EBV-negative Group Research Articles

Sex-dependent different clinicopathological characterization of Epstein-Barr virus-associated gastric carcinoma: a large-scale study.

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) has been reported to account for approximately 5-16% of all GCs with good prognosis compared to EBV-negative GC. We evaluated the clinicopathological characteristics of EBVaGC including survival rate in South Korea. A total of 4,587 patients with GC who underwent EBV in situ hybridization (EBV-ISH) were prospectively enrolled at the Seoul National University Bundang Hospital from 2003 to 2021. Age, sex, smoking status, cancer type and stage, tumor size and location, histological type, molecular features and survival information were analyzed. A total of 456 patients with GC (9.9%) were positive for EBV. The EBVaGC group displayed a higher proportion of males (P < 0.001), a predominant presence in the proximal stomach (P < 0.001), a higher proportion of undifferentiated cancer (P < 0.001), and a lower cancer stage (P = 0.004) than the EBV-negative group. Cox multivariate analyses revealed age (hazard ratio [HR] = 1.025, P < 0.001), tumor size (HR = 1.109, P < 0.001), and cancer stage (stage2 HR = 4.761, P < 0.001; stage3 HR = 13.286, P < 0.001; stage4 HR = 42.528, P < 0.001) as significant risk factors for GC-specific mortality, whereas EBV positivity was inversely correlated (HR = 0.620, P = 0.022). Furthermore, the EBVaGC group displayed statistically significant survival advantages over the EBV-negative cancer group in terms of both overall (P = 0.021) and GC-specific survival (P = 0.007) on the Kaplan-Meier survival curve. However, this effect was evident only in males. EBVaGC patients showed better prognoses despite their association with proximal location and poorly differentiated histology in male, probably due to the difference in immunity between males and females.

Isolation of Epstein-Barr virus-deoxyribonucleic acid in the lower respiratory tract for distinguishing critically ill patients from those with influenza-associated pneumonia: A pilot study.

When influenza A-related pneumonia is complicated by bacteria, aspergillus, and other infections, the disease is aggravated, while there is no research on the role of Epstein-Barr virus (EBV) on patients with influenza A-related pneumonia. This study aimed to evaluate the relationship between the isolation of EBV and influenza A-related pneumonia. This is a clinical study based on the baseline data of a retrospective cohort. A total of 113 cases of influenza A-related pneumonia who underwent polymerase chain reaction test for isolation of EBV-DNA in lower respiratory tract specimens during six influenza seasons from 2013-2014 to 2018-2019 were enrolled. According to the results of EBV-DNA, patients were divided into EBV-positive group and EBV-negative group, and the role of EBV-DNA on patients with influenza A-related pneumonia was analyzed. Regression analysis was used to explore the potential risk factors for the development of moderate-to-severe acute respiratory distress syndrome (ARDS) in patients with influenza A-related pneumonia during hospitalization. Among 113 patients with influenza A-related pneumonia, there were 53 patients with EBV-positive and 60 patients with EBV-negative. The EBV-positive group had higher intensive care unit admission rate, hospital stay, invasive mechanical ventilation rate, extracorporeal membrane oxygenation rate, Sequential Organ Failure Assessment (SOFA) score, and moderate-to-severe ARDS rate. Patients were divided into severe group and mild group. Patients in severe group had lower lymphocyte count, platelet count, and albumin level, while the levels of aspartate aminotransferase, alanine transaminase, creatine kinase, lactic dehydrogenase, and total bilirubin were higher in severe group. The levels of D-dimer, serum ferritin, C-reactive protein, and procalcitonin were higher in severe group than those in the mild group. Multivariate logistic regression analysis revealed that the isolation of EBV (odds ratio = 2.713, 95% confidence interval: 1.094-6.729, P = 0.031) and lymphocyte count (odds ratio = 3.585, 95% confidence interval: 1.157-11.101, P = 0.027) were independent risk factors for moderate-to-severe ARDS in patients with influenza A-related pneumonia. The isolation rate of EBV in the lower respiratory tract was 46.9%. The length of hospital stays, intensive care unit admission rate, invasive mechanical ventilation rate, extracorporeal membrane oxygenation rate, SOFA score, and the proportion of moderate-to-severe ARDS in the EBV-positive group were higher than those in the EBV-negative group, while there was no effect on the death during hospitalization. The isolation of EBV in the lower respiratory tract and low lymphocyte count are independent risk factors for the development of moderate-to-severe ARDS in patients with influenza A-related pneumonia.

No Additional Risk of Ampicillin Rash Among Pediatric Liver Transplant Recipients With Concurrent Epstein-Barr Virus Infection.

