Epstein‐Barr virus‐positive diffuse large B‐cell lymphoma association is not only restricted to elderly patients

Diffuse large B‐cell lymphoma (DLBCL), the most common group of malignant lymphomas, account for 30% of adult non‐Hodgkin lymphomas. The 2008 World Health Organization (WHO) classification included a new entity, Epstein‐Barr virus (EBV)+ DLBCL of the elderly, affecting patients aged 50 years or older. However, some reports of younger EBV+ DLBCL cases, without evidence of underlying immunosuppression, can be found. The role of EBV in tumor microenvironment composition in DLBCL is still not well understood. Our aim was to assess EBV presence and latency pattern as well as tumor T‐cell population in an adult DLBCL series of Argentina. The study was conducted on biopsies from 75 DLBCL patients. EBERs expression was performed by in situ hybridization, while EBV gene expression was analyzed using real‐time polymerase chain reaction. LMP1, LMP2A, EBNA2, EBNA3A, CD4, CD8 and Foxp3 expression was assessed by immunohistochemistry. Nine percent of cases showed EBV expression, with similar frequency among patients younger than 50 years and 50 years or older (13% and 8%, respectively). T‐cell subsets were not altered by EBV presence. Latency type II was the most frequently observed, together with lytic gene expression in EBV+ DLBCL, with ≥20% of EBERs+ cells. These findings suggest that EBV+ DLBCL in our series was similar to the previously described in Asia and Latin‐America, displaying latency II or III expression profile and no age‐specific characteristics. Finally, EBV+ DLBCL may be an entity that is not only restricted to patients who are older than 50 years of age, in consequence the age cutoff revision may be a current goal.     

Clinical outcome of Epstein–Barr virus‐positive diffuse large B‐cell lymphoma of the elderly in the rituximab era

Diffuse large B‐cell lymphoma (DLBCL) is the most common subtype of malignant lymphoma. The incidence of Epstein–Barr virus (EBV)‐positive DLBCL in Asian and Latin American countries ranges from 8 to 10%. The prognosis of patients with EBV‐positive DLBCL is controversial. To compare the clinical outcome of EBV‐positive and EBV‐negative patients with DLBCL in the rituximab era, we analyzed 239 patients with de novo DLBCL diagnosed between January 2007 and December 2011. The presence of EBV in lymphoma cells was detected using EBV‐encoded RNA in situ hybridization, and it was found that 18 (6.9%) of 260 patients with diagnosed DLBCL tested positive. Among the 260 cases, 216 cases were treated with rituximab plus chemotherapy, as were 8 EBV‐positive DLBCL patients. The median overall survival and progression‐free survival times in patients with EBV‐positive DLBCL were 8.7 months and 6.8 months, respectively. The median overall survival and progression‐free survival could not be determined in EBV‐negative DLBCL patients (P = 0.0002, P < 0.0001, respectively). The outcome of patients with EBV‐positive DLBCL remains poor, even in the rituximab era.     

Frequent downregulation of BTB and CNC homology 2 expression in Epstein–Barr virus‐positive diffuse large B‐cell lymphoma

Diffuse large B‐cell lymphoma (DLBCL) is the most common B‐cell lymphoma subtype, and the Epstein–Barr virus (EBV)‐positive subtype of DLBCL is known to show a more aggressive clinical behavior than the EBV‐negative one. BTB and CNC homology 2 (BACH2) has been highlighted as a tumor suppressor in hematopoietic malignancies; however, the role of BACH2 in EBV‐positive DLBCL is unclear. In the present study, BACH2 expression and its significance were studied in 23 EBV‐positive and 43 EBV‐negative patient samples. Immunohistochemistry revealed BACH2 downregulation in EBV‐positive cases (P < 0.0001), although biallelic deletion of BACH2 was not detected by FISH. Next, we analyzed the contribution of BACH2 negativity to aggressiveness in EBV‐positive B‐cell lymphomas using FL‐18 (EBV‐negative) and FL‐18‐EB cells (FL‐18 sister cell line, EBV‐positive). In BACH2‐transfected FL‐18‐EB cells, downregulation of phosphorylated transforming growth factor‐β‐activated kinase 1 (pTAK1) and suppression in p65 nuclear fractions were observed by Western blot analysis contrary to non‐transfected FL‐18‐EB cells. In patient samples, pTAK1 expression and significant nuclear p65, p50, and p52 localization were detected immunohistochemically in BACH2‐negative DLBCL (P < 0.0001, P = 0.006, and P = 0.001, respectively), suggesting that BACH2 downregulation contributes to constitutive activation of the nuclear factor‐κB pathway through TAK1 phosphorylation in BACH2‐negative DLBCL (most EBV‐positive cases). Although further molecular and pathological studies are warranted to clarify the detailed mechanisms, downregulation of BACH2 may contribute to constitutive activation of the nuclear factor‐κB pathway through TAK1 activation.     

