Expression of vascular endothelial growth factor in canine oral malignant melanoma

Serum, plasma and tissue expression of vascular endothelial growth factor (VEGF) was measured in 20 dogs previously diagnosed histologically with oral melanoma. The concentrations of VEGF in serum and plasma were significantly higher in dogs with melanoma than in a control population (P ≤ 0.002). Concentrations of VEGF in the serum and plasma of dogs with melanoma were highly correlated (r = 0.867). Ninety‐five per cent of melanoma tissues expressed VEGF. Two staining patterns were detected: diffuse and granular cytoplasmic staining. High blood concentrations of VEGF were correlated to a shorter survival time in dogs receiving definitive therapy (P = 0.002). Survival times were significantly longer in dogs receiving definitive therapy versus palliative therapy (median 496 versus 97 days, P = 0.007). Blood concentrations of VEGF were associated with stage (P < 0.05). Dogs with oral melanoma have increased serum, plasma and tissue concentrations of VEGF. Increased expression of VEGF may be a reasonable target for future therapy of canine oral melanoma.     

Comparative study of assays for stimulated fluid pinocytosis in canine and human neutrophils

We compared a flow cytometric assay of fluid pinocytosis in stimulated neutrophils with previously used methods, including spectrophotofluorometric assay of fluoresceinated dextran (FD) uptake and beta scintillation spectroscopic assay of radiolabeled sucrose uptake. Neutrophils from human and canine blood were stimulated with formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate, respectively. Flow cytometry was used to quantitate uptake of FD and lucifer yellow, spectrophotometry was used to quantitate uptake of FD, and spectroscopy was used to quantitate uptake of 14C-labeled sucrose. The flow cytometric assay, using either FD or lucifer yellow, was found to be superior on the basis of maximal incremental increase observed, minimal variability within replicate experiments, and maximal sensitivity to detect early stimulation of fluid pinocytosis in neutrophils.     

Comparative analysis of MAPK and PI3K/AKT pathway activation and inhibition in human and canine melanoma

The lack of advanced animal models of human cancers is considered a barrier to developing effective therapeutics. Canine and human melanomas are histologically disparate but show similar disease progression and response to therapies. The purpose of these studies was to compare human and canine melanoma tumours and cell lines regarding MAPK and PI3K/AKT signalling dysregulation, and response to select molecularly targeted agents. Pathway activation was investigated via microarray and mutational analysis. Growth inhibition and cell cycle effects were assessed for pathway inhibitors AZD6244 (MAPK) and rapamycin (PI3K/AKT) in human and canine melanoma cells. Human and canine melanoma share similar differential gene expression patterns within the MAPK and PI3K/AKT pathways. Constitutive pathway activation and similar sensitivity to AZD6244 and rapamycin was observed in human and canine cells. These results show that human and canine melanoma share activation and sensitivity to inhibition of cancer‐related signalling pathways despite differences in activating mutations.     

Induction of dendritic cell-mediated immune responses against canine malignant melanoma cells

To establish the basis for the use of dendritic cells (DC) in the treatment of canine melanoma, dogs were vaccinated using autologous DC pulsed with canine melanoma CMM2 cell lysate in the presence of keyhole limpet haemocyanin (KLH) in vitro (CMM2-KLH-DC), and the induction of immune responses against CMM2 cells in vivo was examined using the delayed-type hypersensitivity (DTH) skin test. The DTH responses against CMM2 cells and KLH were observed in dogs vaccinated with CMM2-KLH-DC, while the responses against KLH but not CMM2 cells were detected with DC pulsed with KLH alone (KLH-DC). Recruitment of CD8 and CD4 T cells was detected in the positively responding sites, suggested that vaccination with CMM2-KLH-DC efficiently elicits T cell-mediated immunity against CMM-2 cells in vivo. These findings demonstrate the potential utility of DC-based tumour vaccination in the treatment of canine malignant melanoma.     

