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The clinical relevance and prognostic significance of HER2/Neu in gastric cancers

Hinnis, Adel R.; Mansour, Sahar F. M.

Egyptian Journal of Pathology: July 2013 - Volume 33 - Issue 1 - p 8–12
doi: 10.1097/
Original Articles

Background Gastric cancer (GC) still represents a major health problem, despite a decrease in its incidence in the last few years. Because of the social impact of GC, there is a need for developing novel biomarkers to stratify patients into appropriate screening, surveillance, or treatment programs. Although histopathology remains the most reliable and less-expensive method, numerous efforts have been made toward identifying and validating novel biomarkers. Recent advances in molecular therapy have identified human epidermal growth factor receptor 2 (HER2) as an important target for anticancer therapy in GC. Although the clinical relevance and prognostic significance of HER2 in breast cancer have been well acknowledged, it remains controversial in GC. The aim of this work was to evaluate the role of HER2 as a prognostic factor in patients with GC.

Methods HER2 expression was investigated in 30 patients with GC by immunohistochemical staining and the results were compared and correlated with clinicopathologic parameters (age, sex, tumor location, histopathological type tumor stage, lymphovascular invasion, and lymph node metastasis).

Results Among the samples that attained a score of 3+, 85% showed staining in 50% or more of the tumor area, 10% that attained a score of 2+ showed staining in 50% or more of the tumor area, whereas only 5% that attained a score of 1+ showed staining less than 50%. There was no statistically significant correlation between HER2 positivity and age (P=0.0601), sex (P=0.3000), or tumor location (P=0.364). HER2-positive tumors were found more often in lesions located in the lower third of the stomach compared with those located in the upper third (83 vs. 71%). According to Lauren’s classification, HER2 overexpression was more often detected in the intestinal histological type (93%) than in the mixed (83%) or diffuse (67%) type, although this difference was not statistically significant. HER2 positivity was statistically significantly correlated with tumor-node-metastasis stage (P=0.0312) and lymph node metastasis (P=0.009).

Conclusion HER2 overexpression was positively correlated with aggressive biological behavior and was an independent poor prognostic factor for GCs. Therefore, HER2-positive GCs should be considered for adjuvant trastuzumab therapy.

Department of Pathology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

Correspondence to Adel R. Hinnis, MD, Department of Pathology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt Tel: +20 122 281 8303; e-mail:

Received February 15, 2013

Accepted March 31, 2013

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Gastric cancer (GC) continues to be a major health problem, despite a decrease in its incidence over the last few years (Bertuccio et al., 2009). According to the most recent estimates, GC accounts for 8% of the total cancer cases and for 10% of cancer-related deaths (Jemal et al., 2010). GC is characterized by a clear geographical distribution, with over 70% of the cases occurring in developing countries. This can be partly attributed to dietary habits as well as to the prevalence of Helicobacter pylori infection. Indeed, the reasons accounting for the decreased GC incidence in most countries are related to changes in dietary habits, amelioration of food preservation, reduction in H. pylori chronic infection (Chen et al., 2007; Tkachenko et al., 2007; Kawakami et al., 2008), and reduction in smoking (Bertuccio et al., 2009). About 90% of GCs are classified as adenocarcinomas, whereas the remaining 10% are represented by non-Hodgkin’s lymphomas, leiomyosarcomas, squamous cell carcinomas, and undifferentiated carcinomas. According to Lauren’s classification, two subtypes of GCs can be distinguished on the basis of their histology: the intestinal type (I-GC) and the diffuse type (D-GC) (Lauren, 1965). The two GC types also display different biological and etiological characteristics. Tumor cells of I-GC form glandular-like structures, a feature that is not found in D-GC, which, in contrast, is characterized by infiltration and thickening of the gastric wall by tumor cells. The two histological subtypes are the result of distinct pathogenetic pathways, well described in the two models, which are proposed to depict the pathogenesis of I-GC (Correa, 1992) and D-GC (Leung and Sung, 2002). I-GC occurrence is preceded by the development of chronic gastritis, which in turn leads to atrophy, and by the subsequent appearance of intestinal metaplasia. Intestinal metaplasia arises from the proliferation of gastric stem cells, whose progeny differentiates into ‘intestinal type’ cells (columnar, goblet, and Paneth cells) because of the persistent irritation of the gastric mucosa caused by H. pylori (Leung and Sung, 2002). D-GC is characterized by reduced or abnormal E-cadherin expression (Mayer et al., 1993). Inactivation of the second CDH1 allele (e.g. the gene encoding E-cadherin) leads to the appearance of an in-situ carcinoma, with the presence of signet-ring cells with a ‘Pagetoid’ pattern of diffusion, which is subsequently followed by the invasion of surrounding tissues. According to this model, the intraepithelial presence of signet-ring cells does not represent a secondary colonization. On the whole, E-cadherin loss/abnormality represents an early event in the cancerogenesis of D-GC (Mayer et al., 1993; Machado et al., 1998; Leung and Sung, 2002), and the dysregulation of the gene is one of the most frequent genetic alterations in D-GC (Machado et al., 1998; Bremm et al., 2008).

