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ORIGINAL ARTICLE
HER2/neu over-expression predicts poor outcome in early gastric cancer without lymph node metastasis Yongjia Yan a, Li Lu b, Chunna Liu b, Weidong Li a, Tong Liu a, Weihua Fu a,∗ a b
Department of General Surgery, Tianjin Medical University General Hospital, Tianjin 300052, China Tianjin Medical University, Tianjin 300070, China
Summary Purpose: Human epidermal growth factor receptor 2 (HER2/neu) is involved in the pathogenesis of several types of cancer, including gastric cancer. However, there remains a paucity of data regarding the prognostic relevance of HER2/neu in early gastric cancer without lymph node metastasis (pN0 EGC). The aim of our study was to analyze whether the over-expression of HER2/neu significantly predicts poor outcomes of pN0 EGC. Patients and methods: Sixty-seven patients who underwent operative resection for pN0 EGC was enrolled. The HER2/neu status was examined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Results: The HER2/neu-positive rate was 16.4%. HER2/neu over-expression showed a significant correlation with histological type (P ≤ 0.001), tumor location (P = 0.022) and Lauren grade (P = 0.012). Multivariate analysis showed HER2/neu serves as a good prognostic marker to predict the risk of poor outcome for pN0 EGC. (HR = 1.384, 95.0% CI: 1.142—1.897 P = 0.005) Conclusion: Considering HER2/neu over-expression significantly predicts poor outcome in pN0 EGC, accurate HER2/neu assessment would be done before endoscopic therapy. For HER2/neupositive patients, radical surgery should be performed. © 2014 Elsevier Masson SAS. All rights reserved.
Introduction
∗
Corresponding author. Department of General Surgery, Tianjin Medical University General Hospital, Tianjin 300052, China. Tel.: +86 1382 1661125; fax: +86 2260 362365. E-mail address: [email protected] (W. Fu).
Gastric cancer (GC) is still a problem of human health all over the world. Despite its incidence rate decreases globally, GC is the fourth most common cancer [1,2]. Early gastric cancer (EGC) is defined as gastric cancer confined to the mucosa and submucosa of the stomach, with/without lymph node metastasis. In recent years, a high participation rate
http://dx.doi.org/10.1016/j.clinre.2014.06.019 2210-7401/© 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Yan Y, et al. HER2/neu over-expression predicts poor outcome in early gastric cancer without lymph node metastasis. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.06.019
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for endoscopic screening has shown that EGC accounts for almost half of all GC [3,4]. The high cure rate of EGC and the low frequency of perigastric lymph node metastases have allowed the development of more limited modified procedure that improve patient quality of life without compromising cure rate [5,6]. Human epidermal growth factor receptor 2 (HER2/neu) is a proto-oncogene encoded by ERBB2 on chromosome 17. The role of HER2/neu is to inhibit cell apoptosis and promote proliferation. This may lead to excessive or uncontrolled cell growth and may cause tumorigenesis [7,8]. Advances in the molecular biology, genomics and proteomics have led to better understanding of the role for HER2/neu. HER2/neu is closely associated with several malignant forms of cancer, notably GC. Anti-HER2/neu antibody trastuzumab has been approved for the treatment of gastric cancer after a successful phase III trial (ToGA) which demonstrated improved survival for HER2/neu-positive advanced GC patients [9]. HER2/neu were connected to both poor outcomes and higher recurrence in breast cancer [10—12]. Studies even have demonstrated trastuzumab was effective against early breast cancer [13—15]. However, the value of HER2/neu serve as a prognosis factor in EGC remains unclear. Therefore, the aim of this study was to analyze whether the over-expression of HER2/neu amplification significantly predicts poor outcomes of EGC without lymph node metastasis.
In IHC 2+ patients, The PathVysion® HER2 DNA Probe kit (LSI® HER2/neu Spectrum OrangeTM/CEP® 17 Spectrum GreenTM) was used to carry out FISH analysis according to the manufacturer’s protocol to detect HER2/neu amplification levels. FISH positive was defined as HER2/neu: CEP17 ratio≥2. IHC 2+ patients would be considered HER2/neupositive only if they are FISH+.
