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.     

Immunohistochemical expression of p63, Ki67 and β‐catenin in canine transitional cell carcinoma and polypoid cystitis of the urinary bladder

Transitional cell carcinoma (TCC) is a urinary bladder tumour associated with high mortality in dogs. In this study, we investigated the feasibility of using p63, Ki67 or β‐catenin as a clinical marker for predicting biological behaviour and prognosis in canine TCC. Expression levels of these proteins in TCC (n = 25), polypoid cystitis (n = 5) and normal urinary bladder (n = 5) were scored after immunohistochemical staining. The staining scores for p63 (P < 0.01) and β‐catenin (P < 0.05) in TCC were significantly lower than those in normal urinary bladder and polypoid cystitis. In contrast, Ki67 (P < 0.01) staining scores in TCC were significantly higher than those in normal urinary bladder and polypoid cystitis. In TCC, low p63 expression was significantly related to the presence of vessel invasion (P < 0.05) and metastasis (P < 0.01) as well as short survival time (P < 0.05). These findings show that p63 could be a reliable marker for predicting prognosis in canine TCC.     

A matched observational study of canine survival with primary intraocular melanocytic neoplasia

A retrospective histopathologic study was performed to evaluate the effect of primary intraocular melanocytic neoplasia on canine survival. Tumor size, location within the globe, extent of infiltration, and mitotic index were analyzed for their potential to predict survival. A total of 244 cases of dogs with melanocytic tumors submitted to the Comparative Ocular Pathology Laboratory of Wisconsin from 1988 to 1998 were evaluated. Histopathologic criteria (mitotic index, cytologic features of anaplasia) were used to differentiate 188 benign melanocytomas from 56 malignant melanomas. Signalment evaluation of age, sex, and breed revealed similarities in both tumor populations, with the majority of tumors discovered in 9-year-old, female/spayed, mixed-breed dogs. A greater percentage of left eyes (66%) vs. right eyes (47%) was found in the melanoma population, but an equal distribution was found in the melanocytoma population (48% and 52%, respectively). The majority of tumors arose from the anterior uveal tract (79% in the melanocytoma and 95% in the malignant melanoma populations). The German Shepherd breed was predisposed in the limbal distribution. At the time of enucleation, most tumors had invaded the sclera, but did not show extrascleral extension (51% in the melanocytoma and 61% in the malignant melanoma populations). Survival analysis showed a significant difference in survival between control and malignant melanoma populations ( P  = 0.0081) and was suggestive of a difference between the melanocytoma and melanoma populations ( P  = 0.031). Tumor extension, tumor size, and mitotic index were not found to be reliable predictors of survival.     

Use of immunocytochemical techniques in canine melanoma

The staining patterns of the monoclonal antibodies S-100 and Melan-A in canine melanoma were assessed based on cytological specimens of six canine melanomas (four benign, two malignant). In addition, eight regional lymph nodes of the two dogs with malignant melanomas were stained using these markers. For reference, all specimens were also evaluated immunohistochemically using S-100 and Melan-A. To assess the immunocytochemical specificity of both antibodies, various canine tumours and normal tissues were stained. The immunocytochemical staining results of the canine melanomas and the regional lymph nodes showed high conformity with the immunohistochemical reactivity patterns for S-100 and Melan-A. The specificity of Melan-A was higher compared with S-100. Melan-A, in particular, may be helpful for the cytological diagnosis of canine melanoma.     

The value of molecular expression of KIT and KIT ligand analysed using real‐time polymerase chain reaction and immunohistochemistry as a prognostic indicator for canine cutaneous mast cell tumours

This study investigated the correlation between KIT gene expression determined by immunohistochemistry and real‐time polymerase chain reaction (RT‐PCR) and the rate of tumour recurrence and tumour‐related deaths in dogs affected with mast cell tumour (MCT). Kaplan–Meier curves were constructed to compare tumour recurrence and tumour‐related death between patients. The log‐rank test was used to check for significant differences between curves. KIT‐I, KIT‐II and KIT‐III staining patterns were observed in 9 (11.11%), 50 (61.73%) and 22 (27.16%) tumours, respectively. Tumour recurrence rates and tumour‐related deaths were not associated with KIT staining patterns (P = 0278, P > 0.05), KIT (P = 0.289, P > 0.05) or KIT ligand (P = 0.106, P > 0.05) gene expression. Despite the lack of association between KIT staining pattern and patient survival time, the results suggest a correlation between aberrant KIT localization and increased proliferative activity of MCTs. RT‐PCR seems to be a sensible method for quantitative detection of KIT gene expression in canine MCT, although expressions levels are not correlated with prognosis.     

