The contribution of genetic and epigenetic changes in granulosa cell tumors of ovarian origin.

PURPOSE: Granulosa cell tumors (GCTs) are relatively rare and are subtypes of the sex-cord stromal neoplasms. A better understanding of the molecular genetics underlying various steps in malignant transformation is critical to success in the battle against this disease. Changes in the status of methylation, known as epigenetic alterations, are one of the most common molecular alterations in human cancers, including GCTs. Chromosomal instability and microsatellite instability (MSI) are common in these GCTs. We tested the hypothesis that C-->T transition polymorphism in the promoter region of cytosine DNA-methyltransferase-3B (DNMT3B) and its altered expression are also associated with hypermethylation of the genes. We also attempted to determine the relationship between MSI of ovarian carcinoma and hMLH1 hypermethylation in these tumors.

EXPERIMENTAL DESIGN: We studied chromosome instability in 25 GCTs by detecting gross chromosome rearrangements in cultured peripheral blood lymphocytes. MSI was assessed using six microsatellite markers (BAT25, BAT26, D2S123, D5S346, D11S1318, and D17S250). Using sensitive methylation-specific PCR, we searched for aberrant promoter hypermethylation in a panel of genes including p16, BRCA1, RASSF1A, ER-alpha, TMS1, TIMP3, Twist, GSTP1, AR, and hMLH1. Polymorphism in the DNMT3B gene was assessed by the PCR-RFLP method, and DNMT3B expression was studied by reverse transcription-PCR assay.

RESULTS: Chromosome instability was indicated by significantly higher frequencies of chromosome aberrations (6.24%; P < 0.001) compared with controls (2.12%). The most frequently observed changes include trisomy 14 and monosomy 22. MSI has been found in 19 of 25 tumors, and loss of heterozygosity has been found in 9 of 25 tumors. Frequencies of methylation in GCTs were 40% for p16 and ER-alpha; 36% for BRCA1 and RASSF1A; 28% for hMLH1; 24% for TIMP3, Twist, and GSTP1; and 20% in TMS1 and AR. TT genotype was found only in two cases; the remainder were either CC or CT type. There was no significant alteration in the expression of DNMT3B in these patients.

CONCLUSIONS: Coexistence of chromosome instability, MSI, and hypermethylation suggests that both genetic and epigenetic mechanisms may act in concert to inactivate the above-mentioned genes in these GCTs. These mechanisms can be an early event in the pathogenesis of these tumors, and it can be a critical step in the tumorigenic process. All these events might play an important role in early clinical diagnosis and in chemotherapeutic management and treatment of the disease. Larger studies may lend further understanding to the etiology and clinical behavior of these tumors.