Aims: Treatment options for advanced urothelial carcinoma (aUC) rapidly evolved: besides immunomodulative therapeutic options and inhibitors targeting Fibroblast growth factor receptor (FGFR) alterations, two new antibody‐drug conjugates (ADC), sacituzumab govitecan (SG) and enfortumab vedotin (EV), have been approved. However, little is known about the associations of specific aUC properties and the surface target expression of TROP2 and NECTIN‐4. Our aim was to characterize associations of TACSTD2/TROP2 and NECTIN‐4/NECTIN‐4 protein and gene expression with morphomolecular and clinicopathological characteristics of aUC in two large independent cohorts.Methods and results: The TCGA BLCA (n = 405) and the CCC‐EMN (n = 247) cohorts were retrospectively analysed. TROP2/TACSTD2 and NECTIN‐4/NECTIN‐4 are highly expressed at the protein and transcript level in aUC, and their expression status did not correlate with patient survival in both cohorts. NECTIN‐4/NECTIN‐4 expression was higher in luminal tumours and reduced in squamous aUCs. NECTIN‐4 was negative in 10.6% of samples, and 18.4% of samples had low expression (H‐score <15). The TROP2 negativity rate amounted to 6.5%. TACSTD2 and NECTIN‐4 expression was reduced in neuroendocrine‐like and/or protein‐based double‐negative tumours. TROP2‐ and NECTIN‐4‐negative tumours included one sarcomatoid and four neuroendocrine aUC. FGFR3 alterations and PD‐L1 expression on tumour and immune cells did not associate with TROP2 or NECTIN‐4 expression.Conclusions: TACSTD2/TROP2 and NECTIN‐4/NECTIN‐4 are widely expressed in aUC, independent of FGFR3 alterations or PD‐L1 expression, thus representing a suitable target for ADC treatment in the majority of aUC. The expression loss was associated with aggressive morphomolecular aUC subtypes, i.e. neuroendocrine(‐like) and sarcomatoid aUC.

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.

Since 2016, large nested urothelial carcinoma (LNUC) has been included within the WHO classification of urothelial tumors. Limited reports with mainly small case series have confirmed the malignant behavior of LNUC despite its bland morphological appearance. We evaluated, for the first time, markers for new immunooncological or targeted therapies including FGFR3 mutational status and PD-L1 status, the frequency of TERT-promoter mutations and the molecular subtype in a cohort of 25 LNUC using SNaPshot analysis and immunohistochemistry. Of the 25 cases, 17 were pure LNUC, with 13 showing an additional exophytic papillary/papillary-like component. Seven mixed LNUCs presented areas of classical nested variant urothelial carcinoma (NVUC) and one showed a component of conventional urothelial carcinoma. Of the 17 evaluable pure LNUCs, 16 were FGFR3-mutated with identical mutations in their concomitant papillary/papillary-like components. An FGFR3 mutation was found in 1/7 evaluable mixed LNUCs combined with NVUC. TERT-promoter mutations were detected in 86.7% pure and 83.3% mixed tumors. Immunohistochemistry revealed a luminal phenotype; PD-L1 was negative in the majority of tumor cells and tumor-associated immune cells. Pure LNUC is a prime example of a luminal, FGFR3-mutated, mostly PD-L1-negative tumor. In contrast, FGFR3 mutations seem to be rare in mixed LNUC, which may indicate a different pathway of tumor development.

Bladder cancer (BCa) is the ninth most commonly diagnosed cancer worldwide. Although there are several well-established molecular and immunological classifications, markers for tumor cells and immune cells that are associated with prognosis are still needed. The chemokine CC motif ligand 2 (CCL2) could be such a marker. We analyzed the expression of CCL2 by immunohistochemistry (IHC) in 168 muscle invasive BCa samples using a tissue microarray. Application of a single cut-off for the staining status of tumor cells (TCs; positive vs. negative) and immune cells (ICs; ≤6% of ICs vs. >6% of ICs) revealed 57 cases (33.9%) and 70 cases (41.7%) with CCL2-positive TCs or ICs, respectively. IHC results were correlated with clinicopathological and survival data. Positive CCL2 staining in TCs was associated with shorter overall survival (OS), disease-specific survival (DSS), and relapse-free survival (RFS) (p = 0.004, p = 0.036, and p = 0.047; log rank test) and appeared to be an independent prognostic factor for OS (RR = 1.70; p = 0.007; multivariate Cox’s regression analysis). In contrast, positive CCL2 staining in the ICs was associated with longer OS, DSS, and RFS (p = 0.032, p = 0.001, and p = 0.001; log rank test) and appeared to be an independent prognostic factor for DSS (RR = 1.77; p = 0.031; multivariate Cox’s regression analysis). Most interestingly, after separating the patients according to their lymph node status (N0 vs. N1+2), CCL2 staining in the ICs was differentially associated with prognosis. In the N0 group, CCL2 positivity in the ICs was a positive independent prognostic factor for OS (RR = 1.99; p = 0.014), DSS (RR = 3.17; p = 0.002), and RFS (RR = 3.10; p = 0.002), whereas in the N1+2 group, CCL2 positivity was a negative independent factor for OS (RR = 3.44; p = 0.019)) and RFS (RR = 4.47; p = 0.010; all multivariate Cox’s regression analyses). In summary, CCL2 positivity in TCs is a negative prognostic factor for OS, and CCL2 can mark ICs that are differentially associated with prognosis depending on the nodal stage of BCa patients. Therefore, CCL2 staining of TCs and ICs is suggested as a prognostic biomarker for BCa patients.