Epstein-Barr virus (EBV) infection frequently develops in children undergoing liver transplantation (LT) because of mandated immunosuppressive therapy. There is a risk of ampicillin rash when penicillin derivatives are used in patients with EBV-associated infectious mononucleosis. Hence, the administration of penicillin derivatives may raise concerns about ampicillin rash in patients with high EBV loads. However, no studies confirmed the risk of administering penicillin derivatives to EBV-infected children after LT. This retrospective study was conducted at the largest pediatric transplantation center in Japan. We investigated all pediatric liver transplant recipients who received penicillin derivatives within 2 years of LT between 2014 and 2020. We separated the cohort into EBV-positive and EBV-negative groups to assess the frequency of ampicillin and antibiotic-associated rash. Two hundred eighty-six liver transplant recipients were enrolled. There were 111 recipients in the EBV-positive group and 175 recipients in the EBV-negative group. In the EBV-positive group, 49 patients had high EBV DNA loads (≥1000 copies/µg DNA). None of the patients in either group developed ampicillin rash, and the frequency of antibiotic-associated rash did not differ [8/111 (7.2%) vs. 10/175 (5.7%), P = 0.797]. Additional subgroup analysis revealed no difference in the frequency of antibiotic-associated rashes regardless of the presence or absence of high EBV loads. In this study, ampicillin rash was not observed, and antibiotic-associated rash was not associated with concurrent EBV infection. Penicillin derivatives can be used safely, even in liver transplant recipients with persistent asymptomatic EBV infection.

Prognostic Significance of ARID1A Expression Patterns Varies with Molecular Subtype in Advanced Gastric Cancer.

AT-rich interactive domain 1A (ARID1A) is frequently mutated in gastric cancer (GC), especially Epstein-Barr virus (EBV)-associated and microsatellite instability high GC. The loss of ARID1A expression has been reported as a poor prognostic marker in GC. However, the relationships between ARID1A alteration and EBV-associated and microsatellite instability high GC, which are known to have a favorable prognosis, has hampered proper evaluation of the prognostic significance of ARID1A expression in GC. We aimed to analyze the true prognostic significance of ARID1A expression by correcting confounding variables. We evaluated the ARID1A expression in a large series (n=1,032) of advanced GC and analyzed the relationships between expression pattern and variable parameters, including clinicopathologic factors, key molecular features such as EBV-positivity, mismatch repair protein deficiency, and expression of p53 and several receptor tyrosine kinases including human epidermal growth factor receptor 2, epidermal growth factor receptor, and mesenchymal-epithelial transition factor. Survival analysis of the molecular subtypes was done according to the ARID1A expression patterns. Loss of ARID1A expression was found in 52.5% (53/101) of mutL homolog 1 (MLH1)-deficient and 35.8% (24/67) of EBV-positive GCs, compared with only 9.6% (82/864) of the MLH1-proficient and EBV-negative group (p<0.001). The loss of ARID1A expression was associated only with MLH1 deficiency and EBV positivity. On survival analysis, the loss of ARID1A expression was associated with worse prognosis only in MLH1-proficient and EBV-negative GC. Multivariate analysis revealed that both loss of ARID1A and decreased ARID1A expression were independent worse prognostic factors in patients with advanced GC. Only in MLH1-proficient and EBV-negative GC, the loss of ARID1A expression is related to poorer prognosis.

CD20 expression is closely associated with Epstein-Barr virus infection and an inferior survival in nodular sclerosis classical Hodgkin lymphoma.

BackgroundNodular sclerosis classical Hodgkin lymphoma (NSCHL) is a rare disease in which Epstein–Barr virus (EBV) and CD20 can be detected. The clinical significance of EBV infection, CD20 expression and their relationship are still unclear in NSCHL presently. The aim of this research was to systematically explore the clinical significance of EBV infection, expression of CD20 and their relationship in NSCHL.Methods109 NSCHL patients diagnosed in Qingdao University’s Affiliated Hospital were chosen from January 2010 to July 2019, and the clinical and survival data of all patients were collected retrospectively.ResultsAmong 109 patients, 33 patients were assigned to the group of EBV-positives, following the results of the EBV-encoded RNA (EBER1). Compared with EBV-negative group patients, those in the group of EBV-positive were older (P=0.004) and their β2-microglobulin (β2-MG) levels were higher (P=0.006). The CD20 positivity rate in the group of EBV-positive was substantially higher than that in the EBV-negative group (54.5% vs 27.6%, P=0.007). Among 109 patients, EBV+ and CD20+ double positive patients acquired the least overall survival (OS), and patients with EBV- and CD20- double negative had the best OS (P < 0.001). Although old age, gender, EBV infection and CD20 positive were the risk factors for OS in NSCHL, multivariate analysis showed that CD20 positivity was the only characteristic that showed to be an independent risk factor for OS in NSCHL patients.ConclusionCD20 was found to be strongly expressed in NSCHL patients who had been infected with EBV, and it was found to be an independent risk factor for NSCHL patients’ survival.

