Tyrosine Kinase Activity in Breast Cancer, Benign Breast ...breast, 21 benign breast diseases, and...

$: Tyrosine Kinase Activity in Breast Cancer, Benign Breast ...breast, 21 benign breast diseases, and 21 normal breast tissues. We measured tyrosine kinase activity in the cytosol fraction$
(CANCER RESEARCH 49, 516-521, February 1, 1989]

Tyrosine Kinase Activity in Breast Cancer, Benign Breast Disease, and NormalBreast TissueA. Hennipman, B. A. van Oirschot, J. Smits, G. Rijksen, and G. E. J. Staal1

Surgical Department Â¡A.H.J and Department of Haematology, Laboratory for Medical Enzymology [B. A. v. O, G. R., G. E. J. S.J, University Hospital, P. O. Box 16250,3500 CG Utrecht, and Institute of Pathology, University of Utrecht [J. S.J, The Netherlands

ABSTRACT

Tyrosine specific protein kinase activity was determined in 70 specimens of the human mammary gland. These included 28 cancers of thebreast, 21 benign breast diseases, and 21 normal breast tissues. Wemeasured tyrosine kinase activity in the cytosol fraction and in themembrane fraction of the homogenates. In addition cytosolic aldolaseactivity was measured. Tyrosine kinase activity was determined usingpoly(glutamic add :tyrosine = 4:1) as an artificial substrate. Cancers ofthe breast exhibited considerable higher tyrosine kinase activities in bothcytosol and membrane fractions, compared to benign breast tumors (P â‚¬¿�0.001). Benign tumors demonstrated increased activities in cytosol incomparison to normal breast tissues (P < 0.001). Furthermore, thereappears to be a strong association of an enhanced expression of activityof tyrosine kinase in cytosol of primary carcinomas and early systemicrelapse. In combination with aldolase activity a nearly complete discrimination is achieved between malignant specimens on one hand and benignand normal tissues on the other.

INTRODUCTION

At present the best indicator of the biological behavior ofbreast cancer is the presence and number of axillary lymphnode mÃ©tastases.In addition several other morphological andbiochemical features with some value as a prognostic factorhave been described. However, there is a need for more andbetter markers of malignancy and prognosis (1-7).

From a clinical point of view attention should be directed (a)to the establishment of additional prognostic factors, (/>) to thepossibility of using markers to differentiate benign from malignant lesions, and (c) to the identification of the benign lesionsthat signify an increased risk for future development of malignancy.

Tyrosine protein kinases are enzymes that phosphorylatetyrosine residues of protein substrates. Tyrosine phosphoryla-tion is now recognized as an important regulatory mechanismin response to a number of processes including the action ofseveral growth factors and oncogenes (8-10). Slamon et al. (11)demonstrated HER 2/neu oncogene amplification in humanbreast cancer. The HER-2/wew oncogene is related to but notidentical with the c-erbB oncogene (11). The HER-2/neu oncogene codes for a protein which has an extracellular domain,a transmembrane domain, and an intracellular tyrosine kinasedomain. Amplification of this gene in human breast cancer isassociated with time to disease relapse and with overall patientsurvival (11). c-erbB codes for the EGF2 receptor. A correlationof c-erbB-2 gene amplification and protein expression in humanbreast cancer with nodal status and nuclear grading has beenobserved (12). EGF has been demonstrated to increase tumorgrowth in cultures of primary human breast cancers (13). Furthermore, the presence of EGF receptors in human breast

Received 1/21/88; revised 8/22/88; accepted 10/20/88.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.

1To whom requests for reprints should be addressed.2The abbreviations used are: EGF, epidermal growth factor; poly(Glu:Tyr,

4:1), poly(glutamic acid:tyrosine, 4:1); DTT, dithiothreitol.

cancers has been demonstrated and is associated with metastaticpotential (13, 14). The internal domain of the EGF receptorhas tyrosine specific protein kinase activity (8, 15). The v-erbBprotein is a truncated EGF receptor, consisting of the trans-membrane and the internal domain (10).

Expression of these oncogenes in breast cancer may be reflected in increased tyrosine kinase activity. Actual measurement of tyrosine kinase activity has been made possible by theuse of artificial substrates. Initially these were composed ofamino acid sequences related to the phosphorylation site of thevarious tyrosine kinases coded for by oncogenes. We determined tyrosine kinase activity in breast tumors as well as innormal breast tissues using poly(Glu:Tyr = 4:1) as an artificialsubstrate (16, 17).

