Digital Multiplexed Analysis of Circulating Nucleic Acids in Small-Volume Blood Specimens

小体积血液样本中循环核酸的数字多重分析

• 批准号: 10467839
• 负责人: Manish Kohli
• 金额: $ 54.71万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10467839
• 关键词: Address; Agreement; Bar Codes; Base Sequence; Biological Assay; Biological Markers; Biometry; Blood; Blood Volume; Blood specimen; Cancer Patient; Caring; Cessation of life; Clinic; Clinic Visits; Clinical; Clinical Oncology; Clinical Trials; Code; Collection; Color; Complex; Devices; Disease; Drops; Enrollment; Evaluation; Failure; Fluorescence; Frequencies; Genetic; Goals; Hematopoietic Neoplasms; Home; Hospitals; Label; Longitudinal cohort; Measurement; Measures; Messenger RNA; MicroRNAs; Microfluidic Microchips; Microfluidics; Molecular; Molecular Probes; Mutation; Neoplasm Metastasis; Nucleic Acids; Oncologist; Oncology; Optics; Outcome; Patients; Pharmaceutical Preparations; Plasma; Prediction of Response to Therapy; Prostate Cancer therapy; Quantitative Reverse Transcriptase PCR; Quantum Dots; RNA; Reagent; Regimen; Reporting; Reproducibility; Sampling; Scheme; Selection for Treatments; Ships; Silicon; Source; Specialist; Specimen; Stream; System; Technology; Testing; Therapeutic; Time; Translational Research; Treatment Protocols; Tumor Biology; Validation; Visit; Work; advanced prostate cancer; base; biomarker discovery; biomarker panel; cancer biomarkers; castration resistant prostate cancer; chemotherapy; companion diagnostics; cost effective; detection limit; digital; docetaxel; drug response prediction; fluorophore; high dimensionality; improved; individual patient; instrument; instrumentation; longitudinal analysis; multiplex assay; new technology; optic flow; pandemic disease; patient response; performance tests; photomultiplier; predictive modeling; prevent; prognostic; prognostic model; prospective; rapid test; ratiometric; response; single molecule; success; survival prediction; treatment response

Abstract Our goal is to develop a technology platform to repeatedly measure cancer biomarkers from a few drops of blood collected from cancer patients at home. Our team identified that blood exosomal microRNA-375 predicts time to survival of patients with metastatic castrate resistant prostate cancer (mCRPC). We succeeded in measuring microRNA-375 in small volume blood samples collected by mCRPC patients at home, which we are now using for longitudinal analysis of individual patients as they undergo treatment. This capacity to collect samples from home has increased clinical value in the current era in which a pandemic necessitates decreased hospital visits and encourages home-based care in oncology. However due to the small sample volume, it is not currently possible to measure a panel of related biomarkers which are needed to address the broad genetic spectrum of mCRPC, including genetic changes that drive therapy-induced clonal selection. Further, we have determined that additional normalization standards are needed to improve validation and reproducibility. Therefore, our goal is to perform high-dimensional multiplexing of well-characterized nucleic acid sequences from these home- collected blood samples using a new assay called single-molecule flow (SiM-Flow). SiM-Flow allows rapid digital counting of nucleic acids that have been extended and fluorescently labeled using a fluorescence-based flow cytometer. We propose to develop and optimize instrumentation and barcoding technologies for quantification of 20 distinct nucleic acid biomarkers that have been shown to be prognostic or predictive of therapy response in mCRPC in addition to 10 normalization sequences, using samples isolated from home-collected fingerstick blood specimens from patients. In particular, we will develop (1) a microfluidic single-molecule counting instrument that evaluates femtoliter volumetric partitions with 5-color readout, (2) 5-color fluorescent labels based on compact, brightness-equalized quantum dots optimized for dispersion profiles of optical prisms and spectral sensitivities of silicon photomultiplier arrays, and (3) nucleic acid coding schemes for diverse exosomal microRNA and mRNA targets. We will validate agreement between this new multiplexed digital assay and digital droplet PCR, optimize normalization probes, and correlate patient survival with the biomarker panel and measurements from at-home samples. Our team has broad expertise needed to accomplish this work, including specialists in molecular probes and single-molecule fluorescence (Andrew Smith), prospective clinical trials and mCRPC biomarker discovery (Manish Kohli), microfluidic devices with optical integration for blood analysis (Rashid Bashir), and clinical biostatistics (Jonathan Chipman). Success in this project will have the potentially transformative technological outcome of an instrument and assay for rapid, longitudinal evaluation of numerous circulating cancer biomarkers in individual patients using biospecimens that are readily collected at home. This platform could fill a void in clinical oncology by reporting molecular changes occurring in metastases in response to therapies, which may be used to match and finely tune treatments to individual patient response profiles.

抽象的 我们的目标是开发一个技术平台，从几滴血液中重复测量癌症生物标志物 从家里的癌症患者那里收集的。我们的团队发现血液外泌体 microRNA-375 可以预测 转移性去势抵抗性前列腺癌（mCRPC）患者的生存率。我们成功测量了 mCRPC 患者在家采集的小体积血液样本中含有 microRNA-375，我们现在正在使用该样本 对接受治疗的个体患者进行纵向分析。这种收集样本的能力 在当前流行病导致医院就诊次数减少的时代，家庭的临床价值增加了 并鼓励肿瘤学的家庭护理。但由于样本量较小，目前还没有 可以测量一组相关的生物标志物，这些生物标志物是解决广泛遗传谱所需的 mCRPC，包括驱动治疗诱导的克隆选择的基因变化。此外，我们还确定了 需要额外的标准化标准来提高验证和再现性。因此，我们的目标 是对来自这些家庭的已充分表征的核酸序列进行高维多重分析 使用一种称为单分子流（SiM-Flow）的新测定法收集血液样本。 SiM-Flow 允许快速数字化 使用基于荧光的流程对已延伸和荧光标记的核酸进行计数 细胞仪。我们建议开发和优化用于量化的仪器和条形码技术 20 种不同的核酸生物标志物已被证明可以预测或预测治疗反应 在 mCRPC 中，除了 10 个标准化序列外，还使用从家庭收集的指尖采血中分离出的样本 患者的血液样本。特别是，我们将开发（1）微流控单分子计数 评估飞升体积分区的仪器，具有 5 色读数，(2) 基于 5 色荧光标记 针对光学棱镜和光谱色散分布进行优化的紧凑、亮度均衡的量子点 硅光电倍增管阵列的灵敏度，以及（3）不同外泌体的核酸编码方案 microRNA 和 mRNA 目标。我们将验证这种新的多重数字测定与数字化之间的一致性 液滴 PCR，优化标准化探针，并将患者生存率与生物标志物组关联起来 家庭样品的测量结果。我们的团队拥有完成这项工作所需的广泛专业知识，包括 分子探针和单分子荧光专家（安德鲁·史密斯）、前瞻性临床试验和 mCRPC 生物标志物发现 (Manish Kohli)，用于血液分析的具有光学集成的微流体装置 （拉希德·巴希尔）和临床生物统计学（乔纳森·奇普曼）。该项目的成功将有可能 用于快速、纵向评估众多物质的仪器和测定的变革性技术成果 使用在家中容易收集的生物样本来检测个体患者的循环癌症生物标志物。这 该平台可以通过报告转移中发生的分子变化来填补临床肿瘤学的空白 治疗，可用于匹配和微调治疗以适应个体患者的反应情况。