BRIEF REPORT |
C-kit Gene Product (CD117) Immunoreactivity in Canine and Feline Paraffin Sections
Department of Veterinary Public Health and Animal Pathology, Alma Mater Studiorum, University of Bologna, Bologna, Italy
Correspondence to: Dott.ssa Maria Morini, Dipartimento di Sanità Pubblica Veterinaria e Patologia Animale, Servizio di Anatomia Patologica, Facoltà di Medicina Veterinaria, Via Tolara di Sopra, 50 40064 Ozzano Emilia (Bologna), Italy. E-mail: bettini{at}vet.unibo.it
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Key Words: CD117 c-kit dog cat immunohistochemistry mast cell tumors GIST
THE PROTO-ONCOGENE c-kit is the cellular homologue of v-kit, an oncogene derived from the feline retrovirus HZ4-FeSV. It encodes a 14.5-kD transmembrane receptor (KIT or CD117, or stem cell factor receptor), belonging to the class III receptor tyrosine kinase family, and the ligand for KIT is the stem cell factor protein, also known as mast cell growth factor. KIT plays a key role during fetal development, and its expression is constitutively maintained in hemopoietic stem cells, mast cells, intraepithelial lymphocytes, germ cells, melanocytes, and interstitial cells of Cajal (ICCs), where it acts as a growth factor receptor (Gibson and Cooper 2002).
In human medicine, the expression of c-kit has been accurately evaluated in normal tissues and in a large number of neoplastic tissues by molecular techniques, flow cytometry, frozen section immunohistochemistry and, more recently, by paraffin section immunohistochemistry (Matsuda et al. 1993; Tsuura et al. 1994
; Arber et al. 1998
). Mast cells, melanocytes, breast ductal epithelium, seminiferous tubules, a subset of glial cells, ovarian stroma, follicles, corpora lutea, oocytes, and ICCs comprise the cellular substrate in which the immunohistochemical expression of CD117 is clearly evident, with a prevailing cytoplasmic distribution of the reactivity (Arber et al. 1998
; Gibson and Cooper 2002
). Among tumors, CD117 expression has been detected in acute myeloid leukemia, malignant melanomas, gastrointestinal stromal tumors (GISTs), small-cell lung carcinomas, germ cell tumors, and systemic mast cell disorders (Izquierdo et al. 1995
; Arber et al. 1998
; Miettinen and Lasota 2001
).
In animal tissues, CD117 expression has been previously demonstrated by IHC in normal and neoplastic canine mast cells (London et al. 1996; Reguera et al. 2000
), in the ICCs of dog, mouse, and rat (Komuro 1999
), and in canine GISTs (Frost et al. 2003
; Bettini et al. 2003
). Nevertheless, CD117 distribution has not been extensively studied in canine tissues and has never been examined in the cat.
The present study was performed to further elucidate the IHC expression and distribution of the KIT protein in normal and neoplastic canine and feline tissues, using a polyclonal rabbit antiserum against c-kit, previously used in human (Tsuura et al. 1994; Arber et al. 1998
) and canine tissues (London et al. 1996
; Bettini et al. 2003
; Frost et al. 2003
) that specifically recognizes the C' terminus of the c-kit intracellular domains, highly conserved among species. Forty-four formalin-fixed, paraffin-embedded samples of normal tissues (30 dogs and 14 cats) from eight different organ sites (skin, mammary gland, gastrointestinal tract, testis, ovary, uterus, adrenal gland, cerebellum) and 104 samples of different kinds of tumors (74 dogs and 30 cats) were selected. Tumors were histologically classified according to WHO criteria on hematoxylineosin-stained sections. IHC was performed with an avidinbiotinperoxidase method. Sections were incubated with 0.3% hydrogen peroxide in methanol for 20 min to block endogenous peroxidase activity and microwave-treated for antigen retrieval in citrate buffer solution at pH 6.0. After overnight incubation with the primary antibody (polyclonal rabbit anti-human KIT protein, CD117, DAKO; Dakopatts, Glostrup, Denmark) at a 1:500 dilution in PBS, the sections were incubated with the secondary antibody (anti-rabbit IgG conjugated with biotin) and then with the streptavidinperoxidase complex. The sections were then immersed for 12 min in DAB chromogenic substrate solution (diaminobenzidine 0.02% and H2O2 0.001% in PBS), counterstained with Papanicolau hematoxylin, dehydrated, and mounted with DPX (Fluka; Riedel-de Haën, Germany). As positive control, the reaction was tested on mast cells in an appropriate tissue section from normal human skin and subcutaneous tissue. As negative control, PBS was used in replicated sections instead of the primary antibody. Tumors were considered positive if more than 5% of neoplastic cells showed immunoreactivity. The immunostaining results were recorded with respect to intensity (strong, moderate, or weak), distribution (focal <30% of cells; intermediate >30% but <70% of cells; and diffuse >70% of cells) and cellular localization of the immunostaining (cytoplasmic, nuclear, membranous, paranuclear, in the luminal side).
