Advances in Cancer Research Volume 81

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Advances in

CANCER RESEARCH Volume 81

This Page Intentionally Left Blank

Advances in

CANCER RESEARCH Volume 81

Edited by

George F. Vande Woude Van Andel Research Institute Grand Rapids, Michigan

George Klein Microbiology and Tumor Biology Center Karolinska Institutet Stockholm, Sweden

San Diego

San Francisco New York Boston London Sydney Tokyo

∞ This book is printed on acid-free paper.

C 2001 by ACADEMIC PRESS Copyright

All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the ﬁrst page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of speciﬁc clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2001 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-230X/01 $35.00 Explicit permission from Academic Press is not required to reproduce a maximum of two ﬁgures or tables from an Academic Press chapter in another scientiﬁc or research publication provided that the material has not been credited to another source and that full credit to the Academic Press chapter is given.

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Contents

Contributors to Volume 81 vii

A Decade of Progress in Allogeneic Hematopoietic Cell Transplantation: 1990–2000 Keith E. Stockerl-Goldstein and Karl G. Blume I. II. III. IV. V. VI. VII. VIII. IX. X.

Introduction 2 Prophylaxis Against Graft-versus-Host Disease 2 Source of Allogeneic Hematopoietic Cells 6 Matched Unrelated Donor Transplantation 8 Transplantation of Umbilical Cord Blood Cells 11 Transplantation from Haploidentical Donors 13 Immunotherapy Following Relapse 15 Allogeneic Transplantation using Nonmyeloablative Regimens 16 Complications of Allogeneic Hematopoietic Cell Transplantation 19 Advances in Allogeneic Hematopoietic Cell Transplantation for Speciﬁc Diseases 25 XI. Conclusions and Future Directions 48 References 48

A Role for Secondary V(D)J Recombination in Oncogenic Chromosomal Translocations? Marco Davila, Sandra Foster, Garnett Kelsoe, and Kaiyong Yang I. II. III. IV. V. VI. VII.

Introduction 62 V(D)J Rearrangement 63 Illicit V(D)J Rearrangement Mediated by Cryptic RSS or RSS-Like Motifs 72 The Germinal Center 73 Chromosomal Translocations in GC-Like Lymphomas 78 Research 83 Conclusions 86 References 87

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Contents

Recombinant Immunotoxins in Targeted Cancer Cell Therapy Yoram Reiter I. II. III. IV. V. VI. VII.

Introduction 94 Design of Recombinant Immunotoxins 98 Construction and Production of Recombinant Immunotoxins 104 Preclinical Development of Recombinant Immunotoxins 105 Application of Recombinant Immunotoxins 107 Other Applications of Recombinant Antibody Fragments 111 Challenges and Future Directions of Recombinant Immunotoxins 115 References 119

Human Herpesvirus-8 and Kaposi’s Sarcoma: Relationship with the Multistep Concept of Tumorigenesis Michael St urzl, ¨ Christian Zietz, Paolo Monini, and Barbara Ensoli I. II. III. IV.

Clinical Presentation of KS 126 Histology of KS and Nature of KS Spindle Cells 127 HHV-8 and KS 128 Conclusion 148 References 150

Reactivation and Role of HHV-8 in Kaposi’s Sarcoma Initiation Barbara Ensoli, Michael St urzl, ¨ and Paolo Monini I. II. III. IV. V. VI. VII.

Kaposi’s Sarcoma 162 Risk Factors Associated with KS Development 164 Histology of KS and Origin of Spindle Cells 166 HHV-8 Infection in KS Lesions 169 KS Initiation: Role of IC in KS Histogenesis and HHV-8 Infection 170 Lack of Control of Reactivated HHV-8 181 KS Progression: Oncogenes, Oncosuppressor Genes, HHV-8 Latency Genes, and the HIV-1 Tat Protein 184 VIII. Concluding Remarks 187 References 188

Index 201

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Karl G. Blume, Division of Bone Marrow Transplantation, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305 (1) Marco Davila, Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710 (61) ` 00161 Barbara Ensoli, Laboratory of Virology, Istituto Superiore di Sanita, Rome, Italy (125, 161) Sandra Foster, Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710 (61) Garnett Kelsoe, Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710 (61) ` 00161 Paolo Monini, Laboratory of Virology, Istituto Superiore di Sanita, Rome, Italy (125, 161) Yoram Reiter, Faculty of Biology, Technion-Israel Institute of Technology, Haifa 3200, Israel (93) Keith E. Stockerl-Goldstein, Division of Bone Marrow Transplantation, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305 (1) ¨ Michael Sturzl, Institute of Molecular Virology, GSF—National Research Center for Environment and Health, 85764 Neuherberg, Germany (125, 161) Kaiyong Yang, Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710 (61) Christian Zietz, Ludwig Maximilians University Munich, Institute of Pathology, 80337 Munich, Germany (125)

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A Decade of Progress in Allogeneic Hematopoietic Cell Transplantation: 1990–2000 Keith E. Stockerl-Goldstein and Karl G. Blume Division of Bone Marrow Transplantation Department of Medicine Stanford University School of Medicine Stanford, CA 94305

I. Introduction II. Prophylaxis Against Graft-Versus-Host Disease A. Graft-Versus-Host Disease Prophylaxis with Drugs B. T-Cell Depletion of the Graft III. Source of Allogeneic Hematopoietic Cells IV. Matched Unrelated Donor Transplantation V. Transplantation of Umbilical Cord Blood Cells VI. Transplantation from Haploidentical Donors VII. Immunotherapy Following Relapse VIII. Allogeneic Transplantation using Nonmyeloablative Regimens IX. Complications of Allogeneic Hematopoietic Cell Transplantation A. Solid Tumors Following Allogeneic Hematopoietic Cell Transplantation B. Posttransplant Lymphoproliferative Disorders C. Cytomegalovirus Infections D. Veno-occlusive Disease of the Liver E. Diffuse Alveolar Hemorrhage X. Advances in Allogeneic Hematopoietic Cell Transplantation for Speciﬁc Diseases A. Aplastic Anemia B. Thalassemia C. Sickle Cell Disease D. Acute Lymphoblastic Leukemia E. Acute Myeloid Leukemia F. Myelodysplastic Syndromes G. Chronic Lymphocytic Leukemia H. Chronic Myeloid Leukemia I. Non-Hodgkin Lymphoma J. Hodgkin Disease K. Multiple Myeloma XI. Conclusions and Future Directions References

1 Advances in CANCER RESEARCH 0065-230X/01 $35.00

C 2001 by Academic Press. Copyright All rights of reproduction in any form reserved.

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Keith E. Stockerl-Goldstein and Karl G. Blume

I. INTRODUCTION The role of hematopoietic cell transplantation (HCT) in the management of malignant and nonmalignant conditions has grown considerably since the initial description of safely infusing bone marrow cells into humans was reported by E. D. Thomas and his colleagues (Thomas et al., 1957). The early studies of marrow transplantation demonstrated the tolerability of myeloablative doses of total body irradiation (TBI) and the infusion of marrow cells with some evidence of clinical beneﬁt (Thomas et al., 1959b). Two children with acute leukemia received TBI followed by syngeneic transplantation, with temporary remissions observed (Thomas et al., 1959a). Later, 16 patients with hematologic malignancies, including 8 patients with acute myeloid leukemia (AML), received cyclophosphamide (CY) and TBI followed by syngeneic grafting (Fefer et al., 1974). The initial studies of allogeneic transplantation were hampered by limited knowledge of the human leukocyte antigen (HLA) system. However, with increased understanding of the HLA groups, along with improvements in immunosuppression and supportive care, there is an enhanced ability to safely perform allogeneic transplants. As the use of marrow transplant procedures became more widespread and experience with managing complications allowed safer transplantation, randomized studies were performed to help deﬁne the exact role for HCT in a variety of diseases. More recently, low-dose, nonmyeloablative regimens followed by allogeneic transplantation are being explored as alternatives to the high-dose therapies which were previously felt to be necessary to make a space available for the new marrow to grow. Over the past 25 years, HCT has moved from a role as a “treatment of last resort” to a standard treatment approach for a variety of disorders, many in their early stages. This chapter will describe some of the signiﬁcant advances in allogeneic HCT that occurred in the last decade between 1990 and 2000. It will also present discussions concerning some disease entities and ongoing controversies in the ﬁeld of transplantation.

II. PROPHYLAXIS AGAINST GRAFT-VERSUS-HOST DISEASE Graft-versus-host disease (GVHD) represents a response of donor T cells against alloantigens present on the surface of recipient cells. The exact antigenic determinants that are recognized are not clearly identiﬁed; however, even in transplants where donor and recipient are matched for the major HLA loci, GVHD will occur if no attempt at immunosuppression is provided.

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Investigations into these minor antigens continue, however, and advances in our understanding of GVHD and the use of newer immunosuppressive agents are allowing safer transplantation of allogeneic hematopoietic cells. GVHD is usually graded on a scale of I to IV, with a grade I toxicity representing minimal GVHD and grades III and IV representing severe GVHD. In addition, GVHD is separated into two clinical entities: acute GVHD, which occurs within the ﬁrst 100 days following infusion of the hematopoietic cell graft, and chronic GVHD, which occurs after that time period. In general, it is better to prevent GVHD from occurring rather than to wait until signiﬁcant GVHD is present and then try to halt the process with further immunosuppression. The incidence of acute GVHD reported depends on many factors, including preparative regimens, graft manipulation, and use of immunosuppressive agents, but usually ranges from 10% to 50% of patients receiving hematopoietic cell grafts from matched sibling donors.

A. Graft-Versus-Host Disease Prophylaxis with Drugs One of the most important agents used currently for GVHD prophylaxis is cyclosporine (CSP), a cyclic peptide isolated from soil fungi. CSP blocks a calcium-dependent signal transduction pathway in T cells that blocks interleukin-2 (IL-2) formation. Over the years, various regimens utilizing CSP along with other agents, most importantly corticosteroids and methotrexate (MTX), have been described for the prevention of GVHD (Storb et al., 1986). A series of consecutive studies performed at Stanford University and the City of Hope National Medical Center compared different combinations of CSP, MTX, and corticosteroids to determine the optimal regimen for GVHD prophylaxis. In the ﬁrst study, the combination of CSP and prednisone (PSE) was compared in a prospective randomized study with CSP/MTX/PSE in a group of patients with good-risk acute leukemia and chronic myeloid leukemia (CML) (Chao et al., 1993). The incidence of acute GVHD was signiﬁcantly lower for patients receiving the three-drug regimen than for patients receiving CSP/PSE alone (9% vs 23%, respectively). There were no differences in chronic GVHD or relapse between the two groups. In a follow-up study, the three-drug regimen of CSP/MTX/PSE was compared in a prospective, randomized fashion with the standard CSP/MTX (Chao et al., 2000). This study demonstrated no signiﬁcant differences in acute GVHD, chronic GVHD, or relapse between the two-drug regimen and the three-drug regimen. Some newer immunosuppressive agents now available are being added to the arsenal to prevent and/or treat GVHD. These agents include FK506 (tacrolimus), rapamycin (sirolimus), and MMF (mycophenolate mofetil). FK506 is a macrolide antibiotic with a mechanism of action almost identical to that of CSP. FK506 has been compared in a prospective randomized

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study of matched sibling donor HCT which evaluated CSP/MTX versus FK506/MTX (Ratanatharathorn et al., 1998). There was a much lower incidence of acute GVHD in patients who received the FK506 combination (31.9% vs 44.4%), although the incidences of grade III–IV acute GVHD were similar. There was an increase in clinically extensive chronic GVHD in the CSP group as well. However, the 2-year disease-free survival (DFS) and overall survival favored the CSP group. One difﬁculty in interpreting this study is the higher percentage of patients with advanced disease randomized to the FK506 arm; it was the poorer survival of these patients that seemed to be associated with the worse survival outcomes for the FK506 group. Further randomized trials will be required to better evaluate whether FK506 may offer an advantage for patients with early disease. At least one Phase II trial evaluated FK506/MTX for GVHD prophylaxis for patients undergoing matched unrelated donor (MUD) transplants and demonstrated a rate of grade II–IV GVHD of 50%, which is lower than reported by most investigators for CSP-based regimens (Devine et al., 1997). Currently, no randomized studies comparing CSP and FK506 have been reported for unrelated donor– recipient pairs. Rapamycin is a macrolide which shares some similarities to FK506 and actually binds to the same FK-binding proteins as FK506; however, the mechanism of action is much different, with rapamycin affecting signaling through the CD28/B7 pathway. Studies of rapamycin for GVHD therapy are ongoing. MMF is a derivative of mycophenolic acid, which is derived from Penicillium molds. The mechanism of action of MMF is distinctly different from that of CSP or FK506, and therefore MMF may have synergistic properties when used in combination with one of these agents. MMF acts by blocking de novo purine synthesis in lymphocytes and prevents T-cell activation. Currently there are limited published data regarding the use of MMF for human HCT; however, studies are being performed to deﬁne the role of this new drug for the prevention and treatment of GVHD. A number of other unique drugs are under investigation for the prevention and treatment of GVHD, including novel agents such as copaxone, CAMPATH, IL-2 receptor antibodies, and various biologic agents directed against T cells; however, outcome data are limited.

B. T-Cell Depletion of the Graft It well accepted that donor T cells are responsible for engraftment , GVHD reaction as well as the graft-versus-leukemia (GVL) effect. One approach that has been used to decrease GVHD is depletion of T cells from the hematopoietic cell graft using physical or immunological methods. Many studies performed in the 1980s demonstrated the feasibility and tolerability

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of T-cell-depleted HCT. Although these studies successfully demonstrated a decreased risk of GVHD compared with historical data using unmanipulated marrow grafts, also noted were apparent increases in graft rejection and relapse. Studies in the past decade continued to expand on these initial reports, often using alternative methods of T-cell depletion or the use of T-cell-subset depletion. In one report, marrow grafts from HLA-identical sibling donors were selectively depleted of CD8+ T cells using an anti-Leu-2 antibody plus complement (Champlin et al., 1990). This approach was undertaken in an attempt to decrease GVHD without affecting the GVL effect. Patients received CSP for GVHD prophylaxis. Thirty-six patients were treated, 3 of whom failed to engraft. The incidence of grade II or higher acute GVHD was 28%. Although the overall relapse rate was 11%, none of the 13 patients transplanted with CML had evidence of leukemic relapse at the time of the report, suggesting that the GVL effect might be intact. An alternative approach taken by some investigators utilizes an anti-CD6 monoclonal antibody for T-cell depletion. In one study, 112 adult patients with hematologic malignancies were treated with HLA-matched sibling donor bone marrow depleted of CD6-positive T cells with no additional posttransplant drug prophylaxis against GVHD (Soiffer et al., 1992). The incidence of graft failure was low (2.7%), with 18% of patients developing grade II–IV acute GVHD. The treatment-related mortality was 14%, with an estimated DFS of 50% at 3 years for standardrisk patients. The same group of investigators subsequently treated 27 adult patients with CD6-positive T-cell depletion using bone marrow from genotypically HLA-nonidentical related donors (Soiffer et al., 1997). In this study, even with most patients receiving grafts that were mismatched at one or two HLA loci, the incidence of grade III–IV acute GVHD was only 8%, with 32% developing grade II acute GVHD. The estimated DFS for patients with chronic-phase CML or acute leukemia in ﬁrst remission was 69% at 2 years. T-cell depletion has the beneﬁt of decreased GVHD, but the increased risks of relapse and graft rejection remain problematic. One method of trying to abrogate the increased risk of relapse associated with T-cell depletion is to deplete the marrow of T cells and then add back speciﬁc quantities of donor T lymphocytes to the graft. One group from the Netherlands treated 70 patients with hematologic malignancies or aplastic anemia with bone marrow depleted of T cells from HLA-identical sibling donors (Verdonck et al., 1994). A deﬁned aliquot of 1 × 105 donor T cells per kilogram was added back to the graft, and short-course cyclosporine was used posttransplant. The incidence of acute GVHD was high at 70% but was limited in all patients to skin GVHD of grade I or II. Chronic GVHD occurred in 31% of patients and was predominately limited to the skin. For patients with standard-risk leukemia, the relapse rate was only 8%, with an estimated 5-year survival of 80%.

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Based on encouraging data concerning GVHD in patients receiving T-cell-depleted grafts from HLA-matched sibling donors, T-cell depletion was investigated in the setting of transplants from MUD. In an early report, data from 115 patients with CML treated with T-cell-depleted grafts from MUD were described (Spencer et al., 1995). These patients received either in vitro or in vivo T-cell depletion. The probability of grade III–IV acute GVHD was 24%, the relapse risk at 3 years was 23%, and leukemia-free survival was 70% for younger patients with CML in chronic phase. The results of randomized studies of T-cell depletion versus unmanipulated marrow from matched sibling donors have been reported. One trial of 38 patients demonstrated a signiﬁcant decreased in GVHD in patients receiving graft depleted of CD8+ T cells but did not show a difference in overall survival or DFS (Nimer et al., 1994). In another study, 48 patients were randomized to receive unmanipulated marrow grafts or T-cell-depleted marrow grafts from matched sibling donors (Ringden et al., 1994). There were no signiﬁcant differences between the two groups with respect to acute or chronic GVHD, relapse, or survival. It is still not clear whether the advantages of decreased GVHD in T-celldepleted marrow transplantation outweigh the increased risks of relapse, graft rejection, or posttransplant lymphoproliferative disorders. Large randomized studies with adequate follow-up are necessary in the related and unrelated donor setting to answer these questions. Attempts to identify speciﬁc T-cell subsets that might allow the dissociation of GVHD and GVL continue, as do studies of T-cell depletion of allogeneic peripheral blood progenitor cell grafts. The debate over the role of T-cell depletion will continue until these issues are resolved.

III. SOURCE OF ALLOGENEIC HEMATOPOIETIC CELLS The use of peripheral blood “stem cells” or progenitor cells has become the standard source of hematopoietic cells in autologous transplantation. These cells are usually collected after administration of chemotherapy, growth factors, or both. Studies have demonstrated that “mobilized” peripheral blood progenitor cells (PBPCs) used for autologous HCT are associated with decreased length of neutropenia and thrombocytopenia as well as decreased costs because of shortened hospital stays. The use of PBPCs in the allogeneic transplant setting is still under investigation. One of the concerns regarding the use of allogeneic PBPCs is the demonstration that the T-lymphocyte content of these grafts is one log greater than that in a bone marrow graft. Serious questions regarding the negative impact of this increased T-cell dose

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on the risk of GVHD were raised (Bensinger et al., 1995). The use of allogeneic PBPCs allows the collection of cells from the donor without the need for an operative procedure or risks associated with anesthesia. The safety of granulocyte colony-stimulating factor (G-CSF) injections and leukapheresis as well as the donor tolerance of the procedure has been acceptable. The initial studies demonstrated the feasibility of obtaining adequate numbers of PBPCs from matched sibling donors without the development of excessive acute GVHD in the recipient. However, studies with longer follow-up suggested that chronic GVHD might be increased in patients who receive allogeneic PBPCs from HLA-matched sibling donors (Storek et al., 1997). More recently, data from randomized trials comparing allogeneic PBPCs to allogeneic bone marrow have been reported. In one small study, 30 patients were randomly assigned to receive PBPC or bone marrow grafts from siblings (Mahmoud et al., 1999). Patients receiving PBPCs had faster recovery of neutrophils and platelets as well as a signiﬁcantly lower incidence of acute GVHD. A different group of investigators described 40 patients randomly assigned to allogeneic PBPCs or bone marrow (Vigorito et al., 1998). They found no difference in the incidence of acute or chronic GVHD, but a higher percentage of patients who received PBPCs developed severe chronic GVHD. Again, no differences in DFS or overall survival were noted. The European Bone Marrow Transplant Group (EBMT) performed a prospective randomized study of 66 patients receiving either allogeneic bone marrow or PBPCs (Schmitz et al., 1998). They also found no differences in measures of GVHD or survival. A randomized study of allogeneic bone marrow versus allogeneic PBPCs involving 101 patients was recently reported from France (Blaise et al., 2000). The time until sustained neutrophil engraftment and the time to reach platelet counts of 25,000/␮l and 50,000/␮l were signiﬁcantly shorter for patients receiving allogeneic PBPCs. There was no difference in acute GVHD between the two groups, but the incidence of chronic GVHD was signiﬁcantly higher for patients receiving PBPCs (50%) than for patients receiving bone marrow (28%), p < 0.03. A larger study, including 174 patients randomized to PBPC or bone marrow grafts from fully matched sibling donors was recently presented (Bensinger et al., 2000). The time to engraftment of neutrophils and platelets was signiﬁcantly shorter for patients receiving PBPCs, and no signiﬁcant difference in the incidence of acute or chronic GVHD was demonstrated. There were reduced transplant-related mortality (TRM) and relapse rates in patients with advanced malignancies, which translated into superior overall survival. One group of investigators compared the outcomes of patients receiving MUD bone marrow and those receiving PBPC grafts (Ringden et al., 1999). The patients receiving PBPCs similarly had a shorter engraftment time, and no differences in acute GVHD compared with the patients receiving bone

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marrow were noted. However, the risk of chronic GVHD was higher for patients receiving bone marrow (85%) than for those receiving PBPCs (59%) or CD34-selected PBPCs (0%). The majority of the reported randomized studies comparing allogeneic hematopoietic cells obtained from the bone marrow or peripheral blood from sibling donors have demonstrated earlier recovery of white blood cells and platelets and no differences in acute or chronic GVHD. Although the ﬁndings of one study indicate that patients with advanced disease may gain a survival beneﬁt with allogeneic PBPCs, longer follow-up is necessary to evaluate differences in chronic GVHD and rates of relapse and survival.

IV. MATCHED UNRELATED DONOR TRANSPLANTATION Bone marrow transplants from unrelated donors were initially reported at least 20 years ago and were facilitated by local blood banks and histocompatability laboratories (Hansen et al., 1980; O’Reilly et al., 1977). Those smaller registries were limited in the number of patients for whom they were able to identify a donor, because of the small numbers of volunteers. The establishment of the National Marrow Donor Program (NMDP) was brought about through the dauntless efforts of many in 1986 to establish a national resource in the United States to locate donors for patients in need (McCullogh et al., 1989), and similar efforts have taken place on almost every continent. Throughout the past decade, increasing numbers of patients with malignant as well as nonmalignant conditions have been treated with high-dose therapy and MUD transplantation (Filipovich et al., 1992). One of the ﬁrst large analyses of the outcomes of patients transplanted under the auspices of the NMDP described the outcome of 462 transplants for acquired and congenital hematolymphoid disorders from unrelated donors (Kernan et al., 1993). In that report, 94% of patients reached engraftment by day 100, with acute GVHD occurring in 64% and a 1-year probability of chronic GVHD of 55%. Most notably, younger patients with good-prognosis leukemias had a 2-year DFS of 40%. The advances that have been achieved in HLA typing are most beneﬁcial for patients receiving transplants from MUD as well as from partially matched related donors (PMRD). Initial matching performed through the NMDP was based on serologic typing of HLA-A, B, and DR groups, along with mixed lymphocyte culture techniques. More recently, the importance of molecular testing of the HLA-DR antigens has become recognized. A retrospective analysis of 364 MUD patient–donor pairs using molecular techniques found that 59 (16%) were actually mismatched for DRB1

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(Petersdorf et al., 1995). The probability of survival at 1 year favored patients who had a molecular DRB1 match, and there was a signiﬁcant decrease in TRM and acute GVHD for these patients as well. Subsequently, DNA analyses of class I and class II HLA types in patients receiving MUD transplants have demonstrated that molecular mismatches at class I are also important predictors of survival (Speiser et al., 1996). The importance of the class I HLA-C loci is still unclear, but recent studies suggest that these loci may need to be considered in choosing an unrelated donor (Grundschober et al., 1997; Prasad et al., 1999). Interestingly, in a study of 440 patients who received MUD transplants, a retrospective analysis of class I typing demonstrated an increase in grade III–IV GVHD for patients mismatched at the HLA-C locus, but also a signiﬁcant decrease in relapse risk for patients who had a C-locus mismatch with their donors (Sasazuki et al., 1998). CML is the malignancy most often treated with HCT from MUD. One analysis of 196 patients transplanted with MUD cells with CML in chronic phase demonstrated a low incidence of relapse of 10%, with 57% overall survival at 5 years (Hansen et al., 1998). A number of factors were found to be predictive of adverse outcome, including a high body-weight index, age > 50, and a mismatch at HLA-DRB1. In addition, treatment in the ﬁrst few years following diagnosis of CML was associated with signiﬁcantly improved survival. More recently, a larger analysis of patients transplanted with CML using unrelated donors was reported (McGlave et al., 2000). It includes data on 1423 patients who received transplants facilitated by the NMDP and were transplanted at 85 centers. The incidence of early graft failure was 9.9%. An additional 6.6% of patients developed late graft failure. Severe acute GVHD occurred in 33%, with 60% of patients developing extensive chronic GVHD at 2 years. DFS at 3 years for patients transplanted in chronic phase was 43% and was signiﬁcantly better than that for those patients transplanted with more advanced stages of CML. A multivariate analysis demonstrated that transplantation in chronic phase, transplant performed within 1 year from diagnosis, recipient being CMV seronegative, and younger patient age were independent predictors of superior DFS. Figure 1 demonstrates the effect of age on survival in patients transplanted during chronic phase of CML less than 12 months from diagnosis. Notably, the relapse rate in the entire patient population was only 8.6%; however, when those patients transplanted in chronic phase were evaluated, the relapse rate was only 3.4% for patients who received unmanipulated marrow grafts, versus 16% for those who received T-cell-depleted grafts ( p = 0.0001). Assessments of performance status 2 years following transplant showed that 79% of the transplant recipients had Karnofsky scores of 90–100%. The procedure-related mortality associated with MUD transplantation appears to be higher than that observed with matched sibling donors; however,

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Fig. 1 Survival of patients with chronic myeloid leukemia transplanted with hematopoietic cells obtained from HLA-matched unrelated and tranplanted less than 12 months from diagnosis. Results are shown for patients less than 20 years of age (n = 49), between 20 and 35 years (n = 108), and other than (n = 137). (Reproduced by permission from McGlave, P. B., Shu, X. O., Wen, W., et al. Unrelated donor marrow transplantation for chronic myelogenous leukemia: 9 years’ experience of the National Marrow Donor Program. Blood 2000;95: 2219–25.)

no direct prospective comparisons have been reported. One study from the International Bone Marrow Transplant Registry (IBMTR) compared the outcomes for patients treated for leukemia using HLA-identical sibling grafts compared with PMRD or unrelated donors (Szydlo et al., 1997). This retrospective analysis of registry data demonstrated an increased relative risk of TRM of 1.8 for patients with leukemia receiving a MUD or PMRD transplant versus a matched sibling donor transplant. One approach to decreasing the toxicities of MUD transplants, including the high incidence of GVHD, consists of T-cell depletion of the graft. Some data suggest that the incidence of acute GVHD is signiﬁcantly lower in HCT recipients of a T-cell-depleted graft from MUD; however, this beneﬁt appears to come at the expense of both increased graft rejection and increased relapse rate. An ongoing prospective, randomized trial facilitated by the NMDP and the National Institutes of Health may answer the question of the overall risk–beneﬁt ratio associated with T-cell depletion following MUD HCT. As the number of unrelated donor transplants increases, it is important to note that the hematopoietic cell donors from these programs continue to have positive experiences. One NMDP-sponsored study evaluated a group of 493 unrelated donors with questionnaires administered pre- and postdonation (Butterworth et al., 1993). In this group, 87% considered the donation “very

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worthwhile,” with 91% stating that they would be willing to donate again at a future time. In addition, in patients with CML transplanted using unrelated donors, an economic analysis found that the cost for quality-adjusted life-year was within the intermediate zone of cost–effectiveness ratios (Lee et al., 1998). Although the morbidity and mortality of MUD transplants are greater than those of patients who have a related donor, this form of treatment is a potentially curative approach which should be considered for appropriate patients.

V. TRANSPLANTATION OF UMBILICAL CORD BLOOD CELLS Umbilical cord blood (UCB) contains hematopoietic progenitor cells similar to those found in bone marrow. In fact, there is evidence that cord blood progenitor cells have some proliferation advantages over cells present in marrow or peripheral blood. In addition, it is being postulated that the immaturity of the cells obtained from the umbilical cord might be associated with decreased GVHD and may allow mismatching of HLA loci with the transplant recipient. The ﬁrst description of an UCB transplant in humans involved the treatment of a child with Fanconi anemia using UCB obtained at the delivery of an unaffected histocompatible sibling (Gluckman et al., 1989). Subsequently, additional reports of cord blood transplants from sibling donors were communicated (Issaragrisil et al., 1995; Kurtzberg et al., 1994; Wagner et al., 1995) and, based on the success of these procedures, banks of cord blood cells were started to allow the possibility of unrelated cord blood transplant procedures (Gluckman et al., 1993; Rubinstein et al., 1995). The initial studies using related UCB units demonstrated that there was reproducible, although delayed, engraftment. Moreover, the risk of GVHD was less than one would have expected using suitably matched bone marrow grafts. As the number of cord blood specimens banked grew, transplants from unrelated cord blood units became feasible. A report of 25 patients, most of whom were children, showed a high level of engraftment, and only 2 of 21 evaluable patients developed severe acute GVHD (Kurtzberg et al., 1996). Remarkably, this low incidence of GVHD was noted even though only one of the patients in this trial received a fully matched UCB graft. Another study of 18 patients who received unrelated UCB transplants reported 100% engraftment with an 11% rate of GVHD (Wagner et al., 1996). With increasing numbers of transplants being performed, additional outcome data regarding survival are becoming available; for example, EBMT reported the outcomes of cord blood transplants from related and unrelated donors (Gluckman et al., 1997). Patients who received related UCB had a 1-year survival of 63%, with a survival of only 29% for unrelated UCB. An association

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between nucleated cell dose per kilogram recipient weight and survival was noted. The largest report of UCB transplants was published recently. It describes the outcome of 562 unrelated UCB transplants facilitated by the Placental Blood Program at the New York Blood Center (Rubinstein et al., 1998). The majority of transplants were performed in children; only 18% of patients were 18 years of age or older. Seventeen percent of recipients weighed ≥60 kg. The majority of patients had leukemia or lymphoma, and 24% had genetic disorders. There was no myeloid engraftment in 28% of patients, and the median time to neutrophil engraftment was 28 days. Grade III–IV acute GVHD was observed in 23%, with 25% of patients developing chronic GVHD. Notably, 218 patients died within the ﬁrst 100 days following transplantation, with almost half of the deaths due to infectious causes. The relapse rate for patients with leukemia was 14%. The number of cells infused per kilogram recipient body weight was signiﬁcantly correlated with the time to neutrophil and platelet engraftment, risk of transplantationrelated events, and event-free survival. Although there is a close association between age and number of cells infused per kilogram recipient body weight, adult patients had signiﬁcantly poorer event-free survival. Attempts at in vitro expansion of UCB cells may eventually increase the number of cells in a graft, but the impact of this maneuver on DFS remains to be demonstrated. A recent report described the outcome of 113 children who received UCB transplants and compared them to a group of 2052 children receiving allogeneic bone marrow in a retrospective fashion (Rocha et al., 2000). All patients received HLA-identical grafts from sibling donors. There was a signiﬁcantly longer time until neutrophil and platelet engraftment for those patients receiving UCB. The risk of both acute and chronic GVHD was signiﬁcantly lower with UCB on multivariate analysis with similar outcomes for survival and mortality between the two groups. Data on relapse were not included in this analysis. These data regarding the use of UCB for transplants are encouraging, but must be viewed in the appropriate context. Most important, the follow-up information regarding relapse are limited and there must be consideration that the decreased risk of GVHD seen after cord blood transplantation, presumably due to immaturity of the donor lymphocytes, may also be associated with higher risks of relapse. In addition, this procedure still is of limited utility in adults, based on weight and cell dose-related factors. Current investigations into expanding cells from cord blood units may help alleviate this issue, but the concerns regarding GVL activity of these cells and the high procedure-related mortality still make this a high-risk procedure. Finally, some unique ethical problems that are associated with using UCB for transplantation must be appreciated and have been discussed elsewhere

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(Burgio and Locatelli, 1997; Sugarman et al., 1997). The issues of ownership, consent, and privacy are not clearly deﬁned. For example, if a child who had his or her cord blood donated develops an infectious disease, genetic disease, or childhood leukemia, should the cord blood bank be notiﬁed in order to remove the unit if it has not been used, or to allow for follow-up of the recipient. Also, routine testing of the cord blood unit for infectious agents and genetic abnormalities may uncover information that would not normally be investigated if the cord blood were not tested. The question regarding disclosure of these results is also problematic. A number of private companies have started storing UCB for autologous or family use. Some companies have relied on skewed or grossly inaccurate information regarding the potential use of these cells to essentially intimidate families into storing UCB (Sugarman et al., 1997). Putting expectant parents into this position based on limited studies on the outcome of UCB transplantation is unacceptable. In fact, the use of autologous UCB to treat childhood leukemias should be questioned, given data suggesting that malignant cells may be present in fetal blood even though a malignancy is not diagnosed for 9 years or more (Ford et al., 1997; Mahmoud et al., 1995; Rowley, 1998). Although there is continued excitement over the use of UCB in the unrelated-donor setting, it is important that clinical studies continue to investigate the optimal method of performing these transplants and further research regarding the possibility of hematopoietic cell expansion be completed.

VI. TRANSPLANTATION FROM HAPLOIDENTICAL DONORS One approach to performing transplants in patients who do not have a fully matched sibling donor is to consider using a haploidentical donor, if one is available. This approach increases the likelihood of ﬁnding a related donor, since the patient will have a haplotype match with both parents as well as a 50% chance of having a single haplotype match with any sibling. In addition, it is fairly likely that close relatives could be identiﬁed who share a haplotype with the patient and all of the patient’s will share a haplotype with the patient. Haploidentical HCT will involve major HLA mismatches which are associated with GVHD, so adequate graft manipulation and/or immunosuppression must be utilized to allow a safe outcome. In a recently published study, 43 patients with high-risk acute leukemia were transplanted from haploidentical donors after no appropriate unrelated donor could be identiﬁed (Aversa et al., 1998). The patients received a preparatory regimen of TBI plus thiotepa, antithymocyte globulin (ATG), and ﬂudarabine. The hematopoietic cell grafts consisted of T-cell-depleted

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Fig. 2 Probability of disease-free survival in patients with acute myeloid leukemia or acute lymphoblastic leukemia and transplanted with haploidentical hematopoietic cells. (Reproduced by permission from Aversa et al., 1998.)

PBPCs with or without T-cell-depleted bone marrow. No posttransplant immunosuppression was given. In 41 of the patients, durable hematopoietic engraftment was attained, with the other 2 patients requiring infusion of T-cell-depleted peripheral cells from a different family member after failure of engraftment. Remarkably, even with major HLA incompatability, no evaluable patients developed acute or chronic GVHD. This type of HCT was associated with a signiﬁcant risk of infection, and two cases of B-cell lymphoproliferative disorder also developed. At the time of the report, with a median follow-up of 18 months, 12 of the 43 patients were alive and free of disease (Fig. 2). Another group of clinical investigators has taken an alternative approach using haploidentical bone marrow grafts which were made anergic to recipient alloantigens. Rather than performing a T-cell depletion technique, these investigators incubated the bone marrow grafts with recipient monocytes in the presence of the fusion protein CTLA-4-Ig (Guinan et al., 1999). CTLA-4Ig is an inhibitor of the B7:CD28 interaction and is used to promote anergy between the donor cells and recipient cells ex vivo. Twelve young patients were treated with this approach and CSP/MTX was used for GVHD prophylaxis. Studies of the bone marrow graft after treatment with CTLA-4-IG demonstrated decreased in vitro reactivity to recipient cells. Only three patients developed acute GVHD with no documented cases of relapse. At the time of the report, ﬁve of the patients were alive and in complete remission. Ongoing studies of haploidentical transplants continue to expand on these early trials. Methods of decreasing the incidence of infectious complications will need to be developed to decrease the risks associated with this maneuver.

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VII. IMMUNOTHERAPY FOLLOWING RELAPSE The relationship between prior clinical GVHD and the decreased risk of subsequent relapse was identiﬁed early in human transplantation experience (Weiden et al., 1979, 1981). The potential importance of a GVL effect was demonstrated most clearly in patients who were transplanted for CML, but was also noted in patients transplanted for other malignancies (Horowitz et al., 1990; Sullivan et al., 1989). In fact, some patients who relapse following allogeneic transplantation may enter another remission with discontinuation of immunosuppression alone (Collins et al., 1992). The potential use of infusions of donor buffy coat cells to treat relapse has been described (Kolb et al., 1990). The initial studies with donor lymphocyte infusions (DLI) demonstrated some responses as well as GVHD. Subsequently, many groups began quantifying the number of infused DLI T cells in attempts to ﬁnd optimal therapeutic doses with decreased risks of GVHD. In one study, eight patients with CML, including six patients in accelerated phase and two in blast crisis, who received HLA-matched sibling marrow grafts and subsequently relapsed were treated with 2.5–5.0 × 108 T cells/kg obtained from the original marrow donor (Drobyski et al., 1993). Seven of these eight patients developed GVHD, with four patients developing marrow aplasia, which in some cases required boosts of bone marrow from the original donor. All six patients who were in accelerated phase attained a cytogenetic remission, with ﬁve of the six even attaining a molecular remission. A larger report from Europe described the outcomes of DLI in 135 patients with CML, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), and polycythemia vera, who had allogeneic transplants which were obtained primarily from HLA-matched sibling donors (Kolb et al., 1995). The highest response rate was demonstrated in CML, with 73% induced into a complete remission, and a complete remission rate of 29% in patients with AML. Notably, there were no responses to DLI for patients with ALL (Fig. 3). Again both GVHD (41%) and myelosuppression (34%) were notable toxicities of this therapy. No association between numbers of infused mononuclear cells and response was identiﬁed; however, the T-cell content of the DLI was not studied. Ideally, one could deﬁne an appropriate T-cell dose of DLI with a high likelihood or remission with an acceptable level of GVHD and myelosuppression. One attempt used escalating doses of DLI with the intent to separate these toxicities from the desired therapeutic effect in 22 patients with CML (Mackinnon et al., 1995). These patients had all relapsed with their underlying disease following a T-cell-depleted HLA-matched marrow transplant procedure. In this study there appeared to be a GVL effect, with limited

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Fig. 3 Probability of survival in patients with recurrent leukemia following allogeneic bone marrow transplantation and subsequent treatment with donor lymphocyte infusions for acute myleoid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS) or polycythemia vera (PVC). (Reproduced by permission from Kolb et al., 1995.)

GVHD in patients receiving relatively low doses of T cells in the range of 1 × 107 /kg; however, patients who receive T-cell-depleted marrow grafts and subsequently receive DLI may behave differently from patients who have received unmanipulated grafts.

VIII. ALLOGENEIC TRANSPLANTATION USING NONMYELOABLATIVE REGIMENS The purpose of myeloablative preparatory regimens used for allogeneic HCT has been to eradicate the malignant clone of cells while also causing immunosuppression of the host to reduce the risk of graft rejection. The myeloablative maneuver was also felt to be necessary in order to create space in the marrow for the hematopoietic cell graft. It was subsequently detected that some patients were not full hematopoietic chimeras as expected, but had attained a state of mixed hematopoietic chimerism, with both host and donor hematopoietic cells present (Branch et al., 1982; Hill et al., 1986). Initially, there were concerns that the identiﬁcation of residual host hematopoietic

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cells would be indicative of subsequent relapse and of a failure of the transplant procedure to cure the patient. However, studies in animal experiments as well as human clinical data suggested otherwise. In fact, retrospective analyses demonstrated that patients who developed some mixed chimerism had a decreased incidence of GVHD and improved survival (Petz et al., 1987). Animal studies were then performed to determine the conditions which might allow the use of a nonmyeloablative regimen in order to purposely develop a mixed chimerism state, at least transiently. This approach might allow tolerance to host tissues to develop, with a decrease in GVHD, and it was hoped would not interfere with a GVL effect. The potential of these transplants to treat a variety of conditions, even in medically inﬁrm patients who might not be able to tolerate the rigors of a myeloablative regimen, has recently been reviewed (McSweeney and Storb, 1999). The general concept of the clinical application of a mixed-chimerism transplant is presented in Fig. 4. The ﬁgure demonstrates changes in microsatellite markers which can be followed in a patient undergoing a nonmyeloablative regimen. Using chemotherapy with or without low-dose radiation and along with immunosuppressive agents such as CSP and MMF, allogeneic hematopoietic cells are infused into a patient. Subsequent evaluation of microsatellite markers demonstrate mixed chimerism, with presence of both donor and recipient bands. Subsequently, by a decrease in the immunosuppressive agents or by the use of DLI, the patient can be converted to full donor hematopoietic

Fig. 4 Concept of mixed chimeric allogeneic transplantation using microsatellite markers of hematopoietic cell DNA to identify recipient and donor bands. (Reproduced by permission from McSweeney and Storb, 1995.)

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chimerism. This method should allow a GVL effect with acceptable GVHD. In fact, for some conditions such as immunodeﬁciency, genetic disorders, or autoimmune diseases, a stable mixed chimerism may be adequate to control the underlying disease. In addition, this method may induce a tolerance to allografted solid organs from the hematopoietic cell donor (Spitzer et al., 1999). Many different terms have been applied to this type of approach, including “mini-transplants,” “transplant-lite,” “mixed-chimerism transplantation,” “nonmyeloablative transplantation,” and “chimerism-induced immunotherapy.” Although there is great interest in this approach, with many names for this maneuver and extensive preclinical data to support these studies, there are few reports in the literature regarding clinical outcomes of humans undergoing such a procedure. In one report, 15 patients with acute myeloid leukemia or MDS were treated with ﬂudarabine-based regimens using CSP and methylprednisolone for immunosuppression (Giralt et al., 1997). This regimen was myelosuppressive but not myeloablative. The time to reach an absolute neutrophil count of >500/␮l was 10 days. There was one treatmentrelated death, and 6 patients were alive at the time of the report. There were only 3 patients who developed severe acute GVHD. Another report using a myelosuppressive regimen using a regimen with busulfan (BU), ATG, and ﬂudarabine has also been described (Slavin et al., 1998). The study describes the outcomes of 26 patients with malignant and nonmalignant conditions. Twenty-four of the patients became neutropenic, with absolute neutrophil counts <500/␮l. Twenty-ﬁve percent of patients developed severe acute GVHD, and 10% died of transplant-related causes. Most notably, the 14-month DFS was 77.5% An alternative approach has been taken in a collaboration between researchers at the Fred Hutchinson Cancer Research Center, Stanford University, and the University of Leipzig. This protocol is based on preclinical data based on a canine model and uses low-dose TBI in a single 200-centigray dose along with posttransplant immunosuppression using CSP and MMF. Patients (n = 45) included in these studies were not considered candidates for a standard allogeneic transplant due to age or concurrent co-morbid conditions (McSweeney et al., 2000). Fifty-three percent of the patients completed the procedure entirely in an outpatient setting. Grade II–III acute GVHD occurred in 47% of patients, and nonfatal graft rejection developed in 20% of patients. Treatment-related mortality was 6.7% and survival was 73.3%, with a median follow-up of 244 days. Fifty percent of patients who had sustained donor engraftment attained a complete remission. Subsequent modiﬁcations of this protocol with the inclusion of ﬂudarabine have decreased the incidence of graft rejection while still allowing a nonmyelosuppressive outpatient procedure. By decreasing the morbidity and mortality of allogeneic transplantation in this way, there will be expansion of the scope of

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patients and medical conditions that can be treated with allogeneic HCT. Ultimately, long-term data from ongoing studies are required to determine the full impact of such chimerism-induced immunotherapy.

IX. COMPLICATIONS OF ALLOGENEIC HEMATOPOIETIC CELL TRANSPLANTATION There are many complications associated with allogeneic HCT related to the direct toxicity and immunosuppression of a myeloablative preparatory regimen and GVHD prophylaxis. This section will concentrate on select complications of allogeneic HCT, and the reader is referred to other sources for in-depth discussions of these topics (Ambinder, 1999; Bowden, 1999; Crawford, 1999; Deeg, 1999; Openshaw and Slatkin, 1999; Strasser and McDonald, 1999; Zaia, 1999).

A. Solid Tumors Following Allogeneic Hematopoietic Cell Transplantation Now that patients have been cured of diseases such as leukemia or severe aplastic anemia (SAA) for a number of decades, data have become available to assess the potential long-term complications of allogeneic HCT. Initial studies in patients with SAA demonstrated a high risk of solid tumors following allografting. In one report from the EBMT evaluating 748 allografted restrictive patients with SAA, the relative risk of cancer was 6.67 compared with the general European population (Socie et al., 1993). In this study, transplant conditioning with radiation-containing regimens was an independent risk factor for secondary tumors, which conﬁrms data from earlier preclinical studies (Deeg et al., 1980). The largest evaluation of solid malignancies occurring following transplantation described the outcome of 19,229 patients transplanted between 1964 and 1992 and composed predominately of allografted patients, although 2.8% of the cohort received syngeneic grafts (Curtis et al., 1997). The median age at the time of transplant was 25.5 years. For patients who survived more than 1 year following transplantation, the median follow-up was 3.5 years, with a maximum follow-up of 25 years. Seventy-ﬁve percent of patients were transplanted for leukemias and 11.2% for SAA. Eighty new cases of invasive solid tumors were observed in this cohort, which was a 2.7-fold increase over the expected incidence. The risks were signiﬁcantly elevated for tumors of the bone, thyroid, connective tissue, central nervous

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system, liver, and buccal cavity and for melanoma. There was little increase in risk for the more common adult tumors. The risk increased with time from transplant, and there was a strong association between patient age at the time of transplant and risk of tumors developing. The risk was highest for patients transplanted at less than 10 years of age and was almost normal for patients transplanted over the age of 29. Patients with acute leukemias had the highest risk of developing tumors. The use of radiation-containing preparatory regimens was associated with a higher risk of solid cancers than the use of chemotherapy-only regimens, and there was a positive relationship between radiation dose and risk of tumors. A report of second cancers developing in children treated with allogeneic HCT for childhood acute leukemias has been published (Socie et al., 2000). This study evaluated 3182 patients up to the age of 16 years, transplanted between 1964 and 1992. In this study population, 3% of patients had received syngeneic grafts. The majority of patients were transplanted for acute lymphoblastic leukemia (ALL). The risk of solid tumors was 11% at 15 years and also showed an association with younger age at HCT. The association of cancers with high-dose TBI was also noted. Although patients who receive allografts have an increased risk of solid cancers as well as an increased risk of late death, often due to GVHD (Socie et al., 1999), it is important to remember that all of these patients were treated for life-threatening diseases. Patients with diseases such as Hodgkin disease (HD) or non-Hodgkin lymphoma (NHL) have an increased risk of late complications such as secondary tumors when treated with nontransplant regimens as well (Doria et al., 1995; Swerdlow et al., 2000; van Leeuwen et al., 2000). Although the information regarding late complications of transplant is valuable to researchers, treating physicians, and patients, one must not overlook the alternative if these patients did not receive a transplant (Thomas, 1999).

B. Posttransplant Lymphoproliferative Disorders Epstein–Barr virus (EBV)-associated posttransplant lymphoproliferative disorders (PTLD) are associated with immunosuppression and can develop in patients following allogeneic HCT as well as after solid organ transplantation (Dror et al., 1999). These disorders typically develop within the ﬁrst 6–12 months following allogeneic HCT, and the use of radiotherapy or chemotherapy is generally unsuccessful in the treatment of these tumors. In order to identify risk factors for PTLD, a retrospective analysis of patients reported to the IBMTR or treated at the Fred Hutchinson Cancer Research Center was performed on 18,014 patients who had received allogeneic HCT (Curtis et al., 1999). There were 78 cases of PTLD which developed in this

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cohort of patients. Multivariate analysis found an increased risk of PTLD associated with MUD or mismatched related donor HCT [relative risk (RR) 4.1], T-cell-depleted marrow (RR 12.7), and the use of either ATG (RR 6.4) or anti-CD3 monoclonal antibody (RR 43.2) for the prevention or treatment of GVHD. There was also an association between the use of radiationcontaining preparatory regimens and the risk of PTLD, although the relative risk was much lower at 2.9. The use of immunotherapeutic maneuvers to treat PTLD are being reported, with some methods showing reasonable success rates. One approach utilized the anti B-cell antibodies CD21 and CD24. In one report, 5 of 14 patients with PTLD following allogeneic HCT were alive and free of PTLD following this treatment (Fischer et al., 1991). The approach of using immunotherapy with DLI can also be applied to posttransplant EBV-associated lymphoproliferative diseases. This situation is different from a relapse following HCT in that the lymphoproliferative disease arises from donor cells rather than from those of the recipient. This intervention is most likely to be effective in patients who received T-cell-depleted grafts. In this case, DLI is used to increase the number of cytotoxic T cells which recognize EBV. In one study, 5 patients with EBV-associated lymphoproliferative disorder were treated with infusions of 1 × 106 CD3+ cells/kg; all attained complete remissions and in some patients was sustained (Papadopoulos et al., 1994). Another group has infused EBV-speciﬁc cytotoxic T lymphocytes (CTLs) into patients for either parallel prophylaxis or treatment of PTLD (Rooney et al., 1998). These CTLs were marked with the neo gene to allow followup evaluations of CTL survival after infusion. No patients developed acute GVHD associated with the CTL infusions, and none of the 39 patients developed PTLD. Two patients who did not receive prophylactic CTL infusions developed PTLD, and both responded completely to the CTL infusions. These approaches using DLI or speciﬁc anti-EBV CTL may offer a reasonable success rate for patients who receive T-cell-depleted allogeneic HCT.

