Histological and proteomic characterization of hyaline material in two dogs with hyalinizing pancreatic adenocarcinoma

Hyalinizing pancreatic adenocarcinoma is a rare subtype of exocrine pancreatic carcinoma in dogs, characterized by abundant extracellular hyaline material within the tumor. In this study, we investigated two canine cases exhibiting this distinctive hyalinizing morphology using histological, proteomic, immunohistochemical, ultrastructural, and molecular approaches. Histological examination revealed amyloid-like eosinophilic hyaline deposits; however, these deposits lacked diagnostic features of amyloid, as confirmed by Congo red and thioflavin S staining and transmission electron microscopy. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified bile salt-activated lipase (BAL) as the predominant component of the hyaline deposits. Immunohistochemistry confirmed strong anti-BAL immunoreactivity in the deposits. In addition to BAL, amyloid-associated proteins, including clusterin, vitronectin, apolipoprotein E, and apolipoprotein A-I, were detected. Together with the negative Congo red and thioflavin S staining and ultrastructural findings, these results support that the hyaline deposits are BAL-rich, non-amyloid extracellular deposits. Some deposits also contained collagen, indicating biochemical heterogeneity of the hyaline material. RT-qPCR analysis suggested higher CEL, and lower GRP94 expression in hyalinizing pancreatic adenocarcinomas than in non-hyalinizing pancreatic carcinomas. These observations raise a hypothesis that excessive BAL production, together with reduced GRP94-mediated chaperone support, may contribute to extracellular accumulation of aggregated BAL. Collectively, this study identifies BAL as the predominant component of hyaline deposits in canine hyalinizing pancreatic adenocarcinoma based on LC-MS/MS analysis and highlights a non-amyloid protein aggregation process associated with this tumor subtype.