Graft-versus-Lymphoma Effects - Core

Biology of Blood and Marrow Transplantation 10:579-590 (2004) 䊚 2004 American Society for Blood and Marrow Transplantation 1083-8791/04/1009-0001$30.00/0 doi:10.1016/j.bbmt.2004.05.008

Graft-versus-Lymphoma Effects: Clinical Review, Policy Proposals, and Immunobiology Andrew Grigg,1 David Ritchie2 1

Department of Clinical Haematology and Medical Oncology, The Royal Melbourne Hospital, Melbourne, Australia; 2Malaghan Institute, Wellington, New Zealand Correspondence and reprint requests: Andrew Grigg, MD, Department of Haematology, Royal Melbourne Hospital, Grattan St., Parkville, Melbourne, Victoria 3050, Australia (e-mail: [email protected]). Received February 23, 2004; accepted May 14, 2004

ABSTRACT The indubitable existence of a graft-versus-lymphoma (GVL) effect is difficult to prove directly. This article reviews the difficulties in interpreting the current literature in this field and, with a number of caveats, argues for the existence of a clinically meaningful GVL effect in follicular, mantle cell, small lymphocytic, and Hodgkin lymphomas. The evidence, however, for a potent GVL effect in diffuse large-cell lymphoma and Burkitt lymphoma is not convincing. Policies for allografting in lymphoma are proposed on the basis of this evidence. The immunobiology of GVL effects is discussed—in particular, the expression of HLA class I and II and co-stimulatory molecules on lymphomas that influence the generation of alloreactive T cells—together with future directions in immunotherapy that may help to eradicate chemoresistant disease. © 2004 American Society for Blood and Marrow Transplantation

KEY WORDS Graft-versus-lymphoma effects

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Immunobiology

INTRODUCTION The recognition that the incidence of relapse after autografting for recurrent or refractory lymphoma is usually substantial and the emerging use of reducedintensity (RI) allogeneic conditioning regimens have resulted in the need to evaluate critically which lymphomas are amenable to graft-versus-lymphoma (GVL) effects. A GVL effect can be convincingly demonstrated only by the durable resolution of biopsy-proven residual progressive disease after allografting in response to immunomodulation such as withdrawal of immunosuppression or donor leukocyte infusion (DLI). Indirect evidence may include eradication of active disease by minimally cytotoxic conditioning regimens and allogeneic cell infusion and statistical association between graft-versus-host disease (GVHD) with a lower risk of relapse and T-cell depletion with a higher risk of relapse. Although a substantial reduction in relapse with a plateau in disease-free survival (DFS) after allogeneic versus autologous transplantation in comparable patient groups would also be consistent with a GVL effect, the possible contribution of reinfused

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autologous tumor cells to relapse [1] means that such observations are not definitive proof. A number of comprehensive reviews of singleinstitution and registry studies of allografting in lymphoma with both myeloablative and RI conditioning regimens have been published [2,3]. The purpose of this article is not to revisit the literature in detail, but to review the difficulties in assessing this literature, to critically evaluate the evidence for a GVL effect in specific histologic subtypes of lymphoma by using the criteria described previously, and to propose allograft policies based on this review. The proposed allograft policies, representing our unit’s current policies, are subject to much debate but provide a framework for discussion. Finally, we discuss the biological characteristics of lymphoma cells and their environment, which may affect their immunologic responsiveness, and we speculate on future directions in harnessing GVL effects to eradicate chemoresistant disease. DIFFICULTIES IN ASSESSING THE LITERATURE There are a number of difficulties in interpreting the literature on GVL effects. 579

