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.     

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.     

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.     

Pathology of the canine and feline uterine tube

Lesions of the uterine tube were described in 37 dogs and nine cats. Of the 52 lesions noted, 18 were developmental abnormalities; four paramesonephric remnants, and 14 mesonephric remnants. Uterine tube cysts were seen in 16 animals, and seven animals had salpingitis. One dog had hyperplastic changes of the infundibular mucosa, and one cat and one dog demonstrated adenomyosis. There were three primary benign uterine tube neoplasms, one adenocarcinoma, two metastatic neoplasms in the uterine tube, and two bursal lipomas, all in dogs. With the exception of neoplasia of the uterine tube, most lesions were unlikely to affect the reproductive performance of these animals and/or were incidental or secondary to disorders elsewhere in the reproductive tract.     

Magnetic resonance imaging of a canine eye with melanoma

An 8-year-old Beagle dog had exophthalmos of the left eye for the last two past months. On ophthalmoscopy, the intraocular lesion could not be evaluated due to the opacity of the cornea. Ultrasonography revealed that the eyeball was distorted in shape and shifted in position, however, the precise lesion could not be identified. On magnetic resonance (MR) imaging, the lesion was observed as hyperintense on T1-weighted and hypointense on T2-weighted images, similar to those reported in human melanoma. The lesion was histologically diagnosed to be malignant intraocular melanoma. Though this is only a case report, canine ocular melanoma may show the similar characteristic MR images as in human uveal malignant melanoma.     

Priapism in a stallion with generalized malignant melanoma

A Thoroughbred stallion developed priapism that was unresponsive to medical treatment and lavage of the corpus cavernosum penis with heparinized 0.9% NaCl solution. Three weeks after onset of priapism, the penis was firm and noncompliant, and penile pain sensation and ability to retract the penis were lost. Ultrasonography confirmed thrombosis of the corpus cavernosum penis. The stallion was euthanatized because of poor prognosis for return to breeding soundness. Necropsy revealed enlargement of numerous lymph nodes. The dorsal penile nerves were demyelinated distal to the crura of the penis. A diagnosis of generalized malignant melanoma was made; however, neither metastasis to the vertebral canal nor compression of spinal nerve roots as they exited the vertebral foramen was found. Priapism is a persistent erection without sexual arousal and is initially unassociated with penile paralysis, but if prolonged, leads to irreversible venous occlusion where collecting veins join the cavernous spaces. Damage to the dorsal penile nerves may explain the long-term penile paralysis and loss of sensation that accompanied priapism in this stallion. Priapism unassociated with the use of phenothiazine-derivative tranquilizers is uncommon in horses.     

Biologic characterization of canine melanoma cell lines

Eight canine melanoma cell lines were established from tissues from 6 dogs with spontaneous primary or metastatic melanomas. Cell lines were characterized for morphologic features and growth patterns on plastic, pigmentation, ultrastructure, cloning efficiency in soft agar, and tumorigenicity in nude mice. Biologic properties of cell lines were distinct and preserved during 40 to 120 passages in vitro. All cell lines were clonogenic and tumorigenic.     

Expression of S100a, vimentin, NSE, and melan A/MART-1 in seven canine melanoma cells lines and twenty-nine retrospective cases of canine melanoma

We evaluated the expression of vimentin, S100a, and Melan A/MART-1 (melanoma antigen recognized by T cells 1) in seven cell lines established independently from dogs with canine melanoma. We also compared routine immunostaining of 29 clinical specimens from melanoma cases using vimentin, S100a, and neuron-specific enolase (NSE) with staining for Melan A/MART-1 as part of a diagnostic panel. All the cell lines were positive for expression of vimentin and S-100a. MelanA/MART-1 expression was seen consistently in only two of the seven cell lines. Staining for Melan A/MART-1 was most intense near areas of heavy melanin pigmentation. All except one of the clinical specimens were positive for vimentin. S 100a was expressed in the majority of both pigmented (15/20, 75%) and amelanotic (8/9, 88.8%) tumors. Seventeen of 29 (58.6%) tumors were positive for NSE. Melan A/MART-1 was expressed in 18/29 (62%) tumors, including 90% of pigmented tumors, but in no amelanotic tumors. Intensity of Melan A/MART-1 staining correlated positively with biologic behavior, with seven malignant tumors showing negative to weak staining and 10 benign tumors showing moderate to strong staining. Three malignant tumors showed moderate to intense staining for Melan A/ MART-1. Our results suggest that expression of Melan A/MART-1 may be unstable in cultured cell lines. Assessment of both S100a and Melan A/MART-1 expression is useful to confirm a diagnosis of canine melanoma, and Melan A/MART-1 may be especially informative regarding the biologic behavior of these tumors.     

