Abstract 7570: Molecular subtyping and immunohistochemistry validation identifies muscle Invasive bladder cancer subgroups with poorer overall survival
Authors: Zhang Jiarui , Feng Mingxiao , Beeharry Neil , Thomas Shibu , Wirtz Ralph , Hartmann Arndt , Eckstein Markus , Choi Woonyoung 
Published: March 2024

Abstract
Introduction: Muscle-invasive bladder cancers (MIBC) are molecularly heterogeneous and have poor clinical outcomes, compared to non-muscle invasive disease. The molecular characterization of subtypes and immune status has shown prognostic value and the potential to guide precision intervention. However, a more comprehensive understanding of the association between subtypes and immune cells is still required. To this end, an integrative multi-omics analysis was performed on patients with MIBC.
Methods: From a cohort of 244 MIBC patients who did not receive neoadjuvant chemotherapy, cystectomy tissue was collected to assess molecular features associated with poor outcomes. Macro-dissected FFPE slides were used to perform whole transcriptome RNA sequencing (N=208, passing quality control) and both the consensus single-sample classifier and TCGA classifier were applied to the RNAseq data to determine molecular subtypes. Immunohistochemistry (IHC) staining for canonical markers of basal (KRT5, 14 and CD44), luminal (KRT20, GATA3, FOXA1) and immune cells (CD3, CD8, PD-L1 (SP263)) was performed, and scoring was assessed by two independent pathologists. Tumor subtypes derived from either RNAseq or IHC were compared and correlated with disease specific survival.
Results: Among the 244 MIBC patients 30.7% were T2, 47.6% were T3 and 21.7% were T4. The consensus molecular classification identified mRNA subtypes and showed agreement with the TCGA molecular classification. Correlations with disease specific survival revealed that luminal subtypes trended towards best outcome while stroma-rich subtype trended towards poorest outcomes compared to other subtypes. IHC markers associated with luminal-like and basal-like tumors recapitulated the molecularly defined luminal/basal subtype assignment. Stroma-rich subtypes were enriched with fibroblast signatures, implying high desmoplastic stromal cell infiltration and low immune cell infiltration. By integrating molecular classification and IHC immune markers (PD-L1, CD3, CD8 - serving as a proxy for ‘immune signature’), we found that the immune signatures themselves stratified the survival benefit, as previously shown. Interestingly, we observed heterogeneity of the IHC-immune signature within mRNA subtypes.
Conclusions: Integrating MIBC subtyping, IHC of immune markers and patient outcomes data provides a biological framework that allows for potential biomarker identification for this lethal disease. The studies presented here underscore the existence of heterogeneity in immune phenotypes within tumor subtypes. A deeper understanding of the association between these tumor subtypes and their immunological states is crucial to guide treatment decisions, particularly for patients facing worse prognostic outcomes.
Citation Format: Jiarui Zhang, Mingxiao Feng, Neil Beeharry, Shibu Thomas, Ralph Wirtz, Arndt Hartmann, Markus Eckstein, Woonyoung Choi. Molecular subtyping and immunohistochemistry validation identifies muscle Invasive bladder cancer subgroups with poorer overall survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7570.