EBV-positive Group Research Articles

Membranous tonsillitis: Aetiology, prevalence and prescribing patterns in patients with upper respiratory tract infection.

Background Membranous tonsillitis is one of the most common forms of acute tonsillitis in childhood. Although many different microorganisms may cause this disease, clinicians generally consider bacterial agents as a possible cause and prescribe a penicillin-group of antibiotic. This study aimed to determine the aetiology of membranous tonsillitis and prescribing errors. In addition, we investigated the effectiveness of epidemiological, clinical and laboratory parameters and their role in guiding treatment. Methods We did this retrospective study at the paediatric emergency department of a tertiary referral hospital including 423 outpatient children aged 0 to 18 years diagnosed with membranous tonsillitis. Results Group A beta-haemolytic streptococcus was found in 132 (31.2%) patients, Epstein-Barr virus (EBV) in 103 (24.3%), and other viral aetiologies in 188 (44.4%). The prescription rate of antibiotics in the EBV-positive group was 27%, and Downey cells were seen at a rate of 98% in this group. Only 7% of patients with a positive throat culture were started on appropriate antibiotics. Conclusion EBV and group A beta-haemolytic streptococcus were the most common causes of membranous tonsillitis. Throat culture and peripheral blood smears are the most useful tests for paediatric emergency clinicians; these are fast and can help ensure correct diagnosis and guide treatment in almost all patients.

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.

Impact of EBV positivity on clinical outcomes in nasopharyngeal cancer.

e18021 Background: Nasopharyngeal carcinoma (NPC) is a rare malignancy with unique geographical distribution. Epstein Barr virus (EBV) plays a key role in the pathogenesis of NPC, with pre and post-treatment serum EBV DNA level as an established prognostic indicator. However, evidence regarding the impact of the tumor cells’ EBV positivity on clinical outcomes is inconsistent. Methods: After IRB approval, we performed a retrospective chart review on NPC patients aged above 18 years seen at Corewell Health William Beaumont University Hospital between July 2015 and December 2020. The Epstein Barr encoding region in-situ hybridization from the biopsy sample determined EBV positivity. We compared progression-free survival (PFS) and overall survival (OS) based on EBV positivity status. Results: A total of 60 NPC patients were included: 19 were EBV-positive, 20 were EBV-negative, and 21 were EBV-unknown. The median age at diagnosis was 50.7 years, and 58.3 years in EBV-positive and negative groups respectively. Most patients in both groups were male (68.4% vs 65%) and had advanced (stage III or stage IV) disease (84.2% vs 75%). The median follow-up duration was 37.3 months. Median PFS and OS were 41 months and 114 months respectively in EBV-negative patients, while median PFS and OS were not reached in EBV-positive patients. There was no significant difference in PFS (0.944) and OS (0.117) among the EBV-positive and negative groups. Among advanced-stage patients, the median PFS was 33 months and 31 months in EBV-negative and EBV-positive groups, respectively, while median OS was not reached in both groups; the difference in PFS (p = 0.821) and OS (0.240) was statistically non-significant. When compared among patients with EBV-positive, negative, and unknown status, PFS was statistically non-significant (p = 0.347) but there was a statistical difference in OS (0.027). Conclusions: Our analysis suggests that the EBV status of the tumor cells does not predict clinical outcomes (PFS or OS) in NPC patients. Even though the OS trend favored EBV-positive patients overall and among those with advanced disease, it was not statistically significant. A difference in OS was seen when patients with unknown status were included and analyzed together with EBV-positive and negative patients, which raises the question of whether the outcome would be different if the majority of patients with unknown status belonged to either the EBV-positive or negative group. With studies in the literature reporting disparate findings, larger prospective studies are needed to better illustrate the impact of tumor cells' EBV positivity on clinical outcomes in NPC.

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.

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.

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.