Patients and methods
Statistical analysis methods
Patients
The Chi2 test and Krushal-Wallis test were carried out to compare the relationship between the HER2/neu status and clinicopathological factors. Overall survival was calculated using the Kaplan-Meier method, and differences between survival rates were analyzed with the long-rank test. The Cox proportional hazard model was used for multivariate analysis. Results were considered significant when P values were less than 0.05. Statistical analyses were performed using SPSS-19 (IBM SPSS Statistics, Chicago, IL).
We chose all patients with EGC who were confirmed between January 2000 and December 2008 by postoperative pathology to have no lymph node metastasis at Tianjin Medical University General Hospital, Tianjin, China. Only patients with gastric cancer confined to the mucosa and submucosa were finally included. Oncological assessment was performed every six months to each patient, and these patients were followed up by phone calls or mail for the status of survival. There were a total of 67 successfully followed up. Formalin-fixed surgical specimens were used. The patients’ clinicopathological characteristics were collected with the help of hospital information system. The pathologic diagnosis was made by two pathologists individually based on Lauren’s classification, and histological differentiated types. HER2/neu status was tested by cutting the samples into 4 m sections then using immunohistochemistry (IHC). An IHC HER2/neu 3+ breast cancer sample was used as a positive control. Ethical and methodological aspects of this study were approved by the Ethical Committee of Tianjin Medical University.
IHC Anti-HER2/neu (4b5) antibody (Ventana Medical Systems, Inc. Tucson, Arizona) was used to carry IHC staining as the primary antibody against HER2/neu on an automatic immmunostainer (Benchmark XT, Ventana Medical Systems Inc. Tucson, Arizona) according to the manufacturer’s instructions.
By following the National Comprehensive Cancer Network (NCCN) guideline, a semiquantitative approach was used to generate a score for each case as follows: no membrane staining or membrane staining in less than 10% of the tumor cells (score 0), faint/barely perceptible partial membrane staining in more than 10% of the tumor cells (1+), weak to moderate staining of the entire membrane in more than 10% of the tumor cells (2+), and strong staining of the entire membrane in more than 10% of the tumor cells (3+). IHC 0, 1+ was considered negative for HER2/neu over-expression, and IHC 3+ were considered positive. In cases with IHC 2+, fluorescence in situ hybridization (FISH) was used to detect HER2/neu amplification levels. IHC scoring was checked by two independent pathologists who were not familiar with the clinical data.
FISH
Result Patients and tumor characteristics Between January 2000 and December 2008 a total of 67 patients underwent curative-intent surgery for EGC and were successfully followed up. The patient characteristics are summarized in Table 1. The patients had an average age of 57.3 years and 43 of them were male, with follow numbers of different Lauren’s tumor types (intestinal: 61.2%; diffuse: 17.9%; mixed: 20.9%). In terms of tumor location, 32.8% of the 67 tumors located at the gastroesophageal junction, 13.4%, 7.5%, 46.3% located at cadia, fundus and body/antrum respectively. The morphological type of patients was type I (16.4%) type II (68.7%) and type III (14.9%). All 67 patients’ specimens were adequate for IHC evaluation of HER2/neu status. Of the 13 IHC 2 + specimens selected for FISH evaluation, and 4 of which were positive. According to the definition, there were 11 patients (16.4%) were HER2/neu-positive. The result of IHC and FISH was shown in Fig. 1.
Please cite this article in press as: Yan Y, et al. HER2/neu over-expression predicts poor outcome in early gastric cancer without lymph node metastasis. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.06.019
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Figure 1 Representative cases of IHC staining and FISH analysis in pN0 EGC. A. HER2/neu IHC staining negative in tumor cells (original magnification, ×200). B. HER2/neu IHC staining positive in tumor cells (original magnification, ×200). C. FISH showed negative for HER2/neu amplification. D. FISH showed positive for HER2/neu amplification.
Correlation of HER2/neu status with clinicopathological factors The correlation between HER2/neu status and the clinicopathological factors is shown in Table 2. There was a significant correlation between HER2/neu status and poorly differentiated type (P < 0.001), tumor location (P = 0.022) and Lauren grade (P = 0.012). There were no significant associations between the positive rate of HER2/neu and gender, age, size of the tumor, morphological type and invasion depth.
