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Granular Cell Tumour in a California Kingsnake (Lampropeltis californiae)
J. Comp. Path. 2020, Vol. 175, 24e28
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DISEASE IN WILDLIFE OR EXOTIC SPECIES
Granular Cell Tumour in a California Kingsnake (Lampropeltis californiae) M. Reifinger*, N. Dinhopl*, M. Gumpenberger†, M. Konecny‡ and P. Cigler‡ * Institute of Pathology, † Diagnostic Imaging Division and ‡ Department of Birds and Reptiles, University of Veterinary Medicine, Vienna, Austria
Summary A 21-year-old female California kingsnake (Lampropeltis californiae) was presented to the University of Veterinary Medicine, Vienna, Austria, with a space-occupying mass in the caudal abdomen. Following clinical, radiological and sonographical evaluation the mass was removed surgically. Histopathology and transmission electron microscopy confirmed the diagnosis of a granular cell tumour, but immunohistochemical labelling for a range of markers was negative. This lesion is rare in mammals and birds, but has not been reported previously in a reptile. Ó 2019 Elsevier Ltd. All rights reserved. Keywords: granular cell tumour; immunohistochemistry; reptile; snake
Granular cell tumours (GCTs) are rare in animals (Robinson and Robinson, 2006) and people (Brown et al., 2015). Initially thought to arise from myoblasts (Abrikossoff, 1926), leading to the term ‘granular cell myoblastoma’, investigations by immunohistology and electron microscopy pointed to a Schwann cell origin (Fletcher, 2007; Caswell and Williams, 2016; Cooper and Valentine, 2016). Nevertheless, a proportion of cases appears to have a different histogenesis (Ordonez, 1999; Becker et al., 2013). In fact, GCT is a descriptive name for a tumour that may be derived from different progenitor cells (Patnaik, 1993; Ordonez, 1999; Becker et al., 2013; Munday et al., 2017). A 21-year-old, female California kingsnake (Lampropeltis californiae) was presented to the Department of Birds and Reptiles, University of Veterinary Medicine, Vienna, Austria. The snake was captive bred and had been owned by the same individual since it was a hatchling. Husbandry was adequate, the activCorrespondence to: M. Reifinger (e-mail: [email protected]. at). 0021-9975/$ - see front matter https://doi.org/10.1016/j.jcpa.2019.11.003
ity of the snake was normal and there was no prior history of health problems. Clinical examination revealed the snake to be in good bodily condition, 190 cm long and 571 g in body weight. In the caudal half of the body there was a 15 cm swelling that was soft on palpation and non-movable. It did not seem to cause any discomfort. Dorsoventral and laterolateral radiographs were taken. An oval mass was visible, caudal to the caudal end of the lung field (Fig. 1). It was well demarcated, homogeneous, soft tissue dense and not associated with the empty gastrointestinal tract. To gain further information ultrasonography was performed using an 8e12 MHz microconvex transducer. The mass was not associated with any coelomic organ. Within the mass, cysts occupied nearly a third of the volume; these had a maximum diameter of 18e23 mm. A wall of moderate echogenicity with variable diameter (1.4e3.1 mm) and irregular inner surface was noted. The imaging findings were suggestive of an abdominal tumour, but an abscess was not excluded. The snake was premedicated with 0.5 mg/kg hydromorphone hydrochloride (HydalÒ; intramuscular Ó 2019 Elsevier Ltd. All rights reserved.
Granular Cell Tumour in a Kingsnake
25
Fig. 1. Laterolateral radiograph showing an oval mass (the tumour) in the coelomic cavity. Bar, 5 cm.
