Bladder cancer detection in urine using DNA methylation markers: a technical and prospective preclinical validation.

BACKGROUND: The development of accurate urinary biomarkers for non-invasive and cost-effective detection of primary and recurrent bladder tumours is recognized as one of the major clinical needs in bladder cancer diagnostics. The purposes of this study were (1) to validate the results of a previous technical comparison by determining the diagnostic performance of nine methylation markers in urine pellet compared to full void urine, and (2) to validate the diagnostic performance of the optimal marker panel GHSR/MAL from a previous exploratory study in a preclinical setting. METHODS: Urine samples of 108 patients with bladder cancer and 100 age- and gender-matched controls were prospectively collected for methylation analysis. Urinary methylation levels of the markers FAM19A4, GHSR, MAL, miR-129, miR-935, PHACTR3, PRDM14, SST and ZIC1 were determined with quantitative methylation-specific PCR in urine pellet. Area under the curves (AUCs) were determined for individual markers and the marker panel GHSR/MAL. The diagnostic performance of the marker panel GHSR/MAL was evaluated in the total study population and in different subgroups of patients with bladder cancer using the Chi-square test. The diagnostic accuracy was assessed by leave-one-out cross-validation. RESULTS: All nine urinary methylation markers (FAM19A4, GHSR, MAL, miR-129, miR-935, PHACTR3, PRDM14, SST and ZIC1) showed significantly higher methylation levels in bladder cancer patients than in controls (p < 0.001). Area under the curves (AUCs) of the nine methylation markers tested in urine pellet were similar to AUCs in full void urine of an independent previous cohort. GHSR/MAL reached an AUC of 0.89 (95% confidence interval [CI] 0.84-0.94), at 80% sensitivity and 93% specificity. Sensitivity of GHSR/MAL increased with higher tumour grades, higher tumour stages, in primary vs. recurrent tumours, and in males vs. females. CONCLUSIONS: This technical validation supports the robustness of DNA methylation analysis in urine pellet and full void urine for the non-invasive detection of bladder cancer. Subsequent preclinical validation confirmed the diagnostic potential of GHSR/MAL. These findings underline the diagnostic potential of the marker panel GHSR/MAL for future bladder cancer diagnostics.

Inter- and Intraobserver Agreement in Measuring Urolithiasis Density on Nonenhanced Computed Tomography.

Introduction: Treatment choice for urolithiasis is partially based on measuring stone density in HU on nonenhanced computed tomography (NECT). Interobserver variability in these measurements could have treatment consequences. This study aims to assess the observer agreement of measuring HU and whether the use of a protocol leads to a better agreement. Materials and Methods: We retrospectively included 155 consecutive NECTs of patients with stones ≥4 mm. Five observers (two radiologists, one urologist, one urology resident, and one radiology resident) assessed all anonymized NECTs four times in randomized order. HU was measured without instruction (rounds 1 and 2) and subsequently using two protocols (A and B, rounds 3 and 4). Protocols comprised using bone setting, zoom, and measuring HU without the penumbra, in either three (A) or one (B) axial plane. The inter- and intraobserver agreement was evaluated using the intraclass correlation coefficient (ICC). Results: Interobserver agreement on HU measurement without protocol was as follows: ICC = 0.84 (confidence interval [CI]: 0.79-0.87). Agreement diminished with protocol A, ICC = 0.62 (CI: 0.37-0.76), and improved with protocol B, ICC = 0.90 (CI: 0.86-0.92). Intraobserver agreement without protocol was ICC = 0.87, with protocol A, ICC = 0.87, and with protocol B, ICC = 0.93. The biggest improvement was seen for urologists' agreement from no protocol to protocol B, where ICC improved from 0.81 (CI: 0.70-0.87) to 0.91 (CI: 0.84-0.94). Conclusions: Observer agreement of HU measurement of urolithiasis without protocol is already good but using zoom, bone setting, and measuring in a representative plane is recommended. This protocol results in higher agreement, especially among urologists. Measuring in three axial planes does not increase agreement.