Prognostic Significance of DNA and Histone Methylation

描述（由申请人提供）： 目前，还没有有效的诊断或预后标准，将确定低度宫颈病变（CIN 1），注定要向CIN 2和3或宫颈癌。我们认为，CIN 1病变的常规组织病理学检查或在常规护理访视时检测高危（HR）-HPV提供的信息不足以预测高级别病变。基于我们最近的研究结果，表明较低的循环叶酸水平与HR-HPV的持续存在和高度宫颈发育不良的发展相关，我们假设这些病变中的叶酸和叶酸相关的表观遗传学改变（整体，CpG岛和DNA和组蛋白的基因特异性甲基化）可能为识别CIN 1病变提供有价值的信息，这些病变注定会发展为CIN 2或3。从2003年1月起，我们机构的所有CIN 1和HR-HPV女性都进行了前瞻性随访，并每年在伯明翰的亚拉巴马大学（UAB）女性健康研究中心（CRWH）进行HPV检测，作为其常规护理的一部分。这为我们提供了一个独特且具有成本效益的机会，招募这些妇女参加前瞻性随访研究，以评估叶酸和叶酸相关表观遗传标记物在识别高风险低级别宫颈病变中的意义。我们假设，在12-24个月后发生CIN 2或3的HR-HPV阳性CIN 1受试者的循环和宫颈细胞叶酸浓度以及DNA和组蛋白甲基化程度将不同于在相似时间段内未发生CIN 2或3病变的相似病变受试者。我们计划招募600名HR-HPV阳性的CIN 1妇女进行为期24个月的随访研究。为了评估叶酸和甲基化是否是CIN 2/3的独立预测因子，我们将结果与宫颈癌的已知流行病学和HPV风险因素以及其他癌症保护维生素相关联。新开发和测试的免疫组织化学技术，其测量完整和特定类型的宫颈细胞中DNA和历史（lys 4和9）的整体甲基化程度，将用于在所提出的研究中评估整体甲基化。胞嘧啶延伸试验和实时PCR试验将分别用于评价CpG岛甲基化和基因特异性甲基化。这将是第一个全面的前瞻性随访研究，旨在评估维生素和相关表观遗传生物标志物对宫颈发育不良的预后意义。由于HR-HPV感染和低度宫颈病变很常见，因此对需要干预的宫颈病变具有更高特异性的新标志物将在未来改善宫颈癌筛查。该研究旨在验证表观遗传生物标志物，这些生物标志物在大规模上是可行的和具有成本效益的。UAB的研究者人才、设备、设施和研究受试者访问是开展本研究的理想选择。

DESCRIPTION (provided by applicant): Currently, there are no validated diagnostic or prognostic criteria that will identify low-grade cervical lesions (CIN 1) that are destined toward CIN 2 & 3 or cervical cancer. We believe that the conventional histopathological examination of CIN 1 lesions or testing for high-risk (HR)-HPV at a routine care visit provides insufficient information to predict high-grade lesions. Based on our recent results which demonstrated that lower circulating levels of folate are associated with HR-HPV persistence and development of high-grade cervical dysplasia, we hypothesize that folate and folate-related epigenetic alterations (global, CpG island and gene specific methylation of DNA and histones) in these lesions may provide valuable information for identifying CIN 1 lesions that are destined toward CIN 2 or 3. From January 2003, all women with CIN 1 and HR-HPV at our institution are followed prospectively and tested annually for HPV at the University of Alabama at Birmingham (UAB) Center for Research in Women's Health (CRWH) as part of their routine care. This gives us a unique and cost-effective opportunity to recruit these women into a prospective follow-up study to evaluate the significance of folate and folate-related epigenetic markers in the identification high-risk low-grade cervical lesions. We hypothesize that the circulating and cervical cell concentrations of folate and the degree of methylation of DNA and histones in HR-HPV-positive CIN 1 subjects who develop CIN 2 or 3 after a 12-24 month period will be different than that of similar lesions ill subjects who do not develop CIN 2 or 3 lesions during a similar time period. We propose to recruit 600 women with HR-HPV positive CIN 1 to a 24-month follow-up study. To evaluate whether folate and methylation are independent predictors of CIN 2/3, we will correlate results with known epidemiological and HPV risk factors for cervical cancer and other cancer-protective vitamins. Newly developed and tested immunohistochemical techniques, which measure the degree of global methylation of DNA and histories (lys4 & 9) in intact and specific types of cervical ceils, will be used to evaluate global methylation in the proposed study. Cytosine extension assay and real-time PCR assays will be used to evaluate CpG island methylation and gene-specific methylation respectively. This will be the first comprehensive prospective follow-up study designed to evaluate the prognostic significance of vitamins and related epigenetic biomarkers for cervical dysplasia. Since HR-HPV infections and low-grade cervical lesions are common, novel markers with higher specificity for cervical lesions requiring intervention will improve cervical cancer screening in the future. The study intends to validate epigenetic biomarkers that are feasible and cost-effective to perform on a large scale. Investigator talent, equipment, facilities and access to study subjects at UAB are ideal for conducting this study.