Non-muscle-invasive bladder cancer molecular subtypes predict differential response to intravesical Bacillus Calmette-Guérin

Abstract

The recommended treatment for patients with high-risk non-muscle-invasive bladder cancer (HR-NMIBC) is tumor resection followed by adjuvant Bacillus Calmette-Guérin (BCG) bladder instillations. However, only 50% of patients benefit from this therapy. If progression to advanced disease occurs, then patients must undergo a radical cystectomy with risks of substantial morbidity and poor clinical outcome. Identifying tumors unlikely to respond to BCG can translate into alternative treatments, such as early radical cystectomy, targeted therapies, or immunotherapies. Here, we conducted molecular profiling of 132 patients with BCG-naive HR-NMIBC and 44 patients with recurrences after BCG (34 matched), which uncovered three distinct BCG response subtypes (BRS1, 2 and BRS3). Patients with BRS3 tumors had a reduced recurrence-free and progression-free survival compared with BRS1/2. BRS3 tumors expressed high epithelial-to-mesenchymal transition and basal markers and had an immunosuppressive profile, which was confirmed with spatial proteomics. Tumors that recurred after BCG were enriched for BRS3. BRS stratification was validated in a second cohort of 151 BCG-naive patients with HR-NMIBC, and the molecular subtypes outperformed guideline-recommended risk stratification based on clinicopathological variables. For clinical application, we confirmed that a commercially approved assay was able to predict BRS3 tumors with an area under the curve of 0.87. These BCG response subtypes will allow for improved identification of patients with HR-NMIBC at the highest risk of progression and have the potential to be used to select more appropriate treatments for patients unlikely to respond to BCG.

Fig. 1.. Study design and gene signatures of patients with high-risk NMIBC according to BCG response subtypes (BRS).

A: RNA-sequencing was performed on Cohort A: n=132 pre-BCG, T1G3 NMIBC tumors (n=64 BCG-responders [R] vs. n=68 BCG non-responders [NR]). From the BCG NR, n=44 post-BCG tumors were also sequenced (n=34 matched pre- and post BCG samples + n=10 non-matched). Paired analysis showed enrichment of BRS3 in post-BCG tumors and identified candidate druggable genes. Cohort B consisted of n=151 pre-BCG, high-risk NMIBC tumors (n=88 BCG-responders vs. n=63 BCG non-responders). For both cohorts, PFS (Kaplan-Meier) is stratified according to BRSs. Last, a qPCR pathway assay had an AUC of 0.87 in identifying BRS3 patients. B: Heatmap of gene signatures and annotation of Cohort A grouped according to BRS. From top to bottom: i) Progression to MIBC; ii) BCG response; iii) carcinoma in situ signature (94); iv) 12-gene progression signature (17); v) UROMOL21 NMIBC subtypes (13); vi) T1BC subtypes (17); vii) TCGA MIBC subtypes (18); viii) Consensus MIBC subtypes (17); ix) UNC MIBC subtypes (95); x) MDA MIBC subtypes (96); xi) Lund BC subtypes (14); xii) Clinicopathological parameters associated to pre-BCG tumors; xiii) BRS signatures based on mean gene expression of selected genes (details in methods). Abbreviations: BCG = Bacillus Calmette-Guérin; (N)MIBC = (non-)muscle-invasive bladder cancer.

Fig. 2.. Gene set enrichment analysis and regulon analysis grouped according to theBRS.

A: Heatmap of all 50 GSEA hallmarks in n=283 pre-BCG, HR-NMIBC patients. Clustering on gene signatures (rows) indicates the existence of different molecular subtypes in HR-NMIBC. B: Boxplots of selected gene signatures in n=283 pre-BCG HR-NMIBC patients grouped according to BRS; p-values are Kruskal-Wallis tests. C: Heatmap of the top 200 most varying regulons using single-sample VIPER analysis on n=283 pre-BCG tumors grouped by BRS (columns). Hierarchical clustering between samples confirms three distinct molecular subtypes; key regulators for which small molecule inhibitors exist are highlighted. Abbreviations: BCG = Bacillus Calmette-Guérin; BRS = BCG response subtypes; GSEA = gene set enrichment analysis; HR-NMIBC = high-risk non-muscle invasive bladder cancer; VIPER = Virtual Inference of Protein Activity by Enriched Regulon Analysis.

