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: 10676313
• 负责人: Manish Kohli
• 金额: $ 51.89万
• 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/10676313
• 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; Evaluation; Failure; Fingers; Fluorescence; Frequencies; Genetic; Goals; 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; Silicon; Source; Specialist; Specimen; Stream; System; Technology; Testing; Therapeutic; Time; Translational Research; Treatment Protocols; Tumor Biology; Validation; Visit; Work; advanced prostate cancer; biomarker discovery; biomarker panel; cancer biomarkers; castration resistant prostate cancer; chemotherapy; clinical translation; companion diagnostics; cost effective; detection limit; digital; docetaxel; drug response prediction; exosome; fluorophore; high dimensionality; improved; individual patient; instrument; instrumentation; longitudinal analysis; multiplex assay; new technology; pandemic disease; participant enrollment; patient response; performance tests; photomultiplier; predictive modeling; prevent; prognostic; prognostic model; prospective; rapid test; ratiometric; response; single molecule; success; survival prediction; technology platform; 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，从几滴血液中反复测量癌症生物标志物 从家里从癌症患者那里收集。我们的团队确定血液外泌体microRNA-375预测时间 转移性castrate抗性前列腺癌（MCRPC）患者的存活率。我们成功地衡量了 McRPC患者在家中收集的小体积血液样本中的microRNA-375，我们现在正在使用 为了对单个患者进行治疗的纵向分析。从中收集样品的能力 房屋在当前时代的临床价值增加，在该时代，大流行需要减少医院就诊 并鼓励肿瘤学家庭护理。但是由于样品量很小，目前不是 可以测量一系列相关的生物标志物，以解决广泛的遗传谱。 MCRPC，包括推动治疗诱导克隆选择的遗传变化。此外，我们已经确定 需要额外的归一化标准来提高验证和可重复性。因此，我们的目标 是从这些家用的核酸序列进行高维的多路复用 使用称为单分子流（SIM-Flow）的新测定法收集了血液样本。 sim-flow允许快速数字 使用基于荧光的流量进行扩展和荧光标记的核酸计数 细胞仪。我们建议开发和优化仪器和条形码技术以进行定量 在20种不同的核酸生物标志物中，这些生物标志物已被证明是预后或预测治疗反应的 除了10个归一化序列外，在MCRPC中使用从家庭收集的指尖分离的样品 患者的血液标本。特别是，我们将开发（1）微流体单分子计数 评估具有5色读数的女性体积分区的仪器（2）5色荧光标签 在紧凑的，亮度平等的量子点上，针对光学棱镜和光谱的色散曲线进行了优化 硅光电塑料阵列的敏感性和（3）核酸编码方案对多种外泌体的编码方案 microRNA和mRNA靶标。我们将验证这种新的多重数字测定和数字之间的一致性 液滴PCR，优化归一化探针，并将患者的生存与生物标志物面板相关联 来自家庭样品的测量。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（安德鲁·史密斯）的专家，前瞻性临床试验和 MCRPC生物标志物发现（Manish Kohli），具有光学整合的微流体设备用于血液分析 （Rashid Bashir）和临床生物统计学（Jonathan Chipman）。这个项目的成功将具有潜在的 仪器的变革性技术结果和测定法，以快速，纵向评估众多 使用容易在家中收集的生物测量的个别患者循环癌症生物标志物。这 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隙。 用于疗法，可用于匹配和细微的疗法与个体患者反应谱。