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Background: Gastric cancer is the fourth leading cause of cancer-related deaths in the world. The identification of gastric cancer subtypes related to recognizable microbial agents may play a pivotal role in the targeted prevention and treatment of this cancer. The current study is conducted to define the frequency of Epstein-Barr virus (EBV) infection in gastric cancers of four major provinces, with different incidence rates of gastric cancers, in Iran., Methods: Paraffin blocks of 682 cases of various types of gastric cancer from Tehran, South and North areas of Iran were collected. Twelve tissue microarray (TMA) blocks were constructed from these blocks. Localization of EBV in tumors was assessed by in situ hybridization (ISH) for EBV-encoded RNA (EBER). Chi-squared test was used to evaluate the statistical significance between EBV-associated gastric cancer (EBVaGC) and clinicopathologic tumor characteristics., Results: Fourteen out of 682 cases (2.1%) of gastric adenocarcinoma were EBER-positive. EBER was positive in 8 out of 22 (36.4%) of medullary carcinomas and 6 out of 660 (0.9%) of non-medullary type, which was a statistically significant difference ( P <0.001). The EBVaGCs were more frequent in younger age ( P =0.009) and also showed a trend toward the lower stage of the tumor ( P =0.075)., Conclusion: EBV-associated gastric adenocarcinoma has a low prevalence in Iran. This finding can be due to epidemiologic differences in risk factors and exposures, and the low number of gastric medullary carcinomas in the population. It may also be related to gastric tumor heterogeneity not detected with the TMA technique., (© 2024 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.)

Background: Epstein-Barr virus (EBV) positivity is associated with better gastric cancer prognosis and is found in a relatively fixed 9% of tumors worldwide., Aim: We aimed to examine the EBV status of gastric adenocarcinomas in a very high-incidence population and to compare prevalence between cardia and non-cardia anatomic subsites., Methods: We evaluated 1035 adult gastric adenocarcinoma cases presenting during 1997-2005 to the Shanxi Cancer Hospital in Taiyuan, Shanxi Province, China. EBV-encoded RNA was detected in alcohol-fixed paraffin-embedded tumor specimens by in situ hybridization. Associations were assessed in case-case comparisons using the Chi-squared test for categorical variables and the Mann-Whitney U test for continuous variables, with p values < 0.05 considered statistically significant. Adjusted odds ratios were calculated using logistic regression, and mortality hazard ratios (HRs) were estimated by Cox proportional hazards regression., Results: Sixty-four percent of the evaluated cancers were found in the cardia. Cardia tumor localization was associated with male sex, advanced primary tumor stage, better differentiated histology, and intestinal-type Lauren classification. Four percent of the non-cardia and only 0.9% of cardia cancers were EBV-positive. EBV positivity was associated with better overall survival (adjusted HR 0.30, 95% CI 0.14-0.63)., Conclusions: Our study highlights unusually low EBV prevalence in gastric adenocarcinoma among a high-incidence population, particularly for cardia cancers. These findings suggest a unique risk factor profile for the high incidence of gastric cancer in this population.

Background: Epstein-Barr virus (EBV)-positive gastric cancers have clinicopathologic differences from EBV-negative tumors and lack TP53 mutation. Serologic profiles may inform viral contribution to carcinogenesis., Methods: We compared humoral responses of EBV-positive ( n = 67) and EBV-negative ( n = 137) patients with gastric cancer from the International EBV-Gastric Cancer Consortium. Serum antibodies against four EBV proteins, nuclear (EBNA), viral capsid (VCA), early-diffuse (EA-D), and Zta replication activator (ZEBRA), and to p53 were assessed by multiplex assays. OR of antibody level tertiles (T1-T3) were adjusted by logistic regression. We also conducted a meta-analysis of reported anti-p53 seropositivity in gastric cancer., Results: Consistent with EBV's ubiquity, 99% of patients were seropositive for anti-EBNA and 98% for anti-VCA, without difference by tumor EBV status. Seropositivity varied between patients with EBV-positive and EBV-negative tumors for anti-EA-D (97% vs. 67%, respectively, P < 0.001) and anti-ZEBRA (97% vs. 85%, respectively, P = 0.009). Adjusted ORs (vs. T1) for patients with EBV-positive versus EBV-negative tumors were significantly elevated for higher antibodies against EBNA (2.6 for T2 and 13 for T3), VCA (1.8 for T2 and 2.4 for T3), EA-D (6.0 for T2 and 44 for T3), and ZEBRA (4.6 for T2 and 12 for T3). Antibodies to p53 were inversely associated with EBV positivity (3% vs. 15%; adjusted OR = 0.16, P = 0.021). Anti-p53 prevalence from the literature was 15%., Conclusions: These serologic patterns suggest viral reactivation in EBV-positive cancers and identify variation of p53 seropositivity by subtype., Impact: Anti-EBV and anti-p53 antibodies are differentially associated with tumor EBV positivity. Serology may identify EBV-positive gastric cancer for targeted therapies., (©2019 American Association for Cancer Research.)