Fig 3.. Immune deconvolution of pre-BCG tumors from Cohort A+B and spatial proteomics of the tumor microenvironment grouped by BRS from Cohort A.

A: Boxplots predicting tumor purity and immune infiltration in n=283 pre-BCG tumors grouped by BRS (25); p-values are Kruskal-Wallis tests. B: Immune cell deconvolution from RNA-sequencing: a single column is the sum of ten immune cell subpopulations and non-characterized cells grouped by BRS; uncharacterized cells are a mixture of malignant and normal cells; p-values are Wilcoxon tests for BRS1/2 vs BRS3; **p.adj<10⁻³; ***p.adj<10⁻⁴. C: Immunohistochemistry of CD8⁺ cytotoxic T cell infiltration in n=36 BRS1, n=48 BRS2 tumors and n=35 BRS3 tumors from Cohort A; p-values are Wilcoxon tests for BRS1/2 vs BRS3. D: Selection of regions for spatial proteomics; on average 20 regions of pre-BCG tumors surrounding the macrodissected areas used for RNA-sequencing were selected. Mean intratumoral protein expression was used for analyses. E: Boxplots of multiplex immunofluorescence results for n=38 BRS1, n=50 BRS2 tumors and n=38 BRS3 tumors; p-values are Wilcoxon tests for BRS1/2 vs BRS3 or Kruskal-Wallis tests (overall). F: Boxplot for immunofluorescence results for only BRS3 patients. BRS3 tumors with progression had higher T regs and macrophages than tumors that did not progress; p-value is a Wilcoxon tests for BRS3-R vs BRS3-P. Abbreviations: BCG = Bacillus Calmette-Guérin; BRS = BCG response subtypes; BRS3-R = BRS3 responder; BRS-P = BRS3 progressor.

Fig. 4.. Kaplan-Meier estimates of survival according to the BRS in pre-BCG HR-NMIBC patients (Cohort A+B) and assay performance to predict BRS3 in Cohort A.

A: Progression-free survival (PFS) stratified according to BRS. B: Recurrence-free survival (RFS) for high-grade tumor recurrences stratified according to BRS. C: Forest plot of multivariable Cox regression analysis; BRS3 vs BRS1 + BRS2. D: PFS stratified for the current EAU guideline recommendations (high-risk [HR] patients vs subgroup of very high-risk [VHR] patients). E: PFS stratified for current EAU risk-stratification and the BRSs. F: Subtype prediction for BRS3 vs BRS1/2 based on the assay results in Cohort A. Figure depicts the ROC with the area under the curve based on a logistic regression model, with corresponding test sensitivity and specificity reported at a threshold of 0.385. Abbreviations: AUC = area under the curve; BCG = Bacillus Calmette-Guérin; BRS = BCG response subtypes; EAU = European Association of Urology; (V)HR-NMIBC = (very) high-risk non-muscle invasive bladder cancer; Progression-free survival (PFS).

Fig. 5.. Pre- vs post-BCG transcriptomic and spatial proteomic comparisons.

A: Frequency of BRS in pre-BGC vs post-BCG tumors in Cohort A+B. Alluvial plot details 34 patients with matched pre- and post-BCG tumor samples. B: Volcano plot of differentially expressed mRNAs in matched post-BCG and pre-BCG tumors from n=34 patients. C: Top regulons with small molecule inhibitors that are up- or down-regulated in pre-BCG tumors and post-BCG tumors depicted. In red: regulators enriched in post-BCG recurrences. In blue: regulators enriched in pre-BCG tumors. VIPER uses a Student’s t-test with 2-tailed p values. D: Selected gene set enrichment hallmarks comparing n=34 paired pre- and post-BCG tumors samples. Dashed lines represent pathway activity scores between paired samples of a single patient; p-values are paired Wilcoxon tests. E: Immune cell deconvolution based on RNA-sequencing: a single column is the sum of ten different immune cell populations and non-characterized cells found in the tumor microenvironment grouped by pre-BCG tumors or post-BCG recurrences; uncharacterized cells are a mixture of malignant and normal cells; p-values are Wilcoxon tests; *p.adj<0.05. F: Heatmap with spatial proteomics grouped by subtype and sorted by z-score. G: Proteomic results of n=30 post-BCG recurrences grouped by post-BCG BRS; p-values are Wilcoxon tests. Abbreviations: BCG = Bacillus Calmette-Guérin; BRS = BCG response subtypes.