Clinico-pathological investigation of inflammatory mammary carcinomas in bitches Nandhini S.1, Madheswaran R.2,*, Balasubramaniam G.A.2, Ramya K.3 1Veterinary Assistant Surgeon, Animal Husbandry Department, Udumalpet, Tiruppur, Tamil Nadu - 642 126 2Department of Veterinary Pathology, Veterinary College and Research Institute, Namakkal - 637 002, Tamil Nadu Veterinary and Animal Sciences University, Tamil Nadu, India 3Department of Veterinary Microbiology, Veterinary College and Research Institute, Namakkal - 637 002, Tamil Nadu Veterinary and Animal Sciences University, Tamil Nadu, India *Address for Correspondence: Dr R. Madheswaran, Department of Veterinary Pathology, Veterinary College and Research Institute (TANUVAS), Namakkal - 637 002, Tamil Nadu, India, E-mail: rmadheswaran2003@gmail.com
Online Published on 03 February, 2023. Abstract A study was conducted to elucidate the altered clinico-pathological parameters in diagnosis and prognosis of inflammatory mammary carcinomas in bitches. Blood and serum samples were collected from 30 each normal and tumor bearing bitches for haemato-biochemical analysis. A significant (P<0.05) reduction of total erythrocyte count and highly significant (P<0.01) reduction of packed cell volume were noticed in tumor affected bitches. A significant (P<0.05) elevation of mean corpuscular haemoglobin and highly significant (P<0.01) elevation of mean corpuscular haemoglobin concentration and total leucocyte count were observed in mammary tumor affected dogs. A significant (P<0.05) increase in total cholesterol and highly significant (P<0.01) increase in alanine transaminase, aspartate transaminase, alkaline phosphatase and potassium values were noticed in tumor bearing bitches. Highly significant (P<0.01) reduction in albumin and significant (P<0.05) reduction in calcium were also noticed. An elevation of superoxide dismutase, reduced glutathione, glutathione peroxidase and lipid peroxidase values whereas reduction of catalase value were noticed in the tumor tissues. Histopathologically, the tumors were classified as benign (2) and malignant (28) tumors with infiltration of inflammatory cells in 18 cases. The haemato-biochemical alterations due to antioxidants and lipid peroxidase were consistent in the diagnosis and management of mammary tumors in bitches. Top Keywords Antioxidants, Biochemistry, Bitches, Haematology, Histopathology, Mammary tumors. Top |
Introduction Mammary tumor represents the most significant gynaecological disorder in canines next to cutaneous neoplasms with approximately 50% as malignant tumors. Advanced age, delayed spaying, purebred and obesity are the major predisposing factors for the development of mammary tumors in dogs1. The hormonal influence along with endocrine disorders plays a crucial role in the development of canine mammary tumors2. The bitches spayed before first oestrus, after first oestrus and after two or any time of oestrus are associated with risk of 0.5, 8.0 and 26% of mammary tumor occurrence in their life time. The cause for the development of mammary tumors in dog is similar to the breast cancer in human1. Haemato-biochemical parameters are used as a prerequisite for the investigation of canine mammary tumors before initiation of treatment or surgeries3. The occurrence of hematologic alterations is more frequently noticed in small animal neoplasms that results from the tumor growth and dissemination or paraneoplastic syndromes. The haemato-biochemical abnormalities in specific tumors will serve as a prompt predictive biomarker for the diagnosis and prognosis4. The exposure to environmental xeno-oestrogen increases the production and bioaccumulation of reactive oxygen species. It causes susceptibility of mammary epithelium to undergo fibroblast proliferation, epithelial hyperplasia and carcinogenesis. The progression of tumor occurs mainly due to the production of reactive oxygen species and infiltration of inflammatory cells4. The role of oxidative stress combated by the antioxidant system in the development of mammary neoplasms is useful for the prognosis and novel therapeutic approaches. Hence, the study has been conducted to unveil the clinicopathological alterations of inflammatory mammary carcinomas in bitches. |
