Cyclin D1 immunohistochemical expression and somatic mutations in canine oral melanoma

Canine oral melanoma (COM) is the most frequent tumour with oral localization in dogs. Copy number gains and amplifications of CCND1, a gene coding for Cyclin D1, are the most frequent chromosomal aberrations described in human non‐UV induced melanomas. Twenty‐eight cases of COM were retrieved from paraffin‐blocks archives. A total of 4 μm thick sections were immunostained with an antibody against human Cyclin D1 and Ki‐67. Cyclin D1 and Ki‐67 expressions were scored through two counting methods. DNA was extracted from 20 μm thick sections of formalin‐fixed paraffin‐embedded blocks. Pathological and surrounding healthy tissue was extracted independently. Cyclin D1 immunolabelling was detected in 69% (18/26) while Ki‐67 was present in 88.5% (23/26) of cases. Statistical analysis revealed correlation between two counting methods for Cyclin D1 (r = 0.54; P = .004) and Ki‐67 (r = 0.56; P = .003). The correlation found between Ki‐67 and Cyclin D1 indexes in 16/26 cases labelled by both antibodies (r = 0.7947; P = .0002) suggests a possible use of Cyclin D1 index as prognostic marker. Polymerase chain reaction analysis on CCND1 coding sequence revealed the presence of nine somatic mutations in seven samples producing synonymous, missense and stop codons. Since none of the single‐nucleotide polymorphisms was found to be recurrent, it is suggested that overexpression of Cyclin D1 may be the consequence of alterations of CCND1 upstream regions or other genetic aberrations not detectable with the methodology used in this study. Future studies are needed to verify the potential use of Cyclin D1 index as prognostic indicator and to highlight the molecular events responsible for Cyclin D1 overexpression in COMs.     

Prognostic value of somatic focal amplifications on chromosome 30 in canine oral melanoma

Canine oral melanoma is the first malignancy of the oral cavity in dogs and is characterized by a local invasiveness and a high metastatic propensity. A better knowledge of genetic alterations is expected to improve management of this tumour. Copy number alterations are known characteristics of mucosal melanomas both in dogs and humans. The goal of this study was to explore the prognostic value of somatic focal amplifications on chromosomes (Canis Familiaris [CFA]) 10 and 30 in canine oral melanoma. The cohort included 73 dogs with oral melanoma confirmed by histology, removed surgically without adjuvant therapy and with a minimal follow‐up of 6 months. Epidemiological, clinical and histological data were collected and quantitative‐PCR were performed on formalin‐fixed paraffin‐embedded (FFPE) samples to identify specific focal amplifications. The 73 dogs included in the study had a median survival time of 220 days. Focal amplifications on CFA 10 and 30 were recurrent (49.3% and 50.7% of cases, respectively) and CFA 30 amplification was significantly associated with the amelanotic phenotype (P = .046) and high mitotic index (MI; P = .0039). CFA 30 amplification was also linked to poor prognosis (P = .0005). Other negative prognostic factors included gingiva location (P = .003), lymphadenomegaly (P = .026), tumour ulceration at diagnosis (P = .003), MI superior to 6 mitoses over 10 fields (P = .001) and amelanotic tumour (P = .029). In multivariate analyses using Cox proportional hazards regression, CFA 30 amplification (Hazard ratio [HR] = 2.08; P = .011), tumour location (HR = 2.20; P = .005) and histological pigmentation (HR = 1.87; P = .036) were significantly associated with shorter survival time. Focal amplification of CFA 30 is linked to an aggressive subset and constitutes a new prognostic factor.     

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.     

