(CANCER RESEARCH 48, 137-141, January 1, 1988|

Expression of Epidermal Growth Factor Receptor in Human Gastric and ColonieCarcinomas1

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 examined immunohistochemically in a total of 122 gastric and 61 coloniecarcinomas, out of which 16 gastric and 8 colonie carcinomas were alsoexamined by I2sl-labeled EGF binding analysis and Western blotting.

The values of EGF binding were 12.68 ±1.98 (SE; n = 16) fmol/mgprotein in gastric carcinomas and 5.72 ±2.15 (n = 8) fmol/mg protein innonneoplastic gastric mucosa, the difference being significant (/' < 0.01).

In the colonie tissue, the binding capacities in carcinomas and nonneoplastic 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 carcinomaswith 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 twoclasses of receptors (A',,= 15.9 and 896 CM).In Western blotting using

monoclonal anti-EGF receptor antibody, various levels of EGF receptorexpression were detected in 12 (85.7%) of the 14 gastric carcinomas and¡n7 (87.5%) of the 8 colonie carcinomas. Immunohistochemically, EGFreceptor immunoreactivity was detected in one (3.8%) of the 26 earlygastric carcinomas, while it was observed in 33 (34.4%) of the 96advanced gastric carcinomas, the incidence between the two being significantly different (/' < 0.01). In the colonie carcinomas, 47 (77.1%) of the

61 cases showed positive immunoreactivity to EGF receptor, which didnot 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) andlater from human urine as urogastrone (2-4). The physiologicalrole of EGF remains to be elucidated, but evidences are accumulating that EGF plays a part in regulating proliferation andfunction of a variety of cells in vitro and in vivo (5-7). EGFstimulates cell growth in the gastrointestinal tract of mice andrats (8, 9) and inhibits gastric acid secretion (3). Recently, wehave found that EGF produced by tumor cells is detected in20-30% of advanced gastric carcinomas and is closely correlated with biological malignancy of gastric carcinoma (10).

The cell surface receptor for EGF and related transforminggrowth factor is a glycoprotein with a molecular weight of170,000 (11). The receptor consists of an external glycosylateddomain capable of recognizing the growth factor and an internaldomain with tyrosine specific protein kinase activity linked bya 23 amino acid transmembrane bridge (12). The activation ofthe protein kinase by a growth factor is considered to initiatethe mitogenic signal (13, 14). The erb-E oncogene of avianerythroblastosis virus codes a product homologous to a portionof the EGF receptor in which the binding domain has beendeleted (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 inaccordance 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 Ministryof 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). Amplification 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 geneamplification and concentration of EGF receptors have beenshown to be directly correlated with the growth of A431 cellsin nude mice (20). Moreover, using an immunohistochemicaltechnique, Sakai et al. (21) reported that EGF receptor wasexpressed in over one-half of gastric carcinomas and that it wasnot correlated with histológica! differentiation or lymph nodemétastases.

In the present study, we examined the expression of EGFreceptor in gastric and colonie carcinomas through 125I-labeledEGF binding analysis, Western blotting, and immunohisto-chemistry in an attempt to clarify the correlation between EGFreceptor expression and tumor staging.

MATERIALS AND METHODS

A total of 122 surgically resected gastric and 61 colonie carcinomaswere used. AH of these carcinomas were fixed in 10% neutral formalinand embedded in paraffin. In some of them, a representative tumorspecimen was frozen in liquid nitrogen shortly after surgical removaland stored at -80°C for I25l-labeled EGF binding assay, Western

blotting, and immunostaining in frozen section. Histological classification and stage grouping of gastric and colonie cancer were madeaccording to the criteria of the Japanese Research Society for GastricCancer and for Cancer of Colon and Rectum (22, 23). Placental tissuefrom 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 agenetically engineered Escherichia coli host was iodinated with Na'25I(New England Nuclear, Inc., Boston, MA) using chloramine-T tospecific activity ranging from 625-675 mCi/^mol. Sample preparationand binding assay were made using the modified method of Fitzpatricket al. (24). Frozen tissues were homogenized in 10 mM Tris-HCl (pH7.6) 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 in25 mM sodium phosphate buffer (pH 7.0) containing 150 mM sodiumchloride and 2 mM magnesium chloride (Buffer A) and then centrifugedat 1500 x g for 5 min. The supernatant was used as the membranefraction and assayed for EGF receptor. Aliquots of the membraneswere incubated in Buffer A containing 0.1 % BSA with 1 nM 12SI-labeled

EGF in the presence (nonspecific binding) or absence (total binding) ofunlabeled 200 nM EGF for 60 min at 20°Cin a final volume of 120 Ml.

