- Email: [email protected]
Cytological, Immunohistochemical and Mutational Analysis of a Gastric Gastrointestinal Stromal Tumour in a Cat
J. Comp. Path. 2011, Vol. 145, 152e157
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
NEOPLASTIC DISEASE
Cytological, Immunohistochemical and Mutational Analysis of a Gastric Gastrointestinal Stromal Tumour in a Cat M. Morini*, F. Gentilini†, M. Pietra†, A. Spadari†, M. E. Turba‡, L. Mandrioli* and G. Bettini* * Department of Veterinary Public Health and Animal Pathology, † Veterinary Clinical Department, Faculty of Veterinary Medicine, Alma Mater Studiorum, University of Bologna, Ozzano Emilia and ‡ Veterinary Practitioner, GeneFast, Castelnuovo Rangone, Modena, Italy
Summary Gastrointestinal stromal tumours (GISTs) represent a distinctive group of primary mesenchymal tumours of the gastrointestinal tract identified immunohistochemically by expression of CD117. A 10-year-old neutered female domestic shorthair cat with a history of recurrent vomiting was examined. The presence of a gastric mass was recognized and a laparotomy was performed. Cytological examination was consistent with a lowgrade malignant mesenchymal tumour and histopathological investigation suggested myogenic differentiation of tumour cells. The diagnosis of GIST was confirmed by immunohistochemical expression of CD117. Sequence analysis of the KIT gene identified a deletion in exon 11. The same mutation is found often in human GISTs. Ó 2010 Elsevier Ltd. All rights reserved. Keywords: cat; CD117; gastrointestinal stromal tumour; KIT
Primary non-lymphoid gastric tumours are rare in cats (Withrow and MacEwen, 1996; Head et al., 2002; Brown et al., 2007). Adenocarcinomas are the most frequently reported of these (Turk et al., 1981; Withrow and MacEwen, 1996) and single cases have been reported of gastric mast cell tumour, extramedullary plasmacytoma, carcinoid and hamartoma (Bortnowski and Rosenthal, 1992; Zikes et al., 1998; Rossmeisl et al., 2002; Smith et al., 2010). Spindle-cell tumours are extremely uncommon: in a series of 5,000 necropsy examinations, there was only one feline gastric leiomyoma (Head et al., 2002). Gastrointestinal stromal tumours (GISTs) are a subset of gastrointestinal (GI) mesenchymal tumours that until their recognition were classified as leiomyomas, leiomyosarcomas, leiomyoblastomas or schwannomas based on their histological pattern and their apparent origin from the muscularis prop-
Correspondence to: G. Bettini (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2010.12.010
ria. Immunohistochemistry (IHC) and electron microscopy eventually permitted recognition of GISTs as a distinct group of mesenchymal tumours of the GI tract, putatively derived from the interstitial cells of Cajal (ICC) or their progenitor stem cells (Hirota et al., 1998; Kitamura, 2008). The diagnostic hallmark of GIST is the immunohistochemical expression of CD117 (KIT) antigen (Hirota et al., 1998; Kitamura, 2008). In human oncology, GISTs account for the vast majority of GI mesenchymal tumours (Hirota et al., 1998; Berman and O’Leary, 2001; Miettinen and Lasota, 2001; Miettinen et al., 2003). In animals, CD117+ GISTs have been described in dogs (Bettini et al., 2003; Frost et al., 2003; Kumagai et al., 2003; Maas et al., 2007; Russell et al., 2007), horses (Hafner et al., 2001; Muravnick et al., 2009), Spanish ibex (Velarde et al., 2008), a ferret (Girard-Luc et al., 2009), a rat (Fujimoto et al., 2006) and non-human primates (Bielefeldt-Ohmann et al., 2005; Saturday et al., 2005; Bommineni et al., 2009). Animal GISTs occur Ó 2010 Elsevier Ltd. All rights reserved.
Gastrointestinal Stromal Tumour in a Cat
most frequently in the jejunum, caecum and colon (Hafner et al., 2001; Bettini et al., 2003; Frost et al., 2003; Maas et al., 2007; Russell et al., 2007), while a majority of human GISTs occur in the stomach (Miettinen et al., 2003). GIST pathogenesis has been linked to activating mutations of the c-kit gene in people (Hirota et al., 1998; Miettinen et al., 2003; Bachet et al., 2009) and dogs (Frost et al., 2003). This report describes the cytological, histological, immunohistochemical and mutational findings of a gastric GIST in a cat. The case was formerly included in a series used to test CD117 expression in canine and feline normal and neoplastic tissues (Morini et al., 2004). A 10-year-old neutered female domestic shorthair cat was presented with a 2-month history of unresponsive, recurrent vomiting. On physical examination, the cat weighed 6 kg and was quiet, alert and reactive. Following general anaesthesia, endoscopic examination of the upper alimentary tract revealed a soft tissue, non-ulcerated mass in the stomach wall measuring 2 1.5 cm and protruding into the lumen at the level of the pyloric antrum. The remaining gastric wall and proximal duodenum were grossly normal. Biopsy samples of the gastric mass, areas of grossly normal gastric wall and proximal duodenum were collected. Samples were fixed in 10% neutral buffered formalin, embedded in paraffin wax and sections (4 mm) were stained with haematoxylin and eosin (HE). Microscopical examination indicated normal gastric mucosa with slight submucosal fibrosis and a moderate number of lymphocytes and plasma cells in the duodenum, consistent with a diagnosis of mild chronic gastritis and mild lymphoplasmacytic duodenitis. Based on the endoscopic images, an intramural gastric body mass was suspected and surgical exploration of the abdomen was performed in order to assess its extent, achieve a diagnosis and evaluate the possibility for excision. At laparotomy a raised, round to oval, firm mass approximately 5 cm in diameter was identified at the lesser curvature, expanding the serosal surface. On visual inspection, the remaining abdominal organs appeared normal. Samples of the mass were collected by fine needle aspiration cytology and stained with the MayeGr€ unwaldeGiemsa stain. These were highly cellular and comprised clusters of spindle cells with elongated nuclei, without prominent nuclear atypia, and a sparse, delicate and wispy cytoplasm with occasional long filamentous extensions (Fig. 1A). The cytological diagnosis was of a potential mesenchymal tumour. The mass was excised with wide margins and the gastric wall defect closed routinely. The cat was normal immediately postoperatively, but died unexpectedly 48 h after discharge. Consent for necropsy examination was not given.
