Analysis of BRAF Gene Mutation and MLH1 Promoter Methylation in MSI-H Colorectal Carcinomas with Loss of MLH1 Protein Expression

Purpose of the study. Hereditary and sporadic colorectal carcinomas (CRCs) with deficit of DNA mismatch repair (MMR-D) should be identified in all patients to ensure accurate treatment and risk assessment for relatives. Almost all MMR-D CRCs can be detected by microsatellite instability (MSI) and immunohistochemical testing. Analysis of MLH1 promoter methylation and evaluation of BRAF gene mutational status can help to differentiate hereditary from sporadic MSI-H MLH1-negative colorectal carcinomas. Methods. The study included a consecutive series of 2248 CRCs surgically resected from January 2004 to March 2012. Immunohistochemical analysis of MLH1, MSH2, MSH6 protein was performed in all tumours, PMS2 protein only in selected cases. MSI status was determined by a fluorescent PCR method using the Bethesda panel markers plus BAT40; tumours were classified as MSI-H, MSI-L and MSS according to the guidelines of Bethesda. In MMR-D tumours, analysis of MLH1 promoter methylation was assessed by methylation specific PCR and evaluation of V600E BRAF mutation was investigated by direct DNA sequencing. Summary of results. Deficit of MMR was observed in 332 tumours (14.7%). Most MMR-D tumours showed loss of MLH1 expression (272/332). V600E BRAF mutation was observed in 118/174 MLH1-negative MMR-D tumours and in only 1/42 MLH1-positive MMR-D cancers. MLH1 methylation was detected in 208/235 MMR-D MLH1-negative carcinomas and in 2/50 MMR-D MLH1-positive carcinomas. BRAF mutation was identified in 115/150 MLH1-negative tumours showing MLH1 methylation and only in 3 MLH1-negative tumours without MLH1 methylation. Conclusions. Our study indicate that MLH1 methylation and BRAF mutation occur frequently in MMR-D CRCs, are closely associated and may be used to identify CRC patients with Lynch syndrome.