Abstract Urothelial carcinoma (UC) of the bladder is one of the most common cancers worldwide. The treatment of patients with metastatic or locally advanced UC consists of platinum-based chemotherapy. Unfortunately, a large number of patients will ultimately develop resistance. Therapeutic options at that point are very limited: the last FDA drug approval for the treatment of bladder cancer dates back more than 2 decades. Identification of activated signaling pathways in UC can provide new targets for treatment. Recent DNA and RNA sequencing projects in invasive UC have revealed somatic mutations in several cancer genes. Some of these mutated genes, such as FGFR3 and PIK3CA, could potentially guide therapy. However, our knowledge of gene rearrangements in bladder cancer remains limited. These fusion genes often involve multiple fusion partners, as was reported for FGFR3 fusions in bladder cancer, which represents a significant challenge for discovery and for subsequent diagnostic screening. Therefore, a global detection method is needed to fully understand the diversity of alterations driving this disease. We used a high-throughput platform to systematically profile kinase fusions through specific enrichment of kinase transcripts. Using this approach, we screened 80 muscle invasive UC specimens and identified a number of activating mutations and novel fusion transcripts. The fusion genes identified in this discovery set will be validated in a second cohort of UC specimens to determine their frequency. Functional validation will be presented for some of these fusion genes. These genetic alterations may provide new avenues for individualized molecular treatment of UC patients. Citation Format: Floris Groenendijk, Iris de Rink, Laura Mertens, Annegien Broeks, Yann Neuzillet, Jeroen de Jong, Bas van Rhijn, Rene Bernards, Michiel van der Heijden. Identification of kinase fusion genes in bladder cancer through kinome-centered RNA sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2767. doi:10.1158/1538-7445.AM2014-2767