153
Gross examination of the surgical specimen indicated a 6 4 4 cm soft, round to oval mass, which expanded the stomach wall and protruded at both the mucosal and serosal surfaces. The overlying gastric mucosa was normal. On cut section, the tumour parenchyma was multilobular, friable, and greyewhite in colour and displayed variably sized foci of necrosis alternating with multiple haemorrhagic areas (Fig. 1B). Samples were collected for histology and processed as described above. Microscopically, a well-circumscribed, non-encapsulated, intramural elevated but intact neoplastic proliferation was evident within the tunica muscularis, extending transmurally close to, but not invading, the serosal layer. The tumour tissue was composed of densely-packed spindle cells arranged in bundles, whorls and interlacing fascicles (Fig. 1C). Tumour cells had indistinct borders with a moderate amount of pale, fibrillary, eosinophilic cytoplasm and small-to medium-sized spindle- or irregularly-shaped single nuclei (Fig. 1D). Mitoses ranged from 4 to 6/10 400 fields. Multifocal and wide areas of haemorrhage and necrosis were also observed. Differential diagnoses included GI mesenchymal tumours, notably leiomyosarcoma, peripheral nerve sheath tumour and GIST. IHC was performed using a modified avidine biotineperoxidase complex (ABC) method (LSAB Kit; Dako, Glostrup, Denmark) according to the manufacturer’s instructions. Antibodies used were cross-reactive and specific for vimentin (VIM, 1 in 40 dilution, Dako, monoclonal), desmin (DES, 1 in 100 dilution, Dako, monoclonal), smooth muscle actin (SMA, 1 in 100 dilution, Dako, monoclonal), S-100 protein (S100, 1 in 200 dilution, Dako, polyclonal), glial fibrillary acidic protein (GFAP, 1 in 1000 dilution, Dako, polyclonal) and CD117/c-kit protein (1 in 500 dilution, Dako, polyclonal). As negative controls, isotype matched irrelevant antibodies (Neomarkers, Fremont, California) were used in replicate sections instead of the primary antibody. Positive controls included normal feline duodenum for VIM, DES, SMA, S-100 and GFAP and a canine GIST for CD117. Immunohistochemically, tumour cells exhibited strong and diffuse cytoplasmic expression of CD117 (Fig. 1E) and VIM and focal cytoplasmic expression of SMA. Tumour cells did not react with antibodies against DES, S-100 and GFAP. The immunohistochemical findings prompted the diagnosis of GIST. DNA was purified from one smear stained for cytological studies. Cells were collected using a cotton swab soaked with phosphate buffered saline by gently rolling the cotton tip on the glass surface. The tip of the swab was transferred into a 1.5 ml tube and was completely immersed in 1 ml of lysis buffer (ChargeSwitchÒ Lysis Buffer; Invitrogen, Milan, Italy) with 10 ml of
154
M. Morini et al.
Fig. 1. Gastric GIST in a cat. (A) Fine needle aspiration cytology. Spindle-shaped cells, with elongated nuclei and sparse delicate and wispy cytoplasm. MayeGr€ unwaldeGiemsa. 200. (B) Cut surface of the neoplastic mass after surgical removal; the tumour tissue is multilobular, friable, and greyewhite in colour. Haemorrhage and necrosis are also evident. (C) Dense proliferation of irregularly arranged spindle cells beneath the gastric mucosa, which appears intact. HE. 100. (D) Tumour cells with indistinct borders and elongated nuclei are arranged in interlacing fascicles. HE. 200. (E) Strong, diffuse and homogeneous cytoplasmic expression of CD117. IHC. 100.
proteinase K added. The swab was gently vortexed in a tube mixer (MixMateÔ; Eppendorf, Milan, Italy) at 500 rpm for 1 h. Thereafter, the digested supernatant was transferred to a new sterile 1.5 ml tube and the genomic DNA purified in accordance with the manufacturer’s instructions (ChargeSwitchÒ Forensic DNA purification Kit; Invitrogen). A negative extraction control was also included. Exons 8, 9 and 11 of the feline KIT gene were amplified by polymerase chain reaction (PCR) using the primers described by Isotani et al. (2010). PCR products were purified using the CloneWell 0.8% SYBR Safe gelsÔ (Invitrogen) and sequenced using the Big Dye Terminator v1.1 kitÔ (Applied Biosystems, Milan, Italy), purified with Centri-SepÔ columns (Applied Biosystems) and separated electrophoretically by an ABI Prism 310 sequencer after denaturation with HiDiÔ formamide (Applied Biosystems). Direct sequencing demonstrated a 6 base pair in-frame deletion c.1672_1677delTGGAAG (GU270865.1),
leading to the loss of tryptophan and lysine in the predicted protein chain (Fig. 2). This single case report represents the first description of an intramural gastric mass in a cat diagnosed as GIST. GISTs are the most common mesenchymal tumours of the human GI tract, and are defined in the World Health Organization classification as primary GI tumours formed by spindle or epithelioid cells that
Fig. 2. Schematic representation of the exon 11 KIT mutation c.1672_1677delTGGAAG. Both wild type (upper) and mutated (bottom) amino-acidic and nucleotide sequences are indicated.
