence of point mutations in the

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DNA binding domain of the RARα gene (48). Finally, patients in second or third relapse generally show in vivo and in vitro ATRA resistance. In ATRA resistance preliminary studies suggest that liposomal ATRA may still induce some responses, possibly because this mode of ATRA administration does not induce hypercatabolism of the drug and reduction of its plasma levels (49). Favorable preliminary results with 9cis retinoic acid, a retinoid that interacts with both RXR and RAR receptors and does not induce its own catabolism to the same extent as ATRA, have been published, but these responses occurred in patients who were possibly not ATRA resistant (50). Am80, a synthetic retinoid more potent than ATRA as an in vitro differentiation inducer, induced CR in 58% of a cohort of patients with APL who were relapsing (51). However, because all those patients had discontinued ATRA for at least 18 mo, it is unclear whether Am80 was superior to ATRA in this situation and whether it was able to overcome ATRA resistance.

8.3. Arsenic Trioxide and Other Approaches In fact, the advent of arsenic trioxide (ATO) (As2O3) has modified the outcome of patients refractory to ATRA (see Chapter 12), and this drug is currently tested earlier in APL treatment. Other new approaches are currently only at an early study stage; they include inhibitors of histone deacetylase (an enzyme associated with transcriptional repression), particularly sodium phenylbutyrate, used with success in animal models and in a few patients with APL resistant to ATRA (52), and anti-CD33 monoclonal antibodies (gemtuzumab ozogamicin) (53).

9. ALL-TRANS-RETINOIC ACID SYNDROME AND OTHER SIDE EFFECTS ATRA therapy is usually well tolerated and its side effects moderate, with the exception of ATRA syndrome.

9.1. All-Trans-Retinoic-Acid Syndrome 9.1.1. INCIDENCE AND CLINICAL SIGNS A precise description of clinical symptoms of ATRA syndrome was made by Frankel et al. (18) and two large series of cases of ATRA syndrome have recently been published (54,55). Clinical signs of ATRA syndrome combine fever, respiratory distress, weight gain, lower extremity edema, pleural or pericardial effusions, hypotension, and occasionally renal failure. These signs are preceded by increasing WBC counts in the majority of cases, but some patients develop symptoms at normal WBC counts (25). Some cases of ATRA syndrome can also occur upon recovery from aplasia in patients who have received early chemotherapy and are still receiving ATRA (54).

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The ATRA syndrome occurred in 6% to 27% of the patients in previous reports, and mortality of the syndrome ranged from 8% to 15% (18,21,23,56,57). In the European APL 93 trial, patients who survived ATRA syndrome had a higher risk of bone marrow relapse than patients who had no ATRA syndrome (32% vs 15% at 2 yr) (54). In another study, the occurrence of ATRA syndrome was associated with a possibly higher risk of subsequent extramedullary relapse (58). Finally, ATRA syndrome has not been reported during maintenance treatment with ATRA. 9.1.2. PATHOPHYSIOLOGY OF HYPERLEUKOCYTOSIS AND ALL-TRANS-RETINOIC ACID SYNDROME The pathophysiology of hyperleukocytosis and ATRA syndrome is still not completely understood. Clinical signs of the ATRA syndrome are reminiscent of those observed in endotoxic shock and in the adult respiratory distress syndrome (ARDS), and a possible stimulatory effect of ATRA on cytokine expression by APL cells has been envisaged: induction of IL-1b and G-CSF secretion by APL cells under ATRA may contribute to hyperleukocytosis in vivo; secretion of IL-1b, IL6, TNFα, and IL-8, which are involved in leucocyte activation and adherence, could have a pathogenetic role in ATRA syndrome (59). It has also been shown that ATRA-induced aggregation of NB4 cells (an APL cell line), a process mediated by LFA1 and ICAM2 adhesion molecules and reversed by addition of methylprednisolone (60). These findings suggest that modification of the adhesive properties of APL cells by ATRA could play a role in ATRA syndrome. 9.1.3. PROPHYLAXIS AND TREATMENT Prevention and early treatment of ATRA syndrome are required to improve its outcome and are currently attempted by two approaches. One of them, mainly used by the European and Japanese groups (17,21,56), consists of adding chemotherapy from the onset of ATRA in patients presenting with high WBC counts (WBC > 5000/mm3 in the European trial or > 3000/mm3 in the Japanese trials) or when increases in the WBC are seen with ATRA. This approach has been associated with a low incidence of fatal ATRA syndrome but leads to early chemotherapy administration in many patients. However, intensive chemotherapy, if not administered early, would have to be administered later as consolidation treatment. In addition, results of the European APL 93 trial suggest that early onset of chemotherapy (ATRA+chemotherapy) reduces the incidence of relapse compared with ATRA followed by chemotherapy (ATRA→chemotherapy). Finally, in the APL 93 trial, there were fewer cases of ATRA syndrome in patients who received ATRA + chemotherapy (11%) compared with those treated by ATRA→chemotherapy (18%) (19).

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The usual US approach is to prevent ATRA syndrome by high-dose intravenous (iv) corticosteroids (dexamethasone, 10 mg iv twice daily for 3 or more d) as soon as the first symptoms occur. This attitude proved effective in the US intergroup study, for both preventing the ATRA syndrome and reducing its mortality (23). In fact, there is consensus that patients presenting with high WBC counts (e.g., > 15,000 to 20,000/mm3) will often develop severe ATRA syndrome with ATRA alone and require chemotherapy and iv dexamethasone from the onset of treatment. Some of these patients even have symptoms analogous to those of the ATRA syndrome at diagnosis (61).

9.2. Coagulopathy and Thrombosis No worsening of the bleeding tendency is observed in patients with APL undergoing ATRA therapy. In the European APL 91 trial, median time to disappearance of significant coagulopathy was 6 d after chemotherapy alone and 3 d in the ATRA group (p = 0.001) (21). ATRA therapy may be especially important in reducing the severity of the bleeding tendency in patients with hyperleukocytic APL, a population still at relatively high risk of early death with chemotherapy alone. On the other hand, treatment with ATRA may lead to a transient period of hypercoagulability, which could explain the few well-documented cases of thromboembolic events in patients with APL treated with ATRA (62).

8.3. Other All-Trans-Retinoic Acid Side Effects ATRA side effects include dryness of lips and mucosae, isolated fever in the absence of other signs of ATRA syndrome (or infection), increases in transaminases and triglycerides (which never required treatment discontinuation in our experience), and headache due to intracranial hypertension, which may be severe in children and associated with signs of pseudotumor cerebri (28). Lower ATRA doses (25 mg/m2/d) can reduce this side effect in children (28). Other side effects, including bone marrow necrosis, hypercalcemia (63), erythema nodosum (64), marked basophilia, severe myositis (65), Sweet syndrome (66), Fournier’s gangrene (necrotizing fasciitis of the penis and scrotum) (67), thrombocytosis (68), and necrotizing vasculitis, have rarely been reported with ATRA treatment.

REFERENCES 1. Bennett JM, Catowsky D, Danier MT, Flandrin G, Galton D, Gralnick M, Sultan C. Proposals for the classification of the acute leukemias. Br J Haematol 1976;33:451. 2. Bennett JM, Catowsky D, Danier MT, et al. A variant form of hypergranular promyelocytic leukemia (M3). Ann Intern Med 1980;92:280. 3. De Botton S, Chevret S, Sanz M, et al. Additional chromosomal abnormalities in patients with acute promyelocytic leukemia (APL) do not confer poor prognosis: results of APL 93 trial. Br J Haematol 2000;111:801–806.

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4. Sanz MA, Martin G, Rayon C, et al. A modified AIDA protocol with anthracycline-based consolidation results in high antileukemic efficacy and reduced toxicity in newly diagnosed PML/RARalpha-positive acute promyelocytic leukemia. Blood 1999;94:3015–3021. 5. Fenaux P, Pollet JP, Vandenbossche L, Dupriez B, Jouet JP, Bauters F. Treatment of acute promyelocytic leukemia: a report on 70 cases. Leukemia Lymphoma 1991;4:237–248. 6. Fenaux P, Tertian G, Castaigne S. A randomized trial of amsacrine and rubidazone in 39 patients with acute promyelocytic leukemia. J Clin Oncol 1991;9:1556–1561. 7. Tallman MS. The thrombophilic state in acute promyelocytic leukemia. Semin Thromb Hemostasis 1999;25:209–215. 8. Kantarjian H, Keating M, Walter R, et al. Role of maintenance chemotherapy in acute promyelocytic leukemia. Cancer 1987;59:1258–1263. 9. Marty M, Ganem G, Fischer J. Leucémie aiguë promyélocytaire: étude rétrospective de 119 malades traités par daunorubicine. Nouv Rev Fr Hematol 1984;24:371–378. 10. Huang M, Yu-Chen Y, Shu-Rong C, et al. Use of all trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood 1988;72:567. 11. Castaigne S, Chomienne C, Daniel MT, Berger R, Fenaux P, Degos L. All trans retinoic acid as a differentiating therapy for acute promyelocytic leukemias. I Clinical results. Blood 1990;76:1704. 12. Warrel RP, Frankel SR, Miller W, et al. Differentiation therapy of acute promyelocytic leukemia with tretinoin (all trans retinoic acid). N Engl J Med 1991;324:1385. 13. Chen ZX, Xue YQ, Zhang R, et al. A clinical and experimental study on all trans retinoic acid treated acute promyelocytic leukemia patients. Blood 1991;78:1413. 14. Chomienne C, Ballerini P, Balitrand N, et al. All trans retinoic acid in promyelocytic leukemias. II. In vitro studies structure function relationship. Blood 1990;76:1710. 15. Elliott S, Taylor K, White S, et al. Proof of differentiating mode of action of all-trans retinoic acid in acute promyelocytic leukemia using X-linked clonal analysis. Blood 1991;79:1916. 16. Cassinat B, Chevret S, Zassadowski F, for the European APL Group, et al. In vitro all-trans retinoic acid sensitivity of acute promyelocytic leukemia blasts: a novel indicator of poor patient outcome. Blood 2001;98(9):2862–2864. 17. Fenaux P, Castaigne S, Dombret H, et al. All-transretinoic acid followed by intensive chemotherapy gives a high complete remission rate and may prolong remissions in newly diagnosed acute promyelocytic leukemia: a pilot study on 26 cases. Blood 1992;80:2176–2181. 18. Frankel SR, Eardley A, Lauwers G, Weiss M, Warrell R. The “retinoic acid syndrome” in acute promyelocytic leukemia. Ann Intern Med 1992;117:292. 19. De Botton S, Coiteux V, Chevret S, et al. Early onset of chemotherapy can reduce the incidence of ATRA syndrome in newly diagnosed acute promyelocytic leukemia (APL). Results of a randomized study. Blood 2001;98(11):766a. 20. Fenaux P, Wattel E, Archimbaud E, et al. Prolonged follow up confirms that all transretinoic acid (ATRA) followed by chemotherapy reduces the risk of relapse in newly diagnosed acute promyelocytic leukemia (APL). Blood 1994;84:666–667. 21. Fenaux P, Le Deley MC, Castaigne S, et al. Effect of all transretinoic acid in newly diagnosed acute promyelocytic leukemia. Results of a multicenter randomized trial. Blood 1993;82:3241–3249. 22. Fenaux P, Chastang C, Chevret S, et al. A randomized comparison of all transretinoic acid (ATRA) followed by chemotherapy and ATRA plus chemotherapy and the role of maintenance therapy in newly diagnosed acute promyelocytic leukemia. Blood 1999;94:1192–1200. 23. Fenaux P, Chevret S, Guerci A, et al. Long term follow up confirms the benefit of all transretinoic acid in acute promyelocytic leukemia. Leukemia 2000;14:1371–1377. 24. Tallman MS, Andersenn JW, Schiffer CA, et al. All trans-retinoic acid in acute promyelocytic leukemia. N Engl J Med 1997;337:1021–1028. 25. Mandelli F, Diverio D, Avvisati G, et al. Molecular remission in PML/RARalpha-positive acute promyelocytic leukemia by combined all-trans retinoic acid and idarubicin (AIDA) therapy. Blood 1997;1014–1021.

