Epstein–Barr virus presence in pediatric diffuse large B‐cell lymphoma reveals a particular association and latency patterns: Analysis of viral role in tumor microenvironment

Non‐Hodgkin's lymphoma represents 6–10% of pediatric malignancies, and diffuse large B‐cell lymphoma (DLBCL) is one of the three major subtypes. The 2008 WHO classification included a new entity, Epstein–Barr virus (EBV)‐positive DLBCL of the elderly, affecting patients >50 years. It has been demonstrated that EBV may play a role in tumor microenvironment composition, disturbing antitumor immune response and disease progression. As most studies were performed in adults, our aim was to assess EBV presence and latency pattern, as well as T‐cell microenvironment in a pediatric DLBCL series of Argentina. The study was conducted on formalin‐fixed paraffin‐embedded biopsies from 25 DLBCL patients. EBV‐encoded small nuclear early regions (EBERs) expression was performed by in situ hybridization, whereas EBV gene expression was analyzed using real‐time PCR. Epstein–Barr virus latent membrane proteins (LMP)1, LMP2A, CD3, CD4, CD8 and Foxp3 expression were assessed by immunohistochemistry (IHC). Forty percent of cases showed EBV expression, with a significantly higher incidence among patients <10 years (p = 0.018), and with immunosuppressed (p = 0.023). T‐cell subsets were not altered by EBV presence. Full EBV latency antigen expression (latency type III) was the most frequently pattern observed, together with BZLF1 lytic gene expression. One patient showed II‐like pattern (LMP1 without LMP2A expression). Based exclusively on IHC, some patients showed latency II/III (EBERs and LMP1 expression) or I (EBERs only). These findings suggest that EBV association in our series was higher than the previously demonstrated for elderly DLBCL and that EBV latency pattern could be more complex from those previously observed. Therefore, EBV could be an important cofactor in pediatric DLBCL lymphomagenesis.     

间变性大细胞淋巴瘤EB病毒基因表达产物的检测及其意义

目的：了解间变性大细胞淋巴瘤与埃泼斯坦－巴尔（ＥＢ）病毒的关系。方法：采用原位杂交及免疫组化ＬＳＡＢ法对１２例间变性大细胞淋巴瘤（ＡＬＣＬ）进行ＥＢＶ编码的ＲＮＡ（ＥＢＥＲｓ）及ＥＢＶ潜伏膜蛋白（ＬＭＰＩ）和潜在膜抗原检测。结果：本组１２例ＡＬＣＬ中ＥＢＥＲｓ阳性率为２５％（３／１２）,１２例ＡＬＣＬ ＬＭＰ１检出率为８．３％,ＥＢＮＡ２检出率为０（０／１２）;１２例ＡＬＣＬ免疫表型,８例为Ｔ源性     

THE DETECTION OF CD56 AND EPSTEIN-BARR VIRUS IN T-CELL LYMPHOMAS

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Establishment and characterization of a novel Hodgkin lymphoma cell line,AM‐HLH,carrying the Epstein‐Barr virus genome integrated into the host chromosome

We describe the establishment and characterization of a cell line, AM‐HLH, obtained from a patient with Epstein‐Barr virus–positive (EBV⁺) nodular sclerosis–type Hodgkin lymphoma (HL). The cells were positive for CD2 and CD30 and negative for CD15. The immunoglobulin heavy‐ and κ light–chain genes were rearranged. The karyotype was of the triploid range. Southern blotting using the EBV terminal repeat probe detected 3 hybridizing bands that were identical to those of the parental HL material. The cells expressed EBV‐encoded RNAs as well as latent genes (EBNA1, EBNA2, LMP1, and LMP2A) and lytic genes (BZLF1 and BALF2). Fluorescence in situ hybridization (FISH) with the cosmid pJB8 clone containing a fragment of EBV DNA as a probe revealed multiple hybridization signals at a marker chromosome. Additional FISH using whole chromosome painting and centromere probes in combination with multicolor FISH determined that multiple EBV copies were clustered within the chromosome 20 materials of the marker chromosome. Culture supernatants of AM‐HLH contained IL‐10 as measured by the bead‐based immunoassay. It is possible that an integrated EBV genome and cellular genes on chromosome 20 were coamplified, leading to the enhanced expression of genes involved in cell growth control. The AM‐HLH cell line will be useful to clarify the role of cytokines in the development of EBV⁺ HL.     