The Prevalence of Epstein-Barr Virus in Plasma Cell Neoplasms is Higher in HIV-Positive Individuals.

Epstein-Barr virus (EBV) is causally associated with many hematolymphoid malignancies. This laboratory-based study aimed to establish the prevalence of EBV in plasma cell neoplasms in a large South African cohort and to determine whether there is any correlation between EBV-positivity and human immunodeficiency virus (HIV) status in patients with plasma cell neoplasms, including plasma cell myeloma and plasmacytoma (solitary plasmacytoma of bone and extraosseous plasmacytoma). This single-institution retrospective study included all patients with a histopathologic diagnosis of plasma cell neoplasm between 2003 and 2020. EBV-expression in the plasma cell neoplasms was assessed by EBV-encoded RNA (EBER) in situ hybridization (ISH) and correlated with HIV status. HIV status was determined by retrieving prior serologic results. Formalin-fixed paraffin-embedded tissue from HIV-unknown patients underwent HIV-1 p24 antibody testing. Sixteen of 89 plasma cell neoplasms (18%) were EBV-positive. There was a significant correlation between EBV and HIV infection in plasma cell neoplasms, with 6/10 tumors from HIV positive patients showing EBV-positivity in tumor cells. The EBV-positive cohort was significantly younger than the EBV-negative group. EBV-positivity in plasma cell neoplasms in this study is higher than previously reported. The significant occurrence of EBV in plasma cell neoplasms from HIV-positive patients suggests a co-carcinogenic relationship between the two viruses.

Association of PD-1/PD-L1 expression and Epstein-–Barr virus infection in patients with invasive breast cancer

PurposeCausative factors of breast cancer include infections, such as Epstein–Barr virus (EBV) infection. The aim of this study was to analyze the clinicopathological features of EBV-positive (IBC) and determine if EBV affects programmed cell death receptor 1 (PD-1)/PD ligand 1 (PD-L1) expression in IBC, similar to other EBV-infected tumors with PD-L1/PD-1 expression.MethodsWe collected 140 samples of IBC tissues and 25 samples of adjacent tissues. All patients were followed-up by telephone from the day of surgery to December 2020. Chromogenic in-situ hybridization was performed to evaluate EBV-encoded RNA (EBER). Immunohistochemistry was performed to evaluate PD-L1 and PD-1 expressions. The correlation between PD1/PDL1 expression and clinicopathological features was also analyzed.ResultsEBER was detected in 57 of 140 (40.7%) IBC tissues and not detected in any adjacent tissue (P < 0.05). Clinicopathologic features of patients were consistent with EBV-associated IBC. EBV infection was correlated with the mass size, menopausal status, axillary lymph node metastasis, vascular invasion, Ki-67 index, clinical stage, and estrogen receptor and progesterone receptor expressions (all P < 0.05), but not with the histological type, invasive ductal carcinoma histological grade, or human epidermal growth factor receptor 2 (HER2) expression (all P > 0.05). The positive rate of PD-1/PD-L1 expression was higher in the EBV-positive group than in the EBV-negative group (P < 0.05). The Kaplan–Meier univariate survival analysis showed that EBV was associated with poor disease-free survival and overall survival in patients with IBC. PD-L1/PD-1 expression could predict a poor prognosis.ConclusionsIn this study, clinicopathologic characteristics of patients were consistent with EBV-infected IBC. Patients with EBV-positive breast cancer were more likely to have elevated PD-1/PDL-1 expression compared to those with EBV-negative breast cancer. This finding could serve as a basis to explore therapeutic targets, particularly immunotherapy, for patients with IBC.

A potential EBV-related classifier is associated with the efficacy of immunotherapy in gastric cancer.