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26. Warrell RP, Maslak P, Eardley A, Heller G, Miller WH, Frankel SR. Treatment of acute promyelocytic leukemia with all-trans retinoic acid: an update of the New York experience. Leukemia 1994;8:926–933. 27. Burnett AK, Grimwade S, Solomon E, Wheatley K, Goldstone AH. Presenting white blood cell count and kinetics of molecular remission predict prognosis in acute promyelocytic leukemia treated with all-trans retinoic acid: result of the randomized MRC Trial. Blood 1999;93:4131–4143. 28. Castaigne S, Lefebvre P, Chomienne C, et al. Effectiveness and pharmacokinetics of lowdose all-trans retinoic acid (25 mg/m2) in acute promyelocytic leukemia. Blood 1993;82:3560–3563. 29. Mahmoud HH, Hurwitz CA, Roberts WM, Santana VM, Ribeiro RC, Krance RA. Tretinoin toxicity in children with acute promyelocytic leukaemia. Lancet 1993;342:1394–1395. 30. Sanz MA, Martin G, Barragan E, PETHEMA group, et al. Risk-adapted treatment of acute promyelocytic leukemia: results of the Spanish PETHEMA trials using ATRA and anthracycline-based chemotherapy. Blood 2001;98(11):765a. 31. Estey E, Thall PF, Pierce S, Kantarjian H, Keating M. Treatment of newly diagnosed acute promyelocytic leukemia without cytarabine. J Clin Oncol 1997;15:483–490. 32. Cornic M, Delva L, Guidez F, Balitrand N, Degos L, Chomienne C. Induction of retinoic acid-binding protein in normal and malignant human myeloid cells by retinoic acid in acute promyelocytic leukemia patients. Cancer Res 1992;52:3329–3334. 33. Muindi JRF, Frankel SR, Huseltion C, et al. Clinical pharmacology of oral all-trans retinoic acid in patients with acute promyelocytic leukemia. Cancer Res 1992;52:2138–2142. 34. Adamson PC, Bailey J, Pluda J, et al. Pharmacokinetics of all-trans-retinoic acid administered on an intermittent schedule. J Clin Oncol 1995;13:1238–1241. 35. Claston DF, Reading CL, Nagarajan L, et al. Correlation of CD2 expression with PML gene breakpoints in patients with acute promyelocytic leukemia. Blood 1992;80:582–586. 36. Paietta E, Andersen J, Gallagher R, et al. The immunophenotype of acute promyelocytic leukemia (APL): an ECOG study. Leukemia 1994;8:1108–1112. 37. Murray CK, Estey E, Paietta E, et al. CD 56 expression in acute promyelocytic leukemia: a possible indicator of poor treatment outcome? J Clin Oncol 1999;17:293–297. 38. Gallagher RE, Willman CL, Slack JL, et al. Association of PML-RARalpha fusion mRNA type with pretreatment hematologic characteristics but not treatment outcome in acute promyelocytic leukemia: an intergroup molecular study. Blood 1997;90:1656–1663. 39. Biondi A, Rambaldi A, Pandolfi PP, et al. Molecular monitoring of the myl/retinoic acid receptor alpha fusion gene in acute promyelocytic leukemia by polymerase chain reaction. Blood 1992;80:492–497. 40. Diverio D, Rossi V, Avvisati G, et al. Early detection of relapse by prospective reverse transcriptase-polymerase chain reaction analysis of the PML/RARalpha fusion gene in patients with acute promyelocytic leukemia enrolled in the GIMENA-AIEOP multicenter AIDA trial. GIMENA-AIEOP multicenter AIDA Trial. Blood 1998;92:784–789. 41. Bolufer P, Barragan E, Sanz MA, et al. Preliminary experience in external quality control of RTPCR PML-RAR alpha detection in promyelocytic leukemia. Leukemia 1998;12:2024–2028. 42. Cassinat B, Zassadowski F, Balitrand N, et al. Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t(15;7) translocations using real-time RT-PCR. Leukemia 2000;14:324–328. 43. Liso V, Specchia G, Pogliani EM, et al. Extramedullary involvement in patients with acute promyelocytic leukemia: a report of seven cases. Cancer 1998;83:1522–1528. 44. Evans GD, Grimwade DJ. Extramedullary disease in acute promyelocytic leukemia. Leukemia Lymphoma 1999;33:219–229. 45. Frankel SR, Eardley A, Heller G, et al. All-trans retinoic acid for acute promyelocytic leukemia. Results of the New York study. Ann Int Med 1994;120:279–286.

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46. Delva L, Cornic M, Balitrand N, et al. Resistance to all-trans retinoic acid (ATRA) therapy in relapsing acute promyelocytic leukemia: study of in vitro ATRA sensitivity and cellular retinoic acid binding protein levels in leukemic cells. Blood 1993;82:2175–2181. 47. Thomas X, Dombret H, Cordonnier C, et al. Treatment of relapsing acute promyelocytic leukemia by all-trans retinoic acid therapy followed by timed sequential chemotherapy and stem cell transplantation. Leukemia, 2000;14:1006–1013. 48. Ding W, Li YP, Nobile LM, et al. Leukemic cellular retinoic acid resistance and missense mutations in the PML-RARalpha fusion gene after relapse of acute promyelocytic leukemia from treatment with all-trans retinoic acid and intensive chemotherapy. Blood 1998;92:1172–1183. 49. Estey EH, Giles FJ, Kantarjian H, et al. Molecular remissions induced by liposomal-encapsulated all-trans retinoic acid in newly diagnosed promyelocytic leukemia. Blood 1999;94:2230–2235. 50. Soignet SL, Benedetti F, Fleischauer A, et al. Clinical study of 9-cis retinoic acid (LGD 1057) in acute promyelocytic leukemia. Leukemia 1998;12:1518–1521. 51. Takeuchi M, Yano T, Omoto E, et al. Relapsed acute promyelocytic leukemia previously treated with all-trans retinoic acid: clinical experience with a new synthetic retinoid, AM-80. Leukemia Lymphoma 1998;31:441–451. 52. Warrell RP, Jr, He LZ, Richon V, Calleja E, Pandolfi PP. Therapeutic targeting of transcription in acute promyelocytic leukemia by use of an inhibitor of histone deacetylase. J Natl Cancer Inst 1998;90:1621–1625. 53. Estey E, Giles F, Cortes J, et al. Gemtuzumab ozogamycin (“Mylotarg”) in untreated acute promyelocytic leukemia (APL). Blood 2001;98(11):766a. 54. De Botton S, Dombret H, Sanz M, et al. Incidence, clinical features, and outcome of alltrans-retinoic acid syndrome in 413 cases of newly diagnosed acute promyelocytic leukemia. Blood 1998;92:2712–2718. 55. Tallman MS, Andersen JW, Schiffer CA, et al. Clinical description of 44 patients with acute promyelocytic leukemia who developed the retinoic acid syndrome. Blood 2000;95:90–95. 56. Kanamaru A, Takemoto Y, Tanimoto M, et al. All-trans retinoic acid for the treatment of newly diagnosed acute promyelocytic leukemia. Blood 1995;85:1202–1206. 57. Vahdat L, Maslak P, Miller WH, Early mortality and the retinoic acid syndrome in acute promyelocytic leukemia: impact of leukocytosis, low-dose chemotherapy, PMN/RAR-alpha isoform, and CD13 expression in patients treated with all-trans retinoic acid. Blood 1994;84:3843–3849. 58. Ko BS, Tang JL, Chen YC, et al. Extramedullary relapse after all-trans retinoic acid treatment in acute promyelocytic leukemia—the occurrence of retinoic acid syndrome is a risk factor. Leukemia 1999;13:1406–1408. 59. Dubois C, Schlageter MH, de Gentile A, et al. Modulation of II-6 and II-1b and G-CSF secretion by all trans retinoic acid in acute promyelocytic leukemia. Leukemia 1994;8:1750–1757. 60. Larson RS, Brown DC, Sklar LA. Retinoic acid induces aggregation of the acute promyelocytic leukemia cell line NB-4 by utilization of LFA-1 and ICAM-2. Blood 1997;90:2747–2756. 61. Stadler M, Ganser A, Hoelzer D. Acute promyelocytic leukemia. N Engl J Med 1994;330:140–141. 62. Forjaz De Lacerda J, Alves Do Carmo J, Lurdes Guerra M, Geraldes J, Forjaz De Lacerda JM. Multiple thrombosis in acute promyelocytic leukaemia after tretinoin. Lancet 1993;342:114. 63. Akiyama H, Nakamura N, Nagasaka S, Sakamaki H, Onozawa Y. Hyper-calcaemia due to all-trans retinoic acid. Lancet 1992;i:308,309. 64. Hakimian D, Tallman MS, Zugerman C, Caro WA. Erythema nodosum associated with all-transretinoic acid in the treatment of acute promyelocytic leukemia. Leukemia 1993;7:758–759. 65. Miranda N, Oliveira P, Frade MJ, Melo J, Marques MS, Parreira A. Myositis with tretinoin. Lancet 1994;344:1096.

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66. Arun B, Berberian B, Azumi N, Frankel SR, Luksenburg H, Freter C. Sweet’s syndrome during treatment with all-trans retinoic acid in a patient with acute promyelocytic leukemia. Leukemia Lymphoma 1998;31:613–615. 67. Levy V, Jeffarbey J, Aouad K, Zittoun R. Fournier’s gangrene during induction treatment of acute promyelocytic leukemia, a case report. Ann Hematol 1998;76:91–92. 68. Kentos A, Le Moine F, Crenier L, et al. All-trans retinoic acid induced thrombocytosis in a patient with acute promyelocytic leukaemia. Br J Haematol 1997;97:685–692.