Top Materials and Methods Animal Details A total of 30 bitches of different breeds having multiple tumor masses in the mammary gland region brought for the treatment to Small Animal Surgery unit of Veterinary Clinical Complex, Veterinary College and Research Institute, Namakkal were included in this study. The control group animals were comprised of healthy, age matched and gynaecologically normal bitches (n = 30) brought for the routine general check-up. Haematology Blood samples were collected in a K3EDTA coated vacutainer by venipuncture of both control and mammary tumor affected bitches. The haematological parameters such as total erythrocyte count (TEC), haemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red cell distribution width (RDW), platelet count (PLT), mean platelet volume (MPV), platelet distribution width (PDW), plateletcrit (PCT) and total leucocyte count (TLC) were analysed using Rayto RT-7600 haematology analyzer. The differential leucocyte count (DLC) like neutrophils, eosinophils, basophils, lymphocytes and monocytes was carried out from the blood smears prepared manually. Serum Biochemistry The blood collected in the clot activator vial was used to extract the serum. Serum biochemical parameters like glucose, total cholesterol, triglycerides, total protein, albumin, blood urea nitrogen (BUN), creatinine, alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total and direct bilirubin, calcium, phosphorus and magnesium were analysed using M/s Biosystems A50, India. The serum electrolytes like sodium, potassium and chloride were analysed in UV-VIS double beam spectrophotometer (M/s Systronics, Model 2202, India) by using kits. Antioxidant Assay The surgically excised mammary tumor masses were stored at -20°C until further analysis. The tissues were homogenised in 10% phosphate buffered saline. Superoxide dismutase (SOD) was estimated based on its ability to inhibit the auto-oxidation of pyrogallol at pH 8.2 with absorbance at 470 nm using spectrophotometer5. The reduced glutathione (GSH) was estimated based on the reduction of 5,5’-dithiobis(2-nitobenzoic acid) (DTNB) or Ellman reagent to 5-thio-2-nitrobenzoate (TNB) and measured at 412 nm6. The glutathione peroxidase (GPx) was estimated based on the reaction of GPx with GSH reagent producing oxidoreductase that reacts with DTNB to form a coloured complex and measured at 412 nm7. Catalase (CAT) was estimated at 240 nm according to the methods of Claiborne8. Lipid peroxidase The lipid peroxidation of tissues was estimated by the production of thiobarbituric acid reactive substances (TBARS) with brilliant pink colour as an end product and measured at 535 nm9. Histopathology The representative pieces of tumors were fixed in 10% neutral buffered formalin for further processing. The tissue samples were trimmed into a thickness of about 3-4 mm. Tissues were processed, embedded in paraffin, sectioned at 3-5 µm thickness and stained with haematoxylin and eosin (H&E) for histopathological examination10. Statistical analysis The values obtained for haemato-biochemical parameters were subjected to independent paired t-test by using Graph Pad Prism software version 5.02. The mean values were calculated for antioxidants and lipid peroxidase. Top Results A total of 30 bitches with mean age of 7.36 years ranged from 2 to 16 years affected with mammary tumors were encountered in this study. Among various breeds affected, pure breeds are most commonly affected than the mongrels. The highest incidence was recorded in the Spid followed by mongrel and other breeds. The mammary tumor occurrence was noticed only in female dogs in this study. Out of 30 animals, 26 bitches were non-neutered and four were spayed when presented with mammary tumors. |
Haematology The mean (±SE) haematological values of both normal and mammary tumor affected bitches are presented in Table 1. A significant (P<0.05) reduction in TEC and highly significant (P<0.01) reduction in PCV were noticed in tumor affected bitches. A significant (P<0.05) elevation of MCH and highly significant (P<0.01) elevation of MCHC and TLC were noticed in tumor affected bitches. A non-significant alteration of total platelet count and its indices were noticed in few cases. Serum Biochemistry The mean (±SE) serum biochemical values of both normal and tumor affected bitches are presented in Table 2. A significant (P<0.05) increase in total cholesterol and highly significant (P<0.01) increase in ALT, AST, ALP and potassium values were noticed in tumor affected bitches. Highly significant (P<0.01) reduction in albumin and significant (P<0.05) reduction in calcium were noticed in tumor bearing dogs. Insignificant alterations of BUN, creatinine and total protein values were noticed in few cases. Antioxidants The mean (±SE) values of SOD, GSH, GPx and CAT of mammary tumor tissues are presented in Table 3. A two-fold