Lipid droplets and perilipins in canine osteosarcoma. Investigations on tumor tissue, 2D and 3D cell culture models.

Lipid droplets were identified as important players in biological processes of various tumor types. With emphasis on lipid droplet-coating proteins (perilipins, PLINs), this study intended to shed light on the presence and formation of lipid droplets in canine osteosarcoma. For this purpose, canine osteosarcoma tissue samples (n = 11) were analyzed via immunohistochemistry and electron microscopy for lipid droplets and lipid droplet-coating proteins (PLINs). Additionally, we used the canine osteosarcoma cell lines D-17 and COS4288 in 2D monolayer and 3D spheroid (cultivated for 7, 14, and 21 days) in vitro models, and further analyzed the samples by means of histochemistry, immunofluorescence, molecular biological techniques (RT-qPCR, Western Blot) and electron microscopical imaging. Lipid droplets, PLIN2, and PLIN3 were detected in osteosarcoma tissue samples as well as in 2D and 3D cultivated D-17 and COS4288 cells. In spheroids, specific distribution patterns of lipid droplets and perilipins were identified, taking into consideration cell line specific zonal apportionment. Upon external lipid supplementation (oleic acid), a rise of lipid droplet amount accompanied with an increase of PLIN2 expression was observed. Detailed electron microscopical analyzes revealed that lipid droplet sizes in tumor tissue were comparable to that of 3D spheroid models. Moreover, the biggest lipid droplets were found in the central zone of the spheroids at all sampling time-points, reaching their maximum size at 21 days. Thus, the 3D spheroids can be considered as a relevant in vitro model for further studies focusing on lipid droplets biology and function in osteosarcoma.

Protein expression of KIT, BRAF, GNA11, GNAQ and RASSF1 in feline diffuse iris melanomas.

Feline iris melanoma, the most common feline intraocular tumour, has a reported metastatic rate of 19-63%. However, there is a lack of knowledge about its molecular biology. Previous studies have reported that feline iris melanomas do not harbour mutations comparable to common mutations found in their human counterpart. Nevertheless, there are differences in the gene expression patterns. The aim of this study was to investigate the protein expression of B-RAF oncogene serine/threonine kinase (BRAF), G protein subunit alpha q (GNAQ) and 11 (GNA11), KIT proto-oncogene receptor tyrosine kinase (KIT), and Ras association family member 1 (RASSF1) in feline iris melanomas. Fifty-seven formalin-fixed paraffin embedded (FFPE) iris melanomas and 25 FFPE eyes without ocular abnormalities were stained with antibodies against the respective proteins using immunofluorescence. Averaged pixel intensities/µm2 and percentage of stained area from total tissue area were measured and the results were compared. Compared to the control group, iris melanomas showed overexpression of BRAF, GNAQ, GNA11 and KIT. The higher expression of BRAF, GNAQ, GNA11 and KIT in feline iris melanomas suggest that these proteins may play a key role in the development of feline iris melanomas and KIT may present a possible target for future therapies in cats with feline iris melanomas.

World Health Organisation Classification of Lymphoid Tumours in Veterinary and Human Medicine: a Comparative Evaluation of Gastrointestinal Lymphomas in 61 Cats.

To diagnose and classify the various entities of lymphomas, the World Health Organisation (WHO) classification is applied in human as well as in veterinary medicine. We validated the concordance of these classification systems by having a veterinary and human pathologist evaluate gastrointestinal lymphoma tissue from 61 cats. In 59% of all cases, there was a match between their respective diagnoses of the lymphoma subtype. A complete consensus between the two evaluators was obtained for all samples with a diagnosis of diffuse large B-cell lymphoma, T-cell anaplastic large cell lymphoma and extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue. A corresponding diagnosis was also made in the majority of samples with enteropathy associated T-cell lymphoma (EATL) type II, although this subtype in cats has similarities to the 'indolent T-cell lymphoproliferative disorder of the gastrointestinal tract', a provisional entity newly added to the revised human WHO classification in 2016. Very little consensus has been found with cases of EATL type I due to the fact that most did not meet all of the criteria of human EATL I. Hence, the human pathologist assigned them to the heterogeneous group of peripheral T-cell lymphomas (not otherwise specified). Consequently, concrete guidelines and advanced immunophenotyping based on the model of human medicine are essential to differentiate these challenging entities in veterinary medicine.

Pretreatment leukocyte ratios and concentrations as predictors of outcome in dogs with cutaneous mast cell tumours.

Leukocyte ratios correlate with outcome in several human cancers. Little is known about their prognostic significance in mast cell tumour (MCT). The aim of the study was to evaluate the prognostic significance of pretreatment leukocyte concentrations and their ratios in dogs with MCT for survival. Medical records of 92 dogs with MCT were retrospectively reviewed. Tumour diagnosis was made by tumour biopsy or fine-needle aspirate. Only dogs without prior treatment were included. Eosinophil, lymphocyte, monocyte and neutrophil concentration were obtained by ADVIA 2120™ (Siemens Healthcare, Vienna, Austria). Neutrophil-to-eosinophil ratio (NER), lymphocyte-to-monocyte ratio (LMR) and neutrophil-to-lymphocyte ratio (NLR) were calculated from collected leukocyte concentrations. Relative eosinophil concentration (REC), NER (P < 0.001), NLR (P = 0.001) and LMR (P < 0.001) were significant prognostic factors for outcome in univariate analysis. REC (P = 0.008) and NER (P = 0.001) remained independent predictors of survival in multiple analyses. Leukocyte concentrations and ratios, especially REC and NER may serve as prognostic indicators in MCT.

