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体细胞改变的超灵敏检测对于影响患者护理具有巨大潜力，例如用于早期发现、建立诊断、改善预后、指导治疗和监测复发。然而，当前的技术不太适合对频率极低（<1%）的体细胞突变进行稳健检测。大规模并行测序代表了一条有利的前进道路，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来克服这一限制。在我们的方法中，样品中存在的目标序列的每个副本在多重捕获反应的第一个循环期间用独特的随机序列进行分子标记。扩增后，目标扩增子及其相应的分子标签进行大规模并行测序。在分析过程中，分子标签用于关联具有共同起源的序列读取。通过过采样，带有相同分子标签的读数可以相互纠错，从而为每个祖分子产生独立的单倍体共识。此外，通常衍生的读数的崩溃本质上纠正了扩增过程中任何等位基因特异性的偏差，从而估计 突变等位基因频率可以伴随精确的置信界限（“分子计数”）。除其他优点外，该方法对 10,000 个未突变拷贝背景中的至少 1 个突变序列敏感。在这里，我们建议对该方法进行高级开发和验证，以用作临床诊断。在我们的第一个目标中，我们将开发一个多重面板，以使用该技术广泛针对常见的癌症相关突变。在我们的第二个目标中，我们将应用该组合来检测急性髓性白血病中的微小残留病，作为疾病复发的预后标志。在我们的第三个目标中，我们将应用该面板来检测循环游离DNA中的超罕见突变，这种DNA是从垂死的肿瘤细胞释放到循环中的，作为一种强大的、非侵入性的癌症诊断。该小组将针对这两个目标的临床使用进行严格验证，并为两种单独的分析物适当设计性能指标。提供稳健、经济高效、定量且通用的工具，用于罕见体细胞事件的超灵敏、多重检测 在临床环境中将提供增强的、变革性的癌症诊断和监测能力。该方法也将应用于基础科学癌症研究。