title: HPV16 methyl-haplotypes determined by a novel next-generation sequencing method are associated with cervical precancer.
abstract: We have developed and evaluated a next-generation bisulfite sequencing (NGS) assay to distinguish HPV16 cervical precancer (CIN2-3; N=59) from HPV16-positive transient infections (N=40). Cervical DNA was isolated and treated with bisulfite and HPV16 methylation was quantified by (i) amplification with barcoded primers and massively parallel single molecule sequencing and (ii) site-specific pyrosequencing. Assays were evaluated for agreement using intraclass correlation coefficients (ICC). Odds ratios (OR) for high methylation vs. low methylation were calculated. Single site pyrosequencing and NGS data were correlated (ICC=0.61) and both indicated hypermethylation was associated with precancer (ORs of 2-37). Concordant NGS and pyrosequencing results yieled ORs that were stronger when compared with using either assay separately. Within the L1 region, the ORs for CIN2-3 were 14.3 and 22.4 using pyrosequencing and NGS assays, respectively; when both methods agreed the OR was 153. NGS assays provide methylation haplotypes, termed methyl-haplotypes from single molecule reads: cases had increased methyl-haplotypes with ≥1 methylated CpG site(s) per fragment compared with controls, particularly in L1 (p=3.0×10(-8)). The maximum discrimination of cases from controls for a L1 methyl-haplotype had an AUC of 0.89 corresponding to a sensitivity of 92.5% and a specificity of 73.1%. The strengthening of the OR when the two assays were concordant suggests the true association of CpG methylation with precancer is stronger than with either assay. As cervical cancer prevention moves to DNA testing methods, DNA based biomarkers, such as HPV methylation could serve as a reflex strategy to identify women at high risk for cervix cancer.
authors: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD.
authors: Lisa
authors: L
authors: Mirabello
authors: Marina
authors: M
authors: Frimer
authors: Ariana
authors: A
authors: Harari
authors: Thomas
authors: T
authors: McAndrew
authors: Benjamin
authors: B
authors: Smith
authors: Zigui
authors: Z
authors: Chen
authors: Nicolas
authors: N
authors: Wentzensen
authors: Sholom
authors: S
authors: Wacholder
authors: Philip E
authors: PE
authors: Castle
authors: Tina
authors: T
authors: Raine-Bennett
authors: Mark
authors: M
authors: Schiffman
authors: Robert D
authors: RD
authors: Burk
mesh_terms: D000328:Adult; D000368:Aged; D016022:Case-Control Studies; D019175:DNA Methylation; D005260:Female; D006239:Haplotypes; D059014:High-Throughput Nucleotide Sequencing; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D008875:Middle Aged; D030361:Papillomavirus Infections / Q000821:virology*; D011230:Precancerous Conditions / Q000821:virology*; D017422:Sequence Analysis, DNA; D002583:Uterine Cervical Neoplasms / Q000821:virology*; D055815:Young Adult
keywords: HPV16; cervical precancer; methylation; next-generation sequencing; pyrosequencing