Canine transmissible venereal tumour (CTVT) is a contagious venereal tumour of dogs, commonly observed in dogs that are in close contact with one another, or in stray and wild dogs that exhibit unrestrained sexual activity. CTVT represents a unique, naturally transmissible, contagious tumour, where the mutated tumour cell itself is the causative agent and perpetuates as a parasitic allograft in the host. Clinical history, signalment and cytological features are often obvious for establishing a diagnosis though biopsy and histological examination may be needed in atypical cases. Most cases are curable with three intravenous injections of vincristine sulphate at weekly intervals. The role of stray and wild dogs makes the disease difficult to control and necessitates sustained animal birth control in stray dogs along with prompt therapy of the affected dogs. This review captures the manifold developments in different areas embracing this fascinating tumour, including its biology, diagnosis and therapeutic alternatives.
Canine transmissible venereal tumour (CTVT), also called transmissible venereal sarcoma, Sticker's sarcoma, venereal granuloma and infectious sarcoma, is a contagious venereal tumour of dogs. Considering the abbreviative similarity with cardio-thoracic vascular surgery (CTVS), it is better to reserve the use of the abbreviation CTVT for this tumour. First described by Hujard in 1820 in Europe, it is most commonly observed in dogs that are in close contact with one another, or in stray and wild dogs that exhibit unrestrained sexual activity. In 1876, first ever in the history of oncology, the Russian veterinarian Novinsky demonstrated the transplantation of the tumour from one dog to another by infecting them with tumour cells. So far, only three naturally transmissible, contagious tumours are known in mammals, viz. CTVT, the Tasmanian devil facial tumour disease (DFTD) and a similar tumour of Syrian Hamsters. Spanning over almost two centuries, CTVT continues to attract the attention of many researchers, as it reflects ever new and often surprising paradigms and offers to have something of everyone's interest. This review captures the manifold developments in different areas embracing this fascinating tumour, including its oncobiology, diagnosis and therapeutic alternatives.
CTVT has been reported from all continents of the world except Antarctica.[1, 2] It is uncommonly reported from North and Central Europe and from North America, mainly because of the control of stray dog populations, thorough prebreeding examinations and effective treatment of clinical cases. Baring few exceptions, CTVT remains enzootic in the rest of the world, including Puerto Rico, Papua New Guinea and south-west France.[4, 5] CTVT is more common in tropical and subtropical regions, particularly in the southern United States, Central and South America, south-east Europe, Ireland, China, the Far East, Middle-East and parts of Africa.[4-6] In Bahamas, Japan and India, it is the most common tumour of dogs.[6-8] Das and Das reported a homogeneous distribution of the neoplasm across varying geo-climatic zones in India.
Aetiology and transmission
Although Novinsky's success at transplanting the tumour in 1876 provided some early cues, it was for Cohen to conclusively prove that the mutated tumour cell itself was the causative agent. The transmission of the tumour needs viable tumour cells as cells that were frozen, heated, treated with glycerin or desiccated failed to produce the disease. An intact epithelial barrier in susceptible dogs disallows the natural transmission of the tumour; the tumour can be naturally transmitted only across abraded mucosa where epithelial continuity has been breached. Tumour cells are exfoliated and transplanted during coitus from animal to animal and perpetuate themselves like any other heterologous, mono-cellular organism. However, Marcos et al. reported a unique case of TVT in a sexually immature, 11-month-old, virgin bitch showing entirely cutaneous lesions without any mucosal involvement, also suggestive of transmission from the dam to the pup during social interactions such as grooming and other maternal behaviour. In natural transmission, the number of tumour cells undergoing transplantation may also be important as it has been shown that only about 13% of the transplanted cells are able to survive until the infection is established in the recipient. The tumour may also spread through licking, scratching, biting or sniffing tumour-affected areas. CTVT, thus, represents an infectious cell line transmissible as a naturally occurring allograft. Experimentally, Karlson and Mann succeeded in passaging the tumour through 40 generations of dogs over a period of 17 years with no changes in the histopathology of tumour during the passage.
