Circulating extracellular vesicles carrying PD-1, PD-L1 and CTLA-4 inform resistance to anti-PD-L1-based therapy in HCC

BACKGROUND: First-line immune checkpoint inhibitor (ICI) therapy has contributed to improved outcomes in hepatocellular carcinoma (HCC). However, only around 30% of patients respond, highlighting the need for biomarker-driven patient selection. Considering the lack of tissue acquirement in HCC in clinical routine, liquid biopsy holds great promise. OBJECTIVE: We aimed at profiling immune checkpoints (IC) on circulating extracellular vesicles (EVs) as potential liquid biopsy-based biomarkers for prediction of treatment response. DESIGN: Three distinct cohorts were analysed in this study: (1) HCC explorer cohort (n=40), testing the presence of membrane-bound ICs on EVs; (2) early-stage HCC cohort (n=37 with paired blood and tissue), assessing the interplay between EV and tissue ICs alongside clinicopathological parameters and (3) treatment cohort (n=202 with 600 sequential blood samples), comprising an ICI treated cohort with a training (n=79, n=402 sequential samples) and a validation group (n=82, n=146 sequential samples), alongside a tyrosine-kinase inhibitor (TKI) treated cohort (n=41, n=52 sequential samples), to identify predictors of immunotherapy response through IC profiling of circulating EVs via multiplex immunoassay. RESULTS: Programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) were enriched in EV fractions compared to EV-depleted serum. Baseline and early dynamics of EV-IC levels significantly discriminated between responders and non-responders in the ICI-treated training and validation cohorts, while simultaneously predicting progression-free survival (PFS) and overall survival (OS). These findings were not reproducible in the TKI-treated cohort. In addition, dynamic changes of EV-IC levels during therapy in patients with initial response predicted acquired therapeutic resistance, approximately 36-42 weeks before progression was traceable on imaging. CONCLUSION: EVs carrying PD-1, PD-L1 and CTLA-4 represent readily quantifiable, non-invasive biomarkers to predict response and survival in patients with advanced HCC undergoing ICI therapy and serve to identify biological ICI resistance much earlier than current standards with imaging.