Evaluation of PD-L1 and other immune markers in bladder urothelial carcinoma stratified by histologic variants and molecular subtypes

Huili Li, Qingzhao Zhang, Lauren Shuman, Matthew Kaag, Jay D Raman, Suzanne Merrill, David J DeGraff, Joshua I Warrick, Guoli Chen, Huili Li, Qingzhao Zhang, Lauren Shuman, Matthew Kaag, Jay D Raman, Suzanne Merrill, David J DeGraff, Joshua I Warrick, Guoli Chen

Abstract

Although advanced bladder cancer overall has a poor prognosis, a subset of patients demonstrate durable response to immune checkpoint inhibitors. Evidence shows that the response to checkpoint inhibitors may be associated with type and degree of immune infiltration in the tumor microenvironment. Here, we evaluated immune markers stratified by molecular subtypes and histologic variants. The study utilized a series of urothelial carcinomas (UCs) by tissue microarray, on which histologic variants and molecular subtypes had previously been established. PD1, CD3, CD8 and CD68 expression was evaluated by immunohistochemistry in tumor infiltrating immune cells, while PD-L1 expression in the tumor microenvironment was assessed. Each marker was scored semi-quantitatively (score 0-3). Tumors were clustered by marker scores using agglomerative methods, and associations among markers, histologies, and molecular subtypes were analyzed. PD-L1 expression in the tumor microenvironment significantly correlated with presence of CD3, CD8 and chronic inflammation. Urothelial carcinoma may be classified as either immune high or low based on marker expression. The immune high group is enriched in higher CD3, PD-L1, and genomically-unstable molecular subtype, suggesting it may respond to checkpoint inhibitors. We also identified a degree of intratumoral heterogeneity in immune markers in bladder cancer.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Immune marker score analysis in association with histological variants and molecular subtypes. (a) Unsupervised hierarchical clustering of all UC cases. Each row is a marker and each column is a patient. Top bar indicates histological variants and molecular subtypes. (b) Distribution of histological variants (CIS, NIPUC, and invasive UC) and immune high and immune low clusters (Chi-square test, p < 0.01); (c) NIPUC is significantly associated with immune low cluster (Fisher’s exact test, p < 0.01); (d) Invasive UC is significantly associated with immune high cluster (Fisher’s exact test, p < 0.01); (e) Distribution of molecular subtypes (urothelial-like, basal-squamous, nontype, genomically-unstable, mesenchymal) in immune high and immune low clusters (Chi-square test, p < 0.05); (f) Genomically-unstable subtype is significantly associated with immune high cluster (Fisher’s exact test, p < 0.01).
Figure 2
Figure 2
Supervised hierarchical clustering with immunohistochemistry staining of 13 proteins representing molecular subtyping genes demonstrates that the immune high and immune low clusters are not associated with the individual gene signatures. Only specimens with available data of the immunohistochemical stains are used for the hierarchical clustering. The dendrogram is determined by unsupervised hierarchical clustering of the immune markers (top panel). Expression levels of the 13 genes are plotted in the bottom panel following the same dendrogram (supervised).
Figure 3
Figure 3
Comparisons of immune marker scores in CIS, NIPUC and invasive UC. (a,b) CIS has significantly higher chronic inflammation and PD1 scores as compared to NIPUC and invasive UC; (c–e) Invasive UC has significantly higher CD3, CD8 and PD-L1 scores as compared to NIPUC and invasive UC. All statistical significance was calculated by one with ANOVA with posttest as described in methods and materials and plotted as Mean ± SD. p < 0.05 is considered significant as *.
Figure 4
Figure 4
Immune scores of PD-L1 and CD3 among invasive UC variants. (a) Sarcomatoid variant shows significantly higher PD-L1 score as compared to the nested, glandular, and conventional UC. (b) Squamous, sarcomatoid, and plasmacytoid variants tend to have higher intra-tumoral CD3 scores. All statistical significance was calculated by one with ANOVA with posttest as described in methods and materials, and plotted as Mean ± SD. p < 0.05 is considered significant as *.
Figure 5
Figure 5
Intra-tumoral immune heterogeneity in invasive urothalial carcinoma. Each row represents a patient and each column is a histologic variant. (a) Distribution of immune low and immune high cluster profile in different histologic variants from the same patient. (b) Distribution of PD-L1 scores in different histologic variants from the same patient. (c) Distribution of molecular subtypes in different histologic variants from the same patient. (d) Distribution of intra-tumoral CD3 scores in different histologic variants from the same patient.

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