| Abstract|| |
Context: Gastric adenocarcinoma (GAC) is a common malignancy with high mortality-rate. Analysis of molecular markers could form a foundation for the future use of targeted therapies to reduce morbidity and mortality. Aims: To find the prevalence and relation of epithelial cadherin (E-cadherin) and human epidermal growth factor receptor 2 (HER-2/neu) protein expression with histological type and grade of GAC using immunohistochemistry (IHC). Materials and Methods: A total of 100 cases of GAC diagnosed over a 2 year period were studied. Expression of E-cadherin and HER-2/neu was analyzed by IHC in relation to the histological type and grade. Results: Of the 100 cases of GAC studied, 11 revealed a loss of E-cadherin and over-expression of HER-2/neu was seen in 17 cases. Loss of E-cadherin was seen in 50% of signet ring-cell carcinomas but only in 8% of tubular and none of papillary and mucin-secreting GAC (P = 0.003). Of all the cases of tubular GAC with loss of E-cadherin expression, majority (71.4%) were Grade III (P = 0.04). Of all the tubular GAC cases with an over-expression of HER-2/neu, 20% and 67% were Grade I and II GAC respectively while only 13% were Grade III (P < 0.001). Conclusions: Although poorly-differentiated tumors show loss of E-cadherin, better-differentiated tumors over-express HER-2/neu protein. Signet ring-cell carcinoma is more likely to exhibit a loss of E-cadherin protein. Targeted therapy toward HER-2/neu in GAC should be considered. Novel therapy to block E-cadherin down-regulation is justified.
Keywords: Epithelial cadherin, gastric adenocarcinoma, human epidermal growth factor receptor 2/neu
|How to cite this article:|
Dewan K, Madan R, Sengupta P, Bharadwaj R. Analysis of epithelial-cadherin and human epidermal growth factor receptor 2/ expression in gastric carcinoma using immunohistochemistry. Indian J Pathol Microbiol 2015;58:154-7
|How to cite this URL:|
Dewan K, Madan R, Sengupta P, Bharadwaj R. Analysis of epithelial-cadherin and human epidermal growth factor receptor 2/ expression in gastric carcinoma using immunohistochemistry. Indian J Pathol Microbiol [serial online] 2015 [cited 2020 Sep 19];58:154-7. Available from: http://www.ijpmonline.org/text.asp?2015/58/2/154/155303
| Introduction|| |
Gastric adenocarcinoma (GAC) is one of the most common cancers worldwide and the second most common cause of cancer-related deaths.  It develops in response to a combination of environmental factors and genetic alterations. Diets rich in salt, smoked or poorly preserved foods, smoking and Helicobacter pylori infection, which has recently been classified as a class I carcinogen by World Health Organization (WHO), increases the risk of subsequent development of GAC.  GAC is preceded by development of sequential changes in gastric mucosa termed as "precancerous cascade," where normal gastric mucosa is transformed to multifocal atrophy, intestinal metaplasia, followed by dysplasia and finally invasive carcinoma. 
According to WHO classification, GACs are classified on the basis of the predominant histological pattern into tubular, papillary, mucin-secreting and signet ring-cell carcinoma.  WHO recommends grading of only tubular GACs and no other histological type of GAC. 
Epithelial cadherin is a cell adhesion molecule, encoded by CDH1 (cadherin 1) gene. Loss of E-cadherin is associated with metastasis thereby providing evidence for its role as an invasion suppressor. ,,,, Mutations in CDH1 have been described in various human cancers including carcinoma of breast, stomach, endometrium, ovary and thyroid. 
Human epidermal growth factor receptor 2 (HER-2)/neu is a tyrosine kinase receptor that is involved in the complex regulation of cell growth, proliferation and survival. In addition to breast carcinoma, over-expression of this protein has been reported in carcinoma of colon, bladder, ovary, endometrium, lung, head and neck and esophagus.  Recent studies have reported an over-expression of HER-2/neu in a subset of 6-30% of patients with GAC. ,,, However, this expression is more heterogenous in GAC when compared with breast carcinoma.
