Abstract
DNA methylation biomarkers are increasingly utilized for the detection, prognosis and monitoring of cancer. Here we use publicly-available whole genome bisulfite sequencing data to identify differentially methylated regions (cDMRs) in diverse tumor types and further define a set of genomic target regions that have optimal characteristics for Methylation Sensitive Restriction Enzyme-PCR (MSRE-PCR)-based detection: conserved hypermethylation in tumors, abundant MSRE sites and low methylation levels in normal tissues. The identified MSRE-PCR target regions (n = 1,294) were primarily encompassed within CpG islands (97%) and promoters (81%) with 39% of the target regions overlapping the transcription start site. Gene set enrichment analysis of the target regions identified significant enrichment of genes involved in neuronal development. A multiplexed MSRE-PCR assay was developed interrogating 47 target regions and was tested on a set of genomic DNAs (n = 100) from diverse tumor and normal tissue types including colon, breast, lung, stomach and blood. A logistic regression model containing seven target region amplicons distinguished between tumor and normal tissue in the training (n = 50) with a ROC AUC of 0.97 (95% CI [0.92, 1]) and independent test set (n = 50) with an AUC of 0.93 (95% CI [0.84, 1]). These findings show that genomic regions with conserved hypermethylation across diverse tumor types, abundant MSRE sites and low methylation levels in normal tissues provide target regions for the detection of tumor DNA via MSRE-PCR. The selective amplification of tumor-derived DNA via MSRE-PCR may have utility in the development of non-invasive cancer detection and surveillance strategies.
Highlights
Global epigenetic changes, including DNA methylation, are widely regarded as a hallmark of cancer [1]
We describe the identification of Differentially Methylated Region in cancer (cDMR) in diverse tumor types using publicly available Whole Genome Bisulfite Sequencing (WGBS) data and further define a set of 1,294 target regions with optimal characteristics for Methylation Sensitive Restriction Enzyme (MSRE)-PCR-based detection of tumor DNA
Despite the diversity of tumor and tissue types in the MSRE-PCR cohort, a logistic regression model using 7 of the amplicons distinguished between tumor versus normal tissue with an area under the curve (AUC) of 0.97 in the training set and 0.93 in the independent test set
Summary
Global epigenetic changes, including DNA methylation, are widely regarded as a hallmark of cancer [1]. Alterations include a decrease in overall CpG methylation levels coupled with discrete regions of hypermethylation, typically localized in the promoterassociated CpG islands. Hypermethylation has been associated with cancer progression [2] and the silencing of growth regulating genes and tumor suppressor genes [3]. A growing number of DNA methylation biomarkers are being utilized in the development of novel assays for monitoring cancer progression [4, 5], treatment response [6] and early detection [7]
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