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.
Journal of the National Cancer Institute (Impact Factor: 12.58). 03/2012; 104(7):556-65. DOI: 10.1093/jnci/djs135
Source: PubMed


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.
    Full-text · Article · Aug 2014 · Journal of Clinical Virology
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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.
    Full-text · Article · Jun 2014 · Frontiers in Genetics
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    • "Methylation of CpG sites within L1 [20-22], the upstream regulatory region (URR) [20,21,23] and/or other regions of the HPV16 genome [24-26] have often, but not always [27,28], been associated with cervical disease. Data on the degree of CpG site methylation for genotypes HPV18 [25,29,30], HPV31 [29] and HPV45 [29] are limited but appear to show a similar trend, suggesting that HPV methylation may be useful as a potential marker for cervical disease [31]. "
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    ABSTRACT: Background Persistent infection with oncogenic Human Papillomavirus (HPV) is associated with the development of cervical cancer with each genotype differing in their relative contribution to the prevalence of cervical disease. HPV DNA testing offers improved sensitivity over cytology testing alone but is accompanied by a generally low specificity. Potential molecular markers of cervical disease include type-specific viral load (VL), integration of HPV DNA into the host genome and methylation of the HPV genome. The aim of this study was to evaluate the relationship between HPV type-specific viral load, integration and methylation status and cervical disease stage in samples harboring HPV16, HPV18, HPV31 or HPV45. Methods Samples singly infected with HPV16 (n = 226), HPV18 (n = 32), HPV31 (n = 75) or HPV45 (n = 29) were selected from a cohort of 4,719 women attending cervical screening in England. Viral load and integration status were determined by real-time PCR while 3’L1-URR methylation status was determined by pyrosequencing or sequencing of multiple clones derived from each sample. Results Viral load could differentiate between normal and abnormal cytology with a sensitivity of 75% and a specificity of 80% (odds ratio [OR] 12.4, 95% CI 6.2–26.1; p < 0.001) with some variation between genotypes. Viral integration was poorly associated with cervical disease. Few samples had fully integrated genomes and these could be found throughout the course of disease. Overall, integration status could distinguish between normal and abnormal cytology with a sensitivity of 72% and a specificity of 50% (OR 2.6, 95% CI 1.0–6.8; p = 0.054). Methylation levels were able to differentiate normal and low grade cytology from high grade cytology with a sensitivity of 64% and a specificity of 82% (OR 8.2, 95% CI 3.8–18.0; p < 0.001). However, methylation varied widely between genotypes with HPV18 and HPV45 exhibiting a broader degree and higher magnitude of methylated CpG sites than HPV16 and HPV31. Conclusions This study lends support for HPV viral load and CpG methylation status, but not integration status, to be considered as potential biomarkers of cervical disease.
    Full-text · Article · May 2014 · BMC Cancer
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