(CANCER RESEARCH 48, 1343-1347, March 1. 19881

with poor prognosis (10). The results, however, are not uniform(1 1). IGF receptors have been identified in four breast cancercell lines (12), but their relation to steroid hormone receptors,EGF receptors or to the prognosis in patients with mammarytumors has not been established.

In this study, we have evaluated the relation between steroidhormone receptors and receptors for EGF and IGF as well asthe relation between tumor differentiation and growth factorreceptors. The presence of growth factor receptors in normaltissue, adjacent to carcinoma tissue, was also studied in anattempt to evaluate the role of growth factor receptors inmalignant transformation of breast tissue.

MATERIALS AND METHODS

Patients and Tissue Samples.Patients who had breast cancer operations were included in the study with approval of the local ethicalcommittee. Approximately 1-cm3samples of tumor tissue were frozenimmediately after excision in liquid nitrogen and stored at —80C untilreceptor analysis. Tissue samples were also fixed in formalin for histological evaluation by a pathologist not aware of the receptor contentof the samples. EGF-R, ER, and PgR were tested in a total of 171breast cancer samples (160 infiltrating ductal carcinoma, one medullarycarcinoma, four lobular carcinoma, three papillary carcinoma, and threecystosarcoma).In 142of thesetissuesIGF-R weredeterminedaswell.The stage of differentiation in 120 ductal carcinomas was divided intothree categories according to WHO classification; low, intermediate,or high. In these120patientsthe tumor sizewasdefined(lessthan 2cm; equal or more than 2 cm), and the presence of axillary metastaseswas evaluated on the basis of surgical reports and histological examination. Thirteen metastases (four local and nine distant soft tissuemetastases) were obtained from patients who underwent a secondoperation. The primary operation had been performed one-half to 14years earlier. In 15 patients carcinoma tissue and samples from normaladjacent tissue were analyzed simultaneously. The normal tissue wasprepared free from fat with scissors, and representative pieces werehistologically verified. In two cases solely normal tissue adjacent tocarcinoma tissue were studied, whereas the tumors were so small thatonly specimens for histological determination were available. In threecases carcinoma tissue and mastopathic surrounding tissue were studied. Biopsies from seven additional patients were also studied, five ofthese biopsies were mastopathic, whereas in two cases the biopsiedmaterial showed completely normal breast tissue.

Processingof BreastTissue.The tissuewasthawed and cut into smallpieces with a razor blade, washed repeatedly with 0.04 MTris-HC1, 1.5mM EDTA, 0.02% NaN3, pH 7.4, and homogenized with an Ultraturrax homogenizer in the same buffer at 4'C. After centrifugation at 800x g for 5 mm, the pellet was discharged and the supemnatantwas furthercentrifuged at 30,000 x g for 15 mm. The 30,000 x g supernatant wasfurther centrifuged at 105,000 x g for 60 mm, and the supernatantfraction, designated cytosol fraction, was immediately used for ER andPgR determination. The 30,000 x g pellet was resuspended in 10 mr@iTris-HC1, 50 mM NaCl, 0.1% bovine serum albumin, 0.02% NaN3(Tris-NaCI-buffer) pH 7.4, and was recentrifuged at 30,000 x g for 15mm. The pellet was rehomogenized in Tris-NaC1-buffer,and its proteincontent was adjusted to 1.7 mg/mI. These membrane samples werestored at —80Cuntil analysis for IGF-R and EGF-R.

Growth Factors. Mouse EGF (Sigma, St. Louis, MO) and humanrecombinant IGF-I (Amgen, Thousand Oaks, CA) were iodinated bychloramine T (13). The specific activities were 50—150@Ci/@g.The

1343

Receptors for Epidermal Growth Factor and Insulin4ike Growth Factor I and TheirRelation to Steroid Receptors in Human Breast Cancer'

Fredrika Pekonen,2 Seppo Partanen, Tuulikki Mäkinen, and Eeva-Marja Rutanen

Minerta Institutefor Medical Research,Helsinki (F. P.. T. M., E-M. R.J,and Department ofPathology, TheJorvi Hospital, Espoo,(S. P.1,Finland

ABSTRACT

Levels of epidermal growth factor receptor (EGF-R) and insulin-likegrowth factor receptor (IGF-R) in breast cancer tissue were evaluated.The bindingof growth factors was compared to the content of estrogenreceptors (ER) and progesterone receptors (PgR).

