A Higher Degree of Methylation of the HPV 16 E6 Gene Is Associated With a Lower Likelihood of Being Diagnosed With Cervical Intraepithelial Neoplasia

Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Cancer (Impact Factor: 4.89). 03/2011; 117(5):957-63. DOI: 10.1002/cncr.25511
Source: PubMed


Although HPV 16 is the most common HPV genotype associated with cancerous lesions of the cervix, only a fraction of HPV 16 infected women are diagnosed with precancerous lesions of the cervix. Therefore, molecular changes in HPV 16, rather than infections per se, may serve as better screening or diagnostic biomarkers. The purpose of the study was to evaluate whether methylation status of specific regions of the HPV E6 gene promoter and enhancer is independently associated with the likelihood of being diagnosed with higher grades of cervical intraepithelial neoplasia (CIN 2+).
The study included 75 HPV 16-positive women diagnosed with CIN 2+ or ≤CIN 1. Pyrosequencing technology was applied to quantify methylation at 6 cytosine guanine dinucleotide (CpG) sites of the HPV 16 E6 promoter and enhancer. CIN 2+ (yes/no) was the dependent variable in logistic regression models that specified the degree of methylation of the CpG sites of the HPV 16 E6 gene as the primary independent predictors. All models were adjusted for demographic, lifestyle, known risk factors for cervical cancer, and circulating concentrations of "cancer-protective" micronutrients.
The odds of being diagnosed with CIN 2+ were 79% lower when the degree of methylation of the HPV 16 enhancer and promoter sites was ≥9.5% (OR = 0.21; 95% CI, 0.06-0.79; P = .02).
Results suggested that CpG methylation is independently involved in the biology of HPV 16 as well as in the development of higher grades of CIN.

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Available from: Chandrika J Piyathilake, Sep 29, 2014
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    • "Invasive cervical cancer (ICC) is the third most common malignant tumor in women and is caused by persistent infection of oncogenic human papillomavirus (HPV) (Jemal et al., 2011), especially type 16, which accounts for greater than 50% of all ICC (Schiffman et al., 2007; Li et al., 2011; Schiffman and Wentzensen, 2013). Recent data indicates that multiple regions of HPV16 and other oncogenic HPV type genomes show increasing CpG methylation patterns among normal, cervical intraepithelial neoplasia (CIN), and cancer tissues, respectively (Badal et al., 2003; Kalantari et al., 2004, 2009, 2010, 2014; Hong et al., 2008; Brandsma et al., 2009, 2014; Ding et al., 2009; Fernandez et al., 2009; Fernandez and Esteller, 2010; Piyathilake et al., 2011; Sun et al., 2011; Wentzensen et al., 2012; Lorincz et al., 2013; Mirabello et al., 2013). Thus, assays for quantitation of CpG methylation of oncogenic HPV genomes in general and HPV16 in particular , indicate that methylation is a promising biomarker for ICC development (Clarke et al., 2012). "
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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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    • "of these studies vary depending on the specific CpGs investigated, the assay used, and the type of material examined [7] [8] [9] [10]. "
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    ABSTRACT: Persistent infection with Human Papillomavirus (HPV) type 16 causes the majority of cervical cancers. Genital HPV infection is very common, but neoplastic progression is uncommon. There is an urgent need to identify biomarkers associated with cervical neoplasia that can be used to triage women who test positive for HPV. To assess the ability of quantitative measurement of HPV16 DNA methylation to separate samples of different cytology grades and cervical cancers, and determine which of the assessed regions of the HPV genome and individual CpGs are most informative. DNA methylation was quantified by pyrosequencing of bisulphite converted DNA from liquid based cytology samples from 17 women with normal cytology and 20 women with severe dyskaryosis, and from fixed tissue from 24 women with cervical cancer. Methylation was assessed in the HPV Long Control Region (LCR), E2 and L1/L2 regions. In cervical cancers, increased HPV DNA methylation was present in all regions. Increased methylation was also observed in severely dyskaryotic relative to normal samples, but only in the E2 and L1/L2 regions. The ability of methylation based classifiers to separate the three classes of material was assessed by ROC curve analyses. The best separation between normal and dyskaryotic samples was achieved by assessment of the L1/L2 CpGs at nucleotide positions 5600 and 5609 (AUC=0.900, 95% CI: 0.793-1). This study demonstrates the potential of quantification of HPV DNA methylation as a biomarker of cervical neoplasia. An algorithm considering methylation at specific L1/L2 CpGs appeared the most promising model.
    Full-text · Article · Nov 2013 · Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology
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    • "Multiple studies have noted aberrant methylation in the promoter regions of biologically relevant genes in cervical cancer (Duenas-Gonzalez et al., 2005; Henken et al., 2007; Huang et al., 2010; Kim et al., 2010a; Lai et al., 2010; Muller et al., 2004; Unger et al., 2004; Wang et al., 2008; Wisman et al., 2006) and in cervical squamous intraepithelial lesions (Apostolidou et al., 2009; Gustafson et al., 2004; Huang et al., 2010; Kahn et al., 2008; Kim et al., 2010a,b; Lai et al., 2010; Lim et al., 2010; Wentzensen et al., 2009). In addition to host cellular gene methylation , studies have reported uneven distribution and clustering of the cytosine–guanine dinucleotide (CpG) pairs within the enhancer region of HPV 16 and HPV 18 genomes (Burnett and Sleeman, 1984; Piyathilake et al., 2011; Rosl et al., 1993). Epigenetic regulation of this region has been recognized for some time, and may play a role in the carcinogenic process by modifying the virulence resulting in increased risk of progression of HPV infection to high-grade preinvasive cervical lesions (Badal et al., 2003; Kalantari et al., 2004; Rosl et al., 1993; Unger et al., 2004). "
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    ABSTRACT: Aberrant promoter methylation of biologically relevant genes in cervical cancer and uneven CpG distribution within the human papillomavirus 16 (HPV 16) enhancer region have been reported. Cervical samples and questionnaires from 151 women screened for cervical cancer in Appalachian Ohio were analyzed. Methylation was measured by bisulfite sequencing in candidate gene sites in ESR1, DCC, p16, and LINE1 elements. Among 89 HPV 16-positive women, CpG sites in the E6 promoter and enhancer regions and the L1 region of the HPV 16 genome were measured. Methylation levels were compared by cervical cytology and HPV 16 status. HPV methylation was low regardless of cytology status, however E6 methylation was significantly higher in women with normal cytology. ESR1 and DCC methylation were significantly higher in HPV 16-positive women. Increased methylation at sites in the E6 promoter region was associated with lower odds of abnormal cytology. Increased methylation in candidate genes was associated with higher odds of abnormal cytology, particularly DCC region 2.4, DCC region 2.6, ESR1 region 3.2, and LINE1 site 1.2. HPV 16 genome CpG methylation was low except for the L1 region. In general, lower HPV 16 methylation and higher candidate gene methylation levels were associated with higher odds of abnormal cytology.
    Full-text · Article · Jun 2012 · Journal of virological methods
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