Optimized size exclusion chromatography demonstrates that extracellular vesicles are the key RNA carriers of ALK translocations in non-small cell lung cancer cell line secretome and patient plasma.

Identification of fusions in non-small cell lung cancer (NSCLC) is key to determining eligibility for treatment with ALK inhibitors that markedly improve patients' quality of life and survival outcomes. Circulating RNA, associated with various carriers including extracellular vesicles (EVs), lipoproteins (LPPs), or protein complexes, presents a viable target for the identification of ALK fusions by liquid biopsy. Our aim was to characterize the specific carrier of fusion RNA, a crucial step in the development of diagnostic methods for clinical use. We employed optimized size-exclusion chromatography (SEC) to separate EVs, LPPs, and protein-enriched fractions from -positive NSCLC cell lines and from pools of plasma obtained from NSCLC patients with translocations. We optimized RNA fusion transcript detection using digital PCR (dPCR). Protein analyses confirmed the successful resolution of EVs, LPPs, and protein fractions by optimized SEC. Our dPCR results indicated that fusions were more prevalent in tetraspanin-enriched SEC fractions from NSCLC cell lines, suggesting that EVs serve as the primary carrier for fusion RNA. After optimization for larger volumes of samples of the RNA isolation protocol, we could also demonstrate that fusion transcripts were found exclusively in EVs from patient plasma. Of note, the circulating number of copies of the transcript was below 5 copies/mL. Our findings underscore the potential of EV-associated RNA as a promising source for detecting fusion variants in plasma samples from NSCLC patients, offering a non-invasive diagnostic approach with significant clinical implications.