Correlation of HER2/neu status and clinicopathological features with survival There were 67 patients successfully followed up till September 2013. During the period of study, 12 patients died of cancer. The rate of 5-year overall survival (OS) in HER2/neupositive patients was 81.8%, and the in HER2/neu-negative patients was 91.1% (Fig. 2). And the Log-rank test show that there was a significant different between HER2/neunegative and HER2/neu—positive patients (P = 0.040). Tumor size, Lauren classification, gender, age, invasion depth, and HER2/neu status were included in multivariate analysis. The HER2/neu status was found to be independent factor negatively affecting OS (hazard ratios 1.384 95% CI 1.142—1.897 P = 0.005).
Figure 2 Kaplan-Meier curves for patient survival. Patients with an HER2/neu-positive tumor showed a significantly shorter survival period after surgery than those with an HER2/neunegative tumor (P = 0.040).
Discussion HER2/neu belongs to the HER family which is composed of four plasma membrane-bound receptor tyrosine kinases. This tyrosine kinase contributes to signal transduction via different pathways, for instance mitogen-activated protein
Please cite this article in press as: Yan Y, et al. HER2/neu over-expression predicts poor outcome in early gastric cancer without lymph node metastasis. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.06.019
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Y. Yan et al. Table 1 Clinicopathologic characteristics.
features
and
Characteristic
Number
Age, years Mean Range
57.3 34—85
Sex Male Female
43 24
tumor %
64.2 35.8
Tumor location Gastroesophageal junction Cardia Fundus Body/antrum
22 9 5 31
32.8 13.4 7.5 46.3
Morphological type Type I Type II Type III
11 46 10
16.4 68.7 14.9
Lauren’s histological type Intestinal Diffuse Mixed
41 12 14
61.2 17.9 20.9
HER2/neu status Positive Negative
11 56
16.4 83.6
Tumour size > 2 cm ≤ 2 cm
38 29
56.7 43.3
Differentiated types Poorly differentiated Moderate or well differentiated Invasion depth Mucous layer Submucosa
24 43 39 28
35.8 64.2 58.2 41.8
kinase, phosphoinositide 3-kinase, phospholipase C and protein kinase C. Many studies have showed [16,17] that HER2/neu is expressed in various tissues, including the heart, kidney, gastrointestinal tract and breast. HER2/neu predicts very poor outcome in breast cancer. However, testing of HER2/neu in early breast cancer is highly controversial in the last decade. In 1986, the expression of HER2/neu was found in GC [18], which was followed by a number of studies to confirm the finding [19]. Consequently, the prognosis value of HER2/neu in GC patients becomes a hot spot of research. However, there remains a paucity of data regarding the prognostic relevance of HER2/neu in EGC. According to the review by Boku, prevalence of HER2/neu positivity in patients with gastric cancer is in the range of 3.8% to 36.6% [20]. In this study, 16.4% of patients were HER2/neu—positive as defined by FISH and IHC. This result is similar to the published report [21—23]. In our study, the positive rate was associated with differentiation types, and the rate of HER2/neu -positive in poorly differentiated cases is significantly higher (P < 0.001) than that in moderate/well
Table 2 Relationship between HER2/neu expression and clinicopathological features. Clinicopathological features
n
HER2/neu status Positive
%
Sex Male Female
43 24
7 4
16.3 16.7
Age, years ≤ 55 > 55
20 47
5 6
25.0 12.8
0.967
0.216
Tumor location Gastroesophageal junction Cardia Fundus Body/antrum
22
8
36.3
9 5 31
1 0 2
11.1 0 6.5
Tumour size > 2.0 ≤ 2.0
38 29
8 3
21.1 10.3
Morphological type Type I Type II Type III Lauren’s histological type Intestinal Diffuse and mixed Differentiated types Poorly differentiated Moderate/well differentiated Invasion depth Mucous layer Submucosa
P
0.022
0.241
0.761 11 46 10
1 8 2
9.1 17.4 20.0 0.012
41 26
3 8
7.3 30.8 < 0.001
24
10
23.3
43
1
4.2
39 28
7 4
17.9 14.3
0.690