Objective: Epstein-Barr virus (EBV)-associated gastric cancer has been proposed to be a distinct gastric cancer molecular subtype. The prognostic significance of EBV infection in gastric cancer remains unclear and needs further investigation. Our study aimed to analyze EBV-positive and EBV-negative gastric cancer patients regarding their personal and tumor-related characteristics, and compare their overall survival., Methods: Gastric cancer patients consecutively treated at the Riga East University Hospital during 2009-2016 were identified retrospectively. Tumor EBV status was determined by in-situ hybridization for EBV-encoded RNA (EBER). Information about clinicopathological characteristics was obtained from patient questionnaires, hospital records. Overall survival was ascertained through 30 July 2017. Cox proportional hazard regression models adjusted for personal and tumor-related covariates compared survival between EBV-positive and EBV-negative patients., Results: There were a total of 302 gastric cancer patients (61% males) with mean and SD age 63.6 ± 11.5 years. EBER positivity was present in 8.6% of tumors. EBV-positive gastric cancer patients had better survival at 80 months [adjusted hazard ratio = 0.37, 95% confidence interval (CI) = 0.19-0.72] compared to EBV-negative patients. Worse survival was observed for patients with stage III (hazard ratio = 2.76, 95% CI = 1.67-4.56) and stage IV (hazard ratio = 10.02, 95% CI = 5.72-17.57) compared to stage I gastric cancer, and overlapping and unspecified subsite (hazard ratio = 1.85; 95% CI = 1.14; 3.00) compared to distal tumors., Conclusion: Tumor EBV positivity is a favorable prognostic factor in gastric cancer.

Background: Geospatial technology has facilitated the discovery of disease distributions and etiology and helped target prevention programs. Globally, gastric cancer is the leading infection-associated cancer, and third leading cause of cancer mortality worldwide, with marked geographic variation. Central and South America have a significant burden, particularly in the mountainous regions. In the context of an ongoing population-based case-control study in Central America, our aim was to examine the spatial epidemiology of gastric cancer subtypes and H. pylori virulence factors., Methods: Patients diagnosed with gastric cancer from 2002 to 2013 in western Honduras were identified in the prospective gastric cancer registry at the principal district hospital. Diagnosis was based on endoscopy and confirmatory histopathology. Geospatial methods were applied using the ArcGIS v10.3.1 and SaTScan v9.4.2 platforms to examine regional distributions of the gastric cancer histologic subtypes (Lauren classification), and the H. pylori CagA virulence factor. Getis-Ord-Gi hot spot and Discrete Poisson SaTScan statistics, respectively, were used to explore spatial clustering at the village level (30-50 rural households), with standardization by each village's population. H. pylori and CagA serologic status was determined using the novel H. pylori multiplex assay (DKFZ, Germany)., Results: Three hundred seventy-eight incident cases met the inclusion criteria (mean age 63.7, male 66.3%). Areas of higher gastric cancer incidence were identified. Significant spatial clustering of diffuse histology adenocarcinoma was revealed both by the Getis-Ord-GI* hot spot analysis (P-value < 0.0015; range 0.00003-0.0014; 99%CI), and by the SaTScan statistic (P-value < 0.006; range 0.0026-0.0054). The intestinal subtype was randomly distributed. H. pylori CagA had significant spatial clustering only in association with the diffuse histology cancer hot spot (Getis-Ord-Gi* P value ≤0.001; range 0.0001-0.0010; SaTScan statistic P value 0.0085). In the diffuse gastric cancer hot spot, the lowest age quartile range was 21-46 years, significantly lower than the intestinal cancers (P = 0.024)., Conclusions: Geospatial methods have identified a significant cluster of incident diffuse type adenocarcinoma cases in rural Central America, suggest of a germline genetic association. Further genomic and geospatial analyses to identify potential spatial patterns of genetic, bacterial, and environmental risk factors may be informative.