Abstract B138: Prognostic value of PTEN loss for second-line paclitaxel treatment efficacy in metastatic gastric cancer

Abstract Introduction: For recurrent and metastatic gastric cancer, systemic chemotherapy is used for palliative purposes, and a paclitaxel- or irinotecan-based regimen is typically used in the second-line setting. Recent advances in research including the TCGA project now enable gastric cancer to be divided into four molecular subgroups, yet whether there are differences in chemotherapy response for each molecular subgroup remain largely unknown. Our aim in this study is to retrospectively compare the efficacy of paclitaxel as second-line chemotherapy according to molecular subtype and to evaluate prognostic value for these markers. Method: A total of 235 metastatic gastric cancer (mGC) patients who have been treated with second-line paclitaxel-containing regimen (paclitaxel monotherapy, FL/paclitaxel) between 2005-2017 were assessed. Archived formalin-fixed, paraffin-embedded tissue blocks obtained before the start of paclitaxel treatment were examined for EGFR, HER2, and PTEN status using immunohistochemistry (IHC). HER2 positivity was defined as either IHC 3+ or 2+ with amplification confirmed by FISH/SISH, and EGFR positivity was defined as IHC 3+. PTEN loss was defined as having complete loss of PTEN expression. MSI-H and EBV-positive group was excluded in this study. Treatment response was based on CT images taken every 2 to 3 cycles of chemotherapy and evaluated according to the RECIST criteria. Overall survival (OS) and progression-free survival (PFS) were calculated based on the starting point of second-line chemotherapy until death or disease progression, respectively. Those negative for all three markers (HER2, EGFR, PTEN loss) were categorized as genome stable (GS)-like molecular group. Result: Patients were classified into subgroups based on HER2, EGFR, and PTEN expression; 19.6% (46/235) were HER2 positive, 15.0% (26/173) were EGFR positive, and PTEN loss was observed in 11.6% (20/173). Expression status for the three markers showed tendency towards mutual exclusivity. When coexpression rates were taken into account, 35.3% (61/173) showed positivity in at least one of the markers, and 64.7% (112/173) were negative for all three. This latter group, categorized as GS-like molecular group, showed a tendency towards poor overall survival compared with patients positive for at least one molecular marker, with median survival of 5.7 vs 8.8 months (P=0.012). There were no significant differences in OS or PFS in terms of HER2 and EGFR status. However, patients with PTEN loss had significantly longer overall survival than those with intact PTEN expression, with median survival time of 12.0 months (95% confidence interval [CI], 7.0 - 21.2) and 6.2 months (95% CI, 4.7-7.2), respectively (P=0.003). Conclusion: In mGC patients treated with second-line paclitaxel chemotherapy, HER2 and EGFR status had no predictive or prognostic value. Contrary to the general association of PTEN loss with poor prognosis in many solid cancers including gastric cancer, PTEN loss group showed better survival outcome for second-line paclitaxel treatment in our study. Indirect association of PTEN with other pathways of multidrug resistance or cell cycle-related molecules that ultimately affect the PI3K/Akt pathway may play a role. Further investigation is needed for specific mechanisms and factors that lead to increased paclitaxel sensitivity in the PTEN loss group. Citation Format: Kyoo Hyun Kim, Jeong Hyun Jo, Sejung Park, Minkyu Jung, Woo Sun Kwon, Jee Hung Kim, Hyo Song Kim, Seung-Hoon Beom, Hyun Cheol Chung, Sun Young Rha. Prognostic value of PTEN loss for second-line paclitaxel treatment efficacy in metastatic gastric cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B138.

Analysis of PD1, PDL1, PDL2 expression and T cells infiltration in 1014 gastric cancer patients

ABSTRACTAlthough immune checkpoint blockade have demonstrated promising results, their effects on gastric cancer (GC) are under investigation. Understanding the clinical significance of PD1 and its ligands' expression, together with T cell infiltration might provide clues for biomarkers screening in GC immunotherapy. Immunohistochemistry were performed on a tissue microarray including 1,014 GC specimens using PD1, PDL1 and PDL2 antibodies. T cell markers CD3 and CD8 were also stained and quantified by automated image analysis. Correlation with clinical features and outcome were analyzed after controlling for potential confounders including EBV infection, HER2, C-met and PCNA expression. 37.8% of the cases showed membranous PD-L1 expression in tumor cells and 74.9% in infiltrating immune cells. PDL1 expression rate was rather higher in patients without metastasis, in EBV positive group and those with C-met and PCNA expression. GC patients with high level PDL1 expression exhibited better survival. GC Patients with higher T cell infiltration also showed elevated PDL1, PDL2 and PD1 expression and predict favorable outcome, indicating an adaptive immune resistance mechanism may exist. The group of patients infiltrated with lower density CD3+ T cells also without PDL1 expression in tumor cells predict the worst outcome in the subgroup of different PTNM stage, which may suggest an inactive immune status. These results highlights the need to assess both PDL1 expression in all tumor context and the characterization of the GC immune microenvironment.

