EBV-associated Gastric Carcinoma Research Articles

High Levels of Class I Major Histocompatibility Complex mRNA Are Present in Epstein-Barr Virus-Associated Gastric Adenocarcinomas.

Epstein–Barr virus (EBV) is responsible for approximately 9% of stomach adenocarcinomas. EBV-encoded microRNAs have been reported as reducing the function of the class I major histocompatibility complex (MHC-I) antigen presentation apparatus, which could allow infected cells to evade adaptive immune responses. Using data from nearly 400 human gastric carcinomas (GCs), we assessed the impact of EBV on MHC-I heavy and light chain mRNA levels, as well as multiple other components essential for antigen processing and presentation. Unexpectedly, mRNA levels of these genes were as high, or higher, in EBV-associated gastric carcinomas (EBVaGCs) compared to normal control tissues or other GC subtypes. This coordinated upregulation could have been a consequence of the higher intratumoral levels of interferon γ in EBVaGCs, which correlated with signatures of increased infiltration by T and natural killer (NK) cells. These results indicate that EBV-encoded products do not effectively reduce mRNA levels of the MHC-I antigen presentation apparatus in human GCs.

EBV-Associated Gastric Carcinoma Arising in the Remnant Stomach: A Case Report and Review of the Literatures

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Potential prognostic impact of EBV RNA-seq reads in gastric cancer: a reanalysis of The Cancer Genome Atlas cohort.

Epstein–Barr virus (EBV)‐associated gastric cancer (EBVaGC), whose prognosis remains controversial, is diagnosed by in situ hybridization of EBV‐derived EBER1/2 small RNAs. In The Cancer Genome Atlas (TCGA) Stomach Adenocarcinoma (STAD) project, the EBV molecular subtype was determined through a combination of multiple next‐generation sequencing methods, but not by the gold standard in situ hybridization method. This leaves unanswered questions regarding the discordance of EBV positivity detected by different approaches and the threshold of sequencing reads. Therefore, we reanalyzed the TCGA‐STAD RNA sequencing (RNA‐seq) dataset including 375 tumor and 32 normal samples, using our analysis pipeline. We defined a reliable threshold for EBV‐derived next‐generation sequencing reads by mapping them to the EBV genome with three different random arbitrary alignments. We analyzed the prognostic impact of EBV status on the histopathological subtypes of gastric cancer. EBV‐positive cases identified by reanalysis comprised nearly half of the cases (49.6%) independent from infiltrating lymphocyte signatures, and showed significantly longer overall survival for adenocarcinomas of the ‘not‐otherwise‐specified’ type [P = 0.016 (log‐rank test); hazard ratios (HR): 0.476; 95% CI: 0.260–0.870, P = 0.016 (Cox univariate analysis)], but shorter overall survival for the tubular adenocarcinoma type [P = 0.005 (log‐rank test); HR: 3.329; 95% CI: 1.406–7.885, P = 0.006 (Cox univariate analysis)]. These results demonstrate that the EBV positivity rates were higher when determined by RNA‐seq than when determined by EBER1/2 in situ hybridization. The RNA‐seq‐based EBV positivity demonstrated distinct results for gastric cancer prognosis depending on the histopathological subtype, suggesting its potential to be used in clinical prognoses.

Epstein-Barr Virus miR-BART17-5p Promotes Migration and Anchorage-Independent Growth by Targeting Kruppel-Like Factor 2 in Gastric Cancer.

