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Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes ( APC ,  CCNA ,  CDH1 ,  CDH13 ,  WIF1 ,  TIMP3 ,  DAPK1 ,  RARB ,  FHIT , and  SLIT2 ). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of  DAPK1 ,  SLIT2 ,  WIF1  and  RARB  with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97–1.00,  p-value  = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as  DAPK1 ,  RARB ,  WIF1 , and  SLIT2 , may also occur early in cervical carcinogenesis and should be evaluated.

Quantitative DNA Methylation Analysis of Candidate Genes in Cervical Cancer