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.

虽然大规模平行测序技术已经相当成熟，但存在一个问题，那就是血浆肿瘤