Brief Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Jun 7, 2013; 19(21): 3316-3323
Published online Jun 7, 2013. doi: 10.3748/wjg.v19.i21.3316
Differences in HER2 over-expression between proximal and distal gastric cancers in the Chinese population
Xiang-Shan Fan, Jie-Yu Chen, Chang-Feng Li, Yi-Fen Zhang, Fan-Qing Meng, Hong-Yan Wu, An-Ning Feng, Qin Huang
Xiang-Shan Fan, Jie-Yu Chen, Yi-Fen Zhang, Fan-Qing Meng, Hong-Yan Wu, An-Ning Feng, Department of Pathology, the Affiliated Drum Tower Hospital, Nanjing University Medical School, Nanjing 210008, Jiangsu Province, China
Chang-Feng Li, Department of Statistics, Nanjing University of Finance and Economics, Nanjing 210046, Jiangsu Province, China
Qin Huang, Department of Pathology, the Affiliated Drum Tower Hospital, Nanjing University Medical School, Nanjing 210008, Jiangsu Province, China
Qin Huang, Department of Pathology and Laboratory Medicine, Veterans Affairs Boston Healthcare System and Harvard Medical School, West Roxbury, MA 02132, United States
Author contributions: Fan XS and Chen JY contributed equally to this work; Fan XS, Chen JY and Huang Q designed the research project; Fan XS, Chen JY, Zhang YF, Meng FQ and Wu HY performed the research; Wu HY performed the immunostaining; Li CF and Feng AN analyzed the data; and Fan XS, Chen JY and Huang Q wrote the paper.
Supported by The National Natural Science Foundation of China, No. 81101815; the Science and Technology Development Project of Medicine in Nanjing, No. YKK08064; Jiangsu Health International Exchange Program and Young Talents Training Project of Health in Nanjing
Correspondence to: Qin Huang, MD, PhD, Department of Pathology and Laboratory Medicine, Veterans Affairs Boston Healthcare System and Harvard Medical School, 1400 VFW Parkway, West Roxbury, MA 02132, United States. qinhuang0122@gmail.com
Telephone: +1-857-2035020 Fax: +1-857-2035623
Received: January 4, 2013
Revised: February 22, 2013
Accepted: April 9, 2013
Published online: June 7, 2013

Abstract

AIM: To investigate HER2 expression and its correlation with clinicopathological variables between proximal and distal gastric cancers (GC) in the Chinese population.

METHODS: Immunostaining of HER2 was performed and scored on a scale of 0-3 in 957 consecutive GC cases, according to the revised scoring criteria of HercepTestTM as used in the ToGA trial. Correlations between HER2 expression and clinicopathologic variables of proximal (n = 513) and distal (n = 444) GC were investigated.

RESULTS: Our results showed that HER2 expression was significantly higher in the proximal than in distal GC (P < 0.05). Overall, HER2 expression was significantly higher in male patients (P < 0.01), the Lauren intestinal type (P < 0.001), low-grade (P < 0.001) and pM1 (P < 0.01) diseases, respectively. There was a significant difference in HER2 expression among some pTNM stages (P < 0.05). In contrast, HER2 expression in the distal GC was significantly higher in male patients (P < 0.001), low-grade histology (P < 0.001), the Lauren intestinal type(P < 0.001), and pM1 (P < 0.001). In the proximal GC, however, higher HER2 expression scores were observed only in tumors with low-grade histology (P < 0.001) and the Lauren intestinal type (P < 0.001).

CONCLUSION: HER2 over-expression in GC of Chinese patients was significantly more common in proximal than in distal GC, and significantly correlated with the Lauren intestinal type and low-grade histology in both proximal and distal GC, and with pM1 disease and male gender in distal GC.

Key Words: HER2, Gastric cancer, Immunohistochemistry, Clinicopathology

Core tip: In this study, immunostaining of HER2 was performed and scored according to the revised scoring criteria of HercepTestTM used in the ToGA trial in a very large cohort of gastric cancers (GC) patients (957 cases). Our results revealed that HER2 over-expression in GC of Chinese patients was significantly more common in proximal than in distal GC, and was significantly correlated with the Lauren intestinal type and low-grade histology in both proximal and distal GC, and with pM1 disease and male gender in distal GC.


