Clinicopathological Spectrum of EBV and MSI in Gastric Adenocarcinomas

Introduction: Gastric carcinoma due to Epstein-Barr virus (EBV) and microsatellite instability (MSI) have distinct clinicopathological characteristics that provide specific treatment options. Material and methods: Patients with gastric adenocarcinomas (GC) diagnosed on treatment-naïve endoscopic biopsy/resection specimens during 19 months were included. The samples were subjected to EBER-ISH and mismatch repair (MMR) protein status by immunohistochemistry and the demographic features, endoscopy/gross features and histologic type were correlated between EBV positive vs negative and MMR-deficient vs proficient groups. Results: EBV was positive in 3.1% (3/97) of GC. Two of the three patients were males with tumor in the proximal part of the stomach showing poorly cohesive NOS (WHO)/diffuse (Lauren) histology. All the three patients had thickening of the gastric wall and lymph node involvement on CECT and received palliative chemotherapy. MMR-d GC constituted 8% (6/75) of GC. There was female predominance with median age of 53 years. All were in the body of the stomach showing3 each of poorly cohesive (NOS) and tubular adenocarcinoma (WHO)/ intestinal (2), indeterminate (1) and diffuse (3) (Lauren). The loss of MMR expression was seen as heterodimers: MLH1+PMS2 in 04 (66.67%) andMSH2+MSH6 in 01 (16.67%) and isolated loss of PMS2 in 01 (16.67%). Patients received immunotherapy/ palliative chemotherapy. EBV positive and MMR-d GC were mutually exclusive. There was no statistically significant difference in EBV positive vs negative or MMR-d vs proficient groups in clinicopathological features. Conclusion: Routine testing for EBV by EBER-ISH and MMR by IHC can identify molecularly distinct gastric carcinoma for therapy and prognosis.

 Introduction

The incidence of gastric carcinoma varies in different parts of the world and among various ethnic groups. Despite advances in the diagnosis and treatment, the 5-year survival rate of gastric carcinoma is about 20% [1-3]. About 95% of the gastric carcinomas are adenocarcinomas [4]. Gastric adenocarcinoma (GC) is multi-factorial and the classifications based on site and morphology, have limited clinical application for stratification of patients and therapeutic options [5-10].

Gastric carcinogenesis involves multiple genetic and epigenetic alterations of oncogenes, tumor suppressor genes, DNA repair genes, cell cycle regulators and signaling molecules [11]. In a comprehensive molecular evaluation of 295 primary GCs as part of The Cancer Genome Atlas (TCGA) project, a molecular classification was proposed dividing gastric cancer into four sub-types: tumors positive for EBV, microsatellite unstable tumors, genomically stable tumors and tumors with chromosomal instability [12]. Asian Cancer Research Group (ACRG) analyzed the mRNA expression level of 300 tumors and classified them as microsatellite instability-high (MSI-H), microsatellite stable/epithelial-mesenchymal transition (MSS/EMT), microsatellite stable/epithelial/ TP53 intact (MSS/TP53+, p53 active) and microsatellite stable/epithelial/TP53 loss (MSS/TP53-, p53 inactive) [13]. MSS/p53+ molecular subtype was closely linked to EBV infection. Additional subtypes of GC have been described [10, 14-16]. Helicobacter pylori (H. pylori) and Epstein-Barr virus (EBV) are the most well-known pathogens in gastric carcinogenesis [17]. H. pylori is an important risk factor found in 65–80% of primary GCs, particularly non-cardia cancers, while EBV causes about 10% of the GC, particularly cardia cancers [11, 17-18].

Epstein–Barr virus-associated GC (EBVaGC) has several distinct genomic or epigenomic features and clinicopathological characteristics compared with other molecular subtypes of GC. It usually occurs in younger patients with male predominance, in the proximal part of stomach, with less differentiated morphology and high content of CD8+tumor infiltrating lymphocytes and high expression of programmed death-ligand 1 (PD-L1) and PD-L2 [19]. GC with MSI constitutes about 10-22% of GC with distinct clinicopathologic characteristics. It is associated with older age, female gender, distal stomach location, and lower number of lymph-node metastases and a significantly better overall and tumor-specific survival [20].

Molecular classification was considered to be more specific than pathological classification as it allows better stratification of patients. Both EBV-associated and MSI-H-GC are considered to have better prognosis and respond to immunotherapy and hence have the possibility to be useful as biomarkers for patient stratification [20-23]. The gold standard technologies include in- situ- hybridization for the detection of EBV in tissue and polymerase chain reaction (PCR) for MSI status respectively. Immunohistochemistry (IHC) for mismatch repair (MMR) protein has excellent concordance with PCR for MSI status [20].

The present study aims to study the EBV status by in situ hybridization and MSI status by IHC for MMR proteins, in treatment-naïve GC and characterize the clinicopathological subtypes.

Materials and Methods

Patients diagnosed as GC on histology of endoscopic biopsy/resected specimens, prior to chemotherapy/ radiotherapy during the period January 2019 and July 2020 were included in the study. Patients who had tumors other than adenocarcinoma, who received prior treatment, and where blocks were unavailable or had inadequate tissue for further studies were excluded. It was a prospective and retrospective observational study. The study was approved by the institutional ethics committee.

The demographic features, site of the tumor, endoscopic features and/or macroscopic features (on resected specimens) were noted. Features on contrast-enhanced computed tomogram (CECT) were collected wherever available. The hematoxylin & eosin (H&E) stained slides were reviewed and the tumor was classified according to the World Health organization (WHO) (5th) edition criteria as well as Lauren classification [8, 9]. Tumor infiltrating lymphocytes, Crohn-like granuloma or desmoplasia in the stroma were noted and graded as mild, moderate or severe. The adjacent mucosa was examined for chronic gastritis, atrophy, intestinal metaplasia (IM) (on alcian-periodic acid Schiff [APAS] stain) and H pylori status (on Giemsa stain). In situ hybridization (ISH) for EBER ISH for EBV was done using the INFORM EBER (Epstein-Barr Virus Early RNA) Probe implementation kit on VENTANA. Briefly, formalin fixed paraffin embedded (FFPE) sections of 4 μm were deparaffinized followed by 10 min incubation in 3% H2O2. Following rinsing in deionized water (DW), target retrieval was achieved using pepsin digestion in humidity chamber for 15 min. Cell conditioning using standard CC1 (P/N 950-110) at 950C for 44 minutes at pH 8.5 was done. One drop of ISH-PROTEASE 2 was added, coverslip applied and incubated for 8 min. Slides were washed in DW and drained off; 100 μl of INFORM EBER Probe on BenchMark XT probe were poured over each slide, and covered with a cover slip using glue for 1 hour. Red counterstain II, ISH iVIEW Blue Plus Detection Kit, probes, and required accessory reagents onto the reagent tray were loaded. The slides were loaded onto the automated slide stainer and at the completion of the run, slides were dehydrated in graded alcohols, air-dried and mounted with DPX. The intensity of staining (weak, moderate or intense) and the percentage of positive cells were recorded. A section from known EBV positive nasopharyngeal carcinoma was used as control in each batch of the procedure. The results were interpreted as positive staining when there was strong nuclear positivity in the majority of the tumor cells. Nuclear staining in < 5% of tumor cells was considered negative for EBER.

