J Clin Pathol 1990;43:752-757

JC70: a new monoclonal antibody that detectsvascular endothelium associated antigen onroutinely processed tissue sections

D V Parums, J L Cordell, K Micklem, A R Heryet, K C Gatter, D Y Mason

AbstractA new monoclonal antibody, JC70, raisedagainst a membrane preparation from a

spleen affected by hairy cell leukaemia,recognises a membrane bound glyco-protein identical with that of the CD31group of monoclonal antibodies. Theantibody stains a fixation resistantepitope on endothelial cells in benignand malignant conditions in a widevariety of paraffin wax embedded tissue.JC70 stained malignant endothelial cellsin 10 angiosarcomas with more consis-tency than monoclonal or polyclonalantibodies to factor VIII related antigen(FVIII-Rag). In four cases of Kaposi'ssarcoma the antibody stained malignantendothelial cells but not spindle cells.

It is concluded that antibody JC70 is ofvalue for studying benign and malignanthuman vascular disorders in routinelyprocessed tissue.

University of Oxford,Nufflield DepartmentofPathology andBacteriology, Level 4,John RadclffeHospital, Headington,Oxford OX3 9DUD V ParumsJ L CordellK MicklemA R HeryetK C GatterD Y MasonCorrespondence to:Dr D V ParumsAccepted for publication15 March 1990

Vascular endothelial cells have an importantrole in coagulation, inflammation, immunity,regulation of vascular tone and in a widevariety of synthetic and metabolic functions.Endothelial cells also have a pivotal role inimmunological diseases, angiogenesis intumours, transplantation rejection, atherogen-esis and placentation.' Their role in healthand disease is therefore of interest to histo-pathologists, and for this reason it is impor-tant to be able to recognise these cells inroutine tissue sections. Morphologically,endothelial cells can be difficult to distinguishfrom other cell types and although a variety ofmarkers is available to diagnostic histopatho-logists all have serious limitations.

Vascular endothelial cells can cause bothbenign and malignant tumours. Angiosar-comas, although rare, pose a diagnosticproblem for histopathologists as they are

tumours with varying morphological pattems.Vascular differentiation may be absent andthey may be mistaken for other soft tissuesarcomas or for undifferentiated carcinomas.Staining of these tumours with antibodies toFVIII-related antigen (FVIII-Rag) has beendisappointing with reports of lack of stain-ing23 and of cross reactions with carcinomas.4Ulex europaeus agglutinin lectin shows vari-able staining of angiosarcoma cells5 and is notspecific for endothelial cells, binding tosquamous cell carcinomas and synovial sar-

comas6 as well as to neoplasms arising fromcolonic epithelium.7

There is therefore a need for a marker ofbenign and malignant endothelial cells of car-diovascular origin which will work reliably onroutinely processed, paraffin wax embeddedtissue. In this paper we report the productionof a new monoclonal antibody JC70 whichrecognises normal and malignant vascularendothelial cells in routinely processed tissueand we evaluate its role in routine diagnosticpractice.

MethodsPREPARATION OF ANTIGENHuman spleen from a patient with hairy cellleukaemia was used as a source of antigen.Crude membranes were prepared by a modi-fication of the method of Standring andWilliams.8 Briefly, frozen tissue (100 g) washomogenised in 2 5% Tween 40, 20 mMTRIS-HCl, pH 7-5 (200 ml), and centrifugedat 3000 x g for 20 minutes. The supernatantwas centrifuged at 40 000 x g for 30 minutesto sediment the membrane fraction which waswashed before being used for immunisation.All buffers contained 1 Mug/ml leupeptin, 1Mug/ml pepstatin, 1 mM phenyl methyl sul-phonyl fluoride and 1 mM iodoacetamide.

CHARACTERISATION OF THE ANTIGENRECOGNISED BY JC70A crude membrane fraction was preparedusing the method described above from aspleen from a patient with idiopathic throm-bocytopenic purpura (which had been foundby immunostaining to be rich in the antigen).Antigen recovery was measured by the alka-line phosphatase-antialkaline phosphatase(APAAP) technique9 10 to material vacuum dotblotted on to nitrocellulose sheets.

Essentially, all of the antigen in a 2% NP40extract of the crude membrane preparationbound to a lentil lectin Sepharose (Pharmacia)column. Antigen in the NP40 extract waseluted with 1 M methyl glucoside in 10%NP40, TRIS-HCI (pH 7-5), and dialysed toremove the methyl glucoside. A secondpreparation from normal human platelets wasprepared in a similar manner.

