Comprehensive analysis of HER2 expression and gene amplification in gastric cancers using immunohistochemistry and in situ hybridization: which scoring system should we use?

Summary

It has been reported that HER2 expression is different in gastric and breast cancers, and a gastric cancer scoring system (GCSS) has recently been suggested. We investigated HER2 protein expression using GCSS and a breast cancer scoring system (BCSS) and correlated it with HER2 gene amplification. HER2 status was evaluated in 1091 cases by analyzing tissue microarrays constructed using 2 different cores from each case. Polyclonal (HercepTest) and monoclonal (Pathway) antibodies were used for immunohistochemistry (IHC), and results were scored by BCSS and GCSS. Gene amplification was evaluated by automated dual-color silver-enhanced in situ hybridization (SISH) in all cases and correlated with the results from fluorescence in situ hybridization (FISH) in 590 cases. The concordance between the IHC results using polyclonal and monoclonal antibodies was high (κ = 0.785). The results of dual-color SISH and FISH showed very high concordance as well (κ = 0.918). GCSS was significantly more sensitive for detecting SISH positivity than was BCSS in both antibodies (polyclonal, P = .003; monoclonal, P < .001), but specificity was higher in BCSS than GCSS (polyclonal, P = .004; monoclonal, P < .001). It has been recently shown that HER2-overexpressing patients with unresectable gastric cancer benefited from trastuzumab therapy. Because IHC is recommended before gene amplification studies in HER2 testing, GCSS should be used for evaluating HER2 expression in gastric cancers.

Introduction

Although the incidence of gastric cancer is slowly decreasing worldwide [1], [2], this disease remains one of the leading causes of cancer-related deaths in Korea. In 2007, there were 25,915 new cases of gastric cancer—accounting for 16% of all cancer cases—and 10,563 gastric cancer–related deaths. The mortality rate of gastric cancer is among the highest of all types of cancer, trailing only those of lung and liver cancer [3]. As is the case for other carcinomas, gastric carcinogenesis is thought to be a multistep process involving numerous genetic or epigenetic alterations, including gene amplification, deletion, or mutation [4].

Overexpression of the human epidermal growth factor receptor 2 (HER2) protein and amplification of the HER2/ERBB2 gene have been identified in various carcinomas, including breast cancer [5], [6] and gastric cancer [7], [8]. HER2 gene amplification and HER2 protein overexpression, which occur in 20% to 25% of breast cancers, have been recognized as prognostic and predictive markers for treatment [9]. The use of humanized mouse monoclonal antibody (trastuzumab) therapy targeting the HER2 protein is now being applied not only in metastatic breast cancer cases but also to localized cases as adjuvant therapy [10], [11].

The use of molecularly targeted therapeutics against HER2 has emerged as an important strategy for advanced gastric cancers as well [12], [13], [14]. Until recently, the role of chemotherapy in gastric cancer has been generally limited; instead, early detection and radical gastrectomy of localized gastric cancer continue to be the most effective strategy for improving patient survival. However, a recent phase III randomized study (ToGA) revealed that the addition of trastuzumab to chemotherapy improved survival in patients with advanced gastric or gastroesophageal junction cancer with HER2 overexpression [15].

In the subgroup analysis of the ToGA study, the patients who benefited from trastuzumab treatment were HER2-positive via both immunohistochemistry (IHC) (3+ or 2+) and fluorescence in situ hybridization (FISH). On the other hand, IHC 0 or 1+ and FISH-positive cases did not show any survival gain. Therefore, selective trastuzumab adminstration has been recommended for IHC 3+ or IHC 2+ and FISH-positive patients; subsequently, the interpretation of IHC has become important in selecting patients for trastuzumab. In the ToGA study, a recently suggested scoring system for gastric cancer [16] was used instead of the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guideline for HER2 IHC interpretation for breast cancer [17]. Although the results of the ToGA study have confirmed that this new gastric cancer scoring system (GCSS) is valid, there has been no comparative study of which scoring system is more adequate for selecting patients for trastuzumab.

In the current study, we evaluated HER2 protein expression by using 2 different IHC methods—HercepTest (rabbit polyclonal antibody) and Pathway (rabbit monoclonal antibody)—that have been approved by the US Food and Drug Administration (FDA) for HER2 assessment in breast cancers [18]. HER2 gene amplification status was evaluated by FISH and automated dual-color silver-enhanced in situ hybridization (SISH) as well. We first estimated the concordance between the 2 IHC staining methods as well as the concordance between the 2 in situ hybridization methods. We then used BCSS and GCSS as IHC scoring criteria and investigated which scoring system showed higher sensitivity or specificity for detecting HER2 gene amplification. We also evaluated which scoring system better correlated with HER2 gene amplification results. The aims of the present study were to compare the IHC results obtained using polyclonal and monoclonal antibodies and to compare the gene amplification status using FISH and dual-color SISH. Finally, we tried to determine which scoring system is more suitable for assessment of HER2 expression in relation to HER2 gene amplification.