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.     

A retrospective analysis of the efficacy of Oncept vaccine for the adjunct treatment of canine oral malignant melanoma

Oral malignant melanoma (OMM) in the dog is often locally aggressive with a high metastatic potential and there are few treatment options that have been demonstrated to improve outcome of this disease. The purpose of this study was to determine whether adjunctive treatment with the Oncept melanoma vaccine affected the outcome of dogs with OMM that had achieved loco‐regional cancer control. Medical records from 45 dogs that presented to the Animal Cancer and Imaging Center were reviewed, including 30 dogs with stage II and III disease. Dogs that received the vaccine did not achieve a greater progression‐free survival, disease‐free interval or median survival time than dogs that did not receive the vaccine.     

Comparative study of anti-oncogenic microRNA-145 in canine and human malignant melanoma

MicroRNA-145 (miRNA-145; miR-145) is aberrantly expressed in most of human cancers and plays a significant role in carcinogenesis and cancer progression. In the current study, we focused on how miR-145 plays a role in canine and human malignant melanomas. MiR-145 was significantly downregulated in canine malignant melanoma tissues and canine melanoma cell lines, as well as human melanoma cell lines tested. The ectopic expression of miR-145 showed a significant growth inhibition in both canine and human melanoma cells tested, and the effect was achieved partly by suppressing c-MYC in canine melanoma LMeC and in human melanoma A2058 and Mewo cells. At the same time, a suppressive tendency on cell migration in canine melanoma KMeC cells and significant suppression of cell migration in human melanoma A2058 cells by suppressing FASCIN1 were also found. These findings suggest that miR-145 acts as a tumor suppressor in both canine and human malignant melanomas.     

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.     

Prognostic evaluation of Ki67 threshold value in canine oral melanoma

Oral melanoma is a common canine cancer with a historically poor prognosis. Recent evidence suggests that a subset of cases may have a more favorable outcome, defined as long-term survival in the absence of intervention other than initial surgery. Traditional histological parameters have had prognostic significance in some studies but not in others, potentially due to interobserver variation. We evaluated the prognostic utility of Ki67 immunohistochemistry in a group of 79 canine oral melanomas using a technique easily applied in a veterinary diagnostic laboratory. A threshold Ki67 value of >19.5 had a sensitivity and specificity of 87.1% and 85.4%, respectively, at predicting death or euthanasia due to melanoma by 1 year postdiagnosis. Threshold values for classical histological parameters were also identified for most cases and were >4 (>30%; sensitivity = 83.9%, specificity = 86.0%) for the nuclear atypia score and >4/10 hpfs (sensitivity = 90.3%, specificity = 84.4%) for the mitotic index. In this study, the percentages correctly classified with respect to death by 1 year postdiagnosis were comparable for Ki67 (86.1%, 68/79), the nuclear atypia score (86.3%, 63/73), and the mitotic index (86.8%, 66/76). High pigmentation (>50%) had a high negative predictive value of 90.9% (18/20), but overall, only 61.0% (47/77) of cases could be correctly classified by this parameter. Based on these results, we recommend a panel of prognostic parameters, including the nuclear atypia score, the mitotic index, Ki67, and pigmentation quantification to more accurately predict the likely outcome of canine oral melanomas.     

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.     

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.     

Evaluation of cisplatin combined with piroxicam for the treatment of oral malignant melanoma and oral squamous cell carcinoma in dogs

OBJECTIVE: To determine the maximum tolerated dose (MTD) of cisplatin administered with piroxicam, the antitumor activity and toxicity of cisplatin combined with piroxicam in dogs with oral malignant melanoma (OMM) and oral squamous cell carcinoma (SCC), and the effects of piroxicam on the pharmacokinetics of cisplatin in dogs with tumors. DESIGN: Prospective nonrandomized clinical trial. ANIMALS: 25 dogs. PROCEDURE: Dogs were treated with a combination of cisplatin (escalating dose with 6 hours of diuresis with saline [0.9% NaCI] solution) and piroxicam (0.3 mg/kg 10.14 mg/lb], PO, q 24 h).The initial cisplatin dose (50 mg/m2) was increased by 5 mg/m2 until the MTD was reached. Tumor stage and size were determined at 6-week intervals during treatment. The pharmacokinetics of cisplatin were determined in dogs receiving a combination of cisplatin and piroxicam during the clinical trial and dogs that were treated with cisplatin alone. RESULTS: 11 dogs with OMM and 9 dogs with SCC were included in the clinical trial. The MTD of cisplatin when administered in combination with piroxicam was 50 mg/m2. Tumor remission occurred in 5 of 9 dogs with SCC and 2 of 11 dogs with OMM. The most common abnormality observed was renal toxicosis. Clearance of cisplatin in dogs that were treated with cisplatin alone was not significantly different from that in dogs treated with a combination of cisplatin and piroxicam. CONCLUSIONS AND CLINICAL RELEVANCE: Cisplatin administered in combination with piroxicam had antitumor activity against OMM and SCC. The level of toxicity was acceptable, although renal function must be monitored carefully.     

