Background: Genomic analyses have identified that bladder cancers can be divided into distinct molecular subtypes: luminal and basal. IHC markers GATA3 and CK5/6 have demonstrated >80% accuracy in assessing the luminal and basal subtypes of primary bladder tumors and may be easily utilized in clinical practice. Correlation of the primary bladder subtype with that of metastatic sites has not been demonstrated and associations with clinical outcomes are uncertain. Methods: We retrospectively identified patients with mBC who were treated with systemic therapy and had biopsies of either primary bladder or metastatic sites. Patient demographic, metastatic sites, treatment patterns, and clinical outcomes were recorded. Tissue microarrays (TMA) were constructed from primary and/or metastatic tumors. IHC was performed using mouse monoclonal antibodies: GATA-3 (L50-823, Pharmingen, 1:200) and cytokeratin 5/6 (XM26, Thermo Fisher, 1:100). Luminal (GATA3+, CK5/6-), basal (GATA3-, CK5/6+), double positive (GATA3+, CK5/6+) or double negative (GATA3-, CK5/6-) subtyping was applied. Concordance of matched bladder and metastatic pairs was quantified using Cohen’s kappa. Wilcoxon rank-sum tests were used for comparison of continuous and ordinal measures and chi-square tests were performed for comparison of categorical measures. Survival was estimated by Kaplan-Meier method; multivariable Cox analysis was performed. Results: Of 62 specimens, 37 bladder and 16 metastatic sites were interpretable. Four IHC subtype patterns were identified, most were luminal (n=20) followed by double-positive (n=12), basal (n=5), and double-negative (n=5). Of 10 pairs of matched primary tumor and metastatic sites, there was near-perfect subtype concordance between primary and metastatic tumors (κ=0.84; 95% CI:0.58-1.00). No association between sites of metastatic progression and subtype were identified, nor was there any difference in overall survival between the subtypes (p = 0.70). The basal subtype had numerically worse survival compared to the luminal subtype, HR =0.164 (95% CI: 0.02-1.58, p=0.12). Conclusions: IHC subtyping by GATA3 and CK5/6 is feasible in the clinical setting and showed strong correlation between primary and metastatic sites. A larger analysis is planned to further investigate associations with clinical features and outcomes.

NAC = Neoadjuvant Chemotherapy, AC = Adjuvant Chemotherapy, LN= Lymph Node, DP = Double Positive, DN = Double Negative.