Survey of Her2-neu Expression and its Correlation with Histology of Gastric Carcinoma and Gastroesophageal Junction Adenocarcinoma

The incidence of primary gastric cancer (GC) and esophageal/gastroesophageal junction (GEJ) adenocarcinomas is increasing, and these tumors are estimated to make up the third and fifth most common cause of cancer deaths worldwide, respectively (Albarello et al., 2011). When a tumor is located at the GEJ, it is often unknown whether the tumor is of esophageal or gastric origin. This group of cancers is therefore called GEJ cancers (Moelans et al., 2011). Esophageal adenocarcinoma incidence has been rapidly increasing in western countries during the past half century, especially in Caucasian males (Hongo et al., 2009). GC is thought to result from a combination of environmental factors and accumulation of specific genetic alterations, and consequently, mainly affects older patients (Moelans et al., 2011). The HER2-neu protein (p185, HER2-neu, ErbB-2) is a 185-kDa transmembrane tyrosine kinase (TK) receptor and a member of the epidermal growth factor receptors (EGFRs) family malignancies(Gravalos and Jimeno, 2008) Patterns are scored as immunohistochemical (IHC) 0 (no staining or staining in<10% of tumor cells, negative), IHC 1+ (faint/barely perceptible incomplete membrane staining in>10% of tumor cells, negative), IHC 2+ (weak to moderate complete membrane staining in >10% of


Introduction
The incidence of primary gastric cancer (GC) and esophageal/gastroesophageal junction (GEJ) adenocarcinomas is increasing, and these tumors are estimated to make up the third and fifth most common cause of cancer deaths worldwide, respectively (Albarello et al., 2011). When a tumor is located at the GEJ, it is often unknown whether the tumor is of esophageal or gastric origin. This group of cancers is therefore called GEJ cancers (Moelans et al., 2011). Esophageal adenocarcinoma incidence has been rapidly increasing in western countries during the past half century, especially in Caucasian males (Hongo et al., 2009). GC is thought to result from a combination of environmental factors and accumulation of specific genetic alterations, and consequently, mainly affects older patients (Moelans et al., 2011).
The aim of this study is evaluation of HER2-neu expression and also some of the clinicopathological features of these neoplasms in the West of Iran.

Patients
In this descriptive and analytical study, we selected 211 adenocarcinoma cases that 193 of them were GC and 18 of them were GEJ. Histological parameters were tumor grade (well, moderately, or poorly differentiated) and growth pattern (intestinal, diffuse, mixed and mucinous).

Clinical and pathologic evaluation
First of all, we provide instruments, apparatus, materials (like Canon Powershot G6) and chemistry compounds (like Hydrogen peroxide, Entelan glue made in Germany (Merk)), kits and colors (c-erbB-2 oncoprotein A*0485, Biotin Blocking System*X0590, EnVision+Dual Link System-HRP*K4063+Liguid DAB+Substrate Chromogen System*K3468, Target Retrieval Solution, pH9(×10)*S2367 made in Denmark and Hematoxylin made in Spain) . After that, we prepared stock buffer (mixing ethylenediaminetetraacetic acid (EDTA) (0.4gr) with tris(hydroxymethyl)aminomethane (Tris) (2gr) and distilled water ( D.W) (1000 ml) and for the IHC staining, first we charged slides with poly-L-lysin glue means that the slides were incubated for an hour in the glue 10 percent (10 ml, 90 ml of distilled water plus glue) and then dried at room temperature overnight. In this study, the patients admitted to pathology laboratory of Imam Reza and Biston Hospitals in Kermanshah (Iran) that were diagnosed GEJ adenocarciunoma and GC for them by pathologist, a sufficient sample size was selected from any patient and the slides were stained by hematoxylin and eosin (H & E) method. Then 4 micron sections were prepared for staining with H & E and also for IHC (Her2-neu) staining.

Statistical analysis
Statistical analyses were performed using the SPSS statistical software package version 19 (SPSS, Chicago, IL, USA). Chi-square test was used to analyze the significance of correlation between the expression of HER2-neu and clinicopathological parameters. P-value<0.05 was considered significant.