The exceedingly poor prognosis of GAC is due to its late presentation, nonspecific symptoms like dyspepsia in early stage and limitations in treatment options. Molecular markers are vital in determining the disease progression and hence disease outcome, survival and prognosis. An association between clinico-pathological features and molecular markers of GAC could give a clue toward the relationship between them and hence provide us an extra tool to combat the high mortality due to these carcinomas. Targeted therapy toward HER-2/neu or E-cadherin can be justified only when sufficient data regarding the role of these molecules in GAC is available.
Our aim was to find the prevalence of E-cadherin and HER-2/neu expression in GAC and to correlate it with clinico-pathological variables.
| Materials and Methods|| |
The study involved the analysis of 100 cases of GAC registered at a tertiary care center from May 2010 to April 2012. The clinical data of all the selected cases were collected from biopsy requisition slips and the records available. Of the 100 cases studied, 78 were endoscopic biopsy specimens and 22 were gastrectomy specimens. After tissue processing, 3-5 μ thick sections were stained with hematoxylin and eosin. The stained slides were examined for WHO histological type (tubular, papillary, mucinous or signet ring carcinoma) and grade (well, moderate or poorly-differentiated). Section of the primary tumor showing the highest grade and a section from normal gastric lining were selected for immunohistochemistry (IHC).
Ready-to-use monoclonal mouse anti-human E-cadherin antibody (Dako) and 1:300 dilutions of Polyclonal rabbit anti-human HER-2/neu oncoprotein antibody were used. 3-5 μ thick sections were dewaxed, rehydrated and epitope retrieval was done by incubating slides in citrate buffer (pH 6.0) in a pressure cooker at 15 psi and 120°C for 10 min. Endogenous peroxidase activity was blocked by hydrogen peroxide treatment. Primary antibody (ready-to-use E-cadherin or 1:300 diluted HER-2/neu) was added and incubated for 1 h in a moist chamber at room temperature. Incubation with biotinylated secondary anti-mouse antibody for 30 min, followed by Streptavidin horse radish peroxidase for 10 min was done. Diaminobenzidine (DAB) was used as the chromogen and counterstaining with hematoxylin was carried out. Staining was characterized by distinct brown color of the nuclei due to oxidation of the DAB chromogen. Controls used were normal human mammary gland tissue for E-cadherin and known case of HER-2/neu positive breast cancer (3+) for HER-2/neu staining.
Expression of E-cadherin was appreciated by the presence of brown color in membranous or cytoplasmic region and measured as percentage positivity. E-cadherin expression in >10% of the tumor cells was considered positive.HER-2/neu was considered positive if complete membranous staining was seen in at least 10% of the tumor cells irrespective of the staining intensity.
Data analysis was performed using SPSS 10.0 for Windows student version (SPSS Inc., 233 South Wacker Drive, 11 th Floor, Chicago, IL 60606-6412). Statistical tools like Chi-square test was applied.
| Results|| |
The patients selected were 29-82 years of age. The mean age was 59 years and peak incidence was noted in 51-60 years age group. Of the 100 patients, 77 were males and 23 were females. Male:female ratio was 3.34:1. The most common presenting symptom was vomiting.
According to WHO histological classification, 87 were tubular, 1 was papillary, 4 were mucin-secreting and 8 were signet ring-cell type. Grading was done as per WHO recommendations. Grading was done for 87 cases of tubular GAC-4 cases (4.5%) were well-differentiated, 34 (39%) moderately-differentiated and 49 (56.5%) were poorly-differentiated.
Controls were put up for all batches of IHC. Normal gastric mucosal glands were taken as internal controls and found membranous positive for E-cadherin and negative for HER-2/neu. Known case of HER-2/neu positive (3+) breast cancer tissue was used as positive control for HER-2/neu immunostaining. Of the 100 cases of GAC, loss of E-cadherin [Figure 1] was seen in 11 cases and HER-2/neu positivity [Figure 2] in 17 cases.
|Figure 1: Loss of epithelial cadherin immunostaining in gastric adenocarcinoma (red arrows) with positively staining normal gastric mucosal glands (H and E ×200)|
Click here to view
|Figure 2: Positive human epidermal growth factor receptor 2 (HER-2)/neu immunostaining (3+) in gastric adenocarcinoma (H and E, ×400). Inset -Normal gastric mucosal glands are negative for HER-2/neu immunostaining (H and E ×400)|
Click here to view
No statistically significant relation was established between WHO histological type and HER-2/neu expression (P = 0.12). We found HER-2/neu expression to be associated with better differentiation of tumors. Of the 87 tubular GAC cases that were graded, 15 exhibited HER-2/neu over-expression. Of the 15 tubular type of GAC that over-expressed HER-2/neu, 3 (20%) cases were Grade I, 10 (67%) cases were Grade II and 2 cases (13%) were Grade III. Of the remaining 72 cases of tubular GAC in which HER-2/neu was negative, 1 case was Grade I, 24 cases were Grade II and 47 cases were Grade III. Hence, of the 04 cases of Grade I tubular GAC, 03 cases (75%) revealed an over-expression of HER-2/neu (Pearson coefficient 18.83, P < 0.001) [Table 1].
|Table 1: Relation between grades of tubular GAC and immunostaining with E-cadherin and HER-2/neu|
Click here to view
Loss of E-cadherin expression was seen in 50% (4/8) of signet ring-cell carcinomas and 8% (7/87) of tubular type but in none of the papillary and mucin-secreting GAC (Pearson coefficient 13.822, P = 0.003) [Figure 3]. A signet ring-cell carcinoma on morphology is, therefore, more likely to show loss of E-cadherin expression than other morphological subtypes. The grade of the tumor increased with the loss in E-cadherin expression. Grading was done for the 87 cases of tubular GAC. 5/7 (71.4%) of tubular GAC cases with loss of E-cadherin expression were Grade III and only 1 case each (14%) was Grade I and II (Pearson coefficient 8.2, P = 0.04) [Table 1].
|Figure 3: Association between histological type of gastric adenocarcinoma and immunostaining with E-cadherin (Pearson coefficient 13.822, P = 0.003)|
Click here to view
| Discussion|| |
Identifying individuals with a genetic predisposition or those at a high risk of developing GAC would considerably help in modifying prevention, management and control strategies. The prognosis is still poor, and the search continues for novel diagnostic markers that may enable diagnosis of GAC in its early stages and novel patient-tailored therapeutic regimens. Rapid progress is being made in understanding the molecular and genetic events that underlie the transformation of a normal cell to a cancer cell, and most of the studies provide conflicting results.
In this study, normal mucosal glands were assessed for expression of E-cadherin and HER-2/neu. Normal gastric mucosal glands were negative for HER-2/neu but expression of E-cadherin was noted in normal gastric crypts and deep gastric glands in a membranous pattern. Loss of E-cadherin expression was seen in 11% cases. Previous studies suggest the range of prevalence of mutations causing loss of E-cadherin as 7-33% of GAC. , Although false-negative E-cadherin staining for normal gastric mucosal glands was not noted in this study, caution is warranted in using loss of E-cadherin immunostaining as the sole criterion for diagnosis of malignancy. E-cadherin expression was found to be strong in well differentiated GAC, which maintain their intercellular adhesiveness and makes them less invasive, but reduced in poorly-differentiated and signet ring-cell cancers, which have lost their cellular adhesions and show a strong invasive tendency. , This is because of the invasion suppressor role that this protein has been endowed with. Shimoyama et al. studied 54 cases of invasive GAC and found loss of E-cadherin in 4 cases (7.4%), all of which were undifferentiated carcinomas.  In the present study, nearly 72% of GAC with loss of E-cadherin expression were poorly-differentiated.
Human epidermal growth factor receptor 2/neu over-expression was seen in 17% cases. Its over-expression has been reported between 6% and 35% in GAC in the studies conducted in the past. ,,,, We found HER-2/neu over-expression to be significantly more in better-differentiated tumors. Of all HER-2/neu positive cases, 20% were well-differentiated, and 67% were moderately-differentiated. This is in agreement with a study by Raziee et al. who found well differentiated tumors to over-express HER-2/neu in 41% cases.  Only two cases revealed loss of E-Cadherin along with over-expression of HER-2/neu. Whereas loss of E-cadherin is seen in poorly-differentiated adenocarcinoma and signet ring-cell adenocarcinoma, HER-2/neu over-expression is seen in well differentiated GAC.