Immunohistochemistry was done using LEICA bio system with monoclonal antibodies: MLH1 (G168-15, prediluted, ready to use), MSH2 (DBM15.82, prediluted, ready to use), MSH6 (44, prediluted, ready to use) and PMS2 (A16-4, prediluted, ready to use). Briefly, FFPE sections of 4µm were deparaffinized, dehydrated and antigen retrieval was done by microwave using citrate buffer/Tris EDTA buffer. After retrieval, slides were cooled to room temperature, washed in distilled water and hydrogen peroxide was added for 10 min. Slides were washed with wash buffer and background snipper was added for 10min. Slides were washed thoroughly thereafter and the sections were treated with primary antibodies against the MMR proteins for 30 min. The reaction product was visualized with diaminobenzidine chromogen (DAB) and counterstaining was done hematoxylin. Only nuclear staining with or without cytoplasmic staining in tumor cells was considered positive (normal expression). Normal gastric mucosa and lymphocytes were used as internal control. Only the complete loss of nuclear staining with positive internal control was considered as loss of MMR protein expression. If any of the MMR protein expression was absent, it was labelled MMR deficient (MMR-d). If all four MMR proteins were expressed, it was labelled as MMR-proficient (MMR-p) [21].

Follow-up details including additional investigations, wherever performed, treatment received and survival were obtained for both EBVaGC and MMR-d GC.

<italic id="italic-1">Statistics<italic id="italic-2"/></italic>

The demographic, site and histological parameters were compared between EBV positive and EBV negative GC, and MMR-d and MMR-p GC using Chi square test or Fisher exact test (whichever is appropriate).

Results

A total of 100 patients with tumors diagnosed as GC were included in the study, which comprised 90 endoscopic biopsies and 10 resected specimens. As per the inclusion and exclusion criteria, analysis was performed on 97 patient samples. The resection specimens included 03 total gastrectomies, 02 partial gastrectomies and 05 distal gastrectomies. The demographic features, site, endoscopic features, histologic type of tumor, changes in the stroma and adjacent gastric mucosa were given in Table 1.

Site Age (median) years Gender Type of Adenocarcinoma(classification)   Tumor stroma   Adjacent gastric mucosa (n=90)      
    M: F WHO Lauren TILs Desmoplasia H pylori IM Chronicgastritis Atro gastritis
Proximal (n =40; 41.24%) 22-71 [58] 21:19 Tubular:13 Intestinal: 08 Mild: 18 Mild: 25 P: 09 P:20 P: 22 P: 20
Fundus (n=6)     Papillary: 04 Indeterminate: 09 Moderate: 11 Moderate: 09 N:28 A:17 A:13 A:15
Body (n=21)     PC(NOS):14 Diffuse: 20 Marked: 11 Marked: 06 NA:03 NA:03 NA: 05 NA:05
Fundus & body (n=13)     PC(SRC): 06 Mixed: 03            
      Mixed:03              
Distal (n=57; 58.76%) 28-77 38:19:00 Tubular: 16 Intestinal: 22 Mild: 26 Mild: 32 P: 14 P:32 P: 30 P:18
Antrum (n=42) [56]   Papillary:05 Indeterminate: 04 Moderate: 14 Moderate:20 N:39 A:21 A:19 A:31
Antrum & body (n=15)     PC(NOS): 12 Diffuse: 28 Marked: 17 Marked:05 NA:04 NA:04 NA:8 NA:08
      PC(SRC): 16 Mixed: 03            
      Mixed: 08              
Total (n=97) 22-77 59:38:00 Tubular:29 Intestinal: 30 Mild: 44 Mild: 57 P:23 P:52 P:52 P:38
  [57]   Papillary: 09 Indeterminate: 13 Moderate: 25 Moderate:29 N: 67 N:38 A:32 A:46
      PC (NOS):26 Diffuse: 48 Marked: 28 Marked: 11 (25.56 %) NA: 07 NA: 13 NA: 13
      PC (SRC): 22 Mixed: 06     NA:07      
      Mixed: 11              

Abbreviations: WHO, World health organization; PC (SRC), Poorly cohesive signet ring cell; PC(NOS), Poorly cohesive not otherwise specified; TILs, tumor infiltrating lymphocytes; NA, Not assessed; P, present; N, negative; A, absent

<italic id="italic-538acb0e8eaf17759c8e758c0e7a5b7f">Demographic features, site and endoscopic features<italic id="italic-6bccad86f8e73be3df3e107a224181dc"/></italic>

There were 59 males and 38 females with age ranging from 22 - 77 (median 57) years. The tumor was located in the proximal part of the stomach in 40 (41.24%; fundus in 06, body in 21 and both fundus and body in 13), distal part in 57 (58.76%; antrum in 42 and antrum and body in 15). The tumor was ulcerated in 46, infiltrative in 6, polypoidal in 4, exophytic in 2, stenotic in 9, gastric wall thickening in 17 and growth (not otherwise specified [NOS]) in 13.

<italic id="italic-3">Histologic type of GC<italic id="italic-4"/></italic>

According to the WHO classification, the adenocarcinoma was tubular in 29 (well differentiated in 09, moderately differentiated in 12 and poorly differentiated in 12), papillary in 09, poorly cohesive in 48 (NOS in 26 and signet ring cell [SRC] type in 22) and mixed in 11. Extracellular mucin was present in 09 (9.2%); however, no tumor was categorized as mucinous adenocarcinoma as majority of the samples were endoscopic biopsies and only one resected specimen showed extra cellular mucin but was less than 50% of the tumor. According to the Lauren classification, GC was intestinal in 30, indeterminate in 13, diffuse in 48 and mixed in 6.

<italic id="italic-5">Changes in the stroma<italic id="italic-6"/></italic>

Desmoplasia was noted in the stroma which was mild in 57 (58.76%), moderate in 29 (29.89%) and marked in 11 (11.34%). Necrosis was observed in 23 (23.71%). Lymphocytic Infiltration was graded as mild in 44 (45.3%), moderate in 25 (25.77%) and marked in 28 (28.86%). However, Crohn-like reaction was not observed in any tumor.

<italic id="italic-7">Changes in the adjacent gastric mucosa<italic id="italic-8"/></italic>

Adjacent gastric mucosa was available in 90 samples. Chronic gastritis was present in 52/84 (61.9%), atrophy in 38/84 (45.2%), IM in 52/90 (53.6%). H.pylori was present in 23/90 (25.56%). Dysplasia was not seen in any sample. Chronic gastritis and atrophy could not be assessed in 6 samples as adjacent mucosa was scant.

EBER-ISH was done in 97 samples and IHC for MMR proteins was done in 75 (as per availability of tissue).

 <italic id="italic-8d821e6311445ca214a23d8980e07ffa">Clinicopathological features of EBV-associated gastric adenocarcinoma (EBVaGC)</italic>

It constituted 3.1% (3/97) of GC. The clinicopathological features were given in Table 2.