Because of the social impact of GC, there is a need to stratify patients into appropriate screening, surveillance, or treatment programs (Bertuccio et al., 2009; Jemal et al., 2010). Although histopathology remains the most reliable and less-expensive method, numerous efforts have been made to identify and validate novel biomarkers to accomplish the above goals. In recent years, several molecules have been identified and tested for their clinical relevance in GC management. Immunohistochemical (IHC) analysis of formalin-fixed and paraffin-embedded tissues is widely used in diagnostic surgical pathology to gather additional information over and above those obtained with classical hematoxylin and eosin (H&E) staining. Different biomarkers detected by IHC are now a common component in many institutional review board protocols for more precise risk stratification and target identification (Teruya-Feldstein, 2010). In this study we have focused on human epidermal growth factor receptor 2 (HER2), a marker whose correlation with prognosis in GC is still controversial.

The HER family comprises four different receptors: HER1 (EGFR or ErbB1), HER2 (ErbB2 or HER2/Neu), HER3 (ErbB3), and HER4 (ErbB4). These receptors cooperate in the regulation of different processes, such as cell proliferation, differentiation, and survival (Normanno et al., 2006; Fornaro et al., 2011). HER family members are implicated in the development of different kinds of tumors and are now recognized targets for biological therapy in breast, colorectal, lung, and head and neck cancers (Ménard et al., 2000). Upon ligand binding, the receptors dimerize, become phosphorylated, and transduce intracellular signals, which ultimately regulate the above-mentioned cellular processes. Receptor dimerization can also occur through the process of receptor pairing, in addition to ligand binding (Sorkin and Goh, 2008). Indeed, HER receptors can either homodimerize or heterodimerize with other HER family members, allowing multiple receptor combinations (Slamon et al., 1989). Dimer formation leads to the phosphorylation of key intracellular proteins, which provide docking sites for a variety of subsiding signaling molecules. The latter then transmit signals to different downstream cascades, including the MAPK and PI3K/AKT pathways (Zhang et al., 2009; Giuffrè et al., 2012).

The purpose of this study was to evaluate the expression of HER2 protein and investigate its prognostic significance in two large, independent series of GC patients.

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Materials and methods

Patients and tissue specimens

The series corresponded to 30 collected cases from January 2008 to December 2011 from the Pathology Lab, Suez Canal University Hospital, Ismailia, Egypt. Clinicopathologic parameters, including age, sex, tumor location, tumor size, histological classification (according to the WHO), Lauren’s classification, depth of invasion, nodal metastasis, pathological-tumor-node-metastasis (pTNM) stage [according to the seventh edition of American Joint Committee on Cancer (AJCC) staging manual], and lymphovascular invasion status were evaluated by reviewing medical charts, pathologic reports, and H&E-stained slides.

