Cytology and Radiology as a Diagnostic Tool to Detect Metastasis in Mammary Tumor Affected Dogs

Incidence of mammary tumor is high as 50 per cent of all neoplasms in female dogs as compared to breast cancer in women which accounts for 27 per cent of all neoplasms (Veena et al., 2014). Malignant mammary gland tumors can metastasize to various organs; of which, lungs and regional lymph nodes are most frequently affected. The sensitivity of radiographic examination for the detection of pulmonary metastases has been determined to be 72 to 95% (Tiemessen, 1988). Regional lymph node metastases are observed in 9-65% of malignant mammary gland tumors and its examination plays an important role in prognosis. Swollen lymph nodes are frequently examined for cytology in dogs with metastatic lymphadenomegaly (Baker and Lumsden, 2000). Fine needle aspiration biopsy of lymph nodes is an important diagnostic tool commonly used by veterinary oncologist and practising field veterinarians to detect metastasis. Cytopathology has been shown to be virtually 100% sensitive in detecting lymph node metastases. This is a much higher sensitivity compared to palpation but also histologic examinations of needle core biopsy specimens, whose sensitivity has been estimated at only 60-70% (Sapierzynski et al., 2017). Chest radiographs and regional lymph node FNAs are important diagnostic tool to detect metastasis as per oncology practice guidelines (Biller et al., 2016). The actual focus of research is to detect the novel biomarkers for canine mammary tumors as like breast cancer. During course of research we found some of the diagnostic tools to detect metastases and hence reported in the present study.

Case study
 
The present study was conducted in the Department of Veterinary Pathology, Veterinary College and Research Institute, Namakkal - 637 002 of Tamil Nadu Veterinary and Animal Sciences University, Tamil Nadu during the period between September 2017 to July 2018. Thirty three (n=33) dogs were affected with mammary tumor in which three bitches (n=3) viz. Dachshund, German Shepherd and Doberman at the age of 6, 8 and 4 years respectively showed swollen regional lymph node and pulmonary metastases.
 
Haematology
 
Blood samples were collected from saphenous vein by venipuncture in K3EDTA coated vacutainer. The haematology parameters, total erythrocyte count, absolute and differential leukocyte counts, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and platelet counts were analysed by using VetScan HM2, USA.
 
Biochemistry
 
Serum samples retrieved from the blood samples were subjected to evaluation of biochemical parameters such as glucose, total protein, albumin, cholesterol, triglycerides, blood urea nitrogen, creatinine, alanine transaminase, aspartate transaminase, alkaline phosphatase, electrolytes like calcium, phosphorus, magnesium, sodium, potassium and chloride by using A50 Biosystems, India.
 
Hormone assay
 
Serum samples were also subjected for analysis of estradiol, progesterone and testosterone by radioimmunoassay (Spoerri et al., 2015) using 125I labelled antigen and antibody coated tubes with standards which were procured from Immunotech, Radiova, Czech Republic and the radioactivity was measured by using Gamma Counter, Stratec, Germany.
 
Radiography
 
Plain thoracic radiographs were taken in left lateral view for each animal to assess metastases in the lungs using Wipro GE 525 DX X ray unit with exposure of 65-80 kVp, 24-30 mAs and source image distance of 100 cm were employed. The exposed film was processed with carestream vitaflex computerised radiography X ray film processor.
 
Cytology
 
Dogs showed lymphadenomegaly on clinical examination were subjected to cytology by Fine needle aspiration biopsy and Impression smears. Fine needle aspirates and impression smears were obtained from swollen accessory lymph nodes to prepare cytology smear and stained with Giemsa stain.
 
Histopathology
 
Mammary gland tumor tissues along with swollen lymph nodes were collected and fixed in 10 per cent neutral buffered formalin for histopathology. They were processed, embedded in paraffin, sectioned at 3-5m thickness and stained with haematoxylin and eosin (H & E) (Bancroft and Gamble, 2008).
 
