J Clin Pathol 1990;43:213-219

Ber-EP4: new monoclonal antibody whichdistinguishes epithelia from mesothelia

U Latza, G Niedobitek, R Schwarting, H Nekarda, H Stein

Department ofPathology, KlinikumSteglitz, FreeUniversity Berlin,Hindenburgdamm 30,1000 Berlin 45,West GermanyU LatzaG NiedobitekR SchwartingH NekardaH SteinCorrespondence to:Dr Ute Latza,Hindenburgdamm 30,1000 Berlin 45,West Germany.Accepted for publication12 October 1989

AbstractA new monoclonal antibody, Ber-EP4,directed against a partially formol resis-tant epitope on the protein moiety of two34 kilodalton and 39 kilodalton glyco-polypeptides on human epithelial cells isdescribed. Immunostaining of a widerange of normal and neoplastic humantissues and cell lines showed that allcarcinomas and all non-neoplastic epith-elial cells, except hepatocytes, parietalcells, and apical cell layers in squamousepithelia, homogeneously expressed Ber-EP4 antigen. As Ber-EP4 does not detectany normal or neoplastic non-epithelialcells, this antibody might prove valuablefor the differentiation of the following(i) non-epithelial tumours from undif-ferentiated carcinomas; (Ui) hepatocytesfrom bile duct cells in certain liverdiseases; (iii) mesothelial cells fromcarcinoma cells in lung biopsy specimens;and (iv) reactive mesothelial cells fromcarcinoma cells in smears of serous

effusions.

Differential diagnosis of anaplastic carcinomaand poorly differentiated tumours of non-

epithelial origin is a common problem insurgical pathology. Advances have been madein this respect by immunohistochemicalmethods'3 using monoclonal antibodies suchas anti-cytokeratin reagents,' but differentintermediate filaments are occasionally co-

expressed in given cells."' In cell separation orflow cytometry, for example, the exclusivelycytoplasmic localisation of cytokeratins causesdifficulties. Monoclonal antibodies directedagainst epithelial surface antigens, such as

carcinoembryonic antigen (CEA) and relatedantigens,12 or epithelial membrane antigen(EMA),'3 are of limited value for the differen-tial diagnosis between epithelial and non-

epithelial tumours. These antigens are notexpressed in all epithelia, their expression in a

given tumour is often heterogeneous, leading tounreliable results in small biopsy specimens,14and they may be expressed in non-epithelialcells. ' In particular, it is often impossible todiscriminate reactive mesothelial cells andmesotheliomas from carcinoma cells withavailable antibodies, though this is of greatdiagnostic importance. As no strictly meso-

thelial cell specific monoclonal antibodies existexpression of cytokeratins and lack of CEAexpression are used as indicators of mesothelialorigin. Not all carcinomas, however, are

detected by anti-CEA monoclonal antibodies,and intermediate filament expression inmesothelia can vary.'6 We describe the use of amonoclonal antibody Ber-EP4 directed againstan epithelium specific membrane antigenabsent from mesothelial cells and compare itwith the monoclonal antibody HEA125."7'8

MethodsCell lines MCF-7, MDA-MB-231, MDA-MB-175, BT-20, MOLT-4, RD and Daudiwere obtained from the American Type Cul-ture Collection (Atlanta, Georgia, USA).ASPC-1, AlAb, and COLO 357 were a kindgift from Dr Knuth, Mainz, West Germany,19Co. and Ho. were kindly supplied by Dr DJones (Southampton, England,202' and L540,L428, and L591 by Dr V Diehl, Cologne, WestGermany.2223Rabbit anti-mouse immunoglobulin sera for

immunocytological staining and monoclonalantibody C3D- 1 were from Dakopatts, Copen-hagen, Denmark, KL1 was from Dianova,Hamburg, West Germany and HEA125 fromProgen Heidelberg, West Germany.