It is well established that malignant cells usually exhibit ahigh glycolytic rate even under aerobic conditions. Recently wedemonstrated significantly increased glycolytic enzyme activities in malignant breast tissues, in comparisons to normal andbenign breast tissues (18). In discriminant analysis the functiongiving the best fit to the histolÃ³gica! data was mainly based onthe activity of the enzyme aldolase and in a lesser degree on theexpression of 7-enolase. The discrimination achieved showed ahigher percentage of correct classifications for both benign andmalignant disease.

Here we present the results of the determination of tyrosinekinase activity in the cytosol fraction and the membrane fractionof homogenates of normal, benign, and malignant breast tissues, in combination with the determination of aldolase activity.With this method it is possible to discriminate normal andbenign breast tissues from breast cancers. Furthermore it appears that tyrosine kinase activity is higher in biologically moreaggressive tumors.

MATERIALS AND METHODS

Patients and Pathology. The patients were grouped according tohistology: juvenile hypertrophy of the breast; benign breast disease; andcarcinomas of the breast. Specimens were obtained when a benignbreast lump was excised. In the case of malignancy, the tumors wereeither biopsied or treated surgically. The specimens of breast tissuefrom patients with juvenile hypertrophy were obtained when a reductionmammoplasty was performed. These specimens did not show anyabnormality on histolÃ³gica! examination and were therefore acceptedto represent the enzyme activities of normal breast tissue.

Two series of patients were investigated. In the second series artificialsubstrate from a different batch had to be used for the tyrosine kinaseassays, resulting in lower figures for the activities measured. The twoseries had to be analyzed separately.

The first series (n = 40) consisted of 10 patients with juvenilehypertrophy of the breast, 10 patients with benign disease, and 20patients with carcinoma of the breast. The histology of the specimenswith benign disease was fibroadenoma (n = 3) and fibrocystic disease(n = 7), all of which demonstrated proliferative features (19). Clinicaland pathological data for the patients with malignant disease aresummarized in Table 1. The 2 patients with systemic disease at presentation were not treated surgically. For the remaining 18 patients theaxillary nodal status was evaluated histopathologically; in 9 of 18 no

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TYROSINE KINASE IN BREAST CANCER

Table 1 Summary of clinical and pathological data of the patients with cancer of the breast

PatientNo.12345678910111213141516171819202122232425262728Series11111111I1111111111122222222Age

(yr)55737939676843734454463963295264576746616545776556735788Clinical

stage"

at diagnosis2B13A43A3A12B2A2A2A2B42B2B12A2A2A2B2A1442A113AMenopause*Post-Post-Post-Pre-Post-Post-Pre-Post-Pre-Post-Pre-Pre-Post-Pre-Pre-Post-Post-Post-Pre-Post-Post-Pre-Post-Post-Post-Post-Post-Post-Estrogen

receptor(fmol/mgprotein)3697363100049610778600261440d17027528539390175166721541756871670HistologyInfiltrating

ductular carcinomaInfiltrating

ductular carcinomaLobularcarcinomaInfiltrating





ductular carcinomaLobularcarcinomaInfiltrating











ductular carcinomaLobularcarcinomaMixed

pattern:infiltratingductularandlobularcarcinomaInfiltrating






ductular carcinomaFollow-up'Disease

free at 15mosDisease

free at 17mosSystemic

relapse at 12mosProgressive

diseaseat7mos, died at22mosDisease


relapse at9mosDisease



free at 28mosDiseasefree at 27mosDisease


relapse at 11mos,died at 22mosWithdrew

herselffromtherapyat 4mosSystemic

relapse at10mosSystemic

relapse at10mosDisease




free at 13mosLocal

and systemic relapse at 12mosDisease

free at 3mosDiseasefree at 3mosPartial

remissionwithtamoxifenat 3mosStable

disease at3mosDisease

free at 3mosDisease

free at 3mosDisease

free at 4mosDisease

free at 1 mo

" Clinical stage according to UICC, revised edition 1987.'' Premenopausal status includes the first 2 years after cessation of menstrual cycle.' Duration of follow-up in months from date of diagnosis.'' No data on steroid receptor content available.

lymph node mÃ©tastaseswere detected. In this first series a medianduration of follow-up of 17.5 months has been reached (range, 11-64months). At present 6 of the 18 patients with locoregional disease atpresentation have relapsed with systemic disease.

