J Clin Pathol 1984;37:14-19

Immunohistochemical detection of Ca antigen innormal, dysplastic and neoplastic squamous epitheliaof the human uterine cervixJM LLOYD, T O'DOWD,t M DRIVER,* DEH TEE

From the Departments ofImmunology and *Morbid Anatomy, King's College Hospital Medical School,Denmark Hill, and the tDepartment of Obstetrics and Gynaecology, Dulwich Hospital & King's CollegeHospital

SUMMARY Immunohistochemical staining was performed on biopsies and cytological samplesfrom normal, dysplastic and neoplastic squamous epithelia using the monoclonal Ca 1 antibody.The results of staining 92 biopsies and 20 cytological samples are described and it is reported thatpositive staining with Ca 1 antibody was detected in normal, dysplastic and neoplastic epithelia.The role of the Ca 1 antibody in the study of cervical cancer is discussed.

One of the most important questions of cancerimmunology is whether human tumour cells expressantigens which are not expressed by normal cells.An antigen described by Ashall, Bramwell and Har-ris', and designated the Ca antigen, was found onthe cell surface in a wide range of malignant humancell lines, but not in non-malignant cell strains. Theantigen was detected by means of a monoclonal IgMantibody, the Ca 1 antibody. Following on from thisdiscovery McGee et a12 used the Ca 1 antibody in animmunohistochemical procedure to detect the Caantigen in tissue sections. These workers showedthat the majority of malignant tumours examinedexpressed the Ca antigen, whereas the only normaltissues to bind the Ca 1 antibody were the transi-tional epithelium of the urinary tract and theepithelium of the fallopian tube.

In the present study the monoclonal Ca 1 anti-body was used in immunofluorescence andimmunoperoxidase procedures on fresh and fixedtissue from the cervix uteri. Carcinoma of the cervixuteri is exceptional in that preinvasive conditionscan be diagnosed cytologically and can be readilybiopsied. Thus biopsies and cytological samplesfrom normal, dysplastic and invasive epithelia wereexamined for the presence of the Ca antigen in anattempt to investigate the relation between malig-nancy and the occurrence of the Ca antigen.

Accepted for publication 21 July 1983

Material and methods

IMMUNOHISTOLOGICAL REAGENTSThe following antibodies were obtained from Well-come Diagnostics, Temple Hill, Dartford, Kent:monoclonal Ca 1 antibody (code RP82); mono-clonal IgM antibodies to meningococcal bacteria(code MB34, MB36) selected for use as controls inorder to demonstrate the specificity of the Ca 1 anti-body; and peroxidase conjugated rabbit antimouseIg antibody.

Fluorescein isothiocyanate (FITC) conjugatedgoat antimouse IgM antibody was obtained fromTCS, 10 Henry Road, Slough, Bucks. Diaminoben-zidine tetrahydrochloride and trypsin were obtainedfrom Sigma Chemical Co, Fancy Road, Poole,Dorset.

TISSUESMost of the specimens were of fresh frozen tissuecollected by biopsy from a referral colposcopy clinicat Dulwich Hospital. Cervical scrapes were also col-lected at this clinic. Other fresh cervical tissue wasobtained from hysterectomy or cone biopsy speci-mens at King's College Hospital or Dulwich Hospi-tal. Normal skin, normal colon, and colon tumourtissues were also collected for use as controls. Allfresh tissue was snap frozen in liquid nitrogen withinone hour of collection, and stored at -80°C prior tocryostat sectioning.

Paraffin embedded material was also collected.14

Ca antigen in normal, dysplastic and neoplastic squamous epithelia of the human uterine cervix

TISSUE SECTION PREPARATIONFresh frozen tissue stored at - 80°C was sectioned at5 ,um in a cryostat. The sections were air dried for30 min and stained immediately using theimmunofluorescence procedure (below).