Context ERBB2 (erb-b2 receptor tyrosine kinase 2 or HER2) is currently the only biomarker established for selection of a specific therapy for patients with advanced gastroesophageal adenocarcinoma (GEA). However, there are no comprehensive guidelines for the assessment of HER2 in patients with GEA. Objectives To establish an evidence-based guideline for HER2 testing in patients with GEA, formalize the algorithms for methods to improve the accuracy of HER2 testing while addressing which patients and tumor specimens are appropriate, and to provide guidance on clinical decision making. Design The College of American Pathologists (CAP), American Society for Clinical Pathology (ASCP), and the American Society of Clinical Oncology (ASCO) convened an Expert Panel to conduct a systematic review of the literature to develop an evidence-based guideline with recommendations for optimal HER2 testing in patients with GEA. Results The Panel is proposing 11 recommendations with strong agreement from the open comment participants. Recommendations The Panel recommends that tumor specimen(s) from all patients with advanced GEA, who are candidates for HER2-targeted therapy, should be assessed for HER2 status before the initiation of HER2-targeted therapy. Clinicians should offer combination chemotherapy and an HER2-targeted agent as initial therapy for all patients with HER2-positive advanced GEA. For pathologists, guidance is provided for morphologic selection of neoplastic tissue, testing algorithms, scoring methods, interpretation and reporting of results, and laboratory quality assurance. Conclusion This guideline provides specific recommendations for assessment of HER2 in patients with advanced GEA while addressing pertinent technical issues and clinical implications of the results.

Context: ERBB2 (erb-b2 receptor tyrosine kinase 2 or HER2) is currently the only biomarker established for selection of a specific therapy for patients with advanced gastroesophageal adenocarcinoma (GEA). However, there are no comprehensive guidelines for the assessment of HER2 in patients with GEA., Objectives: To establish an evidence-based guideline for HER2 testing in patients with GEA, to formalize the algorithms for methods to improve the accuracy of HER2 testing while addressing which patients and tumor specimens are appropriate, and to provide guidance on clinical decision making., Design: The College of American Pathologists, American Society for Clinical Pathology, and American Society of Clinical Oncology convened an expert panel to conduct a systematic review of the literature to develop an evidence-based guideline with recommendations for optimal HER2 testing in patients with GEA., Results: The panel is proposing 11 recommendations with strong agreement from the open-comment participants., Recommendations: The panel recommends that tumor specimen(s) from all patients with advanced GEA, who are candidates for HER2-targeted therapy, should be assessed for HER2 status before the initiation of HER2-targeted therapy. Clinicians should offer combination chemotherapy and a HER2-targeted agent as initial therapy for all patients with HER2-positive advanced GEA. For pathologists, guidance is provided for morphologic selection of neoplastic tissue, testing algorithms, scoring methods, interpretation and reporting of results, and laboratory quality assurance., Conclusions: This guideline provides specific recommendations for assessment of HER2 in patients with advanced GEA while addressing pertinent technical issues and clinical implications of the results., (© 2016 College of American Pathologists, American Society for Clinical Pathology, and the American Society of Clinical Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

MicroRNA expression in formalin-fixed paraffin-embedded tissue (FFPE) or plasma may add value for cancer management. The GastroGenus miR Panel was developed to measure 55 cancer-specific human microRNAs, Epstein-Barr virus (EBV)-encoded microRNAs, and controls. This Q-rtPCR panel was applied to 100 FFPEs enriched for adenocarcinoma or adjacent non-malignant mucosa, and to plasma of 31 patients. In FFPE, microRNAs upregulated in malignant versus adjacent benign gastric mucosa were hsa-miR-21, -155, -196a, -196b, -185, and -let-7i. Hsa-miR-18a, 34a, 187, -200a, -423-3p, -484, and -744 were downregulated. Plasma of cancer versus non-cancer controls had upregulated hsa-miR-23a, -103, and -221 and downregulated hsa-miR-378, -346, -486-5p, -200b, -196a, -141, and -484. EBV-infected versus uninfected cancers expressed multiple EBV-encoded microRNAs, and concomitant dysregulation of four human microRNAs suggests that viral infection may alter cellular biochemical pathways. Human microRNAs were dysregulated between malignant and benign gastric mucosa and between plasma of cancer patients and non-cancer controls. Strong association of EBV microRNA expression with known EBV status underscores the ability of microRNA technology to reflect disease biology. Expression of viral microRNAs in concert with unique human microRNAs provides novel insights into viral oncogenesis and reinforces the potential for microRNA profiles to aid in classifying gastric cancer subtypes. Pilot studies of plasma suggest the potential for a noninvasive addition to cancer diagnostics.