The second series (n = 30) consisted of 11 patients with juvenilehypertrophy, 11 patients with benign disease, and 8 patients withcarcinoma of the breast. The histology of the specimens with benigndisease was fibroadenoma (n = 5) and fibrocystic disease (n = 6) ofwhich 4 specimens showed proliterative features (19). For clinical andpathological data for the patients with carcinoma of the breast, seeTable 1. For 3 carcinoma patients no histopathological axillary nodalstatus is known: the 2 patients with systemic disease at presentationwere not treated surgically; 1 patient underwent mastectomy withoutaxillary dissection. Of the remaining S patients, 1 had tumor positiveaxillary lymph nodes.

HistolÃ³gica!diagnosis was made according to WHO criteria (20). In12 malignant specimens (7 specimens from the first series, 5 specimensfrom the second series) a sufficient amount of tissue was available toallow histopathological evaluation of tumor tissue in juxtaposition tothe enzymologically assayed part. To determine tissue heterogeneity,the amounts of the various tissue components present were estimated(Table 2). Four categories were used: fat; stromal elements; nontumo-

rous epithelium; tumorous epithelium. The proportion of each wasexpressed as percentage of the total. For this purpose a "lattice" on the

microscopic field was used, a method described previously (18). In thisway also the composition of 11 specimens of normal breast tissue wasevaluated (all from the second series) and 9 specimens with benigndisease (2 from the first series and 7 from the second series).

Sample Preparation. The biopsies were divided by the pathologist

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TYROSINE KJNASE IN BREAST CANCER

Table 2 Composition of tissue in juxtaposition to the enzymological assayed partof the specimens

The proportion of each component present was noted as percentage of thetotal. Four categories were used: fat; stromal elements; nontumorous epithelium;tumor epithelium. The mean percentages of the latter 3 categories are presented(range in parentheses).

Stromal Nontumorous Tumorouselements epithelium epithelium

Normal breast tissues (n -11)Benign

breast disease (n â€”¿�9)Breast

cancer (n = 12)72.5%(55-90)72%(5-90)49%(10-75)7%(1-15)11.5%(5-20)2%(0-15)35%(10-60)

directly from surgery. Part of it was used for frozen section and paraffinsections; the remaining parts were stored immediately at â€”¿�80Â°Cfor

estrogen and progesterone receptor assay and enzymological evaluation.Before enzymological assay the specimens were first cleared of fat

and debris. All specimens had a wet weight of 150-250 mg.Samples were homogenized in 4 volumes 10 mM Tris-HCl (pH 7.4)

containing 0.25 M sucrose, 1 mM MgCl2, 1 mM EDTA, 1 mM diisopro-pylfluorophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT,and aprotinin (0.5 mg/ml). All debris and nuclei were removed bycentrifugation at 800 x g for 10 min (4Â°C).The supernatant wascentrifuged at 150,000 x g for 30 min (4Â°C).The 150,000 x g super

natant fraction was used as the cytosolic fraction for assay of tyrosinekinase activity.

The 150,000 x g pellet (membrane fraction) was resuspended in 50mM Tris-HCl (pH 7.5), containing 20 mM magnesium acetate, 5 mMNaF, 1 mM EDTA/ethyleneglycol bis C8-aminoethyl etherJ-tyJV-tetra-acetic acid, 1 mM DTT, 30 MMsodium o-vanadate and 0.5% NonidetP-40. After sonication twice for 15 s (0Â°C),the preparation was centrifuged at 150,000 x g for 30 min (4"C) and this supernatant (solubili/ed

membrane fraction) was also assayed for tyrosine kinase activity.Determination of Tyrosine Kinase Activity. Phosphorylation of en

dogenous cytosolic and solubili/ed membrane protein (3 Mg/assay) wascarried out at 20"C in a total volume of 64 n\ containing 50 mM Tris-

HCl (pH 7.5), 20 mM magnesium acetate, 5 mM NaF, 1.0 mM EDTA/ethyleneglycol bis (0-aminoethyl ether)-Ar,./V-tetraacetic acid, 30 MMsodium o-vanadate, and 1 mM DTT. The reaction was started by adding50 MMATP ([32P]ATP) with a specific activity of 0.75 Ci/mmol. After

20 min the reaction was stopped by the addition of 25 n\ of a solutioncontaining 17.5 mM ATP and 70 mM EDTA.

