hMLH1 promoter hypermethylation is an early event in human endometrial tumorigenesis

Abstract

It has recently been suggested that silencing of the hMLH1 gene by promoter hypermethylation is the mechanism underlying the presence of the microsatellite instability (MSI) phenotype in sporadic colon and endometrial carcinomas. To determine whether hMLH1 promoter hypermethylation is a relatively early event in endometrial tumorigenesis we evaluated endometrial hyperplasia (EH) characterized as simple, complex, and atypical (the direct precursor of endometrial carcinoma) for hMLH1 aberrant methylation. In addition, we studied the hMLH1, hMSH2, hMSH3, and hMSH6 promoter methylation and MSI status of those endometrial carcinomas with synchronous hyperplasias and those without them. We found that 11 of 12 (91%) cases of endometrial carcinoma (EC) displaying MSI had hMLH1 promoter hypermethylation, whereas aberrant methylation of any of the other mismatch repair genes was not observed. All 15 cases of EC without MSI were unmethylated at hMLH1. Abnormal methylation of hMLH1 was also present in 8 of 116 (7%) cases of EH and was restricted primarily to the atypical endometrial hyperplasia (AEH) type with coexisting endometrial carcinoma. In this set, half of EH methylated at hMLH1 displayed MSI, whereas none of the unmethylated EH had MSI. Our data suggest that hypermethylation of hMLH1 can be an early event in the pathogenesis of EC, preceding the development of an apparent MSI phenotype in a subset of cases.

Figure 1.

Methylation-specific PCR (MSP) of MMR genes in endometrial carcinomas. PBR322/Msp digest are shown at left as molecular weight markers. The presence of a visible PCR product in those lanes marked “U” indicates the presence of unmethylated genes; the presence of product in those lanes marked “M” indicates the presence of methylated genes. SW48 DNA is used as positive control for hMLH1 promoter hypermethylation, in vitro methylated DNA (IVD) as positive control for hMSH2, hMSH3, and hMSH6 promoter hypermethylation and normal lymphocytes as negative control for methylation. Water controls for PCR reactions are also shown. A: MSP of hMLH1 in endometrial carcinomas (EC) and matched normal endometrium (NE). MSP of hMSH2 (B), hMSH3 (C), and hMSH6 (D) in primary endometrial carcinomas (EC).

Figure 2.

hMLH1 promoter hypermethylation analysis in endometrial tumorigenesis. A: MSP of hMLH1 in matched endometrial carcinomas (EC) and endometrial hyperplasias (EH) from two patients. The presence of a visible PCR product in those lanes marked “U” indicates the presence of unmethylated genes; the presence of product in those lanes marked “M” indicates the presence of methylated genes. B: Endometrioid carcinoma (right) coexisting with atypical endometrial hyperplasia (left), both methylated at hMLH1 and MSI+. C: Restriction enzyme analysis following bisulfite modification in matched endometrial carcinomas (EC) and endometrial hyperplasias (EH). The colorectal cancer cell lines RKO and SW480 are used as positive methylated and negative unmethylated control for hMLH1, respectively. The colorectal carcinoma cell line HT-29 shows a partial methylation pattern as previously described.