Epstein-Barr Virus-positive Gastric Cancer Research Articles

Epstein-Barr virus positive gastric cancer: the pathological basis of CT findings and radiomics models prediction.

To analyze the clinicopathologic information and CT imaging features of Epstein-Barr virus (EBV)-positive gastric cancer (GC) and establish CT-based radiomics models to predict the EBV status of GC. This retrospective study included 144 GC cases, including 48 EBV-positive cases. Pathological and immunohistochemical information was collected. CT enlarged LN and morphological characteristics were also assessed. Radiomics models were constructed to predict the EBV status, including decision tree (DT), logistic regression (LR), random forest (RF), and support vector machine (SVM). T stage, Lauren classification, histological differentiation, nerve invasion, VEGFR2, E-cadherin, PD-L1, and Ki67 differed significantly between the EBV-positive and -negative groups (p = 0.015, 0.030, 0.006, 0.022, 0.028, 0.030, < 0.001, and < 0.001, respectively). CT enlarged LN and large ulceration differed significantly between the two groups (p = 0.019 and 0.043, respectively). The number of patients in the training and validation cohorts was 100 (with 33 EBV-positive cases) and 44 (with 15 EBV-positive cases). In the training cohort, the radiomics models using DT, LR, RF, and SVM yielded areas under the curve (AUCs) of 0.905, 0.771, 0.836, and 0.886, respectively. In the validation cohort, the diagnostic efficacy of radiomics models using the four classifiers were 0.737, 0.722, 0.751, and 0.713, respectively. A significantly higher proportion of CT enlarged LN and a significantly lower proportion of large ulceration were found in EBV-positive GC. The prediction efficiency of radiomics models with different classifiers to predict EBV status in GC was good.

Effect of Epstein-Barr Virus Infection on the Treatment of Gastric Cancer in Tumor Immunotherapy

Gastric cancer is one of the biggest medical problems in the world today, and it is also a huge medical challenge for human beings. The main challenge is the complexity of the limitations of gastric cancer itself. At present, the efficacy of single-drug immune checkpoint inhibitors in treatment is not clear. It can be found in previous cases that Epstein-Barr virus positive gastric cancer patients have significantly higher response to PD-1 antibodies than the overall patients, from which it can be found that EBV infection can play an important role in gastric cancer immunotherapy. The purpose of this study was to investigate the effect of Epstein-Barr virus infection on gastric cancer and analyze specific clinical cases. The results showed that patients infected with Epstein-Barr virus had obvious advantages in the treatment of gastric cancer, which was significantly correlated with the expression of PD-L1 and EBV (+). This study highlights the potential benefits of Epstein-Barr virus infection in the treatment of gastric cancer, providing new insights into the development of future immunotherapy strategies. These findings will help to improve the treatment of gastric cancer patients, as well as to better understand the immune properties of gastric cancer and develop related vaccines. The significance of this study is that it provides a solid foundation for innovation in the treatment of gastric cancer.

Ceramide promotes lytic reactivation of Epstein-Barr virus in gastric carcinoma.

Epstein-Barr virus (EBV) has a lifelong latency period after initial infection. Rarely, however, when the EBV immediate early gene BZLF1 is expressed by a specific stimulus, the virus switches to the lytic cycle to produce progeny viruses. We found that EBV infection reduced levels of various ceramide species in gastric cancer cells. As ceramide is a bioactive lipid implicated in the infection of various viruses, we assessed the effect of ceramide on the EBV lytic cycle. Treatment with C6-ceramide (C6-Cer) induced an increase in the endogenous ceramide pool and increased production of the viral product as well as BZLF1 expression. Treatment with the ceramidase inhibitor ceranib-2 induced EBV lytic replication with an increase in the endogenous ceramide pool. The glucosylceramide synthase inhibitor Genz-123346 inhibited C6-Cer-induced lytic replication. C6-Cer induced extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB phosphorylation, c-JUN expression, and accumulation of the autophagosome marker LC3B. Treatment with MEK1/2 inhibitor U0126, siERK1&2, or siCREB suppressed C6-Cer-induced EBV lytic replication and autophagy initiation. In contrast, siJUN transfection had no impact on BZLF1 expression. The use of 3-methyladenine (3-MA), an inhibitor targeting class III phosphoinositide 3-kinases (PI3Ks) to inhibit autophagy initiation, resulted in reduced beclin-1 expression, along with suppressed C6-Cer-induced BZLF1 expression and LC3B accumulation. Chloroquine, an inhibitor of autophagosome-lysosome fusion, increased BZLF1 protein intensity and LC3B accumulation. However, siLC3B transfection had minimal effect on BZLF1 expression. The results suggest the significance of ceramide-related sphingolipid metabolism in controlling EBV latency, highlighting the potential use of drugs targeting sphingolipid metabolism for treating EBV-positive gastric cancer.IMPORTANCEEpstein-Barr virus remains dormant in the host cell but occasionally switches to the lytic cycle when stimulated. However, the exact molecular mechanism of this lytic induction is not well understood. In this study, we demonstrate that Epstein-Barr virus infection leads to a reduction in ceramide levels. Additionally, the restoration of ceramide levels triggers lytic replication of Epstein-Barr virus with increase in phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB. Our study suggests that the Epstein-Barr virus can inhibit lytic replication and remain latent through reduction of host cell ceramide levels. This study reports the regulation of lytic replication by ceramide in Epstein-Barr virus-positive gastric cancer.

