Journal of Surgical Oncology 49:209-212 (1992)
Clinicopathological Significance of c-erbB-2 Protein Expression in Human Gastric Carcinoma MASAHIRO TATEISHI,
T O M O H l R O TODA, MD, YOSHIKAZU MINAMISONO, SUSUMU NAGASAKI, MD From the Department of Surgery, The Institute of Gastroenterology of Hofu, Yamaguchi, japan MD,
MD, AND
One hundred seventy-nine primary human gastric tumors not associated with early cancer or noncurative resection were examined immunohistochemically for the expression of c-erbB-2 protein. Positive staining, regarded as an indication of gene amplification, was evident in 22( 12%) of the tumors. Of various clinicopathological factors considered, a statistically significant difference in association with frequency of expression was noted only for histological differentiation, as follows: 39% positive staining in papillary, 17% in well differentiated, 5% in moderately differentiated, and 4% in undifferentiated adenocarcinomas (P > 0.01). The 5-year survival rates of patients with positive and negative c-erbB-2 staining were 57% and 59%, respectively. These findings indicate that, in the case of human gastric adenocarcinoma, expression of c-erbB-2 protein is correlated with tumor histological differentiation. Our results also suggest that the presence or absence of c-erbB-2 protein may not serve as a prognostic indicator, particularly in cases of adenocarcinoma of the stomach. 0 1992 Wiley-Liss, Inc.
KEYWORDS:gastric cancer, differentiation, immunohistochemistry
INTRODUCTION The c-erbB-2 oncogene is related to the neu oncogene and was first identified in a chemically induced rat neuroblastoma [ 1 1. This oncogene codes for a transmembrane glycoprotein [2] of a Mr of 185,000 with tyrosine kinase activity [3,4] and is structurally similar to, but distinct from, epidermal growth factor receptor (EGFR) [2-51. Amplification of the gene has been noted to occur in various types of adenocarcinoma [6], including gastric carcinoma [7]. Recently, several studies have shown that amplification of the c-erbB-2 gene is associated with immunohistological staining for c-erbB-2 protein [8-101. In the case of breast cancer, the level of immunostaining of c-erbB-2 protein is associated with lymph node involvement, relapse, and survival rates [ 1 11. However, the correlation of c-erbB-2 protein expression with such variables for human gastric cancer has been unclear. We have now studied c-erbB-2 protein expression, using an immunohistochemical method, in the cases of advanced gastric adenocarcinoma [ 121 treated with complete and 0 1992 Wiley-Liss, Inc.
curative resection [ 121 in order to determine its clinicopathological significance for human gastric cancer.
MATERIALS AND METHODS We examined paraffin-embedded tissues obtained surgically from 179 patients with primary advanced gastric cancer [12]. All the patients have been diagnosed and treated in the Department of Surgery, the Institute of Gastroenterology of Hofu, between 1980 and 1986. Patients who died within the first postoperative month or who underwent noncurative resection were excluded from consideration in the present study. Stage of disease was classified according to “The general rules for the gastric cancer study in surgery and pathology” [ 121, and a review of the surgical and pathological reports of the resected specimens was included. The present study was Accepted for publication November 26, 1991 Address reprint requests to Masahiro Tateishi, MD, Department of Surgery, The Institute of Gastroenterology of Hofu, 14-33 Eki-minami, Hofu, Yamaguchi 747, Japan.
210
Tateishi et al.
Fig. 1. Immunohistochemistry of adenocarcinoma of the stomach. Note the intense membrane staining for c-erbB-2. X400.
based on results from 35 patients with proper muscular (pm) invasion, 79 with subserosa (ss), 56 with exposes to serosa (se), and 9 with infiltrates to other organs (sei) invasion adenocarcinoma. Of the 179 patients 102 were men and 77 were women; their ages ranged from 2 1 years to 89 years (mean 60 years). The degree of histological differentiation was determined using “The general rules for the gastric cancer study in surgery and pathology” [12]. Clinical charts were reviewed in April 1991 and survival time was taken as that from the time of operation. So the following period of all the cases were more than 5 years. Only cases with tumor-related deaths were considered in the present study. Expression of c-erbB-2 protein was observed in sections of routine 10% formalin-fixed, paraffin-embedded blocks, using a polyclonal antibody and immunization of rabbits with a synthetic peptide (Nichirei Ltd., Tokyo, diluted 1:50) [ 13,141. The specificity was determined using immunoprecipitation, and no cross-reaction was noted to occur with EGFR 113,141. The avidin-biotin peroxidase (ABC) method [ 151 was used to observe the antigen. The staining procedures were as follows. The deparaffinized sections were treated with 0.03% hydrogen peroxidase in methanol for 30 minutes at room temperature in order to eliminate endogenous peroxidase activity. After being washed in phosphate-buffered saline (PBS) and incubated with normal serum (diluted 1:200, 30 minutes, PK-4001; Vector Laboratories Burlingame, CA, U.S.A.), the sections were incubated at room temperature overnight with polyclonal rabbit anti-c-erbB-2 protein. For the second and third phase reagents of the ABC method, Vecstain ABC kit (PK-4001; Vector Laboratories) was used. Following these treatments, visualization of the peroxidase was made feasible using the di-
amino-benzidine method. Counterstaining was done with hematoxylin. Each section was then examined with a transmission light microscope and the findings compared with the those for hematoxylin and eosin (HE) stained sections. Omission of the primary antibody resulted in negative staining. Staining reactions were evaluated as positive or negative, and a positive reaction was considered to be present only when strong brown deposits were visible. Relationships between staining results and various clinicopathological factors were assessed using the chisquare test. Survival curves were prepared using the Kaplan-Meier method [ 161. Comparisons among survival rates were made using the generalized Wilcoxon test [ 171. Differences obtained were considered statistically significant when the P value was less than 0.05.
RESULTS Of the 179 cases of gastric cancer considered, immunoreactivity for c-erbB-2 was obtained in 22 (12%). Positive c-erbB-2 staining was intense, brown, and granular, and located predominantly at the cell membrane (Fig. I ) . Heterogeneity of c-erbB-2 staining was observed in regions with a diffusely stained cytoplasm. Table I presents a comparison of various clinicopathological factors, including patient sex, tumor differentiation, depth of invasion, lymph node metastasis (n), lymphatic permeation (ly), vessel permeation (v), and stage with c-erbB-2 status. Positive c-erbB-2 staining was observed in 9 cases (39%) of papillary adenocarcinoma, 8 cases (17%) of well differentiated, 4 cases ( 5 % ) of moderately differentiated, and only one case (4%) of undifferentiated adenocarcinomas. There was statistically significant differences in histological differentiation ( P
Clinicopathological Significance of c-erbB-2 Protein Expression in Human Gastric Carcinoma MASAHIRO TATEISHI,
T O M O H l R O TODA, MD, YOSHIKAZU MINAMISONO, SUSUMU NAGASAKI, MD From the Department of Surgery, The Institute of Gastroenterology of Hofu, Yamaguchi, japan MD,
MD, AND
One hundred seventy-nine primary human gastric tumors not associated with early cancer or noncurative resection were examined immunohistochemically for the expression of c-erbB-2 protein. Positive staining, regarded as an indication of gene amplification, was evident in 22( 12%) of the tumors. Of various clinicopathological factors considered, a statistically significant difference in association with frequency of expression was noted only for histological differentiation, as follows: 39% positive staining in papillary, 17% in well differentiated, 5% in moderately differentiated, and 4% in undifferentiated adenocarcinomas (P > 0.01). The 5-year survival rates of patients with positive and negative c-erbB-2 staining were 57% and 59%, respectively. These findings indicate that, in the case of human gastric adenocarcinoma, expression of c-erbB-2 protein is correlated with tumor histological differentiation. Our results also suggest that the presence or absence of c-erbB-2 protein may not serve as a prognostic indicator, particularly in cases of adenocarcinoma of the stomach. 0 1992 Wiley-Liss, Inc.
KEYWORDS:gastric cancer, differentiation, immunohistochemistry
INTRODUCTION The c-erbB-2 oncogene is related to the neu oncogene and was first identified in a chemically induced rat neuroblastoma [ 1 1. This oncogene codes for a transmembrane glycoprotein [2] of a Mr of 185,000 with tyrosine kinase activity [3,4] and is structurally similar to, but distinct from, epidermal growth factor receptor (EGFR) [2-51. Amplification of the gene has been noted to occur in various types of adenocarcinoma [6], including gastric carcinoma [7]. Recently, several studies have shown that amplification of the c-erbB-2 gene is associated with immunohistological staining for c-erbB-2 protein [8-101. In the case of breast cancer, the level of immunostaining of c-erbB-2 protein is associated with lymph node involvement, relapse, and survival rates [ 1 11. However, the correlation of c-erbB-2 protein expression with such variables for human gastric cancer has been unclear. We have now studied c-erbB-2 protein expression, using an immunohistochemical method, in the cases of advanced gastric adenocarcinoma [ 121 treated with complete and 0 1992 Wiley-Liss, Inc.
curative resection [ 121 in order to determine its clinicopathological significance for human gastric cancer.