C. Cytomegalovirus Infections Infectious complications following transplantation remain a signiﬁcant cause of morbidity and mortality. Even after engraftment of neutrophils, the immune system remains abnormal for a prolonged period of time following an allogeneic HCT procedure. Aspergillus remains one of the most difﬁcult and life-threatening infectious agents for transplant patients, and little progress has been made in the treatment of this infection (Bowden, 1999). One of the most signiﬁcant advances in the management of infections has been a better ability to treat cytomegalovirus (CMV) infections. Early studies

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performed in the 1980s, before the availability of antiviral therapy for CMV, reported that approximately 30–40% of patients receiving allografts developed CMV disease, predominantly interstitial pneumonia, with a median time of onset of almost 2 months following the HCT procedure. This was almost always a fatal complication. Although ganciclovir became available in the 1980s, the initial studies of treatment of established CMV pneumonia with ganciclovir were not very promising. The addition of intravenous immunoglobulin (IVIG) to ganciclovir ﬁnally demonstrated that established CMV disease could be successfully treated in at least 75% of patients, as reviewed by Zaia (1999). Subsequent studies have focused on preemptive therapy for patients at risk for CMV disease but with no clinical ﬁndings of active CMV. One approach involved the use of bronchoscopy with bronchoalveolar lavage (BAL) on day 35 following transplant for patients who were CMV seropositive pretransplant or who had received grafts from seropositive donors (Schmidt et al., 1991). Patients with positive CMV cultures were randomized to ganciclovir therapy or observation. Patients randomized to preemptive ganciclovir therapy had a signiﬁcantly lower risk of CMV pneumonia or death (25%) than patients randomized to observation (70%). An update in a larger group of patients conﬁrmed the value of preemptive therapy based on BAL (Zaia et al., 1995). Another group of investigators began treatment of patients with ganciclovir once CMV was detected by culture of blood, urine, throat swabs, or BAL (Goodrich et al., 1991). Patients with detectable virus were randomized to treatment with ganciclovir or placebo until day 100 following HCT. CMV disease developed in 43% of patients receiving placebo and in only 3% of patients receiving ganciclovir. More recently, newer methods have allowed earlier detection of CMV using tests of antigenemia or PCR methods. In one comparison of techniques, the median time to ﬁrst detection of CMV was 51 days with the blood culture technique, 42 days for the antigenemia assay, and only 32 days for a leukocyte PCR assay (Boeckh et al., 1997). One study has compared the use of preemptive ganciclovir therapy for patients randomized to a leukocyte PCR method of surveillance or blood cultures (Einsele et al., 1995). The patients followed with PCR had a signiﬁcantly lower risk of developing CMV disease (5% vs 23%), a lower CMV-related mortality, and a lower risk of neutropenia. Most notably, the overall survival for patients monitored by PCR was 94% and only 76% for patients monitored by blood culture methods. The availability of newer antiviral drugs such as foscarnet and cidofovir may allow better prophylaxis and therapy for patients following allogeneic HCT. Vaccination strategies are also being investigated to prevent the development of CMV disease while avoiding the toxicities associated with the

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available antiviral medications. One of the great successes of the past decade for the management of patients undergoing allogeneic HCT is the ability to prevent CMV disease as well as to treat established CMV disease with effective antiviral therapies.

D. Veno-occlusive Disease of the Liver Veno-occlusive disease (VOD) of the liver remains a signiﬁcant complication of allogeneic HCT which is associated with substantial morbidity and mortality. The syndrome involves jaundice, hepatomegaly, ascites, weight gain, and right-upper-quadrant pain. The incidence of this complication in published reports varies considerably depending on the exact deﬁnition used. Diagnosis is predominantly a clinical one with the aid of doppler ultrasound measurements of hepatic blood ﬂow or increases in hepatic “wedge” pressure during transjugular, intrahepatic biopsy procedures (Strasser and McDonald, 1999). Identiﬁed risk factors for the development of VOD include the use of busulfan-containing preparative regimens and prior liver disease including hepatitis B and C infections. It is believed that damage to the vascular endothelium is part of the pathogenesis of this clinical entity, and many of the therapies for prophylaxis and treatment of VOD rely on methods of changing the coagulability of the blood. The mortality rate is high for patients with advanced VOD, and estimates of outcome for patients developing VOD have been proposed (Bearman et al., 1993). Studies of prophylaxis for VOD have been reported. One trial evaluated the effectiveness of ursodeoxycholic acid versus placebo in preventing VOD in patients receiving a BU-containing regimen (Essell et al., 1998). This trial involved 67 patients who received a preparatory regimen of BU/CY with GVHD prophylaxis of CSP/MTX. There was a signiﬁcant decrease in the incidence of VOD for the patients receiving ursodeoxycholic acid. A randomized study of low-dose continuous heparin infusions using a dose of 100 units/kg/day has been reported in a group of patients receiving either an allogeneic or autologous HCT (Attal et al., 1992). The heparin group had a signiﬁcantly lower incidence of VOD, with a beneﬁt in the allogeneic transplant group that was highly signiﬁcant (0% vs 18%). Other studies have not conﬁrmed the beneﬁt of low-dose heparin (Carreras et al., 1998; Marsa-Vila et al., 1991). The ﬁnding of low levels of antithrombin III in patients with VOD led to a study of VOD prophylaxis using antithrombin III infusions; however, no differences in VOD incidence were noted (Budinger et al., 1996). Once VOD is established, the best treatment for this entity is undetermined. Treatment with tissue plasminogen activator is described, with

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signiﬁcant risks of hemorrhage and a response rate in the range of 40% (Bearman et al., 1992; Kulkarni et al., 1999; Leahey and Bunin, 1996). The ﬁbrinolytic agent deﬁbrotide has been used for treatment of VOD, based on encouraging results in one analysis (Richardson et al., 1998). Most patients who develop mild or moderate-severity VOD will have resolution of the process without intervention. Currently, the best methods of VOD prophylaxis and treatment are still to be determined.

E. Diffuse Alveolar Hemorrhage The number of pulmonary complications of allogeneic HCT are substantial, and some centers report the incidence of these problems as high as 60% (Cordonnier et al., 1986). Diffuse alveolar hemorrhage (DAH) was reported as a distinct clinical entity in patients receiving high-dose therapy and ABMT (Robbins et al., 1989). Clinically, DAH is associated with dyspnea, hypoxemia, and pulmonary inﬁltrates and typically occurs in the periengraftment period. Diagnosis requires that BAL be performed with the presence of progressively bloodier return of the ﬂuid on successive lavages of a lung segment. In addition, no evidence of ongoing pulmonary infections should be identiﬁed. Most reports focus on the occurrence of DAH in the autologous transplant setting and have utilized corticosteroids to treat this condition (Chao et al., 1991a). We have previously reported the outcome of 338 adult patients who received an allogeneic HCT at Stanford University (Sinning et al., 1997). Twenty-six patients developed DAH (7.7%). We have now updated a casematched analysis with 4:1 control group matching for this group of patients with a median follow-up of surviving patients of 5.6 years. The median time to diagnosis of DAH was 19 days from the allograft infusion. Patients developing DAH were more likely to be female (65.4% vs 37.5%) and were more likely to have a male donor. In addition, patients who were CMV seropositive had a signiﬁcantly higher risk of DAH. We also noted that the rate of white blood cell engraftment (slope) was signiﬁcantly higher in patients who developed DAH. There was a signiﬁcantly higher incidence of subsequent invasive fungal infections in the DAH population (54% vs 14%). Most notably, patients who developed DAH had a 5-year DFS of 23%, with the control group EFS at 47% ( p = 0.008). The overall survival was also signiﬁcantly different and favored the control population ( p = 0.004). Although the risk of DAH in the allogeneic transplant setting remains substantial and most patients respond to high-dose corticosteroids, the mortality remains high related to infectious complications. The institution of aggressive antifungal prophylaxis in these patients is warranted.

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X. ADVANCES IN ALLOGENEIC HEMATOPOIETIC CELL TRANSPLANTATION FOR SPECIFIC DISEASES The number of diseases being treated with allogeneic HCT has increased over the past two decades as a better understanding of the immunotherapeutic effect of this treatment has been gained. In addition, improvements in prevention and management of complications of allogeneic HCT have allowed the use of this therapy for patients earlier in their disease course. The following sections describe pertinent advances that occurred for select malignant and nonmalignant conditions in the past decade. This is not meant to be a comprehensive list of diseases being treated with allogeneic HCT, and the reader is referred elsewhere for an in-depth discussion on the use of allogeneic HCT for the treatment of inherited disorders (Thomas et al., 1999).

A. Aplastic Anemia The successful treatment of patients with severe aplastic anemia (SAA) was one of the earliest successes of allogeneic HCT (Storb et al., 1977). One unique quality of this disease is that it appears to be an immune-mediated disease without a malignant component. Additionally, the marrow space is essentially empty and available for the bone marrow graft. Thus, the preparatory regimen does not need to be as intense as one necessary to treat acute leukemias or other diseases where a malignant clone must be eradicated. The major problem with the early transplant experience was related to the high incidence of graft rejection, which was in excess of 30% (Storb et al., 1977). For patients transplanted when “untransfused,” the long-term outcome appeared to be improved, with a marked decrease in graft rejection and superior long-term survival (Anasetti et al., 1986). Subsequently, in patients who received their ﬁrst transplant with a conditioning regimen of singleagent CY, second transplants using CY and ATG demonstrated this to be an effective salvage regimen (Storb et al., 1987) while avoiding TBI, which was associated with increased risks of second malignancies in SAA patients (Witherspoon et al., 1989). In a group of 39 patients receiving a preparatory regimen of CY/ATG as an initial transplant approach, only 5% of patients had graft rejection and these patients were successfully retransplanted (Storb et al., 1994). Acute GVHD occurred in only 15% of patients, with a 34% incidence of chronic GVHD. The overall survival rate at 3 years was 92%. An analysis of 1305 patients with SAA reported to the IBMTR evaluated patients who received their allogeneic HCT between 1976 and 1992 (Passweg et al., 1997). When this

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group of patients was evaluated by period of transplantation (1976–1980, 1981–1987, 1988–1992), the 5-year survival increased from 48% in the earliest group to 66% for the most recent group ( p < 0.0001). Multivariate analysis found the most important factor associated with the improvement in outcome to be related to the use of CSP for GVHD prophylaxis. There was no signiﬁcant association between outcome and preparatory regimen utilized for the allografting procedure. The group of investigators at the Fred Hutchinson Cancer Research Center performed an analysis of long-term outcome for 212 patients allografted for SAA (Deeg et al., 1998). The report was restricted to patients transplanted between 1970 and 1993 and who survived for 2 years or greater. The probability of survival at 20 years was 89% for patients without chronic GVHD and 69% for patients with chronic GVHD (Fig. 5). Twelve percent of patients developed a solid tumor. This group of patients demonstrated rapid return to normal levels of function, with 83% returning to school or work at 2 years and 90% by 20 years. Notably, more than 50% of patients had the preserved ability to become pregnant or father children. Over the past two decades, the use of immunosuppressive agents to treat patients with SAA who were not candidates for allogeneic HCT has increased. Studies to determine the effectiveness of immunosuppression compared with allogeneic HCT are reported. The group at the Fred Hutchinson Cancer Research Center compared 227 patients treated with immunosuppressive therapy to 168 patients treated with allogeneic HCT (Doney et al., 1997). The actuarial 15-year survival was 69% for allogeneic HCT patients and only 38% for patients treated with immunosuppression ( p < 0.001). Twenty of the patients receiving immunosuppressive therapy developed MDS, and none of the patients receiving allogeneic HCT developed this serious problem ( p < 0.001). As described above, numerous studies have demonstrated an increased risk of second malignancies in patients undergoing allogeneic HCT, including patients with SAA. One analysis was performed to evaluate the risk of malignancies occurring after either allogeneic HCT or immunosuppressive therapy for SAA (Socie et al., 1993). Eight hundred sixty patients treated with immunosuppression were compared with 748 patients who received an allogeneic HCT. Both groups of patients had an increased relative risk of cancer compared with the general European population, with a relative risk for the combined cohort of 5.5. The 10-year cumulative incidence rate for cancer was 18.8% for patients treated with immunosuppressive therapy and 3.1% for the transplant group. The incidence of solid tumors was similar in the two groups, although patients treated with immunosuppression were at higher risk of myelodysplastic syndrome and leukemia. An analysis of 700 patients who received allogeneic HCT for either SAA or Fanconi anemia showed a 14% incidence of malignancies at 20 years (Deeg et al., 1996). This complication was signiﬁcantly increased

Fig. 5 Survival of patients with severe aplastic anemia receiving allogeneic hematopoietic cells transplanted from a matched related donor by year of transplant: (A) survival of patients with chronic graft-versus-host disease; (B) survival of patients with no chronic graft-versus-host disease. (Reproduced by permission from Deeg et al., 1998.)

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Keith E. Stockerl-Goldstein and Karl G. Blume

in patients with Fanconi anemia. Multivariate analysis demonstrated that the use of azathioprine and radiation-containing preparative regimens were factors which increased the risk of malignancies. Although there appears to be an increased risk of malignancy following immunosuppression as well as better survival for younger patients treated with allogeneic HCT, some patients who are older or who do not have a matched sibling donor may initially be treated with immunosuppressive therapy. These patients can be considered for allogeneic HCT after they have failed immunosuppression. An analysis of 141 patients who failed one or more courses of immunosuppressive therapy and subsequently received MUD transplants has been reported (Deeg et al., 1999). The preparatory regimens varied, but the majority of patients received TBI plus chemotherapy. Most patients received CSP as part of the GVHD prophylaxis, and 32% of patients received T-cell-depleted marrow grafts. Twenty-six percent of the patients received transplants from a mismatched donor. Graft failure occurred in 11% of patients, with a rate of acute GVHD (grade II–IV) of 52% and risk of chronic GVHD of 31%. The 5-year Kaplan-Meier estimate of survival was 34%, and there was no difference in engraftment for patients who received radiation-containing regimens versus those treated with chemotherapy alone. There is evidence to suggest that younger patients with matched sibling donors should receive allogeneic HCT as their initial treatment. For patients who fail immunosuppressive therapy, many can be successfully transplanted from unrelated donors and achieve prolonged survival. The use of nonmyeloablative allogeneic HCT may eventually have a role in the treatment of patients with SAA, since they do not need ablation of a malignant clone. It is hoped that the less intense preparatory regimens will be associated with decreased morbidity in this patient group.

B. Thalassemia In many parts of the world, ␤-thalassemia continues to be a signiﬁcant cause of morbidity and mortality. The standard treatment of this disease requires hypertransfusion along with iron chelation therapy. This approach can prevent some of the complications of the hemolysis associated with the condition, but it requires a regimen of daily infusions of deferoxamine over many hours and exposure to the infectious risks associated with massive amounts of red blood cell transfusions. This regimen of hypertransfusion and iron chelation signiﬁcantly affects the lifestyles of patients with thalassemia, and many patients are unable to comply with this exceedingly expensive treatment. The only available curative treatment is replacement of the hematopoietic system through the use of allogeneic hematopoietic cell

Allogeneic Hematopoietic Cell Transplantation: 1990–2000

29

transplantation. The use of this treatment modality was ﬁrst described in 1982 (Thomas et al., 1982). Subsequently, several hundred patients have been treated with this approach. The initial studies from the group of investigators in Pesaro, Italy, demonstrated the tolerability and efﬁcacy of this approach in children with thalassemia. In one early report, 222 patients less than 16 years old were treated with allogeneic HCT from HLA-identical donors (Lucarelli et al., 1990). DFS was 75%, with overall survival of 82%. A detailed analysis of this patient population demonstrated that those patients without hepatomegaly or portal ﬁbrosis had a DFS of 94%. Unfortunately, most adults with thalassemia have disease characteristics which place them in a high-risk group based on the presence of portal ﬁbrosis, hepatomegaly, and inadequate iron chelation (class 3). Although the initial results in adult patients were poor, changes in preparative regimens have allowed improved outcomes in these patients. In an early study of adult patients who received allogeneic HCT, DFS was 80% (Lucarelli et al., 1992). With increasing experience in treating highrisk patients, studies of class 1 patients were reported with DFS of 85% in a group of 89 patients, with only 7 deaths related to the procedure (Lucarelli et al., 1993). The outcomes of 87 adults with class 2 or class 3 disease were recently reported (Lucarelli et al., 1999). DFS was 62%, with an overall survival of 65% and a rejection rate of only 3%. Patients with no chronic active hepatitis had signiﬁcantly superior survival compared with those patients with chronic active hepatitis. When these 87 patients were evaluated along with a previously reported group of 20 adult patients, the 5-year probability of cure was 62%. With larger numbers of patients treated with allogeneic HCT, it is clear that patients are more likely to beneﬁt from allografting before they are considered class 3, due to decreased mortality and decreased rejection seen in patients transplanted while in a class 1 disease condition (Lucarelli et al., 1996). Allogeneic HCT offers a reasonable chance for cure for both adults and children with thalassemia, with acceptable toxicity. Although no controlled studies have been reported comparing allogeneic HCT to standard supportive therapy, the ability to replace the defective erythropoiesis in patients with thalassemia offers a signiﬁcant improvement in quality of life. The use of nonmyeloablative regimens may allow correction of this disorder with decreased toxicity, especially in patients with more advanced stages of their disease.

C. Sickle Cell Disease Sickle cell disease (SCD) remains a condition that causes signiﬁcant morbidity, with treatment aimed primarily at supportive care of sickle cell crises.

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Keith E. Stockerl-Goldstein and Karl G. Blume

The ﬁrst description of the curative potential of allogeneic HCT for this disorder involved a child who received an allogeneic HCT for acute myeloid leukemia (AML) (Johnson et al., 1984). The theoretical ability to perform allografting for SCD by replacing the hematopoietic system with marrow that does not involve two copies of the hemoglobin S gene has long been recognized; unfortunately, the high morbidity and mortality associated with allogeneic HCT have been prohibitive. The results of a large Belgian report involving 45 patients described an overall survival of 95%, with 86% free of SCD (Vermylen et al., 1998). A number of other reports have conﬁrmed the favorable outcome for younger patients with SCD who were treated with allogeneic HCT as part of a collaborative investigation (Walters et al., 1996, 1997). The most recent update of this effort describes the outcome of 50 children with symptomatic SCD who received bone marrow grafts from matched sibling donors (Walters et al., 2000). Eligible patients were less than 16 years of age with signiﬁcant symptomatic disease and had no extensive end organ damage. Figure 6 demonstrates the results of the 50 patients reported in the most recent analysis, with 6-year estimates of overall survival and event-free survival of 94 and 84%, respectively. Ten percent of patients experienced graft rejection or recurrence of SCD and 3 of the 50 patients died,

Fig. 6 Overall survival, event-free survival, and rejection or recurrence following allogeneic hematopoietic cell transplantation for 50 children with advanced, symptomatic sickle cell disease. (Reproduced by permission from Walters et al., 2000.)

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31

all from transplant-related complications. A signiﬁcant concern for these patients has been the long-term effects of the allotransplantation. Evaluation of long-term complications was performed for patients with at least 2 years of follow-up following the allografting procedure. Patients who had continued graft function and a prior history of SCD-related strokes had no posttransplant strokes, and all had stable or improved MRI scans following transplantation. Most patients had stable pulmonary function, and none of the 8 patients transplanted for recurrent episodes of acute chest syndrome experienced further episodes of this syndrome following allografting. The Karnofsky performance scores of patients with prolonged engraftment were excellent, and improvements in growth were also noted. Currently, the use of myeloablative allogeneic HCT for SCD remains of limited use given the need to identify an appropriate donor in patients with symptomatic but not end-stage SCD. The use of MUD transplants may expand the pool of patients with appropriate donors, but the increased toxicity and mortality expected with MUD transplants will need to be considered. The emerging use of mixed chimeric transplants for hematologic malignancies will likely lead to the expanded use of these transplants for patients with symptomatic SCD. Given the apparent decreased mortality associated with this transplant approach, patients with more end organ damage or with earlier symptomatic disease might be candidates for this approach. In fact, full donor chimerism may not be necessary to prevent sickle cell crises, so that stable mixed hematopoietic chimerism may be an acceptable endpoint for these patients (McSweeney and Storb, 1999).

D. Acute Lymphoblastic Leukemia Children diagnosed with acute lymphoblastic leukemia (ALL) have an excellent chance of attaining a complete remission with standard chemotherapy regimens, with more than 70% of patients being cured of their disease. Adults who develop ALL also respond well to chemotherapy; however, the long-term DFS for this group of patients is considerably lower, with less than one-third of patients cured. The effectiveness of allogeneic HCT for children with recurrent or refractory disease as well as for adults with high-risk, recurrent or refractory disease has been studied extensively. Long-term DFS rates in excess of 50% have been described in children with recurrent ALL treated with allogeneic HCT and, based on these encouraging results, randomized studies were performed. One study compared the outcomes of 376 patients with ALL in second complete remission who received allogeneic HCT and were reported to the IBMTR with a group of 540 patients treated by the Pediatric Oncology Group (Barrett et al., 1994). This registry analysis demonstrated a markedly decreased risk of relapse (45% vs 80%, p < 0.001) for patients receiving an allogeneic HCT, and

32

Keith E. Stockerl-Goldstein and Karl G. Blume

this advantage translated into superior DFS (40% vs 17%). The beneﬁt of transplantation was evident for patients who had high- or low-risk disease. A retrospective single institution study of 75 patients with ALL in second remission also demonstrated improved DFS and a lower relapse rate for patients receiving an allogeneic HCT than for those receiving standard chemotherapy (Boulad et al., 1999). A review of the Italian experience in treating children with ALL in second remission found an advantage for patients receiving an allograft over standard chemotherapy only in patients who had early ﬁrst relapse of disease (Uderzo et al., 1995). Two studies of the treatment of allogeneic HCT compared with chemotherapy for children with high-risk ALL demonstrated a decreased risk of relapse in patients receiving an allograft; however, only in one of the two studies was there a signiﬁcant advantage in DFS (Chessells et al., 1992; Saarinen et al., 1996). The presence of the Philadelphia chromosome in both adults and children with ALL predicts a poor prognosis with standard chemotherapy. A recent analysis compared the outcomes of patients with Philadelphia chromosomepositive (Ph+) ALL who attained a complete remission and were treated with allogeneic HCT from a related or unrelated donor, received ABMT, or received chemotherapy (Arico` et al., 2000). This study described 147 patients treated with chemotherapy only and 120 patients who received HCT. As shown in Fig. 7, a comparison of the patients who received an allograft from a matched related donor to the group of patients treated with chemotherapy alone demonstrates a signiﬁcant advantage for both DFS and overall survival. The 5-year estimates of DFS were 65% for allogeneic HCT recipients and 25% for those patients treated with chemotherapy alone ( p < 0.001). The optimal treatment option for adult patients with ALL has been the subject of many reports comparing allogeneic HCT to conventional chemotherapy. One study describes the outcomes of 484 adults with ALL treated in German ALL trials compared with 234 adults who received matched sibling donor allogeneic HCT and were reported to the IBMTR (Zhang et al., 1995). This study found a signiﬁcantly lower relapse risk of 30% versus 66% in favor of allogeneic HCT; however, because of the higher TRM in allografted patients, no signiﬁcant difference in DFS was reported. Another report from Japan compared 76 patients treated with chemotherapy to 214 allografted patients (Oh et al., 1998). This study also demonstrated decreased relapses and higher TRM for allografted patients; however, DFS was improved in adult patients ≤30 years of age who received allogeneic HCT. A prospective trial was performed in France for adult patients with ALL in ﬁrst remission (Sebban et al., 1994). Only patients with at least one potential sibling donor were included, and patients were assigned to allogeneic HCT if a matched sibling was identiﬁed (n = 116). Patients without a sibling match (n = 141) received standard-dose chemotherapy. There were no statistically signiﬁcant

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Fig. 7 Estimates of overall survival and disease-free survival of children treated with chemotheraphy only or with allogeneic hematopoietic cell transplantation from an HLAmatched related donor for Philadelphhia chomosome-positive acute lymphoblastic leukemia. The p value for overall survival is 0.002, and the p valuse for disease-free survival is <0.001. Numbers below the plot are numbers of patients at risk, with the top line for patients treated with chemotheraphy alone and the bottom line for patients treated with allogeneic transplantation. (Reproduced by permission from Arico` et al., 2000.)

differences in DFS or overall survival when the two groups were compared. However, an analysis of patients with high-risk disease which included Ph+ ALL, null or undifferentiated ALL, age >35, WBC > 30,000/␮l, or time to complete remission > 4 weeks showed a signiﬁcantly better 5-year DFS for allograft recipients (39% vs 14%, p = 0.01). Other studies have also conﬁrmed that patients with high-risk ALL or those patients having induction failure ALL can have prolonged DFS with allogeneic HCT (Biggs et al., 1992; Blume et al., 1980; Chao et al., 1991b; Forman et al., 1991; Snyder et al., 1999). These studies demonstrate that patients with induction failure of ALL, children with recurrent ALL, and adults and children with high-risk ALL should be considered for allogeneic HCT early during the clinical course of their disease.

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Keith E. Stockerl-Goldstein and Karl G. Blume

E. Acute Myeloid Leukemia Approximately 65–80% of patients with newly diagnosed de novo AML will enter a remission with induction chemotherapy, but only 20–30% of them attain long-term remissions. Many Phase II studies have been performed for AML in ﬁrst complete remission (CR), with DFS of 46–62% following allogeneic HCT. Based on the encouraging results of these Phase II studies, a number of trials were performed comparing allogeneic HCT to autologous HCT or standard-dose chemotherapy. In most of the study designs, the allocation to allogeneic HCT was determined by a “genetic randomization”; i.e., those patients who had a suitably matched donor would proceed to allogeneic transplantation, while the others would receive either further chemotherapy or ABMT. Table 1 describes the results of these studies, which compared allogeneic HCT to ABMT or chemotherapy (Appelbaum et al., 1984, 1988; Archimbaud et al., 1994; Cassileth et al., 1998, 1992; Champlin et al., 1985; Conde et al., 1988; Harousseau et al., 1997; Hewlett et al., 1995; Lowenberg et al., 1990; Marmont et al., 1985; Mitus et al., 1995; Powles et al., 1982; Reiffers et al., 1989; Schiller et al., 1992; Zander et al., 1988; Zittoun et al., 1995). Six of these studies reported a signiﬁcant improvement of DFS for patients who received allogeneic HCT compared with either ABMT or standard chemotherapy. In six of the studies, a decreased risk of relapse was evident for patients who received an allogeneic HCT, although this beneﬁt was associated with improved DFS in only one of these studies. When the results of currently available comparative trials are evaluated, there is a trend favoring allogeneic BMT over consolidation chemotherapy or autologous BMT. No study reported a signiﬁcant survival advantage or lower relapse rate for chemotherapy or autologous BMT. Patients who have been treated with standard chemotherapy but subsequently relapse can still proceed to allogeneic HCT with long-term DFS of approximately 25%; however, no prospective randomized studies have investigated the role of allogeneic HCT versus ABMT or standard chemotherapy for patients with AML in second CR. For patients with AML refractory to standard chemotherapy, there is evidence that some patients may still be cured with allogeneic HCT from a matched sibling donor (Copelan et al., 1991; Forman, et al., 1991; Mehta et al., 1994; Zander et al., 1988). Unfortunately, data from the National Marrow Donor Program (NMDP) indicate that very few patients with refractory AML seem to beneﬁt from HCT from an unrelated donor (Stockerl-Goldstein and Blume, 1999). Recent studies have demonstrated the prognostic signiﬁcance of cytogenetic abnormalities for patients with AML. Patients with the chromosomal abnormalities of inversion 16 or the translocations t(8;21) and t(15;17) have a favorable prognosis with induction and standard-dose consolidation

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Fig. 8 Proposed treatment algorithm for adult patients with acute myeloid leukemia based on cytogenetic abnormalities. CR, complete remission; PR, partial remission; IF, induction failure; AlloBMT, allogeneic hematopoietic cell transplantation; AutoBMT, autologous hematopoietic cell transplantation; MUD, matched unrelated donor. (Reproduced by permission from Stockerl-Goldstein and Blume, 1999.)

chemotherapy and may not beneﬁt from transplantation in ﬁrst CR. Patients with intermediate or poor-risk cytogenetics, however, appear to have an unfavorable outcome with standard therapy and are likely to beneﬁt from allogeneic transplantation in ﬁrst CR. Given these data, one possible treatment algorithm for patients with de novo AML is represented in Fig. 8.

F. Myelodysplastic Syndromes The treatment of MDS with standard-dose chemotherapy has generally demonstrated poor long-term outcomes. Although patients can be treated with intensive acute leukemia-type induction regimens, the remission rates are low and remission durations are generally short. There are increasing data indicating that allogeneic HCT is a curative treatment modality for many patients with MDS, although the exact timing of HCT and the need for pretransplant chemotherapy remain undetermined (Anderson et al., 1993; Sutton et al., 1996). Since most patients diagnosed with MDS are over the age of 60, the clinical application of myeloablative allogeneic HCT has been limited. One of the larger analyses published is a retrospective analysis of

36 Table I Allogeneic Bone Marrow Transplantation Versus Autologous Bone Marrow Transplantation Versus Chemotherapy for AML in First Remission Treatment a

Royal Marsden AlloBMT (Powles et al., 1982) ChemoRx a Seattle AlloBMT (Appelbaum et al., 1984; ChemoRx Applebaum et al., 1988) b AlloBMT ChemoRx b UCLA AlloBMT (Champlin et al., 1985) ChemoRx a Genova AlloBMT (Marmont et al., 1985) ChemoRx a M.D. Anderson AlloBMT (Zander et al., 1988) ChemoRx a Spain AlloBMT (Conde et al., 1988) ChemoRx a France AlloBMT (Reiffers et al., 1989) ABMT ChemoRx a Netherlands AlloBMT (Lowenberg et al., 1990) ABMT a UCLA AlloBMT (Schiller et al., 1992) ChemoRx b ECOG AlloBMT (Cassileth et al., 1992) ChemoRx

No. of points

DFS

p value

53 51 33 43

54% 21% 48% 21%

p < 0.005

43 43 23 44 19 18 11 27 14 25 20 12 20 23 32 42 28 54 29

40% 21%

p = 0.07

64% 13%

70% 10% 66% 41% 16% 51% 35% 45% 38% 42% 30%

OS

p value

Relapse

p value

40% 27% 70% 21% 36% 15%

p = NS

40% 71%

p < 0.01

9% 85% 10% 88% 18% 50% 83% 34% 60% 32% 60%

p < 0.01

p < 0.05

p < 0.05

p = NR p = NS

p = NS p < 0.004 p = NS p = NS p = NS

66% 37% 45% 53% 43% 42%

p = 0.05 p = NS p = NS

p < 0.005 p < 0.0002 p = 0.03 p = 0.05

b France (Archimbaud et al., 1994) a Boston (Mitus et al., 1995) b a

SWOG (Hewlett et al., 1995) b EORTC/GIMEMA (Zittoun et al., 1995) a

GOELAM (Harousseau et al., 1997)

b

U.S. Intergroup (Cassileth et al., 1998)

a

AlloBMT ChemoRx AlloBMT ABMT AlloBMT ABMT AlloBMT ChemoRx AlloBMT ABMT ChemoRx AlloBMT ABMT ChemoRx AlloBMT ABMT ChemoRx AlloBMT ABMT

27 31 23 27 31 53 34 110 168 128 126 67 67 61 113 116 117 92 63

41% 27% 62% 62% 56% 45% 38% 28% 55% 48% 30% 45% 47% 53% 43% 34% 34% 47% 48%

p = NS

41% 46%

p = NS

p = NS p = NS

43% 67% 0% 38% 20% 50%

p = 0.1 p = SGNFCT p = 0.04

p = NS

p = SGNFCT

59% 56% 46%

p = NR

p = NS

p = NS p = NR

46% 43% 52% 45% 55%

p = 0.04c p = 0.05d

27% 41% 57% 38% 44% 43% 29% 48% 62%

p = NR p = NS

p = NR

Abbreviations: AML, acute myeloid leukemia; AlloBMT, allogeneic bone marrow transplantation; ABMT, autologous bone marrow transplantation; ChemoRX, chemotherapy; SGNFCT, signiﬁcant; NS, not signiﬁcant; NR, not reported; DFS, disease-free survival; OS, overall survival. a Patients assigned to transplantation based on availability of matched siblings or to chemotherapy and analyzed according to treatment received. a Patients assigned to transplantation based on availability of matched siblings or to chemotherapy and analyzed according to intent-totreat. c p Value reﬂects comparison of allogeneic BMT and chemotherapy. d p Value reﬂects comparison of autologous BMT and chemotherapy.

37

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Keith E. Stockerl-Goldstein and Karl G. Blume

131 patients reported to the EBMT registry (Runde et al., 1998). The majority of the patients had idiopathic MDS, with a small percentage in whom the disease was related to prior cytotoxic chemotherapy for other malignant conditions. These patients were transplanted with various preparatory regimens. Five-year DFS was 34%, with an overall survival of 41%. Multivariate analysis demonstrated that younger age, shorter disease duration, and absence of excess blasts were independent predictors of improved DFS. An analysis of 71 patients with MDS treated with allogeneic HCT and reported to the French Bone Marrow Transplant Registry has been performed (Sutton et al., 1996). With a median follow-up of 6 years, the 7-year estimates of DFS and OS were both 21%. Multivariate analysis demonstrated better treatment outcome for patients with lower numbers of marrow blasts, for patients given a TBI/CY preparative regimen, and for patients who did not receive cytoreductive therapy prior to HCT. The cytogenetic categories deﬁned by the International Prognostic Scoring System (IPSS) (Greenberg et al., 1997) have been applied in a retrospective analysis of 60 adult patients who received an allogeneic HCT for MDS (Nevill et al., 1998). The study described DFS for the good-, intermediate-, and poor-risk cytogenetic groups at 51, 40, and 6%, respectively, and suggested that patients with poor-risk cytogenetic features might not beneﬁt from allografting. An analysis of 251 patients transplanted for MDS in Seattle between 1981 and 1996 demonstrated an increased risk of relapse following allogeneic HCT for patients who had advanced MDS, poor-risk cytogenetics, and an earlier transplant period (Anderson, 1999). The risk of nonrelapse mortality increased with increasing patient age and longer duration of MDS. The actuarial 3-year DFS for this group of patients was 41%, with a risk of relapse of 17%. Applying the IPSS criteria to this group of transplant recipients, the 5-year DFS was 56% for intermediate-1 risk, 32% for intermediate-2 risk, and 24% for high risk. Although it is difﬁcult to compare these transplanted patients to those described in the IPSS analysis, patients appear to beneﬁt from allogeneic HCT. Recently, a report of patients with MDS transplanted between the ages of 55 and 66 described acceptable toxicity and favorable outcomes for this group (Deeg et al., 2000). The data regarding outcomes for patients treated with allogeneic HCT for therapy-related MDS are limited, and most reports describe only a limited number of patients in this category. An analysis of 138 patients with therapyrelated MDS reported in multiple studies has been performed, with DFS of 34%, a relapse rate of 21%, and a transplant-related mortality risk of 45% (Anderson, 1999). Although the toxicity associated with allogeneic HCT for these patients is high, a proportion of patients will be long-term survivors. For patients without sibling donors, transplantation from an unrelated donor remains an option for the treatment of MDS. EBMT reported the outcomes of 118 patients with MDS or secondary AML (Arnold et al., 1998). TRM

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was relatively high at 58%, with a 2-year DFS of 28% and patients under the age of 35 having a better outcome. Another report of 52 patients with MDS or therapy-related MDS treated with MUD HCT described a 2-year DFS of 38% with a nonrelapse mortality rate of 48% (Anderson et al., 1996). The timing of allogeneic HCT still remains controversial, although the availability of the IPSS for MDS can identify patients who are expected to have extended survival without therapy and, given the toxicity of this approach, should not be considered candidates for myeloablative allogeneic HCT. Many studies have determined that patients with higher numbers of blasts have a worse outcome with allogeneic HCT, and transplantation of patients earlier in their disease course should be considered. The use of nonmyeloablative allogeneic HCT might be a consideration for patients with low-risk disease if the long-term data with these transplants continue to demonstrate a low proﬁle of morbidity and mortality. In fact, patients with low-risk MDS have an indolent disease that might be ideally suited to this immunotherapy approach.

G. Chronic Lymphocytic Leukemia Since chronic lymphocytic leukemia (CLL) often has an indolent course and patients are usually of advanced age at diagnosis, few studies of allogeneic HCT for this disorder have been performed. However, there are some patients diagnosed with this disease at a younger age who also have disease characteristics indicative of a more malignant course and who are candidates for allogeneic HCT. Studies that are reported involve a limited number of patients with short follow-up duration, especially when one considers the median survival of patients with CLL who receive standard therapy. In one report, 6 of 8 patients who received allografts achieved a complete remission (Toze et al., 2000). Another group of investigators described the outcome of 15 patients with CLL, most with refractory disease (Khouri et al., 1997). With a median follow-up of 3 years, 53% of patients were alive and in complete remission and no patients had developed visceral GVHD. Another report describes 23 patients with CLL transplanted with allogeneic grafts. In this study, more than half of the patients had refractory disease (Pavletic et al., 2000). Eighty-seven percent of patients attained a complete remission, and 61% were alive and free of disease over 2 years following transplantation. The estimated 5-year relapse rate was only 5%. A registry study characterizing the outcomes of 54 patients with CLL who received allogeneic HCT from matched sibling donors demonstrated a 3-year survival of 46%, with improved outcomes for those transplanted with lower stage disease (Michallet et al., 1996).

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The indolent nature of CLL makes this disorder a candidate for treatment with a nonmyeloablative HCT approach. One report of six patients with CLL treated with this approach using ﬂudarabine and CY demonstrated four responses, including two patients who attained a complete response (Khouri et al., 1998). However, only three of the six patients were alive at the time of the report. Seven patients with CLL have been treated with a nonmyeloablative regimen of 200-centigray TBI (McSweeney et al., 2000). Six of these patients were alive, with ﬁve patients having experienced a GVL effect. These studies using either ablative or nonablative HCT involve limited numbers of patients, and a longer follow-up with larger patient numbers will be necessary to evaluate the potential role of allogeneic HCT for this disease.

H. Chronic Myeloid Leukemia CML is a relatively indolent disease when in a chronic phase, with a median survival of approximately 3 years; however, once the disease enters an accelerated or blastic phase, progression and death occur rapidly. Approximately 15–20% of patients with CML in chronic phase will have a complete hematologic and cytogenetic response to interferon (IFN) therapy, with many of them becoming long-term survivors. However, the only known curative therapy for CML remains allogeneic HCT. Many studies throughout the 1980s conﬁrmed that 60% or more of patients with CML treated with HCT from a matched sibling donor are cured of their disease, and the relapse rate is fairly low (reviewed by Thomas and Clift, 1999). For patients with accelerated-phase CML the success rate of HCT is less than 40% (Clift et al., 1994b), and it is even lower for blastic-phase CML (Bacigalupo et al., 1993). Two prospective analyses comparing TBI-containing HCT regimens to chemotherapy-only HCT regimens for the treatment of CML have been reported. In one study, 120 patients were randomized to receive TBI/CY or BU/CY from an HLA-matched sibling donor for CML in ﬁrst chronic phase (Devergie et al., 1995). Five-year overall survival was greater than 60% for both groups, with DFS above 50% for both groups. There were no statistically signiﬁcant differences in survival between the two groups, but the BU/CY group of patients had a signiﬁcantly lower risk of relapse than patients receiving TBI/CY, p = 0.02. Another prospective randomized study of TBI/CY and BU/CY included 142 patients receiving matched sibling donor HCT (Clift et al., 1994a). This study found no signiﬁcant differences in overall survival, DFS, or relapse, although the toxicity of the BU/CY regimen was lower. As described above, CML is the most common malignancy treated with HCT from MUD. A report from the NMDP of 1423 patients who received

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MUD transplants facilitated by the NMDP demonstrated DFS of 43% for patients in chronic phase (McGlave et al., 2000). One report demonstrated that patients under the age of 50 years who receive a MUD HCT within the ﬁrst year of diagnosis had an overall survival of 74%, essentially the same as that of good-risk patients receiving their transplants from matched sibling donors (Hansen et al., 1998). Since many patients are treated with IFN before proceeding to allogeneic HCT for CML, many investigators have explored the impact of prior IFN exposure on the outcome of allogeneic HCT. One report of 133 patients transplanted with HCT from matched sibling donors or alternative donors evaluated the outcomes of the 50 patients who had received prior IFN therapy versus the 83 patients without prior IFN exposure (Beelen et al., 1995). This study demonstrated a 2.5-fold risk of TRM for patients with prior IFN treatment, due primarily to a higher risk of fatal posttransplant infections. These authors also reported an increased risk of graft failure for IFN-exposed patients with alternative HCT donors compared with nonexposed patients with the same type of donors. These investigators performed an analysis of 184 patients treated with MUD transplants for CML in chronic phase and compared the outcomes of IFN-exposed and -unexposed patients (Morton et al., 1998). In this group of patients, the incidence of acute GVHD and mortality was higher for patients with ≥6 months of IFN therapy. A more recent report of 152 patients transplanted from related or unrelated donors demonstrated that prior IFN exposure did not affect outcome if it was discontinued at least 90 days before the HCT procedure (Hehlmann et al., 1999). The availability of PCR tests for the bcr-abl transcript allows the documentation of a molecular remission of CML following allogeneic HCT. However, some patients will have demonstrable bcr-abl transcripts persisting following allogeneic HCT (Hughes et al., 1991; Radich et al., 1995). One study compared the outcome data of 92 patients with PCR data of 480 samples from this group (Pichert et al., 1995). Researchers were able to classify the patients into three distinct groups: PCR persistently positive (n = 29), PCR intermittently negative (n = 40), and PCR persistently negative (n = 23). The three groups had signiﬁcantly different probabilities of DFS, overall survival, and ability to maintain a remission (Fig. 9). The investigators also found that 95% of the patients who developed GVHD were persistently or intermittently PCR negative, whereas those patients with no GVHD or only grade I GVHD were more likely to be persistently PCR positive. These ﬁndings further support the relationship between GVHD and the GVL effect. The use of mixed-chimeric transplants for CML has been reported. In one study, six of eight patients treated for CML were alive, although one was treated with myeloablative allogeneic HCT for cytogenetic relapse following the nonmyeloablative procedure (Slavin et al., 1998). In another report, ﬁve of nine patients were alive and in molecular complete remission following a

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mixed chimeric procedure (McSweeney et al., 2000). The approach of using a nonmyeloablative allogeneic HCT may be particularly useful for the treatment of chronic-phase CML. No randomized prospective trials have been performed to determine the best therapy for patients with CML in chronic phase. A panel convened by the American Society of Hematology performed an evidence-based analysis of BU, hydroxyurea, IFN, and allogeneic HCT for patients with chronic-phase CML. The ﬁndings of this panel suggested that patients be informed of the treatment options available, as well as the risks and beneﬁts of each treatment modality. The published data demonstrate that at least 50% of patients treated with an allogeneic HCT for chronicphase CML survive for at least 10 years; however, similar long-term data for IFN therapy are lacking. If the TRM associated with myeloablative HCT can be decreased, or if studies of nonmyeloablative HCT demonstrate prolonged DFS with limited TRM, it may make the treatment decisions for patients and their physicians much easier.

I. Non-Hodgkin Lymphoma Patients diagnosed with intermediate-grade non-Hodgkin lymphoma (NHL) have a favorable outcome with standard-dose chemotherapy at diagnosis, with approximately 50% of patients cured of their disease. Unfortunately, for patients with relapsed intermediate-grade NHL, long-term disease control and survival are poor with standard-dose chemotherapy. A Phase III clinical trial has demonstrated a superior outcome for patients with relapsed intermediate-grade NHL who were randomized to ABMT rather than to standard salvage chemotherapy (Philip et al., 1995). Patients in this study randomized to ABMT had DFS of 46%, compared with only 12% for patients randomized to salvage chemotherapy ( p = 0.001). Unfortunately, even in patients who undergo ABMT, the major cause of treatment failure remains relapse. In an attempt to generate a GVL effect to further decrease the rate of treatment failure, many centers have performed Phase II studies evaluating the role of allogeneic HCT for patients with various grades of NHL (Champlin et al., 1999). These studies include patients with many different histologies of NHL, numerous preparatory regimens, and predominantly treat patients with recurrent or refractory disease. Most of these reports demonstrate a high risk of treatment-related mortality, usually in excess of 30%. The largest investigation described 73 patients with aggressive NHL ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Fig. 9 Clinical outcomes following allogeneic hematopoietic cell transplantation for chronic myeloid leukemia based on bcr-abl polymerase chain reaction (PCR) results: (A) probability of maintaining a hematologic remission; (B) probability of disease-free survival; (C) probability of overall survival. (Reproduced by permission from Pichert et al., 1995.)

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who received allogeneic HCT. Patients with low-grade, lymphoblastic, or Burkitt lymphoma were excluded (Dhedin et al., 1999). Most of the patients had refractory disease or were in partial remission. Thirty-two of the 73 patients died of transplant-related complications, and the relapse probability was 30%. The 5-year DFS was 40%, with an overall survival of 41%. A multivariate analysis demonstrated that patients with fewer than three pretransplant regimens and those in CR at transplant achieved superior overall survival. For patients who were in CR at the time of transplantation, the 5-year DFS was 76%, compared with only 23% for patients who were not in CR. Another group of investigators performed a retrospective analysis of 64 patients with refractory or recurrent lymphoma who received an allogeneic HCT (van Besien et al., 1996). A number of different preparatory regimens and combinations to prevent GVHD were utilized. The 2-year DFSs for the different histologies reported were 59% for low-grade NHL, 21% for intermediate-grade NHL, 21% for lymphoblastic lymphoma, and 10% for diffuse small noncleaved-cell NHL. DFS of patients was signiﬁcantly better for patients with low-grade NHL than for the other groups. Unfortunately, the number of long-term survivors reported was limited, and the median follow-up in this report was short. Evidence has emerged that a graft-versus-lymphoma effect exists in patients who have received allogeneic HCT for NHL (Mandigers et al., 1998; van Besien et al., 1997). However, the increased toxicity and mortality associated with allogeneic HCT, especially in patients with extensive prior therapy, demonstrated the need for some comparative studies in allogeneic and autologous HCT. Few prospective, randomized studies comparing allogeneic HCT to ABMT have been reported, but numerous retrospective analyses have been published. EBMT presented a retrospective analysis of 764 patients with either NHL or Hodgkin disease (HD) transplanted with allogeneic HCT and compared these patients to over 9000 patients autografted and reported to the EBMT registry (Peniket et al., 1997). They found a signiﬁcantly lower risk of relapse in patients with low-grade and intermediategrade NHL who received allogeneic grafts; however, because of the high TRM in the allogeneic HCT cohort, the overall survival for both low-grade and intermediate-grade disease favored ABMT. In general, almost all of the reported retrospective studies demonstrate a decreased risk of relapse for patients with NHL who are treated with allogeneic HCT compared with ABMT; however, none of the studies reports a signiﬁcant survival advantage for allogeneic HCT, usually due to the excessive TRM seen in those patients. One report of 118 patients with NHL or HD described a prospective, biologically randomized trial of allogeneic HCT versus ABMT using purged bone marrow (Jones et al., 1991). The risk of relapse for patients with NHL was 29%, lower for patients who received an allogeneic HCT than for those

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in the ABMT group; however, there was no difference in survival, due to the increased TRM for allografted patients. A smaller prospective study evaluated 66 patients with NHL assigned to allogeneic HCT if they were less than 56 years old and had a fully matched or single-locus-mismatched sibling donor (Ratanatharathorn et al., 1994). Patients who received autografts were eligible up to the age of 60. The patients included patients with highrisk disease who failed to achieve a complete remission with initial therapy, who had transformed low-grade lymphoma, or who had poor prognostic features. Again noted in this study was a decreased relapse rate in allotransplanted patients; however, DFS was not signiﬁcantly different between the two groups. Studies of allogeneic HCT for low-grade NHL are more limited also, for reasons similar to those described above for CLL. Although patients with low-grade NHL are of a younger median age, the indolent nature of this disease for many inﬂuences the decision to proceed with a myeloablative allogeneic HCT and the associated risks of the procedure. A case-matched comparison reported by the French Bone Marrow Transplant Group in patients compared 72 patients with low-grade lymphoma receiving allografts with 144 patients who were autografted (Attal et al., 1997). A lower risk of relapse was evident for the allogeneic HCT patients (12% vs 50%, p = 0.001); however, due to a statistically signiﬁcant increase in TRM in patients receiving an allograft (30% vs 4%), the overall survival was not statistically different between the two groups of patients. A report of 38 patients with low-grade NHL transplanted with either allogeneic or autologous HCT also demonstrated a lower risk of relapse in allografted patients (Verdonck, 1999). In this analysis, DFS was also superior in patients receiving an allogeneic HCT, but overall survival was not signiﬁcantly different. Although these studies utilizing allogeneic HCT for low-grade or aggressive NHL demonstrate a signiﬁcant graft-versus-lymphoma effect, the high TRM severely limits the usefulness of the procedure. The use of nonmyeloablative allogeneic HCT, especially in patients with low-grade NHL, may allow the harnessing of the graft-versus-lymphoma effect with acceptable toxicity. One report described 5 patients with refractory NHL who received mixed-chimeric HCT, with two of the patients demonstrating a graft-versuslymphoma effect in the absence of ongoing GVHD (Sykes et al., 1999). Another study of 9 patients with NHL who received a ﬂudarabine-based nonmyeloablative regimen reported 3 patients alive and in complete remission following the procedure (Khouri et al., 1998). A different group of investigators reported the outcomes of 23 patients with malignant lymphomas (19 NHL, 4 HD) treated with a low-intensity regimen of BU and ﬂudarabine followed by allogeneic HCT (Nagler et al., 2000). This group of patients had an actuarial DFS and survival at 37 months of 40%. The exact role for allogeneic HCT in the treatment of NHL will require long-term follow-up of

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patients with low-grade disease, a larger experience using nonmyeloablative techniques, and prospective, randomized studies involving allogeneic HCT.

