Assessment of MLH1 promoter methylation and BRAF gene mutation in colorectal carcinomas with microsatellite instability

Background: Recent studies indicate that analysis of MLH1 promoter methylation and especially evaluation of BRAF gene mutational status can be employed to differentiate hereditary from sporadic MSI-H MLH1-negative colorectal carcinomas. In particular BRAF was demontrated to be frequently mutated in MSI-H sporadic but not in hereditary carcinomas. Design: The study was conducted on a consecutive series of 2162 colorectal adenocarcinomas surgically resected from January 2004 to June 2010. Mismatch repair (MMR) status has been prospectively evaluated by immunohistochemical analysis of MMR protein expression (MLH1, MSH2, MSH6 and, in selected cases, PMS2) and microsatellite instability (MSI) analysis, using a fl uorescent PCR method and the Bethesda panel markers (BAT25, BAT26, D2S123, D5S346, D17S250) plus BAT40. Tumors were classifi ed as MSI-H, MSI-L and MSS according to the guidelines of the International Workshop of Bethesda. In MMR-defi cient (MMR-D) tumors, analysis of MLH1 promoter methylation (C- region) was assessed by methylation specifi c PCR and evaluation of V600E BRAF mutation was investigated by direct DNA sequencing. Results: 316 (14.6%) carcinomas were classifi ed as MMR-D (loss of MMR protein expression and/or MSI-H). Most MMR-D tumors showed loss of MLH1 expression (256, 81%). MLH1 methylation was detected in 196/219 (89%) MLH1-negative carcinomas and in 2/50 (4%) MMR-D MLH1-positive carcinomas. V600E BRAF mutations were observed in 108/158 (68%) MLH1-negative and in only 1/42 (2%) MLH1-positive MMR-D cancers. BRAF mutations were identifi ed only in tumors showing MLH1 promoter methylation (107/142, 75%). All the MLH1-negative carcinomas without MLH1 methylation examined (15 cases) did not demonstrate BRAF mutation. Both MLH1 promoter methylation and BRAF mutation were more frequently observed in older patients. Conclusions: Our results confi rm that MLH1 promoter methylation and BRAF mutation occur in a large fraction of MMR-defi cient MLH1-negative colorectal carcinomas and are closely associated. Furthermore our data indicate that assessment of MLH1 promoter methylation and especially of BRAF mutation might be used in the selection of colorectal cancer patients with presumptive Lynch syndrome.