B-RAF mutation and accumulated gene methylation in aberrant crypt foci (ACF), sessile serrated adenoma/polyp (SSA/P) and cancer in SSA/P

BACKGROUND: Sessile serrated adenomas/polyps (SSA/Ps) are a putative precursor of colon cancer with microsatellite instability (MSI). However, the developmental mechanism of SSA/P remains unknown. We performed genetic analysis and genome-wide DNA methylation analysis in aberrant crypt foci (ACF), SSA/P, and cancer in SSA/P specimens to show a close association between ACF and the SSA/P-cancer sequence. We also evaluated the prevalence and number of ACF in SSA/P patients. METHODS: ACF in the right-side colon were observed in 36 patients with SSA/Ps alone, 2 with cancers in SSA/P, and 20 normal subjects and biopsied under magnifying endoscopy. B-RAF mutation and MSI were analysed by PCR–restriction fragment length polymorphism (RFLP) and PCR–SSCP, respectively, in 15 ACF, 20 SSA/P, and 2 cancer specimens. DNA methylation array analysis of seven ACF, seven SSA/P, and two cancer in SSA/P specimens was performed using the microarray-based integrated analysis of methylation by isochizomers (MIAMI) method. RESULTS: B-RAF mutations were frequently detected in ACF, SSA/P, and cancer in SSA/P tissues. The number of methylated genes increased significantly in the order of ACF<SSA/P<cancer. The most commonly methylated genes in SSA/P were PQLC1, HDHD3, RASL10B, FLI1, GJA3, and SLC26A2. Some of these genes were methylated in ACF, whereas all genes were methylated in cancers. Immunohistochemistry revealed their silenced expression. Microsatellite instability and MLH1 methylation were observed only in cancer. The prevalence and number of ACF were significantly higher in SSA/P patients than in normal subjects. A significant correlation was seen between the numbers of SSA/P and ACF in SSA/P patients. CONCLUSIONS: Our results suggest that ACF are precursor lesions of the SSA/P-cancer sequence in patients with SSA/P, where ACF arise by B-RAF mutation and methylation of some of the six identified genes and develop into SSA/Ps through accumulated methylation of these genes.