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GENE THERAPY

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Gene Therapy Paul J. Orchard, MD and R. Scott McIvor, PhD CONTENTS INTRODUCTION METHODS TO ACHIEVE GENE TRANSFER AND EXPRESSION GENE THERAPY FOR LEUKEMIA/LYMPHOMA FUTURE CHALLENGES IN GENE THERAPY OF LEUKEMIA/LYMPHOMA REFERENCES

1. INTRODUCTION Gene therapy may be defined as the introduction of nucleic acids into a cell with the intention of altering function to achieve a therapeutic benefit (1). Gene therapy approaches can be divided into the manipulation of reproductive cells to maintain a genetic modification in future generations (germ-line gene therapy) or gene transfer into more differentiated tissues, such as cells of the lung, liver, or brain, or hematopoietic cells (somatic gene therapy). Clinical gene therapy protocols for cancer are of this latter category. Cells can be genetically manipulated ex vivo, which has the clear advantage of facilitating control over the gene transfer procedure, and allows testing of the cell product before the administration of the cells therapeutically. However, protocols have been devised to target malignant cells in vivo, with the intention of correcting genetic abnormalities or to result in their eradication. The Journal of Gene Medicine Web site currently lists 596 clinical trials initiated since 1989, including 376 (63%) that are related to the treatment of malignancies, with 2389 patients enrolled in cancer-related gene therapy protocols (2). A total of 46 (8%) of all gene therapy trials focus on the treatment of leukemia or lymphoid malignancies. A review of these approaches and others being considered for therapy can be categorized into several therapeutic strategies (Table 1). However, before discussing these applications, an overview of the current means by which gene transfer and expression can be achieved is warranted. From: Biologic Therapy of Leukemia Edited by: M. Kalaycio © Humana Press Inc., Totowa, NJ

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1) Marking studies to investigate leukemia biology 2) Augment immune responsiveness • Modify leukemia/lymphoma cells; increase immunogenicity, enhance vaccination • Modify effector cells; express cytokines, engineer specificity 3) Chemoprotection; modification of stem cells to provide resistance to myelosuppressive therapy 4) Repair or inactivation of genetic abnormalities • Tumor suppressor genes; repair lost function • Oncogene downregulation; inhibit expression of genes contributing to malignancy 5) Prodrug/prosuicide genes; engineer T-cells to provide graft vs leukemia, allowing selective depletion if graft vs host disease observed

2. METHODS TO ACHIEVE GENE TRANSFER AND EXPRESSION 2.1. Physical Methods of Gene Transfer The transfer of genetic material by physical means can be accomplished by using deoxyribonucleic acid (DNA) alone or with the assistance of carriers such as liposomes. These methods have recently been reviewed (3). The use of naked DNA techniques to obtain expression has shown promise, especially in tissues such as skeletal (4–7) and cardiac (8–11) muscle, and has also been effective in achieving gene transfer in endothelial cells (12) and skin (13–15). Carrier vehicles such as cationic liposomes can increase the association of negatively charged DNA and the cell membrane, a strategy that has been used in several target cells (16–18), including hematopoietic and leukemic cells (19–24). A potential advantage of using liposomes is the opportunity to modify the liposome to enhance delivery to a specific cell target (25–27). In addition, there is great flexibility in the size of the gene that can be physically transferred (28). Electroporation is another methodology that has been used to increase the gene transfer efficacy of naked DNA, and has been used in vaccination experiments in a murine model of multiple myeloma (29,30). The “gene gun” achieves transfer of genetic material by depositing DNA onto gold or tungsten microprojectiles that are accelerated to high velocity for introduction into the nucleus of the recipient cell (31). Unfortunately, physical gene transfer has not yet achieved the level of gene transfer efficiency in hematopoietic cells observed with viral vectors (24,32) but has the advantage of minimizing immunogenicity and infectious risks that are a concern with viruses (1).

2.2. Viral Vectors as Gene Transfer Vehicles Viruses have, throughout millions of years, evolved remarkably efficient means of introducing and expressing DNA or ribonucleic acid (RNA) in

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227 Table 2 Viral Vectors for Gene Therapy

Type of Virus Murine retrovirus Lentivirus Adenovirus Adenoassociated virus (AAV) Herpes Simple Virus (HSV-1)

RNA/ DNA

Mitosis Integration Required

Transgene Size

Titer (virions/mL)

ssRNA ssRNA dsDNA ssDNA

Yes Yes No Yes

Yes No No No

10 kb 10 kb 37 kb 4.9 kb

106–107 106–107 1011 1010

dsDNA

No

No

30–40 kb

109

ss = single strand; RNA = ribonucleic acid; ds = double strand; DNA = deoxyribonucleic acid.

eukaryotic cells. The biologic processes developed by viruses to achieve gene transfer can be harnessed through modification of the viral genome to achieve expression of particular genes of interest. The usefulness of a particular viral vector for gene therapy depends on several factors, including (1) the ability to modify the virus to accommodate the gene of interest while retaining the capacity of the virus to achieve efficient transduction; (2) the ability to design vectors to remain replication incompetent, because in most cases an active infection is to be avoided in the recipient; (3) the immunologic response generated to particular viruses, because these may prove significant and potentially life threatening; (4) the ability to generate reproducibly high concentrations of virus required for efficient gene transfer; and (5) the necessity of long-term expression to realize a therapeutic benefit (33–35). Characteristics of several of the viral vectors currently being used for gene therapy are listed in Table 2. The applicability of a viral vector for a given disorder is therefore related to the goal of therapy, taking into account the characteristics of the gene to be delivered, the susceptibility of different target cells to viral transduction, the proliferative state of the target cell, and the duration of expression necessary for successful intervention (35). Although a large number of viral agents are being developed for clinical gene transfer applications, for the purposes of this discussion the focus is on the vectors that have received the majority of attention in the field of gene therapy for hematologic malignancy. 2.2.1. MURINE RETROVIRAL VECTORS Retroviral vectors, particularly those based on Moloney murine leukemia viruses (MoMLV) have distinct advantages that have resulted in their wide clinical use, including the ability to transduce a broad range of target cells, achieving stable integration into the target cell genome, relatively efficient gene transfer, and, in most cases, high-level expression of the transgene. In

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Fig. 1. Murine leukemia virus and constructs for gene therapy. The wild-type retrovirus 5′ retroviral long terminal repeat (LTR) contains the promoter/enhancer elements for transcription (arrow). The packaging sequence (ψ) is necessary for the recognition of the viral RNA for encapsidation into the virus particle. The gag/pol gene, which encodes the structural proteins of the virus (gag) and the reverse transcriptase and integrase enzymes (pol) are translated from a single gene. The envelope (env) gene is expressed primarily through splicing, using the slice donor (SD) and splice acceptor (SA) sequences (A). In the common types of retroviral constructs used for gene therapy, the gag/pol and env genes are removed and replaced by a transgene that is either expressed from the promoter/enhancer of the LTR using the splicing mechanisms of the virus (B) or without splicing (C). Commonly, an internal promoter is used, which may provide expression of a second gene, such as the neomycin phosphotransferase gene.

addition, their safety record has been encouraging (36). Retroviruses contain two identical single strands of RNA, as well as viral replication enzymes within a protein core and a membrane envelope. Upon infection, the viral RNA is reverse transcribed into DNA, which integrates randomly into the host cell genome. Long terminal repeats (LTRs) located at the 5′ and 3′ ends of the genome regulate viral replication and expression of the gag, pol, and env genes (Fig. 1). In MoMLV, the gag and pol proteins are alternatively translated from the same message. The gag gene provides the structural proteins necessary for the virus, including the capsid and nucleoprotein complex. The pol gene encodes reverse transcriptase, the enzyme that generates DNA from viral RNA, and the integrase enzyme important in providing stable integration of the viral sequences into the host genome. The env gene products are viral envelope glycoproteins that determine receptor binding and cell type specificity. To generate retroviral vectors, the gag, pol, and env gene products are provided in trans, meaning that these genes need not be present in the retroviral genome to

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generate a retroviral particle. Sequences required “in cis” (i.e., within the virus genome) include those necessary for expression, replication, and packaging, including the packaging signal. Retroviral vector constructs are commonly designed in which the gag, pol, and env genes have been substituted by the gene(s) of interest. Beginning in 1983 the development of “packaging” cell lines provided stable expression of the retroviral proteins, facilitating the generation of replication incompetent retroviruses and providing a practical means of generating high titer virus (37,38). Packaging lines have been designed to provide the expression of the necessary gag, pol, and env genes while minimizing potential production of replication competent virus. Because these genes are not present in the vector, the retroviral replicative cycle is interrupted (Fig. 2). Many packaging cell lines have been generated that are capable of producing high titer virus (106–107 virions/mL). The substitution of envelope proteins from different viruses is termed pseudotyping, and has been used as a means to alter the capacity of the virus to transduce specific cell types. For instance, the ecotropic envelope gene of MoMLV mediates gene transfer into rat and murine cells (39,40), while vectors incorporating the amphotropic envelope are capable of transducing several host cell types, including rodent and human cells (41,42). Retrovirus produced from packaging cell lines using the gibbon ape leukemia virus (GALV) envelope gene are efficient in transducing human and simian cells, but do not transduce murine cells (41,43–45). Pseudotyping can also be carried out using envelope protein from viruses other than retroviruses, including the vesicular stomatitis virus G glycoprotein (VSV-G), which has abundant membrane receptors (46–48). Retroviral constructs based on MoMLV vectors were used for the first gene therapy trial, in which a retrovirus containing the adenosine deaminase (ADA) gene was used to transduce lymphocytes from 2 patients with severe combined immunodeficiency (SCID) associated with ADA deficiency in 1990 (49). In addition, encouraging results with a MoMLV-based vector was reported in the treatment of X-linked SCID by ex vivo transduction of hematopoietic stem cells (50). Retroviruses remain the most common vehicles for gene therapy; in the 46 gene therapy trials that targeted leukemia or lymphoma, 32 (70%) used retroviral vectors (51). No significant adverse events have been described thus far. Insertional mutagenesis remains the primary concern in the use of retroviral vectors, because random integration could inactivate a tumor suppressor gene or activate a proto-oncogene (1). In addition, despite the engineering of retroviral packaging lines to minimize recombination events, the potential remains for the generation of replication-competent virus. The development of lymphoma in rhesus macaques after the administration of cells transduced with a retroviral vector contaminated with replication-competent virus is a clear reminder of the theoretic risk of retroviral vectors (52–54). As MoMLV viruses are inactivated in human serum, their use for providing gene transfer in vivo is limited (55). How-

