Epstein-Barr Virus Expression Research Articles

Molecular and pathological features in patients with gastric cancer in Argentina. Association between Epstein Barr Virus, MSI, PD-L1 expression and T-cells infiltration

Introduction: The incidence of gastric cancer (CG) in Argentina is about 3900 cases and 3200 deaths per year. In the recent molecular classification stand out among others, those tumors associated with Epstein Barr virus (EBV) infection and microsatellite instability (MSI). Both are associated with an increase in the lymphoid infiltrate and this tumor microenvironment has been related to an increase in the expression of PD-L1. The clinical relevance of these features remains unclear. The objective of this study is to evaluate the expression of EBV and MSI, to analyze the expression of PD-L1 together with the presence of CD3 and CD8 lymphocytes in the tumor microenvironment and to describe their relationship with clinical-pathological characteristics. Methods: We evaluated a cohort of 47 patients with gastric adenocarcinoma, without neoadjuvant treatment, who underwent curative surgery. Immunohistochemistry (IHC) for MLH1, PMS2, MSH2, MSH6, PD-L1, CD3 and CD8; MSI assay with BAT25, BAT26, NR21, NR24, and MONO27 markers, and qualitative detection of EBV were performed. Results: Qualitative PCR for EVB was positive in 21% (n = 10) of cases. MSI was identified in 8% (N = 4) cases. The status of MSI and EBV positive GC were mutually exclusive. Correlation between MSI and MMR deficiency by IHC was 100% (MLH1 and PMS2 loss in all cases). CG EBV positive and CG MSI were more frequent in male patients, older than 50 years and distal gastric localization. The intestinal histological type was the most frequent in CG MSI, whereas represented 50% in CG EBV cases. 50% of the GC EBV and 75% of the CG MSI showed a high density of tumor-infiltrating lymphocytes (TILs). PDL1 was positive in tumor cells and in infiltrating immune cells in 28% (n = 13) cases (6/10 CG-EVB positive, 2/4 CG-MSI and 5/33 CG-EVB negative / MSS). GCs EBV positive and CG MSI that expressed PD-L1 had a high density of CD3 T-lymphocytes in both intratumoral region and invasion margin but low for CD8 T lymphocytes. Conclusion: In our gastric patients, PD-L1 expression was associated with distinct molecular and histopathological features including a high density of CD3 T lymphocytes, MSI status, and EBV. These data could be relevant to identify potential candidate patients for immunotherapies.

LATENT MEMBRANE PROTEIN 2A (LMP2A) MIMICS B‐CELL RECEPTOR SIGNALING AND PROMOTES IMMUNE ESCAPE IN EPSTEIN‐BARR VIRUS (EBV)‐POSITIVE DIFFUSE LARGE B‐CELL LYMPHOMA (DLBCL)

Background: EBV+ DLBCL is an aggressive malignancy that is largely resistant to current therapeutic regimens. LMP2A is expressed during different latency stages of EBV-infected B cells in which it triggers activation of cytoplasmic protein tyrosine kinases. Early studies have revealed that an immunoreceptor tyrosine-based activation motif in the cytoplasmic N-terminus of LMP2A can trigger a transient increase of the cytosolic Ca2+ concentration similar to that observed in activated B cells. Meanwhile, tumor cells often express PD-L1 in EBV+ DLBCL, providing a possible mechanism for immune escape. We thus explored the correlations between LMP2A expression, B-cell receptor (BCR) signaling, and PD-L1 expression in EBV+ DLBCL. Design: Two cohorts of DLBCL cases, respectively EBV-positive (n=28) and EBV-negative (n=32), were selected. LMP2A, PD-L1 and the BCR signaling-related molecule, phosphorylated form of SYK (pSYK), were immunohistochemically evaluated on formalin-fixed, paraffin-embedded tumor tissues. The expression status of LMP2A, pSYK and BCR were further validated by a flow cytometry analysis using fresh tissues from 3 EBV-positive and 5 EBV-negative DLBCL patients. Results: The EBV-positive cases, with a median age of 61yrs, were more frequently associated with a high-risk IPI score and a non-GCB phenotype (P=0.029). Compared with EBV-negative ones, patients with EBV-positive tumors showed a worse response to the RCHOP therapy and a shorter median survival (P = 0.041). Twenty-one EBV-positive cases (75 %) expressed LMP2A, whereas none of the EBV-negative cohort expressed this protein. The expression level of pSYK was significantly lower (P = 0.0313) in EBV+ DLBCL cases than those EBV-negative ones, and the pSYK level correlated negatively with LMP2A level (P = 0.119). The expression level of PD-L1 was significantly higher (P = 0.042) in EBV+ DLBCL, which correlated positively with the LMP2A level (P=0.184). Flow cytometry confirmed the LMP2A expression in EBV+ DLBCL, which correlated with a down-regulated BCR and pSYK expression. Conclusions: EBV+ DLBCL seems to feature an inactive BCR signaling, which may be related to the expression of LMP2A. Besides, LMP2A may function and promote the PD-L1 expression. These findings indicate that targeting immune checkpoints including PD-1/PD-L1 instead of BCR signaling molecules seems to be more reasonable and promising for the treatment of EBV+ DLBCL. Keywords: diffuse large B-cell lymphoma (DLBCL); Epstein-Barr virus (EBV).

