Clinical effectiveness of a multitarget urine DNA test for urothelial carcinoma detection: a double-blinded, multicenter, prospective trial

Abstract

Urine-based testing is promising for noninvasive diagnosis of urothelial carcinoma (UC) but has suboptimal sensitivity for early-stage tumors. Herein, we developed a multitarget urine tumor DNA test, UI-Seek, for UC detection and evaluated its clinical feasibility. The prediction model was developed in a retrospective cohort (n = 382), integrating assays for FGFR3 and TERT mutations and aberrant ONECUT2 and VIM methylation to generate a UC-score. The test performance was validated in a double-blinded, multicenter, prospective trial (n = 947; ChiCTR2300076543) and demonstrated a sensitivity of 91.37% and a specificity of 95.09%. The sensitivity reached 75.81% for low-grade Ta tumors and exceeded 93% in high-grade Ta and higher stages (T1 to T4). Simultaneous identification of both bladder and upper urinary tract tumors was enabled with sensitivities exceeding 90%. No significant confounding effects were observed regarding benign urological diseases or non-UC malignancies. The test showed improved sensitivities over urine cytology, the NMP22 test, and UroVysion FISH alongside comparable specificities. The single-target accuracy was greater than 98% as confirmed by Sanger sequencing. Post-surgery UC-score decreased in 97.7% of subjects. Overall, UI-Seek demonstrated robust performance and considerable potential for the early detection of UC.

Keywords: Cancer biomarkers; Early detection; Liquid biopsy; Molecular diagnosis; Urine tumor DNA; Urothelial carcinoma.

Fig. 1

Flow diagram of algorithm development and clinical validation of the mt-utDNA test. A total of 1,329 participants were enrolled in this study. The algorithm development cohort included 382 individuals involved in the construction of the integrated prediction model and the determination of appropriate clinical cutoffs of the mt-utDNA test. The performance of the test was validated in the prospective validation cohort (n = 947) for UC detection according to the clinical diagnosis results. Sanger sequencing and postsurgical follow-up were performed to verify the results of the test. UC: urothelial carcinoma; mt-utDNA test: multitarget urine tumor DNA test

Fig. 2

Performance of the mt-utDNA test in the clinical validation study. (A) Distribution of the UC-scores across pathological groups. The median values are depicted as black dots with the line range of the interquartile range. Statistical significance was assessed by the Wilcoxon rank sum test between the UCs and non-UCs. “benign” represented the urological benign disease group and “non-UC” represented the non-UC cancer group. (B) and (C) ROC curve plots of the mt-utDNA test for distinguishing UC patients from non-UC individuals, including those with benign urologic diseases or non-UC cancers (B), or from the subgroup of patients with benign urologic diseases (C). (D) Sensitivity of the mt-utDNA test for the indicated stage (left) or location (right) of UC tumors. Tumors of Ta stage were stratified as Ta-PUNLMP, Ta-LG and Ta-HG. (E) Specificity of the mt-utDNA test for the indicated types of benign urologic diseases (left) and non-UC cancers (right). (F) Sensitivity of the mt-utDNA test in UC patients with the indicated stage (left), grade (center), and location (right), in comparison with urine cytology. Statistical significance was assessed by Pearson’s Chi-squared test. (G) Specificity of the mt-utDNA test in non-UC participants with the indicated type of benign urologic diseases or non-UC cancers, in comparison with urine cytology. Statistical significance was assessed by Pearson’s Chi-squared test. Tumors combined with carcinoma in situ (CIS) were defined as Tis; UC, urothelial carcinoma; PUNLMP, papillary urothelial neoplasm of low malignant potential; LG, low grade; HG, high grade; AUC, area under the ROC curve; CI, confidence interval; BCa, bladder cancer; UTUC, upper tract urothelial carcinoma; *P < 0.05; **P < 0.01; ***P < 0.001; n.s., no significance