Mammary gland tumors are the most commonly diagnosed tumors in sexually intact female dogs¹. They occur most often in middle aged to older dogs with a very low incidence in young dogs. Anatomically, about 70% of all mammary tumors develop in caudal abdominal and inguinal (4^th and 5^th) gland². The majority of mammary-gland tumors in female dogs are ofepithelial origin and approximately 50% are malignant³. Around 50-77 per cent of epithelial mammary-gland tumors express estrogen receptors (ER), andreceptor expression appears to correlate with degree of histologic differentiation. ER positive (ER+) tumors are typically well differentiated and showed a better prognosis⁴. The incidence rate of mammary gland tumors in male dogs rangesfrom 0 to 2.7% (average < 1%)⁵ and the median age at diagnosis was 11.5 years⁶.

Mammary tumors in male dogs occur sporadically and associated with hormonal abnormalities due to estrogen secreting sertoli cell tumor of the testis⁷. Breast cancer in men is rare, reported less than 1% of all diagnosed malignancies.

Possible risk factors for this disease include increasing age, testicular injury, obesity, gynecomastia, sexual hormones, Klinefelter’s syndrome, and a family history of breast cancer^8,9.

Triple-negative breast cancer (TNBC) is a clinical subtype of breast cancer characterized by lack of expression of estrogen receptor (ER), progesterone receptor (PR) or human epidermal growth factor receptor 2 (her2). It is widely considered to have aggressive clinical behavior, poor survival and lack of targeted therapeutic option. Triple negative breast cancers can be further divided into basal-like and nonbasal-like subtypes by IHC, with most categorized as the basal-like subtype¹⁰. Basal-like tumors usually express basal cytokeratins, including cytokeratin (CK5/6, CK5, CK14, and CK17), and epidermal growth factor receptor (EGFR). In particular, CK5/6 and/or EGFR can be helpful in diagnosing basal-like triple negative mammary neoplasm which has a persistently poorer prognosis in the longer term, and screening for those basal markers is important in determining prognosis and therapeutic strategies in patients with triple negative breast cancer¹¹.

Vimentin is an intermediate filament protein normally expressed in cells of mesenchymal origin¹². However, it can also be expressed in epithelial cells if they are undergoing epithelial to mesenchymal transition either under physiological or pathologic conditions¹³. Further, little is known about the expression levels of basal CKs and vimentin in the canine triple negative basal-like immunophenotype and their relation to disease progression in male dogs. Hence in the present study, we investigated the clinical features, existence of triple negative phenotype and basal markers expression in canine mammary tumor of a male dog.

The study was conducted on canine mammary tumour cases brought to the Veterinary Clinical Complex, Veterinary College and Research Institute, Tirunelveli and Namakkal from August 2019 to July 2021. Clinical information like, age, breed, sex and neuter status were collected from mammary tumor affected cases. On physical examination the size and location of the tumour were determined. Fine needle aspiration cytology was performed on firm area and the slides were stained with Leishman stain. Metastasis of tumour in the lung was assessed by lateral thoracic radiographs at the time of presentation. Surgically the tumours were removed and fixed in 10 per cent neutral buffered formalin. The tissues were further routinely processed and embedded in paraffin. Four micron thick sections were stained with hematoxylin and eosin (H&E).

Immunohistochemistry protocols for ER and PR, her2, CK5/6 and vimentin staining was followed as per the standard protocol described by manufacturer (Pathn Situ). Briefly, all the slides were deparaffinized and rehydrated through alcohols to water. Antigen retrieval was performed for 15 minutes in pressure cooker, two whistles with Tris-EDTA buffer, pH between 8.5 to 9.0. Slides were washed in Immuno wash buffer for 2 minutes and 3% hydrogen peroxide block was applied for 10 minutes. After rinse, the rabbit monoclonal ER, PR, Her2, CK5/6, vimentin (three drops per section ready to use antibody) antibodies were applied and incubated for 45 minutes. Slides were washed in Immuno wash buffer for 3 minutes. PolyExcel Target Binder was applied to all the slides and incubated for 12 minutes at room temperature. After washing, PolyExcel HRP was applied to all the slides and incubated for 12 minutes at room temperature. After wash, StunnDAB working solution was applied to all the slides and incubated for 5 minutes at room temperature. After rinsing, all slides were counterstained with hematoxylin dehydrated and cover slipped. The receptors expression was quantified subjectively by determining approximate percentages of neoplastic cells that expressed the receptors at 20x power.

Out of 46 cases diagnosed as mammary tumour, only one case was found in an 11-year-old male nondescript dog. The male dog showed generalized symptoms of anorexia, discomfort in breathing and difficulty in walking due to hanging of large mass from the right caudal abdominal gland (Fig.1). Grossly, the tumour mass was firm and ulcerated with cauliflower like appearance on the edges of the ulcer. Cut surface was gray in colour and lobulated. Thoracic x-ray revealed miliary nodules in the lung.

Cytology revealed the individual and clusters of cuboidal shaped epithelial cells. The epithelial cells showed malignant features like anisocytosis, anisokaryosis with prominent nucleoli (Fig.2). Few spindle shaped cells, necrotic debris along with inflammatory cells were also seen.