Antitumour effects of Liporaxel (oral paclitaxel) for canine melanoma in a mouse xenograft model

Paclitaxel, a member of the taxane family, exhibits antitumour effects by targeting the microtubules in cancer cells. Recently, oral paclitaxel has been developed to overcome the side effects of intravenous paclitaxel administration in human patients. The objective of this study was to investigate the antitumour effects of oral paclitaxel in vitro and in vivo. Three weeks after inoculation, oral paclitaxel (25 and 50 mg/kg) or saline was administered every week for three consecutive weeks. To explore the underlying mechanism, tumour angiogenesis was examined by immunohistochemistry with an anti‐CD31 antibody. Tumour cell apoptosis was detected by Terminal deoxynucleotidyl transferase dUTP Nick‐End Labeling assay, and cell cycle arrest was confirmed by western blot analysis. Oral paclitaxel treatment of canine melanoma cells exerted mediated antiproliferative effects and mediated cell cycle arrest in vitro. In animal experiments, after oral paclitaxel administration, the average tumour size decreased to approximately 30% of that in the control. Histologically, oral paclitaxel showed anti‐angiogenic effects and induced the apoptosis in tumour tissues. Oral paclitaxel also downregulated the intratumoural expression of cyclin D1 and inhibited cell proliferation. The study findings support potential application of oral paclitaxel as a novel chemotherapeutic strategy to treat canine melanoma. This is the first study to investigate the potential of oral paclitaxel as a therapeutic drug against canine tumours.     

Malignant ocular melanoma in a dog

A mongrel male dog of three years old was referred to the Seoul National University Veterinary Teaching Hospital following a one month history of glaucoma. On ophthalmic examination, hyphema, glaucoma, uveitis, iridal mass, and loss of vision were noted in the right eye. Ultrasonography and computed tomography revealed a mass with involvement of the entire uvea. Radiographic evaluation did not reveal any evidence of distant metastasis. The right eye was surgically removed because of the high likelihood of neoplasia. A histologic diagnosis of malignant uveal melanoma was made.     

MIB‐1 immunoreactivity correlates with biologic behaviour in canine cutaneous melanoma

The growth fraction of 68 canine cutaneous melanomas was determined by immunostaining with MIB‐1, a monoclonal antibody to a Ki‐67 epitope that recognizes all proliferating cells. Fifty tumours were classified histologically as benign and 18 as malignant. The Ki‐67 proliferative index (percentage of positive cells over 500 neoplastic cells) was low (< 15%) in 55 cases and high ( 15%) in 13 cases. High Ki‐67 proliferative index and histological malignancy were both associated with significantly poorer 2‐year survival (P < 0.0001). However, the predictive value of the Ki‐67 proliferative index (97%) was higher than the predictive value of classical histology (91%). The evaluation of the growth fraction by the Ki‐67 proliferative index is highly predictive of the biological behaviour of canine cutaneous melanoma.     

A case of conjunctival melanoma in a cat

Since 1985, 5 cases of feline conjunctival melanoma have been reported in the literature. Information on feline conjunctival melanoma epidemiological features, localizations, macroscopic features and histological features is limited. We are describing the clinical, histopathologic features and outcomes in a cat that presented clinically with a slow developing dark brown mass located under the upper eyelid of the left eye. Pertinent literature is reviewed; and the recognizable clinical features and treatment are discussed. The mass was surgically resected. Despite its size, the lesion was easily separated from underlying tissues, making possible a macroscopic complete resection that left intact the adjacent conjunctiva. The tumour histological examination has showed a pigmented melanoma lacking encapsulation, but presenting a clear zone delimiting the lesion. It was exclusively composed of epithelioid cells, and presented mild cellular anaplasia and weak mitotic activity. These features allowed it to be classified as a quite differentiated melanoma with few signs of potential malignancy. In accordance with these histologic features, no recurrence has been registered 34 months after surgery. Thus, a favorable outcome is now reported for two out of six cases of conjunctival melanoma in the cat. This report also confirms the predilection for this neoplasm to arise from the bulbar conjunctiva.     

Suspected latent vertebral metastasis of uveal melanoma in a dog: a case report

A six-year-old intact male Pomeranian was examined because of right eye discomfort. An iris neoplasm was suspected and the eye was enucleated. A uveal melanoma with malignant features was diagnosed. The dog recovered uneventfully from surgery. A general physical examination was performed at 3-month intervals afterwards without any detectable problem, but 18 months after the first presentation the dog suffered a rapid, progressive paraplegia. Radiographic examination and myelography revealed a spinal cord compression at the level of the 8th thoracic (T8) vertebral body. Surgical exploration of the area revealed a potential vertebral neoplasm: histopathology confirmed a melanoma which was suspected to have resulted from metastasis from the previously diagnosed uveal melanoma.     

