Immunohistochemical expression of vimentin, E-cadherin, and CD45 in natural cases of canine cutaneous round tumors.

Round cell tumors are common cutaneous lesions in dogs, with increased occurrence percentages among different skin tumors. This study aimed to investigate the frequency as well as gross and pathological characteristics of round cell tumors in natural cases of tumorous dogs in relation to breed, sex, and age. Moreover, it aimed to evaluate the immunohistochemical expression of a panel of immunohistochemical stains, including vimentin, E-cadherin, and cluster of differentiation (CD45) as an adjunct technique for the differential diagnosis of cutaneous round cell neoplasm. Data were collected from 64 dogs of both sexes (36 females and 28 males), various breeds, and different ages (8 months to 7 years). The histopathological nature of neoplastic growth was reported, and neoplasm prevalence was classified using age, sex, breed, and site on the body. We observed 48 cases of transmissible venereal tumors, 12 cutaneous histiocytomas, and 4 histiocytic sarcoma. Immunohistochemical characterization revealed an intense positive immunoreactivity for vimentin in transmissible venereal tumor cells and moderate positive immunoreactivity for E-cadherin and CD45 in cutaneous histiocytoma and histiocytic sarcoma cells. In conclusion, the canine transmissible venereal tumor was the most frequent form of round cell tumor; thus, a definitive cutaneous neoplasm diagnosis should be based on histopathological morphology and immunohistochemical findings.

Canine transmissible venereal tumor - From general to molecular characteristics: A review.

Cancer is a group of complex diseases resulting from the accumulation of genetic and epigenetic changes affecting control and activity of several genes, especially those involved in cell differentiation and growth processes, leading to an abnormal proliferation. When the disease reaches an advanced stage, cancer can lead to metastasis in other organs. Interestingly, recent studies have shown that some types of cancer spread not only through the body, but also can be transmitted among individuals. Therefore, these cancers are known as transmissible tumors. Among the three types of transmissible tumors that occur in nature, the canine transmissible venereal tumor (CTVT) is known as the oldest cancer in the world, since it was originated from a single individual 11 000 years ago. The disease has a worldwide distribution, and its occurrence has been documented since 1810. The CTVT presents three types of cytomorphological classification: lymphocytoid type, mixed type, and plasmacytoid type, the latter being chemoresistant due to overexpression of the ABCB1 gene, and consequently increase of the P-glycoprotein. More knowledge about the epidemiology and evolution of CTVT may help to elucidate the pathway and form of the global spread of the disease.

Immunophenotypic and molecular profile of cancer stem-cell markers in ex vivo canine transmissible venereal tumour (CTVT).

BACKGROUND: Several studies have attempted to characterise the origin of canine transmissible venereal tumour (CTVT). However, the participation of cancer stem cells (CSC) has not been reported OBJECTIVES: Herein we describe the expression patterns of CSC markers CD44, CD34, CD90 and CD133 in CTVT METHODS: Thirty-eight samples were selected and assessed through flow cytometry and immunohistochemistry RESULTS AND CONCLUSIONS: Twenty-two tumours were classified as plasmacytoid and 16 as mixed. Almost all tumours showed high CD44 and low CD34 levels. CD133 and CD90 expression varied among tumours. Cytological groups did not differ in the proportion of CSC markers. Our results suggest that CSC subpopulations might participate in CTVT.

A retrospective study of canine transmissible venereal tumour in Grenada, West Indies.

BACKGROUND: Canine transmissible venereal tumour (CTVT) is a naturally occurring neoplasia affecting dogs worldwide. Previous CTVT studies in Grenada were limited to case records of dogs with neoplastic conditions at a veterinary diagnostic laboratory. OBJECTIVES: The present retrospective study aimed to determine the occurrence and risk factors of CTVT in a wider population of owned dogs presented to a university-affiliated veterinary hospital between 2008 and 2018. METHODS: Data on the age, breed, gender, and gonadectomy status were retrieved from an electronic database and analyzed using logistic regression. RESULTS: Of the 7180 dogs presented during the period, 102 dogs (1.4%) were diagnosed with CTVT. A higher predisposition was observed in Grenadian pothounds (odds ratio [OR] = 22.8, 95% confidence interval [CI] 10.3-50.4; p < 0.001) and mixed-breed dogs (OR = 9.2, 95% CI 4.1-20.7; p < 0.001) in comparison to the purebreds. Neutered dogs (OR = 2.2, 95% CI 1.4-3.3; p < 0.001) were at an increased risk of CTVT than intact dogs. Age and gender were not identified as significant risk factors. CONCLUSIONS: The percentage of dogs with CTVT in this study represents a crude estimate of the CTVT prevalence in the owned dog population in Grenada. Further studies including both owned and free-roaming dogs are required for a more accurate estimation of the CTVT prevalence in the region. Our results indicate that breed and gonadectomy status are significant risk factors for the occurrence of CTVT in Grenada.