J. Hodgkin Disease Most patients with HD will be cured with standard chemotherapy, radiotherapy, or combined-modality therapy. Those patients who fail to enter a remission or relapse with HD have poor outcomes with standard-dose chemotherapy, but there are many reports of favorable outcomes for patients who received high-dose therapy and ABMT. The importance of ABMT for patients with relapsed HD was demonstrated in two studies comparing ABMT to standard salvage therapies (Linch et al., 1993; Yuen et al., 1997). Based on the favorable outcomes for most patients who receive autografts for recurrent HD (Horning et al., 1997). There have been few studies of allogeneic HCT, due to the higher risk associated with the procedure. A review of 100 patients with advanced HD reported to the IBMTR and treated with allogeneic HCT demonstrated a 3-year survival of 21%, with DFS of only 15% and a relapse rate of 65% (Gajewski et al., 1996); however, the majority of these patients had advanced stages of HD at the time of transplantation. A case-matched analysis of 45 patients with HD who received allogeneic HCT and 45 patients who received ABMT has been reported (Milpied et al., 1996). Patients who received ABMT had superior progression-free survival, overall survival, and nonrelapse mortality. For patients with sensitive disease at the time of transplantation, the 4-year survival was 30% for allogeneic HCT and 64% after ABMT. Although there are limited data available, allogeneic HCT should only be considered for select patients with HD on clinical trials.

K. Multiple Myeloma The median age of patients diagnosed with multiple myeloma (MM) is above 60 years, and initial studies using high-dose chemotherapy were often limited by age. The median survival of patients diagnosed with MM who are treated with standard chemotherapy is approximately 3 years. The superiority of high-dose therapy and ABMT over standard chemotherapy for advanced-stage MM has been demonstrated (Attal et al., 1996), and many Phase II studies conﬁrm favorable survival following ABMT. Unfortunately, there appears to be a continuous rate of relapse following ABMT that suggests that this approach is not curative. Based on the success of allogeneic HCT for acute leukemias and the apparent inability to cure patients using high-dose therapy with autologous HCT, investigators at many institutions

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began trials of allogeneic HCT to allow the use of a graft which had no MM contamination and they hoped to allow a graft-versus-myeloma response to develop. Many of the initial reports of allogeneic HCT for MM demonstrated an unusually high TRM; in fact, many centers reported mortality rates in excess of 40% (Bensinger et al., 1996; Gahrton et al., 1995; Mehta et al., 1997); however, there appeared to be a subset of patients who attained a complete durable remission. These studies demonstrated that patients who entered a complete remission had a 30% or higher DFS. There is some evidence that disease control using allogeneic HCT may be superior to ABMT for this disease. An investigation was performed to determine the frequency of molecular remissions in patients undergoing either type of HCT using patient-speciﬁc PCR assays (Corradini et al., 1999). This nonrandomized study demonstrated that 50% of allografted patients attained a molecular complete remission versus 7% of patients who received an autograft. A retrospective case-matched analysis of 378 patients performed by the EBMT group compared patients with multiple myeloma who received either autologous or allogeneic grafts. This study demonstrated a lower relapse rate in the patients receiving allogeneic transplants (70% ABMT vs 50% allogeneic HCT); however, because of the high transplant-related mortality, the overall survival favored autologous transplantation (Bjorkstrand et al., 1996). Notably, when patients who were alive 1 year after transplantation were evaluated, there was a signiﬁcant beneﬁt in overall survival and DFS for patients who received an allograft, which appears to be due to a decreased relapse rate in allografted patients. For patients who suffer a relapse of MM following allogeneic HCT, there are data demonstrating decreases in detectable myeloma protein following DLI or after the development of GVHD, with some patients entering complete remissions (Lokhorst et al., 1997; Tricot et al., 1996). These studies demonstrate the potential usefulness of inducing a graftversus-myeloma effect if the allogeneic HCT procedure can be performed with acceptable morbidity and mortality. Thus, a nonmyeloablative allogeneic HCT is one way to treat MM which relies on immunotherapy. In fact, one report demonstrated the ability of performing a combined mixedchimeric bone marrow transplant with simultaneous renal allografting from a matched sibling donor (Spitzer et al., 1999). This patient had a functioning renal allograft off all immunosuppression, with evidence of an antimyeloma effect as well. Initial studies of mixed-chimerism transplantation using the regimen of TBI or ﬂudarabine/TBI in patients with MM suggest that there must be adequate control of the underlying disease before the allogeneic HCT, or the risk of graft rejection is too high (McSweeney et al., 2000). Therefore, our current treatment approach for patients with multiple myeloma uses an initial cytoreductive step with high-dose melphalan and autologous hematopoietic cell support followed by a mixed-chimeric

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allogeneic HCT. A limited number of patients have been treated using this schema, with encouraging preliminary results. We hope that this treatment principle will allow us to generate an adequate graft-versus-myeloma effect with acceptable toxicity.

XI. CONCLUSIONS AND FUTURE DIRECTIONS The past decade was one of intensive investigation leading to important advances in the ﬁeld of allogeneic HCT which built upon the initial experiences by the pioneers of transplantation. Throughout this decade, HCT has found an ever-increasing role for the treatment of malignant and inherited disorders, and studies under way are evaluating the potential use of allogeneic HCT for the treatment of autoimmune diseases. Our increasing understanding of the immunology that is part of the allogeneic HCT procedure and a better ability to prevent and treat the complications associated with this form of therapy will allow continued expansion of this treatment modality. Studies of gene therapy may allow treatment of infectious diseases such as HIV or allow infusions of donor lymphocytes that can be killed using “suicide genes.” Continued investigations of UCB and haploidentical transplants will expand the number of patients who might be eligible to receive allogeneic HCT but do not have appropriately matched donors. The most exciting aspect of allogeneic HCT for the coming decade is likely to be the reﬁnement of nonmyeloablative techniques which might allow the ambulatory treatment of patients who are otherwise too ill or too old to tolerate high-dose preparative regimens. The ability to perform such a transplant with limited morbidity and mortality will continue to expand the potential uses of this therapeutic concept. Our expectation is that the technique of performing an allogeneic HCT will be radically different in the next decade.

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A Role for Secondary V(D)J Recombination in Oncogenic Chromosomal Translocations? Marco Davila, Sandra Foster, Garnett Kelsoe,∗ and Kaiyong Yang Department of Immunology Duke University Medical Center Durham, North Carolina 27710

I. Introduction II. V(D)J Rearrangement A. Overview B. Molecular Mechanism of V(D)J Rearrangement C. The V(D)J Recombinase as Transposase III. Illicit V(D)J Rearrangement Mediated by Cryptic RSS or RSS-Like Motifs IV. The Germinal Center A. The Germinal Center Reaction B. The Germinal Center as a Site for Lymphomagenesis V. Chromosomal Translocations in GC-Like Lymphomas A. Burkitt’s Lymphoma B. Follicular Lymphoma VI. Research VII. Conclusions References

Chromosomal translocations are hallmarks of certain lymphoproliferative disorders. Indeed, in many leukemias and lymphomas, translocations are the transforming event that brings about malignancy. Recurrence of the immunoglobulin (Ig) and T-cell receptor (Tcr) loci at the breakpoints of oncogenic chromosomal translocations has led to speculation that the lymphocyte-speciﬁc process of V(D)J rearrangement, which is necessary for the generation of functional Ig and TCR antigen receptors on B and T lymphocytes, mediates translocation. Recent studies have led to a fuller understanding of the molecular mechanisms of V(D)J rearrangement and have revealed that the V(D)J recombinase possesses latent transposase activity. These studies have led to plausible models of illegitimate V(D)J recombination producing chromosomal translocations consistent with those present in lymphomas and leukemias. Errors of V(D)J recombination may even ∗ To whom correspondence should be addressed: Department of Immunology, Box 3010, Duke University Medical Center, Durham, North Carolina 27710. Tel: (919)613-7936; Fax: (919)613-78978; E-mail: [email protected]

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generate lymphomas with the phenotypes of mature cells. For example, follicular and Burkitt’s lymphomas have been classiﬁed by phenotype and somatic genotype as malignant germinal center (GC) B or post-GC B cells. The GC is a site of afﬁnity maturation where B cells undergo V(D)J hypermutation and Ig class switch; in addition, much evidence has accumulated to suggest that GC B cells may also support secondary V(D)J recombination. Interestingly, all three of these elements, genomic plasticity, mutation, and translocation breakpoints near switch sites or recombinational elements, are characteristic of certain lymphomas. The high frequency of lymphomas carrying these GC markers suggests that the GC reaction may play a signiﬁcant role in lymphomagenesis. C 2001 Academic Press.

I. INTRODUCTION The discovery of the Philadelphia chromosome was the ﬁrst cytogenetic demonstration of an association between a speciﬁc chromosomal translocation and malignancy (Nowell and Hungerford, 1960). Subsequent identiﬁcations of recurrent translocations, such as the t(8;14)(q24;q32) translocation of Burkitt’s lymphoma (BL) and the t(14;18)(q32;q21) translocation of follicular lymphoma (FL), strengthened the possibility that chromosomal translocations might cause cancer (Weiss et al., 1987; Zech et al., 1976). Today, oncogenic chromosomal translocations are diagnostic hallmarks for many lymphoproliferative disorders. These cytogenetic studies were followed and complemented by the identiﬁcation of genes ﬂanking translocation breakpoints. Remarkably, the great majority of breakpoints exhibited a pattern of chromosomal fusions with immunoglobulin (Ig) or T-cell receptor (Tcr) loci brought into apposition with a small number of partner genes. Characterization of these translocation partners led to the discovery of several cellular oncogenes, including myc and bcl-2 (Bakhshi et al., 1985; Cleary and Sklar, 1985; Dalla-Favera et al., 1982). Molecular biologic studies demonstrated that these recurrent translocations promote oncogenesis by dysregulating the expression of partnered oncogenes or by the creation of novel fusion proteins (Gauwerky et al., 1988; Nunez et al., 1990; Taub et al., 1984). Although some oncogenic translocations that do not involve the Ig or Tcr loci have been characterized, the consistent involvement of antigen-receptor genes in lymphoid malignancies suggests that the signal event of lymphocyte development, V(D)J rearrangement, might be involved in chromosomal translocations and malignancy (Gilliland, 1998). V(D)J rearrangement is a lymphocyte-speciﬁc process of genome remodeling that is restricted to B and T lymphocytes. The Ig and Tcr loci comprise discrete regions of variable (V), diversity (D), and joining (J) gene segments that are combined and fused to encode functional Ig and TCR proteins (Fig. 1a). Each V, D, and J gene segment is ﬂanked by DNA signal motifs—

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VH region

DH region

JH region

63 switch region

a

b

23

CACAGTG-----------------------ACAAAAACC

12

GGTTTTTGT------------ CACTGTG

Fig. 1 The Igh locus. (a) Schematic of the immunoglobulin heavy-chain locus. Rectangles represent coding units within the different regions of the Igh locus. Triangles represent recombination signal sequences. (b) The 23- and 12-bp RSS.

recombinational signal sequences (RSS)—consisting of conserved heptamer and nonamer sequences separated by less conserved spacers of 12 or 23 base pairs (Fig. 1b). RSS are cis-acting recognition elements that direct V(D)J rearrangement of Ig and Tcr genes by the products of the recombinase activating genes -1 and -2 (Rag-1 and Rag-2). Physiologic V(D)J arrangement occurs only between coding units ﬂanked by RSS with different spacer lengths; this restriction is known as the 12/23 rule (Early et al., 1980; Sakano et al., 1980). Recently, studies of V(D)J rearrangement have provided signiﬁcant insight into the molecular mechanism of recombinase activity and revealed latent transposase activity that suggests a remarkable evolutionary origin for Rag-1 and Rag-2 (Agrawal et al., 1998; Fugmann et al., 2000; Hiom et al., 1998). This new understanding of V(D)J rearrangement and transposition also suggests that illegitimate recombinase activity could play a signiﬁcant role in chromosomal translocation (Hiom et al., 1998; Roth and Craig, 1998).

II. V(D)J REARRANGEMENT A. Overview V(D)J rearrangement drives lymphocyte development by the formation of functional Ig or Tcr genes from diverse menus of V, D, and J gene segments. The recognition of gene segments and the endonucleolytic cleavage reactions necessary to reform genomic structure are wholly dependent on RAG1 and RAG2 and are conﬁned to cells of the lymphoid lineages. In contrast, the fusion of gene segments into functional Ig and Tcr genes is carried out by ubiquitously distributed components that mediate DNA double-strand

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Fig. 2 V(D)J rearrangement. Cleavage occurs between the coding unit and SE of one coding region. The same reaction occurs at another coding region, forming two SEs and two CEs. The SEs fuse together slowly and precisely, while the CEs fuse rapidly with nucleotide addition and deletion. P represents palindrome-nucleotide addition. N represents N-nucleotide addition.

break (dsb) repair (Errami et al., 1996; Frank et al., 1998; Gu et al., 1997; Kirchgessner et al., 1995; Li et al., 1995). The speciﬁcity of V(D)J rearrangement is guided by the RSS that lie adjacent to V, D, and J gene segments. For example, the rearrangement that initiates generation of Ig heavy-chain genes begins with a double-strand cleavage reaction at RSS heptamers ﬂanking the coding regions of DH and JH gene segments (Lewis, 1994). This cleavage separates the RSS DNA (signal ends) from the DH and JH coding sequences (coding ends). The signal ends (SEs) and the intervening genomic region are removed and fused exactly to form a heptamer-to-heptamer signal joint that seals a large, extrachromosomal DNA circle (Lewis, 1994). In contrast, resolution and fusion of the DH and JH coding ends (CE) results in an imprecise coding joint characterized by nucleotide deletions and additions (Fig. 2) (Lewis, 1994). The cell cycle contains stringent checkpoints that detect dsb and halt DNA replication; replication in the presence of dsb can result in inheritable chromosomal abnormalities (Dasika et al., 1999; Jasin and Richardson, 2000). If overwhelmed by dsb, cells opt for apoptosis, presumably to reduce the risk of ﬁxing deleterious chromosomal translocations. The introduction of dsb by RAG1 and RAG2 during V(D)J rearrangement, despite the increased

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potential for genomic reformation, demonstrates the fundamental importance of speciﬁc immunity: the necessity for diverse Ig and Tcr repertoires outweighs the risk of DNA abnormalities induced by recombinase activity. This potential for chromosomal damage is signiﬁcant and clearly demonstrated by the high frequencies of chromosomal translocations and lymphomas present in mice deﬁcient for dsb DNA repair (Diﬁlippantonio et al., 2000; Ferguson et al., 2000; Gao et al., 2000; Vanasse et al., 1999).

B. Molecular Mechanism of V(D)J Rearrangement Transient transfection with Rag-1 and Rag-2 makes mouse ﬁbroblasts competent to support V(D)J rearrangement in extrachromosomal substrates (Schatz et al., 1989). Identiﬁcation and reﬁnement of the recombinase reaction components have lead to in vitro V(D)J recombination that adheres to the 12/23 rule and closely mimics the reaction in cells (van Gent et al., 1996b). In vitro recombination studies demonstrate that rearrangement begins with RAG1 recognizing and binding to the RSS nonamer (Diﬁlippantonio et al., 1996). The heptamer enhances this binding by an unknown mechanism and is critical for V(D)J rearrangement (Diﬁlippantonio et al., 1996). Indeed, V(D)J rearrangement in extrachromosomal templates is virtually lost when any of the ﬁrst three heptamer nucleotides is mutated (Akamatsu et al., 1994; Hesse et al., 1989). RAG2 has been shown to bind not to the RSS but to RAG1/RSS complexes; recruitment is more efﬁcient to RAG1/12 base-pair (bp) RSS than to RAG1/23 bp RSS, a possible basis for 12/23 discrimination (Diﬁlippantonio et al., 1996). In addition, high-mobility-group proteins— HMG1 and HMG2—have been shown to participate in the assembly of RAG1, RAG2, and DNA (van Gent et al., 1997). Their functions appear to overlap, and they can substitute for each other. HMG1 and HMG2 are nonhistone DNA-binding proteins and can induce sharp bends in double-helical DNA (van Gent et al., 1997). Both are known to be involved in the formation of nucleoprotein complexes (van Gent et al., 1997). The requirement for HGM1/2 and the stepwise binding of RAG1 and RAG2 suggest a plausible reaction sequence for the initiation of V(D)J rearrangement: RAG1 recognition of the RSS nonamer, RAG2 recruitment, and stabilization by HGM1/2 of a bent structure arranging 12- and 23-bp RSS in synapsis with the recombinase machinery (Fig. 3). After the assembly of this rearrangement complex, the recombinase proteins perform a single-strand cleavage 5 to the heptamer (Fig. 4) (McBlane et al., 1995). This breaks the phosphodiester bond between the SE and CE, leaving a phosphate on the 5 SE and a hydroxyl group on the 3 CE. Next, the hydroxyl group of the CE acts as a nucleophile and attacks the phosphodiester bond between the CE and SEs of the opposite strand (McBlane

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Fig. 3 Model for recombination synapsis. The RAG1 and RAG2 complex binds to the RSS and introduces a nick at the border of the heptamer. In the presence of HMG proteins, a synaptic complex is formed between 12-bp RSS and 23-bp RSS. Cleavage is completed at both RSS and results in the formation of a blunt-ended signal and hairpinned CE.

Fig. 4 V(D)J rearrangement begins with the recognition of RSS by the V(D)J recombinase and is followed by cutting one strand of the DNA precisely at the end of the heptamer. A hairpinned CE and a blunt SE is then generated by a transesteriﬁcation reaction. Subsequently, two SEs are ligated to form a signal joint. The hairpins are opened by RAG, modiﬁed by TdT, and annealed in the presence of DNA-PK, XRCC4, DNA ligase IV, and other general factors essential for double-strand break repair.

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et al., 1995; van Gent et al., 1996a). This results in a hairpin structure on the CE and a blunt SE. The SE fuses with a SE that has undergone the same reaction. This signal joint is formed precisely and slowly, while the coding joint is formed rapidly and imprecisely (Hagmann, 1997). When cleaved, the hairpin intermediate is the source of the palindromic (P) nucleotides at coding joints (Besmer et al., 1998; Shockett and Schatz, 1999). The coding joint also displays deletions and nontemplate N-nucleotide additions (Lewis, 1994). The resolution of free CE and SEs is performed by the dsb DNA repair system. V(D)J rearrangement and dsb repair have been associated since the discovery of a general dsb repair defect in scid mice, which do not develop B and T cells (Kirchgessner et al., 1995). X-ray cross complementation (XRCC) mutants deﬁne pathways of dsb DNA repair. Four of these, XRCC4–7, were also observed to disable V(D)J rearrangement (Blunt et al., 1996; Danska et al., 1996; Errami et al., 1996; Gu et al., 1997; Li et al., 1995). At the least, this suggests an indirect association between the V(D)J recombinase and lymphomagenesis, because an underlying cause of lymphomas is the induction of dsb, presumably mediated by the V(D)J recombinase (Diﬁlippantonio et al., 2000; Ferguson et al., 2000; Gao et al., 2000; Vanasse et al., 1999). XRCC7, which represents the scid mutation, is defective in coding joint formation, while the XRCC4–6 mutations block the formation of both coding and signal joints (Blunt et al., 1996; Danska et al., 1996; Errami et al., 1996; Gu et al., 1997; Li et al., 1995). Effective dsb DNA repair requires two ubiquitous nuclear proteins, Ku70 and Ku80. These proteins form a heterodimer that binds irregular DNA structures, such as dsb, hairpins, nicks, and gaps (Falzon et al., 1993; Gu et al., 1997; Mimori and Hardin, 1986). The Ku70/80 heterodimer protects DNA ends from degradation and displays both helicase and ATPase activities (Chu, 1997). The fundamental role of Ku80 in V(D)J rearrangement was suggested when Ku80 mutant cells were found to be unable to support V(D)J recombination (Errami et al., 1996). No coding joints could be recovered from Ku80 mutant cells, and the few signal joints that were formed showed large deletions (Chu, 1997). It is now generally believed that Ku70 and Ku80 are the genes identiﬁed by the XRCC6 and XRCC5 mutations, respectively (Gu et al., 1997; Smider et al., 1994; Taccioli et al., 1994). Both Ku proteins recruit a large 450-kDa enzyme—DNA-dependent protein kinase catalytic subunit (PKcs)—which is inactive until the formation of dsb. This recruitment results in a multienzyme complex composed of the Ku heterodimer and PKcs, the DNA-dependent protein kinase (DNA-PK) (Chu, 1997). When Ku binds to dsb and recruits PKcs, it is believed that PKcs in turn phosphorylates Ku, activating its ATPase activity (Cao et al., 1994). Ku−/− mice develop multiple

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chromosomal translocations, while Ku−/− p53−/− mice develop pro-B cell lymphomas before 3 months of age that are characterized by a t(12;15) translocation (Diﬁlippantonio et al., 2000). PKcs is critical for V(D)J rearrangement. The mouse scid mutation is a premature termination codon in the PKcs gene that results in very low levels of gene expression and defective enzyme activity (Blunt et al., 1996; Danska et al., 1996). The XRCC7 mutation is also thought to identify the PKcs gene (Kirchgessner et al., 1995). XRCC7 mutants are complemented by human chromosome region 8q11, where PKcs has been mapped with in situ hybridization (Kirchgessner et al., 1995; Komatsu et al., 1993). Both the scid and XRCC7 mutations result in defective coding joint formation, but support the formation of signal joints (Lieber et al., 1988). Thus, unlike Ku70 and Ku80, which mediate both coding and signal joint formation, PKcs appears necessary only for the generation of coding joints. However, like the studies of Ku−/− p53−/− mice, scid mice that are p53−/− rapidly develop proB-cell lymphomas characterized by a t(12;15) translocation (Vanasse et al., 1999). Ligase IV is the ﬁnal component of the dsb repair machinery known to participate in the resolution of V(D)J CE and SEs. Like the other components of dsb repair, mutation of the ligase IV gene is deleterious to V(D)J recombination (Frank et al., 1998). It is believed to perform the ligation that resolves the dsb of V(D)J rearrangement. Like XRCC4, ligase IV mutation results in a lack of coding and signal joint formation. XRCC4, a novel protein, has been shown to stabilize ligase IV in vitro (Bryans et al., 1999). Both of these dsb repair proteins are associated with chromosomal translocations or lymphomagenesis when knocked out (Ferguson et al., 2000; Gao et al., 2000). This leads to a model of V(D)J recombination where DNA, RAG1, and RAG2 are held in synapsis and dsb at RSS are introduced (Chu, 1997). The freed signal and coding ends become bound by Ku70/80 heterodimers, stabilizing and protecting the DNA breaks from degradation; Ku recruits PKcs to form an active DNA-PK complex on each end. At this point, any proposed mechanism for V(D)J rearrangement must explain why Ku mutations impair the formation of coding and signal joints, while mutations in PKcs affect only the generation of coding joints. A plausible explanation for this dichotomy is that PKcs, bound to SEs, phosphorylates Ku, bound to CEs, in trans (Chu, 1997). This phosphorylation activates the Ku helicase, which processes the CEs and makes them available for hairpin opening and ligation (Chu, 1997). In vitro, DNA-PK bound to DNA hairpins has no phosphorylation activity (Chu, 1997). Thus, the reciprocal activation of helicase activity on SEs cannot take place and they stay protected until some unknown process allows their resolution.

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C. The V(D)J Recombinase as Transposase The ability of RAG1 and RAG2 to introduce strand nicks and then dsb in rearrangement templates is now established (McBlane et al., 1995; Ramsden and Gellert, 1995). Exciting work from the laboratories of Martin Gellert and David Schatz demonstrated that this ability to cleave and hairpin DNA also supports transposase activity in vitro (Agrawal et al., 1998; Hiom et al., 1998). The identiﬁcation of latent transposase activity in RAG1 and RAG2 is signiﬁcant because the Rag genes have been proposed to represent a transposable element enslaved by evolution (Agrawal et al., 1998; Hiom et al., 1998; van Gent et al., 1996a). In fact, the RAG recombinase shares similar mechanisms of action with bacteriophage Mu transposase and the HIV-integrase (van Gent et al., 1996a). Recombinase and transposase reactions involve a mobile gene segment that is ﬂanked and recognized by short, repeated sequences. Both enzymes carry out reactions that are ATP-independent and Mg2+-dependent, and both catalyze transesteriﬁcation reactions. Remarkably, the work of Schatz and Gellert demonstrates that RAG1 and RAG2 can act as a transposase within their normal recombinase activities (Agrawal et al., 1998; Hiom et al., 1998). RAG1 and RAG2 cleave recombination substrates into intermediate signal and coding ends. A 3 hydroxyl group present on the SE can act as a nucleophilic mediator of transposition by allowing cleaved signal DNA to integrate into substrate DNA. This integration is not sequence-speciﬁc, but a preference for GC-rich target sites has been noted (Hiom et al., 1998). Presumably, the transposition reaction supported by RAG1 and RAG2 identiﬁes the original activity of the V(D)J recombinase, but unlike “fossil transposons,” the Rag-1 and Rag-2 genes have retained function through evolutionary domestication (Agrawal et al., 1998; Hiom et al., 1998; Smit and Riggs, 1996; van Gent et al., 1996a). The capacity of RAG1 and RAG2 to transpose DNA offers an intriguing candidate mechanism to effect recombinase-mediated chromosomal translocations, especially those where the Ig or Tcr loci have become fused with a translocation partner that does not contain an identiﬁable RSS (Piccoli et al., 1984; Tsujimoto et al., 1985). Thus intermediates of V(D)J rearrangement are capable of driving transposition reactions that would result in the integration of the Ig or Tcr loci anywhere in the genome. We presume that most of these would be of little consequence to the cell or sufﬁciently deleterious to induce apoptotic death. Such translocation types would be difﬁcult to identify. Innocuous somatic translocations are likely to be sufﬁciently infrequent to arouse little interest, and lethal translocations could exist only brieﬂy. However, the rare translocations that dysregulate cellular oncogenes and avoid apoptosis would be transforming and obvious. M. Gellert has

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Fig. 5 Single end translocation model (Hiom et al., 1998). Free SEs are generated during normal V(D)J recombination. Attacking of a SE to the target chromosome results in a threebranched intermediate. Resolving this structure generates a hairpin and interchromosomal translocation bearing the RSS. Joining of the two hairpin ends yields the reciprocal chromosome translocation.

proposed a one-end transposition model to explain RAG-mediated chromosomal translocations to sites that lack cryptic RSS (Fig. 5) (Hiom et al., 1998). The hydroxyl group on the SE intermediate acts as a nucleophile and attacks a non-Ig or non-Tcr sequence. This results in a branch structure that is resolved by the recombinase machinery into a hairpin and a chromosomal translocation containing the RSS nucleophile. Joining of the

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non-Ig or non-Tcr hairpin with the V(D)J hairpin results in the reciprocal translocation. If the V(D)J recombinase mediates illicit rearrangements between oncogenes and the Ig or Tcr loci, RSS should be present proximal to the breakpoint within the Ig or Tcr locus. However, RSS have not been found at the fusion site in a series of translocations involving Ig loci and oncogenes (Piccoli et al., 1984; Tsujimoto et al., 1985). Roth and Craig (1998) have proposed a two-end insertion model to explain the absence of RSS within translocations. The two-end insertion model proposes that a pair of SEs uses-their two hydroxyl groups to insert into DNA at opposite strands by a transesteriﬁcation reaction (Fig. 6). Once the SE is incorporated into the DNA, the free hydroxyl groups on the opposite strands of the host DNA perform transesteriﬁcation reactions to form hairpins and release the SEs. The resolution of CEs would

Fig. 6 Two-end translocation model (Roth and Craig, 1998). Paired 12- and 23-spacer RSS are generated during D-to-J rearrangement. Both RSS are inserted into a target chromosome, and two three-branched structures are generated on each side of the insertion. Further processing of these intermediates by RAG will break them into two hairpins. The joining of hairpin ends may give rise to reciprocal chromosome translocations.

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result in a translocation, and the loss of the SEs would result in there being no RSS proximal to the translocation breakpoints (Roth and Craig, 1998).

III. ILLICIT V(D)J REARRANGEMENT MEDIATED BY CRYPTIC RSS OR RSS-LIKE MOTIFS Although the Rag genes and V(D)J recombination are necessary for certain lymphoid malignancies, the proximal cause of these malignancies is not fully understood. RAG activity could cause lymphomas and leukemias by destabilizing genomic structure or by creating cell populations susceptible to transformation (Vanasse et al., 1999). However, the observation of canonical RSS at the sites of putative illicit rearrangement is rare. It has been proposed that cryptic RSS and ␹-like sequences mediate illicit V(D)J rearrangement that leads to lymphoid malignancies. It is likely that cryptic RSS are common in the mammalian genome, since RSS are relatively simple recognition motifs. Indeed, S. Lewis (Lewis et al., 1997) has identiﬁed a collection of illegitimate rearrangements and estimates that there might be as many as 10 million cryptic RSS dispersed throughout the human genome. This large number of cryptic RSS must impose a great pressure on the immune system to keep its ﬁdelity. Convincing evidence that cryptic RSS are involved in lymphoid malignancies comes from a site-speciﬁc DNA rearrangement in human T-cell acute lymphoblastic leukemia (T-ALL) (Brown et al., 1990). In approximately 30% of patients with T-ALL, a 90-kb DNA fragment between sil and scl on chromosome 1 is deleted (Janssen et al., 1993). Sequencing of the sil and scl loci suggests that two cryptic RSS in sil and scl mediate the deletion and that the junctions of the deletion bear such ﬁngerprints of V(D)J recombination as nucleotide deletion and N-nucleotide addition (Aplan et al., 1990; Breit et al., 1993; Macintyre et al., 1992). This deletion essentially replaces the 5 regulatory region of scl, which is a basic helix-loop-helix transcription factor, with sil regulatory sequences (Aplan et al., 1997). This dysregulation is believed to be the transforming event (Aplan et al., 1997). In other patients with T-ALL, but no deletion between scl and sil, translocations have been observed between the Tcr locus and the 5 regulatory region of scl (Aplan et al., 1997). Despite the apparent abundance of cryptic RSS and the clear evidence for RAG-mediated genomic rearrangements outside the Ig and Tcr loci, cryptic RSS that are recognized and cleaved by RAG1 and RAG2 are not observed in many translocations. In fact, in many cases no candidate RSS have been found at or near translocation breakpoints (Haluska et al., 1986). Moreover, when potential RSS are found, they are often located some distance from the breakpoint, and it is not clear if the potential RSS fully

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meet the stringent sequence requirements necessary for V(D)J rearrangement in vitro (Akamatsu et al., 1994; Hesse et al., 1989; Piccoli et al., 1984; Tsujimoto et al., 1985). In addition to sequence motifs that resemble RSS, DNA sequences similar to the ␹ prokaryotic activator of recombination have been noted in the chromosomal breakpoint regions that recur in FL (Jaeger et al., 1994; Wyatt et al., 1992). ␹-like sequences are highly overrepresented in the 150-bp region that deﬁnes the major breakpoint region (mbr) of FL, and three clusters of breakpoint hotspots align with ␹-like sequences within the mbr (Jaeger et al., 1994; Wyatt et al., 1992). In fact, one breakpoint cluster is immediately adjacent to a ␹-like sequence. The other two breakpoint clusters are evenly spaced 50 and 100 bp downstream of ␹ -like motifs (Wyatt et al., 1992). In addition, ␹-like motifs are found in all four major partners of the t(14:18) translocation that is virtually diagnostic of FL (Bakhshi et al., 1987; Lee, 1993; Tsujimoto et al., 1985; Wyatt et al., 1992). Wyatt et al. (1992) have proposed that ␹-like sequences represent a distinct class of recognition sites for the V(D)J recombinase. These studies of cryptic RSS and ␹-like sequences suggest a mechanism of illicit V(D)J rearrangement mediated by the recognition of canonical RSS, cryptic RSS, and/or ␹ -like sequences. During V(D)J rearrangement a cryptic RSS within an oncogene would recruit the V(D)J recombinase, which would induce a dsb within the oncogene. The dsb of an Ig CE and the dsb of an oncogene would be repaired by the dsb repair machinery to form a chromosomal translocation. If this is mediated by the V(D)J recombinase, P- and N-nucleotides should also be present within the coding junction. There is evidence for this proposed mechanism. P- and N-nucleotides are present in certain translocations (Cleary and Sklar, 1985; Cotter et al., 1990; Lee, 1993; Piccoli et al., 1984), putative RSS have been located within oncogenes (Breit et al., 1993; Haluska et al., 1986; Tsujimoto et al., 1985), and illicit chromosomal recombination between ␬ and ␭ loci occurs in vitro (Bailey and Rosenberg, 1997).

IV. THE GERMINAL CENTER A. The Germinal Center Reaction The germinal center (GC) reaction is necessary for the generation of highafﬁnity antibody, B-cell memory, and long-lasting serum antibody. B cells that enter the GC become susceptible to activities that alter the genome by hypermutation, Ig class switching, and perhaps secondary V(D)J rearrangement (Fig. 7). The remarkable genomic plasticity of GC B lymphocytes is coincident with rapid proliferation and selection by apoptosis. Thus, the GC microenvironment is a crucible of genetic change and a plausible site for oncogenic errors.

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Fig. 7 The germinal center. Activated B and T cells enter the GC reaction, which is composed of a GC dark zone with large, proliferating, slg centroblasts that become nondividing centrocytes. GC B cells undergo somatic hypermutation, switch recombination, and possibly upregulate V(D)J rearrangement to increase the afﬁnity of their Ig. Centrocytes take up antigen from FDC and then present antigen to T cells. At this step, the centrocytes initiate apoptosis, enter the GC reaction again, or develop into AFC or memory cells.

Complete descriptions of the GC reaction have been published elsewhere (Garside et al., 1998; Kelsoe, 1995, 1996; MacLennan, 1994; Przylepa et al., 1998). Here, we will only outline this crucial component of humoral immune responses. Peripheral lymphoid tissues are divided into regions that are rich in T or B lymphocytes. In the spleen, the T-cell-rich region is known as the periarteriolar lymphoid sheath (PALS), and the region of B cells is the lymphoid follicle. Within a few hours after primary immunization, antigen reaches splenic B cells in the lymphoid follicle and antigen-reactive cells become partially activated and migrate from the follicle to the periphery of the PALS. At the same time, dendritic cells (DC) present antigen to CD4+ helper T cells in the PALS and initiate their local proliferation. By 72 h after immunization, activated, antigen-speciﬁc B cells present in the outer PALS form conjugates with proliferating T cells and begin to divide. This mitotic response continues producing daughter B cells that either differentiate locally into foci of plasmacytes or emigrate back to the follicle to initiate the GC reaction

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(Garside et al., 1998; Jacob et al., 1991a; Kelsoe, 1995; MacLennan et al., 1992). Splenic GCs contain rapidly dividing, antigen-speciﬁc B and T lymphocytes located within a reticulum of follicular dendritic cells (FDCs). The GC can be divided into distinct histologic zones, the dark and light zones (LZ and DZ), which correlate with lymphocyte activity. The DZ contains large, rapidly proliferating B cells called centroblasts, which do not express detectable levels of membrane Ig (mIg−). Centroblasts undergo several rounds of mitosis in the DZ, then exit the cell cycle; subsequently, centroblasts reexpress mIg and migrate to the LZ. The nondividing, mIg+ B cells present in the LZ are known as centrocytes. Several groups (Kelsoe, 1995; Kepler and Perelson, 1993; MacLennan, 1994; Przylepa et al., 1998) have postulated that those centrocytes that survive the genetic modiﬁcations induced by the GC reaction reenter the DZ and initiate new rounds of proliferation as centroblasts. Lymphocytes that cycle between the centroblast and centrocyte states acquire selected mutations that increase afﬁnity for antigen. GCs ﬁrst appear in the spleen 4–5 days after a primary immunization, achieve maximal cell numbers by day 10–11, and end (with nonreplicating antigens) after 4–6 weeks (Garside et al., 1998; Jacob et al., 1991a; MacLennan et al., 1992). In mice, primary GCs develop from 1–3 antigenspeciﬁc B cells (Jacob et al., 1991a, 1991b, 1993) and there is no evidence for B-cell migration between GCs (Fig. 7). Thus, each GC represents an independent “experiment” in somatic evolution (Przylepa et al., 1998). The GC is also a site of active switch recombination, with most, but not all, GC B cells undergoing IgM → IgG/IgA/IgE class switching (Jacob et al., 1993; Toellner et al., 1996). Switch recombination alters the physical properties of antibody and modiﬁes its effector function(s) (Toellner et al., 1996). In some way, the physiology of GC B cells is also altered, in that the progeny of GC B lymphocytes form the long-lived memory cell and bone marrow plasmacyte compartments (Takahashi et al., 1998). Thus, the GC reaction extensively re-forms the B-cell genome and generates cells that become resistant to the death signals that limit the life span of na¨ıve, follicular B cells. There is considerable evidence that secondary V(D)J rearrangement also takes place in a subset of GC B cells. Our laboratory demonstrated immunoreactive RAG1 and RAG2 proteins in the centrocyte compartment of splenic and Peyer’s patch GCs and RAG1 mRNA in cells with a GC phenotype that had been isolated by ﬂow cytometry (Han et al., 1996). Simultaneously, the laboratory of H. Ohmori demonstrated upregulation of RAG1 and RAG2 transcripts in activating cultures of mouse splenic B cells and the expression of RAG proteins in lymph node GCs (Hikida et al., 1996). Subsequent functional studies determined that peripheral B cells with a GC phenotype actively rearranged their Ig light-chain loci (Han et al., 1997; Hertz et al., 1998; Papavasiliou et al., 1997). However, in contrast to

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receptor editing in the bone marrow (BM), avid cross-linking of mIg on peripheral B cells extinguished RAG expression and activity instead of promoting it (Hertz et al., 1998). To emphasize the fundamental distinctions between secondary V(D)J rearrangement in the bone marrow, receptor editing, the process of secondary rearrangement in the periphery is known as receptor revision. The interpretation that mature, peripheral B cells with the phenotype of GC centroblasts could initiate receptor revision was placed into question by a recent study that employed transgenic mice carrying a bacterial artiﬁcial chromosome (BAC). Yu et al. (1999) replaced Rag-2 with green-ﬂuorescent protein (GFP) and incorporated the gene knock-in and approximately 100 kb of ﬂanking DNA into a BAC construct. Thus, ﬂuorescence from the GFP transgene served a surrogate marker for RAG2 expression while recombinase activity was supported by the endogenous loci. T cells and peripheral B cells from BAC transgenic mice were transferred into Rag-2−/− recipients and immunized. Whereas both GFP+ and GFP− splenic B cells were competent to form GC structures in recipient mice, GFP expression was either lost over time or never reexpressed, indicating that RAG expression is not reactivated during the GC reaction (Yu et al., 1999). A similar experiment with mice that carry a functional RAG2:GFP knock-in gene (Monroe et al., 1999) conﬁrmed the induction of splenic B cells that express RAG2 message and protein by imunization. Approximately 20% of the peripheral B cells that expressed the RAG2:GFP fusion protein carried GC markers, but the majority exhibited a phenotype typical of immature and transitional B cells (Monroe et al., 1999). A likely explanation that unites all experimental observations on RAG expression and receptor revision by peripheral B cells is that immunization leads to increased numbers of immature and transitional B cells in peripheral lymphoid tissues. Some of these cells, a small percentage, will be speciﬁc for the eliciting antigen and may enter the GC reaction while retaining the ability to express RAG1 and RAG2. Indeed, a recent study of human, tonsillar GC centrocytes provides strong evidence for coincidental V(D)J hypermutation and receptor revision. Wilson et al. (2000) observed a single genealogy of somatic hypermutation that included a VDJ replacement event typical of Igh receptor editing. If this replacement were mediated by RAG1 and RAG2 recombinase activity, the simplest explanation is that both events took place in the GC (Nemazee and Weigert, 2000).

B. The Germinal Center as a Site for Lymphomagenesis Although a causal relationship between GCs and lymphomas has not been established, there is much evidence for their association. Many lymphomas

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were originally classiﬁed histologically by their resemblance to GCs and GC lymphocytes. The similarity of the lymphoma cell shape, location, and appearance led to the belief that these lymphomas were derived from GC cells (Stein et al., 1982). These histological comparisons were later complemented by the comparative immunophenotyping of normal, differentiated lymphocytes and lymphomas. FL and BL, along with other tumors, possess a variety of surface antigens associated with normal, GC B cells (Anderson et al., 1984; Cossman et al., 1984; Freedman et al., 1987; Gregory et al., 1987; Harris et al., 1984; Kvaloy et al., 1984; Murray et al., 1985; Ritz et al., 1981). Phenotypic similarities between FL, BL, and GC cells were extended by molecular genetic studies demonstrating that BL cells had mutated Ig genes (Klein et al., 1995); V(D)J hypermutation is a hallmark of GC B cells and their progeny (Jacob et al., 1991b, 1993). Thus, BL likely represents transformed GC B cells or lymphocytes that have completed rounds of mutation in the GC reaction. Similar mutational studies indicate that Hodgkin’s lymphoma and large-cell immunoblastic lymphoma are derived from GC cell populations (Kanzler et al., 1996; Kuppers et al., 1997). The remarkable genomic lability that is characteristic of GC lymphocytes makes the GC a plausible site for the malignant transformation of B cells. In GCs, B cells divide extremely rapidly, accumulate high frequencies of point mutations in transcribed Ig and bcl-6 loci, and undergo Ig class switching (Liu et al., 1996; MacLennan, 1994; Pasqualucci et al., 1998; Shen et al., 1998; Toellner et al., 1996). In fact, sporadic BL have characteristic translocation breakpoints in the switch regions of Igh genes (Battey et al., 1983; Pelicci et al., 1986; Piccoli et al., 1984). A variant translocation in endemic BL brings myc in association with the IgJH locus and generally contains point mutations that may dysregulate myc expression (Bentley and Groudine, 1988; Cesarman et al., 1987; Nishikura et al., 1983; Spencer et al., 1990; Taub et al., 1984). Somatic hypermutation and concomitant V(D)J rearrangement appear possible in at least a subset of GC B cells (Han et al., 1996; Hikida et al., 1996; Jacob et al., 1993; Meffre et al., 1998; Papavasiliou et al., 1997; Wilson et al., 2000). Thus, it is possible that Ig class switching or secondary V(D)J recombination and hypermutation in the GC mediates the chromosomal translocations and mutations that recur in lymphomas and leukemias. Although ideal RSS have not been found at translocation breakpoints, the overrepresentation of ␹ -like sequences at breakpoint sites and latent transposase activity of RAG1 and RAG2 argues that typical RSS may not be necessary for recombinase-mediated translocations (Agrawal et al., 1998; Haluska et al., 1986; Hiom et al., 1998; Tsujimoto et al., 1985; Wyatt et al., 1992). The GC may also play a role in the selection of lymphomas. Non-Hodgkin’s lymphoma is often characterized as an indolent disease that sometimes progresses to an aggressive form. Thus, FL transforms into high-grade, diffuse

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large B-cell lymphoma (DLBL) in some 30% of patients (Cullen et al., 1979; Garvin et al., 1983). A recent study has shown the transformation of a clonally diverse FL into a clonally restricted DLBL (Matolcsy et al., 1999). The originating FL was composed of cells that harbored diversely mutated VH rearrangements. These cells were eventually replaced by a clonally related DLBL carrying a single, mutated VH. The mutations present in FL and DLBL cells exhibited a bias for replacement versus silent mutations consistent with antigen-driven selection (Clarke et al., 1985). Interestingly, while FL cells appeared to diversify by active hypermutation, the DLBL variant showed no evidence of continuing hypermutation and could not be isolated from the original FL by sensitive PCR techniques (Matolcsy et al., 1999). These observations support the interpretation that the DLBL variant was derived from the original FL. Presumably, continuing hypermutation [and V(D)J rearrangement?] resulted in the creation of the aggressive DLBL phenotype and the subsequent overgrowth and replacement of the more benign FL. Whereas GCs act as sites for antigen-dependent V(D)J hypermutation and cell selection, the forces that drove mutation in this FL and selected the aggressive DLBL form are unknown. It would be interesting to determine if additional translocations accumulated in the DLBL, a condition similar to lymphomas with independent translocations that involve both myc and bcl-2 separately (Bentz et al., 1996; Brito-Babapulle et al., 1991; Gauwerky et al., 1988; Karsan et al., 1993). This would indicate that the transformation from the FL to DLBL was induced by illegitimate V(D)J recombination within the GC. Mouse models have shown that the deregulation of myc by a BL-like translocation is important for malignant transformation from an indolent follicular hyperplasia derived from a human t(14;18) transgene (McDonnell and Korsmeyer, 1991). This is further evidence that the progression from an indolent lymphoma to an aggressive lymphoma requires a second transforming event, which in the GC could be induced by illegitimate V(D)J recombination.