Diagnostic biopsy specimens were routinelyprocessed or snap frozen in liquid nitrogen andstored at - 80°C. For immunostaining, frozensections were fixed in acetone/chloroform andstained with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) procedure.2425Cytospins and smears were fixed in acetone.Cell lines with endogenous alkaline phos-phatase were stained with immunoperoxi-dase.26Monoclonal antibodies were obtained using

established methods from mice immunisedwith MCF-7 cells.2728 Hybridoma supernatantwere screened for anti-epithelial reactivityimmunocytochemically and absence of anti-cytokeratin reactivity in immunoblottingexperiments (data not shown). The Ig subclasswas determined by double immunodiffusionusing specific anti-mouse Ig reagents (Serotec,Wiesbaden, West Germany).

IMMUNOPRECIPITATIONFor surface radioiodination, MCF-7 cells werelabelled by the lactoperoxidase technique.29For internal labelling, MCF-7 cells wereincubated for two or 24 hours with 35S-meth-ionine (NEN- Dupont, Dreieich, West Ger-many).0 Immunoprecipitation was carried outwith goat anti-mouse agarose beads coated withIg (Sigma).3' Precipitates were analysed on

12-50/o polyacrylamide sodium dodecyl sul-phate gel electrophoresis (SDS-PAGE).32 An

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Figure la Immunoprecipitation of Ber-EP4 antigenfrom 125I surface labelledcell line. Immunoprecipitates were analysed with SDS PAGE under reducingconditions. Negative control precipitate (lane A), Ber-EP4 precipitate (lane (

HEA125 precipitate (lane B); sequential precipitates (lanes D, E, F) ofHEAafter Ber-EP4 precipitation (lane D), of Ber-EP4 after HEAi25 precipitatio;E), of negative control precipitate (lane F).

Figure lb Immunoprecipitation of Ber-EP4 antigenfrom 125I labelled MCF-;under non-reducing conditions. Negative control precipitate (lane A), Ber-EPAprecipitate (lane B).

unrelated antibody of the same subclasas a control.3"For deglycosylation, precipitates wer

in reducing sample buffer withoutphenol blue and digested overnight witml endoglycosidase F (Boehringer, MaxWest Germany) at room temperature.

Figure 2Immunoprecipitation ofBer-EP4 antigen at twohours (lanes A, B, C) and24 hours (lanes D, E, F)3S methionine labelledMCF-7 cell line.Molecular weight wasdetermined with SDSPAGE under reducingconditions. Lanes A and Fshow negative controlprecipitates, lanes C andD Ber-EP4 precipitates,and lanes B and E Ber-EP4 precipitates afteredoglycosidase F cleavage.

CHEMICAL AND ENZYMATIC TREATMENTPeriodate oxidation35 was carried out with0-1 M sodium periodate (Sigma) in 0-05 Msodium acetate buffer, pH 4 5, at 4°C for onehour in the dark on cryostat sections fixed inZamboni's reagent.36 For enzyme digestion,cryostat sections fixed in acetone wereincubated with 0-1 mg/ml pronase E (Sigma) inphosphate buffered saline for one to 60 minutesat 37°C. For dot blot experiments, MCF-7lysates were adsorbed to nitrocellulose mem-brane and treated with periodate or pronase E(10 mg/ml, 37°C, overnight). After incubation,immunostaining was performed. An irrelevantantibody of the same subclass served as anegative control. KL1 monoclonal antibodydirected against a protein epitope and C3D-1

NW * monoclonal antibody directed against a carbo-hydrate (CD 15) epitope served as positivecontrols.

COMPETITIVE INHIBITION OF BER-EP4 ANDHEA125Monoclonal antibodies were purified with

----- Affi-Gel (Bio-Rad, Munich, West Germany).IMCF-7 Purified antibodies (1 mg/ml in 0 1 M sodium

carbonate buffer, pH 9) were fluorescein iso-thiocyanate (FITC) labelled by incubation

1125 with 100o v/v FITC solution (1 mg/ml FITCn (lane (Sigma) in DMSO (Merck)) for three hours in

the dark with agitation and subsequent dialysis.7 cell line Indirect immunofluorescence staining was

performed as described previously.3' For com-petitive inhibition, cells were preincubated

s served with unlabelled antibody at concentrationsranging from 10 to 1 mg/ml, directly stained

re boiled with FITC-labelled primary antibody, andbromo- analysed in an EPICS 752 (Coulter Electron-th 48 U/ ics, Krefeld, West Germany). Irrelevantnnheim, FITC-labelled antibodies and irrelevant cell8 34 lines served as negative controls.