Does Epstein-Barr virus-positive gastric cancer establish a significant relationship with the multiple genes related to gastric carcinogenesis?

Gastric cancer has been categorized into molecular subtypes including Epstein-Barr virus (EBV)-positive tumors, which provide clinicopathological and prognostic information. In this study, we investigated the EBV infection status of patients with gastric cancer and its correlation with the clinicopathological characteristics and multiple genes related to gastric carcinogenesis. The data of 460 gastric cancer patients who underwent curative gastrectomy with D2 lymph node dissection between January 2017 and February 2022 were analyzed. The clinicopathological features and prognosis of the patients with EBV-positive gastric cancers were compared with those of EBV-negative gastric cancers. Immunohistochemistry for epidermal growth factor receptor (EGFR), C-erb B2, Ki-67, and p53 was performed. Additionally, in situ hybridization was conducted to detect EBV, and microsatellite instability (MSI) analysis was used to assess the deficiency in mismatch repair (MMR) genes. EBV-positivity and MSI were identified in 10.4% and 37.3% of gastric cancer patients, respectively. EBV positivity was associated with male gender (P = 0.001), proximal location (P = 0.004), poorly differentiated histological type (P = 0.048), moderate to severe lymphoid stroma (P = 0.006), high Ki-67 expression (P = 0.02), and a shorter resection margin. EGFR was more often expressed in EBV-negative gastric cancers (P < 0.001). MSI tumors were associated with older age (P = 0.01), the presence of lymphatic invasion (P = 0.02), less perineural invasion (P = 0.05), and the presence of H. pylori infection (P = 0.05). EBV positive gastric cancer is associated with increased Ki-67 and decreased EGFR expression and a shorter resection margin due to the prominent lymphoid stroma. However, MMR deficiency is not associated with EBV status even though MSI gastric cancer is related to H. pylori status.

Landscape of EBV-positive gastric cancer.

Epstein-Barr virus-positive gastric cancer [EBV (+) GC] is associated with EBV infection and is one of the GC subtypes defined by the Cancer Genome Atlas. EBV (+) GC has several distinct genomic or epigenomic features and clinicopathological characteristics compared with other molecular subtypes of GC. Here, we summarize the unique features of EBV (+) GC including the clinical and histopathological features, and discuss associated genetic and epigenetic aberrations. We also discuss noncoding RNAs [EBV-encoded RNAs and EBV-encoded microRNAs (miRNAs)] derived from EBV-infected cells, which have not been described in detail previously. These noncoding RNAs are defined by their roles; for example, EBV-encoded miRNAs play pivotal roles in oncogenesis and tumor progression in EBV (+) GC. We also discuss recent advances in therapeutic modalities for EBV (+) GC, as well as the potential of EBV infection as a predictive biomarker of the response to anti-PD-1 therapy with immune checkpoint inhibitors. We introduce our recent studies focusing on AT-rich interactive domain 1A gene mutations and programmed death ligand-1 overexpression/CD274 copy-number amplification, which are recurrently identified in EBV (+) GC. Finally, based on those findings, we propose potential therapeutic options using candidate-targeted therapies against EBV (+) GC.