V. CHROMOSOMAL TRANSLOCATIONS IN GC-LIKE LYMPHOMAS A. Burkitt’s Lymphoma Many human lymphomas are of B-cell origin and contain nonrandom translocations that involve the Igh locus/q32 region of chromosome 14 (Harris et al., 1994; Kirsch et al., 1982). BL was the ﬁrst lymphoid malignancy to be well characterized for chromosomal translocation breakpoints, and most often the breakpoints involve the Igh locus (Battey et al.,

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1983; Dalla-Favera et al., 1982; Haluska et al., 1986; Pelicci et al., 1986). BL is an aggressive B cell malignancy that strikes children and young adults. The developmental stage of the B cells that gives rise to BL is a matter of controversy, but immunophenotyping studies suggest that BL may be derived from GC B cells (Gregory et al., 1987; Kvaloy et al., 1984; Murray et al., 1985; Ritz et al., 1981). However, GC B cells express some surface antigens that are also present on immature B cells, and it is possible that BL arises during lymphopoiesis (Berger et al., 1979; Flandrin et al., 1975; Magrath, 1990; Magrath and Ziegler, 1980; Mitelman et al., 1979; Ritz et al., 1981; Roos et al., 1982). The membrane Ig (mIg) expressed on typical BL is generally IgM without IgD (Gregory et al., 1987). Na¨ıve, mature B cells express IgM and IgD, and post-GC B cells express IgG or IgA; immature and GC B cells express little or no IgD and intermediate levels of IgM, IgG, or IgA (MacLennan, 1994; Tamaru et al., 1995; Toellner et al., 1996). Recent studies on the rearranged Ig genes present in BL have provided strong evidence for the derivation of BL from GC B cells (Klein et al., 1995). The Ig genes present in BL contain numerous point mutations typical of those introduced by V(D)J hypermutation during the GC reaction (Klein et al., 1995). This ﬁnding suggests that BL represents the malignant transformation of GC B lymphocytes or their progeny. BL is classiﬁed as either endemic or sporadic. Endemic BL is usually associated with Epstein–Barr virus (EBV) infection and occurs in Africa, while sporadic BL occurs mostly in Europe and North America and is not associated with EBV. Both lymphomas are characterized by the t(8;14)(q24;q32) translocation between the Igh locus and myc (Fig. 8a). However, the translocation breakpoints differ between endemic and sporadic disease (Fig. 8b) (Barriga et al., 1988; Pelicci et al., 1986). In endemic BL, the upstream region of myc translocates into the Igh J (JH) or -D (DH) loci (Pelicci et al., 1986). In sporadic BL the ﬁrst intron or exon of myc translocates into the switch regions of the Igh constant region (Pelicci et al., 1986). The association between BL and V(D)J rearrangement is not limited to translocations involving Igh; in about 15% of BL cases, a variant translocation joins myc and the Ig␬ or Ig␭ locus (Emanuel et al., 1984; Hollis et al., 1984). In these variant translocations, V␬, J␬, or 5 regions of C␭ translocate into myc at positions 3 of exon 3 (Emanuel et al., 1984; Hollis et al., 1984). These translocations leave the coding unit of myc intact and are associated with increased expression of myc, presumably due to cis-acting enhancers within the Ig loci (Nishikura et al., 1983). As V(D)J rearrangement occurs normally between two RSS, RSS-like motifs were sought near the breakpoint at the Ig loci and myc. Although the breakpoints within the Igh and lightchain loci occur near RSS, no breakpoints were located exactly next to the heptamer, as in V(D)J rearrangement, nor have consensus RSS been located

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Fig. 8 Burkitt’s lymphoma. (a) Schematic of the gross translocation between chromosome 8 at myc and chromosome 14 at the Igh locus. (b) The translocation of BL occurs at the JH or switch regions of the Igh locus and the ﬁrst intron or exon of myc. Endemic BL show a preference for translocations between JH and the ﬁrst intron of myc, while sporadic BL often involves translocations within the ﬁrst exon of myc and the Igh switch region. Dotted lines represent breakpoints and translocations associated with endemic BL, while solid lines represent breakpoints and translocations associated with sporadic BL.

at or near the breakpoints within myc (Battey et al., 1983; Haluska et al., 1986; Pelicci et al., 1986; Piccoli et al., 1984).

B. Follicular Lymphoma FL is a common, indolent lymphoma composed of lymphoid cells that appear mature and contain either small and cleaved or large nuclei (Ersboll et al., 1989). Progression to an aggressive high-grade form is common and accompanied by cytogenetic changes, including a BL-like translocation between myc and Ig (Brito-Babapulle et al., 1991; Cullen et al., 1979; Gauwerky et al., 1988; Oviatt et al., 1984). Historically, FL was thought to derive from GC cells, due to histological similarities in the follicular appearance of the GC and FL. Today, the evidence for the association between FL and the GC is stronger. Like GC cells, FL cells are positive for mIg, CD10, CD19, CD20, CD21, CD71, CD77, B7, and HLA-DR, and they lack CD5 (Anderson et al., 1984; Cossman et al., 1984; Freedman et al., 1987; Harris

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et al., 1984; Kvaloy et al., 1984). More evidence for the relationship of FL with the GC comes from the presence of mutated Ig receptors in FL (Noppe et al., 1999). The hallmark of FL is a t(14;18)(q32;q21) translocation between the bcl-2 and Igh loci (Cleary and Sklar, 1985; Crisan et al., 1993; Lee, 1993). Translocation occurs between the JH region and the major breakpoint region (mbr) of bcl-2 in 60% of translocations, with a minor breakpoint region (mcr) involved in the rest (Weiss et al., 1987). The mbr is located in the untranslated region of bcl-2 exon 3, while the mcr is located approximately 20 kb downstream of the gene (Cleary and Sklar, 1985; Lee, 1993; Tsujimoto et al., 1985). V(D)J rearrangement has been implicated in FL development. The strong selection for breakpoints in these two small regions—the mbr is approximately 150 nucleotides—suggests a mechanism of site-speciﬁc recombination. In support of aberrant V(D)J rearrangement, nontemplate (N)nucleotides are present at the junction between chromosomes 14 and 18 in FL. N-nucleotides are characteristic of terminal-deoxynucleotide transferase (Tdt) nucleotide additions in V(D)J rearrangement (Fig. 9) (Cotter et al., 1990; Kneba et al., 1991). In addition to an Igh bcl-2 translocation, there are variant FL translocations involving bcl-2 and either of the Ig light-chain loci. The consistent appearance of translocated Ig loci, one of only two sites of V(D)J rearrangement, in BL and FL, both B-cell malignancies, suggests that translocation is V(D)J mediated. Candidate RSS have also been sequenced near the mbr of bcl-2 (Fig. 9) (Bakhshi et al., 1987; Tsujimoto et al., 1985; Wyatt et al., 1992). Unlike V(D)J rearrangement, the breakpoints do not appear exactly proximal to the heptamer, and the reciprocal translocation is not with the JH region, but rather with the DH region (Bakhshi et al., 1987; Wyatt et al., 1992). These inconsistencies have led to searches for other possible RSS candidates. ␹-like sequences, conserved sequence motifs that are activators of prokaryotic recombination, have been found at all partners of the FL translocation: the mbr, mcr, the DH, and JH regions (Fig. 9) (Wyatt et al., 1992). Some have proposed that translocation occurs via V(D)J rearrangement mediated by ␹-like sequence recognition (Wyatt et al., 1992). This is supported by research showing ␹-like-mediated recombination in transgenic mice brain cells where RAG1 was expressed (Matsuoka et al., 1991). Bcl-2, an inhibitor of apoptosis in mature B cells, is believed to be dysregulated by translocation to Ig loci. Within the GC, high-afﬁnity B cells upregulate BCL-2 and develop into memory cells, while B cells with poor afﬁnity undergo apoptosis (Nunez et al., 1991). The upregulation of BCL-2 serves as a tool for selection of high-afﬁnity Ig by allowing B cells with highafﬁnity Ig to escape programmed cell death (Liu et al., 1991). Its dysregulation therefore could result in lymphomagenesis. Mice transgenic for the t(14;18) translocation show that dysregulation of bcl-2 does not cause overt lymphomagenesis, but does provide a survival advantage to B lymphocytes

Fig. 9

T(14;18) translocation of follicular lymphoma. (a) All translocation partners for the t(14;18) translocation have ␹ -like sequences, while the mbr has been proposed to harbor a cryptic RSS. (b) Breakpoints occur at the JH locus and mbr or mcr. However, the reciprocal translocation occurs with the DH locus and chromosome 18.

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(McDonnell et al., 1989). This survival beneﬁt leads to outright lymphomagenesis when followed by chromosomal rearrangements involving myc and the Igh locus (McDonnell and Korsmeyer, 1991). This model of lymphomagenesis is consistent with the model of multistep cancer development.

VI. RESEARCH To determine the plausibility of a role for V(D)J rearrangement in the generation of oncogenic chromosomal translocations, we have established a cell model of inducible RAG expression based on the pre-B-cell leukemia 103/BCL-2 (Chen et al., 1994). 103/BCL-2 was created by the transformation of pre-B cells with a temperature-sensitive (ts) mutant Abelson murine leukemia virus (Chen et al., 1994). At 34◦ C, the product of ts v-abl drives rapid cell division and suppresses recombinase activity, but at a nonpermissive temperature, 39◦ C, the v-ABL protein becomes unstable and the cells enter growth arrest. During periods of G0, 103/BCL-2 cells are spared from apoptosis by the active bcl-2 transgene (Chen et al., 1994; Nunez et al., 1990). At 39◦ C, 103/BCL-2 activates V(D)J recombination, as determined by Southern blotting, the expression of RAG message and protein, and the presence of cleaved RSS SEs (Chen et al., 1994; Schlissel et al., 1993). Remarkably, the bcl-2 transgene allows 103/BCL-2 cells to remain viable during growth arrest for as long as 2 weeks (Chen et al., 1994). Thus, prolonged periods of RAG expression and recombinase activity can be achieved, increasing the possibility of observing rare, illegitimate rearrangements. For example, we have used ligation-mediated PCR (LM-PCR) to detect dsb at RSS heptamers, necessary intermediates of V(D)J rearrangement, which form in the Tcr-D␦2 locus of 103/BCL-2 cells at 39◦ C (Schlissel et al., 1993) (Fig. 10). This rearrangement is illegitimate in cells of the B-lymphocyte lineage. Nonetheless, Tcr -D␦2 recombination intermediates were induced at the nonpermissive temperature, although they appeared later (72 h) and were less abundant than J␬2 intermediates. This observation has been extended to other Tcr loci (not shown), demonstrating that recombinase activity is generally available. Similar experiments indicate that interchromosomal rearrangements between the Ig␬ and Ig␭ loci are also possible in 103/BCL-2 (Bailey and Rosenberg, 1997). Therefore, it appears that RAG1 and RAG2 are capable of recognizing, cleaving, and recombining genes ﬂanked by authentic RSS, even when those loci should be inaccessible or located in trans (Bailey and Rosenberg, 1997; McMurry and Krangel, 2000). To determine if the culture conditions that induce normal and illegitimate V(D)J rearrangements in 103/BCL-2 are also associated with the generation of chromosomal translocations, we exposed a cloned subline of

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Fig. 10 (A) Outline of LM-PCR reaction. Cleavage of RSS by RAG gives rise to a blunt SE and hairpined CE. A linker is ligated onto the blunt SE, and two runs of PCR ampliﬁcation are performed with a locus-speciﬁc primer. PCR products are separated on an agarose gel and visualized by blotting with a locus-speciﬁc probe. (B) Detection of SEs in Tcr ␦ locus. 103/BCL-2 cells were cultured at 39◦ C for the indicated period and live cells were harvested. DNA was puriﬁed and ligated to a BW linker. PCR was performed as described previously (Han et al., 1997; McMahan and Fink, 1998). In the experiment, 1 × 106 bone marrow cells were used as the positive control for J␬2, and 1 × 105 thymocytes were used as a positive control for Tcr D␦2.

103/BCL-2 to nonpermissive culture (39◦ C) for 0 (uninduced controls), 24, 48, or 72 h. Surviving cells from each culture were puriﬁed by densitygradient centrifugation and recloned at 34◦ C by limit dilution (1 cell/well) plating. Fewer than 30% of the plated cells gave rise to colonies of dividing cells (data not shown). Iterative chromosome painting was then used to identify the presence of novel, unselected chromosome translocations present in control 103/BCL-2 or sublines that had been cultured at 39◦ C. Chromosome painting speciﬁcally identiﬁes individual chromosomes by the hybridization of chromosomespeciﬁc nucleotide probes that carry a single ﬂuorochrome label (Carter, 1994). Mouse chromosomes are uniformly acrocentric and similar in size, making chromosome identiﬁcation by classical banding techniques problematic (Breneman et al., 1993). Thus, chromosome painting or the comprehensive technique of spectral karyotyping (SKY) is especially valuable in the analysis of mouse chromosomes (Liyanage et al., 1996). Our studies have used a series of chromosome-speciﬁc probes (chromosomes 6, 12, 16, and 17) labeled with ﬂuorescein isothiocyanate (FITC) or indodicarbocyanine (Cy3),

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and a nonspeciﬁc intercalating ﬂuorochrome, 4 ,6-diamidino-2-phenylindole (DAPI). Thus, a normal karyotype consists of green (FITC) and red (Cy3) speciﬁc chromosomes in addition to the remaining 18 chromosome pairs labeled blue by DAPI. Novel blue/red, blue/green, or red/green chromosomes identify any acquired chromosomal translocations. At least two variant karyotypes were present in the 103/BCL-2 cells we obtained from N. Rosenberg, a nonreciprocal t(?;12) translocation present in all cells (B1 karyotype) and another translocation to chromosome 12 (B2 karyotype) present in about 30% of cells (Fig. 11, see color plate). We did not observe translocations involving chromosome 6, 16, and 17; cells subjected to culture under nonpermissive conditions carried the B1 karyotype. No additional translocations involving chromosome 6, 12, 16, and 17 were observed in 103/BCL-2 controls that had been expanded and subcloned but not cultured at 39◦ C; that is, all cells carried the B1 karyotype. In contrast, in 103/BCL-2 sublines exposed to nonpermissive temperature and growth arrest for 24–72 h, we have observed that greater than 15% of the sublines contain novel translocations (Fig. 12 (see color plate) and data not shown). Novel translocations were present in cells that had been exposed to 39◦ C for as little as 24 h, but longer exposures were associated with a higher frequency of novel translocations (data not shown). These observations provide support for an association between induced V(D)J recombinase activity and chromosomal translocation. However, our studies are preliminary. We cannot rule out the possibility that events associated with growth arrest, but unrelated to RAG expression and V(D)J rearrangement, mediate the new translocations we observe. It is also possible that the 103/BCL-2 line is uniquely prone to genomic instability, or that exposure to ≥ 24 h of recombinase activity is so grossly unphysiologic as to be uninformative. Nonetheless, the remarkable frequency of novel translocations that arise in 103/BCL-2 cells during nonpermissive culture is consistent with RAG-mediated chromosomal translocation and merits closer study. When and where in B-cell development might illegitimate recombinase activity produce chromosomal translocations? Cyclic RAG expression takes place during the generation of B lymphocytes in the BM, offering the possibility of transformation in immature cell types. We are also investigating the expression of RAG1 and RAG2 in peripheral lymphocytes and have determined that responses to inﬂammatory antigens or adjuvants, lymphocytes including immature and recirculating B cells, and CD3-positive T cells are lost from the BM (Foster et al., 2001). At least some of these cells migrate to peripheral lymphoid tissues and appear capable of responding to antigen while retaining recombinase function (Foster et al., 2001; Han et al., 1997; Meffre et al., 1998; Wilson et al., 2000). Indeed, immunization and inﬂammation induce the appearance of RAG-positive B cells in the spleen and lymph nodes of mice (Han et al., 1997; Hikida et al., 1996; Papavasiliou et al., 1997) and tonsils of humans (Meffre et al., 1998). These cells bear the

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surface phenotypes of immature and/or GC B cells, and it is currently a matter of debate whether these lymphocytes constitute a signiﬁcant component of the immune response (Nagaoka et al., 2000; Yu et al., 1999). However, Wilson et al. (2000) have recently provided strong evidence for coincident hypermutation and secondary V(D)J rearrangement in human GC B cells. If these observations are conﬁrmed and represent a general phenomenon, immune responses to many natural antigens might establish conditions— hypermutation and V(D)J rearrangement—which are associated with lymphomagenesis (Gregory et al., 1987; Matolcsy et al., 1999; McDonnell and Korsmeyer, 1991; Potter and Wiener, 1992; Taub et al., 1984). This possibility is supported by a large series of studies by M. Potter that demonstrates induction of plasmacytomas by establishing peritoneal inﬂammation with mineral oil (Anderson and Potter, 1969; Muller et al., 1997a, 1997b; Potter and Wiener, 1992). Intraperitoneal administration of mineral oil results in the formation of “granulomas,” sites of aggressive inﬂammation, which harbor B cells carrying oncogenic t(12;15) translocations.

VII. CONCLUSIONS V(D)J rearrangement is suspected to play a fundamental role in generating the chromosomal translocations present in many lymphomas and leukemias. The consistent involvement of translocations within the Ig and Tcr loci in B or T lymphocytes bolsters this notion. Previously, translocations were believed to involve cryptic RSS, including DNA motifs that did not match the consensus RSS necessary for efﬁcient rearrangement in extrachromosomal templates (Haluska et al., 1986; Tsujimoto et al., 1985). However, the discovery of transposase activity by RAG1 and RAG2 provides new and more ﬂexible mechanisms for the generation of chromosomal translocations (Agrawal et al., 1998; Hiom et al., 1998; Roth and Craig, 1998). The formation of dsb during V(D)J rearrangement in Ig and Tcr loci creates a reactive hydroxyl nucleophile on the SE that can integrate into ds DNA via a sequence-independent transposition reaction (Agrawal et al., 1998; Hiom et al., 1998). In principle, then, dsb cleavage at RSS in the Ig and Tcr loci is necessary to generate chromosomal translocations. The characterization of many lymphomas as GC cells by histology and surface antigens has now been complemented by somatic genetic analyses of mutation in Ig and bcl-6 genes. The presence of mutated Ig and bcl-6 genes in a majority of B-cell malignancies deﬁne their origins as GC cells or their progeny (Chaganti et al., 1998; Chen et al., 1998; Klein et al., 1995; Kuppers et al., 1997; Migliazza et al., 1995; Noppe et al., 1999). This leads to the question of how the GC reaction is involved in lymphomagenesis.

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GC B lymphocytes are subject to extensive genomic remodeling by Ig classswitch recombination (Toellner et al., 1996), somatic hypermutation (Jacob et al., 1991b, 1993; Toellner et al., 1996), and, perhaps, secondary V(D)J rearrangement (Han et al., 1996; Hertz et al., 1998; Papavasiliou et al., 1997). These mechanisms of genetic plasticity enhance immunity. Any or all could plausibly effect chromosomal translocations and drive lymphomagenesis. The very mechanisms necessary for effective humoral immunity may be those that generate lymphoid malignancies. Disaster may be the consequence of success.

ACKNOWLEDGMENTS This work was supported in part by U.S. Public Health Service grants AI24335, AG13789, and AG10207, and a Novartis Research Grant to G.K.

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Recombinant Immunotoxins in Targeted Cancer Cell Therapy Yoram Reiter Faculty of Biology Technion-Israel Institute of Technology Haifa 3200, Israel

I. Introduction II. Design of Recombinant Immunotoxins A. The Toxin Moiety B. The Targeting Moiety—Recombinant Antibody Fragments III. Construction and Production of Recombinant Immunotoxins IV. Preclinical Development of Recombinant Immunotoxins V. Application of Recombinant Immunotoxins A. Recombinant Immunotoxins against Solid Tumors B. Recombinant Immunotoxins against Leukemias and Lymphomas VI. Other Applications of Recombinant Antibody Fragments A. Radioimaging and Radioimmunotherapy B. Prodrug Therapy with Fv-Enzyme Fusion Proteins VII. Challenges and Future Directions of Recombinant Immunotoxins A. Immune Responses and Dose-Limiting Toxicity B. Speciﬁcity References

Targeted cancer therapy in general and immunotherapy in particular combines rational drug design with the progress in understanding cancer biology. This approach takes advantage of our recent knowledge of the mechanisms by which normal cells are transformed into cancer cells, thus using the special properties of cancer cells to device novel therapeutic strategies. Recombinant immunotoxins are excellent examples of such processes, combining the knowledge of antigen expression by cancer cells with the enormous developments in recombinant DNA technology and antibody engineering. Recombinant immunotoxins are composed of a very potent protein toxin fused to a targeting moiety such as a recombinant antibody fragment or growth factor. These molecules bind to surface antigens speciﬁc for cancer cells and kill the target cells by catalytic inhibition of protein synthesis. Recombinant immunotoxins are developed for solid tumors and hematological malignancies and have been characterized intensively for their biological activity in vitro on cultured tumor cell lines as well as in vivo in animal models of human tumor xenografts. The excellent in vitro and in vivo activities of recombinant immunotoxins have lead to their preclinical development and to the initiation of clinical trail protocols. Recent trail

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C 2001 by Academic Press. Copyright All rights of reproduction in any form reserved.

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results have demonstrated potent clinical efﬁcacy in patients with malignant diseases that are refractory to traditional modalities of cancer treatment: surgery, radiation therapy, and chemotherapy. The results demonstrate that such strategies can be developed into a separate modality of cancer treatment with the basic rationale of speciﬁcally targeting cancer cells on the basis of their unique surface markers. Efforts are now being made to improve the current molecules and to develop new agents with better clinical efﬁcacy. This can be achieved by development of novel targeting moieties with improved speciﬁcity that will reduce toxicity to normal tissues. In this review, the design, construction, characterization, and applications of recombinant immunotoxins are described. Results of recent clinical trails are presented, and future directions for development of recombinant immunotoxins as a new modality for cancer treatment are discussed. C 2001 Academic Press.

I. INTRODUCTION The rapid progress in understanding the molecular biology of cancer cells has made a large impact on the design and development of novel therapeutic strategies. These are developed because treatment of cancer by chemotherapy is limited by a number of factors and usually fails in patients whose malignant cells are not sufﬁciently different from normal cells in their growth and metabolism. Other limiting factors are the low therapeutic index of most chemotherapeutic agents, the emergence of drug-resistant populations, tumor heterogeneity, and the presence of metastatic disease. The concept of targeted cancer therapy is thus an important means to improve the therapeutic potential of anticancer agents and lead to the development of novel approaches such as immunotherapy. The approach of cancer immunotherapy and targeted cancer therapy combines rational drug design with the progress in understanding cancer biology (1–4). This approach takes advantage of some special properties of cancer cells: many of them contain mutant or overexpressed oncogenes on their surface, and these proteins are attractive antigens for targeted therapy. The ﬁrst cell-surface receptor to be linked to cancer was the EGF receptor, which is present in lung, brain, kidney, bladder, breast, and ovarian cancer (5, 6). Several other members of the EGF family of receptors, the erbB2, erbB3, and erbB4 receptors, appear to be abundant on tumors of breast and ovary and erbB2, for example, is the target for Phase I and II immunotherapy clinical trails (7, 8). Other promising candidates for targeted therapy are differentiation antigens that are expressed on the surface of mature cells but not on the immature stem cells. The most widely studied examples of differentiation antigens which are currently being used for targeted therapy are expressed by hematopoietic

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malignancies and include CD19, CD20, and CD22 on B-cell lymphomas and leukemias and the IL-2 receptor on T-cell leukemias (9–11). Differentiation antigens have also been found on ovarian, breast, and prostate cancer (12–14). Another class of antigens, termed tumor-associated antigens (TAA), are molecules which are tightly bound to the surface of cancer cells and are associated with the transformed cancer cells. An example is the carbohydrate antigen Lewis Y, which is found in many types of solid tumors (15). Another class of TAAs are cancer peptides that are presented by class I MHC molecules on the surface of tumor cells (16, 17). It should be possible to use these molecular cell-surface markers as targets to eliminate the cancer cells while sparing the normal cells. For this approach to be successful, we must generate a targeting moiety which will bind very speciﬁcally the antigen or receptor expressed on the cancer cell surface and arm this targeting moiety with an effector cytotoxic moiety. The targeting moiety can be a speciﬁc antibody directed toward the cancer antigen or a ligand for speciﬁc overexpressed receptor. The cytotoxic arm can be a radioisotope, a cytotoxic drug, or a toxin. One strategy to achieve this is to arm antibodies that target cancer cells with powerful toxins which can originate from both plants and bacteria. The molecules generated are termed recombinant immunotoxins. The goal of immunotoxin therapy is to target a very potent cytotoxic agent to cell surface molecules which will internalize the cytotoxic agent and result in cell death. Developing this type of therapy has attracted much interest in the past years. Since immunotoxins differ greatly from chemotherapy in their mode of action and toxicity proﬁle, it is hoped that immunotoxins will have the potential to improve the systemic treatment of tumors incurable with existing modes of therapy. As shown in Fig. 1, immunotoxins can be divided into two groups: chemical conjugates (or ﬁrst-generation immunotoxins) and second-generation (or recombinant immunotoxins). They both contain toxins that have their cellbinding domains either mutated or deleted to prevent them from binding to normal cells, and that are either fused or chemically conjugated to a ligand or an antibody speciﬁc for cancer cells (Table I). First-generation immunotoxins, composed of whole antibodies chemically conjugated to toxins, demonstrated the feasibility of this concept. Cancer cells cultured in vitro could be killed under conditions in which the immunotoxin demonstrated low toxicity toward cultured normal cells. Clinical trials with these agents had some success; however, they also revealed several problems, such as nonspeciﬁc toxicity toward some normal cells, difﬁculties in production, and, particularly for the treatment of solid tumors, poor tumor penetration owing to their large size.

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Fig. 1 Immunotoxins for targeted cancer therapy. First-generation immunotoxins are whole monoclonal antibodies to which the toxin is chemically conjugated. Second-generation immunotoxins made by recombinant DNA technology by fusing recombinant antibody fragments to the toxin (usually a truncated or mutated form of the toxin). Three types of recombinant antibody fragments are used as the targeting moiety in recombinant immunotoxins. Fabs are composed of the light-chain and the heavy-chain Fd fragments (VH and CH1), connected to each other via the interchain disulﬁde bond between CL and CH1. ScFv fragments are stabilized by a peptide linker which connects the carboxyl terminus of VH or VL with the amino terminus of the other domain. The VH and VL heterodimer in dsFv is stabilized by engineering a disulﬁde bond between the two domains. The biochemical and biological properties described in the ﬁgure are depicted for B3-lysPE38 (LMB-1) (89) (a ﬁrstgeneration antibody–PE chemical conjugate), B3(Fv)-PE38 (LMB-7) (67) (second-generation recombinant scFv-immunotoxin for a scFv-immunotoxin), and B3(dsFv)-PE38 (LMB-9) (74) (for a second-generation recombinant dsFv-immunotoxin).

Second-generation immunotoxins have overcome many of these problems. Progress in the elucidation of the toxins’ structure and function, combined with the techniques of protein engineering, facilitated the design and construction of recombinant molecules with a higher speciﬁcity for cancer cells and reduced toxicity to normal cells. At the same time, advances in recombinant DNA technology and antibody engineering enabled the generation of small antibody fragments. Thus, it was possible to decrease the size of immunotoxins signiﬁcantly and to improve their tumor-penetration potential in vivo. The development of advanced methods of recombinant-protein

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Table I Examples of Recombinant Immunotoxins against Cancer Immunotoxin

Antigen

Toxin

Cancer

Clinical trail References

Anti CD7-dgA

CD7

Ricin Non-Hodgkin’s lymphoma

Phase I

156

DAB389-IL2

IL-2R

DT

T-cell lymphoma, Hodgkin’s disease

Phase III

122, 125

Anti-Tac (Fv)-PE38 (LMB-2)

CD25

PE

B and T lymphoma, leukemias

Phase I

66,106

DT-Anti-Tac(Fv)

CD25

DT

Leukemias, lymphoma

—

RFB4(dsFv)-PE38

CD22

PE

B leukemias

Phase I

76

Di-dgA-RFB4

CD22

Ricin Leukemias, non-Hodgkin’s lymphoma

—

157

B3-lysPE38

Lewis Y

PE

Carcinomas

Phase I

89

B3(Fv)-PE38 (LMB-7)

Lewis Y

PE

Carcinomas

Phase I

67

B3(dsFv)-PE38 (LMB-9)

Lewis Y

PE

Carcinoma

Phase I

74

BR96(sFv)-PE40

Lewis Y

PE

Carcinoma

—

95

e23(Fv)-PE38

erbB2/HER2

PE

Breast cancer

Phase I

68

FRP5(scFv)ETA

erbB2/HER2

PE

Breast cancer

—

96

78

(LMB-1)

Tf-CRM107

Transferrin-R

DT

Glioma

Phase I

103

HB21(Fv)-PE40

Transferrin-R

PE

Various

—

55

MR1(Fv)-PE38

Mutant EGF-R PE

Liver, brain tumors

—

99

SSI(Fv)-PE38

Mesothelin

Ovarian cancer

—

100

PE

production enabled the large-scale production of recombinant immunotoxins of high purity and quality for clinical use in sufﬁcient quantities to perform clinical trials. Another strategy to target cancer cells is to construct chimeric toxins in which the engineered truncated portion of the toxin (PE or DT) gene is fused to cDNA encoding growth factors or cytokines. These include transforming growth factor (TGF)-␣ (18), insuline-like growth factor (IGF)-1 (19), acidic and basic ﬁbroblast growth factor (FGF) (20), IL2 (21), IL4 (22), and IL6 (23). These recombinant toxins (oncotoxins) are designed to target speciﬁc tumor cells that overexpress these receptors. This review will summarize knowledge of the design and application of second-generation recombinant Fv immunotoxins, which utilize recombinant antibody fragments as the targeting moiety, in the treatment of cancer,

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and will also discuss brieﬂy the use of recombinant antibody fragments for other modes of cancer therapy and diagnosis.

II. DESIGN OF RECOMBINANT IMMUNOTOXINS A. The Toxin Moiety The toxins that are most commonly used to make immunotoxins are ricin, diphtheria toxin (DT), and Pseudomonas exotoxin (PE). The genes for these toxins have been cloned and expressed in Eschesichia coli, and the crystal structures of all three proteins have been solved (36, 41). This information, in combination with mutational studies, has elucidated which toxin subunits are involved in their biological activity and, most important, the different steps of the cytocidal process. DT, PE, and ricin, and their derivatives, have all been successfully used to prepare immunotoxin conjugates (3, 24), but only PE- and DT-containing fusion proteins generate active recombinant immunotoxins (1, 25). This is because the toxic moiety must be separated from the binding moiety after internalization (26, 27). PE and DT fusion proteins generate their free toxic moieties by proteolytic processing. Ricin does not possess such a proteolytic processing site, and therefore cannot be attached to the targeting moiety with a peptide bond without losing cytotoxic activity. Recently, proteolytic processing sites were introduced into ricin by recombinant DNA techniques to try to overcome this problem (28).

1. DIPHTHERIA TOXIN AND DT DERIVATIVES DT is a 58-kDa protein, secreted by pathogenic Corynebacterium diphtheria, which contain a lysogenic beta phage (29). DT ADP-ribosylates eukaryotic elongation factor 2 (EF2) at a “diphthamide” residue located at His 415, using NAD+ as a cofactor (30). This modiﬁcation arrests protein synthesis and subsequently leads to cell death (31). Only a few, and perhaps only one, DT molecule needs to reach the cytosol in order to kill a cell. When DT is isolated from the culture medium of C. diphtheria, it is composed of an N-terminal 21-kDa A subunit and a C-terminal 37-kDa B subunit held together by a disulﬁde bond. DT is the expression product of a single gene (29), which, when secreted into the medium, is processed into two fragments by extracellular proteases. When DT is produced as a recombinant singlechain protein in E. coli, it is not cleaved by the bacteria but is instead cleaved by a protease in the target cells (32). The A domain of DT contains its enzymatic activity. The N terminus of the B subunit of DT (or the region between A and B in single-chain DT)

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mediates translocation of the A subunit into the cytoplasm. The B domain, especially its C terminus, is responsible for the binding of DT to target cells. Deletions or mutations in this part of the molecule abolish or greatly diminish the binding and toxicity of DT (33–35). DT enters cells via coated pits and is proteolytically cleaved within the endocytic compartment if it is not already in the two-chain form, and reduced. It also undergoes a conformational change at the acidic pH present in endosomes, which probably assists translocation of the A chain into the cytosol perhaps via a porelike structure mediated by the B chain (36–38). Derivatives of DT that are used to make immunotoxins have the C terminus altered by mutations or partially deleted (DAB486 DAB389, DT388) but retain the translocation and ADP-ribosylation activity of DT (39). Recombinant antibody-fusion proteins with such derivatives target only cells that bind the antibody moiety of the immunotoxin.

2. PSEUDOMONAS EXOTOXIN (PE) AND PE DERIVATIVES Two major research studies have enabled the use and genetic manipulation of PE for the design of immunotoxins (1–3). The ﬁrst is the elucidation of the crystal structure of PE, showing the toxin to be composed of three major structural domains; the second is the ﬁnding that these domains are different functional modules of the toxin. PE is a single-chain 66-kDa molecule secreted by Pseudomonas aeruginosa that, like DT, irreversibly ADP-ribosylates the diphthamide residue of EF2, using NAD+ as cofactor (40). As a consequence, protein synthesis is inhibited and cell death ensues. PE is composed of three major domains (41). Different functions have been assigned to each domain by mutational analysis (42). The N-terminal domain 1a mediates binding to the a2 macroglobulin receptor (43). Domain lb is a small domain that lies between domain II and domain III and has no known function (44). Domain II mediates translocation of domain III, the carboxyl-terminal ADP-ribosylating domain, into the cytosol of target cells (45) (see Fig. 2). Translocation occurs after internalization of the toxin and after a variety of other steps, including a pH-induced conformational change (46–48), proteolytic cleavage at a speciﬁc site in domain II (27), and a reductive step that separates the amino and carboxyl fragments. Ultimately, the carboxyl-terminal portion of PE is translocated from the endoplasmic reticulum into the cytosol. Despite a similar mode of action of PE and DT, which is ADP-ribosylation, and a similar initial pathway of cell entry (internalization via coated pits and endocytic vesicles) and of processing (proteolytic cleavage and a reductive step), they share almost no sequence homology. The only similarity is the spatial arrangement of key residues in their active sites, which are arranged around residue Glu553 in PE and Glu145 in DT (49–52).

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Fig. 2 The biological activity of Pseudomonas exotoxin A. The Fv portion of the immunotoxin targets domains II and III of PE to a cell surface receptor or other target molecule on the tumor cell (A). The immunotoxin enters the cell by internalization and is transferred into the endosome (B). Within the endosome, the molecule unfolds due to a fall in pH. The conformational change exposes a proteolytic site, and a proteolytic cleavage occurs in the translocation domain between amino acid 279 and 280 (C). A disulﬁde bond is then broken, thus creating two fragments: the Fv moiety and a small part of domain II, and the rest of domain II connected to domain III (D). The carboxyl-terminal fragment containing the ADP-ribosylation domain (domain III) and most of the translocation domain (domain II) are carried into the endoplasmic reticulum (E), and translocation occurs from the endoplasmic reticulum into the cytosol (F). The enzymatically active domain ADP-ribosylates elongation factor 2 at a diphtamide residue located at His 415, using NAD+ as a cofactor. This modiﬁcation arrests protein synthesis and subsequently leads to cell death by apoptosis. In DT the poteolytic processing occurs between residues 193 and 194. The catalytic A-chain (amino acids 1–193) then translocates to the cytosol through the endosome with the help of translocation domain residues 326–347 and 358–376, which form an ion channel.

When the whole toxin is used to make an immunotoxin, nonspeciﬁc toxicity occurs mainly due to binding of the toxin portion to cells, mediated by the binding domain. Consequently, the goal of making improved derivatives of PE-based immunotoxins has been to inactivate or remove the binding domain. Molecules in which the binding domain has been retained but inactivated by mutations were made (53); however, a better alternative to

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inactivating the cell-binding domain by mutations is to remove it from PE. The prototype molecule with this sort of deletion is PE40 (aa 253–613, MW 40 kDa). Because PE40 and its derivatives described below lack the binding domain (aa 1–252), they have very low nonspeciﬁc toxicity, but make very active and speciﬁc immunotoxins when fused to recombinant antibodies (54, 55). Currently, almost all PE-derived recombinant immunotoxins are constructed with PE38 (MW 38 kDa), a PE40 derivative that has, in addition to the deletion of domain 1a, a second deletion encompassing a portion of domain Ib (aa 365–379) (44). Another useful mutation is to change the carboxyl-terminal sequence of PE from REDLK to KDEL. This improves the cytotoxicity of PE and its derivatives, presumably by increasing their delivery to the endoplasmic reticulum, where translocation takes place (56, 57).

B. The Targeting Moiety—Recombinant Antibody Fragments The antibody moiety of the recombinant immunotoxin is responsible for speciﬁcally directing the immunotoxin to the target cell, meaning that the usefulness of the immunotoxin depends on the speciﬁcity of the antibody or antibody fragment that is connected to the toxin. Consequently, for the construction of recombinant immunotoxins, the only antibodies that should be used are those that recognize antigens that are expressed on target cancer cells and are not present on normal cells, present at very low levels, or are only present on less essential cells (Table I). Receptors for growth factors such as the epidermal growth factor (EGF), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6(IL-6), or erbB2 are common targets for targeted cancer therapy because they are highly expressed on many cancer cells. Other carcinoma-related antigens include developmental antigens such as complex carbohydrates, which are often highly abundant on the surface of cancer cells. The use of antibodies for immunotoxin production also requires that the antibody–antigen complex be internalized, because the mechanism of PE-toxin killing requires endocytosis as a ﬁrst step in the entry of the toxin into the cell. Recombinant immunotoxins contain antibody fragments as the targeting moiety. These fragments can be produced in E. coli and are the result of intensive research and development in recombinant-antibody technologies (58–60). Several antibody fragments have been used to construct recombinant immunotoxins (Fig. 1). One type contains Fab fragments in which the light-chain and the heavy-chain Fd fragments (VH and CH1) are connected to each other via an interchain disulﬁde bond between CL and CH1. The toxin moiety can be fused to the carboxyl end of either CL or CH1.

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Fabs can be produced in E. coli either by secretion, with coexpression of light chains and Fd fragments, or by expression of the chains in intracellular inclusion bodies in separate cultures; in the latter case, they are reconstituted by a refolding reaction using a redox-shufﬂing buffer system. Several immunotoxins with Fab fragments have been constructed and produced in this way (1–4, 61). The smallest functional modules of antibodies required for antigen binding are Fv fragments. This makes them especially useful for clinical applications, not only for generating recombinant immunotoxins but also for tumor imaging, because their small size improves tumor penetration. Fv fragments are heterodimers of the variable heavy-chain (VH) and the variable light-chain (VL) domains. Unlike whole IgG or Fab, in which the heterodimers are held together and stabilized by interchain disulﬁde bonds, the VH and VL of Fvs are not covalently connected and are consequently unstable; this instability can be overcome by making recombinant Fvs that have the VH and VL ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Fig. 3 Cloning, construction, and composition of scFv- and dsFv-immunotoxins. (A) cloning and construction of recombinant scFv and dsFv immunotoxins. The genes encoding the VH and VL variable domains are cloned usually from hybridoma mRNA by reverse transcription, cDNA synthesis, and subsequent PCR ampliﬁcation using degenerate primers that are complementary to the 5 or 3 end of the VH and VL genes or by primers which are designed according to the amino-terminal amino acid sequence of the MAb to be cloned and conserved sequences at the N terminal of the heavy and light constant regions. The variable genes can be also cloned by constant-domain primers and using the RACE method (rapid ampliﬁcation of cDNA ends). Restriction sites for assembling of the peptide linker sequence which connects the VH and VL domains, and for cloning into the expression vector, are also introduced by PCR. Construction of dsFv involves the generation of two expression plasmids which encode the two components of the dsFv VH-cys and VL-cys. The cysteines are introduced in position 44 in FR2 of VH and position 100 of FR4 of VL or position 105 of FR4 in VH and position 43 of FR2 in VL (numbering system of Kabat et al.) by site-directed mutagenesis using as template a uracil-containing single-stranded DNA of the scFv construct from the F+ origin present in the expression plasmid and cotransfection with M13 helper phage. In addition to the cysteines, cloning sites, ATG translation-initiation codons, and stop codons are introduced at the 5 end and 5 end of the VH and VL genes, as shown by site-directed mutagenesis or PCR. The antibody variable genes are subcloned into an expression vector which contains the gene for a truncated form of Pseudomonas exotoxin. This expression vector is controlled by the T7 promoter and upon induction of the T7 RNA polymerase, which is under the control of the lacUV5 promoter, in E. coli BL21 ␭DE3 by IPTG, large amounts of recombinant protein are produced. (B) composition of recombinant immunotoxins. In PE-derived recombinant Fv immunotoxins the Fv region of the targeting antibody is fused to the N terminus of a truncated form of PE which contains the translocation domain (domain II) and enzymatically active ADP-ribosylation domain (domain III). The cell-binding domain of whole PE (domain I) is replaced by the Fv targeting moiety, thus preserving the relative position of the binding-domain function to the other functional domains of PE. In the dsFv immunotoxins there are two components. In one the VH or VL domains are fused to the amino terminus of the truncated PE, and the other variable domain is covalently linked by the engineered disulﬁde bond. DT-derived immunotoxins are fused to the carboxyl terminus due to the inverse arrangement of the functional modules of PE and DT.

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covalently connected by a peptide linker that fuses the C terminus of the VL or VH to the N terminus of the other domain (Fig. 1). These molecules are termed single-chain Fvs (scFvs) (62, 63), and many retain the speciﬁcity and afﬁnity of the original antibody. The cloning, construction, and composition of recombinant Fv fragments of antibodies and of Fv-immunotoxins are described in Fig. 3. Many recombinant immunotoxins have been constructed using scFvs, in which molecules the scFv gene is fused to PE38 to generate a potent cytotoxic agent with targeted speciﬁcity (1–4, 64–70) (Figs. 1 and 3). Until recently, the construction of scFvs was the only general method available to make stable Fvs. However, many scFvs are unstable or have reduced afﬁnity for the antigen compared with the parent antibody or Fab fragment. This is because the linker interferes with binding or because the linker does not sufﬁciently stabilize the Fv structure, leading to aggregation and loss of activity. This is particularly true at physiological temperatures (37◦ C). To overcome these problems, an alternative strategy has been developed that involves generating stable Fvs by connecting the VH and VL domains by an interchain disulﬁde bond engineered between structually conserved framework residues of the Fv; these molecules are termed disulﬁde-stabilized Fvs (de Fvs) (60, 71–73). The positions at which the cysteine residues were to be placed were identiﬁed by computer-based molecular modeling; as they are located in the framework of each VH and VL, this location can be used as a general method to stabilize almost all Fvs, without the need for any structural information. Many dsFvs have been constructed in recent years (mainly as dsFv immunotoxins, in which the dsFv is fused to PE38), and they show several advantages over scFvs (60, 74–76). In addition to their increased stability (owing to a decreased tendency to aggregate), they are often produced in higher yields than scFvs; in several cases, the binding afﬁnity of the dsFv was signiﬁcantly improved over that of the scFv.

III. CONSTRUCTION AND PRODUCTION OF RECOMBINANT IMMUNOTOXINS In the recombinant immunotoxins derived from PE, the recombinant antibody fragments are fused to the amino terminus of the truncated derivative of PE (with the cell-binding domain deleted), e.g., PE40 or PE38. This restores the original domain arrangement of PE, which consists of an N-terminal binding domain followed by the translocation domain and the C-terminal ADP-ribosylation domain (Fig. 1b). Only fusions of an antigenbinding domain (Fv) to the amino terminus of truncated PE are active; carboxyl-terminal fusions are not active because the bulky antigen-binding domain blocks translocation of the C-terminal fragment into the cytoplasm (1, 25).

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DT immunotoxins are fusions of mutated DT with antigen-binding regions of a recombinant antibody. However, in this case the antigen-binding domain must be fused to the C terminus of DT (77, 78). This corresponds to the inverse arrangement of the functional modules of PE and DT (see Fig. 3). DT immunotoxins are active only when the enzymatically active N-terminal domain is free to translocate into the cytosol. The expression vectors used for DT immunotoxins are very similar to those used with PE, with the exception that the DNA fragments encoding the binding moiety are ligated to the 3 -end of the DT coding region. The cloning of the antibody variable regions is performed using cloning techniques that are now well established (59) (Fig. 3). The plasmid vector for the expression of scFv immunotoxins or the components of dsFv immunotoxins is a high-copy-number plasmid derived from vectors made and described by Studier and Moffatt (79). These contain the T7 promoter, translation-initiation signals, and a transcription terminator, as well as an F+ phage-replication origin to generate single-stranded DNA to be used for site-directed mutagenesis. When these plasmids are transformed into E. coli BL21/DE3 (which contain the T7 RNA-polymerase gene under the control of the lac UV5 promoter), they generate large amounts of recombinant protein upon IPTG induction. The recombinant scFv immunotoxin or the components of the dsFv immunotoxin accumulate in insoluble intracellular inclusion bodies. [dsFv immunotoxins require two cultures, one expressing the VH and one expressing the VL; the toxin moiety (PE38) can be fused to either the VH or the VL.] The inclusion bodies are then isolated, puriﬁed, solubilized, reduced, and subsequently used in a refolding reaction that is controlled for oxidation (redox shufﬂing). In the case of dsFv immunotoxins, solubilized inclusion bodies of VH and VL (with the toxin fused to either) are mixed in 1:1 molar ratio into the refolding solution. The formation of the interchain disulﬁde bond between the VH and VL domains is promoted by inducing oxidation using excess oxidized glutathione or by refolding at high pH. The immunotoxins are then puriﬁed from the refolding mixtures by ion-exchange and size-exclusion chromatography. Approximately 20 mg of clinical-grade active immunotoxin can be obtained from 1 liter of a fermentor culture induced with IPTG.

IV. PRECLINICAL DEVELOPMENT OF RECOMBINANT IMMUNOTOXINS A wide variety of recombinant immunotoxins have been made and tested against cancer target cells. If found active and are considered to be tested in clinical trail, they undergo several years of preclinical development to

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Table II Functional Properties in Vitro and in Vivo of PE-Based Recombinant Fv Immunotoxins

Immunotoxin

Speciﬁcity

Activity in vitro Binding afﬁnity (Kd, nM) (IC50, ng/ml)

Antitumor activity in vivo (xenograft model)

Anti-Tac(Fv)-PE38 CD25 (LMB-2)

0.15

1.4

Complete regressions/ cures (ATAC4)

B3(Fv)-PE38 (LMB-7)

Lewis Y

1.5

1,300

Complete regressions/ cures (A431)

B3(dsFv)-PE38 (LMB-9)

Lewis Y

1.5

24,000

Complete regressions/ cures (A431)

e23(Fv)-PE38 (erb-38)

erbB2/HER2

0.3

RFB4(dsFv)-PE38 (BL22)

CD22

SSI(Fv)-PE38

Mesothelin

MRI(Fv)-PE38 55.1(Fv)-PE38

40

Partial regressions (A431)

10

Partial and some complete regressions (CA46)

0.5

11

Complete regressions/ cures (A431-K5)

Mutant EGF-R

3.0

11

Partial regressions (glioblastoma)

Mucin carbohydrate

0.3

80

Complete regressions (Colo205)

10

determine their efﬁcacy and toxicity in several in vitro and in vivo experimental models (Table II). The initial phase is the characterization of the biological activity of the immunotoxin on cultured tumor cells. These assays include measurement of cell-free enzymatic activity, namely, ADP-ribosylation activity in the case of bacterial toxins; and the binding afﬁnity of the immunotoxin to the target antigen, which can be determined on puriﬁed antigen, on cells by bindingdisplacement assays, or by surface plasmon-resonance assays). Cytotoxicity assays are performed on antigen-bearing cells and measure either inhibition of protein synthesis, proliferation, colony counts, or cell viability. Cytotoxicity assays on malignant, single-cell suspensions obtained directly from patients are a very useful test, if available, since such cells contain the physiological number of receptors or target density, which in many cases is lower than established cell lines (80–82). The stability of recombinant immunotoxins in vitro in various physiologic buffers or human serum is also an important test to predict their stability in vivo (60). In vivo efﬁcacy of recombinant immunotoxins is usually demonstrated in immunodeﬁcient mice bearing xenografts of human tumor cells. The tumor xenografts can be established as subcutaneous solid tumors, orthotopic implants, or disseminated leukemia (83–85).

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Initial toxicity and pharmacokinetics studies are also performed in mice; however, many target antigens are present at some level on some normal tissues and thus toxicology and pharmacokinetics studies should be tested in an animal that has normal cells capable of binding the target antigen. For most immunotoxins, this requires studies in monkeys to test for targeted damage to normal tissues to predict whether such damage will occur in humans (86, 87).

V. APPLICATION OF RECOMBINANT IMMUNOTOXINS A. Recombinant Immunotoxins against Solid Tumors The treatment of solid tumors with immunotoxins is challenging due to their physiological nature of tight junctions between tumor cells, high interstitial pressure within tumors, and heterogeneous blood supply and also antigen expression (88). The greatest need for new therapies is in the treatment of metastatic epithelial cancers, and immunotoxins can be a useful addition to the standard procedures of surgery, radiation, and chemotherapy. As already described, the use of recombinant fragments of antibodies for making recombinant immunotoxins is especially useful for the treatment of solid tumors, because their small size improves tumor penetration. In recent years, several recombinant immunotoxins that target solid tumors have been developed (Table II); targets include breast, lung, gastric, bladder, and central nervous system cancers. They are at different stages of clinical development, and some are already employed in clinical trails (1–4). Monoclonal antibody (MAb) B3 is an antibody that reacts with the Lewis Y (Le Y) antigen present on cancers of the colon, breast, stomach, lung, and bladder (15). Early trials with a ﬁrst-generation immunotoxin (LMB-1) in which an antibody to LeY (MAb B3) was used to make a chemical conjugate with PE38 showed signiﬁcant clinical activity, with responses in colon and breast cancer (89, 90). The 1 CR and 1 PR observed in this trail were the ﬁrst major responses to immunotoxins documented for metastatic breast and colon cancer, respectively. The B3 antibody was then used to make a single-chain immunotoxin termed B3(Fv)-PE38 or LMB-7 (67). LMB-7 has shown good activity against human-cancer xenografts growing in mice (91), and it is also able to cure carcinomatous meningitis in rats when given by the intrathecal route (92). A Phase I clinical trial with LMB-7 began in 1995 and is nearing completion. During the trial, it became evident that LMB-7 lost activity when incubated at 37◦ C because of aggregation (72, 93), which greatly limited its ability to penetrate solid tumors.