injection; Fidelio Healthcare, Limburg, Germany) and anaesthesia was induced with 7 mg/kg alfaxalone (AlfaxanÒ; intravenous injection; Jurox Inc., Kansas City, Kansas, USA). Maintenance was with 1.5% isoflurane (VetfluraneÒ, Virbac Inc., Carros, France) and 1 L/min oxygen flow. A 15 cm skin incision was made on the right flank above the swelling, and the mass was carefully exposed. It was soft and dark blue to light pink in colour. The mass had no connection with the intestinal tract or any other organ. After ligation of three larger vessels the serosal suspension was cut and the mass was removed. The wound was closed by separate sutures of the coelomic membrane, the body wall and the skin. Following surgery, the snake recovered quickly and was returned to the owner’s care after 6 days. After approximately 2 weeks the snake was recovering well, eating and showing no further clinical signs. The tumour was fixed for 24 h in 4% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (4 mm) were stained with haematoxylin and eosin (HE), periodic acideSchiff (PAS), PAS following diastase digestion and Luxol fast blue. Immunhistochemistry (IHC) was performed by the UltraVision LP Detection System HRP Polymer (Fisher Scientific, Vienna, Austria). Primary antibodies against S100 protein (Dako, Glostrup, Denmark), glial fibrillary acidic protein (GFAP; Dako), CD68 (Dako), vimentin (BioGenex, Fremont, California, USA), desmin (Neomarkers, Fremont, California, USA) and actin (Dako) were used. For transmission electron microscopy (TEM) the tumour was cut into 1 mm3 sections and fixed in 5% glutaraldehyde (Merck, Darmstadt, Germany) in 0.1 M phosphate buffer (SigmaeAldrich, Vienna, Austria), pH 7.2, at 4 C for 3 h. Subsequently, samples were post-fixed in 1% osmium tetroxide (Merck) in the same buffer at 4 C for 2 h. After dehydration in
an alcohol gradient series and propylene oxide (Merck), the tissue samples were embedded in glycid ether 100 (Serva, Heidelberg, Germany). Ultrathin sections were cut on a Leica ultramicrotome (Leica Ultracut S, Vienna, Austria), stained with uranyl acetate (SigmaeAldrich) and lead citrate (Merck) and examined with a Zeiss TEM 900 electron microscope (Carl Zeiss, Oberkochen, Germany) operated at 50 kV. Grossly, the mass was spindle shaped, 14 3 3 cm in size, with a smooth surface and a longitudinal, artificial incision exposing a large, longitudinal cavity. It was of firm consistency and predominantly of pale colour. Transection showed the cavity to be delimited by a 0.5e0.8 cm wall of solid white tissue. Histologically, the appearance was of a solid tumour of mesenchymal character with extensive central necrosis and purulent liquefaction. The viable tissue showed some spindle-shaped, fibrocyte-like cells, but the majority of the cells were densely packed, large, swollen, round to polyhedral, histiocyte-like cells with bright, granular to foamy cytoplasm and clear cell borders (Fig. 2A). Some regions showed marked anisocytosis and anisokaryosis (Fig. 2B) as well as binucleation or multiple nuclei. The mitotic count (MC) in these areas was five mitoses per 10 high-power ( 400) fields. The granules were stained by PAS and Luxol fast blue and were diastase resistant (Fig. 2C). The intensity of staining tended to be reduced in the largest, polyhedral cells. IHC for S100, GFAP, CD68, actin, desmin and vimentin was all negative. Positive controls were with dog tissue for CD68 (lung), actin (muscle), desmin (muscle) and vimentin (fibrosarcoma) and cat tissue for S100 (lung) and GFAP (cerebellum). Electron microscopical evaluation of the tumour cells revealed multiple, mid-sized to large heterogeneous, spherical organelles (diameter 0.3e2 mm) of variable electron density, which were bordered by a
26
M. Reifinger et al.
Fig. 2. (A) Large, round cells of varying size and with distinct cell borders. HE. Bar, 40 mm. (B) Area of irregular cell morphology with some macronuclei (arrowheads). HE. Bar, 40 mm. (C) PAS-positive red granules in the tumour cells (arrowheads) after diastase digestion. Bar, 40 mm.
double or unit membrane (Fig. 3). These organelles were identified as phagolysosomes. The tumour showed typical histological and ultrastructural features of GCT. The observed granulation represented secondary lysosomes by TEM. IHC of GCT in published cases is variable, resulting in controversy concerning histogenesis (Patnaik, 1993; Sato et al., 2004; Mandara et al., 2006; Hernandez et al., 2012). There is inconsistent expression of S100, GFAP, NSE, CD68, actin, vimentin, desmin
Fig. 3. Three tumour cells densely packed with phagolysosomes of varying size (arrowheads). Bar, 1,000 nm.