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215Ber-EP4: new monoclonal antibody which distinguishes epithelia from mesothelia

ResultsBIOCHEMICAL CHARACTERISATION OF THE BER-EP4 ANTIGENTwo polypeptides were precipitated underreducing conditions from 125I surface-labelledMCF-7 cells, one chain with an apparentmolecular weight of 34 000 and, with varyingintensity, another chain of 39 000 (figure la).Under non-reducing conditions the molecularweight was slightly higher (39 000 and 44 000)(figure lb). Internal labelling of MCF-7 andK562 cell lines with 35S-methionine for 24hours yielded the same results (figure 2). Aftertwo hours of labelling, however, only one chainof 39 kilodaltons could be precipitated, bothunder reducing and non-reducing conditions.Deglycosylation of the precipitate reduced themolecular weight from 39 000 and 34 000 to36 000 and 31 000, respectively (figure 2).On cryostat sections of breast carcinoma

mild protease treatment before immunostain-ing diminished reactivity with Ber-EP4; stain-ing was unaffected by periodate oxidation.Stronger protein cleavage applied in dot blotexperiments led to a complete loss of reactivitywith Ber-EP4.

Figure 3Immunohistochemicalreactivity of monoclonalantibody Ber-EP4(APAAP, haematoxylincounterstain, frozensections if not indicatedotherwise):(a) Mammary gland:strong cytoplasmic andpreferably basolateralmembrane staining ofductal cells. Myoepthelialcells,fat and connectivetissue components remainunstained.(b) Stomach: staining ofglandular and chromaffincells while parietal cellsremain unstained (arrow).(c) Malignantpnesothelioma ofpleura: nostaining of tumour cells.(d) Same case as c:staining of tumour cellswith comparativemonoclonal antibody KL1.

IMMUNOHISTOCHEMICAL STAINING WITH BER-EP4Immunostaining of a panel of different normaltissues with Ber-EP4 (table 1) showed that allnormal epithelial tissues expressed the Ber-EP4 antigen on the membrane, preferentiallybasolaterally (figure 3), and in the cytoplasm.Only parietal cells in gastric glands and apicalcell layers in squamous epithelia were negative.Adult hepatocytes, in contrast to fetalhepatocytes, did not express the antigen.

Epithelial cells of different origin displayedvarying levels of Ber-EP4 antigen expression,while epithelial cells of one type always expres-sed the antigen homogeneously. Most epitheliawere strongly positive. The Ber-EP4 antigenwas not expressed in non-epithelial cells.Pleura and peritoneum lining cells werenegative, but cells covering the ovary displayedslight positivity.

Cytospins of a total of 37 cell lines of variousorigin were investigated. All carcinoma celllines were homogeneously stained with Ber-EP4 (table 2). All non-epithelial cell lines werenegative except for the erythromyeloid cell lineK562.

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(e) Smear ofpleuraleffusion: staining ofmammary carcinoma cells,no staining of reactivemesothelial cells.(f) Same case as e:staining of carcinoma cellsand reactive mesothelialcells with comparativemonoclonal antibody KLI.(g) Liver: no staining ofhepatocytes and strongstaining of bile duct cells.(h) Fetal liver: staining ofhepatocytes.(i) Liver after hepatitis Binfection: staining ofhepatocytes adjacent toportal tracts and onlyweak staining of bile ductcells (paraffin waxembedded section).

