Extraskeletal osteosarcoma is an uncommon malignant mesenchymal tumor that generates osteoid, bone, and cartilage-like material without being directly attached to the bone or periosteum [4]. Instances in dogs have been documented in the mammary glands, liver, esophagus, jejunum, lungs, and more. This is known for its high metastatic rate, reaching up to 64%, and a poor prognosis, with a median survival span ranging from 26 to 90 days [1,5]. Surgical excision remains the primary treatment approach. However, adding chemotherapy post-surgery can improve outcomes, as seen in some veterinary and human studies that report extended survival and disease-free periods with such adjunctive treatments [1,2,6]. Tyrosine kinase inhibitors (TKIs) have proven effective for metastatic osteosarcoma by blocking receptor tyrosine kinases involved in cancer growth and spread [7]. Despite these advancements, a standardized chemotherapy regimen following surgery for extraskeletal osteosarcoma is still lacking.

This case report highlights an unusual mammary gland osteosarcoma in a dog, initially presenting as a carcinosarcoma. It also aims to detail the treatment process involving adjunctive chemotherapy with toceranib and cyclophosphamide after the tumor's surgical removal. 

A 14-year-old female mixed-breed dog was brought to the Gyeongsang National University Veterinary Teaching Hospital with a solitary mass located in the mammary gland area. The owner had observed the mass a year earlier, noting an increase in size over the past couple of months. The dog also exhibited symptoms of anorexia and lethargy. There was no previous diagnosis or medication history related to the chief complaint.

Upon physical examination, a singular, firm, and fixed 12.4 × 11 cm mass was detected on the left side, positioned between the second and third mammary glands (Fig. 1). The mass was characterized by ulceration, localized heat, and was tender upon palpation. To determine the cell origin and malignancy features, fine-needle aspiration and cytological analysis were conducted before surgical removal. The cytology showed predominant clusters of cells with acinar formations, along with signs of malignancy such as macronucleoli, binucleate cells, anisocytosis, anisokaryosis, coarse chromatin, and nuclear molding. Mesenchymal and multinucleated cells were also noted. The cytological differential diagnosis leaned towards neoplasia of epithelial origin, suspecting mammary gland adenocarcinoma, carcinoma, or a malignant mixed tumor like carcinosarcoma. Non-cancerous conditions such as hyperplasia, dysplasia, and inflammatory lesions like mastitis were excluded based on the malignant cell characteristics observed.

Diagnostic imaging was performed to delineate the mass boundaries and check for local invasion or distant metastasis. Thoracic radiography identified a 13 × 14 cm round mass with extensive radial mineral opacity in the ventral subcutaneous area of the left second and third teats. Abdominal ultrasound showed a hyperechoic lesion with shadowing and an anechoic to hypoechoic area with minimal blood flow. No regional lymph node enlargement or distant metastasis was detected through imaging.

Laboratory tests indicated moderate microcytic normochromic anemia, neutrophilia with a left shift, elevated C-reactive protein, hyperlactatemia, and a hypercoagulable state. These results suggested a paraneoplastic syndrome associated with the mammary gland tumor.

The dog underwent unilateral chain mastectomy and ovariohysterectomy, with imprint cytology of the removed mammary gland mass showing acinar cell clusters and mesenchymal cells with malignant features, as well as osteoblasts and multinucleated cells (Fig. 2). This led to a differential diagnosis of mammary gland carcinosarcoma and osseous metaplasia.

Histopathological analysis revealed neoplastic cells with significant anisocytosis, anisokaryosis, frequent multinucleated cells, and 14 mitoses per 2.37 mm². These cells were supported by fibrovascular stroma and surrounded by narrow bands of pale, eosinophilic, finely fibrillar osteoid material, bone, and cartilage, confirming a definitive diagnosis of extraskeletal osteosarcoma of the mammary gland (Fig. 3).

The owner declined systemic chemotherapy with carboplatin or doxorubicin, opting instead for metronomic and targeted chemotherapy with toceranib phosphate (Palladia; Zoetis, USA), alongside cyclophosphamide, firocoxib, misoprostol, and toceranib at specified dosages, with regular monitoring for side effects and metastasis planned monthly.

Afterwards, the dog continued to do well without any abnormalities, but 8 months after the initial presentation, the following clinical signs appeared: right hindlimb lameness, pain, lethargy, and anorexia. Neurological assessment indicated right hind limb ataxia and absence of postural reactions and reflexes, suggesting lumbosacral disc disease, neurological lesions, and tumor spread to the nervous system and bones. Radiographs showed increased numbers and sizes of soft tissue and mineral opacity nodules in the lungs, with additional nodules in the spinal process and ventral aspect of the 4th thoracic vertebra and left scapula, indicating suspected progressive metastasis of the extraskeletal osteosarcoma. The dog was prescribed prednisolone, tramadol, and gabapentin for neuropathic pain management without further investigation, according to the owner’s request.