The Epstein-Barr virus (EBV)-positive subtype of gastric adenocarcinoma is conventionally identified by in situ hybridization (ISH) for viral nucleic acids, but next-generation sequencing represents a potential alternative. We therefore determined normalized EBV read counts by whole-genome, whole-exome, mRNA and miRNA sequencing for 295 fresh-frozen gastric tumor samples. Formalin-fixed, paraffin-embedded tissue sections were retrieved for ISH confirmation of 13 high-EBV and 11 low-EBV cases. In pairwise comparisons, individual samples were either concordantly high or concordantly low by all genomic methods for which data were available. Empiric cutoffs of sequencing counts identified 26 (9 %) tumors as EBV positive. EBV positivity or negativity by molecular testing was confirmed by EBER-ISH in all but one tumor evaluated by both approaches (kappa = 0.91). EBV-positive gastric tumors can be accurately identified by quantifying viral sequences in genomic data. Simultaneous analyses of human and viral DNA, mRNA and miRNA could streamline tumor profiling for clinical care and research.

Current histopathologic classification schemes for gastric adenocarcinoma have limited clinical utility and are difficult to apply due to tumor heterogeneity. Elucidation of molecular subtypes of gastric cancer may contribute to our understanding of gastric cancer biology and to the development of new molecular markers that may lead to improved diagnosis, therapy, or prognosis. We previously demonstrated that Epstein-Barr virus (EBV)-infected gastric cancers have a distinct human gene expression profile compared with uninfected cancers. We now examine the histopathologic features characterizing infected (n=14) and uninfected (n=89) cancers; the latter of which are now further divided into 2 major molecular subtypes based on expression patterns of 93 RNAs. One uninfected gastric cancer subtype was distinguished by upregulation of 3 genes with neuroendocrine (NE) function (CHGA, GAST, and REG4 encoding chromogranin, gastrin, and the secreted peptide REG4 involved in epithelial cell regeneration), implicating hormonal factors in the pathogenesis of a major class of gastric adenocarcinomas. Evidence of NE differentiation (molecular, immunohistochemical, or morphologic) was mutually exclusive of EBV infection. EBV-infected tumors tended to have solid-type morphology with lymphoid stroma. This study reveals novel molecular subtypes of gastric cancer and their associated morphologies that demonstrate divergent NE features.

A small set of gastric adenocarcinomas (9%) harbor Epstein-Barr virus (EBV) DNA within malignant cells, and the virus is not an innocent bystander but rather is intimately linked to pathogenesis and tumor maintenance. Evidence comes from unique genomic features of host DNA, mRNA, microRNA and CpG methylation profiles as revealed by recent comprehensive genomic analysis by The Cancer Genome Atlas Network. Their data show that gastric cancer is not one disease but rather comprises four major classes: EBV-positive, microsatellite instability (MSI), genomically stable and chromosome instability. The EBV-positive class has even more marked CpG methylation than does the MSI class, and viral cancers have a unique pattern of methylation linked to the downregulation of CDKN2A (p16) but not MLH1. EBV-positive cancers often have mutated PIK3CA and ARID1A and an amplified 9p24.1 locus linked to overexpression of JAK2, CD274 (PD-L1) and PDCD1LG2 (PD-L2). Multiple noncoding viral RNAs are highly expressed. Patients who fail standard therapy may qualify for enrollment in clinical trials targeting cancer-related human gene pathways or promoting destruction of infected cells through lytic induction of EBV genes. Genomic tests such as the GastroGenus Gastric Cancer Classifier are available to identify actionable variants in formalin-fixed cancer tissue of affected patients.

Helicobacter pylori is the primary cause of gastric cancer. However, monoclonal Epstein-Barr virus (EBV) nucleic acid is also present in up to 10% of these tumors worldwide. EBV prevalence is increased with male sex, nonantral localization and surgically disrupted anatomy. To further examine associations between EBV and gastric cancer, we organized an international consortium of 11 studies with tumor EBV status assessed by in situ hybridization. We pooled individual-level data on 2,648 gastric cancer patients, including 184 (7%) with EBV-positive cancers; all studies had information on cigarette use (64% smokers) and nine had data on alcohol (57% drinkers). We compared patients with EBV-positive and EBV-negative tumors to evaluate smoking and alcohol interactions with EBV status. To account for within-population clustering, multilevel logistic regression models were used to estimate interaction odds ratios (OR) adjusted for distributions of sex (72% male), age (mean 59 years), tumor histology (56% Lauren intestinal-type), anatomic subsite (61% noncardia) and year of diagnosis (1983-2012). In unadjusted analyses, the OR of EBV positivity with smoking was 2.2 [95% confidence interval (CI) 1.6-3.2]. The OR was attenuated to 1.5 (95% CI 1.01-2.3) by adjustment for the possible confounders. There was no significant interaction of EBV status with alcohol drinking (crude OR 1.4; adjusted OR 1.0). Our data indicate the smoking association with gastric cancer is stronger for EBV-positive than EBV-negative tumors. Conversely, the null association with alcohol does not vary by EBV status. Distinct epidemiologic characteristics of EBV-positive cancer further implicate the virus as a cofactor in gastric carcinogenesis., (© 2013 UICC.)