EBVaGC Gender/ Age (y) Endoscopy/CECT Histology (classification)   Stroma Adjacent mucosa Follow-up
      WHO Lauren      
Patient 1 M/67 Exophytic, ulcerated bulky growth, 5cm/ circumferential thickening of wall;LN: Paraaortic + Poorly cohesive NOS Diffuse TILs: marked Desmoplasia: mild H. pylori: N Chronic gastritis: AIM: AAtrophy: A Received palliative chemotherapy; alive at 2 years follow-up
Patient 2 M/51 Ulcerated growth along lesser curvature, 1 cm/ asymmetric thickening of wall;LN: Periportal+ Poorly cohesive NOS Diffuse TILs: marked Desmoplasia: mild H. pylori: N Chronic gastritis: AIM: AAtrophy: A Received palliative chemotherapy alive at 2 years follow-up
Patient 3 F/77 Prepyloric encircling growth in antrum, 4x3.5cm/ mild circumferential wall thickening in pylorus;LN: Multiple hepato-gastric, portocaval, peri-pancreatic, retrocaval, aortocaval, pre/para-aortic, mesenteric, bilateral common iliac nodes + Moderately differentiated tubular adenocarcinoma Intestinal TILs: moderate Desmoplasia: mild H. pylori: P Chr gastritis: PIM: PAtrophy: A Received palliative chemotherapy; alive at 3 years follow-up

Abbreviations, EBVaGC, Epstein-Barr virus associated gastric adenocarcinoma; WHO, World health organization; PC(NOS), Poorly cohesive not otherwise specified; TILs, tumor infiltrating lymphocytes; NA, Not assessed; P, present; N, negative; A, absent

Two of the three patients were males with tumor located in the proximal part of the stomach showing poorly cohesive NOS (WHO)/diffuse (Lauren) histology. All the three patients had thickening of the gastric wall and lymph node involvement on CECT. Interestingly, the adjacent mucosa in the moderately differentiated adenocarcinoma (WHO)/intestinal (Lauren) located in the distal part of stomach showed presence of H.pylori, chronic gastritis, IM and atrophy. All the three patients were diagnosed with advanced locoregional disease and received palliative chemotherapy. All patients Please cite figure 1 and 2 in text were alive and at follow-up (2-3 years) and had intact MMR protein expression on IHC.

<italic id="italic-eeb0991ef58aac9628b43fd2f1912d02">Clinicopathological features of MMR deficient-GC<italic id="italic-5dfaccdeaf5c8b1bce69927f29c5de60"/></italic>

MMR- d GC constituted 8% ( 6/ 75) of GC. The clinicopathological features are given in Table 3.

MMR-d GC Gender/Age Site Endoscopy/CECT Histological classification   MMR status Stroma / Adjacent mucosa Additional features Follow-up
        WHO Lauren classification        
Patient 1 F/26 Body & fundus Large cauliflower ulcerated growth/ polypoidal wall thickening; LN: peri-gastric + Tubular, well differentiated Intestinal Loss of MSH2 TIL: Marked Desmoplasia: Mild H. pylori: NChronic gastritis: AIM: AAtrophy: A Significant family history + Stable
Patient 2 M/57 Body & fundus Ulcerated growth along lesser curvature; LN: + Poorly cohesive NOS Diffuse Loss of MLH1, PMS2 TIL: Marked Desmoplasia: Mild H. pylori: NChronic gastritis: A IM: AAtrophy: A NGS: PIK3CA, FGFR2 mt, HER2neu: N On immunotherapy; stable at 2 years;anastomotic site developed recurrence
Patient 3 F/56 Body Ulcerated growth/ wall thickening in body; LN: + Poorly cohesive NOS Diffuse Loss of PMS2 TIL: Marked Desmoplasia: Moderate H. pylori:N Chronic gastritis: P IM: AAtrophy: P PDL1 – TPS Score: 30%; HER2neu: N Palliative chemotherapy; stable at 2 years follow-up
Patient 4 M/59 Body along greater curvature Infiltrating growth/PET-CT: irregular heterogeneously enhancing wall thickening; Omental nodule+ Tubular moderately differentiated Intestinal Loss of MLH1, PMS2 TIL: Moderate Desmoplasia: Mild H. pylori: NChronic gastritis: A IM: A; Atrophy: A PDL-TPS: 5%HER 2 neu: N Palliative chemotherapy, immunotherapy; partial response
Patient 5 F/50 Body along greatercurvature Ulcerated growth 5 cm/ wall thickening;LN: peri-gastric, hepato-gastric, mesentery of epigastrium and along coeliac and left gastric regions Tubular, poorly differentiated Indeterminate Loss of MLH1, PMS2 TIL: Marked Desmoplasia: Mild H. pylori: N Chronic gastritis: PIM: A; Atrophy: A Nil Radical radiotherapy
Patient 6 F/47 Body & antrum Infiltrative growth Poorly cohesive NOS Diffuse Loss of MLH1, PMS2 TIL: Moderate Desmoplasia: Moderate H. pylori: NChronic gastritis: P IM: PAtrophy: P Candida colonization+ Palliative chemotherapy

Abbreviations, MMR-d GC, mismatch repair-deficient gastric adenocarcinoma; CECT, contrast-enhanced computed tomogram; WHO, World health organization. PC (NOS), Poorly cohesive not otherwise specified; TILs, tumor infiltrating lymphocytes; NA, Not assessed; P, present; N, negative; A, absent.

There was female predominance with median age of 53 (26-59) years. All the tumors were in the distal part (body) of the stomach. The WHO category was poorly cohesive (NOS) in three, tubular adenocarcinoma (one each of well, moderately and poorly differentiated) in three. According to Lauren classification, the GC was intestinal in 2, indeterminate in one and diffuse in 3. The stroma showed marked lymphocytic infiltration in 4; moderate desmoplasia in 2 and adjacent mucosa showed chronic gastritis in 04, atrophy in 02 and IM in 01. H.pylori was seen in 01 sample.

Loss of MMR expression was observed in 06 (8%) patients. The loss of expression was seen as heterodimers: loss of MLH1+PMS2 in 04 (66.67%) and loss of MSH2+MSH6 in 01 (16.67%). Isolated loss of PMS2 was seen in 01 (16.67%). EBER-ISH was negative in all 6 patients, which were MMR deficient.

<italic id="italic-687e99d5c0ac7546cc30486175d232a6">Follow-up<italic id="italic-456aee61ac52420820d7b46f33723abf"/></italic>

All the patients were diagnosed with locally advanced disease with regional lymphadenopathy. One patient had significant family history (mother had endometrial carcinoma). Three patients got investigated further; Her2Neu was negative in all 3; PDLI score was 30% and 5% in two patients tested and one showed partial response; next generation sequencing (NGS) done in one patient showed mutations of PIK3CA, FGFR2. Three patients received palliative chemotherapy, one received radical radiotherapy and one had local recurrence. All patients were alive at 1 year follow-up.

<italic id="italic-0f735f6b03ba311a35c99e9a5119eb79">EBVaGC and MMR-d GC<italic id="italic-98ea41be6c5a1dcd09bfcee8546ed08f"/></italic>

The two molecularly defined GC in the present study, EBVaGC and MMR-d GC were mutually exclusive.