Gastrointestinal Stromal Tumour in a Cat
express CD117 (Miettinen et al., 2000; Miettinen and Lasota, 2001; Miettinen et al., 2003). CD117 (KIT) protein is a transmembrane growth factor receptor that belongs to the tyrosine kinase family (Miettinen and Lasota, 2001). In the GI tract, KIT expression is constitutively maintained in the ICC, autonomic nerve-related GI pacemaker cells dispersed between the intestinal smooth muscle cells that regulate the intestinal motility (Komuro, 1999). Because of the immunohistochemical similarity between GISTs and ICC, a histogenic origin from these cells or their progenitor stem cells has been suggested (Hirota et al., 1998; Miettinen and Lasota, 2001). Accordingly, diffuse strong cytoplasmic expression of KIT is a key diagnostic marker for GIST (Berman and O’Leary, 2001). In man, the stomach (60e70%) and the small intestine (20e30%) are the most common sites of origin of GISTs (Miettinen et al., 2003). In contrast to the situation in man, GISTs are less common in the stomach of dogs than in the intestinal tract (no cases out of five described by Bettini et al., 2003; one out of three reported by Kumagai et al., 2003; four out of 21 recorded by Frost et al., 2003; and two out of 28 described by Russell et al., 2007) and the canine GISTs are regarded as a distinctive group of GI mesenchymal tumours (Head et al., 2002, 2003; Brown et al., 2007). Immunohistochemical studies have demonstrated that a significant subset of tumours formerly diagnosed as leiomyosarcoma express CD117 (Bettini et al., 2003; Frost et al., 2003; Kumagai et al., 2003; Maas et al., 2007; Russell et al., 2007) and should be reclassified as GISTs. Microscopically, human GISTs tend to have a higher degree of cellularity than smooth muscle tumours and a more basophilic appearance. Several histological patterns have been identified: the most common is a spindle-cell pattern (60e70%), consisting of elongated or cigar-shaped nuclei with perinuclear vacuoles, followed by the epithelioid (20e30%) and a rare pleomorphic (5%) pattern (Berman and O’Leary, 2001; Miettinen and Lasota, 2001). In addition to expression of CD117, these tumours express VIM (nearly 100%) and CD34 antigens (50e70%) (Miettinen et al., 2003). Human GISTs express SMA in 30e40% of cases, while DES and S-100 are usually not expressed (Miettinen and Lasota, 2001; Miettinen et al., 2003). Canine GISTs are histologically and immunohistochemically similar to human GISTs (Bettini et al., 2003; Frost et al., 2003; Maas et al., 2007; Russell et al., 2007). This feline case is completely consistent with the spectrum of human and canine spindle-cell GISTs as it was a highly cellular expansive tumour composed of spindle cells with strong and diffuse
155
CD117 and VIM expression. The focal expression of SMA seen in this case is also consistent with human and canine GISTs (Bettini et al., 2003; Frost et al., 2003; Miettinen et al., 2003; Maas et al., 2007; Russell et al., 2007) and likely supports the hypothesis that GISTs may develop from a pluripotential ICC or its progenitor cell that can differentiate to smooth muscle cells (Miettinen and Lasota, 2001). Alteration of the gene encoding the tyrosine kinase receptor KIT is considered an early molecular event leading to the development of GIST, as well as other human and animal malignancies (Hirota et al., 1998; Zemke et al., 2002; Miettinen et al., 2003). Gainof-function mutations of the c-kit gene are present in about 80% of human GISTs, causing receptor activation independent of ligand binding and subsequent stimulation of cellular proliferation (Hirota et al., 1998; Kitamura, 2008). Many types of activating mutations of c-kit have been described in human GISTs and 60% of these occur within the juxtamembrane domain of the gene (exon 11; Hirota et al., 1998; Bachet et al., 2009). In canine GISTs, mutational analysis of exon 11 of c-kit has been performed in only four cases (Frost et al., 2003) and mutations were identified in two of these, one with a deletion of Try557 and Lys558 coexisting with duplication of Gln555. KIT mutations have also been studied in canine mast cell tumours and are most often located in the juxtamembrane domain (exon 11) (Ma et al., 1999; London et al., 1999; Zemke et al., 2002; Webster et al., 2008), while in feline mast cell tumours KIT mutations have been found in exons 8 and 9, which encode the fifth immunoglobulin-like domain (Isotani et al., 2010). The KIT juxtamembrane domain mutation identified in this case is similar to those found in human and in canine GISTs. Deletions of TGG AAG coding for Try557 and Lys558 (delWK557-558) in the proximal part of the exon 11 are the most commonly reported mutation in human GISTs, accounting for 8e25% of KIT exon 11 mutations (Miettinen et al., 2003; Bachet et al., 2009). Moreover, a recent study has reported that GISTs with a delWK557-558 mutation occur in the stomach in about 75% of cases, suggesting a possible association between the type of oncogenic event driving KIT mutation and the different parts of the digestive tract where GISTs can grow (Bachet et al., 2009). The biological significance of the KIT mutation documented in this case of feline GIST is likely similar to that of comparable mutations found in man and dogs, and strongly suggests that tumourigenesis of feline, canine and human GISTs follows the same molecular pathway. KIT activating mutations are also potential targets for therapeutic intervention with tyrosine kinase
M. Morini et al.
156
inhibitors (Berman and O’Leary, 2001). Imatinib mesylate (STI571) has been used successfully in the treatment of human GISTs and the interrelationship between KIT gain-of-function mutations and the therapeutic effect has been well characterized. It is difficult to determine by histological examination the grade of malignancy of GISTs. In human oncology, GISTs of the small intestine that are larger than 5 cm or that have >5 mitoses/10 high power fields are considered to carry a poorer prognosis (Miettinen and Lasota, 2001). The biological behaviour of animal GISTs is not well defined. In the survey from Russell et al. (2007), only two out of 28 GISTs had hepatic or mesenteric metastases and although not significant, survival times were longer in animals with GISTs than for those with leiomyosarcomas. However, the correlation between histological appearance and behaviour was not investigated. In the case described here there was a relatively high mitotic rate and extensive necrosis, which are suggestive of aggressive behaviour, but metastases were not evident at the time of surgery. To the authors’ knowledge, this is the first report to describe the cytological, histological, immunohistochemical and genetic features of a feline GIST and provides evidence that GISTs in cats bear KIT mutations similar to those described in people.
References Bachet J-B, Hostein I, Le Cesne A, Brahimi S, Beauchet A et al. (2009) Prognosis and predictive value of KIT exon 11 deletion in GISTs. British Journal of Cancer, 101, 7e11. Berman JJ, O’Leary TJ (2001) Gastrointestinal stromal tumor workshop. Human Pathology, 32, 578e582. Bettini G, Morini M, Marcato PS (2003) Gastrointestinal spindle cell tumours of the dog: histological and immunohistochemical study. Journal of Comparative Pathology, 129, 283e293. Bielefeldt-Ohmann H, Barouch DH, Bakke AM, Bruce AG, Durning M et al. (2005) Intestinal stromal tumors in a simian immunodeficiency virus-infected, simian retrovirus-2 negative rhesus macaque (Macaca mulatta). Veterinary Pathology, 42, 391e396. Bommineni YR, Dick EJ, Hubbard GB (2009) Gastrointestinal stromal tumors in a baboon, a spider monkey, and a chimpanzee and a review of the literature. Journal of Medical Primatology, 38, 199e203. Bortnowski HB, Rosenthal RC (1992) Gastrointestinal mast cell tumors and eosinophilia in two cats. Journal of the American Animal Hospital Association, 28, 271e275. Brown CC, Baker DC, Barker IK (2007) The alimentary system. In: Jubb, Kennedy and Palmer’s Pathology of Domestic Animals, 5th Edit., Vol. 2, MG Maxie, Ed., Elsevier Saunders, Philadelphia, pp. 1e296.