differentiated cases. Alone with the high HER2/neu-positive rate (P = 0.012) in diffuse and mixed Lauren’s type, the HER2/neu shows as a potential prognostic factors to identify more aggressive gastric cancer. And the log-rank test of overall survival between HER2/neu-negative and positive patients confirmed this hypothesis. The multivariate analysis demonstrated HER2/neu can be used as an excellent prognostic factor to predict the risk of poor outcome for EGC (HR = 1.384, 95.0% CI: 1.142—1.897 P = 0.005). However, the study of Okines et al. [24] found the HER2/neu-positive was not relevant to poor prognosis for EGC. Given the fact that outcome of EGC patients was variable due to lymph node metastasis, we rule out all pN1 EGC patients to provide a
Please cite this article in press as: Yan Y, et al. HER2/neu over-expression predicts poor outcome in early gastric cancer without lymph node metastasis. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.06.019
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HER2/neu in early gastric cancer more rational argumentation between HER2/neu status and prognosis. The NCCN guideline suggested that EGC patients without lymph node metastasis remains can be considered adequate endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD) therapy. On the other hand, studies showed that there might be a relationship between the expressions of HER2/neu and higher mortality, higher recurrence/metastasis rate and poor outcomes [25]. Here our study showed that HER2/neu-negative pN0 EGC patients had a significantly higher 5-year overall survival than HER2/neupositive patients. This result agrees with the study that proved HER2/neu-positive may associate with micrometastases in pN0 EGC patients [26]. With the development of special equipment and surgical techniques, application of EMR/ESD to treat EGC is becoming increasingly wide utilization. Given the thought of HER2/neu status may connect with poor outcome of pN0 EGC patients, which is proved by our study, accurate detection of the HER2/neu status is important for selecting the treatment modality in EGC. For the HER2/neu-positive patients, we suggest gastrectomy with lymph node dissection to be considered standard protocol for treating EGC. In conclusion, HER2/neu status is an independent prognostic factor for poor outcome in pN0 EGC patients. For HER2/neu-positive patients, radical surgery should be performed.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
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5 [8] Menard S, Pupa SM, Campiglio M, Tagliabue E. Biologic and therapeutic role of her2 in cancer. Oncogene 2003;22:6570—8. [9] Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki A, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of her2-positive advanced gastric or gastro-oesophageal junction cancer (toga): a phase 3, open-label, randomised controlled trial. Lancet 2010;376:687—97. [10] Toikkanen S, Helin H, Isola J, Joensuu H. Prognostic significance of her-2 oncoprotein expression in breast cancer: a 30-year follow-up. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 1992;10:1044—8. [11] Seshadri R, Horsfall DJ, Firgaira F, McCaul K, Setlur V, Chalmers AH, et al. The relative prognostic significance of total cathepsin d and her-2/neu oncogene amplification in breast cancer. The south Australian breast cancer study group. International journal of cancer Journal international du cancer 1994;56:61—5. [12] Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the her-2/neu oncogene. Science 1987;235:177—82. [13] Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, et al. Trastuzumab after adjuvant chemotherapy in her2-positive breast cancer. The New England journal of medicine 2005;353:1659—72. [14] Romond EH, Perez EA, Bryant J, Suman VJ, Geyer Jr CE, Davidson NE, et al. Trastuzumab plus adjuvant chemotherapy for operable her2-positive breast cancer. The New England journal of medicine 2005;353:1673—84. [15] Spicer J, Harries M, Ellis P. Adjuvant trastuzumab for her2positive breast cancer. Lancet 2005;366:634. [16] Coussens L, Yang-Feng TL, Liao YC, Chen E, Gray A, McGrath J, et al. Tyrosine kinase receptor with extensive homology to egf receptor shares chromosomal location with neu oncogene. Science 1985;230:1132—9. [17] Yamamoto T, Ikawa S, Akiyama T, Semba K, Nomura N, Miyajima N, et al. Similarity of protein encoded by the human c-erb-b-2 gene to epidermal growth factor receptor. Nature 1986;319:230—4. [18] Sakai K, Mori S, Kawamoto