Epstein-Barr Virus-positive Lymphoma Research Articles

Differential antibody response to EBV proteome following EBVST immunotherapy in EBV-associated lymphomas

Epstein-Barr virus (EBV) is associated with a diverse range of lymphomas. EBV-specific T-cell (EBVST) infusions have shown promise in safety and clinical effectiveness in treating EBV-associated lymphomas; however, not all patients respond to T-cell immunotherapies. To identify EBV-antigen specific antibody responses associated with clinical outcomes, we comprehensively characterized the antibody responses to the complete EBV proteome using a custom protein microarray in 56 EBV-associated lymphoma patients who received EBVST infusions enrolled in Phase I clinical trials. Responders (non-progressors) and non-responders (progressors) had distinct antibody profiles against EBV. Twenty-five IgG antibodies were significantly elevated in higher levels in non-responders compared to responders at 3 months post-EBVST infusion. Ten of these remained significant after adjustment for sex, age, and cancer type, including LMP2A (four variants), BGRF1/BDRF1 (two variants), LMP1, BKRF2, BKRF4, and BALF5. Random forest analysis identified these 10-IgG antibodies as key predictors of clinical response. Paired analyses using blood samples collected at both pre-infusion and 3 months post-EBVST infusion indicated an increase in the mean antibody level for six other anti-EBV antibodies (IgG: BGLF2, LF1, BGLF3; IgA: BGLF3, BALF2, BBLF2/3) in non-responders. Overall, our findings suggest that these EBV-directed antibodies as potential serological markers for predicting clinical responses to EBVST infusions and as therapeutic targets for immunotherapy in EBV-positive lymphomas. NCT01555892 - Cytotoxic T-Lymphocytes for EBV-positive Lymphoma, GRALE NCT02973113 - Nivolumab With Epstein Barr Virus Specific T Cells (EBVSTS), Relapsed/Refractory EBV Positive Lymphoma (PREVALE) NCT02287311 - Most Closely Matched 3rd Party Rapidly Generated LMP, BARF1 And EBNA1 Specific CTL, EBV-Positive Lymphoma (MABEL)

A series of four cases on plasmablastic lymphoma: A rare Epstein–Barr virus-positive lymphoma in immunocompetent patients with varied presentation

Plasmablastic lymphoma (PBL) is a rare and aggressive type of non-Hodgkin lymphoma (NHL) which is typically seen in the setting of an immunocompromised state, classically associated with human immunodeficiency virus infection. It is often also associated with Epstein–Barr virus infection. It has an immunophenotype of a terminally differentiated B lymphocyte. The diagnosis of PBL is challenging due to complex overlap with myeloma and other B-cell NHL having a plasmablastic morphology. In the past few years, there are an increasing number of cases being identified in patients with no known immunocompromised state. We hereby report a case series of four such cases in immunocompetent patients with different sites of involvement and varied presentation to add to the literature.

Research Advances on Abnormal Secretion and Function Abnormality of Exosomes Derived from Lymphoma Cells Caused by Latent EB Virus--Review

Abstract The Latent infection cansed by Epstein-Barr virus (EBV) closely relates with the occurrence and development of several kinds of lymphoma. Exosome (EXO) is functional bilayer membrane structural corpuscles which are secreted by cells contain proteins, lipids and nucleic acids. In recent years, researches showed that EXO play an important role in the occurrence and development of tumors. Therefore, the resenrches which compare the differences in quantity and contents of EXO secreted by cells between EBV negative lymphoma and EBV positive lymphoma and the clarify the influence of EXO on biological behaviors of lymphoma cells and immune cells have the important, significance for understanding the mechanisms related with effect of latent EBV on the occurrence and development of lymphoma by exosome pathway. This review focuses on research progress about the effect of latent EBV on amounts, contents and functions of EXO secreted by lymphoma cells.EB病毒潜伏感染致淋巴瘤细胞外泌体分泌和功能异常的研究进展.EB病毒(EBV)潜伏感染与多种淋巴系统肿瘤发生发展有着密切关系。外泌体(EXO)是细胞分泌的包含蛋白质、脂质、核酸等的功能性双层膜结构小体。近几年来有研究表明，EXO在肿瘤发生发展过程中起着重要作用，故对比EBV阴性和EBV阳性淋巴瘤细胞在EXO分泌数量与内容物方面的差异，了解EXO对淋巴瘤细胞和免疫细胞生物学行为的影响，对EBV潜伏感染通过外泌体途径影响淋巴瘤发生发展机制的研究具有重要意义。本综述重点阐述EBV潜伏感染对淋巴瘤细胞EXO分泌数量、内容物及其功能的影响.