EGF-R correlated negativelyto the ER and PgR (Kendallcorrelation,P < 0.001), whereas the IGF-R correlated positively to ER and PgR(analysis ofvariance, P < 0.001). In contrast, no correlation was foundbetween EGF-R and IGF-R. IGF-R binding was higher in tumor tissuesthan in adjacent normal tissues(Wilcoxon rank test, P < 0.001), whereasthe EGF-R binding in normal tissue did not differ from that in cancertissue.The degreeof differentiation in ductal breast cancercorrelated toEGF-R (x2 test, P = 0.018), but not to IGF-R. The bindings of bothgrowth factors were the same in metastases and primary breast tumors.

Our results show that EGF-R and IGF-R are present in normal breasttissue and breast cancer tissue. The growth factor receptors are relatedto steroid receptor content and their presence is associated with m,ligoanttransformation of breast cells and dedifferentiation of breast cancer.

INTRODUCTION

Estrogens are known to play an important role in breastcancer growth (1, 2). The mechanisms by which estrogen induces cell proliferation have recently been a focus of greatinterest. There is substantial evidence for the induction of

autocrine growth factors by estrogen in breast cancer (1). Themammary tumor-promoting role of EGF3 and IGF in vivo havebeen shown in several models. Infusion of EGF and IGF intonude oophorectomized mice injected with MCF-7 breast cancercells induced tumor formation (3). Tumor formation in a mousespecies, highly susceptible to spontaneous mammary tumors,can be reduced by removing the main source of EGF, thesubrnandibulary gland, and can be stimulated by injecting EGFinto the sialoadenectomized mice (4).

Both estrogen and progesterone as well as the growth factorsact by binding to receptors specific for the respective hormonesand growth factors. The estrogen and progesterone receptorcontents of mammary tumors are considered important prognostic indicators. Patients with hormone receptor-positive tumors respond better to hormone therapy and survive longer (5).EGF-receptors, a c-erb B protooncogene related product, havealso been shown to be of importance for the turnorigenicity ofbreast cancer. High concentration of EGF receptors in A43 1cells directly influenced the growth ofthese cells as solid tumorsin nude mice (6). In human breast cancer biopsies high EGF

receptor binding has been found in association with low content

of estrogen receptors (7—9).A high EGF receptor content hasbeen reported in mammary carcinoma rnetastases and tumors

Received6/17/87; revised 10/23/87; accepted11/12/87.The costsof publicationof this article weredefrayedin part by the payment

of page charges.This article must therefore be hereby marked advertisement inaccordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported by grants from the Cancer Society of Finland,

Nordiska Insulinfonden, and Liv och Htlsa.2 To whom requests for reprints should be addressed, at Minerva, P. 0. Box

819, 00101, Helsinki 10, Finland.3 The abbreviations used are: EGF, epidermal growth factor, IGF, insulin-like

growth factor, EGF-R, epidermal growth factor receptor, IGF-R, insulin-likegrowth factor receptor, ER, estrogen; PgR, progesteronereceptors.

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GROWTH FACTOR RECEPTORS IN BREAST CANCER

iodinated growth factor remained stable for at least 2 months, as testedby trichloroacetic acid precipitation.

ReceptorDeterminations.ER and PgR were determined as describedby Vihko et a!. (14). Briefly, the cytosol fractions were incubated withvarious concentrations of estrogen or progesterone for 18 h at 4C.Bound and unbound steroids were separated by dextran-coated charcoal. The affinity and capacity ofthe steroid receptors were determinedby Scatchard analysis (15), and receptor content wasexpressed as fmol/mg cytosol protein.