Background and Objective: About 9% of gastric carcinomas have Epstein-Barr virus (EBV) in the tumour cells, but it is unclear whether viral presence influences clinical progression. We therefore examined a large multicentre case series for the association of tumour EBV status with survival after gastric cancer diagnosis, accounting for surgical stage and other prognostic factors., Methods: We combined individual-level data on 4599 gastric cancer patients diagnosed between 1976 and 2010 from 13 studies in Asia (n=8), Europe (n=3), and Latin America (n=2). EBV positivity of tumours was assessed by in situ hybridisation. Mortality HRs for EBV positivity were estimated by Cox regression models stratified by study, adjusted for distributions of sex (71% male), age (mean 58 years), stage (52% tumour-node-metastasis stages III or IV), tumour histology (49% poorly differentiated, 57% Lauren intestinal-type), anatomic subsite (70% non-cardia) and year of diagnosis. Variations by study and continent were assessed using study-specific HRs for EBV positivity., Results: During median 3.0 years follow-up, 49% of patients died. Stage was strongly predictive of mortality, with unadjusted HRs (vs stage I) of 3.1 for stage II, 8.1 for stage III and 13.2 for stage IV. Tumour EBV positivity was 8.2% overall and inversely associated with stage (adjusted OR: 0.79 per unit change). Adjusted for stage and other confounders, EBV positivity was associated with lower mortality (HR, 0.72; 95% CI 0.61 to 0.86), with low heterogeneity among the study populations (p=0.2). The association did not significantly vary across patient or tumour characteristics. There was no significant variation among the three continent-specific HRs (p=0.4)., Conclusions: Our findings suggest that tumour EBV positivity is an additional prognostic indicator in gastric cancer. Further studies are warranted to identify the mechanisms underlying this protective association.

Gastric adenocarcinoma is the second leading cause of cancer death worldwide. Epstein-Barr virus (EBV) is present in the malignant cells of approximately 10% of cases. It is unclear whether EBV is being missed in some gastric adenocarcinomas due to insensitive test methods or partial EBV genome loss. In this study, we screened 113 gastric adenocarcinomas from low- and high-incidence regions (United States and Central America) for the presence of EBV using a battery quantitative real-time PCR (Q-PCR) assays targeting disparate segments of the EBV genome (BamH1W, EBNA1, LMP1, LMP2, BZLF1, EBER1) and histochemical stains targeting EBV-encoded RNA (EBER), the latent proteins LMP1 and LMP2, and the lytic proteins BMRF1 and BZLF1. EBV DNA was detected by Q-PCR in 48/75 United States cancers (64%) and in 38/38 Central American cancers (100%), which was a significant difference. EBER was localized to malignant epithelial cells in 8/48 (17%) United States and 3/38 (8%) Central American cancers. Viral loads were considerably higher for EBER-positive vs EBER-negative cancers (mean 162 986 vs 62 EBV DNA copies per 100,000 cells). A viral load of 2000 copies per 100,000 cells is recommended as the threshold distinguishing EBER-positive from EBER-negative tumors. One infected cancer selectively failed to amplify the LMP2 gene because of a point mutation, whereas another cancer had an atypical pattern of Q-PCR positivity suggesting deletion of large segments of the EBV genome. Three different viral latency profiles were observed in the cancers based on constant expression of EBER and focal or variable expression of LMP1 or LMP2, without lytic protein expression. We conclude that EBV DNA levels generally reflect EBER status, and a panel of at least two Q-PCR assays is recommended for sensitive identification of infected cancers.

Gastric cancer is a leading cause of cancer deaths, but analysis of its molecular and clinical characteristics has been complicated by histological and aetiological heterogeneity. Here we describe a comprehensive molecular evaluation of 295 primary gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) project. We propose a molecular classification dividing gastric cancer into four subtypes: tumours positive for Epstein-Barr virus, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also known as PD-L2); microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins; genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins; and tumours with chromosomal instability, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies.