BackgroundAssessment of the immune status of muscle-invasive bladder cancer (MIBC) has previously shown to be prognostically relevant after treatment with curative intent. We conducted this study to develop a clinically applicable immune gene expression assay to predict prognosis and adjuvant chemotherapy benefit.Patients and methodsGene expression ofCD3Z,CD8AandCXCL9, immune cell (IC) populations including stromal tumor infiltrating lymphocytes (sTILs), T-cells, natural killer cells (NK-cells), macrophages, Programmed cell death protein 1 positive (PD-1) IC and tumor subtypes (MD Anderson Cancer Center/MDACC-approach) were assessed in 187 MIBC patients (Comprehensive Cancer Center Erlangen-EMN/CCC-EMN-cohort). A gene expression signature was derived by hierarchical-clustering and validated in The Cancer Genome Atlas (TCGA)-cohort. IC populations in the TCGA cohort were assessed via CIBERSORT. Benefit of platinum-containing adjuvant chemotherapy was assessed in a pooled cohort of 125 patients. Outcome measurements were disease specific survival, disease-free survival and overall survival.ResultsThe gene expression signature ofCXCL9,CD3ZandCD8Acorrelates with quantitative amounts of specific IC populations and sTILs (CCC-EMN: ρ-range: 0.44–0.74; TCGA: ρ-range: 0.56–0.82) and allows stratification of three different inflammation levels (inflamed high, inflamed low, uninflamed). Highly inflamed tumors are preferentially basal subtype and show favorable 5-year survival rates of 67.3% (HR=0.27; CCC-EMN) and 55% (HR=0.41; TCGA). Uninflamed tumors are predominantly luminal subtypes and show low 5-year survival rates of 28% (CCC-EMN) and 36% (TCGA). Inflamed tumors exhibit higher levels of PD-1 and Programmed cell death 1 ligand 1 (PD-L1). Patients undergoing adjuvant platinum-based chemotherapy with ‘inflamed high’ tumors showed a favorable 5-year survival rate of 64% (HR=0.27; merged CCC-EMN and TCGA cohort).ConclusionThe gene expression signature ofCD3Z,CD8AandCXCL9can assess the immune status of MIBC and stratify the survival of MIBC patients undergoing surgery and adjuvant platinum-based chemotherapy. Furthermore, the assay can identify patients with immunological hot tumors with particular high expression of PD-L1 potentially suitable for immunotherapy.

Purpose Recently discovered molecular classifications for urothelial bladder cancer appeared to be promising prognostic and predictive biomarkers. The present study was conducted to evaluate the prognostic impact of molecular subtypes assessed by two different methodologies (gene and protein expression), to compare these two approaches and to correlate molecular with histological subtypes in a consecutively collected, mono-institutional muscle-invasive bladder cancer (MIBC) cohort. Methods 193 MIBC were pathologically re-evaluated and molecular subtypes were assessed on mRNA (NanoString technology, modified 21-gene-containing MDACC approach) and protein levels (immuno-histochemical [IHC] analysis of CK5, CK14, CD44, CK20, GATA3 and FOXA1). Descriptive statistical methods and uni-/multi-variable survival models were employed to analyze derived data. Results Neither gene expression nor protein-based subtyping showed significant associations with disease-specific (DSS) or recurrence-free survival (RFS). Agreement between mRNA (reference) and protein-based subtyping amounted 68.6% for basal, 76.1% for luminal and 50.0% for double-negative tumors. Histological subtypes associated with RFS in uni-variable (P = 0.03), but not in multivariable survival analyses. Tumors with variant histology predominantly showed luminal subtypes (gene expression subtyping: 36/55 cases, 65.5%; protein subtyping: 44/55 cases, 80.0%). Squamous differentiation significantly associated with basal subtypes (gene expression subtyping: 44/45 squamous cases, 97.8%; protein subtyping: 36/45 cases, 80.0%). Conclusion In our consecutive cystectomy cohort, neither gene, protein expression-based subtyping, nor histological subtypes associated with DSS or RFS in multi-variably adjusted survival analyses. Application of a limited IHC subtyping marker panel showed high concordance of 83.9% with gene expression-based subtyping, thus underlining the utility for subtyping in pathological routine diagnostics. In addition, histological MIBC subtypes are strong indicators for intrinsic subtypes.