For EGF receptor determination, a 100 @zlmembrane suspensionwas incubated with (‘25IIEGF,30,000 cpm, in the presence or absenceof 1.6 x l0@ M unlabeled EGF in a final volume of 250 @lTris-NaC1buffer, pH 7.4, for 60 mm at 37C. The incubation mixture was layeredon top of 300-edice-coldTris-NaCl buffer and centrifuged at 30,000 xg for 15 mm. The supernatant was aspirated, and the pellet counted inan autogamma counter. The binding depicting EGF-R activity wasexpressed as specificbinding (total [‘25IJEGFbinding minus binding inthe presence of 1.6 x i0@ M EGF) in percentage of total (‘25IIEGFadded (B/T %).

For IGF receptor determination, a 100 @lmembrane suspension wasincubated with [‘251J1GF-1,30,000 cpm, in the presence or absence of0.7 x l0@ M unlabeled IGF-I in a final volume of 250 zl Tris-NaCIbuffer, pH 7.6, for 18 h at 4'C. The binding assay was then processedas described for EGF. The binding depicting IGF-R activity was cxpressed as specific binding (total [‘251J1GF-Ibinding minus binding inthe presence of 0.7 x l0' M IGF-I) in percentage of total [1251JIGF-1added (B/T %).

The presence ofa 34,000 IGF-binding protein, identical with placental protein 12 (16), was measured in the cytosol fraction of breasttissues by a specific radioimmunoassay as previously described (17).The sensitivity of this assay is 9.8 ng/ml. The data were expressed asag/mg cytosol protein.

Analysis of variance and Kendall correlation were used to studycorrelation between steroid receptors and growth factor receptors incancer tissue. For comparison of growth factor binding in cancer tissueand adjacent normal tissue Wilcoxon signed rank test was used. AnEGF and IGF binding higher than 2% was arbitrarily called highbinding and a binding less than that was called a low binding. Comparison of high and low EGF and IGF binding to cancer tissue differentiation, to presence of metastases, to tumor size, to benign mammarylesions, and to normal mammary tissue was carried out by the x2 test.

RESULTS

The EGF binding to breast tumor tissue was specific and hadan apparent affinity of 1—3x iO@M@ (Figs. 1 and 2). Thespecificity of the IGF binding suggests a type I receptor, withless than 10% cross-reactivity by multiplication stimulatingactivity (rat IGF-II) and approximately 0.1% cross-reactivity byinsulin (Fig. 1). The K5 value for the IGF-R was 0.5—1.7 x 10@M' (Fig. 2).

The EGF binding in breast cancer did not correlate to theIGF binding in the same tumor (N = 142, variance analysis, R= 0.1 14, P = 0.1 1 3. Kendall correlation, T = —0.047, P =

0.207). There was, however, a nonlinear negative correlationboth between EGF-R binding and ER (Kendall correlation: N= 170, T = —0.259, P < 0.001), and between EGF-R binding

and PgR (Kendall correlation: N = 170, T = —0.206,P <0.001), indicating an inverse relationship between EGF-R andsteroid receptors in breast cancer (Figs. 3 and 4). There was alinear correlation between IGF-R binding and ER content (N= 142, R = 0.505, P < 0.001), and between IGF-R and PgR

content (N = 142, R = 0.346, P< 0.001), showing that increasedsteroid receptor content is associated with increased IGF-Rbinding (Figs. 5 and 6). The correlation between ER and PgRwas significant (N = 170, R = 0.407, P < 0.001) (data notshown).

There was no correlation between growth factor binding and

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tissue. Breast tumor membranesfrom pooled tumors, 170 @ig/1O0@ilwere incubatedwith (‘25IJEGF(30,000 cpm) in 10 mrs.iTris-HCI, 50 mi@iNaCI, 0.1% BSA,0.02% NaN3, pH 7.4, at 3TC for I h (top), or with [‘251J1GF-I(30,000 cpm) in10 m@.iTris-HCI, 50 mM NaCI, 0.1% BSA, 0.02% NaN,, pH 7.6, at 4C for 18h (bottom) in the presenceof different concentrations of unlabeled proteins.Radioactivity bound to the membraneswas precipitated at 30,000 X g and wascountedin a gammacounter.Nonsaturablebindingmeasuredin the presenceof6 x i0@ M growthfactorIEGF, (top)and IGF (bottom)Jwassubtractedfrom allvalues.Binding wasexpressedasbinding in the presenceofunlabeled protein (B)divided by binding in the absenceof unlabeled protein (Be). The EGF and IGFdisplacementcurvesrepresentmeanfrom sevendifferent experiments in duplicate±SD whereas the cross-reactivity of other proteins represents mean of twodifferentexperimentsin duplicate±SD. Top:•,EGF; Y, transferrin;•,insulin;., thyrotropin;A, humanchorionicgonadotropin.Bottom:•,IGF-I;A, MSA(rat IGF-lI); @,insulin.

age of the patients (mean age, 60 years; range, 28—89years).Neither was any association observed between growth factorbinding and breast tumor size (<2 cm or @2cm), or presenceof axillary rnetastases at the time of the primary operation in120 patients with ductal breast cancer (data not shown). Inductal carcinoma with low differentiation, EGF binding washigh in 26%, and IGF binding was high in 64% (Fig. 7). Inintermediate-differentiated ductal carcinoma, high EGF-Rbinding was observed in 13% and high IGF-R binding in 67%.In highly differentiated cases the EGF-R binding was high in7% and the IGF-R binding was high in 50%. Thus, a trend forhigher EGF binding in less-differentiated tumors was evident.The x2 test resulted in P = 0.01 8 for EGF binding in highversus low-differentiated ductal carcinoma. The IGF bindingwas similar in all three groups.

In metastases, high EGF binding was observed in 7% andhigh IGF binding in 46% (Fig. 7). The growth factor bindingin metastases was not different from that in primary tumors.

In one ofeight cases ofproliferative rnastopathia (14%) EGF1344

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GROWTH FACTOR RECEPTORS IN BREAST CANCER

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tein (jlacental protein 12). The binding protein was detectablein two of 73 breast cancer tissues (6.6 ng/mg cytosol proteinand 2.6 ng/mg cytosol protein) and in one of 19 normal breasttissues (4.0 ng/mg cytosol protein). The latter was adjacent toa cancer tissue without detectable binding protein.

DISCUSSION

20 40 60 80 100 300ESTROGENRECEPTORS(fmol./ing prot&n)

Fig. 3. EGF receptorsversusestrogenreceptorsin 170 breastcancertissues.Kendallcorrelation,T = —0.259,P < 0.001.B, boundtracer,T, total traceradded.

binding exceeded 2%, and in four of eight of the tissues (50%)the IGF binding was above 2%, but lower than 4% (Fig. 8). In5% (one of 19) of normal breast tissues EGF and IGF bindingexceeded 2%. The IGF binding in normal breast tissues displayed in Fig. 8 was significantly lower than that in the ductalcarcinomas in Fig. 7 (x2 test, P< 0.001), whereas no differencewas found between EGF binding to normal and to cancertissues. The only normal tissue with high EGF and IGF bindingwas adjacent to a carcinoma which was so small that onlysamples for histological examination were obtained, but noreceptor determinations were possible.

When growth factor receptor binding was compared in pairedsamples obtained from breast cancer tissue and adjacent normaltissue, the binding of IGF to cancer tissue was higher than thatto normal tissue (Wilcoxon signed rank test, P < 0.001; Fig.9). In the same 15 pairs of tissues, no difference in EGF-Rbinding was evident. Only one cancer tissue showed EGF-Rbinding exceeding 2%, whereas the binding was low in allnormal adjacent tissues.

