Mammary neuroendocrine tumor in a mare

A 10-y-old American Paint Horse mare was presented to the Equine Field Services at Virginia-Maryland College of Veterinary Medicine (Blacksburg, VA, USA) with a presumptive diagnosis of mastitis and an abscess in the right mamma. Palpation of the mamma revealed a non-painful, warm, very firm, 20 × 18-cm swelling that extended through the entire right mamma, with a tract draining purulent material. Based on its location, the tract did not appear to be directly associated with the teat canal or major ducts, although involvement could not be excluded definitively. No other significant clinical abnormalities were observed.

Direct, sediment, and cytocentrifuge smears were prepared from fluid of the right mamma for cytologic analysis. Cellular content was mixed. Microbiologic aerobic culture analyses and susceptibility were inconclusive, with no growth after 3 d, and only mixed flora and no probable pathogens found in 2 separate submissions. The mare was treated with systemic trimethoprim-sulfonamide (30 mg/kg, PO, q12h). After several weeks, the swelling of the mamma was reduced, but the draining tract remained patent. A biopsy was taken, fixed by immersion in 10% neutral-buffered formalin, and processed routinely for the production of 4-µm thick H&E-stained slides.

Microscopically, the mammary gland parenchyma was effaced and replaced by a multilobular, densely cellular, infiltrative, and poorly demarcated and unencapsulated neoplasm ( Fig. 1A ). The neoplastic cells were organized into closely arranged, variably sized islands and packets of polygonal pleomorphic cells, often surrounded by thin stroma ( Fig. 1B ). The neoplastic cells had abundant, highly vacuolated eosinophilic cytoplasm. The nuclei were single, round, often central, and occasionally indented, with stippled-to-clumped chromatin. One or 2 basophilic nucleoli were evident per cell. Anisokaryosis and anisocytosis were marked within the sections analyzed. The mitotic count was 16 mitotic figures in 2.37 mm². Most (~60%) of the neoplasm was necrotic, including the center of some of the neoplastic islands ( Fig. 1C ). Numerous neutrophils and lymphocytes were admixed with cellular debris and fibrin, and the necrotic areas were hemorrhagic.

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Figure 1.

Neuroendocrine mammary neoplasm in a mare. Figure 1A–E are images from the biopsy specimen. A. Multinodular, densely cellular neoplasm replaces the normal mammary parenchyma. H&E. B. Neoplastic cells are closely arranged in packets and islands, surrounded by thin, fine stroma, with markedly eosinophilic and vacuolated cytoplasm and round nuclei. H&E. C. Extensive areas of lytic necrosis admixed with numerous neutrophils span the neoplasm, with intact neoplastic islands surrounding blood vessels. H&E. D. Neoplastic cells have moderate-to-strong cytoplasmic immunolabeling for chromogranin A. Immunohistochemistry (IHC). E. The neoplastic cells do not immunolabel for cytokeratin. IHC. F. Sections taken at autopsy at the mastectomy excision had morphology similar to those in the biopsy submission. The neoplastic cells infiltrated the adjacent skeletal muscle. H&E.

The histologic pattern of packets surrounded by thin fibrovascular septa raised concern for neuroendocrine differentiation. To determine if this was a neuroendocrine tumor (NET), a neuroendocrine immunohistochemistry (IHC) panel was performed on the biopsy sample for chromogranin A, neuron-specific enolase (NSE), S100, and synaptophysin (SYN; School of Veterinary Medicine, University of California–Davis, Davis, CA, USA), and cytokeratin IHC was performed at the University of Florida Veterinary diagnostic laboratories ( Table 1 ). After 3 wk of progression of clinical signs, resection of the right mamma was elected. During the surgical procedure, high vascularization of the neoplasm was observed.

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Table 1.

Antibodies used for neoplasia characterization.

Primary antibody	Host, clone	Vendor	Catalog no.	Dilution	Antigen retrieval
Neuron-specific enolase	Mouse, 1G4	Invitrogen	18-0196	1:2,000	HIER microwave
Chromogranin A	Rabbit	Novus Biologicals	NB100-79914	1:300	HIER microwave
S100 protein	Rabbit (R)	Vector	VP-S276	1:400	HIER steamer
Synaptophysin	Mouse, SY38	Dako	M0776	1:80	HIER steamer
Anti–pan keratin	Mouse	Roche	5266840001	RTU	CC1 HIER

CC1 = cell conditioning 1; HIER = heat-induced epitope retrieval; R = recombinant; RTU = ready to use.

