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Discussion

This study indicates that a proportion of human breastcancers have EGF receptors of high specificity and affinity onthe cell surface. The inverse relation of EGF and oestrogenreceptor status on the primary tumours is highly significant.Tumour cellularity is related to oestrogen-receptor

status," the more cellular tumours having higher receptorlevels. Thus, failure to detect EGF receptors in the majorityof oestrogen-receptor-positive tumours is unlikely to be dueto cell numbers being too low to detect specific binding in thedisplacement experiments. One possibility is that receptorsmay be present but qualitatively or quantitatively abnormal.However, they were not detected by the displacement assayor by immunohistochemistry. Fibroblastic contamination ofthe pellet is unlikely to contribute significantly to the resultsof the displacement assay; even if the stroma consisted purelyof fibroblasts the Kd and number of sites would not maskthose from the tumour cells. Metastatic potential has beenrelated to the presence of plasminogen activator associatedwith tumours. Stimulation of EGF receptors greatlyincreases plasminogen activator activityl2 in A-431 cells andantibodies to human plasminogen activator can block theexperimental metastases of human cancers. 13 3

This study suggests that the growth of a proportion ofoestrogen-receptor-positive breast cancers may be regulatedby EGF or transforming growth factors interacting with theEGF receptor. The presence of EGF receptors on tumoursdoes not indicate that EGF causes malignant transformation,but their qualitative and quantitative expression in differenttumours may be important in the variation of the tumourbiology. It may be possible to regulate the growth of somehuman breast cancers by interfering with EGF-receptorbinding or internalisation.We are now attempting to correlate the presence of EGF

receptors with relapse pattern and prognosis in human breastcancers, and we are looking for activated oncogenesinteracting with EGF and its receptor.We thank Liz Hirst and Fred Carpenter for technical assistance. J. R. C. S. is

funded by the North of England Cancer Research Campaign.

Correspondence should be addressed to A. L. H., Cancer Research Unit,Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP.

REFERENCES

1. Carpenter G. Epidermal growth factor is a major growth promoting hormone in milk.Science 1980; 210: 198-99.

2. Osborne CK, Hamilton B, Titus G, Livingston RB. EGF stimulation of human breastcancer cells in culture. Cancer Res 1980; 40: 2361-66.

3. Carpenter G, Stoscheck CM, Preston YA, De Larco JE. Antibodies to the epidermalgrowth factor block the biological activities of sarcoma growth factor. Proc NatlAcad Sci USA 1983; 80: 5627-30.

4. Waterfield MD, Scrace GT, Whittle N, et al. Platelet-derived growth factor is

structurally related to the putative transforming protein p28sis of simian sarcomavirus. Nature 1984; 304: 35-39.

5. Doolittle RF, Hunkapiller MW, Hood LE, et al. Simian sarcoma virus onc gene, v-sis,is derived from the gene (or genes) encoding a platelet-derived growth factor. Science1983; 221: 275-77.

6. Downward J, Yarden Y, Mayes E, et al. Close similarity of epidermal growth factorreceptor and v-erb-B oncogene protein sequences. Nature 1984; 307: 521-27

7. Stewart JF, King RJB, Sexton SA, et al. Oestrogen receptors, sites ofmetastatic diseaseand survival in recurrent breast cancer. Eur J Cancer 1981; 17: 449-53.

8. Cooke T. The clinical application of oestrogen receptor analysis in early carcinoma ofthe breast Ann Roy Coll Surg Engl 1982; 64: 165-70.

9. Waterfield MD, Mayes ELB, Stroobant P, et al. A monoclonal antibody to the humanepidermal growth factor receptor. J Cell Biol 1982; 20: 149-61.

10. Leeke RE, Laing L, Calman KC, et al. Oestrogen receptor status and endocrine therapyof breast cancers: response rates and status stability. Br J Cancer 1981; 43: 59-66.

11. Haybittle JL, Blamey RW, Elston CW, et al. A prognostic index in primary breastcancer. Br J Cancer 1982; 45: 361-66.

12. Gross JL, Krupp MN, Rifkin DB, Lane MD. Down regulation of EGF receptorcorrelates with plasminogen activator activity in human A-431 epidermal cancercells. Proc Natl Acad Sci USA 1983; 80: 2276-80.

