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Purpose:The BRAF gene encodes a serine/threonine kinase and plays an important role in the mitogen-activated protein kinase signaling pathway. BRAF mutations in sporadic colorectal cancer with microsatellite instability (MSI) are more frequently detected than those in microsatellite stable cancer. In this study, we sought to compare the frequencies of BRAF mutations in sporadic colorectal cancer with MSI with those in hereditary nonpolyposis colorectal cancer (HNPCC).Experimental Design: We analyzed BRAF mutations in 26 colorectal cancer cell lines, 80 sporadic colorectal cancers, and 20 tumors from HNPCC patients by DNA sequencing and sequence-specific PCR. The methylation status of the hMLH1 gene was measured by either sequencing or restriction enzyme digestion after NaHSO 3 treatment.Results: We observed a strong correlation of BRAF mutation with hMLH1 promoter methylation. BRAF mutations were present in 13 of 15 (87%) of the colorectal cell lines and cancers with methylated hMLH1, whereas only 4 of 91 (4%) of the cell lines and cancers with unmethylated hMLH1 carried the mutations (P < 0.00001). Sixteen of 17 mutations were at residue 599 (V599E). A BRAF mutation was also identified at residue 463 (G463V) in one cell line. In addition, BRAF mutations were not found in any cancers or cell lines with K-ras mutations. In 20 MSI؉ cancers from HNPCC patients, however, BRAF mutations were not detectable, including a subset of 9 tumors with negative hMLH1 immunostaining and methylated hMLH1.Conclusions: BRAF mutations are frequently present in sporadic colorectal cancer with methylated hMLH1, but not in HNPCC-related cancers. This discrepancy of BRAF mutations between sporadic MSI؉ cancer and HNPCC might be used in a strategy for the detection of HNPCC families.

Mucinous colorectal cancer (CRC) has been reported to have distinct clinicopathological and genetic characteristics. However, the incidence and the relationship among microsatellite instability (MSI), CpG island methylator phenotype (CIMP) and BRAF and KRAS mutations in mucinous and non-mucinous CRC are not known. Activating mutations of BRAF and KRAS and their relationship with MSI and CIMP were examined in 83 sporadic CRC specimens (26 mucinous and 57 non-mucinous CRC). MSI, CIMP, BRAF and KRAS mutation were observed in 17, 24, 25 and 36% of the tumors, respectively. BRAF mutation was highly correlated with MSI (p < 0.001) and CIMP (p < 0.001). A higher incidence of MSI (27% vs. 12%), CIMP (38% vs. 18%, p < 0.05) and BRAF mutation (46% vs. 16%, p < 0.01) was observed in mucinous CRC. KRAS mutation (27% vs. 40%) was observed more frequently in non-mucinous CRC. Significantly higher percentages of mucinous CRC (54%, p < 0.05) had MSI or CIMP or BRAF mutations. Concordant occurrence of 2 or more of these alterations was observed in 39% of mucinous CRC and only 11% of non-mucinous CRC (p < 0.01). The more frequent occurrence and closer association among MSI, CIMP and BRAF mutation in mucinous CRC observed in our study further supports the idea that its pathogenesis may involve distinct genetic and epigenetic changes. ' 2005 Wiley-Liss, Inc.Key words: mucinous colorectal cancer; microsatellite instability; CpG island methylator phenotype; BRAF mutation; KRAS mutation Colorectal cancer (CRC) develops as a result of progressive accumulation of genetic and epigenetic alterations.1 There are at least 2 major genetic instability pathways involved in colorectal carcinogenesis, chromosomal instability and microsatellite instability (MSI).2,3 The chromosomal instability pathway is found in about 80% of CRCs. 4 This pathway involves chromosomal aberrations such as loss of heterozygosity of 5q, 17p and 18q, with inactivation of APC, p53 and DCC genes.2 MSI, a second form of genetic instability, is found in most cases of hereditary non-polyposis colorectal cancer (HNPCC) and in about 15% of sporadic CRCs. [5][6][7] This pathway involves inactivation of DNA mismatch repair genes followed by mutations in mononuclear tracts in the coding region of genes implicated in tumor progression such as TGFbRII and BAX, which leads to uncontrolled growth and decreased apoptosis.8-10 Chromosomal aberrations are rare in microsatellite unstable tumors. Recently, additional pathways involving epigenetic abnormalities have been described. 11,12 These include CpG island methylator phenotype (CIMP) with hypermethylation of CpG islands in the promoter regions of multiple genes such as p16, p14, MGMT and hMLH1, causing transcriptional inactivation of these genes. 13CIMP is found in 20-32% of sporadic CRC and has been frequently associated with MSI through methylation of hMLH1 promoter region. 12,[14][15][16] The RAS/RAF/MAP kinase cascade is an important pathway that mediates the cellular response to extracellular signals, which regulate cell grow...

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