Epidemiological study of canine transmissible venereal tumor (CTVT) in Brazil, 2000-2020.

Canine transmissible venereal tumor (CTVT) is a contagious neoplasm, mainly transmitted through coitus. This round cell mesenchymal tumor is common in Brazil, often located in the genitalia although extragenital presentations may also occur, such as cutaneous, oral, and nasal forms. The objective of this study was to perform an epidemiological analysis of CTVT from published data in the recent academic literature to systematically demonstrate the distribution of CTVT in Brazil, identify the frequency of this neoplasm and its main diagnostic tests, and characterize its main clinical manifestations in Brazil. For such purpose, it was analyzed the scientific publications with cases of CTVT in Brazil, in English or Portuguese, published between 2000-2020. The CTVT was identified in 19 Brazilian states plus the Federal District, totaling 3,622 cases across the national territory, with the largest number of cases recorded in the Southeast region. The cytological exam was the most used for the diagnosis of CTVT (89.2 %), followed by histopathological (37.8 %) and immunohistochemistry (13.5 %)1 . Predominant epidemiological aspects of CTVT identified in the study were: Mixed breed dogs (75.2 %), females (62.5 %), in adulthood (between 2 and 7 years) and dogs with free extra outdoor access (91.1 %). Genital presentation was the most frequent in the literature (86 %), followed by cutaneous (21.8 %), nasal (10 %), oral and lymph nodes presentations (10-5 %) and less frequent manifestations as ocular and anal/perianal (< 5 %). CTVT is a neoplasm widely distributed in Brazil, highly frequent and with several forms of clinical presentation, which can be underdiagnosed if there is no adequate knowledge of this tumor and its epidemiological characteristics. The extragenital manifestations of the neoplasm need further studies for its better characterization and more precise definition of its frequencies.

Vincristine and ivermectin combination chemotherapy in dogs with natural transmissible venereal tumor of different cyto-morphological patterns: A prospective outcome evaluation.

Vincristine is the first-line drug for the chemotherapy of canine transmissible venereal tumor (CTVT). Drug resistance is related to tumor cyto-morphological patterns of CTVT. There are anti-cancer properties of ivermectin, thus, a combination of ivermectin and vincristine could be an effective chemo-therapeutic treatment regimen for CTVT. Study aims, therefore, were to (1) assess the frequency of CTVT cyto-morphologies, and (2) evaluate treatment efficacy and possible adverse reactions to vincristine compared with a combination vincristine and ivermectin. Dogs (n = 41) with CTVT were characterized by tumor cyto-morphology and disease severity and of those, 20 were randomly allocated into two groups. There was a control group (G-Vin; n = 10) in which there was treatment with vincristine; and an experimental group (G-Iv/Vin; n = 10) in which there was treatment with the ivermectin/vincristine combination. Although dogs in the G-Iv/Vin group had more severe disease at the beginning of the study (P = 0.0031), the number of weeks and chemotherapy sessions until tumor remission were similar among dogs of the two groups, indicating both treatments were effective. There was a decrease in the leukocyte counts (P = 0.0020), related to neutropenia (P = 0.0371) in the G-Vin but not the G-Iv/Vin treatment group. There was no tumor resistance that developed during the study regardless of the treatment regimen used or tumor cytomorphology. In summary, the use of the vincristine/ivermectin combination was well tolerated and efficacious for CTVT treatment.

The role of the multi-drug resistance 1, p53, b cell lymphoma 2, and bcl 2-associated X genes in the biologic behavior and chemotherapeutic resistance of canine transmissible venereal tumors.

