Strategies to Prevent EBV Reactivation and Posttransplant Lymphoproliferative Disorders (PTLD) after Allogeneic Stem Cell Transplantation in High-Risk Patients

Abstract

Epstein-Barr virus (EBV)-associated postallogeneic stem cell transplantation (SCT) lymphoproliferative disorder (PTLD) is often life threatening. The risk of EBV reactivation is highest in older patients, T cell-depleted SCT (in vivo or vitro), and in unrelated or mismatched SCT. Cumulative numbers of patients with EBV reactivation and PTLD are rising as more patients at high risk for EBV reactivation and PTLD are receiving allo-SCT. Novel but easily applicable strategies are needed to prevent EBV reactivation and PTLD to serve the needs of the increasingly enlarging population of high-risk SCT recipients across the globe.

EPSTEIN-BARR VIRUS (EBV)-RELATED B CELL LYMPHOPROLIFERATIVE DISORDER (PTLD) AFTER ALLOGENEIC STEM CELL TRANSPLANTATION

Risk Ractor, Biology, and Pathogenesis

EBV-associated PTLD following allogeneic stem cell transplantation (allo-SCT) is a life-threatening complication resulting predominantly from outgrowth of donor-derived EBV-infected B cells [1]. Most cases of PTLD are associated with EBV infection from B-lymphocytes, which in the setting of immunosuppression can induce a transformation to a lymphoproliferative disorder. Diagnosis, monitoring, and treatment of EBV reactivation and especially PTLD is expensive, and treatments can also carry risk. Patients undergoing unmanipulated allo-SCT have a risk of developing EBV-PTLD of approximately 1%, whereas at the other extreme, T cell depletion using antibodies specific for CD2 and CD3 was associated with very high risk of PTLD (71%) [2]. The factor that conferred the greatest relative risk was the use of anti-CD3 antibody to treat graft-versus-host disease (GVHD). A recently published study evaluated 26,901 allo-SCT recipients to define the risk factors for PTLD. PTLD developed in 127 (0.47%) patients, with more than 80% of cases occurring within the first year after allo-SCT. The authors identified 4 high-risk factors (aged ≥50 years at transplantation, T cell depletion of the graft, antithymocyte globulin [ATG] use, and unrelated or HLA-mismatched grafts) associated with increased risk for PTLD. Patients with no risk factors had a cumulative incidence of 0.2% versus 8.1% for patients in whom 3 or more risk factors were present. In this study, a majority of patients (transplant period 1964 to 1994) received matched related donor SCT [3].

T cell depletion is a risk factor, but the risk associated with various approaches to depletion varied substantially. The use of sheep red blood cell rosetting, anti-T or anti-T, and anti-natural killer (NK) monoclonal antibodies (mAbs) was associated with relative risks of >10-fold. The use of methods that resulted in balanced loss of B cells and T cells was not associated with significantly increased risk. The use of lectins, Campath-1H mAb, or elutriation was not associated with a statistically significant increased relative risk [2,4,5]. It is likely that the protective effect of B cell depletion derived from both decreased numbers of virus-carrying donor lymphocytes and the elimination of the target cell for transformation. In contrast to EBV-PLPD in the solid-organ transplant setting, the recipient’s age, EBV seronegativity, and underlying disease are not risk factors for PTLD in allo-SCT [3,6].

Late PTLD after allo-SCT differ in their risk factors, pathology, and EBV association. These tumors are sometimes of T cell rather than B cell origin, and at times are not EBV associated [7,8]. Late-onset PTLD not associated with EBV have also been described in solid-organ transplant recipients [9]. Hodgkin lymphoma (HL) occurs at increased frequency following allo-SCT generally late onset. A 6-fold increased risk of HL in SCT recipients in comparison with the general population is similar to the increase in risk in human immunodeficiency virus (HIV)-positive patients [10].

