Abstract C17: Hypermethylation frequency of CDKN2A and MGMT genes and its association with protein expression levels and clinical and pathologic features in penile carcinomas

Abstract

Abstract Penile carcinoma is an uncommon disease and the most frequent subtype is squamous cell carcinoma. It represents less than 1% of all cases in developed countries. However, in developing countries it may represent 20% of all male cancers. The main risk factors associated to this tumor are poor hygienic habits, phimosis, HPV infections and high number of sexual partners. Very little is known about penile carcinoma pathogenesis, etiology and molecular biology features. In this study, we evaluated the hypermethylation frequency of two genes that present high frequencies of methylation in various tumor types: CDKN2A e MGMT. CDKN2A plays an important role in cell cycle control, encoding a protein (p16) that inhibits pRB phosphorilation through inactivation of the CDK-cyclin D complex, so blocking cell cycle progression. MGMT encodes a DNA repair protein that removes alkyl and methyl groups from guanines without DNA double strand breaks. Hypermethylation of these genes leads to transcriptional repression and has an important role in tumoregenesis. Unfortunately, scarce data is available about the role of hypermethylation in penile carcinomas. We selected 128 patients with penile squamous cell carcinoma surgically treated in a Brazilian cancer center. DNA from the formalin fixed paraffin embedded samples was obtained using a phenol-chloroform standard protocol. The DNA samples were submitted to bisulfite conversion, amplification and sequencing by the pyrosequencing approach, a quantitative methodology that allows the evaluation of the methylation levels in each CpG site of the target sequence. We also analyzed the expression levels by immunohistochemistry of the proteins p16 and MGMT to verify if the presence of hypermethylation is leading to gene silencing. We observed absence of CDKN2A hypermethylation in all samples and MGMT was hypermethylated in 57.4% of the cases, with levels of methylation varying from 16% to 86%. It was observed p16 positive expression in 66.8% of samples and Mgmt was present in only 10.9%. The decrease in MGMT expression was observed more frequent in tumors thicker than 5 millimeters and in patients with palpable nodes, suggesting a more aggressive tumor behavior in the absence of this protein. MGMT hypermethylation was not associated with reduced protein expression. Among samples with loss of MGMT expression 55.7% were methylated suggesting that only the presence of methyl radicals is not enough to induce gene silencing, it is probable that other mechanisms such as chromatin condensation are also required to repress gene transcription. The high frequency of p16 positivity could be explained by the presence of HPV infection, detected by others in about 30% of penile cancers. It also suggests that other members of the pRb pathway would be altered, such as cyclin D and Cdk amplifications or pRb loss. It cannot be discarded that other pathways of cell cycle control may also be deregulated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C17.