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.

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 in bone marrow of rheumatoid arthritis patients predicts response to rituximab treatment

Objectives. Viruses may contribute to RA. This prompted us to monitor viral load and response to anti-CD20 therapy in RA patients.Methods. Blood and bone marrow from 35 RA patients were analysed for CMV, EBV, HSV-1, HSV-2, parvovirus B19 and polyomavirus using real-time PCR before and 3 months after rituximab (RTX) treatment and related to the levels of autoantibodies and B-cell depletion. Clinical response to RTX was defined as decrease in the 28-joint disease activity score (DAS-28) >1.3 at 6 months.Results. Before RTX treatment, EBV was identified in 15 out of 35 patients (EBV-positive group), of which 4 expressed parvovirus. Parvovirus was further detected in eight patients (parvo-positive group). Twelve patients were negative for the analysed viruses. Following RTX, EBV was cleared, whereas parvovirus was unaffected. Eighteen patients were responders, of which 12 were EBV positive. The decrease in the DAS-28 was significantly higher in EBV-positive group compared with parvo-positive group (P = 0.002) and virus-negative patients (P = 0.04). Most of EBV-negative patients that responded to RTX (75%) required retreatment within the following 11 months compared with only 8% of responding EBV-positive patients. A decrease of RF, Ig-producing cells and CD19+ B cells was observed following RTX but did not distinguish between viral infections. However, EBV-infected patients had significantly higher levels of Fas-expressing B cells at baseline as compared with EBV-negative groups.Conclusions. EBV and parvovirus genomes are frequently found in bone marrow of RA patients. The presence of EBV genome was associated with a better clinical response to RTX. Thus, presence of EBV genome may predict clinical response to RTX.

Clinicopathological differences between EBV-positive and EBV-negative diffuse large B-cell lymphoma.

8083 Background: Epstein Barr virus-positive diffuse large B-cell lymphoma (EBV-positive DLBCL) of the elderly is a new entity considered in the new WHO classification of lymphoid malignancies. EBV-positive DLBCL is a very aggressive entity with a short survival. Few data comparing the clinicopathological features between EBV-positive and EBV-negative DLBCL are available. Our study attempts to compare these characteristics and also identify prognostic factors. Methods: Between January 2002 and December 2009, 102 patients diagnosed with DLBCL were included in the analysis; 21 cases were EBV-positive and 81 cases were EBV-negative. Clinical data were reviewed retrospectively and patient's biopsies were analyzed for the immunohistochemical expression of bcl-6, CD10, CD30 and MUM-1. Samples from patients were also analyzed for the presence of EBV-encoded RNA (EBER). Descriptive statistics were used to assess categorical and continuous variables. Survival curves for OS were estimated using the Kaplan-Meier method and compared using the log-rank test. For the multivariate analysis, the Cox proportional-hazard regression test was used. Results: Mean age for EBV-positive and EBV-negative patients was 73 and 65years, respectively (p=0.043). Sex, LDH levels, performance status, clinical stage, response to initial treatment and extranodal involvement were not significantly different between groups. The EBV status demonstrated a substantially poorer overall survival 3 months (95% CI, 0.0–2.6 months) and 15.5 months (1.0–43.0 months) in EBV-positive and EBV-negative, respectively (p=0.022). At the multivariate level, EBV status was an independent factor compared with the International Prognostic Index (IPI) score (p=0.001) with a 2.4-fold (95% CI, 1.2–4.8) risk for death for EBV-positive cases. In the EBV-positive group, a non-germinal center profile, the Oyama score and the IPI score were associated with survival. In the multivariate analysis only IPI score was significant (p=0.0045). Conclusions: EBV- positive DLBCL correlated with advanced age and a poor survival in comparison with EBV-negative DLBCL. The IPI score was a better prognostic tool than the Oyama score in EBV-positive patients. No significant financial relationships to disclose.