There was no statistically significant difference in the clinicopathological variables between EBVaGC and EBVnGC as well as MMR-d GC and MMR-p GC (Table 4).

Parameter EBVaGC (n=03) EBVnGC p value
    (n=94)  
Age (median) years 67 (51, 67, 77) 53.4  
Gender (M: F) 02:01 1.54:1 0.83 (Chi square test)
Site      
Proximal 2 38  
Distal 1 56 0.567 (Fisher exact test)
Adenocarcinoma histologic type (WHO)      
Tubular 1 28 0.564 (Chi square test)
Papillary 0 9  
Mixed 0 11  
Poorly cohesive SRC 0 22  
Poorly cohesive NOS 2 26  
Adenocarcinoma histologic type (Lauren)      
Intestinal 1 29 0.85 (Chi square test))
Indeterminate 0 13  
Diffuse 2 46  
Mixed 0 6  
Tumor stroma      
TIL Mild: 0 Mild: 44 0.158 (Fisher exact test)
  Moderate: 01 Moderate: 24  
  Marked: 02 Marked: 26  
Desmoplasia Mild: 03 Mild:54  
  Moderate: 0 Moderate: 29 0.686 (Fisher exact test)
  Marked: 0 Marked: 11  
Adjacent mucosa      
Chronic gastritis 1/2 52/82 0.697 (Chi square test)
Atrophy 1/2 38/82 0.918 (Chi square test)
Intestinal metaplasia 1/3 51/87 0.38 (Chi square test)
H.pylori 1/3 22/87 0.75 (Chi square test)

Abbreviations, MMR-d GC, mismatch repair-deficient gastric adenocarcinoma; MMR-p GC, mismatch repair-proficient gastric adenocarcinoma; CECT, contrast-enhanced computed tomogram; WHO, World health organization; PC(NOS), Poorly cohesive not otherwise specified; TILs, tumor infiltrating lymphocytes; NA, Not assessed; P, present; N, negative; A, absent

Though statistically significant differences were not seen, EBVaGC was more frequently seen in males, in proximal part of stomach with poorly cohesive (NOS) histology and MMR-d GC was more frequently seen in females and distal part of the stomach with equal frequency of poorly cohesive (NOS) and tubular adenocarcinoma. Poorly cohesive (SRC), papillary, mixed and mucinous histology were not seen in both. Both showed moderate to marked tumor infiltrating lymphocytes and minimal to moderate desmoplasia. H.pylori was seen in one sample each with EBVaGC and MMR-d GC. Both were diagnosed with locally advanced disease with regional lymphadenopathy.

Discussion

The present study of GC from a tertiary cancer hospital in south India shows a male predominance with a median age of 57 years and a third (32.9%) of the patients in the age group of 51 to 60 years. The most frequent (58.76%) site of involvement was the distal part of the stomach.

These findings were in agreement with other studies from India and other parts of the world [4, 10, 24-28]. Non-cardia GC was reported to be more commonly associated with H. pylori infection and it is a known causative factor for gastric cancer; H pylori induced chronic inflammation leads to chronic gastritis, followed by atrophic gastritis, IM, dysplasia and invasive tumor (intestinal-type GC according to Lauren classification) [29]. With better prevention of H. pylori infection, the proportion of non-cardia GC are declining and the number of cardia GC is on the rise [4, 27, 30]. H. pylori was seen in 25.56% samples in the present study. Adjacent mucosa showed chronic gastritis in 61.9%, atrophy in 45.2%, IM in 53.6%; however, dysplasia was not identified in any sample. Although infection with H. pylori is very common worldwide, only a small fraction of infected individuals develops GC, implying the role of other factors like smoking, alcohol, environmental and genetic factors [17, 31].

Classification of GC was done according to both Lauren and WHO classifications. The most common histology in the present study was poorly cohesive (WHO)/ diffuse (Lauren) type. Differentiated tubular (WHO)/ intestinal (Lauren)/type was reported to be more common [4]; however, relative proportion depends on the risk factors in a particular geographical region.

Association of EBV with GC: EBV is a ubiquitous herpes virus and infects approximately 90% of the world’s population with no endemic areas. It is well documented to be causally associated with various malignant tumors, including gastric carcinoma. It increases the risk of gastric cancer by 18 times [5, 32, 33]. TCGA proposed a classification based on molecular profiling, which included gastric tumors positive for EBV as a distinct entity [12]. EBVaGC is defined by the presence of EBV in neoplastic cells. The different methods to detect EBV include ISH, different types of PCR assays, and IHC. EBV-encoded small RNAs (EBER1/EBER2) are abundantly produced in the nucleus of latently infected cells (10 6–7 copies per cell) and nearly all cancer cells are positive for EBER-ISH, while adjacent nonneoplastic gastric epithelial cells and infiltrating lymphocytes, other normal stromal cells and foci of intestinal metaplasia are negative [34-38]. Hence, EBER-ISH is considered the gold standard technique for the detection of EBV in tissues with high sensitivity and specificity [34-37]. Tavakoli et al reported that EBV was detected more frequently in biopsy samples than in FFPE specimens from surgically resected GC (2.4-fold, P < 0.0001) due to the low yield of extracted nucleic acids and fragmentation of genomes and transcripts resulting from the processing and hence the authors recommended using biopsy samples to prevent false-negative results.5 In the present study, majority (87/97) were endoscopic biopsies and EBER-ISH was used for the detection of EBV. However, intra-tumoral heterogeneity may hamper detection of EBV status in biopsy samples [39].

The reported frequency of EBER positivity varied from 1.7-10% in various series [5, 10, 13, 20, 40-49]. Tavakoli et al in a meta-analysis, showed that the pooled prevalence of EBV in 20,361 gastric cancer patients was 8.77% (95% CI: 7.73–9.92%; I2 = 83.2%) [5]. Frequency of EBER in our study was 3.09%. The intensity of staining (low to strong) and number of cells stained positive (5 to >80%) were reported to vary [20]. Majority cells with strong intensity of positivity only were considered positive in the present study. However, there is no a clear cut-off for defining positivity in the ISH study for EBER, and most published studies consider the nuclear staining in tumor cells as positive without any reference to the intensity or the percentage of cells [50-52]. The EBV genome in patients with GC is reported to vary in different geographical regions; however, when meta-analysis was restricted to studies that used EBER-ISH for EBV detection, no appreciable difference was seen in geographical prevalence rates and hence it was attributed to method of detection rather than geographical differences [5, 26, 53, 54]. An even higher positivity rate has been reported, when EBV was detected by RNA-Seq instead of traditional EBER1/2 in situ hybridization [55].