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Fig. 2. Use of replication-incompetent retrovirus to achieve gene transfer. Retroviral constructs are introduced into the packaging cell line, where clones are identified that have stable expression of the integrated construct, often by resistance to an antibiotic such as the neomycin. The packaging line contains the gag/pol and env genes that have previously been introduced and, using these proteins, the RNA produced from the construct is packaged into viral particles that are released from the cells by budding. The virus binds to specific receptors on the target cell, and it is subsequently internalized, uncoated, and dsDNA produced from the viral RNA using the reverse transcriptase that is a gene product of the pol gene. The dsDNA is transported into the nucleus of an actively dividing cell, where stable integration is achieved. Expression of the gene of interest follows.

ever, the viruses are not adversely affected by cerebral spinal fluid (56), and the clinical use of those viruses in brain tumors has been explored (57–59). 2.2.2. LENTIVIRAL VECTORS Lentiviruses, including the human immunodeficiency virus (HIV), are retroviruses containing additional regulatory sequences (tat and rev) not present in simple murine retroviruses, as well as accessory sequences (vpr, vif, vpu, and

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nef) important in the lentivirus replication cycle (35,60). There is understandable concern regarding the potential for recombination events resulting in the production of replication-competent virus derived from HIV, thus far limiting to the implementation of these viruses clinically. However, the potential of lentiviruses to transduce nondividing cells is an important advantage for many gene therapy applications (61–64). Specifically, lentiviral vectors have great potential in the transduction of human hematopoietic stem cells. There is evidence that human stem cells can be transduced with these vectors in the absence of stimulatory cytokines required for gene transfer with murine retroviruses (65–68). To minimize the possibility of generating infectious virus and to extend the range of cells that can be transduced, lentivirus vectors have, in most cases, been pseudotyped with VSV-G envelope (67,69,70). In a comparative study of acute lymphocytic leukemia (ALL) cell lines, lentivirus vectors compared favorably with MoMLV vectors in achieving high transduction efficiency (24). There also is evidence that lentiviruses may facilitate in vivo transduction, a limitation of murine retroviruses (35,71). Alternative lentiviral constructs make use of HIV-2, which is less pathogenic in humans (72,73), or other nonhuman lentiviruses, such as the simian immunodeficiency virus (SIV) (74,75), feline immunodeficiency virus (FIV) (62,76), or the equine infectious anemia virus (77,78). As in the murine leukemia viruses, the size of the transgene that can be efficiently packaged within a lentivirus is limited; Kumar et al. demonstrated that as the insert size was increased to 12 kb, the titer of a VSVG pseudotyped lentivirus decreased to less than 1% of what was observed in controls, likely due to difficulties in achieving efficient packaging of the virus (69). Nonetheless, despite these concerns, lentiviruses present an alternative for achieving enhanced gene transfer efficiency in nonproliferating cells and for in vivo gene therapy applications. 2.2.3. ADENOVIRAL VECTORS Adenoviruses are double-stranded DNA viruses consisting of genes encoding 11 viral proteins (35). The adenoviral genome is large (36 kb) and is flanked by inverted terminal repeats (ITRs) (79). Adenoviruses do not integrate into the genomes of recipient cells but rather express their gene products from episomal vector genomes, resulting in transient expression (80). High titers (up to 1012 virions/mL) can be achieved, and subsequently efficient gene transfer has been observed (81). Because many individuals have previously been exposed to adenoviruses, those with intact immunologic function have the potential to generate a significant immune response (82,83). First-generation adenoviral vectors, in which the E1 gene has been replaced by the gene of interest, are more likely to generate an immune response directed against transduced cells in a clinical setting (84). Later designs, in which additional adenoviral genes have been removed from the vector, are less immunogenic

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(85–88). In the third-generation adenoviral vectors, the entire adenoviral genome has been removed, except for the ITRs and the packaging signal. This “gutless” strategy has provided ample space for large transgenes (theoretically up to 37 kb); hence the term “high-capacity” adenoviral vectors (82,89). Although these vectors may be successful in diminishing the inflammatory response to transduced cells (90), the most significant challenge is their dependence on the presence of a helper virus to provide the adenoviral proteins for vector packaging, and the elimination of this helper virus from vector preparations (91). The recent death of an individual being treated for ornithine transcarbamylase deficiency has raised considerable concern regarding the use of adenoviral vectors, and for gene therapy in general (34,92,93). In this phase I clinical study, a vector depleted of the E1 and E4 genes was administered intrahepatically to patients. Fevers, myalgias, nausea, and vomiting were commonly observed within 48 h of treatment (94). The final patient on the trial died from respiratory insufficiency that developed after the administration of the virus; marrow aplasia was observed as well (95). Adenoviruses have generated considerable interest for gene therapy applications due to their ability to transduce cells in vivo; clinical trials have been designed to test their use in muscle (96), liver (94,97,98), pulmonary (99–101), and endothelial tissues (85,102), as well as the central nervous system (CNS) (103,104). Several investigators have described a limited capability of adenoviruses to transduce human hematopoietic cells (105,106), but recent techniques such as modification of the adenoviral fiber proteins may enhance binding to hematopoietic targets (107). Myeloid and lymphoid leukemia cells may prove more amenable to transduction than undifferentiated cells (108–110). Additional strategies to enhance the efficacy of adenovirus-mediated gene transfer include the use of agents such as poly-L-lysine (105), polycations, and lipid complexes (111). For example, poly-L-lysines bind well to heparan sulfates, which are expressed at high levels in multiple myeloma, and thus high-efficiency gene transfer can be obtained in myeloma cells using this strategy (112). Adenovirusmediated gene transfer to enhance expression of CD154 (which binds to CD40 on T cells) has been tested clinically in B-lineage malignancies, with the intent of enhancing immune surveillance directed against the tumor, with reported clinical responses (113). Vaccination strategies with adenoviruses in the treatment of lymphoma and multiple myeloma have also been reported (113–118). 2.2.4. ADENOASSOCIATED VIRUS Adenoassociated viruses (AAVs) are parvoviruses that contain a 4.7-kb single-stranded DNA genome with two major open reading frames, Rep and Cap, flanked by 5′ and 3′ ITRs (35). At least six serotypes have been described in primates, but none has been shown to be pathogenic, a clear advantage for the use of AAV as a vehicle for gene therapy (119). An interesting characteristic of wildtype AAV is site-specific integration into a region of the human chromosome 19

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(120,121). However, site-specific integration is not observed in AAV vectors designed for gene transfer if the Rep gene is not included in the vector (122,123). AAV replicates only when the cell is co-infected with helper adenovirus or herpesvirus (124). In the absence of these viruses, AAV is uncoated within the nucleus and integrates into the host cell genome, where it is latent until activated by helper virus infection. Most AAV vectors designed for gene transfer and expression have been derived from serotype 2 (125). The size of the transgene that can be packaged into AAV vectors that have the Rep and Cap genes completely is limited to 4.9 kb (126), although new approaches to increase the size of the gene of interest are being tested (127). AAV vector generation is somewhat cumbersome, requiring cotransfection of vector and AAV helper (Cap and Rep) plasmids, along with adenovirus helper plasmid or coinfection with adenovirus. This latter approach requires removal of contaminating adenovirus by physical methods such as heat inactivation and cesium chloride density gradients (125). This technique, though effective, has for the most part been replaced by 2 and 3 plasmid transfection protocols (128). Several packaging cell lines expressing Cap and Rep proteins have been established, but the titer generated from these lines has not improved upon that achievable by transient transfection. AAV has been tested extensively for delivery and expression of genes in terminally differentiated and nonproliferating cells such as brain, liver, and muscle (129–133), including ongoing trials for hemophilia B targeting muscle (134,135). Transduction of human and murine hematopoietic cells has been reported, with variable degrees of success (136–141). Of interest, a related virus, parvovirus B19, has been used as a vector to transduce human erythroid precursors, achieving superior gene transfer efficiency in comparison with AAV-2 (142). A final yet-unanswered question relating to AAV success as a gene transfer tool, especially in vivo, is the immunologic response to the virus, because a majority of individuals are seropositive to AAV type 2 (143). 2.2.5. HERPES SIMPLEX VIRUS VECTORS The Herpes simplex virus type 1 (HSV-1) is a large and complex virus, with a genome of 152 kb consisting of double-stranded DNA (144). The biology of the virus and its means of transduction have recently been reviewed (145). The virus has the potential to be an important tool in gene therapy based on its ability to transduce nondividing cells and to accommodate large transgenes, and its capacity to transduce a broad range of host cells (146). HSV-1 is capable of infecting and producing a latent infection in nondividing cells of the peripheral nervous system and CNS, which has generated great interest in their use to deliver and express genes in these tissues (145,147–149). A phase I trial for the treatment of malignant glioma using an oncolytic HSV-1 virus was completed with minimal toxicity (150). For hematologic applications, the transduction of human chronic lymphocytic leukemia (CLL) cells has been demonstrated with HSV-1 virus (151), and the success of this approach may relate to the high lev-

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els of herpes virus entry mediator (Hve) A present on human CLL cells (152). Interestingly, the CLL cells were shown to be relatively resistant to the cytopathic effects of the virus, and this was believed to be related to the antiapoptotic effect of bcl-2 expression in these cells. The resistance of hematopoietic progenitors to HSV-1 oncolytic viruses has led to the investigation of HSV-1 virus as a biologic purging agent for solid tumor contamination in marrow or peripheral blood stem cell populations (153).

2.3. Summary: Gene Transfer Methods Physical means of gene transfer for the purposes of gene therapy in leukemia/lymphoma have the potential of achieving: (1) introduction and expression of large or complex genes, (2) in vivo gene transfer and expression, and (3) minimized potential for recombinant events and immunologic responses present with the use of viral vectors. The low incidence of gene transfer remains the primary disadvantage of physical methods, although improving methodology will likely increase gene transfer efficacy and allow the transfer of DNA to specific cell targets. The murine retroviruses have been the primary means of achieving gene transfer and expression in clinical investigations to date, primarily because: (1) packaging and viral production is straightforward, (2) efficient gene transfer is observed in many cell types, (3) they provide stable integration into the host cell, and (4) no significant toxicity or immunologic response has yet been demonstrated. Their use is limited primarily to ex vivo transduction applications targeting proliferating cell populations. In contrast, lentiviruses, commonly based on the HIV-1 virus, can be used in vivo and to transduce cells not actively dividing. However, safety concerns have slowed their integration into clinical gene therapy protocols. Adenoviral vectors can also be used for in vivo applications, but adenoviral vectors are capable of generating a significant inflammatory response, and gene expression is expected to be transient. AAV provides another option for in vivo gene transfer and transduction of nonproliferating cells. However, packaging remains cumbersome, and the size of the gene that can be incorporated into these vectors is limited. The development of HSV-1-based vector systems holds promise, primarily due to their large capacity and ability to transduce nonproliferating cells. Despite limitations in the use of viral vectors for clinical applications, continuing progress in their development suggests that they will continue to be the primary means of gene delivery in the near future.