Despite higher T cell dose in the graft, recipients of allo-peripheral blood stem cell transplantations (PBSCTs) also are at risk for PTLD [11]. The median time to the diagnosis of PTLD is similar to that for bone marrow transplant (BMT) recipients. T cell depletion of the PBSC product and underlying diagnosis of immune deficiency in the recipient were identified as risk factors in multivariate analysis. Reduced intensity conditioning (RIC) transplants have also been associated with EBV-PTLD [12,13]. Fludarabine alone has also been reported to be associated with the development of EBV-PTLD [14], so the contribution of transplantation to the problem is not yet clear. Because EBV does not cross the blood-placenta barrier, risk after cord blood transplantation (CBT) might be anticipated to be lower than other donor sources. Alternately, CBT recipients are anticipated to have a higher incidence of PTLD because they resemble T cell-depleted SCT as they lack EBV-specific cytotoxic T cells. As the incidence of EBV-PTLD in CBT recipients appears not to differ from that in recipients of unmanipulated BM grafts, it may be that these factors balance out [15,16]. High incidence was reported in an RIC CBT recipients study, expected to be related to use of antithymocyte globulin (ATG) in the conditioning regimen [16].

The cellular responses to classes of EBV antigen (especially latent versus lytic) and to particular antigens are well recognized [17–19]. Thus, different peptide epitopes elicit different magnitudes of T cell response. Evidence has been presented suggesting a relationship between EBV CD8⁺ T cell frequencies and viral load in peripheral blood mononuclear cells (PBMC). Whereas EBV reactivation as evidenced by elevated EBV viral loads was not highly predictive of the development of EBV-PTLD, it is impaired EBV specific T cell recovery in conjunction with elevated EBV viral load that was associated with development of EBV-PTLD in all 5 reported cases [20]. Progressive loss of EBV-specific CD4⁺ and CD8⁺ T cells has also been associated with high risk for developing EBV-associated lymphomas in acquired immune deficiency syndrome patients [21]. However, the relative importance of CD8⁺ and CD4⁺ responses, responses to latent versus lytic viral antigens expressed on tumor cells, or responses to specific individual viral antigens remains to be determined. Several investigators have presented evidence for a critical role of CD4⁺ T cells. These may exert direct cytotoxic effects or suppress the outgrowth of EBV-transformed B cell lines [12,22].

EBV-PTLD after auto-SCT

Autologous BMT or peripheral blood progenitor cell (PBPC) transplantation has also been associated with EBV-PTLD, although much less frequently than after allo-SCT. T cell depletion (for the removal of T cell tumor cells) appears to be the major risk factor, but EBV-PTLD has also occurred in association with CD34 cell selection [23–26]. The major determinants of the risk period for PTLD are presumed to be immunologic, and several investigators have presented evidence that reconstitution of CD8⁺ T cell immunity to EBV generally occurs during the 6-month period following allo-SCT. It may occur even more rapidly following autologous PBPC transplantation.

Clinical Presentation and Diagnosis

Fever, generalized lymphadenopathy, respiratory compromise, and rising liver transaminase levels are typical and have usually been associated with a rapidly progressive multiorgan failure and death. Lesions are nodal and extra-nodal, frequently involving Waldeyer’s ring, the gastrointestinal tract, the liver, and the central nervous system. Tumors that arise later after transplantation (>1 year) are more commonly localized and often have an indolent course. On the contrary, patients with EBV infection usually asymptomatic initially. As PTLD may evolve progressively from an EBV-reactivation (infection) to polyclonal disorder to a more aggressive monoclonal variant PTLD, early diagnosis is an important so that preemptive therapy can be started early in the course. Measurement of EBV-DNA viral load by quantitative PCR amplification assays can be a sensitive aid to early diagnosis, but it is not always specific for disease onset. Different assays use whole blood, serum, or PBMC and require differing interpretation. When PBMC are assayed, an elevated EBV-DNA may reflect both EBV in normal B cells (a population that may be expanded in immunosuppressed patients) and EBV in transformed cells. Assays of EBV in serum reflect virus shedding, which occurs intermittently in normal seropositive persons from epithelium and also from lytically transformed B cells as well as virus released from necrotic transformed cells. Assays measuring whole blood will measure EBV-DNA from all these sources. In general, assays using PBMC are the most sensitive; but in all assays, elevated loads may not always reflect PTLD.

The definitive diagnosis of PLTD requires biopsy with in situ hybridization or immunochemistry to define viral association. EBV-encoded RNA in situ hybridization is the most sensitive tool for detecting virus in tumor. LMP-1 staining is also available in most pathology laboratories but is negative in the subset of tumors that do not express the viral antigen. Immunohistochemistry for EBNA-1 could also be broadly applicable.