Citation: Fan XS, Chen JY, Li CF, Zhang YF, Meng FQ, Wu HY, Feng AN, Huang Q. Differences in HER2 over-expression between proximal and distal gastric cancers in the Chinese population. World J Gastroenterol 2013; 19(21): 3316-3323
INTRODUCTION

HER2 gene amplification and over-expression, which possibly represents a negative prognostical factor[1,2] but a potential therapeutic target[3-7], has been found in 6%-53.4% of gastric cancer (GC) in patients from Western countries[3,8]. As such, it is now recommended that all patients with GC should have their tumors tested for the HER2 status at the time of initial diagnosis[9]. At present, the tests for HER2 over-expression by immunohistochemstry (IHC) and HER2 gene amplification by fluorescence in situ hybridization (FISH) are the most common methods. As demonstrated in the phase III ToGA trial, patients with IHC 2+/FISH-positive or IHC 3+ tumors benefited from trastuzumab treatment, but cases with HER2 gene-amplified tumors, revealed by the FISH test, without HER2 over-expression (IHC 0 or 1+) did not show any survival gain[5]. The characteristics of HER2 expression in GC are different from those in breast cancer[5,10]. Therefore, in the phase III ToGA trial, the HER2 expression scoring system for breast cancer, which was proposed by the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP), was modified for evaluation of HER2 expression in GC[11], requiring IHC for HER2 testing before use of the FISH test[9,12,13], because IHC seems to be more predictive of trastuzumab therapeutic responses than the FISH test alone in GC, compared with breast cancers[14].

In China, GC remains one of the leading cancers. Despite recent advances in treatment, the outcome of patients with advanced GCs is poor. Because there exist considerable differences in GC histopathology, environmental factors, and the Helicobacter pylori status between Western and Chinese patients[15], the need for a comprehensive investigation of the HER2 expression profile in GC of Chinese patients is urgent for better clinical management. Therefore, we carried out this study to fully investigate differences in HER2 expression and clinicopathologic features between proximal and distal GC with the same assessment criteria of IHC as used in the ToGA trial[11].

MATERIALS AND METHODS
Case selection

We retrospectively searched a prospectively established electronic pathology database for GC resection cases with HER2 immunostaining results over the period from January 2007 to August 2009 at the affiliated Nanjing Drum Tower Hospital, Nanjing University Medical School in Nanjing, China. A total of 957 consecutive cases were identified, including 513 proximal and 444 distal GC, according to the surgical resection methods of GC and gross specimen description. Because our recent research results in the same group of Chinese GC patients suggested that almost all proximal GC with esophageal involvement also included Siewert II-III GC, which are regarded as esophageal origin and stage-grouped as esophageal cancers[16,17], they might be more accurately staged as gastric rather than esophageal cancers[18,19]. Therefore, the proximal GC in this study included both GC with epicenters entirely below the gastroesophageal junction (GEJ) and those invading through the GEJ into the distal esophagus as a minor component. Distal GC was defined as tumors with epicenters in the region from the incisura angularis to the antrum-pylorus. The Lauren classification of GC was followed to subgroup all cases in histopathology. All cases were staged, according to the staging rules for GC set out in the 7th edition of the American Joint Committee on Cancer Staging[20]. The patient demographics and pathologic information were retrieved from each pathology report. The patient private identification information was deleted and the study protocol was approved by the Medical Ethics Committee of the Hospital.

Immunohistochemistry

A conventional immunostaining protocol was used for all cases. Briefly, paraffin-embedded tumor tissue blocks were cut at 4-μm in thickness. Sections were deparaffinized, dehydrated, subjected to the antigen retrieval procedure, and then incubated with the primary rabbit antihuman HER2 polyclonal antibody (clone A0485, dilution: 1:2500, Dako, Denmark) for one hour at 37 °C. The HER2 immunoreactivity was visualized after a brief treatment with the EnVision Plus system kit (Dako). Both positive and negative controls were included in each run.

Three experienced pathologists independently evaluated HER2-stained slides blindly without the knowledge of patient clinicopathologic information. The HER2 immunoreactivity of neoplastic cells was scored according to the revised ToGA scoring criteria of HercepTestTM for GC[5,11], which was based on the intensity of membrane staining and quantity of positive neoplastic cells on a scale of 0-3. In brief, no membranous reactivity in less than 10% of tumor cells was scored as 0; faint/barely visible complete or basolateral membranous reactivity in 10% or more of tumor cells was scored as 1+; weak-to-moderate complete or basolateral membranous reactivity in 10% or more of tumor cells was scored as 2+; strong complete or basolateral membranous reactivity in 10% or more of tumor cells was scored as 3+. A score of 0 or 1+ was considered negative while scores 2+ and 3+ were positive.

