Comprehensive minimal residual disease tracking in cancer

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微观的 系统或局部治疗后的残留疾病，从那些没有它。目前的成像几乎不能检测 100万个细胞的质量，而只需要一个细胞就可以产生新的肿瘤，当它们被检测到时， 往往是无法治愈的。早期发现微小残留病（MRD）可以使需要进一步治疗的患者 治疗有治愈的机会，并防止过度治疗他人。尽管有希望，MRD检测 基于像数字PCR这样的技术，一次只能检测一种肿瘤标志物， 在早期发现残留的癌症。下一代测序（NGS）可以跟踪许多突变 然而，NGS需要使用分子条形码进行广泛的校正以减少噪音。 检测低水平的突变这一要求必然会降低NGS吞吐量， 开销.目前，对于NGS，它是测序深度或广度，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， 尽管cfDNA材料有限，但仍可追踪血液中的MRD，同时还可保留NGS通量（宽度） 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利用由探针引导的核酸酶 成千上万的DNA靶点，使WT序列不可复制，从而允许突变- 含有要扩增的序列，并以很少的读数进行测序，就好像它们是高丰度的一样。 突变。为了追踪血液中的MRD，我们首先使用整个外显子组为每个患者创建肿瘤指纹， 对原发性肿瘤进行测序，以确定30-100个肿瘤特异性克隆突变， 躯干突变这些将使用NaME-PrO增强的NGS在cfDNA中追踪。NaME-PrO将是 与分子条形码（qNaME-PrO）结合，以实现原始突变的定量 这意味着可以使用较少的测序读段来跟踪患者特异性突变并消除错误。 我们将（a）优化和测试分子条形码与NaME-PrO突变的结合使用， 富集以定量原始突变丰度;（B）进行外显子组测序以获得 突变肿瘤指纹;然后遵循血浆中的指纹和WT血浆中的系列稀释液， 确定定量检测的最低限度;以及（c）对 MRD在黑色素瘤患者中的预后能力。如果项目成功，将通过实践来跟进- 改变临床研究。预期所提出的方法提供高阴性预测 作为主要优势之一，这最终可能使“警惕等待”战略成为可能， 一些病人目前接受了不必要的治疗，从而降低了发病率和保健费用。

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.