Spontaneous Hemangiosarcoma of Bone with Hepatic and Pulmonary Metastasis in a Rhesus Macaque (Macaca mulatta): A Case Report

Introduction

Hemangiosarcoma is a malignant soft tissue neoplasm of blood vessel endothelial cell origin. Hemangiosarcoma, a form of angiosarcoma, is rare in both NHPs and humans and is associated with a poor prognosis. In the human literature, angiosarcoma is commonly used to refer to neoplasms of endothelial cell differentiation, including tumors of both blood vessel and lymphatic endothelial cells.^1,2 The incidence rate of angiosarcoma in humans is 3.3 cases per 1,000,000 person-years, representing only 1%-2% of all soft tissue sarcomas, and ∼0.01% of all cancers.^3,4 Angiosarcoma in people most commonly forms in the skin of the head, neck, and scalp, the breast, the extremities, and less frequently in the liver, right auricle of the heart, bone, and spleen.¹ About 72.3% of angiosarcomas in humans are cutaneous, subcutaneous, or breast angiosarcomas, whereas 24.4% are visceral.³ Dogs have the highest incidence of hemangiosarcomas in animals, representing 5%-7% of all canine neoplasms. A breed predisposition is seen in German shepherds, golden retrievers, and labrador retrievers.^5,6 In dogs, the spleen is the most commonly affected site, followed by skin, right atrium, and liver. Hemangiosarcoma occurs infrequently in multiple other animal species including cats, horses, and ruminants.⁵

Risk factors for the development of angiosarcoma in humans include radiation, chronic lymphedema (Stewart-Treves syndrome), and exposure to toxins such as vinyl chloride, thorium dioxide, arsenic, and anabolic steroids. Genetic mutations in known familial syndromes such as neurofibromatosis NF-1, mutated BRCA1 or BRCA2, Maffucci syndrome, and Klippel-Trénaunay syndrome are also associated with angiosarcoma.^1,7

Next-generation sequencing and whole-exome sequencing revealed that the most common genes mutated in human angiosarcomas were KDR (also known as VEGFR2), TP53, and PIK3CA. Other recurrent alterations were in POT1, RAS, BRAF, PTPRB, PLCG1, and APC genes.⁸ The leading genetic alterations observed in human angiosarcomas from head and neck locations were in TP53 (50.0%), POT1 (40.5%), and ARID1A (33.3%). In angiosarcoma of the breast, the most common genetic alterations were MYC amplification (63.3%), HRAS (16.1%), and PIK3CA (16.1%).^8,9 In a recent study, the genetic landscape of canine hemangiosarcoma was explored. Interestingly, the study found similarities in the predominant mutational signatures between human angiosarcoma and canine hemangiosarcoma.⁷ Mutations in the TP53 gene (59.6%), and 2 genes in the PI3K pathway (PIK3CA [29.8%] and PIK3R1 [8.5%]), were the most frequent in canine hemangiosarcomas.⁷

Hemangiosarcomas are very rare in NHPs; only a few reports have been documented. A literature search recovered 4 reports of hemangiosarcoma,^10–13 and 1 report of epithelioid hemangioendothelioma¹⁴ in rhesus macaques (Macaca mulatta), and 1 report of hepatic hemangiosarcoma in a cynomolgus macaque (Macaca fascicularis).¹⁵ In the rhesus macaque cases, liver and skin were described as the primary sites of hemangiosarcoma, with no mention of bone metastasis.^10–13 The cynomolgus macaque hepatic hemangiosarcoma was reported to have metastasized to the zygomatic arch.¹⁵

This report describes the clinicopathologic features of an experimentally naive, ∼5-year-old rhesus macaque with spontaneous disseminated hemangiosarcoma that originated at the tibial bone. To our knowledge, this is the first report of a spontaneous primary hemangiosarcoma of bone with hepatic and pulmonary metastasis in a rhesus macaque.