Treatment of presumed iris melanoma in dogs by diode laser photocoagulation: 23 cases

A semiconductor diode laser was used to cause remission of isolated presumed iris melanoma in 23 dogs. All cases presented as unilateral areas of raised iris hyperpigmentation, ranging in size from 2× 3 mm to 4×12 mm. Cases were treated using a diode laser delivery system through either an operating microscope adapter (OMA) or a laser indirect ophthalmoscope (LIO) with a 20D lens. Laser treatment was delivered 'to effect' using power ranging from 80 to 1000 mW and cumulative durations up to 14 min, 31 s (14:31). Immediate shrinkage of the mass was noted following treatment. Five cases required more than one laser treatment with three eyes receiving two treatments and two eyes receiving three treatments. Follow-up from the last laser treatment ranged from 6 months to 4.5 years during which time the lesions exhibited no enlargement. Minor complications related to laser treatment were seen, including: dyscoria, iris hyperpigmentation, and corneal edema due to collateral hyperthermia. Glaucoma and cataract formation were not observed. Non-invasive diode laser photocoagulation appears to be a safe and effective method of treatment for isolated, pigmented iris masses in dogs.     

Profiling of plasma metabolites in canine oral melanoma using gas chromatography-mass spectrometry

Malignant melanoma is one of the most common and aggressive tumors in the oral cavity of dog. The tumor has a poor prognosis, and methods for diagnosis and prediction of prognosis after treatment are required. Here, we examined metabolite profiling using gas chromatography-mass spectrometry (GC-MS) for development of a discriminant model for evaluation of prognosis. Metabolite profiles were evaluated in healthy and melanoma plasma samples using orthogonal projection to latent structure using discriminant analysis (OPLS-DA). Cases that were predicted to be healthy using the OPLS discriminant model had no advanced lesions after radiation therapy. These results indicate that metabolite profiling may be useful in diagnosis and prediction of prognosis of canine malignant melanoma.     

Metastatic balloon cell melanoma in a dog

Background: Balloon cell melanoma is a rare variant of amelanotic melanoma that is difficult to differentiate from sebaceous cell carcinoma, liposarcoma, and other clear cell neoplasms without immunohistochemistry or ultrastructural evidence of melanin or melanosomes. Objective: The purpose of this report was to describe the clinical, cytologic, histologic, immunohistochemical, and ultrastructural findings in a dog with metastatic balloon cell melanoma. Methods: A 6‐year‐old female Golden Retriever was evaluated for a white, flocculent infiltrate in the anterior chamber of the left eye and an enlarged left prescapular lymph node. Cytologic evaluation of the eye and lymph node were performed following aqueocentesis and fine‐needle aspiration, respectively. The affected lymph node was examined histologically and stained for cytokeratin, vimentin, S‐100, and Melan A. Following euthanasia a necropsy was performed and samples of the affected lymph node were examined by electron microscopy. Results: Cytologic examination of the lymph node and aqueocentesis sample revealed round neoplastic cells that had abundant clear vacuolated cytoplasm. A tentative diagnosis of metastatic sebaceous cell carcinoma or clear cell neoplasm was made. Histologically, the affected lymph node had similar polygonal clear cells arranged in sheets and packets divided by delicate fibrovascular stroma. Immunohistochemical staining of the cells was negative for cytokeratin but positive for vimentin, weakly positive for S‐100, and strongly positive for Melan A. At necropsy, metastatic lesions were identified in the diaphragm, heart, lung, kidneys, left eye, prescapular and sublumbar lymph nodes, and multiple skin sites. Ultrastructural examination of neoplastic lymph nodes revealed many membrane‐bound vacuoles, myelinlike figures, and rare melanosomes. Conclusion: Immunohistochemical staining and ultrastructural features of the neoplastic cells supported a diagnosis of metastatic balloon cell melanoma.     

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.     

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.     

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 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.