The patients’ ages ranged from 25 to 65 (median, 58) years. There were 20 male and 10 female patients. The tumor was located in the upper-third of the stomach in five patients, in the middle third in 12 patients, the lower third in eight patients, and in the whole stomach in five patients. Sixteen patients were in stages I–II and 14 patients were in stages III–IV according to pTNM staging. None of the patients received neoadjuvant chemotherapy or radiotherapy.

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Immunohistochemical staining

Paraffin blocks that best represented the tumor in each patient were selected after reviewing the H&E slides. Formalin-fixed paraffin-embedded tissue sections were stained using an automatic IHC staining device (Benchmark XT; Ventana Medical System, Tucson, Arizona, USA), as described by the manufacturer. Briefly, 5-μm-thick sections were transferred onto poly-L-lysine-coated adhesive slides and dried at 62°C for 30 min. After epitope retrieval by standard heat treatment for 30 min in ethylene diaminetetraacetic acid (pH 8.0) in the autostainer, the samples were incubated with mouse polyclonal antibodies to HER2 (dilution 1 : 500, clone A0485; Labvision/Neomarker, USA). The sections were subsequently incubated with biotinylated antimouse immunoglobulins, peroxidase-labeled streptavidin (LSAB Kit; Labvision), and 3,3′-diaminobenzidine and counterstained with Harris hematoxylin.

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Evaluation of HER2 immunohistochemical staining

The staining on the slides was examined microscopically and interpreted in a blinded manner by two pathologists. Membrane staining for HER2 was observed. IHC expression of HER2 was scored as follows: 0, no staining or staining in fewer than 10% of tumor cells; 1+, faint/barely perceptible partial staining in more than 10% of tumor cells; 2+, weak to moderate staining of the entire membrane or cytoplasm in more than 10% of tumor cells; and 3+, strong staining in more than 10% of tumor cells. Scores 0 and 1+ were considered negative and scores 2+ and 3+ were considered positive for HER2 overexpression.

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Statistical analysis

All statistical analyses were conducted using the SPSS 15.0 Statistical Software Program (SPSS, Chicago, Illinois, USA). The χ 2-test (two-sided Fisher’s exact test or two-sided linear-by-linear association) was performed to assess the correlation of various clinicopathologic parameters with HER2 positivity. Results were considered statistically significant when P values were less than 0.05.

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Of 30 patients with GC, 25 (83%) were HER2 positive. The association of HER2 positivity and clinicopathological features is summarized in Table 1. There was no significant difference in sex, age, or tumor location with respect to the overexpression of HER2. HER2-positive tumors were found more often in lesions in the lower third than in those in the upper third (83 vs. 71%). According to Lauren’s classification, HER2 overexpression was more often detected in the intestinal histological type (93%) than in the mixed (83%) or diffuse (67%) type; however, the difference was not statistically significant (Fig. 1). Statistically significant correlation was found between HER2 positivity and TNM stage. As shown in Table 1, we can see that a larger proportion of patients with stage III–IV lesions were HER2 positive, compared with those with stage I–II lesions (95 vs. 60%; P=0.0312). Further, a statistically significant correlation was found between HER2 positivity and nodal metastasis, with a higher rate of HER2-positive cases with positive nodal metastasis than with negative nodal metastasis (96 vs. 26%; P=0.0009).

Table 1

Table 1

Figure 1

Figure 1

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In the present study we have evaluated the potential relationship between HER2 expression in GCs and clinicopathologic variables. We found no significant difference in sex, age, or tumor location with respect to the overexpression of HER2. A statistically significant correlation was found between HER2 positivity and TNM stage and nodal metastasis.

