Genetic and epigenetic alterations of Ras signalling pathway in colorectal neoplasia: analysis based on tumour clinicopathological features

Abstract

Activation of RAS signalling induced by K-ras/BRAF mutations is a hallmark of colorectal tumours. In addition, Ras association domain families 1 and 2 (RASSF1 and RASSF2), the negative regulators of K-ras, are often inactivated by methylation of the promoter region in those tumours. However, reports showing differences in the occurrence of these alterations on the basis of tumour characteristics have been scarce. We analysed K-ras/BRAF mutations and the methylation status of RASSF1 and RASSF2 promoter regions in 120 colorectal adenomas with respect to their clinicopathological features. K-ras/BRAF mutations and RASSF2 methylation were observed in 49 (41%) and 30 (25%) of the samples, respectively, while RASSF1 methylation was observed in only 3 (2.5%). Adenomas with RASSF2 methylation often carried K-ras/BRAF mutations simultaneously (22 out of 30, P<0.01). Multivariate analysis revealed that the concomitance of these alterations was frequently observed in serrated adenomas (odds ratio (OR) 11.11; 95% confidence interval (CI) 1.96-63.00), but rarely in adenomas located in the sigmoid or descending colon (OR 0.13; 95% CI 0.03-0.58). A comparison between adenomas and cancers showed a significantly higher prevalence of these alterations in cancers than in adenomas in the proximal colon (58 vs 27%, P=0.02). Frequency and the time point of the occurrence of Ras signalling disorders differ according to colorectal neoplasia's characteristics, particularly the location.

Figure 1

Analysis of methylation status of RASSF1 and RASSF2 promoter CpG island. COBRA was carried out using bisulphite-treated DNA from colorectal adenomas and cancers. A representative result for tumour tissue samples with/without RASSF1 or RASSF2 promoter methylation is shown. A1 is a sample without RASSF1 and RASSF2 methylation. A2 and C2 are samples with RASSF2 methylation, and without RASSF1 methylation. C1 is a sample with RASSF1 methylation, and without RASSF2 methylation. A, adenoma samples; C, cancer samples; M, methylated alleles; HCT116, colon cancer cell line used as a negative control for RASSF1 and RASSF2 methylation; RKO, colon cancer cell line used as a positive control for RASSF1 and RASSF2 methylation; H₂O, sample without DNA.

Figure 2

Locational distributions of adenomas and cancers with K-ras/BRAF mutation and/or RASSF2 methylation. Cancers with K-ras/BRAF mutation and/or RASSF2 methylation exhibit locational distribution similar to that of adenomas. However, the proportion of tumours in the proximal colon with both K-ras/BRAF mutation and RASSF2 methylation was significantly higher in cancers (58%) than in adenomas (27%) (P=0.02). Arrowheads indicate serrated adenomas.