and other markers (Patnaik, 1993; Yoshida et al., 1997; Mandara et al., 2006; Fletcher, 2007; Sunkara et al., 2017), and IHC is not pathognomonic for GCT (Geyer et al., 1992; Patnaik, 1993). In fact, the tumour name is descriptive and does not imply a certain cell origin or line of differentiation (Munday et al., 2017). In the present case, attempts to detect specific antigens were unsuccessful. No labelling for S100 and GFAP (glial and Schwann cells), CD68 (lysosomes, macrophages), actin and desmin (striated and smooth muscle) or vimentin (mesenchymal cells) was identified. This could be due to problems detecting reptilian antigens with mammalian antibodies; however, desmin and actin showed strong positivity in the vessel walls of the tumour. The fact that those two antibodies produced reliable results for blood vessels, but negative labelling of tumour cells, makes a myoid origin unlikely. Regarding the biological behaviour of the tumour, six criteria have been established for malignancy and prognosis of human GCT (Fanburg-Smith et al., 1998): cell necrosis, spindle-shaped morphology of tumour cells, pleomorphism, increased mitotic activity (>2 mitoses per 10 200 fields), vesicular nuclei with large nucleoli, and a high nuclear to cytoplasmic
27
Granular Cell Tumour in a Kingsnake
ratio. Human GCTs are classified as malignant if they meet three or more of these criteria. In the present case, cell necrosis, spindle-shaped morphology, pleomorphism and compatible mitotic activity were present, indicating a malignant variant of GCT. According to the literature, most cases of GCT are benign (Munday et al., 2017; Sunkara et al., 2017), so surgery is generally curative (Munday et al., 2017). In a small number of cases (1e2% in man; Sunkara et al., 2017) GCTs have malignant morphology and may have a fatal outcome (Khansur et al., 1987). GCTs have been reported in man, domesticated and laboratory mammals (including the horse, dog, cat, ferret, rabbit, rat, mouse, guinea pig and djungarian hamster [ Phodopus sungorus]) and birds (Australian parakeet [Melopsittacus undulatus] and Puerto Rican Amazon parrot [Amazona vittata]), where they have been found in various locations. In man, 70 % of GCTs are located in the head and neck (Becker et al., 2013), particularly affecting the tongue (Enzinger and Weiss, 1988), but they can be found in any part of the body (Sunkara et al., 2017). Equine GCTs are found exclusively in the lung (arising from peribronchial tissue) and are often multiple. GCT is the most common primary lung tumour of the horse (Dungworth et al., 1999; Caswell and Williams, 2016). In the dog and cat, GCTs are found mostly in the oral cavity, particularly affecting the tongue, but have also been reported in the lung, pharynx, brain (Higgins et al., 2001; Liu et al., 2004; Mandara et al., 2006), vulva and skin (Patnaik, 1993). In the ferret a cerebral localization (Sleeman et al., 1996) and in a rabbit a testicular localization is described (Irizarry-Rovira et al., 2008). Meningeal GCTs in rats were linked histogenetically with arachnoid cells (Mitsumori et al., 1987; Yoshida et al., 1997) and in the uterus of a rat a neuroectodermal origin was suspected (Toyosawa et al., 1997). In laboratory mice, GCTs are found predominantly in the uterus and less often in the subcutis (Miyajima et al., 2001). A cutaneous location was described in a guinea pig (Willmes et al., 2013), while the uterus und subcutis are known sites in the djungarian hamster (Sato et al., 2004; Golbar et al., 2011). In two Australian parakeets, GCTs were found in the periocular region (Patnaik, 1993) and in the region of the metacarpus, respectively (Hernandez et al., 2012), the latter region being the same site of origin as a GCT in a Puerto Rican Amazon parrot (Quist et al., 1999). In the present case the tumour was located in the coelomic cavity with no direct connection to any organ. Consequently, it may have developed from the subserosal soft tissue, which includes nerves, but not muscle tissue. However, we were not able to identify the cellular origin of the GCT by IHC.
Numerous different neoplasms have been reported in chelonians, crocodiles, lizards and snakes (Frye, 1991; Done, 1996; Ramsay et al., 1996; Catao-Dias and Nichols, 1999; Sassenburg and Zwart, 2005; Zwart and Sassenburg, 2005a,b; Mauldin and Done, 2006; Mitchell, 2008). In one case the term ‘granular cell tumour’ was used in association with a snake (Mitchell, 2008), but in that case GCT appears to have been confused with ‘granulosa cell tumour’. The present case is therefore the first documented GCT in a reptile.
Acknowledgments The authors thank G. Haberl, M. G€artner-Horvath and K. Fragner for histological staining, P. Kodajova and N. Nedorost for IHC and G. Borka for radiography.
