title: Validation of the FAM19A4/mir124-2 DNA methylation test for both lavage- and brush-based self-samples to detect cervical (pre)cancer in HPV-positive women.
abstract: DNA methylation analysis of cancer-related genes is a promising tool for HPV-positive women to identify those with cervical (pre)cancer (CIN3+) in need of treatment. However, clinical performance of methylation markers can be influenced by the sample type utilized. We describe a multiplex quantitative methylation-specific PCR that targets FAM19A4 and mir124-2 loci, to detect CIN3+ using both HPV-positive lavage- and brush self-samples.
  
  
  
  We determined methylation thresholds for clinical classification using HPV-positive training sets comprising lavage self-samples of 182 women (including 40 with CIN3+) and brush self-samples of 224 women (including 61 with CIN3+). Subsequently, independent HPV-positive validation sets of 389 lavage self-samples (including 78 with CIN3+), and 254 brush self-samples (including 72 with CIN3+) were tested using the preset thresholds. Furthermore, the clinical performance of combined methylation analysis and HPV16/18 genotyping was determined.
  
  
  
  Training set analysis revealed similar FAM19A4 and mir124-2 thresholds for both self-sample types to yield highest CIN3+ sensitivity at 70% specificity. Validation set analysis resulted in a CIN3+ sensitivity of 70.5% (95%CI: 60.4-80.6) at a specificity of 67.8% (95%CI: 62.7-73.0) for lavage self-samples, and a CIN3+ sensitivity of 69.4% (95%CI: 58.8-80.1) at a 76.4% (95%CI: 70.2-82.6) specificity for brush self-samples. In combination with HPV16/18 genotyping, CIN3+ sensitivity and specificity were 88.5% (95%CI: 81.4-95.6) and 46.0% (95%CI: 40.4-51.5) for lavage self-samples, and 84.7% (95%CI: 76.4-93.0) and 54.9% (95%CI: 47.7-62.2) for brush self-samples.
  
  
  
  FAM19A4/mir124-2 methylation analysis performs equally well in HPV-positive lavage- and brush self-samples to identify women with CIN3+. In combination with HPV16/18 genotyping, significantly higher CIN3+ sensitivities are obtained, at decreased specificity.
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: l.destrooper@vumc.nl.
authors.forename: Lise M A
authors.initials: LMA
authors.lastname: De Strooper
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: v.verhoef@vumc.nl.
authors.forename: Viola M J
authors.initials: VMJ
authors.lastname: Verhoef
authors.affiliation: Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: h.berkhof@vumc.nl.
authors.forename: Johannes
authors.initials: J
authors.lastname: Berkhof
authors.affiliation: Self-Screen B.V., Amsterdam, The Netherlands. Electronic address: b.hesselink@vumc.nl.
authors.forename: Albertus T
authors.initials: AT
authors.lastname: Hesselink
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: hmedebruin@gmail.com.
authors.forename: Helena M E
authors.initials: HME
authors.lastname: de Bruin
authors.affiliation: Department of Pathology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands. Electronic address: f.vankemenade@erasmusmc.nl.
authors.forename: Folkert J
authors.initials: FJ
authors.lastname: van Kemenade
authors.affiliation: Department of Obstetrics and Gynaecology, Radboud University Medical Center, Nijmegen, The Netherlands. Electronic address: Remko.Bosgraaf@radboudumc.nl.
authors.forename: Remko P
authors.initials: RP
authors.lastname: Bosgraaf
authors.affiliation: Department of Obstetrics and Gynaecology, Radboud University Medical Center, Nijmegen, The Netherlands. Electronic address: Ruud.Bekkers@radboudumc.nl.
authors.forename: Ruud L M
authors.initials: RLM
authors.lastname: Bekkers
authors.affiliation: Department of Obstetrics and Gynaecology, Radboud University Medical Center, Nijmegen, The Netherlands. Electronic address: Leon.Massuger@radboudumc.nl.
authors.forename: Leon F A G
authors.initials: LFAG
authors.lastname: Massuger
authors.affiliation: Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands. Electronic address: Willem.Melchers@radboudumc.nl.
authors.forename: Willem J G
authors.initials: WJG
authors.lastname: Melchers
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: r.steenbergen@vumc.nl.
authors.forename: Renske D M
authors.initials: RDM
authors.lastname: Steenbergen
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: pjf.snijders@vumc.nl.
authors.forename: Peter J F
authors.initials: PJF
authors.lastname: Snijders
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: cjlm.meijer@vumc.nl.
authors.forename: Chris J L M
authors.initials: CJLM
authors.lastname: Meijer
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: dam.heideman@vumc.nl.
authors.forename: Daniëlle A M
authors.initials: DAM
authors.lastname: Heideman
mesh_terms: D000328:Adult; D016207:Cytokines / Q000235:genetics*; D019175:DNA Methylation; 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 / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D030361:Papillomavirus Infections / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000821:virology; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D014626:Vaginal Smears / Q000379:methods; D002578:Uterine Cervical Dysplasia / Q000235:genetics* / Q000378:metabolism / Q000821:virology
chemical_list: D016207:Cytokines; C509207:MIRN124 microRNA, human; D035683:MicroRNAs; C000607939:TAFA4 protein, human
keywords: Cervical cancer; DNA methylation; HPV16/18 genotyping; Human papillomavirus; Molecular screening; Reflex test; Self-sampling