Tumor stroma-infiltrating mast cells predict prognosis and adjuvant chemotherapeutic benefits in patients with muscle invasive bladder cancer

Abstract

Which subgroups patients with muscle-invasive bladder cancer (MIBC) could benefit most from adjuvant chemotherapy (ACT) is blurred. Here we tried to stratify MIBC patients with tumor infiltrating mast cells (TIMs), explore the prognostic and predictive value of TIMs, and provide possible cellular explanations. We selected 259 MIBC patients who underwent radical cystectomy from two independent clinical centers between 2002 and 2014. TIMs were evaluated and prognostic and predictive value was assessed. The CIBERSORT method, Gene Set Enrichment Analysis (GSEA) and differential gene expression analyses were performed to explore the possible cellular mechanisms. TIMs infiltration was distinct between stromal and epithelial area of MIBC specimens. Patients with higher stromal TIMs had a significant worse overall survival and recurrence free survival (HR = 2.228, 95%CI: 1.467-3.550; P = 0.001 and HR = 1.984, 95%CI: 1.105-3.374; P = 0.016). More importantly, pT2 patients with low stromal TIMs tended to have a lower risk of death and recurrence after ACT (HR = 0.233, 95%CI: 0.020-0.814; P = 0.033 and HR = 0.180, 95%CI: 0.022-0.722; P = 0.031). A negative correlativity between TIMs and CD8 + T cells was identified on TCGA-BLCA cohort. Immunohistochemistry results validated that high stromal TIMs were negatively correlated with CD8 + T cells (Spearman's rho = -0.215, P < 0.001). Differential gene expression suggested that low TIMs might represent a state of immune activation in MIBC. To conclude, high stromal TIMs infiltration was an independent unfavorable prognosticator for MIBC patients. Patients with low stromal TIMs might benefit the most from ACT, especially in pT2 stage.

Keywords: adjuvant chemotherapy; bladder cancer; mast cells; overall survival; prognosis; recurrence-free survival.

Figure 1.

Evaluation of TIMs by immunohistochemistry in MIBC patients. (A) Representative immunohistochemistry images of TIMs infiltration in different tumor tissue compartments. Right bottom pictures showed 200X magnification. (B-C) Kaplan-Meier analyses of OS (top) and RFS (bottom) in all (B) and (C) pT2 patients according to TIMs infiltration in tumor stromal area.

Figure 2.

Stromal TIMs density could predict adjuvant chemotherapy effectiveness in different subgroups of pT2 MIBC patients. Kaplan-Meier analyses of OS in (A) all pT2 patients, (B) high stromal TIMs infiltration patients and (C) low stromal TIMs infiltration patients; of RFS in (D) all pT2N0 patients, (E) high stromal TIMs infiltration patients and (F) low stromal TIMs infiltration patients according to adjuvant chemotherapy applications.

Figure 3.

Differences of immune cell infiltration and biological process pathways according to TIMs infiltration in MIBC. (A) Frequency of immune lineages based on TIMs infiltration (left). Barplots (right) showed major abundant immune cells distribution according to TIMs. (*p < 0.05, **p < 0.01, and ***p < 0.001 by Mann-Whitney U test). Barplots show median ± quartile. (B) Heatmap showed Spearman’s correlation coefficients computed from analyses among different cell types and TIMs (left). Charts (right) listed detailed Spearman’s rho and corresponding P values. (C) Top two enriched biology pathways related with immune response in low TIMs infiltration groups. Gene Otology Biological Process gene sets from MSigDB were used. 1000 random sample permutations were performed. NES: Normalized Enrichment Score; NOM-p: Nominal P Value; FDR-q: False discovery rate. (D) Top 20 enriched gene sets in low TIMs group derived from GSEA. Gene Otology Biological Process gene sets from MSigDB were used. 1000 random sample permutations were performed.

Figure 4.

CD8 + T cells were correlated with TIMs infiltration in MIBC. (A) Representative immunohistochemistry images of high (left bottom) and low (right bottom) CD8+ cells infiltration in corresponding low (left upper) and high stromal (right upper) TIMs infiltration patients. Original, 48X; magnification, 200X. (B) Barplots showed frequencies of CD8+ cells density according to stromal TIMs infiltration. (*p < 0.05, **p < 0.01, and ***p < 0.001 by Mann-Whitney U test.). Barplots show median ± quartile. Chart (bottom) listed detailed Spearman’s rho and corresponding P values computed from correlation analyses among CD8+ cell density and stromal or epithelial TIMs density subgroups. (C) Volcano plot presented differential gene expression involved in multiple immune cells reaction between high and low TIMs groups. Gene in red or green were significantly differential expressed (defined as FDR-adjusted p-value ≤ 0.05 and fold change of at least 2x).