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

摘要 我们的目标是开发一种技术平台，从几滴血中重复测量癌症生物标记物 是从家里的癌症患者身上收集的。我们的团队发现，血液外体microRNA-375可以预测 耐去势转移性前列腺癌(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容量血液样本中的microRNA-375，我们现在正在使用 用于对接受治疗的个别患者进行纵向分析。这种收集样本的能力 在大流行导致就诊次数减少的当今时代，家庭具有更高的临床价值 并鼓励肿瘤科的家庭护理。但由于样本量较小，目前还没有。 有可能测量一组相关的生物标记物，这些标记物是解决广泛的遗传谱所需的 MCRPC，包括驱动治疗诱导克隆选择的基因变化。此外，我们已经确定 需要额外的标准化标准来提高有效性和重复性。因此，我们的目标是 是对来自这些家庭的具有良好特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)的新方法采集血液样本。SIM-Flow允许快速数字 使用基于荧光的流动对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 已被证明可预测或预测治疗反应的20种不同的核酸生物标志物 在mCRPC中，除了10个归一化序列外，使用从家庭收集的指棒中分离的样本 病人的血液样本。特别是，我们将发展(1)微流控单分子计数 使用5色读数评估毫微升体积分区的仪器，(2)基于5色荧光标签的 关于优化光学棱镜和光谱的色散分布的紧凑、亮度均衡的量子点 硅光电倍增管阵列的灵敏度，以及(3)不同外体的核酸编码方案 MicroRNA和mRNA靶标。我们将验证这种新的多路数字化验和数字之间的一致性 水滴聚合酶链式反应，优化归一化探针，并将患者生存与生物标志物小组和 来自家庭样本的测量。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控设备 (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 用于快速、纵向评估大量数据的仪器和分析的变革性技术成果 使用在家中很容易收集的生物显微镜在个体患者中循环的癌症生物标记物。这 Platform可以通过报告发生在转移瘤中的分子变化来填补临床肿瘤学的空白 对于疗法，可以用来匹配和微调治疗与个别患者的反应概况。

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.