T, Taniguchi S, Kobori O, Morioka Y, et al. Expression of epidermal growth factor receptors on normal human gastric epithelia and gastric carcinomas. Journal of the National Cancer Institute 1986;77:1047—52. [19] Yonemura Y, Ninomiya I, Yamaguchi A, Fushida S, Kimura H, Ohoyama S, et al. Evaluation of immunoreactivity for erbb2 protein as a marker of poor short term prognosis in gastric cancer. Cancer research 1991;51:1034—8. [20] Boku N. Her2-positive gastric cancer. Gastric cancer: official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association 2014;17:1—12. [21] Yan B, Yau EX, Bte Omar SS, Ong CW, Pang B, Yeoh KG, et al. A study of her2 gene amplification and protein expression in gastric cancer. Journal of clinical pathology 2010;63:839—42. [22] Grabsch H, Sivakumar S, Gray S, Gabbert HE, Muller W. Her2 expression in gastric cancer: rare, heterogeneous and of no prognostic value - conclusions from 924 cases of two independent series. Cellular oncology: the official journal of the International Society for Cellular Oncology 2010;32:57—65. [23] Tanner M, Hollmen M, Junttila TT, Kapanen AI, Tommola S, Soini Y, et al. Amplification of her-2 in gastric carcinoma: association with topoisomerase iialpha gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab. Annals of oncology: official journal of the European Society for Medical Oncology/ESMO 2005;16:273—8. [24] Okines AF, Thompson LC, Cunningham D, Wotherspoon A, ReisFilho JS, Langley RE, et al. Effect of her2 on prognosis and benefit from peri-operative chemotherapy in early oesophagogastric adenocarcinoma in the magic trial. Annals of oncology:
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official journal of the European Society for Medical Oncology/ESMO 2013;24:1253—61. [25] Kim KC, Koh YW, Chang HM, Kim TH, Yook JH, Kim BS, et al. Evaluation of her2 protein expression in gastric carcinomas: Comparative analysis of 1414 cases of whole-tissue sections
and 595 cases of tissue microarrays. Annals of surgical oncology 2011;18:2833—40. [26] Cao L, Hu X, Zhang Y, Huang G. Adverse prognosis of clusteredcell versus single-cell micrometastases in pN0 early gastric cancer. Journal of surgical oncology 2011;103:53—6.
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Clinics and Research in Hepatology and Gastroenterology (2014) xxx, xxx—xxx
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ORIGINAL ARTICLE
HER2/neu over-expression predicts poor outcome in early gastric cancer without lymph node metastasis Yongjia Yan a, Li Lu b, Chunna Liu b, Weidong Li a, Tong Liu a, Weihua Fu a,∗ a b
Department of General Surgery, Tianjin Medical University General Hospital, Tianjin 300052, China Tianjin Medical University, Tianjin 300070, China
Summary Purpose: Human epidermal growth factor receptor 2 (HER2/neu) is involved in the pathogenesis of several types of cancer, including gastric cancer. However, there remains a paucity of data regarding the prognostic relevance of HER2/neu in early gastric cancer without lymph node metastasis (pN0 EGC). The aim of our study was to analyze whether the over-expression of HER2/neu significantly predicts poor outcomes of pN0 EGC. Patients and methods: Sixty-seven patients who underwent operative resection for pN0 EGC was enrolled. The HER2/neu status was examined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Results: The HER2/neu-positive rate was 16.4%. HER2/neu over-expression showed a significant correlation with histological type (P ≤ 0.001), tumor location (P = 0.022) and Lauren grade (P = 0.012). Multivariate analysis showed HER2/neu serves as a good prognostic marker to predict the risk of poor outcome for pN0 EGC. (HR = 1.384, 95.0% CI: 1.142—1.897 P = 0.005) Conclusion: Considering HER2/neu over-expression significantly predicts poor outcome in pN0 EGC, accurate HER2/neu assessment would be done before endoscopic therapy. For HER2/neupositive patients, radical surgery should be performed. © 2014 Elsevier Masson SAS. All rights reserved.
Introduction
∗
Corresponding author. Department of General Surgery, Tianjin Medical University General Hospital, Tianjin 300052, China. Tel.: +86 1382 1661125; fax: +86 2260 362365. E-mail address: [email protected] (W. Fu).
Gastric cancer (GC) is still a problem of human health all over the world. Despite its incidence rate decreases globally, GC is the fourth most common cancer [1,2]. Early gastric cancer (EGC) is defined as gastric cancer confined to the mucosa and submucosa of the stomach, with/without lymph node metastasis. In recent years, a high participation rate
http://dx.doi.org/10.1016/j.clinre.2014.06.019 2210-7401/© 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Yan Y, et al. HER2/neu over-expression predicts poor outcome in early gastric cancer without lymph node metastasis. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.06.019
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for endoscopic screening has shown that EGC accounts for almost half of all GC [3,4]. The high cure rate of EGC and the low frequency of perigastric lymph node metastases have allowed the development of more limited modified procedure that improve patient quality of life without compromising cure rate [5,6]. Human epidermal growth factor receptor 2 (HER2/neu) is a proto-oncogene encoded by ERBB2 on chromosome 17. The role of HER2/neu is to inhibit cell apoptosis and promote proliferation. This may lead to excessive or uncontrolled cell growth and may cause tumorigenesis [7,8]. Advances in the molecular biology, genomics and proteomics have led to better understanding of the role for HER2/neu. HER2/neu is closely associated with several malignant forms of cancer, notably GC. Anti-HER2/neu antibody trastuzumab has been approved for the treatment of gastric cancer after a successful phase III trial (ToGA) which demonstrated improved survival for HER2/neu-positive advanced GC patients [9]. HER2/neu were connected to both poor outcomes and higher recurrence in breast cancer [10—12]. Studies even have demonstrated trastuzumab was effective against early breast cancer [13—15]. However, the value of HER2/neu serve as a prognosis factor in EGC remains unclear. Therefore, the aim of this study was to analyze whether the over-expression of HER2/neu amplification significantly predicts poor outcomes of EGC without lymph node metastasis.
In IHC 2+ patients, The PathVysion® HER2 DNA Probe kit (LSI® HER2/neu Spectrum OrangeTM/CEP® 17 Spectrum GreenTM) was used to carry out FISH analysis according to the manufacturer’s protocol to detect HER2/neu amplification levels. FISH positive was defined as HER2/neu: CEP17 ratio≥2. IHC 2+ patients would be considered HER2/neupositive only if they are FISH+.
Patients and methods
Statistical analysis methods
Patients
The Chi2 test and Krushal-Wallis test were carried out to compare the relationship between the HER2/neu status and clinicopathological factors. Overall survival was calculated using the Kaplan-Meier method, and differences between survival rates were analyzed with the long-rank test. The Cox proportional hazard model was used for multivariate analysis. Results were considered significant when P values were less than 0.05. Statistical analyses were performed using SPSS-19 (IBM SPSS Statistics, Chicago, IL).
We chose all patients with EGC who were confirmed between January 2000 and December 2008 by postoperative pathology to have no lymph node metastasis at Tianjin Medical University General Hospital, Tianjin, China. Only patients with gastric cancer confined to the mucosa and submucosa were finally included. Oncological assessment was performed every six months to each patient, and these patients were followed up by phone calls or mail for the status of survival. There were a total of 67 successfully followed up. Formalin-fixed surgical specimens were used. The patients’ clinicopathological characteristics were collected with the help of hospital information system. The pathologic diagnosis was made by two pathologists individually based on Lauren’s classification, and histological differentiated types. HER2/neu status was tested by cutting the samples into 4 m sections then using immunohistochemistry (IHC). An IHC HER2/neu 3+ breast cancer sample was used as a positive control. Ethical and methodological aspects of this study were approved by the Ethical Committee of Tianjin Medical University.
IHC Anti-HER2/neu (4b5) antibody (Ventana Medical Systems, Inc. Tucson, Arizona) was used to carry IHC staining as the primary antibody against HER2/neu on an automatic immmunostainer (Benchmark XT, Ventana Medical Systems Inc. Tucson, Arizona) according to the manufacturer’s instructions.
By following the National Comprehensive Cancer Network (NCCN) guideline, a semiquantitative approach was used to generate a score for each case as follows: no membrane staining or membrane staining in less than 10% of the tumor cells (score 0), faint/barely perceptible partial membrane staining in more than 10% of the tumor cells (1+), weak to moderate staining of the entire membrane in more than 10% of the tumor cells (2+), and strong staining of the entire membrane in more than 10% of the tumor cells (3+). IHC 0, 1+ was considered negative for HER2/neu over-expression, and IHC 3+ were considered positive. In cases with IHC 2+, fluorescence in situ hybridization (FISH) was used to detect HER2/neu amplification levels. IHC scoring was checked by two independent pathologists who were not familiar with the clinical data.
FISH
Result Patients and tumor characteristics Between January 2000 and December 2008 a total of 67 patients underwent curative-intent surgery for EGC and were successfully followed up. The patient characteristics are summarized in Table 1. The patients had an average age of 57.3 years and 43 of them were male, with follow numbers of different Lauren’s tumor types (intestinal: 61.2%; diffuse: 17.9%; mixed: 20.9%). In terms of tumor location, 32.8% of the 67 tumors located at the gastroesophageal junction, 13.4%, 7.5%, 46.3% located at cadia, fundus and body/antrum respectively. The morphological type of patients was type I (16.4%) type II (68.7%) and type III (14.9%). All 67 patients’ specimens were adequate for IHC evaluation of HER2/neu status. Of the 13 IHC 2 + specimens selected for FISH evaluation, and 4 of which were positive. According to the definition, there were 11 patients (16.4%) were HER2/neu-positive. The result of IHC and FISH was shown in Fig. 1.
Please cite this article in press as: Yan Y, et al. HER2/neu over-expression predicts poor outcome in early gastric cancer without lymph node metastasis. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.06.019
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HER2/neu in early gastric cancer
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Figure 1 Representative cases of IHC staining and FISH analysis in pN0 EGC. A. HER2/neu IHC staining negative in tumor cells (original magnification, ×200). B. HER2/neu IHC staining positive in tumor cells (original magnification, ×200). C. FISH showed negative for HER2/neu amplification. D. FISH showed positive for HER2/neu amplification.
Correlation of HER2/neu status with clinicopathological factors The correlation between HER2/neu status and the clinicopathological factors is shown in Table 2. There was a significant correlation between HER2/neu status and poorly differentiated type (P < 0.001), tumor location (P = 0.022) and Lauren grade (P = 0.012). There were no significant associations between the positive rate of HER2/neu and gender, age, size of the tumor, morphological type and invasion depth.
Correlation of HER2/neu status and clinicopathological features with survival There were 67 patients successfully followed up till September 2013. During the period of study, 12 patients died of cancer. The rate of 5-year overall survival (OS) in HER2/neupositive patients was 81.8%, and the in HER2/neu-negative patients was 91.1% (Fig. 2). And the Log-rank test show that there was a significant different between HER2/neunegative and HER2/neu—positive patients (P = 0.040). Tumor size, Lauren classification, gender, age, invasion depth, and HER2/neu status were included in multivariate analysis. The HER2/neu status was found to be independent factor negatively affecting OS (hazard ratios 1.384 95% CI 1.142—1.897 P = 0.005).
Figure 2 Kaplan-Meier curves for patient survival. Patients with an HER2/neu-positive tumor showed a significantly shorter survival period after surgery than those with an HER2/neunegative tumor (P = 0.040).
Discussion HER2/neu belongs to the HER family which is composed of four plasma membrane-bound receptor tyrosine kinases. This tyrosine kinase contributes to signal transduction via different pathways, for instance mitogen-activated protein
Please cite this article in press as: Yan Y, et al. HER2/neu over-expression predicts poor outcome in early gastric cancer without lymph node metastasis. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.06.019
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Y. Yan et al. Table 1 Clinicopathologic characteristics.
features
and
Characteristic
Number
Age, years Mean Range
57.3 34—85
Sex Male Female
43 24
tumor %
64.2 35.8
Tumor location Gastroesophageal junction Cardia Fundus Body/antrum
22 9 5 31
32.8 13.4 7.5 46.3
Morphological type Type I Type II Type III
11 46 10
16.4 68.7 14.9
Lauren’s histological type Intestinal Diffuse Mixed
41 12 14
61.2 17.9 20.9
HER2/neu status Positive Negative
11 56
16.4 83.6
Tumour size > 2 cm ≤ 2 cm
38 29
56.7 43.3
Differentiated types Poorly differentiated Moderate or well differentiated Invasion depth Mucous layer Submucosa
24 43 39 28
35.8 64.2 58.2 41.8
kinase, phosphoinositide 3-kinase, phospholipase C and protein kinase C. Many studies have showed [16,17] that HER2/neu is expressed in various tissues, including the heart, kidney, gastrointestinal tract and breast. HER2/neu predicts very poor outcome in breast cancer. However, testing of HER2/neu in early breast cancer is highly controversial in the last decade. In 1986, the expression of HER2/neu was found in GC [18], which was followed by a number of studies to confirm the finding [19]. Consequently, the prognosis value of HER2/neu in GC patients becomes a hot spot of research. However, there remains a paucity of data regarding the prognostic relevance of HER2/neu in EGC. According to the review by Boku, prevalence of HER2/neu positivity in patients with gastric cancer is in the range of 3.8% to 36.6% [20]. In this study, 16.4% of patients were HER2/neu—positive as defined by FISH and IHC. This result is similar to the published report [21—23]. In our study, the positive rate was associated with differentiation types, and the rate of HER2/neu -positive in poorly differentiated cases is significantly higher (P < 0.001) than that in moderate/well
Table 2 Relationship between HER2/neu expression and clinicopathological features. Clinicopathological features
n
HER2/neu status Positive
%
Sex Male Female
43 24
7 4
16.3 16.7
Age, years ≤ 55 > 55
20 47
5 6
25.0 12.8
0.967
0.216
Tumor location Gastroesophageal junction Cardia Fundus Body/antrum
22
8
36.3
9 5 31
1 0 2
11.1 0 6.5
Tumour size > 2.0 ≤ 2.0
38 29
8 3
21.1 10.3
Morphological type Type I Type II Type III Lauren’s histological type Intestinal Diffuse and mixed Differentiated types Poorly differentiated Moderate/well differentiated Invasion depth Mucous layer Submucosa
P
0.022
0.241
0.761 11 46 10
1 8 2
9.1 17.4 20.0 0.012
41 26
3 8
7.3 30.8 < 0.001
24
10
23.3
43
1
4.2
39 28
7 4
17.9 14.3
0.690
differentiated cases. Alone with the high HER2/neu-positive rate (P = 0.012) in diffuse and mixed Lauren’s type, the HER2/neu shows as a potential prognostic factors to identify more aggressive gastric cancer. And the log-rank test of overall survival between HER2/neu-negative and positive patients confirmed this hypothesis. The multivariate analysis demonstrated HER2/neu can be used as an excellent prognostic factor to predict the risk of poor outcome for EGC (HR = 1.384, 95.0% CI: 1.142—1.897 P = 0.005). However, the study of Okines et al. [24] found the HER2/neu-positive was not relevant to poor prognosis for EGC. Given the fact that outcome of EGC patients was variable due to lymph node metastasis, we rule out all pN1 EGC patients to provide a
Please cite this article in press as: Yan Y, et al. HER2/neu over-expression predicts poor outcome in early gastric cancer without lymph node metastasis. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.06.019
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HER2/neu in early gastric cancer more rational argumentation between HER2/neu status and prognosis. The NCCN guideline suggested that EGC patients without lymph node metastasis remains can be considered adequate endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD) therapy. On the other hand, studies showed that there might be a relationship between the expressions of HER2/neu and higher mortality, higher recurrence/metastasis rate and poor outcomes [25]. Here our study showed that HER2/neu-negative pN0 EGC patients had a significantly higher 5-year overall survival than HER2/neupositive patients. This result agrees with the study that proved HER2/neu-positive may associate with micrometastases in pN0 EGC patients [26]. With the development of special equipment and surgical techniques, application of EMR/ESD to treat EGC is becoming increasingly wide utilization. Given the thought of HER2/neu status may connect with poor outcome of pN0 EGC patients, which is proved by our study, accurate detection of the HER2/neu status is important for selecting the treatment modality in EGC. For the HER2/neu-positive patients, we suggest gastrectomy with lymph node dissection to be considered standard protocol for treating EGC. In conclusion, HER2/neu status is an independent prognostic factor for poor outcome in pN0 EGC patients. For HER2/neu-positive patients, radical surgery should be performed.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
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