The IHC results for CD117 expression are summarized in Tables 1 and 2.
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Our results have shown that CD117 IHC in normal tissues of dogs and cats has a similar pattern of reactivity. The KIT receptor is expressed by a limited number of cells, with a distribution comparable to that of human tissues in mast cells, ICCs, Purkinje cells, endometrium, and epithelial cells (ducts and acini) of the mammary gland. Nevertheless, epidermal basal cells, germinal cells of the testis, oocytes, and medullary cells of adrenal gland stain moderately to strong in human tissue, whereas in our samples these compartments did not react to CD117 IHC.
Mast cell tumors expressed CD117 with constant and significant intensity. Neoplasms of mast cells are the most common cutaneous malignant tumors of dogs, representing between 721% of all tumors, an incidence much higher than that found in humans (London et al. 1996). In our cases, CD117 immunopositivity was found in both cell membrane and cytoplasm, sometimes with paranuclear aggregates of focal strong accentuation of the positivity. Although further studies are needed to elucidate the correlation between the pattern of IHC expression of KIT and the biological behavior of mast cell tumors, our preliminary data suggest that, in the dog, the cytoplasmicparanuclear staining is correlated with a significantly shortened survival time (personal observation).
In gastrointestinal (GI) tract pathology, a subset of primary mesenchymal tumors, called GISTs, has been recently recognized and defined as CD117-positive spindle or epithelioid neoplasms (Miettinen and Lasota 2001). GISTs have been also documented in dogs, but not yet in cats, by their KIT immunopositivity (Bettini et al. 2003
; Frost et al. 2003
), and histologically they appeared as densely packed spindle cells, quite similar in features to smooth muscle cell tumors and commonly misdiagnosed as leiomyomas or leiomyosarcomas. The ICCs, a complex cellular network postulated to act as pacemaker cells of the GI tract and responsible for slow and rhythmic contractions of the smooth muscle, are believed to represent the cells of origin for GISTs (Miettinen and Lasota 2001
). Therefore, as in humans, CD117 may be a useful diagnostic marker for canine and feline GISTs.
In our study, other sporadic examples of tumors have shown weak to moderate focal staining for CD117 (benign and malignant mammary tumors, amelanotic melanomas, testicular and ovarian tumors). In human pathology, seminoma is another tumor, in addition to GISTs and mast cell disorders that reveals constant strong, diffuse, and membranous KIT positivity, whereas in our results only one case (of two) disclosed weak, focal and cytoplasmic immunoreactivity. However, the meaning of these discrepancies between human and animal findings is not still clear and requires further investigation. In a previous report (Matsuda et al. 1993), pheochromocytoma also showed a significant positivity for CD117 (seemingly nuclear), but this could be related to the use of different antibodies (monoclonal vs polyclonal) because in later studies (Tsuura et al. 1994
; Arber et al. 1998
), by the use of a polyclonal antibody, this neoplasm did not react against CD117.
The results of our study show that CD117 can be considered a reliable and useful marker in canine and feline tissues. Therefore, it may play a decisive role as a diagnostic marker for canine and feline GISTs and as a significant prognostic factor for canine and feline mast cell tumors.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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