Selective modulation of immune transcripts in extracellular vesicles from plasma of renal cell carcinoma patients receiving nivolumab

719 Background: Patients (pts) displaying a full-fledged immune response show high levels of circulating extracellular vesicles (EVs) containing immune transcripts that might be transferred to neighboring cells to amplify immune responses, as in autoimmunity and transplantation. We hypothesized EV immune transcripts could reflect immune activation in cancer pts receiving immunotherapy with immune checkpoint inhibitors (ICI). These RNA-containing EVs may surrogate immune functionality and represent a plasma transcriptional signature of adaptive immunity that can be readily detected by liquid biopsy. Methods: Blood samples from metastatic renal cell cancer pts,treated with ICI nivolumab (n=8) or tyrosine kinase inhibitor cabozantinib (n=9) were collected at baseline, week 4 and 12. EV-RNA was isolated from plasma with membrane affinity spin columns and evaluated for CD274(PD-L1), IFNG, PDCD1 (PD-1), CD3 and GZMB (granzyme B) transcripts by qRT-PCR. Results: All pts showed a statistically significant increase of CD3, IFNG, GZMB, PD-1 and PD-L1 immune transcripts in plasma EVs, evidenced at 4 and 12 weeks of therapy. Treatment subgroup analysis revealed an upregulation of CD3, IFNG, GZMB and PD-1 transcripts only in plasma EVs of patients receiving nivolumab that was more evident in those showing clinical benefit (4/8), while PD-L1 increased significantly during cabozantinib treatment. Upon comparing transcripts with soluble proteins, PD-L1 displayed similar kinetics, while PD-1 increased only as transcript but not as protein, given its potential sequestration by the anti-PD-1 antibody. This effect was detectable only in nivolumab but not in cabozantinib treated pts. Conclusions: Monitoring immune transcripts carried by plasma EVs holds promise as potential tool to assess the entity of the ongoing immune activation during immunotherapy. Plasma EVs may be exploited as indicators of immune response and help to predict clinical efficacy at early on-treatment time points. This approach based on plasma EVs might represent a starting point for the development of a novel strategy to study immune responses in clinical setting.