Discovery and verification of methylated circulating tumor DNA markers for the detection of colorectal cancer in subjects under 50 years of age

摘要 近年来，50岁以下的结直肠癌（CRC）发病率明显上升 目前不向50岁以下的人提供基于人口的筛查。因此，人 早发性（<50岁）CRC（EOCRC）更常在晚期出现症状性疾病， 阶段（III/IV），导致年轻病例的生命损失更大。2020年，美国预防服务工作组 建议通过结肠镜检查或粪便试验进行CRC筛查的年龄降至45岁。但 在所有符合筛查条件的人群中，采用这些方法进行筛查的比例很低，包括50岁以下人群 遗传上处于高风险的人，因此50岁以下的无症状人群的依从性也可能是 低此外，超过一半的EOCRC发生在年龄<45岁的人中，在这些人中没有这样的筛查。 提供。一种微创的、基于血液的EOCRC筛查试验将提供一种具有成本效益和 患者友好的选择，用于分类和识别那些拒绝接受后续结肠镜检查的患者，同时增加 在年轻人群中筛查依从性。癌症特异性甲基化DNA生物标志物非常适合 用于通过检测血浆中的循环肿瘤DNA（ctDNA）进行基于人群的癌症筛查 因为它们在特定癌症类型的患者中比肿瘤突变更普遍， 在血浆中稳定（抗核酸酶）。甲基化SEPTIN 9血浆ctDNA测试，“mSEPT 9”（Epi proColon V2.0，表观基因组学），FDA批准用于年龄≥50岁拒绝结肠镜检查的患者的CRC筛查 和粪便测试这项测试迫切需要在50岁以下的人群中进行评估，以确定其是否适合 检测EOCRC。即便如此，多标志物检测可能比单标志物检测具有上级灵敏度。 因此，我们研究的目的是鉴定和确认一组甲基化的ctDNA标记物， 基于血浆的EOCRC检测，并将该检测组的诊断性能与mSEPT 9进行比较 在50岁以下的人中进行测试。在目标1中，我们将确定最普遍的差异甲基化（肿瘤）。 vs.通过在420万个CpG上进行深度甲基测序， 来自EOCRC病例的配对原发性肿瘤与正常结肠组织中的位点，以及来自健康人的白细胞 对照（过滤掉非特异性标记）。将在独立样本中验证鉴定的标志物 系列.对于排名靠前的标记，我们将开发甲基化特异性实时PCR检测， 分析灵敏度，并在来自转移性CRC病例的合并血浆中测试这些（以选择产生 最强信号）和健康对照（以消除任何产生低水平非特异性信号）来完成 标记面板。在目标2中，我们将评价mSEPT 9检测与 来自结肠镜检查证实的CRC病例和<50岁的对照的血浆中的多标记物组测试。本研究 将产生多标志物甲基化ctDNA组，其诊断性能优于单标志物 mSEPT 9检测用于50岁以下无症状人群的成本效益高、基于血液的CRC筛查。

Abstract In recent years, the incidence of colorectal cancer (CRC) under the age of 50 years has increased significantly and population-based screening is currently not offered to persons under 50 years. Consequently, persons with early-onset (<50 years) CRC (EOCRC) more frequently present with symptomatic disease at advanced stages (III/IV) resulting in greater loss of life in young cases. In 2020, the U.S. Preventive Services Task Force recommended the age for CRC screening by colonoscopy or fecal tests be reduced to 45 years. However, the uptake of screening by these methods in all screen-eligible populations is low, including in those <50 years who are genetically at high risk, so adherence in the asymptomatic population <50 years is also likely to be low. Also, over half of EOCRC occur in persons under <45 years of age, where no such screening would be offered. A minimally invasive, blood-based screening test for EOCRC would provide a cost-effective and patient-friendly option for triaging and identifying those warranting a follow-up colonoscopy, while increasing screening adherence in the younger population. Cancer-specific methylated DNA biomarkers are highly suited for population-based cancer screening via the detection of circulating tumor DNA (ctDNA) in blood plasma because they are more prevalent across patients with a given cancer type than tumor mutations and are more stable (nuclease-resistant) in plasma. The methylated SEPTIN9 plasma ctDNA test, “mSEPT9” (Epi proColon V2.0, Epigenomics), is FDA-approved for CRC screening in persons aged ≥50 years who decline colonoscopy and fecal tests. This test urgently needs to be assessed in persons <50 years to determine its suitability for the detection of EOCRC. Even so, a multi-marker test is likely to have superior sensitivity to a single-marker test. Thus, the objectives of our study are to identify and confirm a panel of methylated ctDNA markers for the plasma-based detection of EOCRC, and to compare the diagnostic performance of this panel to the mSEPT9 test in persons <50 years of age. In Aim 1, we will identify the most prevalent differentially-methylated (tumor vs. normal) DNA markers in CRC cases <50 years by performing deep Methyl-Seq across 4.2 million CpG sites in paired primary tumor vs. normal colon tissues from EOCRC cases, and leukocytes from healthy controls (to filter out non-specific markers). The identified markers will be validated in independent sample series. For the top-ranked markers, we will then develop methylation-specific real-time PCR assays with high analytical sensitivity and test these in pooled plasma from metastatic CRC cases (to select markers producing the strongest signals) and healthy controls (to eliminate any producing low-level non-specific signals) to finalize the marker panel. In Aim 2, we will evaluate the diagnostic performance characteristics of the mSEPT9 test vs. the multi-marker panel test in plasma from colonoscopy-verified CRC cases and controls <50 years. This study will yield a multi-marker methylated ctDNA panel with improved diagnostic performance over the single-marker mSEPT9 test for cost-effective, blood-based, CRC screening in asymptomatic persons <50 years of age.