Cervical Cancer Preventive Measures Based on HPV 16 Epigenome

描述(申请人提供)：HPV16发挥致癌作用的一个主要机制是通过表达两个病毒早期基因E6和E7。因此，调节E6和E7基因表达的因素可能会影响HPV16感染向宫颈上皮内瘤变(CIN2+)或浸润性宫颈癌(ICC)的转化。HPV16DNA甲基化程度(DNaM)影响其基因表达、病毒生命周期、持久性和复制行为。我们最近的研究证实：1)在宫颈脱落细胞(ECs)中检测到较高程度的HPV 16dNaM与被诊断为CIN 2+的可能性降低79%相关。2)与任何致癌HPV类型检测阳性或纸片异常检测阳性的女性相比，HPV16 dNaM检测对CIN 2+女性的阳性预测价值(PPV)最好。3)循环中叶酸浓度较低的HPV16阳性女性被诊断为CIN 2+的可能性是HPV16阴性女性叶酸水平较高的九倍。由于HPV使用人类甲基化机制进行其dNaM，因此影响人类细胞dNaM的相同因素，即由叶酸代谢途径产生的甲基的可用性，也可能具有改变HPV16dNaM的程度的能力。在此背景下，本研究的目的如下：1.利用从宫颈上皮细胞配对样本中提取的DNA和我们发表的研究中确定的HPV16E6增强子的7862位和HPV16E6启动子的第31、37、43、52和58位点与CIN2+之间的相关性，来评估HPV16dNaM与CIN之间的关系，以便在一个单独的、更大的女性群体中重复我们的发现。这一目标将在300名确诊为CIN 2+的妇女(病例)和300名确诊为DCIN 1的妇女(非病例)的样本中进行测试。目的2.评估与目标1相同部位的HPV16dNaM程度与循环叶酸浓度之间的关系，以评估由于美国叶酸强化计划而暴露于不同水平叶酸的影响。这一目的将在300名确诊为DCIN 1的妇女样本中进行测试。这项研究将提供一个独特的机会，以确定检测HPV16dNaM的程度对确定高危妇女患HPV16相关CIN2+的诊断价值。此外，这项研究整合了一个重要的全人群环境暴露(叶酸)和分子数据，通过测试以下假设，即暴露于叶酸可能具有改变HPV16dNaM程度的能力，并改变与HPV16相关的在美国后叶酸强化时代发展为CIN的风险。 公共卫生相关性：这项研究的目的是评估HPV16的特定甲基化变化是否可以作为晚期宫颈上皮内瘤变风险增加的生物标志物，并确定叶酸水平是否可以预测HPV16甲基化的程度。

DESCRIPTION (provided by applicant): A main mechanism by which HPV 16 exerts oncogenic effects is through the expression of two viral early genes, E6 and E7. Therefore, factors that regulate the expression of E6 and E7 are likely to influence transformation of HPV 16 infections to higher grades of cervical intraepithelial neoplasia (CIN 2+) or invasive cervical cancer (ICC). The degree of HPV 16 DNA methylation (DNAm) has been shown to influence its gene expression, viral life cycle, persistence and replicative behavior. Our recent studies demonstrated the following: 1) A higher degree of HPV 16 DNAm assayed in exfoliated cells (ECs) from the cervix was associated with 79% reduced likelihood of being diagnosed with CIN 2+ 2) The degree of HPV 16 DNAm showed the best positive predictive value (PPV) for identifying women with CIN 2+, compared to women tested positive for any carcinogenic HPV type or tested positive for abnormal pap, two tests commonly used by Health Departments to identify high risk women 3) HPV 16-positive women with lower circulating concentrations of folate were nine times more likely to be diagnosed with CIN 2+ compared to HPV 16-negative women with higher folate. Because HPV uses the human methylation machinery for its DNAm, the same factors which influence DNAm in human cells, namely the availability of methyl groups generated by the folate metabolic pathway, may also have the ability to alter the degree of HPV 16 DNAm. With this background, the aims of the study are as follows: Aim 1. To evaluate the association between the degree of HPV 16 DNAm and CIN using DNA extracted from paired samples of ECs and cervical biopsies at sites identified in our published study (position 7862 of the HPV 16 E6 enhancer and positions 31, 37, 43, 52 and 58 of the HPV 16 E6 promoter) and CIN 2+, in order to replicate our findings in a separate, larger group of women. This aim will be tested among samples already collected from 300 women diagnosed with CIN 2+ (cases) and 300 women diagnosed with d CIN 1 (non-cases) Aim 2. To evaluate the association between the degree of HPV 16 DNAm at the same sites as in aim 1 and circulating concentrations of folate, in order to assess the influence of exposure to different levels of folate as a result of the US folic acid fortification program. This aim will be tested among samples already collected from 300 women diagnosed with d CIN 1. The study will provide a unique opportunity to determine the diagnostic value of testing the degree of HPV 16 DNAm for identifying women at high risk for developing HPV 16 associated CIN 2+. Further, the study integrates an important population-wide environmental exposure (folate) and molecular data by testing the hypothesis that exposure to folate may have the ability to alter the degree of HPV 16 DNAm and modify the HPV 16 associated risk of developing CIN in the US post-folic acid fortification era. PUBLIC HEALTH RELEVANCE: The purpose of the study is to assess whether specific methylation changes in HPV 16 can be used as a biomarker for enhanced risk of advanced cervical intraepithelial neoplasia and to determine whether folic acid levels predict the degree of HPV 16 methylation.