Generation of monocyte-derived tumor-associated macrophages using tumor-conditioned media provides a novel method to study tumor-associated macrophages in vitro

BackgroundTumor-associated macrophages (TAM) are expanded and exhibit tumor-promoting properties within the tumor microenvironment. Current methods to study TAM have not been replicated across cancer types and often do not include exogenous growth factors from the tumor, a key factor in TAM differentiation in vivo.MethodsIn this study, an in vitro method to generate monocyte- derived TAM using tumor- conditioned media (TCM) and a cytokine cocktail containing IL-4, IL-10, and M-CSF was utilized to study the phenotype, morphology, and function of TAM across multiple cancer types. TCM was generated from two breast cancer cell lines and an Epstein-Barr virus-positive lymphoma cell line. The properties of in vitro generated TAM were compared to in vitro generated M1 and M2- like macrophages and TAM isolated from patients with cancer.ResultsTAM generated in this fashion displayed an increase in CD163/CD206 co-expression compared to M2- like macrophages (87 and 36%, respectively). TAM generated in vitro exhibited increased transcript levels of the functional markers IL-6, IL-10, CCL2, c-Myc, iNOS, and arginase compared to in vitro generated M2-like macrophages. Functionally, in vitro generated TAM inhibited the proliferation of T cells (47% decrease from M1-like macrophages) and the production of IFN-γ by natural killer cells was inhibited (44%) when co-cultured with TAM. Furthermore, in vitro generated TAM secreted soluble factors that promote the growth and survival of tumor cells.ConclusionsLimited access to patient TAM highlights the need for methods to generate TAM in vitro. Our data confirm that monocyte-derived TAM can be generated reliably using TCM plus the cytokine cocktail of IL-4, IL-10, and M-CSF. Given the ability of TAM to inhibit immune cell function, continued study of methods to deplete or deactivate TAM in the setting of cancer are warranted.

