Comparative Medicine Copyright 2014 by the American Association for Laboratory Animal Science
Vol 64, No 4 August 2014 Pages 309–313
Case Report
Spontaneous Epithelioid Hemangiosarcoma in a Rhesus Monkey (Macaca mulatta) Takayuki Tsuchiya,* Tasha L Gray, Nicholas T Gatto, Thomas Forest, Sam V Machotka, Sean P Troth, and Srinivasa Prahalada Epithelioid hemangiosarcoma is a rare malignant endothelial neoplasia with a unique, predominantly epithelioid morphology. A 4-y-old rhesus monkey from our laboratory had multiple neoplastic nodules in a digit, limb skin, hindlimb muscle, and visceral organs including lung, heart, and brain. The nodules were composed of pleomorphic, polygonal, epithelioid, neoplastic cells that were arranged in sheets, nests, and cords and supported by variably dense fibrovascular connective tissue. The morphologic features of this tumor were predominantly epithelioid. However, some regions contained cystic spaces, clefts, and channel-like structures, all of which were lined with morphologically distinct neoplastic endothelial cells. These neoplastic cells, with or without epithelioid morphology, were positive immunohistochemically for CD31, factor VIII-related antigen, and vimentin. The presence of multiple metastatic nodules, high mitotic rate, and extensive Ki67-positive staining were consistent with malignancy. This report is the first description of epithelioid hemangiosarcoma in a rhesus monkey. Abbreviations: PECAM1, platelet endothelial cell adhesion molecule 1; vWF, von Willebrand factor.
Spontaneous vascular tumors are rare in monkeys and humans13,14,21 but have been described in a variety of species including humans, monkeys, dogs, cats,8,9,13,14,17 horses,19 and rodents.2,22 In humans, epithelioid variants of endothelial tumors were first described in 1982 as a unique morphologic subtype of angiosarcoma with a predominantly epithelioid appearance.20 The epithelioid variant of hemangiosarcoma has rarely been reported in the veterinary literature. Recent reports have described epithelioid hemangioma and hemangiosarcomas in dogs,19 cattle,19 horses,5,19 and African hedgehog.4 Understanding the nature and spectrum of spontaneously occurring neoplasia in nonhuman primates has diagnostic value that may facilitate postmortem assessment in nonhuman primate toxicology studies conducted in support of clinical trials. This report describes the first case of an epithelioid hemangiosarcoma in a rhesus monkey.
Case Report
Animal care and housing before, during, and after treatment conformed to practices established by AAALAC and The Guide for the Care and Use of Laboratory Animals,7 the Animal Welfare Act,1 and European legislation on the protection of animals used for experimental and other scientific purposes.3 All procedures for animal care followed the Merck IACUC guidelines. Veterinary care was provided as required. The macaque was individually housed and provided with chow, drinking water, and daily enrichment ad libitum. A 2-y-old male rhesus macaque (Macaca mulatta) presented initially in June 2010 with a soft-tissue mass (diameter, 1 to 1.5 Received: 09 Aug 2013. Revision requested: 29 Sep 2013. Accepted: 02 Jan 2014. Safety Assessment, Merck Research Laboratories, West Point, Pennsylvania. * Corresponding author. Email: takayuki_tsuchiya @merck.com
cm) along the third digit of the left foot. At this time, treatment involved amputation of the entire toe and evaluation of tissues histomorphologically. The mass was diagnosed as hemangiosarcoma; the slide of the initial biopsy sample was not available for our assessment. There were no significant findings noted regarding results of CBC and serum biochemistry analyses obtained prior to the amputation. The CBC and serum biochemistry assessments obtained approximately 2 wk prior to the time of euthanasia revealed mild hyperalbunemia, hyperkalemia, a slightly elevated WBC with a mild leukocytosis which could be characteristic of either stress or inflammation, and a moderate anemia with marked reticulocytosis. The anemia was characterized as regenerative, according to the reticulocytosis present (Table 1). At 18 mo (January 2012) after the previous mass, similar masses involving the skin of the right forearm and lower left leg were noted. Both masses were biopsied and again were diagnosed as hemangiosarcoma. Over the course of the next 4 wk, the masses increased in size and became ulcerated, resulting in excessive bleeding. Because the macaque’s overall physical condition began to deteriorate, he was euthanized by pentobarbital injection through the saphenous vein. At necropsy, oral mucosal membranes were pale. There was generalized, mild icterus, evident as yellow discoloration of the subcutaneous tissues and the lining of the abdominal cavity. The lower portion of the left hindlimb was swollen and firm on palpation, and there was a region (0.5 cm × 2.0 cm × 2.0 cm) ulcerated skin on the anteriolateral surface (Figure 1). Beneath the ulcer, there was a tan, red, and black, partially circumscribed nodule (1.5 × 2.0 cm) associated with the skeletal muscle. In addition, an ulcerated mass (0.5 cm × 1.5 cm) was noted in the skin on the anterior surface of the right antebrachium. The thoracic cavity contained 50 mL of clear serous fluid. Multifocal to coalescing,
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Table 1. CBC and serum biochemistry Date of blood collectiona 20 Dec 2011
26 Jan 2012
Reference value
ALP (U/L)
145
241
0–603
ALT (U/L)
44
23
1–40
AST (U/L)
38
27
23–68
GGT (U/L)
68
47
21–50
Total protein (g/dL)
7.0
7.5
5.9–7.9
Albumin (g/dL)
4.5
4.6
3.2–4.1
Total bilirubin (mg/dL)
0.2
0.2
0–0.2
Glucose (mg/dL)
56
75
38–74
Cholesterol (mg/dL)
165
145
68–190
Sodium (mEq/L)
147
149
144–152
Potassium (mEq/L)
3.8
5.3
3.1–4.2
Chloride (mEq/L)
109
111
104–112
Phosphous (mg/dL)
5.0
5.0
1.4–6.4
Calcium (mg/dL)
9.4
9.9
8.7–10.8
BUN (mg/dL)
24
17
17–29
Creatinine (mg/dL)
0.9
1.0
0.7–1.4
WBC (K/µL)
5.14
12.49
5.8–10.4
RBC (g/dL)
5.55
3.92
5.7–6.4
Hct (%)
41.6
29.9
42.6–49.6
Reticulocyte (%)
1.4
31.6
not provided
Platelets (K/µL)
225
370
297–499
Neutrophils (K/µL)
2.90
8.48
3.4–5.8
Lymphocytes (K/µL)
1.66
2.68
3.8–6.0
Monocytes (K/uL)
0.44
0.79
0–4
Eosinophils (K/uL)
0.07
0.29
0–4
Values outside the reference range are bolded. Sample analysis was conducted in-house, and reference values were provided by the California National Primate Research Center. a Samples were obtained approximately 6 and 2 wk before euthanasia.
