Background: Our study aims to assess the feasibility and the reproducibility of fluorescent confocal microscopy (FCM) real-time assessment of urethral and ureteral margins during open radical cystectomy (ORC) for bladder cancer (BCa). Methods: From May 2020 to January 2022, 46 patients underwent ORC with intraoperative FCM evaluation. Each specimen was intraoperatively stained for histopathological analysis using FCM, analyzed as a frozen section (FSA), and sent for traditional H&E examination. Sensitivity, specificity, positive predictive value (PPV), and the negative predictive value (NPV) of FCM and FSA were assessed and compared with H&E for urethral and ureteral margins separately. Results: The agreement was evaluated through Cohen’s κ statistic. Urethral diagnostic agreement between FCM and FSA showed a κ = 0.776 (p < 0.001), while between FCM and H&E, the agreement was κ = 0.691 (p < 0.001). With regard to ureteral margins, an overall agreement of κ = 0.712 (p < 0.001) between FCM and FSA and of κ = 0.481 (p < 0.001) between FCM and H&E was found. Conclusions: FCM proved to be a safe, feasible, and reproducible method for the intraoperative assessment of urethral and ureteral margins during ORC. Compared to standard FSA, FCM showed adequate diagnostic performance in detecting urethral and ureteral malignant involvement.

In advanced or metastatic settings, Comprehensive Genomic Profiling (CGP) allows the evaluation of thousands of gene alterations with the goal of offering new opportunities for personalized treatment in solid tumors. This study evaluated the CGP Success Rate in a real-life cohort of 184 patients enrolled in a prospective clinical trial. CGP data were compared with the routine molecular testing strategy adopted in-house. Sample age, tumor area, and the percentage of tumor nuclei were recorded for CGP analysis. We found that 150/184 (81.5%) samples resulted in satisfying CGP reports. The CGP Success Rate was higher in samples from surgical specimens (96.7%) and in specimens that had been stored (sample age) for less than six months (89.4%). Among the inconclusive CGP reports, 7/34 (20.6%) were optimal samples, according to CGP sample requirements. Moreover, with the in-house molecular testing approach, we could obtain clinically relevant molecular data in 25/34 (73.5%) samples that had inconclusive CGP reports. In conclusion, despite the fact that CGP offers specific therapeutical options in selected patients, our data suggest that the standard molecular testing strategy should not be replaced in routine molecular profiling.