Epstein-Barr virus (EBV) infects more than 90% of the global population and is associated with a variety of tumors including nasopharyngeal carcinoma, Hodgkin lymphoma, natural killer/T lymphoma, and gastric carcinoma. In EBV-associated gastric cancer (EBVaGC), highly expressed EBV BamHI A rightward transcripts (BART) miRNAs may contribute to tumorigenesis with limited viral antigens. Despite previous studies on the targets of BART miRNAs, the functions of all 44 BART miRNAs have not been fully clarified. Here, we used RNA sequencing data from the Cancer Genome Atlas to find genes with decreased expression in EBVaGC. Furthermore, we used AGS cells infected with EBV to determine whether expression was reduced by BART miRNA. We showed that the expression of Kruppel-like factor 2 (KLF2) is lower in AGS-EBV cells than in the AGS control. Using bioinformatics analysis, four BART miRNAs were selected to check whether they suppress KLF2 expression. We found that only miR-BART17-5p directly down-regulated KLF2 and promoted gastric carcinoma cell migration and anchorage-independent growth. Our data suggest that KLF2 functions as a tumor suppressor in EBVaGC and that miR-BART17-5p may be a valuable target for effective EBVaGC treatment.

A pilot study of avelumab in Epstein-Barr virus-associated gastric cancer.

TPS473 Background: Gastric Cancer (GC) is the third most common cause of cancer related deaths worldwide. The median overall survival of patients with stage 4 disease is approximately 1 year. Current accepted treatment approach with chemotherapy is applied with little consideration for known genetic or biologic heterogeneity. Whilst immune-based approaches in GC look promising it is clear that single-agent PD1/PDL1 inhibition benefit a minority. We must clarify a means of identifying prospectively those patients who may benefit from this treatment. A recent landmark paper by The Cancer Genome Atlas (TCGA) proposed a classification of GC into four subtypes: Epstein-Barr-virus (EBV)-positive, microsatellite instable (MSI), chromosomal instable (CI), and genomically stable (GS). Two of the four – EBV and MSI subtypes – are likely to be immunogenic and amenable to PD1/PDL1 inhibition. Recent advances have shown EBV-positive tumors to be infiltrated by lymphocytes and be enriched for PDL1. Methods: This single centre single-arm pilot study in gastric or junctional adenocarcinoma will explore the hypothesis that administering anti-PDL1 therapy (Avelumab) in a prospectively identified population enriched for potential responders will result in improved outcomes. The anticipated frequency of EBV associated-GC (c10%) means that approximately N = 100 patients will be screened to identify N = 10 participants. If a positive signal for efficacy is seen this will provide a basis for a larger, multicentre study. Previously treated Patients with confirmation of stage 4 EBV- positive gastric or oesophago-gastric adenocarcinoma meeting eligibility criteria will be enrolled. Avelumab will be administered at a dose of 10mg/kg IV every 14days. Primary endpoint is to determine the 6-month progression free survival (PFS) of Avelumab in EBV-associated GC. Secondary endpoints include overall response rate, overall survival, median PFS time and feasibility/accrual rate at 12 months. Exploratory endpoints will be to evaluate changes in immune parameters in the peripheral blood over time. Kaplan-Meier methods for primary efficacy endpoint with two-tailed one-sample proportion test will be used to evaluate the evidence to reject the null hypothesis. Clinical trial information: 2018-002085-39.

Aberrantly Methylated-Differentially Expressed Genes and Pathways in Epstein–Barr Virus-Associated Gastric Cancer

Aim: To identify the methylated-differentially expressed genes (MDEGs) that may serve as diagnostic markers and therapeutic targets in Epstein-Barr virus-associated gastric cancer (EBVaGC) and to explore the methylation-based pathways for elucidating biological mechanisms of EBVaGC. Materials & methods: Gene expression and methylation profiles were downloaded from GEO database. MDEGs were identified by GEO2R. Pathway enrichment analyses were conducted based on DAVID database. Hub genes were identified by Cytoscape, which were further verified by The Cancer Genome Atlas database. Results: A total of 367 hypermethylated, lowly expressed genes were enriched in specific patterns of cell differentiation. 31 hypomethylated, highly expressed genes demonstrated enrichment in regulation of immune system process. After validation using The Cancer Genome Atlas database, seven genes were confirmed to be significantly different hub genes in EBVaGC. Conclusion: EBVaGC-specific MDEGs and pathways can be served as potential biomarkers for precise diagnosis and treatment of EBVaGC and provide novel insights into the mechanisms involved.