Identification and prevalence of CD8(+) T-cell responses directed against Epstein-Barr virus-encoded latent membrane protein 1 and latent membrane protein 2

Epstein-Barr virus (EBV) is associated with several human malignancies that each show different viral gene expression profiles. In malignancies such as Hodgkin's disease and nasopharyngeal carcinoma only Epstein-Barr nuclear antigen 1 (EBNA1) and varying levels of latent membrane proteins 1 and 2 (LMP1 and -2) are expressed. Since endogenously expressed EBNA1 is protected from CTL recognition, LMP1 and LMP2 are the most likely target antigens for anti-tumor immunotherapy. Therefore, we sought to identify in a systematic way CD8(+) T-cell responses directed against eptitopes derived from LMP1 and LMP2. Using IFNgamma-ELISPOT assays of interferon-gamma release, peripheral blood mononuclear cells (PBMC) of healthy donors were screened with peptide panels (15 mer overlapping by 10) spanning the LMP1 and LMP2 sequences of the prototype EBV strain B95.8. When positive responses were found, CD4(+) or CD8(+) T cells were depleted from donor PBMC to determine the origin of the responder population. We detected CD8(+) T-cell responses to LMP1 in 9/50(18%) donors and to LMP2 in 15/28 (54%) donors. In addition to the already described epitopes, 3 new LMP1- and 5 new LMP2-derived CD8(+) epitopes were identified. In most donors LMP1- and LMP2-specific CD8(+) precursor frequencies were low compared with precursors against immunodominant EBV epitopes from latent (EBNA3A, -3B and -3C) and lytic cycle antigens. These results demonstrate that CD8(+) memory T cell responses to LMP1 and especially to LMP2 do exist in Caucasians, albeit at low levels and could potentially be exploited for therapeutic use.     

Response and survival benefit with chemoimmunotherapy in Epstein‐Barr virus‐positive diffuse large B‐cell lymphoma

Epstein‐Barr virus (EBV)‐positive diffuse large B‐cell lymphoma (DLBCL) is a haematologic malignancy with poor prognosis when treated with chemotherapy. We evaluated response and survival benefits of chemoimmunotherapy in EBV‐positive DLBCL patients. A total of 117 DLBCL patients were included in our retrospective analysis; 33 were EBV‐positive (17 treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone [R‐CHOP] and 16 with CHOP), and 84 were EBV‐negative (all treated with R‐CHOP). The outcomes of interest were complete response (CR) and overall survival (OS) in EBV‐positive DLBCL patients (R‐CHOP versus CHOP) and in DLBCL patients treated with R‐CHOP (EBV‐positive vs EBV‐negative). There were no differences in the clinical characteristics between EBV‐positive and EBV‐negative DLBCL patients. Among EBV‐positive DLBCL patients, R‐CHOP was associated with higher odds of CR (OR 3.14, 95% CI 0.75‐13.2; P = .10) and better OS (hazard ratio 0.30, 95% confidence interval [CI] 0.09‐0.94; P = .04). There were no differences in CR rate (OR 0.52, 95% CI 0.18‐1.56; P = .25) or OS (hazard ratio 0.93, 95% CI 0.32‐2.67; P = .89) between EBV‐positive and EBV‐negative DLBCL patients treated with R‐CHOP. Based on our study, the addition of rituximab to CHOP is associated with improved response and survival in EBV‐positive DLBCL patients. Epstein‐Barr virus status does not seem to affect response or survival in DLBCL patients treated with R‐CHOP.     