Sections (5 ,m) were prepared from cervicalbiopsies or intact cervices which had been fixed inneutral buffered formalin for >24 h and routinelyprocessed to paraffin wax. The sections weremounted on glass slides, dried at 37°C, dewaxed inxylene, rehydrated in alcohols and washed in distil-led water prior to immunoperoxidase staining(below).

CELL PREPARATIONA single cervical scrape was taken at the colposcopyclinic. After preparing a smear for routine cytologi-cal evaluation the scrapings were suspended in20 ml of phosphate buffered saline, pH 7*2 (PBS)used here as a holding solution. The suspended cellswere processed for immunohistochemistry withinthree hours of collection. The suspended cells werewashed twice in PBS and the cell density adjusted toapproximately 100 000 cells/ml; 0 25 ml of suspen-sion was dropped into each chamber of a 12chamber cytospin (Shandon) and after a 5 min spinat 700 rpm, the prepared slides were removed andfixed without drying in 50:50 (chloroform:acetone)for 5 min. The slides thus prepared were stored at- 80°C prior to immunostaining. The technique usedbears similarities to that used on cervical cellimprints3 rather than to that used on unfixed cervicalcells in suspension.45

IMMUNOPEROXIDASE METHODThe immunoperoxidase procedure used was basedon that used by McGee et al.2 Sections and cell prep-arations were incubated with the Ca 1 antibody orthe control antibodies (MB34, MB36) for 60 min.The antibodies were diluted 1/12 in a solution com-posed of 10% fetal calf serum (FCS) and 10%bovine serum albumin (BSA) in PBS, pH 7*4. Thesections were washed three times for 10 min in PBSand incubated for 30 min in rabbit antimouse IgMperoxidase conjugate (Wellcome) at a dilution of1/50 in PBS containing 10% FCS, 10% BSA and5% human AB serum. The sections were washedthree times for 10 min in PBS and reacted for 5 minwith diaminobenzidene tetrahydrochloride at500,ug/ml in PBS containing 0-01% (vol/vol) hyd-rogen peroxide (H202). The sections were washed intap water for 5 min, stained with haematoxylin,dehydrated in alcohols, cleared in xylene andmounted in DPX (BDH Chemicals, Poole, Dorset).To remove endogenous peroxidase the sections

and cell preparations were preincubated with 0.5%

H202 in methanol for 10 min. To remove any"non-specific binding", as described by McGee etal,2 samples of the sections and cell preparationswere incubated for 30 min with 05% (wt/vol) tryp-sin in PBS.

IMMUNOFLUORESCENCESections of tissue were incubated for 60 min with Ca1 or control IgM diluted 1/12 in PBS. The sectionswere washed three times for 10 min in PBS andincubated for 30 min in goat antimouse/FITC con-jugate (Tago) at a dilution of 1/50 in PBS. The sec-tions were then mounted in glycerol/PBS andexamined using a Reichert-Jung Polyvar microscopewith a 200 W mercury vapour lamp, a blue BP455-490 incident light exciter filter, a DS 500 dic-hroic mirror and a blue LP 515 barrier filter.

Results

USE OF CONTROL ANTISERAThe result of using the monoclonal antibodies MB34and MB36 on cervical tissue in both of theimmunochemical techniques showed that there wasa complete absence of staining. For this reasonresults obtained using the Ca 1 antibody were notattributed to non-specific binding.

USE OF CONTROL TISSUESThe normal colon and normal skin tissue did notstain with the Ca 1 antibody. A weak focal reactionwas found in sections of colon tumour.

COMPARISON OF FIXED AND FRESH TISSUEStudies comparing immunohistochemistry on frozenand paraffin embedded sections from adjacentblocks showed similar staining patterns whenstained with Ca 1 antibody. From this it wasdeduced that the Ca antigen was not destroyed byfixation or processing.

COMPARISON OF IMMUNOFLUORESCENCE ANDIMMUNOPEROXIDASEComparison of immunofluorescence andimmunoperoxidase on serial sections of cervical tis-sues showed similar staining patterns. Therefore theresults obtained using both techniques were com-bined.