Statistical analysis

The absolute and relative frequencies of qualitative variables were calculated in percentages. χ2 tests for categorical variables were used and the differences were considered to be statistically significant if P values were less than 0.05. All analyses were performed using the SPSS version 20.0 software for Windows (SPSS Inc., Chicago, IL, United States).

RESULTS
Clinicopathologic features

The mean patient age was 63 years (range: 17-89 years). As shown in Table 1, most patients were male (75%) with a male-female ratio of 3.0; 29% of patients were older than 70 years. Most tumors were the Lauren intestine type (59%), high-grade histology (66%), and at advanced stages of pIII and pIV (61%). About 54% of GC was located in the proximal stomach (Table 2). None of the patients received neoadjuvant therapy before surgical resections.

Table 1 HER2 expression in gastric adenocarcinoma.
nHER2 score
χ2PUnivariate analysis
0123rsP
GenderF24414958191810.1060.001--
M7133661769873
Age (yr) ≤ 7068237016482661.3470.418--
> 70275145703525
pTT1512814637.4640.070--
T21457938199
T37494041799076
T4124323
pNN024313860271813.5910.335--
N114063342320
N2205104503120
N3369210903633
pMM09355062321148319.9840.0010.0770.020
M12292380.1050.002
pTNMI110593216329.9430.0410.0390.187
II267151573029
III5582961436851
IV229238
GLow32413582505751.3600.000--
High6333801526734
LaurenIntestinal568256145838479.5480.000--
Diffuse/mixed38925388347
Table 2 Comparison of HER2 expression between proximal and distal gastric adenocarcinoma.
GroupnHER2 score
χ2P
0123
Proximal51326012672556.6910.011
Distal4442551084536
Overall HER2 immunoreactivity in GC

In general, HER2 immunoreactivity of neoplastic cells in GC was characterized by basolateral, lateral, and/or circumferential membranous, heterogeneous or diffuse, staining patterns (Figures 1-3). HER2 over-expression with score 3, and scores 2 and 3 were found in 9.5% (91/957) and 21.73% (208/957) of cases, respectively. Overall, HER2 expression with score 3 was significantly higher in male patients (10.24% vs 7.38% in females, P < 0.01), the proximal GC (8.11% vs 10.72% in distal GC, P < 0.05), the Lauren intestine type (14.79% vs 1.8% in the Lauren diffuse/mixed type, P < 0.001), histological low-grade histology (17.6% vs 5.37% in high-grade histology, P < 0.001), and pM1 (36.36% vs 8.88% in pM0, P < 0.01). Although there was a significant difference in HER2 expression between advanced and early GC, especially in stage pIV (pI: 2.7%; pII: 10.86%; pIII: 9.12%; pIV: 38.1%; P < 0.05), the correlation between HER2 and pTNM stage was not statistically significant (P > 0.05) (Table 1).

Figure 1
Figure 1 HER2 immunostaining in gastric cancers is scored as 1+, 2+ and 3+ in A, B and C, respectively. EnVision immunohistochemistry stain, × 400.
Figure 2
Figure 2 Heterogeneous (A and B, × 200) or diffuse (C, × 100) expression pattern of HER2 protein is discovered in gastric cancers. EnVision immunohistochemistry stain.
Figure 3
Figure 3 A rare diffuse and strong positive pattern of HER2 expression in the Lauren diffuse type gastric cancers. EnVision immunohistochemistry stain, × 400.
Differences in HER2 immunoreactivity between proximal and distal GCs

HER2 expression was higher in proximal GC than in distal GC (10.7% vs 8.1% with score 3; 25% vs 18.2% with scores 2 and 3; P < 0.05) (Table 2). In the proximal GC, higher expression of HER2 with score 3 was found only in tumors with low-grade histology (17.24% vs 6.45%, P < 0.001), the Lauren intestine type (15.88% vs 0.58%, P < 0.001) than those with high-grade histology and the Lauren diffuse/mixed type (Table 3). In the distal GC, however, HER2 over-expression with score 3 was significantly higher in male patients (9.09% vs 5.88%, P < 0.001), distant metastasis (50% vs 6.94%, P < 0.001), histological low-grade (18.18% vs 4.33%, P < 0.001), the Lauren intestine type (13.16% vs 2.87%, P < 0.001) GC than in female patients, pM0, high-grade histology, and the Lauren diffuse/mixed type (Table 4).