Frost D, Lasota J, Miettinen M (2003) Gastrointestinal stromal tumors and leiomyomas in the dog: a histopathologic, immunohistochemical, and molecular genetic study of 50 cases. Veterinary Pathology, 40, 42e54. Fujimoto H, Shibutani M, Kuroiwa K, Inoue K, Woo G-H et al. (2006) A case report of a spontaneous gastrointestinal stromal tumor (GIST) occurring in a F344 rat. Toxicologic Pathology, 34, 164e167. Girard-Luc A, Prata D, Huet H, Lagadic M, Bernex F (2009) A KIT-positive gastrointestinal stromal tumor in a ferret (Mustela putorius furo). Journal of Veterinary Diagnostic Investigation, 21, 915e917. Hafner S, Harmon BG, King T (2001) Gastrointestinal stromal tumors of the equine cecum. Veterinary Pathology, 38, 24e26. Head KW, Cullen JM, Dubielzig RR, Else RW, Misdorp W et al. (2003) WHO Histological Classification of Tumors of the Alimentary System of Domestic Animals, Vol. X, 2nd Edit., Armed Forces Institute of Pathology, American Registry of Pathology, Washington, DC, p. 81. Head KW, Else RW, Dubielzig RR (2002) Tumors of the alimentary tract. In: Tumors in Domestic Animals, 4th Edit., DJ Meuten, Ed., Iowa State University Press, Ames, pp. 401e481. Hirota S, Isozaki K, Moriyama Y, Hascimoto K, Nischida T et al. (1998) Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science, 279, 577e580. Isotani M, Yamada O, Lachowicz JL, Tamura K, Yagihara H et al. (2010) Mutations in the fifth immunoglobulin-like domain of kit are common and potentially sensitive to imatinib mesylate in feline mast cell tumours. British Journal of Haematology, 148, 144e153. Kitamura Y (2008) Gastrointestinal stromal tumors: past, present, and future. Journal of Gastroenterology, 43, 499e508. Komuro T (1999) Comparative morphology of interstitial cells of Cajal: ultrastructural characterization. Microscopy Research and Technique, 47, 267e285. Kumagai K, Uchida K, Miyamoto T, Uchigusa T, Shinohara S et al. (2003) Three cases of canine gastrointestinal stromal tumors with multiple differentiations and c-kit expression. Journal of Veterinary Medical Science, 65, 1119e1122. London CA, Galli SJ, Yuuki T, Hu ZQ, Helfand SC et al. (1999) Spontaneous canine mast cell tumors express tandem duplications in the proto-oncogene c-kit. Experimental Hematology, 27, 689e697. Ma Y, Longley BJ, Wang X, Blount JL, Langley K et al. (1999) Clustering of activating mutations in c-KIT’s juxtamembrane coding region in canine mast cell neoplasms. Journal of Investigative Dermatology, 112, 165e170. Maas CPHJ, Ter Haar G, Van Der Gaag I, Kirpensteijn J (2007) Reclassification of small intestinal and cecal smooth muscle tumors in 72 dogs: clinical, histologic, and immunohistochemical evaluation. Veterinary Surgery, 36, 302e313.
Gastrointestinal Stromal Tumour in a Cat
Miettinen M, Blay JY, Sobin LH (2000) Mesenchymal tumours of the stomach. In: Pathology and Genetics of Tumours of the Digestive System. World Health Organization Classification of Tumours, SR Hamilton, LA Aaltomen, Eds., IARC Press, Lyon, pp. 62e65. Miettinen M, Kopczynski J, Makhlouf HR, SarlomoRikala M, Gyorffy H et al. (2003) Gastrointestinal stromal tumors, intramural leiomyomas, and leiomyosarcomas in the duodenum: a clinicopathologic, immunohistochemical, and molecular genetic study of 167 cases. American Journal of Surgical Pathology, 27, 625e641. Miettinen M, Lasota J (2001) Gastrointestinal stromal tumors e clinical, histological, immunohistochemical and molecular genetic features and differential diagnosis. Virchows Archives, 438, 1e12. Morini M, Bettini G, Preziosi R, Mandrioli L (2004) C-kit gene product (CD117) immunoreactivity in canine and feline paraffin sections. Journal of Histochemistry and Cytochemistry, 52, 705e708. Muravnick KB, Parente EJ, Del Piero F (2009) An atypical equine gastrointestinal stromal tumor. Journal of Veterinary Diagnostic Investigation, 21, 387e390. Rossmeisl JH Jr., Dru S, Forrester SD, Robertson JL, Wesley T et al. (2002) Chronic vomiting associated with a gastric carcinoid in a cat. Journal of American Animal Association, 38, 61e66. Russell KN, Mehler SJ, Skorupski KA, Baez JL, Shofer FS et al. (2007) Clinical and immunohistochemical differentiation of gastrointestinal stromal tumors from leiomyosarcomas in dogs: 42 cases (1990e2003). Journal of the American Veterinary Medical Association, 230, 1329e1333. Saturday GA, Lasota J, Frost D, Brasky KB, Hubbard G et al. (2005) KIT-positive gastrointestinal stromal tumor
157
in a 22-year-old male chimpanzee (Pan troglodites). Veterinary Pathology, 42, 362e365. Smith TJ, Baltzer WI, Ruaux CG, Heidel JR, Carney P (2010) Gastric smooth muscle hamartoma in a cat. Journal of Feline Medicine and Surgery, 12, 334e337. Turk MAM, Gallina AM, Russell TS (1981) Nonhematopoietic gastrointestinal neoplasia in cats: a retrospective study of 44 cases. Veterinary Pathology, 18, 614e620. Velarde R, Mentaberre G, S anchez J, Ignasi M, Lavın S (2008) KIT-positive gastrointestinal stromal tumors in two Spanish ibex (Capra pyrenaica hispanica). Veterinary Journal, 177, 445e447. Webster JD, Yuzbasiyan-Gurkan V, Thamm DH, Hamilton E, Kiupel M (2008) Evaluation of prognostic markers for canine mast cell tumors treated with vinblastine and prednisone. BMC Veterinary Research, 13, 4e32. Withrow SJ, MacEwen EG (1996) Small Animal Clinical Oncology, 2nd Edit., WB Saunders, Philadelphia. pp. 480e483. Zemke D, Yamini B, Yuzbasiyan-Gurkan V (2002) Mutation in juxtamembrane domain of c-Kit are associated with higher grade mast cell tumors in dogs. Veterinary Pathology, 39, 529e535. Zikes CD, Spielman B, Shapiro W, Roth L, Ablin L (1998) Gastric extramedullary plasmacytoma in a cat. Journal of Veterinary Internal Medicine, 12, 381e383.