3. GENE THERAPY FOR LEUKEMIA/LYMPHOMA 3.1. Marking Studies Although not therapeutic in intent, gene transfer to investigate homing of cells to a particular location or tumor, or to study the persistence of a population of

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Fig. 3. Gene therapy strategies for leukemia/lymphoma. There are currently 46 clinical gene therapy trials for leukemia or lymphoma. Of these, the primary focus of specific trials includes marking (n = 19), enhancing the immune response to leukemia/lymphoma by vaccination strategies (n = 14), or engineering of effector cells such as T cells (n = 2), applications to correct the genetic abnormalities of leukemia (n = 2), providing resistance of hematopoietic precursors to facilitate additional chemotherapy (n = 3) or the use of genes providing the capacity for negative selection such as HSV-tk (n = 6).

cells is of great historic and practical importance in gene therapy (Fig. 3). The first example of clinical gene transfer for the purpose of marking transduced cells was carried out in patients with solid malignancies by Rosenberg et al., who introduced the neomycin phosphotransferase gene (NeoR) into tumor-infiltrating lymphocytes (TIL), providing proof that retroviral-mediated gene transfer could be accomplished in a clinical setting without toxicity (154). Of the 46 gene therapy studies that list leukemia or lymphoma in the primary eligibility criteria for inclusion, 19 (41%) have used molecular techniques to “mark” cells of interest. All of these studies have used replication-incompetent murine retroviruses containing a gene not ordinarily present in human hematopoietic cells, most commonly the NeoR gene. Transduction of a stem cell source, leukemic blasts, or immunologically active cells is performed ex vivo, and the cells are re-infused for subsequent testing. A common strategy that has been employed is the marking of a portion of autologous marrow (typically 10%–30%) before the infusion of the marrow as a rescue in an autologous transplantation setting for acute myeloid leukemia (AML) (155), chronic myelogenous leukemia (CML) (156), follicular lymphoma (157), or multiple myeloma (158,159). Identification of marked cells

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is accomplished using molecular analysis such as a southern blot or the polymerase chain reaction (PCR), which is more sensitive and may provide information if only a small proportion of the cells are marked. In addition, cells expressing the NeoR gene can be selected in the presence of the antibiotic, providing the potential for purification of the transduced cells after their infusion. Using these methods, in an important series of investigations Brenner et al. documented that in children with AML who relapsed after autologous transplantation, several had blasts marked with the NeoR gene, proving that residual marrow disease contributed to the relapse of these patients (155). The rate of gene transfer into hematopoietic progenitors was 2%–15% with the techniques used, and, in some cases, cells containing the gene could be identified 5 yr after marking, suggesting that a stem cell population was successfully transduced (160). Similarly, Deisseroth used a NeoR retroviral vector to transduce CD34 selected cells and documented that in the case of autologous transplantation for CML, the NeoR gene could be identified in residual leukemia (156). These studies suggest that “purging” of marrow was ineffective in these situations. Questions such as the relative contributions of marrow and peripheral blood stem cells to hematologic recovery have also been addressed using gene therapy techniques. Dunbar reported a study in which two distinct NeoR-containing retroviruses (LNL6 and G1Na [161]) were used in an autologous setting, one to transduce marrow and the other to transduce peripheral blood stem cells from the same patient. These vectors could be distinguished molecularly, allowing the determination of the relative number of marrow and nucleated blood cells marked with each vector. Although the overall gene transfer frequency was low, studies suggested superior persistence of marked peripheral blood cells (58). This study design using two marker vectors has the capacity to ask other complex questions, such as comparisons of purging techniques (162). Investigators have demonstrated the ability to genetically mark marrow from patients with myeloma as well as acute leukemia, although low frequencies of gene transfer have remained an obstacle (159,163). In addition to labeling leukemia cells and CD34+ cells, protocols have been designed to follow effector cells infused for the purposes of immunotherapy. Brenner and Heslop developed a procedure to generate donor T cells specific for B cells expressing Epstein-Barr Virus (EBV) proteins, and marked these cells with an NeoR retrovirus to follow the presence of these cells in vivo as treatment for EBV lymphoproliferative disease, documenting persistence of marked T cells for months after infusion (164). Marking is also to be performed for EBV-specific T cells generated for the treatment of EBV-positive Hodgkin’s disease undergoing transplantation or receiving immunotherapy (165). Techniques to provide autologous EBV-specific cells are being developed to facilitate the infusion of EBV-specific T cells for individuals who do not have access to donor cells or for recipients of solid organ transplantations with EBV disease (166,167). Finally, a procedure in which an adenoviral vector is used to transduce dendritic cells with

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the LMP2A gene has been designed to enhance the immune response to EBV antigens, generating specific cytotoxic T cells (168). It has been proposed within this same protocol to mark the EBV-specific T cells produced after interaction to the engineered dendritic cells with an NeoR gene, using a murine retrovirus to follow their persistance and location. Therefore, the design of this protocol uses two independent viral vectors, one an adenovirus and the other a retrovirus, to achieve its aims. As stated, marker genes have primarily been nonfunctional genes such as NeoR, although the β-galactosidase gene has also been tested (169). However, due to concerns about the potential immunogenicity associated with the expression of these nonhuman proteins and rejection of the marked cells, the next generation of vectors to be used for marking studies contain nontranscribed sequences that can be evaluated molecularly through procedures such as quantitative PCR without the production of gene products that may prove immunogeneic (170).

3.2. Modification of the Immune Response The documentation of the graft vs leukemia (GVL) effect in decreasing relapse after allogeneic transplantation and the success of donor lymphocyte infusion (DLI) in producing sustained remission after leukemic relapse are clear indicators of the power of the immune system to control leukemia. Malignant cells may evade immune surveillance through several mechanisms, including actively suppressing the immune response (171–174), alteration of the CD95/Fas system (175) and downregulation of major histocompatibility complex (MHC) class I or II expression (176). The concept of exploiting the immunologic response to provide therapy for leukemia is not new. Mathé in 1965 treated children with ALL in remission with vaccinations consisting of leukemic blasts and BCG, documenting prolonged DFS in 8 of 20 patients, while all 10 controls relapsed (177). Our current ability to transfer and express genes has led to myriad possibilities to manipulate the immune system as a means of therapy. Of these, two primary approaches can be defined: (1) genetic modification of the malignant cell, or a cell to be used with it as a vaccine to augment the immunogenicity of the leukemia, and (2) to engineer an increased capacity of an immunologically active cell population (effector cells) to eradicate cancerous cells otherwise inefficiently identified or killed. These approaches have the common goal of eliminating residual leukemic cells using an immunologic approach, but are distinct and are discussed independently. 3.2.1. ENGINEERING OF LEUKEMIA CELLS TO INCREASE AN IMMUNOLOGIC RESPONSE These applications have generally had the goal of designing a more effective tumor vaccine than was previously possible, and, with few exceptions, this is performed by modification of cells ex vivo. More than a decade ago it was

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shown that the endogenous expression of immunologically active cytokines can decrease tumorigenicity and enhance immunogenicity in several murine malignancies (178–181). Since that time, numerous cytokines, human leukocyte antigen (HLA) molecules, costimulatory proteins, and integrins have been tested and shown to have value in this role. A detailed description of these approaches is beyond the scope of this discussion, and previous reviews have well documented the issues involved (1,182–185). Several investigators have described increased survival of mice vaccinated with leukemia or lymphoma cells engineered to express cytokine genes, such as interleukin (IL)-2 and granulocyte macrophage colony stimulating factor (GM-CSF) (186–190). It is hypothesized that the production of high levels of a cytokine directly stimulatory to cytotoxic T cells or antigen-presenting cells in the immediate environment surrounding the malignant cell may prove more effective in initiating a generalized immune response than systemic administration, which can only hope to achieve modest serum levels. The transfer of the human IL-2 gene stimulates an increased immune response to murine myeloma (189). Clinically, an adenoviral vector encoding the IL-2 gene was used to transduce an intracranial plasmacytoma in a patient with myeloma. Although gene transfer was documented, no apparent clinical response was observed (191). Brenner has tested the combination of human CD40 ligand (hCD40L) to increase the expression of costimulatory factors on the malignant cells and enhance immune recognition and IL-2 in CLL blasts and lymphoma, and has shown that the expression of both molecules provides increased antitumor immunity than either alone (114,118). Vaccination of animals with AML blasts engineered to express IL-12, another cytokine stimulatory to T cells, provides increased survival in mice with previously established leukemia (192,193). The use of agents that can enhance the processing and presentation of antigens, most notably GM-CSF, may also be effective at establishing specific cytotoxic T cells directed against the malignant cell. In a murine model of AML, vaccinations of leukemic cells engineered to produce GM-CSF by retroviral-mediated gene transfer were superior to vaccine strategies in which the B7.2, IL-4, or TNF-α genes were used (194). In a murine Philadelphia chromosome positive (Ph+) ALL model, GM-CSF expression was effective in vaccination experiments but CD80 (B7.1) and GM-CSF coexpression provided the greatest protection, against the administration of wildtype leukemia (195). An interesting approach to GM-CSF production in the local environment for the purposes of leukemia vaccinations uses the K562 cell line engineered to express high GM-CSF levels (196). The development of a cell line producing GM-CSF that can be used instead of transducing autologous cells has the potential to make vaccinations much less complex. In addition, because the K562 line does not express class I or II MHC molecules, the likelihood that the cells will be readily rejected is diminished.