Pathology of canine oral malignant melanoma with cartilage and/or osteoid formation

Of 197 cases of canine oral malignant melanoma, 29 cases with myxoid, cartilage, and osteoid formation were studied pathologically and immunohistochemically. Tumor tissues were classified into spindle cell type (13 cases), epithelioid cell type (1 case), and mixed type (15 cases). Myxoid matrixes (29 tumors) were formed mainly in the tissues of spindle cell type and were positive for Alcian blue (pH 2.5). Cartilaginous matrixes (12 tumors) were formed in the myxoid tumor tissues. The morphology of atrophied neoplastic cells, which were embedded in the cartilage cavities, significantly differed from that of spindle cells proliferating in surroundings. There were reticular areas in the process of transitioning from myxoid to cartilaginous matrixes. Osteoid matrixes were not continuous with myxoid or cartilaginous matrixes, and arose as eosinophilic trabeculae in the dense collagenous connective tissues. A calcified bone trabecula was present among the osteoid trabeculae in a case. Melanin-producing melanocytes were proliferating in the collagenous matrixes, while amelanotic cells were in the osteoid matrixes. Immunohistochemistry demonstrated proliferating neoplastic cells as melanocytes. All cells in/out of these three matrixes were positive for Melan-A, S-100 protein, NSE, and vimentin. From these results, it is suggested that cartilage and osteoid matrixes are produced by dedifferentiated melanocytes.     

Synthetic microRNA‐205 exhibited tumour suppression in spontaneous canine malignant melanoma by intratumoral injection

MicroRNAs (miRNA) are small, noncoding RNA molecules consisting of 18 to 25 nucleotides. Malignant melanomas (MMs) are one of the most common malignancies in both dogs and humans. We previously reported that chemically modified synthetic miRNA‐205 (miR‐205BP/S3) inhibits melanoma growth in vitro and in vivo. The present study aimed to evaluate the efficacy of intratumoral administration of synthetic miR‐205 for spontaneous CMMs and to evaluate its potential as systemic therapy. Ten dogs with various stages of MM were treated with miR‐205BP/S3 injected into tumours. Adverse effects (AEs) were assessed in accordance with the Veterinary Cooperative Oncology Group‐Common Terminology Criteria for Adverse Events (VCOG‐CTCAE) v1.1 guidelines. Five cases attained complete remission (CR), three attained stable disease (SD), and two cases displayed characteristics of progressive disease (PD). In all cases, no changes were observed in the blood parameters upon miRNA administration, and miR‐205BP/S3 administration did not yield any side effects. The present results suggest that intratumoral administration of miR‐205BP/S3 is a potentially applicable treatment for canine melanoma.     

Chondroitin sulfate proteoglycan-4: a biomarker and a potential immunotherapeutic target for canine malignant melanoma

Chondroitin sulfate proteoglycan-4 (CSPG4), also known as high molecular weight-melanoma associated antigen (HMW-MAA), is a membrane-bound chondroitin sulfate proteoglycan highly expressed by human melanoma cells. This phylogenetically conserved tumour antigen plays an important biological role in human melanoma, where it is used as a marker to diagnose forms with unusual characteristics, such as desmoplastic melanoma, and to detect melanoma cells in lymph nodes and peripheral blood, and as a target for immunotherapy because of its restricted distribution in normal tissues. To identify suitable targets to develop novel approaches of treating canine melanoma, CSPG4 was studies to see whether it is expressed in canine malignant melanomas. Immunohistochemical staining of 65 canine malignant melanomas with an anti-human CSPG4-specific antibody detected CSPG4 in 37 cases (56.9%). Positive staining was more frequent, albeit not significantly, in amelanotic compared to melanotic tumours and was statistically associated with tumours having both melanin and the epithelioid histotype. The frequency of CSPG4 expression was similar to that of other melanoma antigens used as diagnostic markers for canine malignant melanoma, such as Melan A and the protein recognized by the PNL2 monoclonal antibody. The results suggest that CSPG4 constitutes a new potential immunohistochemical marker of canine malignant melanoma and may represent an immunotherapeutic target as in humans.     

MicroRNAs as tumour suppressors in canine and human melanoma cells and as a prognostic factor in canine melanomas

Malignant melanoma (MM) is one of the most aggressive cancers in dogs and in humans. However, the molecular mechanisms of its development and progression remain unclear. Presently, we examined the expression profile of microRNAs (miRs) in canine oral MM tissues and paired normal oral mucosa tissues by using the microRNA‐microarray assay and quantitative RT‐PCR. Importantly, a decreased expression of miR‐203 was significantly associated with a shorter survival time. Also, miR‐203 and ‐205 were markedly down‐regulated in canine and human MM cell lines tested. Furthermore, the ectopic expression of miR‐205 had a significant inhibitory effect on the cell growth of canine and human melanoma cells tested by targeting erbb3. Our data suggest that miR‐203 is a new prognostic factor in canine oral MMs and that miR‐205 functions as a tumour suppressor by targeting erbb3 in both canine and human MM cells.     