Epstein-Barr virus-associated gastric carcinomas developed after successful eradication of Helicobacter pylori.

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) can develop in gastric mucosa affected by Helicobacter pylori (H. pylori)-induced atrophic gastritis. However, it is unclear whether EBVaGC can develop in gastric mucosa after successful eradication of H. pylori. We report 3 cases of EBVaGC discovered more than 5years after successful eradication of H. pylori. All 3 cases are men with a history of smoking. The periods from successful eradication to development of EBVaGC were 18, 8, and 9years, respectively. Their tumors were mainly depressed lesions located in the gastric corpus. Two patients had intramucosal cancer and the other had advanced gastric cancer. Mucosal atrophy was present in the background gastric mucosa of all of three, and the anti-H. pylori antibody titer in each patient had fallen to < 3U/mL. These cases indicate that EBVaGC can occur after successful eradication of H. pylori.

Molecular Classification of Gastric Cancer among Alaska Native People.

Gastric cancer is an aggressive and heterogeneous malignancy that often varies in presentation and disease among racial and ethnic groups. The Alaska Native (AN) people have the highest incidence and mortality rates of gastric cancer in North America. This study examines molecular markers in solid tumor samples from eighty-five AN gastric adenocarcinoma patients using next-generation sequencing, immunohistochemistry, and in situ hybridization analysis. AN patients have a low mutation burden with fewer somatic gene mutations in their tumors compared to other populations, with the most common mutation being TP53. Epstein-Barr virus (EBV) was associated with 20% of AN gastric cancers, which is higher than the world average of 10%. The inflammation marker, cyclooxygenase-2 (COX-2), is highly expressed in patients with the lowest survival rates. Mismatch repair deficiency was present in 10% of AN patients and was associated with patients who were female, 50 years or older, gene mutations, and tumors in the distal stomach. Program death-ligand 1 (PD-L1) was expressed in 14% of AN patients who were more likely to have MMR deficiency, EBV-associated gastric cancers, and mutations in the PIK3CA gene, all of which have been linked to clinical response to PD-1 inhibitors. These studies suggest a portion of AN gastric cancer patients could be candidates for immunotherapy. Overall, this study highlights future avenues of investigation for clinical and translational studies, so that we can improve early detection and develop more effective treatments for AN patients.

Observational cohort study of clinical outcome in Epstein–Barr virus associated gastric cancer patients

Background:Epstein–Barr virus-associated gastric cancer (EBVaGC) has unique clinicopathologic features and our present understanding of its treatment outcome is limited. Here, we investigated the clinical outcomes of resectable and metastatic EBVaGC cases with regards to their respective treatment.Methods:We retrieved the data of EBVaGC patients treated at our center from October 2014 to June 2019. The primary endpoint was overall survival (OS). Secondary endpoints were disease-free survival (DFS) for stage I–III patients, progression-free survival (PFS) and objective response rate (ORR) for stage IV patients.Results:Patients classified as stage I–III accounted for 83.7% of the total 197 cases analyzed. Two patients had mismatched repair-deficiency. The 5-year OS rate of the entire cohort was 63.51% [95% (confidence interval (CI): 52.31–72.76%]. Tumor-node-metastasis (TNM) stage and gastric stump cancer were identified as independent prognostic factors for OS. The 3- and 5-year DFS rate for stage I–III patients were 83.72% (95% CI: 75.86–89.19%) and 73.83% (95% CI: 60.39–83.32%), respectively. TNM stage III, neural invasion, lymphovascular invasion, and baseline plasma EBV-DNA positive were correlated with shorter DFS. The ORR and disease control rate (DCR) for metastatic EBVaGC patients to first-line therapy were 29.0% and 90.3% (median PFS: 9.8 months), respectively, and to second-line therapy were 25.0% and 75.0%, respectively. Seven patients received anti-PD1 therapy and had an ORR of 28.5% and a median PFS of 2.8 months.Conclusions:EBVaGC patients have few metastases, long DFS, and high DCR. TNM stage and gastric stump cancer were independent prognostic factors for OS.

