Human epidermal growth factor receptor 2 (HER2) overexpression is increasingly recognized as a frequent molecular abnormality in gastric and gastroesophageal cancer. With the recent introduction of HER2 molecular targeted therapy for patients with advanced gastric cancer, determination of HER2 status is crucial in order to select patients who may benefit from this treatment. This paper provides an update on our knowledge of HER2 in gastric and gastroesophageal cancer, including the prognostic relevance of HER2, the key differences between HER2 protein expression interpretation in breast and gastric cancer, the detection methods and the immunohistochemistry scoring system.

Human epidermal growth factor receptor 2 (HER2), also known as CerbB-2 and ERBB2, is a proto-oncogene located on chromosome 17q21 that encodes a transmembrane protein with tyrosine kinase activity, a member of the HER receptor family and is involved in signal transduction pathways, leading to cell growth and differentiation[1].

Amplification of the HER2 gene and overexpression of its product were first discovered in breast cancer and are significantly associated with worse outcomes[2]. Many studies have demonstrated that HER2 is also present in several other malignancies, including colorectal cancer, ovarian cancer, prostate cancer, lung cancer and, particularly, gastric and gastroesophageal cancer[3].

In gastric and gastroesophageal cancer, the frequency of HER2 overexpression varies widely in the literature; studies have yielded inconsistent findings regarding its prognostic relevance[4-12]. With the recent introduction of trastuzumab for the treatment of patients with advanced gastric cancer, the clinical demand for HER2 assessment is rapidly increasing. However, HER2 testing in gastric cancer differs from testing in breast cancer because of inherent differences in tumor biology, intratumoral heterogeneity of HER2 expression and incomplete membrane staining that are commonly observed in gastric tumors[13].

This paper aims to summarize the current evidence regarding HER2 in gastric and gastroesophageal cancer and to provide a practical update on HER2 testing and scoring that is essential for appropriate selection of patients who are eligible for treatment with trastuzumab.

The frequency of HER2 overexpression in gastric and gastroesophageal cancer ranges from 4.4% to 53.4%, with a mean of 17.9%[4-14].

Although some small-scale studies have not demonstrated the prognostic properties of HER2[4,5,9,12], a larger number of studies indicate that HER2 is a negative prognostic factor, showing more aggressive biological behavior and higher frequencies of recurrence in HER2-positive tumors[1,6-8,11,14].

Given this controversy of HER2 prognostic values, a systematic review of a large number of studies was recently conducted in order to address this issue[14]. Forty-two publications with a total of 12749 patients were reviewed; the majority (71%) of the publications showed that a HER2-positive status was associated with decreased survival and clinicopathological features of tumor progression, such as serosal invasion, metastases and higher disease stage[14]. The results clearly set HER2 as a negative prognostic factor, suggesting that HER2 overexpression/amplification is a molecular abnormality that might be associated with the development of gastric cancer[7,14].

Trastuzumab is a monoclonal antibody directed against HER2; as one of the first molecular-targeted drugs to be developed, it was first introduced for the treatment of HER2-positive advanced breast cancer[2].

There is no consensus on the mechanism in which trastuzumab acts in cancer cells, but the evidence is that in addition to preventing dimerization of HER2 with other HER family members and stimulating endocytosis, it seems to induce cell mediated immunity and inhibit angiogenesis[15].

In the ToGA trial, patients with HER2-expressing unresectable gastric and gastroesophageal tumors were treated with chemotherapy and trastuzumab or with chemotherapy alone. A statistically significant increase in overall survival was observed in patients who received trastuzumab[16].

Although only a modest improvement of 2.7 mo in the median overall survival was observed in HER2-positive patients with the addition of trastuzumab, according to the ToGA trial, there was an improvement of 4.2 mo in the median overall survival in a post-hoc analysis[14,16-18].

Other molecular HER2-targeted agents have been tested or are currently being tested such as pertuzumab, lapatinib, the antibody-drug conjugate trastuzumab-emtansine (TDM-1)[19-23] and afatinib (NIH study trial registration number NCT01522768; ClinicalTrials.gov). However, the efficacy of these agents has been shown to be either unsatisfactory or as modest as trastuzumab[22,24]. Trastuzumab is the first molecular targeted agent approved as a standard treatment in gastric cancer, but it remains under investigation for more potent utilization.

Thus, it is imperative to determine the HER2 status in advanced gastric or gastroesophageal junction adenocarcinoma in order to select patients who may benefit from this promising treatment.

HER2 status is mainly assessed by immunohistochemistry (IHC) or in situ hybridization (ISH) assays. Both methods can be done on formalin-fixed and paraffin-embedded biopsy tissues or surgical specimens and occasionally, cytological samples[25]. Fluorescent in situ hybridization (FISH) is regarded to be the gold standard; however, because of its higher cost and time consumption, as well as the need for a fluorescence microscope, generally only equivocal cases are subjected to this technique. Furthermore, the high concordance between FISH and IHC that is reported in the literature supports the use of IHC, the most familiar and readily accommodated method in most surgical pathology laboratories[26-29].

Thus, IHC should be used as the first screening method for HER2 evaluation and those cases with results considered equivocal for HER2 overexpression (2+) should be referred for FISH analysis or other alternative in situ hybridization method[28] (Figure 1). A simple and practical alternative to FISH for these equivocal cases is provided by the employment of other in situ hybridization techniques such as silver in situ hybridization (SISH), chromogenic in situ hybridization and dual-color dual-hapten in situ hybridization. These three methods can be easily analyzed under a conventional bright field microscope and have shown excellent correlation with results obtained by FISH[30-32].