Ultimately, the owner opted for euthanasia on the 332nd day post-presentation due to tetraparesis attributed to tumor metastasis. Postmortem computed tomography (CT) scans revealed soft tissue nodules and mineral deposits in various organs and bones, including the lungs, heart, liver, kidneys, ribs, scapula, thoracic vertebrae, sacrum, and lymph nodes, confirming widespread metastasis of the extraskeletal osteosarcoma.

The clinical significance of this study lies in the diagnosis and management of extraskeletal osteosarcoma of the mammary gland in dogs. Identifying mammary gland osteosarcomas can be difficult due to the cytological similarities it shares with conditions such as carcinosarcoma, mixed-type carcinoma, and osteoid metaplasia.

Definitive diagnosis for mammary gland mass, along with histopathological classification and prognostic assessment, requires histopathology. The mammary gland comprises luminal epithelial cells and myoepithelial cells, the latter of which are thought to originate from mesenchymal metaplasia or mesenchymal tumors. Immunohistochemistry (IHC) helps differentiate between carcinoma and sarcoma when cytological findings are ambiguous. In some studies, vimentin-supported IHC confirmed sarcoma diagnosis, while Ki67 indicated sarcoma proliferation levels [8]. In this case, histopathology indicated mammary gland osteosarcoma due to the notable osteoid tissue and osteoblast features. Further IHC was not conducted as no epithelial malignancy was evident in cytology.

Distinguishing between mammary gland osteosarcoma, mixed-type carcinoma, and carcinosarcoma is crucial for understanding disease progression, prognosis, and treatment strategies. While the TNM staging system is used for mammary gland carcinoma, it isn't applied to sarcomas. For osteosarcoma, staging involves histopathological grading, anatomical location, and metastasis. Osteosarcomas typically spread hematogenously, leading to frequent lung metastasis, unlike carcinomas that spread via lymphatics. Studies have shown a higher rate of lung metastasis in sarcomas compared to carcinomas. Thus, evaluating distant metastasis is critical in osteosarcoma cases, as demonstrated by CT scans in this case, showing metastasis to bones leading to tetraparesis.

Surgical excision of extraskeletal osteosarcoma is an effective approach, improving survival in both veterinary and human medicine [6,9,10]. However, the role of adjunctive radiotherapy or chemotherapy remains debated due to limited studies and a lack of consensus on protocols, attributed to the rarity of the condition. Therefore, case reports highlighting survival and prognosis in extraskeletal osteosarcoma are vital for developing systemic treatment strategies. In both human and veterinary contexts, adjunctive chemotherapy extends survival and disease-free intervals [11,12]. Studies have shown better outcomes with chemotherapy like carboplatin or cyclophosphamide, compared to surgery alone [13]. Toceranib, a TKI, effectively targets cancer cell pathways, influencing tumor growth and differentiation. It has shown efficacy in treating mast cell tumors, carcinomas, and sarcomas, including canine mammary gland tumors, by inducing apoptosis and cell cycle arrest [14,15].

Numerous research and case reports suggest the potential benefits of toceranib in osteosarcoma treatment, though further studies are needed due to the disease's rarity and current lack of established protocols for extraskeletal osteosarcoma [16-19]. In a separate study on extraskeletal osteosarcoma, chemotherapy emerged as the sole prognostic factor. Dogs that did not undergo chemotherapy faced a 3.62-fold increased risk of dying from tumor-related causes compared to those that did receive chemotherapy [2]. Within this study, out of 14 dogs with extraskeletal osteosarcoma, 2 cases involving mammary gland osteosarcoma were treated with doxorubicin administered 5 times at 3-week intervals following mastectomy. Their survival time were 223 and 185 days, respectively, marking the longest survival among the 14 dogs [2]. In this case, although receptor tyrosine kinase was not confirmed through IHC, an oral chemotherapy agent was chosen due to various reasons such as the owner’s intention, cost, and time constraints. Based on existing literature confirming the efficacy of TKI, toceranib was prescribed. Mammary gland osteosarcoma is aggressive, with a median survival time of 90 days, primarily due to pulmonary metastasis. Despite a generally poor prognosis, this case achieved longer survival (approximately 300 days), likely due to TKI as adjunctive chemotherapy. This case illustrates the practical application of toceranib after tumor removal, providing clinical insight into TKI efficacy in osteosarcoma management.

The limitation of this case was the absence of alkaline phosphatase staining in cytology and IHC staining, which could aid in diagnosing extraskeletal mammary gland osteosarcoma. Additionally, further diagnostics were not conducted, leaving uncertainty about metastasis in the skeletal and nervous systems. Moreover, platinum-based chemotherapy, a highly effective option for osteosarcoma, was not utilized. Future studies are needed to assess the prognostic value of systemic chemotherapy in treating extraskeletal osteosarcoma in veterinary practice. In conclusion, it underscores the importance of histopathology over cytology for accurate diagnosis and the necessity of considering mesenchymal neoplasms in differential diagnoses. Differentiating between sarcoma and carcinoma is crucial for predicting survival, metastasis sites, and treatment options. Based on this diagnosis, although a short survival with potential metastases was anticipated, the dog had survived longer after the use of adjunctive chemotherapy for improved outcomes.