Investigating Human Papillomavirus 16 Methylation As A Biomarker For High-Grade Cervical Intraepithelial Neoplasia

INTRODUCTION: Although type-specific detection of human papillomavirus (HPV) 16 or 18 is recommended to improve specificity of HPV DNA cotesting, further risk stratification is needed. METHODS: Women in two urban clinics were enrolled into a biorepository for cervical cancer biomarker discovery. Total nucleic acids (consisting of both DNA and RNA) or DNA only was extracted from cervical cells collected at colposcopy. TNA was used for HPV typing and determination of HPV 16 viral load and E6–E7 transcript copies by real-time polymerase chain reaction. Methylation at 19 cytosine guanine dinucleotide (CpG) sites in the E6 promoter, LCR, and L1 region was determined by pyrosequencing bisulfite treated TNA or DNA. Cervical disease classification was based on integration of biopsy, colposcopy, and cytology findings. All HPV 16-positive women enrolled within the first year who had methylation data were analyzed (n=109; 54 cervical intraepithelial neoplasia [CIN] 0–1, 25 CIN 2, 30 CIN 3+). Statistical significance of associations was determined with Kruskal-Wallis test and calculating odds ratios. RESULTS: Ratio of E6–E7 transcript copies to viral DNA copy was positively correlated with methylation at all four CpG sites in the L1 region (r=0.363–0.477). Methylation at CpG sites 7091, 7434, 7455, 7553, and 7862 were all significantly associated with CIN 2+ with the strongest association at CpG site 7091 (P