Abstract 4317: Comparison of Single-Molecule Flow (SiM-Flow) and qRT-PCR for detection of plasma miR-375 and miR-1290 in metastatic castrate-resistant prostate cancer (mCRPC)
Authors: Kohli Manish , Nalla Siva , Kuo Chia-Wei , Hanson Claire , Larsen Matt , Neibling Anna , Lloyd Jennifer , Batten Julia , Agarwal Neeraj , Swami Umang , Maughan Benjamin , Gupta Sumati , Smith Rebecca L. , Smith Andrew 
Published: April 2023

Abstract
Background: Plasma microRNA (miR)-375 and miR-1290 levels have been associated with poor survival and resistance to docetaxel chemotherapy. Detection of plasma miRs is typically performed using qRT-PCR, which is time-intensive and laborious. We developed a novel technique, SiM-Flow, that rapidly and accurately quantifies miRs from low blood volumes (~100 uL). SiM-Flow uses a fluorescence-based flow cytometer to digitally count nucleic acids that have been extended and fluorescently tagged. Our goal was to compare plasma miR detection using SiM-Flow with qRT-PCR assays and determine clinical outcomes in mCRPC stage.
Methods: Forty mCRPC patients undergoing state-specific treatment were enrolled prospectively. Uniform processing of plasma was performed on patient blood samples. Total RNA was extracted from 100 µL of plasma and qRT-PCR was performed using TaqMan miRNA assays for targets miR-375 and miR-1290, endogenous controls miR-30a-5p and miR-16, and exogenous control cel-miR-39. SiM-Flow was conducted using a commercial flow cytometer to count fluorescent barcoded rolling-circle-amplification-extended miR nanoparticles for the same targets. Target miR-375 and miR-1290 were normalized using the geometric means of endogenous controls miR-30a-5p, miR-16, and miR-39 and compared across the two assays by Spearman’s rank correlation analysis. Survival analysis was calculated from date of developing mCRPC to death using Cox Proportional Hazard Regression (CPHR) for association with mean of the miR targets normalized by miR-16.
Results: qRT-PCR assays were optimized to detect native targets, endogenous controls, and spike-in miRs from plasma with ≤100 aM limit of detection (LOD), while SiM-Flow was able to detect miRs from plasma with ≤10 aM LOD. After normalization, target miRs in plasma were detected in 26/40 mCRPC patients by qRT-PCR and in 31/40 patients by SiM-Flow. The Spearman Rho for normalized miR-375 was 0.738 and normalized miR-1290 was 0.130. Survival analysis showed a hazard ratio of 1.03 (95% CI 1.01, 1.05) for the mean of miR-375.
Conclusion: Target miR-375 levels detected with SiM-Flow had higher with qRT-PCR, at lower LOD for SiM-Flow. Mean Normalized miR-375 target levels also were associated with poor prognosis in mCRPC.
Citation Format: Manish Kohli, Siva Nalla, Chia-Wei Kuo, Claire Hanson, Matt Larsen, Anna Neibling, Jennifer Lloyd, Julia Batten, Neeraj Agarwal, Umang Swami, Benjamin Maughan, Sumati Gupta, Rebecca L. Smith, Andrew Smith. Comparison of Single-Molecule Flow (SiM-Flow) and qRT-PCR for detection of plasma miR-375 and miR-1290 in metastatic castrate-resistant prostate cancer (mCRPC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4317.