Unveiling mutational dynamics in non-small cell lung cancer patients by quantitative EGFR profiling in vesicular RNA

The mutational status of the epidermal growth factor receptor (EGFR) guides the stratification of non-small cell lung cancer (NSCLC) patients for treatment with tyrosine kinase inhibitors (TKIs). A liquid biopsy test on cell-free DNA is recommended as a clinical decision-supporting tool, although it has limited sensitivity. Here, we comparatively investigated the extracellular vesicle (EV)-RNA as an independent source for multidimensional and longitudinal EGFR profiling in a cohort of 27 NSCLC patients. We introduced and validated a new rapid, highly specific EV-RNA test with wild-type (WT) and mutant-sensitive probes (E746-A750del, L858R, and T790M). We included a cohort of 20 NSCLC patients with EGFR WT tumor tissues and systematically performed molecular EV-RNA and circulating tumor DNA analyses with clinical data statistics and biophysical profiles of EVs. At the single-patient level, we detected variegated tumor heterogeneity dynamics supported by combinations of driver EGFR mutations. EV-RNA-based mutation analysis showed an unprecedented sensitivity of over 90%. The resistance-associated mutation T790M frequently pre-existed at baseline with a gained EV-transcript copy number at progression, while the general mutational burden was mostly decreasing during the intermediate follow-up. The biophysical profile of EVs and the quantitative assessment of T790M revealed an association with tumor size determined by the sum of the longest diameters in target lesions. Vesicular RNA provides a validated tool suitable for use in clinical practice to investigate the dynamics of common driver EGFR mutations in NSCLC patients receiving TKIs.