BackgroundGastric cancer (GC) is a global health problem. As a complicated heterogeneous disease, The Cancer Genome Atlas (TCGA) Research Network recognized four subtypes of GC, including Epstein-Barr virus (EBV)-positive GC (EBVaGC), which accounts for approximately 9% of all GC cases. The response to immunotherapy in GC is limited, and whether EBV status is a predictor of immunotherapy remains controversial.MethodsThe differential gene expression analysis was utilized to compare the gene expression between the EBV-positive group and the EBV-negative group in the TCGA-Stomach Adenocarcinoma (TCGA-STAD) cohort. Weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) network analysis, and gene functional enrichment analysis were used to investigate the most pivotal hub genes and their roles. The “Estimation of Stromal and Immune cells in Malignant Tumours using Expression data” (ESTIMATE) and CIBERSORT algorithms were performed to infer the immune compositions of tissue samples. Furthermore, quantitative real-time polymerase chain reaction (RT-qPCR) and survival analysis were used to validate the expression and prognosis of these hub genes in Sun Yat-sen University Cancer Center (SYSUCC) cohort. A GC cohort that received anti-programmed cell death 1 (PD-1) therapy was used to analyze the predicted efficacy based on the expression of hub genes.ResultsThere is a total of 1,686 differentially expressed genes (DEGs) between the EBV-positive group and EBV-negative group, and WGCNA identified a yellow-colored module that was most related to EBV features. Functional enrichment analysis of 144 genes in this yellow module demonstrated that these genes primarily performed immune-related functions, and PPI network analysis through the CytoHubba plug-in identified 11 hub genes in the network. The RT-qPCR results in the SYSUCC cohort further validated that the hub genes were all increased in the EBV-positive group. Finally, we found that a potential classifier was associated with the efficacy of immunotherapy based on the expression of these 11 hub genes.ConclusionsOur study identified several hub genes associated with EBV status that are closely related to the immune microenvironmental features of EBVaGC and may be used as molecular markers for predicting the immune response in GC.

The effect of Epstein-Barr virus viremia on the progression to severe COVID-19.

Epstein–Barr virus (EBV) is frequently reactivated by coronavirus 2019 (COVID-19), and a high incidence of EBV viremia has been reported in patients with severe COVID-19. However, the impact of EBV viremia on progression to severe COVID-19 is unclear. Therefore, we conducted a study to evaluate the effect of EBV on COVID-19 progression.We investigated EBV viremia at the time of admission in COVID-19 patients hospitalized between February 1, 2020, and April 11, 2021. A cross-sectional study was performed to compare the severity of COVID-19 according to the presence or absence of EBV viremia. However, since it is difficult to analyze the influence of EBV viremia on COVID-19 progression with cross-sectional studies, a retrospective cohort study, limited to patients with mild COVID-19, was additionally conducted to observe progression to severe COVID-19 according to the presence or absence of EBV viremia.Two hundred sixty-nine COVID-19 patients were tested for EBV viremia. In a cross-sectional study that included patients with both mild and severe COVID-19, the EBV viremia group had more severe pneumonia than the EBV-negative group. However, in the cohort study limited to mild cases (N = 213), EBV viremia was not associated with COVID-19 progression.COVID-19 severity may affect EBV viremia; however, there was no evidence that EBV viremia was a factor in exacerbating pneumonia in patients with mild COVID-19.

Identification of differential proteomics in Epstein-Barr virus-associated gastric cancer and related functional analysis

BackgroundEpstein-Barr virus-associated gastric cancer (EBVaGC) is the most common EBV-related malignancy. A comprehensive research for the protein expression patterns in EBVaGC established by high-throughput assay remains lacking. In the present study, the protein profile in EBVaGC tissue was explored and related functional analysis was performed.MethodsEpstein-Barr virus-encoded RNA (EBER) in situ hybridization (ISH) was applied to EBV detection in GC cases. Data-independent acquisition (DIA) mass spectrometry (MS) was performed for proteomics assay of EBVaGC. Functional analysis of identified proteins was conducted with bioinformatics methods. Immunohistochemistry (IHC) staining was employed to detect protein expression in tissue.ResultsThe proteomics study for EBVaGC was conducted with 7 pairs of GC cases. A total of 137 differentially expressed proteins in EBV-positive GC group were identified compared with EBV-negative GC group. A PPI network was constructed for all of them, and several proteins with relatively high interaction degrees could be the hub genes in EBVaGC. Gene enrichment analysis showed they might be involved in the biological pathways related to energy and biochemical metabolism. Combined with GEO datasets, a highly associated protein (GBP5) with EBVaGC was screened out and validated with IHC staining. Further analyses demonstrated that GBP5 protein might be associated with clinicopathological parameters and EBV infection in GC.ConclusionsThe newly identified proteins with significant differences and potential central roles could be applied as diagnostic markers of EBVaGC. Our study would provide research clues for EBVaGC pathogenesis as well as novel targets for the molecular-targeted therapy of EBVaGC.

Occurrence and quantification of Anelloviruses and Herpesviruses in gingival tissue in Chinese Shanghai sub-population