The prognostic significance of EBV DNA load and EBER status in diagnostic specimens from diffuse large B‐cell lymphoma patients

Epstein–Barr virus (EBV)‐encoded small RNA in situ hybridization (EBER‐ISH) is a widely accepted method to evaluate EBV involvement in diffuse large B‐cell lymphoma (DLBCL), although little is known regarding associations between EBV DNA load and the EBER status and whether EBV DNA load data provide additional clinical information. In this study, we quantified EBV DNA load in diagnostic specimens from DLBCL patients diagnosed at our hospital to evaluate clinical implications of EBV DNA load in diagnostic specimens as contrasted with EBER‐ISH. Among 140 DLBCL patients without underlying immunodeficiency, 51 were evaluable for both EBER and EBV DNA load, 83 for EBER only and one for EBV DNA load only. The median EBV DNA load was 708 copies/µg. Although EBV DNA load was significantly higher for EBER‐positive patients than for EBER‐negative patients (p < 0.001), EBV DNA was detected in up to 72% of EBER‐negative patients. Progression‐free survival and overall survival were significantly worse for patients with EBV DNA load above 700 copies/µg than for those with EBV DNA load below 700 copies/µg (p = 0.009 and p = 0.003); they were also significantly worse for EBER‐positive patients than for EBER‐negative patients (p < 0.001 and p = 0.001). Even among EBER‐negative patients, higher EBV DNA load conferred worse progression‐free survival and overall survival (p = 0.041 and p = 0.013). These findings indicate that EBV DNA load in diagnostic specimens is not a simple surrogate for the EBER status and may be a potential biomarker associated with EBV involvement and prognosis in DLBCL. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Prognostic significance of Epstein–Barr virus DNA detection in pretreatment serum in diffuse large B‐cell lymphoma

It is still a matter of debate whether detection of Epstein–Barr virus (EBV) DNA in pretreatment serum has clinical implications for diffuse large B‐cell lymphoma. For this study, we measured EBV DNA load in pretreatment serum from 127 diffuse large B‐cell lymphoma patients without any underlying immunodeficiency to evaluate its effects on clinical manifestations and prognosis. Anthracycline‐based chemotherapy in combination with rituximab was given as initial therapy for 119 patients (94%). Epstein–Barr virus DNA was detected in 15 patients (12%), who were older (P = 0.005) and tended to be at a more advanced disease stage (P = 0.053). They showed significantly worse progression‐free survival (PFS) and overall survival (OS) than other patients (P < 0.001 each). This effect remained significant (P = 0.004 and P = 0.027, respectively) after adjustment for age, lactate dehydrogenase, performance status, stage, and extranodal sites. The status of EBV‐encoded small RNA in situ hybridization was known for 123 patients; 6 of 8 positive patients (75%) and 9 of 115 negative patients (8%) had detectable EBV DNA in pretreatment serum. While patients positive for EBV‐encoded small RNA had significantly worse PFS and OS than negative patients (P = 0.001 and P = 0.029, respectively), EBV DNA detection in pretreatment serum was associated with poorer PFS and OS even for the 115 patients negative for EBV‐encoded small RNA (P < 0.001 each). These findings suggest that EBV DNA detection in pretreatment serum may have an adverse prognostic impact for patients with diffuse large B‐cell lymphoma.     

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.     

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.     

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.     

Expression and clinical value of programmed cell death-ligand 1(PD-L1)in diffuse large B cell lymphoma:a retrospective study

