Intravesical instillations with Bacillus Calmette-Guérin (BCG) is the recommended treatment for T1 high-grade (HG) bladder cancer (BC) patients. Unfortunately, risk stratification is insufficient to identify patients at risk of BCG treatment failure. Another challenge is the worldwide BCG-shortage, which emphasizes the need for alternative therapeutic strategies. Thus, we aimed to investigate molecular signal transduction pathway activities using Philips' OncoSignal consumer-ready test in BCG treated T1HG BC in order to identify differentially expressed pathways that may predict clinical outcome or provide a rationale for alternative therapeutic strategies. Primary T1HG BC patients treated with ≥5/6 BCG induction instillations were retrospectively included. RNA-sequencing (50 million paired-end reads, containing ≥80% tumor) was performed on: 1) BCG-naïve tumors from patients with complete response to BCG at study inclusion (BCG-responders) and 2) BCG-naïve tumors with matching muscle-invasive bladder cancer (MIBC) recurrences during BCG therapy (BCG-failures). Philips OncoSignal analysis was performed to quantify functional signal transduction pathway activities. Molecular subtyping was performed using non-muscle invasive and muscle-invasive classifiers. Clinical and molecular subtyping results were combined with Philips OncoSignal to explore differences between BCG responders and BCG failures and between molecular subtypes. OncoSignal was performed in 20 tumors: 14 BCG-naïve (7 BCG-responders vs 7 BCG-failures) and 6 MIBC recurrences. Median follow-up for BCG-responders was 99 months (IQR 74-120); median follow-up for BCG-failures was 49 months (IQR 25-79), with a median time to progression of 14 months (IQR 10-41). Based on pilot data, MAPK and TGFβ pathway activity was significantly higher in tumors from BCG failures vs BCG-responders (both pCitation Format: Florus C. De Jong, Teemu D. Laajala, Robert F. Hoedemaeker, Sébastien Rinaldetti, Jolien T. Mensink, Angelique C. Van Der Made, Deric K. Van der Schoot, Egbert R. Boevé, Ellen C. Zwarthoff, Joost L. Boormans, Dan Theodorescu, James C. Costello, Tahlita C. Zuiverloon. Differential pathway analyses of BCG-treated T1HG bladder cancer using Philips OncoSignal: A pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 615.