Conflict of Interest Statement The authors declare no conflict of interest with respect to the publication of this manuscript.
References € Abrikossoff A (1926) Uber Myome, ausgehend von der quergestreiften willk€ urlichen Muskulatur. Virchows Archiv f€ur Pathologische Anatomie und Physiologie und f€ur Klinische Medizin, 260, 215e233. Becker C, Maier W, Lutz L, Pfeiffer J (2013) N€ urnberg, Granularzelltumor - eine seltene aber relevante Differentialdiagnose einer zervikalen Raumforderung. 84. Jahresversammlung der Deutschen Gesellschaft f€ur HNO-Heilkunde Kopf- und Hals-Chirurgie, Vol. 08.-12.05.2013. Brown KG, Rao PJ, Oo TH, Fowler A (2015) Granular cell tumors of the spinal canal: intramedullary case report and a review of the literature. Journal of Spine Surgery, 1, 94e98. Caswell JL, Williams KJ (2016) Respiratory system. In: Jubb, Kennedy and Palmer’s Pathology of Domestic Animals, 6th Edit., Vol. 2, MG Maxie, Ed., Elsevier, St. Louis, pp. 465e591. Catao-Dias JL, Nichols DK (1999) Neoplasia in snakes at the national zoological park, Washington, DC (1978e1997). Journal of Comparative Pathology, 120, 89e95. Cooper BJ, Valentine BA (2016) Muscle and tendon. In: Jubb, Kennedy and Palmer’s Pathology of Domestic Animals, 6th Edit., Vol. 3, MG Maxie, Ed., Elsevier, St. Louis, pp. 164e249. Done LB (1996) Neoplasia. In: Reptile Medicine and Surgery, DR Mader, Ed., Saunders, Philadelphia, pp. 125e141. Dungworth DL, Hauser B, Hahn FF, Wilson DW, Haenichen T et al. (1999) Histological classification of tumors of the respiratory system of domestic animals. In: WHO International Histological Classification of Tumors of Domestic Animals, 2nd Series Vol. VI Armed Forces Institute of Pathology, Washington DC, pp. 1e71.
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Enzinger FM, Weiss SW (1988) Benign tumors of peripheral nerve: granular cell tumor. In: Soft Tissue Tumors, 3rd Edit., FM Enzinger, SW Weiss, Eds., Mosby, St. Louis, pp. 864e871. Fanburg-Smith JC, Meis-Kindblom JM, Fante R, Kindblom LG (1998) Malignant granular cell tumor of soft tissue. Diagnostic criteria and clinicopathologic correlation. The American Journal of Surgical Pathology, 22, 779e794. Fletcher CDM (2007) Peripheral neuroectodermal tumors. In: Diagnostic Histopathology of Tumours, 3rd Edit., CDM Fletcher, Ed., Elsevier, St. Louis, pp. 1733e1761. Frye FL (1991) Biomedical and Surgical Aspects of Captive Reptile Husbandry, 2nd Edit., Krieger, Malabar, pp. 576e622. Geyer C, Hafner A, Pfleghaar S, Hermanns W (1992) Immunohistochemical and ultrastructural investigation of granular cell tumors in dog, cat, and horse. Zentralblatt f€ur Veterinarmedizin B, 39, 485e494. Golbar HM, Izawa T, Kuwamura M, Okamura K, Fujita D et al. (2011) A collision tumor consisting of granular cell tumor and adenocarcinoma in the uterus of an aged tsungarian hamster. Journal of Toxicologic Pathology, 24, 233e237. Hernandez V, Carrera E, Mendez A, Morales JC, Morales E et al. (2012) Histopathologic, immunohistochemical and ultrastructural features of a granular cell tumour in an Australian parakeet (Melopsittacus undulatus). Avian Pathology, 41, 437e440. Higgins RJ, LeCouter RA, Vernau KM, Sturges BK, Obradovic JE et al. (2001) Granular cell tumor in the canine central nervous system: two cases. Veterinary Pathology, 38, 620e627. Irizarry-Rovira AR, Lennox AM, Ramos-Vara JA (2008) Granular cell tumor in the testis of a rabbit: cytologic, histologic, immunohistochemical, and electron microscopic characterization. Veterinary Pathology, 45, 73e77. Khansur T, Balducci L, Tavassoli M (1987) Granular cell tumor. Clinical spectrum of the benign and malignant entity. Cancer, 60, 220e222. Liu CH, Liu CL, Liang SL, Cheng CH, Huang SC et al. (2004) Intracranial granular cell tumor in a dog. Journal of Veterinary Medical Science, 66, 77e79. Mandara MT, Ricci G, Sforna M (2006) A cerebral granular cell tumor in a cat. Veterinary Pathology, 43, 797e800. Mauldin GN, Done LB (2006) Oncology. In: Reptile Medicine and Surgery, 2nd Edit., DR Mader, Ed., WB Saunders, St. Louis, pp. 299e322. Mitchell MA (2008) Snakes. In: Manual of Exotic Pet Practice, MA Mitchell, TN Tully, Eds., Saunders Elsevier, St. Louis, pp. 136e163. Mitsumori K, Dittrich KL, Stefanski S, Talley FA, Maronput RR (1987) Immunohistochemical and electron microscopic study of meningeal granular cell tumors in rats. Veterinary Pathology, 24, 356e359. Miyajima R, Hasegawa K, Yasui Y, Kihara T, Hosoi M et al. (2001) Nine cases of granular cell tumors in B6C3F1 mice. Journal of Veterinary Medical Science, 63, 449e452.
