Multi-site Gastrointestinal Cancer Detection by Stool DNA Methylation

项目摘要/摘要： 胃肠道(GI)恶性肿瘤在全球范围内导致癌症死亡，每年导致约300万人死亡。仅在美国， 对结直肠癌(CRC)进行筛查。其他胃肠道癌症由于缺乏准确的检测或 因为流行率被认为太低了，不适合进行成本效益高的筛查。因此，大多数胃肠道疾病患者 癌症出现在晚期，治愈率仍然非常低。有效的早期发现是不顾一切的 需要改善结果。我们的团队在FDA批准的多 大便DNA检测用于结直肠癌筛查。我们已经开始扩展这种方法，显示了检测的可行性 通过粪便DNA检测发现结肠上胃肠道癌。然而，对于非侵入性分子测试来说，关键是 定位原发癌症的部位(“部位预测”)。关键的初步数据表明，这现在可能是 有可能。首先，我们已经完成了严格的下一代测序，以识别差异甲基化 区域(DMR)对于胃肠道肿瘤的普遍和特定部位的检测似乎具有高度的区分性。 其次，我们根据严格的筛选标准对这些DMR进行了优先排序，并进行了具有统计学意义的验证性研究 一组独立的结直肠癌、胃食道癌和胰胆道癌与正常对照的交叉验证 控制组织。这表明，一组由8名选定的DMR组成的小组可以区分癌症和正常(95% 准确性)和指定器官位置(每个类别的准确率为94%-95%)，总体位置预测准确率为 这些发现已经被从粪便标本中检测出的新的DMRS所证实 来自结直肠癌和胰腺癌患者和正常对照组(各30例)。使用两阶段分析，癌症 与对照相比，第一阶段特异性为90%。在阶段2，标记准确地 胰腺癌分类准确率达90%。 88%。第三, 现在我们的中心假设是流明的和 导管腺癌可以通过粪便检测来发现和定位。 这引出了三个关键问题：1)我们新型DMRS的粪便检测是否会显示出高的整体癌症敏感度 以及我们在初步数据中看到的位置预测；2)DMRS是否会针对整个 广泛的患者人口谱和非恶性胃肠道疾病的背景下；以及3)可以 新的检测技术能提高特异性吗？这些问题将按以下并行方式加以解决： 综合但独立的具体目标：1)评估专家小组的敏感度和现场预测准确性 食道、胰腺和结直肠部位腺癌的粪便标本；2)确认和 评估粪便中DMR的特异性；以及3)优化新的检测条件和标记选择 食道、胰腺和结直肠部位的癌症检测和位置预测。带着我们队的 良好的记录，广泛的粪便档案，以及对最先进的测试平台的独特访问，我们期待 以经济高效的方式展示用于检测和现场的新型DMR面板的可行性 特异性胃肠腺癌的预测。结果将为未来的研究设计提供信息，范围从大型- 早期癌症和先兆到关键队列验证(阶段)的大规模病例对照研究(阶段2) 4)非侵入性多消化道肿瘤筛查试验。这对癌症控制的潜在影响是深远的。

PROJECT SUMMARY/ABSTRACT: Gastrointestinal (GI) malignancies lead cancer deaths worldwide, killing ~3 million annually. In the U.S., only colorectal cancers (CRC) are screened. Other GI cancers are not screened due to lack of accurate tests or because prevalence is deemed too low for cost-effective screening. Consequently, most patients with GI cancers present at late-stage and cure rates remain abysmally low. Effective early detection is desperately needed to improve outcomes. Our group was central in development and validation of the FDA-approved multi- target stool DNA test for CRC screening. We have begun to expand this approach, showing feasibility to detect supra-colonic GI cancers by stool DNA testing. However, it is critical for a non-invasive molecular test to localize the site of a primary cancer (“site-prediction”). Key preliminary data suggest that this may now be possible. First, we have completed rigorous next-generation sequencing to identify differentially methylated regions (DMRs) which appear highly discriminant for universal and site-specific detection of GI cancers. Second, we prioritized these DMRs by strict filtering criteria and performed a confirmatory study with statistical cross-validation on an independent set of CRC, gastroesophageal and pancreatico-biliary cancer and normal control tissues. This showed that a panel of 8 selected DMRs could distinguish cancer from normal (95% accuracy) and assign organ site (94-95% accuracy for each category) with overall site-prediction accuracy of these findings have been confirmed with novel DMRs assayed from stool specimens obtained from CRC and pancreatic cancer patients and normal controls (30, each). Using a 2-stage analysis, cancers were distinguished from controls at 90% specificity in the first stage. At stage 2, the markers accurately classified CRCs from pancreatic cancers with 90% accuracy. 88%. Third, It is now our central hypothesis that luminal and ductal adenocarcinomas can be detected and localized by stool assay of universal and site-specific DMRs. This raises 3 key questions: 1) will stool assay of our novel DMRs show the high overall cancer sensitivity and the site-prediction we have seen in preliminary data; 2) will the DMRs be specific for cancer across a wide patient demographic spectrum and in the setting of non-malignant GI diseases; and 3) can sensitivity and specificity be improved by novel assay technology? These will be addressed in the following parallel, integrated, but independent specific aims: 1) Assess panel sensitivity and site-prediction accuracy in stool specimens for adenocarcinoma at esophageal, pancreatic and colorectal sites; 2) Confirm and evaluate DMR specificity in stool; and 3) Optimize novel assay conditions and marker selection for cancer detection and site-prediction at esophageal, pancreatic and colorectal sites. With our team's strong track record, extensive stool archive, and unique access to a state-of-the art assay platform, we expect to demonstrate in a cost-efficient manner the feasibility of a novel DMR panel for the detection and site prediction of specific GI adenocarcinomas. Results will inform designs of future studies ranging from large- scale case-control studies (phase 2) on early-stage cancer and pre-cursors to pivotal cohort validation (phase 4) of a non-invasive multi-GI cancer screening test. The potential impacts on cancer control are far-reaching.