Assessing molecular subtypes of gastric cancer: microsatellite unstable and Epstein-Barr virus subtypes. Methods for detection and clinical and pathological implications

BackgroundThe molecular classification of gastric cancer recognises two subtypes prone to immune checkpoint blockade: the microsatellite unstable and the Epstein-Barr virus (EBV)-related tumours. We aim to assess the concordance between immunohistochemistry and PCR for microsatellite status evaluation, and explore the value of microsatellite instability (MSI) and EBV as predictive survival factors.Material and methodsWe collected 246 consecutively diagnosed gastric cancer cases in all stages and evaluated the microsatellite status using immunohistochemistry for mismatched repair (MMR) proteins and PCR. EBV expression was studied through in situ hybridisation.ResultsForty-five (18%) cases presented MSI and 13 (6%) were positive for EBV. MSI was associated with female sex, older age, distal location and distal non-diffuse type of the modified Lauren classification. EBV expression was most frequent in proximal location and proximal non-diffuse type. The sensitivity, specificity, positive predictive value and negative predictive value of immunohistochemistry for the microsatellite study were 91%, 98%, 91% and 98%, respectively. In the multivariate analysis, MSI was an independent predictor of favourable tumour-specific survival (TSS) in stages I–III (MSI: HR: 0.37, 95% CI 0.12 to 0.95, p=0.04).ConclusionsThe MSI status and the EBV expression should be incorporated in routine pathological report for two reasons. First, MSI defines a different pathological entity with a better outcome. Second, MSI and EBV may be useful biomarkers to identify patients who will respond to immune checkpoint blockade inhibitors. For this purpose, immunohistochemical study for MMR proteins and in situ hybridisation study for EBV evaluation are feasible and cost-effective methods.

Epstein-Barr virus incidental expression in bone marrow cells: a study of 230 consecutive bone marrow biopsy samples

Epstein-Barr virus (EBV) is associated with many neoplastic hematologic conditions, but scattered EBV-positive cells can be detected in lymph nodes of healthy individuals and they usually represent latently infected lymphocytes. The incidence of EBV detection in normal bone marrow samples has not been studied and is largely unknown. The lack of knowledge regarding the true incidence of encountering bystander latent EBV-positive cells in the bone marrow may potentially lead to a diagnostic dilemma when assessing a staging bone marrow for a patient with an EBV-positive B or T/NK-cell lymphoma. The aim of our study was to investigate the rate of detection of EBV expression in bone marrow samples and correlate any positive findings with various clinical parameters including patient's age, sex, clinical history, immune status, and any neoplastic transformation if follow-up data are available. We retrospectively studied 230 consecutive bone marrow biopsies performed in 2013 and found 5 cases (2.17%) with scattered EBV-positive cells by in situ hybridization. The observed scattered EBV-positive cells are largely small in size and likely represent bystander, latently infected cells. The rate of detection of EBV-positive cells in the bone marrow appears to be slightly higher in immunodeficient individuals (3%) than in immunocompetent patients (1%).

Expression and significance of EBV, ARID1A and PIK3CA in gastric carcinoma.

