PBL-PTLD lymphoma cells displayed plasmablastic cytomorphology without maturation toward plasma cells (Fig. 1). Immunohistochemistry of the plasmablast population demonstrated lambda or kappa light chain restriction in six of seven cases tested and expression of CD138 and CD38 (8/8), whereas CD20 was absent in all cases. None expressed the anaplastic lymphoma kinase. CD56 expression was found in one of eight cases and partial positivity for cyclin D1 in another one of eight. With regard to EBV status (Table 1), the one case of primary EBV infection after D+R− living donor renal transplantation had a considerably elevated EBV DNA load in peripheral blood and tumor cells expressed latent membrane protein 1 (LMP1) and Epstein-Barr nuclear antigen 2 (EBNA2) in line with EBV latency type III (20). The other seven patients had low or negative EBV DNA loads in peripheral blood. The remaining four EBER-ISH-positive cases showed no evidence of EBNA2 or LMP1 expression, consistent with latency type 0 or I (20). EBER-ISH showed no EBV association in three of eight cases.
Because of the aggressive nature of the disease, most patients received immunosuppression reduction (IR) combined with systemic chemotherapy as initial treatment (Table 3). Only one patient had IR+irradiation for localized disease. However, when new lesions occurred early after irradiation, systemic chemotherapy was commenced. Even though five of eight patients died from early disease progression, complete remission (CR) was achieved and maintained in PBL-PTLD in localized (2/3) and in disseminated disease (1/5). CR was reached in all three patients with IR followed by CHOP-21-based systemic chemotherapy. The International Prognostic Index (Table 2) of the three survivors ranged from 0 to 4 (median 0), whereas the five patients with early PTLD progression had an International Prognostic Index from 3 to 5. Notably, none of the patients who achieved long-term survival had cytogenetic rearrangements involving the IGH locus and all were EBV-positive by EBER without LMP1 or EBNA2 expression. All patients with gene rearrangements (3/8) and patients with non-EBV-associated PTLD (2/8) were refractory to chemotherapy despite the use of aggressive treatment protocols (escalated BEACOPP, DexaBEAM, CHOP-14; Table 3) and a trial of the proteasome inhibitor bortezomib. Supportive treatment including granulocyte-colony stimulating factor administration notwithstanding, immediate initiation of chemotherapy parallel to reduction of immunosuppression was associated with a high rate of severe infections (5/7) as a consequence of prolonged °III/°IV neutropenia (7/7) in combination with already reduced immune competence.
In the largest case series so far, IR and local therapy were not sufficient to treat PBL-PTLD even in localized disease whereas IR and systemic chemotherapy (CHOP-21) could achieve lasting CRs and may allow a cure even in disseminated disease. In our experience, successful treatment was only possible in lymphomas both EBV-associated and negative for the translocations examined here, MYC/IGH in particular. However, the small numbers of subjects and the observational character of this study make it impossible to draw definitive conclusions regarding the best therapy approach.
Our clinical observation that PBL-PTLD had a poor prognosis if any gene (particularly MYC) rearrangements were present or EBER-ISH (EBV-association) was negative is compatible with findings from other PTLD subtypes, PBL in the nontransplant setting, DLBCL and with cell culture models: MYC translocations have been associated with poor prognosis in DLBCL (21) and a nonsignificant trend toward poorer survival has been noted before in PBL (12, 22). As an explanation, it has been discussed (12), that cytogenetic rearrangements of MYC/IGH might help to overcome the repressor effect of BLIMP1 on proliferation in terminally differentiated plasmablasts as apoptosis induced by ectopic BLIMP1 expression in cell culture models can be partially overcome by ectopic MYC expression (23).
The absence of histological EBV association has long been associated with failure to achieve CR and lower survival in PTLD (24). A recent series of cell culture experiments using a conditionally expressed dominant negative derivative of EBNA1 to evict EBV DNA from Burkitt lymphoma and PTLD cell lines has shown that the degree to which the different malignant cell lines depend on EBV for survival (evasion of apoptosis, permission, and promotion of proliferation) varies correlating to the number of viral genes expressed within them. This was interpreted as evidence for a model of EBV lymphomagenesis whereby proto-tumor cells—rather than expressing defined sets of latency programs—silence or lose viral genes under selective pressure of the immune response as they acquire complementary cellular oncogenic mutations (14). In keeping with this idea and previous observations in PTLD (25), only our one case of early PBL-PTLD (after primary EBV infection) showed expression of EBNA2 and LMP1. The seven late PBL-PTLDs, on the other hand, expressed neither EBNA2 nor LMP1. This and the fact that all three EBV negative cases of PBL-PTLD described here were refractory to chemotherapy can be interpreted in support of the aforementioned hypothesis.