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.

The Human Leukocyte Antigen Class I Region Is Associated with EBV-Positive Hodgkin's Lymphoma: HLA-A and HLA Complex Group 9 Are Putative Candidate Genes

Various studies have indicated that the human leukocyte antigen (HLA) region is associated with Hodgkin's lymphoma. We recently showed a specific association of the HLA class I region with EBV-positive Hodgkin's lymphoma cases. One haplotype of two consecutive microsatellite markers (D6S265 and D6S510) was overrepresented in the patient group, whereas another haplotype was underrepresented. Here, we did fine mapping of this region of approximately 400 kb as a next step to find the causative single-nucleotide polymorphism(s) (SNP). To select candidate SNPs for screening the total study population, several known SNPs were determined by sequencing two individuals homozygous for either of the above-mentioned associated haplotypes. Seven SNPs displayed different alleles in these two individuals and were therefore analyzed in the total study population, including 238 Hodgkin's lymphoma patients and 365 family-based controls. All seven SNPs showed significant association with the EBV-positive patient group. Two of these SNPs were analyzed in a Scottish Hodgkin's lymphoma population and revealed significant associations as well. The associated SNPs are located nearby two putative candidate genes: HLA-A and HLA complex group 9. HLA-A represents the most interesting target because of its consistent expression in EBV-positive Hodgkin's lymphoma cases and its ability to present EBV-derived peptides to cytotoxic T cells.

Association between the Epstein-Barr Virus and Hodgkin’s Lymphoma in the North-Eastern Part of Hungary: Effects on Therapy and Survival

This retrospective study included 109 patients with Hodgkin’s lymphoma (HL; 45 females, 64 males). In 47 of the 109 HL patients (43%), immunohistochemical analysis of their formalin-fixed, paraffin-embedded histologic samples revealed Epstein-Barr virus (EBV) by latent membrane protein (LMP) 1. The highest virus association (50%) was found with the mixed cellularity histologic subtype, especially in patients aged 11–20 and >50 years. Virus positivity in nodular sclerosis was 35% (negative cases accumulated in patients aged 15–30 years). Regarding clinical stages, histologic subtypes, general symptoms, treatments employed and response to treatment, the EBV-positive group was not significantly different from the virus-negative group. During the mean follow-up time of 83 months (9–300 months), the overall or event-free survival of EBV-negative patients was more favorable than that of EBV-positive patients, although the difference was not significant (p = 0.16 and p = 0.24, respectively). EBV infection may be involved in the pathogenesis of HL in our Hungarian study cohort, but it does not significantly affect clinical symptoms, therapeutic results or complete and event-free survival of HL patients.

Haplotype-Based Sequencing To Delineate the Associated HLA Class I Region for EBV Positive Hodgkin Lymphoma.

Various studies have indicated that the human leukocyte antigen (HLA) region is associated with Hodgkin Lymphoma (HL). We recently demonstrated a specific association of the HLA class I region with Epstein-Barr virus (EBV) positive HL cases. The positively associated haplotype was characterized by alleles 126 and 284 of the consecutive microsatellite markers D6S265 and D6S510 while the negatively associated haplotype was characterized by alleles 130 and 302. Further fine mapping of this region is necessary to find the causative SNP(s). To select the most promising candidate SNPs for screening the total study population, known SNPs were sequenced in an individual homozygous for the positively associated haplotype and an individual homozygous for the negatively associated haplotype. Five SNPs (rs2530388, rs2523972, rs4713276, rs2256543, and rs6457110) that were selected on the basis of displaying different alleles in these two individuals were analysed in the total study population. The five SNPs showed significant association with the EBV-positive patient group, three of them revealed a stronger association than the previously identified associations (−log p difference = 1). These five SNPs were also analysed in a Scottish population, and four of them showed association in that population as well. The associated region was narrowed down from 310 kb in the original study to a region comprising less than 60 kb in the present study. Of the genes mapping to this candidate region, HLA-A represents the most interesting target because of its consistent expression in EBV positive HL cases and its ability to present EBV derived peptides to cytotoxic T cells.