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

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.