red to black foci were present on the visceral pleura and within the parenchyma of the lungs. Sections of masses from the skeletal muscle of the left hindlimb and forearm and from major organs including brain, spinal cord, lung, heart, liver, spleen, gastrointestinal tract, lymph node (submandibular and mesenteric), endocrine organs, and testis were examined by light microscopy. The deep skeletal muscle contained an unencapsulated and infiltrative, moderately cellular mass that replaced or surrounded and separated skeletal muscle bundle and extended into the subcutaneous tissue and dermis. The neoplastic mass elevated the overlying epidermis, which was ulcerated and covered by a serocellular crust. Oval to polygonal, neoplastic cells were arranged in sheets, nests, and cords, mimicking an epithelioid proliferation. These cells were supported by dense or fine, fibrovascular connective tissue stroma admixed with plump, active fibroblasts (Figure 2 A). Neoplastic cells were pleomorphic with variably distinct cell borders and variable amounts of eosinophilic to amphophilic, granular cytoplasm. Nuclei were vesicular and round to oval, with 1 or 2 prominent basophilic nucleoli. There was anisokaryosis and anisocytosis and occasional megakaryosis. Mitotic figures were frequent (25 to 30 per 10 high-power fields; Figure 2 B). Occasional neoplastic cells contained cytoplasmic vacuoles with or without RBC
Figure 1. Epithelioid hemangiosarcoma, hindlimb. An ulcerated lesion (0.5 cm × 2.0 cm × 2.0 cm; arrow) was present on the skin on the anteriolateral surface of the swollen hindlimb. Underneath the ulcer is a 1.5 × 2.0 cm nodule in the skeletal muscle (arrow heads).
(Figure 2 C). In some regions, neoplastic cells formed clefts (Figure 2 B), cystic spaces, and variably sized channels filled with RBC or neutrophils (Figure 2 D). There were multifocal areas of necrosis within the neoplastic tissue, with variable combinations of eosinophilic and karyorrhectic cellular debris, neutrophils (occasionally degenerate), fibrin, and RBC (hemorrhage). The forearm skin nodule was morphologically similar to the neoplastic masses found elsewhere. However, in this mass, bloodfilled cystic spaces and slit-like spaces lined by neoplastic cells were more prominent (Figure 2 D). Metastatic masses were widely distributed throughout the lung, brain (cerebrum and medulla oblongata), spinal cord, thyroid, heart, and testis. At metastatic sites, the neoplastic cells were present mainly within vessels, with occasional invasion to adjacent parenchyma; the cells were arranged mainly in sheets and nests with a few areas exhibiting endothelial morphology. The lymphatics and vessels within the neoplastic mass were ectatic and were often invaded and occluded by neoplastic cells. The surrounding fibrovascular stromal tissue was edematous. There was degeneration and regeneration of entrapped muscle fibers. The primary site was presumed to be the skeletal muscle or deep subcutaneous tissues of the left lower hindlimb, in light of the larger size of the associated mass. However, this hindlimb mass was not overt when the soft-tissue mass involving the toe was noted, so the possibility exists that the neoplasm arose in the toe. To further characterize the neoplastic cell population, immunohistochemical staining was performed by using an automated stainer (Dakocytomation Autostainer Universal Staining System, Carpinteria, CA) by means of the streptavidin–biotin immunoperoxidase technique with 3,3-diaminobenzidine–tetrahydrochloride chromogen (Vector Laboratories, Burlingame, CA) and Mayer hematoxylin counterstain (Sigma–Aldrich, St Louis, MO). Antigens were retrieved by using 0.04% pepsin (Sigma–Aldrich) or PT Module (Thermo Fisher Scientific, Waltham, MA) with modified citrate retrieval solution (Lab Vision, Fremont, CA). Antibodies used were rabbit antihuman von Willebrand factor (dilution, 1:1600; vWF [factor VIII-related antigen], Dakocytomation), monoclonal mouse antiCD31 (dilution, 1:100; platelet–endothelial cell adhesion molecule 1 [PECAM-1]; Abcam, Cambridge, MA), and monoclonal mouse antipancytokeratin (dilution, 1:240000; PCK26 Abcam; and dilution, 1:500; AE1/AE3 Abcam), monoclonal