Tyrosine kinase activity was determined in the above system byadding poly(Glu-Tyr, 4:1) (Sigma, Chemical Co., St. Louis, MO) as

substrated (278 MM).After stopping the reaction by adding 17.5 mMATP and 70 mM EDTA samples were spotted on Whatman No. 3 MMpaper, precipitated, and washed several times in 10% trichloroaceticacid and 100 mM NaiPjCh. Paper discs were then washed inethanol:ether (1:1) and 100% ether. Scintillation fluid was added andthe samples were assayed for radioactivity.

Tyrosine kinase activity was corrected for endogenous phosphoryla-tion, measured as described above in the absence of added substrate.Tyrosine kinase activity is expressed as pmol ATP/min/mg protein.The protein content was determined according to the method of Lowryet al. (21).

The protein tyrosine kinase assay is linear as a function of time andprotein up to 30 min and 10 Mgprotein, in the cytosol as well as thepaniculate fractions of normal breast tissues and of benign and malignant breast tumors.

Determination of Aldolase Activity. Aldolase (D-fructose 1,6-diphos-phate D-glyceraldehyde-3-phosphate lyase; EC 4.1.2.13) activity determination was carried out as described previously (18). One unit ofactivity was defined as that amount of enzyme which catalyzes theformation of 1 niuol of product/min in standardized conditions. Specific activities are expressed as units/mg protein.

Statistical Analysis. Differences between groups were tested usingStudent's t test. To normalize the distribution of values, the activities

were first transformed in natural logarithms (22).

RESULTS

Tyrosine kinase activities in the cytosolic as well as in thesolubili/ed membrane fractions of normal breast tissues, benignbreast disease, and carcinomas of the breast are presented inTable 3. Tyrosine kinase activities in tumors, benign and malignant, are higher compared to those of normal tissues of thebreast. Within the group of tumors the cancers have higheractivities compared to benign tumors. For cytosolic tyrosinekinase activity differences between normal and benign as wellas between benign and malignant are highly significant. Incontrast, the difference between membrane bound tyrosine kinase activity in normal and benign breast tissue is less welldeveloped, whereas benign compared with malignant breasttissue gives a highly significant difference.

During the course of our investigations we noticed batch-to-batch differences in the absolute levels of tyrosine kinase activitywhen using different batches of poly(Glu:Tyr, 4:1). The batchesalso varied in their mean molecular weights. We therefore hadto divide our patient material in two series. Although the meanlevels of tyrosine kinase activity were slightly lower in thesecond series, the results of the first series were completelyreproduced.

In both series some overlap exists in the activities observedin normal, benign, and malignant specimens. The tyrosinekinase activity in cytosol produces a better separation betweenthe specimens, compared with the membrane bound tyrosinekinase activity. The 2 series taken together the cytosolic tyrosinekinase activity of 4 of 21 benign specimens overlap with carcinomas. One of these was also demonstrated to be heavilyinfiltrated with lymphocytes. Four of 28 malignant specimensoverlap with benign tumors. Discrimination on the basis ofmembrane bound tyrosine kinase activity shows 9 of 21 benignspecimens in "malignant range," including the specimen dem

onstrating the lymphoid infiltration, whereas 16 of 28 carcinomas overlap with "benign range." In these relatively small

series no significant differences could be observed betweenhistologie-aÃs̄ubgroups of carcinomas, or benign diseases. Al

dolase activities are highest in carcinomas. Benign tumors showan intermediate position, while normal tissues have the lowestactivity levels. All differences are significant (P ss 0.02).

Separation of the three groups according to their histologycan thus be achieved either by tyrosine kinase activity in thecytosolic fraction or by aldolase activity. However, Fig. 1 dem-

Table 3 Tyrosine kinase activity in cytosol ana in the solubilized membranefraction together with aldolase activity of normal breast tissues, benign breast

disease, and carcinomas of the breast of both series

Tyrosine kinase activity is given in pmol ATP/min/mg protein, aldolaseactivity in units/mg protein. All values are mean Â±SE. For aldolase, series 1 and2 are combined.