BACKGROUND: Canine transmissible venereal tumors (CTVTs) generally have different cytomorphologic subtypes and phases of progression. Some tumors have variable biologic behavior including a progressive increase in tumor aggressiveness and variable responses to chemotherapy. This behavior is partially due to high p-glycoprotein expression by tumor cells, which leads to the expulsion of chemotherapeutic drugs. Other possible causes include changes in pro- and anti-apoptotic genes from the BCL-2 family and DNA repair systems, which are associated with the p53 gene family. OBJECTIVES: We aimed to determine the relative expression of the multi-drug resistance 1 (MDR1), p53, b-cell lymphoma 2 (BCL2), and bcl 2-associated X (BAX) genes in CTVT before and after therapy and establish a relationship with treatment responses, cytomorphologic patterns, and tumor progression identified with histopathology. METHODS: RT-qPCR was performed on 21 CTVT tumor samples before and after initiating chemotherapy to determine specific gene expression. Normal canine testicular tissue was used as a negative control for all experiments. RESULTS: MDR1 expression was decreased before and after initiating vincristine therapy in CTVT tumor tissues compared with normal canine testicular tissue; p53 and BAX were overexpressed at both time points compared with normal tissue, and no statistical differences were seen between the different morphologic types. However, BAX expression was decreased in the group with quick therapeutic responses but was still overexpressed compared with normal testicular tissue. In the group with the slowest chemotherapeutic responses, BCL2 was overexpressed. CONCLUSION: The findings of this study showed a relative increase in MDR1 gene expression in response to chemotherapy and higher expression in plasmacytoid CTVTs compared with the other cytomorphologic patterns. BCL2 overexpression was related to a favorable prognosis, and p53, BAX, and BCL2 were expressed independent of the cytomorphologic CTVT type. All of the genes were expressed independent of tumor progression, as noted on histopathology.

Immunogenic potential of three transmissible venereal tumor cell lysates to prime canine-dendritic cells for cancer immunotherapy.

Whole tumor cell lysates consist of a mixture of tumor antigens and danger associated molecular patterns (DAMPs) that can be used for dendritic cell maturation and consequently for the activation of a polyclonal T cell-specific tumor response. We evaluated the in vitro efficacy of three different preparations of canine transmissible venereal tumor (TVT) cell lysates: hypochlorous acid-whole tumor cell lysates (HOCl-L), heat shock-whole tumor cell lysates (HS-L), and freeze-thaw cycles-whole tumor cell lysates (FT-L) for the maturation of canine-derived dendritic cells. Our results showed calreticulin, HSP70, and HSP90 release in the three tumor lysates preparations (HOCl-L, HS-L, and FT-L); however, HMGB1 was detected only in HOCl-L and FT-L. Additionally, the uptake by HOCl-L pulsed dendritic cell (DC) increased compared to HS-L and FT-L pulsed DC; and dendritic cell maturation was confirmed by the appropriate cell surface markers (CD11c, CD80, CD83, and MHCII). Furthermore, dendritic cells pulsed with HOCl-L, HS-L or FT-L were cultured with canine lymphocytes. There was an increase of Th1-type cytokines (IL-12, TNF-α, and IFN-γ), in all the tumor cell lysates co-cultures, this correlates with T lymphocyte activation and cytotoxic response. Our data confirm that TVT cell lysates can induce functional canine-DC and that HOCl-L is the most effective one. This preparation of TVT cell lysates with HOCl is an attractive approach that allows the recognition of neoantigens as potential tumor targets and DC priming and therefore could be used for cancer immunotherapy against TVT.

Computer tomographic imaging in 4 dogs with primary nasal canine transmissible venereal tumor and differing cellular phenotype.

Primary nasal canine transmissible venereal tumor (CTVT) without genital affection is uncommon. The aim of this report was to describe the primary nasal CTVT findings and CT staging in 4 dogs with different cytological phenotypes. Three male dogs and 1 bitch were evaluated for their chronic histories of sneezing, snoring, mucopurulent nasal discharge and nasal deformation. Cytological examination of nasal secretions suggested CTVT, confirmed by histopathological examination and LINE-1/c-myc. Males had the plasmacytoid phenotype of CTVT, and the bitch had the lymphocytoid phenotype. CTVT were staged based on the CT findings using modified Adams staging system. The bitch was classified as stage 1, 2 males were classified as stage 3 and 1 male as stage 4. All dogs had a complete tumoral remission after chemotherapy. Plasmacytoid phenotype was identified in cases with most important damage of the nasal cavity. However, the cytological type did not affect the response to chemotherapy.

Does the tumour microenvironment alter tumorigenesis and clinical response in transmissible venereal tumour in dogs?