AT-rich interaction domain 1A (ARID1A) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) serve important roles in the formation and development of numerous malignancies including gastric cancer. Accumulating evidence has demonstrated that Epstein-Barr virus (EBV) is a pathogenic virus associated with gastric cancer. The present study aimed to investigate the association between EBV infection, and the expression levels of ARID1A and PIK3CA in gastric cancer. EBER in situ hybridization was performed to detect EBV infection. Immunohistochemistry was used to assess the expression levels of ARID1A and PIK3CA in gastric cancer and adjacent normal tissues. A total of 58 gastric cancer and 10 adjacent normal tissues were tested for genetic mutations via single nucleotide polymorphism genotyping assays. Fluorescent polymerase chain reaction was used to detect EBV infection; 9.3% (28/300) of gastric cancer samples were positive for EBV, whereas, all adjacent normal tissues were negative. ARID1A and PIK3CA were negatively correlated in gastric cancer (r=−0.167). The expression levels of ARID1A and PIK3CA in gastric cancer were significantly associated with the depth of invasion of gastric cancer. A total of 62.1% (36/58) of tumor samples exhibited mutations in ARID1A, whereas, 13.8% (8/58) presented mutations in PIK3CA. Notably, EBV-associated gastric cancer (EBVaGC) samples with PIK3CA mutations additionally exhibited ARID1A mutations. Although in the present study it was identified that ARID1A and PIK3CA were negatively correlated in EBVaGC, further studies are required to investigate the association among ARID1A, PIK3CA and EBV in gastric cancer.

Epstein-Barr virus infection and expression of Activation-induced cytidine deaminase/Apolipoprotein B mRNA-editing catalytic polypeptide 3 in adenoids and palatine tonsils

Epstein-Barr virus infection and expression of Activation-induced cytidine deaminase/Apolipoprotein B mRNA-editing catalytic polypeptide 3 in adenoids and palatine tonsils

A novel comparison of Epstein-Barr virus with broad histological spectrum of oral squamous cell carcinoma

Objectives:To evaluate the immunohistochemical expression of Epstein-Barr virus (EBV) in broad spectrum histological subtypes of oral squamous cell carcinoma (OSCC) and to determine the relationship of EBV with clinicopathological parameters of OSCC.Methods:This cross-sectional study included 150 clinically diagnosed OSCC cases from the outpatient of Ziauddin University Hospital from March, 2017 to October, 2018. These were confirmed on histological examination and categorized into conventional squamous cell carcinoma (SCC) and rare variants. Conventional SCC was subcategorized into keratinizing (KSCC), non-keratinizing (NKSCC), and hybrid SCC (HSCC). EBV status was compared among various histological tumor entities and clinicopathological characteristics of OSCC using immunohistochemistry. Chi-square test was used to determine the association of each histological subtype with EBV status with P-value <0.05 considered as statistically significant.Results:Conventional tumor was the most frequent squamous cell carcinoma (n=126; 84%). A significant statistical link of EBV infection was observed with rare histological tumors exhibiting acantholysis (P=0.01), as well as tumors involving buccal mucosa (P=0.03), and habitual smokers (P=0.001).Conclusions:In this study, acantholytic tumor, a rare histological subtype of OSCC, tended to be EBV related. Moreover, OSCC cases bearing EBV infection were more likely smokers favoring buccal mucosa as primary anatomical site for oral cancer.

Prognostic Value of Epstein-Barr Virus (EBV)-Induced Gene 3 (EBI3) in Diffuse Large B Cell Lymphoma

Background: Epstein-Barr virus (EBV)-induced gene 3 (EBI3) as a subunit for heterodimeric cytokines IL-27 and IL-35, plays important roles both in regulation of T cell proliferation and Th1,Th2 and Th17 cells differentiation. EBI3 was closely related with tumor prognosis. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults with cure-rates of 40-60%. However, Relapse and refractory rate remain in 40% or so. In the present study, we aimed to detect the expression of EBI3 in DLBCL and to analyze its relationship with prognosis.Methods: We retrospective reviewed medical records of 51 newly diagnosed DLBCL patients in Sichuan People's Hospital between January 2010 and December 2016. Immunohistochemical (IHC) assay was used to detect the expression of EBI3 and PD-1 in DLBCL. The expression of CD5, CD30, Bcl-2, Bcl-6, C-myc and Epstein-Barr virus (EBV)-encoded RNAs (EBERs) in tumor specimens from 53 patients was also detected by IHC. The Kaplan-Meier method with log-rank test was used for univariate analysis. Cox proportional hazards model was used for multivariate analysis.Results: Of the 51 patients, 40 (78.4%) were EBI3-positive in tumor specimens. And PD-1 expression (39/40) was almost in parallel with EBI3 in tumor specimens. EBI3 expression was common in the non-germinal center B-cell-like (GCB) subtype than in the GCB subtype. Patients with EBI3 expression in tumor were more likely to be resistant to first-line chemotherapy when compared with the patients without EBI3 expression in tumor microenvironment (P <0.05). EBI3 expression in tumor microenvironment was correlated with PD-1 expression (r = − 0.20, P = 0.04). Only one patient with EBI3 expression was no PD-1 expression. No correlations were detected between EBI3 expression and the expression of EBER as well as other markers: ALK, CD5, c-Myc and CD30. The complete remission (CR) rates were 7.5% (3/40) and 71.4% in patients with and without EBI3 expression in tumor cells (P = 0.02). EBI3 expression in tumor cells was an independent risk predictor for ORR (P < 0.05).Conclusions: Our results indicate that EBI3 associated with poor prognosis and EBI3 may be a potential therapeutic target and prognosis marker. DisclosuresNo relevant conflicts of interest to declare.