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A means of providing increased immunologic recognition of B-cell malignancies, which express immunoglobulin idiotype molecules on the cell membrane, is the development of a T-cell response to idiotype antigens using vaccinations. This concept has merit without the use of gene transfer techniques, because vaccinations of idiotype-specific proteins and keyhole limpet hemocyanin (KLH) administered to 16 patients with follicular non-Hodgkin’s lymphoma (NHL) resulted in an increase in the cytotoxic T-cell precursor (CTLp) frequency all patients in vitro, and eight sustained responses were noted (197). However, B-cell neoplasms do not present antigens well, which limits the potency of a T-cell response (113). These cells can be engineered to express costimulatory molecules such as B7.1 and B7.2 or CD40L (CD154) by gene transfer, making them much more likely to generate a T-cell response. This strategy was employed in a clinical trial in which an adenoviral vector was used to express CD154 in CLL cells ex vivo (117). The infusion of engineered cells was in general well tolerated, although fevers, elevated transaminases, and arthralgias were observed. The elevation of plasma cytokine levels, including TNF and IL-12, was observed, and an increase in the number of leukemia-specific T-cells was reported, suggesting that this line of intervention may prove important for B-cell malignancies. An alternative mechanism by which an enhanced antitumor response may be obtained is by increasing the processing and presentation of tumor-associated antigens by cells other than the malignant population. The primary cell target of this approach is the dendritic cell, which was first described in murine tissues by Steinman in the early 1970s (198,199). T-cells can be “educated” to respond to peptide fragments derived from antigens bound to MHC class I (intracellular or endogenous antigens) or class II (exogenous antigens) molecules (200). The dendritic cell has a greater density of MHC, adhesion, and costimulatory molecules on the cell surface than other cells capable of presenting antigen, making them well suited for interacting with T-cells toward the generation of specific cellular responses (201). As techniques for the generation of dendritic cells from peripheral blood have been developed, they have become a primary focus in the engineering of T-cell responses (38,202). To facilitate antigen presentation and increase antileukemia or myeloma T-cell responses, several potential antigen sources can be used, including synthetic peptides, proteins derived from tumor cell extracts, or RNA derived from tumor cells (203–209). The use of a BCR/ABL-derived peptide and dendritic cells markedly increases the number of T-cells directed against murine CML, and vaccination with peptide-pulsed dendritic cells had protective effects against the administration of wildtype leukemia (210). It is possible to obtain dendritic cells directly from myeloid leukemia cells, which appear to present antigens of leukemic origin and increase the specific T-cell response (206). Westermann reported similar findings for dendritic cells derived from patients with CML and that the transduction of dendritic cells

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with the IL-7 gene increased the T-cell response to CML cells (205). To increase antigen specificity, the desired antigen can be produced within the dendritic cell using viral vectors. The engineering of dendritic cells to express LMP2A antigen with an adenoviral vector results in an enhanced T-cell response, which may prove a viable means of increasing the response to Hodgkin’s lymphoma (168). 3.2.2. GENETIC MODIFICATION OF EFFECTOR CELLS An alternative approach to the manipulation of malignant cells or antigenpresenting cells is the direct modification of the effector cell population to increase the recognition, function, or killing of effector cells. Clinical trials were initiated in which TIL were genetically modified to express the TNF gene, with the intention of killing the malignant cells through the local production of providing TNF at the tumor site, resulting in increased control of the malignancy (211,212). The endogenous expression of IL-2 in a human natural killer (NK) cell line or donor-derived human NK cells increases the ability to kill tumor targets (213,214). This approach was also tested by transducing murine T-cells with a retroviral vector containing the IL-2 cDNA. T-cells expressing IL-2 grew independently and maintained antigen specificity (215). Similarly, transduction of hematopoietic precursors has also been reported with IL-2 vectors, and animals receiving transduced cells have improved survival after a leukemia challenge (216). A drawback to this approach, however, is the potential for autocrine stimulation and uncontrolled proliferation of cells transduced with a stimulatory cytokine, which would likely require coupling the expression of the gene designed to enhance function with a “suicide gene” such as the herpes simplex virus thymidine kinase (HSV-tk) gene. Another means of responding to this concern uses constructs designed with alternative cytokine genes to stimulate T-cells; Minamoto designed a fusion gene in which the extracellular moiety of the erythropoietin receptor was combined with genes encoding signaling domains of the IL-2 receptor. The presence of erythropoietin in the environment was sufficient to drive T-cell proliferation (217). In contrast to providing a nonspecific stimulus to T-cells, the capacity to generate effector cell specificity through gene transfer has also been explored. Dembic et al. documented that it is possible to transfer the α and β T-cell receptor genes from a T-cell with the desired specificity into an otherwise naive T-cell and thereby generate specificity (218). However, the ability to recognize antigen after the transfer of T-cell receptors is MHC restricted, because the antigen is presented in association with the MHC molecule, which is a significant drawback to this approach (219). Chimeric genes have been described in which the variable regions of antibodies were fused to T-cell receptors to generate specificity, because this would not require antigen to be expressed in association with MHC molecules and, therefore, cells could be engineered in a non-MHC-

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Fig. 4. Engineering of T-cell specificity. The variable regions of the light and heavy chain (VL and HV) antibody genes are combined with a flexible linker, and joined to the transmembrane and cytoplasmic portions of a gene that can provide signaling within a T-cell, such as the zeta chain of the T-cell receptor. This construct can be introduced into the effector cell by various gene transfer methods. The protein is anchored in the cell membrane with the single chain Fv region (scFv) external to the cell where it can bind the desired antigen of leukemia or lymphoma cells. Binding results in killing of the cell expressing the antigen and activation of the effector cell through signaling pathways.

restricted fashion (219,220). The ability to isolate genes encoding the variable regions of antibodies and combine them into a single gene with maintained antigen specificity allowed further modification of this strategy (221,222). These single-chain Fv (scFv) genes can be combined with genes encoding the transmembrane and intracytoplasmic domains of a protein that signal T-cells to proliferate and become cytotoxic, such as the ζ chain of the T-cell receptor or γ chain of the Fc receptor (223,224) (Fig. 4). The expression of these chimeric genes have been shown to markedly increase the killing of several malignant cells bearing a target antigen and signaling through the intracytoplasmic ζ or γ chain results in activation and cytokine expression (223–228). A clinical trial is underway in which an anti-CD20 scFv/ζ plasmid is transfected into autologous T-cells expanded in the presence of antigen before infusion (229). This method for producing specific effector cells has great promise, but issues such as prolonged function of expanded cells and the potential for rejection of cells with chimeric antibody/T-cell receptor proteins remain to be addressed.

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3.3. Gene Transfer to Provide Drug Resistance A primary limitation of the amount of chemotherapy that can be administered is prolonged myelosuppression. Altering the cellular metabolism of chemotherapeutic agents in normal hematopoietic precursors may provide relative resistance to these toxic agents, potentially allowing chemotherapy dose escalation and subsequently greater killing of the malignant cells. Expression of a dihydrofolate reductase (DHFR) gene modified to alter its sensitivity to methotrexate provides methotrexate resistance in vivo (230–232). A clinical trial for CML was designed using a vector containing both antisense BCR/ABL sequences and a resistant DHFR gene to allow the in vivo selection of transduced hematopoietic precursors (233). The administration of methotrexate after transplantation could therefore result in the selected expansion of precursors expressing antisense BCR/ABL sequences. The human multidrug resistance 1 (MDR1) gene has also been a focus of this type of experiment, primarily to provide protection of the marrow in association with the administration of agents such as paclitaxel, daunomycin, and etoposide (234). Gene transfer of the MDR1 gene into hematopoietic precursors to provide resistance of the marrow to chemotherapy used as treatment of solid tumors has been performed, with documentation of transgene persistence to 1 yr after transplantation (235). In this report several patients had a slight increase in the number of circulating cells containing the transgene after the administration of oral etoposide, suggesting an in vivo selection effect. Similarly, Moscow described a protocol for patients with metastatic breast cancer in which CD34+ cells were exposed to an MDR1- or NeoR-containing retrovirus. After transplantation substrates of the MDR1 gene, including doxorubicin, vinblastine and paclitaxel were administered. In several patients, soon after transplantation the presence of MDR1 could not be detected by PCR, although after therapy it was detectable; in contrast, there was no increase in the ability to detect NeoR-marked cells (236). This suggests that in vivo selection strategies may be possible and that this approach deserves further study. This type of protocol is also being explored for individuals with NHL (237). One potential concern, however, was raised by Bunting et al., who described the development of a myeloproliferative state in murine recipients of MDR1 transduced ex vivo-expanded precursor cells (238). It is unclear if this observation was a result of the culture conditions or the vector, but it was not seen in experiments in which a control DHFR virus was used (238). A final drug-resistance gene that may be useful clinically is cytidine deaminase, which catalyzes the deamination and therefore inactivation of cytosine nucleoside analogues, such as cytosine arabinoside, and could also be used to provide protection of hematopoietic precursors expressing this gene (239,240).

3.4. Strategies to Interfere with the Malignant Genotype Malignant transformation may result from the loss of function of tumor suppressor gene function or from acquired expression of oncogenes. The technical

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difficulties associated with correcting these defects on a molecular basis is daunting, because in either circumstance virtually all of the cells with an abnormal genotype would require successful genetic modification to expect a positive benefit of this type of therapeutic intervention. In addition, because patients with leukemia or lymphoma have widely disseminated disease protocols using gene transfer for leukemia therapy would require high gene-transfer efficiency in vivo, which is clearly beyond our current capability. Nevertheless, because p53 deletions have been described in hematopoietic malignancies (241–243) and expression of the p53 gene by retroviral-mediated gene transfer in a model of murine myeloid leukemia has been shown to inhibit the virulence of the leukemia (244), this approach may theoretically be possible. Another scenario in which gene transfer of the p53 gene could be used is ex vivo purging of leukemia or solid malignancies (245,246). With respect to reversing malignant transformation by targeting oncogenes, specifically inactivating the causative gene within the genome may prove more difficult than other approaches, such as using antisense methods to inhibit transcription of gene products, the destruction of the RNA message after transcription, or blocking the action of the translated gene. There is evidence that antisense oligonucleotides designed to target antiapoptotic genes such as Bcl-2 can inhibit proliferation and increase the sensitivity of AML blasts to cytosine arabinoside (21). Antisense therapies are currently being employed in the treatment of follicular lymphoma by targeting Bcl-2, and could also prove a future target of gene therapy (247,248). Another means of using antisense strategies in leukemia treatment is to inhibit the action of MDR1 genes, leading to increased sensitivity of AML blasts to chemotherapy (249). It has previously been mentioned that the BCR/ABL gene has been a target of antisense approaches (250). In addition, expression of a BCR deletion mutant gene delivered by an adenoviral vector has proven capable of inhibiting proliferation and inducing apoptosis in BCR/ABL-positive cell lines (251). The dependence of malignant cells on a specific growth factor may also be a target of gene therapy. The proliferation of an IL-6 dependent human myeloma line was inhibited through the production of an IL-6 antagonist by another cell line engineered with an adenoviral vector (252). The use of ribozymes in gene therapy for hematopoietic disorders also merits discussion. Ribozymes are RNA molecules that bind specifically to other RNA sequences and have the potential to cleave or alter their targets (253). Several ribozymes exist, but the hammerhead ribozyme has received significant attention for therapeutic applications (254). The hammerhead ribozyme is small (30–40 nucleotides) and consists of three motifs: the first and third are designed to specifically bind to an RNA template by Watson-Crick pairing, while the second domain is catalytic, usually for a GUC site. Hammerhead ribozymes have been designed and tested to cleave MDR1 sequences to inhibit

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drug resistance in AML cell lines (255), AML RTO fusion transcripts (256), and PML/RARα transcripts in acute promyleocytic leukemia (APL) (257,258). In addition, Kuwabara and colleagues have tested the potential of ribozymes or related molecules termed maxizymes in targeting the BCR/ABL RNA template and have demonstrated high activity as well as specificity for the fusion transcript (254,259,260). This group also documented that animals receiving CML cells transduced with a murine retroviral construct containing the maxizyme survived, while all controls died (261). Despite the encouraging nature of these reports, delivery of these molecules in vivo remains a significant hurdle to the implementation of these strategies.