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Spontaneous epithelioid hemangiosarcoma in a rhesus monkey
Figure 2. (A) Epithelioid hemangiosarcoma, hindlimb muscle. Neoplastic cells are arranged in nests or cords, mimicking epithelioid proliferation, and are supported by dense to fine fibrovascular connective tissue that separates and replaces muscle fibers. (B) Epithelioid hemangiosarcoma, hindlimb muscle. Neoplastic cells show frequent mitotic figures (arrow heads). Rarely, neoplastic cells form cystic spaces (asterisk) or clefts (arrows) with or without RBC. (C) Epithelioid hemangiosarcoma, hindlimb muscle. Neoplastic cells are pleomorphic with variably distinct cell borders, contain round to oval vesicular nuclei with 1 or 2 prominent basophilic nucleoli, and have variable amounts of eosinophilic to amphophilic granular cytoplasm that contains occasional cytoplasmic micro to macrovesicles (vacuoles) containing RBC or eosinophilic globules (arrows). (D) Epithelioid hemangiosarcoma, forearm skin. Blood-filled cystic spaces (asterisk) lined by neoplastic cells are prominent. Hematoxylin and eosin stain; bar: 50 μm (panels A though C), 70 μm (panel D).
mouse antivimentin (dilution, 1:10,000; Dako), and monoclonal antiki67 (dilution, 1:200; Abcam). Unaffected skin from a control monkey served as a positive control. Serial sections of the neoplasm from muscle of the left hindlimb and forearm with surrounding tissue were incubated with nonimmune serum to serve as negative controls. Neoplastic cells in the skeletal muscle of hindlimb and in the skin of forearm were strongly positive for CD31 (Figure 3 A), vWF antigen (Figure 3 B), and vimentin. In the neoplastic cells, cytoplasmic membranes were strongly CD31 positive, with relatively weaker diffuse cytoplasmic staining (Figure 3 C). There was fine granular cytoplasmic staining for vWF (Figure 3 D) in approximately 50% to 90% of neoplastic cells, depending on the area evaluated. Almost all neoplastic cells had diffusely positive cytoplasmic vimentin staining. The neoplastic cells were uniformly negative for pancytokeratin. There were frequent Ki67 positive cells (more than 30 positive nuclei in 10 high-power fields), consistent with a high proliferative rate and frequent mitotic figures. According to histomorphology and immunohistochemical
staining, this neoplasm was diagnosed as an epithelioid hemangiosarcoma.
Discussion
Vascular neoplasms are rare in rhesus monkeys, with only a few examples described in the literature. A 2011 reported included only a single hepatic hemangioma (0.09% incidence) in a study describing neoplastic findings of 1170 rhesus monkeys in 2 colonies.16 In our facility, the current case is the first vascular neoplasm that has occurred among approximately 7260 necropsied rhesus monkeys (0.01% incidence). The age of rhesus monkeys diagnosed with spontaneously occurring, endothelial neoplasia in the literatures varies, ranging from 6 mo to 16 y.10,11,13,14,16 In veterinary diagnostic settings, vascular tumors are commonly subdivided into angiomatosis, hemangioma, and hemangiosarcoma.17 Highly malignant epithelioid variants of vascular neoplasms have been diagnosed as epithelioid hemangiosarcoma in animals.14,17,19 Previously, an epithelioid hemangioendothelioma in the right auricle of the heart in a rhesus monkey was
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Figure 3. (A) Epithelioid hemangiosarcoma, forearm skin. The cytoplasm and cytoplasmic membrane of the neoplastic cells lining cystic spaces (asterisk) are positive for CD31. CD31 streptavidin–biotin immunoperoxidase method and Mayer hematoxylin counterstain; bar, 70 μm. (B) The cytoplasm of the neoplastic cells lining cystic spaces (asterisk) are positive for vWF. vWF streptavidin–biotin immunoperoxidase method and Mayer hematoxylin counterstain; bar, 70 μm. (C) Epithelioid hemangiosarcoma, hindlimb muscle. Strongly positive staining of cytoplasmic membranes and relatively weaker diffuse cytoplasmic staining for CD31 are present in most neoplastic cells. CD31 streptavidin–biotin immunoperoxidase method and Mayer hematoxylin counterstain; bar, 50 μm. (D) Strongly positive cytoplasmic staining for vWF is observed. vWF streptavidin–biotin immunoperoxidase method with Mayer hematoxylin counterstain; bar, 50 μm.
