Soluble N-terminal EphA2 fragment as a diagnostic biomarker and functional mediator of tumor progression in triple-negative breast cancer

Triple-negative breast cancer (TNBC) lacks reliable biomarkers for early detection and remains a highly aggressive disease. Ephrin type-A receptor 2 (EphA2), a receptor tyrosine kinase frequently overexpressed in advanced cancers, undergoes MMP14-mediated cleavage that releases soluble N-terminal fragments. Here, we investigated the diagnostic and functional significance of this fragment in TNBC. Immunohistochemical analysis of clinical specimens revealed elevated EphA2 expression in TNBC tissues, with a trend toward higher levels in tumors of advanced stage and grade. To enable non-invasive detection, we established a dual-epitope sandwich ELISA targeting spatially distinct regions of the EphA2 extracellular domain. The assay showed concentration-dependent detection of recombinant EphA2 (EC₅₀, 48 ng/mL) and detected soluble EphA2 in TNBC cell-conditioned media. Serum analysis showed significantly higher EphA2 levels in TNBC patients compared with healthy controls (median 93.3 vs 25.6 ng/mL). In this exploratory cohort, ROC analysis yielded an AUC of 0.912 (95% CI, 0.862-0.962), with a sensitivity of 95.0% (95% CI, 86.3-98.6%) and a specificity of 78.3% (95% CI, 66.4-86.9%), and higher levels were associated with lymph node metastasis and higher histological grade. Functionally, recombinant EphA2-P328 promoted TNBC-cell migration and induced cell context-dependent changes in EMT-associated markers in vitro. Collectively, these findings support the soluble N-terminal EphA2 fragment as a candidate serum biomarker and an in vitro modulator of TNBC cell migration, warranting validation in larger independent cohorts and further mechanistic investigation.