Novel Genetic Subtypes of Urothelial Carcinoma With Differential Outcomes on Immune Checkpoint Blockade

Abstract

Purpose: Immune checkpoint blockade (ICB) therapy has significantly improved clinical outcomes in bladder cancer. Identification of correlates of benefit is critical to select appropriate therapy for individual patients.

Methods: To reveal genetic variables associated with benefit from ICB, we performed whole-exome sequencing on tumor specimens from 88 patients with advanced bladder cancer treated with ICB.

Results: We identified several genetic factors that correlated with progression-free and overall survival after ICB therapy including ARID1A mutation, tumor mutational burden, intratumoral heterogeneity, the ratio of nonsynonymous to synonymous mutations in the immunopeptidome (immune dN/dS), and tumor cell purity. In addition, we noted that neutrophil-to-lymphocyte ratio and smoking history were negatively associated with overall survival. These genetic characteristics define four molecular subtypes demonstrating differential sensitivity to ICB. We validated the association of these four subtypes with clinical benefit from ICB in an independent cohort (IMvigor210). Finally, we showed that these molecular subtypes also correlate with outcome, although with distinct relationships, among patients not treated with ICB using The Cancer Genome Atlas (TCGA) bladder cancer cohort. Using parallel RNA sequencing data, the subtypes were also shown to correlate with immune infiltration and inflammation, respectively, in the IMvigor210 and TCGA cohorts.

Conclusion: Together, our study defines molecular subgroups of bladder cancer that influence benefit from ICB.

FIG 1.

Genomic alterations in 88 patients with advanced bladder cancer treated with ICB. (A) Left, OncoPrint of the 18 most commonly altered genes, color coded by alteration type; right, frequency of each mutation. Top bar plot in OncoPrint shows the total number of mutations in each patient. (B) Frequency of common mutations in our cohort versus TCGA bladder cancer cohort. ICB, immune checkpoint blockade; TCGA, The Cancer Genome Atlas.

FIG 2.

Correlates of survival and response to ICB. (A) Univariate and (B) multivariable analyses of significant predictors of ICB benefit. P values and CIs are corrected for multiple comparisons where grid searches were used to obtain optimal thresholds (TMB, ITH, and INDEL rate). P values derived by the log-rank test. dN/dS, ratio of nonsynonymous to synonymous mutations in the immunopeptidome; HR, hazard ratio; ICB, immune checkpoint blockade; INDEL, insertion or deletion; ITH, intratumoral heterogeneity; NLR, neutrophil-to-lymphocyte ratio; OS, overall survival; PFS, progression-free survival; TMB, nonsynonymous tumor mutational burden.

FIG 3.

Differential outcomes of four molecular subtypes of bladder cancer after ICB therapy. (A) Proposed molecular subtypes of bladder cancer illustrated by OncoPrint and classification schema. (B) Genomic characteristics of bladder cancer subtypes. For each subtype, enrichment or depletion of each molecular feature is measured by z-score. (C) Association between survival and molecular subtypes. Kaplan-Meier curves showing PFS (left) and OS (middle) of each subtype. Right, proportion of patients with a PFS of > 6 months in each molecular subtype. P values calculated by the log-rank test. HR, hazard ratio; ICB, immune checkpoint blockade; Mut, mutated; OS, overall survival; PFS, progression-free survival; purity, tumor cell purity; TMB, tumor mutational burden; wt, wild-type.

FIG 4.

Validation of predictive power of molecular subtypes of bladder cancer in the IMmvigor210 cohort. (A) Multivariable analysis of the association of three biomarkers with OS after ICB therapy. (B) Kaplan-Meier curves showing OS according to the molecular subtype. Association between (C) tumor cell purity, (D) molecular subtype, and (E) T-cell fraction as determined by quantitation of TCRA reads versus immunophenotype as determined by RNA sequencing. In (C) and (E), black lines are median values, box boundaries indicate IQRs, and whiskers represent 1.5 IQR. Kaplan-Meier plots of OS according to (F) TCRA T-cell fraction and (G) molecular subtype using T-cell fraction in place of tumor cell purity. P values calculated by the log-rank test. HR, hazard ratio; ICB, immune checkpoint blockade; Mut, mutated; OS, overall survival; PFS, progression-free survival; purity, tumor cell purity; TMB, tumor mutational burden; wt, wild-type.

FIG 5.

Molecular subtypes also distinguish outcomes and immunophenotypes in non–ICB-treated patients. (A) Kaplan-Meier plots of OS of the TCGA bladder cancer cohort according to the molecular subtype. (B) Association between IFN? score calculated from TCGA RNA sequencing data and molecular subtype. Black lines indicate medians, box boundaries the IQR, and whiskers 1.5 IQR. HR, hazard ratio; ICB, immune checkpoint blockade; OS, overall survival; TCGA, The Cancer Genome Atlas.