*Result*: Whole-Transcriptome Analysis of Gene Expression in Canine Splenic Lymphoid Hyperplasia, Complex Hyperplasia, Histiocytic Sarcoma, and Stromal Sarcoma.

*Simple Summary: Understanding diseases of the spleen in dogs is challenging because different types of splenic nodules have traditionally been grouped under a single name, despite showing very different biological behaviour. These nodules can range from benign tissue changes to aggressive cancers, making accurate diagnosis and treatment decisions difficult. The aim of this study was to improve our understanding of how these splenic nodules differ at a molecular level by examining patterns of gene activity. We compared spleen tissue from dogs with four distinct types of splenic nodules to normal spleen tissue. The results showed consistent differences in gene activity between normal spleen and nodular tissue, as well as clear differences among the four nodule types. Many of the affected genes are known to be involved in cancer development and progression in other diseases. These findings suggest that altered gene activity may contribute to the transition from non-cancerous splenic changes to malignant disease. This study provides new insights into the biological mechanisms underlying splenic tumours in dogs and may support the development of improved diagnostic methods and more targeted treatments, benefiting canine health and veterinary care. Splenic nodules in dogs that were historically classified under the broad term "fibrohistiocytic nodules" are now recognised as distinct entities within likely a biological continuum. These include lymphoid hyperplasia extending to indolent lymphoma and complex hyperplasia to stromal sarcoma. However, the molecular mechanisms underpinning these proposed progressions remain largely unexplored, particularly at the genomic and transcriptomic levels. This study aimed to delineate and compare the transcriptomic landscapes of four distinct canine splenic nodules through differential gene expression profiling. RNA sequencing was performed on twelve formalin-fixed, paraffin-embedded (FFPE) splenic tissue samples obtained from dogs diagnosed with lymphoid hyperplasia, complex hyperplasia, histiocytic sarcoma, and stromal sarcoma, with normal canine spleen serving as a control tissue. Comparative transcriptomic analysis identified 47 differentially expressed genes (DEGs) between splenic nodules and normal spleen, including CSRP1, SLC40A1, C1QA, C1QC, DLA-12, FTL, FXYD6, MPEG1, OAS3, CSF1, and JMJD6. Furthermore, 39 DEGs were significantly altered among the four splenic lesion types, such as MLC1, ERAS, MOV10L1, LOC102152143, COL4A1, COL4A2, COL12A1, NOTCH3, PLOD2, CPXM2, MRC1, GALNT5, TIMP1, and TFPI2. Many of these genes have previously been implicated in tumorigenesis and metastasis in other malignancies. These findings suggest that dysregulated gene expression may contribute to the activation of stromal cells and macrophages within the spleen, facilitating malignant transformation. Overall, these findings deliver novel transcriptomic insights into canine splenic tumorigenesis that may improve diagnostic precision, inform prognostic assessment, and support the development of targeted therapeutic strategies in veterinary oncology. [ABSTRACT FROM AUTHOR]*