Abstract A020: Enhanced cell-free RNA (cfRNA) recovery for liquid biopsy applications: Comparative analysis using controlled RNA samples for early and advanced cancer detection
Authors: Jafari Nafiseh , Saidian Mayer , Saenz Jason , Dunnigan Andrew , Hernandez Carlos , Aghili Leila 
Published: November 2024

Abstract
Introduction: Extracting cfRNA from biological samples is crucial in liquid biopsy applications, particularly for detecting low-frequency mutations like KRAS G12V, essential for early cancer diagnosis and minimal residual disease (MRD) monitoring. Traditional cfRNA extraction methods involve multiple transfer steps, aliquoting, and pooling, leading to cfRNA loss and affecting mutation detection sensitivity and accuracy. The nRichDX Revolution cfTNA Max 20 kit, with its innovative nRicher cartridge, simplifies the extraction process by eliminating these extra steps, potentially enhancing yield and efficiency. This study evaluates the cfRNA recovery efficiency of the nRichDX kit compared to the Qiagen Circulating Total Nucleic Acid kit. Methods: Plasma samples (5 mL each) were spiked with RNA containing KRAS G12V mutation at concentrations ranging from 100 to 2 million copies, mimicking early-stage cancer detection and MRD monitoring, where low copy numbers represent early disease and high copy numbers reflect advanced cancer stages. cfRNA was extracted using the nRichDX and Qiagen kits, with each condition tested in triplicate. cfRNA recovery efficiency was assessed using quantitative reverse transcription PCR (qRT-PCR) to quantify KRAS G12V RNA copies recovered. High-sensitivity RNA ScreenTape Analysis was also used to evaluate cfRNA profile quality. Results: The nRichDX kit recovered significantly higher cfRNA yields than Qiagen across all concentrations. Recovery rates were consistent and reproducible, with over 70% of input RNA recovered, even at lower inputs (100 to 100,000 copies), where recovery rates were approximately 70-85%. High Sensitivity RNA ScreenTape Analysis showed a linear increase in 18S and 28S ribosomal peaks and high RNA Integrity Number (RIN) scores in nRichDX profiles. The ability to robustly recover low copy numbers is significant for early cancer detection and MRD monitoring. In contrast, the kit’s effectiveness in high copy number samples reflects its ability to monitor advanced cancer stages accurately. Conclusion: The nRichDX cfTNA kit demonstrates exceptional efficiency in cfRNA extraction from plasma samples across a broad dynamic range, from as few as 100 copies to as many as 2 million copies of the KRAS G12V mutation. Its versatility makes it suitable for various cancer detection and monitoring applications. The streamlined process offered by the nRicher cartridge minimizes sample handling and reduces cfRNA loss, enhancing performance. This translates to more accurate and sensitive detection of early-stage and high-burden mutations associated with advanced cancer. The high-quality profiles indicated by RNA ScreenTape Analysis further validate the reliability of the nRichDX kit, making it a valuable tool for liquid biopsy applications, with significant advantages in early cancer detection and monitoring disease progression.
Citation Format: Nafiseh Jafari, Mayer Saidian, Jason Saenz, Andrew Dunnigan, Carlos Hernandez, Leila Aghili. Enhanced cell-free RNA (cfRNA) recovery for liquid biopsy applications: Comparative analysis using controlled RNA samples for early and advanced cancer detection [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A020.