Use of adjuvant carboplatin for treatment of dogs with oral malignant melanoma following surgical excision

Melanoma is the most common oral malignancy in dogs. This retrospective study evaluated adjuvant carboplatin chemotherapy (with or without radiation therapy) in 17 dogs with malignant oral melanoma following surgical resection. The median dosage and number of doses of carboplatin administered to the 17 dogs was 300 mg m^?2 (range, 150–300 mg m^?2) and 4 (range, 2–11), respectively. The overall median progression‐free survival for all dogs was 259 days [95% confidence interval (CI₉₅), 119–399 days]. The first progression‐free survival event was local recurrence in seven dogs (41%) and metastases in seven dogs (41%). The median overall survival for all dogs was 440 days (CI₉₅, 247–633 days). The tumour was the cause of death in 10 dogs (59%). On the basis of this study, systemic therapy with carboplatin may be an appropriate adjunct to local treatment for canine malignant melanoma, although future prospective controlled studies are needed to compare treatment modalities for this aggressive neoplasia.     

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.     

Radiation up-regulates the expression of VEGF in a canine oral melanoma cell line

To evaluate radiosensitivity and the effects of radiation on the expression of vascular endothelial growth factor (VEGF) and VEGF receptors in the canine oral melanoma cell line, TLM 1, cells were irradiated with doses of 0, 2, 4, 6, 8 and 10 Gray (Gy). Survival rates were then determined by a MTT assay, while vascular endothelial growth factor receptor (VEGFR)-1 and -2 expression was measured by flow cytometry and apoptotic cell death rates were investigated using an Annexin assay. Additionally, a commercially available canine VEGF ELISA kit was used to measure VEGF. Radiosensitivity was detected in TLM 1 cells, and mitotic and apoptotic cell death was found to occur in a radiation dose dependent manner. VEGF was secreted constitutively and significant up-regulation was observed in the 8 and 10 Gy irradiated cells. In addition, a minor portion of TLM 1 cells expressed vascular endothelial growth factor receptor (VEGFR)-1 intracellularly. VEGFR-2 was detected in the cytoplasm and was down-regulated following radiation with increasing dosages. In TLM 1 cells, apoptosis plays an important role in radiation induced cell death. It has also been suggested that the significantly higher VEGF production in the 8 and 10 Gy group could lead to tumour resistance.     

Canine oral malignant melanoma: Prognostic utility of an alternative staging system

Reports of prognosis based on clinical staging of canine oral malignant melanoma consistently differ. To determine the prognostic utility of the World Health Organization (WHO) tumour, node, metastasis (TNM) system and a proposed alternative staging system, a retrospective study of 41 dogs with naturally occurring oral malignant melanoma was conducted. All the tumours were clinically staged, removed surgically and the tissues histologically reviewed. Treatment responses were correlated with the clinical and histological features reported to have prognostic utility. Dogs presenting with a tumour smaller than 8 cm3, located on the rostral mandible or caudal maxilla, and, or, having a tumour mitotic index of 3 or less had a significantly longer remission length and survival time than did other dogs, regardless of the treatment selected. Treatment by radical surgical excision (eg, hemimandibulectomy) resulted in longer remission lengths and survival times than any other type of treatment regardless of whether or not surgical margins were determined to be free of residual tumour. The WHO system failed to identify a prognostic difference between dogs of a differing clinical stage. An alternative system derived from significant features mentioned above did identify a prognostic difference and is recommended for further evaluation regarding its utility in the pre-treatment evaluation and clinical staging of other dogs with oral -malignant melanoma.     

Analysis of KIT expression and KIT exon 11 mutations in canine oral malignant melanomas

KIT, a transmembrane receptor tyrosine kinase, is one of the specific targets for anti-cancer therapy. In humans, its expression and mutations have been identified in malignant melanomas and therapies using molecular-targeted agents have been promising in these tumours. As human malignant melanoma, canine malignant melanoma is a fatal disease with metastases and the poor response has been observed with all standard protocols. In our study, KIT expression and exon 11 mutations in dogs with histologically confirmed malignant oral melanomas were evaluated. Although 20 of 39 cases were positive for KIT protein, there was no significant difference between KIT expression and overall survival. Moreover, polymerase chain reaction amplification and sequencing of KIT exon 11 in 17 samples did not detect any mutations and proved disappointing. For several reasons, however, KIT expression and mutations of various exons including exon 11 should be investigated in more cases.     

Pathology of canine cataract surgery complications

AIM: To compare pathological complications arising from 2 methods of canine cataract surgery, manual extracapsular cataract extraction (MECCE) and the more automated phacoemulsification and aspiration (PA). METHODS: Case material presented to the Comparative Ophthalmic Pathology Laboratory, University of North Carolina over the previous 20 years (1979-1999) was searched for cases with a history of prior cataract surgery. Data were obtained from the pathology accession forms, and submitting veterinarians were contacted to verify and complete missing data where possible. The slides were re-examined, histological features of surgical complications were noted and secondary changes recorded. RESULTS: Fourteen canine post-cataract-surgery cases were reviewed, the average age of the group being 7.7 years. Of the 14 eyes, 7 had surgery performed using MECCE, and 7 using PA. Of the 7 eyes that underwent MECCE surgery, 5 were enucleated and diagnosed on both clinical and pathological grounds with glaucoma. Of the 7 eyes that underwent PA, 4 were diagnosed with infectious endophthalmitis. CONCLUSIONS: Complications observed were related to the surgical technique performed. Failures of manual extracapsular surgical procedures were more commonly associated with postoperative synechia and glaucoma, compared with failures of phacoemulsification, which were more commonly associated with infection and rhegmatogenous retinal detachments. CLINICAL RELEVANCE: To minimise post surgical complications, MECCE should be accompanied by rigorous efforts to reduce surgically induced inflammation, while PA should be performed using excellent aseptic technique. It is important with both techniques to totally remove cortical material while maintaining the integrity of the posterior capsule.