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: 9269176
• 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 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9269176
• 关键词: Acute Myelocytic Leukemia; Advanced Development; Alleles; Basic Science; Biological Assay; Biological Markers; Blood Circulation; CLIA certified; Cancer Detection; Cancer Diagnostics; Cancer Patient; Catalogs; Cell Line; Cells; Characteristics; Clinical; Consensus; DNA sequencing; Detection; Detection of Minimal Residual Disease; Diagnosis; Diagnostic; Disease; Drug resistance; Early Diagnosis; Event; Exons; Flow Cytometry; Frequencies; Gene Frequency; Generic Drugs; Genes; Genetic screening method; Goals; Gold; Haploidy; Hereditary Disease; Individual; Laboratories; Low Prevalence; Malignant Neoplasms; Massive Parallel Sequencing; Measurement; Measures; Mediating; Methodology; Methods; Mind; Molecular; Molecular Target; Monitor; Monitoring for Recurrence; Mutate; Mutation; Non-Invasive Cancer Detection; Outcome; Patient Care; Patients; Performance; Prognostic Marker; Property; Reaction; Reagent; Recurrence; Recurrent disease; Relapse; Reproducibility; Residual Neoplasm; Residual state; Sampling; Screening for cancer; Site; Somatic Mutation; Specificity; Specimen; Technology; Therapeutic; Time; Validation; actionable mutation; anticancer research; base; cancer genome; cancer recurrence; cancer therapy; cell free DNA; clinical application; clinical diagnostics; clinical practice; clinically relevant; combinatorial; cost; cost effective; cost effectiveness; design; diagnostic assay; exome sequencing; genome sequencing; genomic biomarker; insertion/deletion mutation; molecular scale; multiplex detection; mutant; neoplastic cell; novel; outcome forecast; personalized medicine; progenitor; prognostic; public health relevance; 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）：癌症基因组中临床相关的体细胞改变的超敏感检测具有影响患者护理的巨大潜力，例如为了早期检测，建立诊断，精炼预后，指导治疗和监测复发。但是，当前的技术不适合在非常低的频率（<1％）下对体细胞突变的强大检测。大规模平行的测序代表了一个有利的路径，但其检测非常罕见事件的敏感性从根本上受到测序误差率的限制。我们最近开发了一种新的实验范式，该范式克服了这一限制。在我们的方法中，样品中存在的目标序列的每个副本都是在用独特的随机序列的多重捕获反应的第一个循环中标记的。放大后，靶扩增子及其相应的分子标签进行大规模平行测序。在分析过程中，分子标签用于将共有起源共享读取的序列读取。通过过采样，读取相同的分子标记误差误差彼此，以产生每个祖细胞分子的独立单倍体共识。此外，在放大过程中，常见衍生的读数固有地校正了任何等位基因特异性偏差，以便估计 突变等位基因频率可以伴随精确的置信度界限（“分子计数”）。除其他好处外，该方法在10,000份未经分配的副本的背景下对至少1个突变序列敏感。在这里，我们提出了这种方法的高级开发和验证，以用作临床诊断。在我们的第一个目标中，我们将开发一个多路复用面板，以使用该技术广泛针对常见的癌症相关突变。在第二个目标中，我们将将小组应用于急性髓样白血病中最小残留疾病的检测，作为疾病复发的预后标志。在我们的第三个目标中，我们将在无细胞的DNA中应用小组检测超稀有突变，该突变是一种可靠和非侵入性癌症诊断的疾病肿瘤细胞循环中的循环。该面板将在两个目标中都进行严格验证，以供临床使用，并且针对两个单独的分析物进行了适当设计的性能指标。可用性，具有成本效益，定量且普遍适用的工具，可用于对罕见的体细胞事件的超敏，多重检测 在临床环境中，将在癌症的诊断和监测中提供增强的变革能力。该方法还将应用基础科学癌症研究。