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

开发无细胞循环 DNA 中的 5hmC 和 5mC 生物标志物，用于敏感的结肠癌检测和预后

• 批准号: 10220895
• 负责人: Bruce Marc Bissonnette
• 金额: $ 59.98万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10220895
• 关键词: Achievement; Age; Algorithms; Archives; Bar Codes; Biological Assay; Biological Markers; Blood; Blood Tests; Cancer Control; Cancer Detection; Cancer Etiology; Cancer Prognosis; Cells; Cessation of life; Chemicals; Clinical; Colon Carcinoma; Colonic Neoplasms; Colonoscopy; Colorectal Cancer; Cost efficiency; Custom; Cytosine; DNA; DNA Modification Process; Data; Detection; Development; Diagnosis; Disease; Early Diagnosis; Enhancers; Epigenetic Process; FDA approved; Feces; Future; Gender; Gene Expression; Gene Expression Regulation; Gene Mutation; Genes; Genetic Transcription; Genomics; Goals; Human Genome; Immunoprecipitation; Individual; Label; Location; Malignant Neoplasms; Maps; Modification; Monitoring for Recurrence; Mutation; Neoplasm Metastasis; Outcome; Patients; Pattern; Plasma; Positioning Attribute; Prevalence; Prognosis; Prospective cohort; Public Health; Recurrence; Research Personnel; Resources; Role; Sampling; Study Subject; Technology; Testing; Training; Tumor Biology; Tumor Markers; Tumor Tissue; Unresectable; Validation; Vimentin; base; biobank; bisulfite; blood-based biomarker; cancer biomarkers; cancer diagnosis; cancer prevention; case control; cell free DNA; chemical stability; colon cancer patients; colon cancer screening; colorectal cancer screening; cost efficient; demethylation; differential expression; epigenetic marker; epigenomics; genetic signature; indexing; innovation; liquid biopsy; minimal risk; minimally invasive; nano; neoplasm registry; neoplastic cell; next generation sequencing; oxidation; procedure cost; prognostic; promoter; prospective; rare cancer; recruit; relapse patients; repository; response; sample archive; screening; seal; standard of care; treatment response; tumor; tumor DNA

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.

项目概要/摘要 结直肠癌（CRC）是癌症相关死亡的主要原因。筛查结肠镜检查的标准是 关心 CRC 检测，但由于准备工作和程序成本具有挑战性，合规性仍低于 50% 和潜在的并发症。 CRC 中的特定突变很少见，因此无法用作筛查 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，并且 在 FDA 批准的多项测试中进行了询问，但敏感性有限且没有预后信息。 对循环游离 DNA (cfDNA) 中的生物标志物进行灵敏的血液检测可以提供更大的便利性和 更高的合规性。除了 5mC 之外，5-羟甲基胞嘧啶 (5hmC) 的变化在正常情况下也很重要。 和疾病状态。 5hmC 是一种丰富、稳定的修饰胞嘧啶，由 DNA 去甲基化产生 并经常标记活跃的基因转录。 5hmC 和 5mC 可以作为优越的生物标志物，因为它们的 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。我们开发了 用于捕获 5hmC 碱基的高灵敏度和选择性化学标记技术（nano-hmC-Seal），以及 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 对 47 名对照者和 136 名近期患有癌症的患者的 cfDNA 和配对肿瘤组织中的 5hmC 和 5mC 进行了分析 诊断出癌症，其中 46 例患有结直肠癌。我们开发了一个分类器来识别具有差异 5hmC 的基因 CRC 患者的变化，并在肿瘤和 cfDNA 中确定了一致的癌症特异性表观遗传特征。 由于灵敏度高（~1ng DNA）、稳健性、临床便利性和成本效益，我们 假设独特的无细胞 5hmC 和 5mC 图谱可能可用作检测 CRC 的血浆生物标志物 并最终预测肿瘤的预后。我们建议使用 PLCO 生物样本库和我们自己存档的样本 具有注释临床结果的 CRC，以及新招募的前瞻性 CRC 患者来完善和验证 差异表达的 5hmC 和 5mC 基因特征对 CRC 诊断和预后的敏感性。我们 建议：目标 1 使用 nano-hmC-从 PLCO 样本（CRC 和对照）中分析 cfDNA 中的 5hmC 和 5mC 使用 200 个 CRC 病例和 400 个对照，密封和 NGS 并完善敏感 CRC 血液检测的算法 训练集，以及来自我们前瞻性队列的另外 200 个病例和 400 个对照作为验证集。我们 将开发 5hmC 和 5mC 修饰的鉴别器来诊断 CRC 并通过肿瘤进行控制分析 可能调节 5hmC 和 5mC 分布的地点、阶段、患者年龄和性别；目标 2a 至配置文件 100 PLCO 肿瘤的 5hmC 和 5mC 分布，并将分布与 cfDNA 进行比较（针对 1 个样本）；在目标 2b 中， 我们将从存档样本中研究 150 个具有复发数据的肿瘤（60 名复发患者与 90 名临床患者） 特征匹配的非复发患者），检测肿瘤预后的预测指标。在目标 2c 中，我们将 将肿瘤中确定的标记与 cfDNA 中检测到的标记进行比较，以评估我们是否可以开发一种 用于肿瘤预后的微创、经济高效、临床方便的基于 cfDNA 的标记物。

项目成果

Analysis of genome-wide 5-hydroxymethylation of blood samples stored in different anticoagulants: opportunities for the expansion of clinical resources for epigenetic research.

• DOI: 10.1080/15592294.2023.2271692
• 发表时间: 2023-12
• 期刊: EPIGENETICS
• 影响因子: 3.7
• 作者: Gao, Lu;Zhang, Zhou;Cui, Xiao-Long;West-Szymanski, Diana;Ye, Chang;He, Chuan;Zhang, Wei;Bissonnette, Marc
• 通讯作者: Bissonnette, Marc