Frequency and clinical correlates of elevated plasma Epstein‐Barr virus DNA at diagnosis in peripheral T‐cell lymphomas

Epstein‐Barr virus (EBV)‐encoded RNAs (EBER) in tumor tissue and cell‐free plasma EBV‐DNA (pEBVd) are detected in EBV‐associated lymphomas. Studies have suggested that EBER+ peripheral T‐cell lymphomas (PTCL) have worse prognosis but the role of EBV in these neoplasms remains unclear. pEBVd is quantitative and more easily amenable to standardization than EBER, but frequency of pEBVd detection, clinical impact and agreement with EBER status in PTCL are unknown. We retrospectively assessed frequency of detectable pre‐treatment pEBVd, presence of EBER in tumor tissue, and outcomes in 61 of 135 EBV‐assessable PTCL patients. Fifteen of 61 patients (24.5%, 95% CI: 14–37%) were pre‐treatment pEBVd+, with no significant differences in baseline characteristics or treatment between pEBVd+ and pEBVd? patients. EBER‐ISH was performed on 10 pEBVd+ and 35 pEBVd? tumors. All 10 pEBVd+ patients were EBER+, but 9 pEBVd? patients were also EBER+. With median follow up of 24 months (range 1–96), overall survival (OS) was shorter in pEBVd+ compared to pEBVd? patients (13 vs . 72 months; p = 0.04). In our retrospective study, pre‐treatment pEBVd was elevated in 25% of PTCL patients, was highly specific for EBER+ tumors, and was associated with shorter survival. pEBVd should be further explored as a prognostic variable and tumor biomarker in PTCL.     

Cancer stem cells in Epstein‐Barr virus‐associated gastric carcinoma

Cancer stem cells (CSCs) play a decisive role in the development and progression of cancer. To investigate CSCs in Epstein‐Barr virus (EBV)‐associated carcinoma (EBVaGC), we screened previously reported stem cell markers of gastric cancer in EBV‐infected gastric cancer cell lines (TMK1 and NUGC3) and identified CD44v6v9 double positive cells as candidate CSCs. CD44v6/v9^+/+ cells were sorted from EBVaGC cell line (SNU719) cells and EBV‐infected TMK1 cells and these cell populations showed high spheroid‐forming ability and tumor formation in SCID mice compared with the respective CD44v6/v9^?/? cells. Sphere‐forming ability was dependent on the nuclear factor‐κB (NF‐κB) signaling pathway, which was confirmed by decrease of sphere formation ability under BAY 11‐7082. Small interfering RNA knockdown of latent membrane protein 2A (LMP2A), one of the latent gene products of EBV infection, decreased spheroid formation in SNU719 cells. Transfection of the LMP2A gene increased the sphere‐forming ability of TMK1 cells, which was mediated through NF‐κB signaling. Together, these results indicate that CD44v6v9^+/+ cells are CSCs in EBVaGC that are maintained through the LMP2A/NF‐κB pathway. Future studies should investigate CD44v6/v9^+/+ cells in normal and neoplastic gastric epithelium to prevent and treat this specific subtype of gastric cancer infected with EBV.     

Association of immunoescape mechanisms with Epstein-Barr virus infection in nasopharyngeal carcinoma

We have investigated the association of immunoescape mechanisms in nasopharyngeal carcinoma (NPC) lesions with Epstein-Barr virus (EBV) infection and clinical course of the disease. Tumor biopsy specimens obtained from 36 Japanese NPC patients were examined for antigen processing machinery component and HLA class I antigen expression, CD8(+) T cell infiltration, and Fas, Fas ligand (FasL) and IL-10 expression using immunohistochemical staining. The results were correlated with the histopathological characteristics of the lesions, the clinical course of the disease and EBV infection. LMP2, TAP1, tapasin and HLA class I antigens were downregulated in more than 65% of the lesions tested, while FasL, Fas and IL-10 were expressed in at least 60% of the lesions. Statistical analysis showed that (i) HLA class I antigen expression was significantly correlated with LMP2 and tapasin expression (r = 0.39 and 0.45, respectively); (ii) CD8(+) T cell infiltration into tumor lesions was significantly correlated with HLA class I antigen, LMP2 and Fas expression (r = 0.34, 0.49 and 0.44, respectively); (iii) LMP2 and FasL expression was significantly correlated with IL-10 expression (r = 0.49 and 0.52, respectively); (iv) IL-10 expression was significantly associated with EBERs and EBV oncoprotein LMP1 expression (p = 0.00078 and 0.015, respectively) and (v) FasL overexpression was significantly associated with reduced patients' survival (p = 0.033). Multivariate analysis identified FasL overexpression as an independent unfavorable prognostic marker. These results suggest that NPC cells may utilize multiple immunoescape mechanisms, including dysfunction of HLA class I antigens and Fas/FasL apoptosis pathways. Furthermore, FasL expression appears to be associated with IL-10 upregulation in EBV positive NPC cells.     

