Elsevier

Human Pathology

Volume 38, Issue 9, September 2007, Pages 1386-1393
Human Pathology

Original contribution
Evaluation of HER-2 gene status in gastric carcinoma using immunohistochemistry, fluorescence in situ hybridization, and real-time quantitative polymerase chain reaction

https://doi.org/10.1016/j.humpath.2007.02.005Get rights and content

Summary

HER-2 gene amplification and the overexpression of HER-2 protein have been observed in various solid tumors, including gastric carcinomas. HER-2 gene amplification has attracted research attention since the development of the new therapeutic agent trastuzumab. Here, we evaluated HER-2 status in the surgically resected tissues of 248 gastric carcinoma cases using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and real-time quantitative polymerase chain reaction (q-PCR) and compared the results. In addition, we compared clinicopathologic characteristics with the presence of HER-2 gene amplification and with protein overexpression. Among the 248 cases, 56 (22.6%) cases showed HER-2 overexpression (2+ or 3+) by IHC and 19 cases (7.7%) showed HER-2 gene amplification by FISH. Four (2.1%) of the 192 cases negative (0 or 1+) by IHC showed amplification by FISH, whereas 15 (26.8%) of the 56 cases with HER-2 protein overexpression showed HER-2 amplification by FISH. The correlation between IHC and FISH results was statistically significant (P < .001). HER-2 protein overexpression and HER-2 gene amplification were common in cases with a well- or moderately differentiated histology according to the World Health Organization classification (P < .001) and in cases of intestinal type by the Lauren classification (P < .001). Real-time q-PCR results showed that calculated HER-2/GAPDH ratios were higher in amplified cases with 100.0% sensitivity and 96.9% specificity using FISH results as the standard. Measurements of HER-2 expression by FISH and real-time q-PCR and of HER-2 protein by IHC were found to be highly concordant at determining HER-2 status in gastric carcinoma.

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.

Section snippets

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],

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    This study was supported by grant 03-2004-023 from the Seoul National University Hospital Research Fund, Seoul, South Korea.

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