Gene amplification and mRNA and protein overexpression of c-erbB-2 (HER-2/neu) in human intrahepatic cholangiocarcinoma as detected by fluorescence in situ hybridization, in situ hybridization, and immunohistochemistry
Introduction
The human proto-oncogene c-erbB-2 (also called HER-2/neu) is mapped to chromosome 17q21-22 [1]. It is highly homologous to the epidermal growth factor receptor, and encodes a 185 kDa transmembrane protein (p185) with tyrosine kinase activity [2]. p185 consists of an external domain linked to an internal domain by a transmembrane region [3]. Although p185 is a receptor for a still poorly characterized growth ligand, it is assumed to be involved in the regulation of cell growth and differentiation [4]. The oncogenic activation of c-erbB-2 results in heterodimerizing with large amounts of the non-mutated receptor on the cell surface [5], [6], [7]. Gene amplification and overexpression of this oncogene have been reported in many human carcinomas, including breast [8], stomach [9], [10], bladder [11], ovary [12], prostate [13], endometrium [14], and some neuroendocrine neoplasms [15] such as carcinoid and thyroid tumors. It has been shown that c-erbB-2 gene amplification and/or overexpression in primary breast cancer is an independent biomarker of poor prognosis and has been shown to enhance the malignant potential and metastatic phenotypes [8], [16], [17], [18].
Human intrahepatic cholangiocarcinoma (CC) is a highly malignant epithelial cancer of the biliary tract with high morbidity and mortality, whose cellular and molecular pathogenesis remains unclear. In some CCs, specific epidemiological features were attributed to infection by the liver flukes such as Opisthorchis viverrini and Clonorchis sinensis [19], [20], [21]. Other relevant risks reported to be strongly associated with CC are Thorotrast exposure [22], choledochal cyst [23], primary sclerosing cholangitis [24], Caroli's disease [25], and hepatolithiasis [26], [27]. However, despite the current knowledge of aetiology and pathology of CC, its cellular and molecular pathogenesis is still poorly understood. In CC, c-erbB-2 protein overexpression has rarely been reported [28], [29], [30], [31], [32]. In addition, the results of c-erbB-2 protein overexpression in those studies are conflicting [33]. While 27–73% of tumors were found to express c-erbB-2 in CC of Thai, English, Taiwanese, and American origin [29], [30], [31], Collier et al. [28] were unable to detect the expression of membranous c-erbB-2 in ten cases of human CCs of European Caucasian origin by immunohistochemistry. In Japan, it was also indicated that positive immunohistochemical staining of c-erbB-2-encoded protein in CC correlated with a shorter survival [33]. Overexpressions of the c-erbB-2 protein and amplifications of this gene have been reported by in situ hybridization (ISH) techniques, Southern blotting technique or immunohistochemistry in various human carcinomas, including breast carcinomas [8], [9], [10], [11], [12], [13], [14], [15], [34]. However, the overexpression of the protein in CC was detected only by immunohistochemistry [28], [29], [30], [31], [32], and amplification of this gene and mRNA status of this gene in CC have not been clarified as yet.
Therefore, we aimed to examine the amplification of the c-erbB-2 gene in CC by fluorescence in situ hybridization (FISH), the mRNA status by ISH, and the protein expression by immunohistochemistry. Further, we examined the correlations between these and several clinicopathological features in CC.
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Human tissue specimens
We examined a total of 22 archival CCs retrieved from the surgical and autopsy files at our laboratories (Table 1). The ages of the patients ranged from 19 to 81 years, with an average of 64 years. The male to female ratio was 6:5. The 22 cases of CC were all adenocarcinomas and were classified into well-differentiated CC (ten cases), moderately differentiated CC (seven cases), and poorly differentiated CC (five cases). These tissues were fixed in 10% neutral buffered formalin and embedded in
FISH
FISH analysis was successful in all 22 cases of CC. All the tumors contained cancer cells with c-erbB-2 amplification (Table 1). In all cases, neoplastic cells were found to have red signals as shown in Fig. 1A. In some cases, amplification was detected as distinct red signals. In the other cases, amplification was mostly detected as a cluster signal (CS) or multiple scattered signals (MSS). The copy number ranged from 5.20±2.90 (Case No. 1) to 17.60±7.60 (Case No. 22) (Table 1). Amplification
Discussion
The present study is the first report of c-erbB-2 gene amplification by FISH in CC. Although FISH has become an increasingly popular technique to determine the c-erbB-2 status in carcinomas, it is difficult to compare the results because of the variability in criteria to define amplification. Our results revealed that c-erbB-2 amplification was detected as a CS pattern in most CC cases and they were concordant with the evaluation of gastric carcinomas by Ooi et al. [36]. The CS/MSS pattern was
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