BackgroundHerpesviruses and bacteria and their interplay have long been believed to play important roles in the pathogenesis of periodontitis, but other microbial entities in the oral environment might also be involved. Anelloviruses are commonly detected in human, including in oral samples. The aim of the present study was to explore the occurrence and co-occurrence of human cytomegalovirus (HCMV), Epstein–Barr virus (EBV), and human anelloviruses (HTTVs) in gingival tissue samples collected from participants recruited in Shanghai, China.MethodsGingival tissues were collected from 159 participants (57 with aggressive periodontitis (AP), 59 with chronic periodontitis (CP) and 43 with healthy periodontal status). The presence of HCMV, EBV, torque teno virus (TTV), torque teno mini virus (TTMV) and torque teno midi virus (TTMDV) DNA was detected by nested-PCR. The virus loads were quantified by real-time PCR.ResultsThe detection rates of EBV, TTV, TTMV and TTMDV were significantly higher in the AP and CP groups compared to the healthy group (all P < 0.01). A statistically significant association was found between EBV, TTV and TTMV virus load and periodontitis (all P < 0.05). Participants infected with EBV showed significantly higher infection rates and higher virus loads of TTV and TTMV than the EBV-negative group (all P < 0.05). The coexistence rates of EBV and anelloviruses and the coexistence of three HTTVs were significantly higher in AP and CP groups (all P < 0.01).ConclusionsCollectively, results obtained in this study suggest that HTTVs and the coexistence of EBV and HTTVs in particular, may be associated with periodontitis. Possible mechanisms of the interaction between herpesviruses and anelloviruses in the context of periodontitis require further investigation.

T-lymphocyte subsets and Th1/Th2 cytokines in convalescent patients with Epstein–Barr virus-associated aplastic anemia

ABSTRACT Objective: The aim of this study was to analyze T-lymphocyte subsets and Th1/Th2 cytokines in convalescent patients with Epstein–Barr virus (EBV)-associated aplastic anemia (AA). Methods: Sixty AA patients were enrolled, who were in remission following immunosuppressive therapy, including 34 EBV-negative cases and 26 EBV-positive cases. Their complete blood count (CBC), T-lymphocyte subsets, Th1/Th2 cytokines were analyzed. The correlation between EBV-DNA and T-lymphocyte subsets was evaluated, as well as the relationship between EBV-DNA and Th1/Th2 cytokines. The presence of EBV-DNA in peripheral blood mononuclear cells (PBMCs) was also assessed in 60 normal controls. Results: EBV-DNA was detected in 26/60 (43.33%) patients and 21/60 (35.00%) controls. EBV-DNA copy number in AA patients was higher than in controls (Z = −2.138, P = 0.033). The percentage of CD3+CD4+ T-lymphocytes and the ratio of CD4+/CD8+ T-lymphocytes in the EBV-negative group were higher than in the EBV-positive group (P = 0.001 and 0.001, respectively). EBV was positively correlated with CD3+CD8+ T-lymphocyte percentages (Pearson R: 0.496, P = 0.009). Moreover, EBV was positively correlated with IL-10 and IFN-γ levels (Pearson R: 0.559, P = 0.002 and Pearson R: 0.621, P = 0.001, respectively). Conclusions: EBV-DNA copy number in AA patients was higher than in normal controls. Both AA and EBV infection may cause changes in the levels of T-lymphocyte subsets. We recommend monitoring the changes in the immune function and EBV infection simultaneously in AA patients, especially following immunosuppressive therapy.

A distinctive subgroup of oral EBV+ B-cell neoplasm with polymorphous features is potentially identical to EBV+ mucocutaneous ulcer

Epstein-Barr virus-positive mucocutaneous ulcer (EBVMCU) is a newly recognized provisional entity included in mature B-cell neoplasm in the latest 2016 World Health Organization Classification. It has a self-limited growth potential with a high predilection for oral cavities and occurs in age-related or iatrogenic immunodeficiency with indolent clinical courses. However, it shares histological features with EBV-positive diffuse large B-cell lymphoma (DLBCL), and this often leads to diagnostic challenges and controversies in patients with an oral EBV-positive B-cell neoplasm. The aim of this study was to better characterize and comprehend the pathophysiology of DLBCL and EBVMCU in the oral cavity. We conducted clinicopathologic and recurrent gene mutation analysis of 49 cases (14 EBV positive, 35 EBV negative), including cases diagnosed as DLBCL or B-cell lymphoproliferative disorders with high-grade morphology in the oral cavity. All EBV-positive cases matched the criteria of EBVMCU, with significantly earlier clinical stages than the EBV-negative group (P=.0006). Besides, histological analysis showed that all EBV-positive cases presented polymorphous features, whereas 91.4% (32/35) of the EBV-negative cases showed diffuse and monotonous proliferation (P<.0001). Furthermore, EBV-positive cases presented favorable clinical outcomes without disease-related death or recurrence. Gene mutation analysis (MYD88, CD79A, CD79B, CARD11, and EZH2) revealed that 33.3% (9/27) of EBV-negative cases harbored at least 1 gene mutation, whereas no gene mutation was observed in the EBV-positive group (0/11). These results suggest that oral EBV-positive B-cell lymphoid proliferation with polymorphous features often fulfill the criteria for EBVMCU, with clinicopathologically and genetically distinctive properties.

Epstein-Barr virus BARF1-induced NFκB/miR-146a/SMAD4 alterations in stomach cancer cells.

