J. Comp. Path. 2022, Vol. 199, 81e85 Available online at www.sciencedirect.com ScienceDirect www.elsevier.com/locate/jcpa NEOPLASTIC DISEASE Undifferentiated Wing Sarcoma in a Peach-Faced Lovebird (Agapornis roseicollis) Irene Ruano-Feo*, Bernat Martı́-Garcı́a*, Oriol Jornet-Rius†, Jaume Martorell†,‡,* and Antonio Ramisx * Servei de Diagnostic de Patologia Veterinaria, † Department de Medicina i Cirurgia Animals, ‡ Hospital Clinic Veterinary, Facultat de Veterinaria, Universitat Autonoma de Barcelona and x IRTA, Centre de Recerca en Sanitat Animal, Campus de la Universitat Autonoma de Barcelona, Bellaterra, Barcelona, Spain Summary A 10-year-old peach-faced lovebird (Agapornis roseicollis) was evaluated for an ulcerated and painful mass at the location of a fracture 2 years previously. Whole body radiographs showed a humeral fracture with a presump- tive neoplastic proliferation in the distal diaphysis. Right wing amputation was elected but the animal died during recovery from surgery. Histopathological examination of the amputated wing revealed an infiltrative sarcomatous neoplastic proliferation. Immunohistochemistry (IHC) was carried out to characterize the tumour using antibodies against vimentin, desmin, smooth muscle actin (SMA), S-100, ionized calcium- binding adapter molecule-1 (IBA-1), CD18, cytokeratin and epithelial membrane antigen (EMA). The mesenchymal component of the mass was immunolabelled for vimentin and SMA and sparse epithelial cells were immunopositive for cytokeratin. Very few scattered cells were immunopositive for CD18 and IBA-1. The final diagnosis was consistent with an undifferentiated sarcoma with intralesional hyperplastic epithelium. According to the location, the history of a previous fracture and the histological pattern and IHC profile, the tumour was classified as an undifferentiated sarcoma with entrapped air sac epithelium. Ó 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Keywords: biphasic pattern; immunohistochemistry; lovebird; undifferentiated sarcoma A 10-year-old entire female peach-faced lovebird ated and involved the right humeral area. There were (Agapornis roseicollis) presented with an ulcerated some cutaneous haemorrhages associated with self- mass in the right wing. The bird had been housed mutilation, suggesting a painful process. Whole alone in an indoor metal cage and fed a seed-based body radiographs showed a fracture at the right distal commercial diet. It had suffered a fracture of the right humeral diaphysis, lysis and remodelling of the prox- humerus due to a trauma 2 years previously. At that imal humeral fragment as well as severe osteolysis of time, the owners declined surgical treatment and, the distal fragment, which extended to the proximal therefore, the wing was immobilized with a figure- radial and cubital epiphysis. A round soft tissue opac- of-eight wing bandage combined with wing-body ity at the fracture site and surrounding the elbow was wrap. The fracture healed but a large bony callus also seen (Fig. 1A). formed because bone fragments were not aligned Because of the rapid worsening of the case due to with the humeral axis. self-mutilation, right wing amputation was per- General physical examination was unremarkable formed and samples were submitted for cytological except for the mass in the right wing, which was ulcer- and histological assessment. After removal, cytolog- ical examination of Wright’s-stained impression Correspondence to: J Martorell (e-mail: jaumemiquel.martorell@uab. smears of the mass revealed moderate cellularity cat). and a population of fusiform mesenchymal cells that 0021-9975/$ - see front matter Ó 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the https://doi.org/10.1016/j.jcpa.2022.09.006 CC BY license (http://creativecommons.org/licenses/by/4.0/). 82 I Ruano-Feo et al Fig. 1. Undifferentiated sarcoma, right wing, lovebird. (A) Ventrodorsal radiograph showing fracture of right distal humeral diaphysis, lysis and remodelling of proximal humeral fragment and severe osteolysis of distal fragment, extending to proximal radial and cu- bital epiphyses (arrow). Soft round tissue opacity associated with fracture and elbow joint (star). L, left. (B) Individual fusiform mesenchymal cells have oval nuclei with lacy chromatin and prominent nucleoli. Impression smear. Wright. Bar, 20 mm. were arranged individually or in non-cohesive aggre- bone tissues (Fig. 2A). Neoplastic cells had a fusiform gates, associated with abundant extracellular pink morphology and were arranged in interlacing bun- amorphous material. Nuclei were round to oval dles with a basophilic myxoid matrix. These cells with a finely stippled to lacy chromatin pattern, and were spindle-shaped with