Abstract 4256: Methylation profile in penile carcinomas reveal distinct signatures according to HPV status.
Authors: Kuasne Hellen , de Syllos Cólus Ilce Mara , Vargas Hector Hernandez , Busso Ariane Fidelis , Barros-Filho Mateus Camargo , Marchi Fábio A. , Guimarães Gustavo C. , Scapulatempo-Neto Cristovam , Soares Fernando Augusto , Herceg Zdenco , Hatakeyama Marcia , Rogatto Silvia Regina 
Published: August 2014

Abstract
Background: Penile carcinoma (PeCa) is an important public health problem in poor and developing countries. Despite the unpredictable behavior and aggressive treatment, there are few data on molecular and epigenetic mechanisms reported in PeCa. The aim of this study was to identify epigenetic profile in tumors and surrounding tissue. Patients and Methods: Paired PeCa and surrounding tissue samples were collected from 16 patients. Twenty tumors were also included. To evaluate the methylation profile two independent methods were used: enrichment of methylated region (MeCip) (MethylMiner - Invitrogen) and and enrichment of unmethylated region (digested with restriction enzyme McrBC). Samples were hybridized in a 244K Human DNA Methylation Microarray platform (Agilent Technologies). Genomic Workbench Standard (v 5.0.14) and BRB array tools softwares were used to analyze the data. It was considered only probes with p value&lt;0.001, FDR &lt;0.05 and located inside or in the gene promoter. Global methylation profile of repetitive regions also was evaluated by pyrosequencing (LINE1 and AluYB8). The relationship between the altered probes was analyzed by IPA (Ingenuity Pathways Analysis), Gene Ontology and Biocarta softwares. The validation was accomplished by bisulfite conversion followed by pyrosequencing.
Results: HPV positivity was detected in 43% of cases (Linear Array HPV Test Genotyping - Roche), mainly for 16 and 18 subtypes. Penile carcinoma displayed unique signature related to surround tissue. In the comparison of tumors and controls, 171 probes were methylated and 349 were unmethylated. A differential methylation profile was also found according to HPV status. While HPV positive cases were related with Immunological response and Rb pathway, HPV negative samples were related with regulation of stem cells, development and differentiation. DNA methylation analysis by pyrosequencing for TWIST, CD133 and FOXA1 confirmed the results of microarrays. Furthermore, the global levels of methylation in repetitive regions revealed to be lower in tumor samples (AluYB8: p &lt;0.001). Conclusions: For our knowledge, this is the first study that evaluated methylation profile in penile samples. Our results showed that tumors present a different methylation signature related to surrounding tissue. It was found a differential methylation profile according to HPV status (positive or negative), indicating at least two disrupted pathways, one related to viral infection and other associated with transcriptional regulation of stem cells.
Financial support: FAPESP and CAPES
Citation Format: Hellen Kuasne, Ilce Mara de Syllos Cólus, Hector Hernandez Vargas, Ariane Fidelis Busso, Mateus Camargo Barros-Filho, Fábio A. Marchi, Gustavo C. Guimarães, Cristovam Scapulatempo-Neto, Fernando Augusto Soares, Zdenco Herceg, Marcia Hatakeyama, Silvia Regina Rogatto. Methylation profile in penile carcinomas reveal distinct signatures according to HPV status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4256. doi:10.1158/1538-7445.AM2013-4256