A. Grigg and D. Ritchie

1. Registry analyses. (i). Many have incorporated a number of lymphoma subtypes with distinct histopathologic, molecular, and clinical characteristics into a single group for the purposes of analysis of GVL effects, making it difficult to evaluate responses in specific entities. For example, diffuse small cleaved (often mantle cell [MCL]) and follicular large-cell lymphomas have been regarded by the European Bone Marrow Transplant (EBMT) registry as intermediate-grade lymphomas under the Working Formulation and analyzed together [4], whereas another transplant analysis from this group classified patients as having only low- or high-grade lymphoma [5]. In addition, many of these registry reviews include patients undergoing different conditioning regimens and GVHD prophylaxis. (ii). Lack of centralized histologic review, so that the reported diagnoses have not been independently verified. (iii). Probable imbalances in baseline pretransplantation characteristics in studies comparing the outcome of autografts versus allografts [4], together with potential variability in patient selection and standards of supportive care over long periods of analysis. (iv). Not surprisingly, the results of these studies are often inconsistent. 2. Retrospective single-institution studies comparing autografts with allografts are subject to various potential confounding factors, such as referral bias, physician preference, and improvements in outcome over time, such that definitive conclusions are rarely possible. 3. There are substantial differences in the intensity of RI conditioning regimens. Fludarabine/melphalan 140 to 180 mg/m2, for example, may well be myeloablative, because chimerism early after transplantation is usually exclusively donor and because the risk of GVHD is comparable to that with traditional myeloablative regimens [6]. In contrast, fludarabine/lowdose cyclophosphamide is clearly not ablative, generally results in mixed chimerism, and has a relatively low incidence of GVHD [7]. Moreover, most RI conditioning studies from single institutions or registries have short-term follow-up, so the durability of responses is not established. 4. The importance of documentation by biopsy of viable residual disease after transplantation in assessing the response to DLI or withdrawal of immunosuppression is particularly relevant because functional imaging with positron emission tomography or gallium scanning may not always reliably distinguish inflammatory from neoplastic tissue or indolent from aggressive lymphoma, which may coexist. 580

5. Although responses to DLI have been well documented in some lymphomas, particularly follicular and MCLs, this does not necessarily mean that all such lymphomas are immunologically responsive. Some cases have documented a GVL effect in response to DLI that seems to alter the natural history of previously progressive lymphoma but that is not durable, suggesting that malignant clones may differ in their susceptibility to immunotherapy.

GVL EFFECTS IN HISTOLOGIC SUBTYPES AND PROPOSED ALLOGRAFT POLICIES Data for each subtype will be discussed as follows: (1) DLI, (2) single-institution and registry reviews of RI transplants, and (3) institution and registry reviews of myeloablative transplants, including, where available, an evaluation of the effect of GVHD on relapse. Follicular Lymphoma

A graft-versus–follicular lymphoma (FL) effect was suggested by case reports of resolution of active disease by DLI [8,9] and was confirmed more recently in a British survey in which 8 of 13 patients with overt FL after allografting achieved complete remission (CR) after DLI [10]. A high rate of remission after RI conditioning has been reported [7], although most patients had nonbulky chemosensitive disease at transplantation and although follow-up was relatively short in a disease with a propensity for late relapse. An EBMT registry analysis of RI allografts in low-grade lymphoma (Working Formulation) using a variety of conditioning regimens in heavily pretreated patients, most with chemosensitive disease and with sibling donors, demonstrated a 1-year probability of disease progression of 21% and 2-year progression-free survival (PFS) of 54%; pretransplantation chemosensitivity was the only significant factor predicting for progression [5]. No relapses were seen beyond 1 year, although few patients were followed up beyond 2 years. There are minimal data on the outcome after unrelated donor transplantations for FL and other lymphomas [4,5,11,12]. Single-institution studies with long-term follow-up have reported a low incidence of relapse after myeloablative allografts for refractory or recurrent indolent nonHodgkin lymphoma (NHL), predominantly follicular [13,14], which seems less than after autografts [15]; this is consistent with an EBMT registry analysis [4]. Recently the International Bone Marrow Transplant Registry (IBMTR) and the Autologous Bone Marrow Transplant Registry, in a retrospective study, compared the outcome of myeloablative allogeneic versus purged autologous versus unpurged autologous transplants [16]. Allografts had a higher treatment-related mortality and a lower risk of recurrence. Few recurrences occurred after

