title: Elevated methylation of HPV16 DNA is associated with the development of high grade cervical intraepithelial neoplasia.
abstract: We explored the association of human papillomavirus type 16 (HPV16) DNA methylation with age, viral load, viral persistence and risk of incident and prevalent high grade CIN (CIN2+) in serially collected specimens from the Guanacaste, Costa Rica cohort. 273 exfoliated cervical cell specimens (diagnostic and pre-diagnostic) were selected: (1) 92 with HPV16 DNA clearance (controls), (2) 72 with HPV16 DNA persistence (without CIN2+) and (3) 109 with CIN2+. DNA was extracted, bisulfite converted and methylation was quantified using pyrosequencing assays at 66 CpGs across the HPV genome. The Kruskal-Wallis test was used to determine significant differences among groups, and receiver operating characteristic curve analyses were used to evaluate how well methylation identified women with CIN2+. In diagnostic specimens, 88% of CpG sites had significantly higher methylation levels in CIN2+ after correction for multiple tests compared with controls. The highest area under the ROC curve (AUC) was 0.82 for CpG site 6457 in L1, and a diagnostic sensitivity of 91% corresponded to a specificity of 60% for CIN2+. Prospectively, 17% of CpG sites had significantly higher methylation in pre-diagnostic CIN2+ specimens (median time of 3 years before diagnosis) versus controls. The strongest pre-diagnostic CpG site was 6367 in L1 with an AUC of 0.76. Age-stratified analyses suggested that women older than the median age of 28 years have an increased risk of precancer associated with high methylation. Higher methylation in CIN2+ cases was not explained by higher viral load. We conclude that elevated levels of HPV16 DNA methylation may be useful to predict concurrently diagnosed as well as future CIN2+.
authors: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA. mirabellol@mail.nih.gov
authors: Lisa
authors: L
authors: Mirabello
authors: Mark
authors: M
authors: Schiffman
authors: Arpita
authors: A
authors: Ghosh
authors: Ana C
authors: AC
authors: Rodriguez
authors: Natasa
authors: N
authors: Vasiljevic
authors: Nicolas
authors: N
authors: Wentzensen
authors: Rolando
authors: R
authors: Herrero
authors: Allan
authors: A
authors: Hildesheim
authors: Sholom
authors: S
authors: Wacholder
authors: Dorota
authors: D
authors: Scibior-Bentkowska
authors: Robert D
authors: RD
authors: Burk
authors: Attila T
authors: AT
authors: Lorincz
mesh_terms: D000293:Adolescent; D000328:Adult; D019540:Area Under Curve; D018899:CpG Islands; D019175:DNA Methylation; D004279:DNA, Viral / Q000378:metabolism*; D005260:Female; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D008875:Middle Aged; D060787:Neoplasm Grading; D002583:Uterine Cervical Neoplasms / Q000209:etiology / Q000235:genetics* / Q000473:pathology* / Q000821:virology; D002578:Uterine Cervical Dysplasia / Q000209:etiology / Q000235:genetics* / Q000473:pathology* / Q000821:virology
chemical_list: D004279:DNA, Viral