Studies on diagnosis and management of mammary tumors in canine and proud-flesh in equine
Abstract
The study was conducted on mammary tumors in bitches (n=10) and proud flesh in equine (n=12) which
were admitted to TVCC, LLRUVAS, HISAR. These were divided into 2 groups. In Group 1, All the bitches were
intact females in the range of 5-16.5 years of age (median age 10.7) and was of different breeds. The scanning of
mammary gland was done in lateral respective recumbency using a real time, B-mode machine having a
curvilinear ultrasound transducer of a 7.5 MHz frequency. The ultrasonographic images were used to evaluate the
shape, size and echogenicity of the mammary lesions. On ultrasonogram, benign tumors were homogenous in their
internal echogenic pattern i.e. hypoechoic. Ultrasonograms of malignant tumors have many pockets like structure
of different sizes which have hyperechoic boundary. Out of these 10 cases, 6 were of benign whereas the other 4
cases were of malignant.
These tumors were further confirmed by histopathologically. Excisional biopsy was taken after surgery
of these cases. There were four cases of adenocarcinoma, two cases each of benign mixed tumors and
myoepithelioma and one case of each adenoma and myxoma. Adenocarcinoma, tumor cells were anaplastic,
characterized by lose of their polarity and hyperchromatic nuclei. The acini contained many layers of cells which
were arranged in tubules of irregular size and shape usually lack secretion and often contained detached cells
giving appearance of solid masses of cells. Mitotic figures were present. There was necrotic area in between tumor
cells. In benign mixed tumor, these tumors consist of cartilage tissue, myoepithelial cell and myxomatous tissue. In
Myoepithelioma, tumor cells were spindle shaped and irregularly arranged. Adenoma, there were irregular and
enlarged acini with papillary projection. They were mostly lined by single layer of epithelial cells. In between the
acini, there was fibrous stroma. Myxoma, these tumor cells were of Star shaped/ stellated cells, having cytoplasmic
processes and there was presence of light bluish mass in between the cytoplasmic processes.
For real-time RT-qPCR of slit 2 gene in canine mammary tumors, the quantity of total RNA extracted
from different tissue samples was in the range of 20 Pg/ml-100 Pg/ml from 20-30 mg of different tissues as
determined by Qubit fluorometer or Biophotometer. Amplification plots and dissociation curves of RT-qPCR were
indicative of specific amplification of the canine slit 2 gene in all the samples. Further, amplification product of a
160 bp slit2 gene and 120 bp β-actin gene in all the RT-qPCR tubes was visible by 2% agarose gel electrophoresis.
DNA sequence of a 96 nucleotide long segment of slit 2 gene further confirmed specific amplification of the gene
in the present study. Transcript levels of slit 2 gene in canine mammary tumors as determined by real-time RTqPCR
were 2 to 85 fold. Out of 12 tissue samples studied, transcript levels of slit 2 gene in 11 different samples
ranged from 1.90-84. It was 98-fold higher than the mean level of normal tissues (n=5). One of the samples did not
perform in RT-qPCR. Sample ID no. 3 showing 1.92-fold higher than normal levels was from a bitch treated with
Vincristine sulphate injection @ 1.4 mg/m², I/V slowly.
For management of these cases, surgical removal of the affected tissue was an essential treatment for
these mammary tumors. Skin sutures were removed after 2 weeks. Surgical excision of mammary tumors allowed
the histological examination of the patients, increased survival time and the patient’s quality of life. For
chemotherapy, Vincristine sulphate injection was administered thrice via slow intravenous injection @ 1.4 mg/m²,
at weekly intervals. Homecare was done with the help of antibiotics, analgesics, B-Complex and Vit-C. After
chemotherapy and homecare, these bitches recovered and become normal. Most of these dogs do not lose their hair
and usually have only mild side effects of the medication, such as transient loss of appetite and vomiting.
In group II, study was conducted on 12 cases of proud flesh of equine. There was history of injury in
some of these cases resulting in formation of proud flesh. Some of these cases, bleeding from the lesion also
occurred. Lesions were 15 days to 6 months old in these cases. Mainly limbs were affected because of being more
prone to injury.
Biopsy was taken before or after surgical excision according to the possibility. In 10 out of 12 cases,
surgical removal of this proud-flesh was done and the biopsy samples were taken. Histopathological examination
of these cases revealed that 8 cases were of granulation tissue and 4 cases were of fibroma. Granulation tissue was
characterized by proliferation of fibrous tissue along with blood vessels and inflammatory cells. In one case
excessive numbers of eosinophills were present. Fibroma was characterized by whorls or interlacing bundles of
fibroblasts. Tumor cells are stellete shape having large ovoid or elongated nuclei.
BPV-PCR was optimal at 50°C primer annealing temperature. None of the samples showed PCR product
of the expected size of 314bp/307bp as a major specific band of BPV-1/2. However, at least in 5 samples, a band
of approx. 370bp was visible in agarose gels.
Out of 12, in 10 cases surgical removal of the exuberant granulation tissue was performed. In this
process, the granulation tissue was excised to level with the skin edges. For chemotherapy, Anthiomaline
injections @ 15 ml total dose I/M on alternate day for six times and Vincristine sulphate @ 5mg/m² administered
every one week’s by slow intravenous injection for three times. Homecare was done with the help of antibiotics,
analgesics, B-Complex and Vit-C. These cases become nearly normal after 4 week of this treatment.
From the present study, the following conclusions can be drawn. Ultrasonographic characteristics appear
to be correlated with histopathologic changes in canine mammary tumors. Histopathology has an important role in
the evaluation of tissue composition of mammary tumors in canine. The study further provided evidence of
association of slit 2 up-regulation in canine mammary malignancy. Slit 2 up-regulation in malignancies agreed
with histopathology and ultrasonography. Histopathological changes in the proud-flesh in horses revealed either
granulation tissue or fibroma. BPV1/2-DNA was not detectable by PCR in any of these proud-flesh cases
including the fibroma. The proud-flesh in horses responded well to surgical removal of the tissue in combination
with chemotherapy using Anthiomaline and Vincristine sulphate.
Description
Keywords
Canis familiaris; Mammary gland; Ultrasonography; Histopathology; Quantitative real-time PCR; Equidae familiaris; Proud-flesh; Histopathology; PCR