A murine hybridoma monoclonal antibody (MAB), IBF9, was generated by fusing myeloma cells (P3X63Ag8.653) with spleen cells from a BALB/c mouse immunized with the canine melanoma cell line CML-10c7. Initial screening of hybridoma antibodies was performed by use of an indirect immunoperomidase assay on formalin-fixed CML-10c7 cells. The isotype of MAB IBF9 was IgG1 as determined by radial gel immunodiffusion. The antibody was tested for reactivity against a panel of formalin-fixed, paraffin-embedded normal and neoplastic canine tissues, using immunoperoxidase staining. Immunostaining was observed in melanomas (24 of 38), a few carcinomas, basal cell tumors, and cutaneous lymphosarcomas. Immunostaining was not observed in fibrosarcomas, hemangiosarcomas, hemangiopericytomas, or histiocytomas. Staining of normal adult canine tissues was limited to a few epithehal tissues and a small percentage of lymphocytes. Fetal tissues were not reactive with MAB IBF9. There were statistically significant differences in frequency of reactivity among melanomas with regard to oral vs non-oral, malignant vs benign, and mitotic indices greater than or equal to 1 vs mitotic indices < 1. Differences were not significant when tumors were compared for degree of pigmentation or histologic type. On the basis of these findings, we suggest that MAB IBF9 may be of assistance in diagnosis of nonpigmented melanomas and in assessing the malignant potential of melanomas.

OBJECTIVE To characterize the distribution and intensity of cyclooxygenase (COX)-2 expression in the eyes of cats with and without uveitis and to determine whether COX-2 expression is correlated with severity of inflammation. SAMPLES Archived ocular tissue specimens from 51 cats with and 10 cats without ocular disease. PROCEDURES Specimens from only 1 eye were evaluated for each cat. Specimens were stained with H&E stain or immunohistochemical stain for detection of COX-2 and reviewed. For each eye, the type, severity, and distribution of inflammation and the distribution and intensity of COX-2 expression were determined for the uvea and other ocular tissues. Correlation between COX-2 expression and inflammation severity was also assessed. RESULTS COX-2 was not expressed in any nondiseased eye. Of the 51 diseased eyes, 20 had histologic evidence of lymphocytic-plasmacytic uveitis, 13 had neutrophilic uveitis, 11 had diffuse iris melanoma with uveitis, and 7 had diffuse iris melanoma without uveitis. Of the 44 eyes with uveitis, COX-2 was detected in the uvea of 16, including 11 eyes with lymphocytic-plasmacytic uveitis, 4 with neutrophilic uveitis, and 1 with diffuse iris melanoma–induced uveitis. Inflammation was severe, moderate, or mild in 10, 5, and 1 of those eyes, respectively. Cyclooxygenase-2 was detected in the cornea of 21 eyes with uveitis and 1 eye with diffuse iris melanoma without uveitis. Uveitis severity was positively correlated with COX-2 expression in both the uvea and cornea. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that COX-2 is an inflammatory mediator in feline uveitis but not diffuse iris melanoma.

A murine hybridoma monoclonal antibody (MAB), IBF9, was generated by fusing myeloma cells (P3X63Ag8.653) with spleen cells from a BALB/c mouse immunized with the canine melanoma cell line CML-10c7. Initial screening of hybridoma antibodies was performed by use of an indirect immunoperomidase assay on formalin-fixed CML-10c7 cells. The isotype of MAB IBF9 was IgG1 as determined by radial gel immunodiffusion. The antibody was tested for reactivity against a panel of formalin-fixed, paraffin-embedded normal and neoplastic canine tissues, using immunoperoxidase staining. Immunostaining was observed in melanomas (24 of 38), a few carcinomas, basal cell tumors, and cutaneous lymphosarcomas. Immunostaining was not observed in fibrosarcomas, hemangiosarcomas, hemangiopericytomas, or histiocytomas. Staining of normal adult canine tissues was limited to a few epithehal tissues and a small percentage of lymphocytes. Fetal tissues were not reactive with MAB IBF9. There were statistically significant differences in frequency of reactivity among melanomas with regard to oral vs non-oral, malignant vs benign, and mitotic indices greater than or equal to 1 vs mitotic indices < 1. Differences were not significant when tumors were compared for degree of pigmentation or histologic type. On the basis of these findings, we suggest that MAB IBF9 may be of assistance in diagnosis of nonpigmented melanomas and in assessing the malignant potential of melanomas.

Objective—To evaluate expression of matrix metalloproteinase (MMP)-2 and -9 and membrane-type 1 MMP (MT1-MMP) in melanocytomas and malignant melanomas of dogs, analyze in vitro production of MMPs by canine melanoma cell lines and primary dermal fibroblasts, and investigate mutual communication between tumor cells and fibroblasts and the influence of collagen on MMP regulation. Sample—35 biopsy specimens from melanocytic tumors and primary dermal fibroblasts of dogs and 3 canine melanoma cell lines (CML-1, CML-10c2, and CML-6M). Procedures—MMP-2, MMP-9, and MT1-MMP were detected in tumor samples by use of unohistochemical analysis. In vitro production was analyzed via reverse transcriptase-PCR assay, immunocytochemical analysis, zymography, and immunoblotting. Results—MMP-9 was overexpressed in malignant melanomas, compared with expression in melanocytomas, whereas no significant differences in MMP-2 and MT1-MMP immunostaining were detected. Stromal cells also often had positive staining results. In vitro, all 3 melanoma cell lines and dermal fibroblasts had evidence of MMP-2 and MT1-MMP, but only melanoma cells had evidence of MMP-9. Coculture of CML-1 or CML-10c2 cells and dermal fibroblasts induced an increase in expression of the active form of MMP-2. Culture of melanoma cells on type I collagen increased the activation state of MT1-MMP. Conclusions and Clinical Relevance—MMP-9 expression was increased in malignant melanomas of dogs. Stromal cells were a source for MMPs. Stromal cells, in combination with matrix components such as type I collagen, can interact with tumor cells to regulate MMP production. Information about MMP production and regulation could help in the development of new treatments.