reported.11 The author described local invasion, but no metastasis, as well as myxohyaline stroma, minimal anisocytosis and anisokaryosis, and a low mitotic rate (3 mitotic figures per 10 high power fields). These features were consistent with intermediate malignancy as defined for hemangioendotheliomas18 and different from those in the current case. In humans, angiosarcoma is the term used to denote a malignant tumor of blood vessel or lymphatic endothelial origin.12 Endothelial vascular tumors in humans are categorized into 3 major groups: hemangiomas, hemangioendotheliomas, and angiosarcomas, with respectively increasing malignancy.9 Hemangiomas are considered benign, hemangioendotheliomas are typically of intermediate malignancy with local recurrence and metastatic potential, whereas angiosarcomas generally demonstrate highly malignant behavior with frequent metastasis and cellular pleomorphism.9 In humans, the epithelioid variant of cutaneous endothelial tumors was first described in 1982 as a unique morphologic subtype of angiosarcoma with a predominantly epithelioid neoplastic cell population.20 The current case shared many gross and histomorphologic similarities with the description of epithelioid angiosarcoma in
humans.6 The solid epithelioid structure of neoplastic cells was a prominent feature in most of the organs affected in the current case. Obvious vascular structures were limited to some of the skin masses. In addition, the immunohistochemical profile of the current case, positive for vimentin, CD31, and vWF, as well as the high proliferate rate (frequent Ki67-positive cells) observed, were consistent with the features of epithelioid angiosarcoma described in humans.6,20 Vascular tumors in horses, dogs, and monkeys are commonly positive for vWF and CD31.11,15,19 Human epithelioid angiosarcoma is CD34 positive in 40% to 100% of cases, but typically only areas with readily apparent vessel formation are stained. Therefore, the utility of CD34 in making the diagnosis of epithelioid angiosarcoma in poorly differentiated tumors composed primarily of solid sheets is limited.6 In the current case, CD31 staining was used as an adjunct to staining for vWF, given that in the human literature the combination of stains is considered to be sufficient for the definitive diagnosis of the epithelioid variant of vascular tumors and has been used as an aid in differentiating epithelial malignancies in rhesus monkeys.
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Spontaneous epithelioid hemangiosarcoma in a rhesus monkey
The neoplastic cells in the current case did not stain positively for cytokeratin. To our knowledge, there have been no reports of cytokeratin-positive epithelioid variants of endothelial tumors in the veterinary literature. Among 9 reported cases of epithelioid hemangiosarcoma in dogs and horses, none was positive for cytokeratin.19 However, it should be noted that epithelioid hemangiosarcoma has been described as variably positive for cytokeratin in the human literature.6,9 Due to the paucity of case reports in the veterinary literature, it is difficult to conclude that lack of cytokeratin staining is a typical feature of epithelioid vascular neoplasm in animals. Differential diagnoses to consider for epithelioid hemangiosarcoma would be epithelial or histiocytic neoplasms, or lymphangiosarcoma. However, the identification of the cyst-like spaces or clefts recapitulating vascular structures containing RBC and immunohistochemical staining of CD31 and vWF are reasons to rule out those differential diagnoses. In summary, the current report describes a case of spontaneous epithelioid hemangiosarcoma in a rhesus monkey. The features of this case were consistent with epithelioid hemangiosarcoma as described in the veterinary literature and shared many of the unique characteristics of epithelioid angiosarcoma in humans. The diagnosis of epithelioid hemangiosarcoma is consistent with the observation of vessel-like structures lined with neoplastic cells and positive staining for CD31 and vWF. To our knowledge, this report is the first description of epithelioid hemangiosarcoma in a nonhuman primate.
Acknowledgments
We thank Christine Reichard, Caron Cunningham, Debora Harner, and Adam Smith for technical assistance with the immunohistochemical staining and image capturing.
References
1. Animal Welfare Act as Amended. 2008. 7 USC §2131–2159. 2. Elwell MR, Mahler JF, Ruecker FA. 2004. Proliferative and nonproliferative lesions in the heart and vasculature in mice. In: Guides for toxicologic pathology. Washington (DC): Society of Toxicologic Pathologists. 3. The European Parliament and the Council of the European Union. 2010. Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the Protection of Animals Used for Scientific Purposes. Off J Eur Communities L276:33–79. 4. Finkelstein A, Hoover JP, Caudell D, Confer AW. 2008. Cutaneous epithelioid variant hemangiosarcoma in a captive African hedgehog (Atelerix albiventris). J Exot Pet Med 1:49–53.