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2 years, although the maximum follow-up of 5 years is still relatively short. Intriguingly, the relapse rate was higher in unpurged versus purged autografts, and there was no association between acute or chronic GVHD and recurrence. The EBMT registry analysis also found no effect of acute GVHD on relapse, although in the British review, the responses of FL to DLI correlated with both acute and chronic GVHD [10]. Overall, these data suggest that the major benefit of allografting in FL may relate to effective high-dose chemoradiotherapy followed by infusion of uncontaminated stem cells rather than a GVL effect. The absence of a significant difference in relapse risk after syngeneic versus allografts for FL and the reduction in recurrence rate after syngeneic versus autologous unpurged transplantation is consistent with this [17], although a syngeneic GVL effect cannot be excluded. To complicate the issue, however, a prospective randomized autograft study found no benefit in PFS or overall survival between purged and unpurged marrow [18]. Long-term results of minimally cytotoxic conditioning regimens in patients with active disease are needed to clearly evaluate the clinical effect of a graftversus-FL effect. Additional questions include the durability of responses after RI allografts, the effect of the histologic grade in FL on the incidence of relapse, the outcome after unrelated donor allografts, and the identification of tumor antigens that are immunologically relevant for allogeneic responses. Because of a relatively high risk of both early and late relapse after autografting, in general an allograft is the transplant option recommended if a compatible sibling is available. We consider patients up to age 60 to 65 years, depending on their general fitness and level of donor compatibility. A well-matched unrelated donor transplant is considered in selected younger patients, generally younger than 45 years and with a good performance status. The indications for transplantation are at least 1 of (1) progressive symptomatic disease within a year of CHOP-like (cyclophosphamide, hydroxydaunomycin, vincristine, and prednisone) or purine analog– based therapy either as induction or for relapse or (2) multiply relapsed disease. Our preference is to attain a minimal residual disease state (usually with fludarabine with or without cyclophosphamide with or without rituximab, depending on prior therapy), and we use a very-lowintensity conditioning regimen such as fludarabine/ low-dose cyclophosphamide or fludarabine/low-dose total body irradiation [12], with the addition of rituximab if not used previously [7]. This is based on our acceptance that a graft-versus-FL effect exists to a degree. The intention is to achieve mixed chimerism early after transplantation to reduce the risk of severe acute GVHD and to gradually convert to donor chimerism either from the natural course of the transplantation or with DLI. More intensive conditioning

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with increased organ toxicity and a higher risk of GVHD due to early establishment of full donor chimerism (fludarabine/melphalan or cyclophosphamide/ total body irradiation) is reserved for patients with bulky, aggressive, or chemorefractory disease. Mantle Cell Lymphoma

The role of allografting in MCL has been reviewed by Sweetenham [19]. A graft-versus-MCL effect has been unequivocally documented in case reports of resolution of progressive disease after withdrawal of immunosuppression [20,21] or DLI [22]. This is broadly consistent with single-institution studies reporting a low relapse rate after RI [23] and myeloablative [21] conditioning for advanced or recurrent chemosensitive disease, although the follow-up in both of these studies was relatively short. This contrasts with a poor outcome in an EBMT registry survey in 22 older patients undergoing RI allografts [5]: this was due to a high early transplantrelated mortality and a substantial rate of relapse. The ability of a GVL effect to eradicate chemoresistant MCL or to improve the poor outcome observed in patients autografted in CR1 with high ␤2-microglobulin [24] is not proven. Moreover, although a GVL effect may occur in the diffuse form of the disease [21], its activity against the more aggressive blastoid variant is not known. The effect of GVHD on relapse in MCL has not, to our knowledge, been evaluated. Although there is some conflicting evidence, we believe that an allograft is appropriate therapy for patients with relapsed MCL, because the autograft results with readily available conditioning regimens are poor [25]. The intensity of conditioning used depends on disease status and on patient age and performance status. Ideally, the allograft should be offered in first relapse rather than subsequent relapses and limited to patients with chemosensitive disease. The optimal approach to patients in CR1 is controversial. Options include an autograft [26] or observation, the latter based on promising results with short-term follow-up in patients receiving aggressive induction therapy, including rituximab, without an autograft [27]. This has to be balanced against the strong evidence for an allogeneic graft–versus-MCL effect. Currently our preferred option is a very-low-intensity allograft in CR1 in patients younger than 60 years with a sibling donor, particularly in those with increased ␤2microglobulin at diagnosis. A randomized study addressing observation versus autograft versus RI allograft in CR1 is needed. Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma

A durable response to DLI has been reported in chronic lymphocytic leukemia (CLL) [28], although 581


British data suggest that the response may be less frequent than in FL [10]. Promising early results of RI conditioning allografts have been reported by the EBMT registry [29] and Schetelig et al. [30]. The former study reported a 2-year probability of relapse of 31%, with no relapses beyond 12 months in patients receiving T cell–replete grafts, contrasting with ongoing late relapses in those receiving T cell– depleted grafts. The development of chronic GVHD was very significantly associated with a lower risk of relapse. In the latter study, a graft-versus-CLL effect was suggested by the late occurrence of remissions and the effectiveness of DLI in early relapse; DLI was ineffective in patients with a high tumor burden. Also consistent with, but not proof of, a graft-versus-CLL effect are registry data suggesting a lower relapse rate after allografts than autografts, with a plateau in DFS after allografting [31]. Our policy is similar to that with FL. Grafting is offered to suitable patients with disease relapsing early after, or refractory to, purine analog– based therapy. The intensity of conditioning depends on the chemosensitivity and bulk of disease before transplantation. Hodgkin Lymphoma

