Mesothelin gene expression and promoter methylation/hypomethylation in gynecological tumors.

PURPOSE: Mesothelin is a cell surface glycoprotein that is present on normal mesothelial cells and overexpressed in several cancers. In this study, we investigated the methylation/hypomethylation status in the promoter region of the mesothelin gene in gynecological tumors. METHODS: Forty-four ovarian tumor specimens and 16 cases of uterine endometrial carcinoma, and normal tissue specimens were used. Monoclonal antibody (5B2) was employed for the immunohistochemical analysis. The methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) technique was used to quantify the methylation/hypomethylation status at 20 CpG sites in the mesothelin promoter region. RESULTS: Mesothelin was expressed in 100% of serous cystadenocarcinoma and 100% of serous borderline tumor of the ovary. None of the germ cell tumors and sexcord-stromal tumors was immunoreactive. Fifty percent of endometrial carcinoma was immunoreactive for mesothelin. The average methylation of CpG sites in ovarian tumors ranged from 6-56% (median: 31%) in mesothelin-positive and 13-79% (median: 43%) in mesothelin-negative samples. In endometrial tumors, the average methylation ranged from 5-52% (median: 28%) in mesothlin-positive and from 15-67% (median: 22%) in mesothlin-negative samples. A correlation was found between mesothelin expression and the average methylation/hypomethylation status as well as methylation/hypomethylation status at four of 20 CpG sites in ovarian samples. No correlation was found in endometrial samples. CONCLUSION: We detected diverse levels of methylation/hypomethylation at CpG sites in the mesothelin promoter region in ovarian and endometrial tumors. We speculate that, although methylation/hypomethylation changes may affect its transcription, other mechanisms may synergically operate in tissue-specific expression and tumor-related mesothelin overexpression.

Birt-Hogg-Dubé gene mutations in human endometrial carcinomas with microsatellite instability.

Birt-Hogg-Dubé (BHD) syndrome is a rare form of autosomal dominantly inherited genodermatosis characterized by benign hamartomatous skin lesions named fibrofolliculomas, and an increased risk for developing pulmonary cyst/pneumothorax and various forms of renal cell carcinoma. Many of the patients harbour insertion/deletion mutations in the hypermutable poly(C)8 tract in exon 11 of the BHD gene. This mutational hot spot is also reported to be a target of mutation in microsatellite instability (MSI) sporadic colorectal cancer. To test the hypothesis that the BHD gene is also a mutational target in sporadic endometrial carcinoma with microsatellite instability, 139 cases of sporadic endometrial carcinoma were screened for MSI status, and mutations of the poly(C)8 tract in exon 11 as well as other coding exons of the BHD gene. The poly(G)8 tract of the BAX gene, the poly(C)8 tract of MSH6, and methylation status of hMLH1 were also assessed. Thirty-nine of 139 cases (28%) showed MSI. Mutations in the poly(C)8 tract of BHD were detected in five of the 39 MSI cases (12.8%). Of these, one showed additional mutation in exon 4, possibly satisfying the two-hit hypothesis of tumour suppressor genes. BAX gene mutation was detected in ten of the 39 MSI cases (25.6%). Four tumours showed both BAX and BHD mutations, and a significant positive association was found between mutations of the two genes. No association was found between BHD status and MSH6 mutation or hMLH1 methylation. When multiple foci were microdissected and individually screened for mutation, BHD mutations were shown to have been acquired during tumour progression, after mutation of the BAX gene, in three of five cases. Taken together, these findings show that the BHD gene is a target gene in MSI endometrial carcinoma. However, its mutational frequency is lower than that of BAX, and BHD mutation tends to occur during neoplastic progression after the acquisition of mutations in another MSI target gene, BAX.