PD-1 antibody (Camrelizumab) for metastatic EBV positive gastric cancer: A prospective single-arm, open-label, phase 2 trial.

e16014 Background: Programmed cell death 1 (PD-1) inhibitors have been approved as third line therapy for patients with metastatic gastric cancer (mGC). Epstein–Barr virus (EBV)-positive is a potential predictor to response of PD-1 inhibitors. The aim of this study was to assess the efficacy and safety of camrelizumab as salvage treatment in metastatic EBV positive GC patients and explored the potential biomarkers associated with response (NCT03755440). Methods: In this prospective single-arm, phase II study, stage IV EBV positive GC patients received 200 mg camrelizumab by intravenous infusion every 2 weeks until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR), which were based on RECIST V.1.1. Secondary endpoints were progression free survival (PFS), overall survival (OS), disease control rate (DCR), durationn of response (DoR), and toxicity. Exploratory analysis included association between response and tumor mutational burden (TMB) and cell death ligand-1 (PD-L1) expression. Results: According to Simon’s optimum two-stage design with α = 0.05 and β = 0.20. P0 = 15% (null hypothesis), the presence of at least 1 success in the 6 patients enrolled in the first stage allowed the trial to proceed to the second stage in which 13 more patients were needed to be enrolled for the minimum total of 20 patients (5% lost to follow-up calculated). Six eligible patients were enrolled at the first stage. All the patients were HER2 negative and pMMR. None of the patients had an objective response. The DCR was 66.7%. The median PFS was 2.2 months (95% CI: 1.5 months-not reached (NR)) and median OS was 6.8 months (95% CI: 1.7 months-NR). The most common treatment-related adverse event (TRAE) was reactive cutaneous capillary endothelial proliferation. No grade 3 or worse TRAEs were found. Only one patient had PD-L1 combined positive score (CPS) ≥1 but got disease progression. Three patients were TMB &gt; 10Mb and two of them got stable disease while one got disease progression. Conclusions: EBV positive is not a good predictor for response to PD-1 inhibitors. Potential biomarkers are needed to identify EBVaGC patients who might respond to PD-1 inhibitors. Clinical trial information: NCT03755440.

PD-L1 overexpression in EBV-positive gastric cancer is caused by unique genomic or epigenomic mechanisms

Epstein-Barr virus-positive gastric cancer [EBV (+) GC] is a distinct GC subtype with unique genetic and epigenetic aberrations. Here, we examined resected GC samples and publicly available microarray data and The Cancer Genome Atlas (TCGA) database to identify the mechanism underlying overexpression of PD-L1 in EBV (+) GC. We found that high levels of PD-L1 overexpression in EBV (+) GC were caused by focal amplification of CD274. By contrast, relatively high expression of PD-L1 in tumor tissue and infiltrating immune cells correlated with CD8 lymphocyte infiltration and IFN-γ expression via IRF3 activation. Since we reported previously that PD-L1 expression is associated both with the presence of CD8 T cells in the tumor microenvironment and with IFN-γ expression in GC, we examined a database to see whether IFN-γ-associated overexpression of PD-L1 plays a significant role in EBV (+) GC. Immunohistochemical staining showed that expression of the IRF3 signature in clinical GC samples was higher in EBV (+) than in EBV (−) cases. The data presented herein reveal a unique dual mechanism underlying PD-L1 overexpression in EBV (+) GC: high focal amplification of CD274 or IFN-γ-mediated signaling via activation of IRF3.

Epstein–Barr Virus Positive Gastric Cancer: A Distinct Subtype Candidate for Immunotherapy

