Abstract PR08: Identification of novel methylated genomic regions in invasive cervical cancer using a genome-wide approach

Abstract

Abstract Introduction: In the US, an estimated 35,000 cancer cases per year are attributable to Human papillomavirus (HPV), including cancers of the cervix, anus and oropharynx. Despite the availability of an HPV-targeted vaccine, issues with low uptake and significant lag time from vaccination to cancer prevention will result in cervical cancer continuing to be a substantial burden to US and world-wide populations. It is becoming increasingly clear that epigenetic events play a critical role in HPV-associated carcinogenesis, including changes in host genome DNA methylation patterns. In the cervix, methylation patterns have been shown to be altered; however, there have been inconclusive findings as to the most informative methylation targets in cervical cancer. We hypothesize that there are novel epigenetic alterations in HPV-associated cervical cancer and these changes may be early detection biomarkers in pre-invasive cervical lesions. In this study, we sought to identify epigenetic differences in HPV-positive cervical cancers compared to HPV-positive normal tissue using genome-wide methylation arrays and to explore if these differences can be identified in cervical intraepithelial neoplasia (CIN) 3 lesions. Methods: DNA was extracted from 24 frozen cervical tissues (9 normal, 5 CIN 3 and 10 invasive cancers) following pathology review and macrodissection. Bisulfite-modified DNA was interrogated for methylation at over 450,000 CpG loci using the Illumina HumanMethylation450 BeadChip array. HPV genotyping was conducted using SFP10 LiPA HPV-typing system. Differential methylation between normal and tumors at individual CpG loci was determined by t-test with significance set by a false discovery rate (or q-value) and final sets were restricted to those with a mean difference in β-values≥0.3. Next, using a window-based approach, we examined clusters of CpG loci within gene coding regions or CpG islands. Finally, a binary classification strategy was used for each gene set of probes, where a probe was defined as methylated if the β-values were above the minimum point in the bi-modal histogram. Sensitivity and specificity for each gene at the set methylation cut-point was determined. Results: A total of 24 HPV positive tissues were examined in this study (9 normal, 5 CIN 3 and 10 invasive cancers). A total of 321 CpG loci displayed differential methylation with mean difference ≥0.5 in tumors compared to normal (FDR corrected q<0.0005), of which 310 were hypermethylated and 11 were hypomethylated in tumors. We examined CpG site clusters within gene-coding regions among a larger set of 13,515 loci differentially methylated using less stringent criteria (q<0.01, β-value difference≥0.3). A total of 369 genes demonstrated differential methylation across clusters of CpG loci (12-1293 CpG sites per gene) between normal and tumors. Of these 369 genes, 80 were differentially methylated in ≥50% of CpG loci evaluated within the gene. Methylation status of 19 genes had both a sensitivity and specificity for distinguishing tumor from normal of 100%, including MIR129-2, protocadherin 10 (PCDH-10), and POU class 3 homeobox 3 (POU3F3). All 19 genes were also differentially methylated in at least one CIN 3 lesion. Conclusions: We have identified several novel methylated CpG loci and genomic regions in HPV-associated cervical cancer, some of which were also found to be altered in CIN 3 lesions. Validation of these novel CpG loci in a larger population of women with cervical dysplasia and cancer is warranted. A number of these novel methylated sites may emerge as promising biomarkers for the early detection of cervical cancer and possibly, for other HPV-associated malignancies. This abstract is also presented as Poster C24. Citation Format: Erin M. Siegel, Anders Berglund, Bridget Riggs, Steven Eschrich, Maddox Kristen, Abidemi Ajidahun, David Shibata, Kevin D. Brown. Identification of novel methylated genomic regions in invasive cervical cancer using a genome-wide approach. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr PR08.