The canine transmissible venereal tumour (CTVT) is a transmissible cancer that is spread naturally between dogs, with the ability to develop and evade the immune system, despite strict immune surveillance of the host. Furthermore, molecular signalling between cells of the immune system and the tumour microenvironment appear to influence the behaviour and development of the tumour. Thus, this study aimed to quantify the expression of genes related to the immune system such as IL-6, IFN-γ, and TGF-ß, as well as angiogenic factors (VEGF, CXCR4), in CTVT cells in vivo and in vitro (primary culture), correlating with the clinical response of the animals treated with vincristine. As expected, the most prevalent subtype was plasmacytoid cells, although lymphocytic cells were also found, indicating the possibility of polyclonality. When we compared the gene expressions of IFN-γ and IL-6, we mostly found low expression, concluding that MHC expression was probably not occurring in tumour cells, and no activation of immune cells to eliminate the tumour. The TGF-ß gene was normal in the majority of animals but demonstrated decreased expression in vincristine resistant animals, leading to the hypothesis that the concentration of tumour-derived TGF-ß was affecting and even suppressing the real TGF-ß expression, favouring tumour proliferation and progression in these cases. VEGF expression was extremely high, demonstrating its angiogenic role in tumour growth, while CXCR4 was decreased, possibly because of CTVT's low metastatic potential. Thus, we concluded that the tumour microenvironment, together with the immune system of the host, influences CTVT, presumably altering its tumorigenesis and the animal's clinical response to treatment.

Evaluation of a genetic assay for canine transmissible venereal tumour diagnosis in Brazil.

The canine transmissible venereal tumour (CTVT) is a transmissible cancer that is spread between dogs by the allogeneic transfer of living cancer cells. The infectious agents in CTVT are the living cancer cells themselves, which are transmitted between dogs during coitus. CTVT first arose several thousand years ago and the disease has a global distribution and is frequently observed in dogs from Brazil. We evaluated the utility of a LINE-MYC quantitative polymerase chain reaction for diagnosis of CTVT cases in Brazil. Our analysis indicated that the LINE-MYC rearrangement was detectable in all CTVT samples but not in their corresponding hosts. This genetic assay proves to be a useful tool for providing a definitive molecular diagnosis of CTVT, which presents with varying degrees of aggressiveness and invasiveness in different host dogs and can therefore be a diagnostic challenge in some specific cases.

Identification of mitochondrial DNA transfer in canine transmissible venereal tumours obtained from dogs in Mexico.

Canine transmissible venereal tumour (CTVT) has been transmitted by cell transplantation from dog to dog, for over 10 000 years. Although initial studies report a single genetic origin for CTVT, recent samples from around the world reveal high genetic diversity. An elevated number of polymorphisms have been determined in mitochondrial DNA (mtDNA) of CTVT. The recent discovery of mtDNA transference from the host into tumoural cells could be a novel source of genetic diversity in CTVT. The aim of this study was to determine the presence of host mtDNA in samples of CTVT in Mexican dogs. Genotyping of 49 samples of CTVT and 49 samples of blood cells pertaining to affected dogs was performed by direct sequencing from the mtDNA D-loop region. Exogenous mtDNA was observed in 6% of the analysed tumours. This is the first investigation reporting the prevalence of exogenous mtDNA in CTVT in the Mexican dog population.

Cell cycle kinetics, apoptosis rates and gene expressions of MDR-1, TP53, BCL-2 and BAX in transmissible venereal tumour cells and their association with therapy response.

Transmissible venereal tumour (TVT) generally presents different degrees of aggressiveness, which makes them unresponsive to conventional treatment protocols. This implies a progressive alteration of their biological profile. This study aimed to evaluate the cytotoxicity, cell survival, apoptosis and cell cycle alterations in TVT cell cultures subjected to treatment with vincristine. Similarly, it assessed possible implications of MDR-1, TP53, BCL-2, and BAX gene expressions in eight TVT primary cultures for both resistance to chemotherapy and biological behaviour. When comparing TVT cells receiving vincristine to those untreated, a statistical difference related to increased cytotoxicity and decreased survival rates, and alterations in G1 and S cell cycle phases were found but without detectable differences in apoptosis. Increased MDR-1 gene expression was observed after treatment. The groups did not differ statistically in relation to the TP53, BAX and BCL-2 genes. Although preliminary, the findings suggest that such augmented expression is related to tumour malignancy and chemotherapy resistance.