moderate pale eosinophilic one to two nucleoli. Cytoplasm was moderately pale cytoplasm and poorly defined cell borders. Nuclei blue with indistinct cell borders. Moreover, spindle- were large, oval, eccentric with coarsely stippled shaped cells had a moderate degree of anisocytosis chromatin and a single nucleolus. There were 1e3 and anisokaryosis. Binucleation, multinucleation mitotic figures in 2.37 mm2 and occasional aberrant and multiple nucleoli were also commonly observed mitoses were observed. Marked pleomorphism was (Fig. 1B). Cytological findings were consistent with seen with moderate anisocytosis and anisokaryosis, a malignant mesenchymal cell neoplasm. as well as scarce multinucleated giant cells. Moreover, Post-surgical recovery was appropriate but soon af- there was an occasional proliferation of well- ter wards the bird became lethargic with wing flap- differentiated epithelial cells organized in either small ping and died suddenly. Unfortunately, post- clusters or tubular structures lined by a simple mortem examination was declined by the owners. cuboidal epithelium. These epithelial cells were large The right wing that had been removed during sur- and polygonal with moderate eosinophilic cytoplasm gery was submitted for histopathology. Grossly, the and well-defined cytoplasmic borders (Fig. 2B). distal humeral diaphysis contained a firm, brownish Nuclei were round and centrally located, with two mass (2 cm diameter), which invaded and effaced evident nucleoli. Multifocally, scattered small foci of the humeroradial joint. Multiple sagittal sections of necrosis were seen. the mass were prepared, fixed in 4% formalin, Immunohistochemical labelling of formalin-fixed routinely processed and stained with haematoxylin paraffin-embedded serial tissue sections was per- and eosin (HE) for histopathological evaluation. In formed to determine the phenotype of the cells. The addition, sagittal sections of the whole joint were de- antibody panel included vimentin 3B4 (M7020; calcified for 48 h in fast decalcifier with saline Dako, www.agilent.com), desmin (M0760; Dako), corrector, fixed in formalin and processed as for histo- smooth muscle actin (SMA) (M0851; Dako), S100 pathological evaluation. (Z0311; Dako), ionized calcium-binding adapter Examination of the samples from the whole joint molecule-1 (IBA-1) (019e19741; Wako, https:// and the associated mass revealed the same histological labchem-wako.fugifilm.com), CD18 (CA1.4E9; pattern, which consisted of a highly cellular non- University of California, Davis, USA), cytokeratin encapsulated and infiltrative neoplastic proliferation AE1/AE3 (PA0909; Leica Biosystems, www. that distorted the adjacent dermal, muscular and leicabiosystems.com) and epithelial membrane Undifferentiated Wing Sarcoma in a Lovebird 83 Fig. 2. Undifferentiated sarcoma, right wing, lovebird. (A) Longitudinal section of right humeral mass. Infiltrative mesenchymal neoplastic cell proliferation associated with abundant myxoid matrix and distortion of adjacent dermal, muscle and bone tissues. HE. Bar, 1 mm. (B) Higher magnification of A. Proliferation of spindloid cells and occasional epithelial cells organized in tubules. HE. Bar, 50 mm. antigen (EMA) GP1.4 (PA0035; Leica Biosystems). tered cells, probably of inflammatory origin were im- Immunohistochemistry (IHC) was performed using munopositive with these markers). We also excluded an Autostainer Plus Immunostaining System (Dako) rhabdomyosarcoma, which is SMA negative and des- with 40 min of primary antibody incubation for des- min positive, and myoepithelial or myofibroblastic min (1:40), SMA (1:500), S100 (1:500), IBA-1 tumours, which are desmin positive (Ramos-Vara (1:250) and CD18 (1:100); manually with 60 min of and Borst, 2017). Lastly, peripheral nerve sheath primary antibody incubation for vimentin (1:300); tumour, which is S100 positive, and fibrosarcoma, and with Leica platform with 15 min of primary anti- which is S100 positive and SMA negative body incubation for cytokeratin ready-to-use (RTU) (Hendrick, 2017), were also discarded as possible di- and EMA RTU. Antigen retrieval was performed agnoses. with high pH for desmin, SMA and vimentin and Because of the location of the tumour and the low pH for S100, IBA-1, CD18, cytokeratin and simultaneous presence of mesenchymal and epithe- EMA. As a control, histological sections of a non- lial polygonal cells, we could hypothesize about the affected wing of a psittacine bird (Amazona albifrons) diagnosis of a biphasic synovial sarcoma. In avian were used including skin, striated muscle, synovial species, synovial cell sarcomas have been reported membrane, cartilage and bone. in a pigeon (Columba livia) (Liu and Moroff, 1993), In