Histopathologically the tumour mass was unencapsulated and exhibited stromal invasion in some locations. The tumour contained two types of cell populations with moderate amount of fibrous stroma. The epithelial component composed of cuboidal to columnar cells arranged in irregular tubules, acinar pattern and solid nests. These cells had scant to moderate cytoplasm and vesicular nuclei with marked anisokaryosis. The glandular epithelium contained little secretions. The connective tissue, peripheral to the tubules and nests were separating the spindle cells from epithelial components. The second population consists of spindle shaped cells. They are arranged in solid sheets. The spindle cells have poorly demarcated borders, moderate homogeneous eosinophilic cytoplasm with round to ovoid central nuclei (Fig.3). Necrosis with infiltration of inflammatory cells was also observed in some places. In some areas the epithelial and spindle cell populations are equally distributed (Fig.4) and other areas showed predominant spindle cells with few epithelial components.

Immunohistochemical studies revealed that expression of estrogen receptor (ER), progestron receptor (PR) and human epidermal growth factor receptor 2 (her2) was negative. Immunohistochemistery of cytokeratin 5/6 showed moderate positivity in the epithelial components which indicates basal cell involved in the epithelial components (Fig.5 & 7). However, Cytokeratin 5/6 revealed negative reaction in the spindle cell populations. To confirm the presence of mesenchymal cells, the serial sections were stained with vimentin. The vimentin marker expression was very strong in the spindle cells (Fig.6 & 8). The expression of both cytokeratin 5/6 and vimentin in the same tumour indicates the presence of both epithelial and mesenchymal components.

Mammary tumours are uncommon in male dogs, and very few reports documented in male dogs as benign tumours^{5, 6}. Clinical conclusions, regarding the behavior of mammary tumors in male dogs are limited because of their rarity⁶. The present work documented an incidence of 2.17 % mammary tumour in male dog, which is in accordance with previous report in that the incidence rate of mammary gland tumors in male dogs ranged from 0 to 2.7%⁵ while in another study the incidence was reported of 5.26% in male dogs¹⁴. The controversy in the incidence both in lower and upper side may be due the variation in the sample size. The average age of onset for the mammary tumors in male dog was 9 (5-12) years¹⁵. In the present study, the age of the animal was 11 years. Almost similar finding was reported by earlier study⁶.

Like in female dogs, in the present case the tumour was located in the caudal abdominal gland. The earlier reports recorded mammary gland tumors in male dogs mostly in the 4th (caudal abdominal) and 5th (inguinal) mammary glands¹⁶. The dog in the present study was sexually intact. A previous study evaluating 8 male dogs with mammary tumors showed that 50% of the patients were sexually intact when diagnosed, and that 3 of the 4 were then neutered at the time of the mammary mass removal. No difference in the relative risk of mammary tumor development between neutered and intact male dogs was identified⁶. Contrary to this other study reported that mammary tumors in male dogs are associated with hormonal abnormalities such as estrogen secreting sertoli cell tumor of the testis⁷. In the present observation, the dog had a history of no testicular neoplasia or other testicular abnormalities except obesity.

According to the published literature the mammary tumours and testicular tumours can occur in male dogs without relationship to female sexual hormone¹⁷. This phenomenon is very well correlated with the present study, where the expression of estrogen receptor, progesterone receptor was negative. The exact cause of mammary cancer in male dogs is still obscure and is under debate. The cytological finding in the present work was well correlating with earlier study¹⁸.

On histological examination of the tumour mass in the present case revealed both epithelial and mesenchymal components. The epithelial cells were cuboidal to columnar, they were arranged in irregular tubules, acinar and solid sheets. The tubules contained two to three layers of pleomorphic cells with vesicular nuclei. The mesenchymal cell population contained plumpy cells with elongate to oval nucleus. Some areas in the histological sections were very difficult to differentiate the epithelial components from mesenchymal cells. The results showed the malignancy character of the tumour. This is very well correlating with the previous report¹⁹. Cytokeratin 5/6 and vimentin were used to differentiate both the components. Cytokeratin was expressed only in the epithelial cells and not in spindle cells and other components. Whereas vimentin was expressed in the spindle cells and other stromal portion and not in epithelial cells. The high-level expression of CK5/6 may have a role in the development of nodal or distant metastases in basal-like triple negative breast cancers (TNBC) and may be predictive of metastatic disease¹¹. Epidermal growth factor receptor and CK5/6 are adverse prognostic markers in TNBC. EGFR and CK5/6 expression could serve as biomarkers for identifying TNBC patients with poor survival that are unlikely to benefit from neoadjuvant chemotherapy²⁰. In the present case the animal had miliary metastatic nodules in the lung at the time of diagnosis. After surgical removal the animal showed no recurrence of tumour for 6 months period.

Vimentin is considered a canonical marker for epithelial mesenchymal transition which plays a role in tumour invasive and progression²¹. In the present study, tumour contained an equal amount of epithelial and mesenchymal components in some areas. This finding suggests that the possibility of myoepithelial cells transformed into mesenchymal cells in the tumour mass. A number of IHC studies support a myoepithelial cell histogenesis based on immunophenotype characteristics and have found that myoepithelial cells can undergo metaplastic transformation to mesenchymal cells by mechanisms that are not completely understood. Whereas supra-basal myoepithelial cells retain their immunoprofile, interstitial myoepithelial cells lose the expression of epithelial markers (keratins) and acquire a fibroblastic-like phenotype with increased vimentin expression ^{22, 23}.

The present study recorded the hormone receptor expression pattern and clinical behavior of the triple negative basal like tumour in a male dog. The incidence of mammary tumour in male dogs is very low. The role of sex hormone in the tumour development in male dogs could not be ascertained. Further, large scale studies required to identify the role of sex hormones in mammary tumour development in male dogs.

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