On-treatment dynamics of circulating extracellular vesicles (EVs) in the first-line setting of patients with advanced non-small cell lung cancer (NSCLC): the LEXOVE prospective study

Abstract

Introduction: The evaluation of extracellular vesicles (EVs) might be a complementary tool to assess response in the clinic. We aimed to describe whether the serial characterization of EVs could longitudinally reflect response and resistance to first-line treatments in advanced NSCLC. Methods Treatment-naïve patients with advanced NSCLC receiving osimertinib (osi), alectinib, pembrolizumab (pembro), or platinum-based chemotherapy (CT) ± pembro were prospectively enrolled at the University Hospital of Palermo, Italy. Isolated EVs were characterized by Static and Dynamic Light Scattering (DLS) to assess the size distribution and amount of vesicles (R90, Dz and PDI). EV protein amount was evaluated by Bradford assay (BA) through the quantification of circulating cell-free EV protein content (cfEV). According to the radiologic response, cfEV and R90 kinetics were evaluated in patients from baseline (T0) to the first radiologic restaging (T1) with a 20% cfEV increase being used as the cut-off point for median progression-free survival (mPFS) analysis. Results Among 27 consecutive patients, a total of 135 plasma samples were collected both at T0 and T1 to isolate EVs. Purified EVs were characterized by WB for ALIX and TSG-101. EV size was determined by DLS showing an average size ranging from 183 to 260 nm. The mean cfEV value at T0 and at T1 time was 1.26 and 1.49 µg/ml, respectively (p = 0.02). Within the cfEV responsive group, 13 patients had a clinically improved mPFS (25.2 months, 95% CI: 14.9–35.5) when compared to 11 cfEV non-responders (8.3 months, 95% CI: 3.6–12.9) (p = 0.07). Namely, cfEV responders receiving single-agent pembro experienced a significantly improved mPFS (25.2 months, 95% CI: 11.7–38.8; p = 0.04) compared to patients receiving CT plus pembro (6.1 months, 95% CI: 1.1–11.1; p = 0.9). EGFR-positive cfEV responders showed a clinically improved mPFS (35.1 months, 95% CI: 14.9–35.5) as compared to cfEV non-responders (20.8 months, 95% CI: 11.2–30.4) (p = 0.06). In the EGFR-mutated subgroup, four patients with R90 decreasing values are still responding whereas one patient with R90 increasing value had a rapidly progressive disease. Conclusions This study showcased the feasibility of the serial on-treatment monitoring of plasma EVs in the first-line setting of NSCLC, mostly in those patients receiving single-agent pembro or osi. The increased amount of circulating EVs (R90) and the higher level of associated proteins (cfEV) warrant larger controlled studies to explore EVs as novel promising liquid biopsy biomarkers.