September 3rd, 2010 ½ Received, Accepted, December 14th, 2010
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
NEOPLASTIC DISEASE
Cytological, Immunohistochemical and Mutational Analysis of a Gastric Gastrointestinal Stromal Tumour in a Cat M. Morini*, F. Gentilini†, M. Pietra†, A. Spadari†, M. E. Turba‡, L. Mandrioli* and G. Bettini* * Department of Veterinary Public Health and Animal Pathology, † Veterinary Clinical Department, Faculty of Veterinary Medicine, Alma Mater Studiorum, University of Bologna, Ozzano Emilia and ‡ Veterinary Practitioner, GeneFast, Castelnuovo Rangone, Modena, Italy
Summary Gastrointestinal stromal tumours (GISTs) represent a distinctive group of primary mesenchymal tumours of the gastrointestinal tract identified immunohistochemically by expression of CD117. A 10-year-old neutered female domestic shorthair cat with a history of recurrent vomiting was examined. The presence of a gastric mass was recognized and a laparotomy was performed. Cytological examination was consistent with a lowgrade malignant mesenchymal tumour and histopathological investigation suggested myogenic differentiation of tumour cells. The diagnosis of GIST was confirmed by immunohistochemical expression of CD117. Sequence analysis of the KIT gene identified a deletion in exon 11. The same mutation is found often in human GISTs. Ó 2010 Elsevier Ltd. All rights reserved. Keywords: cat; CD117; gastrointestinal stromal tumour; KIT
Primary non-lymphoid gastric tumours are rare in cats (Withrow and MacEwen, 1996; Head et al., 2002; Brown et al., 2007). Adenocarcinomas are the most frequently reported of these (Turk et al., 1981; Withrow and MacEwen, 1996) and single cases have been reported of gastric mast cell tumour, extramedullary plasmacytoma, carcinoid and hamartoma (Bortnowski and Rosenthal, 1992; Zikes et al., 1998; Rossmeisl et al., 2002; Smith et al., 2010). Spindle-cell tumours are extremely uncommon: in a series of 5,000 necropsy examinations, there was only one feline gastric leiomyoma (Head et al., 2002). Gastrointestinal stromal tumours (GISTs) are a subset of gastrointestinal (GI) mesenchymal tumours that until their recognition were classified as leiomyomas, leiomyosarcomas, leiomyoblastomas or schwannomas based on their histological pattern and their apparent origin from the muscularis prop-
Correspondence to: G. Bettini (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2010.12.010
ria. Immunohistochemistry (IHC) and electron microscopy eventually permitted recognition of GISTs as a distinct group of mesenchymal tumours of the GI tract, putatively derived from the interstitial cells of Cajal (ICC) or their progenitor stem cells (Hirota et al., 1998; Kitamura, 2008). The diagnostic hallmark of GIST is the immunohistochemical expression of CD117 (KIT) antigen (Hirota et al., 1998; Kitamura, 2008). In human oncology, GISTs account for the vast majority of GI mesenchymal tumours (Hirota et al., 1998; Berman and O’Leary, 2001; Miettinen and Lasota, 2001; Miettinen et al., 2003). In animals, CD117+ GISTs have been described in dogs (Bettini et al., 2003; Frost et al., 2003; Kumagai et al., 2003; Maas et al., 2007; Russell et al., 2007), horses (Hafner et al., 2001; Muravnick et al., 2009), Spanish ibex (Velarde et al., 2008), a ferret (Girard-Luc et al., 2009), a rat (Fujimoto et al., 2006) and non-human primates (Bielefeldt-Ohmann et al., 2005; Saturday et al., 2005; Bommineni et al., 2009). Animal GISTs occur Ó 2010 Elsevier Ltd. All rights reserved.
Gastrointestinal Stromal Tumour in a Cat
most frequently in the jejunum, caecum and colon (Hafner et al., 2001; Bettini et al., 2003; Frost et al., 2003; Maas et al., 2007; Russell et al., 2007), while a majority of human GISTs occur in the stomach (Miettinen et al., 2003). GIST pathogenesis has been linked to activating mutations of the c-kit gene in people (Hirota et al., 1998; Miettinen et al., 2003; Bachet et al., 2009) and dogs (Frost et al., 2003). This report describes the cytological, histological, immunohistochemical and mutational findings of a gastric GIST in a cat. The case was formerly included in a series used to test CD117 expression in canine and feline normal and neoplastic tissues (Morini et al., 2004). A 10-year-old neutered female domestic shorthair cat was presented with a 2-month history of unresponsive, recurrent vomiting. On physical examination, the cat weighed 6 kg and was quiet, alert and reactive. Following general anaesthesia, endoscopic examination of the upper alimentary tract revealed a soft tissue, non-ulcerated mass in the stomach wall measuring 2 1.5 cm and protruding into the lumen at the level of the pyloric antrum. The remaining gastric wall and proximal duodenum were grossly normal. Biopsy samples of the gastric mass, areas of grossly normal gastric wall and proximal duodenum were collected. Samples were fixed in 10% neutral buffered formalin, embedded in paraffin wax and sections (4 mm) were stained with haematoxylin and eosin (HE). Microscopical examination indicated normal gastric mucosa with slight submucosal fibrosis and a moderate number of lymphocytes and plasma cells in the duodenum, consistent with a diagnosis of mild chronic gastritis and mild lymphoplasmacytic duodenitis. Based on the endoscopic images, an intramural gastric body mass was suspected and surgical exploration of the abdomen was performed in order to assess its extent, achieve a diagnosis and evaluate the possibility for excision. At laparotomy a raised, round to oval, firm mass approximately 5 cm in diameter was identified at the lesser curvature, expanding the serosal surface. On visual inspection, the remaining abdominal organs appeared normal. Samples of the mass were collected by fine needle aspiration cytology and stained with the MayeGr€ unwaldeGiemsa stain. These were highly cellular and comprised clusters of spindle cells with elongated nuclei, without prominent nuclear atypia, and a sparse, delicate and wispy cytoplasm with occasional long filamentous extensions (Fig. 1A). The cytological diagnosis was of a potential mesenchymal tumour. The mass was excised with wide margins and the gastric wall defect closed routinely. The cat was normal immediately postoperatively, but died unexpectedly 48 h after discharge. Consent for necropsy examination was not given.
