Validation studies of circulating methylation biomarkers

超出提供的空间。R21期我们的初步数据表明，在食道癌患者的血浆中可以发现自由循环的甲基化DNA。具体地说，这些数据表明，在食道癌患者的血流中可以检测到APC、HPP1和Pi6基因的甲基化等位基因。此外，我们的初步数据显示，血浆APC DNA甲基化水平高的患者存活率降低，这也与恶性肿瘤的复发平行。因此，这些循环中的甲基化核酸有望成为预测和监测食道癌患者的生物标志物。这一拟议项目的总体范围将是将这些循环生物标记物从实验室转移到临床。在最初的R21阶段，将以已建立的甲基化靶标Pi6和APC为模板，利用实时定量甲基化特异的聚合酶链式反应来开发和精炼基因特异性甲基化血浆DNA的精确分析。此外，在这个探索性的R21阶段，还将在食道肿瘤组织和血浆中发现其他新的甲基化靶点。在第二个阶段，R33阶段，在R21阶段确定的新的甲基化目标将通过与组织和血浆甲基化水平的临床相关性在更大的独立队列上进行临床验证。目的1.利用已建立的组织和血浆甲基化靶点p！6和APC，建立和验证准确、可靠、标准化和可扩展的实时定量血浆DNA甲基化检测方法。检测将用来自正常受试者的已知标准的血浆进行分析验证，这些血浆中添加了已知水平的基因组DNA，以及已知但盲目的阳性和阴性患者血液样本。目的2.鉴定和检测50例食管癌和50例正常食道组织中的20个新的甲基化事件。一组已知与癌症和结果相关的候选基因，以及据报道在胃肠道和其他人类肿瘤中频繁甲基化的候选基因，将使用实时定量甲基化特异性聚合酶链式反应(MSP)进行评估。在目标3中，将进一步研究至少20%的肿瘤中甲基化的基因，但在不到5%的正常标本中，将进一步研究目标3。目标3.研究同一试点组50名患者在目标2期间在组织中确定的目标基因的血浆甲基化。在当前提议的R33阶段，超过10%的患者血浆中甲基化的基因将成为临床验证的血浆靶标。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. R21Phase Our preliminary data indicate that freely circulating methylated DNA can be found in the plasma of patients with esophageal cancer. Specifically, these data show that methylated alleles of the APC, HPP1 and pi6 genes can be detected in the bloodstream of esophageal cancer patients. Furthermore, our preliminary data show decreased survival in patients with high plasma levels of methylated APC DNA, which also parallel relapse of malignancy. Thus, these circulating methylated nucleic acids show promise as biomarkers for the prognostication and monitoring of esophageal cancer patients. The overall scope of this proposed project will be to move these circulating biomarkers from the laboratory into the clinic. In the initial R21 phase, precise assays of gene-specific methylated plasma DNA will be developed and refined using quantitativereal-time methylation-specific PCR, with the established methylation targets pi6 and APC serving as templates. In addition, during this exploratory R21 phase, additional novel methylation targets will be identified in neoplastic esophageal tissues and plasma. In the second, R33 phase, novel methylation targets identified in the R21 phase will be clinically validated on a larger, independent cohort by performing clinical correlations with tissue and plasma methylation levels. Aim 1. To develop and validate accurate, robust, standardizable, and scalable quantitative real-time plasma DNA methylation assays using the established tissue and plasma methylation targets p!6 and APC. Assays will be analytically validated with known standards of plasma from normal subjects spiked with known levels of genomic DNA, as well as known but blinded positive and negative patient blood samples. Aim 2. To identify and measure 20 novel methylation events in 50 primary esophageal carcinomas and 50 normal esophageal tissues. A panel of candidate genes with known cancer and outcome relevance and reported frequent methylation in gastrointestinal and other human tumors will be evaluated using real-time quantitativemethylation- specific PCR (MSP). Genes methylated in at least 20% of tumors, but in less than 5% of normal specimens, will be further pursued in Aim 3. Aim 3. To study the same pilot group of 50 patients forplasma methylation of target genes identified in tissues during Aim 2. Genes methylated in the plasma of greater than 10% of these patients will constitute plasma targets for clinical validation during the R33 phase of the current proposal. PERFORMANCE SITE ========================================Section End===========================================