Abstract 3026: Concurrent gene promoter hypermethylation and reduced immunohistochemical expression of potential biomarkers in cervical cancer: A Phase I biomarker development trial
Authors: Brebi Priscilla , Ili Carmen , Sandoval Alejandra , Leal Pamela , Garcia Patricia , Saavedra Kathleen , Tapia Oscar , Guzman Pablo , Soudry Ethan , Pérez Jimena , Sidransnky David , Muñoz Sergio , Guerrero-Preston Rafael , Roa Juan Carlos 
Published: June 2012

Abstract
Introduction: Cervical cancer (CC) is the second most common neoplasia in women and it is the fifth cause of death by cancer in this population. Pap smears and HPV infection tests have been the most widely used tools to detect cytological abnormalities and women's risk of developing cervical cancer. However, they cannot distinguish between lesions that will progress to an invasive carcinoma and those that will not. Methylation biomarkers could be useful in early detection and progression for cervical carcinoma. Several studies have shown an association between promoter DNA hypermethylation and decreased gene expression.
Aim: To identify potential epigenetic biomarkers for diagnosis and clinical management of cervical cancer.
Materials and Methods: Isolated DNA from 12 normal samples and 7 cervical cancers was enriched with Methylated DNA Immunoprecipitation (MeDIP) and hybridized to Nimblegen 385K CpG Islands plus Promoter arrays (MeDIP-chip). Bioinformatics strategies were used for background correction, array normalization and data analysis of differentially methylated genomic regions between tumor and normal tissue. Methylation Specific PCR (MSP) validated promoter methylation frequency in the 221 samples: 25 normal, 66 Low Grade Lesions (LSIL), 91 High Grade Lesions (HSIL) and 39 cervical cancers (CC). Immunohistochemistry (IHQ) was performed to identify expression of genes at protein level in a separate cohort of 122 samples: 59 normal and 63 CC. Immunoreactivity score was determined by tabulating the percentage of cells with different levels of reactivity to the target antibodies.
Results. Two hypermethylated genes were identified as potential biomarkers after validation by MSP: GGTLA4 and ZNF516. The methylation frequency for the two genes was higher in tumor: GGTLA4 (100%) and ZNF516 (96%); than in normal samples: GGTLA4 (12%) and ZNF516 (16%). The methylation status of GGTLA4 showed a progression in methylation frequency from normal samples to invasive carcinoma. The immunohistochemical expression for the two genes was lower in tumor: GGTLA4 (50.8 %) and ZNF516 (66.2%); than in normal samples: GGTLA4 (71.2%) and ZNF516 (88.1%) (p&lt;0.05).
Conclusions: We have identified two potential methylation markers for cervical cancer, GGTLA4 and ZNF516, with the use of MeDIP-chip and confirmed the results with IHQ in an independent cohort. These genes may be useful tools for diagnosis and clinical management cervical cancer. These results need to be confirmed in an independent laboratory before the genes are examined in a Phase II Biomarker Development Trial.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3026. doi:10.1158/1538-7445.AM2011-3026