5. Gumber S, Baia P, Wakamatsu N. 2011. Vulvar epithelioid hemangiosarcoma with solar elastosis in a mare. J Vet Diagn Invest 23:1033–1036. 6. Hart J, Mandavilli S. 2011. Epithelioid angiosarcoma: a brief diagnostic review and differential diagnosis. Arch Pathol Lab Med 135:268–272. 7. Institute for Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National Academies Press. 8. Jennings RN, Miller MA, Ramos-Vara JA. 2012. Comparison of CD34-, CD31-, and factor VIII-related antigen immunohistochemical expression in feline vascular neoplasms and CD34 expression in feline nonvascular neoplasms. Vet Pathol 49:532–537. 9. Kempson RL, Fletcher CDM, Evans HL, Hendrickson MR, Sibley RK. 2001. Tumors of the soft tissues (Atlas of Tumor Pathology [AFIP], 3rd series), fascicle 30, p 325–328. Washington (DC): Armed Forces Institute of Pathology. 10. Kent SP, Pickering JE. 1958. Neoplasms in monkeys (Macaca mulatta): spontaneous and irradiation-induced. Cancer 11:138–147. 11. Lombardini ED, Virmani R, Blanchard TW, Lafond JF, Ménard S, Doré M. 2010. Epithelioid hemangioendothelioma in the right auricle of an adult, male rhesus macaque (Macaca mulatta). J Med Primatol 39:315–317. 12. Mankey CC, McHugh JB, Thomas DG, Lucas DR. 2010. Can lymphangiosarcoma be resurrected? A clinicopathological and immunohistochemical study of lymphatic differentiation in 49 angiosarcomas. Histopathology 56:364–371. 13. Mejia AF, Gierbolini L, Jacob B, Westmoreland SV. 2009. Pediatric hepatic hemangiosarcoma in a rhesus macaque (Macaca mulatta). J Med Primatol 38:121–124. 14. Myers DD Jr, Dysko RC, Chrisp CE, Decoster JL. 2001. Subcutaneous hemangiosarcomas in a rhesus macaque (Macaca mulatta). J Med Primatol 30:127–130. 15. Shor S, Helfand SC, Gorman E, Lohr CV. 2009. Diagnostic exercise: epithelioid hemangiosarcoma mimicking metastatic prostatic neoplasia in a dog. Vet Pathol 46:548–552. 16. Simmons HA, Mattison JA. 2011. The incidence of spontaneous neoplasia in 2 populations of captive rhesus macaques (Macaca mulatta). Antioxid Redox Signal 14:221–227. 17. Smith AN. 2003. Hemangiosarcoma in dogs and cats. Vet Clin North Am Small Anim Pract 33:533–552. 18. Tsang WY, Chan JK. 1993. The family of epithelioid vascular tumors. Histol Histopathol 8:187–212. 19. Warren AL, Summers BA. 2007. Epithelioid variant of hemangioma and hemangiosarcoma in the dog, horse, and cow. Vet Pathol 44:15–24. 20. Weiss SW, Enzinger FM. 1982. Epithelioid hemangioendothelioma: a vascular tumor often mistaken for a carcinoma. Cancer 50:970–981. 21. Woodruff JM, Johnson DK. 1968. Hepatic hemangioendothelioma in a rhesus monkey. Pathol Vet 5:327–332. 22. Zwicker GM, Eyster RC, Sells DM, Gass JH. 1995. Spontaneous vascular neoplasms in aged Sprague–Dawley rats. Toxicol Pathol 23:518–526.
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Case Report
Spontaneous Epithelioid Hemangiosarcoma in a Rhesus Monkey (Macaca mulatta) Takayuki Tsuchiya,* Tasha L Gray, Nicholas T Gatto, Thomas Forest, Sam V Machotka, Sean P Troth, and Srinivasa Prahalada Epithelioid hemangiosarcoma is a rare malignant endothelial neoplasia with a unique, predominantly epithelioid morphology. A 4-y-old rhesus monkey from our laboratory had multiple neoplastic nodules in a digit, limb skin, hindlimb muscle, and visceral organs including lung, heart, and brain. The nodules were composed of pleomorphic, polygonal, epithelioid, neoplastic cells that were arranged in sheets, nests, and cords and supported by variably dense fibrovascular connective tissue. The morphologic features of this tumor were predominantly epithelioid. However, some regions contained cystic spaces, clefts, and channel-like structures, all of which were lined with morphologically distinct neoplastic endothelial cells. These neoplastic cells, with or without epithelioid morphology, were positive immunohistochemically for CD31, factor VIII-related antigen, and vimentin. The presence of multiple metastatic nodules, high mitotic rate, and extensive Ki67-positive staining were consistent with malignancy. This report is the first description of epithelioid hemangiosarcoma in a rhesus monkey. Abbreviations: PECAM1, platelet endothelial cell adhesion molecule 1; vWF, von Willebrand factor.
Spontaneous vascular tumors are rare in monkeys and humans13,14,21 but have been described in a variety of species including humans, monkeys, dogs, cats,8,9,13,14,17 horses,19 and rodents.2,22 In humans, epithelioid variants of endothelial tumors were first described in 1982 as a unique morphologic subtype of angiosarcoma with a predominantly epithelioid appearance.20 The epithelioid variant of hemangiosarcoma has rarely been reported in the veterinary literature. Recent reports have described epithelioid hemangioma and hemangiosarcomas in dogs,19 cattle,19 horses,5,19 and African hedgehog.4 Understanding the nature and spectrum of spontaneously occurring neoplasia in nonhuman primates has diagnostic value that may facilitate postmortem assessment in nonhuman primate toxicology studies conducted in support of clinical trials. This report describes the first case of an epithelioid hemangiosarcoma in a rhesus monkey.