Munday JS, L€ ohr CV, Kiupel M (2017) Tumors of the alimentary tract. In: Tumors in Domestic Animals, 5th Edit., DJ Meuten, Ed., Wiley Blackwell, Ames, pp. 499e601. Ordonez NG (1999) Granular cell tumor: a review and update. Advances in Anatomic Pathology, 6, 186e203. Patnaik AK (1993) Histologic and immunohistochemical studies of granular cell tumors in seven dogs, three cats, one horse and one bird. Veterinary Pathology, 30, 176e185. Quist CF, Latimer KS, Goldade SL, Rivera A, Dein FJ (1999) Granular cell tumour in an endangered Puerto Rican Amazon parrot (Amazona vittata). Avian Pathology, 28, 345e348. Ramsay EC, Munson L, Lowenstine L, Fowler ME (1996) A retrospective study of neoplasia in a collection of captive snakes. Journal of Zoo and Wildlife Medicine, 27, 28e34. Robinson WF, Robinson NA (2006) Cardiovascular system. In: Jubb, Kennedy and Palmer’s Pathology of Domestic Animals, 6th Edit., Vol. 3, MG Maxie, Ed., Elsevier, St. Louis, pp. 1e101. Sassenburg L, Zwart P (2005) Schildkr€ oten. In: Krankheiten der Heimtiere, 6th Edit., K Gabrisch, P Zwart, Eds., Schl€ utersche, Hannover, pp. 653e738. Sato T, Takada M, Shibuya H, Shirai W, Matsuo K (2004) A malar granular cell tumor in a djungarian hamster. Journal of Veterinary Medicine A, Physiology, Pathology and Clinical Medicine, 51, 23e26. Sleeman JM, Clyde VL, Brenneman KA (1996) Granular cell tumour in the central nervous system of a ferret (Mustela putorius furo). The Veterinary Record, 138, 65e66. Sunkara T, Nagaraj SV, Gaduputi V (2017) Granular cell tumor of rectum: a very rare entity. Case Reports in Gastrointestinal Medicine, 2017, 3795482. Toyosawa K, Okimoto K, Koujitani T, Kikawa E, Irie M et al. (1997) Granular cell tumors of the female reproductive tract in Wistar rats. Journal of Toxicologic Pathology, 10, 63e65. Willmes A, Leuw ND, Wohlsein P (2013) Fallbericht: kutaner Granularzelltumor bei einem Meerschweinchen. Der Praktische Tierarzt, 94, 198e205. Yoshida T, Mitsumori K, Harada T, Maita K (1997) Morphological and ultrastructural study of the histogenesis of meningeal granular cell tumors in rats. Journal of Toxicologic Pathology, 25, 211e216. Zwart P, Sassenburg L (2005a) Schlangen. In: Kranheiten der Heimtiere, 6th Edit., K Gabrisc, P Zwart, Eds., Schl€ utersche, Hannover, pp. 739e793. Zwart P, Sassenburg L (2005b) Echsen. In: Krankheiten der uterHeimtiere, 6th Edit., K Gabrisch, P Zwart, Eds., Schl€ sche, Hannover, pp. 795e847.