Characterization of a PCR-based lymphocyte clonality assay as a complementary tool for the diagnosis of feline lymphoma.

Differentiation between resident mature lymphocyte populations and small cell lymphoma cannot be made by cytological review alone and remains challenging in histopathological review. These cases warrant application of complementary tools like PCR-based immunoglobulin (IG) and T-cell receptor (TCR) clonality testing for confirmation. In this prospective study, diagnostic sensitivity and specificity of different primer sets for routine diagnosis of feline TCR gamma (TCRG) and complete IG heavy chain (IGH) gene rearrangements were assessed. Fine needle aspirates from 20 feline lymphoma cases and lymph node material from 10 cats without hematopoietic neoplasia were subjected to clonality testing. Feline lymphoma cell lines and previously confirmed patient material served as positive control. Detection limits for clonal populations within a polyclonal background was 90% for B-cells and 50% for T-cells. Diagnostic sensitivity and specificity of the clonality assay were 70% and 90%. Overall diagnostic accuracy was 77%, positive predictive value 93% and negative predictive value 60%.

Encephalitozoon cuniculi causes focal anterior cataract and uveitis in dogs.

Three mongrel dogs, aged 10 months (case 1), 14 months (case 2) and 7.5 years (case 3), were presented because of ophthalmologic disorders of 4 months, 6 months and 7 years duration, respectively. All three dogs were offspring of stray dogs from Hungary and Serbia and had positive serum antibody titres against Encephalitozoon (E.) cuniculi. The two young dogs showed unilateral, the older dog bilateral chronic anterior uveitis with posterior synechia and focal anterior cortical cataract. The fundi that could be evaluated developed focal tapetal hyporeflective lesions in the course of the disease. Dogs 1 and 2 underwent removal of the lens via phacoemulsification. PCR of the lens material was positive for E. cuniculi strains IV and II, respectively. In dog 2 findings suggestive of microsporidia were detected underneath the anterior lens capsule by immunohistochemical staining. In all cases medical treatment consisted of systemic fenbendazole, prednisolone, and topical anti-inflammatory drugs, and additional brinzolamid/timolol for dog 3. For the time being all cases (follow up 23 months, 6 months and 3 months, respectively) are still on topical anti-inflammatory therapy. It is concluded that E. cuniculi infections can cause cataract and chorioretinal lesions in dogs.

Murine xenograft model demonstrates significant radio-sensitising effect of liposomal doxorubicin in a combination therapy for Feline Injection Site Sarcoma.

Therapy of cats suffering from feline injection site sarcomas (FISS) is still a challenging problem, as the recurrence rate after surgery is up to 70%. Four FISS-derived primary tumour cell lines and corresponding xenograft tumour mouse models were established to evaluate the efficacy of a concomitant chemo-/radiation therapy with doxorubicin. In vitro, strongly depending upon the timing of administration, pre-treatment with 0.25 µmol doxorubicin resulted in a significant enhancement of radiation-induced (3.5 Gy) tumour cell death. This result was confirmed in vivo, where only the combined chemo-/radiation therapy resulted in a significant reduction in tumour growth compared to the respective mono-therapies with either doxorubicin or radiation. These results support the use of the concomitant chemo-/radiation therapy for adjuvant treatment of FISS, particularly in advanced or recurrent disease where surgery alone is no longer feasible.

Angiogenic markers in canine lymphoma tissues do not predict survival times in chemotherapy treated dogs.

Angiogenesis, which is essential for malignancies to progress, depends on various signalling proteins including vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors 1 and 2 (VEGFR-1 and VEGFR-2). Microvessel density (MVD) is frequently used to evaluate angiogenesis. This study assessed the relationship between expression of VEGF, VEGFR-1 and VEGFR-2, MVD and the survival time in dogs with lymphoma. VEGF, VEGFR-1 and VEGFR-2 expression was evaluated immunohistochemically and microvessel profiles were counted in 34 lymphoma samples. Seventy-nine percent of the samples showed high VEGF expression and 62% were highly positive for VEGFR-1; VEGFR-2 immunoreactivity was mostly negative. Dogs treated with chemotherapy had a median survival time of 266days, but no significant relationships were found between overall survival time, MVD and expression of VEGF, VEGFR-1 or VEGFR-2. In this study, VEGF its receptors and the MVD were no prognostic factors in dogs with lymphoma.

Sudden death in a dog with lymphoplasmacytic hypophysitis.

A 10-year-old dog with a history of progressive anorexia and weight loss died suddenly despite treatment. Histopathological examination revealed severe follicular lymphoplasmacytic adenohypophysitis and atrophy of the zona fasciculata and zona reticularis of the adrenal cortex. It is likely that lack of production of adrenocorticotropic hormone and cortisol was the cause of death of this dog.

Severe chemosis caused by nephrotic syndrome in a goat: a case report.

A 3-year old female goat with nephrotic syndrome was presented because of severe bilateral chemosis. The diagnosis was based on clinical findings, decreased packed cell volume, increased serum urea and creatinine, decreased serum protein and protein loss via the urinary tract. At post mortem examination, a chronic glomerulonephritis and interstitial nephritis were found. In addition to severe conjunctival oedema, ascites and subcutaneous oedema in the region of the larynx were noted. Immunohistochemistry for the detection of chlamydial antigen in the conjunctiva was negative. This is the first report of severe chemosis caused by nephrotic syndrome in a goat.