Neoplastic cells had moderate-to-strongly positive cytoplasmic immunolabeling for chromogranin A ( Fig. 1D ) and did not immunolabel for cytokeratin ( Fig. 1E ). The neoplastic cells also did not immunolabel for NSE or SYN. Scattered neoplastic cells had moderate nuclear-to-cytoplasmic immunolabeling for S100. Histologic and IHC results confirmed a diagnosis of neuroendocrine neoplasia.

The mass was extensive and infiltrative. Given the highly invasive nature of the mass and severe presumptive infection, the surgical approach involved an elliptical incision encompassing the mass, with the goal of excising the entire mamma and affected overlying skin to achieve clean margins. However, complete excision created a substantial closure deficit, and only the caudal portion of the incision could be primarily apposed. Consequently, >50% of the wound would be left to heal by second intention, carrying a high risk of dehiscence and postoperative infection. Also, despite extensive attempts at hemostasis, severe intraoperative blood loss (~4 L) occurred. Considering these complications and the overall severity of the condition, euthanasia was elected.

The surgically removed, firm, multilobular, 40-cm wide mass was submitted with the body of the mare. On cut surface, multiple, variably sized, firm, yellow-to-gray-to-tan nodules coalesced and were surrounded by firm tan connective tissue. Numerous cavitations containing hemorrhage and necrotic debris were observed. Close to the surgical margin, many similar firm nodules extended into the surrounding subcutis. The superficial inguinal, mammary, and iliac lymph nodes were 2–4 times their normal size but retained identifiable corticomedullary architecture. Samples from the mastectomy excision site had morphology similar to those in the biopsy submission. The neoplastic cells infiltrated the adjacent skeletal muscle ( Fig. 1F ) and were found rarely within blood vessels and lymphatics. However, metastases were not found in regional lymph nodes.

Various cytokeratin markers of high- and low-molecular-weight and smooth-muscle markers are routinely used to distinguish luminal epithelial cells from basal or myoepithelial cells, respectively. However, in mammary tumors, ⁷ a panel of markers—including chromogranin A,^8,16,22 NSE, ¹⁰ S100, ⁴ and synaptophysin—can assist in the diagnosis of NETs. Chromogranin A ³ is widely utilized to identify neuroendocrine origin, such as gastroenteropancreatic NETs and pheochromocytomas.^19,22 NSE and S100 are additional supportive NET markers; positivity for NSE often indicates undifferentiated or metastatic tumors,^2,10 and S100 is immunolabeled in well-differentiated tumors, such as pancreatic NETs.^4,18

Mammary gland disease in mares is uncommon and often inflammatory; mammary neoplasia is rare and frequently mimics mastitis, complicating and delaying diagnosis. Equine mammary neoplasms are sparsely reported and typically carry a poor prognosis. Neuroendocrine neoplasia of the mammary gland is exceedingly rare in veterinary medicine; a thorough search of PubMed, Google Scholar, and CAB abstracts for mammary NEC in mares did not yield any reports. We have classified mammary gland neoplasms in mares as those originating from the mammary gland (primary) and those of non-mammary origin (originating from tissues adjacent to the mammary gland or as a result of metastatic disease; Table 2). Carcinomas are the most common primary and metastatic mammary neoplasms in mares,^1,20 and include solid carcinoma, ductal carcinoma, ⁹ papillary ductal adenocarcinoma, ⁶ and metastatic squamous cell carcinoma. ¹¹ Additional neoplasms of non-mammary origin reported in the mammary region in mares include mammary sarcoma,^21,23 melanoma, ¹³ lymphoma, ¹⁵ and sarcoid. ¹³ Histopathology, supported by IHC, remains the most reliable approach for diagnosis and prognostic assessment of mammary neoplasia.

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Table 2.

Summary of primary and secondary mammary gland neoplasms reported in horses.