Multi-layered control of PD-L1 expression in Epstein-Barr virus-associated gastric cancer.

Gastric cancer (GC) is the fifth most common cancer worldwide. In approximately 10% of GC cases, cancer cells show ubiquitous and monoclonal Epstein-Barr virus (EBV) infection. A significant feature of EBV-associated GC (EBVaGC) is high lymphocytic infiltration and high expression of immune checkpoint proteins, including programmed death-ligand 1 (PD-L1). This highlights EBVaGC as a strong candidate for immune checkpoint blockade therapy. Indeed, several recent studies have shown that EBV positivity in GC correlates with positive response to programmed cell death protein 1 (PD-1)/PD-L1 blockade therapy. Understanding the mechanisms that control PD-L1 expression in EBVaGC can indicate new predictive biomarkers for immunotherapy, as well as therapeutic targets for combination therapy. Various mechanisms have been implicated in PD-L1 expression regulation, including structural variations, post-transcriptional control, oncogenic activation of intrinsic signaling pathways, and increased sensitivity to extrinsic signals. This review provides the most recent updates on the multilayered control of PD-L1 expression in EBVaGC.

CXCR4 induces cell autophagy and maintains EBV latent infection in EBVaGC.

Rationale: Epstein-Barr virus (EBV) is found in ~7% of gastric carcinoma cases worldwide, and all tumour cells harbour the clonal EBV genome. EBV can regulate pathways and protein expression to induce gastric carcinoma; however, the molecular mechanism underlying EBV-associated gastric carcinoma (EBVaGC) remains elusive.Methods: GEO microarray and molecular experiments were performed to compare CXCR4 expression between EBV-positive and EBV-negative gastric carcinoma (EBVnGC). Transfections with LMP2A plasmid or siRNA were carried out to assess the role of LMP2A in CXCR4 expression. The effects and mechanisms of CXCR4 on cell autophagy were analysed in vitro using molecular biological and cellular approaches. Additionally, we also determined the regulatory role of CXCR4 in latent EBV infection.Results: CXCR4 expression was significantly upregulated in EBVaGC tissues and cell lines. LMP2A could induce AKT phosphorylation to increase NRF1 expression, thereby binding to the CXCR4 promoter to increase its transcriptional level. Moreover, CXCR4 promoted ZEB1 expression to upregulate ATG7 synthesis, which could then activate autophagy. Moreover, CXCR4 increased the number of cells entering the G2/M phase and inhibited cell apoptosis via the autophagy pathway. Finally, CXCR4 knockdown was associated with elevated BZLF1 expression, but this effect was not influenced by autophagy.Conclusions: Our data suggested new roles for CXCR4 in autophagy and EBV replication in EBVaGC, which further promoted cell survival and persistent latent infection. These new findings can lead to further CXCR4-based anticancer therapy.

A novel vaccine candidate based on chimeric virus-like particle displaying multiple conserved epitope peptides induced neutralizing antibodies against EBV infection.

