Gastrointestinal Stromal Tumour in a Guinea Pig : a Case Report

The objective of this contribution was to present a case report of gastrointestinal stromal tumour (GIST) in one guinea pig: a pet animal, male, 3.5 year of age, tricolour. Approximately for five weeks before death wasting, in spite of good appetite, decreased locomotor activity, soft and malodorous faeces were observed by the owner. The animal spontaneously died and at necropsy a grey-pink coloured tumour of walnut size located in the terminal segment of the ileum was observed. A solid malignant tumour of mesenchymal appearance was diagnosed histologically. One part of the sample consisted of tightly packed spindle cells arranged in interlacing fascicles, in the other part epithelioid cells predominated with slightly myxoid intercellular matrix. Lymphocytic sheaths were observed around some blood capillaries. Immunohistochemistry revealed positivity for actin, CD 117, neuron-specific enolase, glial fibrillary acidic protein, and p53 protein; however, desmin, S100 protein and synaptophysin were negative. On the basis of histological and immunohistochemical examinations GIST was diagnosed. Intestine, neoplasia, interstitial cells of Cajal, GIST Ramon y Cajal described special cells located in the vicinity of the neuronal plexuses at first in 1889 and later in 1893 and 1911 (Kobayashi et al. 1989). He supposed their function in the regulation of gut peristalsis. Later these cells were called interstitial cells of Cajal (ICC). By the end of 1980s and during 1990s some authors, e.g. Kobayashi et al. (1989), Ward et al. (1994), Huizinga (1995), Ordög et al. (1999) proved that ICC generate impulses of motoric automatism similarly as in the myocardium. The most recent results demonstrate that ICC located in the myenteric plexuses and in submucosa really possess a pacemaker function, whereas ICC situated in the interstitium of the lamina muscularis transmit signals from neurons to smooth muscle cells (Mitsui and Komuro 2003; Iino and Horiguchi 2006; Lee et al. 2007). A tumour arising from ICC is named the gastrointestinal stromal tumour (GIST), and is considered as a low-grade sarcoma harbouring mutation of c-kit. GIST was first described in humans in the 1980s, and the term GIST was first coined by Mazur and Clark in 1983 (Ozcan et al. 2007). Banerjee et al. (1991) have described GIST in two Rhesus macaques (Macaca mulatta), Del Piero et al. (2001) and Hafner et al. (2001) diagnosed it in horses, and Frost et al. (2003) characterized and analyzed 29 cases of GIST in dogs. To the best of our knowledge, the histogenesis of a tumour of ICC has not been described in a guinea pig until now. For this reason we publish our observation. Case description The animal Guinea pig, male, 3.5 year of age, tricolour, was kept as a pet animal in a household. Approximately for five weeks before death wasting, in spite of good appetite, decreased locomotor activity, soft and malodorous faeces were observed by the owner. Veterinary clinician revealed by palpation a globoid formation of walnut size in the abdominal cavity. Some days later the animal died spontaneously. ACTA VET. BRNO 2009, 78:287–291; doi:10.2754/avb200978020287 Address for correspondence: Prof. MVDr. František Jelínek, CSc. Veterinary Histopathological Laboratory Sojovická 16 197 00 Prague 9, Czech Republic E-mail: jelinekvet@seznam.cz http://www.vfu.cz/acta-vet/actavet.htm Gross pathology Necropsy was performed by the veterinary clinician. Apart from mild hydrothorax, ascites and atelectasis in the part of the pulmonary lobes, a tumour of grey-pink colour that grew from the terminal segment of the ileum was observed (Plate IX, Fig. 1). Inside the neoplastic formation a cavity communicating directly with lumen of the bowel was found, filled with intestinal contents. No other pathological changes were macroscopically obvious. A sample of the neoplasia was submitted to histopathological examination. Methods of histopathological examination The sample was fixed in 10% buffered formalin and processed by the common paraffin wax method. Histological sections 5 μm thick were stained with haematoxylin and eosin. Immunohistochemical examination was performed by means of a common immunoperoxidase method on paraffin wax sections attached to slides treated with 3-aminopropyltriethoxysilane (Fluka Chemie AG). After the usual deparaffinisation the antigens were unmasked by boiling in 0.1 mol/l citrate buffer, pH 6.0 for 10 min, and endogenous peroxidase acitivity was eliminated with 3% hydrogen peroxide for 15 min at room temperature. Non-specific binding was suppressed with Protein Blocking Agent (Immunotech) for 5 7 min at room temperature. The list of primary antibodies manufactured by DakoCytomation is given in Table 1. Histological sections were incubated with primary antibodies in a humidified chamber for 60 min at room temperature. Visualisation of the reaction was done by means of UltraTech HRP set (Immunotech) and UltraTech DAB set (Immunotech). The slides were counterstained with haematoxylin, dehydrated and mounted in Canada balsam. Results of histopathological examination A solid tumour of mesenchymal appearance was observed. One part of the sample consisted of tightly packed spindle cells arranged in interlacing fascicles. In the other part epithelioid cells predominated with slightly myxoid intercellular matrix. Marked anisokaryosis, oval, cigar-shaped or irregular nuclei with fine chromatin were found in the neoplastic cells. In a majority of the nuclei, especially in the large and light ones, the nucleoli were clearly apparent. On average four mitotic figures were found per one high-power field (in the range of 2-7) and a number of them were atypical. Cytoplasm of the neoplastic cells was lightly basophilic, reticular or finely vacuolated with indistinct cellular boundaries. There were also many neoplastic cells that exhibited signs of apoptosis. Lymphocytic sheaths were observed around some blood capillaries (Plate IX, Fig. 2, Plate X, Fig. 3, 4) Results of immunohistochemical examination are summarized in Table 2 and demonstrated in Figs Plate XI, Figs 5, 6, Plate XII, Figs. 7, 8.