Proliferation activity in oral and cutaneous canine melanocytic tumours: correlation with histological parameters,location, and clinical behaviour

A total of 62 canine melanocytic tumours (10 melanocytomas and 52 primary malignant melanomas) were investigated to compare the accuracy of prognosis provided by MIB-1 proliferation index (MIB-1-PI) with classical histological criteria and location. MIB-1-PI was assessed by means of quantitative image analysis of sections immunostained with MIB-1 monoclonal antibody. Tumour location, histological cell type, stromal or lymphatic vessel invasion, maximum tumour thickness, and presence of inflammation or necrosis were recorded for each case. Thirty-eight dogs were submitted to a 1-year follow-up and the clinical outcome of the disease determined. MIB-1-PI in melanocytomas differed significantly from that detected in primary malignant melanomas (P=0.0001). A significant difference in MIB-1-PI was revealed between oral and cutaneous malignant melanomas (P=0.015), and between presence and absence of lymphatic vessel invasion (P=0.05). MIB-1-PI was not correlated with the other parameters. In univariate analysis, only tumour location (oral vs cutaneous), presence of lymphatic vessel invasion, and MIB-1-PI were associated with decreased overall survival (P=0.0001,P=0.0144, and P=0.0489, respectively). In conclusion, the results of our study confirm that the assessment of the MIB-1-PI may be of additional prognostic value for dogs with primary malignant melanomas.     

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.     

Chondroitin sulfate proteoglycan-4: a biomarker and a potential immunotherapeutic target for canine malignant melanoma

Chondroitin sulfate proteoglycan-4 (CSPG4), also known as high molecular weight-melanoma associated antigen (HMW-MAA), is a membrane-bound chondroitin sulfate proteoglycan highly expressed by human melanoma cells. This phylogenetically conserved tumour antigen plays an important biological role in human melanoma, where it is used as a marker to diagnose forms with unusual characteristics, such as desmoplastic melanoma, and to detect melanoma cells in lymph nodes and peripheral blood, and as a target for immunotherapy because of its restricted distribution in normal tissues. To identify suitable targets to develop novel approaches of treating canine melanoma, CSPG4 was studies to see whether it is expressed in canine malignant melanomas. Immunohistochemical staining of 65 canine malignant melanomas with an anti-human CSPG4-specific antibody detected CSPG4 in 37 cases (56.9%). Positive staining was more frequent, albeit not significantly, in amelanotic compared to melanotic tumours and was statistically associated with tumours having both melanin and the epithelioid histotype. The frequency of CSPG4 expression was similar to that of other melanoma antigens used as diagnostic markers for canine malignant melanoma, such as Melan A and the protein recognized by the PNL2 monoclonal antibody. The results suggest that CSPG4 constitutes a new potential immunohistochemical marker of canine malignant melanoma and may represent an immunotherapeutic target as in humans.     

Surgical treatment of epibulbar melanocytomas by complete excision and homologous corneoscleral grafting in dogs: 11 cases

Objective The aim of the study was to evaluate the efficacy, outcome, and complications following penetrating sclerokeratoplasty and frozen homologous corneoscleral grafting for the management of extensive canine epibulbar melanocytomas. Methods Medical records of canine patients treated at three different veterinary hospitals between 1999 and 2010 were reviewed. Signalment, location and extent of melanocytomas, recurrence rate, and early and late complications were reported. Patients were re‐examined postoperatively to provide follow‐up information. Results Patients included one intact male, three castrated males, six intact females, and one spayed female, with a median age of 5 years (range, 3–9). German Shepherds were overrepresented. Tumors extended from 2 to 4 clock hours at the limbus and up to 17 mm from clear cornea to globe equator. One case showed iridocorneal angle invasion; corneal involvement was present in all cases, and lipid keratopathy was present in four cases. In two cases, there was incomplete resection owing to tumor extent. Follow‐up time ranged from 3 to 72 months (median, 17 months), with one case of intraocular tumor progression. Early complications included anterior uveitis (11/11), intracameral fibrin (5/11), hyphema (4/11), corneal edema (4/11), exuberant corneal granulation tissue (2/11), focal retinal edema (1/11), dyscoria (1/11), and partial suture dehiscence (1/11). Late complications included corneal fibrosis and/or pigmentation (11/11), faint anterior cortical cataracts (3/11), and lipid keratopathy (1/11). Vision was retained in all cases. Conclusions This technique offers a surgically challenging but effective treatment for extensive epibulbar melanocytomas. In this case series, complications were mild and transient, with preservation of ocular anatomy and function.     