The largest series documenting the response to DLI for Hodgkin lymphoma (HL) relapsing after an allograft has been reported recently [32]. Of 7 patients with progressive (n ⫽ 6) or residual (n ⫽ 1) disease who received DLI in the absence of chemotherapy, 3 achieved a CR, 2 of whom remained in remission with follow-up more than 12 months (K. Peggs, personal communication). No response was seen in 2 patients, and a partial remission was seen in the other 2. Responses correlated with the development of GVHD. Of note, most patients had nodular sclerosing histology, had undergone a prior autograft, had disease that had run a relatively indolent course over a number of years, and did not have a rapidly progressive recent relapse (K. Peggs, personal communication). Updated data from this group, reported in abstract form, are consistent with a response rate in approximately half of this selected patient cohort [33]. Responses with and without GVHD after DLI have been reported by other authors, but concomitant chemotherapy, absence of histologic detail, and only short-term follow-up make these data difficult to interpret [34,35]. Longer follow-up of various recently published single-institution and registry series of RI conditioning allografts [36-38] may provide useful information. The results of the largest of these studies, published in abstract form [37], argue against the existence of a profound graft-versus-HL effect, because the outcome was poor in chemoresistant disease and there was no plateau in PFS in the second year after transplantation. A smaller single-institution study demon582

strated little effectiveness of this approach in patients with rapidly progressive disease relapsing early after autografting [39]. Another study used fludarabine/ low-dose cyclophosphamide followed by DLI or peripheral blood stem cells in 8 patients with HL relapsing after autografting [36]; 5 did not respond, 2 died of acute GVHD, and only 1 patient was alive in CR, notably, without evidence of donor engraftment. Results of myeloablative allografts for HL are disappointing. The IBMTR reported a 3-year probability of relapse of 65% and a DFS rate of 15% in 100 patients with advanced HL undergoing sibling allografts [40]. There are conflicting data about the effect of GVHD on relapse and whether allografts have a lower rate of relapse than autografts. A trend for a lower probability of relapse after allografting compared with autografting in patients with chemosensitive disease has been reported [41]. An early EBMT registry analysis found a significantly lower risk of relapse with acute GVHD grade II or higher [42]. A more recent EBMT analysis, however, reported no influence of acute GVHD on the rate of relapse and in fact reported a higher risk of relapse after allografts versus autografts for HL, although the former group almost certainly contained a higher proportion of patients with advanced chemoresistant disease [6]. A collaborative international effort is under way among various investigators examining the outcome of patients undergoing RI allografts for HL relapsing after autografts. The effect of factors such as histology (classic versus lymphocyte predominant; the latter clinically behaves as a low-grade B-cell lymphoma and is likely to be immunologically responsive), different conditioning regimens, and acute and chronic GVHD on relapse and survival will be examined in addition to DLI/withdrawal of immunosuppression in biopsyproven active disease. Prospective studies under consideration include an autograft followed by an RI allograft in poor-prognosis patients, the feasibility of which has been established [43]. An allograft for classic histology is considered only if all of the following conditions apply: 1. Biopsy-proven relapsed disease after autografting in which the duration of remission after autografting is ⬎6 months. 2. The relapse is not rapidly progressive, is not chemosensitive, is not amenable to local radiotherapy (eg, pulmonary relapse), and is preferably not associated with “B” symptoms. 3. A second autograft is not feasible (eg, insufficient stem cells) [44]. The choice of conditioning regimen and whether to consider an unrelated donor depends on factors such as patient age, time since previous high-dose therapy, organ function, and degree of donor match. Ideally, these patients should be enrolled on a prospective study (see below).