Case Report
Animal care and housing before, during, and after treatment conformed to practices established by AAALAC and The Guide for the Care and Use of Laboratory Animals,7 the Animal Welfare Act,1 and European legislation on the protection of animals used for experimental and other scientific purposes.3 All procedures for animal care followed the Merck IACUC guidelines. Veterinary care was provided as required. The macaque was individually housed and provided with chow, drinking water, and daily enrichment ad libitum. A 2-y-old male rhesus macaque (Macaca mulatta) presented initially in June 2010 with a soft-tissue mass (diameter, 1 to 1.5 Received: 09 Aug 2013. Revision requested: 29 Sep 2013. Accepted: 02 Jan 2014. Safety Assessment, Merck Research Laboratories, West Point, Pennsylvania. * Corresponding author. Email: takayuki_tsuchiya @merck.com
cm) along the third digit of the left foot. At this time, treatment involved amputation of the entire toe and evaluation of tissues histomorphologically. The mass was diagnosed as hemangiosarcoma; the slide of the initial biopsy sample was not available for our assessment. There were no significant findings noted regarding results of CBC and serum biochemistry analyses obtained prior to the amputation. The CBC and serum biochemistry assessments obtained approximately 2 wk prior to the time of euthanasia revealed mild hyperalbunemia, hyperkalemia, a slightly elevated WBC with a mild leukocytosis which could be characteristic of either stress or inflammation, and a moderate anemia with marked reticulocytosis. The anemia was characterized as regenerative, according to the reticulocytosis present (Table 1). At 18 mo (January 2012) after the previous mass, similar masses involving the skin of the right forearm and lower left leg were noted. Both masses were biopsied and again were diagnosed as hemangiosarcoma. Over the course of the next 4 wk, the masses increased in size and became ulcerated, resulting in excessive bleeding. Because the macaque’s overall physical condition began to deteriorate, he was euthanized by pentobarbital injection through the saphenous vein. At necropsy, oral mucosal membranes were pale. There was generalized, mild icterus, evident as yellow discoloration of the subcutaneous tissues and the lining of the abdominal cavity. The lower portion of the left hindlimb was swollen and firm on palpation, and there was a region (0.5 cm × 2.0 cm × 2.0 cm) ulcerated skin on the anteriolateral surface (Figure 1). Beneath the ulcer, there was a tan, red, and black, partially circumscribed nodule (1.5 × 2.0 cm) associated with the skeletal muscle. In addition, an ulcerated mass (0.5 cm × 1.5 cm) was noted in the skin on the anterior surface of the right antebrachium. The thoracic cavity contained 50 mL of clear serous fluid. Multifocal to coalescing,
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Table 1. CBC and serum biochemistry Date of blood collectiona 20 Dec 2011
26 Jan 2012
Reference value
ALP (U/L)
145
241
0–603
ALT (U/L)
44
23
1–40
AST (U/L)
38
27
23–68
GGT (U/L)
68
47
21–50
Total protein (g/dL)
7.0
7.5
5.9–7.9
Albumin (g/dL)
4.5
4.6
3.2–4.1
Total bilirubin (mg/dL)
0.2
0.2
0–0.2
Glucose (mg/dL)
56
75
38–74
Cholesterol (mg/dL)
165
145
68–190
Sodium (mEq/L)
147
149
144–152
Potassium (mEq/L)
3.8
5.3
3.1–4.2
Chloride (mEq/L)
109
111
104–112
Phosphous (mg/dL)
5.0
5.0
1.4–6.4
Calcium (mg/dL)
9.4
9.9
8.7–10.8
BUN (mg/dL)
24
17
17–29
Creatinine (mg/dL)
0.9
1.0
0.7–1.4
WBC (K/µL)
5.14
12.49
5.8–10.4
RBC (g/dL)
5.55
3.92
5.7–6.4
Hct (%)
41.6
29.9
42.6–49.6
Reticulocyte (%)
1.4
31.6
not provided
Platelets (K/µL)
225
370
297–499
Neutrophils (K/µL)
2.90
8.48
3.4–5.8
Lymphocytes (K/µL)
1.66
2.68
3.8–6.0
Monocytes (K/uL)
0.44
0.79
0–4
Eosinophils (K/uL)
0.07
0.29
0–4
Values outside the reference range are bolded. Sample analysis was conducted in-house, and reference values were provided by the California National Primate Research Center. a Samples were obtained approximately 6 and 2 wk before euthanasia.
red to black foci were present on the visceral pleura and within the parenchyma of the lungs. Sections of masses from the skeletal muscle of the left hindlimb and forearm and from major organs including brain, spinal cord, lung, heart, liver, spleen, gastrointestinal tract, lymph node (submandibular and mesenteric), endocrine organs, and testis were examined by light microscopy. The deep skeletal muscle contained an unencapsulated and infiltrative, moderately cellular mass that replaced or surrounded and separated skeletal muscle bundle and extended into the subcutaneous tissue and dermis. The neoplastic mass elevated the overlying epidermis, which was ulcerated and covered by a serocellular crust. Oval to polygonal, neoplastic cells were arranged in sheets, nests, and cords, mimicking an epithelioid proliferation. These cells were supported by dense or fine, fibrovascular connective tissue stroma admixed with plump, active fibroblasts (Figure 2 A). Neoplastic cells were pleomorphic with variably distinct cell borders and variable amounts of eosinophilic to amphophilic, granular cytoplasm. Nuclei were vesicular and round to oval, with 1 or 2 prominent basophilic nucleoli. There was anisokaryosis and anisocytosis and occasional megakaryosis. Mitotic figures were frequent (25 to 30 per 10 high-power fields; Figure 2 B). Occasional neoplastic cells contained cytoplasmic vacuoles with or without RBC
Figure 1. Epithelioid hemangiosarcoma, hindlimb. An ulcerated lesion (0.5 cm × 2.0 cm × 2.0 cm; arrow) was present on the skin on the anteriolateral surface of the swollen hindlimb. Underneath the ulcer is a 1.5 × 2.0 cm nodule in the skeletal muscle (arrow heads).