BackgroundEpstein–Barr virus (EBV) positive gastric cancer (GC) has been described as a distinct molecular subtype of the disease, especially associated with gastric carcinoma with lymphoid stroma (GCLS). The possibility that EBV associated GC (EBVaGC) had better prognosis and may be susceptible to immunotherapy has increased the interest in this subtype. However, immune checkpoint and survival of EBVaGC are still controversial, especially with regard to GCLS and conventional gastric adenocarcinoma (CGA). This study aimed to evaluate the clinicopathological characteristics, immunohistochemical profiles and prognosis of EBVaGC according to the histological type GCLS and CGA. Methodswe retrospectively evaluated a series of EBVaGC who underwent gastrectomy with D2-lymphadenectomy. Biomarkers and tumor-infiltrating cells were evaluated by immunohistochemistry. PD-L1 was evaluated using a combined positive score (CPS). ResultsFrom a total of 30 EBVaGC, 14 (46.7%) were identified as GCLS and 16 (53.3%) as CGA (9 Intestinal, 6 diffuse, 1 undetermined). There were no significant differences in age, sex, and pTNM between GCLS and CGA. CPS-positivity and high-CD8+ was significantly higher in GCLS compared with CGA (P = 0.007 and P = 0.005, respectively). Diffuse EBVaGC had worse survival than intestinal type (P = 0.020). There was no difference in survival between GCLS and intestinal CGA (P = 0.260). In multivariate analysis, CPS and pN status were related with survival in EBVaGC. ConclusionsCGLS was associated with a predominance of CD8+ cell infiltration and PD-L1 expression. CPS and lymph node metastasis were independent factors associated with prognosis in EBVaGC. These results suggest that specifically EBV-positive GCLS may be prime candidates for PD-1 directed therapy.

ARID1A deficiency in EBV-positive gastric cancer is partially regulated by EBV-encoded miRNAs, but not by DNA promotor hypermethylation

AT-rich interactive domain 1A (ARID1A), which is a tumor suppressor gene, is frequently mutated in Epstein-Barr virus-positive gastric cancer [EBV (+) GC]. While most ARID1A mutations in GC are truncating mutations, leading to loss of ARID1A protein expression, epigenetic modifications appear to contribute to ARID1A deficiency in EBV (+) GC harboring wild-type ARID1A. Based on the significant role of epigenetic modifications in EBV (+) GC that contributes to ARID1A deficiency, the methylation status of ARID1A was evaluated in EBV-infected cells and GC patients using a publicly available microarray and the Cancer Genome Atlas (TCGA) database. EBV-encoded miRNAs that potentially target ARID1A were identified as an additional epigenetic modulator by computational prediction. In vitro experiments were conducted to evaluate how EBV-encoded miRNAs affected ARID1A mRNA and protein levels. In clinical GC samples, the expression of predicted miRNAs and ARID1A and the mutation status of ARID1A was evaluated. As results, ARID1A was not hypermethylated in EBV (+) GC samples or EBV-infected GC cells. EBV infection did not alter ARID1A mRNA levels, suggesting that ARID1A protein deficiency was caused by post-transcriptional gene silencing in ARID1A-WT EBV (+) GC. Overexpression of miR-BART11-3p and miR-BART12, which were identified as miRNAs that potentially bind ARID1A, suppressed ARID1A protein expression in MKN7 and NCI-N87 cells. Highly expressed miR-BART11-3p and miR-BART12 were correlated with decreased ARID1A levels in GC tumors which did not harbor ARID1A mutations. The present findings revealed that ARID1A expression was epigenetically regulated by miR-BART11-3p and miR-BART12 in EBV (+) GC.

152P ARID1A deficiency in EBV-positive gastric cancer is partially regulated by EBV-encoded miRNAs, but not by DNA promotor hypermethylation

AT-rich interactive domain 1A (ARID1A) is a tumor suppressor gene, which is frequently mutated in Epstein-Barr virus-positive gastric cancer (EBV (+) GC). While most ARID1Amutations in GC are truncating mutations, leading to loss of ARID1A protein expression, epigenetic modifications appear to contribute to ARID1A deficiency in EBV (+) GC harboring wild-type ARID1A. Based on the hypothesis that DNA promotor hypermethylation is a significant epigenetic modification in EBV (+) GC that contributes to ARID1A deficiency, the methylation status of ARID1Awas evaluated in EBV-infected cells and GC patients using a publicly available microarray database and the Cancer Genome Atlas (TCGA) database. EBV-encoded miRNAs that potentially target ARID1Awere identified as an additional epigenetic modulator by computational prediction. In vitro experiments were conducted to evaluate how EBV-encoded miRNAs affected ARID1A mRNA and protein levels. In clinical GC samples, expression of the predicted miRNAs was evaluated by qRT-PCR and correlated with ARID1A expression, as assessed by IHC staining. ARID1Awas not hypermethylated in EBV (+) GC samples or EBV-infected GC cells. EBV infection did not alter ARID1AmRNA levels, suggesting that ARID1A protein deficiency was caused by post-transcriptional gene silencing in ARID1A-WT EBV (+) GC. Overexpression of miR-BART-X and miR-BART-Y, which were identified as miRNAs that potentially bind ARID1A, suppressed ARID1A protein expression in MKN7 and NCI-N87 cells. Highly expressed miR-BART-X and miR-BART-Y were correlated with decreased ARID1A levels in GC tumors. The present findings revealed a pivotal role for epigenetic modifications that EBV-encoded miRNAs contribute to gastric carcinogenesis via ARID1A suppression.

