Because there was an association be

Because there was an association between age and the prevalence of mutation in the UTUC cohort, the difference in age between the UTUC and he- maturia cohorts could have influenced the positive rate of detecting mutant cfDNA. However, there are no reports of an increased rate of TERT promoter mutation in healthy elderly compared with healthy young persons. Further prospective large-scale studies and a longer follow-up period are warranted to confirm our findings. TP53 is well known as one of the key players in carcinogenesis and is frequently mutated in UC. Although our urinary cfDNA ddPCR assay targeting three hotspot mutations has low sensitivity for detecting UTUC, mas- sive parallel sequencing of multiple genes could be applicable to the analysis of urinary cfDNA and might increase sensitivity, thus provid- ing more benefit to patients.16 In combination with the results of cy- tology, our assay for urinary cfDNA may lead to an earlier diagnosis of UTUC and optimized follow-up strategy and may help the physician avoid an invasive procedure to determine a definitive diagnosis. In conclusion, we could detect TERT promoter and FGFR3 hotspot muta- tions in urinary cfDNA from UTUC patients. In combination with cytology results, the sensitivity of our non-invasive urinary test was high enough to apply this assay to the clinical setting. Liquid biopsy analy- sis of TERT promoter and FGFR3 mutations in urinary cfDNA could be a novel diagnostic biomarker and a reliable factor for staging UTUC.