Comprehensive minimal residual disease tracking in cancer

癌症的全面微小残留病追踪

• 批准号: 9920128
• 负责人: G. Mike Makrigiorgos
• 金额: $ 37.96万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9920128
• 关键词: Admixture; Alleles; Bar Codes; Biological Assay; Biopsy; Blood; Cancer Patient; Cells; Clinical; Clinical Research; Clinical Sensitivity; Clinical Trials; Computing Methodologies; Consensus Sequence; DNA; Data; Detection; Detection of Minimal Residual Disease; Disease; Early Diagnosis; Fingerprint; Genes; Health Care Costs; Image; Intervention; Local Therapy; Malignant Neoplasms; Methods; Microscopic; Molecular; Molecular Profiling; Monitor; Morbidity - disease rate; Mutation; Mutation Detection; Names; Noise; Patient observation; Patients; Plasma; Primary Neoplasm; Probability; Public Health; Relapse; Residual Cancers; Residual Neoplasm; Residual Tumors; Sampling; Sensitivity and Specificity; Systemic Therapy; Technology; Testing; Time; Tumor Markers; Tumor-Derived; Variant; base; cancer therapy; cell free DNA; circulating DNA; cost effective; digital; exome sequencing; follow-up; individual patient; melanoma; mutant; next generation sequencing; nuclease; overtreatment; prevent; prognostic value; screening; side effect; tumor

Identifying the right amount of therapy – no more and no less – for patients with early stage cancer remains a challenge because there is no reliable method by which to separate those with microscopic residual disease after systemic or local therapies, from those without it. Current imaging can barely detect a mass of 1 million cells, while it takes just one cell to spawn new tumors that, by the time they are detected, are often incurable. Early detection of minimal residual disease (MRD) could give patients who need further treatment a chance at a cure, and prevent over-treatment of others. Despite its promise, MRD detection based on technologies like digital PCR that detect a single tumor marker at a time has inadequate ability to detect residual cancer at early stages. Next generation sequencing (NGS) can track many mutations simultaneously; however NGS requires extensive corrections using molecular barcoding to reduce noise and detect low-level mutations. This requirement invariably diminishes NGS throughput and increases expense. Currently, for NGS it is either sequencing depth or breadth, but not both. Here we propose to refine and apply a transformative technology that enables highly sensitive tracing of MRD in blood despite limited cfDNA material, while also retaining NGS throughput (breadth) and depth. We recently developed NaME-PrO, a simple and powerful technology that enables NGS to track extremely low-level mutations in circulating DNA. NaME-PrO utilizes a nuclease guided by probes to thousands of DNA targets, to render WT sequences non-amplifiable thereby allowing mutation– containing sequences to amplify and be sequenced with few reads as if they were high abundance mutations. To track MRD in blood, we first create a tumor fingerprint for each patient using whole exome sequencing of the primary tumor to define 30-100 tumor-specific clonal mutations and encompassing truncal mutations. These will be tracked in cfDNA using NaME-PrO-enhanced NGS. NaME-PrO will be combined with molecular barcoding (qNaME-PrO) to enable quantification of the original mutation fraction with few sequencing reads for the patient-specific mutations tracked and elimination of errors. We will (a) optimize and test the use of molecular barcoding in conjunction with NaME-PrO mutation enrichment for quantification of original mutation abundance; (b) Perform exome sequencing to derive mutational tumor fingerprints; then follow fingerprints in plasma and serial dilutions in WT plasma to determine the lowest limit of quantitative detection; and (c) perform a preliminary assessment of the prognostic ability of MRD in melanoma patients. If the project is successful, it will be followed by practice- changing clinical studies. The proposed method is anticipated to provide a high negative predictive power, as one of the main advantages. This could eventually enable `watchful waiting' strategies for some patients currently treated unnecessarily, thus reducing morbidity and health care costs.

为早期癌症患者确定正确的治疗量——不多也不少 仍然是一个挑战，因为没有可靠的方法来分离那些与微观 全身或局部治疗后残留的疾病，来自那些没有接受治疗的人。目前的成像技术几乎无法检测到 一百万个细胞组成的团块，而只需要一个细胞就能产生新的肿瘤，当它们被发现时， 往往无法治愈。早期检测微小残留病 (MRD) 可以为需要进一步治疗的患者提供帮助 治疗有治愈的机会，并防止他人过度治疗。尽管有这样的承诺，MRD 检测 基于数字PCR等一次性检测单一肿瘤标志物的技术能力不足 以便在早期发现残余癌症。下一代测序（NGS）可以追踪许多突变 同时地;然而 NGS 需要使用分子条形码进行大量校正以减少噪音 并检测低水平突变。这一要求总是会降低 NGS 吞吐量并增加 费用。目前，对于 NGS 来说，要么是测序深度，要么是测序广度，但不能两者兼而有之。 在这里，我们建议完善和应用一种变革性技术，使高度敏感 尽管 cfDNA 材料有限，仍可追踪血液中的 MRD，同时还保留 NGS 吞吐量（广度） 和深度。我们最近开发了 NaME-PrO，这是一种简单而强大的技术，使 NGS 能够 追踪循环 DNA 中极低水平的突变。 NaME-PrO 利用探针引导的核酸酶 到数千个 DNA 目标，使 WT 序列不可扩增，从而允许突变 – 包含要扩增的序列并以很少的读数进行测序，就好像它们是高丰度一样 突变。为了追踪血液中的 MRD，我们首先使用全外显子组为每位患者创建肿瘤指纹 对原发肿瘤进行测序以确定 30-100 个肿瘤特异性克隆突变并涵盖 树干突变。这些将使用 NaME-PrO 增强的 NGS 在 cfDNA 中进行跟踪。 NaME-PrO 将是 与分子条形码 (qNaME-PrO) 结合以实现原始突变的量化 跟踪患者特异性突变的测序读数很少，并且消除了错误。 我们将 (a) 优化并测试分子条形码与 NaME-PrO 突变结合的使用 富集以量化原始突变丰度； (b) 进行外显子组测序以获得 突变肿瘤指纹；然后跟踪血浆中的指纹和 WT 血浆中的系列稀释 确定定量检测的下限； (c) 进行初步评估 MRD 对黑色素瘤患者的预后能力。如果该项目成功，接下来将进行实践—— 改变临床研究。预计所提出的方法将提供高阴性预测 功率，作为主要优点之一。这最终可能会启用“观察等待”策略 目前一些患者接受了不必要的治疗，从而降低了发病率和医疗费用。