MSI and EBV Positive Gastric Cancer's Subgroups and Their Link With Novel Immunotherapy.

Gastric cancers have been historically classified based on histomorphologic features. The Cancer Genome Atlas network reported the comprehensive identification of genetic alterations associated with gastric cancer, identifying four distinct subtypes— Epstein-Barr virus (EBV)-positive, microsatellite-unstable/instability (MSI), genomically stable and chromosomal instability. In particular, EBV-positive and MSI gastric cancers seem responsive to novel immunotherapies drugs. The aim of this review is to describe MSI and EBV positive gastric cancer’s subgroups and their relationship with novel immunotherapy.

Clinical impact of Epstein-Barr virus status on the incidence of lymph node metastasis in early gastric cancer.

Epstein-Barr virus-positive gastric cancer (EBVGC) comprises approximately 9% of all gastric cancers and is associated with a low prevalence of lymph node metastasis (LNM). Given that limited data concerning LNM in EBV-related early GC are available, EBV status is not considered an indicator for endoscopic submucosal dissection (ESD). In this review, we focused on pT1 EBVGC and on gastric carcinoma with lymphoid stroma (GCLS), and discuss expanded ESD indications and curative resection criteria. In pT1b EBVGC, the incidence of LNM was low (6/180 patients, 3.3%; 95% confidence interval [CI] 1.2-7.1), especially in lymphovascular invasion-negative EBVGC (1/109 patients, 0.9%). No patients with pT1a EBVGC had LNM (0/38 patients, 0%; 95% CI 0-7.6), even those who did not meet the current curative ESD criteria. Although the frequency of LNM in GCLS was low (5.0-10.6%), the incidence of LNM in non-EBV GCLS was relatively high (10.0-20.0%); therefore, EBV status can be considered a more important factor than GCLS. In summary, the clinicopathological characteristics of EBVGC differ from those of conventional GC, and EBV negativity is a risk factor for LNM in early GC. Therefore, patients in this group are likely to be promising candidates for ESD, and we recommend that EBV status evaluation be included in early GC treatment guidelines.

Clinicopathological characteristics of Epstein-Barr virus-positive gastric cancer in Latvia.

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. 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. 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. Tumor EBV positivity is a favorable prognostic factor in gastric cancer.

Epigenetic alternate promoter utilization and association with PD-L1 expression in Epstein–Barr virus positive gastric cancer.

e15509 Background: We recently elicited the role of epigenetic promoter alterations as a mechanism of immune-evasion and primary resistance to immune checkpoint inhibition in gastric cancer. High prevalence of epigenetic modifications are known to occur in Epstein-Barr virus associated gastric cancer (EBVaGC). EBVaGC has high response rates to anti-PD-1 immune checkpoint inhibitors and is associated with high levels of PD-L1 expression. However, not all EBVaGC express PD-L1 and mechanisms that mediate these phenomena are unknown. Methods: We performed NanoString profiling and PD-L1 immunohistochemistry (using Dako PD-L1 IHC 22C3) on tissue from gastric cancer patients undergoing primary tumor resections at Samsung Medical Centre, South Korea. NanoString panel was designed for 90 recurrent somatic alternate promoter-related genes, and immune-related genes including PD-L1. EBV status was determined using EBV-encoded RNA in situ hybridization and categorized as EBVaGC and EBV-negative. Samples in the top-quartile of alternate promoter utilization were classified as APhigh and the remaining APlow. Results: A total of 272 samples (EBVaGC n = 79; EBV-negative n = 193) were included in this study. EBVaGC had significantly higher PD-L1 expression (p &lt; 0.001) compared to EBV-negative samples. APhigh group (n = 67) consisted of 61 EBV-negative and 6 EBVaGC samples. EBVaGC APhigh tumors had significantly lower PD-L1 transcript expression compared to EBVaGC APlow tumors (p = 0.011, Wilcoxon-rank sum). Similar correlation was also found with PD-L1 IHC combined positive score (CPS)(median CPS score 1 vs 8, p = 0.047). There was a trend towards poorer survival for EBVaGC APhigh tumors (vs EBVaGC APlow; HR 0.23, 95% CI: 0.046 – 1.23, p = 0.087). EBV-negative APhigh tumors also had lower PD-L1 expression (vs EBV-negative APlow; p = 0.046, Wilcoxon-rank sum). Conclusions: Increased utilization of epigenetic alternate promoter isoforms correlates with lower transcriptomic and protein expression of PD-L1 in EBVaGC. Here we describe a potential mechanism of immune-evasion to explain low immune-infiltration and PD-L1 expression that occurs in a group of EBVaGC that is traditionally considered highly immunogenic.

