Lack of CD117 and rare bcl-2 expression in stomach cancer by immunohistochemistry. An immunohistochemical study with review of the literature

Gastric carcinoma is the second most frequent malignancy in the world constituting about 10% of newly diagnosed malignancies and accounting for more than 12% of cancer deaths [8]. It is the disease of the elderly with a median age of 72 years in American patients at the time of diagnosis [8]. Immunohistochemically (IHC), almost all tumors are positive for cytokeratin and epithelial membrane antigen [43]. Furthermore, gastric carcinomas have been shown to be positive for following factors in different proportions: Pancreatic Secretory Trypsin Inhibitor (76% and 89% in intestinal and diffuse types respectively) [19], carcinoembryonic antigen (30–55 %), Villin (47 %), Beta Catenin (63%), CD44 (55–58%), [22, 43] and Placental Alkaline Phosphatase (23%) [49]. Mutations of the p53 protein and abnormalities of the Adenomatous Polyposis Coli (APC) gene were found in about 50% of cases [1]. In the literature, only one study examining the expression of CD117 in gastric carcinomas exists to our knowledge until now [18]. This study reports an expression of the protooncogene c-kit and its ligand stem cell factor (SCF) in gastric carcinoma cell lines [18]. In vivo, the potential expression of the protein in human gastric carcinoma has not been analyzed, and no statement about the potential clinical significance of the in vitro study has been given to our knowledge.

Classically gastric carcinomas are classified to intestinal and diffuse type according to the Lauren classification [29]. Although the role of adjuvant therapies in advanced gastric cancer is under debate, generally surgical resection is considered as the standard therapy and response to other methods of treatment is poor [46].

Gastric carcinomas are usually detected at advanced tumor stages. The factors involving the miss an early diagnosis comprise anatomical and clinical reasons. Anatomically, the stomach has a very large luminal space and the tumors can reach quite a large size prior to inducing pain or symptoms of obstruction. From the clinical point of view, symptoms of gastric carcinoma are equivalent to benign lesions such as chronic gastritis or peptic ulcer. Often, these lately diagnosed tumors are no longer amenable to surgery. Population based gastric cancer screening programs have not been implemented in most countries due to lack of sensitive and specific serum tumor markers, high economic burden, and poor compliance of routine gastroscopy.

Thus, it seems reasonable to investigate in new therapeutic modalities for advanced gastric cancer. Immunotargeting and blocking specific tumor growth pathways is one of the new promising strategies. Successful examples include regimes against CD20 and Her-2-neu in non-Hodgkin's B cell lymphoma (NHL) and advanced stage breast carcinoma, respectively [5, 23]. Chimeric anti-CD20 antibody (rituximab) sensitizes B cells in NHL to inducing apoptosis by cytotoxic substances, for example, by diminishing the Bcl-x expression [23]. Inhibitor of Her-2-neu protein (trastuzumab) has been reported to induce a 19% response rate in metastatic breast carcinoma patients [5]. An additional agent is associated with the expression and inhibition of CD117 (c-kit), and has been effectively used in various neoplasms such as hematological malignancies and gastrointestinal stromal tumors (GIST). Thus, these inhibitors could be potentially considered as a new target for therapeutic regimes in gastric carcinomas; however, only if a significant CD117 expression could be demonstrated, i.e., by use of IHC.

Paraffin blocks of 38 cases with gastric carcinoma operated on between 11^th March 1999 and 5^th September 2003 were collected from the archive of the Pathology Laboratory of the Afzalipour Hospital. Representative tumor blocks were selected for immunohistochemistry. A paraffin block of a case with a gastrointestinal stoma tumor (GIST) was used for positive control. Immunohistochemistry was performed by application of the Avidin-Biotin Complex (ABC) method for CD117 and bcl-2. Both primary antibodies and LSAB visualization kit were commercially available from DAKO, Copenhagen, Denmark. The heat – induced method (HIER) was applied for antigen retrieval by tris-EDTA buffer PH9. The primary antibody dilution was set to 1/400 for CD117 and to 1/40 for bcl-2 respectively. The deparaffinized and rehydrated slides were incubated with the primary antibodies for 20 minutes, after thorough washing with the secondary antibody for 10 minutes, with streptavidin for 10 minutes, and liquid Diaminobenzydine (DAB) for 5 minutes. Cases were grouped positive if a dark brown reaction in the tumor cells was noted.

