Methylation of human papillomavirus type 16 genome and risk of cervical precancer in a Costa Rican population

Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, 6120 Executive Blvd, EPS/7101, Rockville, MD 20892, USA.
CancerSpectrum Knowledge Environment (Impact Factor: 15.16). 03/2012; 104(7):556-65. DOI: 10.1093/jnci/djs135
Source: PubMed

ABSTRACT Previous studies have suggested an association between human papillomavirus type 16 (HPV16) genome methylation and cervical intraepithelial neoplasia grade 3 (CIN3) (ie, cervical precancer) and cancer, but the results have been inconsistent.
We designed a case-control study within a large prospective cohort of women who underwent multiple screenings for cervical cancer in Guanacaste, Costa Rica. Diagnostic specimens were collected at the time of CIN3 diagnosis (n = 30 case subjects) and persistent HPV16 infection (persistence; n = 35 case subjects), prediagnostic specimens at the first HPV16-positive screening visit (n = 20 CIN3 case subjects; n = 35 persistence case subjects), and control specimens from women with infection clearance within 2 years (n = 34 control subjects). DNA extracted from specimens (cervical cells) was analyzed for methylation levels at 67 CpG sites throughout the HPV16 genome using pyrosequencing. Benjamini-Hochberg method was used to account for multiple testing. Associations between methylation levels and risk of CIN3 or persistence were assessed using logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals (CIs).
Increased methylation in diagnostic vs control specimens at nine CpG sites, three in each L1, L2, and E2/E4 genomic regions, was associated with an increased risk of CIN3 (third tertile [high] vs first and second tertiles combined [low], OR = 3.29 [95% CI = 1.16 to 9.34] to 11.12 [95% CI = 2.29 to 76.80]) and persistence. High methylation at three of these CpG sites was associated with a much higher risk when combined compared with low methylation at these sites (OR = 52, 95% CI = 4.0 to 670). In prediagnostic vs control specimens, increased methylation at a CpG site (nucleotide position 4261) in L2 was associated with an increased risk of CIN3.
In this HPV16-infected cohort, increased methylation of CpG sites within the HPV16 genome before diagnosis and at the time of diagnosis was associated with cervical precancer.