PTLD includes a heterogeneous group of lymphoproliferative disorders ranging from reactive, polyclonal hyperplasia to aggressive non-Hodgkin lymphomas. A revised classification was published in 2008 by the World Health Organization and recommends classifying PTLD into 4 categories: (1) early lesions, (2) polymorphic PTLD, (3) monomorphic PTLD, and (4) classic HL-type PTLD [27]. All types are associated with EBV. There is no consensus on the prognostic predictive value of either morphology or clonality. However, it is clear that, on occasions, patients with polyclonal disease may progress despite aggressive therapy including DLI [28]. Whereas in solid organ transplant recipients, PTLD most commonly arises in host B cells, in allo-SCT recipients EBV-PTLD usually arises in donor B-lymphocytes (exceptions auto-SCT or auto recovery).

Earlier studies in recipients of allo-SCT that were selectively T cell-depleted to prevent GVHD, suggesting that an elevated EBV-DNA load was highly predictive of EBV-PTLD [20,29,30]. Follow-up studies, however, which also included non-T cell-depleted allo-SCT recipients, showed that small population of patients with elevated EBV-DNA subsequently developed PTLD [1,17,31–33]. Recent evidence-based review guidelines from the European Conference in Infections in Leukemia recommend weekly screening of EBV-DNA for at least 3 months in high-risk allo-SCT recipients [31]. Serial monitoring is important to distinguish patients with a stable-elevated EBV-DNA load from those with increasing EBV-DNA, which may indicate preemptive therapy with (eg, rituximab) to prevent developing PTLD. Combined monitoring of EBV-DNA and EBV-specific CTL responses appears to better predict individual patients at risk for PTLD development [20,28]. Unfortunately, EBV-specific CTL assay is not routinely available commercially and available mainly in the research setting. The context and risk factors are also important in deciding when to intervene; preemptive intervention in high risk early post-allo-SCT recipients is preferred (eg, older adults who received URD allo-SCT with severe GVHD who have received in vivo or ex vivo T cell depletion).

EBV Reactivation and PTLD Rates Are Expected to Increase

At present, 15,000–20,000 patients receive allo-SCT annually throughout the world, and more than half of all allo-SCT are performed from nonmatched related donor (MRD) stem cell sources [34]. With continued improvement in SCT outcome, the indications for SCT continue to grow. Furthermore, the sourced of donor stem cell and the number of suitable matches are expanding. At the same time, modified transplantation regimens have facilitated safer procedures despite an increase in patient’s age and comorbidies. An aging population is increasing the proportion of individuals susceptible to diseases for which SCT is indicated. Moreover, proposed national health insurance reforms in the United States may expand the number of insured patients and reduce economic barriers for allo-SCT in more patients [35–38]. Because EBV reactivation and PTLD risk is reported to be high among CBT, mismatched (MM), and unrelated donor (URD) allo-SCT, and as most centers use ATG with URD SCT, more high-risk patients will receive an allo-SCT every year and cumulative numbers of patients with EBV-reactivation and PTLD are likely to rise [3,16,39,40].

Current Therapeutic Options to Control EBV Reactivation and PTLD

Reduced immunosuppression

Reducing immunosuppression (IS) to restore immune responses to EBV is not usually a useful approach for treating PTLD early after allo-SCT because the patients are profoundly immunosuppressed and the regenerating immune system cannot recover rapidly enough to eradicate the lymphoproliferative process [17,31]. Although data is limited as to the safety and efficacy of this approach in treating PTLD and there is a rational fear that reducing IS will induce or exacerbate GVHD and/or EBV replication, careful modulation and reduction of IS has been successfully used early post-SCT in select cases to prevent PTLD at the time of EBV reactivation [41].

EBV-specific cytotoxic T cell line (CTL)

The problem of alloreactivity can also be overcome by infusing EBV-specific cytotoxic T cell lines (CTLs) generated using EBV-transformed lymphoblastoid B cell lines (EBV-LCL) which, as professional antigen-presenting cells (APCs), efficiently present the viral antigens. Because of the underlying latency of EBV and the highly immunogenic nature of EBV disease, adoptive transfer of EBV-CTL is extremely effective [1,17,42–46]. The use of adoptive immunotherapy for PTLD is increasing in the clinic and a large quantity of recent basic science literature investigating the ideal approach has been recently reported. There are number of different approaches to develop EBV-specific CTL, for example, using EBNA-1-specific T cells or peptide-selected T cells [19,47]. Also, there are preclinical studies using CD8⁺CTL cells or CD4⁺ cytotoxic T cells, or both [48–50].

