Background: We previously reported the clinical outcomes of a positive multi-center phase II trial of neoadjuvant gemcitabine (G) and cisplatin (C) plus atezolizumab (A) in patients with muscle-invasive bladder cancer (Funt, et al. JCO 2022). In this and another trial of neoadjuvant GC with pembrolizumab (Rose et al, JCO 2021), PD-L1 positivity by immunohistochemistry was not predictive of non–muscle-invasive downstaging ( < pT2N0). Therefore, we investigated other pre-treatment tissue-based genomic and gene expression biomarkers of response and resistance. Methods: 36 pts had pre-treatment tissue available for genomic analysis. We performed targeted hybridization capture DNA sequencing using the CLIA-certified MSK-IMPACT platform and whole transcriptome RNA sequencing. We examined genomic and gene expression biomarkers which have been previously investigated in the context of neoadjuvant cisplatin-based chemotherapy or anti-PD-1/L1 immunotherapy for MIBC, including tumor mutation burden (TMB), a DNA damage response (DDR) 9-gene panel (NCT03609216) associated with response to neoadjuvant chemotherapy, and an 8-gene cytotoxic T cell transcriptional signature associated with response to neoadjuvant A (tGE8; Powles et al, Nature Medicine 2019). We also evaluated TGF-β pathway activation, which was associated with resistance to A in pts with metastatic BC (Mariathasan et al, Nature 2018). Putative biomarkers were assessed for correlation with < pT2N0, the trial’s primary endpoint. Results: DNA was available from all 36 pts, and RNA met quality control metrics for 29 pts. TMB was significantly higher in pts with < pT2N0 (median 16 mut/Mb, IQR 12-25) versus ≥ pT2N0 (median 10 mut/ Mb, IQR 8-10; p < 0.01). A single patient had a TMB > 200 Mut/Mb with a POLE hotspot mutation and achieved pT0N0; TMB was still significantly higher in responders after omission of this patient (p < 0.01). Nine of 25 pts (36%) with < pT2N0 had a deleterious DDR mutation versus 1 of 10 pts (10%) with ≥ pT2N0 (p = 0.13). While tGE8 was significantly increased in patients with < pT2N0 compared to those without (p = 0.01), TGF-β pathway activation was not increased in pts with ≥ pT2N0 (p = 0.99). Conclusions: TMB and the tGE8 cytotoxic T cell transcriptional signature were associated with response to combination GC+A in muscle-invasive bladder cancer. More detailed molecular analyses will be reported. Clinical trial information: NCT02989584.