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

先进的开发和验证靶向分子计数方法，用于低流行体细胞突变的精确和超灵敏定量

• 批准号: 9061644
• 负责人: Stephen J Salipante
• 金额: $ 37.59万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9061644
• 关键词: Acute Myelocytic Leukemia; Address; Advanced Development; Alleles; Basic Science; Biological Assay; Biological Markers; Blood Circulation; CLIA certified; Cancer Detection; Cancer Diagnostics; Cancer Patient; Cataloging; Catalogs; Cell Line; Cells; Characteristics; Clinical; Consensus; DNA Sequence; Detection; Detection of Minimal Residual Disease; Diagnosis; Diagnostic; Disease; Drug resistance; Early Diagnosis; Event; Exons; Flow Cytometry; Frequencies; Gene Frequency; Genes; Genetic screening method; Goals; Gold; Haploidy; Health; Hereditary Disease; Individual; Laboratories; Low Prevalence; Malignant Neoplasms; Massive Parallel Sequencing; Measurement; Measures; Mediating; Methodology; Methods; Mind; Molecular; Molecular Analysis; Molecular Target; Monitor; Monitoring for Recurrence; Mutate; Mutation; Non-Invasive Cancer Detection; Outcome; Patient Care; Patients; Performance; Prognostic Marker; Property; Reaction; Reading; Reagent; Recurrence; Recurrent disease; Relapse; Reproducibility; Residual Neoplasm; Residual state; Sampling; Screening for cancer; Site; Somatic Mutation; Specificity; Specimen; Technology; Time; Validation; actionable mutation; anticancer research; base; cancer genome; cancer recurrence; cancer therapy; cell free DNA; clinical application; clinical practice; clinically relevant; combinatorial; cost; cost effective; cost effectiveness; design; diagnostic assay; exome sequencing; genome sequencing; genomic biomarker; insertion/deletion mutation; meetings; multiplex detection; mutant; neoplastic cell; novel; outcome forecast; personalized medicine; progenitor; prognostic; research clinical testing; resistance mutation; targeted sequencing; technology validation; tool; tumor; tumor DNA; whole genome

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.

描述(由申请人提供)：对癌症基因组中临床相关的体细胞变化的超灵敏检测在影响患者护理方面具有巨大的潜力，例如，在早期发现、确定诊断、改善预后、指导治疗和监测复发方面。然而，目前的技术不太适合对存在于极低频率(&lt；1%)的体细胞突变进行稳健的检测。大规模并行测序代表着一条有利的前进道路，但其检测非常罕见的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，存在于样本中的靶序列的每个拷贝在与唯一随机序列的多重捕获反应的第一个周期中被分子标记。扩增后，对靶扩增产物及其相应的分子标签进行大规模平行测序。在分析过程中，分子标签被用来关联共享共同起源的序列读数。通过过度采样，带有相同分子标签的读数相互纠错，为每个前体分子产生独立的单倍体共识。此外，共同派生的读数的折叠本质上纠正了扩增过程中任何等位基因特定的偏见，这样估计 突变等位基因频率可以伴随着精确的置信限(“分子计数”)。除其他优点外，该方法对10,000个未突变拷贝的背景中至少1个突变序列敏感。在这里，我们建议进一步开发和验证这一方法，以用于临床诊断。在我们的第一个目标中，我们将开发一个多路小组，使用这项技术来广泛地针对常见的癌症相关突变。在我们的第二个目标中，我们将该小组应用于检测急性髓系白血病的微小残留病，作为疾病复发的预后标志。在我们的第三个目标中，我们将应用该小组来检测循环中无细胞DNA的超罕见突变，作为一种强大的非侵入性癌症诊断方法，该DNA从濒死的肿瘤细胞释放到循环中。该小组将在两个目标的临床使用中进行严格的验证，并为两个单独的分析物设计适当的性能指标。用于罕见躯体事件的超灵敏、多重检测的可靠、经济、定量和普遍适用的工具的可用性 在临床环境中，将在癌症诊断和监测方面提供增强的、变革性的能力。该方法也将应用于基础科学癌症研究。