SODIUM DODECYL SULPHATE POLYACRYLAMIDEGEL ELECTROPHORESIS AND IMMUNOBLOTTINGThe membrane glycoprotein preparation (100pl from 200 mg of tissue) was precipitated with10% trichloroacetic acid, the precipitatewashed with acetone and solubilised by boilingin SDS-PAGE sample buffer"' containing 4Murea. This material was analysed on a 7-5%

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Antiendothelial antibody JC70

gel11 and the separated proteins were elec-troeluted on to a nitrocellulose sheet andstained for JC70 antigen by the APAAP tech-nique.

MONOCLONAL ANTIBODY PRODUCTION

Tween membrane preparations were washed ina 150 mM sodium chloride solution and thepellet was resuspended in 10 ml of 10% formolsaline and left rotating at room temperature for30 minutes. Membranes were spun in a micro-fuge at 13 000 rpm for five minutes and thenwashed twice in physiological saline. Themembrane pellet was resuspended in 20 ml ofphysiological saline, aliquoted, and stored at- 20°C until required for use.

Balb/c mice were immunised three timesintraperitoneally at 10 day intervals with 50 pgof antigen in physiological saline. Cell fusionwas performed by conventional techniques.'2For cytological preparations, routine peri-

pheral blood smears were obtained from thehaematology department. Slides were air-driedovernight and stored at - 20°C. They were

fixed before use in buffered formol acetone.

TISSUE SECTIONS

Fresh tissueFrozen samples of tonsil and spleen were

retrieved from storage at - 70°C, sectioned on a

cryostat, fixed and immunostained as describedpreviously.'0

Fixed tissueTissue sections were prepared from routineblocks stored in the Nuffield Department of

Pathology, John Radcliffe Hospital, Oxford.

All tissues were fixed in unbuffered formol

saline before paraffin wax embedding. For

convenience in screening paraffin wax embed-

ded specimens, some multiblock preparationswere prepared as previously described.'3

Sections from a range of benign vascular

lesions were examined together with 10 angio-sarcomas and four Kaposi's sarcomas (table).Normal tissues examined were skin and its

appendages, placenta, testis, thyroid, cervix,uterus, stomach, small intestine, colon, lung,spleen, tonsil, lymph nodes, bone marrow,liver, bladder, kidney, adrenals, myocardium,aortic valve, vein and aorta. Sections of benign,non-vascular tumours included uterine

leiomyoma, meningioma, and neurofibroma.

Malignant tumours examined were squamouscell carcinoma of skin, malignant melanoma,carcinoma of lung (24 cases), adrenal carci-

noma, transitional cell carcinoma of bladder,hepatoblastoma, non-Hodgkin's lymphomas(eight T cell and 20 B cell), leiomyosarcomaand fibrosarcoma.

IMMUNOENZYMATIC LABELLING

Tissue sections were stained with the mouse

monoclonal antibody F8/86/3 raised againsthuman FVIII-Rag'4 and with antibody JC70using the APAAP method. Before immuno-

staining paraffin wax sections were dewaxed,hydrated, and then incubated for about 30

minutes in 0I1% trypsin solution in 0I1%calcium chloride solution (pH 7 8).

Tissue sections were stained with rabbit

polyclonal antibody to human FVIII-Rag

Reactions of endothelial markers on routinely processed tissue sections

AntibodyFVIII-Rag

Diagnosis Site No JC70 Monoclonal Polyclonal UEA

Vascular tissuesAcutethrombus Vein 1 + + + + + + +Organising thrombus Vein 1 + + + + + + +Pyogenic granuloma Skin 2 + + + ± + + ++Capillary haemangioma Skin 1 + + + + + + ++Cavemous haemangioma Skin, liver 2 + + + + + + ++Angiofibroma Nose, lung 3 + + + + + ++Angiokeratoma Skin 2 + + + + + ++Angioleiomyoma Thigh 1 + + + + + + ++Angiolipoma Skin 4 + + + + + + ++Glomustumour Skin 2 + + + + +Haemangiopericytoma Skin I + + + + + +Chemodectoma Carotid 1 + + + +Atrial myxoma Atria 3 + + + + NDLymphangioma Skin 4 - + + + +Lymphoepithelial cyst Skin 1 - + + NDPneumatosis coli Rectum 1 - + + + ++Submucosal venous dilatation Rectum 1 + + + ++Atherosclerosis Carotid artery 1 ++ + ± ++ ++Cystic hygroma Neck 2 + + + + +Intravascular papillary Vein 1 ± + + ± ++ ++

endothelial hyperplasiaAngiodysplasia Colon 2 + + + + + + ++

AngiosarcomaLiver 1 + + + + +Rectum 1 ++ + + +Rectum I + - + +Rectum 1 + + + + +Breast 1 + + + + +Scalp 1 ± + + +Axilla 1 + + + + +Vein 1 + + + + +Skinofleg 1 + + + ±Skin 1 ++ + + +