Epstein - Barr virus association with plasma cell neoplasms.

Epstein-Barr virus (EBV) expression has been reported in several hematopoietic and non-hematopoietic disorders but its expression in plasma cell neoplasms has been largely limited to immunodeficiency-related cases such as in the setting of post-organ transplantation or human immunodeficiency virus (HIV) infection. The aim of this study is to evaluate the association of EBV with plasma cell neoplasms, mainly in immunocompetent patients. We retrospectively studied 147 cases of patients with different plasma cell neoplasms (109 plasma cell myelomas, 22 plasmacytomas, and 16 monoclonal gammopathy cases). Six patients were immunocompromised. EBV was positive in 6 cases; 3 immunocompromised (2 patients with HIV infection and 1 patient was post-renal transplant) and 3 immunocompetent patients with plasmacytoma and variable plasmablastic features. Our data shows that EBV was negative in all plasma cell myeloma cases in immunocompetent patients and has an overall low association with the different plasma cell neoplasms in the immunocompetent setting. When expressed, it is usually associated with variable plasmablastic features.

Epstein-Barr Virus-Positive Cancers Show Altered B-Cell Clonality

Epstein-Barr virus (EBV) is convincingly associated with gastric cancer, nasopharyngeal carcinoma, and certain lymphomas, but its role in other cancer types remains controversial. To test the hypothesis that there are additional cancer types with high prevalence of EBV, we determined EBV viral expression in all the Cancer Genome Atlas Project (TCGA) mRNA sequencing (mRNA-seq) samples (n = 10,396) from 32 different tumor types. We found that EBV was present in gastric adenocarcinoma and lymphoma, as expected, and was also present in >5% of samples in 10 additional tumor types. For most samples, EBV transcript levels were low, which suggests that EBV was likely present due to infected infiltrating B cells. In order to determine if there was a difference in the B-cell populations, we assembled B-cell receptors for each sample and found B-cell receptor abundance (P ≤ 1.4 × 10-20) and diversity (P ≤ 8.3 × 10-27) were significantly higher in EBV-positive samples. Moreover, diversity was independent of B-cell abundance, suggesting that the presence of EBV was associated with an increased and altered B-cell population. IMPORTANCE Around 20% of human cancers are associated with viruses. Epstein-Barr virus (EBV) contributes to gastric cancer, nasopharyngeal carcinoma, and certain lymphomas, but its role in other cancer types remains controversial. We assessed the prevalence of EBV in RNA-seq from 32 tumor types in the Cancer Genome Atlas Project (TCGA) and found EBV to be present in >5% of samples in 12 tumor types. EBV infects epithelial cells and B cells and in B cells causes proliferation. We hypothesized that the low expression of EBV in most of the tumor types was due to infiltration of B cells into the tumor. The increase in B-cell abundance and diversity in subjects where EBV was detected in the tumors strengthens this hypothesis. Overall, we found that EBV was associated with an increased and altered immune response. This result is not evidence of causality, but a potential novel biomarker for tumor immune status.

EBV infection determines the immune hallmarks of plasmablastic lymphoma

ABSTRACTDespite recent therapeutic progress, plasmablastic lymphoma (PBL), a distinct entity of high grade B cell lymphoma, is still an aggressive lymphoma with adverse prognosis. PBL commonly occurs in patients with HIV infection and PBL cells frequently express Epstein Barr virus (EBV) genome with type I latency. Occasionally however, PBL may develop in patients with an immunodepressed status without EBV and HIV infection. The aim of this study was to determine which PBL patients may benefit from the emerging strategies of immune checkpoint blockade. Here, we produced and analyzed the transcriptomic profiles of such tumors to address this question. Unsupervised hierarchical clustering analysis of PBL samples revealed they segregate according to their tumor EBV-status. Moreover, EBV+ PBL displays abundant leucocyte infiltrates and T-cell activation signatures, together with high expression levels of mRNA and protein markers of immune escape. This suggests that EBV infection induce an anti-viral cytotoxic immunity which progressively exhausts T lymphocytes and promotes the tolerogenic microenvironment of PBL. Hence, most EBV+ PBL patients presenting an early stage of cancer immune-editing process appear as the most eligible patients for immune checkpoint blockade therapies.