The reaction was terminated by adding 3 ml of Buffer A containing0.5% BSA and filtered through a cellulose nitrate filter (0.2-Mm poresize; Toyo Roshi, Tokyo, Japan). The filter was washed with 10 ml ofBuffer A containing BSA and counted in a gamma counter. Specificbinding was calculated by subtracting nonspecific binding from totalbinding. The binding activity was expressed as fmol per mg membraneprotein as measured by the method of Lowry et al. (25). Specificity ofI25l-labeled EGF binding to membrane sample was evaluated using

competition binding of unlabeled hormones (gastrin, glucagon, somatostatin, ferritin, transferrin, insulin, and mouse EGF). Each of fivegastric and colonie carcinomas was used for Scatchard analysis. Scat-chard analysis of binding of EGF to membrane sample was performedusing the concentration of '"I-labeled EGF ranging from 10 pM-100

nM (26). The samples were prepared by the methods described above.

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EGF RECEPTOR IN GASTRIC AND COLONIC CANCER

Western Blotting. The crude membranes prepared for the 125I-labeled

EGF binding study were disrupted in 100 m\i potassium phosphatebuffer (pH 7.5) containing 1% Triton X-100, 0.01% sodium deoxycho-late, and 1 HIM phenylmethylsulfonyl fluoride, and the 30,000 x gsupernatant was used as the lysate. Western blotting was performed asdescribed previously (27). The lysate equivalent to 100 ng of proteinwas loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel. Afterelectrophoresis, the lysates were electroblotted onto a nitrocellulosefilter (0.45-^m pore size; Schleicher and Shuell, Dassel, West Germany). Mouse monoclonal antibody to EGF receptor developed byusing A431 epidermoid carcinoma cells as immunogen was obtainedfrom Oncor. Inc., Gaithersburg, MD. The treatment of the gastriccancer cells (TMK-1), which shows EGF-dependent cell growth andhas EGF-stimulated t\ rosine kinase activity, with this antibody resultedin inhibition of '"I-labeled EGF binding.4 Therefore, this antibody was

thought to react specifically with the binding domain of EGF receptors.The filter was incubated with anti-EGF receptor antibody at a dilutionof 1:50. An avidin-biotin system (Vectastain ABC kit; Vector Lab.,Inc., Burlingame, CA) using secondary biotinylated anti-mouse IgGhorse antiserum (diluted 1:100) was used. Peroxidase staining was donefor 15 min using a solution of 50 mg of 4-chloro-l-naphthol in 100 mlof phosphate-buffered saline containing 17% methanol and 0.05%hydrogen peroxide.

Inununohistochemistry. A modification of the immunoglobulin enzyme bridge technique (ABC method) was used as described previously(28). Deparaffinized (4 /im) and cryostat (6 »im )sections were immersedin methanol containing 0.03% hydrogen peroxide for 20 min to blockthe endogenous peroxidase activities and incubated with nonimmunizedhorse serum (diluted 1:20) for 20 min to block the nonspecific Fc-receptor in tissue. The sections were treated consecutively for morethan 30 min with (a) anti-EGF receptor antibody which was the sameas that used in Western blotting (diluted 1:20) at room temperature,(b) biotinylated anti-mouse IgG horse antiserum (diluted 1:100; Vector,and (r) avidin dehydrogenase-biotinylated horseradish peroxidase complex (Vectastain ABC kit. Vector). Peroxidase staining was performedfor 10-20 min using a solution of 30 mg of 3,3'-diaminobenzidine-

tetrahydrochloride in 100 ml of 20 mM Tris-HCl (pH 7.6) containing0.001% hydrogen peroxide. The sections were counterstained with 3%methylgreen. The specificity of the reaction was determined as follows:(a) nonimmune mouse serum was used as the first layer; and (/>)3,3'-

diaminobenzidine-tetrachloride or hydrogen peroxide was omitted fromthe incubation mixture for the peroxidase reaction. The EGF receptor-immunoreactivity in tumor tissue was graded as follows: tissue withmore than 50% of tumor cells having immunoreactivity was graded+++; 25-50%, ++; less than 25%, +; and negative, -. The dataobtained were evaluated by Student's / and x2 tests.

