(CANCER RESEARCH 48, 137-141, January 1, 1988| Expression of Epidermal Growth Factor Receptor in Human Gastric and Colonie Carcinomas1 Wataru Yasui, Hiromichi Sumiyoshi, Jotaro Hata, Takashi Kameda, Atsushi Ochiai, Hisao Ito, and Eiichi Tahara2 First Department of Pathology, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan ABSTRACT The expression of epidermal growth factor (EGF) receptor was ex amined immunohistochemically in a total of 122 gastric and 61 colonie carcinomas, out of which 16 gastric and 8 colonie carcinomas were also examined by I2sl-labeled EGF binding analysis and Western blotting. The values of EGF binding were 12.68 ±1.98 (SE; n = 16) fmol/mg protein ingastric carcinomas and 5.72 ±2.15 (n = 8) fmol/mg protein in nonneoplastic gastric mucosa, the difference being significant (/' < 0.01). In the colonie tissue, the binding capacities in carcinomas and nonneo plastic mucosa were 13.29 ±4.17 (n = 8) and 10.68 ±0.41 (n = 3)fmol/ mg protein, respectively. Scatchard analysis of I25l-labeled EGF binding indicated a single class of receptors in gastric and colonie carcinomas with an apparent A"dvalue of from 111 to 277 (n = 4) and from 87.4 to 341 IM (n = 5), respectively, except for one gastric carcinoma having two classes of receptors (A',,= 15.9 and 896 CM).In Western blotting using monoclonal anti-EGF receptor antibody, various levels of EGF receptor expression were detected in 12 (85.7%) of the 14 gastric carcinomas and ¡n7 (87.5%) of the 8 colonie carcinomas. Immunohistochemically, EGF receptor immunoreactivity was detected in one (3.8%) of the 26 early gastric carcinomas, while it was observed in 33 (34.4%) of the 96 advanced gastric carcinomas, the incidence between the two being signif icantly different (/' < 0.01). In the colonie carcinomas, 47 (77.1%) of the 61 cases showed positive immunoreactivity to EGF receptor, which did not differ by histológica!type. INTRODUCTION EGF3 is a single polypeptide chain of 53 amino acids which was first purified from male mouse submaxillary glands (1) and later from human urine as urogastrone (2-4). The physiological role of EGF remains to be elucidated, but evidences are accu mulating that EGF plays a part in regulating proliferation and function of a variety of cells in vitro and in vivo (5-7). EGF stimulates cell growth in the gastrointestinal tract of mice and rats (8, 9) and inhibits gastric acid secretion (3). Recently, we have found that EGF produced by tumor cells is detected in 20-30% of advanced gastric carcinomas and is closely corre lated with biological malignancy of gastric carcinoma (10). The cell surface receptor for EGF and related transforming growth factor is a glycoprotein with a molecular weight of 170,000 (11). The receptor consists of an external glycosylated domain capable of recognizing the growth factor and an internal domain with tyrosine specific protein kinase activity linked by a 23 amino acid transmembrane bridge (12). The activation of the protein kinase by a growth factor is considered to initiate the mitogenic signal (13, 14). The erb-E oncogene of avian erythroblastosis virus codes a product homologous to a portion of the EGF receptor in which the binding domain has been deleted (15). The expression of EGF receptor is observed in Received 3/16/87; revised 9/4/87; accepted 9/30/87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare for a Comprehensive 10-Year Strategy for Cancer Control, Japan. 2To whom requests for reprints should be addressed. 'The abbreviations used are: EGF. epidermal growth factor; BSA, bovine serum albumin; ABC, avidin-biotin-peroxidase complex. cells of various normal tissues and carcinomas (16, 17). Ampli fication of EGF receptor, increased mRNA level, and over- expression of EGF receptor are mainly found in human squa- mous carcinoma cell lines (18,19). Recently, the degree of gene amplification and concentration of EGF receptors have been shown to be directly correlated with the growth of A431 cells in nude mice (20). Moreover, using an immunohistochemical technique, Sakai et al. (21) reported that EGF receptor was expressed in over one-half of gastric carcinomas and that it was not correlated with histológica! differentiation or lymph node métastases. In the present study, we examined the expression of EGF receptor in gastric and colonie carcinomas through 125I-labeled EGF binding analysis, Western blotting, and immunohisto- chemistry in an attempt to clarify the correlation between EGF receptor expression and tumor staging. MATERIALS AND METHODS A total of 122 surgically resected gastric and 61 colonie carcinomas were used. AH of these carcinomas were fixed in 10% neutral formalin and embedded in paraffin. In some of them, a representative tumor specimen was frozen in liquid nitrogen shortly after surgical removal and stored at -80°C for I25l-labeled EGF binding assay, Western blotting, and immunostaining in frozen section. Histological classifi cation and stage grouping of gastric and colonie cancer were made according to the criteria of the Japanese Research Society for Gastric Cancer and for Cancer of Colon and Rectum (22, 23). Placental tissue from normal delivery was used as a positive control. Measurement of I25I-labeled EGF Binding. Highly purified human EGF (Wakunaga Pharm. Co., Hiroshima, Japan) prepared from a genetically engineered Escherichia coli host was iodinated with Na'25I (New England Nuclear, Inc., Boston, MA) using chloramine-T to specific activity ranging from 625-675 mCi/^mol. Sample preparation and binding assay were made using the modified method of Fitzpatrick et al. (24). Frozen tissues were homogenized in 10 mM Tris-HCl (pH 7.6) containing 10% glycerol, 1.5 mM EDTA, and 10 mM monothio- glycerol using a Polytron homogenizer, and the homogenate was cen- trifuged at 40,000 x g for 60 min. The precipitate was resuspended in 25 mM sodium phosphate buffer (pH 7.0) containing 150 mM sodium chloride and 2 mM magnesium chloride (Buffer A) and then centrifuged at 1500 x g for 5 min. The supernatant was used as the membrane fraction and assayed for EGF receptor. Aliquots of the membranes were incubated in Buffer A containing 0.1 % BSA with 1 nM 12SI-labeled EGF in the presence (nonspecific binding) or absence (total binding) of unlabeled 200 nM EGF for 60 min at 20°C in a final volume of 120 Ml. The reaction was terminated by adding 3 ml of Buffer A containing 0.5% BSA and filtered through a cellulose nitrate filter (0.2-Mm pore size; Toyo Roshi, Tokyo, Japan). The filter was washed with 10 ml of Buffer A containing BSA and counted in a gamma counter. Specific binding was calculated by subtracting nonspecific binding from total binding. The binding activity was expressed as fmol per mg membrane protein as measured by the method of Lowry et al. (25). Specificity of I25l-labeled EGF binding to membrane sample was evaluated using competition binding of unlabeled hormones (gastrin, glucagon, soma tostatin, ferritin, transferrin, insulin, and mouse EGF). Each of five gastric and colonie carcinomas was used for Scatchard analysis. Scat- chard analysis of binding of EGF to membrane sample was performed using the concentration of '"I-labeled EGF ranging from 10 pM-100 nM (26). The samples were prepared by the methods described above. 137 on July 12, 2015. © 1988 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from