Abstract 4210: HP and EBVaGC in Peruvian patients

Abstract BACKGROUND: Gastric cancer (GC) is the second most common cancer and first leading cause of cancer-related deaths in Peru. Infection by Helicobacter pylori (HP) and Epstein-Barr virus (EBV) are accepted carcinogenic and infection is widely spread in the Peruvian population. The aims of this study were to evaluate HP and EBV-associated GC (EBVaCG), to assess the prevalence rate and to define the characteristics of Peruvian patients. METHODS: GC samples were prospectively collected from patients who underwent gastroscopy or surgical resection with no preoperative treatment at INEN between 2015 and 2017. Tumor tissue (T), proximal healthy tissue (P) and distal healthy tissue (D) samples were assessed for HP and EBV by quantitative PCR (qPCR) using specific primers. HP and EBV status were analyzed along with clinicopathologic parameters of the tumor. Kaplan-Meier estimation curves overall survival (OS) was applied. Comparison between qPCR detection and current standard methods of detection was also performed. All tests were two sided, and a p≤0.05 was considered statistically significant. RESULTS: A total of 150 patients were studied with a mean age 65 years and predominance of males (51.3%). Intestinal-mixed histological subtype rate was 72.5% in cases. Most frequent clinical stage was III-IV (74.6%). HP+ patients were determinate with at least one ureA/hspA gene result and EBV+ patients were divided in high viral load (&amp;gt;100copy/μl) and low viral load (&amp;lt;100copy/μl) groups. Results found HP in 87.3% (n=131/150) and EBV in 10% (n=15/150) of the population. Cases and HP concentration were higher in D (ureA: 62.8%, [703.58±245.47pg]; hspA: 73.8%, [42.77±10.17pg]) than P (ureA: 59.0%, [539.69±121.32pg]; hspA: 71.0%, [31.90±8.64pg]) and T (ureA: 49.7%, [296.32±164.98pg]; hspA: 70.5%, [4.6±1.33pg]) (p&amp;lt;.001). High viral load EBV was found in 40% (n=6/15) of cases. Co-infection detection rate was 7.3% (n=11/150). Correlation between qPCR and H&amp;E evaluation of HP was found in only 72.9% (n=19/57) of cases without evidence of HP (p&amp;lt;.001) and no detectable expression of EBV was found by EBER in qPCR positive cases (n= 15). Cases were defined in 4 groups: HP+EBV+ (n=9), HP+EBV- (n=98), HP-EBV+ (n=6), and HP-EBV- (n=37). Results showed median age (67, 66, 67 and 66), most frequent III-IV clinical stage (75%, 69.5%, 72.7% and 62.1%), histologic grade found was GII (50%, 28.8%, 50% and 43.3%) and intestinal-mixed histological subtype (66.6%, 56.6%, 100% and 62.1%) in four groups respectively. EBVaGC was associated to OS (38.3% vs 72.3%) at 1 year, p=0.033). Based on a mean follow-up of 24 months, the 1-year survival was lower in EBV+ [45.5% (HP+ EBV+) and 25% (HP- EBV+)] than in EBV- [73.1% (HP+ EBV-) and 61.5% (HP- EBV-)] groups. CONCLUSION: HP infection is frequent in Peruvian CG patients. Presence of HP and EBV infection evaluated by qPCR define 4 groups with different features and survival. Study supported by CIENCIACTIVA-CONCYTEC, under the contract #196-2015-FONDECYT. Citation Format: Carolina Belmar López, Miluska Castillo Garcia, Carlos A. Castañeda Altamirano, Luis F. Barreda Bolaños, Daniel Valdivia Leonardo, Eduardo Payet Meza, Valeria Villegas Bernaola, Jais Nieves Prado, Joselyn R. Sanchez Sifuentes, Luis A. Bernabé Monsalve, Manuel A. Chumpitaz La Rosa Sanchez, Omar Mejía Dionisio, Ivan Chavez Passiuri. HP and EBVaGC in Peruvian patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4210.

Correction for Borozan et al., "Analysis of Epstein-Barr Virus Genomes and Expression Profiles in Gastric Adenocarcinoma".

Correction for Borozan et al., "Analysis of Epstein-Barr Virus Genomes and Expression Profiles in Gastric Adenocarcinoma".