Diffuse Large-Cell Lymphoma

To our knowledge, there is no published systematic review of the role of DLI in diffuse large-cell lymphoma (DLCL). There are only 2 reports of a durable response to DLI after allografting: 1 in a patient with relapsed mediastinal B-cell NHL [2] and the other with Richter transformation of CLL [45]. A remission lasting ⱖ140 days after cessation of tacrolimus for relapsed T cell–rich B-cell NHL has been reported; 3 other patients with relapsed DLCL did not respond to withdrawal of cyclosporine and DLI [46]. Failure of DLI despite GVHD has been documented in anaplastic large-cell NHL [47] and in lymphomatoid granulomatosis and B-cell DLCL [48]. Two patients with high-grade NHL relapsing after a nonmyeloablative allograft did not respond to DLI [35]. Single-institution and registry data in this area are difficult to interpret for the reasons outlined previously. Of note, many RI conditioning studies have been published as abstracts only (without peer review). Most studies have varied with respect to histology, status at transplantation, and T-cell depletion. The EBMT registry reported the results of RI conditioning regimens (mainly fludarabine based and varying from low-dose cyclophosphamide to high-dose melphalan) in 62 patients with high-grade lymphoma, including transformed low-grade disease [7]. The results were disappointing despite most patients having chemosensitive disease before transplantation: the probability of disease progression at 2 years was 79%, with a PFS of 13%. The myeloablative allograft data are inconclusive. There were no survivors in a series of 14 patients who received allografts with a myeloablative regimen for advanced intermediate-grade NHL (predominantly DLCL) reported by the M.D. Anderson group in the mid 1990s; all died of progression or toxicity [49]. A French review found no effect of acute or chronic GVHD (although the data were not provided) in a series of allografts for aggressive NHL, predominantly DLCL [50]. In this series, the 5-year survival was 23% in 48 patients not in CR at transplantation— results not obviously different from those of autografting in a similar patient group. A more recent survey from the EBMT registry reported a lower relapse rate after myeloablative allografting compared with autografting for intermediate-grade NHL by using the Working Formulation [6]. As discussed previously, this category includes follicular large-cell, diffuse large-cell, and diffuse small cleaved (most likely mantle cell) histologies, so the specific GVL effects in these individual histologies cannot be elucidated. Acute GVHD was associated with a reduced rate of relapse, but insufficient data were available to analyze the effect of chronic GVHD.

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In contrast, the recent review by Bierman et al. [17] showed no difference in relapse rate for intermediateor high-grade NHL between syngeneic, allogeneic T cell–replete or– depleted, or autologous transplants, although the mix of histologies, imbalances in pretransplantation characteristics, and small numbers (particularly in the syngeneic and T cell– depleted groups) make interpretation of these results difficult. There are few specific data on the outcome of allografting for peripheral T-cell lymphomas. The Milan group have reported in abstract form the outcome of RI conditioning in 8 patients with relapsed nodal peripheral T-cell lymphomas, most with chemorefractory disease and in half of whom a previous autograft had failed [51]. With a short median follow-up of 18 months, all were alive and in remission. Questions that need to be addressed in properly designed studies include the effect of immunophenotype (B versus T cell), histology (de novo B-cell DLCL versus follicular large cell versus transformed low grade), and location (nodal versus site specific, eg, mediastinal or bony) on the outcome after transplantation. We are not convinced that a clinically relevant graft-versus– de novo B-cell DLCL effect commonly exists. Hence, an allograft is not generally offered to patients with primary refractory disease, chemorefractory relapse, or postautograft relapse. Possible exceptions include those with transformed low-grade NHL (based hypothetically on a higher chance of a GVL effect in the presence of an underlying low-grade component) relapsing more than 6 months after autografting without rapid disease progression or mediastinal lymphoma [2]. An allograft is also considered in patients with chemosensitive first relapse in whom sufficient autologous stem cells cannot be collected or in whom the collection is overtly contaminated with lymphoma. Rituximab is offered after allografting to patients with CD20⫹ B-cell tumors who have not previously received this drug, on the basis of promising preliminary experience with this approach [52]. Lymphoblastic Lymphoma

A single report of late-onset cutaneous relapse of T-cell lymphoblastic lymphoma (LBL) that responded durably to withdrawal of cyclosporine and a flare of cutaneous GVHD has been published [46]. A recent publication from the IBMTR and the Autologous Bone Marrow Transplant Registry compared autologous versus myeloablative non–T cell– depleted allografts for LBL [53]. The relapse rate beyond 6 months after transplantation was significantly lower in the allograft patients and was independent of disease status at the time of transplantation. A graft-versusLBL effect cannot be concluded unequivocally from these data, however, because infusion of contaminated 583


Table 1. Lymphoma Type and Evidence for GVL Effect

Lymphoma Type

Durable* Response to DLI/Withdrawal of IS

Durable* Response to Very-Low-Dose-Intensity Conditioning Allo

Less Relapse with GVHD and/or More Relapse with T-Cell Depletion

Plateau on DFS Curve after Allo and/or Less Relapse with Allo versus Auto

Follicular Mantle cell CLL/diffuse small cell Nodular sclerosing Hodgkin Diffuse large B cell Lymphoblastic Burkitt Mycosis fungoides

