Microsatellite Instability and Altered Expressions of MLH1 and MSH2 in Gastric Cancer

Abstract

Introduction: Microsatellite instability (MSI) is a hallmark of defective DNA mismatch repair (MMR) of genes especially MLH1 and MSH2. It is frequently involved in the carcinogenesis of various tumours including gastric cancer (GC). However, MSI in GCs have not been reported in Malaysia before. Objective: This study was conducted to determine the microsatellite instability (MSI) status in gastric cancer by microsatellite analysis, sequencing, its association with MLH1 and MSH2 protein expression and H.pylori infection by immunohistochemistry. Method: A total of 60 gastric cancer cases were retrieved. DNA was extracted from paired normal and tumour tissues while MLH1 and MSH2 protein expression as well as H. pylori status were determined by IHC staining. For microsatellite analysis, polymerase chain reaction (PCR) was performed for paired tissue samples using a panel of five microsatellite markers. MSI-positive results were subjected for DNA sequencing to assess mutations in the MLH1 and MSH2 genes. Results: Microsatellite analysis identified ten MSI positive cases (16.7%), out of which only six cases (10.3%) showed absence of MLH1 (n=3) or MSH2 (n=3) protein expression by IHC. The most frequent microsatellite marker in MSI positive cases was BAT26 (90%). Nine of ten MSI positive cases were intestinal type with one diffuse and all were located distally. H. pylori infection was detected in 13 of 60 cases (21.7%) including in three MSI positive cases. All these results however were not statistically significant. Our sequencing data displayed novel mutations. However these data were not statistically correlated with expression levels of MLH1 and MSH2 proteins by IHC. This may be due to small sample size to detect small or moderately sized effects. Conclusion: The frequency of MSI in this study was comparable with published results. Determination of affected MMR genes by more than two antibodies may increase the sensitivity of IHC to that of MSI analysis.

Keywords: Gastric cancer; microsatellite instability; immunohistochemistry; microsatellite analysis.

Figure 1

Disease Free Survival of Gastric Cancer Patients in Association with Transcriptomic MLH1 and MSH2 Expression Levels. (A) High MLH1 expression is significantly associated with worst overall survival in gastric cancer patients (HR = 1.43; p-value = 0.00069). Inverse correlation observed in high MSH2 gene expression displaying good overall survival in gastric cancer patients (HR = 0.58; p-value = 7.6e-08)

Figure 2

(A),Well differentiated intestinal type gastric cancer with positive MSI showing total loss of MLH1 protein expression in tumour cells (T). Normal gastric glands (N) as internal control showing positive staining; (B), Diffuse type gastric cancer with positive MSI showing negative staining for MSH2 protein in tumour cells (T). Stromal cells (S) with positive staining as internal control. (x 200 magnification)

Figure 3

Electropherograph from a Case of Gastric Cancer with MSI Showing Additional Peaks in Tumour Compared to Matched Normal Tissue in Two Markers, (A) BAT25 and (B) BAT26

Figure 4

Presence of H. pylori Indicated by Brownish-Stained Organisms Located at the Luminal Surface of Gastric Mucosa Cells (Magnification x 400)