Tyrosine kinaseTyrosine kinase activity

activity in membranecytosol" bound* Aldolase

Normal breast tissueSeries 1 (n = 10)Series 2 (n = 11)Benign

breast diseaseSeries 1 (n = 10)Series 2 (n =11)Breast

cancerSeries 1 (n = 20)Series 2 (n = 8)2.44

Â±0.491.24 Â±0.3910.89

Â±2.514.09 Â±1.2658.21

Â±6.8433.84 Â±10.6053.11

Â±11.3024.04Â±7.2584.50

Â±19.3066.44 Â±18.42215.52

Â±32.49214.58 Â±55.000.005

Â±0.0006(1 =21)0.01

5 Â±0.003(n =21)0.107

Â±0.017(n = 28)

â€¢¿�Series 1: normal/benign, P < 0.001; benign/carcinoma, P < 0.001. Series 2:

normal/benign, P < 0.05; benign/carcinoma, P < 0.001.* Series 1: normal/benign, not significant; benign/carcinoma, P = 0.001. Series

2: normal/benign, P = 0.05; benign/carcinoma, P < 0.01.

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Aldolase activity0.500-

0.100

o.ao

I 0.8-I/I

O

|o.e-

0.2

1.00 10.00 100.00T.K. cylosol activity

Fig. 1. Discrimination of normal breast tissue, benign breast disease, andbreast cancer by plotting cytosol tyrosine kinase (T.K.) activity against aldolaseactivity of each specimen separately. Cytosol tyrosine kinase activity is presentedlogarithmically on the X-axis, aldolase activity is presented logarithmically onthe Y-axis. â€¢¿�,normal breast tissue; A, benign breast disease; A, breast cancer.

onstrates an improved discrimination of the groups of the firstseries, by plotting cytosol tyrosine kinase activity of each specimen against its aldolase activity. In this way a fair discrimination is reached. Normal, benign, and malignant specimensconcentrate each in 1 of 3 zones with, in this small series,relatively few overlaps. Of clinical interest is distinguishingmalignant from benign specimens. The benign specimen inproximity of the main group of carcinomas is a specimen withthe histology of fibroadenoma exhibiting a high aldolase activity. The carcinoma in the benign range shows low expressionof activity of both cytosolic tyrosine kinase and aldolase. Apossible contamination with nontumorous epithelium accountable for low activities in this specimen can be neither provednor denied because a histolÃ³gica! evaluation of tissue in juxtaposition to the enzymological assayed part is not available.Plotting the specimens of the second series (graph not shown),only one benign specimen overlapped with the carcinomas. Thehistology observed in this specimen was a fibrocystic diseasewith proliferative features; in addition it proved to be heavilyinfiltrated with lymphocytes. This contamination with lymphocytes readily explains the high activities measured, inasmuch asnormal lymphocytes demonstrate extremely high activities fortyrosine kinase in both cytosol and membrane bound (resultsnot shown).

We considered the possibility that the level of tyrosine kinaseactivity reflects the grade of malignancy of breast cancer. Weanalyzed the natural history of the disease in the patients of thefirst series. Because of the relatively short duration of follow-up only disease free survival was used as end point of thisanalysis. At a median follow-up of nearly 18 months 6 of 18patients with operable disease have relapsed. Fig. 2 displays thedisease free survival curves for the population stratified intoone group having tyrosine kinase activities in cytosol lower thanor equal to the mean of all patients in the first series and asecond group with activities higher than the mean. The onepatient who relapsed with a tyrosine kinase activity in cytosollower than the mean is the same patient that had to be plottedin a benign range (Fig. 1). The median follow-up in the groupwith lower or equal activities for tyrosine kinase in cytosol isapproximately 10 weeks shorter compared to the median follow-up for the group with the higher activities. Nevertheless,even this shorter median duration of follow-up exceeds thelongest disease free survival of the patients that relapsed. It

6 12 18 months

Fig. 2. Disease free survival curves of the patients with operable cancer of thebreast from the first series (n = 18). The patients are stratified into a group withtyrosine kinase activity in cytosol below or equal to the mean of the total numberof breast cancer patients of the first series and a second group with tyrosine kinaseactivity in cytosol higher than the mean. , group of patients with lowactivities for tyrosine kinase in cytosol; , patient group with high activities.The curves for each group are drawn for the period of median duration of follow-up; in the remaining patients of each group no events occurred afterwards.

therefore would appear that a high activity of tyrosine kinasein cytosol is accompanied by a more aggressive biology of thetumor.

