Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas

Abstract

Colorectal cancer is a multi-step process characterized by a sequence of genetic alterations in cell growth regulatory genes, such as the adenomatous polyposis coli, KRAS, p53 and DCC genes. In the present study mutation analysis was performed with SSCA/direct sequencing of the hot-spot regions in exons 11 and 15 for the BRAF gene and exons 1-2 for the KRAS gene in 130 primary colorectal cancer tumors and correlated with clinico-pathological and mutational data. We also performed mutation analysis of the corresponding conserved regions in the ARAF and RAF-1 genes. Mutations in the BRAF and KRAS genes were found in 11.5 and 40% of the tumors, respectively. One germline exonic and nine germline intronic genetic variants were found in the ARAF and RAF-1 genes. All of the BRAF mutations were located in the kinase domain of the conserved region 3 in exon 15 of the BRAF gene. One novel somatic mutation was also identified in the BRAF gene. The majority of the BRAF mutations were found in colon compared with rectal tumors (P = 0.014). In agreement with others, a statistically significant correlation between BRAF mutations and microsatellite instability could be found. A negative correlation was also evident between mutations in the BRAF and KRAS genes, which supports earlier studies where somatic mutations in these genes are mutually exclusive. Collectively, our results provide support for the idea that activation of the MAP kinase pathway, especially via BRAF and KRAS mutations, is of critical importance for the development of colorectal cancer.

A gene marker panel covering the Wnt and the Ras-Raf-MEK-MAPK signalling pathways allows to detect gene mutations in 80% of early (UICC I) colon cancer stages in humans

“…The CTNNB1 gene was only found to be mutated in 2 out of 50 tumours, which is in agreement with previously published data reporting that CTNNB1 mutations were present in up to 3% of the sporadic CRC analyzed [13][14][15][16] and that they are mainly associated with HNPCC [17]. B-RAF mutations were found to be present in four MSI-L/H and in three MSS tumours, the mutation frequency being in the range of that reported by Fransé n et al [33].…”

“…Table 8 shows that the percentage of CRC carrying certain gene mutations in previous publications varies between 55% and 87% [16,[29][30][31][32][33]. If one takes into account that three out of six gene marker combinations [16,30,32] detect mutations in 60% of the tumours analyzed and that in one study [31] the CTNNB1 status was analyzed by immunohistochemistry, the primer panel [24] used in the present study shows an excellent performance.…”

Raf kinases: Oncogenesis and drug discovery

“…A couple of recent publications addressing the mutational status of A-RAF and C-RAF genes in more than 600 cancer cell lines and 500 primary human samples obtained from colorectal and gastric carcinoma, acute leukemias, gliomas, as well as lung, ovarian and testis tumors suggest that mutations in both RAF genes are very rare or nonexistent events. [83][84][85] The most detailed analysis of 545 cancer cell lines and about 80 primary tumor samples by Marais and coworkers detected no A-RAF mutations and only 4 rare polymorphisms in C-RAF corresponding to a mutational rate of 0.7%. 85 The identified 4 C-RAF mutants (P207S, V226I, Q335H, E478K) displayed differences with respect to basal and Ras induced kinase activities but were similar active in standard 3T3 colony formation assays.…”

Ultrasensitive Detection of Unknown Colon Cancer-Initiating Mutations Using the Example of the Adenomatous Polyposis Coli Gene

“…In addition, APC and KRAS are the most frequently mutated genes in sporadic colorectal cancer being affected in about 40% (KRAS) and up to 60% (APC) of patients with colorectal cancer, respectively (14)(15)(16)(17). Commercially available kits for KRAS and BRAF mutation analysis (e.g., Signature KRAS/BRAF Mutations assay, Asuragen) possess an average detection limit of 1% mutated DNA in mixtures with wild-type DNA which is satisfactory for prognostic purposes rather than for early cancer screening, when taken into account that the ratio of mutated human DNA to total feces DNA is assumed to be in the range of 1:10,000-1:100,000 (18)(19)(20).…”