Prognostic Significance of DNA and Histone Methylation

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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.