**3. Unique features of CTVT and its diagnostic methods**

The CTVT mass frequently manifests in the external genitalia of the dog after transmission through coitus. However, other parts of the body can be affected by this cancer. CTVT can be classified into two types according to its anatomical location: lesions typically located on the external genital area of both male and female dogs are called genital TVT (GTVT), while those found in extragenital areas (including subcutaneous, the mucosa of eyes, and in nasal and oral cavities) are called extragenital TVT (ETVT). The GTVT mass at the external genital area is observed as a cauliflower-like mass feature that is friable tissue with hemorrhage or presents with serosanguinous and hemorrhagic discharge and possible secondary bacterial infection [1, 2, 11, 14–18]. ETVT may be related to social behaviors among dogs because of the means of species communication—for example, licking, sniffing, fighting during the breeding season, and routine socialization. As such, the ETVT type is found more frequently in males than in females due to natural behaviors (**Figure 1**) [13].

CTVT has remarkable cytogenetic features. There is an aberration in the number of chromosomes of CTVT. Normally, the normal number of chromosomes in the somatic cells of dogs is 78, 76 acrocentric chromosomes, and couple of metacentric sex chromosomes [7, 21]. Conversely, the number of chromosomes in the CTVT cells varies from about 58–59, with 13–17 metacentric and 42 acrocentric chromosomes and no sex chromosome [1, 7, 13]. These cytogenetic features are consistent and unique and are found in both GTVT and ETVT. This chromosome pattern also appears in CTVT cell cultures and in experimental transplantation [24].

CTVT is one of the round cell tumors, according to its cytomorphologic features. GTVT cases are easier to diagnose according to the location (genital areas) and shape (oozing cauliflower-like mass) of their gross lesions [25]. The CTVT mass can be 0.5 to 10 cm in diameter. Histologic examination of the predictable growth pattern of CTVT reveals numerous round-to-ovoid-shaped cancer cells arranged in a diffuse pattern with an abundance of mitotic figures and few TILs, supported by thin trabeculae of fibrovascular tissue [23]. There are some neutrophils, lymphocytes, macrophages, and plasma cells. The CTVT cell passaging tumor showed no change in the histology of the tumor during the experimental passage [13]. However, the atypical anatomical lesions in ETVT cases are more ambiguous to diagnose based on their location and gross lesion because their features depend on their affecting sites.

**Figure 1.** *Gross lesion of genital TVT (1A, 1B) and extragenital TVT (1C, 1D).*

For example, they perform like a button mass with ulceration when they are located subcutaneously or on the skin. On the other hand, they display irregular shapes in the conjunctiva and oral and nasal cavities. The ETVT tumor must be differentiated from other types, including mast cell tumor, histiocytic tumor, lymphoma, amelanotic melanoma, and poorly differentiated carcinoma, which depends on the anatomical site of the lesion and the characteristics of the histologic examination [25].

Cytologic diagnosis is of great value for easy and rapid on-site diagnosis [26]. As mentioned before, CTVT is one of the round cell tumors, so the main populations of CTVT cells are round-to-ovoid-shaped cells that may originate from the histiocytic system. To improve cytologic knowledge, researchers found that there are three types of cytomorphologic classification of CTVT, which are categorized by the cell morphology of the majority population: (1) lymphocytic type, characterized by more than 60% of round cells, with fine granular cytoplasm, central nuclei, and few intracytoplasmic vacuoles; (2) plasmacytic type, characterized by containing more than 60% of cells with broad cytoplasm, eccentric nuclei, and large amount of vacuoles; and (3) mixed type, presenting both lymphocytoid and plasmacytoid cells, neither of which exceeds 59% [26, 27]. Recently, a study with computerized cytomorphometric analysis of round cells revealed that CTVT had the largest cellular and nuclear size which followed by the histiocytic tumor cell, mast cell tumor, and lymphoma cell. CTVT cell from GTVT case had the largest cellular and nuclear size followed by CTVT cell from ETVT case, histiocytic tumor cell, mast cell tumor cell, and lymphoma cell. According to the CTVT cytomorphologic type, the mixed type had the largest cellular and nuclear size followed by the plasmacytic and lymphocytic type. The researchers have revealed that the plasmacytic type is the most common cytomorphologic type [27, 28]. The plasmacytic type [26–30] and the mixed type [26] are related to malignant behaviors and chemotherapeutic drug resistance.

### *Canine Transmissible Venereal Tumor: An Infectious Neoplasia in Dogs DOI: http://dx.doi.org/10.5772/intechopen.106150*

The lymphocytic type shows aggressive behavior less than other types [26]. So, cytomorphologic classification can provide a prognostic for treatment in each CTVT case (**Figure 2**).

Most canine round cell tumors have been immunocharacterized using several tumor markers—for example, cluster of differentiation 3 (CD3), CD79, paired box-5 protein (PAX-5), and protein-tyrosine kinase (c-kit and CD117). Diagnosis and classification using the immunophenotype are more accurate than routine histopathologic examination. However, the cell origin of CTVT is unclear. CTVT has been previously described as lymphosarcoma, a round cell sarcoma, a histiocytoma, and a tumor of neuroectodermal or reticuloendothelial origin [23]. Immunohistochemistry studies revealed that CTVT cells are negative for keratins, α-smooth muscle actin, desmin, CD3, immunoglobulins G and M, γ-light chains, and κ-light chains. These panels ruled out epithelial, smooth muscle, and T- and B-lymphocytes. CTVT cells are positive for vimentin, ACM1, lysozyme, and alpha-antitrypsin (AAT). Lysozyme and alpha-antitrypsin are not expressed by other mesenchymal cells. Moreover, ACM1 is a canine-specific antibody recognized in canine mononuclear phagocyte stem cells. This panel immunophenotypic expression suggests that CTVT has a histiocytic origin because this set of antigens is not expressed by other mesenchymal round cells [23].

CTVT has unique molecular characteristics. CTVT has the rearrangement of the c-*myc* gene, which is related to the LINE-1 [12, 31]. The LINE-1 is a retrotransposon or jumping gene localized at the 5′ region to the exon of the c-*myc* locus of CTVT cells. This jumping gene causes destabilization in the entire cellular genome, leads to cellular proliferation and differentiation, and performs the malignant transformation of cells. A rearrangement of the LINE-1–c-*myc* gene sequence has been used with polymerase chain reaction (PCR) and *in situ* PCR to diagnose CTVT [6]. The localization of the LINE-1 positive was in the nuclei of CTVT cells; this was not present in other inflammatory cells or CTVT connective tissue. The LINE-1 is inserted in all cases in the same position and presented in the same PCR product size. This method can be performed using fine-needle aspiration (FNA) samples with only 10 nanograms of

#### **Figure 2.**

*Cytomorphology of CTVT cells presents the lymphocytic cell type (arrow) and plasmacytic cell type (star); H&E (40X).*

sample DNA for conventional PCR. The LINE-1–c-*myc* PCR revealed a diagnostic sensitivity of 100% and specificity greater than 80% [25]. As mentioned before, the CTVT somatic cell lineage is remarkably stable and lacks subclonal heterogeneity despite many genome rearrangements, copy number changes, and retrotransposon insertion [8]. The unique, specific, and constant molecular feature of CTVT is utilized as a definitive diagnostic marker and for applicability in veterinary clinical routines because this feature is found in normal CTVT and vincristine-resistant CTVT cells. Also, the PCR method using FNA samples can be used as a surveillance protocol during and after chemotherapy to determine complete remission status and the appropriate times to stop or restart chemotherapy [11].
