Single-nucleus and Spatially Resolved Intratumor Subtype Heterogeneity in Bladder Cancer

Sia V Lindskrog; Sofie S Schmøkel; Iver Nordentoft; Philippe Lamy; Michael Knudsen; Frederik Prip; Trine Strandgaard; Jørgen Bjerggaard Jensen; Lars Dyrskjøt

doi:10.1016/j.euros.2023.03.006

Single-nucleus and Spatially Resolved Intratumor Subtype Heterogeneity in Bladder Cancer

Eur Urol Open Sci. 2023 Apr 7:51:78-88. doi: 10.1016/j.euros.2023.03.006. eCollection 2023 May.

Authors

Sia V Lindskrog^{1

2}, Sofie S Schmøkel^{1

2}, Iver Nordentoft², Philippe Lamy², Michael Knudsen², Frederik Prip^{1

2}, Trine Strandgaard^{1

2}, Jørgen Bjerggaard Jensen^{1

3}, Lars Dyrskjøt^{1

2}

Affiliations

¹ Department of Clinical Medicine, Aarhus University, Aarhus, Denmark.
² Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.
³ Department of Urology, Aarhus University Hospital, Aarhus, Denmark.

Abstract

Background: Current bulk transcriptomic classification systems for bladder cancer do not consider the level of intratumor subtype heterogeneity.

Objective: To investigate the extent and possible clinical impact of intratumor subtype heterogeneity across early and more advanced stages of bladder cancer.

Design setting and participants: We performed single-nucleus RNA sequencing (RNA-seq) of 48 bladder tumors and additional spatial transcriptomics for four of these tumors. Total bulk RNA-seq and spatial proteomics data were available from the same tumors for comparison, along with detailed clinical follow-up of the patients.

Outcome measurements and statistical analysis: The primary outcome was progression-free survival for non-muscle-invasive bladder cancer. Cox regression analysis, log-rank tests, Wilcoxon rank-sum tests, Spearman correlation, and Pearson correlation were used for statistical analysis.

Results and limitations: We found that the tumors exhibited varying levels of intratumor subtype heterogeneity and that the level of subtype heterogeneity can be estimated from both single-nucleus and bulk RNA-seq data, with high concordance between the two. We found that a higher class 2a weight estimated from bulk RNA-seq data is associated with worse outcome for patients with molecular high-risk class 2a tumors. The sparsity of the data generated using the DroNc-seq sequencing protocol is a limitation.

Conclusions: Our results indicate that discrete subtype assignments from bulk RNA-seq data may lack biological granularity and that continuous class scores may improve clinical risk stratification of patients with bladder cancer.

Patient summary: We found that several molecular subtypes can exist within a single bladder tumor and that continuous subtype scores can be used to identify a subgroup of patients with poor outcomes. Use of these subtype scores may improve risk stratification for patients with bladder cancer, which can help in making decisions on treatment.

Keywords: Intratumor heterogeneity; Muscle-invasive bladder cancer; Non–muscle-invasive bladder cancer; RNA sequencing; Single-nucleus RNA sequencing; Spatial transcriptomics; Subtyping.