Rationale: Epstein-Barr virus (EBV) is the causative pathogen for infectious mononucleosis and many kinds of malignancies including several lymphomas such as Hodgkin's lymphoma, Burkitt's lymphoma and NK/T cell lymphoma as well as carcinomas such as nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBV-GC). However, to date no available prophylactic vaccine was launched to the market for clinical use.Methods: To develop a novel vaccine candidate to prevent EBV infection and diseases, we designed chimeric virus-like particles (VLPs) based on the hepatitis B core antigen (HBc149). Various VLPs were engineered to present combinations of three peptides derived from the receptor binding domain of EBV gp350. All the chimeric virus-like particles were injected into Balb/C mice for immunogenicity evaluation. Neutralizing titer of mice sera were detected using an in vitro cell model.Results: All chimeric HBc149 proteins self-assembled into VLPs with gp350 epitopes displayed on the surface of spherical particles. Interestingly, the different orders of the three epitopes in the chimeric proteins induced different immune responses in mice. Two constructs (149-3A and 149-3B) induced high serum titer against the receptor-binding domain of gp350. Most importantly, these two VLPs elicited neutralizing antibodies in immunized mice, which efficiently blocked EBV infection in cell culture. Competition analysis showed that sera from these mice contained antibodies to a major neutralizing epitope recognized by the strong neutralizing mAb 72A1.Conclusion: Our data demonstrate that HBc149 chimeric VLPs provide a valuable platform to present EBV gp350 antigens and offer a robust basis for the development of peptide-based candidate vaccines against EBV.

Epstein-Barr virus miR-BART1-3p suppresses apoptosis and promotes migration of gastric carcinoma cells by targeting DAB2.

Although Epstein-Barr virus (EBV) is known to encode over 40 different miRNAs of its own, the roles of most EBV miRNAs remain unknown. Disabled homolog 2 (DAB2) is a putative tumor suppressor, but its role in gastric carcinoma (GC), especially in EBV-associated GC, needs to be clarified. Our qRT-PCR and mRNA microarray results showed that DAB2 expression was down-regulated in EBV-positive GC cells compared to EBV-negative cells. Four BART miRNAs that might target DAB2 were predicted, and we found, using a luciferase reporter assay, that miR-BART1-3p directly targeted the 3'-UTR of DAB2. The miR-BART1-3p transfection decreased DAB2 expression at both mRNA and protein levels, while transfection of an inhibitor of miR-BART1-3p, miR-BART1-3p(i), increased DAB2 expression. In addition, miR-BART1-3p as well as siDAB2 increased migration and decreased apoptosis. Meanwhile, miR-BART1-3p(i) or pcDNA3.1-DAB2 transfection decreased migration and increased apoptosis in EBV-infected GC cells. Furthermore, decreased migration by miR-BART1-3p(i) was abrogated by co-transfected siDAB2, while decreased migration by miR-BART1-3p(i) was further suppressed by a co-transfected DAB2 over-expression vector. Our data suggest that miR-BART1-3p plays an important role in the tumorigenesis of EBV-associated GC by directly targeting DAB2.

Thirty years of Epstein-Barr virus-associated gastric carcinoma.

Thirty years have passed since a possible association of Epstein-Barr virus (EBV) with gastric carcinoma was reported. We now know EBV-associated gastric carcinoma to be a specific subtype of gastric carcinoma. Global epigenetic methylation and counteraction of the antitumour microenvironment are two major characteristics of this subtype of gastric carcinoma. Recent development of therapeutic modalities for gastric carcinoma, such as endoscopic mucosal dissection and immune checkpoint inhibitor therapy, has made the presence of EBV infection a biomarker for the treatment of gastric carcinoma. This review presents a portrait of EBV-associated gastric carcinoma from initiation to maturity that we define as the 'gastritis-infection-cancer sequence', followed by its molecular abnormalities and interactions with immune checkpoint molecules and the microenvironment. EBV non-coding RNAs (microRNA and circular RNA) and exosomes derived from EBV-infected cells that were previously behind the scenes are now recognized for their roles in EBV-associated gastric carcinoma. The virus utilizes cellular machinery skilfully to control infected cells and their microenvironment. We should thus strive to understand virus-host interactions more fully in the following years to overcome this virus-driven subtype of gastric carcinoma.

