title: Methylation marker analysis and HPV16/18 genotyping in high-risk HPV positive self-sampled specimens to identify women with high grade CIN or cervical cancer.
abstract: OBJECTIVE
  Methylation marker analysis using bi-marker panel MAL/miR-124-2 is a promising triage test for identifying cervical (pre)cancer in high-risk human papillomavirus (hrHPV) positive women. Bi-marker panel MAL/miR-124-2 can be applied directly on self-sampled cervico-vaginal material and its sensitivity is non-inferior to that of cytology, yet at the cost of more colposcopy referrals. Our objective was to increase specificity of MAL/miR-124-2 methylation analysis by varying the assay thresholds and adding HPV16/18 genotyping.
  
  
  METHODS
  1019 hrHPV-positive women were selected from a randomized controlled self-sampling trial (PROHTECT-3; 33-63 years, n=46,001) and nine triage strategies with methylation testing of MAL/miR-124-2 and HPV16/18 genotyping were evaluated. The methylation assay threshold was set at four different predefined levels which correspond with clinical specificities for end-point cervical intra-epithelial grade 3 or worse (CIN3+) of 50%, 60%, 70%, and 80%.
  
  
  RESULTS
  The CIN3+ sensitivity of methylation analysis decreased (73.5 to 44.9%) while specificity increased (47.2 to 83.4%) when increasing the assay threshold. CIN3+ sensitivity and specificity of HPV16/18 genotyping were 68.0% and 65.6%, respectively. Combined methylation analysis at threshold-80 and HPV16/18 genotyping yielded similar CIN3+ sensitivity as that of methylation only at threshold-50 (77.6%) with an increased specificity (54.8%).
  
  
  CONCLUSIONS
  Combined triage by MAL/miR-124-2 methylation analysis with threshold-80 and HPV16/18 genotyping reaches high CIN3+ sensitivity with increased specificity to identify women with cervical (pre)cancer among HPV self-sample positive women. The combined strategy is attractive as it is fully molecular and identifies women at the highest risk of cervical (pre)cancer because of strongly elevated methylation levels and/or HPV16/18 positivity.
authors.affiliation: Department of Pathology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
authors.forename: Viola M J
authors.initials: VM
authors.lastname: Verhoef
authors.affiliation: Department of Pathology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
authors.forename: Daniëlle A M
authors.initials: DA
authors.lastname: Heideman
authors.affiliation: Department of Pathology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
authors.forename: Folkert J
authors.initials: FJ
authors.lastname: van Kemenade
authors.affiliation: Department of Pathology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
authors.forename: Lawrence
authors.initials: L
authors.lastname: Rozendaal
authors.affiliation: Department of Obstetrics and Gynaecology, Radboud University Medical Center, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
authors.forename: Remko P
authors.initials: RP
authors.lastname: Bosgraaf
authors.affiliation: Department of Pathology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
authors.forename: Albertus T
authors.initials: AT
authors.lastname: Hesselink
authors.affiliation: Department of Obstetrics and Gynaecology, Radboud University Medical Center, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
authors.forename: Ruud L M
authors.initials: RL
authors.lastname: Bekkers
authors.affiliation: Department of Obstetrics and Gynaecology, Radboud University Medical Center, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
authors.forename: Leon F A G
authors.initials: LF
authors.lastname: Massuger
authors.affiliation: Department of Pathology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
authors.forename: Renske D M
authors.initials: RD
authors.lastname: Steenbergen
authors.affiliation: Department of Pathology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
authors.forename: Peter J F
authors.initials: PJ
authors.lastname: Snijders
authors.affiliation: Department of Epidemiology and Biostatistics, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
authors.forename: Johannes
authors.initials: J
authors.lastname: Berkhof
authors.affiliation: Department of Pathology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Electronic address: cjlm.meijer@vumc.nl.
authors.forename: Chris J L M
authors.initials: CJ
authors.lastname: Meijer
mesh_terms: D002584:Cervix Uteri / Q000821:virology*; D019175:DNA Methylation; D004279:DNA, Viral / Q000378:metabolism; D059026:Diagnostic Self Evaluation; D005260:Female; D005838:Genotype; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000302:isolation & purification; D052161:Human papillomavirus 18 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D035683:MicroRNAs; D060787:Neoplasm Grading; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis / Q000473:pathology* / Q000821:virology*; D002578:Uterine Cervical Dysplasia / Q000175:diagnosis / Q000473:pathology* / Q000821:virology
chemical_list: D004279:DNA, Viral; C509207:MIRN124 microRNA, human; D035683:MicroRNAs
keywords: Cervical intraepithelial neoplasia; Cervical screening; DNA methylation; HPV genotyping; Human papillomavirus; Self-sampling