Advanced development and validation of targeted molecular counting methods for precise and ultrasensitive quantitation of low prevalence somatic mutations

描述（由申请人提供）：对癌症基因组中临床相关体细胞改变的超灵敏检测具有巨大的潜力，可以影响患者护理，例如早期发现，建立诊断，改善预后，指导治疗和监测复发。然而，目前的技术不适合在非常低的频率（<1%）下检测体细胞突变。大规模并行测序是一条有利的发展道路，但其检测非常罕见事件的灵敏度从根本上受到测序错误率的限制。我们最近开发了一种新的实验范式，克服了这一限制。在我们的方法中，在多重捕获反应的第一个循环中，样品中存在的目标序列的每个拷贝都被分子标记为具有唯一随机序列。扩增后，靶扩增子及其对应的分子标签进行大规模平行测序。在分析过程中，分子标签用于关联具有共同起源的序列读数。通过过采样，具有相同分子标签的读数相互纠正错误，从而为每个祖分子产生独立的单倍体一致性。此外，共同衍生reads的崩溃固有地纠正了扩增过程中任何等位基因特异性偏差，例如估计

DESCRIPTION (provided by applicant): The ultrasensitive detection of clinically relevant somatic alterations in cancer genomes has great potential for impacting patient care, e.g. for early detection, establishing diagnoses, refining prognoses, guiding treatment, and monitoring recurrence. However, current technologies are poorly suited to the robust detection of somatic mutations present at very low frequencies (<1%). Massively parallel sequencing represents an advantageous path forward, but its sensitivity to detect very rare events is fundamentally constrained by the sequencing error rate. We have recently developed a new experimental paradigm that overcomes this limitation. In our approach, each copy of a target sequence that is present in a sample is molecularly tagged during the first cycle of a multiplex capture reaction with a unique random sequence. After amplification, target amplicons and their corresponding molecular tags are subjected to massively parallel sequencing. During analysis, the molecular tags are used to associate sequence reads sharing a common origin. Through oversampling, reads bearing the same molecular tag error-correct one another to yield an independent haploid consensus for each progenitor molecule. Furthermore, the collapsing of commonly derived reads inherently corrects for any allele-specific bias during amplification, such that estimates of mutant allele frequency can be accompanied by precise confidence bounds ("molecular counting"). Among other benefits, the approach is sensitive to at least 1 mutated sequence in a background of 10,000 unmutated copies. Here we propose the advanced development and validation of this approach for use as a clinical diagnostic. In our first aim, we will develop a multiplexed panel to broadly target common cancer associated mutations using this technology. In our second aim, we will apply the panel to the detection of minimal residual disease in acute myeloid leukemia as a prognostic marker of disease relapse. In our third aim, we will apply the panel to detection of ultra-rare mutations in circulating cell-free DNA, which is released into circulation from dying tumor cells, as a robust and non-invasive cancer diagnostic. The panel will be rigorously validated for clinical use in both aims, with performance metrics appropriately designed for the two separate analytes. The availability of robust, cost-effective, quantitative, and generically applicable tools for the ultrasensitive, multiplex detection of rare somatic events in the clinical setting will provide enhanced, transformative capabilities in the diagnosis and monitoring of cancers. The methodology will also have application to basic science cancer research.