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B3(dsFv)-PE38 (LMB-9) is the dsFv version of LMB-7 (93), with improved stability over that of LMB-7. This improved stability also allowed it to be used in a continuous-infusion mode in mice bearing human-tumor xenografts; this route of administration showed an improved therapeutic window over a bolus injection (94). Clinical trials with LMB-9 started in the middle of 1998. A different recombinant single-chain immunotoxin, BR96(sFv)-PE40, was derived from the anti-Lewis Y monoclonal antibody BR96 and is also currently undergoing clinical testing (95). MAb e23 is directed at erbB2 (Her2/neu), which is highly expressed in many breast, lung, ovarian, and stomach cancers. e23(dsFv)-PE38 is a dsFv immunotoxin composed of the Fv portion of the e23 antibody and PE38 (68). This dsFv immunotoxin has a signiﬁcantly improved binding afﬁnity and stability compared with its scFv analog, e23(Fv)-PE38 (75). FRP5scFvETA is also a recombinant immunotoxin targeting erbB2 (96). Clinical trials with e23(Fv)-PE38 were initiated in early 1998. An example of the antitumor activity of e23(dsFv) immunotoxin and a comparison with its scFv analog is given in Fig. 4. In a Phase I study on breast cancer patients, hepatotoxicity was observed in all patients. Immunohistochemistry showed the presence of erbB2 on hepatocytes, explaining the liver toxicity of the immunotoxin. This study demonstrated that targeting of tumors with antibodies to erbB2 armed with toxic agents or radioisotopes may result in unexpected organ toxicity due to the expression of the target antigen on normal cells (97). Other recombinant immunotoxins that have been constructed and have antitumor activities in vitro and in mouse models in vivo include Bl (Fv)PE38, also directed against the Le Y antigen (98); 55.1(Fv)-PE38 and 55.1 (dsFv)-PE38, which are directed at a carbohydrate mucin antigen overexpressed in colon cancers (70); MR1 (Fv)-PE38, constructed by antibody phage display technology and directed to a mutant EGF receptor overexpressed in liver and brain tumors (99); and SS(Fv)-PE38, a new recombinant immunotoxin speciﬁc for mesothelin, a differentiation antigen present on the surface of ovarian cancers, mesotheliomas, and several other types of human cancers (100). SS(Fv)-PE38 was constructed from an Fv fragment that was isolated by antibody phage display from mice that underwent DNA immunization with a plasmid expressing the cloned antigen (100). This approach to antibody formation eliminates the need for the production of proteins for immunization. Immunotoxins were also used to target tumors of the central nervous system. Since the transferrin receptor is expressed on tumor and normal hepatic cells but not in normal brain, several trails have targeted anti-transferrin receptor immunotoxins to brain tumors. These include a conjugate of

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Fig. 4 Antitumor activity of anti-erbB2 immunotoxins. (a) In vitro activity on cultured tumorcell lines of e23(Fv)-PE38K and e23(dsFv)-PE38K. They contain a mutant peptide KDEL sequence at their carboxyl termini that increases their cytotoxic activity. Cytotoxic assays were performed by measuring incorporation of tritiated leucine into cell proteins. IC50 values are the concentration of immunotoxin that causes 50% inhibition of protein synthesis after a 24-h incubation with immunotoxin. (b) Antitumor activity of anti-erbB2 immunotoxins in a mouse model of human A431 tumor xenograft. Mice were injected on day 0 with 3 × 107 A431 cells. By day 4 the tumor size was approximately 50 mm3 , and animals were treated three times (days 4, 6, and 8) with two versions of the anti-erbB2 immunotoxins. One is the single-chain Fv immunotoxin [e23(Fv)-PE38KDEL] and the second is the disulﬁde-stabilized Fv immunotoxin [e23(dsFv)-PE38KDEL]. Note that mice treated with the dsFv immunotoxin had complete regressions and even cures of their tumors, while the scFv immunotoxin gave only a partial response.

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monoclonal antibody 454A12 with a recombinant form of ricin A (plant toxin) (101), a conjugate of human transferrin with a mutant form of DT (102, 103), and chimeric toxin of recombinant IL4-PE38 fusion (104, 105).

B. Recombinant Immunotoxins against Leukemias and Lymphomas Conventional immunotoxins, in which IgGs or Fabs are coupled to toxins, have also been used to target leukemias and lymphomas. This approach should be quite effective because many of the tumor cells are in the blood and bone marrow, where they are readily accessible to the drug. Moreover, fresh cells from patients may be easily tested for immunotoxin binding and cytotoxic activity. Immunotoxins have also been developed for indirect treatment of malignancies by their killing of T cells that mediate graft-versus-host disease (GVHD) in the setting of allogeneic transplantation. Clinical trials using ricin-based immunoconjugates for treatment of leukemias have shown some promising results, but dose escalation has been limited by the side effects of the toxin (2). In addition, it is important to eliminate not only easily accessible tumor cells but also malignant cells that are less accessible. Therefore, even for leukemias, there is a need to develop small recombinant immunotoxins that will reach cells outside the circulation. Recombinant immunotoxins targeted at leukemia and lymphoma antigens have been made with antibody fragments speciﬁc for the subunit of the IL-2 receptor (CD25) and for CD22. In addition, growth-factor fusion proteins have been made that target the IL-2, IL-4, IL-6, and granulocyte-macrophage colony-stimulating factor (GMCSF) receptors. The most potent immunotoxin produced against leukemia cells is antiTac(Fv)-PE38 (LMB-2); this targets CD25, which is overexpressed on many T-cell leukemias (66, 106). LMB-2 is very active against leukemia cell lines in vitro and has very good activity in animal models (107). It also selectively kills cells obtained from patients with adult T-cell leukemia (ATL) in vitro, without harming hematopoietic stem cells (81, 82). Phase I clinical trails with LMB-2 are showing promising results (108, 109). The immunotoxin was administered to 35 patients for a total of 59 treatment cycles. One hairy-cell leukemia (HCL) patient achieved a complete remission, which was ongoing at 20 months. Seven partial responses were observed in cutaneous T-cell lymphoma, HCL, chronic lymphocytic leukemia, Hodgkin’s disease, and adult T-cell leukemia. Responding patients had 2 to 5 log reduction of circulating malignant cells, improvement in skin lesions, and regression of lymphomatous mass and splenomegally. All four patients with HCL responded to the treatment (one

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with CR and three with 98 to 99.8% reductions in malignant circulating cells). A Phase II trail is planned in patients with CD25+ hematologic malignancies, and Phase I trails are planned for the prevention of GVHD in patients undergoing high-risk allotransplantation (110). The conventional immunotoxin RFT5-SMPT-dgA has also been developed to target CD25 and has resulted in several responses in Hodgkin’s disease, one of which lasted over 2 years (111, 112). It is already undergoing testing for the prevention of GVHD in patients undergoing allotransplantation and has recently been shown ex vivo to remove all reactive donor T cells while preserving antileukemia and antiviral T-cell responses (113). A new agent, RFB4(dsFv)-PE38 (BL22), is a new dsFv immunotoxin directed at the CD22 differentiation antigen present on most B-cell leukemias (76). It has high cytotoxic activity on cultured tumor cells as well as in animal models, and preclinical tests have been completed. This recombinant immunotoxin recently entered clinical trials in patients with leukemias (114). Initial Phase I trails in 7 HCL patients resulted in 2 CRs (4 + and 2 + months) and 3 PRs (two 3 + months and one 2 + months) including 2 PRs in patients ineligible for LMB-2 because of CD25-negative HCL cells. Responses to BL22 were associated with at least 99.5% reduction in circulating HCL cells (114). BL22 also induced responses in chronic lymphocytic leukemia. These recent results demonstrate that recombinant Fv immunotoxins containing truncated Pseudomonas exotoxin are particularly effective in patients with chemotherapy-refractory HCL and other hematological malignancies. Other targets for the development of B-cell-leukemia-speciﬁc recombinant immunotoxins include the CD19 and CD20 differentiation antigens in B-cell tumors and CD30 in Hodgkin’s lymphoma. The B-cell lymphoma markers CD22 and CD19 were also targeted using conventional ﬁrst-generation immunotoxins of deglycosylated ricin A chain, IgG-RFB4-dgA (targeting CD22), and IgG-HD37-dgA (targeting CD19) (115–121). Leukemias and lymphomas were also targeted with recombinant fusions of IL-2 with truncated DT (122–125).

VI. OTHER APPLICATIONS OF RECOMBINANT ANTIBODY FRAGMENTS The new wave of antibody fragments which are about to enter the clinic for cancer therapy and diagnosis are the result of enormous progress made in recent years in the ﬁeld of antibody engineering technology. It is now possible to select high-afﬁnity antibody fragments directly from phage display libraries rather than from a live mouse (126). It is possible now to isolate

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antibodies against highly conserved refractory antigens, thereby avoiding the limitations inherent in the mammalian immune response in the conventional hybridoma technology. Recombinant antibodies can be reduced in size into their minimal binding sites (usually Fvs), rebuilt into multivalent, high-avidity reagents by making them bi-, tri-, or even tetravalent (127). Antibody fragments can be fused not only to toxins, as described for recombinant immunotoxins, but also been to a wide range of molecules including enzymes for prodrug therapy, viruses for gene therapy, cationic tails for DNA delivery, and liposomes for improved drug therapy. Not only fragments but also whole recombinant antibodies are entering the drug market for effective cancer treatment. Recombinant antibodies that have recently been approved by the Food and Drug include Administration Rituxan (IDEC Pharmaceuticals, San Diego, CA) and Bexxar (Beckman Coulter, Fullerton, CA), both targeting CD20 for therapy of non-Hodgkin’s lymphoma (128), and Herceptin (Genentech, San Francisco, CA), targeting the HER2 antigen in breast cancer (129). We will review here very brieﬂy two additional major application of recombinant antibody fragments: using radioactive recombinant antibodies for cancer imaging and therapy and for making fusion proteins for prodrug therapy.

A. Radioimaging and Radioimmunotherapy Radioactive recombinant antibodies are becoming widely used for cancer imaging and therapy. This technique relies on a positive ratio between the amount of radioactivity in the target tumor and the amount in adjacent normal tissues and circulating blood. For therapy with radiolabeled antibodies, it is necessary to achieve a high radiation dose to tumor sites and yet maintain acceptable toxicity levels in normal tissues, especially in bone marrow, to prevent myelotoxicity (130–132). As described for the ﬁrst generation of immunotoxins (conjugates made with whole antibodies), various physical characteristics limit antibody targeting to tumor cells. Because of variations in tumor vasculature, antibodies may only reach well-perfused areas of the tumor, and a high interstitial back pressure within the tumor can oppose the inﬂux of the antibody. Because of the large size of antibodies, diffusion within sites of bulky disease is slow. Among the most promising strategies to overcome these physical barriers is the use of engineered low-molecular-weight antibody fragments such as recombinant Fabs or scFvs, which may have higher transport rates. Studies have been performed to investigate the biodistribution of antibody fragments in tumors and compare their distribution with the use of a whole

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antibody. For example, the biodistribution of an IgG and a Fab-fragment mixture of 99mTc-labeled anti-carcinoembryonic-antigen (anti-CEA) antibodies were compared in colorectal-cancer patients. It was found that the fragment cleared more rapidly from normal tissues and provided an earlier, more accurate diagnosis than whole IgG (133). Recently, an imaging and Phase I trial was performed to study the ability of11 In-mAb B3 targeting the Lewis Y carbohydrate antigen to image known metastasis and determine the maximum tolerated dose, dose-limiting toxicity, kinetics, and biodistribution (134). Because of their smaller size, Fvs show improved kinetics of localization, much faster than for intact IgGs, and their distribution in tumors is more uniform. This allows the use of short-lived single-photon emitters such as99mTc or 123I. In addition, small fragments are less immunogenic than intact IgG. The use of stabilized forms of Fv fragment, such as dsFvs, may contribute to improved biodistribution and tumor localization. A dsFv fragment of the anti-Tac antibody was constructed and radiolabeled with 18F and 125I. As described above, this antibody is directed to the subunit of the IL-2 receptor, which is overexpressed by many leukemias. The afﬁnity of this dsFv fragment is very similar to that of humanized anti-Tac IgG, and it is substantially more stable than the scFv fragment. Biodistribution studies showed rapid and speciﬁc uptake of anti-Tac(dsFv) by IL-2-receptor-bearing tumors. Results in this case also show improved tumor penetration of small Fv fragments compared with the whole antibody (135, 136). Using these new constructs, radioimmunodetection has the potential for detecting small tumors that cannot be detected by other means and for distinguishing between abnormal radiographic images that are benign from those that are malignant and contain a tumor-associated antigen. For radioimmunotherapy, there is a need for more studies to be performed with recombinant fragments. There are promising and signiﬁcant clinical responses using whole radiolabeled antibodies, mainly in the radioimmunotherapy of hematologic malignancies (132, 133). The use of antibody fragments for radioimmunotherapy may be especially suited to the elimination of minimal residual disease, as their delivery to tumor sites is more easily accomplished after tumor reduction. The use of radiolabeled antibodies with emitting radionuclides is particularly attractive for minimal disease because they may allow more speciﬁc tumor destruction with less toxicity to normal cells. Despite the advantages of recombinant antibody fragments such as scFvs and dsFvs for immunotargeting and immunodetection (mainly as a result of improved tumor penetration), there are some important factors that must be addressed. Faster serum clearances, decreased binding avidities, and, for some scFvs, decreased molecular stability will have effects on their ultimate efﬁcacy as agents for therapeutic and diagnostic use.

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B. Prodrug Therapy with Fv-Enzyme Fusion Proteins The concept of selective targeted delivery of therapeutic agents to tumor sites has been extended by the introduction of the antibody-directed enzyme prodrug therapy (ADEPT) technique (137, 138). This mode of therapy has several stages: ﬁrst, an antibody or antibody fragment that is conjugated or fused to an enzyme that is unique in the human extracellular environment is administered and allowed to localize to the tumor target; once localized to the tumor, the antibody–enzyme complex can locally activate a subsequently administered “inactive” prodrug at the tumor site; ﬁnally, the prodrug is converted to an active drug that is capable of penetrating into adjacent tumor cells and causing their death. This method enables a small, highly toxic drug to accumulate within the tumor at concentrations that are signiﬁcantly higher than in other tissues. The enzymes, which should have a high substrate turnover, are capable of generating many drug molecules for each antibody–enzyme conjugate bound at the tumor site, amplifying their effect. A possible additional step is the application of a clearing reagent that will remove circulating antibody–enzyme conjugates, thereby minimizing prodrug activation at sites away from the tumor site. The antibodies that have been used in ADEPT experiments are those commonly used in the targeted-therapy ﬁeld, such as antibodies against CEA, growth-factor receptors (e.g., EGF, erbB2), and some other antigens on carcinoma and melanoma cells. The repertoire of target tumor-associated antigens that can be used for ADEPT therapy is larger and more diverse than the antigen targets available for immunotoxin therapy or for direct targeting of drugs to cancer cells: the latter agents have to be internalized into the tumor target cells after binding of the targeting antibody and, owing to the absence of bystander effects, the antigen must be expressed on the surface of each target cell; on the other hand, for ADEPT therapy, a noninternalizing antibody is an advantage, and the antigen used as a target for ADEPT need not be expressed on every target cell because of the bystander effect. In such cases, the antibody–enzyme conjugates that are bound to antigen-positive cells convert the prodrug to the active drug, which accumulates at a high local concentration and can also kill antigen-negative cells that are close to the antigen-positive cells. Most ADEPT systems described to date use enzymes conjugated by chemical means to whole gig molecules or FAA fragments that are generated by proteolytic cleavage, similar to ﬁrst-generation immunotoxins; the limitations of these molecules are described above. Recent research has been directed toward the use of recombinant antibody fragments for ADEPT. Examples include the construction of a fully humanized recombinant

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fusion protein consisting of the FAA fragment of the high-afﬁnity anti-CEA antibody BW431 fused to human glucoronidase gene (139) and Fab fragments of monoclonal antibody L6 fused to lactamase. The latter was shown to be able to eliminate tumor cells bearing the L6 antigen (140). Wels et al. (96) constructed an scFv speciﬁc to the extracellular domain of erbB2 that was fused to alkaline phosphatase. Recently, a dsFv-lactamase fusion protein for use in ADEPT was constructed to be used with a cephalosporin-based prodrug. The dsFv was derived from the humanized anti-erbB2 antibody humAb4D5-8 (141). The dsFv-lactamase fusion protein, which was targeted to tumor cells overexpressing erbB2 (p185HER2), efﬁciently enhanced the killing of antigenpositive target cells by converting a cephalosporin–doxorubicin prodrug into doxorubicin (141). The ﬁrst pilot-scale clinical trail of ADEPT was carried out using an antiCEA F(ab )2 antibody conjugated to the bacterial enzyme carboxypeptidase G2 (142). ADEPT is a valuable addition to other immunotherapeutic approaches that are currently being evaluated, but more studies are required to advance this concept. Antibody technology and engineering will have a key role in this process.

VII. CHALLENGES AND FUTURE DIRECTIONS OF RECOMBINANT IMMUNOTOXINS Although some of the problems, including design, large-scale production, and stability, associated with the initial recombinant immunotoxins have been solved, other fundamental problems need to be addressed that are relevant to much of the immunotherapy ﬁeld. Speciﬁcity, toxicity, and immunogenicity are major factors that will determine the usefulness and success of recombinant immunotoxins.

A. Immune Responses and Dose-Limiting Toxicity As with any cytotoxic agent, side effects such as nonspeciﬁc toxicity and immunogenicity) can occur when large amounts of immunotoxins are given. One class of side effects is due to inappropriate targeting of the immunotoxin to normal cells because of the poor speciﬁcity of the antibody. In addition, the toxin or the Fv portion of the antibody can bind nonspeciﬁcally to various tissues. For example, in mice, which usually do not contain target antigens, liver damage occurs when large amounts of immunotoxins are given (89).

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Molecular modeling combined with site-directed mutagenesis may help in the design of new versions of the toxin with decreased toxicity caused by nonspeciﬁc binding. The development of neutralizing antibodies usually occurs after 10 days and limits the therapeutic application of immunotoxins to this 10-day period (4). Recent data from clinical trails indicate that patients with solid tumors develop antibodies much more readily than those with hematologic tumors. It is speculated that some hematologic tumors may be associated with less immunogenicity than others. For example, none of 14 patients with chronic lymphocytic leukemia treated with LMB2 or BL22 have shown any evidence of antibodies (108, 109, 114). Several approaches have been taken to reduce immunogenicity. One is to make small molecules, which appear to be less immunogenic; another is to use immunosuppressive agents such as deoxyspergualin (143) or CTLA4 Ig (144), an inhibitor of the costimulation pathways required for T-cell help and activation through the CD28/CTLA4–CD80/CD86 complex. Another approach is to use the anti-CD20 monoclonal antibody Rituximab, which induces B-cell depletion in the majority of patients and is itself nonimmunogenic (145). The dose-limiting toxicity of many immunotoxins is vascular leak syndrome (VLS). Recent studies indicate that recombinant toxins, including those containing mutated forms of PE, produce VLS in rats and that inﬂammation, which can be suppressed by steroids or nonsteroidal anti-inﬂammatory agents, mediates the VLS. The VLS can also be mediated indirectly by the activation of endothelial cells and/or macrophages via cytokines such as TNF-␣ and IFN-␥ . The activated cells produce nitric oxide, which then can mediate oxidative damage to the endothelial cells and result in increased permeability (146). ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Fig. 5 A recombinant immunotoxin targeting MHC/peptide complex. Phage display technology was used to isolate an antibody that has T-cell receptor-like speciﬁcity. It recognizes mouse MHC class I H-2Kk molecules complexed with a H-2Kk -restricted inﬂuenza virus-derived hemagglutinin peptide (Ha255-262) but does not bind to class I H-2Kk alone, peptide alone, or H-2Kk complexed with other peptides. A recombinant immunotoxin with this antibody was constructed by fusion with truncated PE38. (A) The recombinant immunotoxin speciﬁcally kills antigen-presenting cells in a peptide-dependent manner and with T-cell receptor-like speciﬁcity. Killing is determined as described for Fig. 3. (B and C) The recombinant immunotoxin is killing speciﬁcally inﬂuenza virus-infected target cells in a peptide-speciﬁc manner. The inﬂuenza virus strain A2/Japan/305/57 presents the hemagglutinin peptide 255-262 FESTGNLI, while the stain PR8 makes after processing the hemagglutinin peptide 255-262 FEANGNLI (two mutations in position 3 and 4 compared to the A2/Japan strain). The immunotoxin was cytotoxic to cells infected with the A2/Japan stain but not with PR8, which presents a different HA peptide in complex with H-2Kk that is not recognized by the T-cell receptor-like antibody.

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Some studies demonstrate direct endothelial cell damage caused by binding the toxin to the cells. The direct damage to the cell is mediated by the enzymatic activity of the toxin (147, 148), while others show indirect damage that is mediated by binding of the targeting moiety. For example, experiments with human umbilical-vein endothelial cells exposed to LMB 1 (antibody conjugate with truncated PE38) indicated that the monoclonal antibody B3 rather than PE38 was binding to the Lewis Y antigen on endothelial cells (149). Recent experiments using an in vivo model composed of human neonatal foreskin xenografts in SCID-immunodeﬁcient mice identiﬁed a 3-amino acid motif present in protein toxins and in IL-2 that causes VLS without other toxin activity (150–152). Thus, VLS can be blocked in future trails with antiinﬂammatory agents to block cytokine action or by mutations or peptide inhibitors that will prevent the binding of the toxin or the targeting moiety to endothelial cells.

B. Speciﬁcity Speciﬁcity of the recombinant immunotoxin is determined by the distribution of the target antigens; several target antigens are relatively cancer speciﬁc but are present on some normal cells in small amounts. For example, erbB2, although overexpressed on tumor cells, is also expressed in a limited number of normal cells. This reactivity with normal cells may cause side effects during immunotoxin therapy. It was discovered during a clinical trial that small amounts of the LeY antigen are expressed on the surface of endothelial cells and that damage to these cells caused vascular-leak syndrome. To overcome such problems, new speciﬁc targets and new reagents against the cancer antigens that will recognize only the tumor-associated molecules must be identiﬁed and developed. The construction of large phage-displayedantibody libraries may result in the isolation and characterization of new reagents with improved speciﬁcity and afﬁnity for cancer-targeted therapy. The phage-display approach has been used to isolate a scFv that binds with high afﬁnity to a mutant form of the EGF receptor in which a deletion of a portion of the extracellular domain of the receptor generates a tumor-speciﬁc epitope (99). Another novel target for cancer therapy could be cancer-speciﬁc peptides presented on human leukocyte antigen (HLA) molecules on the surface of tumor cells. To accomplish this, it will be necessary to isolate antibodies that recognize tumor-speciﬁc peptides associated with class I major histocompatibility complex (MHC) molecules on tumor cells. As a ﬁrst step in this direction, a recombinant immunotoxin has been constructed using an antibody that was isolated by phage display and that binds speciﬁcally to peptide–MHC complexes found on virally infected cells

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(153–155). This recombinant immunotoxin was cytotoxic only to cells speciﬁcally expressing hemagglutinin peptide Ha 255-262 in complex with H-2Kk (mouse class I MHC), and was not cytotoxic to cells that express other peptides associated with H-2Kk, nor to cells not expressing H-2Kk. Example of the activity of this unique recombinant immunotoxin is shown in Fig. 5. These studies indicate that if antibodies that recognize tumor-speciﬁc peptides in the context of class I MHC molecules can be developed, they should be very useful agents for targeted cancer immunotherapy. Early trials with ﬁrst-generation immunotoxins have shown signiﬁcant antitumor activity. Several second-generation recombinant immunotoxins with improved properties have been developed and are currently in clinical trials. Several of these show already clinical activity and promising results in Phase I trails (4, 108, 109, 114). The outcome of these clinical trials demonstrate that the promising preclinical results with these new agents can be translated into more substantial clinical responses and that similar agents that target other cancer antigens merit further clinical development.

NOTE ADDED IN PROOF While this manuscript was in press it has been reported that LANA can interact with the retinoblastoma protein, and in cooperation with Hras can transform primary ﬁbroblasts (Radkov, S. S., Kellam, P., and Boshoff, C. (2000) Nat. Med. 10, 1121–1127), and that the vFLIP protein is expressed in HHV-8 infected pleural effusion lymphoma-derived cells (Low, W., Harries, M., Ye, H., Du, M. Q., Boshoff, C., and Collins, M. (2001) J. Virol. 75, 2938–2945.)

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Human Herpesvirus-8 and Kaposi’s Sarcoma: Relationship with the Multistep Concept of Tumorigenesis Michael Stu¨ rzl,1,∗ Christian Zietz,2 Paolo Monini,3 3 and Barbara Ensoli 1

Institute of Molecular Virology GSF—National Research Center for Environment and Health 85764 Neuherberg, Germany 2 Ludwig Maximilians University Munich Institute of Pathology 80337 Munich, Germany 3 Laboratory of Virology Istituto Superiore di Sanita` 00161 Rome, Italy

I. Clinical Presentation of KS II. Histology of KS and Nature of KS Spindle Cells III. HHV-8 and KS A. Epidemiology of HHV-8 Infection B. HHV-8 Reactivation C. Dissemination of HHV-8 from the Blood into Tissues D. HHV-8 Infection and Gene Expression in KS at the Single-Cell Level IV. Conclusion References

Kaposi’s sarcoma (KS) develops through discrete inﬂammatory-angiogenic stages of polyclonal nature (early-stage lesions) to monomorphic nodules of spindle-shaped cells that can be clonal (late-stage lesions) and resemble true sarcomas. Molecular and epidemiological studies indicate that development of KS is tightly associated with infection by the human herpesvirus-8 (HHV-8). However, only individuals with speciﬁc conditions of immunodysregulation develop KS. In these individuals the systemic and tissue increase of Th-1–type cytokines (IC) reactivate HHV-8 infection, ∗ Address correspondence to Priv. Doz. Dr. rer. nat. Michael Sturzl, ¨ GSF—National Research ¨ Center for Environment and Health GmbH, Institute of Molecular Virology, Ingolstadter Landstrasse 1, 85764 Neuherberg, Germany. E-mail: [email protected]

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C 2001 by Academic Press. Copyright All rights of reproduction in any form reserved.

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leading to increased viral load, antibody titers, and an expanded cell tropism that precedes the clinical appearance of KS. Recruitment of the virus into tissues by infected monocytes and other cell types is facilitated by the endothelial cell activation due to IC. In clinical lesions, HHV-8 infection increases with lesion stage and in late-stage lesions most of the spindle cells are latently infected, whereas only few lytically infected cells are present, suggesting that latent genes may have a role in the transformation of the early inﬂammatory-hyperplastic lesion into a real sarcoma. The development of tumors, however, is regulated through a multistep process based on the acquisition by cells of several different capabilities leading to malignant growth. Here we review the available data on the expression of HHV-8-encoded genes in primary KS lesions and, in view of their biological activity, analyze their potential function in different steps of tumorigenesis. By this pragmatic approach interesting insights into potential key functions of HHV-8-encoded genes are found and steps of potential cooperativity with other viral factors (HIV-1-Tat) in the pathogenesis of KS are identiﬁed. C 2001 Academic Press.

I. CLINICAL PRESENTATION OF KS Kaposi’s sarcoma (KS) is characterized by multiple purple-blue or reddishbrown lesions, often arising on the skin of the extremities but involving also mucosas and visceral organs. Lesions evolve from ﬂat discolorations or patches (early/patch stage) to plaques (plaque stage), and then to nodules that can coalesce (late/nodular stage) (Ackerman and Gottlieb, 1988). KS occurs in 4 epidemiological forms: classical KS (CKS) was the ﬁrst clinically recognized form, is a milder form of KS, and affects elderly men of the Eastern-Mediterranean area with a male-to-female ratio up to 15:1 (Friedman-Kien and Saltzman, 1990; Kaposi, 1872). Acquired immunodeﬁciency syndrome (AIDS)-associated KS (AIDS-KS) is the most frequent tumor of human immunodeﬁciency virus type I (HIV-l)– infected homo–bisexual men and is the most aggressive form of KS. AIDS-KS is at least 20,000 times more common in HIV-1–infected patients as compared to the general population and represents the most common AIDSassociated cancer (Friedman-Kien, 1981; Gottlieb and Ackerman, 1982; Haverkos and Drotman, 1985; Safai et al., 1985). African KS (AKS) was already frequent in certain areas of Africa before the AIDS pandemic, representing up to 10% of the total tumors in Uganda. An aggressive lymphadenopathic form of KS also affects African children (Slavin et al., 1969; Taylor et al., 1972). With the outbreak of AIDS, a signiﬁcant increase in the incidence of KS has been observed in Central Africa, where KS now accounts for 50% of the tumors reported in men. In Eastern and Southern Africa, KS represents 25–50% of soft-tissue sarcomas and 2–10% of all cancers in childhood, respectively (Athale et al., 1995; Wabinga et al.,

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1993). By contrast, KS is rare in West Africa, where HIV-2 infection is more prevalent than HIV-1 (Ariyoshi et al., 1998). Posttransplant KS (PKS) occurs in transplanted individuals after therapy with cyclosporin and corticosteroids, particularly in certain ethnic groups of Ashkenazi or Sephardi Jewish descent and in geographic areas such as Italy and Saudi Arabia (Penn, 1979; Trattner et al., 1993). The median interval from organ transplantation to KS diagnosis is 30 months, with a maleto-female ratio ranging from 2:1 to 4:1 (Lesnoni La Parola et al., 1997; Montagnino et al., 1994). In about half of the patients this type of KS has a mild clinical course; in others it can be aggressive.

II. HISTOLOGY OF KS AND NATURE OF KS SPINDLE CELLS Early-stage lesions are characterized by an inﬂammatory-granulation-type reaction with activated proliferating endothelial cells forming new vessellike structures, often abnormal, either incomplete or dilated, associated with extravasation of red blood cells and edema (Ackerman and Gottlieb, 1988). This precedes the appearance of the typical “spindle cells” [KS cells (KSC)], which are ﬁrst recognizable in the plaque stage and are considered to be the tumor cells of KS. In the nodular stage the KSC become the predominant cell type and the lesions acquire a more monomorphic aspect, resembling a ﬁbrosarcoma, although angiogenesis always remains a prominent feature (Dorfman, 1984; Ensoli et al., 1991; McNutt et al., 1983; Roth et al., 1992; ¨ Sturzl et al., 1992). The nature of the inﬂammatory cell inﬁltrate of KS is important since it is the ﬁrst to appear and precedes spindle cell formation. Immunohistochemical studies indicate a prevalent inﬁltration of monocyte-macrophages [CD68+ (Fig. 1A, see color plate), MAC387+ (Fig. 1B, see color plate), CD4+, CD14+, CD45+, PAM-1+], often with a spindle-like morphology and subendothelial localization together with CD4-positive and CD8positive T cells (Fig. 1C, see color plate), and dendritic cells (FXIIIa+), whereas B cells (CD19+, CD20+, or CD30+) are rare or absent (Fiorelli et al., 1998; MacPhail et al., 1996; Nickoloff and Grifﬁths, 1989; Regezi et al., 1993; Tabata et al., 1993; Uccini et al., 1997). The same features are also observed by analyzing tumor-inﬁltrating lymphocytes and monocytic cells isolated from the lesions (Sirianni et al., 1998). The monocytic cells in KS appear to inﬁltrate the tissues from the blood and to differentiate in loco into macrophages and dendritic cells. This is facilitated by the high levels of adhesion molecule expression in resident vessels (MacPhail et al., 1996).

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The nature of the KSC has been debated for many years, but recent studies indicate that spindle cells are a heterogeneous cell population of endothelial-derived KSC (E-KSC) mixed with macrophagic spindle-shaped cells (see earlier). E-KSC express markers of both lymphatic vessel and blood vessel endothelial cells and resemble an undifferentiated vasculoblastic precursor cell that expresses CD34 (Figs. 1D and 1E, see color plate), VEGFR-3, podoplanin, VE-cadherin, ULEX, CD36, ICAM-1, V-CAM-1, ELAM-1, CD40, DR, and, moderately, CD31 (Fig. 1F, black arrow, see color plate) and FVIII-RA (Dupin et al., 1999; Fiorelli et al., 1998; MacPhail et al., 1996; Pammer et al., 1996; Ramani et al., 1990; Regezi et al., 1993; Roth et al., 1988; Weninger et al., 1999). E-KSC can be distinguished from normal endothelial cells surrounding vessels (EC) by morphologic and immunophenotypic criteria—for example, the latter express higher levels of FVIII-RA ¨ and CD31 (Figs. 1F and 1G, white arrows, see color plate) (Sturzl et al., 1992). In addition to these histologic features, all forms of KS reveal low proliferation rate, spontaneous regression, and lack of chromosomal abnormalities (Bisceglia et al., 1992; Casado et al., 1988; Kaaya et al., 1992; Kondo et al., 2000). This, together with the appearance of the lesions at several different sites without evidence of a primary lesion or metastasis, suggested the reac¨ tive nature of KS in its early stages (Sturzl et al., 1992). This is supported by more recent studies on the X-chromosome inactivation pattern of the human androgen receptor gene, which indicated that most KS lesions are polyclonal, whereas advanced lesions can be monoclonal (Gill et al., 1998; Rabkin et al., 1997). In addition, a more recent study looking at the clonality of KS tumors by determining the size heterogeneity of HHV-8–fused terminal repeats (TR) revealed oligoclonality in 4 cases and monoclonality in 2 of the 6 examined (Judde et al., 2000). Altogether, these observations and the results from numerous studies both in situ and with primary EKSC cultures, including experimental animal models (reviewed elsewhere: ¨ ¨ Ensoli and Sturzl, 1998; Sturzl et al., 1992), indicate that early-stage lesions are polyclonal and cytokine-driven, whereas advanced lesions may represent true sarcomas.

III. HHV-8 AND KS Epidemiological studies suggested earlier that a sexually transmitted infectious agent is involved in KS development (Beral et al., 1990). However, human viruses including human papillomaviruses, hepatitis B virus, cytomegalovirus, human herpesvirus (HHV)-6, and BK virus were only sporadically identiﬁed in KS tissues (Adams et al., 1995; Kempf et al., 1994;

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Monini et al., 1996; Siegal et al., 1990). More recently, Chang and Moore identiﬁed a new DNA virus named HHV-8 or Kaposi sarcoma-associated herpesvirus (KSHV), which is consistently associated with all forms of KS (Chang et al., 1994). Sequence analysis of the 165-kb HHV-8 genome revealed that HHV-8 is a ␥ -herpesvirus closely related to herpesvirus saimiri (HSV) and Epstein–Barr virus (EBV) (Moore et al., 1996b; Neipel et al., 1997; Russo et al., 1996). As for other herpesviruses, HHV-8 infection can be lytic or latent. During lytic replication, virions are produced and released from the cell, and this results in cell death. In contrast, latent infection does not kill the cell and is characterized by the persistence of the viral genome as a covalently closed circular episome with a limited and speciﬁc viral gene expression.

A. Epidemiology of HHV-8 Infection Epidemiological studies show a large variability of HHV-8 infection in different geographic areas or ethnic groups. The most striking differences are between African or Amerindian populations, where the virus is widespread, and Northern Europe, where HHV-8 prevalence is very limited (Andreoni et al., 1999; De The et al., 1999; Gao et al., 1996b; Lennette et al., 1996; Melbye et al., 1998; Rezza et al., 2000; Simpson et al., 1996). An intermediate prevalence is found in Saudi Arabia, Mediterranean countries, and Central America. Recent studies indicate that the prevalence of HHV-8 infection in Asia is also highly variable (Ablashi et al., 1999; Calabro et al., 1998; Gao et al., 1996b; Huang et al., 2000; Lennette et al., 1996; Qunibi et al., 1998; Rezza et al., 1998, 2000; Simpson et al., 1996; Whitby et al., 1998), and the same is found in North America, where serologic determination of HHV-8 prevalence has yielded results ranging from 0 to 20% in different studies (Chatlynne et al., 1998; Davis et al., 1997a; Gao et al., 1996a, 1996b; Kedes et al., 1996; Lennette et al., 1996; Smith et al., 1997; Zhu et al., 1999). A higher HHV-8 seroprevalence is observed in countries where KS is endemic, suggesting that the geographic distribution of HHV-8 infection may mirror the incidence of KS (Franceschi and Geddes, 1995; Geddes et al., 1994, 1995; Simpson et al., 1996; Whitby et al., 1998; Calabro et al., 1998). However, this association is not found in the African countries. In fact, HHV8 infection is widely diffused in Egypt and Gambia, where the incidence of KS is very low (Andreoni et al., 1999; Ariyoshi et al., 1998; Lennette et al., 1996). Moreover, recent studies have also shown that HHV-8 seroprevalence remained unchanged in Africa during the sharp increase of KS associated with the HIV-1 epidemic (Olsen et al., 1998; De The et al., 1999). Thus, infection by HHV-8 appears to be required but is not sufﬁcient for KS development, which requires additional factors.

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Earlier studies have shown that the prevalence of HHV-8 infection and HHV-8 antibody titers are signiﬁcantly higher in patients with KS or at risk for KS, including HIV-1–positive homosexual men as compared to the general population or low-risk groups (Gao et al., 1996a; Kedes et al., 1996; Lennette et al., 1996; Moore et al., 1996c; Rezza et al., 1998; Simpson et al., 1996; Whitby et al., 1995). Furthermore, individuals with KS or at risk for KS have a high viral load in peripheral blood mononuclear cells, uninvolved tissues, and body ﬂuids including plasma, serum, nasal secretions, saliva, and show evidence of HHV-8 productive replication in PBMC (Corbellino et al., 1996; Decker et al., 1996; Fiorelli et al., 1998; Howard et al., 1997; LaDuca et al., 1998; Monini et al., 1999a, 1999b; Moore et al., 1996c; Whitby et al., 1995). PBMC-associated viremia or high antibody titers against HHV-8 latencyassociated or lytic antigens are predictive of KS onset in HIV-1–infected people (Gao et al., 1996a; Jacobson et al., 1940; Martin et al., 1998; Moore et al., 1996c; Renwick et al., 1998; Rezza et al., 1999; Whitby et al., 1995). However, longitudinal studies have shown that the progression rate to KS is signiﬁcantly lower in individuals showing HHV-8 seroconversion before HIV-1 infection as compared to HIV-1–infected individuals (Renwick et al., 1998; Jacobson et al., 2000). In addition, in these studies KS onset was never observed before co-infection, further suggesting that KS development requires additional factors in addition to HHV-8 infection (Renwick et al., 1998; Jacobson et al., 2000).

B. HHV-8 Reactivation The observation that PBMC-associated HHV-8 viremia and increasing antibody titers against HHV-8 lytic or latent antigens are predictive of disease development in risk individuals (Gao et al., 1996a; Jacobson et al., 1940; Martin et al., 1998; Moore et al., 1996c; Renwick et al., 1998; Rezza et al., 1999; Whitby et al., 1995) indicates that reactivation of HHV-8 and, perhaps, expansion of the HHV-8 latently infected cell pool may be required for KS development. For other herpesviruses, lytic infection is controlled by the immune system, whereas a decrease of immune surveillance is associated with virus reactivation. The latter is likely to be required also for HHV-8 viremia and virus spread into tissues. In fact, immunosuppression is a common trait of HIV-1–infected and posttransplant patients, and decreased CD4+ T-cell counts have also been described in patients with AKS in the absence of HIV-1 infection (Urassa et al., 1998). In addition, an impaired cytotoxic activity of CTLs and NK cells to certain HHV-8–encoded antigens has been found in CKS and AIDS-KS patients (Osman et al., 1999; B. Ensoli, unpublished results).

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Additional mechanisms can be provided by the virus. HHV-8, in fact, encodes two zinc-ﬁnger membrane proteins, K3 and K5, that downregulate major histocompatibility complex (MHC) class I molecules from the cell surface reducing cytotoxic T-lymphocyte (CTL)-speciﬁc function against infected cells (Ishido et al., 2000). In addition, the vMip-II protein (see Section III,D,2,c) has been shown to prolong graft survival in mice by reducing donor-speciﬁc CTL inﬁltration into the grafts and by inhibiting autoantibody production (DeBruyne et al., 2000). These results suggest that immunosuppression and viral-mediated immune escape mechanisms may both be important for HHV-8 reactivation and spread, as occurs for other herpesviruses. However, virus reactivation also requires stimuli that induce the lytic gene expression program and virus replication. Recent data indicate that inﬂammatory cytokines (IC) of the Th-1 type are increased in blood and tissues of patients with KS and at risk of KS due to immune activation and dysregulation, and that this may be key to both HHV-8 reactivation and KS development. Speciﬁcally, patients with all forms of KS and individuals at risk of KS, including homosexual men and HIV-1–infected individuals or elderly people of Mediterranean origin, show increased levels of IC [␥ -interferon (␥ -IFN), interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor (TNF)-␣], neopterin, soluble ICAM-1, soluble CD8, or oligoclonal expansion of CD8+ T cells (Fagiolo et al., 1993; Fagnoni et al., 1996; Fan et al., 1993; Hober et al., 1989; Honda et al., 1990; Schlesinger et al., 1994; Sirianni et al., 1998; B. Ensoli, unpublished results). The administration of ␥ -IFN or TNF␣ to HIV-1–infected patients can induce KS development or leads to KS progression (Aboulaﬁa et al., 1989; Krigel et al., 1989). In addition, KS progression is also observed in HIV-1–infected patients during opportunistic infections that are associated with IC production (Mitsuyasu, 1993), and KS can occur in homosexual men in the absence of HIV-1 infection and apparent immunosuppression (Friedman-Kien et al., 1990). Moreover, AIDS-KS and CKS patients and HIV-1–infected homosexual men have increased serum and tissue levels of IC, possibly due to immunoactivation (Fiorelli et al., 1998; Sirianni et al., 1998; B. Ensoli, unpublished results). AKS is also associated with Th-1–type immunoactivation, probably due to the frequent exposure to different infections (Rizzardini et al., 1996, 1998), and it is feasible that both immunosuppression and immunoactivation may also occur in PKS, where allogeneic stimulation may establish local foci of activated immune cells producing factors activating HHV-8 in the immunocompromised patient. Recent data indicate that the IC increased in KS reactivate HHV-8 infection. Speciﬁcally, IC treatment of PBMC from HHV-8–infected patients increases lytic gene expression and viral load, and allows the long-term maintenance of the virus which otherwise is lost shortly after cultivation

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(Monini et al., 1999b). Furthermore, IC have been shown to increase the expression of the HHV-8–encoded K8.1 virion glycoprotein (orfK8.1), vCycD (orf72), and processivity factor-8 (orf59) in latently infected primary effusion lymphoma (PEL)-derived cell lines (Blackbourn et al., 2000; Chang et al., 2000; Yu et al., 1999), conﬁrming that the predominant effect of IC may be activation of HHV-8 replication. Furthermore, IC increase Bcell survival and induce growth of circulating KSC-like cells, and both have been shown to be infected by HHV-8 (Monini et al., 1999b; Sirianni et al., 1997). In addition to IC, immunodeﬁciency may contribute to virus spread to various cell types, as also indicated by the presence of HHV-8 in T cells of KS patients and posttransplanted individuals under therapy, as compared to groups at risk of KS where HHV-8 can only be detected in B cells and/or monocytes (Monini et al., 1999b; B. Ensoli, unbublished results). Therefore, both IC and immunodeﬁciency may contribute to the increase of HHV-8 load and virus dissemination to tissues.

C. Dissemination of HHV-8 from the Blood into Tissues In KS patients, HHV-8 infection can be detected in several different circulating cell types, including B cells, T cells, and monocytes (Monini et al., 1999b). This suggests that HHV-8 infection may spread through these cells into the tissues and even into KS, as suggested by the low or absent HHV-8 infection of early-stage KS lesions as compared to late-stage lesions, where most spindle cells are infected (see Section III.D.1). This is supported by several observations. 1. In 80% of HIV-1–infected patients the aortic endothelium reveals a dramatic disruption and activation of the endothelial cell monolayer, with expression of VCAM-1, ELAM-1, and HLA-DR and increased adhesion of monocytes, regardless of HHV-8 infection (Fig. 2A, see color plate) (Zietz et al., 1996). These alterations are generalized, as indicated by increased endothelial cell permeability and vascular leakage in the capillaries of the eye and the brain and by the increased concentrations of FVIII-RA in the blood of HIV-1–infected individuals (Gariano et al., 1993; Petito and Cash, 1992; Pober and Cotran, 1990; Rhodes, 1991). This appears to be due to increased levels of IC. In fact, CD8+ T-cell activation and increased IC are found in all KS forms and in individuals at risk of KS, both in blood and tissues, and induce production of angiogenic growth factors (AGF) (Barillari et al., 1999b; Cornali et al., 1996; Fiorelli et al., 1998; Samaniego et al., 1995, 1997, 1998; Sirianni et al., 1998). IC induce endothelial cell activation and adhesiveness for monocytes (Bevilacqua et al., 1985; Cavender et al., 1991;

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Gamble et al., 1985; Zimmerman and Hill, 1984), whereas AGF trigger endothelial cell proliferation, angiogenesis, and edema (Connolly et al., 1989; Detmar et al., 1995; Ferrara and Henzel, 1989; Folkman and Klagsburn, 1987; Gospodarowicz et al., 1989; Keck et al., 1989). Concerted activity of both factors may lead to disseminated and clinically inapparent reactive foci of activated endothelial cells and inﬁltrating inﬂammatory cells, which may represent the germinating areas of KS or areas prone to KS development ¨ (Ensoli and Sturzl, 1998). In fact, some early KS lesions (AIDS-KS and CKS) have been identiﬁed that express detectable IC and HLA-DR prior to HHV-8 detection by PCR (Fiorelli et al., 1998). 2. IC reactivate HHV-8 infection (see Section III.B), increase viral load, and promote in PBMC the transmission of HHV-8 to other cells including monocytes (Monini et al., 1999b). HHV-8–infected monocytes have been detected by PCR and at the single-cell level in PBMC from HHV-8–infected patients (Fig. 2B, see color plate) (Monini et al., 1999b), while adhering to ¨ et al., 2000), the tumor vessels in KS lesions (Fig. 2C, see color plate) (Sturzl and as inﬁltrating cells in KS tissues (Fig. 2D, see color plate) (Blasig et al., 1997; Parravicini et al., 2000). In addition, HHV-8 is lost after culture of E-KSC from the lesions (Dictor et al., 1996; Lebbe et al., 1995), but it is maintained in monocyte cultures derived from the lesions (Sirianni et al., 1998). 3. Chemoattractants for monocytes including monocyte chemoattractant protein 1 are highly expressed in KS lesions (Sciacca et al., 1994). 4. In AIDS-KS the Tat protein of HIV-1 appears to increase all these effects by a synergistic activity with IC and AGF (Barillari et al., 1993, 1999a, 1999b; Ensoli et al., 1994; Fiorelli et al., 1998), possibly explaining the higher aggressiveness of the disease in HIV-1–infected patients. Altogether, these ﬁndings suggest that in KS risk groups increased serum concentrations of IC and AGF may induce a generalized vasculopathy with disseminated reactive foci of activated endothelial cells and adherent inﬂammatory cells. In the presence of HHV-8 infection, IC may reactivate the virus in its latently infected reservoirs and in monocytes1 which may carry the virus into the reactive foci of the very early stage of KS. In the lesions, HHV8–infected cells may differentiate into spindle cells and/or release HHV-8 during lytic infection, supporting transmission to resident endothelial cells (see Section III.D). 1 Circulating spindle cell progenitors have been found in patients with all forms of KS and in individuals at high risk to develop KS. These cells are infected by HHV-8 and express the same markers (CD68, MAC 387) that were used to identify HHV-8–infected monocytes in KS tissues (Browning et al., 1994; Monini et al., 1999b; Sirianni et al., 1997). In the following, these cells will be included in the term “monocytes.”

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D. HHV-8 Infection and Gene Expression in KS at the Single-Cell Level The mechanisms by which HHV-8 participates in KS development and exerts tumorigenic activity are unknown as yet. In addition, since only some lesions are monoclonal, this may occur only rarely and could be mediated either by lytic or latency-associated viral genes. As for other herpesviruses, the HHV-8 genome has incorporated several host genes during its evolution. Homologs of cellular genes that encode proteins with function in cell transformation, chemoattraction, cell growth, and cell survival have been suggested as playing a role in KS development. However, to understand their potential tumorigenic role in KS, it is necessary to know (1) the type of HHV-8–infected cells, (2) the type of infection (lytic or latent), and (3) the expression of the respective gene in the lesions. An overview of the location of the HHV-8–encoded genes discussed in this review as well as their expression in KS is presented in Fig. 3 (see color plate).

