Molecular classification of gastric cancer based on immunohistochemistry and in-situ hybridization, determination of the role of tropomyosin receptor kinase (TRK) signaling pathway in gastric cancer classification and their association with clinicopathological parameters

Aim: The aims of this study are; 1) to reclassify primary gastric adenocarcinoma (G-ACa), by using more common, accessible and cost-effective techniques such as immunohistochemistry (IHC) and in- situ hybridization (ISH) in accordance with the identified molecular subtypes, to evaluate their distribution in our population, to evaluate their morphological reflections and their relationship with the clinical and pathological parameters; 2) to determine the ratio of NTRK gene fusion in G-ACa by IHC (pan-NTRK) method, to investigate its relationship with our molecular classification subtypes and to investigate its independent prognostic role in individual subtypes. Material and Metods: A total number of 240 gastrectomy specimens, which had been diagnosed as primary G-ACa in the Pathology Department of Marmara University School of Medicine, between 2013 and 2018 were included in the study. For tissue microarray (TMA) analysis, 4 samples, which are thought to best represent the tumor in tumor areas of different morphology, were placed in 3 mm diameter cores in the recipient blocks. In our study, 240 G-ACa cases were divided into five groups by EBER-chromogenic ISH (CISH) as well as, MLH1, MSH2, MSH6, PMS2, E-cadherin and p53 IHC, similar to the classification proposed by Setia et al., considering the previously published molecular classifications. In our study, the prevalence of NTRK gene fusions in G-ACa and its relationship with G-ACa groups based on molecular studies were evaluated by applying the IHK (pan-TRK-EPR17341) method. NTRK 1/2/3 fluorescence-ISH (FISH) analysis was performed for confirmation in cases with pan-TRK expression. In order to create a control group among the Pan-TRK negative cases, five cases with advanced stage were selected and NTRK 1/2/3 FISH analysis was applied. The routine HER2 ISH and ISH (FISH / SISH) analyzes of the cases were also included in our study, and the results were obtained from the pathology reports recorded in the hospital information management system. Findings: The mean follow-up period of the cases in our study was 19,97 months. In overall survival analysis; tumor macroscopic type 1 (polypoid), well differentiation, early gastric wall invasion level (pT1 + pT2) and lymph node involvement (pN0 + pN1), lymphatic / angioinvasion negativity and surgical margin negativity were associated with long survival. In relapse-free survival analysis; advanced stage lymph node metastasis (pN3), lymphatic invasion, angioinvasion, perineural invasion, tumor positivity at the surgical margin and desmoplastic response were associated with short recurrence-free survival. In our study, 240 cases were divided into 5 groups by using IHC and ISH analysis; 3,7% (n = 9) EBV (+) group, 12,1% (n = 29) dMMR group, 12,9% (n = 31) E-cadherin aberrant group, 47,9% (n = 115) P53 aberrant group and 23,4% (n = 56) P53 normal group. In our study, due to the limited number of EBV (+) cases, no statistically significant difference was found in terms of histomorphological and survival characteristics compared to other molecular groups. However, it is remarkable that all of the cases were male, 6 cases (66%) were proximally localized, and lymphocytic response was detected in 7 cases (77%). When the histological type of 9 cases with EBV (+) in our study was examined; 2 cases (22%) medullary carcinoma, 5 cases (55%) mixed type carcinoma (poorly cohesive + tubular), 1 case (11%) poorly cohesive carcinoma (containing signet ring cell component), 1 case (11%) tubular carcinoma (poorly differentiated / solid pattern dominant) was observed. In all EBV (+) cases, mismatch repair proteins (MLH1, MSH2, MSH6, PMS2) were detected as intact (mutually exclusive pattern) as IHC. The average age seen in the dMMR group was found to be significantly higher than the other molecular groups (mean age 71 years). The presence of lymphocytic response was detected at a higher rate with the p53 aberrant group compared to other molecular groups. As histological type, with p53 aberrant group, Lauren intestinal type / WHO tubular type were found to be significantly higher than other molecular groups. Perineural invasion was found to be statistically significantly less when compared with other molecular groups. In our study, except for 18 tubular G-ACa cases in the dMMR group, 3 cases had medullary carcinoma, 3 cases had weak cohesive G-ACa, 4 cases had mixed carcinoma and 1 case had mucinous carcinoma. In one case, micropapillary pattern was found to accompany tubular morphology at a rate of 10%. In the dMMR group, loss of MLH1 and PMS2 was found to be the dominant pattern (86.2%). Although there was a higher rate of Lauren diffuse type / WHO weak cohesive type in the E-cadherin aberrant group compared to other molecular groups, no statistically significant difference was found in terms of age compared to the other groups. It was also observed that E-cadherin aberrant group had significantly higher differeantiation grade compared to the other groups. P53 aberran group was found to be statistically significantly correlated with Lauren intestinal type (62,6%) and WHO tubular type (54.8%). The HER2 positivity rate (HER2 IHC score 3) in the p53 aberrant group was found to be statistically significantly higher than the other groups. No statistically significant difference was found between the 5 defined molecular groups in terms of overall and recurrence-free survival rates. In 3 out of 240 cases, weak immunoexpression was observed in a small number of neoplastic cells with pan-TRK. In the NTRK 1/2/3 FISH analysis applied for confirmation, the split ratio in NTRK 1/2/3 genes was found to be less than 5% in all 3 cases. The NTRK1 gene split ratio is more than 10% (10% and 14%) in two of the five Pan-TRK IHC negative cases. Conclusion: In our study, with IHC and ISH analyzes, groups formed on the basis of molecular-based studies in the literatüre, the distribution of G-ACa's in our own population was determined. Although there was a significant difference between the distribution of cases in the groups according to histological classifications, no specific histological type was detected for any of the groups. In this respect, this study has shown that it is important to routinely apply IHC and ISH methods in order to determine the G-ACa groups that can be effective in directing target therapies. Although the results of the FISH analysis performed in our cases with pan-TRC IHC expression are considered negative according to current criterias, we believe that the FISH results obtained in the study and control groups should be investigated in terms of predictive and / or prognostic significance with extensive molecular studies in the future. Keywords: Gastric adenocarcinoma, molecular classification, TCGA, ACRG, microsatellite instability, EBV, neurotrophic- tropomyosin receptor tyrosine kinase (NTRK) gene fusion, pan-TRK, prognosis.