Original contributionEvaluation of HER-2 gene status in gastric carcinoma using immunohistochemistry, fluorescence in situ hybridization, and real-time quantitative polymerase chain reaction☆
Introduction
Gastric carcinoma is one of the leading causes of global cancer mortality and is surpassed only by lung cancer [1]. Genetic alterations including gene amplification and deletion commonly lead to neoplastic transformation, but the underlying molecular events that are critical to the development of gastric carcinoma are largely undetermined. Development of new diagnostic, preventive, and therapeutic measures requires a thorough understanding of the mechanisms of tumorigenesis in gastric carcinoma.
The HER-2 gene, also called ERBB2, is located in chromosome 17 (17q12-q21) and encodes a 185-kd transmembrane tyrosine kinase receptor (p185), which is a member of the epidermal growth factor receptor family [2], [3], [4]. Amplification of the HER-2 gene and overexpression of the HER-2 protein have been observed in various solid tumors, including breast and gastric carcinomas [5], [6], [7]. In the case of breast carcinoma, HER-2 amplification is observed in 15% to 30% of cases and is associated with adverse clinicopathologic features and outcomes [8]. Recently, HER-2 gene amplification has attracted a great deal of attention because of the development of the new therapeutic agent trastuzumab (Herceptin), which is a monoclonal antibody to the HER-2 gene product. This agent has been shown to provide effective adjuvant therapeutic option in the breast carcinoma patients with HER-2 gene amplification [9], [10], [11], [12].
A number of studies have been conducted on HER-2 gene amplification and/or overexpression in gastric carcinoma [7], [12], [13], [14]. Although the frequency of HER-2 amplification has been variously reported, recent studies show amplification rates of 8.2% to 15% [7], [13], [14]. HER-2 gene amplification in gastric carcinoma is associated with an intestinal-type histology and poor survival [12], [13], [14]. However, no large-scale clinical study has been performed to determine response to trastuzumab therapy in gastric carcinoma patients with HER-2 gene amplification. A case report and cell line and xenograft model studies show that trastuzumab has a growth inhibitory effect in HER-2 amplified and/or overexpressed gastric carcinomas [14], [15], [16]. Therefore, the accurate evaluation of HER-2 gene amplification status in gastric carcinoma patients has become important in determining eligibility for trastuzumab therapy.
HER-2 status in tumor samples can be determined using various methods. Immunohistochemistry (IHC), Western blotting, and enzyme-linked immunosorbent assay are currently used to measure HER-2 protein expression; and HER-2 gene amplification can be detected by Southern blotting, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization, and real-time quantitative polymerase chain reaction (q-PCR). In practice, IHC and FISH methods are routinely used in cases of breast carcinoma. IHC is faster and more economical, but is highly dependent on quality control and is difficult to standardize. FISH is regarded as the criterion standard for the determination of the presence of HER-2 gene amplification, but it is expensive in terms of equipment and is labor intensive. Furthermore, FISH is not valuable when tissue samples are not well fixed or fixed in the Bouin fixative. To overcome these limitations, alternative methods such as real-time q-PCR are used experimentally in breast carcinoma cases; and the reported results show relatively good concordance rates [17], [18], [19]. However, comparative multimethodological measurements of HER-2 status, including real-time q-PCR, have not been studied in cases of gastric carcinoma.
In the present study, we evaluated HER-2 status in 248 cases of gastric carcinoma using IHC, FISH, and real-time q-PCR. Initially, we compared the IHC and FISH methods to clarify whether the mechanism of gene amplification and protein overexpression in gastric carcinoma is the same as in breast carcinoma. Afterward, we examined the clinicopathologic characteristics of HER-2 amplified and/or overexpressed cases for the certification of the previous reports. Finally, to assess the accuracy and usefulness of the alternative method, the result of real-time q-PCR methods was compared with the result of the criterion standard FISH method.
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Cell lines
Ten human gastric cancer cell lines—SNU-1, SNU-5, SNU-16, SNU-216, SNU-484, SNU-601, SNU-620, SNU-638, SNU-668, and SNU-719—were included in this study. All cell lines were originated from Korean gastric carcinoma patients. Cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea) and maintained in RPMI-1640 (GIBCO, Grand Island, NY) containing 10% fetal bovine serum (HyClone, Logan, UT), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma Chemicals, St Louis, MO) at 37°C in a
HER-2 protein expression status and clinicopathologic findings in 248 gastric carcinoma cases
HER-2 protein expression status was determined by IHC for the 248 gastric carcinoma tissues (Fig. 1A-C). Of these, 91 cases (36.7%) scored 0; 101 cases (39.1%), 1+; 46 cases (16.5%), 2+; and 10 cases (4.0%), 3+. Scores of 0 and 1+ were regarded as negative for HER-2 overexpression and scores of 2+ and 3+ as positive. Table 2 summarizes HER-2 protein expression results and clinicopathologic findings. Gastric carcinomas with HER-2 overexpression showed a predominantly well- or moderately
Discussion
Carcinogenesis in the stomach, like that in other organs, is a multistep process involving the accumulation of several genetic alterations, which include the activations of oncogenes and the inactivations of tumor suppressor genes [20], [21]. The several genetic alterations associated with gastric carcinomas showing HER-2 overexpression at the DNA or protein levels may form a specialized subgroup in terms of their clinicopathologic characteristics and optimal therapeutic approaches [7], [12],
References (28)
- et al.
Localization of the human erbB-2 gene on normal and rearranged chromosomes 17 to bands q12-21.32
Genomics
(1989) - et al.
HER2 overexpression in various tumor types, focusing on its relationship to the development of invasive breast cancer
Ann Oncol
(2001) - et al.
Targeting HER2 in other tumor types
Ann Oncol
(2001) - et al.
Amplification of HER-2 in gastric carcinoma: association with topoisomerase IIα gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab
Ann Oncol
(2005) - et al.
A genetic model for colorectal tumorigenesis
Cell
(1990) - et al.
Prognostic significance of p53, nm23, PCNA and c-erbB-2 in gastric cancer
Jpn J Clin Oncol
(2003) - et al.
Alterations of the CCND1 and HER-2/neu (ERBB2) proteins in esophageal and gastric cancers
Cancer Genet Cytogenet
(2006) - et al.
Estimates of the worldwide incidence of 25 major cancers in 1990
Int J Cancer
(1999) - et al.
The product of the human c-erbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity
Science
(1986) - et al.
Characterization of epidermal growth factor receptor gene expression in malignant and normal human cell lines
Proc Natl Acad Sci U S A
(1984)
Status of c-erbB-2 in gastric adenocarcinoma: a comparative study of immunohistochemistry, fluorescence in situ hybridization and enzyme-linked immuno-sorbent assay
Int J Cancer
HER-2/neu (c-erb-B2) gene and protein in breast cancer
Am J Clin Pathol
Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2
N Engl J Med
Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer
N Engl J Med
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This study was supported by grant 03-2004-023 from the Seoul National University Hospital Research Fund, Seoul, South Korea.