Kaposi's sarcomaSkin 1 + + + +Lymphnode 1 + + + + +Skin 1 + + + + +Rectum I + - + +

- No detection of endothelial antigen; + = focal endothelial antigen staining; + + = uniform endothelial antigen staining;+ = antigen staining on normal endothelium but not malignant cells; ND = not done.

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Figure 1 Western blothe antigen recognisedantibody JC70 is showhave a relative molecumass of 100 000fromspleen (track 1) and130 000from platelets(track 2).

(Dakopatts A/S) using an indirect peroxidasemethod, as described previously.'" Tissue sec-

5 tions were stained with Ulex europaeusagglutinin lectin according to the method of

(kD) Holthofer.'6

ResultsBIOCHEMICAL CHARACTERISATION OF THEJC70 ANTIGENEssentially all of the antigen recognised byantibody JC70 was found in the NP40 soluble

7t of membrane protein which bound to lentil lectin.tbyo This preparation contained about 43% of thelar antigen and 0-6% ofthe total protein present in

the tissue homogenate (constituting an enrich-ment of 70-fold). This indicates that the anti-gen is a membrane glycoprotein. On Westernblotting the antigen was shown to have arelative molecular mass of 100 000 from thespleen (fig 1, track 1) and 130 000 from theplatelet preparation (fig 1, track 2). This pat-tern was identical with a blot using an anti-CD31 monoclonal antibody.

REACTIONS OF JC70 ON BLOOD SMEARS ANDCRYOSTAT SECTIONSOn peripheral blood smears (fig 2) antibodyJC70 labelled several different cell types,including neutrophil polymorphs, 50% of thelymphocytes, and all of the monocytes andplatelets. On tonsil and spleen the strikingfeature was the intense labelling of endothelialcells, but some non-endothelial cells wereweakly labelled including mantle zone B cellsand T cells (fig 3).

REACTIONS OF JC70 ON PARAFFIN WAX EMBEDDEDTISSUE SECTIONSNormal tissuesAntibody JC70 reacted with endothelial cells ina wide range of normal tissues (figs 4-7).Staining was generally comparable with thatusing polyclonal and monoclonal antibodies toFVIII-Rag, but JC70 also stained endothelium

in renal glomerular capillaries and the endo-thelium of vasa vasorum. JC70 also labelledmegakaryocytes and occasional plasma cells inbone marrow. Staining of plasma cells was notseen in any other sections. Ulex europaeusagglutinin lectin stained normal endothelialcells but also a wide variety of other cells andparticularly connective tissue.

Vascular proliferationsBenign: Antibody JC70 stained endothelialcells in a variety of benign vascular lesionsmore consistently than the monoclonal andpolyclonal antibodies to FVIII-Rag. It did notstain lymphatic endothelium in the cases oflymphangioma, lymphoepithelial cyst andpneumatosis coli (table); anti-FVIII-Rag andUlex europaeus agglutinin lectin gave variable,focal staining. JC70 stained endothelial cells incapillaries in a case of haemangiopericytoma aswell as tumour cells outside vessels. Theantibody stained numerous endothelial cells aswell as "myxoma" (stromal) cells in three casesof atrial myxoma. The endothelial cells liningsinusoids of the liver, spleen, and lymph nodeswere stained more reliably with JC70 thanwith antibodies to FVIII-Rag or with Ulexeuropaeus agglutinin lectin.Angiosarcomas: Antibody JC70 stained malig-nant vascular endothelial cells in 10 cases ofangiosarcoma (table) (figs 7A-C). Staining ofmalignant endothelial cells was more uniformwith JC70 than with antibodies to FVIII-Ragor with Ulex europaeus agglutinin lectin andseemed to be more intense than on normalendothelium. Moreover, JC70 showed little ofthe intense background staining exhibited byantibodies to FVIII-Rag and Ulex europaeusagglutinin lectin in areas of haemorrhage andnecrosis, which tended to mask any positivestaining of tumour cells.Kaposi's sarcomas: Antibody JC70 gave vari-able staining of small vessels in four cases ofKaposi's sarcoma but spindle cells in thestroma were unlabelled (table). Occasionalstromal spindle cells were labelled by poly-clonal and monoclonal antibodies to FVIII-Rag.