This ‘naturally occurring allograft’ has become a true pathogen that defies natural transplantation barriers. Murgia et al. employed microsatellite and dog leukocyte antigen (DLA) analysis to conclude that the CTVT cells arose around 200–2500 years back in wolves or in some old East Asian dog breed such as the Shih Tzu. Cancer cells rapidly accumulate mutations and become genetically instable; CTVT presents a new paradigm as the study showed that soon after emergence, the tumour cell underwent some drastic genetic re-arrangements following which the tumour cell has been passed on with little genetic variation. The study also suggested that CTVT must be regarded as a special case of somatic chimerism. However, Rebbeck et al. employed microsatellite analysis to conclude that TVT is more than 6000 years old, and using sequence analysis of RPPH1 gene they also suggested its origin from a dog or wolf during the first domestication of dogs. Rebbeck et al. further showed that the cancer has periodically acquired mitochondria from its host as the TVT mitochondria tend to degenerate because of high mutations.
Intact dogs form the high-risk group during periods of maximal sexual activity when females exhibit the signs of oestrus. The disease usually (80%) occurs in animals of reproductive age (2–8 years old) and less often at older ones.[1, 6, 19, 20] Females are infected more often than males (64.5 and 35.5%, respectively) because one infected male often mates with numerous females, in free range or stray group and also in kennels.[1, 21, 22] On the basis of social behaviour, the high-risk groups include the habitually yard-escaping dogs (75.5%), guard dogs (41.3%) and hunting dogs (41.5); CTVT is rare in well-supervised home-kept companion animals (4%), occurring only after an escape eventuating into an unwanted copulation event. Incidence of extra-genital tumours may be higher in males owing to the breeding behaviour of the male. Metastatic events are more numerous in the male dogs.[23, 24]
The tumour occurs naturally only in dogs; however, experimental infection (heterotransplantation) has been established in other canine species, such as foxes, coyotes and jackals as well as in immunodeficient mice models, such as athymic and NOD/LtSz-SCID mice.[12, 16, 25, 26] Any well-established heritable breed-related prevalence is not known although Kabuusu et al. observed an increased incidence of TVT in Pothounds as compared with other dog breeds in Grenada, West Indies. Molecular studies suggest that the insertion of a transposable element viz. long interspersed nuclear element (LINE) -1 near the 5′ end of the first exon of the c-myc oncogene in the germline might represent a predisposition to develop CTVT after exposure to an oncogenic agent.[16, 28]
The tumour is cauliflower-like, pedunculated, nodular, papillary or multilobulated. It ranges from a small nodule (5 µm) to a large mass (up to 15 cm) and is firm, though friable. The surface is often ulcerated and inflamed and may be haemorrhagic and infected (Figs 1 and 2A). The tumour may be solitary or multiple and is almost always located on the external genitalia, although it may occur in adjacent skin and oral, nasal and conjunctival mucosae. Incidence varies from relatively high in some parts of the body to rare in others. The tumour may arise deep in the prepuce or vagina and be difficult to see. This may lead to misdiagnosis if bleeding is confounded with oestrus, urethritis, cystitis or prostatitis.[30, 31] In female dogs, the neoplastic lesions are usually located at vestibule (95.6%) and less often at the vagina (44.5%) or invading the vulvar lips (18.6%). Main lesions are almost always present at the junction of the vestibule and vagina, perhaps because of the high pressure exerted on this area during the mating act. In male dogs, neoplastic lesions are located on the more caudal part (bulbus glandis, 81.5%; Fig. 2A) and less often on the shaft (pars longa glandis, 25.9%) or the tip (9.9%) of the glans penis. Occasionally lesions may be present in other parts of the body without any genital involvement;[6, 32, 33] primary intranasal transmissible venereal tumour in the dog has been reported by Papazoglou et al. In cases with extra-genital localization of the TVT, clinical diagnosis is usually more difficult because TVTs cause a variety of signs depending on the anatomical localization of the tumour, e.g. sneezing, epistaxis, epiphora, halitosis and dental fistula, exophthalmos, skin bumps, facial or oral deformation along with regional lymph node enlargement. Infrequently, the tumour cells may be parasitized by Leishmania, representing an alternative mode of transmission of canine leishmaniasis where the two diseases co-exist.[35-37]
Initially, the tumour grows rapidly presenting a brightly sanguine colouration, commonly associated with the oozing of a haemorrhagic fluid (94.6%), to be followed by protrusion of the neoplastic lesions (31.3%) and deformation of the external genitalia (30.4%). The peculiar odour of the neoplastic lesions discharge (27.2%), which after secondary bacterial infection becomes particularly unpleasant, and the excessive licking (5.8%) of the genitalia are also commonly noticed. Less commonly observed symptoms include dysuria (5.4%), weakness (4.6%), ulcers in the perineum area (2.1%), anorexia (1.7%), constipation (0.8%), paraphimosis (0.8%), mating refusal (0.4%) and weight loss (0.4%). Dogs with transmissible venereal tumours are at a high risk of having bacteriuria likely owing to obliteration of the urethral orifice by the tumour leading to urine retention. Few cases have also been recorded where this neoplasm has caused actual mechanical obstruction to the flow of urine or has produced dystocia in whelping females.