Epstein-Barr virus status is a promising biomarker for endoscopic resection in early gastric cancer: proposal of a novel therapeutic strategy.

Epstein-Barr virus-positive gastric cancer (EBVGC) is associated with a low prevalence of lymph node metastasis (LNM); however, EBV status is not considered in the indication of endoscopic resection (ER). We aimed to clarify the implication of EBV status for ER of pT1b GC. Consecutive cases of pT1b GCs treated with surgery between 2005 and 2014 were retrospectively analyzed. Clinicopathological factors and LNM status were compared between EBVGC and non-EBVGC groups. EBVGC accounted for 7.9% (71 of 898) cases. Compared to non-EBVGC, EBVGC was more frequent in males (p = 0.0055), the upper third region (p < 0.0001), showed elevated growth features (p = 0.0059), and was associated with a lower frequency of accompanying ulceration (p = 0.002), greater depth of submucosal invasion (p = 0.017), and lower frequency of lymphatic invasion (p < 0.0001). Frequency of LNM was significantly lower in EBVGC than in non-EBVGC (4.2% vs. 21.9%, p < 0.0001). In EBVGC, tumors without lymphovascular invasion showed significantly lower frequency of LNM than those with lymphovascular invasion (0 of 50, 0%; vs 3 of 21, 14.3%; p = 0.023). Histologically, 84.5% (60 of 71) of EBVGC included carcinomas with lymphoid stroma and/or lace pattern components. pT1b EBVGC is a convincing candidate for ER, regardless of risk factors other than lymphovascular invasion.

Histone modification alteration coordinated with acquisition of promoter DNA methylation during Epstein-Barr virus infection.

Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer. Epstein-Barr virus positive gastric cancer is the most frequently hypermethylated tumor among human malignancies. Herein, we performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIP-sequencing for histone modification alteration during EBV infection into gastric cancer cell line MKN7. Among 7,775 genes with increased DNA methylation in promoter regions, roughly half were “DNA methylation-sensitive” genes, which acquired DNA methylation in the whole promoter regions and thus were repressed. These included anti-oncogenic genes, e.g. CDKN2A. The other half were “DNA methylation-resistant” genes, where DNA methylation is acquired in the surrounding of promoter regions, but unmethylated status is protected in the vicinity of transcription start site. These genes thereby retained gene expression, and included DNA repair genes. Histone modification was altered dynamically and coordinately with DNA methylation alteration. DNA methylation-sensitive genes significantly correlated with loss of H3K27me3 pre-marks or decrease of active histone marks, H3K4me3 and H3K27ac. Apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation-resistant genes. Genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Our data show that orchestrated epigenetic alterations are important in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at transcription start site.

Molecular Testing for Gastrointestinal Cancer.

With recent advances in molecular diagnostic methods and targeted cancer therapies, several molecular tests have been recommended for gastric cancer (GC) and colorectal cancer (CRC). Microsatellite instability analysis of gastrointestinal cancers is performed to screen for Lynch syndrome, predict favorable prognosis, and screen patients for immunotherapy. The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor has been approved in metastatic CRCs with wildtype RAS (KRAS and NRAS exon 2–4). A BRAF mutation is required for predicting poor prognosis. Additionally, amplification of human epidermal growth factor receptor 2 (HER2) and MET is also associated with resistance to EGFR inhibitor in metastatic CRC patients. The BRAF V600E mutation is found in sporadic microsatellite unstable CRCs, and thus is helpful for ruling out Lynch syndrome. In addition, the KRAS mutation is a prognostic biomarker and the PIK3CA mutation is a molecular biomarker predicting response to phosphoinositide 3-kinase/AKT/mammalian target of rapamycin inhibitors and response to aspirin therapy in CRC patients. Additionally, HER2 testing should be performed in all recurrent or metastatic GCs. If the results of HER2 immunohistochemistry are equivocal, HER2 silver or fluorescence in situ hybridization testing are essential for confirmative determination of HER2 status. Epstein-Barr virus–positive GCs have distinct characteristics, including heavy lymphoid stroma, hypermethylation phenotype, and high expression of immune modulators. Recent advances in next-generation sequencing technologies enable us to examine various genetic alterations using a single test. Pathologists play a crucial role in ensuring reliable molecular testing and they should also take an integral role between molecular laboratories and clinicians.