(Figure 2 C). In some regions, neoplastic cells formed clefts (Figure 2 B), cystic spaces, and variably sized channels filled with RBC or neutrophils (Figure 2 D). There were multifocal areas of necrosis within the neoplastic tissue, with variable combinations of eosinophilic and karyorrhectic cellular debris, neutrophils (occasionally degenerate), fibrin, and RBC (hemorrhage). The forearm skin nodule was morphologically similar to the neoplastic masses found elsewhere. However, in this mass, bloodfilled cystic spaces and slit-like spaces lined by neoplastic cells were more prominent (Figure 2 D). Metastatic masses were widely distributed throughout the lung, brain (cerebrum and medulla oblongata), spinal cord, thyroid, heart, and testis. At metastatic sites, the neoplastic cells were present mainly within vessels, with occasional invasion to adjacent parenchyma; the cells were arranged mainly in sheets and nests with a few areas exhibiting endothelial morphology. The lymphatics and vessels within the neoplastic mass were ectatic and were often invaded and occluded by neoplastic cells. The surrounding fibrovascular stromal tissue was edematous. There was degeneration and regeneration of entrapped muscle fibers. The primary site was presumed to be the skeletal muscle or deep subcutaneous tissues of the left lower hindlimb, in light of the larger size of the associated mass. However, this hindlimb mass was not overt when the soft-tissue mass involving the toe was noted, so the possibility exists that the neoplasm arose in the toe. To further characterize the neoplastic cell population, immunohistochemical staining was performed by using an automated stainer (Dakocytomation Autostainer Universal Staining System, Carpinteria, CA) by means of the streptavidin–biotin immunoperoxidase technique with 3,3-diaminobenzidine–tetrahydrochloride chromogen (Vector Laboratories, Burlingame, CA) and Mayer hematoxylin counterstain (Sigma–Aldrich, St Louis, MO). Antigens were retrieved by using 0.04% pepsin (Sigma–Aldrich) or PT Module (Thermo Fisher Scientific, Waltham, MA) with modified citrate retrieval solution (Lab Vision, Fremont, CA). Antibodies used were rabbit antihuman von Willebrand factor (dilution, 1:1600; vWF [factor VIII-related antigen], Dakocytomation), monoclonal mouse antiCD31 (dilution, 1:100; platelet–endothelial cell adhesion molecule 1 [PECAM-1]; Abcam, Cambridge, MA), and monoclonal mouse antipancytokeratin (dilution, 1:240000; PCK26 Abcam; and dilution, 1:500; AE1/AE3 Abcam), monoclonal
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Figure 2. (A) Epithelioid hemangiosarcoma, hindlimb muscle. Neoplastic cells are arranged in nests or cords, mimicking epithelioid proliferation, and are supported by dense to fine fibrovascular connective tissue that separates and replaces muscle fibers. (B) Epithelioid hemangiosarcoma, hindlimb muscle. Neoplastic cells show frequent mitotic figures (arrow heads). Rarely, neoplastic cells form cystic spaces (asterisk) or clefts (arrows) with or without RBC. (C) Epithelioid hemangiosarcoma, hindlimb muscle. Neoplastic cells are pleomorphic with variably distinct cell borders, contain round to oval vesicular nuclei with 1 or 2 prominent basophilic nucleoli, and have variable amounts of eosinophilic to amphophilic granular cytoplasm that contains occasional cytoplasmic micro to macrovesicles (vacuoles) containing RBC or eosinophilic globules (arrows). (D) Epithelioid hemangiosarcoma, forearm skin. Blood-filled cystic spaces (asterisk) lined by neoplastic cells are prominent. Hematoxylin and eosin stain; bar: 50 μm (panels A though C), 70 μm (panel D).
mouse antivimentin (dilution, 1:10,000; Dako), and monoclonal antiki67 (dilution, 1:200; Abcam). Unaffected skin from a control monkey served as a positive control. Serial sections of the neoplasm from muscle of the left hindlimb and forearm with surrounding tissue were incubated with nonimmune serum to serve as negative controls. Neoplastic cells in the skeletal muscle of hindlimb and in the skin of forearm were strongly positive for CD31 (Figure 3 A), vWF antigen (Figure 3 B), and vimentin. In the neoplastic cells, cytoplasmic membranes were strongly CD31 positive, with relatively weaker diffuse cytoplasmic staining (Figure 3 C). There was fine granular cytoplasmic staining for vWF (Figure 3 D) in approximately 50% to 90% of neoplastic cells, depending on the area evaluated. Almost all neoplastic cells had diffusely positive cytoplasmic vimentin staining. The neoplastic cells were uniformly negative for pancytokeratin. There were frequent Ki67 positive cells (more than 30 positive nuclei in 10 high-power fields), consistent with a high proliferative rate and frequent mitotic figures. According to histomorphology and immunohistochemical
staining, this neoplasm was diagnosed as an epithelioid hemangiosarcoma.