T Cell Dysfunction in Epstein-Barr Virus-Associated Lymphoma

The Epstein-Barr virus (EBV) is thought to be one of tumorigenic viruses, which can be detected in many kinds of lymphomas, such as extranodal NK/T-cell lymphoma and Burkitt lymphoma. Those EBV positive lymphomas are also called EBV-associated lymphomas. Some studies showed that patients with EBV-associated lymphoma had a worse prognosis. Dysfunction of T cells may contribute to failure of immune surveillance and tumorigenesis. Therefore, this study was aimed to investigate T cell dysfunction in patients with EBV-associated lymphoma. Peripheral blood was collected from 20 patients with EBV-associated lymphoma and 10 healthy donors (healthy EBV seropositive donors) at the Peking University First Hospital. First, the phenotypes and ratios of PBMCs were analyzed using flow cytometry. Compared with healthy donors, the proportion of CD3+ T cells were little lower in the patients, but had no significantly difference (healthy donors vs patients, 47.82±6.85% vs 34.43±3.63%, p=0.09). The ratios of CD4+ T cells / CD8+ T cells were significantly decreased in the patients, the proportion of CD4+ T cells in CD3+ T cells were decreased (healthy donors vs patients, 59.40±3.84% vs 29.46±2.84%, p<0.0001), and CD8+T cells in CD3+T cells were increased (healthy donors vs patients, 33.94±3.60% vs 57.36±2.59%, p=0.0003) in the patients. NK cells had no significantly difference between the two groups (healthy donors vs patients, 9.74±3.68% vs 10.37±2.16%, p=0.88). Further, an enzyme-linked immunospot assay revealed that the production of IFN-γ in T cells was remarkably reduced upon stimulation with EBV mixed peptides, suggesting EBV specific T responses were insufficient in the patients with EBV-associated lymphoma. To study the mechanism of T cells dysfunction in EBV-associated lymphoma, multiple inhibitory receptors were analyzed. Compared with healthy donors, we found that CD4+ T cells and CD8+ T cells expressed higher levels of PD-1, LAG-3, TIM-3 and CTLA-4 in the patients, suggesting T cells were exhausted in the patients with EBV-associated lymphoma. Furthermore, the T-bethi PD1mid exhausted T cells which can be rescued by blockade of PD1 pathway and the EOMEShi PD1hi exhausted T cells which can not be rescued by the blockade of PD1 pathway were both higher in the patients with EBV-associated lymphoma. CD57+KLRG-1+CD160+CD28- were markers of T cell senescence, we found this subset cells were also a little higher in patients, suggesting that T cell senescence might be another contributor of T cell dysfunction in the patients with EBV-associated lymphoma. In addition, some studies have demonstrated that exhausted T cells do not derive from the ‘senescent’ KLRG-1+ subset of effector T cells but rather from the CD127+ subset of memory precursors that are present in the effector phase. We found the proportion of CD127+ T cells reduced significantly in the patients, further suggesting memory T cells decreased and exhausted T cells increased in the patients with EBV-associated lymphoma. All together, we demonstrated here that T cell dysfunction was the predominant feature of the patients with EBV-associated lymphoma, including the T cells ratio imbalance and T cell exhaustion. Our study contributes to understand the pathogenesis of EBV-associated lymphoma and suggests that immune checkpoint blockade may be warranted. DisclosuresNo relevant conflicts of interest to declare.

Methylation status and AP1 elements are involved in EBV-mediated miR-155 expression in EBV positive lymphoma cells

The relationship between Epstein Barr Virus (EBV) and miR-155 is well established. EBV infection induces miR-155 expression, which is expressed at higher levels in EBV latency type III cells compared to EBV latency type I cells. However, the mechanism by which EBV latency genes activate miR-155 expression is still unclear. Here we present data showing that DNA methylation regulates miR-155 expression. We also provide evidence that the AP1 signaling pathway is involved in EBV-mediated miR-155 activation, and that Bay11 influences signaling of the miR-155 promoter AP1 element. Lastly, we show that LMP2A, LMP1 and EBNAs cannot activate miR-155 expression alone, indicating that the regulation of miR-155 by EBV is dependent on more than one EBV gene or cell signaling pathway. We conclude that the regulation of miR-155 in EBV-positive cells occurs through multiple cell signaling processes involving EBV-mediated chromatin remodeling, cell signaling regulation and transcription factor activation.

Sequence analysis of Epstein-Barr virus (EBV) early genes BARF1 and BHRF1 in NK/T cell lymphoma from Northern China.

BackgroundNK/T cell lymphoma is an aggressive lymphoma almost always associated with EBV. BamHI-A rightward open reading frame 1 (BARF1) and BamHI-H rightward open reading frame 1 (BHRF1) are two EBV early genes, which may be involved in the oncogenicity of EBV. It has been found that V29A strains, a BARF1 mutant subtype, showed higher prevalence in NPC, which may suggest the association between this variation and nasopharyngeal carcinoma (NPC). To characterize the sequence variation patterns of the Epstein-Barr virus (EBV) early genes and to elucidate their association with NK/T cell lymphoma, we analyzed the sequences of BARF1 and BHRF1 in EBV-positive NK/T cell lymphoma samples from Northern China.MethodsIn situ hybridization (ISH) performed for EBV-encoded small RNA1 (EBER1) with specific digoxigenin-labeled probes was used to select the EBV positive lymphoma samples. Nested-polymerase chain reaction (nested-PCR) and DNA sequence analysis technique were used to obtain the sequences of BARF1 and BHRF1. The polymorphisms of these two genes were classified according to the signature changes and compared with the known corresponding EBV gene variation data.ResultsTwo major subtypes of BARF1 gene, designated as B95-8 and V29A subtype, were identified. B95-8 subtype was the dominant subtype. The V29A subtype had one consistent amino acid change at amino acid residue 29 (V → A). Compared with B95-8, AA change at 88 (L → V) of BHRF1 was found in the majority of the isolates, and AA79 (V → L) mutation in a few isolates. Functional domains of BARF1 and BHRF1 were highly conserved. The distributions of BARF1 and BHRF1 subtypes had no significant differences among different EBV-associated malignancies and healthy donors.ConclusionThe sequences of BARF1 and BHRF1 are highly conserved which may contribute to maintain the biological function of these two genes. There is no evidence that particular EBV substrains of BARF1 or BHRF1 is region-restricted or disease-specific.