The adoptive immunotherapy is reported to be highly effective as prophylaxis in high-risk patients with a past history of PTLD or patients receiving selective T cell depletion [51,52]. However, several of these approaches remain experimental and additional drawbacks are the time and facilities required for CTL production. Another strategy is to develop a bank of partially HLA-matched allogeneic lines, which can be readily and rapidly available [53,54]. However, limited numbers of stem cell allograft recipients have been treated in this way, and further studies are needed before unrelated EBV-specific CTLs can be widely offered and available to all allo-SCT recipients.

Anti-CD20 mAb

Rituximab has been used as prophylaxis (preemptive therapy for EBV-reactivation after allo-SCT) and treatment for PTLD after allo-SCT, with initial response rates between 55% and 100% in small case series [1,17,31,32,55,56]. Because CD20 expression is not confined to malignant cells, normal B cells are also destroyed. This can be a significant concern in patients who are already immunosuppressed, and fatal viral infections have been reported after rituximab therapy [57]. Rituximab can deplete B cells (both donor and host) for 6 to 9 months in these already immunosuppressed patients [58,59]. An additional concern is that when used as therapy, it does not restore the cellular immune response to EBV, which is a crucial requirement if EBV-mediated B cell proliferation is to be controlled long term [60]. Additionally, anti-CD20 antibodies are poorly effective against CNS disease because of low penetrance across the blood-brain barrier.

Antiviral therapy

The limited data available on antiviral therapy in patients with EBV reactivation and PTLD following allo-SCT does not support their use. Long-term prophylaxis with antiviral agents or IVIG may decrease the incidence of EBV reactivation and PTLD by limiting intercellular virus transmission [61], but EBV reactivation continues to be reported in high-risk transplants such as CBT or URD, despite long-term antiviral prophylaxis with or without IVIG in the first 100 days or longer posttransplantation. The main cause of PTLD is the proliferation of the latently infected EBV + B cells (and not lytic replication), which is why antiviral pharmacotherapy is not expected to be effective in this setting.

Chemotherapy

Treatment of PTLD with chemotherapy appears to be rarely effective, and carries the risk of further IS. One concern is that PTLD patients may be more susceptible to chemotherapy toxicity after allo-SCTs following intensive conditioning. There are insufficient reports supporting chemotherapy for PTLD following an allo-SCT in the immediate posttransplant setting.

Time to Explore Novel But Easily Available Strategies to Prevent EBV Reactivation Early Posttransplantation

The highest risk of developing PTLD is during the first 6 months following a transplant. Similarly, EBV-reactivation also occurs very early posttransplant at a median interval of 45 to 51 days in 25% to 50% of patients [1,3,62–64]. EBV reactivation leads to development of PTLD in some patients. Removing EBV-infected memory B cells or EBV-transformed B cells early posttransplant might prevent EBV reactivation and therefore PTLD.

We hypothesized that incorporating agents that can remove memory B cells or EBV transformed B cells with conditioning regimen would significantly reduce or prevent the EBV reactivation and PTLD.

