METHYLATION BIOMARKER DEVELOPMENT FOR NONINVASIVE DETECTION OF COLORECTAL CANCER

描述（由申请人提供）：结直肠癌（CRC）是美国第三大常见癌症，估计每年有15万新病例发生。早期发现临床相关结直肠肿瘤（CRN），即CRC和晚期结直肠腺瘤（A-CRA），是提高生存率的最佳途径。然而，由于侵入性和费用（如结肠镜检查）或诊断准确性不足（如基于粪便的血液检查），现有的筛查方式不切实际。一种更好的筛查和监测方法将是非常可取的。异常DNA甲基化是一种重要的表观遗传机制，与结直肠癌的发病和进展密切相关。异常DNA甲基化在血清中可检测到，使其成为非侵入性筛查的理想候选生物标志物物种。先前的研究在开发用于CRC筛查的明确的甲基化生物标志物方面取得了有限的成功，由于方法不全面，并且未能将所有crn作为临床发现的有意义的靶标。此外，在基于血清的研究中使用的队列没有足够的动力，并且缺乏独立的验证集。在本提案中，创新的策略将应用于使用CRN患者的组织和血清进行基因组尺度的甲基化定位，并与非CRN个体的组织和血清进行比较。一种新颖而强大的新方法正在被提出，以最高的灵敏度和特异性来鉴定新的生物标志物和甲基化靶点，并将通过以下特定目标使用大型，表征良好的样本集进行验证。目标1：在CRN患者和正常结肠患者的匹配组织和血清中，使用下一代亚硫酸氢盐测序将发现候选DNA甲基化生物标志物。目标2：将发现候选血清甲基化生物标志物，以区分CRN患者和非CRN患者，并使用定量PCR检测验证生物标志物。目标3:Aim 2b中确定的优先血清甲基化生物标志物的临床验证将在无症状平均风险个体中进行。该项目的创新之处在于首次使用了一种新的、下一代的基于测序的方法，该方法使用基因组规模的测定方法来鉴定与结直肠肿瘤相关的甲基化位点，并通过大量具有良好特征的CRN患者和对照组来验证这些新发现的生物标志物。该项目的长期目标和潜在影响是开发一种敏感、特异、无创和廉价的早期结直肠肿瘤诊断检测方法。

DESCRIPTION (provided by applicant): Colorectal carcinoma (CRC) is the third most common cancer in the United States, with an estimated 150,000 new cases occurring annually. Early detection of clinically relevant colorectal neoplasia (CRN), i.e., CRC and advanced colorectal adenomas (A-CRA), is the best way to improve survival. However, available screening modalities are impractical due to invasiveness and cost (i.e., colonoscopy) or insufficient diagnostic accuracy (i.e., fecal-based blood tests). A better approach to screening and surveillance would be highly desirable. Abnormal DNA methylation is an important epigenetic mechanism that has been strongly implicated in the pathogenesis and progression of colorectal neoplasia. Aberrant DNA methylation is detectable in serum making it an ideal candidate biomarker species for non-invasive screening. Previous studies have achieved limited success in developing definitive sets of methylation biomarkers for CRC screening due non-comprehensive approaches, and a failure to include all CRNs as meaningful targets for clinical discovery. Also, cohorts used in serum-based studies have been insufficiently powered and lacked independent validation sets. In this proposal, innovative strategies will be applied to permit genome-scale methylation mapping using tissues and sera from patients with CRN, and compared with tissues and sera from individuals without CRN. A novel and powerful new approach is being proposed to identify new biomarkers and methylation targets with the highest sensitivity and specificity, and will be validated using large, well- characterized sample sets through the following Specific Aims. Aim 1: Candidate DNA methylation biomarkers will be discovered using next generation bisulfite sequencing in matched tissue and serum from patients with CRN and individuals with a normal colon. Aim 2: Candidate serum methylation biomarkers will be discovered that distinguish patients with CRN vs. those without CRN, and validated biomarkers using quantitative PCR assays. Aim 3: Clinical validation of prioritized serum methylation biomarkers identified in Aim 2b will be performed in asymptomatic average risk individuals. The innovation of this project is based upon the first use of a novel, next generation sequencing-based approach for identifying methylation-based biomarkers using a genome-scale assay for methylation sites linked to colorectal neoplasia, and validating these newly discovered biomarkers using a large, well-characterized cohort of patients with CRN and controls. The long-term goal and potential impact of this project is to develop a sensitive, specific, non-invasive and inexpensive diagnostic test for early colorectal neoplasia.