Table 3 HER2 expression in proximal gastric adenocarcinoma.
nHER2 score
χ2PUnivariate analysis
0123rsP
GenderF108572813100.8840.232--
M405203985945
Age (yr) ≤ 703711899248421.6470.476--
> 7014271342413
pTT110431215.0260.472--
T2613016114
T34392261075947
T430012
pNN0132713415126.0710.524--
N17031161310
N212460292213
N318798472220
pMM050325512570531.9590.269--
M1105122
pTNMI432014636.7900.133--
II12974261613
III331161854837
IV105122
GLow2038452323519.9240.000-0.1790.000
High310176744020
LaurenIntestinal34015085515439.3510.000-0.2100.000
Diffuse/mixed17311041211
Table 4 HER2 expression in distal gastric adenocarcinoma.
nHER2 score
χ2PUnivariate analysis
0123rsP
GenderF13692306811.5100.0000.1480.000
M308163783928
Age (yr) ≤ 703111817234242.4430.446--
> 7013374361112
pTT1412411513.9830.131--
T284492285
T3310178723129
T494311
pNN011167261269.6450.255--
N17032181010
N281442197
N3182112431413
pM0432251107443029.7280.0000.1330.000
1124116
pTNMI67391810039.7020.164--
II13877311416
III227135582014
IV124116
GLow1215130182231.2860.000-0.2310.000
High323204782714
LaurenIntestinal22810660323040.3520.000-0.2090.000
Diffuse/mixed20914347136
DISCUSSION

In this retrospective study using the revised IHC scoring criteria of HercepTestTM for HER2 expression in GC of Chinese patients, we compared HER2 protein expression profiles along with clinicopathologic features between 513 proximal and 444 distal GC in Chinese patients. The data showed that HER2 over-expression was significantly more common in proximal GC than in distal GC, and significantly correlated with the Lauren intestine type and low-grade histology in both proximal and distal GC, and with pM1 disease and male gender in distal GC.

In the recent literature, the frequency of HER2 over-expression in GC, determined by IHC, ranges widely from 6.8% to 26.8%[21]. In this study, HER2 over-expression with score 2 and 3 was found in 21.73% of cases, which was higher than that (15.9%) in another recent study using the same antibody and scoring criteria in Korean patients[8]. The discrepancy appears to result from the differences in tissue preparations because they used tissue microarray for HER2 immunostaining, which has a much lower sensitivity for HER2 immunoreactivity because of the well-known heterogeneous expression of HER2 in GC[14,21-24]. In addition, different primary antibody clones, various immunostaining protocols, and diverse immunoreactivity scoring schemes may also contribute to variations described among recent studies[8,12,25]. This inconsistency indicates an urgent need for standardized HER2 immunostaining in GC for better reporting and comparison of HER2 IHC results among different centers.

HER2 expression is known to differ among various clinicopathologic factors, such as patient gender, age, ethnicity, tumor location, type, and differentiation, etc., as shown in this and previous studies[26-28]. Our data showed high HER2 expression in GC with low-grade histology and advanced pTNM stages. Similar to our results, a recent study in Japanese patients also described higher HER2 expression in male patients, tumors with the Lauren intestine type, and pM1 stage[26]. With a multivariate analysis, Janjigian et al[29] reported a significantly higher frequency of HER2 immunopositivity in GC with liver metastasis and the Lauren intestine histology, but did not find significant differences in HER2 immunopositivity between resections and biopsies, or primaries and metastases. In that study, approximately 20% (78/381) of distant metastatic GC or GEJ cancers in Western patients were HER2-immunopositive (score 3+ or FISH-positive)[29], a figure which is much lower than ours (36.4%, 8/22). However, the number of GC cases with distant metastasis was limited in this study and our results should be verified in studies with more qualified cases. Nonetheless, our data suggested that HER2 over-expression was more often seen in GC with the Lauren intestine type, low-grade histology, advanced pTNM stage, and in male patients.