The clinical findings are less striking in male dogs and the disease is consequently less easily perceptible than in females. Clinical and laboratory findings suggest that the general health of affected animals is not impaired unless the lesions turn necrotic and infected or occlude the urethral orifice, or metastasis occurs. Haematocrit values are slightly lower than normal in less than 10% of the affected dogs, but no severe anaemia is found. In about 30% of the cases a mild-to-moderate leukocytosis may be apparent, probably caused by the inflammation of the tumour surface.
CTVT undergoes a predictable cycle: an initial growth phase of 4–6 months (P phase), a stable phase and a regression phase (R phase), although not all tumours will regress. Progressing tumours are characterized by higher mast cell counts and microvessel counts at the invasive edges of the tumours. Regressing tumours contain higher numbers of lymphocytes, most of which are T cells. P-phase tumours consist of round cells with microvilli, whereas those in stable phase consist of cells undergoing transition from round cells to spindle-shaped fibroblasts. Regressing tumours have spindle-shaped cells with intracellular collagen bundles within vacuoles. Mitotic figures, telomerase activity, proliferating cell nuclear antigen (PCNA) positive rate and argyrophilic nucleolar organizer region (AgNOR) content is higher during P phase. Metastasis is uncommon (5%); when it occurs, it is usually to the regional lymph nodes including inguinal and external iliac lymph nodes but may also be seen in the kidney, spleen, liver, eye, tonsils, brain, pituitary, skin and subcutis, maxillary bone, mesenteric lymph nodes and peritoneum.[1, 2, 43, 44]
Diagnosis is established based on anamnesis, signalment, clinical findings, cytology and histology; cytogenetic and molecular techniques may also be applied. Paterson et al. compared the uptake of 67Gallium and 45Calcium in lactating bitches and found that concentrations in individual pieces of a lactating mammary gland at 5 h after injection were correlated; however, no such correlation was seen in multiple samples of a transmissible venereal tumour. Increased concentration and longer retention of 67Gallium citrate in mammary CTVT was observed. The differential uptake of such radionuclides by CTVT tissue may be of diagnostic importance. Also, the use of a contrast agent (NC100100) during transrectal ultrasonography has been shown to increase the accuracy of lesion detection from 67 to 87%.
TVT cells may be collected by fine-needle aspiration of the tumour mass; cells may also be detected in free-catch samples of urine. Cytologically, TVT cells have a very distinct appearance (Fig. 3). They are round to oval in shape and often contain mitotic figures, with chromatin clumping and one or two prominent nucleoli. The most striking cytological finding is the presence of multiple clear cytoplasmic vacuoles. Vacuolation increases during early stages of regression as TVT cells undergo degeneration. During degeneration, amounts of endoplasmic reticulum and ribosomes also increase, as does swelling and vacuolation of mitochondria. Degenerating cells often contain numerous membrane-bound granules and clusters. Cytology provides a better evidence of CTVT as compared with histology. The histologic appearance of transmissible venereal tumour may be difficult to distinguish from other round cell tumours such as histiocytoma, lymphosarcoma or mast cell tumours; especially so when the tumours involve extra-genital locations. Upon reticulin staining, TVT cells show invasion of the inter-alveolar spaces by reticulum fibres, characteristic of alveolar soft-part sarcoma.
