Abstract 4248: Methylation specific digital karyotyping of human papillomavirus type 16 E6E7 expressing keratinocytes identifies novel methylation targets in cervical carcinogenesis.

Persistent infection with high-risk human papillomavirus (hrHPV) types is causally related to the development of cervical cancer and a subset of other anogenital and head and neck cancers. HrHPV-induced malignant transformation of epithelial cells is associated with (epi)genetic aberrations in host cell genes, including alterations in DNA methylation often affecting tumor suppressor gene expression. This study aimed to comprehensively unravel genome-wide DNA methylation events linked to a transforming hrHPV-infection, which is characterized by deregulated expression of the viral oncogenes E6 and E7 in dividing epithelial cells. Hereto, primary human keratinocytes transduced with HPV16E6E7 and their untransduced counterparts were subjected to methylation-specific digital karyotyping (MSDK) to screen for genome-wide DNA-methylation changes at different stages of HPV-induced transformation. Integration of the obtained methylation karyotype with genome-wide expression profiles of cervical carcinomas identified 34 genes with increased methylation in HPV-transformed cells and reduced expression in cervical carcinomas. For 12 genes (CLIC3, CREB3L1, FAM19A4, LFNG, LHX1, MRC2, NKX2-8, NPTX-1, PHACTR3, PRDM14, SOST and TNFSF13) specific methylation in HPV-containing cell lines was confirmed using a real-time methylation specific PCR (MSP) detection platform. Except from NPTX-1, methylation of none of these genes has to the best of our knowledge been linked to hrHPV-induced cancers before. Subsequent analysis of FAM19A4, LHX1, NKX2-8, NPTX-1, PHACTR3 and PRDM14 in cervical tissue specimens revealed an increase in methylation of all six genes with disease progression. All genes were commonly methylated in cervical carcinomas, with highest frequencies of up to 100% detected for FAM19A4, PHACTR3 and PRDM14. NKX2-8, PHACTR3 and PRDM14 were already methylated in approximately one third of precancerous lesions, indicating that their alterations represent relatively early events in cervical carcinogenesis. In conclusion, MSDK analysis of HPV16 transduced keratinocytes at different stages of HPV-induced transformation resulted in the identification of novel methylation events in cervical carcinogenesis, including methylation of FAM19A4, LHX1, NKX2-8, PHACTR3 and PRDM14 gene promoters. These genes may provide promising triage markers for assessment of the presence of (pre)cancerous cervical lesions in hrHPV-positive women. Citation Format: Renske D M Steenbergen, Mate Ongenaert, Suzanne Snellenberg, Chris JLM Meijer, Kornelia Polyak, Noga Bloushtain-Qimron, Peter JF Snijders, Wim van Criekinge. Methylation specific digital karyotyping of human papillomavirus type 16 E6E7 expressing keratinocytes identifies novel methylation targets in cervical carcinogenesis. [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 4248. doi:10.1158/1538-7445.AM2013-4248