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Available from: Robert D Burk, May 19, 2014
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    • "HPV testing is likely to replace cytology as the primary screen for cervical neoplasia [6], however the majority of HPV infections are transient and clinically inconsequential, hence a method to identify clinically significant infections is urgently required. Methylation of HPV16 DNA correlates with disease grade [7] [8] [9] [10] [11], and has shown considerable promise as a triage marker in preliminary clinical studies [10] [12] [13]. Previous studies have established the importance of integration of viral DNA into the host genome [14] and of up-regulation of the E6 and E7 oncogenes in driving neoplasia [15], but how viral DNA methylation relates to these aspects of viral oncogenesis remains to be established. "
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    ABSTRACT: Background Methylation of HPV16 DNA is a promising biomarker for triage of HPV positive cervical screening samples but the biological basis for the association between HPV-associated neoplasia and increased methylation is unclear. Objectives To determine whether HPV16 DNA methylation was associated with viral integration, and investigate the relationships between viral DNA methylation, integration and gene expression. Study design : HPV16 DNA methylation, integration and gene expression were assessed using pyrosequencing, ligation-mediated PCR and QPCR, in biopsies from 25 patients attending a specialist vulval neoplasia clinic and in short-term clonal cell lines derived from vulval and vaginal neoplasia. Results Increased methylation of the HPV16 L1/L2 and E2 regions was associated with integration of viral DNA into the host genome. This relationship was observed both in vivo and in vitro. Increased methylation of E2 binding sites did not appear to be associated with greater expression of viral early genes. Expression of HPV E6 and E7 did not correlate with either integration state or increased L1/L2 methylation. Conclusions The data suggest that increased HPV DNA methylation may be partly attributable to viral integration, and provide a biological rationale for quantification of L1/L2 methylation in triage of HPV positive cervical screening samples.
    Journal of Clinical Virology 08/2014; 61(3). DOI:10.1016/j.jcv.2014.08.006 · 3.47 Impact Factor
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    • "For each CpG site, the methylation ratio is calculated by the formula: number of C reads divided by the sum of C and T reads at each CpG site. The pyrosequencing result for each site was determined on a PSQ96 ID Pyrosequencer (Qiagen, Valencia, CA) at the Albert Einstein College of Medicine, Genomics Core Facility (Bronx, NY) and the readout was percent methylation, as previously described (Mirabello et al., 2012). The correlation between NG sequencing and pyrosequencing was performed by linear regression in SPSS 16.0 (SPSS Inc., Chicago, IL) and the null hypothesis was rejected when P < 0.05. "
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    ABSTRACT: Invasive cervix cancer (ICC) is the third most common malignant tumor in women and human papillomavirus 16 (HPV16) causes more than 50% of ICC. DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides and increased methylation across the HPV16 genome is strongly associated with ICC development. Next generation (Next Gen) sequencing has been proposed as a novel approach to determine DNA methylation. However, utilization of this method to survey CpG methylation in the HPV16 genome is not well described. Moreover, it provides additional information on methylation "haplotypes." In the current study, we chose 12 random samples, amplified multiple segments in the HPV16 bisulfite treated genome with specific barcodes, inspected the methylation ratio at 31 CpG sites for all samples using Illumina sequencing, and compared the results with quantitative pyrosequencing. Most of the CpG sites were highly consistent between the two approaches (overall correlation, r = 0.92), thus verifying that Next Gen sequencing is an accurate and convenient method to survey HPV16 methylation and thus can be used in clinical samples for risk assessment. Moreover, the CpG methylation patterns (methylation haplotypes) in single molecules identified an excess of complete-and non-methylated molecules and a substantial amount of partial-methylated ones, thus indicating a complex dynamic for the mechanisms of HPV16 CpG methylation. In summary, the advantages of Next Gen sequencing compared to pyrosequencing for HPV genome methylation analyses include higher throughput, increased resolution, and improved efficiency of time and resources.
    Frontiers in Genetics 06/2014; 5:150. DOI:10.3389/fgene.2014.00150
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    • "Intensive research in a variety of cancers shows great promise in quantification of DNA methylation as diagnostic and prognostic biomarkers. In cervical cancer many studies have shown that elevated methylation of CpG sites in the HPV16 L1 and L2 genes are associated with CIN2/3 and cancers [10] [11] [12] [13] [14]. We recently reported that a classifier score S1 developed in a study of Central American women based on HPV16 methylation of 7 CpG sites in L1 and L2 had similar differentiating potential to identify CIN2/3 in HPV16 infected European women, with 92% sensitivity and 40% specificity [13]. "
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    ABSTRACT: High risk human papillomavirus (HR-HPV) infection is common and only a small minority of infections become persistent and lead to cervical cancers. Women positive for HR-HPV usually require a second test to avoid unnecessary colposcopies and over treatment. Elevated DNA methylation of HR-HPV L1 and L2 genes in high grade disease has emerged as a promising molecular triage tool. Our aim was to accurately measure methylation levels at selected CpG positions in the HPV18, HPV31 and HPV33 genomes. We focused on the L2, L1, URR and E6 regions because these were previously shown to be interesting areas for study. Pyrosequencing was used to measure methylation in 208 HPV18, 207 HPV31, and 126 HPV33 positive women selected from a London colposcopy referral population. After adjustment for multiple testing, at FDR 5%, elevated methylation was significantly associated with cervical intraepithelial neoplasia grades 2 or worse (CIN2+) in all investigated CpGs in HPV18 L2 and L1. Two of 6 L2 and 12 of 15 L1 sites in HPV31 and 6 of 8 L2 and 3 of 13 L1 sites in HPV33 showed significantly elevated methylation in CIN2+. Methylation of CpG sites in the URR and E6 region of the HPV types was low and most differences were not significant. Elevated methylation of CpG sites in the L1 and L2 regions of HPV18, HPV31 and HPV33 is associated with CIN2+ and a panel test may be useful for triage of women with HR-HPV infections.
    Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 01/2014; 59(3). DOI:10.1016/j.jcv.2013.12.014 · 3.47 Impact Factor
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