July 12th, 2019 ½ Received, Accepted, November 12th, 2019
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DISEASE IN WILDLIFE OR EXOTIC SPECIES
Granular Cell Tumour in a California Kingsnake (Lampropeltis californiae) M. Reifinger*, N. Dinhopl*, M. Gumpenberger†, M. Konecny‡ and P. Cigler‡ * Institute of Pathology, † Diagnostic Imaging Division and ‡ Department of Birds and Reptiles, University of Veterinary Medicine, Vienna, Austria
Summary A 21-year-old female California kingsnake (Lampropeltis californiae) was presented to the University of Veterinary Medicine, Vienna, Austria, with a space-occupying mass in the caudal abdomen. Following clinical, radiological and sonographical evaluation the mass was removed surgically. Histopathology and transmission electron microscopy confirmed the diagnosis of a granular cell tumour, but immunohistochemical labelling for a range of markers was negative. This lesion is rare in mammals and birds, but has not been reported previously in a reptile. Ó 2019 Elsevier Ltd. All rights reserved. Keywords: granular cell tumour; immunohistochemistry; reptile; snake
Granular cell tumours (GCTs) are rare in animals (Robinson and Robinson, 2006) and people (Brown et al., 2015). Initially thought to arise from myoblasts (Abrikossoff, 1926), leading to the term ‘granular cell myoblastoma’, investigations by immunohistology and electron microscopy pointed to a Schwann cell origin (Fletcher, 2007; Caswell and Williams, 2016; Cooper and Valentine, 2016). Nevertheless, a proportion of cases appears to have a different histogenesis (Ordonez, 1999; Becker et al., 2013). In fact, GCT is a descriptive name for a tumour that may be derived from different progenitor cells (Patnaik, 1993; Ordonez, 1999; Becker et al., 2013; Munday et al., 2017). A 21-year-old, female California kingsnake (Lampropeltis californiae) was presented to the Department of Birds and Reptiles, University of Veterinary Medicine, Vienna, Austria. The snake was captive bred and had been owned by the same individual since it was a hatchling. Husbandry was adequate, the activCorrespondence to: M. Reifinger (e-mail: [email protected]. at). 0021-9975/$ - see front matter https://doi.org/10.1016/j.jcpa.2019.11.003
ity of the snake was normal and there was no prior history of health problems. Clinical examination revealed the snake to be in good bodily condition, 190 cm long and 571 g in body weight. In the caudal half of the body there was a 15 cm swelling that was soft on palpation and non-movable. It did not seem to cause any discomfort. Dorsoventral and laterolateral radiographs were taken. An oval mass was visible, caudal to the caudal end of the lung field (Fig. 1). It was well demarcated, homogeneous, soft tissue dense and not associated with the empty gastrointestinal tract. To gain further information ultrasonography was performed using an 8e12 MHz microconvex transducer. The mass was not associated with any coelomic organ. Within the mass, cysts occupied nearly a third of the volume; these had a maximum diameter of 18e23 mm. A wall of moderate echogenicity with variable diameter (1.4e3.1 mm) and irregular inner surface was noted. The imaging findings were suggestive of an abdominal tumour, but an abscess was not excluded. The snake was premedicated with 0.5 mg/kg hydromorphone hydrochloride (HydalÒ; intramuscular Ó 2019 Elsevier Ltd. All rights reserved.
Granular Cell Tumour in a Kingsnake
25
Fig. 1. Laterolateral radiograph showing an oval mass (the tumour) in the coelomic cavity. Bar, 5 cm.
