Flow-cytometric profiling of large extracellular vesicles as immunophenotypic biomarkers in head and neck squamous cell carcinoma

BACKGROUND: Large extracellular vesicles (large EV) released from tumor and benign cells are detectable in blood and hold potential as non-invasive biomarkers. While their diagnostic relevance has been shown in several malignancies, their role in head and neck squamous cell carcinoma (HNSCC) remains insufficiently characterized. METHODS: Large EV were isolated from peripheral blood of patients with HNSCC (n = 66), non-small cell lung cancer (NSCLC; n = 52; adenocarcinoma n = 39, squamous cell carcinoma (SQCCL) n = 11), and healthy controls (n = 11) by differential centrifugation. Flow cytometry quantified surface expression of EGFR, EPCAM, MUC1, and PD-L1. Associations with clinical parameters were analyzed using t-tests, Spearman correlations, logistic regression, and random forest modeling. RESULTS: HNSCC-derived large EV showed significantly higher EGFR and MUC1, but lower EPCAM expression compared to controls. PD-L1 expression increased with advancing tumor stage and was positively associated with metastatic status, whereas EGFR levels declined in metastatic disease. Combined ROC analysis of EGFR, EPCAM, and PD-L1 yielded an AUC of 0.785 (p = 0.003), distinguishing HNSCC from controls. Comparative profiling revealed higher EGFR and EPCAM expression in HNSCC versus NSCLC, while MUC1 predominated in NSCLC, particularly in SQCCL; notably, the NSCLC cohort was predominantly adenocarcinoma. A marker panel comprising EGFR, EPCAM, and MUC1 differentiated HNSCC from SQCCL in this limited subgroup (n = 11) with 96.97% sensitivity, 45.45% specificity, 92.75% positive predictive value and 75.00% negative predictive value. CONCLUSION: Flow-cytometric profiling of circulating large EV provides a feasible liquid biopsy approach for tumor characterization in HNSCC. PD-L1 expression reflects tumor burden, and combined large EV marker analysis enables differentiation between HNSCC and primary squamous lung carcinoma, supporting its diagnostic utility in clinical oncology.