Exploratory analysis of extracellular vesicle Profile changes during pembrolizumab treatment in NSCLC

Predictive biomarkers for pembrolizumab resistance and durable clinical benefit in advanced non-small cell lung cancer (NSCLC) remain insufficient.

Serum extracellular vesicles (EVs) were isolated from 98 patients with advanced NSCLC treated with pembrolizumab monotherapy. Isolation was performed using CD9/CD63 antibodies, and EV proteins were identified using liquid chromatography-tandem mass spectrometry. Patients were classified into primary resistance and clinical benefit groups; patients with clinical benefit were further divided into acquired resistance and long-term responder groups. Differential protein abundance, pathway enrichment, progression-free survival (PFS), and receiver operating characteristic (ROC) analyses were performed.

The cohort included 40 patients with primary resistance and 58 with clinical benefit. Proteomic analysis identified 305 differentially abundant EV proteins between these groups. Proteins enriched in primary resistance were associated with extracellular matrix organization, adhesion, complement activation, and chemotaxis, whereas response-enriched proteins were related to mitochondrial metabolism, fatty acid oxidation, and vesicle/lysosome-associated transport. TNXB and SMIM1 showed moderate discriminatory performance for primary resistance, with area under the curve (AUC) values of 0.712 and 0.706, respectively. A four-protein panel comprising TNXB, KRT12, TFPI2, and HLA-E improved discrimination between primary resistance and clinical benefit, achieving an AUC of 0.788. Among patients with clinical benefit, ITLN1, ABCG5, NPC1L1, MME, and NOP2 distinguished acquired resistance from long-term response, and a four-protein panel comprising ITLN1, ABCG5, NPC1L1, and NOP2 achieved an AUC of 0.808.

Circulating EV proteomic analysis identified candidate biomarkers for pembrolizumab resistance and durable clinical benefit in advanced NSCLC.