MiR-BART1-5p targets core 2β-1,6-acetylglucosaminyltransferase GCNT3 to inhibit cell proliferation and migration in EBV-associated gastric cancer

GCNT3 (core 2β-1,6-acetylglucosaminyltransferase) is a novel core mucin synthase. It is known that abnormal expression of GCNT3 promotes the progression of several human cancers. However, its relationship with Epstein-Barr virus (EBV) has not been comprehensively studied. We found GCNT3 expression in EBV-associated gastric cancer cells and tissues to be lower than in EBV-negative gastric cancer cells and tissues, and high expression was significantly associated with advanced tumor-lymph node metastasis. Luciferase reporter assay revealed that miR-BART1-5p directly targeted GCNT3. In addition, miR-BART1-5p mimics transfection was observed to reduce cell proliferation and migration, while miR-BART1-5p inhibitor increased cell proliferation and migration following transfection. In conclusion, both miR-BART1-5p and knockdown of GCNT3 inhibited cell proliferation and migration. In addition, EBV may regulate GCNT3 by affecting the NF-kB signaling pathway. E-cadherin, N-cadherin, vimentin, and p-ERK were found to be downstream molecules of the miR-BART1-5p/GCNT3 pathway.

Prevalence and characteristics of Epstein-Barr virus associated gastric carcinoma in Gansu Province, Northwest China with mRNA expression of glycoprotein BMRF2.

Gansu province is a region with the highest gastric cancer incidence and mortality in Northwest China. Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is an important subtype of gastric cancer which shows specific clinicopathological features such as older-age bias, male predominance, lower lymph-node-metastasis, and a better cancer-related survival comparing to EBV-negative gastric cancers. However, the prevalence of EBVaGC has never been studied in Gansu Province, Northwest China. The present study investigated the incidence, characteristics, and EBV messenger RNA (mRNA) profile of EBVaGC in this area. We have collected 270 stomach samples from gastric cancer patients and analyzed the presence of EBV DNA and EBV-encoded small RNAs (EBERs) by nested polymerase chain reaction (PCR) and in situ hybridization, respectively. The EBV mRNA profiling was investigated by quantitative reverse transcription PCR (qRT-PCR). EBV DNA was detected in 51/95 patients (53.7%), while EBER transcripts were detected in 18/270 patients (6.7%). EBER positivity was significantly associated with older age and less lymph node metastasis, but no obvious association with gender or histological type of tumors. The expression of EBV genes was observed with different patterns, and the mRNA of glycoprotein BMRF2 was detected in EBVaGC. The present study showed unique clinicopathological features and mRNA expression patterns of EBVaGC in Gansu Province, Northwest China, suggesting that geographic variation can contribute to new epidemiological features in EBVaGC. The transcript of glycoprotein BMRF2 was observed consistently in EBVaGC, which may serve as a biomarker and play a role in the pathogenesis of EBVaGC in Gansu Province, Northwest China.

Establishment of a Screening Method for Epstein-Barr Virus-Associated Gastric Carcinoma by Droplet Digital PCR.

Background: Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is classified as one of the molecular subtypes of gastric cancer. We used droplet digital polymerase chain reaction (ddPCR) to enable highly sensitive and quantitative detection of EBV. Methods: EBV-DNA load was calculated based on the copy number of the BamH1-W fragment of EBV by ddPCR, and the cut-off value of EBV-DNA load was set. We conducted both ddPCR and EBER1 ISH to examine whether their results coincided in 158 gastric cancer specimens of unknown EBV status. We prepared 26 biopsy specimens and 49 serum samples including EBVaGC and assayed them by ddPCR. Results: The median values of EBV-DNA load for EBVaGC and EBV-negative control were 17.0 and 0.00308, respectively. A cut-off value of 0.032 was determined for which the sensitivity was 1. Among the 158 gastric cancer specimens, 14 lesions were judged as EBV-positive by the 0.032 cut-off value determined by ddPCR. The results of ddPCR and EBER1 ISH were in complete agreement. Even when using a biopsy specimen as a sample for ddPCR, the EBV-DNA load of all EBVaGCs was larger than the cut-off value. Conclusions: We established a new method of diagnosing EBVaGC from tissue samples by ddPCR.

