Identification of a Muscle-Invasive Bladder Carcinoma Molecular Subtype of Poor Responders to Neoadjuvant Chemotherapy and High Expression of Targetable Biomarkers

Neoadjuvant chemotherapy (NACT) is the standard treatment for muscle-invasive bladder carcinoma (MIBC), but its efficacy varies significantly among patients. The aim of this study is the identification of biomarkers and biological processes related to the response to neoadjuvant chemotherapy (NACT) in muscle-invasive bladder carcinoma (MIBC). Fifty-eight transurethral resection (TURBT) samples and thirty cystectomy samples from NACT non-responders were analyzed using mass spectrometry. Samples were classified with sparse k-means and consensus clustering. Protein networks were built using probabilistic graphical models, grouped into functional nodes, and analyzed for activity differences. Gene set enrichment analysis was applied to identify resistance mechanisms, and results were validated using The Cancer Genome Atlas (TCGA) cohort. Proteomic analysis revealed two independent classifications in TURBT samples. The first (Layer1) divided tumors into three groups, including one NACT non-responder subtype not aligned with traditional luminal or basal classifications but characterized by high expression of targetable markers NECTIN4 and Her2. The second (Layer3) separated luminal-papillary tumors from luminal-infiltrated/luminal and basal tumors. While Layer3 groups did not differ in NACT response, they showed distinct disease-free survival outcomes. Importantly, complete response to NACT was linked to improved survival in luminal subgroups but not in basal tumors, suggesting subtype-specific prognostic implications. Finally, analysis of cystectomy samples identified unique mechanisms of resistance for each subgroup, suggesting tailored therapeutic approaches. Two classification systems were defined as follows: one identified a proteomics-based non-responder group with actionable targets, and the other linked tumor subtype to prognosis. Distinct resistance mechanisms suggest opportunities for subtype-specific therapies, supporting improved management and treatment development for MIBC patients.