Flow Cytometric Panel and Liquid Biopsy to Identify Neoplastic Conditions in Canine Effusions

Serous effusions in dogs are a frequent clinical finding that may reflect inflammatory, infectious, or neoplastic diseases. However, differentiating between benign and malignant effusions, particularly the non-hematopoietic (NH) cell populations, including epithelial and mesothelial cells, remains one of the most significant diagnostic challenges. While cytology remains the gold standard for effusion analysis, its sensitivity is frequently limited, especially when distinguishing reactive from neoplastic mesothelium or determining the tumour origin. The aim of this PhD project was to improve the diagnosis of canine cavitary effusions by applying a multimodal approach involving flow cytometry (FC), immunohistochemistry (IHC) on cell blocks, next-generation sequencing (NGS), and droplet digital PCR (ddPCR). The first phase aimed to evaluate FC as a potential alternative to IHC on cell blocks for the immunophenotyping of NH cells, demonstrating a high agreement between the two methods using a panel including cytokeratin, vimentin, and desmin. Further markers—CD44, CALB2, WT1, PDPN and EpCAM—were tested to optimize the diagnostic accuracy and facilitate the discrimination between epithelial and mesothelial cells and between the benign and malignant populations. Subsequently, the first genomic data on canine mesothelioma, a rare and genomically unexplored tumor, were obtained and analyzed. The sequencing of tumor biopsies revealed potentially relevant mutations for diagnostic and therapeutic use, paving the way for future veterinary oncogenomic studies. Finally, the project addressed diagnostic challenges through liquid biopsy approach by developing a ddPCR assay to detect a recurrent mutation in canine pulmonary carcinomas, using the effusion fluid as a diagnostic matrix. This method showed both high sensitivity and specificity, highlighting the potential of minimally invasive approaches in molecular diagnosis. Overall, this project enables and provides the basis for the incorporation of cytometric and molecular advanced techniques into veterinary diagnosis, contributing to overcome the main diagnostic challenges related to the analysis of canine effusions, particularly in NH neoplasms.