Lipid-rich carcinoma is a malignant mammary gland tumour characterized by neoplastic cells with abundant vacuolated cytoplasm containing a large amount of neutral lipid¹. According to the World Health Organization (WHO) tumour classification system of domestic animals, this type of tumour is extremely rare and classified into special types of mammary tumours, which may be found in younger bitches² and has an unfavourable biological behaviour with lymphatic invasion, nodal and distant metastasis^1,3. Similarly, human lipid-rich carcinoma is frequently classified as histological grade III with triple negative phenotype, nodal metastasis at presentation and with a high first-year mortality rate. Lipid-rich carcinoma of the human breast is diagnosed when no fewer than 90% of the cells contain abundant, cytoplasmic neutral lipid, either on histochemical or ultrastructural evaluation⁴. However, in dogs, a diagnosis of lipid-rich carcinoma should only be made when more than 50% of the neoplastic cells have vacuolated cytoplasm⁵. The present work was carried out to study the pathomorphological features, immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER 2), cytokeratin 5/6 (CK 5/6), vimentin and clinical behaviour of lipid-rich mammary carcinoma in a dog.

A seven years old Labrador dog was brought to the Veterinary Clinical Complex, Veterinary College and Research Institute, Tirunelveli with the history of subcutaneous mass in the left inguinal mammary gland. Clinical examination was performed to collect the clinical information like, age, breed, sex, neuter status, size and location of the tumour. Fine needle aspiration cytology was performed on the firm area and the slides were stained with Leishman stain. Metastasis of tumour in the lungs was assessed by lateral thoracic radiographs at the time of presentation. Surgically the tumour was removed, fixed in 10 per cent neutral buffered formalin. The paraffin embedded tissue sections were cut into 4 micron thickness and stained with Hematoxylin and Eosin (H&E).

Immunohistochemical staining for ER, PR, HER2, CK5/6 and vimentin was performed as per the standard protocol as per the manufacturer’s instructions (Pathn Situ). Briefly, the sections were deparaffinized and rehydrated through descending grades of isopropyl alcohol and brought to water. Antigen retrieval was performed for 15 minutes in pressure cooker, with Tris-EDTA buffer (PS 009, Pathn Situ), pH between 8.5 to 9.0. Slides were washed in immuno wash buffer (PS 006, Pathn Situ) for 2 minutes and then 3% hydrogen peroxide block was applied for 10 minutes. After rinsing, the rabbit monoclonal ER (PR042), PR (PR068), HER2 (PR047), CK5/6 (PR106) and vimentin (PR 075) antibodies from Pathn Situ (three drops per section ready to use antibody) were applied and incubated for 45 minutes at room temperature. Slides were washed with immuno wash buffer for 3 minutes. Polyexcel target binder (PHE 002, Pathn Situ) was applied to all the slides and incubated for 12 minutes at room temperature. After washing, HRP polymer based secondary antibody (PEH 002, Pathn Situ) was applied to all the slides and incubated for 12 minutes at room temperature. After washing, StunnDAB (PHE 002, Pathn Situ) working solution was applied to all the slides and incubated for 5 minutes at room temperature. After rinsing, slides were counterstained with hematoxylin for 30 seconds, dehydrated and cover slip was applied. The slides were examined by light microscopy and immunoreactivity was evaluated.

Clinical examination revealed a single subcutaneous mass on the left inguinal mammary gland of an intact female Labrador dog. Grossly, the tumour mass was solitary, non-ulcerated and round to oval in shape (Fig.1). Cut surface appeared uneven, grayish-white in colour and showing multi-lobulations (Fig.2). Thoracic x-ray revealed no metastatic nodules in the lungs.

The cytology revealed clusters of neoplastic cells with high cellularity. The neoplastic cells had small to moderate amount of basophilic cytoplasm with variably-sized vacuoles and peripherally placed nucleus. The nuclei were round to oval and had coarse chromatin, and single to multiple nucleoli. Moderate to marked anisocytosis and anisokaryosis were noticed. Abundant lipid droplets were scattered in the background (Fig. 3).

Histopathologically, the tumour was encapsulated and the tumour cells were arranged in small to medium sized solid nests which were separated by delicate fibrovascular stroma. Most of the neoplastic cells, contained multiple, small or large vacuoles in the cytoplasm, with peripherally placed round to flattened nuclei (Fig. 4). Neoplastic cells exhibited moderate to marked anisocytosis and anisokaryosis. Nuclear atypia and occasional mitotic figures were observed. Lymphocytes, plasma cells, and eosinophils were diffusely infiltrated in the tumor growth especially in the stroma.

Examination of IHC slides revealed negative expression for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6 (Fig.5) and vimentin in the vacuolated tumour cells. However, vimentin revealed strong positive reaction in the cytoplasm of stromal cells running in-between the nests of tumour cells (Fig.6). The negative expression of vimentin in the vacuolated cells ruled out the liposarcoma and confirmed that the tumour arise from epithelial tissue.

The current classification system for dogs, lipid-rich mammary carcinoma was the only mammary carcinoma histotype that can be diagnosed by the presence of cytoplasmic vacuolation in the neoplastic cells⁵. However, in humans, breast cancers with clear cells include several types of tumours viz. lipid-rich carcinoma, glycogen-rich carcinoma, apocrine carcinoma, secretory carcinoma and myoepithelial neoplasms⁴. The usual form of presentation of canine lipid-rich mammary carcinoma was that of a single large tumor mass affecting one mammary gland and mostly in intact pure breeds³. This is coinciding with the present case. In the present case, a single tumour mass was recorded in the left inguinal mammary gland of an intact female Labrador dog. The mean age of occurrence of the lipid rich mammary carcinoma in the dogs was 7 years⁶ as observed in the present case and it was reported to be varied between 4 and 13 years³. The cytological findings of high cellularity of neoplastic cells in clusters with vacuolated cytoplasm and background lipid droplets were similar to earlier report⁷.

The histological appearance of the present tumour was consistent with lipid-rich carcinomas according to the current morphologic descriptions of human and canine cases⁸. The lipid droplets present in the neoplastic cells might have produced by the tumour cells because lipid droplets were uniformly present in the cytoplasm of neoplastic cells and also found in the metastasized lymph nodes^3,9. It has been suggested that intracellular accumulation of lipids permits the survival of the circulating tumour cells in the blood by inducing membrane phase separation, which reduces cell to cell contact and promotes tissue invasion¹⁰.

Published literature indicated that the epithelial cells of lipid-rich carcinoma can express both luminal and basal cytokeratins¹¹ and usually do not express estrogen and progesterone receptors³. Absence of expression of ER alpha in human and canine mammary carcinomas is related to an unfavourable prognosis^2,12. No ER alpha expression was detected in any of 49 human patients with lipid-rich carcinoma¹³. In addition, all 10 lipid-rich carcinomas reported in dogs also lacked ER alpha expression^4,14. In the present case the tumour cells were negative for estrogen and progesterone receptors as well as negative for cytokeratin 5/6 and HER2, hence the tumour was classified as triple negative non basal type. Further the vacuolated tumour cells were negative for vimentin which ruled out the liposarcoma and the stromal cells showed positivity indicated the presence of mesenchymal cells in-between the nests of epithelial cells.

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