Subcutaneous leiomyosarcoma in a cynomolgus macaque (Macaca fascicularis)

Abstract Leiomyosarcoma, a malignant tumour originating from smooth muscle cells, has rarely been documented in non‐human primates. In this case study, a 7‐year‐old female cynomolgus macaque (Macaca fascicularis) presented with a rapidly growing mass overlying the left elbow joint. Radiographs indicated the presence of a soft tissue neoplasm without any associated bone involvement. The mass was surgically resected. Histological and immunohistochemical analyses revealed spindle‐shaped cells with eosinophilic cytoplasm that resembled smooth muscle cells, exhibiting positive immunoreactions for vimentin, desmin and smooth muscle actin and a negative reaction for pan‐cytokeratin. This is the first reported case of subcutaneous leiomyosarcoma in a cynomolgus macaque and provides important insights into the incidence and characteristics of this condition in this species.


INTRODUCTION
Leiomyosarcoma is a type of malignant tumour derived from smooth muscle cells; it represents a significant clinical challenge owing to its aggressive behaviour and limited treatment options (Ahuja et al., 2017;Gladdy et al., 2013;Sbaraglia et al., 2021).The tumour is distinguished by wide interlacing fascicles composed of spindle-shaped or ovoid neoplastic cells (Miettinen, 2016).These neoplastic cells retain many features characteristic of normal smooth muscle cells, such as elongated nuclei and eosinophilic cytoplasm (Weiss et al., 2019).This type of neoplasm appears relatively frequently in the visceral organs, particularly in the gastrointestinal tract and female reproductive system, This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.© 2024 The Authors.Veterinary Medicine and Science published by John Wiley & Sons Ltd. of humans and most other animals (Birkebak et al., 1996;Cook et al., 2004;George et al., 2018;Meuten, 2020;Weiss et al., 2019).
Despite its relatively high prevalence in the visceral organs of humans and animals, leiomyosarcoma is also known to occur in less common anatomical sites, such as the oral cavity, oesophagus, blood vessels and bladder, as documented in a limited number of studies (Abed et al., 2009;Liu & Mikaelian, 2003;Meuten, 2020;Weiss et al., 2019).Although subcutaneous leiomyosarcoma in animals is rare, it has been reported in some species, such as cows, hamsters, wolves and squirrel monkeys (Bock et al., 2007;Brunnert et al., 1990;de Castro Pires et al., 2017;Hanzaike et al., 1995;Nakamura et al., 2010;Park et al., 2010;Yi et al., 2008).The cynomolgus macaque (Macaca fascicularis), a highly relevant species in both biomedical and zoological research (Gardner & Luciw, 2008;Kaplan & Manuck, 1999;Kaplan et al., 1984;Lin et al., 2009), is known to have relatively few reported tumour cases.Malignancies, such as malignant lymphoma and squamous cell carcinoma, as well as benign tumours, such as leiomyomas and polyps in various organs, have been described in previous reports (Bright et al., 2019;Kaspareit et al., 2007;Schmelting et al., 2011).
Here, we report a case of leiomyosarcoma in the left elbow of a cynomolgus macaque, providing important information regarding the incidence and characteristics of this tumour in the species.

CASE DESCRIPTION
A 7-year-old female cynomolgus macaque, weighing 4.86 kg, with an abnormality in her left elbow was observed at the Primate Resources Center (Jeongeup, South Korea) (Figure 1A).The macaque had normal food intake, faecal condition and vitality.However, a mass developed over her left elbow joint and rapidly increased in size.No signs of necrosis or ulceration were observed on the skin above the mass.
Radiography indicated that the mass had not invaded the elbow joint or nearby bones, and that there was calcification in some portions of the tissue (Figure 1B).
The mass was surgically resected; it was present in the subcutaneous tissue of the left elbow joint and measured 7.5 × 6.0 × 4.5 cm 3 (Figure 2A).The mass was in contact with the skeletal muscles but did not infiltrate them.The cut surface of the mass was solid, appeared white or greyish-white and showed signs of haemorrhage (Figure 2B).Histologically, the tumour exhibited extensive proliferation of eosinophilic smooth muscle bundles.The proliferation of tumour cells exhibited partial density, and neoplastic cells were observed interweaving in various directions, occasionally forming a herringbone pattern (Figure 3A).The proliferation area was represented by smallto medium-sized spindle-shaped cells that formed bundles resembling a smooth muscle.Some of these cells had pleomorphic nuclei, multiple and indistinct nucleoli, and moderate-to-abundant eosinophilic cytoplasm (Figure 3B).The mitotic count within a 2.37 mm 2 field of observation was low, with one mitotic figure.Additionally, focal areas of fibrosis and infiltration of lymphocytic inflammatory cells were observed.
Immunohistochemical analysis revealed strong positive staining for vimentin (approximately 50%-70% positive cells with intense reaction) and for smooth muscle actin (greater than 90% positive cells with intense reaction), as well as positive staining for desmin (approximately 30%-50% positive cells with moderate reaction) in neoplastic cells.
However, it was negative for pan-cytokeratin (Figure 4A-D).
One year has elapsed since the resection of the tumour without any confirmed case of recurrence or metastasis to the removal or other sites, indicating the effectiveness of the surgical procedure employed.