Breast tissues were also tested for 34,000 IGF-binding pro1345

The observation that breast tumor cell lines produce growthfactors in response to estrogen stimulation (1), and that breasttumor cell lines have receptors for growth factors includingEGF receptors (1, 18) and IGF receptors (12), raised thequestion of a possible relationship between steroid receptorsand growth factor receptors. In this work we show that suchrelations exist, both between ERs, EGF-Rs, and IGF-Rs as wellas between PgRs, EGF-Rs, and IGF-Rs. The relation betweensteroid receptors and EGF-Rs is, however, distinct from thatbetween steroid receptors and IGF-Rs, and no relation betweenEGF-Rs and IGF-Rs was observed.

Our results on an inverse relation between EGF receptorsand steroid receptors are consistent with previous reports (7—10, 19). The reason why high ER and PgR excludes the presence

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GROWTH FACTOR RECEPTORS IN BREAST CANCER

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tissues. Analysis of variance, R = 0.346, P < 0.001. B, bound tracer, T, totaltracer added.

patient numberFig.9. TheIGF receptors(top)andEGFreceptors(bottom)in15breastcancer

tissues(I) and adjacent normal tissues(0). B, bound tracer T, total tracer added.

of high EGF-R and vice versa@ is not evident. The relation isfurther complicated by observations that both estrogen (20) andprogesterone (21) increase EGF-R content in hormone-responsive tissue, and that EGF (22) induces its own receptor.

Recently, the HER-2/neu oncogene which is related to theEGF receptor was shown to be amplified in human breasttumors, and the gene amplification was a significant predictorof relapse and survival (23). Similarly, the content of EGFreceptors which is related to the c-erb B oncogene has been

reported to be correlated to metastases and dedifferentiation(6, 10). In support of such a dedifferentiating role for EGF-Rwe observed a higher EGF-R binding in tumors with lowhistological differentiation than in tumors with high differentiation. Walker et a!. (1 1), in contrast, found no correlationbetween tumor differentiation or DNA ploidy and EGF-R.Similarly, no correlation between EGF-R and differentiationwas seen by Skoog et aL (19). Sainsbury et a!. (9) reported thattumors with a higher EGF binding have a higher potential formetastases and that metastases have a higher EGF-R binding.Our results, however, do not support such a conclusion; wefound an EGF-R binding in metastases similar to that inprimary breast tumors, and we found no correlation betweenEGF-R binding in breast tumors and the presence of metastasesat primary operation.

Comparing the EGF-R binding in normal breast tissue andadjacent carcinoma tissue no statistically significant differencewas seen, although the highest EGF-R binding in the pairedsamples was seen in tumor tissue. The only normal tissue withhigh EGF-R binding was adjacent to a carcinoma tissue whichdue to its small size was not available for receptor determination. Therefore, in this particular case it remained unclearwhether the binding in the tumor exceeded that in the normaltissue. The presence of EGF-R in normal murine breast cellshas been reported (24), supporting our observation on measurable EGF-R in normal human breast tissue.

The presence of IGF-R in breast cancer, as shown in thisstudy, and their presence in breast tumor cell lines (12), as wellas the production of IGF by breast tumors (1), suggest a rolefor IGF in breast tumor growth. The IGF-R expression was

metastases

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GROWTH FACTOR RECEPTORS IN BREAST CANCER

different from that of the EGF-R in breast tumors, and correlated positively to both ER and PgR. The mechanisms possiblyregulating the IGF-R expression in breast tumors are not

known, but the observed relation to steroid receptors suggestthat there might be a common regulator for these receptors.Estrogen, which induces the progesterone receptor, has beenshown to increase the IGF-I production in breast cancer (1),but the effect of estrogen on the IGF receptor has not, to ourknowledge, been evaluated.

The IGF binding in breast tumors was significantly increasedcompared to that in the adjacent normal tissue, suggesting thatthe expression of IGF-R is associated with malignant transformation ofbreast epithelial cells. The difference between normaland tumor tissue was much more consistent as regards to IGFR than to EGF-R. Interestingly, the only normal breast tissueexhibiting high IGF binding was the same tissue sample exhibiting high EGF binding. It is tempting to speculate that thetissue in close proximity to mammary cancer tissue, in spite ofits normal histological appearance, possesses malignant biochemical features. The growth factor receptors in normal breasttissue need, however, to be studied in more detail before thenormal range for growth factor binding in breast tissue can beestablished.

