Urine extracellular vesicles are a valuable low-invasive source of information, especially for the cells of the genitourinary tract. In the search for biomarkers, different techniques have been developed to isolate and characterize the cargo of these vesicles. In the present work, we compare five of these different isolation methods (three commercial isolation kits, ultracentrifugation, and lectin-based purification) and perform miRNA profiling using a multiplex miRNA assay. The results showed high correlation through all isolation techniques, and 48 out of 68 miRNAs were detected above the detection limit at least 10 times. The results obtained by multiplex assay were validated through Taqman qPCR. In addition, using this technique combined with a clinically friendly extracellular vesicle (uEV)-enrichment method, we performed the analysis of selected miRNAs in urine from patients affected with bladder cancer, benign prostate hyperplasia, or prostate cancer. Importantly, we found that those miRNAs could be detected in almost 100% of the samples, and no significant differences were observed between groups. Our results support the feasibility of analyzing exosomes-associated miRNAs using a methodology that requires a small volume of urine and is compatible with a clinical environment and high-throughput analysis.

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To address the needs for microRNA (miRNA) biomarker discovery and verification, we developed the FirePlex® miRNA assay. This assay enables the detection of 5-400 miRNA targets per sample in 96-well format, with readout on standard flow cytometers and analysis with an included bioinformatics software package. The FirePlex miRNA assay combines particle-based multiplexing, using patented FirePlex® hydrogel particles, with single step RT-PCR signal amplification using universal primers. Thus, the FirePlex miRNA assay leverages PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target-specific qPCR. The miRNA assay can reliably detect as few as 1000 miRNA copies per sample with a linear dynamic range of ~5 logs, and without the need of prior RNA purification, making the assay ideally suited for profiling in serum, plasma, exosomes, urine, and directly from FFPE tissues. Furthermore, the ability to multiplex targets in each well eliminates the need to split valuable samples into multiple reactions. Results from the FirePlex miRNA assay are displayed and interpreted using the integrated FirePlex Analysis Workbench, which allows visualization, normalization, and export of experimental data with only a few mouse clicks. To aid biomarker discovery studies, we offer pre-designed panels of 400 miRNAs based on a research-curated list of miRNAs published to be found in circulating biofluids. For studies focused on specific research areas of interest, we offer pre-designed focus panels for Oncology, Cardiology, Neurology, Immunology, and Organ Toxicity. These carefully curated panels include hemolysis markers to assess sample quality, as well as critical normalization factors. Here we present the data from several studies investigating circulating miRNA profiles, as well as miRNA profiles obtained directly from FFPE tissues, using the FirePlex miRNA Assay Panels. Together, this novel combination of bioinformatics tools and multiplexed, high-sensitivity assays enables rapid discovery and verification of miRNA biomarker signatures from biofluid samples. Citation Format: Elnaz Atabakhsh, Ivona Kelley, Michael Tackett, Izzuddin Diwan, Gensis Tejada, Conor Rafferty, Daniel Pregibon. Multiplex profiling of miRNA biomarkers for discovery and verification using the FirePlex® platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5405.

Background Non-endoscopic cell collection devices combined with biomarkers can detect Barrett's intestinal metaplasia and early oesophageal cancer. However, assays performed on multi-cellular samples lose information about the cell source of the biomarker signal. This cross-sectional study examines whether a bespoke artificial intelligencebased computational pathology tool could ascertain the cellular origin of microRNA biomarkers, to inform interpretation of the disease pathology, and confirm biomarker validity. MethodsThe microRNA expression profiles of 110 targets were assessed with a custom multiplexed panel in a cohort of 117 individuals with reflux that took a Cytosponge test. A computational pathology tool quantified the amount of columnar epithelium present in pathology slides, and results were correlated with microRNA signals. An independent cohort of 139 Cytosponges, each from an individual patient, was used to validate the findings via qPCR.Findings Seventeen microRNAs are upregulated in BE compared to healthy squamous epithelia, of which 13 remain upregulated in dysplasia. A pathway enrichment analysis confirmed association to neoplastic and cell cycle regulation processes. Ten microRNAs positively correlated with columnar epithelium content, with miRNA-192À5p and -194À5p accurately detecting the presence of gastric cells (AUC 0.97 and 0.95). In contrast, miR-196a-5p is confirmed as a specific BE marker.Interpretation Computational pathology tools aid accurate cellular attribution of molecular signals. This innovative design with multiplex microRNA coupled with artificial intelligence has led to discovery of a quality control metric suitable for large scale application of the Cytosponge. Similar approaches could aid optimal interpretation of biomarkers for clinical use.