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The antibody reacted with 142 of 144 malig-nant epithelial tumours of various origin, irre-spective of their differentiation, reflecting thestaining pattern of their non-malignant coun-terparts (table 3). There were some exceptions,however: (1) hepatocellular carcinomas showedheterogeneity (table 3); (2) in squamous cellcarcinomas tumour cells were generallypositive, although only the basal cell layerswere stained in normal tissue; (3) some carcin-omas, such as gastric carcinomas, expressedmore Ber-EP4 antigen than normal tissue,especially on the membrane.No reaction was seen with non-epithelial

tumours (table 3), and 20 cases of leukaemia(T-ALL, B-ALL, c-ALL, pb-ALL, Non-ALL, O-ALL, AML). Two cases ofmalignantmesotheliomas were negative with monoclonalantibody Ber-EP4, whereas they strongly reac-ted with anti-cytokeratin antibody KL1(figures 3c and d). Immunostained smears from36 serous effusions were also analysed (table 4).Reactive transformed mesothelial cells werenot detected by Ber-EP4, whereas all cases withcarcinoma cells displayed strong reactivity(figure 3e). In contrast, KL1 stained bothreactive transformed mesothelial cells and car-cinoma cells (figure 3f).

In general, the antibody gave identical

results on cryostat and paraffin wax embeddedsections, irrespective of pronase digestion ofparaffin wax embedded sections beforeimmunostaining (table 1). There were somedifferences, however: reactions were oftenstronger on cryostat sections; prolonged fixa-tion of tissue (more than 48 hours) led to asometimes complete reduction of reactivitywith Ber-EP4.

Epithelial cells containing large amounts of

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Ber-EP4: new monoclonal antibody which distinguishes epithelia from mesothelia

Table 1 Reactivity of Ber-EP4 with normal humanfrozen (F) and paraffin wax embedded (P) tissues

Reactivity

F PTissue

Epithelia glands:Salivary glandsPancreas: ducts, endocrine + exocrine glandsThyroid gland: thyrocytes, C cellsParathyroid glandAdrenal gland: cortical epitheliumPituitary gland: adenohypophyseal cellsMammary gland: acini, ductsProstate: glandular epithelia

duct cellsGastrointestinal tract:Stomach: glandular cells

parietal cellsSmall intestine: enterocytes, Paneth cells, goblet cells, Brunner's glandsColon: enterocytes, goblet cellsLiver: hepatocytes

bile duct cellsGall bladder: mucosal cellsRespiratory and urogenital tract:Trachea: tracheal epithelium, ciliated cellsLung: pneumocytes, bronchial epitheliumKidney: distal tubules, collecting ducts

proximal tubules, Bowman's capsuleBladder: transitional epitheliumUterus: endometrial glandPortio uteri: squamous epitheliumTestis: rete testis

germ epitheliumPlacenta: amnion epitheliumLymphoid tissues and skin:Tonsils: crypt epithelia

squamous epitheliaThymus: cortical epithelia Hassall bodies

medullary epitheliaSkin: epidermis

sweat glandsOther tissues:SpleenPeripheral bloodBone marrowBrainConnective tissueMuscle: smooth

striatedHeartEndotheliaMyoepitheliaMesothelia: pleura, peritoneum

Results obtained with APAAP immunostaining. + positive; (+) weak; + / - inhomogeneous; - negative; ND not detected.

the Ber-EP4 antigen, such as duct cells inbreast, liver, and rete testis, constantly stainedstrongly in paraffin wax embedded sections; inother tissues reactivity was weak (lung andstomach) or completely absent (adrenal gland,prostate, tonsil and testis).On paraffin wax sections of 49 epithelial

tumours, Ber-EP4 gave consistent results for

Table 2 Reactivity ofBer-EP4 with human cell lines

Origin Cell line Reactivity

Epithelial cells:Breast carcinoma: MCF-7, MDA-MB-175, MDA-MB-231* +

BT-20,* AlAb +Colon carcinoma: HT-29 +Pancreas carcinoma: ASPC-1,* COLO 357* +Vulva carcinoma: A-431* +Ovary carcinoma: CAOV-3 +Total 10/10 +Non-epithelial cellsB cells: IM-9, CESS, Daudi, NALM 12 -

U266, REH-6 -

Hairy cell leukemia: JOK-1T cells: Jurkat, HSB-2, MOLT-4, MT-2 -

HPB-ALL, HUT-102 -

Hodgkin's disease: L540, L591, L428KMH2, Co, Ho -

Myeloid cells: HL-60, U937, THP-1, KG-1, DHL-1 -

K562 +Neuroblastoma: LAN-5Rhabdomyosarcoma: RDTotal 26/27 -

Results determined with APAAP immunostaining. + positive; - negative.*Tested with immunoperoxidase because of endogene alkaline phosphatase.