Richter transformation driven by Epstein-Barr virus reactivation during therapy-related immunosuppression in chronic lymphocytic leukaemia.

The increased risk of Richter transformation (RT) in patients with chronic lymphocytic leukaemia (CLL) due to Epstein-Barr virus (EBV) reactivation during immunosuppressive therapy with fludarabine other targeted agents remains controversial. Among 31 RT cases classified as diffuse large B-cell lymphoma (DLBCL), seven (23%) showed EBV expression. In contrast to EBV- tumours, EBV+ DLBCLs derived predominantly from IGVH-hypermutated CLL, and they also showed CLL-unrelated IGVH sequences more frequently. Intriguingly, despite having different cellular origins, clonally related and unrelated EBV+ DLBCLs shared a previous history of immunosuppressive chemo-immunotherapy, a non-germinal centre DLBCL phenotype, EBV latency programme type II or III, and very short survival. These data suggested that EBV reactivation during therapy-related immunosuppression can transform either CLL cells or non-tumoural B lymphocytes into EBV+ DLBCL. To investigate this hypothesis, xenogeneic transplantation of blood cells from 31 patients with CLL and monoclonal B-cell lymphocytosis (MBL) was performed in Rag2-/- IL2γc-/- mice. Remarkably, the recipients' impaired immunosurveillance favoured the spontaneous outgrowth of EBV+ B-cell clones from 95% of CLL and 64% of MBL patients samples, but not from healthy donors. Eventually, these cells generated monoclonal tumours (mostly CLL-unrelated but also CLL-related), recapitulating the principal features of EBV+ DLBCL in patients. Accordingly, clonally related and unrelated EBV+ DLBCL xenografts showed indistinguishable cellular, virological and molecular features, and synergistically responded to combined inhibition of EBV replication with ganciclovir and B-cell receptor signalling with ibrutinib in vivo. Our study underscores the risk of RT driven by EBV in CLL patients receiving immunosuppressive therapies, and provides the scientific rationale for testing ganciclovir and ibrutinib in EBV+ DLBCL. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Analysis of Epstein-Barr Virus Genomes and Expression Profiles in Gastric Adenocarcinoma.

Epstein-Barr virus (EBV) is a causative agent of a variety of lymphomas, nasopharyngeal carcinoma (NPC), and ∼9% of gastric carcinomas (GCs). An important question is whether particular EBV variants are more oncogenic than others, but conclusions are currently hampered by the lack of sequenced EBV genomes. Here, we contribute to this question by mining whole-genome sequences of 201 GCs to identify 13 EBV-positive GCs and by assembling 13 new EBV genome sequences, almost doubling the number of available GC-derived EBV genome sequences and providing the first non-Asian EBV genome sequences from GC. Whole-genome sequence comparisons of all EBV isolates sequenced to date (85 from tumors and 57 from healthy individuals) showed that most GC and NPC EBV isolates were closely related although American Caucasian GC samples were more distant, suggesting a geographical component. However, EBV GC isolates were found to contain some consistent changes in protein sequences regardless of geographical origin. In addition, transcriptome data available for eight of the EBV-positive GCs were analyzed to determine which EBV genes are expressed in GC. In addition to the expected latency proteins (EBNA1, LMP1, and LMP2A), specific subsets of lytic genes were consistently expressed that did not reflect a typical lytic or abortive lytic infection, suggesting a novel mechanism of EBV gene regulation in the context of GC. These results are consistent with a model in which a combination of specific latent and lytic EBV proteins promotes tumorigenesis.IMPORTANCE Epstein-Barr virus (EBV) is a widespread virus that causes cancer, including gastric carcinoma (GC), in a small subset of individuals. An important question is whether particular EBV variants are more cancer associated than others, but more EBV sequences are required to address this question. Here, we have generated 13 new EBV genome sequences from GC, almost doubling the number of EBV sequences from GC isolates and providing the first EBV sequences from non-Asian GC. We further identify sequence changes in some EBV proteins common to GC isolates. In addition, gene expression analysis of eight of the EBV-positive GCs showed consistent expression of both the expected latency proteins and a subset of lytic proteins that was not consistent with typical lytic or abortive lytic expression. These results suggest that novel mechanisms activate expression of some EBV lytic proteins and that their expression may contribute to oncogenesis.

Mutation analysis of the EBV-lymphoblastoid cell line cautions their use as antigen-presenting cells.