The mean age of gastric carcinoma cases in this study (56 years) is significantly lower than that of population based incidence (70 years). Most of our cases had been operated on in advanced tumor stages, and account to a percentage of 86% in stage three and higher.

Obviously, a potential curative surgical and chemotherapeutic treatment of such advanced, poorly differentiated, and aggressive tumors is not possible. Therefore, new therapeutic approaches should be searched for.

One potential regime could be the treatment with imantinib, a substance that originally served for the inhibition of the abl-bcr fusion gene product in chronic myelogenous leukemia. It can also act as a kit tyrosine kinase inhibitor, and, therefore, its expression has been analyzed in GIST and a broad variety of other malignancies [34]. In fact, some oncologists request kit IHC stains when they are confronted with mesenchymal tumors in order to potentially apply imatinib for treatment [34]. Surprisingly, the CD117 expression in gastric carcinomas was not clearly evident although it has been investigated in numerous tumors, such as germ cell or melanocytic malignancies. We could only evaluate one article that describes an over expression of the CD117 gene in gastric carcinoma cell cultures [18].

In our study, none of the studied 38 cases displayed a CD117 expression immunohistochemically. Therefore, it seems to be unlikely, that CD117 plays a remarkable role in gastric carcinoma formation and progression. In addition, its potential application as a new target for therapeutic strategies is questionable. This statement holds true, even if more sensitive methods like fluorescent in situ hybridization (FISH) may detect a CD117 expression in gastric carcinoma tumor cells without corresponding IHC results. Miettinen et al. have examined kit-positive angiosarcomas for kit mutations and could not detect any abnormalities in the region typically mutated in GIST [34]. Thus, a positive IHC reaction cannot imply the application of anti kit drug regimes per se. On the other hand, mutations of CD117 genes might be present in the development of gastric carcinomas, and cannot be excluded by our study, as the applied antibodies are only directed against the "wild type" protein formation.

A similar situation was found for bcl-2 expression, which could be demonstrated intra-cytoplasmatically in only one case of a diffuse gastric carcinoma. Thus, the majority of gastric carcinomas (if not all) are immunohistochemically negative for bcl-2. These results are in agreement with the data reported by Wang and his colleagues. The authors reported only 2 bcl-2 positive cases out of 30 gastric carcinomas [47]. Fricke et al. reported a higher percentage of immunohistochemical reactivity (5/24) for bcl-2 in a study performed on 24 cases of diffuse type gastric carcinomas [15]. All of these cases were concurrently positively associated with E-cadherin mutations [15]. But there are increasing reports on Bcl-2 reactivity in gastric cancer with much higher frequency (11, 14). Chen and colleagues reported 80.56% of immunohistochemical reactivity of gastric cancer cells to bcl-2 protein which was irrespective to tumor location, histologic type of cancer, and lymph node metastasis status (11). A report from Brazil shows 45% of IHC reactivity of tumor cells for bcl-2 protein (14). In both studies different types of antibody were used. In Chen study a specific method of assessment was considered for quantitative and qualitative evaluation of IHC reaction for bcl-2 (11). Considering low frequency of reactivity of gastric cancer cells in many reports including that of us, it seems to be rational to perform a large scale and inter-institutional study using specific measurements as electronic measurements to verify the exact rate of bcl-2 reactivity in gastric cancer.

Summarizing these data and including our own results the expression of CD117 in gastric carcinoma seems to be a very unlikely event and can not practically considered as a potential target of therapy. The expression of bcl-2 in gastric carcinoma seems to be quite rare and currently its clinical significance is unclear. But further large studies needed to perform in order to verify the exact rate of bcl-2 reactivity in gastric carcinoma.