From the perspective of posttransplant epidemiology, patients receiving heart transplants (5/8) for DCM (4/8) are overrepresented among PBL-PTLD. Heart transplants usually account for only 10% to 30% of PTLD (26–28) and DCM in turn accounts for only approximately 30% of heart transplants (29). One potential explanation is that the combination of prolonged survival of these young patients with few cardiovascular risk factors and the long interval from transplantation to diagnosis of PBL-PTLD (more than 10 years in 7/8 cases in this series) leads to selection bias. However, no association with heart transplantation has been described in plasmacytoma-like PTLD, which occurs similarly late (median time to PTLD 8.3 years (30). Another causative factor could be the relatively high level of immunosuppression after heart transplantation. In contrast to HIV-associated PBL (31), however, CD4 T-cell counts were not significantly reduced, suggesting that potent long-term iatrogenic immunosuppression causes a functional inhibition of the immune system similar to massive T-cell depletion.
It has been suggested in the past that immunosuppression with the mammalian target of rapamycin-inhibitor rapamycin might prevent EBV-induced B-cell PTLD development on the basis of in vitro data showing inhibition of LMP1-induced IL-10 production (32). Seven of eight PBL did not express LMP1 and we have previously shown that serum IL-10 levels in PTLD, whereas elevated at diagnosis, do not correlate with treatment response (33). In our case series, long-term rapamycin did not prevent PBL-PTLD development.
Finally, several aspects of PBL-PTLD lend themselves to comparison with PBL overall—in particular with the 42 case cohort by Valera et al. (12) including predominately HIV-associated cases. Epidemiologically, median age and sex at diagnosis of PBL-PTLD were remarkably similar (47 years, 75% male for PBL-PTLD vs. 48 years, 81% male for PBL). The rate of EBV-association (EBER) was also nearly identical at 63% (5/8) vs. 59%. With the exception of one case associated with primary EBV infection, the pattern of EBV latency type was identical (EBNA2 negative throughout and LMP1 0% vs. 6%). MYC rearrangements were found in a slightly lower percentage (2/6, 33%) of cases in our series versus 49% in the series of Valera et al. (12).
To assess the clinical features, treatment options and outcome of rare PTLD subtypes, a prospective PTLD registry has been initiated in Germany in 2006. Here, we present our data on solid organ transplant recipients diagnosed with PBL-PTLD from 2006 to 2011. Treatment was at the discretion of the local physician. Clinical data on the patients in the registry are collected before, during and at least 4 weeks, 6 months, 12 and 24 months after treatment. The responsible local ethics committee approved the registry, and all patients gave written informed consent according to the Declaration of Helsinki. Follow-up data were reviewed for all patients up to November 2011. Tumor response to treatment was defined according to the WHO criteria. Disease-free survival (DFS) was defined from first evidence of CR to disease progression or to death from PTLD, whereas overall survival was defined from diagnosis of PTLD to death from any cause.
The diagnosis of PTLD was based on the examination of histological material, obtained by open biopsy or core needle biopsy. Diagnostic tissue samples were reviewed independently by two expert pathologists (I.A. and W.K.) and classified morphologically according to the WHO classification. Histological EBV association was confirmed by ISH for EBER; latency type was assessed by immunohistochemical staining for LMP1 and EBNA2. The extent of existing disease was determined through a complete patient history; physical examination; laboratory investigations (including full blood count, lactate dehydrogenase [upper limit, 240 U/L]; renal and liver function tests and determination of the EBV DNA load in peripheral blood); bone marrow biopsy; and computed tomography scans of the chest, abdomen, and pelvis.
Immunohistochemistry was performed using standard commercially available antibodies for the Bond-autostainer (CD20, CD38, CD138, CD56, cyclin D1, Alk1, kappa, lambda, EBNA2, LMP1). ISH for EBV-encoded mRNAs was performed using a commercially available probe (Menarini, Bond, Leica). Staining for EBER-ISH, cyclin D1 and Ki-67 was assessed in percent of positive tumor cells. CD38 and CD138 were scored positive when more than 25% of the tumor cells reacted positive. Interphase fluorescence ISH for the detection of breakpoints affecting the IGH, BCL2, BCL6, and MYC loci or fusions of MYC and IGH was carried out on paraffin sections of tumor tissues using commercially available probes (Abbott/Vysis, Downers Grove, IL) and a recently published protocol (34).
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