elevation of SOD, ten-fold elevation of GSH and three-fold elevation of GPx and 5.5 fold reduction of CAT values were noticed in tumor tissues when compared with normal mammary tissues11. Lipid Peroxidase The mean (±SE) values of TBARS of mammary tumor tissues are presented in Table 3. A five-fold elevation of lipid peroxidase value was noticed in tumor tissues when compared with normal mammary tissues11. Histopathological Classification The mammary tumors of this study were classified according to the Goldschmidt et al.12. The histological evaluation of tumor tissues revealed 2 benign and 28 malignant tumors and infiltration of inflammatory cells were noticed in 18 out of 30 tumors. The fibro-adenoma (n=2) showed glandular acini with epithelial cells and stromal fibroblast with necrotic areas and inflammatory cells infiltration (Fig. 1). The fibrosarcoma (n=4) revealed a comedo pattern of tumor necrosis contained eosinophilic material mixed with necrotic debris and inflammatory cells (Fig. 2). The cystic-papillary carcinoma (n=5) showed the cyst filled with eosinophilic material covered by multi-layered neoplastic cells and few mast cells (Fig. 3). The tubular carcinoma (n=2) showed multiple tubules lined by neoplastic cells supported by fine fibrovascular connective tissue and inflammatory cells. The solid carcinoma (n=2) exhibited the neoplastic cells in solid nests surrounded by fibrovascular stroma (Fig. 4). The ductal carcinoma (n=1) revealed ductular acini filled with exfoliated neoplastic cells, plasma cells and lymphocytes (Fig. 5), tubulopapillary carcinoma (n=1) showed numerous tubules lined by multi-layered columnar cells surrounding the pink eosinophilic material mixed with inflammatory cells (Fig. 6) and complex carcinoma (n=1) revealed both epithelial and myoepitheilal components and phagocytic macrophages. Histopathological grading revealed benign tumors with grade I category and malignant neoplasms with grade I (n=7), II (n=12) and III (n=9). Top Discussion The occurrence of mammary tumor is more in pure breeds than mongrels of this study is agreed with earlier reports1,2. The frequency of tumor occurrence is more in spid when compared to other breeds might be due to predominant breed population in the location. The tumor incidence in intact and spayed bitches observed in the present study is in agreement with previous reports13. |
The decreased TEC in tumor affected bitches of this study could be due to hepatic dysfunction which reflected as increased AST and ALT enzymes. The oxidative stress to red blood cells alters the membrane composition and then destruction14. The reduced PCV value noticed in this study is in agreement with earlier reports on human breast cancer. It might be due to chronic disorders and metastasis of bone marrow that resulted in suppression of erythropoiesis15. The MCH and MCHC values were increased in this study is contradicted to the previous reports in dogs and humans where the values tend to be decreased16,17. |
The insignificant reduction of platelet count, MPV, PDW and PCT noticed in the current study is different from the earlier observations in human18. Either increased or decreased platelet values were also reported in mammary tumor affected dogs15,19,20. Slight thrombocytopenia observed in the present study might be due to increased splenic platelet sequestration. It could be also caused by interaction of immune-mediated cells with cancer cells or hypercoagulability of platelets. Ucmak et al. reported an insignificantly decreased MPV values in mammary tumor affected dogs17 and Mantas et al. observed increased MPV values with distant organ metastasis in human breast cancer cases18. |
The increased TLC in tumor affected bitches in the present study might be associated with acute or chronic inflammation/stress of the animals or tumor necrosis15. The increased leucocyte count is correlated with infiltration of inflammatory cells in the tumor tissues18. The hallmark of inflammatory carcinoma is invasion of tumor cells into neighbouring tissues were also observed in the current study12. |
The hypercholesterolemia recorded in this study could be due to lipid-rich feed inclusion in the diet of bitches. The increased susceptibility of cholesterol to oxidative stress results generating larger amount of lipid peroxidation products4. The insignificant increase in triglycerides of this study is in the normal range of the dogs. |