13. Ossowski L, Reich E. Antibodies to plasminogen activator inhibit human tumourmetastases. Cell 1983; 33: 616-19.

EPIDERMAL-GROWTH-FACTOR RECEPTORSIN HUMAN BLADDER CANCER: COMPARISONOF INVASIVE AND SUPERFICIAL TUMOURS

DAVID E. NEALMARK K. BENNETTREGINALD R. HALL

COLIN MARSHPAUL D. ABEL

J. R. C. SAINSBURYADRIAN L. HARRIS

Departments of Urology and Pathology, Freeman Hospital;University Departments of Surgery and Clinical Oncology,

University of Newcastle upon Tyne

Summary The presence of epidermal-growth-factor(EGF) receptors in normal and neoplastic

human urothelium was studied in 12 control patients and in48 patients with transitional cell carcinoma of the bladder, 24with invasive (pT3) and 24 with superficial tumours (9 pT1,15 pTa). EGF receptors were identified on frozen sections bymeans of an indirect immunoperoxidase technique with amonoclonal antibody against the EGF receptor. Significantlymore invasive tumours (21 of 24) than superficial (7 of 24)were stained positively for the EGF receptor (&khgr;2=14&middot;49;p<0&middot; 001). Significantly more poorly differentiated tumours(18 of 21) than moderately differentiated tumours (10 of 27)were EGF-receptor positive (&khgr;2=9&middot;6; p<0&middot;01). No control

sample stained positively for the EGF receptor. These

findings suggest that the presence of a high intensity ofstaining for the EGF receptor in human bladder tumours isassociated with poor differentiation and with invasion.

Introduction

AN important factor in predicting survival after treatmentof patients with bladder cancer is the pathological stage of thetumour. In the long term 70-80% of superficial bladdertumours can be controlled satisfactorily by means of regularcystoscopic treatment.’ In 10-15% of patients invasivetumours will develop during follow-up, and in 10%-15% thesuperficial tumours will become difficult to control. Survivalof patients with invasive bladder cancer is poor; only about30% of patients are alive 5 years after radical local treatment.2 2

Epidermal growth factor (EGF) is a low-molecular-weightpolypeptide found in high concentration in human milk andurine.3 Its physiological function is unclear, although it

promotes the growth of hair and teeth in immature mice.4Also some cultured tumour cell lines (including bladdertumours) exposed to EGF respond by synthesising DNA,RNA, and protein and undergoing cell division.5,6 The EGFreceptor is a large molecule in the cell membrane. The EGFbinding site faces the outside of the cell7 and the internal partof the receptor has protein kinase activity; this has theunusual property of phosphorylating intracellular protein ontyrosine residues,8 a property shared with the proteinproducts of some oncogenes. Activated oncogenes have beenfound in certain human tumours, including a bladder cellline.9 Similarities have also been noted between the EGFreceptor and oncogene proteins.7,10EGF receptors have been demonstrated on cultured breast

carcinoma cell lines," non-neuronal brain tumours,12 andbiopsy samples from human breast cancers.r3,14 The growthof three bladder cancer cell lines has also been shown to bestimulated by EGF.6 We decided to see whether EGF

receptors could be detected in human bladder cancer.

Because metastasis in breast carcinoma appears to beassociated with presence of the EGF receptor, 14 we wished to

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test the hypothesis that a greater proportion of invasivebladder tumours than superficial bladder tumours, was

positive for the EGF receptor.

Patients and Methods

We studied 48 patients with bladder cancer; 40 were male, 8female. The median age was 62 years (range 35-90). 24 patientshad superficial transitional cell carcinoma (15 pTa, 9 pTl);5 were poorly differentiated (2 pTa, 3 pTI) and 19 moderatelydifferentiated. 24 patients had invasive transitional cell carcinoma(pT3); 16 were poorly differentiated and 8 moderatelydifferentiated.Bladder biopsy samples were taken from 12 control patients with

no evidence of bladder carcinoma (median age 64 years, range30-70), 10 of whom had bladder outflow obstruction andunderwent prostatectomy.Biopsy samples were taken by means of a resectoscope using the

diathermy cutting current. A sample of the resected tumour wasplaced in cold. saline before being snap-frozen in isopentaneprecooled to -150&deg;C with liquid nitrogen. The delay beforefreezing was 30 min on average and was never more than 1 h. Thespecimens were wrapped in metal foil to prevent dehydration andstored in liquid nitrogen.The EGF receptor was identified by means of an indirect

