Development of 5hmC and 5mC biomarkers in cell-free circulating DNA for sensitive colon cancer detection and prognosis

项目总结/摘要 结直肠癌（CRC）是癌症相关死亡的主要原因。筛查结肠镜检查是 护理CRC检测，但由于具有挑战性的准备，程序成本， 和潜在的并发症。特定的突变在CRC中是罕见的，排除了它们作为筛查的实用性 生物标志物。表观遗传变化，包括DNA 5-甲基胞嘧啶（5mC），有助于CRC， 在几个FDA批准的测试中询问，但灵敏度有限，没有预后信息。 循环无细胞DNA（cfDNA）中生物标志物的灵敏血液检测可以提供更大的便利性， 更高的依从性。除了5mC，5-羟甲基胞嘧啶（5hmC）的变化在正常人中也很重要。 和疾病状态。5hmC是由DNA去甲基化产生的丰富、稳定的修饰胞嘧啶 并经常标记活跃的基因转录。5hmC和5mC可以作为上级生物标志物，因为它们 广泛的基因组流行，在基因调控中的独特作用，以及强大的化学稳定性。我们开发 一种高灵敏度和选择性的化学标记技术（nano-hmC-Seal），用于捕获5hmC碱基， 使用下一代测序（NGS），绘制它们的基因组位置。在初步研究中，我们捕获并 在来自47名对照和136名最近患有癌症的患者的cfDNA和配对肿瘤组织中分析了5hmC和5mC。 确诊的癌症，包括46例CRC。我们开发了一个分类器来识别具有差异5hmC的基因， 研究人员发现，CRC患者中的癌症特异性表观遗传特征发生了变化，并在肿瘤和cfDNA中鉴定了一致的癌症特异性表观遗传特征。 由于其高灵敏度（~1ng DNA）、稳健性、临床方便性和成本效益，我们 假设独特无细胞5hmC和5mC谱可能用作检测CRC的血浆生物标志物 并最终预测肿瘤预后。我们建议使用PLCO生物储存库样品和我们自己的存档 CRC与注释的临床结果，以及新招募的前瞻性CRC患者，以完善和验证 差异表达的5hmC和5mC基因标记对CRC诊断和预后的敏感性。我们 目的1：使用纳米hmC-1，分析来自PLCO样品（CRC和对照）的cfDNA中的5hmC和5mC。 Seal和NGS并完善敏感CRC血液检测算法，使用200例CRC病例和400例对照， 训练集，以及来自我们前瞻性队列的另外200例病例和400例对照用于验证集。我们 将开发5hmC和5mC修饰的鉴别器，以诊断CRC并通过肿瘤控制分析 可能调节5hmC和5mC分布的位置、分期、患者年龄和性别;目标2a至轮廓100 PLCO肿瘤的5hmC和5mC分布，并将分布与cfDNA（目标1样品）进行比较;在目标2b中， 我们将研究150例来自存档样本的具有复发数据的肿瘤（60例复发患者与90例临床患者）， 特征匹配的非复发患者），以检测肿瘤预后的预测指数。在目标2c中，我们将 将肿瘤中确定的标志物与cfDNA中检测到的标志物进行比较，以评估我们是否可以开发出一种新的标记物。 本发明提供了一种用于肿瘤预后的微创、成本有效、临床上方便的基于cfDNA的标记物。

PROJECT SUMMARY/ABSTRACT Colorectal cancer (CRC) is a major cause of cancer-related deaths. Screening colonoscopy is the standard of care for CRC detection, but compliance remains below 50% because of challenging preps, procedural costs and potential complications. Specific mutations are rare in CRC, precluding their utility as screening biomarkers. Epigenetic changes, including DNA 5-methylcytosine (5mC), contribute to CRC and are interrogated in several FDA-approved tests, but have limited sensitivity and no prognostic information. Sensitive blood tests for biomarkers in circulating cell-free DNA (cfDNA) could offer greater convenience and higher compliance. In addition to 5mC, changes in 5-hydroxymethylcytosine (5hmC) are important in normal and disease states. 5hmC is an abundant, stable modified cytosine that is generated by DNA demethylation and frequently marks active gene transcription. 5hmC and 5mC could serve as superior biomarkers, given their widespread genomic prevalence, distinct roles in gene regulation, and robust chemical stability. We developed a highly sensitive and selective chemical labeling technology (nano-hmC-Seal) to capture 5hmC bases, and using next generation sequencing (NGS), map their genomic positions. In preliminary studies, we captured and profiled 5hmC and 5mC in cfDNA and paired tumor tissues from 47 controls and 136 patients with recently diagnosed cancers, including 46 with CRC. We developed a classifier to identify genes with differential 5hmC changes in CRC patients and identified consistent cancer-specific epigenetic signatures in tumors and cfDNA. Because of the high sensitivity (~1ng DNA), robustness, clinical convenience and cost-efficiency, we hypothesize that unique cell-free 5hmC and 5mC profiles might be useful as plasma biomarkers to detect CRC and ultimately predict tumor prognosis. We propose to use PLCO biorepository samples and our own archived CRC with annotated clinical outcomes, and newly recruited prospective CRC patients to refine and validate the sensitivity of differentially expressed 5hmC and 5mC gene signatures for CRC diagnosis and prognosis. We propose: Aim 1 to profile 5hmC and 5mC in cfDNA from PLCO samples (CRC and control), using nano-hmC- Seal and NGS and refine algorithms for sensitive CRC blood test, using 200 CRC cases and 400 controls for training set, and an additional 200 cases and 400 controls from our prospective cohort for validation set. We will develop discriminators of 5hmC and 5mC modifications to diagnose CRC and control analyses by tumor location, stage, patient age and gender that likely modulate 5hmC and 5mC distributions; Aim 2a to profile 100 PLCO tumors for 5hmC and 5mC distributions and compare distributions to cfDNA (aim 1 samples); In aim 2b, we will study 150 tumors from archived samples with recurrence data (60 relapsed patients vs. 90 clinical feature-matched non-relapsed patients), to detect a predictive index for tumor prognosis. In aim 2c, we will compare markers determined in tumors to markers detected in cfDNA to assess whether we can develop a minimally-invasive, cost-efficient, clinically convenient cfDNA-based marker for tumor prognosis.