English Cocker Spaniels (ECSs) are about 7.3 times more predisposed to anal sac carcinoma (ASC)development than other dog breeds. Hence, there is a genetic base for ASC development in ECSs and this study aims to identify genetic factors that are causative. Funded by AKC CHF, previous studies were performed including whole genome sequencing (WGS) and RNA-seq with ECS dogs with ASC. This led to the discovery of three causative germline mutation candidates: PRPF3 R277C, ABCF3 K616N, and the PKD2-COIL gene fusion. Moreover, analysis reveals a very different landscape for the transcriptome of ASC of ECS, compared to other canine cancer types, with numerous canonical cancer genes and pathways (e.g., MYC network) downregulated. ECS ASC tumors also harbor increased endothelial cells and CD4 T cells. To validate these findings and expand the study, this work will include both in silico and wet lab analyses. First expanding the sequencing study of ECS dogs to determine if the alterations are ECS-specific and/or ASC-specific. Second, to perform experimental assays to determine if the ECS- and ASC-specific alterations promote tumorigenesis. This study will discover germline alterations that predispose ECS dogs to ASC development, providing targets for future research on prediction, prevention, and treatment of ASC in ECS dogs