The total protein value of the tumor affected bitches is similar to the normal dogs. The flutteration of hyperproteinemia associated hypergammaglobulinaemia commonly noticed in the small animal cancers might be due to acute or chronic inflammatory stimulus15. The hypoalbuminaemia observed in this study suggested that the albumin is a negative acute phase protein that gets reduced after inflammatory stimulus16. |
The insignificant elevation of BUN in few cases and normal values in most of the cases of this study is contradicted to the earlier observations in mammary tumor affected bitches16. A slight increase in creatinine value of two cases attributed to the renal damage in this study is different from the earlier observations21. |
The elevation of ALT in this study could be due to the involvement of primary liver disease and geriatric animals aff ected with mammary tumors. The elevation of ALP in the present study might be due to hepatic dysfunction associated with bone metastasis22. The bilirubin levels were within the normal range even though the liver enzymes were elevated in the present study. The hyperbilirubinemia associated with poor survival rate was reported previously in metastatic breast cancer patients3. |
The hypocalcaemia observed in the present study was also reported in human breast cancer that might be due to abnormal calcitonin secretion by the tumors. It is also correlated with reduced albumin that is essential to carry the calcium16. The phosphorous, magnesium, sodium and chloride values were within the normal limits. An increased potassium value of this study might be due to metastasis of tumor cells into the visceral organs like heart. |
The increased values of SOD in tumor tissues in the present study are in accordance with earlier reports on malignant human breast cancers. The elevation of SOD is believed to be the first line of defence against the damage produced by reactive oxygen species11. The increased SOD and decreased CAT activity reflects the anti-apoptotic intracellular environment that favoured the DNA mutation23. The superoxide anion cleaved by SOD to hydrogen peroxides that further catalysed by GPx involving lipoxygenase and cyclooxygenase pathway of tumorigenesis11. The GSH is suggested to maintain the redox status of the cell and enzymes essential for DNA synthesis. The elevated levels of GSH contribute the cellular proliferation and antioxidant defence status24. The extensive lipid peroxidation found in the mammary tumor tissues of this study is correlated with high cholesterol levels in serum suggesting the obesity which is a risk factor for canine mammary tumors1. |
The alterations of clinico-pathological parameters associated with inflammatory cell infiltrations indicating the tumor progression and tumorigenesis. The changes of haemato-biochemistry, antioxidants and lipid peroxidase in mammary tumor affected bitches are the evidence of paraneoplastic syndrome. Hence, the evaluation of clinicopathological alterations due to antioxidants in relation to histological grades of mammary tumors will be useful for the diagnosis, treatment and prognosis in dogs. |
How to cite this article : Nandhini, S., Madheswaran, R., Balasubramaniam, G.A. and Ramya, K. 2022. Clinico-pathological investigation of inflammatory mammary carcinomas in bitches. Indian J. Vet. Pathol., 46(4) : 277-282. Top Figures | Fig. 1.: Fibroadenoma showing proliferating glandular epithelial cells and stromal fibroblast with necrosis and inflammatory cells. (arrow) H&E x100;
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| | Fig. 2.: Fibrosarcoma showing comedo pattern of central necrosis surrounded by proliferating fibrous tissue and inflammatory cells. H&E x100;
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| | Fig. 3.: Cystic-papillary carcinoma showing dilated cyst with eosinophilic secretion surrounded by multiayered columnar cells. H&E x400;
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| | Fig. 4.: Tubular carcinoma showing multiple tubules lined with neoplastic columnar cells surrounded by fibrovascular stroma and mononuclear cells. H&E x400;
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| | Fig. 5.: Ductal carcinoma showing the duct lined by cuboidal epithelial cells occluded with neoplastic cells, plasma cells and lymphocytes. H&E x400;
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| | Fig. 6.: Tubulopapillary carcinoma showing the tubules lined by multilayered columnar neoplastic cells surrounding eosinophilic material with inflammatory cells. (arrow) H&E x400
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Tables | Table 1.: Mean (±SE) haematological values of normal and mammary tumor affected bitches.