When differentiation of ductal carcinoma was compared toIGF binding, no trend for increased receptor binding withdecreasing differentiation was evident. The total number ofmammary tumors with high IGF binding was, however, greaterthan the total number of tumors with high EGF binding. Thus,the increased expression of IGF-R seems to be a more generalproperty for breast tumors than the expression of EGF-R.

It has been reported that IGF-R determinations might behampered by the presence of secreted nonreceptor bindingproteins. A 34,000 IGF-binding protein (identical to placentalprotein 12) has indeed been shown in breast cancer tissue (25).It was, therefore, ofinterest to determine what role this bindingprotein might play. Our result, however, show that the amountof 34,000 IGF-binding protein in breast tissue is low andexceeded the detection limit of our assay only in three out of80 breast tissue samples.

In conclusion, we have shown that the EGF-R and IGF-R inbreast cancer are both correlated to the ER and the PgR. TheEGF-R is negatively and nonlinearly related to the steroidreceptors so that a high EGF-R binding excludes high ER andPgR contents, and high level of ERs and PgRs excludes highEGF-R binding. In contrast, IGF-R is positively correlated toboth ER and PgR in breast cancer. Both EGF-R and IGF-Rare also present in normal breast tissue.

REFERENCES

1. Dickson, R. B., and Lippman, M. E. Estrogenicregulationof growth andpolypeptide growth factor secretion in human breast carcinoma. EndocrineRev.,8: 29—43,1987.

2. Soto, A. M., and Sonnenschein, C. Cell proliferation of estrogen-sensitivecells:thecasefor negativecontrol.EndocrineRev.,8: 44-52, 1987.

3. Dickson, R. B., Huff, K. K., Spencer,E. M., and Lippman, M. E. Inductionofepidermal growth factor related polypeptides by 17 fi-estradiol in MCF-7

humanbreastcancercells.Endocrinology,118: 138—142,1985.4. Kurachi,H., Okamoto, S., andOka, 1. Evidencefor the involvementof the

submandibular gland epidennal growth factor in mouse mammary tumorigenesis.Proc.Nati Acad.Sci.USA,82:5940—5943,1985.

5. Howell, A., Barnes,D. M., Harland, R. N. L., Redford, J., Bramwell, V. H.C., Wilkinson, M. J. S., Swindell, R., Crowther, D., and Sellwood,R. A.Steroid-hormone receptors and survival after first relapse in breast cancer.Lancet, 1: 588—591,1984.

6. Santon, J. B., Cronin, M. T., MacLeod, C. L., Mendelsohn, J., Masui, H.,andGill, G. N. Effectsofepidermalgrowthfactorreceptorconcentrationontumorigenicity of A43l cells in nude mice. Cancer Res., 46: 4701—4705,1986.

7. Fitzpatrick, S. L., Brightwell, J., Wittliff, J. L., Barrows, G. H., and Schultz,G. S. Epidermalgrowthfactorbindingbybreasttumorbiopsiesandrelationship to estrogen receptor and progestin receptor levels. Cancer Res., 44:3448—3453,1984.

8. Perez, R., Pascual,M., Macins, M., and Lage, A. Epidermalgrowthfactorreceptors in human breast cancer. Breast Cancer Res. Treat., 4: 189—193,1984.

9. Sainsbury,J. R. C., Farndon, J. R., Sherbet, G. V., and Harris, A. L.Epidermal-growth-factor receptors and oestrogenreceptors in human breastcancer. Lancet, 1: 364—366,1985.

10. Sainsbury, J. R. C., Malcolm, A. J., Appleton, D. R., Farndon, J. R., andHarris, A. L. Presenceof epidermal growth factor receptor as an indicatorof poor prognosis in patients with breast cancer.J. Clin. Pathol., 38: 1225—1228, 1985.