DISCUSSION
Leiomyosarcoma is a malignant mesenchymal tumour characterized by interlacing bundles of smooth muscle fibres (Miettinen, 2016).It predominantly arises in deep soft tissue sites, such as the retroperitoneum; however, its occurrence in superficial soft tissue is uncommon.
Subcutaneous leiomyosarcomas are rare and account for approximately 4%-6.5% of all soft tissue sarcomas in humans (Jena et al., 2014).Similarly, cases of subcutaneous leiomyosarcoma in animals are rare.In humans, subcutaneous leiomyosarcoma tends to have a high rate of recurrence (37%) and metastasis (21%-62%), which makes the prognosis less favourable (Soares et al., 2021).The situation is similar for animals with leiomyosarcoma, as they generally have a poor prognosis and a low chance of long-term survival (Cohen et al., 2003;Kapatkin et al., 1992;Patnaik et al., 1986).
The inclusion of IHC in tumour evaluation significantly contributes to enhancing diagnostic accuracy.
In this case, histologically, within certain regions of welldifferentiated neoplastic cells, neoplastic cells displayed nuclear pleomorphism, variable nuclear sizes and rare mitotic figures, meeting the criteria for malignant diagnosis.Immunoreactivity for vimentin, desmin and smooth muscle actin was observed in the neoplastic cells, indicating a mesenchymal and smooth muscle origin (Hollmig et al., 2012;Leader et al., 1987;Painter et al., 2010), which is suggestive of the possibility of leiomyosarcoma.The absence of a positive reaction to pan-cytokeratin, a marker for tumour cells of epithelial origin (Hollmig et al., 2012;Menz et al., 2022;Painter et al., 2010), further supports the diagnosis of leiomyosarcoma.These findings are also consistent with previous research conducted on both human and animal subjects (Carvalho et al., 2009;Cook et al., 2004;de Castro Pires et al., 2017;Nakamura et al., 2010), thereby ruling out other tumour types, including osteosarcoma, synovial sarcoma, fibrosarcoma, malignant schwannoma and rhabdomyosarcoma.
Together these findings led to a diagnosis of subcutaneous leiomyosarcoma.The tumour was located in the subcutaneous tissue of the elbow and showed the typical features of leiomyosarcoma on histological examination.The use of IHC was critical in distinguishing leiomyosarcoma from other tumours with similar morphological features, enabling an accurate diagnosis.
In macaques, the occurrence of neoplasms in superficial soft tissue is rare; however, there are few reports of liposarcoma, mast cell tumour, basal cell tumour, hemangiosarcoma and melanoma (Doane et al., 2017;Myers et al., 2001;Pellegrini et al., 2009;Tsugo et al., 2017;Yanai et al., 1995).To the best of our knowledge, there have been no reported cases of subcutaneous leiomyosarcoma in any macaque species, with the majority of leiomyosarcomas typically found in deep soft tissues such as the uterus (Birkebak et al., 1996;Cook et al., 2004).
The present case report sheds new light on this subject as it is the first reported case of subcutaneous leiomyosarcoma in a cynomolgus macaque.Moreover, the absence of tumour recurrence or any adverse health effects for more than a year after neoplasm removal underscores the remarkable features of the health status of the animal compared with that in other reported cases of leiomyosarcoma in animals.
Further studies are needed to better understand the aetiology and pathogenesis of leiomyosarcoma in animals.The study was approved by the Institutional Animal Care and Use Committee (IACUC) of the KRIBB (KRIBB-AEC-22028).

F
Gross and radiographic findings of leiomyosarcoma: (a) anterior view of the mass on the left elbow joint and (a) radiographic image of the left elbow.The mass did not invade the elbow joint and nearby bones.

F
Surgical excision of the subcutaneous mass on the left elbow joint: (a) intraoperative photograph showing the surgical incision of the mass using an electric surgical knife and (b) postoperative photograph of a greyish-white or white solid mass with haemorrhage in the subcutaneous tissue, which was located medial to the elbow joint.F I G U R E 3 Histopathological analysis of the subcutaneous mass: (a) low-magnification haematoxylin and eosin (H&E) stained image showing highly cellular neoplastic tissue with pleomorphic cells and an atypical pattern of muscle fibres.Scale, 500 µm.(b) High-magnification H&E-stained image showing individual neoplastic cells, ranging from small and round to large and polygonal, with eosinophilic cytoplasm.A rare mitotic figure is indicated by the arrow.Scale, 50 µm.F I G U R E 4 Immunohistochemical staining of the tumour tissue for vimentin, desmin, smooth muscle actin and pan-cytokeratin: (a) Vimentin immunostaining revealed strong positive staining in neoplastic cells, indicating mesenchymal differentiation; (b) desmin immunostaining demonstrated positive staining in neoplastic cells, indicating muscle differentiation; (c) smooth muscle actin immunostaining exhibited strong positive staining in neoplastic cells, suggesting smooth muscle differentiation; and (d) pan-cytokeratin immunostaining showing negative staining, indicating the absence of epithelial differentiation.Scale, 100 µm.