Background: The programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) pathway inhibits the activation of T cells and plays a crucial role in the negative regulation of cellular and humoral immune responses. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. In the present study, we aimed to detect the expression of PD-L1 in DLBCL and to analyze its relationship with prognosis. Methods: We reviewed medical records of 204 newly diagnosed DLBCL patients in Sun Yat-sen University Cancer Center between October 2005 and August 2012. The expression of PD-L1 in tumor tissues from these 204 patients was detected using immunohistochemical (IHC) assay. The expression of anaplastic lymphoma kinase (ALK), CD5, CD30, and C-Myc in tumor specimens from 109 patients was detected using IHC, and Epstein–Barr virus (EBV)-encoded RNAs (EBERs) were detected using fluorescence in situ hybridization. The Spearman method was used for correlation analysis. The Kaplan–Meier method with log-rank test was used for univariate analysis. Cox proportional hazards model was used for multivariate analysis. Results: Of the 204 patients, 100 (49.0%) were PD-L1-positive in tumor cells and 44 (21.6%) were PD-L1-positive in tumor microenvironment. PD-L1 expression in tumor cells and tumor microenvironment were more common in the non-germinal center B-cell-like (GCB) subtype than in the GCB subtype (P = 0.02 and P = 0.04). Patients with PD-L1 expression in tumor microenvironment were more likely to be resistant to first-line chemotherapy when compared with the patients without PD-L1 expression in tumor microenvironment (P = 0.03). PD-L1 expression in tumor micro-environment was negatively correlated with C-Myc expression (r = ? 0.20, P = 0.04). No correlations were detected between PD-L1 expression and the expression of ALK, CD5, and CD30 as well as EBERs. The 5-year overall survival (OS) rates were 50.0% and 67.3% in patients with and without PD-L1 expression in tumor cells (P = 0.02). PD-L1 expression in tumor cells was an independent risk predictor for OS (P < 0.01). Conclusions: PD-L1 expression is more common in the non-GCB subtype than in the GCB subtype. PD-L1 expres-sion in tumor microenvironment has a negative correlation with C-Myc. PD-L1 positivity predicts short survival in DLBCL patients. For patients with PD-L1 expression, more strategy such as anti-PD-L1 antibody treatment should be recommended.     

The impact of EBV and HIV infection on the microenvironmental niche underlying Hodgkin lymphoma pathogenesis

The pathogenesis of classical Hodgkin lymphoma (cHL) is still enigmatic, largely because its tumor cells, the so‐called Hodgkin and Reed–Stenberg (HRS) cells, invariably reside in a prominent reactive microenvironment, are rare and therefore difficult to analyze. On the other hand, the broadly investigated cHL‐derived cell lines are not unequivocally considered as suitable and representative models for this puzzling disease. Based on current knowledge, it appears that the cross talk between the tumor cells and the reactive infiltrate of the microenvironment is complex and that multiple mechanisms occur, making cHL a very heterogeneous disease. In 20–40% of cHL cases, HRS cells carry a monoclonal infection by Epstein Barr virus (EBV), which is considered a tumor‐initiating factor. In these cases, EBV shows a latency type II infection pattern with the expression of latent membrane protein‐1 (LMP‐1), a viral oncoprotein that mimics CD40 activation. This scenario is particularly intriguing for the pathogenesis of cHL arising in HIV‐infected patients, which, for still obscure reasons, is invariably EBV‐associated with LMP‐1 expression in HRS cells. Recent evidences are consistent with the occurrence of different pathogenic pathways variably triggered by virus infections (EBV and HIV), genetic alterations, and interactions with critical microenvironmental components. This review focuses on the different microenvironmental niches that characterize cHL of the general population as well as cases of HIV‐infected patients. A more comprehensive understanding of the complex interplay existing between HRS and tumor microenvironment is pivotal for the development of more effective treatments, particularly for relapsed or refractory diseases.     

Epstein–Barr virus‐induced epigenetic alterations following transient infection

Epstein–Barr virus (EBV) is a known tumor virus associated with an increasing array of malignancies; however, the association of the virus with certain malignancies is often erratic. To determine EBV's contributions to tumorigenesis in a setting of incomplete association, a transient model of infection was established where a clonal CCL185 carcinoma cell line infected with recombinant EBV was allowed to lose viral genomes by withdrawal of selection pressure. Global gene expression comparing EBV‐negative, transiently infected clones to uninfected controls identified expression changes in more than 1,000 genes. Among downregulated genes, several genes known to be deoxyribonucleic acid (DNA) methylated in cancer were identified including E‐cadherin and PYCARD. A cadherin switch, increased motility and enhanced cellular invasiveness present in EBV‐positive cells were retained after viral loss, indicating an epigenetic effect. Repression of PYCARD expression was a result of increased promoter CpG methylation, whereas loss of E‐cadherin expression after transient EBV infection did not correlate with increased DNA methylation of the E‐cadherin promoter. Rather, repression of E‐cadherin was consistent with the formation of a repressive chromatin state. Decreased histone 3 or 4 acetylation at the promoter and 5′ end of the E‐cadherin gene was observed in an EBV‐negative, transiently infected clone relative to the uninfected controls. These results suggest that EBV can stably alter gene expression in a heritable fashion in formerly infected cells, whereas its own contribution to the oncogenic process is masked.     