Rituximab use in the peritransplant period

After autologous SCT, there is a significant deficiency of CD 27⁺ memory B cells for up to 2 years after a single-dose rituximab [65]. Rituximab has been evaluated as preemptive therapy in patients with a rising EBV viral load [1,17,56]. However, use of rituximab posttransplant risks delaying recovery of a donor-derived B cell immune response. Rituximab therapy 2 months after allo-SCT prevents donor B cell reconstitution up to 1 year following HCT [58,59]. The half-life of rituximab, despite the influence of the tumor burden, could range from several days to months. However, if rituximab is used during the conditioning regimen, it should theoretically have less of an impact on donor derived B cell reconstitution compared to posttransplantation administration for EBV reactivation or PTLD where effects are long lasting. When patients received rituximab before (within 6 months of) allo-SCT for B cell lymphoid malignancies (n = 38), we observed no EBV reactivation reported even in patients with 3 risk factors for PTLD (including CBT recipients) and no increased infection [66]. In a another study, the European Group for Blood and Marrow Transplantation (EBMT) Working Party on Severe Aplastic Anemia (SAA) added rituximab on day 5 of their fludarabine, cyclophosphamide, low-dose total body irradiation (TBI), and ATG conditioning regimen for unrelated donor transplants in acquired SAA [67]. Vianna et al. [68] recently reported no cases of PTLD following a single dose of rituximab (150 mg/m²), in a cohort receiving intensive IS including rabbit ATG (10 mg/kg) and 6 months of methylprednisone and tacrolimus for patients receiving multivisceral transplantation (n = 29). Rejection was treated with an increase in the baseline tacrolimus levels combined with 1 to 3 doses of intravenous methylprednisone (500 mg), depending on initial severity of the rejection episode and clinical response to treatment. For steroid resistant rejection, patients were dosed with additional thymoglobulin or with Campath. In this study, 13 patients (48%) experienced 19 episodes of acute rejection, 8 (30%) episodes occurred in the first 90 days posttransplant. Despite intense IS early post-graft, no patient developed PTLD; higher prevalence of EBV reactivation and PTLD expected after multivisceral transplantation. In almost 4 years since the initiation of their intestinal transplant program, the authors did not document a single case of PTLD.

A study has shown low-dose subcutaneous rituximab effectively depletes low burdens of CD20-positive B cells [69], a situation comparable to most SCT recipients (excluding some with B cell malignancies) at the time of conditioning.

Moreover, in patients treated with rituximab close to the time of transplantation or given as a part of the conditioning regimen, the additional depletion of donor B cells in the stem cell graft may result in greater protection from aGVHD (aGVHD) [70–72].

Sirolimus—a dual role as a GVHD prophylaxis and prevention of EBV reactivation

Sirolimus (rapamycin), a macrolide antifungal antibiotic isolated from Streptomyces hygroscopicus, has potent immunosuppressive properties. Besides its inhibitory effects on normal cells of the immune system, rapamycin also inhibits proliferation of transformed cell lines. Experimental studies suggest that rapamycin inhibits growth of human Epstein-Barr virus-transformed B lymphocytes [73,74]. The drug had a profound inhibitory effect on the growth of PTLD-like EBV + B cells xenotransplanted into severe combined immunodeficiency (SCID) mice. In this in vivo xenotransplant model, rapamycin markedly delayed growth and induced regression of the established tumors. Cell death induced by rapamycin in BKS-2 lymphoma was found to be via apoptosis induction [73–75]. Rapamycin has been shown to be effective in GVHD prophylaxis after SCT [76]. However, the prevalence of PTLD following sirolimus use has not been reported. A recent report describes 2 patients with PTLD successfully treated with rituximab and rapamycin after renal transplantation [77]. Similarly, in pediatric transplant patients in whom the immunosuppressive therapy was converted to sirolimus, PTLD remained in remission for as long as 23 months [78,79]. Another benefit is that mTOR inhibitors are effective against a number of malignancies and they may also add antitumor activity that could be helpful in eliminating any residual disease post-SCT in certain settings [80].

Implications for EBV PTLD Prevention

We propose that elimination of host and donor memory B cells by low-dose rituximab in the conditioning regimen could reduce the incidence of EBV reactivation and PTLD post-SCT. Furthermore, rituximab may attenuate donor T cell activation in the early phase of transplantation via depletion of host B cells and reduce the risk of aGVHD. In addition, rituximab might help in vivo tumor depletion in patients with minimal residual disease present pretransplantation. Similarly sirolimus, an effective GVHD prophylactic agent, by reducing EBV transformed B cells will reduce EBV reactivation and therefore PTLD. Combining rituximab low-dose (100–150 mg/m²) and sirolimus GVHD prophylaxis (with other immunosuppressive agents) might significantly reduce the expense, morbidity, and mortality associated with EBV-reactivation and PTLD in high-risk populations, especially older patients receiving unrelated or mismatched T-depleted (in vivo or vitro) allo-SCT. The optimal dose of rituximab during conditioning regimen to induce maximum benefit and prevent EBV reactivation remains to be determined, but a single low dose with conditioning regimen appears attractive.