Molecular Triage of Premalignant Lesions in Liquid-Based Cervical Cytology and Circulating Cell-Free DNA from Urine, Using a Panel of Methylated Human Papilloma Virus and Host Genes

Cancer Prev Res (Phila). 2016 Dec;9(12):915-924. doi: 10.1158/1940-6207.CAPR-16-0138. Epub 2016 Sep 26.


Clinically useful molecular tools to triage women for a biopsy upon referral to colposcopy are not available. We aimed to develop a molecular panel to detect cervical intraepithelial neoplasia (CIN) grade 2 or higher lesions (CIN2+) in women with abnormal cervical cytology and high-risk HPV (HPV+). We tested a biomarker panel in cervical epithelium DNA obtained from 211 women evaluated in a cervical cancer clinic in Chile from 2006 to 2008. Results were verified in a prospective cohort of 107 women evaluated in a high-risk clinic in Puerto Rico from 2013 to 2015. Promoter methylation of ZNF516, FKBP6, and INTS1 discriminated cervical brush samples with CIN2+ lesions from samples with no intraepithelial lesions or malignancy (NILM) with 90% sensitivity, 88.9% specificity, 0.94 area under the curve (AUC), 93.1% positive predictive value (PPV), and 84.2% negative predictive value (NPV). The panel results were verified in liquid-based cervical cytology samples from an independent cohort with 90.9% sensitivity, 60.9% specificity, 0.90 AUC, 52.6% PPV, and 93.3% NPV, after adding HPV16-L1 methylation to the panel. Next-generation sequencing results in HPV+ cultured cells, and urine circulating cell-free DNA (ccfDNA) were used to design assays that show clinical feasibility in a subset (n = 40) of paired plasma (AUC = 0.81) and urine (AUC = 0.86) ccfDNA samples obtained from the prospective cohort. Viral and host DNA methylation panels can be tested in liquid cytology and urine ccfDNA from women referred to colposcopy, to triage CIN2+ lesions for biopsy and inform personalized screening algorithms. Cancer Prev Res; 9(12); 915-24. ©2016 AACR.

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Aged, 80 and over
  • Biomarkers, Tumor / genetics*
  • Biopsy
  • Cohort Studies
  • Colposcopy
  • DNA Methylation*
  • DNA, Viral / genetics
  • DNA, Viral / urine
  • DNA-Binding Proteins / genetics
  • Female
  • Human Papillomavirus DNA Tests
  • Human papillomavirus 16 / genetics
  • Human papillomavirus 16 / isolation & purification*
  • Humans
  • Middle Aged
  • Papillomavirus Infections / blood
  • Papillomavirus Infections / diagnosis*
  • Papillomavirus Infections / urine
  • Papillomavirus Infections / virology
  • Prospective Studies
  • Retrospective Studies
  • Sensitivity and Specificity
  • Tacrolimus Binding Proteins / genetics
  • Uterine Cervical Dysplasia / diagnosis*
  • Uterine Cervical Dysplasia / pathology
  • Uterine Cervical Dysplasia / virology
  • Uterine Cervical Neoplasms / diagnosis*
  • Uterine Cervical Neoplasms / pathology
  • Uterine Cervical Neoplasms / virology
  • Vaccines, Virus-Like Particle / genetics
  • Vaginal Smears
  • Wnt1 Protein / genetics


  • Biomarkers, Tumor
  • DNA, Viral
  • DNA-Binding Proteins
  • FKBP6 protein, human
  • L1(deltaN26)-E7(deltaC38) protein, HPV16
  • Vaccines, Virus-Like Particle
  • WNT1 protein, human
  • Wnt1 Protein
  • ZNF516 protein, human
  • Tacrolimus Binding Proteins