Methylation of tumor suppressor gene p16 and prognosis of epithelial ovarian cancer

Abstract

Objective. Methylation of p16 promoter was evaluated in ovarian cancer to determine the role of p16 methylation in ovarian cancer prognosis.

Methods. Two hundred and forty-nine patients with primary epithelial ovarian cancer were selected for the study; these patients were followed for a median of 31 months. Genomic DNA extracted from fresh frozen tumor tissues were treated with sodium bisulfite and were analyzed for p16 methylation using methylation-specific PCR (MSP). Cox regression survival analysis was performed to examine the associations of p16 methylation with progression-free and overall survivals.

Results. Of the 249 patients, 100 (40%) were tested positive for p16 promoter methylation. The status of p16 methylation did not change significantly with patient age, disease stage, histological grade, residual tumor size, and debulking results, although p16 methylation seemed to occur more often in patients with advanced diseases or aggressive tumors. Compared to those without p16 methylation, patients with p16 methylation had significantly higher risk for disease progression (P = 0.01). The relative risk for progression was 1.69 (95% CI: 1.12–2.54), and the association remained statistically significant (RR = 1.54, 95% CI: 1.01–2.34) after adjusting for clinical and pathological variables. The risk for death was also higher in methylation positive patients than in methylation negative patients (RR = 1.33, 95% CI: 0.88–2.00), but the difference was not statistically significant.

Conclusion. The study suggests that promoter methylation in the p16 gene is associated with ovarian cancer progression, and evaluation of p16 methylation may have values in predicting ovarian cancer prognosis.

Introduction

Tumor suppressor gene p16 (INK4A/MTS1/CDKN2) encodes a 15.8-kDa protein that interacts with cyclin-dependent kinase CDK4 and CDK6. CDK4 and CDK6 bind to cylcin D regulating cell cycle progression from G1 to S phases. Binding of p16 to CDKs inhibits the formation of CDK/cyclin D complex, resulting in cell cycle arrest at G1 phase. The role of p16 in cell cycle control is believed to be involved in cancer development and progression [1], [2], [3], [4], [5]. Loss of p16 expression has been observed in many types of cancer [2]. Genetic and epigenetic alterations, including homozygous deletion, point mutation, and promoter methylation, are considered to be responsible for the loss of p16 activity [5], [6].

Promoter methylation has been recognized as an important mechanism in regulation of gene expression [7], [8]. Aberrant methylation of p16 promoter has been found in many forms of cancer, including ovarian cancer [5]. Clinical studies indicate that p16 expression is undetectable in about a third of ovarian cancer cases [9], [10], [11], [12] and that patients with low p16 expression have poor responses to chemotherapy and unfavorable survival outcome [11], [13]. Cell culture experiments demonstrate that reintroducing functional p16 into p16-null ovarian cancer cells results in inhibition of cell growth and increases in apoptosis, suggesting that p16 plays a role in ovarian cancer progression [14].

Epithelial ovarian cancer is one of the most lethal malignancies; 5-year survival of patients with the disease is only 50% [15], [16]. To improve our understanding of ovarian cancer progression and to search for molecular markers for prediction of ovarian cancer prognosis, we analyzed p16 promoter methylation in epithelial ovarian cancer tissue and examined its association with patient progression-free and overall survival.