tumours containing large amounts of the Ber-EP4 antigen-for example, breast and cholan-giolar carcinomas (table 3). In carcinomasarising from tissues with no or smaller amountsof the Ber-EP4 antigen-hepatocellular andlung carcinoma-the antibody Ber-EP4 did notwork satisfactorily. No reactivity was observedin non-epithelial tumours.As no frozen material was available evalua-

tion of diseased liver tissue with Ber-EP4immunostaining was performed on paraffinwax embedded specimens of 15 livers withhepatitis of varying aetiology. In these cases

pseudoductules and some hepatocytes adjacentto portal tracts showed a strong reaction withBer-EP4, while bile ducts stained only weakly.

COMPARISON OF BER-EP4 and HEA125MONOCLONAL ANTIBODIESSequential immunoprecipitation from 1251I or

35S-methionine labelled MCF-7 lysates withBer-EP4 and HEA125 monoclonal antibodiesshowed that both precipitated the same antigen(figure 1). In cross-blocking experiments usingK562 cells, HEA125 inhibited the binding ofthe FITC-labelled Ber-EP4 and vice versa. Incell and tissue binding studies HEA125produced staining results identical with thoseseen with Ber-EP4.

+

+

+

(+)

++

++-(+)

ND

+

++

(+)

+

+

(+)+

+

+D+D

+ +

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Table 3 Reactivity of Ber-EP4 with neoplasticfrozen (F) and paraffin waxembedded (P) tissues

No ofpositive cases/Total No of cases

F PTissue

Epithelial tumoursPrimary carcinomas:Breast: infiltrating ductal

infiltrating lobularOesophagus: squamousStomach: adenocarcinomaColon: adenocarcinomaRectum: adenocarcinomaPancreas: adenocarcinomaKidney: renal cellLiver: hepatocellular

cholangiolarLung: adenocarcinoma

squamousoat cell

Thyroid gland: adenocarcinomaSalivary gland: adenocarcinomaVagina: adenocarcinomaOvary: adenocarcinomaCervix uteri: squamousMetastatic carcinomas:Oesophagus: squamousStomach: adenocarcinomaColon: adenocarcinomaNasopharynx: undifferentiatedPancreas: adenocarcinomaOvary: adenocarcinomaOthers:Appendix: appendix carcinoidPancreas: insulinomaTotalNon-epithelial tumoursB cell lymphomaT cell lymphomaHodgkin's diseaseLeiomyomaSarcomatMalignant melanomaMalignant fibrous histiocytomaMalignant mesotheliomaPhaeochromocytomaTotal

9/9nd

4/4*59/5919/191/1

10/101/14/6t5/53/31/1

nd3/31/11/11/12/2

1/110/103/31/11/11/1

nd1/1

142/144

0/340/260/40/30/60/110/10/20/10/88

22/221/1

nd1/12/2ndndnd1/62/23/61/14/5ndndndnd1/1

ndndndndndnd

1/11/1

40/49

0/3ndndndnd0/1nd0/12nd0/16

Results determined with APAAP immunostaining.*In one case of a carcinosarcoma reactivity only with the epithelial differentiated tumourcomponent.tTwo cases negative, two cases partially negative.$Fibrosarcoma, rhabdomyosarcoma, neurogen sarcoma and Ewing's sarcoma.

Table 4 Immunostaining of smears of serous effusions from pleura and peritoneum

No of positive casesl Total No of cases

Diagnosis Ber-EP4 KLI

Carcinomatous effusions 11/11 7/7*Reactive effusions 0/25 23/25t

Results determined with APAAP immunostaining.*Only seven cases due to lack of material.;Positive reaction due to labelling of mesothelial cells.

