Osteosarcoma (OSA) represents the most common primary bone tumor in dogs and is characterized by a highly aggressive behavior. Cell lines represent one of the most suitable and reproducible pre-clinical models, and therefore the knowledge of their molecular landscape is mandatory to investigate oncogenic mechanisms and drug response. The present study aims at determining variants, putative driver genes, and gene expression aberrations by integrating whole-exome and RNA sequencing. For this purpose, eight canine OSA cell lines and one matched pair of primary tumor and normal tissue were analyzed. Overall, cell lines revealed a mean tumor mutational burden of 9.6 mutations/Mb (range 3.9–16.8). Several known oncogenes and tumor suppressor genes, such as ALK, MYC, and MET, were prioritized as having a likely role in canine OSA. Mutations in eight genes, previously described as human OSA drivers and including TP53, PTCH1, MED12, and PI3KCA, were retrieved in our cell lines. When variants were cross-referenced with human OSA driver mutations, the E273K mutation of TP53 was identified in the Wall cell line and tumor sample. The transcriptome profiling detected two possible p53 inactivation mechanisms in the Wall cell line on the one hand, and in D17 and D22 on the other. Moreover, MET overexpression, potentially leading to MAPK/ERK pathway activation, was observed in D17 and D22 cell lines. In conclusion, our data provide the molecular characterization of a large number of canine OSA cell lines, allowing future investigations on potential therapeutic targets and associated biomarkers. Notably, the Wall cell line represents a valuable model to empower prospective in vitro studies both in human and in dogs, since the TP53 driver mutation was maintained during cell line establishment and was widely reported as a mutation hotspot in several human cancers.

Osteosarcoma is the most common primary malignant bone tumour in dogs, characterized by a locally aggressive and highly metastatic behaviour. Despite the current standards of care, most dogs succumb to the disease, indicating the need for novel treatment strategies. Polo-like kinase 1 (PLK1) is dysregulated in a variety of human cancer types, including osteosarcoma, and induces c-Myc accumulation. The crosstalk between the two molecules coordinates cell proliferation, differentiation, self-renewal and apoptosis. Therefore, PLK1 has recently emerged as a potential therapeutic target, mainly in tumours overexpressing c-Myc. BI 2536 is a selective PLK1 inhibitor promoting mitotic arrest and apoptosis in a variety of cancer cells. This research aimed at evaluating PLK1 and c-Myc protein expression in 53 appendicular canine osteosarcoma (cOSA) samples and the in vitro effects of BI 2536 on a c-Myc and PLK1-overexpressing cOSA cell line (D17). PLK1 and c-Myc expression in cOSA samples showed no correlation with clinicopathological data. However, c-Myc overexpression was associated with a significantly reduced overall survival (p = .003). Western Blot and RT-qPCR assays revealed that D17 expressed high protein and transcript levels of both PLK1 and MYC. When treated with BI 2536 (range 2.5-15 nM) for 24 h, D17 showed a substantial decrease in cell growth, inducing apoptosis and G(2)/M cell cycle arrest. Interestingly, under BI 2536 treatment, D17 showed decreased c-Myc protein levels. Consistent with human OSA, these preliminary data outline the prognostic value of c-Myc expression in cOSA and highlight the potential role of PLK1 as an antiproliferative therapeutic target for tumours overexpressing c-Myc.

We report the pathological and molecular findings in an adult male Himalayan red panda (Ailurus fulgens fulgens) whose death was attributed to parenchymal brain haemorrhage (PBH) of the thalamus. Post-mortem examination revealed severe, acute PBH and intraventricular haemorrhage with major involvement of the thalamus, as well as scattered chronic microinfarctions. Vascular disease in the brain and other organs was suggestive of systemic hypertension. Histological lesions included arteriolar hyalinosis and varying degrees of arteriosclerosis, arterial tunica media hypertrophy and hyperplasia and infiltration of arterial walls by lipid-laden macrophages. Other relevant findings included marked myocardial fibrosis, lymphoplasmacytic tubulointerstitial nephritis, lymphoplasmacytic meningoencephalitis and chronic mitral valve degeneration. The changes in the cerebral vasculature were consistent with hypertensive encephalopathy and a cerebrovascular accident, specifically PBH, which has not been previously reported in this species. Additionally, polymerase chain reaction analysis for red panda amdoparvovirus (RPAV) was positive in the brain and kidneys. Preceded by hypertensive vascular changes and brain microinfarctions, sudden death in this animal likely resulted from fatal PBH with intraventricular haemorrhage. The clinicopathological role of RPAV infection is unknown in this case, although its contribution to the chronic renal disease is considered possible in the context of our current understanding of RPAV-associated pathology.