Epstein-Barr virus (EBV)-encoded BamHI-A rightward frame 1 (BARF1) is a putative viral oncogene in EBV-infected stomach cancer. The aim of the present study was to investigate BARF1-induced cellular protein and microRNA alterations. In this study, BARF1-expressing stomach cancer cells showed a high rate of proliferation, high levels of NFκB, and miR-146a upregulation, which was reversed by NFκB knockdown. During BARF1-induced NFκB upregulation, hCSF1 receptor level was unchanged. Knockdown of BARF1 in the naturally EBV-infected YCCEL1 stomach cancer cells suppressed cell proliferation, and downregulated NFκB and miR-146a. SMAD4 was identified as a miR-146a target and was downregulated in BARF1-expressing cells, whereas SMAD4 expression was restored by anti-miR-146a. Knockdown of BARF1 in YCCEL1 cells upregulated SMAD4, and this effect was reversed by miR-146a overexpression. Transfection of BARF1-expressing cells with pCEP4-SMAD4 abolished the cell proliferating effect of BARF1. In stomach cancer tissues, miR-146a was expressed at higher levels, and more frequent NFκB nuclear positivity immunohistochemically, but not of SMAD4 nuclear loss was found in the EBV-positive group compared with the EBV-negative group. In conclusion, EBV-encoded BARF1 promotes cell proliferation in stomach cancer by upregulating NFκB and miR-146a and downregulating SMAD4, thereby contributing to EBV-induced stomach cancer progression.

Survival analysis of patients with advanced-stage nasopharyngeal carcinoma according to the Epstein-Barr virus status.

PurposeThe main aim of this study is to analyze the prognostic differences in nasopharyngeal carcinoma (NPC) patients who are positive and negative for Epstein-Barr virus (EBV).ResultsOf the 1106 patients, 248 (22.4%) had undetectable pre-treatment plasma EBV DNA levels. The total distant metastasis rate for EBV-negative group vs. EBV-positive group were 3.6% (9/248) vs. 15.0% (128/858) (P < 0.001). The estimated 4-year disease-free survival (DFS), overall survival (OS), distant metastasis-free survival (DMFS) and locoregional relapse-free survival (LRRFS) for EBV-negative group vs. EBV-positive group were 88.9% vs. 76.9% (P < 0.001), 93.6% vs. 85.9% (P = 0.001), 96.7% vs. 84.8% (P < 0.001) and 94.1% vs. 90.0% (P = 0.1), respectively. Multivariate analysis revealed that the EBV status was an independent prognostic factor for DFS (HR, 1.813; 95% CI, 1.219-2.695; P = 0.003), OS (HR, 1.828; 95% CI, 1.075-3.107; P = 0.026) and DMFS (HR, 3.678; 95% CI, 1.859-7.277; P <0.001), and overall stage still remained the most important prognostic factor in patients with stage III-IVB NPC.Methods and MaterialsData on 1106 patients with non-metastatic, histologically proven advanced-stage (III-IVB) NPC who underwent intensity-modulated radiotherapy (IMRT) were retrospectively reviewed. Patient survival between different EBV status groups were compared.ConclusionsEBV status was an independent prognostic factor for patients with stage III–IVB NPC. Neoadjuvant chemotherapy (NCT) plus concurrent chemoradiotherapy (CCRT) should be better treatment regimen for EBV-positive patients since distant metastasis was the main failure pattern, and CCRT may be enough for EBV-negative patients.

High Rates of HTLV-1 Seropositivity in Patients with EBV-Positive Diffuse Large B-Cell Lymphoma