injection; Fidelio Healthcare, Limburg, Germany) and anaesthesia was induced with 7 mg/kg alfaxalone (AlfaxanÒ; intravenous injection; Jurox Inc., Kansas City, Kansas, USA). Maintenance was with 1.5% isoflurane (VetfluraneÒ, Virbac Inc., Carros, France) and 1 L/min oxygen flow. A 15 cm skin incision was made on the right flank above the swelling, and the mass was carefully exposed. It was soft and dark blue to light pink in colour. The mass had no connection with the intestinal tract or any other organ. After ligation of three larger vessels the serosal suspension was cut and the mass was removed. The wound was closed by separate sutures of the coelomic membrane, the body wall and the skin. Following surgery, the snake recovered quickly and was returned to the owner’s care after 6 days. After approximately 2 weeks the snake was recovering well, eating and showing no further clinical signs. The tumour was fixed for 24 h in 4% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (4 mm) were stained with haematoxylin and eosin (HE), periodic acideSchiff (PAS), PAS following diastase digestion and Luxol fast blue. Immunhistochemistry (IHC) was performed by the UltraVision LP Detection System HRP Polymer (Fisher Scientific, Vienna, Austria). Primary antibodies against S100 protein (Dako, Glostrup, Denmark), glial fibrillary acidic protein (GFAP; Dako), CD68 (Dako), vimentin (BioGenex, Fremont, California, USA), desmin (Neomarkers, Fremont, California, USA) and actin (Dako) were used. For transmission electron microscopy (TEM) the tumour was cut into 1 mm3 sections and fixed in 5% glutaraldehyde (Merck, Darmstadt, Germany) in 0.1 M phosphate buffer (SigmaeAldrich, Vienna, Austria), pH 7.2, at 4 C for 3 h. Subsequently, samples were post-fixed in 1% osmium tetroxide (Merck) in the same buffer at 4 C for 2 h. After dehydration in
an alcohol gradient series and propylene oxide (Merck), the tissue samples were embedded in glycid ether 100 (Serva, Heidelberg, Germany). Ultrathin sections were cut on a Leica ultramicrotome (Leica Ultracut S, Vienna, Austria), stained with uranyl acetate (SigmaeAldrich) and lead citrate (Merck) and examined with a Zeiss TEM 900 electron microscope (Carl Zeiss, Oberkochen, Germany) operated at 50 kV. Grossly, the mass was spindle shaped, 14 3 3 cm in size, with a smooth surface and a longitudinal, artificial incision exposing a large, longitudinal cavity. It was of firm consistency and predominantly of pale colour. Transection showed the cavity to be delimited by a 0.5e0.8 cm wall of solid white tissue. Histologically, the appearance was of a solid tumour of mesenchymal character with extensive central necrosis and purulent liquefaction. The viable tissue showed some spindle-shaped, fibrocyte-like cells, but the majority of the cells were densely packed, large, swollen, round to polyhedral, histiocyte-like cells with bright, granular to foamy cytoplasm and clear cell borders (Fig. 2A). Some regions showed marked anisocytosis and anisokaryosis (Fig. 2B) as well as binucleation or multiple nuclei. The mitotic count (MC) in these areas was five mitoses per 10 high-power ( 400) fields. The granules were stained by PAS and Luxol fast blue and were diastase resistant (Fig. 2C). The intensity of staining tended to be reduced in the largest, polyhedral cells. IHC for S100, GFAP, CD68, actin, desmin and vimentin was all negative. Positive controls were with dog tissue for CD68 (lung), actin (muscle), desmin (muscle) and vimentin (fibrosarcoma) and cat tissue for S100 (lung) and GFAP (cerebellum). Electron microscopical evaluation of the tumour cells revealed multiple, mid-sized to large heterogeneous, spherical organelles (diameter 0.3e2 mm) of variable electron density, which were bordered by a
26
M. Reifinger et al.
Fig. 2. (A) Large, round cells of varying size and with distinct cell borders. HE. Bar, 40 mm. (B) Area of irregular cell morphology with some macronuclei (arrowheads). HE. Bar, 40 mm. (C) PAS-positive red granules in the tumour cells (arrowheads) after diastase digestion. Bar, 40 mm.
double or unit membrane (Fig. 3). These organelles were identified as phagolysosomes. The tumour showed typical histological and ultrastructural features of GCT. The observed granulation represented secondary lysosomes by TEM. IHC of GCT in published cases is variable, resulting in controversy concerning histogenesis (Patnaik, 1993; Sato et al., 2004; Mandara et al., 2006; Hernandez et al., 2012). There is inconsistent expression of S100, GFAP, NSE, CD68, actin, vimentin, desmin
Fig. 3. Three tumour cells densely packed with phagolysosomes of varying size (arrowheads). Bar, 1,000 nm.