Immunohistochemistry
 
Formalin fixed paraffin embedded tissue sections were subjected to immunohistochemical staining. Mounted sections were incubated, antigen retrieved with the help of 1M Tris EDTA (pH 9) in pressure cooker for 15 min at 120°C, incubated with peroxide block (3 per cent peroxide in water) for 30 min followed by incubation with primary antibody, HRP polymer based secondary antibody for 30 min at room temperature, DAB + substrate buffer for 10 min, counter stained with mayer’s haematoxylin for 30 sec and examined (Ramos-vara and Miller, 2013).

The affected dogs were adult pure breeds, non-spayed which showed tumors in caudal pairs of mammary gland. The Dachshund and Doberman showed single tumor in left and right inguinal mammary gland respectively (Fig 1 and 3) whereas the German shepherd showed multiple tumors in left caudal abdominal and left inguinal mammary glands (Fig 2). The tumor occurrence in caudal pairs might be due to greater proliferative change in response to estrogen and presence of larger mass of tissue which are subjected to physiologic change make susceptible to tumors (Anjan Kumar et al., 2011). Breeding history showed animals were exposed to various hormonal changes due to puberty, pregnancy and abortion. Soremno et al., (2011) reported that estrus irregularity, pseudo pregnancy and pregnancy may result in physiological changes in endogenous hormone levels which may predispose to tumor development. The affected dogs showed clinical signs such as coughing, dyspnoea, abnormal percussion and auscultation of the chest. Pulmonary metastases may produce clinical signs when 70% of the lung parenchyma is replaced or obstructed by tumor (Tiemessen, 1988). TNM system of clinical staging revealed all the three dogs were affected with stage IV tumors and showed distant pulmonary metastases. Those falling into the category of stage IV were usually malignant with regional lymph node involvement and frequent metastases.



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The mean haematological, serum biochemical and serum sex hormonal values in the dogs affected with mammary tumors is represented in Table 1. The blood picture from German shepherd showed anemia, leucocytosis and reduced platelet count (PCV - 33.6%, WBC - 26.7×103/cmm and Platelet - 52000/mm3). This anemic changes could be due to secretion of cytokines which sequester iron, reduce erythrocyte half-life (IL-1) and also reduce erythropoietin (TNF and IL-6) release. Leukocytosis might be attributed to inflammation or tissue necrosis associated with mammary tumor or triggered by chronic stress (Duda et al., 2017). Reduced platelet count might be due to paraneoplastic effect or metastasis in bone marrow. Serum biochemistry of German shepherd showed reduced protein and electrolyte levels (Albumin - 1.5 g/dl, chloride - 62.5 mmol/L, Potassium -1.1 mmol/L, Sodium - 33.5 mmol/L) with no abnormality in mean values. Hypoproteinemia was not in accordance with findings of Baithalu et al., (2010) who reported hyperproteinemia which might be due to toxaemia induced hepatocellular damage. Electrolyte disturbances might be related to failure in calcium homeostasis in tumor affected dogs. Serum hormones such as estradiol and progesterone levels were increased into many-fold with no abnormality in testosterone level. This could be due to canine neoplastic mammary gland act as an endocrine organ and it can synthesize and secrete prolactin and steroid hormone in an autocrine/ paracrine manner (Queiroga et al., 2005). This many-fold elevation clearly indicates the hormonal dependency of canine mammary tumors and the risk of tumor occurrence greatly increases after each estrus cycle. 



Upon radiographic examination, the dogs presented with swollen lymph node usually showed pulmonary metastases. Veena et al., (2014) reported that most common target organ for metastases is the lungs which could be evaluated always in any tumor affected dogs. Metastatic nodules were diffuse ill-defined with poor demarcation completely obliterating the entire surface of lungs (Fig 4). Demarcation, definition and diffuse nature of nodules indicates that dogs were affected with advanced stage of cancer which clearly correlated with TNM system of tumor staging. This visible radiographic lesion in the lungs might be due to replacement of 70 per cent of lung parenchyma by metastatic nodules of the tumor (Tiemessen, 1988). On clinical presentation itself, swelling of accessory inguinal lymph nodes were noticed in three dogs (n=3) from thirty three dogs (n=33) affected with mammary tumors. Metastatic lymphadenomegaly was detected only in inguinal lymph node and there was no retrograde metastasis. This might be due to the tumor metastasis to the accessory lymph node through lymphatics. Retrograde metastasis from inguinal gland via the lymphatic plexus in the subcutis of inner thigh and to the popliteal lymph node was reported earlier (Soremno et al., 2011). Based on the location of tumor, the draining accessory lymph node should be identified and examined on clinical examination (Anjan Kumar et al., 2011). 