Immunohistochemistry has been used extensively to evaluate protein expression in clinical and research settings. However, immunohistochemistry is not always successful in veterinary medicine due to the lack of reliable antibody options, poor tissue preservation, labor-intensive staining, and antigen-retrieval optimization processes. RNAscope in-situ hybridization (ISH) is a powerful technology that uses a specific sequence probe to identify targeted mRNA. In this study, we demonstrate RNAscope ISH in 4 common canine malignancies, which are traditionally diagnosed by histopathology and immunohistochemistry. Probes were designed for commonly targeted mRNA markers of neoplastic tumors; these included c-kit in mast cell tumor, microphthalmia-associated transcription factor in malignant melanoma, ionized calcium-binding adapter molecule(-1) in histiocytic sarcoma, and alkaline phosphatase in osteosarcoma. A strong staining signal was obtained by these 4 targets in each canine malignancy. These results support the use of RNAscope ISH for definitive diagnosis in canine malignancies.

Piroxicam and other nonsteroidal anti-inflammatory drugs (NSAID) have antitumor activity against naturally acquired cancer in dogs and human beings, and against experimentally induced tumors in rodents. We are investigating potential mechanisms of NSAID anti-tumor activity. The direct cytotoxicity of piroxicam, indomethacin, and aspirin against 4, canine tumor cell lines (transitional cell carcinoma, squamous cell carcinoma, melanoma, and soft tissue sarcoma) was determined in short-term growth rate assays and in clonogenic assays. Piroxicam was evaluated alone and in combination with the lipoxygenase inhibitor zileuton, and in combination with the chemotherapeutic agents cisplatin and carboplatin. The 50% inhibitory concentrations (IC50) against melanoma cells in short-term growth rate assays were: 530 micromolar piroxicam, 180 micromolar indomethacin, and greater than 1 mM aspirin. These IC50 values were over 10 times greater than serum concentrations of these drugs that could safely be achieved in vivo. The IC50 of zileuton combined with piroxicam (280 micromolar) was not different from the IC50 of zileuton alone (230 micromolar; ANOVA P = 0.47) in melanoma cells. Similarly, addition of piroxicam did not alter the IC50 of either cisplatin (1.6 micromolar) or carboplatin (6.1 micromolar). These results suggest that NSAID, at serum concentrations achievable in vivo, do not have direct cytotoxicity against canine tumor cells tested. It is unlikely that the in vivo antitumor activity of NSAID is attributable to a direct cytotoxic effect.

Specimens of neoplastic tissues from 19 dogs and 4 cats were examined immunohistochemically for intermediate filament expression, using commercially available antibodies. Staining was observed in a wide range of tumor tissues and in normal internal controls by use of antibodies to vimentin, desmin, glial fibrillary acidic protein, and low and high molecular weight cytokeratins. Intermediate filament expression was found to be consistent with light and/or electron microscopic findings, and hence believed to be an accurate indicator of tumor histogenesis in cats and dogs. Three fixatives were evaluated for their relative abilities to preserve antigenicity. Absolute alcohol was superior to B5 fixative and both were superior to formalin. Some tissues that clearly displayed intermediate filament antigens with alcohol and B5 fixative failed to stain when fixed in formalin.

The effect of Fusarium-produced T-2 toxin on tumor growth was evaluated in ICR, CFW, and C57B6/6 mice inoculated with murine sarcoma, Ehrlich ascites carcinoma, or B16F1 melanoma tumor cell lines. Mice were given T-2 toxin intragastrically either at the rate of 2 mg of toxin/kg of body weight daily for 5 days or a single dosage of 4 mg of toxin/kg and were inoculated SC with tumor cells 1 or 2 days after administration of toxin. Tumor growth was assessed 15 to 41 days after tumor challenge by determining the frequency of tumor development and tumor weights. Significant increases in the frequency of development of murine sarcoma (P < 0.005). Ehrlich ascites carcinoma (P < 0.01), and B16F1 melanoma tumors (P < 0.05) were detected in toxin-treated mice, compared with control mice. Murine sarcoma and B16F1 melanoma tumor weights also were significantly (P < 0.01) higher in toxin-treated mice. The effect of T-2 toxin on tumor growth was more marked after 5 daily treatments than after a single dose.

A one and half year old heifer was presented with a hard mass hanging from brisket region. The mass was oval shaped, 10 x15 cm in size, pedunculated with the base at the level of carpal joint. Cytological examination revealed melanocytes with the presence of numerous blackish melanin pigments in the cytoplasm. Histopathological examination revealed that the tumor was comprised of heavily pigmented spindle shaped cells, arranged in sheets with a large nuclei and abundant granular black pigment in the cytoplasm. Following surgical removal of the mass, the animal recovered with no recurrence. Based on clinical examination, cytology and histopathological findings, the growth was diagnosed as cutaneous melanoma.