Case Report

An experimentally naive, 4.8-year-old, female Indian-origin rhesus macaque presented to the Tulane National Biomedical Research Center (TNBRC) clinic with right hindlimb lameness and poor body condition. On physical examination, significant muscle atrophy of the right leg and pelvis were present. The initial clinical differentials included neoplasia (osteosarcoma, hemangiosarcoma, chondrosarcoma, and fibrosarcoma), infectious disease caused by a bacterial or fungal agent, trauma, and malnutrition. Complete blood count (XN-1000V hematology analyzer; Sysmex USA, Lincolnshire, IL) showed nonregenerative anemia; the hematocrit (Hct) was 31.5% (reference interval, 34.8%-55.2%). Blood smear evaluation showed mild poikilocytosis (+1). Blood chemistry analysis (Olympus AU480 chemistry analyzer; Beckman Coulter, Brea, CA) showed hypoalbuminemia (2.7 g/dL; reference interval, 3.0-5.95 g/dL), combined with hypocalcemia (8.5 g/dL; reference interval, 9.4-12.212 g/dL). Radiographs demonstrated severe, diffuse degeneration and lysis of the right tibia, accompanied by a small lytic lesion in the proximal diaphysis of the right fibula (Figure 1A). Chest radiographs showed multiple pulmonary nodules (Figure 1B). The alkaline phosphatase enzyme serum level was within the normal range (ie, 230 U/L; reference interval, 55-649 U/L). After conducting diagnostic testing and imaging, the list of clinical differentials was revised to include neoplasia (osteosarcoma, hemangiosarcoma, chondrosarcoma, and fibrosarcoma) and infectious disease caused by a bacterial or fungal agent. Based on clinical findings, as well as the list of clinical differential diagnoses, the animal was humanely euthanized due to poor prognosis.

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On necropsy, the right tibia was markedly thickened 2-fold the normal thickness (Figure 2A). Multifocal 1- to 7-mm-diameter dark red, moderately firm nodules were present in the periosteum, compact cortical bone, and medullary cavity (Figure 2A). Multifocal dark red nodules measuring 1-10 mm were present in the lung (Figure 2B) and liver (Figure 2C).

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Histologically, the nodules were composed of neoplastic endothelial cells forming irregular clefts and anastomosing vascular channels (Figure 3). Neoplastic cells were spindled to polygonal with plump nuclei, and amphophilic to light basophilic cytoplasm with distinct borders. Anisocytosis and anisokaryosis were moderate; however, anaplastic cells were present in some areas of the tumor. Neoplastic endothelial cells were wrapped around a fibrovascular stroma. The nucleus-to-cytoplasm ratio was high. The neoplasm had very high mitotic activity; the mitotic count was 27 mitotic figures in the standard area of 2.37 mm².¹⁶ Large areas of necrosis were present in the tibial sections of tumor. Necrosis was not seen in the lungs or liver neoplastic nodules. Neoplastic cells exhibited positive staining with CD31, as well as von Willebrand factor immunohistochemical markers (Figure 4). More specifically, neoplastic endothelial cells in both the bone and lung masses showed strong membranous positive staining for CD31, with occasional, weak cytoplasmic CD31 staining (Figure 4A [bone] and B [lung]). Von Willebrand factor immunoreactivity in the neoplastic endothelial cells of both the bone and lung masses showed positive, coarse granular cytoplasmic staining (Figure 4C and D). The primary antibodies used for immunohistochemistry and their dilution, source and catalogue numbers are listed in Table S1.

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Based on the gross findings, histopathology, and immunohistochemistry results, this rhesus macaque had a hemangiosarcoma originating at the right tibial bone with metastasis to lung and liver.