Canine osteosarcoma (OSA) is an aggressive and highly malignant tumor of bone with a poor prognosis and it mirrors the disease in humans. Angiogenesis, the formation of new blood vessels, is driven by hypoxia-induced factors such as HIF-1α and VEGF, both of which play a crucial role in tumor growth and metastasis. However, the role of angiogenesis in OSA remains a topic of ongoing debate. This study aimed to investigate the relationship between angiogenesis, measured by intratumoral microvessel density (MVD), hypoxic markers, and clinical outcomes in 28 dogs diagnosed with appendicular OSA. Clinicopathological data such as age, breed distribution, tumor localization, histopathological subtypes, and metastatic behavior were consistent with reported epidemiologic characteristics of canine OSA, though no significant correlation was found among these variables. The results indicated a significant association between higher MVD and high-grade OSA (p = 0.029), suggesting that increased tumor vascularization is linked to more aggressive tumor behavior. Additionally, elevated VEGF expression was strongly correlated with disease-free interval DFI), with a p-value of 0.045. Although HIF-1α positivity showed a trend towards poorer survival, the results did not reach statistical significance (p = 0.07). These findings highlight the potential role of VEGF as a valuable prognostic marker in canine OSA, which could have potentially important implications for therapeutic targeting and clinical management of the disease. This study advances the understanding of angiogenesis in canine OSA, while emphasizing the need for continued research into the complex mechanisms regulating the interplay between hypoxia, angiogenesis and tumor progression.

Formaldehyde-based fixation is the most used chemical system for histopathological examination worldwide. However, its toxicity is well known, and preservation of gross features, proteins, and nucleic acids is not optimal. Alternative fixatives resulting in similar morphological tissue quality and costs, but with reduced toxicity and with better preservation of gross features, proteins, and nucleic acids would increase operator safety and application possibilities in pathology. This multi-institutional study aimed to compare the morphological, histochemical, immunohistochemical (IHC), and molecular outcomes of fixation with a newly patented, non-toxic, acid-free glyoxal (GAF) fixative with neutral-buffered formalin (NBF). Fifty-nine tissue biopsies and 21 necropsies of different animal species were analyzed. Gross features were preserved after GAF fixation, with no tissue hardening or discoloration. Cellular ultrastructure was better preserved with GAF. Histology, histochemistry, and in situ hybridization results from GAF-fixed samples were mainly equal when compared to NBF-fixed samples, except for the loss of mast cell granules in GAF-fixed samples compared to NBF. IHC analyses showed comparable results with slight and rare protocol adjustment. DNA yields were higher and amplification of selected genes (ie, TP53 and COX1 ) was more efficient in GAF-fixed biopsies ( P < .05). DNA and RNA yields were higher also in necropsy GAF-fixed tissues, but no difference was detected for selected gene amplification (ie, COX1, GAPDH, β-actin ). Based on these data, despite not yet being economically competitive, GAF could represent a valuable alternative to NBF for standard laboratory applications, while also improving on-field sampling and teaching applications.

Formaldehyde fixation is worldwide the most used system for histopathological examination. However, its toxicity is well known, and preservation of proteins and nucleic acids is not optimal. Alternative fixatives warranting similar morphological quality of tissues and costs, but lacking toxicity and allowing better preservation of proteins and nucleic acids would therefore increase both safety of operators and quality of molecular analysis in pathology. This multi-institutional study aimed to compare the morphological, histochemical, immunohistochemical (IHC), and molecular analyses outcomes of a newly patented, non-toxic, acid-free Glyoxal (GAF) fixative with neutral buffered formaldehyde (NBF). Tissues from a total of 73 subjects were analyzed, including 13 necropsies. Gross features were preserved after GAF fixation, with no tissue hardening or discoloration. Cellular ultrastructure was also better preserved with GAF and histology and histochemistry on GAF-fixed samples showed good results when compared to NBF-fixed samples, with the exception of loss of tinctorial affinity of erythrocytes and mast cell granules. IHC analyses also showed comparable results with only slight and rare protocol adjustment. DNA and RNA yields were higher from GAF-fixed samples (P<0.05) and the tested genes ( p53 and COX1 ) were better amplified. RNA scope showed positive results for c-KIT expression in GAF-fixed mast cell tumors. Based on these data, the non-toxic GAF fixative allows good macroscopical, histological and immunohistochemical analyses of tissue samples, including on-field application, and better molecular analyses when compared to NBF. This represents a promising possibility for teaching, diagnostic, and research in veterinary pathology.