EndoGenus Toolkit: A Biometric Method for Absolute Quantification of Tumor Markers by Massive Parallel Sequencing

EndoGenus 工具包：通过大规模并行测序对肿瘤标志物进行绝对定量的生物识别方法

• 批准号: 10248332
• 负责人: Margaret L Gulley
• 金额: $ 15.35万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10248332
• 关键词: Algorithms; Alleles; Apoptosis; Bioinformatics; Biological; Biological Assay; Biological Factors; Biometry; Blood; Blood Cells; Buffers; Cancer Patient; Clinical; Clinical Management; Clinical Trials; Clonal Evolution; Collection; Conscious; Cytolysis; DNA; DNA Primers; DNA sequencing; Disease; Drug resistance; Edetic Acid; Future; Genomics; Gold; Health Personnel; Human Engineering; Infection; Inflammation; Informatics; Intervention; Kinetics; Laboratories; Leukocytes; Libraries; Malignant Neoplasms; Massive Parallel Sequencing; Measurement; Measures; Medical; Methods; Monitor; Mutation; Needles; Normal Cell; Numerical value; Patients; Performance; Plasma; Preparation; Procedures; Proliferating; Reagent; Recovery; Recurrence; Reporting; Reproducibility; Research; Scientist; Sensitivity and Specificity; Somatic Mutation; Specimen; Specimen Handling; Technology; Time; Tissues; Tumor Burden; Tumor Markers; Variant; Viral Genome; Whole Blood; base; chemoradiation; cost; digital; genetic variant; human DNA; improved; new technology; novel; novel strategies; rate of change; sample collection; synthetic construct; tool; tumor DNA; tumor heterogeneity; virus related cancer

While massive parallel sequencing technology is quite mature, a problematic aspect is that plasma tumor markers are measured relative to total DNA, and total DNA levels vary with biologic factors like inflammation and with pre-analytic interferences such as leukocyte lysis during blood collection and handling ex vivo. This research aims to develop a novel strategy to quantify tumor markers in ‘copies per mL of plasma’, thus harmonizing the massive parallel sequencing assay with gold standard values generated by quantitative PCR. First, we will spike plasma with synthetic DNAs (called “EndoGenus Spikes”) which are then targeted for enrichment during library preparation and are quantified using informatic scripts after massive parallel sequencing. By normalizing levels of each tumor marker to the fractional recovery of spiked DNAs, numerical values are reportable in units of “copies per mL of plasma”, which we will show reflect clonal abundance. This new capability for absolute quantification of clonal abundance is likely to benefit basic scientists studying tumor heterogeneity and clonal evolution, and is likely to benefit patients and healthcare providers who seek more informative ways to monitor tumor burden, to evaluate the impact of medical interventions, and to find emerging drug resistance clones so that alternate therapy may be considered in a timely fashion. At the conclusion of this study, we will have developed and validated the ‘EndoGenus Toolkit’, comprised of synthetic DNAs, reagents to enrich for them during library preparation, and bioinformatic scripts to convert tumor marker levels from fractions to absolute concentrations. We will show that applying the ‘Toolkit’ to mock plasma specimens yields sensitive, specific, linear and reproducible sequencing results for multiple tumor markers. In blood from active cancer subjects, we will show that the ‘Toolkit’ helps overcome pre-analytic problems associated with blood storage. These tools should facilitate future clinical trials aimed at setting numeric thresholds for changing patient management.

虽然大规模平行测序技术已经相当成熟，但一个问题是血浆肿瘤细胞的基因测序技术还不成熟。 标记物是相对于总DNA测量的，总DNA水平随生物因素如炎症而变化 以及分析前的干扰，例如在血液收集和离体处理期间的白细胞溶解。这 研究旨在开发一种新的策略，以“每毫升血浆的拷贝数”量化肿瘤标志物， 使大规模平行测序测定与定量PCR产生的金标准值相协调。 首先，我们将用合成DNA（称为“EndoGenus Spikes”）刺入血浆，然后靶向 在文库制备期间富集，并在大量平行后使用信息脚本进行定量 测序通过将每种肿瘤标志物的水平标准化为加标DNA的回收分数， 数值以“拷贝/mL血浆”为单位报告，我们将显示其反映克隆丰度。这 克隆丰度绝对定量的新能力可能会使研究肿瘤的基础科学家受益 异质性和克隆进化，并可能有利于患者和医疗保健提供者谁寻求更多的 监测肿瘤负荷、评估医疗干预的影响以及发现 新出现的耐药克隆，以便可以及时考虑替代疗法。在 本研究的结论，我们将开发和验证'EndoGenus工具包'，包括合成 DNA，在文库制备期间富集它们的试剂，以及转化肿瘤标志物的生物信息学脚本 从分数到绝对浓度。我们将证明，应用'工具包'模拟血浆 样本产生敏感、特异、线性和可重复的多肿瘤标志物测序结果。在 从活跃的癌症受试者的血液，我们将表明，'工具包'有助于克服分析前的问题 与血液储存有关。这些工具应该有助于未来的临床试验，旨在设置数字 改变患者管理的阈值。

项目成果

Use of Spiked Normalizers to More Precisely Quantify Tumor Markers and Viral Genomes by Massive Parallel Sequencing of Plasma DNA.

• DOI: 10.1016/j.jmoldx.2020.01.012
• 发表时间: 2020-02
• 期刊: The Journal of molecular diagnostics : JMD
• 作者: M. Gulley;Sandra H. Elmore;G. Gupta;Sunil Kumar;Matthew Egleston;Ian J. Hoskins;A. Garnett