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Lymph node aspiration cytology revealed mixed population of lymphocytes and neoplastic cells (Fig 5). The lymph node impression cytology exhibited lymphocytes with few neoplastic cells (Fig 6). This cytological findings in metastatic lymph nodes were not reported earlier. The clinically enlarged or palpable lymph nodes in the dogs affected with mammary tumor can be considered for detecting metastatic disease (Sorenmo et al., 2011). The swollen lymph nodes were excised along with tumor excision during surgical intervention and subjected to gross and histopathology. Grossly, the lymph nodes were found to be enlarged, blood tinged and covered with subcutaneous adipose tissue (Fig 2). Histopathology of lymph nodes revealed lymphoid follicles which were surrounded by epithelial cells and metastatic neoplastic cells (Fig 7). In some cases, they revealed depletion of lymphoid cells, lymphocytolysis and neoplastic cells. In few nodes, replacement of lymphoid cells by infiltration of neoplastic cells was noted (Fig 8 and 9). This is in agreement with the findings of Kim et al., (2011) and Thangapandiyan et al., (2013). This might be due to most frequent metastasis of mammary tumors into the regional lymph nodes through lymphatics.

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Histopathology of tumor masses from three dogs revealed cystic papillary carcinoma (Dachshund) (Fig 10), ductal carcinoma (German Shepherd) (Fig 11) and anaplastic carcinoma (Doberman) (Fig 12). All the three were carcinoma which falls into class of malignant tumors. Sarkar et al., (2021) reported that 10-50 % of the histologically malignant neoplasms had distant metastases. Further for advanced diagnosis and prognosis of mammary tumors we employed immunohistochemistry in which we found that the dogs were affected with triple negative tumors (Estrogen receptor - negative (Fig 13), Progesterone receptor - negative (Fig 14), Human epidermal growth factor - negative (Fig 15) and Pancytokeratin receptor - positive (Fig 16)). Triple negative mammary cancer are usually associated with larger size, grade 3 category, pushing margin, recurrence, lymphovascular invasion, distant metastasis, necrosis, high mitotic index, resistance to molecular targeted therapies and shorter survival rate (Abadie et al., 2011). Larger size, grade 3 category, lymphovascular invasion and distant metastasis were recorded in this study. The hormonal dependency of canine mammary tumors generally decreases with increase in malignancy and higher cellular proliferation occurs in malignant tumors with low expression of hormonal receptors. Hormonal receptor negative tumors generally have a poor prognosis than hormonal receptor positive tumors (Leena Rajathy et al., 2019). In present study, all the three dogs showed only swelling of accessory inguinal lymph node. This is due to the anatomy that caudal pairs of mammary gland have their lymphatic drainage into inguinal lymph node whereas cranial pairs into axillary lymph node. Epithelial tumor which might be metastasized through lymphatics to regional lymph node was evident. By convention most malignant epithelial neoplasms (carcinomas) metastasize via lymphatics whereas malignant mesenchymal neoplasms (sarcomas) metastasize via capillaries and veins (Sorenmo et al., 2011). Radiology, cytology and histology of tumor masses confirmed that the bitches were affected with malignant tumors. In the present study, dogs presented with swollen accessory lymph nodes usually showed metastasis in lungs. Gupta et al., (2013) stated that distant metastases can be confirmed via lymph node palpation, chest radiography and ultrasonography. In course of further study we are going to employ ultrasonography for detection of distant metastases. Hence, the above said diagnostic aids such as palpation, cytology and radiology helped us to confirm metastasis in mammary tumor affected dogs.

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When mammary tumor is identified, the subsequent draining lymph node should be examined clinically by palpation and subjected to cytological examination. Those dogs presented with swollen lymph nodes should be subjected to radiological examination. Hence, cytology and radiology played a vital role in the diagnosis of metastasis in mammary tumor affected dogs. The authors are very grateful to the Tamil Nadu Veterinary and Animal Sciences University, Chennai and the Dean, Veterinary College and Research Institute, Namakkal for the facilities provided.