Other factors known to be important in the prognosis ofbreast cancer are the size of the tumor, the axillary nodal status,hormonal receptor status, and age. In the first series no significant differences were found between groups when stratified ondiameter of the tumor (smaller than or equal to 2 cm and largerthan 2 cm) or between the groups with and without axillarylymph node mÃ©tastases;also no differences were noted betweenpremenopausal and postmenopausal patients and no correlation was seen between tyrosine kinase activity and level ofestrogen receptor binding. However, this present series is small.In this respect it is noteworthy that a clear prognostic separationappears when a stratification on tyrosine kinase activity incytosol is used.

DISCUSSION

Tyrosine specific protein kinase activity is associated withproducts of several viral transforming genes and their cellularhomologues, as well as with a number of growth factor receptors(8-10). This paper is an attempt to assess the relevance ofoverall tyrosine kinase activity for the diagnostic and/or prognostic classification of human mammary tumors. Recently ithas been demonstrated that protein tyrosine kinases phospho-rylate synthetic random copolymers of tyrosine and glutamicacid (16, 17). In this study poly(Glu:Tyr, 4:1) was used as anartificial substrate.

Our data show a 25-fold increase of tyrosine kinase activityÂ¡nthe cytosol fraction of breast cancer specimens, when compared to normal breast tissue. For membrane bound tyrosinekinase the increase in breast cancer specimens compared tonormal breast tissue is 4- to 8-fold. Benign tumors take anintermediate position: compared to normal the increase intyrosine kinase activity in the cytosolic fraction is 4-fold; formembrane bound tyrosine kinase activity the increase is 2-fold.In the first series, at a median follow-up of nearly 18 months,a strongly increased risk of relapse is demonstrated for thegroup with higher activities for tyrosine kinase in the cytosol.

Tissue heterogeneity has been recognized to influence thelevel of activity that is measured Â¡nenzymological assays inbreast cancer specimens (18, 23). The best method at hand toassess the composition of the enzymological assayed part of the

519




tumor is to evaluate histologically the parts in juxtaposition. Inthis study in nearly half of the specimens a sufficient amountof tissue was available to allow this evaluation (Table 2). Comparing benign with malignant tumors the increase in tyrosinekinase activity exceeds the increased presence of epithelium,normal and malignant. Therefore, the increased tyrosine kinaseactivity in cytosol in malignant breast tissues is not in alllikelihood explained by the increased presence of epithelium inthe malignant specimens. Moreover, we could not establish asignificant correlation between the amounts of nontumorousand/or tumorous epithelium present and the activities of tyrosine kinase that were measured.

It is tempting to speculate on the origin of tyrosine kinaseactivity in breast cancers. In human breast cancer specimensamplification of an oncogene that codes for tyrosine kinaseactivity has been described. Slamon et al. (11) examined 189breast cancers; 53 demonstrated amplification of the HER-2/neu oncogene. In an earlier study he demonstrated the expression of c-fes oncogene in 3 of 4 breast cancer specimens examined (24). The c-fes transcript also has tyrosine kinase activity(8, 9). Correlation of HER-2/neu gene amplification with several disease parameters demonstrated a highly significant correlation with overall survival and time to relapse (11). It isnoteworthy that only 4 specimens of 189 exhibited EGF receptor gene amplification.

Berger et al. (12) analyzed 51 primary human breast cancersfor amplification of the c-erbB-2 protooncogene. In 13 of 51DNA isolates a 2- to 15-fold amplification of this protooncogene was demonstrated. Also c-erbB-2 protein specific antibodies were used to stain paraffin embedded slides of these tumors.In 10 of 12 tumors demonstrating amplification, the c-erbB-2protein was observed. In addition, 13 of 35 specimens with asingle copy c-erbB-2 stained positively for this protein. Positivestaining was significantly associated with nodal status andnuclear grade of malignancy.

