Discovery and validation of candidate host DNA methylation markers for detection of cervical precancer and cancer |
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| Authors: | Megan A Clarke Patricia Luhn Julia C Gage Clara Bodelon S Terence Dunn Joan Walker Rosemary Zuna Stephen Hewitt J Keith Killian Liying Yan Andrew Miller Mark Schiffman Nicolas Wentzensen |
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| Affiliation: | 1. Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD;2. Genentech Inc, South San Francisco, CA;3. Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK;4. Center for Cancer Research, National Cancer Institute, Bethesda, MD;5. EpigenDx Inc, Hopkinton, MA |
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| Abstract: | Human papillomavirus (HPV) testing has been recently introduced as an alternative to cytology for cervical cancer screening. However, since most HPV infections clear without causing clinically relevant lesions, additional triage tests are required to identify women who are at high risk of developing cancer. We performed DNA methylation profiling on formalin‐fixed, paraffin‐embedded tissue specimens from women with benign HPV16 infection and histologically confirmed cervical intraepithelial neoplasia grade 3, and cancer using a bead‐based microarray covering 1,500 CpG sites in over 800 genes. Methylation levels in individual CpG sites were compared using a t‐test, and results were summarized by computing p‐values. A total of 12 candidate genes (ADCYAP1, ASCL1, ATP10, CADM1, DCC, DBC1, HS3ST2, MOS, MYOD1, SOX1, SOX17 and TMEFF2) identified by DNA methylation profiling, plus an additional three genes identified from the literature (EPB41L3, MAL and miR‐124) were chosen for validation in an independent set of 167 liquid‐based cytology specimens using pyrosequencing and targeted, next‐generation bisulfite sequencing. Of the 15 candidate gene markers, 10 had an area under the curve (AUC) of ≥ 0.75 for discrimination of high grade squamous intraepithelial lesions or worse (HSIL+) from <HSIL cytology using at least one assay. Overall, SOX1, DCC, and EPB41L3 showed the best discrimination with AUC values of ≥0.80, irrespective of methylation detection assay. In addition to verifying candidate markers from the literature (e.g., SOX1 and EPB41L3), we identified novel markers that may be considered for detection of cervical precancer and cancer and warrant further validation in prospective studies. |
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| Keywords: | methylation cervical cancer human papillomavirus next‐generation sequencing pyrosequencing |
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