Genome-wide methylome analysis using MethylCap-seq uncovers 4 hypermethylated markers with high sensitivity for both adeno- and squamous-cell cervical carcinoma

Abstract

Background: Cytology-based screening methods for cervical adenocarcinoma (ADC) and to a lesser extent squamous-cell carcinoma (SCC) suffer from low sensitivity. DNA hypermethylation analysis in cervical scrapings may improve detection of SCC, but few methylation markers have been described for ADC. We aimed to identify novel methylation markers for the early detection of both ADC and SCC.Results: Genome-wide methylation profiling for 20 normal cervices, 6 ADC and 6 SCC using MethylCap-seq yielded 53 candidate regions hypermethylated in both ADC and SCC. Verification and independent validation of the 15 most significant regions revealed 5 markers with differential methylation between 17 normals and 13 cancers. Quantitative methylation-specific PCR on cervical cancer scrapings resulted in detection rates ranging between 80% and 92% while between 94% and 99% of control scrapings tested negative. Four markers (SLC6A5, SOX1, SOX14 and TBX20) detected ADC and SCC with similar sensitivity. In scrapings from women referred with an abnormal smear (n=229), CIN3+ sensitivity was between 36% and 71%, while between 71% and 93% of adenocarcinoma in situ (AdCIS) were detected; and CIN0/1 specificity was between 88% and 98%. Compared to hrHPV, the combination SOX1/SOX14 showed a similar CIN3+ sensitivity (80% vs. 75%, respectively, P>0.2), while specificity improved (42% vs. 84%, respectively, P < 10

Host-cell DNA methylation patterns during high-risk HPV-induced carcinogenesis reveal a heterogeneous nature of cervical pre-cancer

“…DNA extraction, quality assessment, sodium bisulfite-treatment, (multiplex) qMSP conditions, and DNA methylation ratio calculations were described previously for ANKRD18CP, C13orf18, EPB41L3 , and JAM3 [8,9]; SOX1 and ZSCAN1 [9,10]; GHSR, SST and ZIC1 [11]; and FAM19A4, PHACTR3 , and PRDM14 [12]. Quality control was routinely performed using a methylation-independent assay for the housekeeping gene ACTB [13].…”

“…Using both targeted and genome-wide approaches, we previously identified 12 genes, including ANKRD18CP, C13orf18, EPB41L3 , and JAM3 [8,9]; SOX1 and ZSCAN1 [9,10]; GHSR, SST , and ZIC1 [11]; and FAM19A4, PHACTR3 , and PRDM14 [12]. These genes were considered promising biomarkers to identify hrHPV-positive women and/or women with abnormal cytology at risk for cervical cancer [1,7–14].…”

DNA methylation markers as a triage test for identification of cervical lesions in a high risk human papillomavirus positive screening cohort

“…Over the course of several years we sought to develop and validate sensitive and specific DNA methylation assays in order to further improve the early detection of cervical cancer . In cohorts of women referred to the outpatient clinic with an abnormal cytology result, a combination of specific methylation markers as a triage method showed similar sensitivity of ~75% for detection of CIN2+ compared to hrHPV testing, but better specificity.…”

“…The purpose of the present study was to assess the diagnostic potential of our previously established CIN2+ specific methylation markers (i.e., sensitivity, specificity and positive and negative predictive value (PPV and NPV)) in hrHPV positive women attending the routine national cervical cancer screening program with high‐coverage in Slovenia. The diagnostic performance was additionally compared to previously acquired data from a Dutch cohort, which was primarily used to validate our methylation markers …”

Sex-determining region Y box-containing genes: regulators and biomarkers in gynecological cancers

“…Higher methylation level of these genes was accompanied by more severe cervical squamous cell lesions 14,41-46,55 . Moreover, the sensitivity and specificity of this early screening method was increased by the combined detection of the methylation level of more than one SOX gene, such as SOX1 and SOX14 , or SOX8 and SOX17 53,65 . Further studies are therefore needed to determine if analyzing the expression level of all SOX genes combined might increase their sensitivity as screening biomarkers for CC.…”