title: Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3.
abstract: BACKGROUND
  Assessment of DNA promoter methylation markers in cervical scrapings for the detection of cervical intraepithelial neoplasia (CIN) and cervical cancer is feasible, but finding methylation markers with both high sensitivity as well as high specificity remains a challenge. In this study, we aimed to identify new methylation markers for the detection of high-grade CIN (CIN2/3 or worse, CIN2+) by using innovative genome-wide methylation analysis (MethylCap-seq). We focused on diagnostic performance of methylation markers with high sensitivity and high specificity considering any methylation level as positive.
  
  
  RESULTS
  MethylCap-seq of normal cervices and CIN2/3 revealed 176 differentially methylated regions (DMRs) comprising 164 genes. After verification and validation of the 15 best discriminating genes with methylation-specific PCR (MSP), 9 genes showed significant differential methylation in an independent cohort of normal cervices versus CIN2/3 lesions (p < 0.05). For further diagnostic evaluation, these 9 markers were tested with quantitative MSP (QMSP) in cervical scrapings from 2 cohorts: (1) cervical carcinoma versus healthy controls and (2) patients referred from population-based screening with an abnormal Pap smear in whom also HPV status was determined. Methylation levels of 8/9 genes were significantly higher in carcinoma compared to normal scrapings. For all 8 genes, methylation levels increased with the severity of the underlying histological lesion in scrapings from patients referred with an abnormal Pap smear. In addition, the diagnostic performance was investigated, using these 8 new genes and 4 genes (previously identified by our group: C13ORF18, JAM3, EPB41L3, and TERT). In a triage setting (after a positive Pap smear), sensitivity for CIN2+ of the best combination of genes (C13ORF18/JAM3/ANKRD18CP) (74 %) was comparable to hrHPV testing (79 %), while specificity was significantly higher (76 % versus 42 %, p ≤ 0.05). In addition, in hrHPV-positive scrapings, sensitivity and specificity for CIN2+ of this best-performing combination was comparable to the population referred with abnormal Pap smear.
  
  
  CONCLUSIONS
  We identified new CIN2/3-specific methylation markers using genome-wide DNA methylation analysis. The diagnostic performance of our new methylation panel shows higher specificity, which should result in prevention of unnecessary colposcopies for women referred with abnormal cytology. In addition, these newly found markers might be applied as a triage test in hrHPV-positive women from population-based screening. The next step before implementation in primary screening programs will be validation in population-based cohorts.
authors.affiliation: Department of Gynecologic Oncology, internal postal code DA13, Cancer Reserch Center Groningen, University of Groningen, University Medical Center Groningen, PO box 30.001, 9700 RB Groningen, The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: Boers
authors.affiliation: Department of Gynecologic Oncology, internal postal code DA13, Cancer Reserch Center Groningen, University of Groningen, University Medical Center Groningen, PO box 30.001, 9700 RB Groningen, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Wang
authors.affiliation: Department of Gynecologic Oncology, internal postal code DA13, Cancer Reserch Center Groningen, University of Groningen, University Medical Center Groningen, PO box 30.001, 9700 RB Groningen, The Netherlands.
authors.forename: R W
authors.initials: RW
authors.lastname: van Leeuwen
authors.affiliation: Department of Gynecologic Oncology, internal postal code DA13, Cancer Reserch Center Groningen, University of Groningen, University Medical Center Groningen, PO box 30.001, 9700 RB Groningen, The Netherlands.
authors.forename: H G
authors.initials: HG
authors.lastname: Klip
authors.affiliation: Department of Epidemiology, Cancer Reserch Center Groningen, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: G H
authors.initials: GH
authors.lastname: de Bock
authors.affiliation: Department of Pathology, Cancer Reserch Center Groningen, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: H
authors.initials: H
authors.lastname: Hollema
authors.affiliation: Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: W
authors.initials: W
authors.lastname: van Criekinge
authors.affiliation: Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: T
authors.initials: T
authors.lastname: de Meyer
authors.affiliation: Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: S
authors.initials: S
authors.lastname: Denil
authors.affiliation: Department of Gynecologic Oncology, internal postal code DA13, Cancer Reserch Center Groningen, University of Groningen, University Medical Center Groningen, PO box 30.001, 9700 RB Groningen, The Netherlands.
authors.forename: A G J
authors.initials: AGJ
authors.lastname: van der Zee
authors.affiliation: Department of Pathology, Cancer Reserch Center Groningen, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: Schuuring
authors.affiliation: Department of Gynecologic Oncology, internal postal code DA13, Cancer Reserch Center Groningen, University of Groningen, University Medical Center Groningen, PO box 30.001, 9700 RB Groningen, The Netherlands.
authors.forename: G B A
authors.initials: GBA
authors.lastname: Wisman
mesh_terms: D000328:Adult; D016022:Case-Control Studies; D002584:Cervix Uteri / Q000473:pathology; D019175:DNA Methylation / Q000235:genetics; D005260:Female; D052138:Genes, Neoplasm / Q000235:genetics; D005819:Genetic Markers; D055106:Genome-Wide Association Study; D006801:Humans; D065006:Papanicolaou Test; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D002578:Uterine Cervical Dysplasia / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology
chemical_list: D005819:Genetic Markers
keywords: Cervical cancer screening; Cervical precancerous lesions; Cervical scraping; DNA methylation; Human papillomavirus (HPV); MethylCap-seq; Quantitative methylation-specific PCR (QMSP)