1. NATURE OF THE HHV-8-INFECTED CELLS AND TYPE OF INFECTION IN KS The presence of HHV-8 genomic DNA, transcripts, or proteins in KS has been investigated at the single-cell level on KS tissue sections by in situ polymerase chain reaction, in situ hybridization, and/or immunohistochemistry, respectively. In early-stage lesions HHV-8 is often undetectable by in situ hybridization or is present only in a few cells. Speciﬁcally, in three different studies the expression of lytic genes [orfK7 (nut-1/T1.1), orf25 (major capsid protein), and orf26 (VP23, capsid protein)] has not been detected in early-stage KS lesions (Blasig et al., 1997; Staskus et al., 1997). In addition, kaposin RNA (orfK12), which is expressed in both lytically and latently infected cells, and the latency-associated nuclear antigen [LANA, LNA, LNA-1 (orf71)] are either absent (Rainbow et al., 1997) or expressed by only a small fraction of ¨ et al., 1997; Cathomas et al., 2000). Only the cells (Dupin et al., 1999; Sturzl two studies reported a high expression of LANA in CD34-positive E-KSC (Parravicini et al., 2000) or in spindle cells around blood vessels (Katano et al., 2000) of early-stage KS. However, the presence of numerous spindle cells in these lesions suggests that later stages might have been evaluated. The determination of the cell type infected by HHV-8 in early lesions is difﬁcult due to uncertain histological classiﬁcation of the earliest KS-associated tissue alterations, and the results may also vary from patient to patient. For example, we recently detected high expression of kaposin RNA in a large ¨ number of cells in one early-stage KS lesion (M. Sturzl, unpublished results), whereas 12 other early-stage lesions analyzed were either negative or revealed only very few infected cells.

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In late-stage lesions HHV-8 infection can be detected in almost all E-KSC and is predominantly latent. HHV-8 DNA has been detected in E-KSC and EC by in situ polymerase chain reaction (PCR) (Boshoff et al., 1995; Foreman et al., 1997). Kaposin RNA and the LANA protein have also been detected in the majority (70–90%) of spindle cells [Fig. 4A, kaposin (bright ﬁeld); Fig. 4B, corresponding dark ﬁeld; Fig. 4C, LANA, see color plate] (Cathomas et al., 2000; Dittmer et al., 1998a; Dupin et al., 1999; Katano et al., 1999b, 2000; Kellam et al., 1999; Linderoth et al., 1999; Parravicini et al., ¨ 2000; Rainbow et al., 1997; Staskus et al., 1997; Sturzl et al., 1997, 1999a; ¨ Sturzl and Ensoli, 1999; Sun et al., 1999). Co-expression of CD34 (Katano et al., 1999b; Parravicini et al., 2000; Staskus et al., 1997) or VEGFR3 molecules (Dupin et al., 1999) and LANA proteins or kaposin RNA transcripts conﬁrmed that E-KSC are the predominantly infected cell type. By contrast, LANA is not expressed in CD31-positive EC (Fig. 4D). However, sporadic expression of kaposin RNA has been detected in CD31-positive EC ¨ lining normal vessels (Bobroski et al., 1998; Linderoth et al., 1999; Sturzl ¨ et al., 1997; Sturzl and Ensoli, 1999). In late-stage KS tissues lytic infection is restricted to only a few cells (on average ≤ 1%) (Fig. 4E, see color plate), as indicated by the expression in the lesion of transcripts and proteins speciﬁcally associated with lytic infection, including T1.1, MCP, VP23, Rta (orf50), K8 (orfK8), K10 (orfK10), K11 (orfK11), and processivity factor-8 (PF-8) (orf59) (Blasig et al., 1997; Dittmer et al., 1998b; Katano et al., 1999a, 1999b, 2000; Linderoth et al., 1999; Parravicini et al., 2000; Staskus et al., 1997; Sun et al., 1999), and by the ultrastructural detection of viral particles (Orenstein et al., 1997; Said et al., 1997). The type of lytically infected cells is still debated. Viral lytic gene expression and/or viral particles have been found in CD68-positive monocytic cells (Blasig et al., 1997) (Fig. 4F, see color plate) that have been recently shown to express also LANA (Parravicini et al., 2000), in cells resembling lymphocytes (Orenstein et al., 1997), in CD31-positive EC (Linderoth et al., 1999), and in CD34-positive E-KSC (Staskus et al., 1997). However, not all these different cells have been consistently detected in every study. Nevertheless, the majority of the available data show that viral load is low in early-stage lesions, increases with lesion stage, and is highest in late-stage KS, where the virus is mostly in latent form and in E-KSC.

2. HHV-8 GENE EXPRESSION IN KS AND THE MULTISTEP CONCEPT OF TUMORIGENESIS As presented above, there is a compelling epidemiological and histological evidence that HHV-8 is involved in KS pathogenesis, but whether and by which molecular mechanism(s) HHV-8 may contribute to KS development is as yet unknown. However, robust infection in late-stage lesions as

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compared to early-stage lesions suggests that the main role of HHV-8 is in the transformation of KS into a true sarcoma. Novel concepts of cancer research indicate that cancerogenesis in humans is a multistep process requiring four to seven rate-limiting genetic alterations that drive the progressive transformation of normal cells into highly malignant tumor elements (Renan, 1993). Speciﬁcally, at least six essential alterations of the cell physiology are required for malignant growth: independence from activating growth signals, insensitivity to growth-inhibitory signals, evasion of programmed cell death (apoptosis), induction of angiogenesis, limitless replicative potential, and tissue invasion and metastasis (for a review, see Hanahan and Weinberg, 2000). Additionally, paracrine activities may also play a role, for example, in the recruitment of blood-derived cells that produce cell growth and invasion stimuli. It has been suggested that this multistep concept of tumorigenesis may be applied to the growth of all human tumors. Therefore, since late-stage KS can be monoclonal, it also may follow this pathway. In this case, HHV-8 may represent the key factor. However, to this end the KS pathogenic genes of HHV-8 have to fulﬁll at least two criteria: (1) they should regulate at least one of the steps mentioned above, and (2) they should be expressed in E-KSC during KS development. Summarized in the following are published data on the expression of speciﬁc HHV-8–encoded genes with potential effect in the multistep tumorigenesis process.

a. HHV-8 and Proliferation of E-KSC: vIL-6, vIRF, vCyc Normal cells require exogenous growth signals in order to proliferate. Tumor cells generate many of these growth signals by themselves, reducing their dependence on the tissue microenvironment. In addition, in normal conditions, antiproliferative signals maintain cellular quiescence and tissue homeostasis. At the molecular level, many antiproliferative signals converge at the level of the retinoblastoma protein (pRb). In a hypophosphorylated state, pRb blocks cell proliferation by sequestering and altering the function of E2F transcription factors that control the expression of genes essential for the G1-to-S cell cycle progression (Weinberg, 1995). Disruption of the pRb pathway renders cells insensitive to antigrowth signals. In certain DNA virus-induced tumors, pRb function is eliminated through sequestration by viral oncoproteins such as the E7 protein of human papillomavirus (Dyson et al., 1989). Among HHV-8–encoded genes whose expression has been investigated in KS, three have mitogenic activity or may abrogate inhibition of cell growth, namely, orfK2 (vIL-6), orfK9 [viral interferon regulatory factor (vIRF)], and orf72 [viral cyclin (vCyc)] (Table I).

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i. orfK2 (vIL-6). The viral cytokine interleukin-6 (vIL-6) is secreted from infected cells during lytic infection and can activate proliferation of IL-6–dependent myeloma cell lines (Burger et al., 1998; Moore et al., 1996a; Nicholas et al., 1997). Similar to human IL-6, vIL-6 activates STAT1, STAT3, and Jak1 phosphorylation, but at the receptor level this requires only the transducing subunit gp130 and not the IL-6 receptor (IL-6R) as for human IL-6 activity (Molden et al., 1997; Nicholas et al., 1997). Both IL-6 and gp130 are highly expressed by E-KSC, whereas no IL-6R is expressed in KS ¨ et al., 1995). Thus, vIL-6 may activate E-KSC despite the large lesions (Sturzl molar excess of IL-6 in KS lesions. However, in KS lesions vIL-6 RNA and protein can be detected only rarely and in a few cells that are productively infected (Figs. 5A and 5B, arrows, see color plate) (Moore et al., 1996a; ¨ Parravicini et al., 2000; Staskus et al., 1999; Sturzl and Ensoli, 1999; YenMoore et al., 2000). Thus, it is unlikely that vIL-6 plays a role in E-KSC proliferation. ii. orfK9 (vIRF). orfK9 encodes a homolog of the interferon regulatory factor (IRF) family and is expressed during lytic infection (Moore et al., 1996a). IRFs act either as transcriptional activators or as repressors on class I interferon receptor signaling (reviewed by Taniguchi et al., 1995). vIRF heterodimerizes with members of the IRF family and binds competitively with cellular transcription factors to p300 (Burysek et al., 1999), and to the transcriptional co-activator CREB-binding protein (CBP) (Seo et al., 2000). In addition, vIRF inhibits IFN-induced gene transcription, blocks TNF-induced apoptosis, and confers resistance to the antiproliferative effect of IFN-␣ (Burysek et al., 1999; Flowers et al., 1998; Gao et al., 1997; Li et al., 1998; Zimring et al., 1998). Since IFN-␣ inhibits proliferation of cultivated ¨ KSC that are not infected by HHV-8 (Koster et al., 1996; Reiter et al., 1992), these data suggest that vIRF may abrogate the antiproliferative signals of this factor on E-KSC in the lesions. In addition, it has been shown that vIRF can transform rodent ﬁbroblasts to a tumorigenic phenotype (Gao et al., 1997; Li et al., 1998). However, expression of vIRF is low in KS tissues and detected only by RT-PCR (Yen-Moore et al., 2000), but not by Northern blot (Gao et al., 1997) or immunohistochemistry (Parravicini et al., 2000). The lytic expression pattern of vIRF in HHV-8–infected lymphoma cells (Sarid et al., 1998) explains this low expression level, since lytically infected cells are rare in KS (see Section III.D.1). iii. orf72 (vCyc). orf72 is a latent HHV-8 gene. The encoded protein, vCyc, shares 29.8% and 32.5% sequence identity with cellular D cyclins and the cyclin homolog of herpesvirus saimiri (HVS), respectively (Cesarman et al., 1996; Chang et al., 1996; Jeffrey et al., 1995; Kobayashi et al., 1992). vCyc associates and activates the kinase activity of cyclin-dependent kinase (Cdk)-6 and weakly of Cdk-2 and Cdk-4 (Godden-Kent et al., 1997; Li et al., 1997). The vCyc/Cdk-6 complex phosphorylates the retinoblastoma protein

138 Table I Activity and Expression of HHV-8–Encoded Genes in KS Lesions and Potential Role in the Multistep Process of Tumorigenesis Gene mode of expressiona

Activity

Expression in KS lesions

Possible step-regulating role in KS

Proliferation vIL-6 (orfK2) Lytic

Activates proliferation of myeloma cell lines

ISH and IHC: negative or positive in only a few cells

Unlikely

vIRF (orfK9) Lytic

Inhibits IFN-induced gene transcription, TNF-induced apoptosis, and confers resistance to anti-proliferative effects of IFN-␣

RT-PCR: positive NB: negative IHC: negative

Unlikely

vCyc (orfK72) Latent

Associates with Cdk-6 and Cdk-4 and induces Rb phoshorylation; vCyc/Cdk complexes are resistant to CdkIs p21 and 27

RT-PCR: positive ISH: positive in the majority of E-KSC of late-stage lesions

Proliferation of E-KSC

Inhibits Fas-mediated apoptosis in murine B-lymphoma cells

ISH: positive in the majority of E-KSC and some EC of late-stage lesions

Stage-related increase of expression of vFLIP-RNA correlates with reduced apoptosis in late-stage KS lesions, may act as a KS progression factor

Apoptosis vFLIP (orfK13) Latent

LANA (orf73) Latent

Regulates HHV-8 genome segregation, binds to RING and p53, inhibits p53-mediated apoptosis in osteosarcoma and lymphoma cells

IHC: positive in almost all E-KSC of late-stage lesions

Stage-related increase of expression of LANA protein correlates with reduced apoptosis in late-stage KS lesions; likely an anti-apoptotic KS progression factor

vBcl-2 (orf16) Lytic

Prevents Bax-mediated apoptosis in ﬁbroblasts and various other cells

RT-PCR: positive ISH: positive in few cells

Unlikely

K 15/LAMP (orfK15) Lytic

Signiﬁcant homology to EBV LMP1. LMP1 induces cellular Bcl-2 expression

Unknown

May act anti-apoptotic in E-KSC through induction of cellular Bcl-2, which has been suggested to inhibit apoptosis in late-stage KS lesions

vGPCR (orf74) Lytic

Transforms NIH3T3 ﬁbroblasts and upregulates VEGF expression; transgenic mice expressing vGPCR in hematopoietic cells develop angioproliferative KS-like lesions

RT-PCR: positive in AIDS-KS but not in CKS ISH: positive in a few cells

Unlikely

vIL6 (orfK2) Lytic

vIL-6–transfected ﬁbroblasts induce vascularized tumors in mice due to increased VEGF expression by these cells

ISH and IHC: negative or positive in only a few cells

Unlikely

vMip-I-II-III (orfK6, orfK4, orfK4.1) Lytic

All three vMips are angiogenic in the chorioallantoic assay

RT-PCT: vMip-I positive, vMip-II negative ISH: vMip-II positive in a few cells WB: vMip-III positive in late-stage lesions

Angiogenic activity of vMip-III is lower as compared to bFGF and VEGF; both bFGF and VEGF are highly expressed in KS lesions and have synergistic activity

Angiogenesis

139

(continues)

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Table I (continued ) Gene mode of expressiona Unlimited replication K1 (orfK1) Lytic Kaposin-A,-B-C (orfK12) Latent

Activity

Expression in KS lesions

Possible step-regulating role in KS

Immortalizes marmoset T cells in an STP-deleted herpesvirus saimiri vector

Unknown

Unknown

Weak transforming activity in immortalized rodent cells

ISH: expressed in almost every E-KSC (T0.7 mRNA), IHC: positive in a few cells

Despite robust mRNA expression in almost all E-KSC, kaposin protein can be detected in only a few cells of KS lesions; the specific effect of kaposin on limitless proliferation needs further investigation

Unknown

Presently, no HHV-8−encoded gene has been identified that may regulate this step

IHC: positive in almost all E-KSC of late-stage lesions

Genomic instability has not been reported for KS

Invasion Unknown Genome instability LANA (orf73) Latent

Besides other activities (see above), binds to p53 and can inhibit transcription of p53-activated genes

Abbreviations: Methods for the detection of the respective RNA transcripts or proteins in KS tumor extracts or at the single-cell level, respectively: IHC, immunohistochemistry; ISH, in situ hybridization; NB, Northern blot; RT-PCR, reverse transcriptase polymerase chain reaction; WB, Western blot. Cell types; E-KSC, endothelial KS spindle cells; EC, differentiated normal endothelial cells surrounding tumor vessels. Others: bFGF, basic fibroblast growth factor; Cdk, cyclin-dependent kinase; CdkI, Cdk inhibitory protein; Cyc, cyclin; FLIP, FLICE [FADD (Fas-associated death domain)-like interleukin-1␤−converting enzyme] inhibitory protein; GPCR, G-protein−coupled receptor; IL, interleukin; IFN, interferon; IRF, interferon regulatory factor; LANA, latency-associated nuclear antigen (LNA, LNA-1); VEGF, vascular endothelial growth factor. a The classification of lytic and latent gene expression has been adopted from studies on HHV-8 gene expression with pleural effusion lymphoma-derived cell lines (BC-1 .. cells) (Sarid et al., 1998). It has to be considered that expression of HHV-8 genes may vary in different cell types and tissues, as suggested recently (Sturzl et al., 1999a).

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and other substrates regulating the transition through the G1 and possibly other restriction points of the cell cycle (Godden-Kent et al., 1997; Li et al., 1997). In addition, the vCyc/Cdk-6 complex is resistant to the cyclindependent kinase inhibitory proteins (CdkIs) p21 and p27 (Godden-Kent et al., 1997; Swanton et al., 1997). It has been shown that vCyc/Cdk phosphorylates p27, which induces its proteolytic degradation and progression of cells arrested in G1 to S (Ellis et al., 1999; Mann et al., 1999). Impairment of p27 is also observed with the E1a and E7 proteins of adenovirus and human papillomavirus, respectively (Mal et al., 1996; Zerfass-Thome et al., 1996). In addition, EBV downregulates p27 by a posttranscriptional mechanism, suggesting that p27 may be a common target of inactivation by oncogenic viruses (Cannell et al., 1996).RNA expression studies support a role of vCyc in KS development. vCyc RNA expression is consistently detected by RTPCR in AKS (6/6) and CKS (3/3) lesions (Yen-Moore et al., 2000), and by in situ hybridization in the majority (70%) of spindle cells in nodular KS lesions (Figs. 5C and 5D, arrows, see color plate) (Davis et al., 1997b; Dittmer et al., ¨ ¨ 1998b; Sturzl and Ensoli, 1999; Sturzl et al., 1999a, 1999b). By contrast, only a few positive cells are detected in a minority (1/4) of early-stage KS ¨ lesions (Davis et al., 1997b; Sturzl et al., 1999a). Activity of vCyc and its expression in E-KSC both suggest that this gene may induce an increased proliferative capability of E-KSC, although it is not transforming in the classical assays (Cannell and Mittnacht, 1999). However, protein expression studies are required in order to determine the role of vCyc in KS development.

b. HHV-8 and Apoptosis of E-KSC: vFLIP, LANA, vBcl-2, K15/LAMP, vIRF Tumor growth is determined not only by the rate of cell proliferation but also by the rate of programmed cell death (apoptosis). Apoptotic stimuli activate intracellular proteases termed caspases, which execute the death program (Thornberry and Lazebnik, 1998). Apoptosis can be induced by extracellular factors such as the Fas ligand (FasL) or TNF binding to their receptors (Ashkenazi and Dixit, 1999), or by intracellular signals following DNA damage, signaling imbalance, survival factor insufﬁciency, or hypoxia (Evan and Littlewood, 1998). Members of the Bcl-2 family including pro-apoptotic (Bax, Bak, Bid, Bim) or antiapoptotic (Bcl-2, Bcl-XL, Bcl-W) proteins and p53 regulate apoptosis. Resistance to apoptosis through the loss of p53 function is seen in more than 50% of human cancers (Harris, 1996). Additionally, the PI3 kinase–AKT/PKB pathway, decoy receptors for FasL as well as FLICE [FADD (Fas-associated death domain)-like interleukin–1␤–converting enzyme] inhibitory proteins (FLIPs), inhibit apoptosis (Pitti et al., 1998). Viruses have developed a range of strategies to defend the infected cell against apoptosis (reviewed by Roulston et al., 1999). Notably, in KS a signiﬁcant reduction of apoptosis is observed in late stages as compared to early

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¨ stages (Sturzl et al., 1999a). The reduction of apoptosis correlates with an increasing number of HHV-8 latently infected E-KSC in the lesions. HHV8 encodes at least 5 genes [orfK13(vFLIP), orf73(LANA), orf16(vBcl-2), orfK15(K15/Lamp), and orfK9(vIRF) (discussed earlier)] which may block the apoptotic program of the infected cell (Table I). i. orfK13 (vFLIP). vFLIP is a latent HHV-8 gene encoded by orfK13 and a viral homolog of cellular FLIPs, which have been recently identiﬁed in muscle and lymphoid tissues. vFLIPs are encoded by several ␥ -herpesviruses and by the tumorigenic human molluscipoxvirus (Bertin et al., 1997; Thome et al., 1997). FLIPs block early signaling events of the death receptors Fas (APO-1/CD95), TRAMP (WSL/DR-3/APO-3), TRAIL-R1 (DR-4), TRAILR2 (DR5), and TNFR (Bertin et al., 1997; Goltsev et al., 1997; Hu et al., 1997a, 1997b; Irmler et al., 1997; Thome et al., 1997). HHV-8-vFLIP contains two death-effector domains (DED) (Thome et al., 1997). One of these may bind to death-effector domains (DED) of FADD (MORT-1) or FLICE (Caspase 8/MACH/Mch-5) and interfere with the FADD–FLICE interaction, hereby inhibiting the recruitment and activation of FLICE by Fas (Thome et al., 1997). Recent data have shown that transcripts of HHV-8-vFLIP are expressed at high levels by the majority of E-KSC (Figs. 5E and 5F, see color plate) and in some EC (Figs. 5E and 5F, arrows) in nodular KS lesions. By contrast, these transcripts cannot be detected in most early lesions ¨ (Sturzl et al., 1999a). The increase of the vFLIP expression in late-stage KS lesions correlates with the reduction of apoptosis in progressed tumor stages ¨ (Sturzl et al., 1999a). In addition, expression of HHV-8-vFLIP in murine B-lymphoma cells allows the growth of aggressive tumors in mice due to the inhibition of Fas-mediated cytotoxic T-cell responses (Djerbi et al., 1999). Thus, vFLIP may act as a KS progression factor for KS through inhibition of receptor-mediated apoptosis. ii. orf73 (LANA). orf73 encodes a high-molecular-weight (224–234 kDa) nuclear protein (LANA, LNA, or LNA-1) which is a component of the HHV-8 latency-associated nuclear antigen (Kellam et al., 1997; Rainbow et al., 1997; Russo et al., 1996). LANA accumulates in nuclear bodies in interphase nuclei, producing a characteristic stippled pattern (Rainbow et al., 1997; Szekely et al., 1999). Recently, it has been shown that this protein regulates the segregation of HHV-8 episomes in cell progeny (Ballestas et al., 1999). It has also been shown that LANA interacts with RING3, a member of the Drosophila female sterile homeotic (fsh) family that is implicated in controlling gene expression and in modulating chromatin structure (Platt et al., 1999). Most interestingly, LANA can interact with p53 and by this inhibits p53-mediated apoptosis in osteosacoma cells and in HHV-8–infected lymphoma cells (Friborg et al., 1999). p53 is highly expressed in E-KSC of late-stage KS (Hodak et al., 1999; Noel et al., 1997; Pillay et al., 1999). This suggests that LANA may contribute to the survival or transformation of

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E-KSC by blocking p53-mediated apoptosis. Although LANA RNA expression in KS lesions is below the detection threshold of in situ hybridization ¨ (Sturzl et al., 1999a), the LANA protein can be detected in almost 90% of E-KSC in late-stage KS lesions (Figs. 5G and 5H) (Dupin et al., 1999; Katano et al., 1999b; 2000; Kellam et al., 1999; Parravicini et al., 2000; Rainbow et al., 1997). Therefore, LANA fulﬁls the two basic criteria for an HHV-8–encoded progression factor and may represent the most likely candidate HHV-8 gene to contribute to the multistep tumorigenesis process via antiapoptotic effects. iii. orf16 (vBcl-2). orf16 is a lytic HHV-8 gene. The encoded protein, vBcl-2, shares 15–20% amino acid identity with members of the Bcl-2 family including Bcl-2, Bcl-XL, Bak, Bax, and other viral Bcl-2 homologs such as those encoded by EBV and HVS (Cheng et al., 1997; Russo et al., 1996; Sarid et al., 1997). No homodimerization of vBcl-2 or heterodimer formation of vBcl-2 with human Bcl-2 family members has been observed in mammalian cells (Cheng et al., 1997), whereas yeast two-hybrid experiments indicated heterodimer formation with human Bcl-2 (Sarid et al., 1997). Functional studies indicate that vBcl-2 prevents Bax-mediated toxicity or apoptosis in yeast, in transfected ﬁbroblasts, and in Sindbis virus-infected cells (Cheng et al., 1997; Sarid et al., 1997). Transcripts of the v-Bcl-2 gene can be detected in KS lesions by RT-PCR (Sarid et al., 1997; Yen-Moore et al., 2000), but vBcl-2 can only be detected in the few lytically infected cells by in situ hybridization (Figs. 5I and 5J, arrows) (Ascherl et al., 1999; Sarid et al., ¨ 1997; Sturzl et al., 1999b). This suggests that vBcl-2 may not prevent apoptotic death of latently infected E-KSC although, similar to the EBV homolog (Murray et al., 1996), it may prolong the survival of lytically infected cells. Furthermore, the cellular Bcl-2 is highly expressed by E-KSC in progress¨ ing KS stages (Dada et al., 1996; Morris et al., 1996; Sturzl et al., 1999a), suggesting that vBcl-2 may not be required for E-KSC survival. iv. orfK15 (K15/LAMP). orfK15 has been described as a lytic gene in HHV-8–infected lymphoma cells (Sarid et al., 1998). The expression of this gene in KS lesions has not yet been investigated, but there is indirect evidence that it may be involved in the regulation of apoptosis in KS. The gene encoding K15/LAMP produces a family of alternatively spliced transcripts of approximately 7.5 kb (Glenn et al., 1998), encoding proteins with up to 12 transmembrane domains and a hydrophilic C terminus. These proteins localize on the cell surface or intracellular membranes with an orientation of the C terminus toward the cytoplasm. This, the peculiar splicing pattern, the presence of sequence with homology to known TRAF-binding sites, and the interaction with members of the TRAF family are highly reminiscent of the LMP1 protein of EBV (Devergne et al., 1996; Eliopoulos et al., 1999; Fennewald et al., 1984; Mosialos et al., 1995). Since EBV/LMP1 induces the expression of cellular Bcl-2 (Henderson et al., 1991) and in KS Bcl-2

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cellular expression increases with lesion stage (Dada et al., 1996; Morris ¨ et al., 1999a), it is tempting to speculate that HHV-8 may et al., 1996; Sturzl be responsible for the high cellular Bcl-2 expression in KS through the action of K15/LAMP.

c. HHV-8 and Angiogenesis in KS: vGPCR, vMIP-I,-II,-III, v-IL6 In order to progress to a size larger that 2 mm2, tumors must develop angiogenic ability (Bouck et al., 1996; Folkman, 1995; Hanahan and Folkman, 1996). Tumor-associated angiogenesis is regulated by the coordinated expression of positive [basic ﬁbroblast growth factor (bFGF), vascular endothelial growth factor (VEGF)] and negative (thrombospondin-1) soluble mediators and their receptors (expressed on endothelial cells), and by integrins and adhesion molecules mediating cell-to-cell and cell–matrix interactions (Singh et al., 1995; Volpert et al., 1997). In KS, differently from solid tumors, angiogenesis precedes KS spindle cell formation and KS lesions are always highly vascularized. Therefore, angiogenesis is a characteristic and important component of the lesions. The induction of angiogenesis in KS by IC, AGF, and in AIDS-KS also by the HIV1-Tat protein, have been extensively studied and reviewed elsewhere (Ensoli ¨ and Sturzl, 1998). It should be mentioned, however, that bFGF and VEGF are highly expressed in all forms of KS, at both the RNA and protein levels, and act synergistically to induce endothelial cell growth and angiogenesis (Cornali et al., 1996; Samaniego et al., 1998; Xerri et al., 1991). HHV8–encoded gene products including G-protein–coupled receptor (vGPCR), vIL-6, and three homologs of host chemokines [viral macrophage inﬂammatory protein (vMip)-I, -II, -III] have been shown to exert angiogenic activitiy in some model systems (Table I). i. orf74 (vGPCR). The orf74 gene of HHV-8 is a lytic gene with a high sequence homology to the IL-8R (Cesarman et al., 1996). The encoded protein binds to several CXC and CC chemokines and is also constitutively active in the absence of ligands (Arvanitakis et al., 1997). vGPCR has been shown to transform NIH3T3 cells, and this is accompanied by the secretion of VEGF which may regulate the “angiogenic switch” required for tumor progression (Bais et al., 1998). Recently, it has been shown that transgenic mice expressing vGPCR within hematopoietic cells develop angioproliferative lesions in multiple organs that resemble KS lesions (Yang et al., 2000). Within these murine lesions both vGPCR and VEGF are expressed only in a subset of cells (Yang et al., 2000). In contrast, VEGF-transgenic animals expressing VEGF in numerous cells develop only mild angiogenic changes that do not evolve in KS-like lesions (Detmar et al., 1998; Larcher et al., 1998). This raises the question of whether it is the upregulation of VEGF expression by vGPCR that induces the angioproliferative murine lesions. In addition, vGPCR RNA expression can be detected by in situ hybridization

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in only few lytically infected cells in AIDS-KS lesions (Kirshner et al., 1999), but not in CKS lesions (Yen-Moore et al., 2000), suggesting that vGPCR may not contribute signiﬁcantly to KS-associated angiogenesis. ii. orfK2 (vIL-6). It has been recently shown that ﬁbroblasts expressing vIL-6 induce highly vascularized tumors in mice, due to the production of VEGF by these cells (Aoki et al., 1999). However, vIL-6 is a lytic gene expressed at a very low level in KS (see Section III.D.2.a), arguing against a role of this viral cytokine in KS angiogenesis. iii. orfK6, orfK4, orfK4.1 (vMip-I,-II,-III). HHV-8 encodes three lytic proteins with signiﬁcant sequence similarity to the cellular CC chemokines vMip-I (orfK6), vMip-II (orfK4), and vMip-III (orfK4.1) (Kledal et al., 1997; Moore et al., 1996a; Neipel et al., 1997; Nicholas et al., 1997). All three vMips induce angiogenesis in the chorioallantoic membrane (CAM) assay, which suggested a possible role for these molecules in the neoangiogenesis of KS (Boshoff et al., 1997; Stine et al., 2000). By RT-PCR, vMip-I expression is found in almost all AKS and CKS lesions, whereas no expression of vMip-II has been detected (Yen-Moore et al., 2000). In situ hybridization reveals that vMip-I RNA is expressed only in a few lytically infected cells of KS tissues ¨ et al., 1998; Sun et al., 1999), indicating that its con(Figs. 5K and 5L) (Sturzl centration in KS tissue is low. However, a signiﬁcant expression of the vMIPIII protein is detected by Western blot in late-stage KS lesions (Stine et al., 2000), suggesting higher concentrations for this factor in KS tissues. However, vMIP-III is four- to ﬁvefold less potent than bFGF in the stimulation of angiogenesis (Stine et al., 2000). Considering that both bFGF and VEGF mRNA and proteins are highly expressed in KS lesions by numerous E-KSC (Cornali et al., 1996; Samaniego et al., 1998; Xerri et al., 1991), it is unlikely that vMip-III inﬂuences signiﬁcantly the angiogenic milieu in KS lesions.

d. HHV-8 and the Limitless Replicative Potential of E-KSC: K1, vGPCR, vIRF, Kaposin Human cells stop growing after they have progressed through a certain number of doublings—a process termed senescence. This is due to the progressive erosion of telomers, which causes the loss of their ability to protect the ends of the chromosomal DNA, resulting in the death of the cell (Counter et al., 1992). Telomere maintenance due to increased expression of the telomerase or to recombination-based interchromosomal exchanges is a typical trait of malignant cells and a key factor for unlimited cell replication (Bryan and Cech, 1999; Shay and Bacchetti, 1997). Considering that KS may not originate from a single transformed cell but grows mostly as a polyclonal proliferation, it is unclear whether the limitless replication is a general feature or even required for E-KSC. Only a few studies have addressed the potential of HHV-8 to induce limitless replicative capability of infected cells. In one study, infection of primary human endothelial cells

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with HHV-8 resulted in cell immortalization (Flore et al., 1998); however, these effects were not conﬁrmed by a similar study (Panyutich et al., 1998). Studies aiming to determine the transforming capability of HHV-8–encoded vIRF (Gao et al., 1997), vGPCR (Bais et al., 1998), and kaposin (Muralidhar et al., 1998) have been carried out with cell lines that were already immortalized, and the results are difﬁcult to interpret. However, studies on HHV-8 orfK1 showed that this viral gene may confer unlimited growth capability to some cell types (Table I). i. orfK1 (K1). orfK1 encodes a nonconserved transmembrane glycoprotein that has been shown to act like a constitutively activated immunoreceptor tyrosine-based activation motif (ITAM)-bearing signaling molecule (Lagunoff et al., 1999). Positional homology of orfK1 with transformationrelevant genes of other ␥ -herpesviruses [STP-C and -A (saimiri-transforming protein); tip (tyrosine kinase-interacting protein)] suggested that it may have transforming activity (Neipel and Fleckenstein, 1999). In fact, replacement of STP with orfK1 in the HVS genome leads to a recombinant virus that can immortalize marmoset CD8 T cells in vitro and induce lymphoma development in vivo (Lee et al., 1998). However, the recombinant virus still contained tip, the other major HSV–transforming gene. Thus, the role of K1 in cell immortalization is not yet completely clariﬁed. Expression of K1 in KS tissues has not been investigated as yet, but in preliminary in situ hybridization ¨ experiments we did not detect K1 expression in KS tissues (M. Sturzl, unpublished observation), and other authors have suggested that K1 may not be expressed in latently infected cells (Neipel and Fleckenstein, 1999). Among the other potentially transforming genes of HHV-8, the vIRF protein is not detected in KS lesions (Parravicini et al., 2000), and vGPCR RNA expression is restricted to a few lytically infected cells of AIDS-KS but is not expressed in CKS lesions (Kirshner et al., 1999; Yen-Moore et al., 2000). By contrast, kaposin mRNA is expressed in almost all E-KSC of late-stage lesions (see Section III.D.1). Recently, it has been shown that transcripts originally ascribed to orfK12 more frequently encompass upstream sequences (Sadler et al., 1999) and may encode three proteins termed kaposin A, B, and C (Sadler et al., 1999). However, by immunohistochemical studies the kaposin proteins were detected in only a few cells of KS lesions (Sadler et al., 1999). The ﬁndings described above suggest that among the potentially transforming genes of HHV-8, orfK1 is the most likely to induce limitless replication in E-KSC.

e. HHV-8 and Tissue Invasion and Metastasic Capability of E-KSC During the development of most types of human neoplasia, cells invade adjacent tissues and spread via the circulation to distant sites, where they

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establish metastatic colonies. Activation of extracellular proteases and alteration of both cell-to-cell and cell–matrix interactions are key for invasion and metastatic tumor spread (Christofori and Semb, 1999; Fogar et al., 1997; Johnson, 1991; Kaiser et al., 1996). In many tumors, proteases are not produced by the tumor cells, but rather by resident stromal cells or inﬁltrating inﬂammatory cells (Werb, 1997), and docking of active proteases on the cell surface promotes tumor cell invasion into the stroma (Chambers and Matrisian, 1997; Coussens and Werb, 1996). The activation of extracellular proteases and the altered binding properties of cadherins, CAMs, and integrins are central to the acquisition of the invasiveness and metastatic capability. No studies have been performed to investigate whether HHV-8–encoded proteins can induce cell invasiveness and/or cell metastatic potential. Nevertheless, cell invasiveness may be an important step in late stages of KS development. This is indicated by earlier reports that have shown that primary cells isolated from KS tissues have an increased invasive capability as compared to cells isolated from adjacent normal skin (Albini et al., 1988; Schirren et al., 1990). These cells were not infected by HHV-8, suggesting that the increased invasiveness was due to long-lasting stimulating factors present in the KS tissue. In this framework, it is interesting that AIDS-KS has a more aggressive clinical course than the other epidemiological variants of KS and that the HIV-1-Tat protein can induce the migration, invasion, and the expression of collagenase IV of the 72-kDa type in cultivated KSC and IC-activated endothelial cells (Albini et al., 1995; Barillari et al., 1992; Ensoli et al., 1990, 1994; Fiorelli et al., 1999). In addition, Tat mimics the effect of ECM proteins such as ﬁbronectin and vitronectin which are known to play a key role in cell invasion (Ensoli et al., 1994; Varner and Cheresh, 1996). These data suggest that the HIV-1-Tat protein may regulate the invasive capability in AIDS-KS. Therefore, invasiveness of KS may be a possible step for cooperation of HIV-1 and HHV-8, leading to the more aggressive clinical course of AIDS-KS compared to other forms of the disease.

f. HHV-8 and Genome Instability of E-KSC: LANA In nonvirally induced tumors the cells acquire the different capabilities leading to transformation that have been described above through mutations in their genomes. These are rare events due to mistakes of cellular control mechanisms (DNA monitoring, repair, checkpoints). Increased mutability often is linked to malfunction of speciﬁc components of the genomic “caretaker” systems (Lengauer et al., 1998). For example, p53 together with other sensory or reparatory genes of DNA damage is often lost in most human cancers (Lengauer et al., 1998; Levine, 1997).

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As described earlier, LANA has been shown to inhibit the transcriptionactivating ability of the p53 protein. Through functional deactivation of p53, LANA may induce decreased genomic stability of E-KSC. However, genomic instability does not appear to be a common characteristic of E-KSC and may occur only in late-stage KS. In fact, aneuploidy or protooncogene activation has not yet been observed in KS (Roth et al., 1992). Therefore, it is unclear whether the LANA/p53 interaction that was observed in cultured cell lines may have similar consequences in E-KSC of KS lesions.

3. PARACRINE ACTIVITIES OF HHV-8: vMIP-I,-II,-III, vGPCR, vIL-6 Novel concepts in cancer biology underscore the contributions of inﬁltrating blood-derived ancillary cells for tumor cell proliferation. There is, in fact, evidence that heterotypic signaling among the diverse cell types within a tumor may be as important as the autocrine mechanisms regulating the capabilities of tumor cells. Especially, inﬂammatory cells attracted to the sites of neoplasia may promote tumor cell growth and survival (Cordon-Cardo and Prives, 1999; Coussens et al., 1999; Hudson et al., 1999). Since KS lesions contain in all stages lymphocytes and monocytes, potential paracrine effects of HHV-8 gene products on cell recruitment should be considered. Possible candidates to attract lymphocytes and monocytes into the lesions are the HHV-8–encoded vMIP-I, -II, -III proteins. However, it has been shown that these proteins inhibit monocyte chemotaxis and that they are potent Th-2 T-cell chemoattractants (Endres et al., 1999; Kledal et al., 1997; Sozzani et al., 1998; Stine et al., 2000). Both activities are, however, in contrast with the cellular composition of KS, which includes numerous monocytes and a prevalent Th-1 T-cell inﬁltration. The lack of effect of the Mips indicates that these proteins may not be produced in biologically relevant amounts in the KS lesions. However, further studies on other HHV8 genes are required to determine whether HHV-8 infection may contribute to the cell inﬁltration of KS lesions.

IV. CONCLUSION In this review we summarized data on the expression of HHV-8 genes and gene products in KS lesions and their respective biological activities, speciﬁcally addressing their lytic and latent expression, cell type-related localization, and their tumorigenic potential.

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Localization and pattern of infection: HHV-8 infection is low in early KS lesions, and the type of infected cells in these stages is unclear. By contrast, in late-stage lesions almost all endothelial KS spindle cells are infected by the virus, and this infection is predominantly latent. Productive infection is almost exclusively observed in late-stage lesions and restricted to a few cells including monocytes. These results, in combination with previous data, suggest that HHV-8 is recruited by circulating inﬂammatory cells into disseminated reactive foci of activated endothelial cells and monocytes that may either represent very early-stage KS lesions or sites prone to develop KS. In these foci HHV-8 may infect and, perhaps, induce the formation of E-KSC, whose appearance in later-stage lesions coincides with the increase of HHV-8 infection. Tumorigenic potential: Although early KS is of polyclonal nature, latestage KS can be a monoclonal tumor, and therefore should undergo the multistep process of tumorigenesis. We analyzed the capability of HHV-8 genes to regulate these steps, according to gene function and expression in KS lesions. This pragmatic approach indicated that HHV-8 may regulate some steps, whereas for other steps there is no indication that HHV-8 may be involved. Possible HHV-8–regulated steps may be cell proliferation (vCyc), inhibition of apoptosis (vFLIP and LANA), and induction of genome instability (LANA). Although, several HHV-8–encoded genes have angiogenic activity (vGPCR, v-Mips, vIL-6), only vMip-III is expressed at considerable levels in KS tissues. However, bFGF and VEGF are highly expressed in KS tissues, have higher angiogenic activity than vMip-III, and can interact synergistically in the induction of angiogenesis. Therefore, a role of vMip-III in KS angiogenesis is unlikely. K1 has been shown to induce limitless replication in cells, but it likely is not expressed in KS. As yet, no data are available on whether and how HHV-8 may regulate cell invasiveness of KS. Furthermore, no data are available on potential paracrine activities of HHV-8 gene products, for example, on cell recruitment. Notably, the inhibitory activities of vMips on monocyte chemotaxis are clearly in contrast with the presence of a rich monocytic inﬁltration in KS. The apparent incapability of HHV-8 to regulate some steps of tumorigenesis may suggest that either these activities have not been discovered yet, or that HHV-8 may be unable to regulate them. However, some capabilities may be acquired by E-KSC independently of HHV-8 infection, either by mutation in their genome or in cooperation with other viruses. For example, the activation of invasiveness of cultivated KSC and endothelial cells by the HIV-1-Tat protein suggests that HIV-1 may compensate the missing capability of HHV-8 to induce cell invasiveness, at least in this aggressive form of KS. This concept supports epidemiological data suggesting that HHV-8 is necessary but not sufﬁcient to induce KS. However, HHV-8 clearly has the capability to regulate certain steps of tumorigenesis (proliferation,

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apoptosis). In this case, HHV-8 may play in KS the same role as activated proto-oncogenes in other tumors and may be considered as a human tumor virus.

ACKNOWLEDGMENT ¨ Bildung und This work was funded by the BioFuture program of the Bundesministerium fur Forschung (BMBF), the Deutsche Forschungsgesellschaft (SFB 464), the Deutsche Krebshilfe, and the Bavarian Nordic Research Institute AS (Martinsried, Germany) (grants to M.S.), and by the Associazione Italiana per la Ricerca sul Cancro (AIRC) and the IX AIDS project from the Ministry of Health (grants to B.E.).

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Reactivation and Role of HHV-8 in Kaposi’s Sarcoma Initiation 2 ¨ Barbara Ensoli,1,∗ Michael Sturzl, and Paolo Monini1 1 Laboratory of Virology Istituto Superiore di Sanita` 00161 Rome, Italy 2 Institute of Molecular Virology GSF—National Research Center for Environment and Health 85764 Neuherberg, Germany

I. II. III. IV. V.

Kaposi’s Sarcoma Risk Factors Associated with KS Development Histology of KS and Origin of Spindle Cells HHV-8 Infection in KS Lesions KS Initiation: Role of IC in KS Histogenesis and HHV-8 Infection A. Role of IC in KS Histogenesis B. Effects of IC on HHV-8 Infection VI. Lack of Control of Reactivated HHV-8 VII. KS Progression: Oncogenes, Oncosuppressor Genes, HHV-8 Latency Genes, and the HIV-1 Tat Protein A. Oncogenes and Oncosuppressor Genes B. HHV-8 Latency Genes C. The HIV-1 Tat Protein VIII. Concluding Remarks References

Kaposi’s sarcoma (KS) is an angioproliferative disease occurring in several clinicalepidemio-logic forms but all associated with infection by the human herpesvirus-8 (HHV8). At least in early stages, KS is a reactive disease associated with a state of immune dysregulation characterized by CD8+ T-cell activation and production of Th1-type inﬂammatory cytokines (IC) that precedes lesion development. In fact, evidence indicates that IC can trigger lesion formation by inducing the activation of endothelial cells that leads to adhesion and tissue extravasation of lymphomonocytes, spindle cell formation, and angiogenesis, and HHV-8 reactivation that, in turn, leads to virus spread to all circulating cell types and virus dissemination into tissues. Due to virus escape mechanisms and deﬁcient immune responses toward HHV-8, virus reactivation and spread are not controlled by the immune system but induce immune responses that may paradoxically ∗ Address correspondence to Barbara Ensoli, M.D., Ph.D., Laboratory of Virology, Istituto ` Viale Regina Elena, 299, 00161 Rome, Italy. E-mail: [email protected] Superiore di Sanita,

161 Advances in CANCER RESEARCH 0065-230X/01 $35.00

C 2001 by Academic Press. Copyright All rights of reproduction in any form reserved.

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exacerbate the reactive process. The virus is recruited into “activated” tissue sites where it ﬁnds an optimal environment for growth. In fact, viral load is very low in early lesions, whereas almost all spindle cells are infected in late-stage lesions. Although early KS is a reactive process of polyclonal nature that can regress, in time and in the presence of immunodeﬁciency, it can progress to a true sarcoma. This is likely due to the long-lasting expression of HHV-8 latency genes in spindle cells associated with the deregulated expression of oncogenes and oncosuppressor genes and, for AIDS-KS, with the effects of the HIV-1 Tat protein. C 2001 Academic Press.

I. KAPOSI’S SARCOMA Kaposi’s sarcoma (KS) is a tumor of vascular origin arising with multiple independent lesions in the form of angiogenic maculae or plaques that can ¨ progress into nodular lesions (Ensoli and Sturzl, 1998). KS arises as different clinical-epidemiologic forms. An indolent form is usually seen in elderly men of Mediterranean or Eastern Europe origin (classic KS, CKS) (Franceschi and Geddes, 1995; Geddes et al., 1995; Safai and Good, 1981) or in posttransplant patients (PT-KS) treated with cyclosporin (Civati et al., 1988; Penn, 1979; Trattner et al., 1993). More aggressive forms involving visceral and/or lymphatic organs occur in young adults and children of sub-Equatorial Africa (African KS, AKS) (Slavin et al., 1969; Taylor et al., 1971; Taylor et al., 1972) and in HIV-1–infected homo-bisexual ¨ man (AIDS-KS) (Beral et al., 1990; Ensoli and Sturzl, 1998; Friedman-Kien, 1981; Gottlieb and Ackerman, 1982; Haverkos and Drotman, 1985; Safai et al., 1985). AIDS-KS is aggressive, disseminated, and fatal, and represents the most frequent tumor of HIV-1–infected individuals (FriedmanKien, 1981; Gottlieb and Ackerman, 1982; Haverkos and Drotman, 1985; Safai et al., 1985). Several lines of evidence indicate that, at least in its early stage, KS may not be a true sarcoma but rather a hyperplastic reactive-inﬂammatory process. This is suggested by the polyclonal nature of early lesions (Gill et al., 1998), by the simultaneous appearance of multiple symmetrical lesions with a dermatome distribution developing in the absence of metastasis (Brooks, 1986), and by the spontaneous regressions of PT-KS or AIDS-KS upon withdrawal of the immunosuppressive therapy or after treatment with HIV-1 protease inhibitors, respectively (Akhtar et al., 1984; Bencini et al., 1993; Brooks, 1986; Conant et al., 1997; Hoshaw and Schwartz, 1980; Janier et al., 1985; Lebb`e et al., 1998; Real and Krown, 1985; Real et al., 1986; Rizzieri et al., 1997; Wit et al., 1998; Zisbrod et al., 1980). Evidence indicates two major risk factors for KS development, namely, the reactivation of human herpesvirus 8 (HHV-8) (Chang et al., 1994) infection and a dysregulation state of the immune system characterized by CD8+

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T-cell activation and by a deﬁcient immune response to the virus. Compared to HHV-8–infected healthy subjects, in fact, patients with KS or individuals at risk for KS show a higher HHV-8 load in blood and tissues and higher anti-HHV-8 antibody titers that precede KS onset and are highly predictive of disease development (Corbellino et al., 1996; Decker et al., 1996; Fiorelli et al., 1998; Gao et al., 1996a, 1996b; Jacobson et al., 2000; Martin et al., 1998; Moore et al., 1996a; Renwick et al., 1998; Rezza et al., 1999; Whitby et al., 1995). In addition, a CD8+ T-cell activation leading to production of Th-1–type inﬂammatory cytokines (IC) is found in individuals with KS or at risk of KS, including homo-bisexual men (even prior to HIV infection), PT patients, African individuals, and elderly men of Mediterranean origin (Fagiolo et al., 1993; Fagnoni et al., 1996; Fan et al., 1993; Hober et al., 1989; Honda et al., 1990; Rizzardini et al., 1996; Schlesinger et al., 1994; Sirianni et al., 1998; B. Ensoli, unpublished data). IC promote key events in KS initiation, including the activation of the vascular system and the reactivation of HHV-8 infection (Monini et al., 1999a; Pober and Cotran, 1990). The activation of endothelial cells (EC), in turn, leads to the production of chemokines and angiogenic factors that mediate recruitment of circulating cells into tissues and angiogenesis, respectively (Cotran and Pober, 1988; Cornali et al., 1996; Fiorelli et al., 1998; Folkman and Klagsburn, 1987; Folkman; 1995; Mantovani et al., 1997; Samaniego et al., 1995; Samaniego et al., 1997; Samaniego et al., 1998; ¨ Sirianni et al., 1998; Sciacca et al., 1994; Sturzl et al., 1997a). IC, in addition, activate EC to acquire the features of KS spindle cells, including the responsiveness to the mitogenic and angiogenic properties of the HIV-1 Tat protein (Albini et al., 1995; Barillari et al., 1992, 1993, 1999a, 1999b; Faris et al., 1998; Fiorelli et al., 1995, 1998, 1999; Samaniego et al., 1997, 1998). A deﬁcient immune response to HHV-8 alone or in the context of a more compromised immune system, likely associated with mechanisms of virus escape, may be required for disease progression, since it hampers the control of HHV-8 reactivation that is induced by IC, leading to the spread of HHV8 infection to all circulating cell types and to the dissemination of HHV-8– infected cells in tissues, as is usually found in KS patients (Coscoy et al., 2000; Djerbi et al., 1999; Haque et al., 2000; Ishido et al., 2000a; Sirianni et al., 1999, 2000; Touloumi et al., 1999; Urassa et al., 1998). Both activation of HHV-8 infection and a deﬁcient immune control of the virus appear to be key also for the spread of the virus in lesions. In fact, HHV-8 load is low and sometimes undetectable in early-stage lesions, whereas IC production and interferon-␥ (IFN-␥ )–producing CD8 T cells are already present (Fiorelli et al., 1998). However, HHV-8 load increases progressively, and in latenodular lesions most or all KS spindle cells are infected. In these cells infection is mostly latent, whereas only a very small fraction of infected cells including

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lympho-monocytes undergoes HHV-8 productive replication, suggesting a prevalent role of HHV-8 latency genes in KS spindle cells transformation and progression (Blasig et al., 1997; Davis et al., 1997; Dupin et al., 1999; ¨ Orenstein et al., 1997; Staskus et al., 1997; Sturzl et al., 1997b, 1999a, ¨ 1999b) (see also the chapter by Sturzl et al. in this volume). In fact, although early KS is polyclonal (Gill et al., 1998), late-stage KS can be transformed and monoclonal, as indicated by aneuploidy, microsatellite instability, and clonality of KS spindle cells from some late-stage KS lesions (Bedi et al., 1995; Delabesse et al., 1997; Rabkin et al., 1995, 1997). The long-term expression of HHV-8 latency genes associated with the deregulated expression of oncogenes or oncosuppressor genes such as c-myc, Bcl-2, and p53 that is found in late-stage KS (Bohan-Morris et al., 1996; Dada et al., 1996; Koster et al., 1996) may play a key role in transformation of KS ¨ into a true sarcoma (see the chapter by Sturzl et al. in this volume). Overt immune deﬁciency may facilitate this process, due to the lack of control of tumor growth leading to chromosomal changes, as suggested by the presence of microsatellite instability in AIDS-KS but not in CKS (Bedi et al., 1995).