Sero‐epidemiology of Canine Leptospirosis in Trinidad: Serovars,Implications for Vaccination and Public Health

A sero‐epidemiological study on canine leptospirosis was conducted in house, stray, farm and hunting dogs, as well as in suspect cases of clinical canine leptospirosis. Serum samples were collected from apparently healthy (vaccinated and non‐vaccinated), house dogs. A questionnaire was administered to the owners to elicit information on risk factors for leptospirosis. The microscopic agglutination test was used to screen for leptospirosis using 17 international serovars. Reciprocal titres of between 100 and <800 were considered as evidence of past exposure while reciprocal titres of 800 or greater were classified as suggestive of acute/current infection. Of a total of 419 serum samples tested, 61 (14.6%) were seropositive for Leptospira agglutinins, 23 (5.5%) had mixed infections and 16 (3.8%) had current infection. Amongst 50 suspected cases of clinical leptospirosis, 24 (48.0%) were seropositive and only 13 (26.0%) had current infection compared with 10 (6.3%) and three (1.9%) of 160 apparently healthy house dogs respectively. The difference was statistically significant (P < 0.05; χ²). Twelve (25.5%) of 47 hunting dogs, 10 (20.4%) of 49 farm dogs and five (4.4%) of 113 stray dogs were seropositive (P < 0.05; χ²). Overall, a total of nine serovars were detected with serovars mankarso, icterohaemorrhagiae RGA, autumnalis and copenhageni being involved in 29 (47.5%), 20 (32.8%), 25 (41.0%) and 10 (16.4%) respectively in 61 seropositive dogs (P < 0.05; χ²). Serovar mankarso was most predominant in seropositive apparently healthy dogs, 37.8% (14/37), suspected clinical cases of leptospirosis, 62.5% (15/24) compared with serovar icterohaemorrhagiae with a frequency of 21.6% (8/37) and 50.0% (12/24), the difference being statistically significant (P < 0.05; χ²). Although all vaccines used for prevention of canine leptospirosis in the country contain serovars canicola and icterohaemorrhagiae, serovar mankarso was mostly associated with infection and disease and may be a good candidate for inclusion in the vaccine used locally. The public health risk posed to owners of dogs infected with Leptospira cannot be over‐emphasized considering the zoonotic nature of the disease.     

VEGF and MMP‐9: biomarkers for canine lymphoma

Vascular endothelial growth factor (VEGF) and metalloproteinase (MMP) 2 and 9 are useful biomarkers in human lymphoma. During cancerogenesis, transforming growth factor beta (TGF‐β) stimulates VEGF and MMPs production. VEGF and TGF‐β plasma levels were tested by ELISA, MMP‐2 and MMP‐9 by gelatine zymography in 37 dogs with lymphoma, 13 of which were also monitored during chemotherapy. Ten healthy dogs served as control. Lymphoma dogs showed higher act‐MMP‐9 (P < 0.01) and VEGF (P < 0.05), and lower TGF‐β than controls, and a positive correlation between act‐MMP‐9 and VEGF (P < 0.001). Act‐MMP‐9 and VEGF were significantly higher in T‐cell lymphomas, and in stage V compared with stages III–IV disease, regardless of immunophenotype. VEGF was higher in high‐grade compared with low‐grade T‐cell lymphomas. No correlation was found between cytokines levels at presentation and outcome. During chemotherapy, act‐MMP‐9 and VEGF decreased in B‐cell lymphomas (P < 0.01), suggesting a possible predictive role in this group of dogs.     

All‐trans retinoic acid improves goat oocyte nuclear maturation and reduces apoptotic cumulus cells during in vitro maturation

All‐trans retinoic acid (t‐RA) is a natural component and representative physiologically active metabolite of vitamin A, having multiple physiologic functions. The objective of this study was to evaluate the effect of t‐RA on goat oocyte maturation and cumulus cell apoptosis during in vitro maturation (IVM). Immature goat cumulus‐oocyte complexes (COCs) were matured in vitro in the absence or presence of t‐RA at concentrations of 10 nmol/L, 100 nmol/L and 1000 nmol/L. Oocyte maturation and embryo development were assessed by polar body formation and parthenogenetic activation, respectively. Oocyte survival was checked by Trypan blue staining. Apoptosis of cumulus cells was analyzed by terminal deoxynucleotidyl transferase nick end labeling staining and quantitative real‐time PCR. In comparison with the control group, 100 nmol/L and 10 nmol/L t‐RA significantly improved goat nuclear oocyte maturation and survival (P < 0.05). Addition of 1000 nmol/L t‐RA improved nuclear maturation (P < 0.05), but had no effect on survival of goat oocytes. t‐RA had no positive effect on goat parthenogenetic embryonic cleavage, blastocyst formation or total cell numbers. However, t‐RA inhibits the apoptosis of cumulus cells (P < 0.01). t‐RA treatment up‐regulated the expression of B‐cell lymphoma 2 (BCL‐2), catalase (CAT) (P < 0.05) and down‐regulated the expression of Caspase‐8 (P < 0.05). In conclusion, t‐RA has positive effects on goat oocyte nuclear maturation and reduces apoptotic cumulus cells during IVM.     

