Genetic and epigenetic aberrations occurring in colorectal tumors associated with serrated pathway

Abstract

To clarify molecular alterations in serrated pathway of colorectal cancer (CRC), we performed epigenetic and genetic analyses in sessile serrated adenoma/polyps (SSA/P), traditional serrated adenomas (TSAs) and high-methylation CRC. The methylation levels of six Group-1 and 14 Group-2 markers, established in our previous studies, were analyzed quantitatively using pyrosequencing. Subsequently, we performed targeted exon sequencing analyses of 126 candidate driver genes and examined molecular alterations that are associated with cancer development. SSA/P showed high methylation levels of both Group-1 and Group-2 markers, frequent BRAF mutation and occurrence in proximal colon, which were features of high-methylation CRC. But TSA showed low-methylation levels of Group-1 markers, less frequent BRAF mutation and occurrence at distal colon. SSA/P, but not TSA, is thus considered to be precursor of high-methylation CRC. High-methylation CRC had even higher methylation levels of some genes, e.g., MLH1, than SSA/P, and significant frequency of somatic mutations in nonsynonymous mutations (p < 0.0001) and insertion/deletions (p = 0.002). MLH1-methylated SSA/P showed lower methylation level of MLH1 compared with high-methylation CRC, and rarely accompanied silencing of MLH1 expression. The mutation frequencies were not different between MLH1-methylated and MLH1-unmethylated SSA/P, suggesting that MLH1 methylation might be insufficient in SSA/P to acquire a hypermutation phenotype. Mutations of mismatch repair genes, e.g., MSH3 and MSH6, and genes in PI3K, WNT, TGF-β and BMP signaling (but not in TP53 signaling) were significantly involved in high-methylation CRC compared with adenoma, suggesting importance of abrogation of these genes in serrated pathway.

Keywords: DNA methylation; colorectal cancer (CRC); gene mutation; sessile serrated adenoma/polyp (SSA/P); traditional serrated adenoma (TSA).

Figure 1

Macroscopic and histopathological appearance of serrated polyps. Endoscopic images after administration of 0.1% indigo carmine solution (left) and HE staining (middle and right). (a) SSA/P without dysplastic change. (b) SSA/P with low‐grade dysplasia. (c) TSA with low‐grade dysplasia. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

Heatmap of methylation levels of six Group‐1 markers and 14 Group‐2 markers. The color scale is presented as average methylation levels of each marker. Methylation data of CRC and LSTs were quoted from our previous reports.3, 4 As previously described, high‐methylation CRC, LST granular type and LST nongranular type could be classified into high‐, intermediate‐ and low‐methylation epigenotypes, respectively. SSA/P exhibited generally high methylation levels of both Group‐1 and Group‐2 markers, similar to the results of high‐methylation CRC; thus, SSA/P was considered to be high‐methylation epigenotype. TSA exhibited high methylation levels of Group‐2 markers but low methylation levels of Group‐1 markers similar to the results of LST granular type; thus, TSA was considered to be intermediate‐methylation epigenotype. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

Comparison of the methylation levels of each marker. Methylation data of CRC and LSTs were quoted from our previous reports.3, 4 *p < 0.05 between high‐methylation CRC and SSA/P (Student's t‐test). †p < 0.05 for comparison of SSA/P and TSA (Student's t‐test). Group‐2 markers were highly methylated in both SSA/P and TSA. For Group‐1 markers, SSA/P showed significantly higher methylation rate than TSA in four of the six markers. High‐methylation CRC showed even higher methylation rate than SSA/P in most of the genes. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4

Average number of somatic mutations. The frequency of somatic mutations in high‐methylation CRC was significantly higher than those in SSA/P and TSA, both for nonsynonymous mutations (p < 0.0001 and p = 0.001, respectively) and indels (p < 0.0001 and p < 0.0001, respectively). *p < 0.05 between high‐methylation CRC and SSA/P (Student's t‐test). †p < 0.05 for comparison of high‐methylation CRC and TSA (Student's t‐test).

Figure 5

Genetic alterations in individual genes and carcinogenic signaling pathways. Black circle, nonsynonymous mutations. Black rhombus, frame‐shift indels. For MLH1 expression, MLH1‐loss(+), ‐loss(±) and ‐loss(−) were shown by black, gray and blank, respectively. The alterations that are shown were predicted to be “disease causing” by MutationTaster.28 *p < 0.05 for comparison of high‐methylation CRC and SSA/P. †p < 0.05 for comparison of SSA/P and TSA. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]