Disseminated transmissible venereal tumour associated with Leishmaniasis in a dog.

This report addresses an atypical transmissible venereal tumour in an 8-year-old bitch that was pluriparous and seropositive for leishmaniasis. There were ascites and a serosanguineous discharge from the vulva, but no lesions on the external genital mucosa. An aspirate of the peritoneal fluid showed mononuclear round cells characteristic of transmissible venereal tumour (TVT). Exploratory laparotomy revealed light red, granulomatous structures in the peritoneum, omentum, spleen, liver and uterine horns. Cytological and histopathological tests confirmed the diagnosis of intra-abdominal TVT. Dissemination of the TVT to several organs inside the abdominal cavity probably resulted from immunosuppression caused by leishmaniasis, which favoured the presence and aggressiveness of TVT.

Detection of the tumour suppressor gene TP53 and expression of p53, Bcl-2 and p63 proteins in canine transmissible venereal tumour.

Canine transmissible venereal tumour (CTVT) is a neoplasm transmitted among healthy dogs by direct contact with injured skin and/or mucous tissue. This study aimed to identify the TP53 gene, messenger RNA (mRNA) as well as the expression of p53, Bcl-2 and p63 proteins in histological sections of 13 CTVT samples at different stages of evolution. The in situ hybridization (ISH) and in situ reverse transcriptase polymerase chain reaction (RT-PCR) assays were used, which showed the DNA homologous to TP53 and its respective mRNA in 92.3% of the samples. We detected p53, p63 and Bcl-2 proteins in most of the cell samples in different grades of intensity. In addition, 46% of the samples were in the progressive and 54% in the regression phase. This is the first description of these proteins and a detailed study of their role in CTVT cells needs to be addressed in or to verify how these cells undergo apoptosis.

Analysis of canine transmissible veneral tumor genotypes using the D-loop region of mitochondrial DNA.

Canine transmissible venereal tumor (CTVT) is the only neoplasm that can be spread among dogs through cell transplantation. Therefore, this tumor does not originate from host cell transformation. Although CTVT has a monophyletic origin, several studies have shown the presence of genetic diversity which was probably acquired after the development of its original clone. To investigate the genetic diversity of CTVT in Mexico and its relation with CTVTs disseminated worldwide, we sequenced a fragment of mitochondrial DNA in 50 tumor samples and matched blood samples from dog hosts from Mexico. We found ten new haplotypes in tumor samples, which were all distinct from their matched host. The TVT1 haplotype was the most frequent in our samples, suggesting that it could be the origin of the others. We found that haplotypes in Mexico and other countries are distributed in two well-defined clusters. Our data also suggest a close relationship among American haplotypes (Mexico, USA, Chile and Brazil). Interestingly, these American haplotypes were also closely related to Asian haplotypes. Taking into account the estimated timing of the origin of CTVT, we propose that CTVT might have originated in Asia; consequently, haplotypes currently present in America could descend from Asiatic lineages.

Risk factors and characteristics of canine transmissible venereal tumours in Grenada, West Indies.

We studied risk factors and characteristics of canine transmissible venereal tumours (TVTs) in Grenada. We abstracted data for 38 TVT cases and 114 TVT-free dogs submitted to a veterinary diagnostic laboratory between 2003 and 2006. Occurrence profiles, odds ratios (ORs), and logistic regression models for TVT were determined using a significance level of alpha = 0.05. TVT was found in 20 (52.6%) female and 18 (47.4%) male dogs. Of the TVT cases, 32 (84.2%) were between 1 and 7 years old, 20 (52.6%) were mixed breeds of dogs, 14 (36.8%) were Grenadian pothounds, while 4 (10.6%) were pure-bred dogs. Characteristic TVT lesions were genital growths [OR = 96.7; 95% CI (27,461), P < 0.001], genital bleeding [OR = 12.7; 95% CI (4.6, 39.2), P < 0.001] and secondary inflammation of TVT lesion [OR = 4.3; 95% CI (2, 10), P < 0.001]. Extragenital TVT lesions were observed in 23% (9/38) of dogs. An increased risk for TVT was associated with age as adult (1-7 years) dogs [OR = 12; 95% CI (1.6, 94), P < 0.001] and status as a Grenadian pothound [OR = 8.6; 95% CI (3, 25), P < 0.001]. Clinicians should educate dog owners about increased risk of TVT for Grenadian pothounds and consider TVT as a possibility for some extragenital tumours.