Associations between canine benign and malignant neoplasms

Associations between benign and malignant neoplasms of the dog were quantitatively evaluated. Using histologically confirmed submissions and population-based methodology, canine benign tumors were significantly associated with both the unordered and subsequent development of additional distinct malignancies in the same dogs. A detailed examination of the methods indicated that these results are not likely due to methodologic artifact. The implication is that benign and malignant tumor development are associated and that the occurence of a benign neoplasm is an indicator of a parallel predisposition to malignancy.     

Cloning and characterization of a canine oral papillomavirus

A papillomavirus, isolated from oral papillomas in young Beagles, was used to produce a live-virus vaccine. After the IM use of this vaccine, some dogs developed squamous cell carcinomas at the inoculation site. The virus was isolated from the original vaccine and was cloned into pBR322. A detailed restriction map of the viral genome was generated.     

A case of atypical canine lymphoma with oral mass and multiple osteolysis

An 8-year-old female Golden Retriever had an oral mass and lameness. Multiple osteolysis of the systemic skeleton without monoclonal gammopathy was shown on electrophoresis of serum and urine samples. Cytological and histopathological examinations of the oral mass revealed atypical polymorphic cells similar to myeloid cells, and bone marrow aspiration indicated that these abnormal cells also might have invaded the bone marrow. These cells were negative to peroxidase and non-specific esterase staining, and clonal expansion of B lymphocytes could be detected by polymerase chain reaction (PCR) assay for antigen receptor gene rearrangement. The case was diagnosed as atypical lymphoma and treated by multi-drug chemotherapy. On the 142nd day after the first admission, the case had remission and the oral mass and multiple osteolysis were improved.     

Pathology in dogs with experimental canine H3N2 influenza virus infection

Avian-lineage H3N2 canine influenza virus (CIV)-associated respiratory disease, which can be fatal, emerged in South Korean dogs in 2007. We show here that dogs experimentally infected with CIV only developed respiratory tract diseases, as no extrapulmonary lesions and virus antigens were detected. This differs from the multiorgan diseases that avian influenza H5N1 induces in small experimental animals. However, the CIV-infected dogs developed a distinctively severe, long-persistent bronchointerstitial pneumonia, which differs from the acute but short-term bronchopneumonia that human (H1N1 and H3N2) influenza cause in rodents and ferrets. Histopathology and in situ TUNEL assays revealed that the neutrophils infiltrating the lesions were undergoing apoptosis, which probably reflects the attempts by the body to maintain appropriate numbers of neutrophils for defense against secondary bacterial infections. Our observations suggest that neutrophils along with the related chemoattractant cytokines (TNF-α, IL-1 and IL-8, etc.) may play a key role in the pathogenesis of H3N2 CIV in dogs.     

First report of a malignant collision skin tumour with malignant melanoma and anaplastic sarcoma components in a dog

This paper describes the occurrence of a rare skin tumour that has been removed surgically from the upper lip of a 13-year-old Tibetan spaniel. The tumour was 0.5 cm in diameter and macroscopically appeared as a single dermal mass, but histopathological analysis identified it as a biphasic collision mixed tumour. In the anatomically uniform tumour, 70% (4 mm in diameter) of the total parenchyma was formed by a high-grade sarcoma (with the presence of giant cells), and about 30% of it (1 mm in diameter) was a malignant melanoma (again with the presence of giant cells). The histologically distinct, but anatomically uniform tumour parts were separated by a macroscopically invisible, non-neoplastic epithelial process originating from the overlying hyperplastic epidermis. The two malignant components did not infiltrate the peritumoural vessels and each other's substance. In the sarcoma part, the mitotic and apoptotic indexes were 32 and 8, respectively, whereas in the melanoma part the same parameters were 10 and 6, respectively. During the immunohistochemical investigations anti-α-SMA, anticytokeratin AE1-AE3, anti-Melan-A, anti-Ki-67 and anti-claudin-5 antibodies were applied. In conclusion, this is the first report of a primary cutaneous malignant biphasic collision mixed tumour formed by an anaplastic sarcoma with giant cells and a malignant melanoma.