Comparative histopathological study of the canine thyroid gland in London and Munich

The thyroid glands of two series of ninety-nine unselected dogs from consecutive autopsies in London and in Munich were examined histologically. The prevalence of goitre and of neoplasia was distinctly higher in dogs in the Munich series.
Résumé. On a examiné de façon histologiyue les glandes thyroïdes de deux séries de quatrevingt-dix-neufs chiens d'après des autopsies consécutives à Londres et à Munich. La prédominance de goîtres et de néoplasie était nettement plus élevée chez les chien de la série de Munich
Zusammenfassung. Die Schilddrüsen von zwei Serien von 99 Hunden Wurden von darauf-folgenden Autopsien in London und München histologisch untersucht. Das Vorkommenwon Kropf und Neoplasia war entschieden höher in den Hunden der Munchener Serie.     

Use of immunocytochemical techniques in canine melanoma

The staining patterns of the monoclonal antibodies S-100 and Melan-A in canine melanoma were assessed based on cytological specimens of six canine melanomas (four benign, two malignant). In addition, eight regional lymph nodes of the two dogs with malignant melanomas were stained using these markers. For reference, all specimens were also evaluated immunohistochemically using S-100 and Melan-A. To assess the immunocytochemical specificity of both antibodies, various canine tumours and normal tissues were stained. The immunocytochemical staining results of the canine melanomas and the regional lymph nodes showed high conformity with the immunohistochemical reactivity patterns for S-100 and Melan-A. The specificity of Melan-A was higher compared with S-100. Melan-A, in particular, may be helpful for the cytological diagnosis of canine melanoma.     

Cytology of canine malignant histiocytosis

Cytologic features of bone marrow, tissue, and abdominal fluid in seven cases of malignant histiocytosis in dogs are described, and histopathology, hematology, and serum biochemistry of the cases are reviewed. Diagnosis of malignant histiocytosis was confirmed by tissue morphology and immunohistochemistry; neoplastic cells in all cases had positive immunoreactivity to lysozyme. This stain can be used to definitively establish the diagnosis of malignant histiocytosis on cytology specimens as well as tissue sections. Cytologic findings included numerous pleomorphic, large, discrete mononuclear cells with abundant, lightly basophilic, vacuolated, granular cytoplasm. Nuclei were round to oval to reniform with marked anisocytosis and anisokaryosis; nucleoli were prominent. Mitotic figures, often bizarre, were occasionally seen. Multinucleated giant cells and phagocytosis of erythrocytes and leukocytes were prominent features in cytologic preparations in four cases. Four dogs were anemic, five dogs were thrombocytopenic, and three dogs were hypercalcemic. Breeds affected included Doberman Pinscher (1), Golden Retriever (2), Flat Coated Retriever (3), and mixed-breed dog (1).     

Efficacy and side effects of radiation therapy in comparison with radiation therapy and temozolomide in the treatment of measurable canine malignant melanoma

Prognosis for unresectable canine malignant melanoma (MM) is typically poor, and therapeutic approaches remain largely palliative. A bi‐institutional trial was conducted to compare efficacy and safety of radiation therapy (RT) and RT with post‐radiation temozolomide in dogs with chemotherapy‐naïve, measurable MM. RT consisted of 5 × 6 Gy fractions over 2.5 weeks. Dogs whose owners wished to pursue chemotherapy received adjuvant oral temozolomide (60 mg m^?2 for 5 days every 28 days). Fifteen dogs were treated with RT only (Group 1) and 12 dogs subsequently received temozolomide (Group 2). Overall response rate was similar between Group 1 (86.7%) and Group 2 (81.1%). Median time to progression (TTP) was significantly longer in Group 2 (205 days) compared to Group 1 (110 days; p = 0.046). Survival time was not significantly different between groups. Both treatments were well tolerated. Post‐radiation temozolomide has a good safety profile, and may improve TTP in MM when compared to coarse fractionated RT.