3.5. Use of “Suicide Genes” to Provide Negative Selection Genes that alter function to facilitate specific killing of engineered cells have been termed “suicide genes.” Most commonly these gene products provide an enzymatic function that converts a relatively nontoxic “prodrug” into an agent that is toxic to the cell. The best tested of these is the HSV-tk gene, which converts acyclovir or ganciclovir (GCV) into a monophosphate that can be converted by the cell into the triphosphate form, which is toxic to actively dividing cells. The HSV-tk gene has been used in clinical trials in which the virus is used to transduce intracranial tumors or localized solid tumors in vivo (58,103,262–265). In the treatment of hematopoietic malignancies, however, the HSV-tk gene has been used to enhance the safety of cellular immunotherapy (266,267). Although allogeneic transplantation remains an important therapeutic modality for hematologic malignancies, in part due to the immunologic process termed the GVL effect, the morbidity and mortality associated with graft vs host disease (GVHD) remains high (268,269). One approach proposed to address these issues is the expression of a gene such as HSV-tk providing the potential for negative selection in donor T-cells, either in association with the initial transplant to assist in engraftment or with DLI administered after relapse (270,271). There have been reports suggesting that this is feasible in murine models (272,273) and that the approach may prove advantageous in a clinical setting (271,274). However, it remains unclear if HSV-tk is the optimal T-cell suicide gene, because standard cytomegalovirus (CMV) prophylaxis and treatment regimens rely on acyclovir or GCV; in addition, a cell-mediated response to T-cells expressing the HSV-tk/hygromycin fusion gene has been identified in patients with AIDS (275). This suggests that the expression of HSV-tk will be immunogenic and may lead to eradication of HSV-tk+ cells. Alternative genes for negative selection include cytosine deaminase, which facilitates the conversion of the substrate 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) (276,277). Another gene undergoing testing as a means of eliminating cells in vivo is a chimeric gene constructed to include the nerve growth factor (NGFR) extracellular and transmembrane domains and an intra-

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cellular domain derived from Fas; when exposed to a chemical dimerizer, cells expressing this gene are induced to undergo apoptosis (278,279). The potential use of other suicide genes is being explored, including the Escherichia coli nitroreductase, which converts CB1954 (a mustard prodrug) to an intermediate, which can be activated into an alyklating agent, and the cytochrome p450 enzyme to enhance hydroxylation of cyclophosphamide (280,281).

4. FUTURE CHALLENGES IN GENE THERAPY OF LEUKEMIA/LYMPHOMA There has been healthy skepticism regarding the current and potential usefulness of gene therapy in the treatment of malignancies. Although many gene therapy protocols have enrolled patients, it has in most cases been difficult to provide clear demonstration of a therapeutic effect. Primary limitations have included the difficulty in obtaining efficient gene transfer, especially into nondividing cells, and the ability to maintain adequate expression of a desired product once gene transfer is achieved. In some circumstances this has been complicated by the use of genes producing proteins that are immunogenic. Other concerns relate to safety issues; although the murine retroviruses used in many trials have been well tolerated, the death of an individual after administration of cells transduced with an adenoviral vector have raised concern in investigators in the field, regulatory groups, and the public. It is clear that protocol design needs to incorporate clear means of defining outcomes to further develop the field. Nevertheless, there is reason to be optimistic regarding the future of gene therapy. Continuing progress is being made in achieving more efficient gene transfer and expression using both physical and viral strategies. Exciting developments have been described in the use of gene therapy for immunodeficiencies and hemophilia. There has been evidence of persistence of gene modified cells in patients with leukemia, as well as in other disorders. Indeed, evidence of a therapeutic effect has been suggested in the treatment of GVHD after the infusion of HSV-tk-expressing donor lymphocytes. Further development and refinement of gene transfer as a therapeutic tool seems inevitable but will require continuing evaluation for safety and efficacy.

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Index

261

INDEX A AAV, see Adeno-associated virus ABC transporters, see Multidrugresistance associated protein; P-glycoprotein Activating mutations, cytokine receptors, 130, 131 Acute lymphocytic leukemia (ALL), apoptosis dysregulation, 172 drug resistance relationship, 173, 174 interleukin-2 therapy, see Interleukin-2 relapse and prognosis, 93, 94 Acute myeloid leukemia (AML), apoptosis dysregulation, 171, 172 Bcl-2, expression, 115, 116 G3139 trials, 117–119 chemotherapy, 146 chromosomal translocations, 129 dendritic cell characteristics, 5, 6 gemtuzumab ozogamicin therapy, see Gemtuzumab ozogamicin HuM195 trials, 30, 31 interleukin-2 therapy, see Interleukin-2 markers, 44 P-glycoprotein reversal trials, biricodar, 154 cyclosporine, 150–152, 154 gemtuzumab ozogamicin combination therapy, 154, 155 prospects, 156 PSC833, 150–153, 155

quinine, 149, 150 verapamil, 149, 150 zosuquidar, 154 prognosis, 146 radioimmunotherapy, alpha-particle emitters, 72, 73 beta-particle emitters, 65–71 relapse and prognosis, 93, 94 Acute promyelocytic leukemia (APL), all-trans retinoic acid therapy, see All-trans retinoic acid arsenic trioxide trials and alltrans retinoic acid combination therapy, 192–194, 200 chemotherapy response, 206, 207 chromosomal translocations, 128, 244 HuM195 trials, 30 prognosis, 148 Adeno-associated virus (AAV), gene therapy vectors, 232, 233 Adenovirus, gene therapy vectors, 231, 232 Adult T-cell leukemia/lymphoma (ATL), radioimmunotherapy, 73, 74 AG490, apoptosis induction in leukemia management, 177 Alemtuzumab, CD52 targeting, 35 development, 35 leukemia trials, B-cell chronic lymphocytic leukemia, 36–38

261

262

T-cell prolymphocytic leukemia, 36 ALL, see Acute lymphocytic leukemia All-trans retinoic acid (ATRA), acute promyelocytic leukemia treatment, arsenic trioxide combination therapy, 192–194, 200, 216 chemotherapy combination therapy, AraC role, 211, 212 dosing and duration during induction treatment, 210, 211 maintenance treatment, 212 nonrandomized studies, 208 overview, 205, 206 prognostic factors, complete remission achievement factors, 213 extramedullary relapse, 214 minimal residual disease monitoring, 214 pretreatment factors, 213, 214 randomized studies, 208, 210 scheduling, 211 coagulopathy and thrombosis induction, 218 monotherapy response, 207, 208 rationale, 128, 129 relapse management, 214, 215 side effects, 218 syndrome, clinical signs, 216, 217 hyperleukocytosis pathophysiology, 217 incidence, 216, 217 prevention, 217, 218

Index

treatment, 217, 218 synthetic retinoids, 215, 216 TRAIL induction, 174, 175 AML, see Acute myeloid leukemia AML1-ETO, chromosomal translocation, 129 tumorigenesis role, 131, 132 Angiogenesis, arsenic trioxide inhibition, 192, 195 Antisense therapy, bispecific antisense oligonucleotides in apoptosis targeting, 176 DNA modifications, 110 mechanisms of action, 110, 111 oligonucleotide features, 109, 110 ribonuclease H conjugation, 110 ribozyme utilization, 243, 244 targets, Bcl-2 and G3139 targeting, 113–120 BCR-ABL, 111, 112, 243 Myb, 112, 113 Myc, 113 p53, 113 APL, see Acute promyelocytic leukemia Apoptosis, arsenic trioxide induction, 190, 191 Bcl-2, anti-apoptotic mechanisms, 113, 114 cytochrome c release regulation, 168, 169, 173 caspases, regulation by inhibitor of apoptosis proteins, 169 role in apoptosis, 164 death receptor pathway,

Index

activation, 174, 175 overview, 164, 165 regulation, 167, 168 drug resistance relationship, 173, 174 leukemia dysregulation, acute lymphocytic leukemia, 172 acute myeloid leukemia, 171, 172 chronic lymphocytic leukemia, 169, 170 chronic myelogenous leukemia, 170, 171 large granular lymphocyte leukemia, 170 mitochondrial pathway, inhibition reversal in therapy, 175–179 overview, 165 regulation, 168, 169 morphological features, 163 therapeutic induction, AG490, 177 all-trans retinoic acid induction of TRAIL, 174, 175 Bcl-2 antisense targeting, 175, 176 bispecific antisense oligonucleotides, 176 caspase activation, 173 CI-1040, 177 DR5 activation, 174 flavopiridol, 177, 178 inhibitor of apoptosis protein inhibitors, 179 PD98059, 177 prospects, 179, 180 protein kinase C inhibitors, 178 STI571, 177 TRAIL therapy, 174

263

Arsenic trioxide (ATO), acute promyelocytic leukemia trials and all-trans retinoic acid combination therapy, 192–194, 200, 216 chronic myelogenous leukemia trials, 196 history of use, 189, 190 mechanisms of action, angiogenesis inhibition, 192, 195 apoptosis induction, 190, 191 mitochondrial injury, 191 PML/RAR-α transcript product relocalization, 191, 192 miscellaneous hematologic malignancy trials, 196, 197 multiple myeloma trials, 195 solid tumor trials, 197 toxicity, 198, 199 ATL, see Adult T-cell leukemia/ lymphoma ATO, see Arsenic trioxide ATRA, see All-trans retinoic acid B BC8, acute myeloid leukemia radioimmunotherapy, 70, 71 B-cell chronic lymphocytic leukemia (B-CLL), alemtuzumab trials, 36–38 rituximab trials, 31–34 Bcl-2, anti-apoptotic mechanisms, 113, 114 cytochrome c release regulation, 168, 169, 173 expression in leukemias, acute myeloid leukemia, 115, 116 chromosomal translocations, 128

264

chronic lymphocytic leukemia, 114, 115 G3139, acute myeloid leukemia trials, 117–119 chronic lymphocytic leukemia trials, 117 chronic myelogenous leukemia trials, 119, 120 combination therapy, 176 non-Hodgkin’s lymphoma trials, 116, 175 pharmacology, 116 inhibition for apoptosis induction in therapy, AG490, 177 antisense targeting, 175, 176 bispecific antisense oligonucleotides, 176 CI-1040, 177 flavopiridol, 177, 178 PD98059, 177 protein kinase C inhibitors, 178 STI571, 177 B-CLL, see B-cell chronic lymphocytic leukemia BCR-ABL, antisense targeting, 111, 112, 243 chromosomal translocation, 130 STI571 targeting in chronic myelogenous leukemia, applications for other molecular targets and cancers, 140 clinical trials, 135, 136 combination therapy, 138 development, 133, 134 dose selection, 136, 137 preclinical studies, 134, 135 prospects, 137 rationale, 133

