Clinicopathological features of tumor mutation burden, Epstein-Barr virus infection, microsatellite instability and PD-L1 status in Chinese patients with gastric cancer

Abstract

Objectives: Gastric cancer (GC) is the 4th most common type of cancer worldwide. Different GC subtypes have unique molecular features that may have different therapeutic methods. The aim of the present study was to investigate Epstein-Barr virus (EBV) infection, microsatellite instability (MSI) status, the expression of programmed death-ligand 1 (PD-L1) and gene mutations in GC patients.

Methods: The data of 2504 GC patients, who underwent curative gastrectomy with lymphadenectomy at Peking University Cancer Hospital between 2013 and 2018, were reviewed. We analyzed the clinicopathological factors associated with the immunohistochemistry (IHC) profiles of these patients, and genetic alterations were analyzed using next generation sequencing (NGS).

Results: Mismatch repair-deficient (d-MMR) GC patients were found to have a higher probability of expressing PD-L1 (p = 0.000, PD-L1 cutoff value = 1%). In addition, 4 and 6.9% of the 2504 gastric cancer patients were EBV-positive and d-MMR, respectively. The number of MLH1/PMS2-negative cases was 126 (6%), and the number of MSH2/MSH6-negative cases was 14 (0.9%). d-MMR status was associated with a intestinal group (p = 0.012), but not with tumor differentiation. Furthermore, MSI and d-MMR GC status (detected by NGS and IHC, respectively) were consistently high, and the rate of MSI was higher in patients with d-MMR GC. A number of genes associated with DNA damage repair were detected in GC patients with MSI, including POLE, ETV6, BRCA and RNF43. In patients with a high tumor mutation burden, the most significantly mutated genes were LRP1B (79.07%), ARID1A (74.42%), RNF43 (69.77%), ZFHX3 (65.12%), TP53 (58.14%), GANS (51.16%), BRCA2 (51.16%), PIK3CA (51.16%), NOTCH1 (51.16%), SMARCA4 (48.84%), ATR (46.51%), POLE (41.86%) and ATM (39.53%).

Conclusions: Using IHC and NGS, MSI status, protein expression, tumor mutation burden (TMB) and genetic alterations were identified in patients with GC, which provides a theoretical basis for the future clinical treatment of GC.

Keywords: EBV-encoded RNA; Gastric cancer; MSI; Next generation sequencing; PD-L1; TMB.

Fig. 1

Strong PD-L1 staining in patients with d-MMR GC. a Poorly differentiated GC; H&E staining, × 200 magnification. b The area of positive PD-L1 staining in tumor cells was > 90% (moderate- to strong-positive); × 200 magnification. c MLH1 expression-negative IHC staining, × 200 magnification; stromal cells with positive staining were used as the internal control; d PMS2 expression-negative IHC staining, × 200 magnification; stromal cells were used as internal positive control. PD-L1, programmed death-ligand 1; d-MMR, mismatch repair-deficient; GC, gastric cancer; H&E, hematoxylin and eosin; IHC, immunohistochemical

Fig. 2

EBER-positive patients primarily exhibit diffused/mixed Lauren type and poor tumor differentiation. a Poorly differentiated GC, diffuse type; H&E staining, × 200 magnification. b Poorly differentiated GC; EBER ISH-positive staining, 200x magnification. c Moderately differentiated GC, intestinal type; H&E staining, × 200 magnification. d Moderately differentiated GC, EBER ISH-negative staining, 200x magnification. EBER, EBV-encoded RNA; GC, gastric cancer; H&E, hematoxylin and eosin; ISH, in situ hybridization

Fig. 3

TSO500 MSI scores of patients with d-MMR GC are higher than those with p-MMR GC. The results of MSI detection (by next generation sequencing) were highly consistent with the immunohistochemistry results of those with d-MMR GC. d-MMR patients had higher MSI scores. MSI, microsatellite instability; d-MMR, mismatch repair-deficient; p-MMR, mismatch repair-proficient; GC, gastric cancer

Fig. 4

Overview of the frequency of DDR gene mutations in d-MMR GC samples. Various DDR genes were detected, including POLE, ETV6, ATR, TMPRS52, BRCA1, BRCA2, CHEK2, CCND3, FANCA and NSD1. DDR, DNA damage repair; d-MMR, mismatch repair-deficient; GC, gastric cancer

Fig. 5

Oncoplot reporting the most recurrently mutated genes across the d-MMR GC samples analyzed in this study. TMB, MSI status, gene mutation frequency and mutation type were detected by next generation sequencing. In high-TMB and MSI tumors, the deletion mutation of certain genes (such as RNF43, BCORL1 and ATR) is apparent. d-MMR, mismatch repair-deficient; GC, gastric cancer; TMB, tumor mutation burden; MSI, microsatellite instability