The heterogeneity of bladder cancer (BlCa) prognosis and treatment outcome requires the elucidation of tumors’ molecular background towards personalized patients’ management. tRNA-derived fragments (tRFs), although originally considered as degradation debris, represent a novel class of powerful regulatory non-coding RNAs. In silico analysis of the TCGA-BLCA project highlighted 5′-tRF-LysCTT to be significantly deregulated in bladder tumors, and 5′-tRF-LysCTT levels were further quantified in our screening cohort of 230 BlCa patients. Recurrence and progression for non-muscle invasive (NMIBC) patients, as well as progression and patient’s death for muscle-invasive (MIBC) patients, were used as clinical endpoint events. TCGA-BLCA were used as validation cohort. Bootstrap analysis was performed for internal validation and the clinical net benefit of 5′-tRF-LysCTT on disease prognosis was assessed by decision curve analysis. Elevated 5′-tRF-LysCTT was associated with unfavorable disease features, and significant higher risk for early progression (multivariate Cox: HR = 2.368; p = 0.033) and poor survival (multivariate Cox: HR = 2.151; p = 0.032) of NMIBC and MIBC patients, respectively. Multivariate models integrating 5′-tRF-LysCTT with disease established markers resulted in superior risk-stratification specificity and positive prediction of patients’ progression. In conclusion, increased 5′-tRF-LysCTT levels were strongly associated with adverse disease outcome and improved BlCa patients’ prognostication.

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Background Tumor heterogeneity and lack of personalized prognosis leads to bladder cancer (BlCa) patients’ lifelong surveillance with invasive interventions, highlighting the need for modern minimally invasive tools for disease management. Herein, we have evaluated the clinical utility of preoperative serum cell-free DNA (cfDNA) in ameliorating patients’ risk-stratification and prognosis. Methods cfDNA was purified from 190 preoperative BlCa patients and 26 healthy individuals’ serum samples and quantified by 2 assays: an in-house quantitative real-time PCR (qPCR) assay using LEP as reference control and a direct fluorometric assay using Qubit HS dsDNA. Capillary electrophoresis was performed in 31 samples for cfDNA fragment profiling. Tumor relapse/progression and metastasis/death were used as clinical endpoints for non-muscle-invasive bladder cancer and muscle-invasive bladder cancer (MIBC), respectively. Results cfDNA profiling by capillary electrophoresis highlighted that total and fragment-related cfDNA levels were significantly increased in BlCa and associated with advance disease stages. Evaluation of cfDNA levels by both Qubit/qPCR displayed highly consistent results (rs = 0.960; P < 0.001). Higher cfDNA was correlated with MIBC and stronger risk for early metastasis (Qubit:hazard ratio [HR] = 3.016, P = 0.009; qPCR:HR = 2.918, P = 0.004) and poor survival (Qubit:HR = 1.898, P = 0.042; qPCR:HR = 1.888, P = 0.026) of MIBC patients. Multivariate cfDNA-fitted models led to superior risk stratification and net benefit for MIBC prognosis compared to disease established markers. Conclusions Elevated preoperative cfDNA levels are strongly associated with higher risk for short-term metastasis and poor outcome of MIBC, supporting modern noninvasive disease prognosis and management.