Gross pathology
Necropsy was performed by the veterinary clinician.Apart from mild hydrothorax, ascites and atelectasis in the part of the pulmonary lobes, a tumour of grey-pink colour that grew from the terminal segment of the ileum was observed (Plate IX, Fig. 1).Inside the neoplastic formation a cavity communicating directly with lumen of the bowel was found, filled with intestinal contents.No other pathological changes were macroscopically obvious.A sample of the neoplasia was submitted to histopathological examination.

Methods of histopathological examination
The sample was fixed in 10% buffered formalin and processed by the common paraffin wax method.Histological sections 5 µm thick were stained with haematoxylin and eosin.Immunohistochemical examination was performed by means of a common immunoperoxidase method on paraffin wax sections attached to slides treated with 3-aminopropyltriethoxysilane (Fluka Chemie AG).After the usual deparaffinisation the antigens were unmasked by boiling in 0.1 mol/l citrate buffer, pH 6.0 for 10 min, and endogenous peroxidase acitivity was eliminated with 3% hydrogen peroxide for 15 min at room temperature.Non-specific binding was suppressed with Protein Blocking Agent (Immunotech) for 5 -7 min at room temperature.The list of primary antibodies manufactured by DakoCytomation is given in Table 1.Histological sections were incubated with primary antibodies in a humidified chamber for 60 min at room temperature.Visualisation of the reaction was done by means of UltraTech HRP set (Immunotech) and UltraTech DAB set (Immunotech).The slides were counterstained with haematoxylin, dehydrated and mounted in Canada balsam.

Results of histopathological examination
A solid tumour of mesenchymal appearance was observed.One part of the sample consisted of tightly packed spindle cells arranged in interlacing fascicles.In the other part epithelioid cells predominated with slightly myxoid intercellular matrix.Marked anisokaryosis, oval, cigar-shaped or irregular nuclei with fine chromatin were found in the neoplastic cells.In a majority of the nuclei, especially in the large and light ones, the nucleoli were clearly apparent.On average four mitotic figures were found per one high-power field (in the range of 2-7) and a number of them were atypical.Cytoplasm of the neoplastic cells was lightly basophilic, reticular or finely vacuolated with indistinct cellular boundaries.There were also many neoplastic cells that exhibited signs of apoptosis.Lymphocytic sheaths were observed around some blood capillaries (Plate IX, Fig. 2, Plate X, Fig. 3

