Soluble immune checkpoints as diagnostic biomarkers in bladder cancer

Bladder cancer (BC) is a common malignancy with high recurrence and substantial monitoring costs. Limitations of current diagnostic tools: cystoscopy is invasive and expensive, urine cytology lacks sensitivity and existing urine biomarkers cannot replace cystoscopy, underscoring the need for improved non-invasive methods. Immune checkpoints (ICs) regulate immune activity, and newer ICs: T-cell immunoglobulin and mucin domain 3 (TIM-3), galectin-9 (Gal-9), B- and T-lymphocyte attenuator (BTLA), herpesvirus entry mediator (HVEM), cluster of differentiation 160 (CD160), and lymphocyte-activation gene 3 (LAG-3), are involved in cancer immune evasion. This study demonstrated that levels of soluble ICs (sICs) are markedly elevated in patients with early-stage BC. Serum levels of sTIM-3, sGal-9, sBTLA, sHVEM, sCD160, and sLAG-3 were quantified in BC and controls. sTIM-3, sGal-9, and sBTLA were significantly elevated in BC, while their concentrations didn’t vary by tumor stage or grade, supporting diagnostic rather than prognostic utility. Whereas sHVEM, sCD160, and sLAG-3 showed no diagnostic value. Age, sex, and body mass index (BMI) had minimal influence on sIC levels. Receiver operating characteristic (ROC) analyses showed strong performance: sGal-9 was the best classifier (area under the curve (AUC) = 0.98; 91% sensitivity; 100% specificity), followed by sTIM-3 (AUC = 0.91) and sBTLA (AUC = 0.78). sHVEM, sCD160, sLAG-3 performed poorly. A multivariate model incorporating sGal-9, sTIM-3, and sBTLA achieved a cross-validated AUC = 0.982, with sGal-9 remaining independently predictive. Correlation analyses revealed two clusters: sGal-9/sTIM-3/sBTLA and sHVEM/sCD160, that relationships align with known receptor-ligand biology and may reflect shedding during T-cell exhaustion. Overall, sGal-9, supported by sTIM-3 and sBTLA, constitutes promising serum biomarker panel associated with early-stage BC, however, as these markers are not disease-specific and may be affected by inflammatory or other pathological conditions, further validation is required.