153
Gross examination of the surgical specimen indicated a 6 4 4 cm soft, round to oval mass, which expanded the stomach wall and protruded at both the mucosal and serosal surfaces. The overlying gastric mucosa was normal. On cut section, the tumour parenchyma was multilobular, friable, and greyewhite in colour and displayed variably sized foci of necrosis alternating with multiple haemorrhagic areas (Fig. 1B). Samples were collected for histology and processed as described above. Microscopically, a well-circumscribed, non-encapsulated, intramural elevated but intact neoplastic proliferation was evident within the tunica muscularis, extending transmurally close to, but not invading, the serosal layer. The tumour tissue was composed of densely-packed spindle cells arranged in bundles, whorls and interlacing fascicles (Fig. 1C). Tumour cells had indistinct borders with a moderate amount of pale, fibrillary, eosinophilic cytoplasm and small-to medium-sized spindle- or irregularly-shaped single nuclei (Fig. 1D). Mitoses ranged from 4 to 6/10 400 fields. Multifocal and wide areas of haemorrhage and necrosis were also observed. Differential diagnoses included GI mesenchymal tumours, notably leiomyosarcoma, peripheral nerve sheath tumour and GIST. IHC was performed using a modified avidine biotineperoxidase complex (ABC) method (LSAB Kit; Dako, Glostrup, Denmark) according to the manufacturer’s instructions. Antibodies used were cross-reactive and specific for vimentin (VIM, 1 in 40 dilution, Dako, monoclonal), desmin (DES, 1 in 100 dilution, Dako, monoclonal), smooth muscle actin (SMA, 1 in 100 dilution, Dako, monoclonal), S-100 protein (S100, 1 in 200 dilution, Dako, polyclonal), glial fibrillary acidic protein (GFAP, 1 in 1000 dilution, Dako, polyclonal) and CD117/c-kit protein (1 in 500 dilution, Dako, polyclonal). As negative controls, isotype matched irrelevant antibodies (Neomarkers, Fremont, California) were used in replicate sections instead of the primary antibody. Positive controls included normal feline duodenum for VIM, DES, SMA, S-100 and GFAP and a canine GIST for CD117. Immunohistochemically, tumour cells exhibited strong and diffuse cytoplasmic expression of CD117 (Fig. 1E) and VIM and focal cytoplasmic expression of SMA. Tumour cells did not react with antibodies against DES, S-100 and GFAP. The immunohistochemical findings prompted the diagnosis of GIST. DNA was purified from one smear stained for cytological studies. Cells were collected using a cotton swab soaked with phosphate buffered saline by gently rolling the cotton tip on the glass surface. The tip of the swab was transferred into a 1.5 ml tube and was completely immersed in 1 ml of lysis buffer (ChargeSwitchÒ Lysis Buffer; Invitrogen, Milan, Italy) with 10 ml of
154
M. Morini et al.
Fig. 1. Gastric GIST in a cat. (A) Fine needle aspiration cytology. Spindle-shaped cells, with elongated nuclei and sparse delicate and wispy cytoplasm. MayeGr€ unwaldeGiemsa. 200. (B) Cut surface of the neoplastic mass after surgical removal; the tumour tissue is multilobular, friable, and greyewhite in colour. Haemorrhage and necrosis are also evident. (C) Dense proliferation of irregularly arranged spindle cells beneath the gastric mucosa, which appears intact. HE. 100. (D) Tumour cells with indistinct borders and elongated nuclei are arranged in interlacing fascicles. HE. 200. (E) Strong, diffuse and homogeneous cytoplasmic expression of CD117. IHC. 100.
proteinase K added. The swab was gently vortexed in a tube mixer (MixMateÔ; Eppendorf, Milan, Italy) at 500 rpm for 1 h. Thereafter, the digested supernatant was transferred to a new sterile 1.5 ml tube and the genomic DNA purified in accordance with the manufacturer’s instructions (ChargeSwitchÒ Forensic DNA purification Kit; Invitrogen). A negative extraction control was also included. Exons 8, 9 and 11 of the feline KIT gene were amplified by polymerase chain reaction (PCR) using the primers described by Isotani et al. (2010). PCR products were purified using the CloneWell 0.8% SYBR Safe gelsÔ (Invitrogen) and sequenced using the Big Dye Terminator v1.1 kitÔ (Applied Biosystems, Milan, Italy), purified with Centri-SepÔ columns (Applied Biosystems) and separated electrophoretically by an ABI Prism 310 sequencer after denaturation with HiDiÔ formamide (Applied Biosystems). Direct sequencing demonstrated a 6 base pair in-frame deletion c.1672_1677delTGGAAG (GU270865.1),
leading to the loss of tryptophan and lysine in the predicted protein chain (Fig. 2). This single case report represents the first description of an intramural gastric mass in a cat diagnosed as GIST. GISTs are the most common mesenchymal tumours of the human GI tract, and are defined in the World Health Organization classification as primary GI tumours formed by spindle or epithelioid cells that
Fig. 2. Schematic representation of the exon 11 KIT mutation c.1672_1677delTGGAAG. Both wild type (upper) and mutated (bottom) amino-acidic and nucleotide sequences are indicated.