immunoperoxidase technique with a murine monoclonal antibody(EGFR1; donated by Dr M. Waterfield). The antibody was raisedfrom an epidermoid carcinoma line (A431) that expresses a highconcentration of EGF receptors. 15 5 &micro;m cryostat sections were cutand picked up on lysine-coated slides. After drying with a fan for30 min, they were fixed in acetone for 20 min and washed twice for5 min in saline buffered to pH 7.6 6 with "tris". The sections werethen covered with normal rabbit serum (diluted 1/5 with tris-buffered saline) as a blocking agent for 10 min. The sections wereincubated at room temperature with primary antibody for 30 min.After 2 washes in tris-buffered saline, the blocking step was

repeated. The sections were then incubated with peroxidase-conjugated rabbit anti-mouse immunoglobulin (Dakopatts) for 30min. After 2 further 5 min washes peroxidase activity was developedby means of a solution of diaminobenzidine incorporatingimidazole, 16 the sections were washed in water, counterstained withhaematoxylin, dehydrated, and mounted.The positive control was human placenta, which contains large

numbers of EGF receptors.7 For negative controls we omitted theprimary antibody or used primary antibody that had been absorbedwith purified EGF receptor derived from A431 cells (P. Parker,Imperial Cancer Research Fund).Assessment of the staining was carried out by one of us (M. K. B.)

without knowledge of the tumour stage or grade. The sections weregraded as positive if cells that exhibited a similar intensity ofstaining to the positive control were present. Grading of the tumourwas carried out by means of paraffin sections of the remainingresected material and parallel slices of the frozen biopsy material.

Statistical analysis was done by means of the chi-square test withcorrection for continuity. 17

Results

The cytotrophoblast and syncytiotrophoblast of thechorionic villi showed dense staining. This positive stainingwas absent when the absorbed antibody was used.Urothelium from the 12 control patients did not stain

positively for the EGF receptor. Weak staining, ofinsufficient intensity to be graded positive, was identified inthe basal layers of the urothelium of 4 control patients. Inanother control patient weak staining was noted in an area ofcystitis cystica. Weak background staining of the detrusormuscle was observed both in controls and in patients withbladder tumours.

7 of the 24 superficial tumours (29%) were graded positivefor EGF receptors. 3 were pTa and 4 were pTl tumours; 4were moderately and 3 poorly differentiated. In 4 of the

remaining 17 tumours weak staining, of similar intensity tothe background, was observed.21 of the 24 invasive tumours (87-5%) were positively

stained for EGF receptors. Thus the proportion of patientspositive for EGF receptors was significantly greater for thosewith invasive than for those with superficial tumours

(X2 =14.49; p<0’001). The stain in positive tumours,invasive or superficial, was in the cytoplasm in all but 2; inthese the stain was membranous. The distribution of the

positively stained cells throughout the tumours was notuniform, but focal positivity was observed in only 4 of the 28positive tumours.

Significantly more of the poorly differentiated tumours (18of 21) than the moderately differentiated tumours (10 of 27)were positively stained (X2 = 9’ 6; p<0. 01).

Discussion

We have shown that human transitional cell tumourscontain EGF receptors. A greater proportion of invasive thanof superficial tumours was graded positive for the receptors.Identification of these receptors on human urothelialtumours has not been reported previously, although EGF isknown to stimulate growth of some human bladder cancercell lines. 6

The specificity of the monoclonal antibody (EGFR1) weused to demonstrate the EGF receptor has been described

previously. It is derived from a human epidermoid carcinomacell line (A431) which, though not dependent on EGF forgrowth, contains large quantities ofrece.ptor.15 This antibodydoes, however, recognise normal EGF receptors on

placenta.7 Definition of the presence or absence of EGFreceptors by an immunoperoxidase technique requires that acertain intensity of staining be chosen as a cut-off point. Sinceit is unlikely that the distribution of receptors on a cell willfollow an all or none pattern, some of the weakly stainingbladder tumours that were graded as negative might containEGF receptors.The identification of EGF receptors on tumour cells does,

not necessarily imply that such cells will be dependent onEGF for growth. For example, the EGF receptor recognisedby the antibody may be abnormal and may be active whetheror not EGF is bound to the receptor. The aminoacid sequenceof the v-erb-B oncogene protein of the avian erythroblastosisvirus closely resembles a truncated EGF receptor. Despitethe lack of a domain for binding EGF and a tyrosine-specificprotein kinase on this oncogene protein, this virus cantransform cells; the property is probably due to the presenceof the v-erb-B oncogene.Our finding that invasive bladder tumours were more likely

than superficial tumours to be EGF-receptor positive showsthat possession of the EGF receptor is associated withinvasion. In human breast carcinomas the presence of EGF

receptors is associated positively with metastasis and

negatively with oestrogen receptor status.14 These findingssuggest that breast and bladder tumours which are positivefor EGF receptors are likely to have a poor prognosis. ,