Discussion
Vascular neoplasms are rare in rhesus monkeys, with only a few examples described in the literature. A 2011 reported included only a single hepatic hemangioma (0.09% incidence) in a study describing neoplastic findings of 1170 rhesus monkeys in 2 colonies.16 In our facility, the current case is the first vascular neoplasm that has occurred among approximately 7260 necropsied rhesus monkeys (0.01% incidence). The age of rhesus monkeys diagnosed with spontaneously occurring, endothelial neoplasia in the literatures varies, ranging from 6 mo to 16 y.10,11,13,14,16 In veterinary diagnostic settings, vascular tumors are commonly subdivided into angiomatosis, hemangioma, and hemangiosarcoma.17 Highly malignant epithelioid variants of vascular neoplasms have been diagnosed as epithelioid hemangiosarcoma in animals.14,17,19 Previously, an epithelioid hemangioendothelioma in the right auricle of the heart in a rhesus monkey was
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Figure 3. (A) Epithelioid hemangiosarcoma, forearm skin. The cytoplasm and cytoplasmic membrane of the neoplastic cells lining cystic spaces (asterisk) are positive for CD31. CD31 streptavidin–biotin immunoperoxidase method and Mayer hematoxylin counterstain; bar, 70 μm. (B) The cytoplasm of the neoplastic cells lining cystic spaces (asterisk) are positive for vWF. vWF streptavidin–biotin immunoperoxidase method and Mayer hematoxylin counterstain; bar, 70 μm. (C) Epithelioid hemangiosarcoma, hindlimb muscle. Strongly positive staining of cytoplasmic membranes and relatively weaker diffuse cytoplasmic staining for CD31 are present in most neoplastic cells. CD31 streptavidin–biotin immunoperoxidase method and Mayer hematoxylin counterstain; bar, 50 μm. (D) Strongly positive cytoplasmic staining for vWF is observed. vWF streptavidin–biotin immunoperoxidase method with Mayer hematoxylin counterstain; bar, 50 μm.
reported.11 The author described local invasion, but no metastasis, as well as myxohyaline stroma, minimal anisocytosis and anisokaryosis, and a low mitotic rate (3 mitotic figures per 10 high power fields). These features were consistent with intermediate malignancy as defined for hemangioendotheliomas18 and different from those in the current case. In humans, angiosarcoma is the term used to denote a malignant tumor of blood vessel or lymphatic endothelial origin.12 Endothelial vascular tumors in humans are categorized into 3 major groups: hemangiomas, hemangioendotheliomas, and angiosarcomas, with respectively increasing malignancy.9 Hemangiomas are considered benign, hemangioendotheliomas are typically of intermediate malignancy with local recurrence and metastatic potential, whereas angiosarcomas generally demonstrate highly malignant behavior with frequent metastasis and cellular pleomorphism.9 In humans, the epithelioid variant of cutaneous endothelial tumors was first described in 1982 as a unique morphologic subtype of angiosarcoma with a predominantly epithelioid neoplastic cell population.20 The current case shared many gross and histomorphologic similarities with the description of epithelioid angiosarcoma in
humans.6 The solid epithelioid structure of neoplastic cells was a prominent feature in most of the organs affected in the current case. Obvious vascular structures were limited to some of the skin masses. In addition, the immunohistochemical profile of the current case, positive for vimentin, CD31, and vWF, as well as the high proliferate rate (frequent Ki67-positive cells) observed, were consistent with the features of epithelioid angiosarcoma described in humans.6,20 Vascular tumors in horses, dogs, and monkeys are commonly positive for vWF and CD31.11,15,19 Human epithelioid angiosarcoma is CD34 positive in 40% to 100% of cases, but typically only areas with readily apparent vessel formation are stained. Therefore, the utility of CD34 in making the diagnosis of epithelioid angiosarcoma in poorly differentiated tumors composed primarily of solid sheets is limited.6 In the current case, CD31 staining was used as an adjunct to staining for vWF, given that in the human literature the combination of stains is considered to be sufficient for the definitive diagnosis of the epithelioid variant of vascular tumors and has been used as an aid in differentiating epithelial malignancies in rhesus monkeys.
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The neoplastic cells in the current case did not stain positively for cytokeratin. To our knowledge, there have been no reports of cytokeratin-positive epithelioid variants of endothelial tumors in the veterinary literature. Among 9 reported cases of epithelioid hemangiosarcoma in dogs and horses, none was positive for cytokeratin.19 However, it should be noted that epithelioid hemangiosarcoma has been described as variably positive for cytokeratin in the human literature.6,9 Due to the paucity of case reports in the veterinary literature, it is difficult to conclude that lack of cytokeratin staining is a typical feature of epithelioid vascular neoplasm in animals. Differential diagnoses to consider for epithelioid hemangiosarcoma would be epithelial or histiocytic neoplasms, or lymphangiosarcoma. However, the identification of the cyst-like spaces or clefts recapitulating vascular structures containing RBC and immunohistochemical staining of CD31 and vWF are reasons to rule out those differential diagnoses. In summary, the current report describes a case of spontaneous epithelioid hemangiosarcoma in a rhesus monkey. The features of this case were consistent with epithelioid hemangiosarcoma as described in the veterinary literature and shared many of the unique characteristics of epithelioid angiosarcoma in humans. The diagnosis of epithelioid hemangiosarcoma is consistent with the observation of vessel-like structures lined with neoplastic cells and positive staining for CD31 and vWF. To our knowledge, this report is the first description of epithelioid hemangiosarcoma in a nonhuman primate.
Acknowledgments
We thank Christine Reichard, Caron Cunningham, Debora Harner, and Adam Smith for technical assistance with the immunohistochemical staining and image capturing.
References
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