EBVaGC is reported to have distinct clinicopathological features; more predominant in men, tends to be located proximally, often a diffuse histologic subtype, and shows a lower frequency of lymph node metastasis than conventional adenocarcinoma [10, 33, 45, 47, 48, 52, 54, 56-58]. Though the number of patients with EBVaGC was small in the present study, 2/3 patients were male and the tumor was in the proximal part of stomach with poorly cohesive NOS/diffuse histology and lymphocytic infiltration in the stroma. Camargo et al. observed that the average age of EBV-positive gastric cancer patients was 58 years old, and 71% of them were men [47]. EBVaGC in its early stage shows a characteristic histology called a ‘lace pattern’ in an intramucosal lesion, which shows irregularly anastomosing tubules and cords associated with moderate to dense lymphocytic infiltration as seen in one of the samples. When this pattern is observed in biopsy specimens, EBER-ISH is recommended for diagnostic purposes [36]. Apart from poorly differentiated/diffuse histology, lymphoepithelioma-like histology and gastric carcinoma with lymphoid stroma were reported to be frequently associated with EBV [57, 59]. The patient who had tumor in distal part of the stomach showed tubular/intestinal histology and was associated with chronic gastritis and H. pylori in the adjacent mucosa. Simultaneous infection with EBV and H pylori can occur and infection with H pylori is related to the occurrence and development of EBV-positive gastric cancer [56]. Similar findings were reported earlier [60, 61]. The distribution of histological subtypes of gastric cancer and the frequencies of H. pylori and EBV associated gastric cancer vary across the globe [62]. EBVaGC were reported to have lymphoid stroma and minimal fibrosis as seen in the present study[63]. Prominent inflammatory infiltrate, particularly CD8-positive or CD4-positive T cells accompanied by CD68-positive histiocytes in the tumor is one of the characteristic features of EBVaGC [36]. This feature reflects the immunogenicity of EBV. EBVaGC was found to express high levels of PD-L1 in cancer and infiltrating immune cells [64, 65]. EBVaGC is reported to have less lymph node involvement and favorable outcome[66]. All three patients in the present study had locally advanced disease with lymph node involvement. It is probably related to presentation at an advanced stage. There was no statistically significant difference between EBVaGC and EBVnGC. Similar observations were made earlier [8, 9, 20, 59].

Tumors positive for EBV, display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also known as PD-L2) and EBVaGCs seem to be particularly suitable for an anti-PD-L1/PD-1 immune therapy [39, 56]. Thus, detecting EBVaGCs may have clinical implications for considering immune checkpoint therapy [39, 56].

MMR-d GC: Carcinogenesis being a multistep process driven by multiple genetic and epigenetic changes, genetic instability is an important factor. Microsatellites (MS) are short tandem repeats (1–6 nucleotides) scattered through the whole genome, that are prone to a high mutation rate [67]. MSI results from abnormal function of one or more mismatch repair genes (MLH1, PMS2, MSH2, MSH6). Across the gastrointestinal tract, the role of MSI in tumorigenesis has been studied more often in colorectal cancer; however, MSI is one of the pathways implicated in gastric carcinoma. The molecular classification by both TCGA and ACRG identify the MSI subgroup as a specific and well-defined GC entity, associated with favorable outcome [67].

MSI evaluation may be done by IHC, PCR (Fluorescent multiplex PCR and capillary electrophoresis (CE), NGS, single-molecule molecular inversion probes (smMIPs) [68, 69]. PCR testing currently is the most direct, accurate, and cost-effective measurement method. IHC detection of MMR proteins is based on specific antibody recognition of MLH1, MSH2, MSH6, and PMS2 in tumor cell nuclei and is simple, rapid, inexpensive, and requires minimal specialized instrumentation. MSI PCR testing and MMR IHC show an excellent concordance [20]. MMR protein IHC was done in the present study. All samples were treatment-naïve (endoscopy biopsies and resection specimens prior to NACT) as tumors from patients exposed to preoperative chemotherapy or radiation therapy are more difficult to assess using IHC due to artifactual loss of MSH6 protein expression. Disadvantage for IHC is that it does not cover all MMR genes and sample large enough to perform IHC for all the proteins. Hence IHC for MMR proteins could be performed on 75 samples only. However, both techniques can be considered to be equally proficient tests for establishing MMR/MSI status, when there is awareness of the potential pitfalls of either method [70]. Martinez-Ciarpaglini et al showed the sensitivity, specificity, positive predictive value and negative predictive value of the MMR immunostaining for the MSI status were 91%, 98%, 91% and 98%, respectively [20]. MSI status was divided into three categories: MSI-High (MSI-H), MSI-Low (MSI-L), or MS-Stable

(MSS). In general, MMR-d is considered equivalent to MSI-High21. Currently, it is recommended to classify the tumors as MSI (MMR-d) and MSS (including MSI-L and MS-stable) [71].

The frequency of MSI varies from 8.2–37% [20, 32, 48, 72, 73]. In a meta-analysis of 48 studies with a total of 18 612 patients, MSI was found in 9·2% of patients [74]. In the present study, MMR-d GC constituted 8%. The loss of expression was seen as heterodimers: loss of MLH1+PMS2 in 04/06 (66.67%) and loss of MSH2+MSH6 in 01/06 (16.67%). Isolated loss of PMS2 was seen in one (16.67%). Martinez-Ciarpaglini et al reported 18% of GC to be MMR-d by IHC which showed loss of MLH1+PMS2 in 91% and loss of MSH2+MSH6 in 9% [20]. Tsai et al in a series of 329 GC, reported PMS2/ MLH1-deficiency in12% and MSH2+MSH6 in none.10 Most MSI-GCs demonstrate loss of MLH1-expression as seen in the present study [10, 20, 64].

GC with MSI constitutes a small but relevant subgroup associated with older age, female sex, distal stomach location, and lower number of lymph-node metastases [48, 73]. In the present study, there was predominance with median age being 58 years. The tumor was predominantly located in the body in all patients. These features were in agreement with other studies [20, 48, 73] Marked to moderate lymphocytic infiltrate was seen in all the tumors. Lymph node involvement/omental nodules were present at the time of diagnosis. MSI status is reported to be associated with a better overall survival across all classifications in resectable stages of GC [48, 67, 74].

The molecular sub-typing has therapeutic implications, indicating a potential individulized treatment. The present study showed that both EBVaGC and MSI status were mutually exclusive subtypes associated with distinct clinicopathological characteristics. EBVaGC with a low mutation burden is a subset of MSS-GC and may respond to immune checkpoint therapy [20, 56]. The hypermutated nature of sporadic MSI-GC and the amplification of PD-L1 in EBVaGC make them liable for immune checkpoint blockade [74-77]. Both EBV and MSI status can be incorporated in the routine laboratory evaluation of GC, which would lead to significant progress in the treatment of gastric cancer.

Summary box: Molecular sub typing has potential benefit for individual treatment. Both EBV-associated and MSI-H-GC are considered to have better prognosis and respond to immunotherapy and hence have the possibility to be useful as biomarkers for patient stratification. EBVaGC and MMR-d GC were mutually exclusive in our study. There was no statistically significant difference in the clinicopathological variables between EBVaGC and EBVnGC as well as MMR-d GC and MMR-p GC.

Acknowledgments

Statement of Transparency and Principals:

• Author declares no conflict of interest

• Study was approved by Research Ethic Committee of author affiliated Institute.

• Study’s data is available upon a reasonable request.

• All authors have contributed to implementation of this research.