DiscussionImmunobiochemical investigations on theradiolabelled MCF-7 cell line showed thatmonoclonal antibody Ber-EP4 is directedagainst the protein moiety of two 34 kilodaltonand 39 kilodalton glycopolypeptide chains,which are not covalently bound. Metaboliclabelling with 35S-methionine suggests a 39kilodalton primary gene product for bothchains.Based on biochemical data, the Ber-EP4

antigen is different from all other known epi-thelial specific antigens except the HEA125antigen (see below). The molecular weight andthe basolateral localisation on 'the cell mem-brane distinguish the Ber-EP4 antigen fromother surface antigens such as EMA, CEA, andrelated antigens.'2 13 Glycosylation andpresence on the cell surface distinguish theBer-EP4 antigen from cytokeratins. The reac-

tivity of monoclonal antibody Ber-EP4 resem-bles that of the recently described HEA125'7 18and MH9937 antibodies. MH99, however,precipitates a molecule of different size. Ourimmunobiochemical investigations with Ber-EP4 and HEA125 antibodies showed identicalresults in sequential immunoprecipitations.Furthermore, cross-blocking experiments withflow cytometry on K562, an erythromyeloidcell line atypically expressing epithelialantigens such as cytokeratins,38 39 and the Ber-EP4 antigen, showed that both antibodies aredirected against a neighbouring or identicalepitope of the same antigen. The strongpositivity of the K562 cell line with bothmonoclonal antibodies is at variance with theresults obtained with HEA125 in previousreports. '8

In conclusion, the Ber-EP4 monoclonalantibody may be of value in: (i) the differentialdiagnosis of undifferentiated primary or meta-static tumours: (ii) the identification of bileduct cells in the diagnosis of liver diseases; (iii)the distinction between epithelial cells andnormal, reactive, or neoplastic mesothelialcells.

All carcinomas, except some hepatocellularcarcinomas, were positive with the Ber-EP4monoclonal antibody, unlike all non-epithelialtumours tested. The antibody works reliablyon frozen sections fixed in acetone, cytospins,and cell smears, whereas the epitope recognisedby Ber-EP4 and HEA125 monoclonalantibodies can be destroyed by prolonged for-malin fixation. Thus in paraffin wax embeddedsections only a positive reaction with Ber-EP4monoclonal antibody can be deemed a reliableresult.The lack of Ber-EP4 antigen expression in

hepatocytes is particularly interesting. In nor-mal liver Ber-EP4 stains only bile duct cells butnot hepatocytes, similar to antibodies againstcytokeratins 7 and 19.4 This should proveuseful in the diagnosis of innate bile ducthypoplasia and diseases accompanied by eitherdestruction or proliferation of bile ducts.Most importantly Ber-EP4 monoclonal

antibody does not react with normal or reactivemesothelial cells. The antibody can thereforebe used for the differential diagnosis betweenmesothelial cells and carcinoma cells, par-ticularly in smears of serous effusions. Anti-cytokeratin monoclonal antibodies are nothelpful in this respect because of their reac-tivity with mesothelial cells. Anti-CEAantibodies and Leu-Ml and B72.3 have beenreported to help in the discrimination ofcarcinoma cells and reactive or malignanttransformed mesothelial cells because of theirunreactivity with the latter cell type.4' Theseantibodies, however, are not consistently reac-tive with all carcinomas and Leu-Ml also stainsnon-epithelial cells.Ber-EP4 might also be useful in the differen-

tial diagnosis ofmalignant mesotheliomas. Theseries investigated in this paper, however (twocases on cryostat sections), is too small toanswer conclusively, this question.The function of the Ber-EP4 antigen is

unknown. The broad reactivity of monoclonal

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Ber-EP4: new monoclonal antibody which distinguishes epithelia from mesothelia

antibody Ber-EP4 with almost all carcinomas,irrespective of the grade of differentiation,suggests an essential functional role for thisantigen in epithelial cells.

We thank Dr Horst Durkop for advice in internal labellingexperiments, Dr Barbara Fleige, and Dr Carl-Alexander Hart-mann for help in selecting lung biopsy specimens and smears ofserous effusions, and Dr Hebst for critical reading of themanuscript.

This work is part of the doctoral thesis of Ute Latza and wassupported by a postgraduate grant (NaFoG) and by "DeutscheKrebshilfe".

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