Post-Transplantation Lymphoproliferative Disorders (PTLD) Management in Solid Organ Transplantation (SOT) Recipients

Abstract 4941 Introduction:PTLD are lymphoid proliferations that develop as a consequence of immunosuppression in 1% of SOT recipients. The majority of PTLD are associated with Epstein-Barr virus (EBV) infection, as therapeutic immunosuppression causes decreased T-cell surveillance, increasing the proliferative potential of EBV in latently infected B-cells. PTLD may manifest as early lesions including polyclonal plasmacytic hyperplasia and infectious mononucleosis-like PTLD as well as monoclonal polymorphic PTLD. Patients who develop polymorphic PTLD, universally associated with EBV and occurring early after transplant, usually have good prognosis and respond well to reduction of immunosuppression and antiviral therapy. Monomorphic PTLD is indistinguishable from a subset of B-cell and much less frequently T-cell lymphomas that occur in immunocompetent individuals. Monomorphic PTLD in SOT patients frequently involve extranodal sites as well as the allograft and causes significant morbidity and mortality in this group of patients. There is no universally accepted treatment strategy for monomorphic PTLD as randomized trials are lacking with most of the data coming from prospective and retrospective cohort trials evaluating heterogeneous populations of patients. Standard therapy consists of a stepwise treatment approach, aimed at partially restoring cellular immunity by reduction of immunosuppression (RI) sometimes in combination with or followed by rituximab. If there is no response chemotherapy is initiated. However, this strategy is vague as there are no clear rules for how much and for how long immunosuppression is reduced and may be associated with both graft loss and disease progression. We believe that aggressive monomorphic PTLD patients can be successfully treated by employing aggressive chemo-immunotherapy and withdrawal of standard immunosuppressive agents. We prefer to use “dose dense” administration schedule to 1) provide intensive therapy and 2) provide strong enough immunosuppression to prevent rejection. This retrospective study was designed to assess the outcome of such a strategy in monomorphic PTLD patients treated at the Yale Cancer Center (YCC) over a 15 year period. Patients and Methods:We identified patients with the PTLD after SOT by searching Yale Tumor Registry. Patients were eligible for selection if they were diagnosed with PTLD after SOT between January 1st of 1995 and December 31st of 2009 provided they were 18 years of age or older at the time of diagnosis (diagnosis criteria). We planned to analyze the outcomes among patients treated with combined approach (treatment criteria). Sixteen patients met inclusion criteria. Results:Out of 16 identified patients 11 received kidney, 1 kidney and pancreas, 3 heart and 1 liver transplants. Thirteen patients (81%) were diagnosed with diffuse large B cell lymphoma (DLBCL), 3 with Burkitt or Burkitt-like lymphoma (19%). All patients were treated with a combined approach with most of the patients (n=11, 69%) receiving CHOP-R every 2 weeks (“dose dense”). 10 out of the 16 patients had EBV positive lymphoma (62%). Only one patient had early PTLD (< 1 year after SOT) which was EBV positive. 6 out of 15 patients with late PTLD had EBV negative tumors. 9 (56%) patients had an advanced stage disease and 13 (81%) had extranodal involvement. One patient who developed PTLD after kidney transplantation had graft involvement with PTLD. Complete response (CR) was seen in all but one patient (94%). Median overall survival and median progression free survival were 5.39 years. Only 3 patients died due to PTLD and median cause specific survival time has not been reached. Out of the 16 patients, 4 had graft rejection and graft loss due to PTLD. Both the PTLD related-graft-rejection rate and graft loss rate were 25% with 95% CI (0.07-0.52). Conclusion:Combined therapy approach utilized at the YCC yields excellent results for patients with monomorphic PTLD after SOT. High CR rate, low number of PTLD-related deaths and low graft rejection /graft loss rate make this strategy an appealing option in the treatment armamentarium for this disease. Disclosures:No relevant conflicts of interest to declare.