Yes Yes Yes Yes† Yes† Yes† No Yes

Yes† Yes† Yes Probably no Probably no NE NE NE

No†‡ NE Yes Conflicting data NE Conflicting data NE NE

Yes NE Yes Conflicting data No Yes No NE

NE indicates not evaluable, ie, insufficient data or duration of follow-up; Allo, allogeneic transplantation; Auto, autologous transplantation; IS, immunosuppression. *In most of the studies, follow-up was less than 2 or 3 years. †See text for caveats. ‡Studies have not shown a reduction in relapse.

autologous cells may have contributed to a higher risk of relapse. GVHD had no significant effect on the risk of relapse, but the study was not powered adequately to detect a small effect. EBMT studies have also demonstrated a lower risk after allografting for LBL and suggested a reduced risk with acute [6] and chronic [54] GVHD. A myeloablative allograft is offered to suitable patients with high-risk LBL in CR1 or those with chemosensitive relapse. Immunosuppression is tapered early in the absence of GVHD [55]. Burkitt Lymphoma

There is a paucity of data in this area and no convincing evidence that allografting can cure adult patients with Burkitt lymphoma relapsing after, or refractory to, intensive induction regimens. A response to withdrawal of immunosuppression has been reported, but this was not durable [56]. The M.D. Anderson group reported the results of myeloablative allografts in 10 patients with diffuse small noncleaved lymphoma and noted a very high rate of rapid disease progression after transplantation [49]. No difference in relapse rate between autologous and allogeneic transplants for Burkitt lymphoma and no effect of acute GVHD on the rate of relapse have been reported by the EBMT [6]. Allografts are not offered to patients with Burkitt lymphoma, because most patients are either cured by aggressive induction regimens [57] or relapse early with rapidly progressive chemoresistant disease. Cutaneous T-Cell NHL

A graft-versus–mycosis fungoides effect has been reported by a number of investigators. In one case, histologically and molecularly documented persistent disease at day ⫹60 after a nonmyeloablative allograft regressed after withdrawal of cyclosporine and devel584

opment of cutaneous GVHD. Remission was maintained for ⱖ24 months of follow-up [58]. In a second case, mycosis fungoides recurring 9 months after a myeloablative allograft subsequently resolved after cessation of cyclosporine and development of lichenoid chronic GVHD. However, low-grade cutaneous disease subsequently recurred over the next 4 years and responded temporarily to DLI [59]. The latter case points out the necessity of long-term follow-up in evaluating GVL effects. We have observed a patient with large-cell transformation of mycosis fungoides in whom the low-grade cutaneous disease regressed with the onset of chronic GVHD but in whom the largecell component progressed. Resolution after withdrawal of immunosuppression of a cutaneous CD30⫺ large T-cell lymphoma persisting after a nonmyeloablative allograft has been reported [60]. A GVL effect was suggested by infiltration of the skin tumors by donor lymphocytes. Suitable patients with relapsed mycosis fungoides are considered for an allograft. Large-cell transformation is a contraindication. Summary

Table 1 summarizes, to the best of our knowledge, each of the GVL criteria for various types of lymphoma. There are insufficient data to comment meaningfully for histologies such as peripheral T-cell NHL, hepatosplenic lymphoma, and nodular lymphocyte-predominant HL. A number of collaborative groups have phase II trials under way evaluating the role of RI conditioning regimens in diseases such as HL, in which the existence of a GVL effect is controversial. Enrollment of patients in these prospective studies is strongly encouraged because without a collaborative systemic approach in a large number of patients, it is unlikely that the questions raised in this article will be answered.


Ultimately, however, randomized trials will be required for definitive conclusions.

GVL IMMUNOBIOLOGY In the following section, we review the immunobiology of B cells as both initiators and targets of T-cell cytotoxicity. By examining the mechanisms by which malignant B cells may be induced to function as effective antigen-presenting cells (APC) to allogeneic T cells, we may better understand (1) the observed variations in sensitivity of lymphoma subtypes to GVL effects and (2) how to successfully manipulate interactions between T cells and lymphoma to maximize eradication of residual disease while minimizing GVHD. The induction of a GVL effect after allogeneic stem cell transplantation (SCT) is dependent on tumor-antigen recognition, subsequent cytotoxic T lymphocyte (CTL) generation, and sensitivity of the lymphoma to cytotoxicity effector mechanisms. Activation of resting donor T cells follows recognition of alloantigens or tumor-specific antigens expressed on the surface of either professional APCs, such as dendritic cells (DC), or lymphoma cells themselves. Host APCs are central to donor T-cell activation, as demonstrated in animal models in which GVHD and GVL effects diminish and are eventually lost as initial mixed APC chimerism evolves to full donor chimerism [61-63]. Once activated, CTLs mediate the effector phase of GVHD/GVL via cytotoxicity produced either by cell/cell contact mechanisms (Fas, perforin, and tumor necrosis factor-␣) or by the production of soluble mediators, including interferon-␥ and soluble tumor necrosis factor-␣ [64]. B-Cell Lymphoma