Discussion
Gastrointestinal stromal tumour (GIST) was first described in humans in the 1980s as a non-lymphomatous, non-epithelial tumour of the gut and the term GIST was first coined by Mazur and Clark in 1983 (Ozcan et al. 2007).LaRock and Ginn (1997) included in GIST the diagnosis of leiomyoma, leiomyosarcoma, neurofibrosarcoma and undifferentiated sarcomas.In the opinion of Cooper and Valentine (2002), the term GIST, of which leiomyoma and leiomyosarcoma are the most common subsets, is the most appropriate designation.On the other hand, by means of immunohistochemical examination, Sircar et al. (1999) classified 43 gastrointestinal mesenchymal tumours in humans into eight myoid tumours, one schwannoma, and 34 GIST.Head et al. (2003) characterize GIST as a neoplasm presumed to originate from primitive mesenchymal cells capable of pluripotent differentiation; neoplastic cells have smooth muscle, neural, or dual differentiation.GIST is considered as a low-grade sarcoma arising from the interstitial cells of Cajal and harbouring mutation of c-kit.It may be demarcated, growing expansively, usually forming exophytic nodule on the serosal surface of the intestine, or it may be transmural and invasive.In our case, a nodule on the serosal surface was prominent but transmural growth of the tumour was present.GISTs are usually comprised of spindle cells arranged as interlacing fascicles or in a storiform pattern.A minority of cases may have somewhat epithelioid tumour cells arranged in trabecule or solid sheets, sometimes with a somewhat myxoid intercellular matrix.Epithelioid tumours are thought to reflect some degree of neural differentiation.Haemorrhage or necrosis may be present, and lymphocytic and/or eosinophilic infiltrates can occur.In our case, a part of the tumour consisted of tightly packed spindle cells arranged in interlacing fascicles, and in the remaining part epithelioid cells prevailed with slightly myxoid intercellular matrix.We did not find haemorrhages and necrotic foci but lymphocytic sheaths were observed around of some blood capillaries.
In humans GIST most commonly arises in the stomach (51%), followed by the small intestine (36%), colon (7%) and rectum (1%) or esophagus (1%) (Tran et al. 2005).In domestic and farm animals GIST occurs predominantly in the intestine.Banerjee et al. (1991) described gastric stromal tumours in two Rhesus macaques (Macaca mulatta).Del Piero et al. (2001) diagnosed 11 GIST in 10 aged horses and one pony.Clinical signs were associated with two neoplasms (22%); the remaining nine tumours were incidental findings at laparotomy or necropsy.Serosal or mural masses were located in the caecum (5), ileum (3), colon (2) and one in the stomach and in the jejunum.Histology revealed spindle cells arranged in fascicles and whorls or cribriform pattern with fascicles, often with myxoid interstitial matrix.Frost et al. (2003) described and analysed 29 GIST in dogs.The mean age of the animals was 11 years.Only 24% neoplasias were associated with clinical symptoms.Majority of the tumours were located in the large intestine.In the small intestine and stomach the GIST occurred with decreasing frequency.Histologically they were highly cellular spindles, less commonly epitheloid with mitotic rates from 0 to 19 per 10 HPF.In our guinea pig clinical symptoms were present, the tumour was located in the terminal part of the ileum, histologically it was similar to GISTs described in dogs, and rather high mitotic activity with numerous atypical figures was recorded.However, no metastases were found.
In human beings GISTs express in an overwhelming majority the tyrosine kinase KIT oncoprotein, having activating mutations in the KIT gene.However, around 10 -15% of GIST lack KIT mutations and show absence of CD 117 expression as detected by immunohistochemistry, although chromosomal analysis showed losses of chromosomes 14, 22, and 1p, which is a characteristic feature of GISTs (Debiec-Rychter et al. 2004).
In a collection of 55 human GISTs by Romagnoli et al. (2005), CD117 was expressed in 96% tumours, CD34 was positive in 76% cases and the same quantity of neoplasias expressed both CD117 and CD34 antigens (Romagnoli et al. 2005).Ozcan et al. (2007) found some degree of expression of CD117, CD34, smooth muscle actin and Ki-67 in all 20 analyzed GISTs of humans.No case was reactive for desmin or S-100 protein.Feakins (2005) analyzed the expression of p53 and bcl-2 in human GISTs.Only positivity p53, but not bcl-2, showed association with the clinical outcome or risk category.Yamaguchi et al. (2003) proved that the prognosis of GIST in humans correlated with c-kit, Ki-67 and p53 positivity.
Although GISTs in animals are similar microscopically, they are a heterogeneous group of lesions via immunohistochemistry (Cooper nad Valentine 2002).Of 11 GISTs from horses three were S-100 protein positive, two were muscle actin positive, no neoplasms were positive for glial fibrillary acidic protein (GFAP), desmin, factor VIII, chromogranin, or neuron-specific enolase (Del Piero et al. 2001).In dogs, only 52% were positive for CD117, none for desmin and S-100 protein (Frost et al. 2003).In our case the immunohistochemistry revealed positivity for actin, CD 117, neuronspecific enolase, GFAP and p53 protein; however, desmin, S100 protein and synaptophysin were negative.In literature, the results of GFAP examination are not given, with the exception of Del Piero et al. (2001).In contrast to horses, GFAP in our case was positive.
On the basis of histological and immunohistochemical examinations we diagnosed GIST in the terminal ileum in one guinea pig.Although morphological properties, marked mitotic activity and p53 positivity were indicators for malignancy, no metastases or generalisation were recorded.
Fig. 1.Globoid tumourous formation located in the terminal segment of the small intestine

Table 1 .
Antibodies, their specification and dilution

Table 2 .
Results of immunohistochemical examination