Epstein-barr virus-positive diffuse large B-cell lymphoma of the elderly: what we know so far

Epstein-Barr virus-positive (EBV-positive) diffuse large B-cell lymphoma (DLBCL) of the elderly is a newly described lymphoproliferative disorder recently included as a "provisional" entity in the most current WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. The objective of this review is to provide a thorough and current summary of the existing knowledge of this subtype of DLBCL. We will review and discuss the incidence of EBV expression in DLBCL, the pathogenesis behind EBV-driven malignant transformation of B cells, the different EBV latency patterns associated with DLBCL, the distinct pathologic characteristics of EBV-positive DLBCL, the potential predictive and prognostic value of EBV tumoral status in patients with DLBCL, and potential strategies for the treatment of this rare entity, which is characterized by a suboptimal response to therapy and poor survival rate.     

EBV primary infection in childhood and its relation to B‐cell lymphoma development: A mini‐review from a developing region

In most underdeveloped countries, the initial contact with Epstein Barr virus (EBV) usually happens in the first decade of life and results in an asymptomatic infection, whereas in developed areas, primary infection in adolescence or adulthood is accompanied by infectious mononucleosis in 50% cases. Although it is generally a harmless passenger, in some individuals, it is associated with B‐cell lymphoma. In Argentina, EBV primary infection shows the classical pattern observed in developing populations, given that nearly 70% of patients are seropositive by the age of 2 years. However, EBV association with pediatric Hodgkin and Burkitt lymphoma resembles that observed in developed regions. Concerning diffuse large B‐cell lymphoma, our series demonstrated higher EBV association than other adult ones from either developed or underdeveloped countries. Interestingly, the early EBV primary infection observed, characteristic of an underdeveloped population, together with the statistically significant EBV association with patients ≤10 years old demonstrated in all types of lymphoma studied, suggest a relationship between low age of EBV seroconversion and B‐cell lymphoma development risk.     

Epstein‐Barr virus‐negative extranodal “true” natural killer‐cell lymphoma harbouring a KDM6A mutation

Extranodal natural killer (NK)/T‐cell lymphoma, nasal type (ENKTL) is an extranodal aggressive T or NK‐cell lymphoma that is characteristically associated with Epstein‐Barr virus (EBV) infection and cytotoxic tissue‐destructive features. Although ENKTL is described as a distinct entity according to the 2008 WHO classification, a considerable complexity is associated with the differential diagnosis of other T‐cell lymphomas with respect to tumour cell origins, locations, and the presence of EBV infection, as well as molecular and cytogenetic abnormalities. Here, we report a rare case of EBV‐negative ENKTL, where the absence of EBV in the true NK‐lineage cells was confirmed by extensive phenotypic and genotypic analyses. Furthermore, using the next‐generation sequencing approach, we identified mutations in the tumour suppressor genes KDM6A and TP53. The clinicopathological characteristics were almost similar to those of EBV‐positive ENKTL, except for the absence of EBV and histologically apparent angioinvasiveness. This is the first reported ENKTL case with mutations in the KDM6A gene. KDM6A is one of the histone‐modifying genes that are mutated in many human diseases including haematological cancers. Epigenetic regulation of gene expression has recently been demonstrated in ENKTL, and a similar pathway is thought to play an oncogenic role in EBV‐negative ENKTL. Our report shows the extent of comprehensive examination required before making a definitive diagnosis for NK‐ and T‐cell neoplasms and broadens the therapeutic options for potential targets.     