II. RISK FACTORS ASSOCIATED WITH KS DEVELOPMENT Previous and recent results indicate that both infection by HHV-8 and a disturbance of the immune system are major risk factors for KS development. A variety of polymerase chain reaction (PCR) based and serological studies indicate that HHV-8 infection acts as a strong risk factor for KS development (Gao et al., 1996a; Jacobson et al., 2000; Moore et al., 1996a; Renwick et al., 1998; Rezza et al., 1999; Whitby et al., 1995). HHV-8 infection is also more prevalent in countries with a high incidence of KS, such as certain areas of Africa, Eastern Europe, Greece, and Italy, as compared to other geographical areas where both HHV-8 infection and KS incidence are low (Andreoni et al., 1999; Bestetti et al., 1998; Calabro` et al., 1998; De-Th`e, 1999; Franceschi and Geddes, 1995; Gao et al., 1996b; Geddes et al., 1994, 1995; Gessain et al., 1999; He et al., 1998; Kedes et al., 1996; Lennette et al., 1996; MacKenzie et al., 1997; Marcelin et al., 1997; Mayama et al., 1998; Melbye et al., 1998; Olsen et al., 1998; Perna et al., 2000; Rezza et al., 1998, 2000; Simpson et al., 1996; Whitby et al., 1998). In addition, an increased HHV-8 seroprevalence and load are found in population groups at risk for KS, including homo-bisexual men and HIV-1–infected individuals (Andreoni et al., 1999; Bestetti et al., 1998; Calabro` et al., 1998; De-Th`e, 1999; Gao et al., 1996a, 1996b; Gessain et al., 1999; He et al., 1998; Kedes et al., 1996; Lennette et al., 1996; Mayama et al., 1998; Melbye et al., 1998;

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Olsen et al., 1998; Perna et al., 2000; Rezza et al., 1998, 2000; Whitby et al., 1998). However, other data indicate that HHV-8 requires additional factors to exert its effects in KS development. In fact, in Mediterranean regions where KS is endemic, the prevalence of HHV-8 infection in the general population is exceedingly high compared to KS incidence (Calabro` et al., 1998; Franceschi and Geddes, 1995; Geddes et al., 1994, 1995; Perna et al., 2000; Whitby et al., 1998). Moreover, recent studies have indicated that HHV-8 seroprevalence in Africa remained unchanged upon the sharp increase of KS incidence associated with the HIV-1 epidemic (de-Th`e et al., 1999; Olsen et al., 1998; Wabinga et al., 1993), pointing to HIV-1 infection as a strong risk factor for KS development. Several lines of evidence indicate that, in addition to HHV-8 infection, immune dysregulation is a key factor for KS initiation. Speciﬁcally, patients with all forms of KS and individuals at risk of KS, including homobisexual men and HIV-1–infected individuals, African subjects, and elderly man of Mediterranean origin, show one or more signs of CD8 T-cell activation, including increased levels of IC [IFN-␥ , interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-␣], neopterin, soluble ICAM-1, soluble CD8, or an oligoclonal expansion of CD8+ T cells (Caruso et al., 1990; Fagiolo et al., 1993; Fagnoni et al., 1996; Fan et al., 1993; Hober et al., 1989; Honda et al., 1990; Lahedvirta et al., 1988; Lepe-Zanuga et al., 1987; Master et al., 1970; Rizzardini et al., 1996, 1998; Schlesinger et al., 1994; Sirianni et al., 1998, Touloumi et al., 1999; Vyakarnam et al., 1991; B. Ensoli, unpublished data). The administration of IFN-␥ or TNF-␣ to HIV-1–infected patients can induce KS development or KS progression (Aboulaﬁa et al., 1989; Krigel et al., 1989). This is also observed in HIV-1–infected patients during opportunistic infections that are associated with IC production (Mitsuyasu, 1993). In addition, KS can occur in homo-bisexual men in the absence of HIV-1 infection or overt immunosuppression (Friedman-Kien et al., 1990; Maurice et al., 1982). Moreover, AIDS-KS and CKS patients and HIV1–infected homosexual men have increased serum and tissue levels of IC (Fiorelli et al., 1998; Sirianni et al., 1998; B. Ensoli, unpublished data). AKS is also associated with Th-1–type immunoactivation, probably due to the frequent exposure to different infections (Rizzardini et al., 1996, 1998). Finally, both immunosuppression and immunoactivation may also occur in PT-KS, where allogeneic stimulation may induce local foci of activated immune cells. IC play a key role in KS initiation by inducing several events associated with KS development since (1) they provide a major stimulus for HHV-8 reactivation in circulating cells that increases virus load and spreading; (2) they induce EC to express adhesion molecules and chemokines for lympho-monocytes, leading to recruitment of HHV-8–infected cells into tissues; (3) they induce EC to acquire the phenotypic and functional

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properties of KSC including the responsiveness to the effects of extracellular HIV-1 Tat protein; and (4) they induce production of angiogenic factors and chemokines that mediate EC and KS spindle cells proliferation, angiogenesis, edema, and lesion growth (see below). Patients that develop KS, however, also present in a variable degree a state of immunosuppression (Sirianni et al., 1999, 2000; Touloumi et al., 1999; Urassa et al., 1998) that may hamper the control of HHV-8 reactivation and may allow the spread of HHV-8 infection to all circulating cell types and in tissues. In addition, HHV-8 itself possesses genes encoding for viral products allowing infected cells to escape host immune responses (Coscoy et al., 2000; Djerbi et al., 1999; Haque et al., 2000; Ishido et al., 2000a). As discussed later, these mechanisms may be key to the progression of KS in a true cancer, due to the lack of control of HHV-8 infection and KS cell proliferation.

III. HISTOLOGY OF KS AND ORIGIN OF SPINDLE CELLS KS lesions are characterized by inﬁltration of lymphomononuclear cells, activation and proliferation of EC forming abnormal blood vessels (slitlike vessels), neoangiogenesis, edema, and by the growth of spindle-shaped cells (KS spindle cells, KSC) that are considered to be the tumor cells of KS. In early lesions KSC are few and intermingled with stromal cells; however, in time KSC ﬁll the stroma between vascular spaces and KS lesions acquire a more monomorphic aspect, resembling a ﬁbrosarcoma (Dorfman et al., 1984; Ensoli et al., 1991; Ensoli and Gallo, 1995; McNutt et al., ¨ 1983; Regezi et al., 1993; Ruszczak et al., 1987a, 1987b; Sturzl et al., 1992). The ﬁrst histological change of KS is the appearance of an inﬂammatory cell inﬁltrate that precedes the spindle cell formation (Ackerman and ¨ Gottlieb, 1988; Dorfman et al., 1984; Ensoli et al., 1991; Ensoli and Sturzl, 1998; Fiorelli et al., 1998; McNutt et al., 1983; Sirianni et al., 1998). Immunohistochemical analysis of early KS lesions shows the presence of T cells, particularly CD8+ T cells, monocyte-macrophages, and dendritic cells (FXIIIa+), whereas B cells are few or absent (Fig. 1A, see color plate) ¨ (Ensoli and Sturzl, 1998; Fiorelli et al., 1998; MacPhail et al., 1996; Nickoloff and Grifﬁths, 1989; Regezi et al., 1993; Sirianni et al., 1998; Tabata et al., 1993; Uccini et al., 1994, 1997). Inﬁltrating cells obtained from KS biopsies (tumor-inﬁltrating lymphocytes, TIL) and lesional macrophagic spindle cell cultures derived from the lesions show the same features of

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Table I Effects of the Major Inﬂammatory Cytokines, Angiogenic Factors, and Chemotactic Factors Expressed in KS A. Systemic effects of IC 1. Vessel activation; extravasation of inﬂammatory cells (combined IC, IL-1␣/␤, TNF-␣, IFN-␥ ) 2. Proliferation and differentiation into endothelial macrophages of circulating KSC progenitors (combined IC) 3. HHV-8 reactivation in PBMC, increase of viral load in circulating cells, virus spread to tissues, expansion of HHV-8 latently infected cell reservoire (combined IC, IFN-␥ ) B. Local (tissue/KS lesion) effects of IC, angiogenic factors, and chemotactic factors 1. Recruitment of inﬂammatory cells; activation and differentiation of moncytic cells into macrophages, endothelial macrophages, and dendritic cells (combined IC, TNF-␣/␤, IFN-␥ , GM-CSF, IL-8, MCP-1) 2. Maintenance of TIL phenotype; TIL survival (combined IC) 3. KSC proliferation (combined IC, bFGF, VEGF, PDGF-B, SF/HGF) 4. Enhancement of E-KSC angiogenic factor production and angiogenic activity (combined IC, IFN-␥ , IL-1␤, TNF-␣) 5. Angiogenic activity (bFGF, VEGF, IL-8) 6. Activation of endothelial cells and induction of the KSC phenotype (combined IC, IFN-␥ , IL1-␤, TNF-␣): induction of a spindle morphology; downregulation of FVIII-RA (due to its release); expression of activation markers and upregulation of adhesion molecules; expression of bFGF, IL-6, IL-8, MCP-1, GM-CSF, IL-1, PDGF-A, and induction of bFGF release; induction of responsiveness to the angiogenic effects of the HIV-1 Tat protein; acquisition of an angiogenic phenotype and induction of KS-like lesions upon inoculation in nude mice 7. Induction of KS-like lesions after injection in nude mice (mediated by induction of angiogenic factors) (combined IC, IFN-␥ ) EC, endothelial cells; TIL, tumor-inﬁltrating lymphocytes; KSC, KS spindle cells; IC, inﬂammatory cytokines; IL-1, interleukin 1; TNF, tumor necrosis factor; IFN-␥ , interferon-gamma; GM-CSF, granulocyte/macrophage colony-stimulating factor; MCP, monocyte chemotactic protein; bFGF, basic ﬁbroblast growth factor; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; SF/HGF, scatter factor/hepatocyte growth factor; FVIII-RA, factor VIII-related antigen. The table summarizes the prominent effects of inﬂammatory cytokines, angiogenic factors, and chemotactic factors in KS. Some of these effects are elicited by single factors; however, these factors are all present at the systemic and/or tissue level and show synergistic effects.

in situ T cells and macrophages (Sirianni et al., 1998). As peripheral blood mononuclear cells (PBMC) from patients with KS or at risk of KS, these cells produce Th-1-type cytokines including IFN-␥ (Fig. 1A), TNF, IL-1, IL-6, and others (Fiorelli et al., 1998; Miles et al., 1990; Oxholm et al., 1989; Sirianni ¨ et al., 1998; Sturzl et al., 1995). These cytokines activate EC to acquire the KS cell phenotype and to produce angiogenic factors such as basic ﬁbroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), which are also highly expressed in primary lesions (Fig. 1B) and mediate angiogenesis, edema, and KS cell growth (see below) (Table I) (Barillari et al., 1992,

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1999a; Cornali et al., 1996; Faris et al., 1998; Fiorelli et al., 1995, 1998; Samaniego et al., 1995, 1997, 1998). Most studies indicate that lesional KSC consist of a heterogeneous cell population dominated by activated vascular and lymphatic EC mixed with cells of macrophagic origin (Corbeil et al., 1991; Dupin et al., 1999; Fiorelli et al., 1995, 1998; Huang et al., 1993a; Kaaya et al., 1995; Kraffert et al., 1991; Jussila et al., 1998; Little et al., 1986; MacPhail et al., 1996; Marchio et al., 1999; Nadji et al., 1981; Pammer et al., 1996; Ramani et al., 1990; Regezi et al., 1993; Roth et al., 1988; Sirianni et al., 1998; Yang et al., 1994; Weninger et al., 1999; Zhang et al., 1994). Furthermore, due to the co-expression of markers of macrophages and vascular endothelial cells such as the vascular-endothelial-cadherin (Uccini et al., 1994, 1997), some KSC show a phenotype similar to the so-called endothelial-macrophages of the lymph nodes (Lampugnani, 1992). Spindle-shaped cells can also be cultured from PBMC of patients with KS or at risk for KS (Browning et al., 1994; Monini et al., 1999a; Sirianni et al., 1997; Uccini et al., 1997). In the presence of IC these cells differentiate in culture into a cell type with a phenotype similar to endothelial macrophages that is also expressed by some lesional KSC (Monini et al., 1999a; Uccini et al., 1994, 1997). These data suggest, therefore, that KSC and endothelial macrophages are related cell types, that circulating KSC may be the cell progenitors of lesional KSC, and that the recruitment of circulating KSC into tissues may lead to the appearance of multiple lesions at independent sites. Evidence indicates that the reactive or hyperplastic KSC of endothelial cell origin are polyclonal and not transformed cells (Bisceglia et al., 1992; Gill et al., 1998; Kaaya et al., 1992; Roth et al., 1988). However, these cells have an activated phenotype, and after inoculation in nude mice they promote the formation of highly angiogenic KS-like lesions of mouse cell origin that is mediated by the paracrine action of cytokines and angiogenic factors produced by the cells (Ensoli et al., 1989, 1994a, 1994b; Samaniego et al., 1995; Salahuddin et al., 1988). This supports the concept that KSC, at least in early stage, are reactive cells and not tumor cells. However, in late stage KS can transform into a true sarcoma and KSC can become monoclonal (Judde et al., 2000; Rabkin et al., 1995, 1997), as also indicated by the establishment of a few transformed KS cell lines from late-stage lesions (Albini et al., 1997; Herndier et al., 1994; Lunardi-Iskandar et al., 1995). As discussed below EC present in KS lesions, lesional KSC, and the circulating KSC from patient’s PBMC are all infected by HHV-8 (Boshoff et al., 1995; Davis et al., 1997; Dupin et al., 1999; Foreman et al., 1997; Monini ¨ et al., 1999a; Sirianni et al., 1997; Staskus et al., 1997; Sturzl et al., 1997b, 1999a, 1999b).

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IV. HHV-8 INFECTION IN KS LESIONS HHV-8 infection of EC and KSC is a speciﬁc trait of KS lesions (Blasig et al., 1997; Boshoff et al., 1995; Davis et al., 1997; Dupin et al., 1999; ¨ Foreman et al., 1997; Sturzl et al., 1997b, 1999a, 1999b). However, most studies indicate that only a small fraction of the cells is infected in earlypatch lesions, whereas both viral load and gene expression increase as lesions progress to more advanced stages and are maximal in late-nodular lesions (Blasig et al., 1997; Davis et al., 1997; Dupin et al., 1999; Katano et al., 1999a, 1999b, 2000; Kellam et al., 1999; Linderoth et al., 1999; Parravicini et al., 2000; Rainbow et al., 1997; Reed et al., 1998; Staskus et al., 1997; ¨ ¨ Sturzl et al., 1997b, 1999a, 1999b) (see also the chapter by Sturzl et al. in this volume). HHV-8 DNA is detected in both EC and KSC present in advanced lesions (Boshoff et al., 1995; Foreman et al., 1997). Both these cell types express HHV-8 latency genes at the RNA and protein level (Blasig et al., 1997; Davis et al., 1997; Dittmer et al., 1998; Dupin et al., 1999; Katano et al., 1998a, 1999b, 2000; Kellam et al., 1999; Linderoth et al., 1999; Parravicini et al., 2000; Rainbow et al., 1997; Reed et al., 1998; Staskus et al., 1997, 1999; ¨ Sturzl et al., 1997b, 1999a, 1999b; Sun et al., 1999). Evidence indicates, however, that EC and KSC may express different HHV-8 latency programs. In fact, compared to KSC, normal EC do not express the HHV-8 latencyassociated nuclear antigen (LANA) and show low and rare expression of other HHV-8 latency-associated products, including viral cyclin D (vCycD), viral FLICE inhibitory protein (vFLIP), and kaposin (Bobroski et al., 1998; ¨ Davis et al., 1997; Dupin et al., 1999; Linderoth et al., 1999; Sturzl et al., ¨ 1997b, 1999a) (see also the chapter by Sturzl et al. in this volume). In contrast, lytic infection is always low and conﬁned to a very small fraction of cells including monocytes and T cells inﬁltrating KS tissue. These cells express the whole spectrum of HHV-8 genes, including viral genes endowed with potential paracrine actions (Blasig et al., 1997; Katano et al., 1999a, 1999b, 2000; Orenstein et al., 1997; Parravicini et al., 2000; Said et al., 1997; Staskus et al., 1997, 1999). Replication of HHV-8 in these cells may provide a reservoir of virus that may be required for persistent infection of KSC, as suggested by the loss of HHV-8 infection upon culture of KSC from lesions (Dictor et al., 1996; Lebb`e et al., 1995; Monini et al., 1996). However, as dicussed above, KSC may be recruited into KS lesions from the peripheral blood (Browning et al., 1994; Monini et al., 1999a; Sirianni et al., 1997; Uccini et al., 1997). These circulating KSC are also latently infected by HHV-8 (Davis et al., 1997; Monini et al., 1999a; Sirianni et al., 1997), and their extravasation into tissues is facilitated by the activated endothelium present in individuals with KS or at risk of KS (Fiorelli et al, 1998;

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MacPhail et al., 1996; Yang et al., 1994; Zhang et al., 1994; Zietz et al., 1996).

V. KS INITIATION: ROLE OF IC IN KS HISTOGENESIS AND HHV-8 INFECTION A. Role of IC in KS Histogenesis Inoculation of IC in mice induces KS-like lesions of mouse cell origin that closely resemble primary KS lesions in humans (Barillari et al., 1999a) and regress as KS lesions do. These data indicate that IC can trigger a cascade of events leading to KS lesion formation. IC, in fact, participate in KS histogenesis through several mechanisms (Table I). They activate EC, leading to adhesion molecule expression and production of chemokines that mediate the extravasation and tissue recruitment of inﬂammatory cells (Bevilacqua et al., 1985; Cavender et al., 1991; Cotrand and Pober, 1988; Gamble et al., 1985; Kerbel and Hawley, 1995; ¨ Mantovani et al., 1997; Pober and Cotran, 1990; Sciacca et al., 1994; Sturzl et al., 1997a; Zimmerman and Hill, 1984); activate EC to acquire the phenotypic and functional features of KSC (Albini et al., 1995; Barillari et al., 1992, 1993, 1999a, 1999b; Fiorelli et al., 1995, 1998, 1999; Samaniego et al, 1997, 1998); and induce EC and KSC to produce and release angiogenic factors including bFGF and VEGF that, in turn, mediate EC and KSC proliferation, angiogenesis, and edema (Cornali et al., 1996; Ensoli et al., 1989; Faris et al., 1998; Samaniego et al., 1995, 1997, 1998). IC also have proliferative and survival effects on both lesional and circulating KSC. In fact, primary cultures of KSC of both endothelial (E-KSC) or macrophagic (M-KSC) cell origin have been established from KS lesions by utilizing the same combination of IC as found in the lesions (Barillari et al., 1992; Nakamura et al., 1988; Sirianni et al., 1998). Similarly, IC promote the differentiation of circulating KSC from PBMC cultures of patients with KS or at risk of KS (Table I) (Browning et al., 1994; Monini et al., 1999a). Together, these results indicate that the IC produced in KS lesions are capable of triggering a cascade of events leading to lesion development.

1. ACTIVATION OF THE VASCULAR ENDOTHELIUM BY IC AND ROLE IN CELL RECRUITMENT The systemic and local production of IC found in individuals at risk of KS or with KS activate the vascular endothelium and induces the extravasation and tissue recruitment of inﬂammatory cells (Table I). In fact, HIV-1–infected patients show a generalized activation of the vessel

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endothelium, with expression of adhesion molecules (ICAM-1, ELAM-1, V-CAM-1), increased adhesion and extravasation of lympho-monocytes, increased endothelial cell permeability, and increased serum levels of FVIII-RA, which is released by activated EC (Gariano et al., 1993; Petito and Cash, 1992; Pober and Cotran, 1990; Rhodes, 1991; Zietz et al., 1996). EC lining lesional vessels have the same phenotype. These “activated tissue sites” are characterized by enhanced extravasation of circulating cells, which also produce IC, and may represent sites prone to KS lesion development (see the ¨ et al. in this volume). This view is supported by recent data chapter by Sturzl showing that uninvolved skin from KS patients, but not skin from subjects with other dermatologic disorders, is characterized by the presence of tissue foci expressing IFN-␥ and human leukocyte antigen (HLA)-DR (Fiorelli et al., 1998). These tissue changes induced by IC may be key to KS development; in fact, as discussed later, IC produced at these tissue sites induce EC to acquire the phenotype of KSC and to reactivate HHV-8 infection in resident infected cells.

2. INDUCTION OF SPINDLE CELL FORMATION BY IC The same IC increased in KS and in individuals at risk of KS activate normal EC to acquire the phenotypic and functional features of E-KSC. These include the typical spindle cell morphology, the expression of the same markers (downregulation of factor VIII-related antigen (FVIII-RA), activation of ELAM-1, ICAM-1, VCAM-1, DR, ␣5␤1, ␣v␤3 integrin expression), and the angiogenic phenotype (Barillari et al., 1992, 1999a; Faris, 1998; Fiorelli et al., 1995, 1998; Samaniego et al., 1995, 1997, 1998) (Table I). IC, in fact, induce EC to promote the formation of KS-like lesions after inoculation in nude mice as E-KSC do (Fig. 1C) (Table I) (Ensoli et al., 1989, 1994a, 1994b; Fiorelli et al., 1995, 1998; Salahuddin et al., 1988; Samaniego et al., 1997, 1998). As discussed later, IC also induce normal EC to become responsive to the adhesive, mitogenic, and invasive effects of extracellular HIV-1 Tat protein (Table I) that is a typical feature of E-KSC (Albini et al., 1995; Barillari et al., 1992, 1993, 1999a, 1999b; Chang et al., 1997; Ensoli et al., 1990, 1993; Fiorelli et al., 1995, 1999). This leads to augmented angiogenesis and spindle cell growth in AIDS-KS (Ensoli et al., 1994a). It should be pointed out, however, that a few differences still exist between IC-activated EC and E-KSC. In fact, unlike EC, E-KSC produce VEGF in large amounts but do not proliferate in response to VEGF, despite the fact that the level of expression of VEGF receptors is similar in these two cell types (Brown et al., 1996; Cornali et al., 1996; Samaniego et al., 1998). In addition, KSC, but not IC-activated EC, proliferate in response to Tat peptides carrying the RGD sequence (Barillari et al., 1999b), which is known to mediate Tat binding to surface integrins expressed by IC-activated EC and KSC (Barillari et al., 1993, 1999a, 1999b; Ensoli et al., 1994a). These data indicate that

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E-KSC have acquired a “trans-differentiated” phenotype, although they are not transformed nor tumorigenic.

3. INDUCTION OF ANGIOGENESIS, EDEMA, AND LESION FORMATION BY IC IC induce EC and KSC to produce angiogenic molecules, growth factors, and chemokines (Table I). These molecules have been shown to be produced at high levels in all lesion stages, to mediate KSC growth, angiogenesis, and edema, and to recruit circulating cells that transmigrate through the activated endothelia. bFGF and VEGF are potent angiogenic factors that are induced by IC (Cornali et al., 1996; Faris et al., 1998; Fiorelli et al., 1998; Samaniego et al., 1995, 1997, 1998). Both are present at high levels in sera from patients with ¨ KS or at risk of KS (Ascherl et al., 1999; B. Ensoli and M. Sturzl, unpublished data) and are highly expressed in KS lesions (Figs. 1B and 1C) (Cornali et al., 1996; Samaniego et al., 1998; Xerri et al., 1991). Evidence indicate that these angiogenic factors are the major mediators of KSC growth, angiogenesis, and edema present in KS. bFGF is released by KSC and IC-activated EC in the absence of cell death or cell permeability changes (Ensoli et al., 1989; Fiorelli et al., 1998; Samaniego et al., 1995, 1997, 1998), has autocrine and paracrine growth and chemotactic activities, and stimulates angiogenesis (Table I) (Ensoli et al., 1989, 1994a; Faris et al., 1998; Samaniego et al., 1995, 1997, 1998). Due to the production of bFGF, E-KSC or IC-activated EC are highly angiogenic in the chorioallantoic membrane assay and, upon inoculation in nude mice, they induce angiogenic lesions of mouse cell origin that closely resemble KS and regress as human lesions can regress (Fig. 1C) (Ensoli et al., 1989, 1994a, 1994b; Fiorelli et al., 1995, 1998; Salahuddin et al., 1988; Samaniego et al., 1995, 1997, 1998). The expression of bFGF is detected at both the RNA and protein level in both primary KS lesions (Fig. 1B), KS-like mice lesions (Fig. 1C), and primary KS cells or IC-activated EC (Barillari et al., 1992; Ensoli et al., 1989, 1994a; Faris et al., 1998; Fiorelli et al., 1995, 1998; Samaniego et al., 1995, 1997, 1998; Xerri et al., 1991). In addition, the inoculation of bFGF in nude mice results in the formation of KS-like lesions (Ensoli et al., 1994a; Samaniego et al., 1998). Although KSC produce other angiogenic factors (see later), inhibition studies with neutralizing antibodies or antisense oligodeoxynucleotides directed against bFGF have shown that bFGF is the autocrine growth factor for KSC and it is necessary and sufﬁcient for the formation of KS-like lesions in mice (Ensoli et al., 1999b). VEGF, another angiogenic factor highly expressed in KS lesions (Fig. 1B) (Table I), is also produced by cultured KSC and synergizes with bFGF in inducing EC growth, angiogenesis, and edema (Table I) (Cornali et al., 1996;

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Samaniego et al., 1998; Weindel et al., 1992). VEGF production is induced by IC in E-KSC but, as discussed earlier, these cells do not proliferate in response to VEGF although they express the VEGF receptors KDR/FLK-1 and ﬂt-1 in vitro and in vivo (Brown et al., 1996; Cornali et al., 1996; Samaniego et al., 1998). VEGF, however, mediates the growth of two transformed KS cell lines established from KS lesions (Masood et al., 1997). Other angiogenic molecules are found in KS lesions, including the scatter factor/hepatocyte growth factor (SF/HGF), which also induces EC to acquire a spindle morphology and stimulates proliferation of cultured KS spindle cells (Naidu et al., 1994; Rosen and Goldberg, 1995), and platelet-derived growth factor (PDGF)-B (Brown et al., 1995), which is a potent mitogen ¨ for cultured E-KSC (Roth et al., 1989; Sturzl et al., 1995; Werner et al., 1990) and is expressed in vivo by cells that are intermingled with the spindle ¨ cells (Sturzl et al., 1992, 1995) (Table I). E-KSC, in addition, express both the protein c-met, which functions as receptor for SF/HGF, and the PDGF ¨ et al., 1995; Werner ␤-receptor (Maier et al., 1996; Naidu et al., 1994; Sturzl et al., 1990), suggesting that SF/HGF and PDGF may also play a role in KS development. IC produced in KS lesions also induce the expression of several chemokines that mediate cell recruitment into tissues. In particular, the monocyte chemotactic protein-1 (MCP-1) and IL-8 are expressed by E-KSC or IC-activated EC in vitro and in vivo and may contribute to the recruitment of monocytes ¨ into KS lesions (Table I) (Mantovani et al., 1997; Sciacca et al., 1994; Sturzl et al., 1997a). IL-8 also shows migratory effects on E-KSC and EC and thus may also contribute to angiogenesis (B. Ensoli, unpublished data). IFN-␥ inducible protein-10 (IP-10) and Mig are expressed in AIDS-KS lesions by inﬁltrating inﬂammatory cells, EC, and KSC (Ensoli, unpublished data), most likely in response to IFN-␥ , which is produced in large amounts in KS lesions (Liao et al., 1995; Sgadari et al., 1996, 1997). In addition, the macrophage inﬂammatory protein-1 (MIP-1) alpha and beta and RANTES are expressed by lesional TIL and PBMC from KS patients and are increased in sera from patients at risk of KS (B. Ensoli, unpublished data). Of note, some of these chemokines also modulate the constitutive signaling by the HHV-8–encoded (viral) G-protein–coupled receptor/IL-8 receptor (v-GPCR/vIL-8R), which, in turn, has been suggested to have a role in KS angiogenesis and lesion development (Bais et al., 1998; Geras-Raaka et al., 1998a, 1998b; Gershengorn et al., 1998; Rosenkilde et al., 1999).

B. Effects of IC on HHV-8 Infection Recent evidence indicates that IC induce HHV-8 reactivation in latently infected cells (Monini et al., 1999a). This leads to viremia and to virus spread to

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all circulating cell types including monocytes and T cells, which are numerous in KS lesions whereas B cells are few or absent (Fiorelli et al., 1998; Monini et al., 1999a; Sirianni et al., 1998). Reactivation of HHV-8 in inﬂammatory cells inﬁltrating KS lesions may also be key for the infection of resident EC and KSC and may lead to the increased virus load that is observed as lesions progress from early to late-nodular stages (Dupin et al., 1999; Monini et al., ¨ 1999a; Sturzl et al., 1997b). IC also promote the recruitment of HHV-8– infected cells into tissues via the activation of the vascular endothelium and ¨ et al., 2000). Finally, the induction of chemokines (Blasig et al., 1997; Sturzl during lytic gene expression, viral proteins with potential paracrine effects on KS lesion formation are produced (see below).

1. EVIDENCE OF HHV-8 REACTIVATION IN INDIVIDUALS WITH KS AND AT RISK OF KS DEVELOPMENT Several lines of evidence indicate that HHV-8 infection is reactivated in individuals with KS or at risk of KS in response to IC and that this leads to virus spreading and tissue recruitment of infected cells which is enhanced by the lack of immune control of the virus. As compared to HHV-8–infected healthy individuals, patients with KS or at risk of KS show increased levels of Th-1–type cytokines in sera and tissues (Caruso et al., 1990; Fagiolo et al., 1993; Fan et al., 1993; Fiorelli et al., 1998; Hober et al., 1989; Honda et al., 1990; Lahdevirta et al., 1988; LepeZanuga et al., 1987; Rizzardini et al., 1996; Sirianni et al., 1998; Vyakarnam et al., 1991; B. Ensoli, unpublished data) and higher anti-HHV-8 antibody titers and viral load in PBMC, uninvolved tissues, and body ﬂuids, including plasma, serum, nasal secretions, saliva, and sperm, or show evidence of HHV-8–productive replication in PBMC (Blackbourn et al., 1998; Cattani et al., 1999; Corbellino et al., 1996; Decker et al., 1996; Fiorelli, 1998; Harrington et al., 1996; Howard et al., 1997; Koelle et al., 1997; LaDuca et al., 1998; Lucht et al., 1998; Monini et al., 1999a, 1999b; Moore et al., 1996a; Vieira et al., 1997; Whitby, 1995) (Table II). Reactivation of HHV-8 infection is frequently found in kidney transplant recipients, who show increased titers of antibodies directed against HHV-8 lytic antigens (antilytic antibodies) compared to healthy people (Hudnall et al., 1998; Mendez et al., 1999) (Table II). In regions with a high HHV-8 seroprevalence, PT-KS development is associated with virus reactivation rather than with primary infection through donor organ allograft (Farge et al., 1998, 1999; Franc`es et al., 1999; Mendez et al., 1999; Nocera et al., 1998; Parravicini et al., 1997; Regamey et al., 1998, 1999), and the degree of HHV-8 viremia correlates with the degree of iatrogenic immunosuppression (Moosa et al., 1998). Antilytic antibody titers and PBMC-associated viremia are found to be particularly high in patients with CKS (Brambilla et al., 1996;

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Table II Human Herpesvirus 8 Reactivation and Virus Spreading upon Kaposi’s Sarcoma Development A. Evidence of HHV-8 activation in patients with KS or at risk of KS 1. High HHV-8 load in PBMC 2. Presence of viral DNA in plasma, serum, nasal secretions, saliva, sperm 3. Presence of viral DNA in uninvolved tissues including all lymphoid organs and prostate 4. Evidence of virus-productive replication in PBMC 5. Presence of high anti latent and antilytic antibody titers 6. Evidence of HHV-8 reactivation in PT-KS B. Factors involved in HHV-8 reactivation in PBMC or PEL cell lines 1. IC (combined), IFN-␥ , IL-1␤, OSM, SF/HGF 2. HIV infection: HIV Tat (?) 3. Allogeneic stimulation (?) C. Immunologic conditions promoting HHV-8 spreading upon virus reactivation 1. Deﬁcient immunity (HIV infection, iatrogenic immunosuppression) 2. Inefﬁcacy of CTL responses due to expression of vFLIP or KIR upregulation 3. Deﬁcient NK-cell cytotoxic activity 4. Virus immune evasion (expression of HHV-8 K3 and K5 ORFs) 5. Deﬁcient IFN-␣ response (?) PBMC, peripheral blood mononuclear cells; PT-KS, posttransplant KS; IFN-␥ , interferongamma; IL-1, interleukin-1; OSM, oncostatin-M; SF/HGF, scatter factor/hepatocyte growth factor; CTL cytotoxic T lymphocyte; NK, natural killer; KIR killing inhibitory receptor; FLIP, ﬂice inhibitory peotein; IFN-␣, interferon-alpha. The table summarizes the major features indicating HHV-8 reactivation in KS, the factors involved or inducing HHV-8 reactivation and virus spreading in blood and tissues.

B. Ensoli, unpublished results). These data suggest that HHV-8 reactivation is not due primarily to immune suppression, which however, allows virus dissemination due to the impaired clearance of productively infected cells (see later). A condition of persistent immune activation followed by severe immune deﬁciency characterizes HIV-1–infected individuals (Caruso et al., 1990; DePaoli et al., 1994; Fan et al., 1993; Hober et al., 1989; Honda et al., 1990; Kalinkovich et al., 1993; Lahdevirta et al., 1988; Lepe-Zanuga et al., 1987; Schlesinger et al., 1994; Vyakarnam et al., 1991). These subjects also show a persistent or intermittent PBMC-associated HHV-8 viremia (Harrington et al., 1996; Decker et al., 1996; Monini et al., 1999a, 1999b; Moore et al., 1996a; Whitby et al., 1995) high antilytic HHV-8 antibody titers, (Gao et al., 1996a, 1996b; Jacobson et al., 2000; and high levels of antibodies directed agaist HHV-8 latency-associated antigens (antilatent antibodies). Kedes et al., 1996; Martin et al., 1998; Renwick et al., 1998; Rezza et al., 1999). In these individuals, the risk of KS development further increases with increasing antilytic antibody titers, while low CD4+ T-cell counts are an additional independent risk factor for KS development (Rezza et al., 1999).

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However, HHV-8 viremia and the rise in antibody titers against HHV-8 occur months or years before KS development. These data are strongly suggestive of early HHV-8 reactivation upon immune activation, followed by virus dissemination due to the intervening immune deﬁciency. This is also suggested by recent longitudinal studies showing that in individuals at risk for HIV-1 and HHV-8 infection, KS onset is never observed prior to co-infection (Jacobson et al., 2000; Renwick et al., 1998; Rezza et al., 1999), and that the progression rate to KS is signiﬁcantly lower in individuals who become infected by HHV-8 prior to HIV-1 infection, compared to those already infected by HIV-1 (Jacobson et al., 2000; Renwick et al., 1998).

2. REACTIVATION OF LATENT HHV-8 IN CIRCULATING CELLS BY IC Recent work indicates that the high HHV-8 load that is observed in patients with KS or at risk of KS is due to reactivation of latent HHV-8 by the IC that are increased in KS. Evidence for this comes from the observation that PBMC from KS patients lose the virus upon culture but maintain HHV-8 DNA or increase HHV-8 DNA load when cultured in the presence of the same IC increased in KS (Fig. 2A) (Monini et al., 1999a). These effects on virus load are accompanied by the activation of HHV-8 lytic gene expression that is found both in lymphocytes and in cells of monocytic origin upon culture with IC (Tables I and II, Figs. 2B and 2C, see color plate) (Monini et al., 1999a). In addition, in KS patients or at-risk individuals, PBMC, which are negative for HHV-8 DNA prior to or after short-term culture, can become PCRpositive upon several weeks of culture in the presence of IC (Fig. 2A) (Monini et al., 1999a). This is strongly suggestive of virus reactivation upon chronic exposure to IC and is similar to the reactivation of human cytomegalovirus that is observed in PBMC after prolonged allogeneic stimulation (Table II) ¨ (Soderberg-Naucl´ er et al., 1997). IFN-␥ appears to be key for these effects (Fig. 2D), although other IC may contribute to it (Table II) (Monini et al., 1999a). Since IFN-␥ and other IC are produced at high levels during HIV-1 infection, these data may explain why HHV-8 infection is associated with a faster progression to KS in the setting of HIV-1 infection as compared to HIV-1–negative individuals (Jacobson et al., 2000; Renwick et al., 1998). However, part of these effects may also be related to the expansion of a pool of HHV-8 latently infected cells, since the same IC increased in KS can also prolong the survival of B cells (Monini et al., 1999a), which are the circulating cell type infected by HHV-8 in most individuals including normal subjects (Ambroziack et al., 1995; Blackbourn et al., 1997; Monini et al., 1999a). In addition, IC induce the growth and differentiation of circulating KSC (Browning et al., 1994; Monini et al., 1999a), which are also latently infected by HHV-8 (Davis et al., 1997; Monini et al., 1999a; Sirianni

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et al., 1997). Noteworthy, PBMC cultures exposed to IC show expression of HHV-8 lytic genes after short-term culture, but, unlike control cultures, they subsequently maintain viral DNA in the absence of detectable gene expression, suggesting that in the presence of IC, virus reactivation is followed by a persistent, latent infection with a very low gene expression (B. Ensoli, unpublished data). Although additional studies are required, these data indicate that IC induce HHV-8 lytic replication in PBMC and may also expand the pool of latently infected cells (Table I). In support of this concept are also recent data showing that recombinant IC or IC from HIV-1–infected cells including IFN-␥ , IL-1␤, oncostatin M (OSM), or HGF/SF increase the expression of both HHV-8 lytic and latency genes in chronically infected primary effusion lymphoma (PEL)-derived cell lines (Tables I and II) (Blackbourn et al., 2000; Chang et al., 2000; Mercader et al., 2000; Yu et al., 1999).

3. SPREAD OF HHV-8 INFECTION TO CIRCULATING CELLS AND TISSUE RECRUITMENT OF INFECTED CELLS The data reviewed above indicate that circulating cells of hematopoietic origin are a site of virus reactivation in patients with KS or at risk of KS. However, limited studies have been performed to identify the circulating cell types infected by HHV-8 in KS patients as compared to risk individuals and healthy donors. These studies have indicated that HHV-8 DNA is present in B cells from normal donors, but that in patients with KS, T cells and monocytes are also infected (Ambroziack et al., 1995; Blackbourn et al., 1997; Harrington et al., 1996; Howard et al., 1998; Sirianni, 1997). In our studies we have found that HHV-8 DNA is present in all mononuclear cell types, including B cells, CD4+ and CD8+ T cells, and monocytes in patients with all forms of KS (Monini et al., 1999a) (Fig. 3 and Table III). In contrast, in atrisk individuals HHV-8 is detected only in B cells and/or monocytes, whereas T cells are not infected except for PT patients under immunosuppressive therapy (B. Ensoli, unpublished data). HHV-8 is also found in circulating KSC from KS patients but not in healthy donors or patients with other dermatologic disorders (Monini et al., 1999a; Sirianni et al., 1997). These data suggest that HHV-8 has a broad tropism for hematopoietic cells but that infection of all blood mononuclear cell types, and particularly T cells, may occur only upon KS development. The factors responsible for the spread of HHV-8 infection to all circulating cell types, particularly T cells, are not completely understood. However, these likely include both IC-induced HHV-8 reactivation and immune deﬁciency, as indicated by the infection of T cells in PT patients who are immunosuppressed as, in various degree, are also patients with other forms of KS. In addition, virus immune evasion mechanisms allowing HHV-8–infected cells

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Fig. 3 Semiquantitative PCR analysis of HHV-8 DNA load in PBMC, and puriﬁed B cells

(CD19+), T cells (CD3+CD4+ or CD3+CD8+), and monocytes (CD14+) from a PT-KS patient. Cell extracts from the same number of cells were serial diluted and analyzed for HHV-8 DNA by PCR. FACS analysis (see Table III) showed that CD4 and CD8 positive cells did not contain CD19-or CD24-positive cells, indicating that the PCR signals from T cells were not due to contaminant cells. NC, negative controls made without the addition of DNA template; PC, positive controls consisting of 5 ng of DNA puriﬁed from a KS lesion (KS) and/or the indicated number of molecules of a plasmid containing the HHV-8 target sequences.

to escape the control of cytotoxic T cells or natural killer (NK) cells may also be involved (see later). Spread of viral infection to all circulating cell types appear to be key for virus spread to tissues (Tables I, II, and IV). In particular, since B cells are rare or absent in KS lesions (Fiorelli et al., 1998; Sirianni et al., 1998), infected circulating monocytes may recruit the virus into tissues and, upon exposure to IC, may undergo lytic infection and transmit the virus to neighbor cells

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Table III Cytoﬂuorimetric Analysis of PBMC and Puriﬁed Cell Populations from a PT-KS Patient Analyzed by DNA-PCR for the Presence of HHV-8 DNA Cytoﬂuorimetric analysis (% of positive cells) Cell fraction

CD3+CD4+

CD3+CD8+

CD19+

CD14+

PBMC CD4+ CD8+ CD14+ CD19+

36 99 0.5 0 ND

25 0 99 15 ND

0.4 0 0 0 69

16 0 0 47 10

PT-KS, posttransplant KS; PCR, polymerase chain reaction; NA, not available. Representative cytoﬂuorimetric analysis from a PT-KS patient showing the percentage of cells expressing markers of B, T, or monocytic cells.

(Table III) (Blasig et al., 1997; Monini et al., 1999a). This is also supported by data showing that monocyte-macrophages are present in KS lesions and are productively infected by HHV-8 (Blasig et al., 1997; Fiorelli et al., 1998; ¨ Orenstein et al., 1997; Sirianni et al., 1998; Sturzl et al., 2000; Uccini et al., 1994). Latently infected circulating KSC may also be recruited into lesions (Davis et al., 1997; Monini et al., 1999a; Sirianni et al., 1997; Uccini et al., 1997), as indicated by the presence in lesions of resident KSC expressing the endothelial-macrophage phenotype (Uccini et al., 1994, 1997) (Tables I and IV). The activated endothelium present in subjects at risk for KS is an important target for the homing of HHV-8–infected lympho-monocytes. In this context, it is of interest that HHV-8 encodes two lytic gene products, including a Table IV Effects of Human Herpesvirus 8 Reactivation in Kaposi’s Sarcoma Development 1. Spread of HHV-8 infection to all circulating cell types 2. Localization and dissemination of HHV-8 infection to tissues via transmigration of infected lymphocytes/monocytes 3. Transmission of viral infection to lesional EC and KSC, or circulating KSC with the endothelial-macrophage phenotype 4. Induction of chronic, nonefﬁcacious immunoresponses against HHV-8 at the systemic and local level (CTL, CD8+ and CD4+ IFN-␥ –expressing tissue-inﬁltrating cells) with further IC induction 5. Paracrine effects (?) 6. Exacerbation of all KS reactive processes KSC, KS spindle cells; EC, endothelial cells; CTL, cytotoxic T lymphocytes; IFN-␥ , interferon-gamma; IC, inﬂammatory cytokines. The major effects of HHV-8 reactivation in KS development are found in association with lack of immunologic control of the virus.

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chemokine receptor (vGPCR) (Bais et al., 1998; Cesarman et al., 1996; Russo et al., 1996) and a homolog of adhesion molecules (viral adhesion molecule/ viral neural cell adhesion molecule, vAdh/vNCAM) (Russo et al., 1996) that may be speciﬁcally involved in the homing and adhesion of HHV-8–infected cells to activated EC. However, recent work has indicated that EC activation and production of IC, in particular IFN-␥ , can be detected in early KS lesions and uninvolved tissue even prior to detection of HHV-8 by PCR (Fiorelli et al., 1998). This, and the low HHV-8 load in early lesions as compared to nodular-tumor stage, indicates that HHV-8 reactivation and virus dissemination participates in the reactive processes that precede KS development but may not be sufﬁcient to initiate KS.

4. PARACRINE EFFECTS OF HHV-8 REACTIVATION IN KS INITIATION Lytic HHV-8 infection may also contribute to viral pathogenesis due to paracrine effects of viral gene products. In fact, HHV-8 encodes viral lytic products with homology to cytokines and chemokines such as vIL-6 (Aoki et al., 1999; Cannon et al., 1999; Moore et al., 1996b; Staskus et al., 1999) and viral MIP I, II, and III (vMIP I, II, and III) (Boshoff et al., 1998; Kledal ¨ et al., 1997; Moore et al., 1996b; Stine et al., 2000; Sturzl et al., 1998), or endowed with indirect paracrine actions such as vGPCR (Bais et al., 1998). These viral products have been suggested to play a role in KS (Bais et al., 1998; Boshoff et al., 1997; Kledal et al., 1997; Stine et al., 2000). However, this is yet unclear, because paracrine effects such as induction of angiogenesis by vMIPs (Boshoff et al., 1997; Stine et al., 2000) have been observed only at very high molecule concentration, which is unlikely to be achieved in the few lesional cells undergoing virus reactivation. Similarly, in KS lesions vIL6 is undetectable or expressed at very low levels (Cannon et al., 1999; Staskus et al., 1999) (Table V). The HHV-8 MIP homologs show multiple actions including inhibition or promotion of cell chemotaxis, but they are also potent Th-2 cell chemoattractants and inhibitors of monocyte chemotaxis (Endres et al., 1999; Kledal et al., 1996; Sozzani et al., 1998; Stine et al., 2000). These effects, however, are in contrast with the immunophenotype of inﬁltrating cells in KS (Table V) (Fiorelli et al., 1998; Sirianni et al., 1998). vGPCR can induce KS-like lesions in transgenic mice due to the production and release of VEGF in tissues by vGPCR-expressing circulating cells (Yang et al., 2000); however, in the natural host, vGPCR expression can only be limited and transient as it only occurs in a few productively infected cells undergoing cell lysis. In addition, vGPCR expression is associated with the expression of vMIP-II, which acts as a vGPCR inhibitor (Geras-Raaka et al., 1998b)

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Table V Activity of Human Herpesvirus 8-Encoded Factors with Paracrine Effects

Factor

Effects suggesting of a role in KS initiation

Features in contrast with a role in KS initiation

vIL-6

Angiogenic effects (mediated by VEGF); signaling independent from IL-6R (only gp 130 is required)

Absent or very low-level expression in KS lesions; transient expression due to lytic HHV-8 infection

vMIPs

Recruitment of cells into tissues; angiogenic in the chrioallantoic membrane assay

Chemotactic for Th-2 but not Th-1 cells; potent inhibitors of monocyte chemotaxis; role in KS angiogenesis unclear, due to high-level expression of host angiogenic factors

vGPCR

Angiogenic effects (mediated by VEGF); upregulated by IL-8; development of KS-like lesions in transgenic mice (due to VEGF production)

Transient expression due to HHV-8 lytic infection; down-modulated by IP-10, vMIP-II; high-level expression of VEGF by KSC negative for vGPCR expression

VIL-6, viral interleukin-6; vMIP, viral macrophage inﬂammatory protein; vGPCR, viral G-protein–coupled receptor; Th-1/2, T-helper type 1/2; IP-10, interferon-gamma-inducible protein-10; VEGF, vascular endothelial growth factor; KSC, KS spindle cells; IL-6R, IL-6 receptor; gp 130, signaling molecule required for IL-6 activity. The table summarizes the most relevant actions and features of HHV-8 lytic gene products endowed with paracrine actions.