Lymphoblastoid cell lines (LCLs) have been widely used as professional antigen-presenting cells (APCs). However, neoantigen-loaded LCLs could induce nonspecific T-cell response, which could be due to expression of both Epstein-Barr virus (EBV) antigens and nonsynonymous mutations arising in LCLs. Since the number of passages could influence mutational characteristics of LCLs, and moreover extensive proliferation of LCLs in vitro is necessary to activate T cells for immunotherapy, we comprehensively profiled mutational characteristics by comparing eight sets of B cells and matched high-passage LCLs using whole-exome sequencing in order to assess the effect of nonsynonymous mutations arising in LCLs on nonspecific T-cell response. We found 315 nongermline mutations (approximately 40mut/subject) randomly distributed across all chromosomes including 18 mutations in immunoglobulin V and J genes in eight LCLs, of which 137 candidate neoantigens (approximately 17mut/subject) were identified. The underlying mutational processes linked to EBV-transformed LCLs could be attributed to activation induced cytidine deaminase gene expression which contributes to cytosine mutation clusters in LCLs through cytosine deamination. Pathways significantly enriched by nonsilent mutations of each LCL were totally different among all LCLs. In conclusion, high-passage LCLs may not be suitable to serve as APCs due to random nonsilent mutations, particularly for presentation of neoantigens of low immunogenicity, although further experimental proofs are needed.

Abstract 53: Expression of EBV in the non-neoplastic cell population in trephine biopsies of patients with multiple myeloma is associated with a significant survival advantage

Abstract Introduction: Multiple myeloma (MM) is a plasma cell (PC) disorder of the elderly, affecting predominantly males. Therapeutic advancement during the last decade has improved overall survival. Crosstalk between malignant PCs and the microenvironment is considered to play an important role in the development of resistance to treatment and relapse. Epstein Barr virus (EBV) is the first B-lymphotropic herpes virus identified for its association with human tumors. Reports relating an association of EBV to MM are few and inconsistent. It has been proposed that initiation of viral lytic cycle in EBV infected B cells is preceded by X-Box binding Protein-1 (XBP-1) mediated differentiation, resulting in a subset of EBV expressing infected PCs. Increased incidence of EBV expressing MM cases have been observed in immune-compromised patients. However, the significance of EBV in the bone marrow (BM) of MM patients has not been fully elucidated. The aim of our study was to evaluate prevalence of EBV in association with MUC1, XBP-1, ALDH1, and B-cell lineage markers (CD20, CD45, CD56, and CD138) in bone marrow trephine biopsies of patients with MM and to assess if there was any association with prognosis in a cohort of 151 MM patients at a single tertiary teaching hospital institution in Pakistan. Methodology: Formalin-fixed, paraffin-embedded trephine blocks of 151 MM cases diagnosed from 2007-2015 were evaluated for immunohistochemical (IHC) expression of MUC1, XBP-1, and EBV along with stem cell marker (ALDH1) and B-cell lineage markers (CD20, CD45, CD56, and CD138). Clinicopathologic details and follow-up data were collected from patients' medical records. Data was analyzed on SPSS 19. Five-year survival was estimated by Kaplan Meier curve for patients with follow-up available for 5 years or more (n=75). Results: Mean age at diagnosis was 57 years (range 30-89) with male preponderance (68.2%). Women (60.4%) with MM presented at an earlier age (median age 55 years, range 42-78) and difference in age of presentation was found to be significant (p value 0.04). Bone lesions were identified in 68.9% of patients at presentation. Stage I, II, and III disease was identified in 32.4%, 23.4%, and 44.1 % of patients, respectively (ISS staging system). Relapse was observed in 79.6% of patients and was significantly correlated with the expression of XBP-1 (p value 0.035). CD56 was expressed in 60.3% of cases and was significantly associated with cases diagnosed after the age of 57 years (p value 0.003). Expression of MUC1 was observed in 62.3% of tumors and 70% of MUC1-positive tumors also expressed XBP-1 (p value 0.06). Although the number of cases expressing EBV in the MM tumor cell population was only 7.2%, we detected EBV in 80.2% of the cases in the adjacent non-neoplastic cells with conspicuous expression in megakaryocytes. Five-year survival analysis (n=75) showed that expression of EBV+ in non-neoplastic cells was associated with significantly improved outcome with an advantage of 1.8 years (95% CI 3.32-4.4) as compared to EBV- cases (p value &amp;lt; 0.005). Among the XBP-1+ tumors, lack of EBV expression in non-neoplastic cells significantly correlated with adverse outcome (95%CI, 0.70-2.6) (p value &amp;lt; 0.005). Conclusions: To the best of our knowledge, this is the first study reporting expression of EBV in non-neoplastic cell population in BM trephines of MM patients. Salient conclusions of our study are as follows: 1. Expression of EBV in the surrounding tumor microenvironment of trephine biopsies of patients with MM is associated with a significant survival advantage; 2. Patients with MM in Pakistan present at a comparatively earlier age; 3. XBP1 expression was associated with relapse. Citation Format: Sheerien Rajput, Khurram Minhas, Iqbal Azam, Sadia Habib, Azhar Hussain, El-Nasir Lalani. Expression of EBV in the non-neoplastic cell population in trephine biopsies of patients with multiple myeloma is associated with a significant survival advantage [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 53.

