Abstract
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.
Highlights
Epigenetic gene silencing via dense DNA methylation within CpG islands has been demonstrated to occur in many tumor types including human papillomavirus (HPV)-associated cervical cancer [1]
CDH1 gene hypermethylation has been observed in the majority of primary cervical tumors and a substantial number of high-grade cervical intraepithelial neoplasia-3 (CIN3), suggesting that the epigenetic status of this gene has a potential application as a biomarker of cervical malignancy [6]
We examined a set of normal cervical formalin-fixed paraffin embedded (FFPE) blocks from women archived from women who were diagnosed with either benign soft tissue uterine leiomyoma (N = 15) to examine methylation levels across by tissue preservation methods (LBC vs. FFPE)
Summary
Epigenetic gene silencing via dense DNA methylation within CpG islands has been demonstrated to occur in many tumor types including human papillomavirus (HPV)-associated cervical cancer [1]. Tumor suppressor genes (TSGs) that are of clear importance in the pathogenesis of cervical cancer are common targets for gene silencing in this disease [2,3]. Genes encoding several key regulators of the oncogenic Wnt/β-catenin pathway, such as CDH1 (E-cadherin), APC, and WIF1, are frequently silenced via dense methylation of their promoter regions in cervical cancer [5]. Other genes reportedly hypermethylated in cervical cancer with little to no methylation in normal or low-grade CINs include DAPK1[7,8], RARB [8,9], TIMP3 [10], CCNA[11] and FHIT [7]. There have been inconsistencies in the reported prevalence of DNA hypermethylation of several TSG within cervical cancers [2,12], which may be due to the use of non-quantitative methods to detect methylation
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