the mesenchymal neoplastic population, most of a moluccan cockatoo (Cacatua molucensis) (Kennedy the cells had intense immunoreactivity for vimentin et al, 1994), a sulphur crested cockatoo (Cacatua galer- and about 80% were immunopositive for SMA ita) (Van Der Horst et al, 1996) and a peach-faced (Fig. 3A); occasionally, sparse histiocytic cells were lovebird (Agapornis roseicolis) (Nakano and Une, weakly labelled for CD18 and IBA-1. In the epithelial 2016). On the contrary, in dogs (Fox et al, 2002; cell population, the lining epithelium of the tubules Loukopoulos et al, 2004) and cats (Liptak et al, and some epithelial clusters labelled for cytokeratin 2004; Cazzini et al, 2015) it usually has been (Fig. 3B). No cells express positivity for EMA, desmin described as a monophasic synovial cell sarcoma. or S-100 (Table 1). Despite all the existing literature on synovial cell sar- The neoplasm in the present report was consistent coma in veterinary medicine, this tumour is with a mesenchymal neoplastic proliferation without currently considered not to exist in animals (Craig the presence of osteoid material and with the presence and Thompson, 2017). Furthermore, in our case of small clusters and tubules lined by epithelial cells. the epithelial proliferation did not show any malig- Considering only the mesenchymal neoplastic popu- nant histological features, which indicates that these lation and the results of IHC, which showed vimentin epithelial clusters and follicles are not of neoplastic positivity, we discarded histiocytic sarcoma as a diag- origin (Craig and Thompson, 2017). Therefore, nosis because neoplastic mesenchymal cells were biphasic synovial cell sarcoma was discarded as a dif- CD18 negative and IBA-1 negative (only a few scat- ferential diagnosis. 84 I Ruano-Feo et al Fig. 3. Undifferentiated sarcoma, right wing, lovebird. (A) Neoplastic mesenchymal cells immunopositive for smooth muscle actin. Epithelial cells immunonegative. IHC. Bar, 50 mm. (B) Well-differentiated intratumoural epithelial cells immunopositive for cy- tokeratin. IHC. Bar, 50 mm. Table 1 usually directly related with orthopaedic implants. Immunolabelling pattern of neoplastic cells In avian species, Nakano and Une (2016) described Antigen Mesenchymal cells Epithelial cells a synovial cell sarcoma in a peach-faced lovebird that had been treated with an intramedullary pin af- Vimentin +++  ter a bone fracture. However, the present case is defi- S100   SMA +++  nitely related to a prior bone fracture but without any Desmin   surgical implant. IBA-1   CD18   Cytokeratin  +++ Funding EMA   This research did not receive any specific grant from SMA, smooth muscle actin; IBA-1, ionized calcium-binding adapter funding agencies in the public, commercial or not- molecule1; EMA, epithelial membrane antigen; , no positivity; , for-profit sectors. positivity in <5% of the cells; ++, positivity in >50% of the cells; +++, positivity in >90% of the cells. Acknowledgments The authors thank Dr. Monica Hilbe of the Institute Interestingly, in a case report of a monophasic sy- of Veterinary Pathology of the University of Zurich novial cell sarcoma in the humerus of a pigeon (C. li- for performing the vimentin 3B4 immunohistochem- via) (Liu and Moroff, 1993), the authors described the istry. The authors also thank Blanca Perez and Aida presence of epithelial cells that were consistent with Neira of Servei de Diagnostic de Patologia Veterin a- hyperplasia of humeral air sacs and concluded that ria, Universitat Autonoma de Barcelona for labora- epithelial proliferation might be compatible with en- tory technical assistance and Caroline Idowu for trapped air sacs within the mesenchymal neoplastic revising the English language in the manuscript. growth. Therefore, to the best of the authors’ knowl- edge, the present tumour should be classified as an un- differentiated sarcoma with the presence of Conflict of Interest Statement intralesional epithelial proliferation. The authors declared no potential conflicts of interest Independently of the histological classification of with respect to the research, authorship or publica- this tumour, it is interesting to remark that it was asso- tion of this article. ciated with a previous humeral fracture at the same location without any surgical implant involved. Fracture-associated sarcomas have been reported in References different mammalian species, including dogs (Isaka Baum JI, Skinner OT, Boston SE (2018) Fracture-associ- et al, 2021), cats (Sonnenschein et al, 2012; Baum ated osteosarcoma of the femur in a cat. The Canadian et al, 2018) and goats (Steinberg and George, 1989), Veterinary Journal, 59, 1096e1098. 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