Antagonistic effects of c-myc and Epstein-Barr virus latent genes on the phenotype of human B cells

Epstein-Barr virus (EBV) immortalized cells and Burkitt lymphoma cells have a completely different growth pattern and phenotype. EBV immortalized cells express a set of 11 viral genes to accommodate B cell activation and proliferation, whereas EBV-positive Burkitt lymphoma cells highly express the c-myc oncogene that is activated through translocation into 1 of the immunoglobulin loci and EBNA1 as the only viral protein. We have developed a primary human B cell line conditionally immortalized by Epstein-Barr virus in which the viral gene program responsible for the induction of proliferation can be switched on and off by the addition or withdrawal of estrogen (cell line EREB2-5). Starting from this cell line we have generated 2 daughter cell lines that proliferate in a c-myc dependent fashion, 1 with a highly active exogenous c-myc gene (cell line A1) and 1 with a regulatable c-myc gene that can be switched on by withdrawal and switched off by addition of tetracycline (cell line P493-6). The comparison of the 3 cell lines has allowed us to dissect the contribution of c-myc and EBV genes to the regulation of the growth pattern and expression of cell surface molecules. We show that MYC and EBNA2 (and their respective target genes) have opposing effects on the expression of several surface markers involved in B cell activation. We show that MYC contributes to the phenotype of Burkitt lymphoma cells by upregulating CD10 and CD38 and downregulating activation markers. The phenotype of the cells is determined on one hand by the absence of the viral gene products EBNA2 and LMP1 that mediate the phenotype of activated lymphoblasts and to a lesser extent by an active contribution of the c-myc gene.     

Reappraisal of nodal Epstein‐Barr Virus‐negative cytotoxic T‐cell lymphoma: Identification of indolent CD5+ diseases

Nodal cytotoxic molecule (CM)‐positive peripheral T‐cell lymphoma (CTL) has recently been recognized as a clinicopathologically distinct disease. To further characterize this disease, here we compared 58 patients with Epstein‐Barr virus (EBV)‐negative CTL to 48 patients with EBV‐positive CTL. The two groups did not differ in histopathology, T‐cell receptor (TCR) expression or rearrangement incidences, or survival curves. However, patients with EBV‐negative CTL less frequently showed hepatic involvement (P = .007), B symptoms (P = .020), hemophagocytosis (P = .024), and detectable CD4 (P = .002) and CD5 (P = .009). Univariate and multivariate analyses identified three factors that independently predicted favorable survival, onset age <60 years (P = .002), CD5 expression (P = .002), and mixed morphology (P = .013), TCRαβ was not an independent predictor (P = .30), but was strongly linked with long survivorship among patients younger than 60 years old. A prognostic model incorporating these factors worked well for prognostic delineation, independently of the International Prognostic Index (P = .007 vs P = .082) and Prognostic Index for PTCL (P = .020 vs P = .15). Moreover, this constellation of findings indicated two nodal indolent diseases: CD5⁺TCRαβ (n = 13), and CD5⁺ NK‐cell type lacking TCR expression or clonal TCRγ rearrangement (n = 4). The survival curves for these two groups were significantly superior to others (n = 29, P < .001). These diseases appear to be unique in their indolent clinical behavior, and should be managed differently from other diseases.     

Age cutoff for Epstein-Barr virus-positive diffuse large B-cell lymphoma–is it necessary?

Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly (EBV+ DLBCL-e) is a molecularly distinct variant of DLBCL, characterized by a monoclonal B-cell proliferation that occurs in patients >50 years of age without a history or clinicopathologic evidence of immunodeficiency. However, patients with EBV+ DLBCL younger than 50-years-old also exist in Western countries. We evaluated the clinicopathologic, immunophenotypic and genetic features in Cacausian patients with EBV+ DLBCL who are ≤50 years of age and compared this patient group to patients who are >50 years. In patients who are ≤50 years, less frequent expression of BCL6 and a trend of more frequent expression of CD30 and pSTAT3 were found in patients with EBV+ DLBCL. In patients who are >50 years, common expression of CD30, p50, pSTAT3 and less frequent expression of BCL6 were observed. Older patients also more commonly had a poor performance status (ECOG≥2). Comparing EBV+ DLBCL patients in ≤50 years versus >50 years, both groups had similar clinicopathologic, immunophenotypic and genetic features. Gene expression profiling, microRNA profiling and treatment outcome of the younger patients with EBV+ DLBCL was not distinctive from tumors in older patients. Based on our data, we suggest that the arbitrary age cutoff for EBV+ DLBCL is unnecessary and should be eliminated in the WHO lymphoma classification scheme.