RESULTS'"I-Labeled EGF Binding

The time course study of I25l-labeled EGF binding at 20°C

to membrane sample showed that the equilibrium of bindingwas reached after approximately 30 min and it remained constant for 120 min. The analysis of I25l-labeled EGF binding tomembranes was performed at 20°Cfor 60 min. Specificity of125I-labeledEGF binding to membranes was evaluated by com

petitive displacement using unlabeled hormones. Only humanand mouse EGFs at a high dose competed effectively for 125I-

labeled EGF binding to membranes, while other hormonesdescribed in "Materials and Methods" were not affected (data

not shown).Bindings of >2SI-labeledEGF to individual gastric and colonie

tissues are summarized in Table 1. All of the membrane samplesshowed 2 fmol or greater EGF bound/mg of membrane proteinspecifically. EGF specific binding of gastric carcinoma membranes was 12.68 ±1.96 (SE) fmol/mg protein which was

significantly higher than that of nonneoplastic gastric mucosa(P < 0.01). There was no difference in binding capacities ofgastric carcinomas by histológica! type. In colonie tissues, thebinding capacities of carcinomas were slightly higher than thoseof nonneoplastic mucosa but the difference was not significant.

Membranes from gastric and colonie tumor tissues wereincubated with increasing concentrations of I25l-labeled EGF,

and the specific binding was calculated. Representative resultsare shown in Fig. 1. Scatchard analysis of I25l-labeled EGF

binding generated a straight line, suggesting a single class ofnoncooperative binding sites for 125I-labeled EGF, with the

exception of one gastric carcinoma having two classes of binding sites (Kd = 15.9 and 896 fivt).KAvalues in a single class ofEGF receptor were from 111-277 fM in four gastric and from124-341 fM in five colonie carcinomas, respectively. The binding capacities of the gastric and colonie carcinomas were almostthe same as those of the standard binding assay described above.

Expression of EGF Receptor Using Western Blotting. Theexpressions of EGF receptor in representative gastric and co-Ionic carcinomas using Western blotting are shown in Fig. 2.EGF receptor was expressed as M, 170,000 in 12 (85.7%) ofthe 14 gastric carcinomas and the levels varied by case (Table2). The expression of EGF receptor was also detected in seven(87.5%) of eight colonie carcinomas with various levels.

Immunohistochemical Staining of EGF Receptor. The resultsof immunohistochemical staining of EGF receptor in paraffinand frozen sections in gastric carcinomas are summarized inTable 2. There was a good correlation in EGF receptor immunoreactivity between the paraffin and frozen sections; agreement between the two types of sections was found in 11 (84.6%)of 13 tumors.

Table 3 shows the EGF receptor immunoreactivity in tumorcells of 122 gastric carcinomas and 61 colonie carcinomas inparaffin sections. EGF receptor-positive tumor cells were detected in 33 (34.4%) of 96 advanced gastric carcinomas (Fig. 3)but in only one (3.8%) of 26 early gastric carcinomas, theincidence being significantly different (P < 0.01). In the sametumor, however, EGF receptor immunoreactivity was not always expressed more frequently in deeply invasive tumors thanin superficially invasive tumors. Histologically, its incidence inwell-differentiated advanced adenocarcinomas showed a highertendency than that in poorly differentiated advanced adenocarcinomas but it was not significant. There was no difference inthe incidence of EGF receptor between scirrhous and nonscir-rhous poorly differentiated adenocarcinoma. EGF receptor wasdetected in 47 (77.1%) of 61 colonie carcinomas (Fig. 4) andits incidence was not different by histológica! types.

The relationship between EGF receptor immunoreactivity

Table 1 Binding of1" ¡-labeledEGF to membranes of gastric and colonie tissues

TissueStomachCarcinoma

(16)°Well

differentiated(6)Poorlydifferentiated(10)Nonneoplastic

mucosa(8)Fundus(6)Annum

(2)ColonCarcinoma

(8)Nonneoplasticmucosa(3)PlacentaEGF-binding

capacity(fmol/mgprotein)12.68±

1.98»'c12.45

±2.6512.98±2.805.72±2.15C5.63

±1.035.9613.68

±2.8310.68±0.41764.5

4 Unpublished data.

* Numbers in parentheses, number of experiments.* Mean ±SE.' Significantly different (P < 0.01).

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EGF RECEPTOR IN GASTRIC AND COLON1C CANCER

Gastric cancer

200.0IO .