Dysregulation of FOXO transcription factors in Epstein-Barr virus-associated gastric carcinoma

Epstein-Barr virus (EBV) infection is associated with the development of gastric cancer (GC). Forkhead box class O (FOXO) transcription factors play important roles in tumor suppression. This study aims to investigate the interplay between EBV and FOXOs in EBV-associated GC (EBVaGC). The results showed that EBV infection of GC cells led to the downregulation of FOXO1 by the inhibition of its mRNA and protein expression. FOXO3 protein is repressed by EBV infection. FOXO4 mRNA is upregulated in EBV-positive cell lines, while its protein expression is downregulated. FOXO1, FOXO3 and FOXO4 proteins are upregulated following PI3K inhibition in GT39 cells, confirming that they are partially suppressed by the PI3K/AKT pathway. However, the upregulation of FOXO1 and FOXO3 by single transfection with LMP1 or LMP2A implies that the dysregulation of FOXOs in EBVaGC is affected by various EBV latent genes and that PI3K/AKT signaling is not the only mechanism of FOXO regulation.

Quercetin Synergistically Inhibit EBV-Associated Gastric Carcinoma with Ganoderma lucidum Extracts.

Mycotherapy has been shown to improve the overall response rate during cancer treatment and reduce some chemotherapy-related adverse events. Ganoderma lucidum is a traditional mushroom used for pharmaceutical purposes. G. lucidum extracts (GLE) showed potential antitumor activities against several cancers. These tumor inhibitory effects of GLE were attributed to the suppression of the proliferation and metastasis of cancer cells. Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is defined as the monoclonal proliferation of carcinoma cells with latent EBV infection. The inhibitory effects of GLE against EBVaGC are questionable. The aim of this study was to investigate GLE as potential antitumor agents and a counterpart of quercetin (QCT) for the cotreatment in suppressing EBVaGC development. Therefore, this study conducted antitumor assays using a EBVaGC xenograft mice model and found that GLE could suppress tumor development. These inhibitory effects were significantly augmented by the low concentration of the quercetin (QCT) cotreatment in the xenograft mice. The addition of GLE in low concentrations synergistically reinforced QCT-induced apoptosis and EBV lytic reactivation. GLE contains various polysaccharides and triterpenes, such as ganoderic acid. Interestingly, the addition of ganoderic acid A (GAA) could produce similar bioactive effects like GLE in QCT-mediated antitumor activity. The GAA addition in low concentrations synergistically reinforced QCT-induced apoptosis and EBV lytic reactivation. GAA was sufficiently effective as much as GLE. Therefore, our results suggested that QCT-supplemented GLE could be a potential food adjunct for the prevention of EBVaGC development.

A single nucleotide polymorphism in the BART promoter region of Epstein-Barr virus isolated from nasopharyngeal cancer cells

Epstein-Barr virus (EBV) encodes BamHIA rightward transcript (BART) microRNAs (miRNAs). These miRNAs are expressed at high levels in epithelial tumors, such as nasopharyngeal carcinoma (NPC). BART miRNAs play important roles in EBV-associated malignancies, however, the reason for their high expression in NPC is unclear. We performed multiple sequence alignment of six completely sequenced EBV strains: Akata, YCCEL1, SNU719, C666-1, Mutu I, and M81. A single-nucleotide deletion was identified at the promoter region of BART. The luciferase assay suggested that this single-nucleotide polymorphism (SNP) significantly increased BART promoter activity. In addition to deletion, substitution at the same site also increased BART promoter activity. Analysis of the 170 EBV genome sequences from NPC and EBV-associated gastric cancers revealed that the frequency of this SNP was associated with NPC incidence and this SNP was found to be accumulated in the BART promoter region. Overall, our results suggested that this SNP should enhance BART promoter activity and thus, might contribute to the development of EBV-associated epithelial malignancies.