title: Credentialing of DNA methylation assays for human genes as diagnostic biomarkers of cervical intraepithelial neoplasia in high-risk HPV positive women.
abstract: OBJECTIVE
  Testing for high risk human papillomavirus (HR-HPV) is increasing; however due to limitations in specificity there remains a need for better triage tests. Research efforts have focused recently on methylation of human genes which show promise as diagnostic classifiers.
  
  
  METHODS
  Methylation of 26 genes: APC, CADM1, CCND2, CDH13, CDKN2A, CTNNB1, DAPK1, DPYS, EDNRB, EPB41L3, ESR1, GSTP1, HIN1, JAM3, LMX1, MAL, MDR1, PAX1, PTGS2, RARB, RASSF1, SLIT2, SOX1, SPARC, TERT and TWIST1 was measured by pyrosequencing in cytology specimens from a pilot set of women with normal or cervical intraepithelial neoplasia grade 3 (CIN3) histology. Six genes were selected for testing in Predictors 1, a colposcopy referral study comprising 799 women. The three genes EPB41L3, DPYS and MAL were further tested in a second colposcopy referral study, Predictors 2, comprising 884 women.
  
  
  RESULTS
  The six genes selected from the pilot: EPB41L3, EDNRB, LMX1, DPYS, MAL and CADM1 showed significantly elevated methylation in CIN2 and CIN3 (CIN2/3) versus ≤CIN1 in Predictors 1 (p<0.01). Highest methylation was observed in cancer tissues. EPB41L3 methylation was the best single classifier of CIN2/3 in both HR-HPV positive (p<0.0001) and negative samples (p=0.02). Logistic regression modeling showed that other genes did not add significantly to EPB41L3 and in Predictors 2, its classifier value was validated with AUC 0.69 (95% CI 0.65-0.73).
  
  
  CONCLUSIONS
  Several methylated genes show promise for detecting CIN2/3 of which EPB41L3 seems the best. Methylated human gene biomarkers used in combination may be clinically useful for triage of women with HR-HPV infections.
authors: Queen Mary University of London, Centre for Cancer Prevention, Wolfson Institute of Preventive Medicine, Barts and The London School of Medicine, London, EC1M 6BQ, United Kingdom.
authors: Nataša
authors: N
authors: Vasiljević
authors: Queen Mary University of London, Centre for Cancer Prevention, Wolfson Institute of Preventive Medicine, Barts and The London School of Medicine, London, EC1M 6BQ, United Kingdom.
authors: Dorota
authors: D
authors: Scibior-Bentkowska
authors: Queen Mary University of London, Centre for Cancer Prevention, Wolfson Institute of Preventive Medicine, Barts and The London School of Medicine, London, EC1M 6BQ, United Kingdom.
authors: Adam R
authors: AR
authors: Brentnall
authors: Queen Mary University of London, Centre for Cancer Prevention, Wolfson Institute of Preventive Medicine, Barts and The London School of Medicine, London, EC1M 6BQ, United Kingdom.
authors: Jack
authors: J
authors: Cuzick
authors: Queen Mary University of London, Centre for Cancer Prevention, Wolfson Institute of Preventive Medicine, Barts and The London School of Medicine, London, EC1M 6BQ, United Kingdom. Electronic address: a.lorincz@qmul.ac.uk.
authors: Attila T
authors: AT
authors: Lorincz
mesh_terms: D000581:Amidohydrolases / Q000235:genetics; D014408:Biomarkers, Tumor / Q000235:genetics*; D019175:DNA Methylation; D005260:Female; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics; D030361:Papillomavirus Infections / Q000235:genetics* / Q000473:pathology; D015203:Reproducibility of Results; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000821:virology*; D002578:Uterine Cervical Dysplasia / Q000235:genetics* / Q000821:virology*
chemical_list: D014408:Biomarkers, Tumor; C116887:EPB41L3 protein, human; D008840:Microfilament Proteins; D000581:Amidohydrolases; C020047:dihydropyrimidinase
keywords: Cervical intraepithelial neoplasia; Diagnostic biomarker; EPB41L3; Human gene methylation