(Table V). As large amounts of VEGF are also produced by lesional KSC (Cornali et al., 1996; Samaniego et al., 1998; Weindel et al., 1992), which are negative for vGPCR expression (Kirshner et al., 1999), most of the VEGF production in KS lesion may be substained by cells that do not express the HHV-8–encoded GPCR. These data argue, therefore, against a paracrine effect of HHV-8 lytic genes in KS initiation (Table V) (see also the chapter ¨ by Sturzl et al. in this volume).

VI. LACK OF CONTROL OF REACTIVATED HHV-8 Sporadic herpesvirus reactivation can occur in healthy individuals; however, this is controlled by the immune system and produces asymptomatic or modest and self-limiting pathological, conditions. By contrast, in the immunocompromised host, herpesvirus reactivation may cause lifethreatening illness (Arvin, 1996; Whitley, 1996; Britt and Alford, 1996). Evidence indicates that the lack of control of HHV-8 reactivation plays a role in KS development since it allows virus dissemination in blood and tissues (Table II).

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Impaired clearance of HHV-8–infected circulating cells may be key for KS development, as indicated by recent studies showing that KS regression or remission is associated with clearance of HHV-8 DNA from the circulation. For example, regression of AIDS-KS in response to HAART is associated with clearance of HHV-8 DNA from PBMC (Conant et al., 1997; Lebb`e et al., 1998; Murphy et al., 1997; Rizzieri et al., 1997; Sirianni et al., 1999; Wit et al., 1998; B. Ensoli, unpublished data), whereas persistent viremia is observed in patients showing KS worsening upon HAART (Sirianni et al., 1999; B. Ensoli, unpublished data). KS regression upon systemic administration of IFN-␣ is also accompanied by clearance of HHV-8 from PBMC and by a decrease of antilytic HHV-8 antibody titers (Monini et al., 1999c; B. Ensoli, unpublished data). These data suggest that the efﬁcacy of HAART or systemic IFN-␣ in inducing KS regression is due to improved clearance of HHV-8 from circulating cells that, in KS patients, is reduced and insufﬁcient (see later). Several factors appear to be responsible for a decreased or impaired immunological control of HHV-8 infection in KS. These include overt immune deﬁciency as in AIDS-KS, iatrogenic immune suppression as in PT-KS, or virus escape mechanisms and impaired NK cytotoxic activity likely present in all forms of KS (Table II) (see later). Recent data have indicated the presence of HHV-8–speciﬁc CTLs and T-helper cells in peripheral blood from KS patients (Osman et al., 1999; Strickler et al., 1999). KS lesions themselves are inﬁltrated by numerous activated (i.e. IFN-␥ producing) CD4+ and CD8+ T cells and NK cells (Fiorelli et al., 1998; Sirianni et al., 1998). These cells, however, are unable to clear HHV-8–infected cells from circulation or tissues. The reasons for this are poorly understood, but both virus and/or host mechanisms may be involved. In particular, recent data point to the expression of v-FLIP as a mechanism to avoid killing of latently infected cells by HHV-8–speciﬁc CTLs due to the inhibition of apoptosis mediated by the FAS pathway (Table II) (Djerbi ¨ et al. in this volume). Furthermore, et al., 1999; see also the chapter by Sturzl HHV-8 products encoded by the viral open reading frames (ORFs) K3 and K5 act by downregulating major histocompatibility class I (MHC-I) and coactivation molecules, and inhibit killing of HHV-8–infected cells by speciﬁc CTLs or by NK cells (Table II) (Coscoy et al., 2000; Haque et al., 2000; Ishido et al., 2000a, 2000b). These viral products are expressed upon virus reactivation (Rimessi et al., 2000) and may allow circulating or inﬁltrating lympho-monocytes to undergo a complete virus replication cycle despite the presence of effector cells. In addition to the overt immune suppression that is found in AIDS and PT patients, a trend to a decline in CD4+ T-cell counts is found in patients with C-KS and A-KS (Touloumi et al., 1999; Urassa et al., 1998). Our recent work, moreover, indicates the presence of decreased NK cell cytotoxic responses

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in patients with KS as compared to matched controls (Sirianni et al., 1999; B. Ensoli, unpublished data). Finally, our recent data indicate that the killing inhibitory receptors, (KIRs) are upregulated in both T cells and NK cells from KS patients or HIV-infected individuals without KS, suggesting that this is another mechanisms of altered immune control of HHV-8 that may contribute to the lack of killing of infected target cells in individuals at risk of KS or with KS in the absence of a manifest immunosuppression (Table II) (Sirianni et al., 2000; B. Ensoli, unpublished results). Recent studies have shown that recombinant IFN-␣ is a potent inhibitor of HHV-8 reactivation, replication, and assembly in infected PEL cells and is

Fig. 4 Effect of neutralizing anti-IFN-␣ Ab on HHV-8 DNA load in PBMC cultured from patients with AIDS-KS (A, D), HIV-1+ (B), or C-KS (C). Cells were analyzed prior to culture (PBMC day 0) or after short-term culture in the absence (RPMI) or presence of neutralizing anti-IFN-␣ Ab (Ab). In A and B, ﬂoating and adherent cells were harvested separately; in C and D, cells were harvested as a bulk. Cells were analyzed for viral DNA by PCR. The ﬁgure shows that neutralizing anti-IFN-␣ Ab increased HHV-8 DNA load from cells cultured in the absence (A, B, C) or presence (D) of IC (RTCM), suggesting that production of IFN-␣ may be a major mechanism for the control of HHV-8 reactivation by IC. NC, negative control made without template; PC, positive-control PCR reactions performed with the indicated number of molecules of a plasmid containing the target sequences or with DNA (1 ng) from a KS lesion. (Modiﬁed in part from Monini et al., 1999b.)

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capable of reducing HHV-8 load in cultured PBMC from patients with KS or at risk of KS (Chang et al., 2000; Monini et al., 1999b). In addition, HHV-8 infection is inhibited by endogenous IFN-␣ released by PBMC from patients with KS or at risk of KS, as indicated by the increase of viral load in PBMC cultured in the presence of IFN-␣–neutralizing antibodies (Monini et al., 1999b) (Fig. 4). These effects of IFN-␣ antibodies are also observed when cells are cultured in the presence of IC (Fig. 4), suggesting that production of IFN-␣ may be a major mechanism for the control of HHV-8 reactivation. Therefore, the investigation of the integrity of this natural response to HHV-8 infection in patients with KS or at risk of KS and of its efﬁcacy in inhibiting HHV-8 infection may be key to understanding HHV-8 control and requires further studies (Table II). Altogether, these results suggest that a variable degree of immunosuppression leading to virus spreading may be present in different settings or stages of KS development (Table II).

VII. KS PROGRESSION: ONCOGENES, ONCOSUPPRESSOR GENES, HHV-8 LATENCY GENES, AND THE HIV-1 Tat PROTEIN Although in early stages KS behaves as a reactive process, it can evolve into a real tumor and KSC can become monoclonal (Delabesse et al., 1997; Rabkin et al., 1995, 1997). In addition to the deregulated expression of oncogenes (Bcl-2, c-myc, c-int, ras) and oncosuppressor genes (p53) (Dada et al., 1996; Hodak et al., 1999; Huang et al., 1993b; Koster et al., 1996; Bohan-Morris et al., 1996; Noel et al., 1997; Pillay et al., 1999; Scincariello et al., 1994), this may de due to the long-lasting expression of HHV-8 latency genes (LANA, vCycD, vFLIP, kaposine) that are all expressed by KSC in the nodular-late stage of KS (Davis et al., 1997; Dupoin et al., 1999; Parravicini ¨ et al., 2000; Reed et al., 1998; Staskus et al., 1997; Sturzl et al., 1997b, ¨ et al. in this volume). In fact, although 1999a) (see also the chapter by Sturzl HHV-8 terminal repeats have a polyclonal proﬁle in KS lesions, some advanced lesions show an oligoclonality or clonality (Judde et al., 2000). In addition, as discussed later, the HIV-1 Tat protein acts as a progression factor for AIDS-KS due to its effects on KSC growth and angiogenesis.

A. Oncogenes and Oncosuppressor Genes Among the several oncogenes that have been found to be expressed in KS, Bcl-2 appears to play a major role. Bcl-2, an antiapoptotic protein, is expressed in lesional EC and KSC, and its expression increases with lesion

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stage in all forms of KS and thus may represent a progression factor for KS (Dada et al., 1996; Bohan-Morris et al., 1996). In fact, regression of KS is observed in patients treated with taxol (Saville et al., 1995), which is known to inhibit Bcl-2 function (Haldar et al., 1995). Recent data also indicate that taxol blocks KSC growth and migration, and KS-like lesion formation induced by the inoculation of KSC in nude mice (Sgadari et al., 2000). This is due to a downregulation of Bcl-2 expression and induction of apoptosis of KS cells (Sgadari et al., 2000). Thus, the inhibition of KSC apoptosis by the combined effect of bcl-2 and vFLIP (see later) may be key to KS lesion growth, which generally shows only a few mitotic ﬁgures (Kaaya et al., 1992).

B. HHV-8 Latency Genes HHV-8 latency-associated products including vCycD, vFLIP, LANA, and kaposin may be involved in KS progression due to their capability to promote cell growth by direct effects or antiapoptotic effects. Only a few KSC appear to express these genes in early-stage KS, but their expression increases with lesion stage and most KSC in nodular lesions express these viral genes at the RNA or protein level (Davis et al., 1997; Dupin et al., 1999, Parravicini et al., ¨ 2000; Reed et al., 1998; Staskus et al., 1997; Sturzl et al., 1997b; 1999a). This suggests that the continuous expression of these genes may be required ¨ for KS progression to a true cancer (see the chapter by Sturzl et al. in this volume). vCycD is likely to play a role in KS progression due to effects on cell growth. In fact, this viral protein mediates Rb phosphorylation (GoddenKent et al., 1997; Li et al., 1997) in a cdk-inhibitor independent manner (Swanton et al., 1997) and downregulates p27(Kip 1) (Ellis et al., 1999; Mann et al., 1999). Consistent with these features, overexpression of CycD induces cell cycle progression. The HHV-8 kaposin locus may be involved in the progression of KS to the nodular-tumor stage by transforming KSC. This is suggested by the transformation capacity of kaposin A and by the increased expression of kaposin transcripts in nodular lesions as compared to early lesions (Muralidhar et al., ¨ 1998; Sturzl et al., 1997b). By interfering with the recruitment of FLICE that is driven by the TNF receptor family members (Bertin et al., 1997; Djerbi et al., 1999; Thome et al., 1997), vFLIP may inhibit both apoptosis of KSC and render these cells resistant to lysis by killer cells. Recent data, in fact, have shown that expression of vFLIP in late-nodular lesions correlates with reduction of apoptosis of KSC ¨ in lesions (Sturzl et al., 1999a). In addition, overexpression of HHV-8 FLIP

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(vFLIP) in murine B-lymphoma cells allows the growth of aggressive tumors in mice due to the inhibition of FAS-mediated cytotoxic T-cell responses (Djerbi et al., 1999). HHV-8 LANA is another HHV-8 latency gene product that may contribute to the survival or transformation of KSC by the inactivation and downregulation of p53 that is associated with antiapoptotic effects and by targeting the Rb-EzF complex (Friborg et al., 1999; Radkov et al., 2000). In addition, LANA has been shown to interact with RING3, a homolog of the Drosophila female sterile homeotic gene, which may be involved in the modulation of host gene expression (Platt et al., 1999). Together these data support the concept that HHV-8 latency genes may play a key role in the progression of KS by providing KSC with growth and/ ¨ or anti-apoptotic signals (see the chapter by Sturzl et al. in this volume).

C. The HIV-1 Tat Protein The HIV-1 Tat protein appears to be responsible for the higher incidence and aggressiveness of KS in HIV-1–infected people. Tat, a transcriptional activator of HIV-1 gene expression, is released in an active form by HIV-1 acutely infected T cells in the absence of cell death (Chang et al., 1997; Ensoli et al., 1990, 1993). Extracellular Tat can induce the growth, migration, and invasion of KSC and EC (Albini et al., 1995; Barillari et al., 1992, 1993, 1999a, 1999b; Ensoli et al., 1990, 1993, 1994a; Fiorelli et al., 1995, 1998, 1999). However, these effects of Tat on normal EC require a previous exposure of the cells to the same IC increased in KS (Barillari et al., 1992, 1993; Fiorelli et al., 1995, 1998, 1999). These IC induce EC to express both the ␣5␤1 and ␣v␤3 integrins and bFGF (Barillari et al., 1993, 1999a; 1999b; Fiorelli et al., 1995, 1999; Samaniego et al., 1997, 1998). bFGF, in turn, ampliﬁes the expression of these integrins ¨ (Barillari et al., 1999a; Friedlander et al., 1995; Stromblad and Cheresh, 1996). Recent data indicate that the RGD sequence and the basic region of Tat cooperate to induce Tat angiogenic effects by different pathways (Barillari et al., 1999b). The RGD sequence of Tat mediates EC adhesion, migration, and invasion by binding to the ␣5␤1 and ␣v␤3 integrins. This interaction also provides EC with the adhesion signal required for growth in response to mitogens. In turn, the Tat basic sequence, which binds heparan sulfate proteoglycans (HPSG) (Chang et al., 1997), retrieves into a soluble form extracellular bFGF bound to HPSG by competing for heparin-binding sites (Barillari et al., 1999b). This soluble bFGF mediates Tat-induced vascular cell growth (Barillari et al., 1999b). This explains why bFGF is required for the angiogenic effect of Tat (Ensoli et al., 1994a). Consistent with these

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data, KS lesion formation in nude mice is induced in a synergistic way by bFGF and Tat (Ensoli et al., 1994a) and is inhibited by competitors RGD peptides that block the interaction of Tat with ␣5␤1 and ␣v␤3 integrins (Barillari et al., 1999a). Since these effects resemble those of extracellular matrix proteins, these data suggest that Tat enhances angiogenesis and promotes KS progression by a molecular mimicry of these molecules (Ensoli et al., 1999a). These data are supported by in vivo data indicating that these integrins and bFGF are highly expressed by KSC and activated EC of primary lesions and that extracellular Tat is present in AIDS-KS lesions (Ensoli et al., 1994a). In addition, recent work has showed that transgenic mice expressing HIV-1 Tat develop larger and more sever KS-like lesions upon inoculation of a KS-derived endothelial cell line as compared to mice transgenic for a RGD-deleted Tat or control mice (Prakash et al., 2000). Tat has also been reported to activate HHV-8 infection in PBMC from patients with KS or at risk of KS and in PEL cells (Harrington et al., 1997; Varthakavi et al., 1999), suggesting that the effects of HIV-1 infection on HHV-8 may be, at least in part, due to the effects of the Tat protein.

VIII. CONCLUDING REMARKS In this article we reviewed data indicating that early-stage KS is a reactive hyperplastic disease initiated by IC. IC activate vessels and induce chemotactic factors that recruit lymphocytes, monocytes, and circulating KSC into tissues, and induce these cells to differentiate in macrophages, dendritic cells, or KSC with an endothelial-macrophage phenotype. IC also promote spindle cell formation, EC and KSC proliferation, angiogenesis, and edema via the induction of angiogenic factors and chemokines. IC reactivate HHV-8 infection, leading to viremia and virus spread to all circulating cell types including monocytes and T cells that are present in infected tissues and KS lesions. In early KS, HHV-8 may further increase the reactive processes by inducing systemic and local immune responses that, however, are not effective in controlling the virus and, paradoxically, exacerbate IC production and lesion growth. This is indicated by the presence of HHV-8–speciﬁc CTL and T-helper responses in patients with KS and by the presence of activated and IFN-␥ –producing CD4+ and CD8+ T cells in KS lesions that, however, do not kill virus-infected cells and do not clear HHV-8 infection from tissues and circulation. A decreased NK cell cytotoxic activity, a deﬁcient IFN-␣ response, KIR upregulation, virus-escape mechanisms, or a more compromised immune response appear to be responsible for the lack of control of HHV-8 infection and may allow uncontrolled virus spread upon reactivation. These data

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suggest, therefore, that HHV-8–infected cells may be recruited into tissues in response to the KS reactive processes, rather than initiate KS. This is supported by the detection of IC in KS lesions and uninvolved tissues prior to HHV-8 detection and by the increase of HHV-8 load by IC and with lesion progression. Several lines of evidence also indicate that HHV-8 products expressed during lytic infection and capable of paracrine effects are unlikely to play a role in KS initiation, although they may participate in KS progression. In contrast, HHV-8 latency genes may play a key role in progression of reactive KS to a true sarcoma, due to their long-lasting expression and their growth and anti-apoptotic properties. The effects of HHV-8 latency gene products may be facilitated by the dysregulated expression of host oncogenes and oncosuppressor genes that is found in late-stage KS and by the immune deﬁciency that is present in those cases in which real tumor growth has been found. In this respect, the Tat protein of HIV-1 acts as a progression factor for AIDS-KS, and may be responsible for the higher frequency and aggressiveness of KS in the setting of HIV-1 infection. Although a complete understanding of the pathogenesis of KS is required to establish a pathogenetic therapy of KS, different factors including HHV-8, HIV-1 Tat, molecules involved in the homing of circulating KSC, oncogens, and oncosupressor genes should be investigated to identify therapeutic agents against the hyperplastic-early stage of KS or the nodular-tumor stage of the disease, which may likely require different inhibitory agents.

ACKNOWLEDGMENTS We thank Ms. A. Lippa, Mrs. F. M. Regini, and Ms. A. Carinci for editorial assistance. The authors’ research was supported by Italian grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), the AIDS project from the Italian Ministry of Health (to B. Ensoli), the Biofuture program of the German Ministry of Education and Research, and the Deutsche ¨ Krebshilfe (to M. Sturzl).

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Yu, Y., Roan, F., and Offermann, M. K. (1999). AIDS 13, 2178–2180. Zhang, Y. M., Bachmann, S., Hemmer, C., van Lunzen, J., von Stemm, A., Kern, P., Dietrich, M., Ziegler, R., Waldherr, R., and Nawroth, P. P. (1994). Am. J. Pathol. 144, 51–59. ¨ Zietz, C., Hotz, B., Sturzl, M., Rauch, E., Penning, R., and Lohrs, U. (1996). Am. J. Pathol. 149, 1887–1898. Zimmerman, G. A., and Hill, H. R. (1984). Thromb. Res. 35, 203–217. Zisbrod, Z., Haymov, M., Schanzer, H., Ambinder, E., and Burrows, L. (1980). Transplantation 30, 383–384.

Index

A ABMT. See Allogeneic bone marrow transplant Activated tissue sites characteristics, 171 Activation HHV-8, 163 Acute graft-versus-host disease, 3 Acute lymphoblastic leukemia (ALL) ABMT, 32 allogeneic HCT, 31–33 chemotherapy, 32 disease-free survival, 14f DLI, 15–16 Philadelphia chromosome, 32 Acute myeloid leukemia (AML) allogeneic HCT, 30, 34–35 vs. ABMT, 34–35 disease-free survival, 14f DLI, 15–16 ADEPT technique, 114–115 Administration Rituxan, 112 ADP-ribosylation, 106 African KS (AKS), 126–127, 162 risk factors, 165 African populations HHV-8 infection, 129 KS risk factor, 165 AGF, 132–133 AIDS-KS, 126, 127, 133, 162 AKS, 126–127, 129, 162, 165 ALL. See Acute lymphoblastic leukemia (ALL) Allogeneic bone marrow vs. allogeneic PBPC, 7 Allogeneic bone marrow transplant (ABMT) vs. allogeneic HCT AML, 34–37 recurrent leukemia DLI, 16f

vs. UCB transplants children, 12 Allogeneic hematopoietic cells source, 6–8 Allogeneic hematopoietic cell transplantation vs. ABMT AML, 34–37 complications, 19–24 vs. immunosuppression SAA, 26 nonmyeloablative regimens, 16–19 Allogeneic peripheral blood progenitor cells vs. allogeneic bone marrow, 7 Amerindian populations HHV-8 infection, 129 AML. See Acute myeloid leukemia (AML) Angiogenesis effects KS, 167t induction, 172–174 Angiogenic growth factors (AGF), 132 serum, 133 Antibody-directed enzyme prodrug therapy (ADEPT) technique, 114–115 Anti-CD6 monoclonal antibody T-cell depletion, 5 Anti-erbB2 immunotoxins antitumor activity, 109f Anti-IFN-␣ HHV-8 DNA, 183f Anti-Leu-2 antibody T-cell depletion, 5 AntiTac(Fv)-PE38 (LMB-2) leukemia, 110 Antithymocyte globulin (ATG), 13–14 Aortic endothelium disruption, 132 Aplastic anemia allogeneic HCT, 25–28

201

202 Apoptosis E-KSC, 141–144 HHV-8, 141–144 resistance, 141 Aquired immunodeﬁciency syndrome-associated KS (AIDS-KS), 126, 127, 133, 162 Aspergillus, 21 Associated posttransplant lymphoproliferative disorders EBV following allogeneic HCT, 20–21 ATG, 13–14 Autologous hematopoietic cell transplantation PBPCs, 6–7

B B3, 96f, 101, 102f, 103, 107 BAC, 77–78 Bacterial artiﬁcial chromosome (BAC), 77–78 Basic ﬁbroblast growth factor (bFGF), 144, 167 detection, 172 KSC, 186 B-cells lymphomas, 95 spleen, 76 Bcl-2, 82, 141 lesional EC and KSC, 184 Bexxar, 112 BFGF. See Basic ﬁbroblast growth factor Binding-displacement assays, 106 BK virus, 128 BL. See Burkitt’s lymphoma BL22, 111 Bladder cancer LeY, 107 B lymphocytes cyclic RAG expression, 85 Bone marrow. See Allogeneic bone marrow MUD vs. PBPC grafts, 7–8 BR96, 108 Brain tumors immunotoxins, 108–109 MR1, 108 Breast cancer ergB2, 108

Index

LeY, 107 recombinant antibodies, 112 B-thalassemia allogeneic HCT, 28–29 Burkitt’s lymphoma (BL) chromosomal translocation, 80–81 GC, 78–80 translocation, 72f

C Cancerogenesis humans multistep process, 246 Capsid protein, 134 CBP, 137 CD19, 95, 111 CD20, 95 CD22, 95, 110 CD25, 110, 111 CD34, 128 Cdk, 137 CdkIs, 141 CD6 monoclonal antibody anti T-cell depletion, 5 CD31-positive EC, 135 CD68-positive monocytic cells, 135 CD8+ T-cell activation, 163 CD4+ T-cells C-KS and A-KS, 182 Cell death. See Apoptosis Cell invasiveness induced, 147 Cell metastatic potential induced, 147 Cell permeability bFGF, 172 Cell recruitment IC, 170–171 Central America HHV-8 infection, 129 Central nervous system tumors immunotoxins, 108–109 CH1, 96f, 101, 102f, 103 Chemical conjugates, 95–96 Chemoattractants, 133 Chemokine receptor, 178 Chemotactic factors effects KS, 167t

203

Index

Chemotherapy vs. allogeneic HCT AML, 34–35 Children allogeneic HCT ALL, 31–33 ␤-thalassemia, 29 SCD, 30 solid tumors following allogeneic HCT, 20 sub-Equatorial Africa AKS, 162 UCB transplants vs. ABMT, 12 Chimeric toxins, 97, 101 Chimerism-induced immunotherapy, 18 Chromosomal translocations GC-like lymphomas, 80–83 Chronic graft-versus-host disease, 3 Chronic lymphocytic leukemia (CLL) allogeneic HCT, 39–40 nonmyeloablative allogeneic HCT, 40 Chronic myeloid leukemia (CML) HCT allogeneic, 40–44 MUD, 9 survival, 10f mixed-chimeric transplants, 41–43 PCR, 41 TBI, 40 Cidofovir CMV, 22–23 Circulating cells HHV-8 in KS, 177–179 Circulating spindle cell progenitors, 133 Classical KS (CKS), 126 Eastern Europe, 162 CLL, 39–40 CML. See Chronic myeloid leukemia (CML) CMV, 21–23, 128 Colon cancer LeY, 107 Copaxone GVHD, 4 Corynebacterium diphtheria, 98 CREB-binding protein (CBP), 137 Cryptic recombinational signal sequences illicit V(D)J rearrangement, 73–75 CSP, 3, 18 CTL. See Cytotoxic T lymphocyte CTLA-4-Ig, 14

Cultured cell lines in vitro, 109f CY, 2, 25, 40 CY3, 84 Cyclic RAG expression B lymphocytes, 85 Cyclin-dependent kinase (Cdk), 137 Cyclin-dependent kinase inhibitory proteins (CdkIs), 141 Cyclophosphamide (CY), 2, 25 CLL, 40 CML, 40 Cyclosporine (CSP) GVHD, 3 posttransplant immunosuppression, 18 Cytokines, 167 Cytomegalovirus (CMV), 21–23, 128 Cytotoxicity assays, 106 Cytotoxic T cells, 177, 182 Cytotoxic T lymphocyte (CTL) EBV, 21 HHV-8 reactivation, 131 HHV-8-speciﬁc, 182

D DAH, 24 Dark zone (DZ) GC, 76 Death-effector domains (DED), 142 Deﬁcient immune response HHV-8, 163 Differentiation antigens, 94 Diffuse alveolar hemorrhage (DAH), 24 Diffuse large-B-cell lymphoma (DLBL), 79 Diphtheria toxin (DT), 98–99 immunotoxins expression vectors, 105 DLBL, 79 Donor lymphocyte infusions (DLI) CML, 15–16 PTLD, 21 DR-4, 142 DR5, 142 Dsb DNA repair, 68–70 LM-PCR, 83 DsFv-immunotoxin cloning, construction, composition, 102f

204 DsFv-lactamase fusion protein, 115 DT, 98–99 immunotoxins expression vectors, 105 DZ GC, 76

E E23 antibody, 108 Eastern Europe CKS, 162 EBV. See Epstein Barr virus EC activation, 166 monolayer, 132 recombinant immunotoxin, 118 Edema induction, 172–173 EF2, 98 Egypt HHV-8 infection, 129 E-KSC. See Endothelial-derived KSC ELAM-1, 132 Elongation factor 2 (EF2), 98 Endemic BL, 81 Endothelial cells (EC) activation, 166 monolayer, 132–133 recombinant immunotoxin, 118 Endothelial-derived KSC (E-KSC), 128, 133, 170 apoptosis, 141–144 HHV-8, 135, 145–146 genome instability, 147–148 metastatic capability, 146–147 human herpesvirus-8 (HHV-8), 148–149 Endothelial-macrophages lymph nodes, 168 Epstein Barr virus (EBV), 129 associated PTLD following allogeneic HCT, 20–21 speciﬁc CTL, 21 speciﬁc cytotoxic T lymphocytes (CTLs), 21 ErbB2 immunotoxins anti antitumor activity, 109f ErgB2 mAb 323, 108 Escherichia coli, 98

Index

Ethics UCB transplantation, 12–13 Ethnic groups HHV-8 infection, 129

F FADD, 141 FADD-like interleukin-1␤-converting enzyme (FLICE), 141, 185 Fas-associated death domain (FADD), 141 FDCs, 76 Fd fragments, 96f, 101, 102f, 103 Fibroblast growth factor (FGF), 97 First-generation immunotoxins, 95–96 FITC, 84–85 FK 506 GVHD, 3–4 FL. See Follicular lymphoma FLICE, 141, 185 FLICE inhibitory proteins (FLIPS), 141–142, 169–170 KS progression, 184–185 lesional EC and KSC, 184 Fludarabine, 13–14 CLL, 40 Fluorescein isothiocyanate (FITC), 84–85 Follicular dendritic cells (FDCs), 76 Follicular lymphoma (FL) chromosomal translocation, 83–83 DLBL, 79 GC, 78–80 translocation, 74f Foscarnet CMV, 22–23 FRP5scFv-ETA, 108 Fv-enzyme fusion proteins, 114–115

G Gambia HHV-8 infection, 129 Ganciclovir CMV, 22 GC. See Germinal center Geographic areas HHV-8 infection, 129 Germinal center (GC) like lymphomas chromosomal translocations, 80–83

Index

lymphocytes genomic lability, 79 lymphomagenesis, 78–80 NHL, 79–80 reaction, 76–78 splenic, 76 V(D)J rearrangement, 77 GFP, 77–78 Glu145, 99 Glu553, 99 GMCSF, 110 G-protein-coupled receptor (GPCR), 178 KS initiation, 180 paracrine activities, 148 Graft-versus-host disease (GVHD) acute, 3 chronic, 3 immunotoxins, 110, 111 prophylaxis, 2–4 Graft-versus-leukemia (GVL), 4 Graft-versus-lymphoma effect allogeneic HCT NHL, 44 Graft-versus-myeloma, 47 Granulocyte-macrophage colony-stimulating factor (GMCSF), 110 Green-ﬂuorescent protein (GFP), 77–78 GVHD. See Graft-versus-host disease GVL, 4

H HAART KS regression, 181 Haploidentical hematopoietic cell transplantation, 13–14 HCT. See Hematopoietic cell transplantation HD allogeneic HCT, 46 Hematopoietic cells DNA microsatellite markers, 17, 17f source, 6–8 Hematopoietic cell transplantation (HCT), 2 vs. ABMT AML, 34–37 complications, 19–24 haploidentical, 13–14 vs. immunosuppression SAA, 26

205 MUD CML, 9 nonmyeloablative regimens, 16–19 PBPCs, 6–7 T-cell depleted, 4–6 Hepatitis B virus, 128 Herceptin, 112 Herpesvirus saimiri (HVS), 129, 137 HHV-6, 123 HHV-8. See Human herpesvirus-8 HIV-1. See Human immunodeﬁciency virus type-1 (HIV-1) HLA. See Human leukocyte antigen (HLA) Hodgkin disease (HD) allogeneic HCT, 46 Homo-bisexual men KS risk factor, 164–165 Homosexual men KS, 131 Human herpesvirus-6 (HHV-6), 123 Human herpesvirus-8 (HHV-8), 128–149 apoptosis, 141–144 dissemination, 132–133 DNA detection, 169 E-KSC, 135 semiquantitative PCF, 178f E-KSC, 145–146, 148–150 genome instability, 147–148 metastatic capability, 146–147 proliferation, 136–141 encoding KS lesions, 138t–140t lytic gene products, 178 paracrine effects, 180t epidemiology, 129–130 gene expression KS, 135–148 IC, 173–180 infected cells nature, 134–135 infected monocytes PCR, 133 infection HHV-8 reactivation, 179–180 IC, 173–180 KS, 169–170 KS risk factor, 164 spread, 177–179 kaposin locus KS progression, 185

206 Human herpesvirus-8 (continued) KS, 169–170 angiogenesis, 144–145 gene expression, 134–148 latent reactivation, 176–177 lytic infection, 169 MIP homologs, 180 paracrine activities, 148 prevalence, 130 reactivation, 130–132 KS, 174–176, 175t, 179t tat protein, 186 Human immunodeﬁciency virus type-1 (HIV-1) infection KS, 131 KS risk factor, 164–165 tat protein, 133, 185–186 Human leukocyte antigen (HLA), 118–119 DR, 132 identical sibling grafts vs. PMRD leukemia, 10 typing MUD, 8–9 PMRD, 8–9 Human papillomaviruses, 128 HVS, 129, 137 Hyperplastic KSC endothelial cell, 168 Hyperplastic reactive-inﬂammatory process, 162

I IBMTR, 10 IC. See Inﬂammatory cytokines (IC) Idiopathic MDS, 38 Ig loci, 62–63, 72 IL2, 97, 101 receptor, 95 IL4, 97, 101 IL6, 97, 101 paracrine activities, 148 IL8, 173 Illicit V(D)J rearrangement cryptic RSS, 73–75 Immune activation, 175 Immune deﬁciency, 175

Index

Immune dysregulation KS initiation, 165 Immunodeﬁciency KS, 132 Immunoglobulin (Ig) loci, 62–63, 72 Immunoreceptor tyrosine-based activation motif (ITAM), 146 Immunosuppression vs. allogeneic HCT SAA, 26 KS, 166 Immunotargeting, 113 Immunotherapy, 94 following relapse, 15–16 MM, 47 PTLD, 21 Immunotoxins groups, 95–96, 96f Immunotoxin therapy goal, 95 Indodicarbocyanine, 84 Inﬂammatory cell ﬁltrate KS, 127–128 Inﬂammatory cytokines effects KS, 167t Inﬂammatory cytokines (IC), 131, 163 activated EC, 171–172 angiogenic molecules, 172 activated KSC, 171–172 angiogenic molecules, 172 HHV-8 infection, 173–180 KS histogenesis, 170–183 KS initiation, 165–166 latent HHV-8 reactivation, 176–177 serum, 133 spindle cell morphology, 171–172 vascular endothelium activation, 170–171 Interferon CML, 40–41 Interferon-␣ anti HHV-8 DNA, 183f neutralizing antibodies, 183 systemic KS regression, 181 Interferon regulatory factor (IRF), 136, 137 Interlukin. See IL International Bone Marrow Transplant Registry (IBMTR), 10

207

Index

International Prognostic Scoring System (IPSS), 38 Intravenous immunoglobulin (IVIG) CMV, 22 IPSS, 38 IRF, 136–137 ITAM, 146 Iterative chromosome painting, 84–85 IVIG CMV, 22

J Jak1, 137

K K1, 140t, 146 Kaposin, 140t, 145–146, 169 KS progression, 184–185 Kaposi’s sarcoma cells (KSC), 127–129 hyperplastic endothelial cell, 168 lesional, 168 Kaposi’s sarcoma (KS), 126–135, 162–163 angiogenesis HHV-8, 144–145 characteristics, 126 clinical presentation, 126–127 epidemiological forms, 126 HHV-8 gene expression, 134–148 HHV-8 gene products lesions, 148 HHV-8-infected circulating cells, 181 HHV-8 infection, 169–170 histology, 127–128 infection type, 134–135 lytic infection, 135 progression, 184–186 risk factors, 162–166, 174 spindle cells, 166 Kidney transplant recipients HHV-8 infection, 174 Killer-cell inhibitory receptor (KIR) KS and HIV, 182 K15/LAMP, 138t, 143–144 KS. See Kaposi’s sarcoma KSC, 127–129, 168 KSHV, 129

Ku70 protein, 68–70 Ku80 protein, 68–70

L Lac UV5 promoter, 105 Latency-associated nuclear antigen (LANA), 134–136, 138t, 140t, 142–143 KS progression, 184–185 Latent HHV-8. See also Human herpesvirus-8 reactivation, 176–177 Lesional KSC, 168. See also Kaposi’s sarcoma cell (KSC) Lesion formation induction, 172–173 Leu-2 antibody anti T-cell depletion, 5 Leukemia HLA-identical sibling grafts vs. PMRD, 10 Lewis Y (Le Y) antigen, 107 Ligase IV, 70 Ligation mediated PCR (LM-PCR) dsb, 83 Light zone (LZ) GC, 76 Liver VOD, 23–24 Liver tumors MR1, 108 LMB-1, 107 LMB-2 anti leukemia, 110 leukemia, 110 LMB-9, 108 LM-PCR dsb, 83 LMP1 protein, 143 LNA, 134. See also Latency-associated nuclear antigen LNA-1, 134. See also Latency-associated nuclear antigen Low-dose TBI allogeneic HCT, 18 Lung cancer ergB2, 108 LeY, 107 Lymphoid follicle, 76

208 Lymphomagenesis GC, 78–80 Lymphomononuclear cells activation, 166 Lytic genes, 134, 135 Lytic HHV-8 infection HHV-8 reactivation, 179–180 Lytic infection HHV-8, 169 KS, 135 vIL-6, 137 LZ GC, 76

M MAb, 107–108 Macrophagic (M-KSC), 170 Major capsid protein, 134 Major histocompatibility complex (MHC) HHV-8 reactivation, 131 recombinant immunotoxin, 116f Matched unrelated donor (MUD) bone marrow vs. PBPC grafts, 7–8 hematopoietic cell transplantation CML, 9 HLA typing, 8–9 transplantation, 8–11 mortality, 9–10 MCP-1, 173 MDS allogeneic HCT, 35, 38–39 DLI, 15–16 Mediterranean countries HHV-8 infection, 129 KS, 162, 165 Melphalan, 47–48 Mesotheliomas SS(Fv)-PE38, 108 Methotrexate (MTX) GVHD, 3 MHC HHV-8 reactivation, 131 peptide complex recombinant immunotoxin, 116f Microsatellite markers hematopoietic cell DNA, 17, 17f Mini-transplants, 18 MIP-I,-II,-III viral, 138t, 145, 148

Index

Mixed-chimerism transplantation, 18 M-KSC, 170 MM allogeneic HCT, 46–48 MMF GVHD, 3–5 posttransplant immunosuppression, 18 Mobilized peripheral blood progenitor cells autologous HCT, 6–7 Monoclonal antibody (MAb) 323 ergB2, 108 Monoclonal antibody (MAb) B3, 107–108 Monocyte chemotactic protein-1 (MCP-1), 173 Monocyte chemotaxis KS initiation, 180 Monocytes adhesion, 132 MR1 (Fv)-PE38, 108 MTX GVHD, 3 MUD. See Matched unrelated donor Multiple myeloma (MM) allogeneic HCT, 46–48 Mycophenolate mofetil (MMF) GVHD, 3–5 posttransplant immunosuppression, 18 Myeloablative maneuver, 16–19 Myelodysplastic syndrome (MDS) allogeneic HCT, 35, 38–39 DLI, 15–16

N National Marrow Donor Program (NMDP) establishment, 8 Neoangiogenesis, 166 NHL. See Non-Hodgkin lymphoma NK cells, 177, 182 NMDP establishment, 8 Non-Hodgkin lymphoma (NHL) allogeneic HCT, 43–45 GC, 79–80 recombinant antibodies, 112 Nonmyeloablative regimens allogeneic HCT, 16–19 Nonmyeloablative transplantation, 18 Northern Europe HHV-8 infection, 129

Index

O Oncotoxins. See Recombinant toxins Orf26, 134 OrfK1 (K1), 140t, 146 OrfK12 (Kaposin), 140t OrfK15 (K15/LAMP), 138t OrfK25 (K15/LAMP), 143–144 OrfK13 (vFLIP), 138t OrfK2 (vIL-6), 136–137, 138t, 145 OrfK9 (vIRF), 136–137, 138t, 145–146 OrfK6 (vMip-I), 138t, 145 OrfK4 (vMip-II), 138t, 145 OrfK4.1 (vMip-III), 138t, 145 Orf3 (LANA), 142–143 Orf73 (LANA), 138t, 140t Orf16 (vBcl-2), 138t, 143 Orf72 (vCyc), 136–141, 138t Orf74 (vGPCR), 138t, 144–145 Ovarian cancer ergB2, 108 SS(Fv)-PE38, 108

P P21, 141 P27, 141 Palindromic nucleotides, 67 PALS, 76 Paracrine activities HHV-8, 148 Paracrine effects HHV-8 reactivation KS initiation, 179–180 human herpesvirus 8-encoded factors, 180t Partially matched related donors (PMRD), 8 vs. HLA-identical sibling grafts leukemia, 10 HLA typing, 8–9 PBMC. See Peripheral blood mononuclear cells PBPCs. See Peripheral blood progenitor cells PCR HHV-8 infection, 135 PE. See Pseudomonas exotoxin Peptide complex MHC recombinant immunotoxin, 116f Periarteriolar lymphoid sheath (PALS), 76

209 Peripheral blood mononuclear cells (PBMC) associated viremia, 130 cultures IC, 176–177 cytoﬂuorimetric analysis, 181t spindle-shaped cells, 168 Peripheral blood progenitor cells (PBPCs) vs. allogeneic bone marrow, 7 grafts vs. MUD bone marrow, 7–8 mobilized autologous HCT, 6–7 Peripheral blood stem cells, 6 Philadelphia chromosome ALL, 32 discovery, 62 PKCS, 70 PKS. See Posttransplant KS (PKS) PMRD, 8–10 Polycythemia vera DLI, 15–16 Polymerase chain reaction (PCR) HHV-8 infection, 135 Posttransplant KS (PKS), 126, 127, 133, 162, 178, 181 Posttransplant lymphoproliferative disorders (PTLD), 20–21 EBV following allogeneic HCT, 20–21 Prednisone (PSE) GVHD, 3 Programmed cell death E-KSC, 141–144 HHV-8, 141–144 resistance, 141 Protein kinase catalytic subunit (PKcs), 70 PSE GVHD, 3 Pseudomonas aeruginosa, 98 Pseudomonas exotoxin (PE), 98–100, 108–112 based recombinant Fv immunotoxin functional properties, 106t chemical conjugate, 96f, 101, 102f, 103 PT-KS. See Posttransplant KS PTLD, 20–21 Puriﬁed cell populations cytoﬂuorimetric analysis, 181t

210

R RACE method, 102f Radioactive recombinant antibodies, 112–113 Radioimmunodetection, 113 RAG cyclic, 85 RAG1. See Recombinase activating gene-1 RAG2. See Recombinase activating gene-2 Rapamycin GVHD, 3–4 Rapid ampliﬁcation of cDNA ends (RACE) method, 102f Reactivated HHV-8 control, 181–183 Reactive KSC endothelial cell, 168 Recombinant antibody fragments, 101–104 types, 96f Recombinant IFN-␣ HHV-8 reactivation, 182 Recombinant immunotoxins, 93–96 construction, 104–105 dose-limiting toxicity, 115–118 examples, 97t future, 115–119 immune response, 115–118 leukemias, 110–111 lymphomas, 110–111 molecular modeling, 117 preclinical development, 105–107 solid tumors, 107–110 speciﬁcity, 118–119 targeting moiety, 101–104 Recombinant toxins, 97 toxin moiety, 98–99 Recombinase activating gene (RAG) cyclic, 85 Recombinase activating gene-1 (RAG1) cryptic RSS, 75 transposase activity, 71–73 V(D)J rearrangement, 65–66 Recombinase activating gene-2 (RAG2) cryptic RSS, 75 transposase activity, 71–73 V(D)J rearrangement, 65–66 Recombinational signal sequences (RSS), 62–63 cryptic illicit V(D)J rearrangement, 73–75 V(D)J rearrangement, 63

Index

Replapse immunotherapy following, 15–16 RFB4(dsFv)-PE38 (BL22), 111 RGD sequence, 171, 186 Ricin, 98 leukemia, 110 RSS, 62–63 cryptic illicit V(D)J rearrangement, 73–75

S SAA. See Severe aplastic anemia Saimiri-transforming protein A, 146 Saimiri-transforming protein C, 146 Saudi Arabia HHV-8 infection, 129 Scatter factor/hepatocyte growth factor (SF/HGF), 173 SCD, 29–31 ScFv-immunotoxin cloning, construction, composition, 102f Second-generation immunotoxins, 95–97 Severe aplastic anemia (SAA) allogeneic HCT, 25–28 vs. immunosuppression, 26 survival, 27f solid tumors following HCT, 19–20 Sexually transmitted infectious agent KS, 128 SF/HGF, 173 Sickle cell disease (SCD) allogeneic HCT, 29–31 myeloablative allogeneic HCT, 31 Sirolimus GVHD, 3–4 SKY, 84 Slitlike vessels, 166 Solid tumors following allogeneic HCT, 19–20 Spectral karyotyping (SKY), 84 Spindle cells circulating, 133 formation induction, 171–172 KS, 127–128, 166 morphology IC, 171–172 Spindle-shaped cells PBMC, 168

211

Index

Spleen B-cells, 76 T-cell-rich region, 76 Splenic GC zones, 76 Sporadic BL, 81 Sporadic herpesvirus reactivation, 181 SS(Fv)-PE38, 108 STAT1, 137 STAT3, 137 Stomach cancer ergB2, 108 LeY, 107 STP-A, 146 STP-C, 146 Sub-Equatorial Africa children AKS, 162 Surface plasmon-resonance assays, 106 Systemic IFN-␣ KS regression, 181

T TAA, 95 Tac(Fv)-PE38 (LMB-2) anti leukemia, 110 Tacrolimus GVHD, 3–4 T-ALL, 73–75 Targeted cancer therapy, 94–95 Tat angiogenic effects, 186 HHV-8, 186 peptides, 171 protein HIV-1, 133 TBI, 2–3, 18, 40 T-cell acute lymphoblastic leukemia (T-ALL), 73–75 T-cell depleted hematopoietic cell transplantation, 4–6 risks, 5 T-cell leukemia, 95 T-cell-rich region spleen, 76 Tcr loci, 62–63, 72 TGF-␣, 97 Thalassemia allogeneic HCT, 28–29

T-helper cells HHV-8-speciﬁc, 182–183 T-helper-1 cells type cytokines. See Cytokines T-helper-2 cells chemoattractants KS initiation, 180 TIL, 166 Tip, 146 Tissue plasminogen activator VOD, 23–24 Tissue recruitment HHV-8 in KS, 177–179 TNF, 137, 142, 165 TNRF, 142 Tonsillar GC centrocytes, 78 Total body irradiation (TBI), 2–3, 18 CML, 40 TRAF-binding sites, 143 TRAIL-R1 (DR-4), 142 TRAIL-R2 (DR5), 142 TRAMP (WSL/DR-3/APO-3), 142 Transforming growth factor (TGF)-␣, 97 Transplant-lite, 18 Transposase v(D)J recombinase, 71–73 T7 RNA-polymerase gene, 105 Tumor-associated antigens (TAA), 95 Tumorigenesis, 135–148 Tumor necrosis factor (TNF), 165 induced apoptosis, 137 receptor, 142 Two-end translocation model V(D)J rearrangement, 68f Tyrosine kinase-interacting protein (tip), 146

U Umbilical cord blood (UCB) cell transplantation, 11–13 vs. ABMT children, 12 ethics, 12–13 Ursodeoxycholic acid VOD, 23

V Variable heavy chain (VH), 96f, 101, 102f, 103

212 Variable light-chain (VL), 96f, 101, 102f, 103 Vascular-endothelial-cadherin, 168 Vascular endothelial growth factor (VEGF), 144–145, 167, 172 IC-activated EC, 171 KS initiation, 180 Vascular endothelium activation, 170–171 Vascular leak syndrome (VLS), 117 VBcl-2, 138t, 143 VCAM-1, 132 VCyc. See Viral cyclin (vCyc) V(D)J rearrangement, 62–73 BL, 81 cleavage, 63–64 FL, 82–83 GC, 77 molecular mechanism, 65–71 RSS, 63 V(D)J recombinase transposase, 71–73 VEGF. See Vascular endothelial growth factor Veno-occlusive disease (VOD) liver, 23–24 prophylaxis, 23 treatment, 23–24 VFLIP. See Viral FLICE inhibitory protein (vFLIP) VGPCR, 138t, 144–145, 178 KS initiation, 180 paracrine activities, 148 VH, 96f, 101, 102f, 103 VIL6, 136–137, 138t, 145 paracrine activities, 148

Index

Viral cyclin (vCyc), 136–141, 138t, 169 KS progression, 184–185 Viral FLICE inhibitory protein (vFLIP), 169–170 KS progression, 184–185 lesional EC and KSC, 184 Viral interferon regulatory factor (vIRF), 136. See also Interferon regulatory factor Viral load, 133 Viral lytic gene expression, 135 VIRF, 136–137, 138t, 145–146 Virus spreading KS, 175t Vital infection spread, 178 VL, 96f, 101, 102f, 103 VLS, 117 VMIP-I, 138t, 145 paracrine activities, 148 VMIP-II, 138t, 145 paracrine activities, 148 VMIP-III, 138t, 145 paracrine activities, 148 VOD liver, 23–24 VP23, 134

W WSL/DR-3/APO-3, 142

X X-ray cross complementation (XRCC) mutants, 68–69