Elevated expression of EBV and TLRs in the brain is associated with Rasmussen's encephalitis.

Rasmussen's encephalitis (RE) is a rare pediatric neurological disorder, the etiology of which remains unclear. It has been speculated that the immunopathogenesis of RE involves damage to neurons, which eventually leads to the occurrence of RE. Viral infection may be a critical factor in triggering RE immunopathogenesis. In this study, we analyzed the expression of Epstein-Barr virus (EBV) antigens as well as of Toll-like receptor 3 (TLR3), TLR9, and downstream adapter TIRdomain-containing adapter-inducing interferon-β (TRIF) in the brain tissues of 26 patients with RE and 16 control individuals using immunohistochemistry (IHC). In the RE group, EBV antigens were detected in 53% of individuals at various expression levels. In contrast, there was no detectable EBV antigen expression in control brain tissues. Moreover, we found marked increases in the expression of TLR3, TLR9, and TRIF in the brain tissues of RE patients compared with levels in the control group. Furthermore, among RE cases, EBV expression and high TLR3 expression were associated with more severe brain atrophy. Our results suggest that the elevated expression of EBV and TLRs may be involved in RE occurrence through the activation of downstream molecules.

Molecular diversity of IgG responses to Epstein-Barr virus proteins in asymptomatic Epstein-Barr virus carriers.

The Epstein-Barr virus (EBV) is a ubiquitous pathogen that infects over 90 % of adults. EBV is the primary etiological agent of infectious mononucleosis and is closely associated with nasopharyngeal carcinoma, gastric carcinoma, Hodgkin lymphoma and Burkitt lymphoma. Clinical serological assays for EBV diagnosis only survey a small portion of the viral proteome, which does not represent the total antigenic breadth presented to the immune system during viral infection. In this study, we have generated an expression library containing the majority of EBV ORFs, and have systematically evaluated IgG responses to those EBV proteins in sera from EBV carriers. In addition to confirming previously recognized dominant EBV antigens, this study has identified additional immunodominant antigens, and has revealed a more expansive antigenic profile of the humoral responses to EBV in asymptomatic carriers. This EBV expression library will be deposited in a public repository with the goal of disseminating this new research tool for the application of identifying potential new biomarkers for EBV-associated diseases.

The Expression of mRNA LMP1 Epstein-Barr Virus from FFPE Tumour Biopsy: a Potential Biomarker of Nasopharyngeal Carcinoma Diagnosis

Nasopharyngeal carcinoma (NPC) is a multifactorial disease that is endemic geographically in the world. Indonesian population has a highly incidence rate that is 6.2/100,000 people year. The pathogenesis of NPC is more directly reflected by carcinoma-specific viral transcriptional activity at the site of primary tumour. Epstein-Barr virus (EBV) infection in NPC is reflected by the expression of EBV latent and lytic gene. In fact, mRNA Latent Membrane Protein 1 (LMP1) EBV expression was an important latent infection biomarker. The aim of this study was to determine a potential use of relative expression of mRNA LMP1 EBV from formalin-fixed paraffin embedded (FFPE) tumour biopsy in NPC as a tumour biomarker. This reseach design was a cross sectional study. The samples were the archived specimens of FFPE tumour biopsy from NPC WHO-3 patient which were collected from untreated patients from 2014 in the Department of Pathology Anatomy, Prof. dr. Margono Soekarjo Hospital, Purwokerto. The expression of mRNA LMP1 EBV expression was determined by RT-PCR technique. The positivity of mRNA LMP1 EBV expression was 51.9%, indicating a moderate positivity. The result proved that the expression of mRNA LMP1 EBV from FFPE NPC WHO-3 tumour biopsy was a potential biomarker of NPC diagnosis. The molecular methods would improved the management of NPC, particularly in the histopathological diagnosis of NPC.