The question arises whether B lymphoma cells represent competent APCs for direct activation of alloresponsive T cells or whether B lymphoma cells are subsequent targets for CTLs generated initially by the allostimulatory effects of host DCs. Normal B cells constitutively express both major histocompatibility complex (MHC) class I and II, along with costimulatory molecules, including CD80 and CD86, which are essential for generation of CTLs from naive T cells. It is important to note that the activation status of B cells is central to their ability to act as APCs. Resting normal B cells have been variously shown to induce direct tolerance of antigen-specific CD8⫹ T cells [65], induce T-cell anergy via TGF-␤ production [66], downregulate interleukin-12 production by DCs [67,68], and influence T-helper type 1 and 2 differentiation via the production of regulatory cytokines, including interleukin-10 [69]. Similarly, resting B cells

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exert a regulatory function in in vivo models of T-cell immunity, including tumor rejection [70,71]. Conversely, B cells activated via ligation of CD40 are potent inducers of T-cell activation, which in turn can deliver antitumor immune responses against both non–B-cell [72] and B-cell [73-75] malignancies in vivo. The expression of co-stimulatory surface-bound molecules is significantly enhanced after B-cell activation by a range of stimuli, including CD40L and lipopolysaccharide [76]. B cells activated by lipopolysaccharide and interleukin-4, however, fail to induce effector T-cell responses [75], and this may lead to T-cell tolerance [66], thus underlining the importance of the different outcomes of B-cell activation depending on the stimulating signal used. These studies indicate that normal B cells can potentially be induced to act as effective APCs for the generation of T-cell immune responses. Follicular Small-Cell Lymphoma and MCL

Parallel to findings in normal B cells, some malignant lymphoma cells may also demonstrate an APC phenotype. B cells isolated from follicular NHL and small lymphocytic lymphoma/CLL share phenotypic similarities to normal resting B cells in both their expression of MHC and co-stimulatory molecules and in their responses to CD40L. These lymphomas consistently show high levels of CD80, CD86, and CD40 expression [77,78]. Similarly, the B cells from MCL express CD40 and show intense upregulation of CD80 and CD86 after treatment with CD40L [79]. The sensitivity of small-cell lymphomas to T-cell cytotoxicity has been demonstrated by studies of autologous vaccination with either DCs [80,81] or killed autologous lymphoma cells [82]. In these studies of autologous immunotherapy, CTL generation against B-cell idiotype was successfully achieved from within the patient’s naive-T-cell population. In the post– allogeneic SCT setting, there is potential not only for a greater range of CTLs to be generated from the donor T-cell repertoire, but also for residual lymphoma B cells to act as functional APCs. Activation of resting lymphoma B cells may occur in response to CD40L provided by donor CD4⫹ T cells alloreactive to host major or minor histocompatibility antigens. When activated by CD40L, the lymphoma B cells, in turn, can drive the subsequent activation of allogeneic CD8⫹ T cells to CTLs. Once generated, both activated CD8⫹ T cells [83] and subsets of CD4⫹ T cells [84] are capable of inducing the cytotoxicity of the stimulatory APCs and eradicating residual disease. This hypothesis is supported by the observation that these lymphoma subtypes are those that show convincing responses to an allogeneic GVL effect. 585


DLBCL and B-Cell LBL

In contrast to small-cell lymphoma, DLBCL and B-cell LBL express significantly less CD86 [77] and demonstrate MHC class I downregulation in both animal models [85] and clinical samples [86] of B-cell DLCL. These biological features of large-cell lymphomas lead to failure of recognition by CTLs and likely escape from immune attack. There may, however, not be uniformity in the apparent inability of B-cell DLCL to induce T-cell activation. Lymphochip technology has demonstrated that B-cell DLCL can be separated on the basis of gene expression into either a normal germinal center phenotype or an activated peripheral blood B-cell phenotype [87,88]. Although the latter group shows a poorer prognosis when treated with conventional chemotherapy, these genotypic differences may indicate a greater ability to initiate a cytotoxic response by allogeneic T cells. This is countered by the finding that the antiapoptotic genes PDCE4B and PKC␤ are overexpressed in these subgroups. Clinical studies are required to clarify the effect of genotypic differences on susceptibility to GVL effects.