Discussion

An ∼5-year-old, Indian-origin rhesus macaque presented to the TNBRC clinic with right hindlimb lameness, significant hindlimb muscle atrophy, and poor body condition. Radiographs of the right tibia showed diffuse areas of radiolucency within cortical and medullary bone. A small focal area of radiolucency was present in the proximal diaphysis of the right fibula. Several radiopaque nodules were scattered throughout the lung and liver parenchyma. Telangiectatic osteosarcoma was one of the clinical differentials. Grossly, telangiectatic osteosarcomas are dark red and soft due to multiple large blood-filled spaces comprising the tumor. Histologically, these large blood-filled spaces are lined by neoplastic osteoblasts. Telangiectatic osteosarcoma can be histologically differentiated from hemangiosarcoma by the deposition of osteoid material among neoplastic cells.^17,18 No osteoid deposition was noted histologically within the neoplasm in this case. In the bone, lung, and liver of this animal, neoplastic cells formed small blood-filled clefts and anastomosing vascular channels characteristic of neoplastic endothelial cells in hemangiosarcomas. The histologic organization of neoplastic cells in the lungs had an interesting microcapillary pattern in some areas of the neoplasm.

CD31, also known as PECAM-1 (platelet endothelial cell adhesion molecule-1), is a transmembrane adhesion molecule found primarily on endothelial cells as well as platelets. Von Willebrand factor is primarily synthesized and stored in endothelial cells and functions in platelet adhesion and clotting. Both CD31 and von Willebrand factor are key markers for endothelial cells.^5,17 Neoplastic cells in this case stained positively for both CD31 and von Willebrand factor, confirming the endothelial origin of this neoplasm. Telangiectatic osteosarcomas are immunonegative for von Willebrand factor.¹⁸

A combination of indicators strongly suggested that the hemangiosarcoma in this monkey originated at the right tibial bone and metastasized to lung and liver. These indicators include the location of the largest tumor, the gross and histopathologic characteristics of the tumor’s growth pattern, and the primary clinical signs.

The largest tumor in this animal was found in the right tibia. The nodules in the lungs were significantly smaller, and the nodules in the liver were also smaller and significantly fewer. Also, the tumor in the tibia filled the medullary cavity and infiltrated into and invaded cortical bone and periosteum (Figures 1 and 2A). The nodules in the lung and liver had a disseminated, spread-out pattern (Figure 2B and C), strongly suggestive of hematogenous spread. In addition, the tumor in the tibia had ill-defined margins, whereas the lung nodules were well-demarcated from the surrounding normal lung tissue (Figure 3C).

In dogs, the most commonly reported primary sites for hemangiosarcoma are the spleen, right atrium of the heart, and liver. Common metastatic sites in dogs include lungs and liver. In cats, skin and subcutaneous tissues are reported as common primary sites for hemangiosarcoma. Metastatic foci are often seen in visceral organs in cats.^5,17 Hemangiosarcoma is very rare in NHPs, and established disease prevalence and tumor progression data are unavailable. Only one case in the literature was identified describing metastatic hemangiosarcoma to bone from a primary tumor in the liver, in a cynomolgus monkey.¹⁵

Dogs with hemangiosarcoma of the right atrium often present clinically with signs of right-side heart failure. Many dogs with splenic hemangiosarcoma present with acute collapse due to a ruptured splenic hemangiosarcoma, or with hemoabdomen. The animal in the case described here was presented to the TNBRC clinic with the primary clinical sign of right hindlimb lameness and poor body condition. Considering the above indicators, our assessment with high confidence is that the hemangiosarcoma in this rhesus monkey originated at the right tibial bone and metastasized to lung and liver.

As mentioned previously, multiple environmental factors can contribute to the development of hemangiosarcoma in humans, including toxins. The rhesus macaque in this report was experimentally naive and was not exposed to known toxins.

In summary, hemangiosarcomas are very rare in NHPs; however, they should be considered among the primary differentials for neoplastic processes affecting bone in NHPs. To our knowledge, this report documents the first case of spontaneous metastatic hemangiosarcoma originating in bone in an NHP.

Supplementary Material

Table S1. Primary antibodies used for immunohistochemistry.