Histopathologic and spatial transcriptomic analyses of en bloc resections of de novo and recurrent bladder tumors.

Abstract

864 Background: En bloc transurethral resection of bladder tumors (ERBT) is a safe and effective treatment for non-muscle invasive bladder cancer (NMIBC). ERBT additionally preserves the relationship of the tumor, margin, and surrounding urothelium, enabling better evaluation of lateral extension (LE) of atypia and molecular changes in nearby tissues. Analyses of these specimens may provide unique insights into mechanisms of tumorigenesis and recurrence. Methods: En bloc specimens from de novo and recurrent high-grade papillary NMIBC were obtained via ERBT. H&E slides were prepared and reviewed to characterize LE, which was defined as atypical urothelium extending > 1 mm from the tumor base. Targeted next generation sequencing (NGS) of cancer-related genes was performed on tumor and LE samples. Spatial transcriptomic analyses were performed with the 10X Genomics Xenium platform. Results: 37 specimens from 26 patients (10 de novo, 16 recurrent) were obtained for analysis. 13 patients had multifocal tumors, and 35% of tumors had LE. 9/11 cases with LE were de novo tumors, whereas 14/15 cases without LE were recurrent tumors. Of those with LE, the surrounding atypia was generally low-grade (77%). 70% of LE extended to the ERBT resection margin with a mean length of 4 mm. NGS demonstrated overlap between tumor mutations and LE mutations in 8 samples (72%). 3 LE samples (all low-grade) had no identified mutations. Preliminary spatial transcriptomic analyses on 8 tumors (6 with LE) demonstrated differences with and without LE in terms of margin cell types and organization. With LE, there are cells that cluster with tumor cells throughout, compared to distinct cell-type clusters in margins without LE. Furthermore, ranking cells from basal to luminal subtypes showed normal urothelial organization in margins without LE and a disorganized structure in LE. Conclusions: ERBT preserves spatial relationships between NMIBC and surrounding tissue, permitting more informed analyses. These results suggest significant differences in the surrounding urothelium for patients with and without LE. The pattern of prevalence vs absence of LE in de novo vs recurrent cases suggests distinct biology between these entities, such as a field effect for initial tumorigenesis compared to reimplantation in recurrence. Clinically, the frequency of LE extending to the margin in de novo tumors argues for obtaining wider margins during initial resections. Total cohortN = 2637 tumors Lateral extension absentN = 15 (58%)24 tumors (65%) Lateral extension presentN = 11 (42%)13 tumors (35%) De novo 10 (38%) 1 (7%) 9 (82%) Multifocal 13 (50%) 9 (60%) 4 (36%) Mean tumor size (range) 1.1 (0.1-2.6) 0.9 cm (0.1-2.5) 1.4 cm (0.3-2.6) Mean LE size (range) - - 4.1 mm (1-10.8) LE at margin - - 9 (70%) NGS overlap (tumor vs LE) - - 8 (72%) Cell-clustering - Margin and tumor distinct LE and tumor overlap Basal to Luminal organization - Organized Disorganized