Frequent CpG island methylation in serrated adenomas of the colorectum

Abstract

Serrated adenomas are characterized by a saw-toothed growth pattern with epithelial dysplasia (intraepithelial neoplasia). The CpG island methylator phenotype (CIMP) is a recently described mechanism for tumorigenesis in colorectal carcinomas and adenomas characterized by methylation of multiple CpG islands. The role of these epigenetic alterations in the pathogenesis of serrated adenomas is not clear. We therefore evaluated CIMP in 22 sporadic serrated adenomas and 6 serrated adenomas with multiple (6 to 10) hyperplastic polyps, including 5 with admixed hyperplastic glands and adenomatous glands, and compared the results with 34 conventional adenomas. Bisulfite methylation-specific polymerase chain reaction was used for the p16 and hMLH1 genes, and three MINT (methylated in tumor) loci (MINT1, MINT2, and MINT31). Patients with sporadic serrated adenomas had a higher frequency of hyperplastic polyps (1.3 +/- 1.6) as compared to patients with tubular adenomas (0.4 +/- 0.9, P = 0.02). Mean number of methylated sites was significantly higher in sporadic serrated adenomas (2.0 +/- 1.7) than in tubular adenomas (0.8 +/- 0.9, P = 0.00001). Sporadic serrated adenomas had significantly more frequent methylation of MINT1 (48%, 10 of 22) and MINT2 (71%, 15 of 21) than tubular adenomas (9%, 3 of 34, P = 0.001; and 18%, 6 of 34, P = 0.0001), respectively. Concordant methylation of two or more sites (CIMP-high) was also more frequent in sporadic serrated adenomas (68%, 15 of 22) than in tubular adenomas (18%, 6 of 34, P = 0.0005). All five serrated adenomas with admixed hyperplastic glands and adenomatous glands were CIMP-high. Our results indicate that CpG island methylation is common in sporadic serrated adenomas and may play an important role in their pathogenesis.

Figure 1.

Histopathological appearance of serrated adenoma and serrated adenoma with admixed hyperplastic glands and adenomatous glands. A: Serrated adenoma with saw-toothed architecture and epithelial dysplasia. B: Serrated adenoma with admixed hyperplastic glands (arrowhead) and adenomatous glands (arrow) from case 2. Both hyperplastic and adenomatous glands showed methylations of all five loci (p16, MINT1, MINT2, MINT31, and hMLH1) and allelic shifts of BAT25, BAT26, and TGF-βRII.

Figure 2.

Examples of methylation-specific PCR (MSP) at p16, MINT1, MINT2, and MINT31 in serrated adenomas, tubular adenomas, and nonlesional mucosa. Methylation of p16, MINT1, MINT2, and MINT31 was evaluated by MSP using primers for methylated (M) and unmethylated (U) alleles of bisulfite-treated DNA. Gene or loci examined are indicated on the left of each gel and samples on top of the first gel. L, designates size marker; SA1, SA2, and SA3, three serrated adenomas; TA, tubular adenoma; nonlesional, nonlesional colonic mucosa; RKO, a colon cancer cell line used as a positive control; and dH₂O, samples without DNA used as negative control. Methylation of p16 is present in all of the adenomas. Serrated adenomas (SA1 and SA2) have methylation of MINT1 and MINT31 and all three serrated adenomas have methylation of MINT2. The upper band of the double bands shown in MINT1 and MINT31 is a spurious band appearing only on 6% acrylamide gels but not on 2% agarose gels.

Figure 3.

Summary of histology; methylation status at p16, MINT1, MINT2, MINT31, and hMLH1; CIMP status; and MSI in sporadic serrated adenomas, serrated adenomas with multiple (6 to 10) hyperplastic polyps, and tubular adenomas.

Figure 4.

The numbers of gene or loci methylated in nonlesional colonic mucosas, sporadic serrated adenomas, serrated adenomas with multiple (6 to 10) hyperplastic polyps, and tubular adenomas. Sporadic serrated adenomas showed more concordant methylation sites as compared to tubular adenomas (P = 0.002) and serrated adenomas with multiple hyperplastic polyps (P = 0.03).

Figure 5.

CIMP status of serrated adenomas and tubular adenomas. CIMP-high was more frequent in sporadic serrated adenomas than in tubular adenomas (P = 0.0005) and serrated adenomas with multiple hyperplastic polyps (P = 0.03).

Figure 6.

CIMP status and distribution of hyperplastic polyps in patients with sporadic serrated adenomas and serrated adenomas with multiple (6 to 10) hyperplastic polyps (SA no. 6, 7, 11, 17, 27a, and 27b). Serrated adenomas (SA) no. 27a and 27b were from the same patient. The majority of the hyperplastic polyps were located in the distal colorectum (rectum and sigmoid colon).