Urinary and Serum ENOX2 As a Diagnostic Biomarker for Non-Muscle-Invasive Bladder Cancer: An Original Investigation

Bladder cancer (BCa) diagnosis and surveillance rely heavily on invasive cystoscopy and urine cytology, which has limited sensitivity, underscoring the need for reliable non-invasive biomarkers. ENOX2 (tumor-associated NOX) is a candidate pan-cancer biomarker shed from tumor cell membranes into biological fluids, but its utility in BCa remains unexplored. This prospective pilot study enrolled 33 non-muscle-invasive bladder cancer (NMIBC) cases, 32 non-cancer controls for the primary analysis, and 38 other cancer controls. Serum and urinary ENOX2 were measured by ELISA, and ENOX2 gene expression by qPCR. Diagnostic performance was assessed using receiver operating characteristic (ROC) curve analysis and the area under the curve (AUC). The urinary ENOX2/creatinine ratio was significantly higher in NMIBC cases than controls (median 17.95 vs 0.74 ng/mg, p = 0.018), achieving the highest diagnostic performance among studied markers (AUC 0.670, with sensitivity 53% and specificity 91% at a cutoff of ≥17.85 ng/mg), while serum ENOX2 and qPCR expression showed no diagnostic utility (AUC 0.435 and 0.417, respectively). Further exploratory analyses showed that the urinary ENOX2/creatinine ratio demonstrated comparable AUC across low- and high-grade tumors and correlated with tumor size. Additionally, the levels did not vary across benign urological conditions. The urinary ENOX2/creatinine ratio demonstrates modest but statistically significant diagnostic utility in NMIBC, outperforming serum ENOX2 measures in this pilot study. Further evaluation of ENOX2 in larger, multicenter cohorts, including its potential role within multimarker diagnostic panels, is recommended.