Studies to date analyzing HER2 expression in GCs have yielded conflicting results with respect to the correlation between HER2 positivity and clinicopathologic variables (García et al., 2003; Park et al., 2006; Gravalos and Jimeno, 2008; Giuffrè et al., 2012). These inconsistencies can be attributed to multifactorial causes, but the most important aspects are probably the use of nonstandardized IHC staining methods and the application of different scoring criteria (Hofmann et al., 2008; Barros-Silva et al., 2009; Rüschoff et al., 2010). It has been generally reported that HER2 overexpression is correlated with aggressive biological behavior and poor prognosis, despite some contradictory results (Allgayer et al., 2000; Tanner et al., 2005; Ding et al., 2010; Zhao et al., 2012). However, virtually all studies have indicated that HER2 overexpression is strongly associated with differentiated or I-GCs, which generally have a better prognosis than undifferentiated or diffuse-type cancers (Gravalos et al., 2007; Yamashita et al., 2008; Ross and Mulcahy, 2011).

High correlation between HER2 expression and intestinal histological type was reported by several authors (Tanner et al., 2005; Park et al., 2006; Kang et al., 2008). Our group observed a higher rate of HER2 overexpression in intestinal than in diffuse type (93 vs. 67%). In the study by Park et al. (2006), amplification of HER2 was strongly associated with poor carcinoma-specific survival, particularly evident in the subgroup of intestinal type of cancers (P=0.0019), which is usually considered to associate with more favorable prognosis compared with the diffuse type of gastric adenocarcinoma (Kang et al., 2008). I-GC also exhibited higher rates of HER2 amplification compared with D-GC (P<0.05) in the study by Tanner et al. (2005). Finally, in the ToGA trial, HER2 positivity differed significantly by histological subtype (intestinal 34%, diffuse 6%, mixed 20%). The reasons for the selective overexpression of HER2 in I-GC are complex and it is necessary to investigate them in depth. The association of this oncogene with a specific histological tumor type indicates that certain characteristics (e.g. HER2 overexpression and intestinal phenotype) may be expressed together preferentially. However, as not all tumors of the intestinal type overexpress HER2, this cannot be the only factor involved (Van Cutsem et al., 2009). The reasons for the selective overexpression of HER2 in I-GC are complex and unclear at present. In breast carcinomas, HER2 gene amplification is a common feature of invasive ductal carcinomas and an uncommon feature in lobular carcinomas (Ménard et al., 2000). In GC, HER2 gene amplification associates inversely with E-cadherin mutations (Yuan et al., 2009; Zhou et al., 2010); E-cadherin mutations are typical for diffuse gastric and lobular invasive breast carcinomas but rare in intestinal gastric and ductal invasive breast carcinomas (Ménard et al., 2000; Chen et al., 2003; Graziano et al., 2004; Tan and Yu, 2007). Gene amplification and protein overexpression of the growth factor receptors HER2 and K-sam may be prognostic factors for I-GC and D-GC, respectively (Li et al., 2012). Recently, germline mutations of E-cadherin have been identified that are responsible for a dominantly inherited form of D-GC (Zhang et al., 2009).

Depending on the depth of invasion (T), involvement of lymph nodes (N), and the presence of distant metastasis (M), the TNM stage is the most important prognostic parameter for GC in clinical practice (Yamashita et al., 2008). Previous studies including all pathological stages have also reported no correlation between pathological stage and HER2 overexpression (Yamashita et al., 2008; Yuan et al., 2009). In a series of 260 GCs, Okegawa et al. (2006) found that HER2 overexpression was an independent factor and correlated with serosal invasion and lymph node metastases. Aggressive biological behavior, such as lymphovascular invasion and lymph node metastasis, usually leads to poor prognosis. Our study confirmed that HER2 positivity was associated with lymphovascular invasion and lymph node metastases (including pN stage) in agreement with previous studies. Our results demonstrated that HER2 overexpression was statistically higher in phase III–IV lesions than in phase I–II lesions (P<0.05).

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Conflicts of interest

There are no conflicts of interest.

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