I Non-vascular tumoursIn both benign and malignant tumours ofnon-vascular origin antibody JC70 stained endo-thelial cells in normal vessels but not tumourcells. The same pattern of staining was seenwith antibodies to FVIII-Rag and with Ulexeuropaeus agglutinin lectin.

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Figure 2 Peripheral blood smear showing labelling ofplatelets and many mononuclearcells. Note that not all lymphocytes are stained (arrow).

DiscussionFactor VIII related antigen is commonly usedas a marker for vascular endothelial cells.'7 It isnot uniformly distributed among species. Inman it is found in endothelial cells or arterioles,capillaries, and venules of many tissues in-cluding high endothelial vessels in lymphatictissue and liver sinusoids. It is absent fromglomerular capillaries, and splenic sinusoidsexpress it only weakly.'8 FVIII-Rag is alsofound in platelets, megakaryocytes, mast cellsand the mesangial region of renal glomeruli.

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Figure 3 Cryostat sectionof tonsil illustratingmonoclonal antibody JC70staining vascularendothelium (arrow), butalso lymphocytes in themantle zone (MZ).Lymphocytes elsewhere,including the germinalcentre (GC) areunlabelled.

*.4.

It is not reliably detectable in routinely pro-cessed material and has been reported by some

workers to show lack of specificity, sensitivity,and reproducibility, particularly when usingpolyclonal preparations.419

Ulex europaeus agglutinin is a lectin thatreacts with c-L-fucose and was originallythought to be selective for vascular endothelialcells. It is now known to bind to L-fucoseresidues in other cell types.'6 It has beenclaimed to be a more sensitive marker thanFVIII-Rag and works well in paraffin wax

sections.' Its limitations include lack ofspecificity and reproducibility.20

Antibodies directed against blood group

antigens such as ABO have been suggested as

markers of blood vessels, though they are not

suitable for routine use as prior knowledge of a

patient's blood group is required which is notalways available.2' Antibodies against cyto-skeletal antigens like laminin, found in base-ment membrane22 and vimentin, cannot im-

,*

w~ ~ ~~~4

A1.

ef f i 4' b*<84 e'q~~~~~~ Mb

A'

Figures 4-7 Labelling of a variety of normal and neoplastic endothelial cells intrypsinised, paraffin wax sections with antibody JC70 using the APAAP technique.Figure 4 Aortic adventitial vasa vasorum.

prove the immunohistochemical diagnosis ofvascular lesions because the antigens are notrestricted to endothelial cells.Monoclonal antibodies, which bind to

epitopes of endothelial cell specific antigens,are an alternative to these markers. Thosemonoclonal antibodies to endothelial antigenswhich have been reported to date are restrictedto frozen or specially fixed tissue because theirantigens are not detectable in routinely proces-sed tissue. Monoclonal antibodies in this cate-gory include E9223 and PAL-E.24 More re-

cently, a monoclonal antibody, BMA 120, hasbeen described which is directed against a

formalin resistant endothelial antigen with a

molecular weight of 200 kilodaltons.6 Thismolecular weight is similar to that of FVIII-Rag, and it is not yet known whether thisantibody shares the lack of specificityassociated with antibodies to FVIII-Rag.Antibody JC70 recognises an antigen that is

resistant to formalin fixation and which ispresent on a wide variety of benign vascularendothelial cells. Although JC70 labels a

variety of non-endothelial cells in blood smearsand cryostat sections, its reactions are essen-

tially restricted to endothelium when fixedtissue is stained. One interesting observationis that JC70 does not stain lymphatic endo-thelium in benign lesions. Previously reportedmarkers ofendothelial cells have not shown thisdegree of specificity. For example, antibodiesto FVIII-Rag have been shown to stain at leasta proportion of lymphatics.2 Stephenson andMills have shown that monoclonal antibodiesto A, B, and H blood group isoantigens gavestrong staining of both lymphatic and vascularendothelium.3 On the basis of the results in ourstudy, endothelial cells might be identified as

being of lymphatic origin if they are positivefor A, B, and H blood group isoantigens andFVIII-Rag but unstained by antibody JC70.Further studies on a wider range of lesionscontaining cells of lymphatic origin are re-

quired, however, to support these preliminaryfindings.Antibody JC70 also stained occasional