The presence of EGF receptors on a cell may be partly areflection of the means by which it undergoes rapid division.For example, three chemically transformed cell lines showedgreater binding ofEGF when they were rapidly dividing thanwhen they were arrested in GI phase by deficiency ofnutrients.18 However, the three non-transformed parent celllines showed no increase in EGF binding when they wererapidly dividing.18 Furthermore, in our study the activelydividing basal cells of the urothelium of the controls were not

368

intensely stained for the EGF receptor, and over a third of themoderately differentiated tumours were EGF-receptorpositive. It is unlikely therefore, that the relation between celldivision and the presence of EGF receptors is simple.The presence ofEGF receptors probably represents simply

one feature of the genetic alteration that orchestrates thebehaviour of the malignant cell. Nevertheless, thedemonstration on human bladder tumours of receptors for a

growth factor found in high concentration in urine may beimportant if future studies demonstrate that the receptors arefunctional and therefore susceptible to modulation.We thank Dr M. Waterfield, Imperial Cancer Research Fund, for the

primary antibody. We also thank the North of England Cancer ResearchCampaign for support for J. R. C. S.

Correspondence should be addressed to D. E. N., Department of Urology,Freeman Hospital, Freeman Road, Newcastle upon Tyne NE7 7DN.

REFERENCES

1. England HR, Paris AMI, Blandy JP. The correlation of T1 bladder tumour historywith prognosis and follow-up requirements. Br J Urol 1981, 53: 593-97.

2. Bloom HJG, Hendry WF, Wallace DM, Skeet RG. Treatment of T3 bladder cancer:controlled trial of pre-operative radiotherapy and radical cystectomy versus radicalradiotherapy. Second report and review. Br J Urol 1982; 54: 136-51

3. Gregory H. Isolation and structure of urogastrone and its relationship to epidermalgrowth factor. Nature 1975; 257: 325-27.

4. Cohen S. The epidermal growth factor (EGF). Cancer 1983; 51: 1787-91.

5. Osborne CK, Hamilton B, Titus G, Livingstone RB. Epidermal growth factorstimulation of human breast cancer cells in culture. Cancer Res 1980; 40: 2361-66.

6. Messing E. Growth factors and human bladder tumours. J Urol 1984; 131: 111A.7 Downward J, Yarden Y, Mayes E, et al. Close similarity of epidermal growth factor

receptor and v-erb-B oncogene protein sequences. Nature 1984; 307: 521-27.8. Soderquist AM, Carpenter G. Developments in the mechanism of growth factor action:

activation of protein kmase by epidermal growth factor. Fed Proc 1983, 42:2615-20.

9. Capon DJ, Chen EY, Levinson AD, Seeburg PH, Goeddel DV. Complete nucleotidesequences of the T24 human bladder carcinoma oncogene and its normal

homologue. Nature 1983; 302: 33-37.10. Chinkers M, Cohen S. Purified EGF receptor-kinase interacts specifically with

antibodies to Rous sarcoma virus transforming protein. Nature 1981; 290: 516-1911. Osborne CK, Hamilton B, Nover M. Receptor binding and processing of epidermal

growth factor by human breast cancer cells. J Clin Endocrinol Metab 1982, 55:86-93.

12. Libermann TA, Razon N, Bartal AD, Yarden Y, Schlessinger J, Soreq H. Expression ofepidermal growth factor receptors in human brain tumors. Cancer Res 1984; 44:753-60.

13. Fitzpatrick SL, Brightwell J, Wittliff JL, Barrows GH, Schulz GS. Epidermal growthfactor binding by breast tumor biopsies and relationship to estrogen receptor andprogestin receptor levels. Cancer Res 1984, 44: 3448-53.

14. Sainsbury JRC, Farndon JR, Sherbet GV, Harris AL. Epidermal-growth-factorreceptors and oestrogen receptors in human breast cancer. Lancet 1985; i: 364-66.