For the diagnosis of TVT by immuno-histochemistry a panel of antibodies is required. TVT shows the immuno-histochemical staining characteristics of histiocytic cells and the differentiation between these two tumour types should, therefore, be based on clinical and histopathologic criteria. The tumours stain with antibodies against vimentin, lysozyme, α-1-antitrypsin and glial fibrillary acidic protein, and are negative for keratins, S100 and muscle markers.[50, 51]
Cytogenetics is of decisive advantage in the definitive diagnosis of the CTVT because of the highly significant karyotypic differences that exist between normal and cancerous cells. Although the normal chromosome number for the dog is 78 and all but two are acrocentric, the chromosome count in TVT cells is 59 (range 57–64) of which 15 or 17 are metacentric and 42 or 40 are acrocentric whereas the total amount of DNA remains close to normal.[51, 52] The same chromosomal pattern is also maintained in cell culture. The sex chromosome complement is XO. Microarray-based comparative genomic hybridization analysis suggests that TVT has accumulated these non-random chromosome copy number aberrations that are naturally distributed widely throughout the dog genome.
Molecular biology can also be useful in the diagnosis of canine TVT. Palker and Yang succeeded in identifying and characterizing a 70 kDa, temperature and pH labile TVT-specific antigen. Later, Yang et al. also reported a TVT-associated antigen from the sera of affected dogs. The c-myc oncogene is re-arranged in this tumour by insertion of a 1.5 kbp transposable sequence, known as the long interspersed element (LINE), 5′ to the first exon. Analysis of TVT samples from around the world has revealed that in all tumours the insertion of the same LINE occurs in the vicinity of the c-myc gene, and a PCR amplification of the DNA segment covering the mutation area is of diagnostic importance.[28, 57] Heat shock proteins (HSPs) 60 and 70 are potential markers for TVT, and HSP 60 appears to be involved in CTVT regression. Choi and Kim identified a point mutation of the tumour suppressor protein p53 (T963C) resulting in the change of amino acid (Phe–Ser) in TVT cases. Vázquez-Mota et al. concluded that this mutation did not participate in the clonal origin of the tumour, but was acquired at a later stage. Further, TVT tissue from bitches lacks surface oestrogen receptor-α expression.
The prognosis for total remission is good, unless metastatic involvement of the central nervous system or eye is present. TVT is antigenic in the dog and the course of the disease is markedly influenced by the immune status of the dog. In healthy, immuno-competent, adult dogs, the tumour regresses spontaneously after a period of logarithmic growth, and the development of tumour immunity prevents successive occurrences. Das and Das recorded that the chances of self-regression in tumours over 9 months of age were remote. The poor prognosis in TVT correlates with an increase of the Ag-NOR proteins in the nucleus of TVT cells. A strong intensity of stromal hyaluronan and a high proportion of hyaluronan-positive tumour cells are significantly associated with progressing tumours.
Immunological studies have established that TVT is antigenic in the dog as specific antibodies can be demonstrated in circulation as well as on the surface of the tumour cells, and the immune response against the tumour plays a major role in determining the course of the disease.[63, 64] In most adult dogs, the tumour regresses spontaneously after a period of logarithmic growth; after complete regression, the development of tumour immunity prevents successive occurrences. Spontaneous regression is due to the formation of immunoglobulin (Ig) G in the sera of dogs after about 40 days of tumour growth. Upon experimental rechallenge, tumours are detected on the third day, begin to regress on the 9th day and disappear on approximately the 15th day after the secondary transplantation. Newborn pups from immune dams are more resistant towards development of the tumour and exhibit faster remission. In contrast, the tumour progresses to ulceration and metastasis in the immunologically incompetent or compromised host. Lymphocytes from dogs in which TVT has regressed are cytotoxic to TVT cells; sera of such dogs mediate antibody-dependent cellular cytotoxicity with normal dog lymphocytes on TVT cells.