Gastrointestinal Stromal Tumour in a Cat
express CD117 (Miettinen et al., 2000; Miettinen and Lasota, 2001; Miettinen et al., 2003). CD117 (KIT) protein is a transmembrane growth factor receptor that belongs to the tyrosine kinase family (Miettinen and Lasota, 2001). In the GI tract, KIT expression is constitutively maintained in the ICC, autonomic nerve-related GI pacemaker cells dispersed between the intestinal smooth muscle cells that regulate the intestinal motility (Komuro, 1999). Because of the immunohistochemical similarity between GISTs and ICC, a histogenic origin from these cells or their progenitor stem cells has been suggested (Hirota et al., 1998; Miettinen and Lasota, 2001). Accordingly, diffuse strong cytoplasmic expression of KIT is a key diagnostic marker for GIST (Berman and O’Leary, 2001). In man, the stomach (60e70%) and the small intestine (20e30%) are the most common sites of origin of GISTs (Miettinen et al., 2003). In contrast to the situation in man, GISTs are less common in the stomach of dogs than in the intestinal tract (no cases out of five described by Bettini et al., 2003; one out of three reported by Kumagai et al., 2003; four out of 21 recorded by Frost et al., 2003; and two out of 28 described by Russell et al., 2007) and the canine GISTs are regarded as a distinctive group of GI mesenchymal tumours (Head et al., 2002, 2003; Brown et al., 2007). Immunohistochemical studies have demonstrated that a significant subset of tumours formerly diagnosed as leiomyosarcoma express CD117 (Bettini et al., 2003; Frost et al., 2003; Kumagai et al., 2003; Maas et al., 2007; Russell et al., 2007) and should be reclassified as GISTs. Microscopically, human GISTs tend to have a higher degree of cellularity than smooth muscle tumours and a more basophilic appearance. Several histological patterns have been identified: the most common is a spindle-cell pattern (60e70%), consisting of elongated or cigar-shaped nuclei with perinuclear vacuoles, followed by the epithelioid (20e30%) and a rare pleomorphic (5%) pattern (Berman and O’Leary, 2001; Miettinen and Lasota, 2001). In addition to expression of CD117, these tumours express VIM (nearly 100%) and CD34 antigens (50e70%) (Miettinen et al., 2003). Human GISTs express SMA in 30e40% of cases, while DES and S-100 are usually not expressed (Miettinen and Lasota, 2001; Miettinen et al., 2003). Canine GISTs are histologically and immunohistochemically similar to human GISTs (Bettini et al., 2003; Frost et al., 2003; Maas et al., 2007; Russell et al., 2007). This feline case is completely consistent with the spectrum of human and canine spindle-cell GISTs as it was a highly cellular expansive tumour composed of spindle cells with strong and diffuse
155
CD117 and VIM expression. The focal expression of SMA seen in this case is also consistent with human and canine GISTs (Bettini et al., 2003; Frost et al., 2003; Miettinen et al., 2003; Maas et al., 2007; Russell et al., 2007) and likely supports the hypothesis that GISTs may develop from a pluripotential ICC or its progenitor cell that can differentiate to smooth muscle cells (Miettinen and Lasota, 2001). Alteration of the gene encoding the tyrosine kinase receptor KIT is considered an early molecular event leading to the development of GIST, as well as other human and animal malignancies (Hirota et al., 1998; Zemke et al., 2002; Miettinen et al., 2003). Gainof-function mutations of the c-kit gene are present in about 80% of human GISTs, causing receptor activation independent of ligand binding and subsequent stimulation of cellular proliferation (Hirota et al., 1998; Kitamura, 2008). Many types of activating mutations of c-kit have been described in human GISTs and 60% of these occur within the juxtamembrane domain of the gene (exon 11; Hirota et al., 1998; Bachet et al., 2009). In canine GISTs, mutational analysis of exon 11 of c-kit has been performed in only four cases (Frost et al., 2003) and mutations were identified in two of these, one with a deletion of Try557 and Lys558 coexisting with duplication of Gln555. KIT mutations have also been studied in canine mast cell tumours and are most often located in the juxtamembrane domain (exon 11) (Ma et al., 1999; London et al., 1999; Zemke et al., 2002; Webster et al., 2008), while in feline mast cell tumours KIT mutations have been found in exons 8 and 9, which encode the fifth immunoglobulin-like domain (Isotani et al., 2010). The KIT juxtamembrane domain mutation identified in this case is similar to those found in human and in canine GISTs. Deletions of TGG AAG coding for Try557 and Lys558 (delWK557-558) in the proximal part of the exon 11 are the most commonly reported mutation in human GISTs, accounting for 8e25% of KIT exon 11 mutations (Miettinen et al., 2003; Bachet et al., 2009). Moreover, a recent study has reported that GISTs with a delWK557-558 mutation occur in the stomach in about 75% of cases, suggesting a possible association between the type of oncogenic event driving KIT mutation and the different parts of the digestive tract where GISTs can grow (Bachet et al., 2009). The biological significance of the KIT mutation documented in this case of feline GIST is likely similar to that of comparable mutations found in man and dogs, and strongly suggests that tumourigenesis of feline, canine and human GISTs follows the same molecular pathway. KIT activating mutations are also potential targets for therapeutic intervention with tyrosine kinase
M. Morini et al.
156
inhibitors (Berman and O’Leary, 2001). Imatinib mesylate (STI571) has been used successfully in the treatment of human GISTs and the interrelationship between KIT gain-of-function mutations and the therapeutic effect has been well characterized. It is difficult to determine by histological examination the grade of malignancy of GISTs. In human oncology, GISTs of the small intestine that are larger than 5 cm or that have >5 mitoses/10 high power fields are considered to carry a poorer prognosis (Miettinen and Lasota, 2001). The biological behaviour of animal GISTs is not well defined. In the survey from Russell et al. (2007), only two out of 28 GISTs had hepatic or mesenteric metastases and although not significant, survival times were longer in animals with GISTs than for those with leiomyosarcomas. However, the correlation between histological appearance and behaviour was not investigated. In the case described here there was a relatively high mitotic rate and extensive necrosis, which are suggestive of aggressive behaviour, but metastases were not evident at the time of surgery. To the authors’ knowledge, this is the first report to describe the cytological, histological, immunohistochemical and genetic features of a feline GIST and provides evidence that GISTs in cats bear KIT mutations similar to those described in people.
References Bachet J-B, Hostein I, Le Cesne A, Brahimi S, Beauchet A et al. (2009) Prognosis and predictive value of KIT exon 11 deletion in GISTs. British Journal of Cancer, 101, 7e11. Berman JJ, O’Leary TJ (2001) Gastrointestinal stromal tumor workshop. Human Pathology, 32, 578e582. Bettini G, Morini M, Marcato PS (2003) Gastrointestinal spindle cell tumours of the dog: histological and immunohistochemical study. Journal of Comparative Pathology, 129, 283e293. Bielefeldt-Ohmann H, Barouch DH, Bakke AM, Bruce AG, Durning M et al. (2005) Intestinal stromal tumors in a simian immunodeficiency virus-infected, simian retrovirus-2 negative rhesus macaque (Macaca mulatta). Veterinary Pathology, 42, 391e396. Bommineni YR, Dick EJ, Hubbard GB (2009) Gastrointestinal stromal tumors in a baboon, a spider monkey, and a chimpanzee and a review of the literature. Journal of Medical Primatology, 38, 199e203. Bortnowski HB, Rosenthal RC (1992) Gastrointestinal mast cell tumors and eosinophilia in two cats. Journal of the American Animal Hospital Association, 28, 271e275. Brown CC, Baker DC, Barker IK (2007) The alimentary system. In: Jubb, Kennedy and Palmer’s Pathology of Domestic Animals, 5th Edit., Vol. 2, MG Maxie, Ed., Elsevier Saunders, Philadelphia, pp. 1e296.