974 K»

_<X ,.V rfo

ff & &

0.01 0.1 1 10125I-EGF concentration ( nM I

Fig. 1. Scatchard analysis of'"I-labeled EGF binding in gastric (a) and colonie(ft) carcinomas. One hundred-/*! aliquots containing 200 or 300 ng of membraneprotein are incubated with increasing concentrations of '"I-labeled EGF for 60min at 20°Cand then filtered as described in "Materials and Methods." Specific

binding was calculated by substracting the nonspecific binding from specificbinding. A',,values were 136 and 246 I'Mand maximum binding capacities were

20.1 and 22.1 fmol/mg protein, respectively. Inset, Scatchard plot of specificbinding.

and tumor staging in gastric and colonie carcinomas is shownin Table 4. In gastric carcinomas, the incidence of EGF receptorimmunoreactivities in stages I, II, HI, and IV was 12.7, 13.3,33.3, 41.3%, respectively; that is, the expression of EGF receptor increased as the stage progressed, the correlation beingsignificant (P < 0.05). However, there was no significant difference in the incidence of EGF receptor in the colonie carcinomasamong stages I-V. Moreover, the incidence of EGF receptor incolonie carcinomas did not differ by tumor localization.

In nonneoplastic gastric mucosa, parietal cells were positiveto EGF receptor. EGF receptor immunoreactivity was occasionally observed in the mucosa! epithelium showing regenerative changes and in the intestinal metaplastic gland. In thecolonie mucosa, the regenerative epithelium sometimes showedpositive immunoreactivity to EGF receptor.

Most cases showing high levels of EGF receptor expressionin Western blotting were positive to EGF receptor in immu-nohistochemistry (Table 2), but the results of the binding studydid not always agree.

DISCUSSION

The present study demonstrated the expression of EGF receptor in gastric and colonie carcinomas through I25l-labeledEGF binding assay. Western blotting, and immunohistochemis-

Colonie cancer

•£

200.0K»-

EGF-R|

974KH

Fig. 2. Expression of EGF receptor (-Ä) in representative 9 of 16 gastriccarcinomas and 8 colonie carcinomas by Western blotting using monoclonal anti-EGF receptor antibody. K. thousands.

Table 2 Comparison of immunohistochemical staining of EGF receptor inparaffin and frozen sections, level of EGF receptor in Western blotting, and

binding capacity in advanced gastric carcinomas

Case6173624767159015642081442517391767296533659651842421311111841Histology"Tub,'PapPorTub:PorTub¡PorPorPorPorPorSigMucTub:Tub:SigEGF

receptorimmunoreactivityParaffin

FrozenNS+'+

+NS+++-—-+++——+—--——-----

—+++++-

-Level

of EGFreceptor in

WesternblottingLowNDLowHighLowHighTraceLowHighNDTraceNSLowHighHighNSBinding

capacity(fmol/mgprotein)9.5619.5435.5919.2418.3815.3713.68ND10.987.729.36ND8.496.114.858.69

" According to the classification of the Japanese Research Society for Gastric

Cancer.*Tubi, well-differentiated tubular adenocarcinoma; NS, not studied. Pap,

papillary adenocarcinoma; ND, not detectable; Por, poorly differentiated adenocarcinoma; Tub2, moderately differentiated tubular adenocarcinoma; Sig, signetring cell carcinoma; Muc, mucinousadenocarcinoma.

' +, less than 25 % of tumor cells stained; ++, more than 25% of tumor cellsstained; —,negative.

try. Our results of EGF binding analysis revealed the presenceof a single class of specific, saturable, high-affinity binding sitesfor EGF in membranes from homogenates of surgically removed gastric and colonie carcinoma tissues. Many studiesshowed that EGF receptor levels of human squamous cellcarcinomas were higher than that of normal tissue (18-20, 29).In fact, the binding capacity in squamous cell carcinoma of theesophagus was 100-200 fmol/mg of membrane protein.4 In the

present study, the binding capacities to EGF in gastric andcolonie carcinomas were lower when compared to esophagealcarcinomas and placenta! tissue, the values being 12-13 fmol/mg protein. There may be differences in EGF receptor levelsby type of tissues and tumors. The binding capacity in gastriccarcinomas was significantly higher than that in nonneoplastic

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EGF RECEPTOR IN GASTRIC AND COLONIC CANCER

Table 3 Incidence of EGF receptor immunoreactivity in gastric and coloniecarcinomas

EGF receptorimmunoreactivityClassification"Gastric

carcinomaEarlyWellPoorlyTotalAdvancedWellPoorlyScirrhousTotalColonie

carcinomaWellModeratelyPoorlyTotalNo.

ofcases22426363624962431661Positivecases1011896331923547Incidence(%)4.53.8*50.025.025.034.4*79.274.283.377.1

* According to the criteria of the Japanese Research Society for Gastric Cancer

and for Cancer of Colon and Rectum.* Significantly different (P < 0.01).