11. Walker,R. A., and Camplejohn,R. S. DNA flowcytometryof human breastcarcinomas and its relationship to transferrin and epidermal growth factorreceptors.J. Pathol., 150: 37—42,1986.

12. Furlanetto, R. W., and DiCarlo, J. N. Somatomedin-C receptors and growtheffects in human breast cells maintained in long-term tissueculture. CancerRes.,44: 2122—2128,1984.

13. Mäkinen,T., Pekonen, F., Franssila, K., and Lamberg, B-A. Receptors forepidermalgrowthfactorand thyrotropinin thyroidcarcinoma.Acta Endocrinol. (Copenh),in press,1988.

14. Vihko, R., Jänne,0., Kontula, K., and Syrjalá,P. Femalesexsteroid receptorstatusin primary and metastaticbreastcarcinomaand its relationshiptoserum steroid and peptide hormone levels. Int. J. Cancer, 26: 13—21,1980.

15. Scatchard,G. The attractionof proteinsfor small moleculesand ions.Ann.NY Acad. Sci., 51: 660-672, 1949.

16. Koistinen,R., Kalkkinen, N., Huhtala, M-L, SeppSla,M., Bohn, H., andRutanen,E-M. Placentalprotein 12 is a decidualprotein that bindssomatomedinand hasan identicalN-terminal aminoacidsequencewith somatomedin-binding protein from human amniotic fluid. Endocrinology, 118:1375—1378,1986.

17. Rutanen, E-M., Bohn, H., and SeppSlh,M. Radioimmunoassayof placentalprotein 12: levelsin amnioticfluid, cordblood,andserumofhealthy adults,pregnantwomen, and patientswith trophoblasticdisease.Am. J. Obstet.Gynecol., 144: 460—463,1982.

18. Real, F. X., Rettig, W. J., Chess, P. G., Melamed, M. R., Old, L. J., andMendelsohn,J. Expressionof epidermalgrowth factor receptorin humancultured cells and tissues:relationship to cell lineageand stageof differentialion. Cancer Res., 46: 4726—4731, 1986.

19. Skoog, L., Macias, A., Azavedo, E., Lombardero, J., and Klintenberg, C.Receptors for EGF and oestradiol and thymidinekinase activity in differenthistological subgroupsof human mammary carcinomas. Br. J. Cancer., 54:271—276,1986.

20. Mukku, V. R., and Stancel, G. M. Regulation of epidermal growth factorreceptor by estrogen.J. Biol. Chem., 260: 9820—9824,1985.

21. Murphy, L. J., Sutherland, R. L., and Lazarus, L. Regulation of growthhormone andepidermal growth factor receptorsby progestins in breastcancercells.Biochem.Biophys.Res.Commun.,131: 767—773,1985.

22. Kudlow, J. E., Cheung, C-Y. M., and Bjorge, J. D. Epidermal growth factorstimulates the synthesis of its own receptor in a human breast cancer cellline.J. Biol. Chem.,261: 4134—4138,1986.

23. Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A., andMcGuire,W. L Humanbreastcancercorrelationof relapseandsurvivalwith amplification of the her-2/neu oncogene.Science(Wash. DC), 235:177—182,1987.

24. Taketani, Y., and Oka, T. Biological action of epidermal growth factor andits functional receptors in normal mammary epithelial cells. Proc. NatI.Acad. Sci. USA, 80: 2647—2650,1983.

25. Inaba, N., Renk, T., Wurster, K., Rapp, W., and Bohn, H. Ectopic synthesisof pregnancyspecific @Il-glycoprotein(SP1) and placental specifictissueproteins (PP,. PP,@,PP,1, PP,2) in nontrophoblastic malignant tumours.Possiblemarkersin oncology.Klin. Wochenschr.,58: 789—791,1980.

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1988;48:1343-1347. Cancer Res   Fredrika Pekonen, Seppo Partanen, Tuulikki Mäkinen, et al.   Breast CancerFactor I and Their Relation to Steroid Receptors in Human Receptors for Epidermal Growth Factor and Insulin-like Growth

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