Results
The mean age for the patients at diagnosis was 65.59 ±12.03 years (range, 24-98 years). One hundred and fifty patients (71.1%) were male and sixty-one patients ( DOI:http://dx.doi.org/10.7314/APJCP.2015.16.17.7755 Her2-neu Expression and its Correlation with Histology of Gastric Carcinoma and Gastroesophageal Junction Adenocarcinoma of HER-2 protein for score 1+, score 2+ and score 3+ has been shown in Figures 1, 2 and 3, respectively. The Table 2 shows the correlation between Her2-neu scores and a number of variables in 206 patients (5 patients lost HER2-neu scores), that HER2-neu positivity rates is higher in GEJ adenocarcinomas (P<0.05). There is the correlation between HER2-neu scores with the intestinal type of adenocarcinoma and also with well differentiated tumor (P<0.005). Therefore, HER2-neu (3+) is more in intestinal subtype and well differentiated tumor.
We divided the patients to two age groups (<60 or ≥60 years). The Table 3 shows the correlation between age groups and a number of variables in 211 patients. Well differentiated tumor happens more in patients with age<60 years and also that this correlation was statistically significant (P<0.05).

Discussion
There is increasing evidence that HER2-neu is an important biomarker in GC and GEJ tumors; (Gravalos and Jimeno, 2008) analysis of HER2-neu status by IHC and in situ hybridization techniques, using different scoring methods or assays, suggests that HER2-neu is overexpressed in ~7-34% of GC (Hofmann et al., 2008) and this percentage increases to 33% in GEJ tumors (Albarello et al., 2011). Also, a review study reported that Up to a fifth of gastric carcinomas and up to a third of GEJ carcinomas are positive for HER2 overexpression amplification (Hechtman and Polydorides, 2012 ). Almost all primary tumors with an IHC grade of 0 or 1+ showed no amplification by FISH, whereas all IHC 3+ cases showed amplification. Of the IHC2+ cases, only about one-sixth showed HER2-neu amplification (Kim et al., 2011). Although FISH shows a high sensitivity and specificity and remains a criterion for determining gene amplification status, IHC for HER-2 protein expression may be a good alternative when FISH can't be performed (Yan et al., 2011). Therefore, reliable separation of IHC 1+/0 and IHC 2+ may be difficult in biopsy samples, and FISH analysis should be used for definitive classification (Shan et al., 2013). Detecting HER2-neu status has important significance in diagnosis of GC and GEJ, (Wolff et al., 2007) and pathologists now routinely evaluate HER2neu protein expression in gastroesophageal tumors to identify patients who may benefit from the addition of trastuzumab(molecular targeted therapy) (Jeung et al., 2012). A number of studies, (Wang et al., 2011;Wu et al., 2011;Yan et al., 2011;Shan et al., 2013) reported that in gastric adenocarcinoma patients, HER2-neu (3+) rate is between 5-10%, but in this study is more (10.6%). In addition, HER2-positivity varied by tumor site, with higher rates of HER2-positivity in GEJ adenocarcinoma than in GC in this study (27.8 % vs. 10.6% respectively; P<0.05), which is consistent with the results of other studies (Hofmann et al., 2008;Ruschoff et al., 2010;Kunz et al., 2012;Shan et al., 2013). A study reported 145 gastric carcinoma tissue samples, 98 (67.6%) were scored as 0, 25 (17.2%) as 1, 12 (8.3%) as 2, and 10 (6.9%) as 3 (Yan et al., 2011). In our study for GC and GEJ (211 cases), 74 (35.1%) were scored as 0, 53 (25.1%) as 1, 54 (25.6%) as 2, and 25(11.8%) as 3. Analyses demonstrated a statistically significant correlation between the expressions of HER2-neu with the pathological grade of the gastric tumors (P<0.05) (Tanner et al., 2005;Rakhshani et al., 2014). Our study had 17.5% poorly differentiated cancers, 11.7% moderately differentiated cancers and 3.8% well differentiated cancers in score 3+ from HER2-neu  overexpression and another study 46.2%, 30.7%, 23.1%, respectively (Rakhshani et al., 2014). Some studies showed that HER2-neu overexpression associated with the intestinal-type carcinomas (Lauren classification) (Gravalos and Jimeno, 2008;Shan et al., 2013;Rakhshani et al., 2014). A study reported that intestinal-type carcinoma for HER-2/neu positivity prevalence rate was 16.8% in vs. 2.3/8.4% in diffuse-/mixed-type cancers, (Shan et al., 2013) and also in our study, intestinal subtype was higher than other subtypes. There was no significant relation between clinicopathologic variables (sex and age of the patients, with tumor diameter, differentiation or location), (Chen et al., 2006) but we found that well differentiation tumor happened more in patients with age<60 years (P<0.05).
In patients with advanced cancer of GC and GEJ, surgery alone is not curative and other forms of therapy (molecular targeted therapy) may be required to prolong patient survival. Also, HER2-neu overexpression was more associated with intestinal cancer subtype that this can be guideline for new complementary therapy in these patients.