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

摘要 我们的目标是开发一个技术平台，从几滴血中反复测量癌症生物标志物 从癌症患者的家中收集。我们的团队发现，血液外泌体microRNA-375预测了 转移性去势抵抗性前列腺癌（mCRPC）患者的生存率。我们成功地测量了 mCRPC患者在家中采集的小体积血液样本中的microRNA-375，我们现在正在使用 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 在当前流行病需要减少医院就诊的时代，家庭增加了临床价值 并鼓励肿瘤学的家庭护理。但由于样本量小，目前还没有 可以测量一组相关生物标志物，这些生物标志物是解决广泛的遗传谱所需的 mCRPC，包括驱动治疗诱导的克隆选择的遗传变化。此外，我们已确定 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充分表征的核酸序列进行高维多路复用， 使用一种名为单分子流（SiM-Flow）的新检测方法收集血液样本。SiM-Flow允许快速数字化 使用基于荧光的流动对已经延伸和荧光标记的核酸进行计数 细胞计数器。我们建议开发和优化用于定量的仪器和条形码技术 20种不同的核酸生物标志物，已被证明是预后或预测治疗反应 在mCRPC中，除了10个标准化序列外，使用从家庭采集的手指针刺分离的样本 患者的血液样本。特别是，我们将开发（1）微流控单分子计数 一种评估具有5色读数的毫微微升体积分区的仪器，（2）基于5色荧光标记的 在紧凑的，亮度均衡的量子点优化光学棱镜和光谱的色散分布， 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靶点。我们将验证这种新的多重数字检测和数字检测之间的一致性。 液滴PCR，优化标准化探针，并将患者生存与生物标志物组相关联， 在家里的样品测量。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），用于血液分析的光学集成微流体装置 （Rashid Bashir）和临床生物统计学（Jonathan奇普曼）。这个项目的成功将有可能 仪器和分析的变革性技术成果，用于快速、纵向评估许多 使用在家中容易收集的生物样本在个体患者中循环癌症生物标志物。这 该平台可以通过报告转移瘤中发生的分子变化来填补临床肿瘤学的空白， 涉及可用于将治疗与个体患者的反应曲线相匹配和精细调节的治疗。

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.