Expression of EBV Encoded viral RNA 1, 2 and anti-inflammatory Cytokine (interleukin-10) in FFPE lymphoma specimens: a preliminary study for diagnostic implication in Pakistan

BackgroundEpstein Barr Virus (EBV) plays a significant role as a cofactor in the process of tumorigenesis and has consistently been associated with a variety of malignancies. EBV encoded RNAs (EBER1 and EBER2) are the most abundant viral transcripts in latently EBV-infected cells and their role in viral infection is still unclear. Formalin Fixed Paraffin Embedded (FFPE) tissues of surgically removed carcinoma biopsies are widely available form but have never been exploited for expressional studies previously in Pakistan. Immunohistochemistry (IHC) and in situ hybridization (ISH) in FFPE biopsy tissues remains the gold standard for proving EBV relationship in a histopathological lesion but their reagents associated limitations confines their reliability in some applications. Recently introduced targeted drug delivery systems induce viral lytic gene expression and therefore require more sensitive method to quantify viral as well as cellular gene expression.MethodsEight (8) lymphoma samples were screened to detect the EBV genome. Qualitative and quantitative expression of EBV Encoded RNAs (EBER1, EBER2) and anti-inflammatory cytokine (interleukin-10) in FFPE EBV positive lymphoma tissue samples were then analysed by using Reverse transcriptase Polymerase Chain Reaction (RT-PCR) and Real Time Polymerase Chain Reaction (qRT-PCR), respectively.ResultsIn this study we have successfully quantified elevated expressional levels of both cellular and viral transcripts, namely EBER1, EBER2 and anti-inflammatory cytokine (IL-10) in the FFPE Burkitt's lymphoma (BL) specimens of Pakistani origin.ConclusionsThese results indicate that FFPE samples may retain viral as well as cellular RNA expression information at detectable level. To our knowledge, this is first study which represents elevated expressional levels of EBER1, EBER2 and IL-10 in FFPE tissue samples of Burkitt's lymphoma in Pakistan. These observations will potentially improve current lacunas in clinical as well as diagnostic practices in Pakistan and can be further exploited to develop new strategies for studying cellular and/or viral gene expression.

Primary cutaneous lymphomas: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Primary cutaneous lymphomas: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Predictors of Crohn’s Disease

Early intensive therapy in Crohn's disease should be considered only in patients with disabling disease. The aim of our study was to identify at diagnosis factors predictive of a subsequent 5-year disabling course. Among the 1526 patients seen at our unit with Crohn's disease diagnosed between 1985 and 1998, we excluded patients operated on within the first month of the disease, patients with inadequate data, and patients with severe chronic nondigestive disease. In the 1188 remaining patients, Crohn's disease course within the first 5 years of the disease was categorized as disabling when at least 1 of the criteria of clinical severity, conventionally predefined, was present. Among the 1123 patients with follow-up data allowing full 5-year course classification, the rate of disabling disease was 85.2%. Independent factors present at diagnosis and significantly associated with subsequent 5-year disabling were the initial requirement for steroid use (OR 3.1 [95% CI: 2.2-4.4]), an age below 40 years (OR 2.1 [95% CI: 1.3-3.6]), and the presence of perianal disease (OR 1.8 [95% CI: 1.2-2.8]). The positive predictive value of disabling disease in patients with 2 and 3 predictive factors of disabling disease was 0.91 and 0.93, respectively. These values were 0.84 and 0.91, respectively, when tested prospectively in an independent group of 302 consecutive patients seen at our institution from 1998. At diagnosis of Crohn's disease in a referral center, factors predictive of subsequent 5-year disabling course are an age below 40 years, the presence of perianal disease, and the initial requirement for steroids.