allogeneic T cells within the secondary lymphoid tissues, has been used to promote a GVL effect while preventing the development of GVHD in the peripheral tissues in a haploidentical mouse model [92]. This agent has not, however, been successful in treating established GVHD in a nonidentical canine model [93]. The differences in outcome observed between these models likely reflect the fundamental differences between preventing the onset of GVHD and attempting to suppress activated circulating T cells. T-Cell Lymphoma

The ability of T lymphoma cells to function as APCs in the activation of antilymphoma CTLs is less well described than for B cells. Normal T cells express MHC class I and, when activated, high levels of MHC class II in addition to low levels of co-stimulatory molecules. In vitro studies demonstrate that T cells may also be targeted by CTL clones [94]. Low-grade cutaneous T-cell lymphomas express high levels of MHC class II, which initiate allogeneic cytotoxicity sufficient to eradicate the lymphoma [95].

CTL Trafficking

FUTURE DIRECTIONS

To take advantage of the potential APC function of B cells, T cells must first gain access to the sites of lymphoma involvement. Although naive T cells can freely access the secondary lymphoid tissues, activated CTLs are excluded from doing so by the downregulation of the chemokine receptor CCR7 [89]. As a consequence, T cells activated by exposure to alloantigens expressed on APCs outside of the lymph nodes, such as DCs resident within epithelial tissues, are actively excluded from “seeing” residual lymphoma cells within a lymph node. Thus, for a GVL effect to occur, both the activation and effector phases of the GVL effect must occur within the lymphomatous node, as occurs in the lymphoid hypoplasia associated with GVHD [90]. T-cell trafficking to sites of lymphomatous involvement may also vary between lymphoma subtypes. For example, CLL B cells actively attract CD4⫹ T-cell help via the expression of chemokine ligand 22 [91]. The implication of these variations in T-cell trafficking is that after allogeneic BMT, residual CLL may actively recruit allogeneic T cells and be activated into APC function, as outlined previously. The requirement for both activation and effector responses to occur within the lymph node may limit the application of adoptive transfer of ex vivo–activated CTLs and may necessitate in vivo CTL generation either by allogeneic transplantation or vaccination strategies. To this end, manipulation of allogeneic T-cell trafficking by using the sphingosine1-phosphate receptor agonist FTY720, which traps

The challenges faced in the application of allogeneic SCT to the treatment of lymphomas center on limiting GVHD and identifying those subtypes of lymphoma that are sensitive to CTL-mediated cytotoxicity. It is likely that the B-cell malignancies that are genotypically or phenotypically similar to normal B cells able to differentiate into an effective APC phenotype, sensitive to apoptosis induction and permissive to T-cell trafficking, will be more sensitive to GVL effects. Whether genotype analyses with microarray technology can help in identifying immunoresponsive lymphoma subgroups is unknown. To date, lymphoma-specific antigenic targets for the GVL effect remain limited; hence, it is not yet possible to achieve adoptive immunotherapy with highly selected, ex vivo– expanded GVL-specific CTLs. As a result, means of controlling allogeneic T-cell function are the current focus of research. It has been suggested that delaying T-cell infusions until after resolution of the cytokine storm may maintain the antitumor efficacy of engrafted T cells while limiting nonspecific alloreactivity. This is an attractive hypothesis and has been effectively demonstrated in animal models [96,97], but it is hindered by the dual observations that maximal GVL effect is dependent on a state of mixed APC chimerism and that prediction of GVHD risk at any given dose of DLI is imprecise [98]. Given the difficulties of minimizing GVHD by manipulating T-cell doses or timing, there has been increased interest in the use of regulatory cell populations to restrict the action of allogeneic CTLs.

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Both regulatory T cells [99] and regulatory DCs [100] are capable of limiting GVHD while preserving the GVL effect. These phenomena are yet to be tested in the clinical setting. Given the central role of host APCs in the induction of GVL after SCT, vaccination with either autologous DCs or irradiated, ex vivo–activated lymphoma cells may lead to enhanced re-stimulation of engrafted allogeneic T-cell responses against minimal residual disease and result in lower rates of recurrence. Mouse models of pretransplantation vaccination of donors [101-103] and posttransplantation vaccination [104,105] of recipients have been shown to result in enhanced graft-versus-tumor effects without exacerbation of GVHD. These strategies have not yet been demonstrated in a clinical setting, but they offer great potential in the delivery of enhanced immunotherapy as an adjunct to allogeneic SCT.

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