Background: Epstein-Barr virus (EBV)-positive diffuse large B cell lymphoma (DLBCL) of the elderly is a novel entity included in the most recent edition of WHO Classification of hematologic neoplasms. It usually affects patients older than 60 years of age and carries a poor survival. Human T-lymphotrophic virus type 1 (HTLV-1) is a retrovirus that induces immunosuppression, has been associated with the development of adult T-cell leukemia/lymphoma, and has a high prevalence in Peruvian population.Aim: The goal of this retrospective study was to evaluate the HTLV-1 serological status in EBV-positive DLBCL Peruvian patients.Methods: We identified untreated patients with a diagnosis of DLBCL between January 2002 and June 2015 from the medical records of our institution. IRB approval was obtained prior to data collection. HTLV-1 positivity was defined based on Western Blot analysis. EBV-positivity was defined by identifying at least 10% EBV-encoded RNA (EBER) expression by chromogenic in situ hybridization in the pathological sample. Clinical data were reviewed retrospectively and patient's biopsies were analyzed for the immunohistochemical expression of CD20, PAX5, BCL6, CD10, CD30 and MUM-1/IRF4 for re-classification purposes.Results: A total of 210 patients with DLBCL were identified. Of these, 11 patients (5%) were HTLV-1 seropositive and 199 (95%) were HTLV-1 seronegative. Thirty-two patients (15%) were EBV-positive by expression of EBER and 178 (85%) were EBV-negative. HTLV-1 positivity was found in 4/32 (12.5%) of EBV-positive DLBCL patients, and in 7/178 (4%). The odds ratio for HTLV-1 positivity in EBV-positive DLBCL was 3.5 (95% CI 0.7-14.7; p=0.045) when compared with the EBV-negative group. The median age of the HTLV-1-positive DLBCL patients was 65 years (range 45-85 years). There were 5 (45%) men and 6 women (55%). ECOG was >1 in 5 patients (45%), LDH was elevated in 8 (73%), extranodal disease in 7 (64%), and advanced clinical stage (III and IV) in 7 (64%). High IPI score (3-5 factors present) was seen in 8 patients (73%). Interestingly, all the patients who were EBV-positive and HTLV-1-positive presented with early stage (I and II), normal LDH levels and low IPI scores (0-2 factors).Conclusions: There is a high prevalence of HTLV-1 seropositive status in patients with EBV-positive DLBCL. Patients with double EBV and HTLV-1 positivity appear to present at earlier stages and with lower risk disease. Larger studies are needed to confirm these findings. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2716: Epstein-Barr virus-encoded BARF1 downregulates SMAD4 and increases miR-146a in gastric carcinoma cells

Abstract Recently, molecular analytic data from ‘The Cancer Genome Atlas’ (TCGA) (http://www.cbioportal.org) has re-confirmed that Epstein-Barr virus (EBV)-infected gastric carcinoma is a specialized subset of gastric carcinomas. However, viral oncogene has not yet been established in gastric carcinomas. Several reports have suggested that EBV-encoded BARF1 may be a putative viral oncogene in gastric carcinoma. The aim of this study was to investigate alterations of cellular microRNA and cellular protein by BARF1 in gastric carcinoma cells. We cloned BARF1 from naturally EBV-infected gastric carcinoma cell line, and transfected BARF1 into EBV-negative gastric carcinoma cells, thus produced a stable cell line of BARF1-transfected gastric carcinoma cells. BARF1-transfected gastric carcinoma cells revealed significantly increased cellular proliferation than its original cells. The MicroRNA array (Agilent release 16.0) data and Taqman RT-PCR results showed that cellular miR-146a was highly expressed in BARF1-transfected gastric carcinoma cells, comparing to that in original cells. In BARF1-transfected gastric carcinoma cells, transfection with BARF1-siRNA offset the increment effect of miR-146. Using bioinformatics analyses for miR-146a target (RNA hybrid 2.2 and TargetScan 5.2 sites), we found that miR-146a target seed sequence existed in the 3′ UTR of SMAD4 mRNA. The Western blot assay demonstrated that SMAD4 was remarkably reduced by BARF1 transfection. When immunohistochemistry for SMAD4 was performed on 343 cases of surgically resected, human gastric carcinoma tissues, 32 (9%) EBV-infected gastric carcinoma group disclosed more frequent loss of SMAD4 than EBV-negative group. In conclusion, EBV-encoded BARF1 could promote proliferation of gastric carcinoma cells, thereby contributing to EBV-induced cancer progression, in which SMAD4 downregulation, probably through miR-146a increase, may be entailed. Citation Format: Dong Ha Kim, Chan Jin Yoon, Jin, Kyung Rho, Jaap M. Middeldorp, Jun Hee Woo, Mee Soo Chang. Epstein-Barr virus-encoded BARF1 downregulates SMAD4 and increases miR-146a in gastric carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2716. doi:10.1158/1538-7445.AM2015-2716

The expression of renal Epstein-Barr virus markers in patients with lupus nephritis.

The aim of this study was to investigate the role of renal Epstein-Barr virus (EBV) infection in the pathogenesis of lupus nephritis (LN). A total of 58 renal tissue samples from patients with LN, seven normal renal tissue samples from patients with non-glomerular hematuria and 37 renal tissue samples from patients with minimal change nephropathy were collected. The expression of EBV-latent membrane protein-1 (EBV-LMP1) and EBV-encoded RNA 1 (EBER-1) in the renal tissue was examined by immunohistochemistry (IHC) and in situ hybridization (ISH), respectively. The sera levels of anti-nuclear antibody as well as antibodies to extractable nuclear antigen in patients with LN were also measured. An equivalence test showed that the results from the IHC and the ISH analyses had strong agreement. The positive rates of renal EBER-1 and EBV-LMP1 in the LN patients were significantly higher than those of the normal and minimal change nephropathy patients (P<0.001), while no significant difference was identified between those of the normal and minimal change nephropathy groups (P>0.05). The positive rates of EBV-LMP1 and EBER-1 in the renal tissues of patients with LN were not determined to be significantly different between the relapse (immunosuppressant-treated) and initial onset (non-treated) patients, between the patients with and without concurrent infection, and among the patients with different age ranges (P>0.05). The proportion of LN patients positive for anti-Sm antibody was significantly higher in the renal EBV-positive group than in the EBV-negative group (P<0.05), while the proportions of LN patients positive for the other autoantibodies that were examined were not identified to be significantly different between these two groups (P>0.05). The present study shows that renal EBV infection may contribute to the pathogenesis of LN by inducing anti-Sm antibody production.