References 3 05 209-249 71 2021 10.3322/caac.21660 Sung H Ferlay J Siegel RL Laversanne M Soerjomataram I Jemal A Bray F CA: a cancer journal for clinicians Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries 17 10125 03 1023-1075 391 2018 10.1016/S0140-6736(17)33326-3 Allemani C Matsuda T Di Carlo V Harewood R Matz M Nikšić M Bonaventure A et al Lancet (London, England) Global surveillance of trends in cancer survival 2000-14 (CONCORD-3): analysis of individual records for 37 513 025 patients diagnosed with one of 18 cancers from 322 population-based registries in 71 countries 11 11 1493-1505 20 2019 10.1016/S1470-2045(19)30456-5 Arnold M Rutherford MJ Bardot A Ferlay J Andersson TML Myklebust TA Tervonen H et al The Lancet. Oncology Progress in cancer survival, mortality, and incidence in seven high-income countries 1995-2014 (ICBP SURVMARK-2): a population-based study 1 26-38 14 2019 10.5114/pg.2018.80001 Rawla P Barsouk A Przegla̜d Gastroenterologiczny Epidemiology of gastric cancer: global trends, risk factors and prevention 01 1 06 493 20 2020 10.1186/s12885-020-07013-x Tavakoli A Monavari SH Solaymani Mohammadi F Kiani SJ Armat S Farahmand M BMC cancer Association between Epstein-Barr virus infection and gastric cancer: a systematic review and meta-analysis 15 2 01 487-490 136 2015 10.1002/ijc.28999 Plummer M Franceschi S Vignat J Forman D Martel C International Journal of Cancer Global burden of gastric cancer attributable to Helicobacter pylori 6 11 394-424 68 2018 10.3322/caac.21492 Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A CA: a cancer journal for clinicians Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries 31-49 64 1965 10.1111/apm.1965.64.1.31 Lauren P. Acta Pathologica Et Microbiologica Scandinavica The Two Histological Main Types Of Gastric Carcinoma: Diffuse And So-Called Intestinal-Type Carcinoma. An Attempt At A Histo-Clinical Classification 2 01 182-188 76 2020 10.1111/his.13975 Nagtegaal ID Odze RD Klimstra D Paradis V Rugge M Schirmacher P Washington KM Carneiro F Cree IA Histopathology The 2019 WHO classification of tumours of the digestive system 8 08 1017-1030 44 2020 10.1097/PAS.0000000000001521 Tsai J Jeng Y Chen K Lee C Yuan C Liau J The American Journal of Surgical Pathology An Integrative Morphomolecular Classification System of Gastric Carcinoma With Distinct Clinical Outcomes 15 7 07 156-169 4 2012 10.4251/wjgo.v4.i7.156 Nagini S World Journal of Gastrointestinal Oncology Carcinoma of the stomach: A review of epidemiology, pathogenesis, molecular genetics and chemoprevention 11 7517 09 202-209 513 2014 10.1038/nature13480 Cancer Genome Atlas Research Network Nature Comprehensive molecular characterization of gastric adenocarcinoma 5 05 449-456 21 2015 10.1038/nm.3850 Cristescu R Lee J Nebozhyn M Kim K Ting JC Wong SS Liu J et al Nature Medicine Molecular analysis of gastric cancer identifies subtypes associated with distinct clinical outcomes 6 12 275-282 12 2019 10.14740/gr1187 Wang Q Liu G Hu C Gastroenterology Research Molecular Classification of Gastric Adenocarcinoma 7 07 772-784 29 2016 10.1038/modpathol.2016.55 Setia N Agoston AT Han HS Mullen JT Duda DG Clark JW Deshpande V et al Modern Pathology: An Official Journal of the United States and Canadian Academy of Pathology, Inc A protein and mRNA expression-based classification of gastric cancer 1 01 106-115 41 2017 10.1097/PAS.0000000000000756 Ahn S Lee S Kim Y Kim A Shin N Choi KU Lee C et al The American Journal of Surgical Pathology High-throughput Protein and mRNA Expression-based Classification of Gastric Cancers Can Identify Clinically Distinct Subtypes, Concordant With Recent Molecular Classifications 14 8 04 1878 13 2021 10.3390/cancers13081878 Palrasu M Zaika E El-Rifai W Que J Zaika AI Cancers Role of Bacterial and Viral Pathogens in Gastric Carcinogenesis 14 6 02 1503-1509 20 2014 10.3748/wjg.v20.i6.1503 Misra V Pandey R Misra SP Dwivedi M World Journal of Gastroenterology Helicobacter pylori and gastric cancer: Indian enigma 2021 de Sousa HML Ribeiro JPC Timóteo MB Epstein-Barr Virus-Associated Gastric Cancer: Old Entity with New Relevance. In: Drouet, E. , editor. Epstein-Barr Virus - New Trends [Internet]. London: IntechOpen 3 e000470 4 2019 10.1136/esmoopen-2018-000470 Martinez-Ciarpaglini C Fleitas-Kanonnikoff T Gambardella V Llorca M Mongort C Mengual R Nieto G et al ESMO open Assessing molecular subtypes of gastric cancer: microsatellite unstable and Epstein-Barr virus subtypes. Methods for detection and clinical and pathological implications 15 22 11 5248-5257 58 1998 5555 Boland C. R. Thibodeau S. N. Hamilton S. R. Sidransky D. Eshleman J. R. Burt R. W. Meltzer S. J. et al Cancer Research A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer 01 15 08 4441-4449 23 2017 10.1158/1078-0432.CCR-16-2211 Sohn BH Hwang J Jang H Lee H Oh S Shim J Lee K et al Clinical Cancer Research: An Official Journal of the American Association for Cancer Research Clinical Significance of Four Molecular Subtypes of Gastric Cancer Identified by The Cancer Genome Atlas Project 19 41 09 69888-69905 8 2017 10.18632/oncotarget.19435 Kim Y Cho M Kim J Kim SN Oh SC Lee K Oncotarget Profiling cancer-associated genetic alterations and molecular classification of cancer in Korean gastric cancer patients 1 01 12-16 32 2011 10.4103/0971-5851.81884 Sharma A Radhakrishnan V Indian Journal of Medical and Paediatric Oncology: Official Journal of Indian Society of Medical & Paediatric Oncology Gastric cancer in India 2 04 142-147 5 2014 10.3978/j.issn.2078-6891.2014.003 Barad AK Mandal SK Harsha HS Sharma BM Singh TS Journal of Gastrointestinal Oncology Gastric cancer-a clinicopathological study in a tertiary care centre of North-eastern India 3 11 384-389 68 2002 10.1002/jmv.10215 Kattoor J Koriyama C Akiba S Itoh T Ding S Eizuru Y Abraham EK Chandralekha B. Amma N. S. Nair MK Journal of Medical Virology Epstein-Barr virus-associated gastric carcinoma in southern India: A comparison with a large-scale Japanese series 3 03 534-542 18 2020 10.1016/j.cgh.2019.07.045 Thrift AP El-Serag HB Clinical Gastroenterology and Hepatology: