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.     

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.     

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.     

Cyclooxygenase-2 (COX-2) expression in canine intracranial meningiomas

Meningiomas are the most common canine intracranial tumour. Neurologic disability and death from treatment failure remain problematic despite current surgical and radiotheraputic treatments for canine intracranial meningiomas. Cyclooxygenase-2 (COX-2) over-expression has been demonstrated in multiple canine malignancies, and COX-2 inhibitory treatment strategies have been shown to have both preventative and therapeutic effects in spontaneous and experimental models of cancer. The purpose of this study was to evaluate COX-2 expression in canine intracranial meningiomas. Immunohistochemical and Western blot (WB) analyses showed COX-2 expression in multiple tissues of the normal canine brain, and 87% (21/24) of intracranial meningiomas studied were immunoreactive to COX-2. No significant associations between COX-2 immunoreactivity and tumour grade were identified. Further studies are required to elucidate the physiologic roles of constitutive COX-2 expression in the central nervous system as well as its participation in meningioma tumourigenesis.     

Combination CCNU and vinblastine chemotherapy for canine mast cell tumours: 57 cases

The purpose of this study was to evaluate the efficacy and toxicity of a CCNU and vinblastine chemotherapy protocol for canine mast cell tumours. Fifty-seven tumours in 56 dogs were evaluated, 37 had macroscopic disease and 20 had microscopic disease. A 57% response rate was seen in dogs with macroscopic disease for a median duration of 52 weeks. Dogs with macroscopic disease had a median progression free survival time (PFST) of 30 weeks and a median overall survival time (OST) of 35 weeks. Dogs with microscopic disease had a median PFST of 35 weeks and a median OST of 48 weeks. Toxicity was recorded in 54% of the dogs treated, with the majority of events being mild. This chemotherapy protocol appears to be well tolerated and should be considered for canine mast cell tumours.     

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.     

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.     

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.     

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.     

Canine intranasal tumours treated with alternating carboplatin and doxorubin in conjunction with oral piroxicam: 29 cases

Few veterinary studies have evaluated the response to chemotherapy treatment of canine intranasal tumours, while many have focused on the efficacy of radiation therapy. Given the higher costs and limited access to radiation therapy, alternative treatment options are needed. The study describes a cohort of dogs with histologically confirmed intranasal tumours treated with chemotherapy as a sole therapy. This retrospective study was conducted using data from the Melbourne Veterinary Specialist Centre (MVSC) database between 2004 and 2017. Dogs with a histologically confirmed intranasal tumour who received chemotherapy treatment were included. Signalment, presenting signs, tumour type, chemotherapy details, adverse events (AEs) and survival times were reviewed. Twenty‐nine dogs met the inclusion criteria. Overall median survival time for dogs in the study was 234 days (range 12‐1698 days). Median survival for dogs with adenocarcinoma or carcinoma (n = 12) was 280 days, transitional cell carcinoma (n = 6) 163 days, squamous cell carcinoma, anaplastic carcinoma or undifferentiated carcinoma (n = 7) 59 days and all sarcomas (n = 4) 448 days. Adverse events were reported following 28% of treatments and 69% of dogs experienced at least one AE. Twenty four per cent of all dogs experienced grade 3 or 4 toxicities. The chemotherapy protocol was generally well tolerated. The study suggests potential benefit in the use of chemotherapy for dogs with adenocarcinoma, carcinoma and sarcoma.     

Phase II study of oral docetaxel and cyclosporine in canine epithelial cancer

The goal of the current study was to determine the efficacy of oral docetaxel in combination with cyclosporine in the treatment of canine epithelial cancer. Requirements for eligibility were histological confirmation of epithelial neoplasia, measurable disease, no chemotherapy treatment within 2 weeks, and a life expectancy of ≥3 months. Fifty‐one dogs were enrolled. All dogs received 1.625 mg kg^?1 of docetaxel with 5 mg kg^?1 of cyclosporine (DT/CSA) by gavage. Ten dogs had progressive disease at 2 weeks, one dog died, and one dog was withdrawn from the study. Thirty‐nine dogs were given a second dose of DT/CSA, three each receiving a third or fourth dose. Eight dogs had a dose reduction (1.5 mg kg^?1) and six dogs had treatment delays primarily for gastrointestinal toxicity. The overall response rate was 16.7% (8/48 had a partial response there were no complete responses). The highest response rate was seen in dogs with oral squamous cell carcinoma (50%; 6/12).     

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.     

Prevalence of elevated alanine transaminase activity in dogs treated with CCNU (Lomustine)*

The purpose of this study was to evaluate prevalence of serum alanine transaminase (ALT) elevation in dogs receiving lomustine (CCNU) and to analyse the pattern of occurrence and potential risk factors. Serum ALT activity in 109 dogs during single‐agent CCNU chemotherapy was retrospectively analysed. The median initial dose, dose‐intensity and cumulative dose of CCNU were 64 mg m^?2, 21 mg m^?2 week^?1 and 171 mg m^?2, respectively. The overall prevalence of major ALT elevation [> 5‐fold upper reference limit (URL)] was 29% (32/109) and developed most commonly after one to three doses of CCNU. These ALT elevations occurred without preceding mild ALT elevation in 53% (17/32) of the cases. Three dogs (2.8%) developed clinical hepatopathy. For severe ALT elevation (>10‐fold URL), age ≤5‐year‐old was associated with higher risk. The findings of this study showed that elevation of ALT is common during CCNU chemotherapy in dogs and severe elevation can develop on a sudden onset.     

Calculation of dose intensity and comparison of published methods using a cohort of canine T-cell lymphoma patients undergoing CHOP-based chemotherapy

Methods of calculating and reporting dose intensity (DI) of CHOP-based protocols in the veterinary literature vary. The goal of this retrospective study is to examine the prognostic significance of the average percentage of planned DI received in a cohort of canine T-cell lymphoma patients treated with a modified CHOP protocol with corresponding toxicity and efficacy data. Our data set of 40 dogs was analysed using various previously published methods for calculating DI. Median progression-free survival and overall survival were 91 and 196 days, respectively. Receiving a higher percentage of planned DI was not found to be associated with patient outcome. Outcomes remain poor for dogs with T-cell lymphoma treated with CHOP-based chemotherapy irrespective of received DI. Standard methods of DI calculation and reporting should be adopted in veterinary oncology to enable repeatable and rigorous comparisons of published chemotherapy protocols and to ascertain the potential prognostic relevance of DI in canine lymphoma patients.     

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.