Combining circulating tumor cells and tumor-derived extracellular vesicles for prognosis stratification in patients with metastatic breast cancer

Background: Treatment strategies for patients with metastatic breast cancer (MBC) are becoming increasingly complex, underscoring the need for reliable biomarkers to inform clinical decision making. Circulating tumor cells (CTCs) are well-established prognostic biomarkers in MBC, while tumor-derived extracellular vesicles (tdEVs) have recently demonstrated complementary significance in patients undergoing first-line chemotherapy. This study seeks to validate the complementary role of tdEVs in a larger patient population and establish a clinically relevant cut-off. Materials and methods: Archived baseline CellSearch® images from 355 patients with MBC (NU16B06 study) were reanalyzed using the Automated CTC Classification, Enumeration and PhenoTyping (ACCEPT) software to quantify CTCs and tdEVs. Multiple tdEV cut-offs were evaluated for association with overall survival (OS), alone and with CTCs (≥5, CTChigh). The selected cut-off was validated in a subcohort of 385 patients from the phase III STIC CTC trial. Results: In the NU16B06 cohort, higher tdEV levels were consistently associated with shorter OS, regardless of cut-off. As models performed similarly (Brier Score difference <0.005), a cut-off of >20 tdEVs/7.5 ml (tdEVhigh) was selected based on prior literature and clinical relevance. Patients with tdEVhigh had nearly doubled risk of death compared with tdEVlow. Combining tdEVs with CTCs identified distinct prognostic subgroups, with CTChigh/tdEVhigh patients showing the shortest OS. Among CTClow patients, tdEVhigh was associated with increased mortality risk. In the STIC CTC cohort, the cut-off was validated for OS and progression-free survival, with tdEVhigh consistently associated with poorer surivial. When combined, patients with CTChigh/tdEVhigh had the poorest outcomes, while tdEVhigh remained associated with worse prognosis among CTClow patients. Conclusion: These findings support the use of tdEVs as complementary prognostic biomarkers to CTCs, potentially enhancing clinical trial design and informing treatment decisions in MBC.