The Official Clinical Practice Journal of the American Gastroenterological Association Burden of Gastric Cancer 05 101404 47 2022 10.1016/j.eclinm.2022.101404 Morgan E Arnold M Camargo MC Gini A Kunzmann AT Matsuda T Meheus F et al EClinicalMedicine The current and future incidence and mortality of gastric cancer in 185 countries, 2020-40: A population-based modelling study 01 13 07 3554-3560 48 1988 555 Correa P. Cancer Research A human model of gastric carcinogenesis 07 21 06 2901-2912 13 2007 10.3748/wjg.v13.i21.2901 Rocco A Nardone G World Journal of Gastroenterology Diet, H pylori infection and gastric cancer: evidence and controversies 07 9 03 1346-1351 12 2006 10.3748/wjg.v12.i9.1346 Singh K Ghoshal UC World Journal of Gastroenterology Causal role of Helicobacter pylori infection in gastric cancer: an Asian enigma 2 04 150-157 49 2016 10.1590/0037-8682-0270-2015 Jácome AADA Lima EM Kazzi AI Chaves GF Mendonça DC Maciel MM Santos JSD Revista Da Sociedade Brasileira De Medicina Tropical Epstein-Barr virus-positive gastric cancer: a distinct molecular subtype of the disease? 3 03 353-365 476 2020 10.1007/s00428-019-02724-4 Fukayama M Abe H Kunita A Shinozaki-Ushiku A Matsusaka K Ushiku T Kaneda A Virchows Archiv: An International Journal of Pathology Thirty years of Epstein-Barr virus-associated gastric carcinoma 5 11 1250-1254 143 1993 Tokunaga M. Land C. E. Uemura Y. Tokudome T. Tanaka S. Sato E. The American Journal of Pathology Epstein-Barr virus in gastric carcinoma 5 05 337-350 60 2010 10.1111/j.1440-1827.2010.02533.x Fukayama M Pathology International Epstein-Barr virus and gastric carcinoma 4 04 1421-1434 46 2015 10.3892/ijo.2015.2856 Shinozaki-Ushiku A Kunita A Fukayama M International Journal of Oncology Update on Epstein-Barr virus and gastric cancer (review) 29 6 05 167 10 2018 10.3390/cancers10060167 Nishikawa J Iizasa H Yoshiyama H Shimokuri K Kobayashi Y Sasaki S Nakamura M et al Cancers Clinical Importance of Epstein⁻Barr Virus-Associated Gastric Cancer 5 10 255-261 53 2000 10.1136/mp.53.5.255 Takada K. Molecular pathology: MP Epstein-Barr virus and gastric carcinoma 01 5 05 1005-1014 28 2017 10.1093/annonc/mdx047 Böger C. Krüger S. Behrens H. M. Bock S. Haag J. Kalthoff H. Röcken C. Annals of Oncology: Official Journal of the European Society for Medical Oncology Epstein-Barr virus-associated gastric cancer reveals intratumoral heterogeneity of PIK3CA mutations 15 4 02 664-670 22 2004 10.1200/JCO.2004.08.061 Beek J Hausen A Klein Kranenbarg E Velde CJH Middeldorp JM Brule AJC Meijer CJLM Bloemena E Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology EBV-positive gastric adenocarcinomas: a distinct clinicopathologic entity with a low frequency of lymph node involvement 04 5 03 702-704 86 2002 10.1038/sj.bjc.6600107 Burgess D. E. Woodman C. B. Flavell K. J. Rowlands D. C. Crocker J. Scott K. Biddulph J. P. Young L. S. Murray P. G. British Journal of Cancer Low prevalence of Epstein-Barr virus in incident gastric adenocarcinomas from the United Kingdom 21 39 10 6096-6103 11 2005 10.3748/wjg.v11.i39.6096 Herrera-Goepfert R Akiba S Koriyama C Ding S Reyes E Itoh T Minakami Y Eizuru Y World Journal of Gastroenterology Epstein-Barr virus-associated gastric carcinoma: Evidence of age-dependence among a Mexican population 11 11 767-770 17 2014 Faghihloo E Saremi MR Mahabadi M Akbari H Saberfar E Archives of Iranian Medicine Prevalence and characteristics of Epstein-Barr virus-associated gastric cancer in Iran 9 09 1001-1007 21 2009 10.1097/MEG.0b013e32831f1f53 Trimeche M Ksiâa F Ziadi S Mestiri S Hachana M Gacem RB Sriha B Korbi S European Journal of Gastroenterology & Hepatology Prevalence and characteristics of Epstein-Barr virus-associated gastric carcinomas in Tunisia 3 09 824-833 137 2009 10.1053/j.gastro.2009.05.001 Murphy G Pfeiffer R Camargo MC Rabkin CS Gastroenterology Meta-analysis shows that prevalence of Epstein-Barr virus-positive gastric cancer differs based on sex and anatomic location 4 04 769-774 140 1992 555555 Shibata D. Weiss L. M. The American Journal of Pathology Epstein-Barr virus-associated gastric adenocarcinoma 2 04 676-681 19 2016 10.1007/s10120-015-0508-x Camargo MC Bowlby R Chu A Pedamallu CS Thorsson V Elmore S Mungall AJ Bass AJ Gulley ML Rabkin CS Gastric Cancer: Official Journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association Validation and calibration of next-generation sequencing to identify Epstein-Barr virus-positive gastric cancer in The Cancer Genome Atlas 9980410 2021 2021 10.1155/2021/9980410 Nshizirungu JP Bennis S Mellouki I Sekal M Benajah D Lahmidani N El Bouhaddouti H et al Disease Markers Reproduction of the Cancer Genome Atlas (TCGA) and Asian Cancer Research Group (ACRG) Gastric Cancer Molecular Classifications and Their Association with Clinicopathological Characteristics and Overall Survival in Moroccan Patients 2 02 e15-e21 26 2018 10.1097/PAI.0000000000000575 Chang L Chang M Chang HM Chang F Applied immunohistochemistry & molecular morphology: AIMM Microsatellite Instability: A Predictive Biomarker for Cancer Immunotherapy 7 07 641-651 16 2003 10.1097/01.MP.0000076980.73826.C0 Grogg KL Lohse CM Pankratz VS Halling KC Smyrk TC Modern Pathology: An Official Journal of the United States and Canadian Academy of Pathology, Inc Lymphocyte-rich gastric cancer: associations with Epstein-Barr virus, microsatellite instability, histology, and survival 3 09 197-211 455 2009 10.1007/s00428-009-0813-z Solcia E Klersy C Mastracci L Alberizzi P Candusso ME Diegoli M Tava F et al Virchows Archiv: An International Journal of Pathology A combined histologic and molecular approach identifies three groups of gastric cancer with different prognosis 4 10 1041-1051 19 2016 10.1007/s10120-015-0565-1 Park JH Kim EK Kim YH Kim J Bae YS Lee YC Cheong J Noh SH Kim H Gastric Cancer: Official Journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association Epstein-Barr virus positivity, not mismatch repair-deficiency, is a favorable risk factor for lymph node metastasis in submucosa-invasive early gastric cancer 6 583-590 15 2011 Shukla SK Prasad K. N. Tripathi A