Epstein-Barr virus-positive gastric cancer: a distinct molecular subtype of the disease?

Approximately 90% of the world population is infected by Epstein-Barr virus (EBV). Usually, it infects B lymphocytes, predisposing them to malignant transformation. Infection of epithelial cells occurs rarely, and it is estimated that about to 10% of gastric cancer patients harbor EBV in their malignant cells. Given that gastric cancer is the third leading cause of cancer-related mortality worldwide, with a global annual incidence of over 950,000 cases, EBV-positive gastric cancer is the largest group of EBV-associated malignancies. Based on gene expression profile studies, gastric cancer was recently categorized into four subtypes; EBV-positive, microsatellite unstable, genomically stable and chromosomal instability. Together with previous studies, this report provided a more detailed molecular characterization of gastric cancer, demonstrating that EBV-positive gastric cancer is a distinct molecular subtype of the disease, with unique genetic and epigenetic abnormalities, reflected in a specific phenotype. The recognition of characteristic molecular alterations in gastric cancer allows the identification of molecular pathways involved in cell proliferation and survival, with the potential to identify therapeutic targets. These findings highlight the enormous heterogeneity of gastric cancer, and the complex interplay between genetic and epigenetic alterations in the disease, and provide a roadmap to implementation of genome-guided personalized therapy in gastric cancer. The present review discusses the initial studies describing EBV-positive gastric cancer as a distinct clinical entity, presents recently described genetic and epigenetic alterations, and considers potential therapeutic insights derived from the recognition of this new molecular subtype of gastric adenocarcinoma.

Validation and calibration of next-generation sequencing to identify Epstein-Barr virus-positive gastric cancer in The Cancer Genome Atlas

Validation and calibration of next-generation sequencing to identify Epstein-Barr virus-positive gastric cancer in The Cancer Genome Atlas

Abstract 4621: Validation and calibration of next-generation sequencing to identify Epstein-Barr Virus-positive gastric cancer

Abstract Background: Gastric adenocarcinoma was recently classified by a large genomic study into four molecular subtypes, including one defined by Epstein-Barr virus (EBV)-positivity. EBV infection in tumors is conventionally assessed by in situ hybridization (ISH), but detection of viral nucleic acids by next-generation sequencing represents a potential alternative. Methods: Normalized EBV read counts were determined 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 selected at random. Results: By each sequencing method, numbers of EBV reads were bimodally distributed across tumors, with a minority having much higher counts. In pairwise comparisons, individual samples were either consistently high or consistently low by all genomic methods for which data were available. Empiric cut-offs based on molecular data of 1000 normalized reads for whole genome, 100 for exome, 4 for mRNA and 5000 for miRNA had perfect concordance identifying 26 (9%) tumors as EBV-positive. One sample tested by ISH lacked tumor cells. For the 23 tumors with EBV status determined by both approaches, EBV-positivity or -negativity by molecular testing was confirmed by EBER-ISH in all but one case (kappa = 0.91). The exception was a microsatellite instability-type cancer EBV-negative by both mRNA and miRNA sequencing, with equivocally positive ISH interpretation. Conclusions: EBV-positive gastric tumors may 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. Citation Format: M. Constanza Camargo, Reanne Bowlby, Andy Chu, Chandra S. Pedamallu, Vesteinn Thorsson, Sandra Elmore, Andrew Mungall, Adam Bass, Margaret L. Gulley, Charles S. Rabkin. Validation and calibration of next-generation sequencing to identify Epstein-Barr Virus-positive gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4621. doi:10.1158/1538-7445.AM2015-4621