15. Waterfield MD, Mayes ELV, Stroobant P, et al. A monoclonal antibody to the humanepidermal growth factor receptor. J Cell Biol 1982; 20: 149-61.

16 Stuart AE, Warford A. Staining of human splenic sinusoids and demonstration ofunusual banded structures by monoclonal antisera. J Clin Pathol 1983; 36:

1176-80.

17. Siegal S. Non-parametric statistics for the behavioural sciences. London: McGraw-Hill, 1956.

18. Robinson RA, Branum EL, Volkenant ME, Moses HL. Cell cycle variation in 125I.labelled epidermal growth factor binding in chemically transformed cells CancerRes 1982; 42: 2633-38.

LONG-TERM OUTCOME FOR CHILDREN WITHMINIMAL-CHANGE NEPHROTIC SYNDROME

R. S. TROMPETERJ. HICKS

B. W. LLOYDR. H. R. WHITE

J. S. CAMERON

Evelina Children’s Department, Guy’s Hospital, London, andThe Children’s Hospital, Birmingham

Summary A retrospective study was undertaken toassess the outcome of a cohort of 183

unselected children who presented with the nephroticsyndrome between 1963 and 1969. All subjects showedminimal glomerular changes in biopsy samples and weregiven conventional steroid therapy. Information was

available on 152 children, now aged 14-32 years. Activitypersisted longer in patients presenting at an early age. Theoutcome for most of the children was favourable. Only 10patients (5&middot; 5%), all of whom presented with initial symptomsbefore their 6th birthday, continued to have steroid-

responsive relapses in adult life. There were 11 deaths, ofwhich 7 (4% of the series) were from avoidable complicationsof the disorder.

Introduction

THE clinical course of the nephrotic syndrome in

childhood, both before and after corticosteroid therapy, hasbeen described.1-8 In most of these studies the patients were aheterogeneous group of nephrotic children with variousforms of underlying glomerular pathology, some of whomdied from renal failure and, in the early days, many frominfection. Complete histopathological data are lacking, and inmany cases the period of follow-up is inadequate.We studied the long-term outcome of a cohort of mainly

unselected children with biopsy-proven minimal-changenephrotic syndrome (MCNS) who had presented more than13 years previously. We also investigated the relationbetween the duration of the disorder and age at onset of

symptoms.

Patients and Methods

During the period 1963-69 nearly every child presenting withnephrotic syndrome had a renal biopsy as a part of the initialevaluation, and 183 were classified as having minimal-changehistology.9 These patients came mainly from two areas of the UK,the south-east of England and the Midlands, and were identifiedfrom the clinical and histopathology records.

All subjects were contacted by post (unless they were still beingregularly followed up at hospital) and their consent was obtained forthe study. Patients were then seen either by us at hospital or by theirgeneral practitioners. The date of the last nephrotic relapse and thedate when corticosteroid therapy was discontinued were obtained.Blood-pressure and height were measured, and a urine sample wastested with ’Albustix’.

If a patient could not be traced a death certificate was sought at theoffices of the Registrar of Deaths, but if no evidence of death couldbe found then the patient was presumed to be alive.

Results

Of 183 children identified from the records follow-up datawere available on 152. 7 of the other 31 patients were alive andin remission from their nephrotic syndrome but wereunavailable for follow-up examination. 24 patients, including2 who had emigrated from the UK, were presumed alive.In the group of 152 there were 108 males and 44 females

(ratio 2 4:1). The age at onset of symptoms ranged from 11 months to 15 years. The modal age of onset was 3-4 years, asobserved in previous studies.1,10 The age at follow-up rangedfrom 14 to 32 years. Renal biopsy was carried out at onset in99 (65%) patients and from 6 months to 13 years after onset(mean 4.5 5 years) in the other 53. All biopsy specimensindicated minimal-change nephrotic syndrome (classified byR. H. R. W.). The initial corticosteroid dosage varied but wasusually up to 60 mg prednisolone/24 h for 8-12 weeks. Allpatients were steroid responsive at presentation and remainedso throughout the course of the disorder.

’

At follow-up 127 patients were in remission as defined bythe absence of proteinuria. Another 4 patients (2 male and 2female) were in remission but had hypertension (blood-pressure>150/90 mmHg); all 4 had normal renal function