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: 9767750
• 负责人: Margaret L Gulley
• 金额: $ 22.63万
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9767750
• 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.

虽然大规模平行测序技术已经相当成熟，但有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水平随着炎症等生物因素的变化而变化 以及分析前的干扰，例如在血液采集和体外处理过程中的白细胞溶解。这 研究的目标是开发一种新的策略，以每毫升血浆的拷贝数来量化肿瘤标记物，因此 使大规模平行测序分析与定量聚合酶链式反应产生的金标准值相协调。 首先，我们将在血浆中加入合成DNA(称为内源性DNA刺激物)，然后将其定位于 在文库准备过程中进行丰富，并在大规模并行后使用信息脚本进行量化 测序。通过将每个肿瘤标记物的水平归一化到添加的DNA的恢复分数，数字 这些值以“每毫升血浆的拷贝数”为单位进行报告，我们将显示该值反映了克隆丰度。这 克隆丰度绝对量化的新能力可能有利于研究肿瘤的基础科学家 异质性和克隆进化，并可能使患者和寻求更多 监测肿瘤负担，评估医疗干预的影响，并找到 新出现的耐药克隆，以便及时考虑替代治疗。在 本研究的结论是，我们将开发并验证由合成的 DNA、在文库准备过程中用于丰富它们的试剂，以及用于转换肿瘤标记物的生物信息学脚本 从分数到绝对浓度的水平。我们将演示如何应用“工具包”来模拟等离子体 对于多个肿瘤标志物，标本产生敏感、特异、线性和可重复性的测序结果。在……里面 来自癌症活动性受试者的血液，我们将展示“工具包”帮助克服分析前的问题 与血液储存有关。这些工具应该会促进未来的临床试验，目的是设置数字 改变病人管理的门槛。