title: Quantitative measurement of Human Papillomavirus type 16 L1/L2 DNA methylation correlates with cervical disease grade.
abstract: BACKGROUND
  Persistent infection with Human Papillomavirus (HPV) type 16 causes the majority of cervical cancers. Genital HPV infection is very common, but neoplastic progression is uncommon. There is an urgent need to identify biomarkers associated with cervical neoplasia that can be used to triage women who test positive for HPV.
  
  
  OBJECTIVE
  To assess the ability of quantitative measurement of HPV16 DNA methylation to separate samples of different cytology grades and cervical cancers, and determine which of the assessed regions of the HPV genome and individual CpGs are most informative.
  
  
  METHODS
  DNA methylation was quantified by pyrosequencing of bisulphite converted DNA from liquid based cytology samples from 17 women with normal cytology and 20 women with severe dyskaryosis, and from fixed tissue from 24 women with cervical cancer. Methylation was assessed in the HPV Long Control Region (LCR), E2 and L1/L2 regions.
  
  
  RESULTS
  In cervical cancers, increased HPV DNA methylation was present in all regions. Increased methylation was also observed in severely dyskaryotic relative to normal samples, but only in the E2 and L1/L2 regions. The ability of methylation based classifiers to separate the three classes of material was assessed by ROC curve analyses. The best separation between normal and dyskaryotic samples was achieved by assessment of the L1/L2 CpGs at nucleotide positions 5600 and 5609 (AUC=0.900, 95% CI: 0.793-1).
  
  
  CONCLUSIONS
  This study demonstrates the potential of quantification of HPV DNA methylation as a biomarker of cervical neoplasia. An algorithm considering methylation at specific L1/L2 CpGs appeared the most promising model.
authors.affiliation: School of Medicine, Cardiff University, UK.
authors.forename: Dean
authors.initials: D
authors.lastname: Bryant
authors.affiliation: School of Medicine, Cardiff University, UK.
authors.forename: Amanda
authors.initials: A
authors.lastname: Tristram
authors.affiliation: Department of Molecular and Clinical Cancer Medicine, University of Liverpool, UK.
authors.forename: Triantafillos
authors.initials: T
authors.lastname: Liloglou
authors.affiliation: School of Medicine, Cardiff University, UK.
authors.forename: Samantha
authors.initials: S
authors.lastname: Hibbitts
authors.affiliation: School of Medicine, Cardiff University, UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Fiander
authors.affiliation: School of Medicine, Cardiff University, UK. Electronic address: powellng@cf.ac.uk.
authors.forename: Ned
authors.initials: N
authors.lastname: Powell
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000032:analysis; D036022:Capsid Proteins / Q000235:genetics*; D019175:DNA Methylation; D004279:DNA, Viral / Q000378:metabolism*; D005260:Female; D006801:Humans; D008875:Middle Aged; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D030361:Papillomavirus Infections / Q000150:complications* / Q000821:virology; D002583:Uterine Cervical Neoplasms / Q000473:pathology* / Q000821:virology
chemical_list: D015415:Biomarkers; D036022:Capsid Proteins; D004279:DNA, Viral; C063316:L2 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; C063317:L1 protein, Human papillomavirus type 16
keywords: AUC; Area Under Curve; CIN; Cervical cancer; HPV; Human Papillomavirus; LBC; LCR; Methylation; ROC; Receiver Operating Characteristic; cervical intraepithelial neoplasia; liquid based cytology; long control region