Index

resistance, 137, 138 specificity of receptor inhibition, 138–140 tyrosine kinase inhibition, 128 tumorigenesis role, 131 tyrosine kinase activation, 130 BCRP, drug transport, 148 prognostic value, 149 Biricodar, P-glycoprotein reversal and clinical trials, 154 BMT, see Bone marrow transplantation Bone marrow transplantation (BMT), autologous transplantation, 95, 96 graft vs host disease, 14, 15 graft vs leukemia effect, see Graft vs leukemia effect interleukin-2 combination therapy in acute leukemia, 98–100 nonmyeloablative allogeneic transplantation, 19–23 rationale in cancer therapy, 13 Burkitt’s lymphoma, gene mutations, 132 C CAMPATH-1H, see Alemtuzumab Caspases, apoptosis role, 164 arsenic trioxide activation, 190 regulation by inhibitor of apoptosis proteins, 169 therapeutic activation, 173 CBF, see Core binding factor CD5 antibody, see T101 CD20 antibody, see Rituximab, CD25, adult T-cell leukemia/lymphoma radioimmunotherapy targeting, 73, 74 CD33 antibody, see HuM195; p67

Index

CD45 antibody, see BC8 CD52 antibody, see Alemtuzumab CD66, acute myeloid leukemia radioimmunotherapy targeting, 71 Chronic lymphocytic leukemia (CLL), alemtuzumab, 36–38 apoptosis dysregulation, 169, 170 Bcl-2, expression, 114, 115 G3139 trials, 117 rituximab, 31–34 Chronic myelogenous leukemia (CML), apoptosis dysregulation, 170, 171 arsenic trioxide trials, 196 chromosomal translocation, see BCR-ABL dendritic cell, characteristics, 5, 6 clinical trials, 6, 8 prospects, 8–10 epidemiology, 133 G3139 trials, 119, 120 interferon-α therapy, 86–88 phases, 133 prognosis, 133 STI571 therapy, see STI571, CI-1040, apoptosis induction in leukemia management, 177 C-kit, activating mutations, 130, 131 STI571 inhibition of receptor tyrosine kinase, 139 CLL, see Chronic lymphocytic leukemia CML, see Chronic myelogenous leukemia Core binding factor (CBF), components, 129 therapeutic targeting, 129, 131, 132

265

Cyclin D1, chromosomal translocation and overexpression, 128 Cyclosporine, P-glycoprotein reversal and clinical trials, 150–152, 154 D DC, see Dendritic cell Dendritic cell (DC), ex vivo differentiation, 4 leukemia therapy, chronic myelogenous leukemia trials, 6, 8 prospects, 8–10 rationale, 3, 4 myeloid leukemia cell characteristics, 5, 6 DLI, see Donor leukocyte infusion Donor leukocyte infusion (DLI), clinical trials in leukemia, 18, 19, 95 gene therapy utilization, 244 graft vs host disease, 18 graft vs leukemia effect, 17–19 DR5, activation, 174 F Flavopiridol, apoptosis induction in leukemia management, 177, 178 Flt3, activating mutations, 130, 131 G G3139 acute myeloid leukemia trials, 117–119 Bcl-2 targeting, 116 chronic lymphocytic leukemia trials, 117 chronic myelogenous leukemia trials, 119, 120

266

combination therapy, 176 non-Hodgkin’s lymphoma trials, 116, 175 pharmacology, 116 Gemtuzumab ozogamicin, acute myeloid leukemia treatment rationale and response, 44, 53, 54 clinical trials, 48–50, 54–56 design and structure, 45–47 immune response, 52 P-glycoprotein reversal combination therapy, 154, 155 pharmacokinetics, 50, 52 toxicity, 52, 53 Gene therapy, see also Antisense therapy, clinical trials, 225 definition, 225 drug resistance gene transfer to hematopoietic precursors, 242 effector cell genetic modification, cytokine expression, 240 T-cell receptor genes, 240, 241 leukemia cell genetic modification, antigen expression, 239, 240 costimulatory molecule expression, 239 cytokine gene expression, 238 rationale, 237 malignant transformation interference, 242–244 marking studies, 234–237 prospects, 245 suicide genes and negative selection, 244, 245 vectors, adeno-associated virus, 232, 233 adenovirus, 231, 232

Index

criteria for successful virus vectors, 227 herpes simplex virus, 233, 234 lentivirus, 230, 231 Moloney murine leukemia virus, 227–230 physical gene transfer, 226, 234 prospects, 234 GM-CSF, see Granulocyte-macrophage colony-stimulating factor Graft vs leukemia (GVL) effect, definition, 13, 14 donor leukocyte infusion, 17–19 graft vs host disease, gene therapy utilization in prevention, 244 relationship, 14, 15, 23 lymphocyte-activated killer cells, 94, 95 T-cell depletion effects in bone marrow transplantation, 15–17 Granulocyte-macrophage colonystimulating factor (GM-CSF), leukemia cell engineering for expression, 238 GVL effect, see Graft vs leukemia effect H Hairy cell leukemia, interferon-α therapy, 88, 89 rituximab trials, 34, 35 Herpes simplex virus (HSV), gene therapy vectors, 233, 234 HSV, see Herpes simplex virus Hu1D10, leukemia therapy, 38 HuM195 CD33 targeting, 30 leukemia trials,

Index

267

acute myeloid leukemia, 30, 31 acute promyelocytic leukemia, 30 radioimmunotherapy trials of acute myeloid leukemia, 65, 68, 69, 72, 73 I IAPs, see Inhibitor of apoptosis proteins IL-2, see Interleukin-2 Inhibitor of apoptosis proteins (IAPs), caspase regulation, 169 inhibitors in apoptosis induction, 179 Interferons, antiviral activity, 83, 84 biosynthesis, 81, 82 classification, 81 interferon-α therapy, antitumor mechanisms, 84, 85 chronic myelogenous leukemia trials, 86–88 hairy cell leukemia trials, 88, 89 pharmacokinetics, 86 toxicity, 86 receptor and signaling, 82, 83 subtypes, 81, 82 Interleukin-2 (IL-2), acute leukemia trials, outcomes, 100, 101 single-agent trials, 98 stem cell transplantation combination, 98–100 function, 96 gene, 96 immune marker effects, 97 leukemia cell engineering for expression, 238

lymphocyte-activated killer cell effects, 96 solid tumor management, 97, 98 toxicity, 96, 97 L Large granular lymphocyte leukemia, apoptosis dysregulation, 170 Lentivirus, gene therapy vectors, 230, 231 LRP, see Lung-resistance protein Lung-resistance protein (LRP), assays, 148 prognostic value, 147 M MM, see Multiple myeloma Moloney murine leukemia virus, gene therapy vectors, 227–230 Monoclonal antibody therapy, alemtuzumab, 35–38 gemtuzumab ozogamicin, acute myeloid leukemia treatment rationale and response, 44, 53, 54 clinical trials, 48–50, 54–56 design and structure, 45–47 immune response, 52 pharmacokinetics, 50, 52 toxicity, 52, 53 Hu1D10, 38 HuM195, 30, 31 human antimouse antibody response development, 29, 30, 54 prospects for unconjugated antibody therapy, 39 radiolabeled antibodies, acute myeloid leukemia radioimmunotherapy, alpha-particle emitters, 72, 73

268

beta-particle emitters, 65–71 adult T-cell leukemia/lymphoma radioimmunotherapy, 73, 74 antigenic targets, 60, 61 conjugation methods, 63 dosimetry, 64, 65 mechanism of action, 60 pharmacokinetics, 63, 64 prospects, 74 radioisotope selection, 61–63 rationale, 29, 43, 59, 60 rituximab, 31–35 T101, 38, 39 MRP, see Multidrug-resistance associated protein Multidrug-resistance associated protein (MRP), ABC transporter, 147 genetic engineering of hematopoietic precursors, 242 prognostic value, 147, 149 Multiple myeloma (MM), arsenic trioxide trials, 195 Myb, antisense targeting, 112, 113 Myc, antisense targeting, 113 chromosomal translocation and overexpression, 128 N NHL, see Non-Hodgkin’s lymphoma Non-Hodgkin’s lymphoma (NHL), G3139 trials, 116, 175 P p53, antisense targeting, 113 gene therapy, 243

Index

p67, acute myeloid leukemia radioimmunotherapy, 70 PD98059, apoptosis induction in leukemia management, 177 PDGF, see Platelet-derived growth factor P-glycoprotein (PgP), ABC transporter, 146, 147 assays, 147 prognostic value, 145, 146, 148, 149 resistance, 155 reversal trials in acute myeloid leukemia, biricodar, 154 cyclosporine, 150–152, 154 gemtuzumab ozogamicin combination therapy, 154, 155 prospects, 156 PSC833, 150–153, 155 quinine, 149, 150 verapamil, 149, 150 zosuquidar, 154 therapeutic targeting rationale, 145, 146 PgP, see P-glycoprotein PKC, see Protein kinase C Platelet-derived growth factor (PDGF), STI571 inhibition of receptor tyrosine kinase, 138–140 PML/RARα, transcript product relocalization with arsenic trioxide, 191, 192 Protein kinase C (14K), inhibitors in apoptosis induction, 178 PSC833, P-glycoprotein reversal and clinical trials, 150–153, 155 Q Quinine, P-glycoprotein reversal and clinical trials, 149, 150

Index

R Radiolabeled antibodies, see Monoclonal antibody therapy Rituximab, CD20 targeting, 31 leukemia trials, B-cell chronic lymphocytic leukemia, 31–34 hairy cell leukemia, 34, 35 S STI571, apoptosis induction in leukemia management, 177 applications for other molecular targets and cancers, 140 chronic myelogenous leukemia management, clinical trials, 135, 136 combination therapy, 138 dose selection, 136, 137 preclinical studies, 134, 135 prospects, 137 rationale, 133 resistance, 137, 138 development, 133, 134 specificity of receptor inhibition, 138–140 tyrosine kinase inhibition, 128 Suicide gene, negative selection, 244, 245

269

T T101, leukemia therapy, 38, 39 T-cell, depletion effects in bone marrow transplantation, 15–17 genetic modification for leukemia therapy, 240, 241 T-cell prolymphocytic leukemia, alemtuzumab trials, 36 TRAIL, antileukemia therapy, 174 death receptor pathway, activation, 174, 175 overview, 164, 165 regulation, 167, 168 V Verapamil, P–glycoprotein reversal and clinical trials, 149, 150 Z Zosuquidar, P-glycoprotein reversal and clinical trials, 154

270

Index