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: 10001973
• 负责人: Margaret L Gulley
• 金额: --
• 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 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10001973
• 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

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.

虽然大规模并行测序技术已经相当成熟，但一个问题是血浆肿瘤 标记物是相对于总 DNA 进行测量的，总 DNA 水平随炎症等生物因素而变化 以及分析前的干扰，例如血液采集和离体处理过程中的白细胞裂解。 研究旨在开发一种新的策略来量化“每毫升血浆的拷贝数”中的肿瘤标志物，从而 将大规模平行测序测定与定量 PCR 生成的黄金标准值相协调。 首先，我们将用合成 DNA（称为“EndoGenus Spikes”）掺入血浆，然后将其靶向 在文库制备过程中进行富集，并在大规模并行后使用信息脚本进行量化 通过将每个肿瘤标志物的水平标准化为掺入 DNA 的回收分数，进行数值测序。 值以“每毫升血浆的拷贝数”为单位报​​告，我们将显示该值反映克隆丰度。 克隆丰度绝对定量的新功能可能有利于研究肿瘤的基础科学家 异质性和克隆进化，并可能使寻求更多的患者和医疗保健提供者受益 监测肿瘤负荷、评估医疗干预措施的影响并发现问题的信息方法 出现耐药性克隆，以便及时考虑替代疗法。 这项研究结束后，我们将开发并验证“EndoGenus 工具包”，其中包括合成的 DNA、在文库制备过程中富集 DNA 的试剂以及转换肿瘤标志物的生物信息学脚本 我们将展示如何将“工具包”应用于模拟血浆。 样本可对多种肿瘤标志物产生灵敏、特异、线性且可重复的测序结果。 来自活跃癌症受试者的血液，我们将证明“工具包”有助于克服分析前的问题 这些工具应该有助于未来旨在设定数字的临床试验。 改变患者管理的阈值。