and other markers (Patnaik, 1993; Yoshida et al., 1997; Mandara et al., 2006; Fletcher, 2007; Sunkara et al., 2017), and IHC is not pathognomonic for GCT (Geyer et al., 1992; Patnaik, 1993). In fact, the tumour name is descriptive and does not imply a certain cell origin or line of differentiation (Munday et al., 2017). In the present case, attempts to detect specific antigens were unsuccessful. No labelling for S100 and GFAP (glial and Schwann cells), CD68 (lysosomes, macrophages), actin and desmin (striated and smooth muscle) or vimentin (mesenchymal cells) was identified. This could be due to problems detecting reptilian antigens with mammalian antibodies; however, desmin and actin showed strong positivity in the vessel walls of the tumour. The fact that those two antibodies produced reliable results for blood vessels, but negative labelling of tumour cells, makes a myoid origin unlikely. Regarding the biological behaviour of the tumour, six criteria have been established for malignancy and prognosis of human GCT (Fanburg-Smith et al., 1998): cell necrosis, spindle-shaped morphology of tumour cells, pleomorphism, increased mitotic activity (>2 mitoses per 10 200 fields), vesicular nuclei with large nucleoli, and a high nuclear to cytoplasmic
27
Granular Cell Tumour in a Kingsnake
ratio. Human GCTs are classified as malignant if they meet three or more of these criteria. In the present case, cell necrosis, spindle-shaped morphology, pleomorphism and compatible mitotic activity were present, indicating a malignant variant of GCT. According to the literature, most cases of GCT are benign (Munday et al., 2017; Sunkara et al., 2017), so surgery is generally curative (Munday et al., 2017). In a small number of cases (1e2% in man; Sunkara et al., 2017) GCTs have malignant morphology and may have a fatal outcome (Khansur et al., 1987). GCTs have been reported in man, domesticated and laboratory mammals (including the horse, dog, cat, ferret, rabbit, rat, mouse, guinea pig and djungarian hamster [ Phodopus sungorus]) and birds (Australian parakeet [Melopsittacus undulatus] and Puerto Rican Amazon parrot [Amazona vittata]), where they have been found in various locations. In man, 70 % of GCTs are located in the head and neck (Becker et al., 2013), particularly affecting the tongue (Enzinger and Weiss, 1988), but they can be found in any part of the body (Sunkara et al., 2017). Equine GCTs are found exclusively in the lung (arising from peribronchial tissue) and are often multiple. GCT is the most common primary lung tumour of the horse (Dungworth et al., 1999; Caswell and Williams, 2016). In the dog and cat, GCTs are found mostly in the oral cavity, particularly affecting the tongue, but have also been reported in the lung, pharynx, brain (Higgins et al., 2001; Liu et al., 2004; Mandara et al., 2006), vulva and skin (Patnaik, 1993). In the ferret a cerebral localization (Sleeman et al., 1996) and in a rabbit a testicular localization is described (Irizarry-Rovira et al., 2008). Meningeal GCTs in rats were linked histogenetically with arachnoid cells (Mitsumori et al., 1987; Yoshida et al., 1997) and in the uterus of a rat a neuroectodermal origin was suspected (Toyosawa et al., 1997). In laboratory mice, GCTs are found predominantly in the uterus and less often in the subcutis (Miyajima et al., 2001). A cutaneous location was described in a guinea pig (Willmes et al., 2013), while the uterus und subcutis are known sites in the djungarian hamster (Sato et al., 2004; Golbar et al., 2011). In two Australian parakeets, GCTs were found in the periocular region (Patnaik, 1993) and in the region of the metacarpus, respectively (Hernandez et al., 2012), the latter region being the same site of origin as a GCT in a Puerto Rican Amazon parrot (Quist et al., 1999). In the present case the tumour was located in the coelomic cavity with no direct connection to any organ. Consequently, it may have developed from the subserosal soft tissue, which includes nerves, but not muscle tissue. However, we were not able to identify the cellular origin of the GCT by IHC.
Numerous different neoplasms have been reported in chelonians, crocodiles, lizards and snakes (Frye, 1991; Done, 1996; Ramsay et al., 1996; Catao-Dias and Nichols, 1999; Sassenburg and Zwart, 2005; Zwart and Sassenburg, 2005a,b; Mauldin and Done, 2006; Mitchell, 2008). In one case the term ‘granular cell tumour’ was used in association with a snake (Mitchell, 2008), but in that case GCT appears to have been confused with ‘granulosa cell tumour’. The present case is therefore the first documented GCT in a reptile.
Acknowledgments The authors thank G. Haberl, M. G€artner-Horvath and K. Fragner for histological staining, P. Kodajova and N. Nedorost for IHC and G. Borka for radiography.
Conflict of Interest Statement The authors declare no conflict of interest with respect to the publication of this manuscript.
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July 12th, 2019 ½ Received, Accepted, November 12th, 2019