Epstein-Barr virus-positive lymphoma in patients with inflammatory bowel disease treated with azathioprine or 6-mercaptopurine

Background & Aims:The use of azathioprine and 6-mercaptopurine for inflammatory bowel disease increased in the early 1990s. We sought to determine the effect of this change in therapy on the risk of lymphoma in patients with inflammatory bowel disease. Methods: All patients with inflammatory bowel disease at a single tertiary care medical center who developed lymphoma between 1985–2000 were identified and the pathologic features of the lymphoma including presence of Epstein-Barr virus were determined. The patients were divided into two 8-year periods (1985–1992, 1993–2000) corresponding with the introduction of azathioprine and 6-mercaptopurine in 1993. Methods:Eighteen patients with lymphoma were identified, 6 between 1985–1992 and 12 between 1993–2000. Six of 18 lymphomas occurred in patients treated with azathioprine or 6-mercaptopurine, all between 1993-2000. Seven patients developed Epstein-Barr virus-positive lymphoma (1 from 1985–1992, 6 from 1993–2000). Five of 7 Epstein-Barr virus-positive lymphomas occurred in patients treated with azathioprine or 6-mercaptopurine compared with 1 of 11 Epstein-Barr virus-negative lymphomas (P = 0.01). Approximately 1200 patients with inflammatory bowel disease were treated with these agents between 1993–2000. Conclusions:Treatment of inflammatory bowel disease with azathioprine or 6-mercaptopurine appears to be associated with a small increased risk of Epstein-Barr virus-positive lymphoma.

Molecular characterization of HHV-8 positive primary effusion lymphoma reveals pathogenetic and histogenetic features of the disease.

Primary effusion lymphoma (PEL) associates with HHV-8 infection, preferentially develops in immunodeficient patients and grows in the serous body cavities. PEL derives from post-germinal center, pre-terminally differentiated B-cells. The pathogenesis of PEL is unclear and the sole identified genetic lesions are human herpesvirus type-8 (HHV-8) infection in all cases and EBV infection in 70% of cases. Epstein-Barr virus (EBV) infection in PEL displays a latency I phenotype. To clarify the pathogenesis and histogenesis of PEL by investigating (1) the lymphoma karyotype; (2) the expression status of the Met tyrosine kinase receptor and of its ligand hepatocyte growth factor (HGF); (3) the molecular profile of EBV, with particular focus on mutations of EBNA-1 genes, which are thought to affect viral tumorigenicity in EBV-infected neoplasms displaying the latency I phenotype. Twenty-four PEL (nine cell lines and 15 primary specimens) formed the basis of the study. Karyotypes were investigated by conventional cytogenetics and fluorescent in situ hybridization (FISH) in selected cases. The expression status of Met and HGF was defined by multiple techniques, including RT-PCR, FACS analysis, immunocytochemistry, Western blot studies and ELISA. The molecular profile of EBNA-1 genes of EBV were investigated by DNA direct sequencing. Trisomy 7, trisomy 12 and breaks at 1q21-q25 are recurrently associated with PEL. PEL consistently co-express Met and HGF both at the mRNA and protein level. Among aggressive B-cell lymphomas, Met/HGF co-expression appears to be relatively specific for PEL. The EBNA-1 gene of EBV displays a high degree of genetic heterogeneity in PEL, with no preferential association with one specific variant. PEL associates with recurrent chromosomal alterations, suggesting that viral infection is not sufficient for tumor development and that lesions of cellular genes may be required. The expression of Met/HGF by PEL cells may bear implications for the lymphoma proliferation and growth pattern, since Met/HGF interactions influence cell mitogenesis and motogenesis. EBV infection in PEL displays a latency I phenotype and fails to associate with specific EBNA-1 variants, suggesting that the role of EBV in PEL is not mediated by the major transforming pathways currently known in EBV positive lymphomas.