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Figure 5 Organising thrombus in a vessel wall with adjacent vasa vasorum.

plasma cells and megakaryocytes in formalinfixed sections. The latter observation may beexplained by the fact that endothelial cellscontain several proteins, mainly glycoproteins,which are antigenically related to proteinsisolated from platelet membranes.25 The JC70antigen is a 100 kilodalton membrane glyco-protein which seems to be identical with theantigen recognised by CD31 antibodies. Thishas been confirmed by the cloning of cDNAencoding JC70 antigen, which has the samenucleotide sequence as CD31 clones (D Sim-mons, personal communication). Cells trans-fected with either CD31 or JC70 cDNA clonesare also reactive with both CD31 and JC70 (DSimmons, personal communication). Theorigin ofan antigen band smaller than expectedfrom splenic preparations (100 rather than the130 kilodaltons of classic CD31) is not knownbut may be due to proteolytic breakdown or tovariations in glycosylation.

Figure 6 New vesselformation in granulation tissue.

The reaction with atrial myxoma cells sup-ports the endothelial origin of some of thesecells.26 The staining of both capillary endo-thelial cells and tumour cells in haeman-giopericytoma with the same antibody has notpreviously been reported and suggests that theendothelial cell and the neoplastic pericyteshare at least one antigen in common.Antibody JC70 stained sinusoidal endo-

thelial cells in liver, lymph node, and spleen.Other workers have shown variable staining ofthese endothelial cells with Ulex europaeusagglutinin lectin'6 and antibodies to FVIII-Rag,20 indicating that sinusoidal endotheliumdiffers phenotypically from other vascularendothelium. Some workers, however, haveshown that hepatic sinusoidal endothelium isUlex europaeus agglutinin lectin positive infetal and cirrhotic liver, suggesting a phen-otypic change during growth and disease.27 Thepresent findings with monoclonal antibodyJC70 suggest that the results with these othermarkers reflect no more than changes in theamount of endothelial antigen present.Antibody JC70 was more reliable than other

markers of malignant endothelial cells becauseit labelled each of the 10 cases of angiosarcomatested. JC70 also gave clear, strong staining ofthese cells unlike the focal and backgroundstaining frequently seen with antibodies toFVIII-Rag and Ulex europaeus agglutinin lec-tin, particularly in areas of necrosis andhaemorrhage. The amount of JC70 antigenmay be increased on malignant endothelial cellsof angiosarcomas, as indicated by the greaterintensity of staining of these cells comparedwith normal endothelial cells.

Kaposi's sarcoma is a spindle cell tumour inwhich the spindle cells enclose endothelial andnon-endothelial lined vascular spaces contain-ing red cells. The lack of staining of spindlecells with JC70 (some of which show positivitywith antibodies to FVIII-Rag and Ulex euro-paeus agglutinin lectin) in sections of Kaposi'ssarcoma supports the view that the spindle cellsoriginate from lymphatic vessels.'Most tissue samples examined by diagnostic

histopathologists are fixed and paraffin waxembedded. The availability of an antibodywhich can distinguish endothelial cells in thistype of material is potentially of great diagnos-tic value. At present, the most commonly usedendothelial markers are the lectin Ulexeuropaeus agglutinin lectin and antibodies toFVIII-Rag. As this study has shown, Ulexeuropaeus agglutinin lectin and FVIII-Rag areless reliable as markers of malignant endo-thelial cells and may be less specific for endo-thelial cells of cardiovascular origin.The antigen recognised by JC70 is resistent

to formalin fixation and is present on benignendothelial cells ofcardiovascular origin and onmalignant endothelial cells. This is apparenteven when there is tissue necrosis andhaemorrhage. JC70 is therefore of value in thediagnosis of a range of vascular conditions,including angiosarcomas, in routinely pro-cessed tissue sections. The range of potentialapplications of this antibody are likely to bewide. They include the investigation of vas-

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cularisation of inflammatory lesions and oftumours, investigation of the development ofthe placental bed and investigation of the vasavasorum and neovascularisation of the aorticand arterial intima in relation to atherogenesis.

This work was supported by grants from the LeukaemiaResearch Fund and the Cancer Research Campaign.

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Figure 7 Angiosarcomashown at low power (A)with two different highpowerfields (B and C).

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