Digital profiling of tumor-derived extracellular vesicle-RNA: A sensitive approach for early detection of EGFR mutations in osimertinib-resistant non-small cell lung cancer.

e20076 Background: Osimertinib resistance in EGFR-mutant non-small cell lung cancer (NSCLC) patients presents a significant challenge due to limited post-osimertinib treatment options. The C797S mutation is a common tertiary EGFR mutation conferring osimertinib resistance. Current diagnostic methods lack sensitivity for early detection of C797S and T790M mutations, necessitating novel approaches. This study explores the use of extracellular vesicle RNA (EV-RNA) from plasma as a diagnostic tool, utilizing a new assay that facilitates membrane fusion between EVs and liposomes. Methods: The study introduces a digital EV-RNA profiling assay (LiquiDyne) that employs charge-mediated fusion between extracellular vesicles and charged liposomes (CLIPs) carrying molecular beacons. This process occurs on a droplet microfluidic chip, where the surface charge of CLIPs is optimized for fusion efficiency. The assay was validated analytically using EVs from NSCLC cell lines (H1975, SL777) with known EGFR mutations (L858R, T790M, C797S). Clinical validation involved testing retrospective patient samples with a history of osimertinib treatment. Results: The assay requires only 20 µL of plasma, eliminating the need for prior EV isolation or RNA preparation, thus minimizing sample loss. It successfully detected EGFR mutations using a digital EV-RNA profiling approach, enhanced by an artificial intelligence algorithm, in both cell lines and patient samples with high accuracy, demonstrating its potential as a reliable diagnostic tool. Conclusions: This innovative digital assay presents a groundbreaking method for early detection of acquired EGFR mutations, enabling precise quantification of rare EV subpopulations. This approach demonstrates significant potential beyond initial diagnosis, offering a powerful tool for monitoring minimal residual disease and treatment response. By addressing critical limitations in current NSCLC diagnostics, this digital assay could substantially enhance patient care through more timely and accurate molecular profiling, potentially leading to improved treatment strategies and outcomes in EGFR-mutant NSCLC.