Expression and functionality of TRPV1 receptor in human MCF‐7 and canine CF.41 cells

As canine mammary tumours (CMT) and human breast cancer share clinical and prognostic features, the former have been proposed as a model to study carcinogenesis and improved therapeutic treatment in human breast cancer. In recent years, it has been shown that transient receptor potential vanilloid 1 (TRPV1) is expressed in different neoplastic tissues and its activation has been associated with regulation of cancer growth and progression. The aim of the present research was to demonstrate the presence of TRPV1 in human and canine mammary cancer cells, MCF‐7 and CF.41, respectively, and to study the role of TRPV1 in regulating cell proliferation. The images obtained by Western blot showed a signal at 100 kDa corresponding to the molecular weight of TRPV1 receptor. All tested TRPV1 agonists and antagonists caused a significant decrease (P < 0.05) of cell growth rate in MCF‐7 cells. By contrast, in CF.41 cells capsaicin and capsazepine induced a significant increase (P < 0.05) in cell proliferation, whereas resiniferatoxin (RTX) and 5‐iodo‐resiniferatoxin (5‐I‐RTX) had no influence on CF.41 cell proliferation. Further studies are needed to elucidate the underlying molecular mechanism responsible for the different effects evoked by TRPV1 activation in MCF‐7 and CF.41 cells.     

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.     

Immunohistochemical expression of MDR1‐Pgp 170 in canine cutaneous and oral melanomas: pattern of expression and association with tumour location and phenotype

Canine melanoma (CMM) more commonly affects the oral mucosa and the cutis. CMM shares several features with human melanomas (HMM), included resistance to a broad variety of antineoplastic chemotherapy agents. P‐glycoprotein 1 (Pgp) expression is a well‐recognized feature of multi‐drug resistance and the purpose of this study was to investigate its expression in treatment naïve CMM. We also investigated Pgp association with tumour location and histological features. Histology records of CMM were retrieved, including patients from 2012–2014. Twenty‐five cases of CMM were included in this study. Results revealed that Pgp is expressed in CMM and oral tumours were more likely to have a membranous Pgp expression (100%) than cutaneous tumours (66.6%) (P = 0.010). Cytoplasmic and nuclear Pgp expression could also be identified. Results of this study bring useful data that help in understanding one of the possible mechanisms responsible of intrinsic chemotherapy resistance in canine CMM.     

Effect of synchronization of donor cells in early G1‐phase using shake‐off method on developmental potential of somatic cell nuclear transfer embryos in cattle

In this study, we compared the developmental ability of somatic cell nuclear transfer (SCNT) embryos reconstructed with three bovine somatic cells that had been synchronized in G0‐phase (G0‐SCNT group) or early G1‐phase (eG1‐SCNT group). Furthermore, we investigated the production efficiency of cloned offspring for NT embryos derived from these donor cells. The G0‐phase and eG1‐phase cells were synchronized, respectively, using serum starvation and antimitotic reagent treatment combined with shaking of the plate containing the cells (shake‐off method). The fusion rate in the G0‐SCNT groups (64.2 ± 1.8%) was significantly higher than that of eG1‐SCNT groups (39.2 ± 1.9%) (P < 0.05), but the developmental rates to the blastocyst stage of SCNT embryos per fused oocytes were similar for all groups. The overall production efficiency of the clone offspring in eG1‐SCNT groups (12.7%) per recipient cow was higher than that in G0‐SCNT groups (3%) (P < 0.05). The mean birth weight of cloned calves and the average calving score in the G0‐SCNT groups (48.1 ± 3.4 kg and 3.3 ± 0.3, respectively) was significantly higher (P < 0.05) than those of eG1‐SCNT groups (37.2 ± 2.1 kg and 2.3 ± 0.2, respectively). Results of this study indicate that synchronization of donor cells in eG1‐phase using the shake‐off method improved the overall production efficiency of the clone offspring per transferred embryo.