Sainsbury et al. (14) assayed by radioligand binding andmonoclonal antibody techniques for the presence of EGF receptors in a series of 104 primary breast cancers and 14 lymphnode mÃ©tastases.Of the primary cancers 35 were EGF receptorpositive and 10 lymph node mÃ©tastaseswere EGF receptorpositive. They demonstrated a significant inverse relationshipbetween the presence of EGF receptors and the presence ofestrogen receptors in primary tumors. Also EGF receptors werefound in a greater proportion of the mÃ©tastasesthan in primarytumors (14).

Fitzpatrick et al. (25) used specific binding of I25I-EGF to the

membrane fraction of tissue homogenates of breast cancerspecimens and found that 48% of 137 primary cancers and 42%of 24 metastatic cancers were EGF receptor positive (EGFreceptor positive defined as >1 fmol EGF binding per mgprotein).

In vitro studies show enhancement of growth in cell culturesof human breast cancers when EGF was added to the medium( 13). Other mitogenic growth factors have been documented tohave a growth stimulating effect on breast cancer cell lines (26).Several of these receptors have tyrosine kinase activity (8). It isto be noted that all tyrosine kinase activity due to growth factorreceptors is plasma membrane bound. Also the HER-2/neuprotein is plasma membrane bound. Only the c-fes protein hasactivity both in the cytosol fraction and in the membranefraction (9).

The rise in expression of tyrosine kinase activity occurs inboth the cytosol and the membrane fractions. However, theincrease in activity has its greatest extent in the cytosol. Nearly

all of the known sources for tyrosine kinase activity are boundto the plasma membrane. At this moment the explanation forthis clearly developed difference is only speculative. Perhapsthe role of c-fes expression in breast cancer is more importantthan is understood at present. There is a distinct possibilitythat oncogen products not yet described do contribute to thetyrosine kinase activity that is measured. It cannot be excludedthat the major part of the activity measured in the cytosoloriginates from the plasma membrane and has been detachedinto the cytosol during sample preparation. Perhaps the cytosolfraction partially stems from internalized membrane receptorsor from newly synthesized receptors in transit to the membrane.

Our experience in earlier work on glycolytic enzymes in breastcancer prompted us to include determination of aldolase activityin the present research. Using discriminant analysis on a panelof glycolytic enzymes to separate benign from malignant lesions, the aldolase activity proved to be the major contributorto the discriminant function (18). Combining aldolase activityand tyrosine kinase activity in cytosol produces a nearly complete separation of the 3 groups (Fig. 1).

An analysis of events during follow-up of the first series ofpatients was performed at a median duration of follow-up of

18 months. A strongly increased risk of relapse is associatedwith a high activity of tyrosine kinase in cytosol. It is to benoted that an inverse correlation with the estrogen receptormeasurements could not be demonstrated. This result contrastswith observations on the correlation between EGF receptorsand estrogen receptors (14). However, our study deals with 28patients with cancer of the breast, divided over 2 series. Perhapslarger patient numbers are required before significant correlations are to be observed.

From the data quoted it follows that in tumorigenesis ofhuman breast cancer multiple protooncogenes and growth factor receptors associated with tyrosine kinase activity might beinvolved. Without completely knowing the exact origin of thetyrosine kinase activity that is measured, it may well be that thetotal of tyrosine kinase activity is associated with the biologicalpotential of a tumor of the breast. We attempted a first analysisof the clinical relevance of our approach in 2 series with stillrather limited patient numbers. The follow-up study excepted,all results obtained in the first series were completely reproduced in the second series. As no patient of the second seriescould be followed for more than only a few months, diseasefree survival curves were not calculated for the second series.

In conclusion we present data demonstrating an enhancedexpression of tyrosine kinase activity in breast cancers, compared to benign breast tumors, whereas benign disease showshigher levels of activity than normal breast tissue does. Theincrease in activity is more clearly developed in the cytosolfractions of the homogenates. There appears to be an enhancedexpression of activity of tyrosine kinase in primary carcinomasthat cause systemic disease. In combination with aldolase activity a nearly complete discrimination is achieved between malignant specimens on one hand and benign and normal tissues onthe other. It is attractive to research the diagnostic and prognostic aspects of tyrosine kinase activity expression in breastcancer in a larger series of patients.

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1989;49:516-521. Cancer Res A. Hennipman, B. A. van Oirschot, J. Smits, et al. Disease, and Normal Breast TissueTyrosine Kinase Activity in Breast Cancer, Benign Breast

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