Comprehensive minimal residual disease tracking in cancer

确定早期癌症患者的适量治疗--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带有显微镜的 全身或局部治疗后的残留疾病，来自那些没有治疗的人。目前的成像几乎不能检测到 100万个细胞的质量，虽然只需要一个细胞就能产生新的肿瘤，当它们被检测到时， 通常是不治之症。早期发现微小残留病(MRD)可以为需要进一步治疗的患者提供 治疗提供了治愈的机会，并防止过度治疗他人.尽管有希望，但MRD检测 基于数字聚合酶链式反应等技术，一次检测一个肿瘤标记物的能力不足 以便在早期阶段发现残留的癌症。下一代测序(NGS)可以跟踪许多突变 同时；然而，NGS需要使用分子条形码进行广泛的校正以降低噪声 并检测低水平的突变。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，它要么是测序深度，要么是广度，但不是两者兼而有之。 在这里，我们建议改进和应用一种变革性技术，使高度敏感 尽管cfDNA材料有限，但仍可追踪血液中的MRD，同时还能保持NGS吞吐量(广度) 和深度。我们最近开发了NAME-PRO，这是一项简单而强大的技术，使NGS能够 追踪循环DNA中极低水平的突变。NAME-PRO利用由探针引导的核酸酶 使WT序列不可扩增，从而允许突变- 含有要扩增和测序的序列，读数很少，就好像它们是高丰度的一样 突变。为了跟踪血液中的MRD，我们首先使用整个外显子组为每个患者创建一个肿瘤指纹 对原发肿瘤进行测序以确定30-100个肿瘤特异性克隆性突变 主干突变。这些将在cfDNA中使用NAME-PRO增强的NGS进行跟踪。名称-PRO将是 与分子条码(qNaME-PRO)相结合，能够对原始突变进行量化 很少有测序读数的片段，用于跟踪患者特定的突变并消除错误。 我们将(A)优化和测试分子条形码与名字前突变的结合使用 用于量化原始突变丰度；(B)进行外显子组测序以得出 突变肿瘤指纹；然后跟随血浆中的指纹和WT血浆中的连续稀释来 确定定量检测的最低限度；和(C)对 黑色素瘤患者MRD的预后能力。如果项目成功了，接下来就是实践-- 不断变化的临床研究。所提出的方法预计将提供高度的负面预测。 电力，作为主要优势之一。这最终可能会为以下几个方面启用“警惕等待”策略 一些患者目前接受了不必要的治疗，从而降低了发病率和医疗成本。

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.