CYTOLOGICAL AND IMMUNOHISTOCHEMICAL DIAGNOSIS OF CANINE TRANSMISSIBLE VENEREAL TUMOURS

T. Chandravathi1, A. Anand Kumar, Y. Narasimha Reddy2 and V. Samatha

1Assistant professor, Department of Pathology, 2 Professor, Department of Microbiology, College of Veterinary Science, PVNRT Veterinary University, Rajendranagar, Hyderabad-500 030 (Telangana). [Received: 07.4.2017; Accepted: 24.10.2017]
{DOI 10.29005/IJCP.2017.9.2.169-171}

The present study includes tumour samples collected from 8 dogs including 5 males and 3 females. The samples
were collected after surgical removal for both cytological and histopathological examination. Cytological examination of impression smears revealed a more cellularity with a predominant population of homogeneous, discrete round cells with mild anisocytosis and anisokaryosis. These cells had a moderate amount of pale-blue cytoplasm with few small vacuoles and prominent nucleolus. Histopathologically, tumours were composed of loose sheets of uniform to round ovoid cells with indistinct borders showing loose round hyper chromatic nucleus and centrally placed nucleoli. Proliferation markers of the samples was estimated by silver staining like AgNORS and immunohistochemistry like PCNA revealed the mean counts as 12.46±1.76 and 364.12±12.96 indicating that transmissible venereal tumour was highly proliferative tumour.
Key words: AgNORs, Cytology, Immunohistochemistry, PCNA, Transmissible tumour.

Canine transmissible venereal tumor (TVT) has been reported from many regions of the world and is a naturally occurring cont -agious round cell tumor of dogs. The tumor is seen most commonly in young, sexually act
-ive, intact dogs allowed to roam freely (Calv -ert et al., 1982). Due to the unique nature of transmission by sexual contact, the external genitalia of either sex are most commonly affected. The biological behavior of TVT is
quite variable and depends on the host immun -e response. Metastasis of TVT usually occur -s in suboptimal physiological conditions of the dog, such as immunosuppression, malnutrition, or young age.
In this report, TVT was described in 8 case and the diagnosis was supported by cytology, histopathology, immunohistochemical stain.

Materials and Methods
A total of 8 tumour samples were collected from dogs including 5 males and 3 females af -ter surgical removal. Impression smears were made from neoplastic masses, air dried, and stained with papanicolas and H&E stains. The
tumours were collected, fixed in 10% neutral buffered formalin, routinely processed, embed in paraffin, sectioned at 4mm, and stained with hematoxylin and eosin for microscopic examination. Replicate sections from the neoplastic
masses were also stained with toluidin -e blue to rule out canine mast cell tumor.
Special staining techniques like argyrophi -li cnucleolar organizer regions (AgNORs) and proliferating cell nuclear antigen (PCNA) were performed to evaluate the prognosis of tumours. The tissue sections were stained by modified silver colloid staining by AgNORs. The AgNOR dots in 100 non overlapping nuc -lei were counted under oil immersion objecti -tive (1000x magnification) and mean number of AgNOR dots per nucleus (AgNOR index)
was calculated for each specimen. Immunohis -tochemistry of proliferating cell nuclear anti -gen was done using the staining procedure. Formalin fixed paraffin embedded sections of 4 μm thickness were taken on to poly-l-lysine
coated slides. Endogenous peroxidase was blocked by placing the sections in 3% hydrogen peroxide for 10 min and rinsed with 0.01M phosphate buffer saline (PBS) at pH 7.4 and incubated with 5% normal goat serum (Sigma,
G9023), then with PCNA monoclonal antibodies. The immunoreactivity for PCNA was done by counting the positive cells in 10 randomly selected high power fields (x200) and the mean values were calculated.

Results and Discussion
Three tumour samples were collected from vulva in female dogs aged 4-8 years and 5 tumour samples were collected from the base of penis in male dogs with age 8-10years. Gro -ssly tumours were pink cauliflower like and
size ranged from 2 – 4 cm, with firm const.
Cytological examination of impression smears revealed a markedly cellular sample with a predominant population of homogeneo us, discrete round cells with mild anisocytosis and anisokaryosis. Tumor cells were present
in sheets with occasional individual cells. They were round with distinct cytoplasmic borders. These cells showed moderate amount of pale-blue granular cytoplasm often contain -ing a few small punctate vacuoles (Fig.1).
Oval to round nuclei were usually centrally located and had a prominent nucleolus and finely stippled chromatin.The nuclei were round with coarse chromatin and a single prominent nucleolus. Moderate mitotic activit
-y was observed. The nuclei were large in relation to cell size as also reported by Henson (2001) and Goldschmidt and Hendrick (200 -2). Occasional neutrophils, lymphocytes, pla -sma cells, and squamous cells were noted in
the background, as were variable numbers of of erythrocytes. The results were in confirmit- -y with previous reports Shilpa et al. (2007). Histopathologically, tumours composed of loose sheets of uniform to round ovoid cells
with indistinct borders showing loose round nucleus and centrally placed nucleoli (Fig.2). Scanty amount of connective-tissue stroma was present. Individual neoplastic cells had a round hyper chromatic nucleus with coarsely stippled chromatin and distinct eosinophilic nucleolus. The cytoplasm was moderate and granular with indistinct cytoplasmic borders. Necrosis and hemorrhage were present in som -e areas. Diffuse lymphocytes, plasma cells and macrophage infiltration also observed. Ne oplastic cells within the submucosa of the vag
ina elevated the overlying mucosa with attenu -ated epithelial cells. Toluidine blue staining did not reveal metachromaticgranules in the tumor cells.Our findings were in conformity with Shilpa et al. (2007) and George et al. 2008).

The nucleolar organizer regions (NORs) being the DNA loops are transcribed to ribosomal RNA (rRNA), a vital need for ultimate synthesis of proteins; thus their small size and high numbers are correlated with aggressive proliferative activity of the cell, while large-sized AgNORs in low numbers indicate less or minimum proliferative
activity of the cell as also recorded by Hall et al. (1990). The PCNA in normal cell functions as co-factor for DNApolymerasedelta which is essential for DNA replication during S-phase of the cell-cycle as mentioned
by Braco et al. (1987). Its expression increases rapidly from late G1 phase, is most abundant during the S phase and declines to
undetectable level in G2/M phase of cellcycle Thus, the results of strong nuclear immunolabelling for PCNA obtained in a majority of tumour cells suggested the higher growth fraction of the tumour, as the proportion of the proliferative cells within the total tumour cell population was referred to as growth fraction which was chiefly
responsible for the rate of tumour growth as also mentioned y Pawaiya et al. (2006).
AgNOR counts and PCNA indices were used as proliferation markers in canine tumours. The mean value of AgNOR countsb and PCNA indices were 12.46±1.76 and 264.12±12.96 respectively (Fig.3 and 4). Our findings were in correlation with the observations of Karademir et al. (1998) who recorded high proliferative activity in transmissible venereal tumour on the basis of high mean AgNOR counts (9.65) and Pawaiya et al. (2006) who observed for
AgNOR counts and PCNA indices as 12.01±0.06 and 205.82±21.55. There exists
linear correlation between the two indices. The PCNA and AgNORs have been reliably used to differentiate several benign and malignant neoplasms of humans and animals and adjunct tools in determining the biological behavior of round cell tumours as also recorded by Tiwari et al. (2016).

The study concludes that the application of PCNA and AgNOR in tumour cells gives proliferative activity, differentiation of benign from malignant tumours and helps in determining the biological behavior of round
cell tumours.

References
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