In this study, we found a significantly higher frequency of HER2 over-expression in proximal GC than in distal GC. The result is similar to those reported recently in most other studies[30]. It must be pointed out that the vast majority of GEJ cancers in Chinese patients are not Barrett’s esophagus-related and originate primarily in the proximal stomach, invading into the distal esophagus with clinicopathologic features of GC, as we reported previously[31]. Despite similar HER2 over-expression characteristics between Western GEJ cancers and Chinese proximal GC, there exist a number of differences in HER2 over-expression features between proximal and distal GC of Chinese patients in the present study. For example, in distal rather than proximal GC, HER2 over-expression in GC was more common in male patients and in tumors staged at pM1 than in female patients and in pM0 cases, respectively.

A major limitation in this observational study is the absence of the confirmatory FISH test for HER2 gene amplification. However, unlike in breast cancer, HER2 expression with an IHC score of 0 or 1+ but with FISH positivity in GC tumors does not play a statistically significant role with regard to trastuzumab therapy[5]. GC with IHC 3+ HER2 status responds well to this treatment. Thus, the ToGA trial recommended testing HER2 gene amplification by the FISH method only in GC cases with an IHC score of 2+[5]. Therefore, a conformation FISH test might not be performed in GC with IHC 0, 1+ or 3+. However, our cohort is large with 957 GC resection cases and differences in HER2 expression are dramatically significant in many important clinicopathologic parameters. Therefore, the validity of our results should be reasonably sound.

In summary, our data showed a significantly higher frequency of HER2 over-expression in proximal GC than in distal GC. HER2 over-expression was significantly correlated with low-grade histology and the Lauren intestine type in both proximal and distal GC, and with male gender and distant metastasis in distal GC.

ACKNOWLEDGMENTS

This work was presented as a poster in an abstract form at the annual meeting of United States and Canadian Academy of Pathology in 2011 and Digestive Disease Week in 2011.

COMMENTS
Background

HER2 gene amplification and over-expression is a potential therapeutic target and is found in 6%-53.4% of gastric cancer (GC) in patients from Western countries. As such, it is now recommended that all patients with GC should have their tumors tested for HER2 status at the time of initial diagnosis. In China, GC remains one of the leading cancers. Because there exist considerable differences in GC between Western and Chinese patients, the need for a comprehensive investigation of the HER2 expression profile in GC of Chinese patients is urgent for better clinical management.

Research frontiers

The purpose of the present study was to investigate HER2 expression with the same assessment criteria of IHC as used in the ToGA trial and its correlation with clinicopathological variables between proximal and distal GC in the Chinese population.

Innovations and breakthroughs

Our study represented a very large cohort of GC. In this study, HER2 expression in the overall GC was significantly higher in male patients, the Lauren intestinal type, low-grade and pM1 diseases. There was a significant difference in HER2 expression among some pTNM stages. Similar to some Western study results, our data showed that HER2 expression was significantly higher in proximal GC than in distal GC. Also, HER2 expression in the distal GC was significantly higher in male patients, low-grade histology, the Lauren intestinal type, and pM1. In the proximal GC, however, higher HER2 expression scores were observed only in tumors with low-grade histology and the Lauren intestinal type.

Applications

Our data, derived from a comprehensive investigation of HER2 expression profile with the same ToGA criteria in GC resection specimens in a large cohort of homogeneous Chinese patients, provide pathologists and oncologists with more accurate study results than tissue microarray regarding HER2 over-expression characteristics in both proximal and distal GC, which is essential for better clinical management of patients with GC in the Chinese population.

Terminology

ToGA trial: An international, open-label, randomized, controlled, phase III trial of Herceptin (Trastuzumab) in combination with chemotherapy compared with chemotherapy alone in patients with HER2-positive advanced gastric cancer, which was undertaken in 122 centers in 24 countries; Lauren classification: the Lauren classification is based on the histological features of gastric adenocarcinomas, and divides gastric adenocarcinomas into 3 types: intestinal type (the tumor consists of neoplastic glands arranged in tubules, acini, and papillae), diffuse type (the tumor cells are discohesive and many show the signet-ring morphology) and mixed type.

Peer review

The authors reported statistically more frequent HER2 over-expression in proximal than distal GC. HER2 over-expression was also associated with some clinicopathological characteristics, such as gender, the Lauren intestinal type, low-grade dysplasia, and pM1 diseases in GC. This is a valuable paper which represents a very large cohort of cancers (957 cases). This provides some real strength and confidence in the HER2 expression values. The data are consistent with but extend (for the Chinese population) the information already available.

Footnotes

P- Reviewers Langdon S, Zhang HT S- Editor Wen LL L- Editor Logan S E- Editor Zhang DN

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