Epstein-Barr virus (EBV) positive diffuse large B cell lymphoma of the elderly—Experience of a single center from Turkey

In the 2008 WHO lymphoma classification, ‘EBV-positive diffuse large B cell lymphoma (DLBCL) of the elderly is included as a new provisional entity. We aimed to evaluate the morphological, immunophenotypic, and clinical characteristics of the cases diagnosed as ‘EBV-positive DLBCL of the elderly’ in our center and compared them with the ‘EBV-negative DLBCL’ patients older than 50 years of age. EBV status was detected by Epstein-Barr early RNA (EBER) in situ hybridization analysis. By immunohistochemistry, a panel of antibodies for CD10, Bcl-2, Bcl-6, IRF4/MUM1, CD30, and Ki67 was performed. Out of 149 DLBCL patients older than 50 years, without any known history of immunodeficiency or prior lymphoma, eight patients who fulfill the criteria were re-evaluated. Five patients were male and three were female, with a median age of 67.6 years. Four patients presented with nodal involvement; others presented with bone and soft tissue, bone marrow, and spleen infiltrations. Five cases revealed predominantly monomorphic morphology, one also contained focal areas consistent with polymorphous subtype; and three patients revealed a polymorphous infiltrate. When classified according to ‘Hans criteria’, five were non-GCB, and three were of the GCB cell phenotype. All cases with polymorphous morphology were revealed to be of the non-GCB cell phenotype, and all expressed IRF4/MUM1. Two patients died with disease, four patients are alive and in complete remission following R-CHOP therapy, and two patients have just recently been diagnosed.When compared with the EBV-negative group, there are no reliable morphological and immunohistochemical features indicating EBV positivity. Therefore, EBER in situ hybridization analysis is necessary to identify ‘EBV-positive DLBCL of the elderly’.Further studies are needed to fully understand the details of this disease, which can lead to new treatment modalities.

Relationship Between EBV Infection and Expression of Cellular Proteins c-Myc, Bcl-2, and Bax in Gastric Carcinomas

Epstein-Barr virus (EBV) has been related to tumorigenesis in about 10% of all gastric carcinomas. Several studies have demonstrated strong evidence of its involvement in this process, but most of the mechanisms used by the virus to control this process are still unknown. Previous studies in vitro have indicated a relationship between the virus and some cellular genes involved in processes such as proliferation and apoptosis. The aim of the present study was to investigate a possible EBV-induced tumorigenic pathway involving the cellular proteins Bcl-2, Bax, and c-Myc. One hundred patients of gastric carcinoma, obtained from 2 hospitals in Fortaleza, Brazil were assessed for the presence of EBV by in situ hybridization, for the expression of Bcl-2, Bax, and c-Myc (nuclear and cytoplasmic staining) proteins by immunohistochemistry techniques, and for the apoptotic index. EBV was detected in 8 (8%) patients showing strong staining situated in the nuclei of the tumor cells, 6 of them displaying a diffuse pattern, and 2 demonstrating a focal pattern of staining. The correlation with the immunohistochemistry results demonstrated that none of the EBV-positive cases exhibited Bcl-2 staining. On the other hand, Bax and c-Myc (nuclear) proteins demonstrated a significant positivity index and staining scores (labeling index and H-score) in the EBV-positive group; however, the values were lower than those obtained in the EBV-negative group, notably for c-Myc nuclear protein. In contrast, the cytoplasmic staining of c-Myc protein revealed slightly higher staining values in the EBV-positive group. The balance between Bcl-2 and Bax proteins demonstrated that the majority of the evaluated cases exhibited apoptosis orientation; however, in 62.5% of the EBV-positive cases neither protein was observed. The average apoptotic index was 4.58%, demonstrating a slightly lower average in the EBV-positive group. EBV is not related to the overexpression of Bcl-2 and c-Myc (nuclear) in gastric carcinomas; however, the results point to a possible EBV involvement with the transport mechanisms of the nuclear membrane, resulting in cytoplasmic c-Myc accumulation. The suppression of Bax expression could represent an alternative viral mechanism for inhibition of apoptosis.