- . , ;i

y^V

Fig. 3. Immunostaining of EGF receptor in gastric carcinomas, a. well-differentiated tubular adenocarcinoma of the stomach: b, poorly differentiatedadenocarcinoma of the stomach. ABC method, x 250.

gastric mucosa, while no significant difference in capacity wasnoted between colonie carcinomas and nonneoplastic coloniemucosa. It is possible that the higher expression of EGF receptor in gastric carcinomas in comparison with nonneoplasticmucosa may be related to biological malignancy of gastriccarcinomas.

The present Western blotting study revealed that the EGFreceptor was expressed with a high frequency in the gastric andcolonie carcinomas but that the levels of expression varied.Mouse monoclonal anti-EGF receptor antibody used in thepresent study, which specifically reacted with EGF receptor ofA431 cells, reacted with the protein of M, 170,000. Therefore,

' •x•>;'v ••_. ^.

-' :^ • ;»"

It- . '

tlUìT'" •

Fig. 4. Immunostaining of EGF receptor in colonie carcinomas, a, well-differentiated adenocarcinoma of the colon: h. poorly differentiated adenocarci-noma with mucinous stroma of the colon. ABC method, x 250.

Table 4 Relationship between EGF receptor immunoreactivity and tumor stagingin gastric and colonie carcinomas

EGF receptorimmunoreactivityStage'Gastric

carcinoma*

InIHIVNo.

ofcases31

153046Positive

cases4

21019Incidence12.7

13.333.341.3

Colonie carcinomaInmIVv

61915138

41412116

66.773.780.084.675.0

* According to the criteria of the Japanese Research Society for Gastric Cancer

and for Cancer of Colon and Rectum.* Correlation between EGF receptor immunoreactivity and staging was signif

icant in x2 test (P < 0.05).

this antibody was considered to be usable for immunohisto-chemical analysis of EGF receptor.

Immunohistochemically, there was a good correlation inEGF receptor expression between the paraffin and frozen sections but two cases were positive to EGF receptor in the paraffinsections and negative in the frozen sections. In the frozen tissue,the limited scope in comparison with the paraffin section mighthave caused this discrepancy. In most of the gastric carcinomas,the levels of EGF receptor expression in Western blottingcoincided with the immunohistochemical reactivities to EGFreceptor. However, only one case showing a high level of EGFreceptor could not be detected by immunohistochemistry. Thisfailure might be due to the heterogeneity of EGF receptor

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expression in tumor cells. The EGF binding capacity and receptor expression in Western blotting and immunohistochemistrydid not necessarily agree. Some cases with a large EGF bindingcapacity showed a low level of the EGF receptor expression inWestern blotting and immunohistochemistry and vice versa.The discrepancy between the binding analysis and immunohis-tochemistry might account for the heterogeneity of tumor cellswith heterogeneous levels of receptor and various amount ofstromal elements. If so, the immunohistochemical study issuitable for clarifying the expression of EGF receptor in tumorcells although it is a semiquantitative analysis.

The incidence of EGF receptor immunoreactivity was about4% in early gastric carcinomas and 34% in advanced gastriccarcinomas regardless of histological type. It is very interestingto note that a correlation evidently exists between EGF receptorimmunoreactivity and staging of gastric carcinomas. This finding is compatible with the observation on human bladder carcinomas made by Neal et al. (30). We have reported previouslythat production of EGF in gastric carcinomas is closely relatedto the depth of tumor invasion, metastasis, and the prognosis(10). Therefore, it can be conceived that some of the gastriccarcinomas show "autocrine secretion" for self-stimulation,

whereby a tumor cell secretes EGF for which the tumor cellitself has the receptor. The expression of EGF receptor ingastric carcinomas may be correlated with tumor progressionand may serve as a biological marker for high malignancy aswell as EGF. We have also observed that the incidence of EGFreceptor in metastatic carcinomas in perigastric lymph node ishigher than that of primary carcinomas.4 On the other hand,

EGF receptor immunoreactivity was observed in 77% of coloniecarcinomas with no correlation with tumor staging. The biological significance of EGF receptor expression in colonie carcinomas could not be elucidated in the present study. The synchronous expression of EGF and its receptor in individual colontumors and the relationship between the expression of EGFreceptor and prognosis need to be studied in the near future.

ACKNOWLEDGMENTS

We are grateful to M. Takatani and M. Imada for their excellenttechnical assistance.

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1988;48:137-141. Cancer Res   Wataru Yasui, Hiromichi Sumiyoshi, Jotaro Hata, et al.   Gastric and Colonic CarcinomasExpression of Epidermal Growth Factor Receptor in Human

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