These results show that in patients with resectable GAC, biomarkers could provide important prognostic information. Testing for these genetic alterations in early lesions may help us in identifying patients with greater risk for progression. The method of blocking E-cadherin down-regulation in tumors is one of the future approaches in gene therapy due to its potential to prevent metastasis of almost any epithelial tumor including GAC. Trastuzumab is the targeted biologic therapy against HER-2/neu receptor protein. The first successful phase III trial of Anti-HER-2/neu in combination with chemotherapy in advanced HER-2/neu positive GAC is a major advancement in systemic therapy of this disease. The TOGA (trastuzumab with chemotherapy in HER-2/neu positive advanced gastric cancer) study showed a significant improvement in overall survival compared with chemotherapy alone.  In reference to our study, this suggests that around 17% of GAC patients in the Indian setting could benefit from combined chemotherapy and trastuzumab.
| Conclusion|| |
The study highlights the significance of E-cadherin and HER-2/neu in GAC. Loss of E-cadherin expression was seen in 11% and HER-2/neu over-expression in 17% cases of GAC. Loss of E-cadherin was seen in poorly-differentiated tubular type and signet ring cell carcinomas. HER-2/neu over-expression was significantly associated with well differentiated tubular GAC. No correlation of both the markers with the stage of disease or with each other was observed. Delays in diagnosis along with the limitation of therapeutic options are the factors that contribute to the extremely poor prognosis of GAC. Hence, the contribution of these genetic markers toward early diagnosis, prediction of progression and prognosis along with newer therapeutic modalities could be of immense benefit in GAC patients.
| References|| |
Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74-108.
Correa P. Human gastric carcinogenesis: A multistep and multifactorial process - First American Cancer Society Award Lecture on Cancer Epidemiology and Prevention. Cancer Res 1992;52:6735-40.
Bosman FT, Carneiro F, Hruban RH, Theise ND. WHO Classification of Tumours of the Digestive System. 4 th
ed. Lyon, France: IARC Press; 2010. p. 45-64.
Zanghieri G, Di Gregorio C, Sacchetti C, Fante R, Sassatelli R, Cannizzo G, et al.
Familial occurrence of gastric cancer in the 2-year experience of a population-based registry. Cancer 1990;66: 2047-51.
Pecina-Slaus N. Tumor suppressor gene E-cadherin and its role in normal and malignant cells. Cancer Cell Int 2003;3:17.
Saad AA, Awed NM, Abd Elkerim NN, El-Shennawy D, Alfons MA, Elserafy ME, et al.
Prognostic significance of E-cadherin expression and peripheral blood micrometastasis in gastric carcinoma patients. Ann Surg Oncol 2010;17:3059-67.
He Q, Chen J, Lin HL, Hu PJ, Chen MH. Expression of peroxisome proliferator-activated receptor gamma, E-cadherin and matrix metalloproteinases-2 in gastric carcinoma and lymph node metastases. Chin Med J (Engl) 2007;120:1498-504.
Shimoyama Y, Hirohashi S. Expression of E- and P-cadherin in gastric carcinomas. Cancer Res 1991;51:2185-92.
Whitney-Miller CL, Hicks DG. HER-2/neu testing in gastric and esophageal adenocarcinoma: Emerging therapeutic options and diagnostic challenges. Connection 2010;15:47-50.
Liang Z, Zeng X, Gao J, Wu S, Wang P, Shi X, et al.
Analysis of EGFR, HER2, and TOP2A gene status and chromosomal polysomy in gastric adenocarcinoma from Chinese patients. BMC Cancer 2008;8:363.
Hilton DA, West KP. C-erbB-2 oncogene product expression and prognosis in gastric carcinoma. J Clin Pathol 1992;45:454-6.
Tapia C, Glatz K, Novotny H, Lugli A, Horcic M, Seemayer CA, et al.
Close association between HER-2 amplification and overexpression in human tumors of non-breast origin. Mod Pathol 2007;20:192-8.
Chen Y, Kingham K, Ford JM, Rosing J, Van Dam J, Jeffrey RB, et al.
A prospective study of total gastrectomy for CDH1-positive hereditary diffuse gastric cancer. Ann Surg Oncol 2011;18:2594-8.
Raziee HR, Kermani T, Ghaffarzadegan K, Shageri T, Ghavamnasiri R. HER-2/neu express
ion in resectable gastric cancer and its relationship with histopathologic subtype, grade and stage. Iran J Basic Med Sci 2007;10:139-45.
Dr. Khushboo Dewan
Department of Pathology, Army Hospital Research and Referral, Delhi Cantt, New Delhi
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]