CTVT has evolved several mechanisms to dodge the host immune response. The tumour cells downregulate the expression of their DLA genes that encode for markers which identify the cell. While DLA class II expression is completely inhibited, some expression of DLA class I genes is allowed up to 10%. This is of strategic importance for the tumour to evade the host immunity. Complete masking of the class I DLA tends to activate natural killer cell activity; a low background level of class I expression effectively prevents this activation while making the cells virtually invisible to the host immune system. CTVT cells also secrete transforming growth factor (TGF)-β1 to suppress the host immune system. The TGF-β1 induced immunosuppression is antagonized by interleukin (IL)-6 and IL-15.[68, 69] During tumour regression host IL-6, derived from tumour infiltrating lymphocytes, works in combination with host interferon (IFN)-γ to restore DLA expression. Further, TVT is known to decrease the number of monocytes in the peripheral blood by 40%, and substantially impair the efficiency with which dendritic cells (DCs) are generated from monocytes and with which mature DCs are generated from immature DCs.
High rate of spontaneous regression warrants proper caution in the evaluation of the success achieved with different therapeutic approaches.[2, 5] Treatment can be attempted by excisional surgery, chemotherapy, radiotherapy, immunotherapy or a combination thereof.
Surgical treatment has been applied since the last century[1, 2] with a low rate of efficacy resulting in a high recurrence rate (between 18 and 60%), influenced by the location and extent of disease; up to 50–68% relapses occurring solely owing to tumour cell transplantation into the surgical wound during traditional surgical intervention.[19, 24, 72, 73] During operating male dogs, care should be taken not to damage the urethra of penis. If the urethral orifice is involved, an in-dwelling catheter should be used till complete healing is accomplished. Scalpel surgery has been condemned as impracticable in cases of generalized CTVT. The use of electrocautery makes the operation easier and seems to be a little more effective. Therefore, surgical excision should be applied to those dogs that present solitary, small, easily accessible and non-invasive tumour nodules. Even in such resectable tumours electrosurgical or cryosurgical excision seems to have an edge over traditional surgical approaches. Voorhees and Babbs advocated adjuvant treatment with hydralazine (0.5 mg/kg i.v.) to increase the temperature difference between TVT and surrounding normal tissues during inductive diathermy. Badylak et al. found CTVT to be resistant to microwave-induced hyperthermia damage and Hazle et al. reported limited success of magnetic resonance imaging-guided high temperature thermal therapy of transplanted TVT in the canine prostate using a directional transurethral ultrasound applicator. However, Schwartz et al. could demonstrate selective and effective photothermal ablation of TVT in brains of mixed breed hounds using near-infrared absorbing nanoshells.
Chemotherapy gives more promising results and up to 100% remission can be achieved. Trials with new generation antineoplastic drugs that started during the 1950s established antimitotic agents as the preferred agents for the treatment of the tumour. An array of chemotherapeutic agents has been tried against CTVT such as cyclophosphamide,[20, 24] methotrexate, cyclophosphamide with prednisone, vinblastine with cyclophosphamide or methotrexate, vincristine, vincristine with doxorubicin, cyclophosphamide with methotrexate and vincristine.[1, 20, 74] Vincristine, which has emerged as the drug of choice for TVT therapy, was included in treatment protocols since the eight decade of the last century.[20, 74, 80, 81]
Many studies suggest a better response with vincristine alone as compared to combined chemotherapy, and currently, the intravenous administration of vincristine sulphate at the dose of 0.025 mg kg−1 of body weight, once a week, for 2 weeks beyond the complete resolution of the gross tumour mass, is the treatment of choice, irrespective of the neoplasm size and extent, the presence of metastases and the duration of the disease (Fig. 2). The animals recover fully, with no impact on behaviour and reproductive ability. Scarpelli et al. observed that larger tumour mass, increased age and therapy during hot and rainy months were unfavourable predictive factors retarding the success of vincristine therapy.