Tumor classification	Type	Etiology, origin	Gross presentation	Histologic presentation	Disease progression	Ref.
Mammary origin	Solid mammary carcinoma	Mammary ductal epithelial cells	Enlarged mammary gland with well-demarcated nodules; lymphadenopathy	Solid nests of neoplastic cells, with abundant fibrous stroma, frequent mitotic figures, and epithelial units with central necrosis; metastases to LN	Hematogenous or lymphatic metastasis possible	Prendergast et al. 20
	Mammary ductal carcinoma	Mammary ductal epithelial cells	Firm mass with ulcerated surface and purulent discharge; metastases can be found	Irregular sheets and islands of epithelial cells separated by fibrous connective tissue	Invasion into ducts, acini, and interstitium, leading to metastasis	Hirayama et al. 9
	Mammary papillary ductal adenocarcinoma	Mammary ductal epithelial cells in a micropapillary pattern	Enlarged mammary gland with ulcerations and multinodular masses that can be found in the LN	Arranged in nests in stromal cystic spaces; fibrosis, necrosis, hemorrhage, and elevated mitotic count	Invasive micropapillary carcinoma of the mammary gland; extensive LN metastasis	Gamba et al. 6
	Mammary adenoma	Benign epithelial proliferation	Enlarged mammary gland with presence of firm mass	Tubules lined by up to 2–3 layers of cuboidal epithelial cells, without myoepithelial cell growth	Usually have a good prognosis after resection	Spadari et al. 23
	Mammary sarcoma (previously malignant fibrous histiocytoma)	Pleomorphic spindle cells consisting of histiocytes, fibroblasts, and multinucleate giant cells arranged in a storiform pattern	Multifocal, discrete, homogeneous, firm, tan-white nodules on the mammary gland, and LNs	Poorly demarcated, pleocellular neoplastic atypical spindle cells arranged in storiform or interlacing patterns; neoplastic cells are often pleomorphic	Locally invasive with high local recurrence	Reesink et al. 21
Non-mammary origin	Melanoma	Melanocytes	Single–multiple raised firm nodules; typically found superficially, but can occur as a primary visceral mass	Neoplastic melanocytes with variable pigmentation, moderate-to-marked anisocytosis and anisokaryosis	Tumor growth can infiltrate muscle and compromise blood supply; can metastasize by implantation, lymphatic or hematogenous spread	MacEachern et al. 13
	Lymphoma	Lymphocytes	Increased parenchymal volume of mammary glands with firm, white, soft, lobulated, well-demarcated mass; enlarged LNs	Tissue effaced by round cell neoplasm, marked anisocytosis and anisokaryosis; multiple areas of necrosis are common	Multiple presentation types, including primary LNs, skin, thymus, or alimentary tract; most common hematopoietic malignancy	Mendes et al 15
	Squamous cell carcinoma	Epithelial (epidermis) cells	Large primary squamous cell carcinoma with LN metastases	From well-differentiated tumors to poorly differentiated anaplastic squamous cells with frequent mitotic figures, and prominent nucleoli	Locally invasive with slow metastasis	Karcher et al 11
	Sarcoid	Fibroblasts	Presentation depends on the type of sarcoid, but ranges from areas of alopecia, crusting, raised nodules, lichenified areas, to wart-like nodules, or largely ulcerated masses	Epidermal acanthosis, hyperkeratosis and hyperplasia with long rete pegs into dermal fibroblastic tissue; immature fibroblasts containing mitotic figures	Associated with bovine papillomavirus; locally invasive fibroblastic skin tumor, but mixed and malevolent forms have poorer prognosis	McGladdery 14

LN = lymph node.

Mammary neuroendocrine carcinomas (NEC) are the least common neuroendocrine neoplasm in humans and thus have been challenging to diagnose as they differ morphologically from NECs that arise from other parts of the body. The current World Health Organization classification defines neuroendocrine breast neoplasms as those with histologic evidence of neuroendocrine differentiation in >90% of cells. Given the low prevalence and lack of studies, cases of mammary NECs in people are typically treated as invasive breast cancer of no special type.

Mammary NETs are exceedingly rare and have been documented predominantly in women; loss of cytokeratin expression has been reported in up to 10% of the cases. ¹² The very few reports in veterinary species include only dogs ¹⁷ and a tiger. ⁵ The reported canine cases share some histopathologic features with human mammary NECs. Although the neoplasm in our equine case had negative immunolabeling for cytokeratin, we cannot rule out a NEC.

Standardized diagnostic criteria and comprehensive immunohistochemical characterization of NETs are lacking in veterinary medicine, and meaningful assessments of biological behavior and prognostic significance are not possible. Given the distribution of the neoplasm within deep sections of the mamma in our case, and the extensive replacement of the mammary tissue leaving only a few small normal glands, we favored a neuroendocrine mammary tumor. However, we cannot rule out a neuroendocrine tumor of cutaneous origin with infiltration of the mamma. Our case highlights the importance of considering NET as a differential diagnosis when evaluating mammary gland tumors in horses.