Epstein Barr virus in relation to apoptosis markers and patients' outcome in pediatric B-cell non-Hodgkin lymphoma

In this study, we investigated Epstein Barr virus (EBV) presence, associated to proliferation and apoptosis proteins in pediatric B-cell Non-Hodgkin lymphoma (B-NHL). EBERs, Ki67, active caspase 3, Bax and Bcl2 were analyzed on B-NHL tissue from 40 patients. Forty percent showed EBV expression, significantly higher among patients ?10 years (P = 0.027), and associated with immunosuppression (P = 0.020), but not associated apotosis markers. However, EBV was associated with a worse event-free survival (P = 0.016), particularly under immunosuppression. Even though EBV did not seem to alter apoptotic pathways, it exhibited survival disadvantage and could be an important cofactor in B-cell lymphomagenesis in younger children.     

Spatiotemporal homogeneity and distinctness of the T‐cell receptor β‐chain repertoires in Epstein–Barr virus‐associated primary and metastatic nasopharyngeal carcinomas

Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus (EBV)‐associated lymphoepithelioma. The aim of this study was to characterize the homogeneity and distinctness of the T‐cell repertoires within and between primary and metastatic NPCs. We used ultra‐deep sequencing of the hypervariably rearranged antigen‐binding CDR3 regions of T‐cell receptor beta (TCR_beta) to comprehensively profile the T‐cell repertoires in NPC patients receiving definitive chemoradiotherapy with long‐term follow‐up. We observed not only various spatially heterogeneous patient‐specific TCR_beta clone compositions that changed with time but also several commonly enriched TCR_beta subclones that were constantly shared between primary NPCs in the head and neck regions, locally recurrent tumors after treatment and later‐developed distant metastatic tumors in the liver, lung and bone. Comparison of the overlap frequency of the T‐cell clonality between TCR_beta repertoires enabled us to calculate the pairwise genetic distance between primary NPCs of different patients and different sites of metastatic or recurrent NPCs. The constructed NPC phylogeny clearly differentiated the low‐risk patients without relapse from the high‐risk patients with distant metastasis after chemoradiotherapy. In contrast to the rather low frequency of nonsilent somatic mutations in NPC cells, the degrees of similarity and divergence of NPC‐infiltrating lymphocyte TCR_beta repertoires among different patients showed prognostication. Moreover, the persistent presence of commonly NPC‐shared in‐frame TCR_beta CDR3 gene sequences spatiotemporally identified in the NPC‐infiltrating lymphocytes within varied EBV‐positive NPCs and their metastases suggest the existence of frequently shared epitopes of neoantigens virally or nonvirally displayed on cancer cells, thereby providing opportunities for the development of precisely tumor‐targeted immunotherapy for distant metastasis.     

Malaria,Epstein–Barr virus infection and the pathogenesis of Burkitt's lymphoma

A geographical and causal connection has long been recognized between malaria, Epstein–Barr virus (EBV) infection and Burkitt's lymphoma (BL), but the underlying mechanisms remain obscure. Potential clues are that the malaria parasite Plasmodium falciparum selectively absorbs vitamin A from the host and depends on it for its biological activities; secondly, alterations in vitamin A (retinoid) metabolism have been implicated in many forms of cancer, including BL. The first author has proposed that the merozoite‐stage malaria parasite, emerging from the liver, uses its absorbed vitamin A as a cell membrane destabilizer to invade the red blood cells, causing anemia and other signs and symptoms of the disease as manifestations of an endogenous form of hypervitaminosis A (Mawson AR, Path Global Health 2013;107(3):122–9). Repeated episodes of malaria would therefore be expected to expose the tissues of affected individuals to potentially toxic doses of vitamin A. It is proposed that such episodes activate latent EBV infection, which in turn activates retinoid‐responsive genes. Expression of these genes enhances viral replication and induces germinal center (GC) B cell expansion, activation‐induced cytidine deaminase (AID) expression, and c‐myc translocation, which in turn predisposes to BL. Thus, an endogenous form of retinoid toxicity related to malaria infection may be the common factor linking frequent malaria, EBV infection and BL, whereby prolonged exposure of lymphatic tissues to high concentrations of retinoids may combine to induce B‐cell translocation and increase the risk of Burkitt's lymphoma.