Temporary side effects (partial anorexia and mild depression) may be reported in less than 20% of the treated dogs, usually 1–2 days after vincristine administration. Chemotherapy may cause transient leucopenia, but less than 2% subjects present such a leucopenia that might deserve an adjunctive antibiotic treatment or impose discontinuation of one or more chemotherapeutic administrations. Dogs undergoing therapy with vincristine also exhibit a transient deterioration of semen quality that rapidly returns to normal within 15 days of last injection.[83, 84] Before initiating vincristine chemotherapy it is important to assess the general health condition of the animal, while during therapy it is necessary to follow-up the total number of leukocytes, at least at weekly intervals. Novel approaches for improved and targeted drug delivery have also been attempted. Vilensky et al. evaluated vascular-targeted photodynamic therapy (VTP) using Pd-bacteriopheophorbide (WST09) as an alternative to conventional chemotherapy, and concluded that VTP with WST09 can efficiently treat CTVT in a single session, as compared with four to six sessions of chemotherapy.
TVT has already been proven to be highly sensitive to irradiation from the last century. Solacroup proposed radiation therapy for TVT in Europe in 1950, which has been employed since, mainly in France resulting in a sufficient reduction of TVT cases. Dosage recommendations range from 1500 to 2500 rads (depending on the chronicity and the extension of neoplastic lesions), divided in sessions of 400–500 rads over a period of 1–2 weeks, or a single dose of 1000 rads which, if not curative, can safely be repeated one to four times. However, radiotherapy requires trained personnel, specialized equipment, high expenses and, also, the foregoing necessity to chemically immobilize the dog. Therefore, its use is recommended only in cases where other treatments fail. Intensive radiotherapy or chemotherapy may lead to fatal leukopenia and immunosuppression.
Immunotherapy has also been reported as a method for the treatment of this tumour. While traditional approaches employed convalescent sera and whole blood for transfer of passive immunity, or tumour homogenates as autochthonous vaccine and bacterial toxins for active immunization, modern approaches have used interleukins and viral agents for stimulating active cellular immunity.[88, 89] Hess et al. found intralesional BCG therapy of canine venereal tumours to be highly effective in causing regression. Mukaratirwa et al. reported faster remission with combined BCG and vincristine therapy than vincristine or BCG alone. Co-infection with canine parvovirus influences the course of the disease; when inoculated simultaneously with the tumour at a distant site, a modified-live parvoviral vaccine prevents tumour transplantation. However, the vaccine has no effect if inoculated 3 days after tumour transplantation. Therapy of TVT with plasmid encoded IL-6, IL-12 and IL-15 genes delivered through electroporation has also been suggested.[69, 92, 93] CTVT cells in culture are subject to a growth-repressing effect by sulphur-containing amino acids; therefore, supplementation of diet with such amino acids may also be of value towards faster remission of the tumour. Pai et al. evaluated fused DC/TVT hybrids as a vaccine against TVT and reported substantial inhibition of tumour progression and accelerated regression in the vaccinated group. Successful protection against TVT has also been reported with a Chicken HSP70 DNA vaccine.
CTVT represents the oldest known malignant cell in continuous propagation whereby a single malignant clone of cells has colonized dogs worldwide. Clinical history, signalment and cytological features are often sufficient for making diagnosis for CTVT. Biopsy and histological examination are not necessary except cases with an atypical presentation. CTVT is curable in almost all cases with three cycles of vincristine sulphate intravenous injection at the dose rate 0.025 mg kg−1 body weight. The disease is difficult to control owing to the role of stray and wild dogs as reservoirs of infection. Proper examination of animals prior to mating to eliminate animals with patent infection from the breeding population and strict measures against mingling with stray animals will control the disease. Sustained animal birth control programme of stray dogs with chemotherapy of the affected dogs shall definitely decrease CTVT cases to a great extent.
Conflict of interest
The authors declare no conflict of interest.
The authors acknowledge the anonymous reviewers for their constructive criticism of the manuscript in advance. Financial support received by B. G. in the form of a Junior Research Fellowship from Council of Scientific and Industrial Research (CSIR, India) is also duly acknowledged.