Abstract New biomarkers are needed to improve cervical cancer screening technologies, which are mostly based on cytological examination since the 1940's. HPV testing has been adopted for the triage of patients after a cervical cytology-screening test. HPV testing is now also increasingly used for screening in conjunction with cervical cytology. However, a percentage of HPV-positive and inconclusive-Pap women have negative biopsies when refered to colposcopy clinics. The aim of this study was to use a genome-wide discovery approach to identify novel epigenetic biomarkers for cervical cancer, which could be used as companion diagnostic panels to HPV and PAP. DNA from twelve normal and seven cervical cancer samples was enriched with Methylated DNA Immunoprecipitation (MeDIP), hybridized to Nimblegen 385K CpG Islands plus Promoter arrays and validated by quantitative Methylation Specific PCR in discovery (n=49) and prevalence cohorts (n=108). After correction and normalization performed using Nimblegen algorithms genes methylated in tumor and not in normal samples were ranked by methylation peak values and sequence homology. The most significant loci were verified against a report by Ongenaert et al. (BMC Med Genomics, 2008) that uses a relaxation ranking algorithm to identify re-expressed genes in cervical cancer cell lines after treatment with demethyalting agents. Two genes, ZNF516 and FKBP6, were identified as candidate epigenomic biomarkers after rigourous bioinformatics and in-silico analyses. These genes were also found to be re-expressed by Ongenaert et al. Using the most optimal cut-off as determined by ROC, ZNF516 promoter methylation had 90% sensitivity and 95% specificity (AUC=0.95) in the discovery cohort. FKBP6 promoter methylation had a sensitivity of 73% and a specificity of 79% in the same cohort. Promoter methylation of FKBP6 (OR=4.51, 95%C.I.=2.04-9.97, P<0.001) and ZNF516 (OR=11.84, 95%C.I.=4.59-30.57, P<0.001) was associated to HPV infection in the prevalence cohort. FKBP6 (OR=7.15, 95%C.I.=1.45-35.34, P=0.01) and ZNF516 (OR=26.72, 95%C.I.=2.61-273.05, P <0.01) promoter methylation was associated with histological diagnosis of cervical cancer in the overall cohort, after controlling for age and HPV infection. Promoter methylation of ZNF516 and FKBP6 performed better than HPV at identifying normal from tumor tissue in the overall cohort. Our results suggest that a genome-wide approach using MeDIP-Chip and qMSP is useful for identifying novel screening and diagnostic markers. ZNF516 and FKBP6 were identified as a potential companion diagnostic panel for HPV-positive and inconclusive-Pap women. Examination of these biomarkers in a larger, independent cohort is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5574. doi:1538-7445.AM2012-5574