Singh A Saxena A Ghoshal UC Krishnani N Husain N The Brazilian Journal of Infectious Diseases: An Official Publication of the Brazilian Society of Infectious Diseases Epstein-Barr virus DNA load and its association with Helicobacter pylori infection in gastroduodenal diseases 5 212-223 82 2015 10.1159/000434683 Abe H Kaneda A Fukayama M Pathobiology: Journal of Immunopathology, Molecular and Cellular Biology Epstein-Barr Virus-Associated Gastric Carcinoma: Use of Host Cell Machineries and Somatic Gene Mutations 3 03 455-467 10 2020 10.1002/2211-5463.12803 Sadato D Ogawa M Hirama C Hishima T Horiguchi S Harada Y Shimoyama T et al FEBS open bio Potential prognostic impact of EBV RNA-seq reads in gastric cancer: a reanalysis of The Cancer Genome Atlas cohort 583463 10 2020 10.3389/fonc.2020.583463 Sun K Jia K Lv H Wang S Wu Y Lei H Chen X Frontiers in Oncology EBV-Positive Gastric Cancer: Current Knowledge and Future Perspectives 10 10 1738-1744.e2 13 2015 10.1016/j.cgh.2015.04.015 Lim H Park YS Lee JH Son DH Ahn JY Choi K Kim DH et al Clinical Gastroenterology and Hepatology: The Official Clinical Practice Journal of the American Gastroenterological Association Features of Gastric Carcinoma With Lymphoid Stroma Associated With Epstein-Barr Virus 3 03 354-365 24 2009 10.1111/j.1440-1746.2009.05775.x Lee J Kim S Han S An J Lee E Kim Y Journal of Gastroenterology and Hepatology Clinicopathological and molecular characteristics of Epstein-Barr virus-associated gastric carcinoma: a meta-analysis 3 07 852-859 19 2016 10.1007/s10120-015-0524-x Min B Tae CH Ahn SM Kang SY Woo S Kim S Kim K Gastric Cancer: Official Journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association Epstein-Barr virus infection serves as an independent predictor of survival in patients with lymphoepithelioma-like gastric carcinoma 1 07 39-43 29 1999 10.1097/00004836-199907000-00010 Yanai H. Murakami T. Yoshiyama H. Takeuchi H. Nishikawa J. Nakamura H. Okita K. et al Journal of Clinical Gastroenterology Epstein-Barr virus-associated gastric carcinoma and atrophic gastritis 2-3 87-94 34 2003 10.1385/IJGC:34:2-3:087 Hirano A Yanai H Shimizu N Okamoto T Matsubara Y Yamamoto K Okita K International Journal of Gastrointestinal Cancer Evaluation of epstein-barr virus DNA load in gastric mucosa with chronic atrophic gastritis using a real-time quantitative PCR assay 01 3 08 666-673 125 2009 10.1002/ijc.24290 Bertuccio P Chatenoud L Levi F Praud D Ferlay J Negri E Malvezzi M La Vecchia C International Journal of Cancer Recent patterns in gastric cancer: a global overview 9 10 633-645 23 2015 10.1097/PAI.0000000000000122 Speck O Tang W Morgan DR Kuan PF Meyers MO Dominguez RL Martinez E Gulley ML Applied immunohistochemistry & molecular morphology: AIMM Three Molecular Subtypes of Gastric Adenocarcinoma Have Distinct Histochemical Features Reflecting Epstein-Barr Virus Infection Status and Neuroendocrine Differentiation 1 01 467-481 149 2023 10.1007/s00432-022-04408-0 Röcken C. Journal of Cancer Research and Clinical Oncology Predictive biomarkers in gastric cancer 31 22 05 32925-32932 7 2016 10.18632/oncotarget.9076 Derks S Liao X Chiaravalli AM Xu X Camargo MC Solcia E Sessa F et al Oncotarget Abundant PD-L1 expression in Epstein-Barr Virus-infected gastric cancers 3 09 173-177 54 2018 10.4068/cmj.2018.54.3.173 Baek DW Kang BW Kim JG Chonnam Medical Journal The Predictive Value of Epstein-Barr Virus-Positivity in Patients Undergoing Gastrectomy Followed by Adjuvant Chemotherapy 04 102175 95 2021 10.1016/j.ctrv.2021.102175 Puliga E Corso S Pietrantonio F Giordano S Cancer Treatment Reviews Microsatellite instability in Gastric Cancer: Between lights and shadows 18 1 06 12880 11 2021 10.1038/s41598-021-91974-x Dedeurwaerdere F Claes KB Van Dorpe J Rottiers I Van der Meulen J Breyne J Swaerts K Martens G Scientific Reports Comparison of microsatellite instability detection by immunohistochemistry and molecular techniques in colorectal and endometrial cancer 16 20 2020 10.1186/s12935-019-1091-8 Li K Luo H Huang L Luo H Zhu X Cancer Cell International Microsatellite instability: a review of what the oncologist should know 3 02 401-413 78 2021 10.1111/his.14233 Loughrey MB McGrath J Coleman HG Bankhead P Maxwell P McGready C Bingham V et al Histopathology Identifying mismatch repair-deficient colon cancer: near-perfect concordance between immunohistochemistry and microsatellite instability testing in a large, population-based series 4931-4940 19 2021 10.1016/j.csbj.2021.08.037 Yu F Makrigiorgos A Leong KW Makrigiorgos GM Computational and Structural Biotechnology Journal Sensitive detection of microsatellite instability in tissues and liquid biopsies: Recent developments and updates 3 09 147-157 6 2020 10.1016/j.cdtm.2020.03.004 Zhu Z Gong Y Xu H Chronic Diseases and Translational Medicine Neoadjuvant therapy strategies for advanced gastric cancer: Current innovations and future challenges 22 11 4151-4162 75 2018 10.1007/s00018-018-2906-9 Ratti M Lampis A Hahne JC Passalacqua R Valeri N Cellular and molecular life sciences: CMLS Microsatellite instability in gastric cancer: molecular bases, clinical perspectives, and new treatment approaches 3 02 159-167 105 2018 10.1002/bjs.10663 Polom K. Marano L. Marrelli D. De Luca R. Roviello G. Savelli V. Tan P. Roviello F. The British Journal of Surgery Meta-analysis of microsatellite instability in relation to clinicopathological characteristics and overall survival in gastric cancer 8 1460-1465 8 2017 10.7150/jca.18470 Kim BJ Jang HJ Kim HS Kim JH Journal of Cancer Current Status of Immune Checkpoint Inhibitors in Gastrointestinal Cancers 9 09 2510-2515 16 2009 10.1245/s10434-009-0580-8 Oki E Kakeji Y Zhao Y Yoshida R Ando K Masuda T Ohgaki K Morita M Maehara Y Annals of Surgical Oncology Chemosensitivity and survival in gastric cancer patients with microsatellite instability 15 2 07 505-511 131 2012 10.1002/ijc.26399 An JY J Kim H Cheong J Hyung WJ Kim H Noh SH International Journal of Cancer Microsatellite instability in sporadic gastric cancer: its prognostic role and guidance for 5-FU based chemotherapy after R0 resection