Abstract 3868: Distinct tumor microenvironmental landscapes define molecular subtypes in non-muscle-invasive bladder cancer.

Abstract Background: Non-muscle-invasive bladder cancer (NMIBC) are biologically heterogeneous tumors that are classified based on stage/grade alone, which does not provide an accurate separation as it misses underlying molecular phenotypes. The UROMOL molecular classification, derived from large-scale transcriptomic profiling of NMIBC (Lindskrog SV, et al. Nat Commun. 2021;12:2301), stratifies tumors into prognosis-associated biology with distinct molecular subtypes. The relationship between molecular states and tumor microenvironment (TME) composition remains poorly defined. Methods: We performed transcriptomic subtyping and single-cell-level TME profiling of NMIBC tumors (patient n=16, sample n=34, cell n = 108108). Tumors were classified into UROMOL subtypes using NMIBC classifier (Lindskrog SV, et al. Nat Commun. 2021;12:2301) based on aggregated gene expression profiles. We quantified epithelial, stromal, and immune populations, and assessed pathway activity using curated immune and stromal gene module scores. Results: 3 UROMOL subtypes were observed: Class 1 (luminal papillary), Class 2a (basal-inflamed), and Class 2b (luminal-inflamed). Class 1 tumors (n=6, 17.6%) exhibited an epithelial-dominant, immune-cold TME characterized by ∼80.1% epithelial cells (95% CI: 74-86.2%) and minimal immune infiltration, including T cells (∼1.9%, CI: 0.5-3.3%), macrophages (∼1.4%, CI: 0.5-3.3%), and fibroblasts (∼8.4%, CI: 4.3-12.5%). This composition is consistent with differentiated luminal biology and may have favorable baseline prognosis. Class 2a tumors (n=9, 26.5%) displayed a distinct TME composition pattern, with an increase in T-cell (∼4.8%, CI: 1.7-8.0%) and macrophage (∼1.9%, CI: 0.6-3.1%) infiltration compared to Class 1, alongside increased fibroblast (∼17.5%, CI: 9.6-25.3%) and endothelial (∼8.1%, CI: 4.9-11.4%) content. These tumors showed increased effector and checkpoint activity, reflecting a more active immune microenvironment than class 1. Class 2b tumors (n=19, 55.9%) showed a heterogeneous immune-stromal landscape with pronounced fibroblast enrichment (∼27%, CI: 19.7-34.4%) and extracellular matrix remodeling, alongside elevated immune infiltration, including T cells (∼17.5%, CI: 15-20.0%) and macrophages (∼11.2%, CI: 7.4-14.9%), this reflects an immune-active, fibroblast-rich TME architecture. Conclusions: UROMOL subtypes are defined by distinct TME architectures with differences in cellular composition from immune-cold, epithelial-rich Class 1 tumors to highly immune infiltrated Class 2b tumors enriched with T cells and fibroblasts. Given the limited sample size, these observations should be validated in a larger cohort. This study demonstrates that UROMOL molecular classes of NMIBC are associated with distinct TMEs, revealing biologically meaningful differences in immune infiltration and stromal composition. Citation Format: Songjun Xu, Matthew H. Byrne, Jiarui Zhang, Ahmad Alalti, Devika Agarwal, Neil Beeharry, Olesya Chornoguz, Kyla Dooley, Kin Cheung Lee, Shuwei Li, Guneet Walia, Tommaso Mansi, Joel Greshock, Calliope Dendrou, Freddie Hamdy, Lisa Browning, Dan J. Woodcock, Patrick Wilkinson, Shibu Thomas. Distinct tumor microenvironmental landscapes define molecular subtypes in non-muscle-invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3868.