Frost D, Lasota J, Miettinen M (2003) Gastrointestinal stromal tumors and leiomyomas in the dog: a histopathologic, immunohistochemical, and molecular genetic study of 50 cases. Veterinary Pathology, 40, 42e54. Fujimoto H, Shibutani M, Kuroiwa K, Inoue K, Woo G-H et al. (2006) A case report of a spontaneous gastrointestinal stromal tumor (GIST) occurring in a F344 rat. Toxicologic Pathology, 34, 164e167. Girard-Luc A, Prata D, Huet H, Lagadic M, Bernex F (2009) A KIT-positive gastrointestinal stromal tumor in a ferret (Mustela putorius furo). Journal of Veterinary Diagnostic Investigation, 21, 915e917. Hafner S, Harmon BG, King T (2001) Gastrointestinal stromal tumors of the equine cecum. Veterinary Pathology, 38, 24e26. Head KW, Cullen JM, Dubielzig RR, Else RW, Misdorp W et al. (2003) WHO Histological Classification of Tumors of the Alimentary System of Domestic Animals, Vol. X, 2nd Edit., Armed Forces Institute of Pathology, American Registry of Pathology, Washington, DC, p. 81. Head KW, Else RW, Dubielzig RR (2002) Tumors of the alimentary tract. In: Tumors in Domestic Animals, 4th Edit., DJ Meuten, Ed., Iowa State University Press, Ames, pp. 401e481. Hirota S, Isozaki K, Moriyama Y, Hascimoto K, Nischida T et al. (1998) Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science, 279, 577e580. Isotani M, Yamada O, Lachowicz JL, Tamura K, Yagihara H et al. (2010) Mutations in the fifth immunoglobulin-like domain of kit are common and potentially sensitive to imatinib mesylate in feline mast cell tumours. British Journal of Haematology, 148, 144e153. Kitamura Y (2008) Gastrointestinal stromal tumors: past, present, and future. Journal of Gastroenterology, 43, 499e508. Komuro T (1999) Comparative morphology of interstitial cells of Cajal: ultrastructural characterization. Microscopy Research and Technique, 47, 267e285. Kumagai K, Uchida K, Miyamoto T, Uchigusa T, Shinohara S et al. (2003) Three cases of canine gastrointestinal stromal tumors with multiple differentiations and c-kit expression. Journal of Veterinary Medical Science, 65, 1119e1122. London CA, Galli SJ, Yuuki T, Hu ZQ, Helfand SC et al. (1999) Spontaneous canine mast cell tumors express tandem duplications in the proto-oncogene c-kit. Experimental Hematology, 27, 689e697. Ma Y, Longley BJ, Wang X, Blount JL, Langley K et al. (1999) Clustering of activating mutations in c-KIT’s juxtamembrane coding region in canine mast cell neoplasms. Journal of Investigative Dermatology, 112, 165e170. Maas CPHJ, Ter Haar G, Van Der Gaag I, Kirpensteijn J (2007) Reclassification of small intestinal and cecal smooth muscle tumors in 72 dogs: clinical, histologic, and immunohistochemical evaluation. Veterinary Surgery, 36, 302e313.
Gastrointestinal Stromal Tumour in a Cat
Miettinen M, Blay JY, Sobin LH (2000) Mesenchymal tumours of the stomach. In: Pathology and Genetics of Tumours of the Digestive System. World Health Organization Classification of Tumours, SR Hamilton, LA Aaltomen, Eds., IARC Press, Lyon, pp. 62e65. Miettinen M, Kopczynski J, Makhlouf HR, SarlomoRikala M, Gyorffy H et al. (2003) Gastrointestinal stromal tumors, intramural leiomyomas, and leiomyosarcomas in the duodenum: a clinicopathologic, immunohistochemical, and molecular genetic study of 167 cases. American Journal of Surgical Pathology, 27, 625e641. Miettinen M, Lasota J (2001) Gastrointestinal stromal tumors e clinical, histological, immunohistochemical and molecular genetic features and differential diagnosis. Virchows Archives, 438, 1e12. Morini M, Bettini G, Preziosi R, Mandrioli L (2004) C-kit gene product (CD117) immunoreactivity in canine and feline paraffin sections. Journal of Histochemistry and Cytochemistry, 52, 705e708. Muravnick KB, Parente EJ, Del Piero F (2009) An atypical equine gastrointestinal stromal tumor. Journal of Veterinary Diagnostic Investigation, 21, 387e390. Rossmeisl JH Jr., Dru S, Forrester SD, Robertson JL, Wesley T et al. (2002) Chronic vomiting associated with a gastric carcinoid in a cat. Journal of American Animal Association, 38, 61e66. Russell KN, Mehler SJ, Skorupski KA, Baez JL, Shofer FS et al. (2007) Clinical and immunohistochemical differentiation of gastrointestinal stromal tumors from leiomyosarcomas in dogs: 42 cases (1990e2003). Journal of the American Veterinary Medical Association, 230, 1329e1333. Saturday GA, Lasota J, Frost D, Brasky KB, Hubbard G et al. (2005) KIT-positive gastrointestinal stromal tumor
157
in a 22-year-old male chimpanzee (Pan troglodites). Veterinary Pathology, 42, 362e365. Smith TJ, Baltzer WI, Ruaux CG, Heidel JR, Carney P (2010) Gastric smooth muscle hamartoma in a cat. Journal of Feline Medicine and Surgery, 12, 334e337. Turk MAM, Gallina AM, Russell TS (1981) Nonhematopoietic gastrointestinal neoplasia in cats: a retrospective study of 44 cases. Veterinary Pathology, 18, 614e620. Velarde R, Mentaberre G, S anchez J, Ignasi M, Lavın S (2008) KIT-positive gastrointestinal stromal tumors in two Spanish ibex (Capra pyrenaica hispanica). Veterinary Journal, 177, 445e447. Webster JD, Yuzbasiyan-Gurkan V, Thamm DH, Hamilton E, Kiupel M (2008) Evaluation of prognostic markers for canine mast cell tumors treated with vinblastine and prednisone. BMC Veterinary Research, 13, 4e32. Withrow SJ, MacEwen EG (1996) Small Animal Clinical Oncology, 2nd Edit., WB Saunders, Philadelphia. pp. 480e483. Zemke D, Yamini B, Yuzbasiyan-Gurkan V (2002) Mutation in juxtamembrane domain of c-Kit are associated with higher grade mast cell tumors in dogs. Veterinary Pathology, 39, 529e535. Zikes CD, Spielman B, Shapiro W, Roth L, Ablin L (1998) Gastric extramedullary plasmacytoma in a cat. Journal of Veterinary Internal Medicine, 12, 381e383.
September 3rd, 2010 ½ Received, Accepted, December 14th, 2010