HLA-G expression associates with immune evasion muscle-invasive urothelial cancer and drives prognostic relevance

Abstract

Introduction: Urothelial bladder cancer is frequent and exhibits diverse prognoses influenced by molecular subtypes, urothelial subtype histology, and immune microenvironments. HLA-G, known for immune regulation, displays significant membranous expression in tumor tissues.

Methods: We studied the protein expression of Human Leucocyte Antigen G (HLA-G) in 241 Muscle-Invasive Bladder Cancer (MIBC) patients, elucidating its potential clinical and biological significance. Protein expression levels were evaluated and correlated with molecular subtypes, histological characteristics, immune microenvironment markers, and survival outcomes.

Results: High HLA-G expression associates with poor overall survival (OS) and diseasespecific survival (DSS), independent of clinicopathological parameters. HLA-G expression varies among molecular subtypes and Urothelial Subtype Histology, e.g., elevated expression levels in basal/squamous MIBC and those with sarcomatoid differentiation. Notably, HLA-G is increased in MIBC with an immune evasive microenvironment (high PD-L1 tumor cell expression, NK cell depletion, granzyme B (GZMB)/CD8 ratio reduction, MHC class I (MHCI) expression reduction) that are characterized by immunosuppressive features and poor prognosis. Furthermore, HLA-G correlates with elevated levels of other immune checkpoint proteins (TIGIT, LAG3, CTLA-4), indicating its role in immune evasion.

Discussion: Our findings underscore HLA-G's role as a potential prognostic marker and interesting immunotherapeutic target in MIBC. Its impact on immune evasion mechanisms and broad expression, coupled with associations withpoor survival and distinct tumor phenotypes, positions HLA-G as a promising protein for further exploration in developing targeted immunotherapies for MIBC patients.

Keywords: HLA-G; immune checkpoints; immune evasion; immune microenvironment; urothelial cancer.

Figure 1

HLA-G expression patterns in MIBC: (A) Histogram of HLA-G expression in H-score scale and logarithmic scale. The Shapiro-Wilk test indicates that the expression of HLA-G is not normally distributed (P=0.01, significance level of 0.05). (B) Expression levels of HLA-G at the invasion front (upper row) and tumor center (lower row) in MIBC. (C) Spearman rank correlation and not significant Mann-Whitney test (P=0.30) of HLA-G expression of tumor center and evasion front. (D) Kaplan-Meier analysis of overall (OS) and disease specific survival (DSS) based on HLA-G expression levels (low, intermediate, high). Univariable log-rank P value is depicted in the lower left corner of the survival plots. Table shows the number of patients at risk in 12 months increments. Statistical significance at P<0.05; ***significance at P<0.0001.

Figure 2

Distribution of continuous HLA-G expression by (A) molecular subtypes (n=230), (B) urothelial subtype histology (n=241), and (C) immune phenotypes (n=241). P values are derived by Dunn´s multiple comparisons test. Distribution of HLA-G low, intermediate, and high expressing tumors in (A) Urothelial Subtype Histology, (B) Molecular subtypes, and (C) immune phenotypes in absolute number and percentage. Statistical significance at P<0.05; *significance at P<0.01; **significance at P<0.001; ***significance at P<0.0001.

Figure 3

(A) Spearman-rank Correlation of HLA-G expression and GZMB-CD8 ratio and FOXP3 expression in the evasion phenotype. (B) Characterization of TIME (CD56, MHCI, CD68-CD8 Ratio, PD1-CD8 ratio, HLA-G-MHCI Ratio) across immune phenotypes. P-values are derived by Dunn´s multiple comparisons test. (C) Correlation matrix for pairwise associations between ICP protein expression. Correlation plots are presented in the bottom left part, histograms of logarithmic protein expression levels in the diagonal, the corresponding absolute correlation coefficients, and the level of statistical significance (Spearman correlation test) in the upper right part. Statistical significance at P<0.05; *significance at P<0.01; **significance at P<0.001; ***significance at P<0.0001.