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Figure 1 Human epidermal growth factor receptor 2 testing algorithm. HER2: Human epidermal growth factor receptor 2; IHC: Immunohistochemistry; FISH: Fluorescent in situ hybridization; CEP17: Chromosome 17.

Because IHC is the easiest, least expensive and most widespread method of HER2 assessment, this paper focuses on IHC. Table 1 shows the different HER2 methods and their advantages and disadvantages.

Differences between HER2 expression in breast and gastric cancer

The key differences between HER2 expression in breast and gastric and gastroesophageal cancer are listed[17,30]: (1) the membranous distribution of the antibody in the neoplastic cells of breast cancer is predominantly circumferential, whereas in gastric cancer, it is generally incomplete, predominantly basolateral (“U”-shaped) or lateral (parallel lines) (Figure 2). Thus, unlike for breast cancer, circularity of IHC staining is not a criterion for HER2 IHC scoring in gastric cancer; (2) intratumoral heterogeneity, defined as the presence of areas with different HER2 scores within the same tumor, i.e., focal or patchy positivity, is a common pattern encountered in gastric tumors but is only rarely seen in breast cancer (Figure 3). It may cause sampling errors when randomly sampled biopsies are examined (see below). Although the causes of intratumoral heterogeneity of HER2 expression are not yet fully understood, some studies indicate that it could be explained merely by tumor inherent genetic heterogeneity[33,34]. Since Helicobacter pylori (H. pylori) is widely accepted as the main causative agent of gastric cancer[35], we speculate whether among the diverse bacterial factors, concomitant infection with different strains and diverse host responses there could be a reasonable link with HER2 intratumoral heterogeneity. Interestingly, Tegtmeyer et al[36] showed that some H. pylori strains could in fact activate HER2, while infection with other strains suppressed HER2 activity. However, this correlation of the bacterium with HER2 intratumoral heterogeneity is still a matter of debate and requires further studies; and (3) variation of the incidence of HER2 expression with anatomic location does not occur in breast cancer, whereas it is more frequent in the proximal stomach, including the esophageal-gastric junction, than in the distal stomach. With the introduction of the seventh edition of TNM classification, a large number of tumors that were formerly categorized as gastric are now considered as esophageal and gastroesophageal junction tumors instead, with relatively high HER2-positivity rates in these primary neoplasms[37].

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Figure 2 Human epidermal growth factor receptor 2 expression in gastric and breast tumors. A: A HER2-positive (3+) case of gastric adenocarcinoma; the cytoplasmic membranous immunostaining is incomplete and predominantly basolateral (× 400); B: A HER2-positive (3+) case of invasive ductal carcinoma of the breast; the cytoplasmic membranous staining is fully circumferential (× 400). HER2: Human epidermal growth factor receptor 2.

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Figure 3 Representative image of the intratumoral heterogeneity of HER2 expression. Arrows indicate areas with strong continuous membranous staining (score 3+) and arrowheads indicate negative areas (score 0) (× 100). HER2: Human epidermal growth factor receptor 2.

IHC score system

Given these differences between HER2 expression in breast and gastric cancer, an appropriate scoring system, exclusive for gastric tumors, was developed, because just transferring the breast cancer IHC scoring roles to gastric cancer could lead to a significant loss of patients. The system proposed by Hofmann et al[38] that has been assimilated by CAP and FDA, besides being specific for gastric tumors, also distinguishes biopsies from surgical specimens[17]. Table 2 shows the IHC score system for HER2 in gastric cancer and Figure 4 illustrates it.

Score	Surgical specimen	Biopsy	HER2 overexpression assessment
0	No membranous staining or staining of < 10% of the tumor cells	No membranous staining or staining only in rare cells (less than 5 cohesive cells)	Negative
1+	Staining is weak or detected in only one part of the membrane in ≥ 10% of the cells	Staining is weak or detected in only one part of the membrane of at least 5 cohesive cells	Negative
2+	Moderate/weak complete or basolateral membranous staining in ≥ 10% of the cells	Moderate/weak complete or basolateral membranous staining of at least 5 cohesive cells	Equivocal
3+	Strong complete or basolateral membranous staining in ≥ 10% of the neoplastic cells	Strong complete or basolateral membranous staining of at least 5 cohesive cells	Positive

HER2: Human epidermal growth factor receptor 2.

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Figure 4 Human epidermal growth factor receptor 2 protein expression in gastric and gastroesophageal tumors. A: A negative (0) case; B: A negative (+1) case; C: An equivocal (2+) case; D: A positive (3+) case. HER2: Human epidermal growth factor receptor 2.

Given the recent introduction of trastuzumab for the treatment of patients with advanced gastric cancer, assessment of HER2 status is now mandatory for selecting patients eligible for this treatment. Although the development of automated platforms and image analysis should broaden the availability of in situ hybridization technologies, immunohistochemistry continues to play an essential role in HER2 status assessment. The overall reliability of HER2 evaluation by IHC, however, can be affected by diverse pre-analytical, analytical and post-analytical variables. Therefore, gastric and gastroesophageal cancer requires a unique scoring system, but above all, it requires expertise in interpretation.