MATERIALS AND METHODS We examined paraffin-embedded tissues obtained surgically from 179 patients with primary advanced gastric cancer [12]. All the patients have been diagnosed and treated in the Department of Surgery, the Institute of Gastroenterology of Hofu, between 1980 and 1986. Patients who died within the first postoperative month or who underwent noncurative resection were excluded from consideration in the present study. Stage of disease was classified according to “The general rules for the gastric cancer study in surgery and pathology” [ 121, and a review of the surgical and pathological reports of the resected specimens was included. The present study was Accepted for publication November 26, 1991 Address reprint requests to Masahiro Tateishi, MD, Department of Surgery, The Institute of Gastroenterology of Hofu, 14-33 Eki-minami, Hofu, Yamaguchi 747, Japan.
210
Tateishi et al.
Fig. 1. Immunohistochemistry of adenocarcinoma of the stomach. Note the intense membrane staining for c-erbB-2. X400.
based on results from 35 patients with proper muscular (pm) invasion, 79 with subserosa (ss), 56 with exposes to serosa (se), and 9 with infiltrates to other organs (sei) invasion adenocarcinoma. Of the 179 patients 102 were men and 77 were women; their ages ranged from 2 1 years to 89 years (mean 60 years). The degree of histological differentiation was determined using “The general rules for the gastric cancer study in surgery and pathology” [12]. Clinical charts were reviewed in April 1991 and survival time was taken as that from the time of operation. So the following period of all the cases were more than 5 years. Only cases with tumor-related deaths were considered in the present study. Expression of c-erbB-2 protein was observed in sections of routine 10% formalin-fixed, paraffin-embedded blocks, using a polyclonal antibody and immunization of rabbits with a synthetic peptide (Nichirei Ltd., Tokyo, diluted 1:50) [ 13,141. The specificity was determined using immunoprecipitation, and no cross-reaction was noted to occur with EGFR 113,141. The avidin-biotin peroxidase (ABC) method [ 151 was used to observe the antigen. The staining procedures were as follows. The deparaffinized sections were treated with 0.03% hydrogen peroxidase in methanol for 30 minutes at room temperature in order to eliminate endogenous peroxidase activity. After being washed in phosphate-buffered saline (PBS) and incubated with normal serum (diluted 1:200, 30 minutes, PK-4001; Vector Laboratories Burlingame, CA, U.S.A.), the sections were incubated at room temperature overnight with polyclonal rabbit anti-c-erbB-2 protein. For the second and third phase reagents of the ABC method, Vecstain ABC kit (PK-4001; Vector Laboratories) was used. Following these treatments, visualization of the peroxidase was made feasible using the di-
amino-benzidine method. Counterstaining was done with hematoxylin. Each section was then examined with a transmission light microscope and the findings compared with the those for hematoxylin and eosin (HE) stained sections. Omission of the primary antibody resulted in negative staining. Staining reactions were evaluated as positive or negative, and a positive reaction was considered to be present only when strong brown deposits were visible. Relationships between staining results and various clinicopathological factors were assessed using the chisquare test. Survival curves were prepared using the Kaplan-Meier method [ 161. Comparisons among survival rates were made using the generalized Wilcoxon test [ 171. Differences obtained were considered statistically significant when the P value was less than 0.05.
RESULTS Of the 179 cases of gastric cancer considered, immunoreactivity for c-erbB-2 was obtained in 22 (12%). Positive c-erbB-2 staining was intense, brown, and granular, and located predominantly at the cell membrane (Fig. I ) . Heterogeneity of c-erbB-2 staining was observed in regions with a diffusely stained cytoplasm. Table I presents a comparison of various clinicopathological factors, including patient sex, tumor differentiation, depth of invasion, lymph node metastasis (n), lymphatic permeation (ly), vessel permeation (v), and stage with c-erbB-2 status. Positive c-erbB-2 staining was observed in 9 cases (39%) of papillary adenocarcinoma, 8 cases (17%) of well differentiated, 4 cases ( 5 % ) of moderately differentiated, and only one case (4%) of undifferentiated adenocarcinomas. There was statistically significant differences in histological differentiation ( P
