Primary Posttransplantation Lymphoproliferative Disorder Research Articles

Expression and Targeted Inhibition of the Immunoregulatory Carbohydrate-Binding Lectin, Galectin 1, in EBV-Driven Post-Transplant Lymphoproliferative Disorders.

Abstract 96EBV-associated post-transplant lymphoproliferative disorders (PTLD) are potentially fatal EBV-driven B cell proliferations that develop in immunocompromised solid organ or bone marrow allograft recipients. Proliferation of EBV-infected B cells in PTLD is maintained by expression of EBV latent genes such as latent membrane protein 1 (LMP1) and LMP2A, viral immune evasion strategies, and impaired host immune surveillance. Management of early PTLD lesions is currently based on reduction or withdrawal of immunosuppression which increases the risk of graft rejection. For these reasons, alternative treatments that specifically boost host anti-EBV immune responses are needed.To identify novel T-cell inhibitory molecules induced by EBV and expressed in PTLDs, we compared the transcription profiles of a series of EBV-transformed lymphoblastoid B-cell lines (LCLs) and diffuse large B-cell lymphoma (DLBCL) cell lines. The LCLs overexpressed galectin-1 (Gal-1), a carbohydrate-binding lectin that induces tolerogenic dendritic cells and triggers the selective apoptosis of CD4+ Th1 and CD8+ cytotoxic T cells. Gal-1 protein expression was also uniformly high in LCLs and low or undetectable in DLBCL lines by western blotting. Immunohistochemical staining of primary tumors confirmed abundant Gal-1 expression in PTLD B-cells, whereas DLBCLs were negative.To elucidate the mechanisms responsible for Gal-1 overexpression in EBV-associated PTLD, we performed transcriptional reporter assays using cotransfected FLAG-tagged LMP1 and/or LMP2A and a luciferase reporter vector driven by Gal-1 promoter. LMP2A and LMP1 each increased Gal-1-driven luciferase expression 3- to 6-fold, respectively, and the combination of LMP2A and LMP1 was synergistic. In complementary experiments, siRNA-mediated depletion of LMP2A decreased Gal-1 protein abundance in EBV-transformed lymphoblastoid cells. The mechanism of LMP1 and LMP2A-induced Gal-1 expression in LCLs was both AP-1 and PI3K-dependent; the PI3K selective inhibitor, LY294002, markedly inhibited Gal-1 expression in these cells.Given the tolerogenic and immunosuppressive function of Gal-1 and the EBV-driven overexpression of the lectin in LCLs and primary PTLDs, we postulated that Gal-1 might be a targetable immune escape mechanism in EBV-associated malignancies. For these reasons, we assessed the neutralizing activity of a recently developed series of anti-Gal1 monoclonal antibodies. After confirming that these reagents selectively identified both recombinant and native Gal1, we asked whether certain Gal-1 monoclonal antibodies neutralized recombinant Gal-1-mediated apoptosis of EBV-specific polyclonal, largely CD8+, T-cells. Preincubation of human recombinant Gal-1 (hrGal-1) with Gal-1 monoclonal antibodies, but not with isotype-matched controls, selectively inhibited hrGal-1 induced apoptosis of EBV-specific CD8+ T-cells.Taken together, these studies indicate that EBV-transformed LCLs and primary EBV-associated PTLDs express Gal-1 in a LMP-1 and LMP2A-dependent manner. Given the acknowledged role of Gal-1 in mediating an immunopriviledged tumor microenvironment, the lectin likely inhibits the host anti-tumor response in these tumors. Antibody-mediated neutralization of secreted Gal-1 may represent a novel immunotherapeutic strategy in PTLD and other Gal1-expressing tumors. Disclosures:No relevant conflicts of interest to declare.

Lack of BIC and microRNA miR-155 Expression in Primary Cases of Burkitt Lymphoma.