| | S. No. | Haematological parameters | Normal dogs (n = 30) | Tumor affected dogs (n = 30) | | 1. | TEC (x106/µL) | 6.44a±0.24 | 5.66b±0.23* | | 2. | Hb (g/dL) | 13.38±0.54 | 12.33±0.53 | | 3. | PCV (%) | 43.60a±1.58 | 38.10b±1.51** | | 4. | MCV (fL) | 68.13±0.98 | 67.51±1.06 | | 5. | MCH (pg) | 20.62a±0.33 | 21.83b±0.46* | | 6. | MCHC (g/dL) | 30.28a±0.25 | 32.40b±0.61** | | 7. | RDW (%) | 14.02±0.35 | 14.40±0.34 | | 8. | PLT (x103/µL) | 497.53±25.97 | 474.03±28.57 | | 9. | MPV (fL) | 7.89±0.23 | 7.69±0.11 | | 10. | PDW (fL) | 19.05±1.81 | 17.94±1.46 | | 11. | PCT (%) | 0.42±0.04 | 0.34±0.02 | | 12. | TLC(x103/µL) | 16.56a±1.10 | 24.32b±2.24** | | 13. | Neutrophils (%) | 71.83±1.03 | 71.35±1.83 | | 14. | Eosinophils (%) | 4.90±0.34 | 5.00±0.28 | | 15. | Basophils (%) | 0.50±0.09 | 0.53±0.09 | | 16. | Lymphocytes (%) | 18.06±0.88 | 18.18±1.68 | | 17. | Monocytes (%) | 4.71±0.32 | 4.92±0.32 |
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| Means with same superscripts within a row do not differ (P > 0.01) from each other. | Significant (P < 0.05) | Highly significant (P < 0.01) | | | Table 2.: Mean (±SE) serum biochemical values of normal and mammary tumor affected bitches.
| | S. No. | Parameters | Normal dogs (n = 30) | Tumor affected dogs (n = 30) | | 1. | Glucose (mg/dL) | 96.70±3.85 | 99.87±10.23 | | 2. | Total cholesterol (mg/dL) | 196.40a±10.44 | 236.48b±14.67* | | 3. | Triglycerides (mg/dL) | 110.80±5.97 | 127.45±7.63 | | 4. | Total protein (g/dL) | 7.33±0.18 | 7.46±0.18 | | 5. | Albumin (g/dL) | 3.38a±0.12 | 2.95b±0.10** | | 6. | BUN (mg/dL) | 10.67±1.30 | 12.54±1.64 | | 7. | Creatinine (mg/dL) | 1.00±0.05 | 1.11±0.08 | | 8. | ALT (U/L) | 36.58a±2.84 | 74.90b±13.14** | | 9. | AST (U/L) | 36.51a±3.63 | 51.75b±6.15** | | 10. | ALP (U/L) | 110.47a±9.67 | 170.58b±28.42** | | 11. | Total bilirubin (mg/dL) | 0.69±0.03 | 0.66±0.05 | | 12. | Direct bilirubin (mg/dL) | 0.27±0.01 | 0.30±0.05 | | 13. | Calcium (mg/dL) | 13.05a±0.39 | 11.75b±0.55** | | 14. | Phosphorus (mg/dL) | 5.06±0.31 | 4.85±0.30 | | 15. | Magnesium (mg/dL) | 2.13±0.17 | 2.23±0.15 | | 16. | Sodium (mmol/L) | 128.70±3.79 | 134.67±3.92 | | 17. | Potassium (mmol/L) | 3.48a±0.16 | 4.51b±0.35** | | 18. | Chloride (mmol/L) | 109.14±3.78 | 108.29±2.62 |
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| Means with same superscripts within a row do not differ (P > 0.01) from each other. | Significant (P < 0.05) | Highly significant (P < 0.01) | | | Table 3.: Mean (±SE) antioxidants and lipid peroxidase values of mammary tumor affected bitches.
| | S. No. | Parameters | Tumor affected dogs (n = 30) | Reference values | | 1. | Superoxide dismutase (SOD) | 18.14±0.73 U/mg of tissue protein | 11.82±1.12 U/mg of tissue protein5 | | 2. | Reduced glutathione (GSH) | 104.38±5.77 mg/100g of tissue protein | 10.07±0.94 mg/100g of tissue protein5 | | 3. | Glutathione peroxidase (GPx) | 34.92±1.47 U/mg of tissue protein | 10.96±0.98 U/mg of tissue protein5 | | 4. | Catalase (CAT) | 4.248±0.385 U/mg of tissue protein | 22.2±6.6 U/mg of tissue protein12 | | 5. | Lipid peroxidase | 5.76±0.57 nmol/mg of tissue protein | 1.52±0.35 nmol/mg of tissue protein5 |
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