We hypothesize that the urinary immune checkpoint protein profile differs across various stages of bladder cancer and healthy individuals. This study aims to characterize this profile in bladder tumors predominantly subjected to different intravesical therapies and to compare its expression in recurrent bladder tumors with that in healthy controls.

A cohort comprising 30 patients diagnosed with bladder cancer, referred from the Urology Division of the Tri-Service General Hospital (TSGH), and 20 healthy control subjects aged 20 years or older, was recruited for this study. Urine samples were collected, processed, and preserved for subsequent analysis. Concurrent demographic data and blood biochemical parameters were recorded. Quantitative measurements of immune checkpoint proteins, including IDO, PD-1, PD-L1, PD-L2, TIM-3, CD27, CD28, CD80, CD135, and CD137, were performed using ProcartaPlex® Multiplex Immunoassays. Statistical analyses were carried out employing Dunn's multiple comparison test, with significance defined as P<0.05. The study protocol was approved by the Joint Institutional Review Board of the Tri-Service General Hospital (TSGH IRB No. 2-106-05-077).

Significant differences in the quantitative measurement of immune checkpoint proteins were observed, including CD137, CD28, and TIM-3 (P<0.01), IDO and PD-L1 (P<0.05), as well as CD27 (P=0.000). Furthermore, PD-L2 expression was elevated compared to the control group, warranting further investigation into potential influencing factors such as medication, therapeutic strategies, and disease status. Urinary proteomic analyses demonstrated notable associations between IDO, PD-L1, TIM-3, CD28, CD80, CD137, CD27, CD152, and bladder cancer prognosis. Correlation analysis revealed a negative correlation between CD28 and CD137 (P=0.045). This preliminary study is limited by a small sample size and high heterogeneity among subjects, necessitating further research to confirm its validity.

Urine represents a highly promising biological fluid for biomarker research, attributed to its noninvasive collection method and anatomical proximity to bladder tumors. PD-L2, akin to PD-L1, is hypothesized to contribute to the regulation of T cell-mediated immune responses and immune tolerance, although its precise functional role remains a topic of ongoing debate. To optimize the application of immune checkpoint inhibitors, further investigation is warranted into the prognostic significance of biomarkers in the management of bladder cancer, particularly the potential integration of urinary protein profiling.