We previously demonstrated a high expression of primary-microRNA BIC (pri-miRNA-155) in Hodgkin lymphoma (HL) and lack of expression in most non-Hodgkin lymphoma subtypes including some Burkitt lymphoma (BL) cases. Recently, a high expression of BIC was reported in BL in comparison to pediatric leukemia and normal peripheral blood samples. In this study we extended our series of BL cases and cell lines for BIC expression by RNA in-situ hybridization (ISH) and quantitative (q)RT-PCR. Both BIC RNA-ISH and qRT-PCR revealed no or only low levels of BIC in 25 BL tissues, including 7 Epstein-Barr virus (EBV) positive cases, compared to HL and normal controls. In agreement with these findings, Northern blotting revealed absence of miR-155 in BL tissues. EBV negative and EBV latency type I BL cell lines also showed very low BIC and miR-155 expression levels as compared to HL cell lines. Higher levels of BIC and miR-155 were detected in in vitro transformed lymphoblastoid EBV latency type III BL cell lines. An association of latency type III infection and induction of BIC was supported by consistent expression of BIC in 11 and miR-155 in 2 posttransplantation lymphoproliferative disorder (PTLD) cases. In summary, we demonstrated that expression of BIC and miR-155 is not a common finding in BL. Expression of BIC and miR-155 in 3 latency type III EBV positive BL cell lines and in all primary PTLD cases suggests a possible role for EBV latency type III specific proteins in the induction of BIC expression.

Lack of BIC and microRNA miR‐155 expression in primary cases of Burkitt lymphoma

We previously demonstrated high expression of primary-microRNA BIC (pri-miR-155) in Hodgkin lymphoma (HL) and lack of expression in most non-Hodgkin lymphoma subtypes including some Burkitt lymphoma (BL) cases. Recently, high expression of BIC was reported in BL in comparison to pediatric leukemia and normal peripheral-blood samples. In this study, we extended our series of BL cases and cell lines to examine expression of BIC using RNA in situ hybridization (ISH) and quantitative RT-PCR (qRT-PCR) and of miR-155 using Northern blotting. Both BIC RNA ISH and qRT-PCR revealed no or low levels of BIC in 25 BL tissue samples [including 7 Epstein-Barr virus (EBV)-positive cases] compared to HL and normal controls. In agreement with these findings, no miR-155 was observed in BL tissues. EBV-negative and EBV latency type I BL cell lines also showed very low BIC and miR-155 expression levels as compared to HL cell lines. Higher levels of BIC and miR-155 were detected in in vitro transformed lymphoblastoid EBV latency type III BL cell lines. An association of latency type III infection and induction of BIC was supported by consistent expression of BIC in 11 and miR-155 in 2 posttransplantation lymphoproliferative disorder (PTLD) cases. In summary, we demonstrated that expression of BIC and miR-155 is not a common finding in BL. Expression of BIC and miR-155 in 3 latency type III EBV-positive BL cell lines and in all primary PTLD cases suggests a possible role for EBV latency type III specific proteins in the induction of BIC expression.

Post-Transplant Lymphoproliferative Disorders (PTLD) after Renal Transplantation: Management and Evolution of Seven Cases Among 1002 Renal Transplants in Sao Paulo, Brazil

We reported seven cases (0.7%) of PTLD among 1002 renal transplants performed at Renal Transplant Service from Hospital Sao Paulo - Universidade Federal de Sao Paulo/Escola Paulista de Medicina, Sao Paulo, Brazil, between 1976 and 1997. There were three male and four female patients with median age of 37 year-old. According to Ann Arbor staging system there were four localized extra-nodal intermediate-grade NHL, one disseminated low-grade NHL and two polyclonal lymphoid hyperplasia. Four patients received cadaveric, two received related and one received unrelated renal transplant. PTLD occurred after a median latency period of 36 months (ranging from 5 to 84 months). In situ hybridization for EBER1 was performed in five patients and molecular evidence of EBV was found in 3 cases (two DLCL and one lymphoplasmocytoid lymphoma). All patients were treated with immunosuppression withdrawal, four patients received anthracyclin-based chemotherapy for control of localized or systemic clonal disease and three were treated with resection of primary PTLD. Four of seven patients (57%) are in complete remission 11, 20, 25 and 79 months after PTLD onset. One patient lost follow-up and two patients died due to lymphoma relapse, respectively 4 and 10 months after completion of treatment. In conclusion, our experience with this small group of patients showed that: 1) immunosuppression withdrawal is not necessarily associated with loss of renal transplant and can be used as the only treatment for polyclonal PTLD; 2) chemotherapy can simultaneously lead to clonal PTLD remission and periodic immunosuppression, avoiding graft rejection after immunosuppression withdrawal.