USE OF TRYPSINTrypsin incubation as a pretreatment for theimmunoperoxidase method did not alter the pat-terns of staining. The observed staining pattemswere considered to be due to the presence of the Caantigen and not to the occurrence of "non-specificbinding" which could be removed with trypsin.2

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Table 1 Immunohistochemical staining with Ca I antibody of biopsies from the cervical transformation zone

Histological diagnosis No of biopsies No staining with Ca 1examined anibody

Normal mature and immature metaplastic squamous epithelium 27 25Mildly dysplastic epithelium 15 15Moderately dysplastic epithelium 22 22Severely dysplastic epithelium 8 8Carcinoma in situ of epithelium 8 8Invasive carcinoma of epithelium 15* 15Total 95 93

*Fourteen of these biopsies were paraffin embedded and stained by the immunoperoxidase technique.

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Ca antigen in normal, dysplastic and neoplastic squamous epithelia of the human uterine cervix

Fig. 2 Invasive squamous cell-carcimoma labelled with theimmunoperoxidase method and counterstained withMayer's haematoxylin showing positive staining with the

monoclonal Ca I antibody. (a) Staining predominantly on

cell membranes (arrow) x 225. (b) Staining predominantlyin the cytoplasm (arrow) x 525.

CERVICAL BIOPSIESSections from 81 fresh biopsies were stained by theimmunofluorescence technique and sections from 14fixed biopsies were stained by the immunoperoxid-ase method. Immunohistochemical staining with theCa 1 antibody was seen in almost all the materialexamined (Table 1). This staining was seen in nor-

mal, dysplastic and invasive epithelia (Fig. 1). Thestaining pattern was uneven. In some areas all thecells of the epithelium were intensely stained and inother areas only isolated cells or groups of cells werestained.

Although this variation in staining was found inrepresentatives from all the histological groups,there was a tendency for normal and metaplasticepithelia to exhibit least widespread staining. In themajority of sections the variation in histochemicallocalisation did not correlate with a variation in his-topathology. Exceptions to this general observationwere seen in a small number of cases in which stain-ing coincided with the appearance of an area of dys-plasia.

In almost all sections of dysplastic and normalepithelia the basal layer did not stain with the Ca 1antibody. This distinction was not seen in invasiveepithelia or in four sections showing carcinoma insitu or in four sections showing severe dysplasia.The distribution of the Ca antigen within the cells

was variable. The antigen was generally detectedboth in the cytoplasm and on the cell membrane,although some cells were apparently stained eitherin the cytoplasm or on the membrane (Fig. 2). Boththe cytological distribution and the intensity of stain-ing of the Ca 1 antibody varied within each section.

CELL PREPARATIONThe results of the immunoperoxidase staining of thecell preparations showed positive staining in all buttwo of the 20 samples examined (Table 2). Thenormal cell preparations contained a number ofpositively stained cells. The dyskaryotic cell prep-arations contained a proportion of both positivenormal cells and positive dyskaryotic cells. Similarlymalignant cell preparations contained proportionsof both positive normal cells and positive malignantcells. Examples of normal and dyskaryotic cellsdemonstrating positive membrane staining areshown in Fig. 3.

DISCUSSION

Histological and cytological samples of normal dysp-lastic and neoplastic cervical epithelia examined inthis study showed expression of the Ca antigen.These results are at variance with those obtained byMcGee et a12 who found staining in squamous cellcarcinoma of the cervix but not in normal cervical

Table 2 Immunoperoxidase staining with Ca I antibodyof cervical cell preparations

Cytological diagnosis No No stainingexamined with Ca I antibody

Normal 4 3Mild dyskaryosis 4 4Mode,rate dyskaryosis 4 3Severe dyskaryosis 4 4Malignant 4 4Total 20 18

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Lloyd, O'Dowd, Driver, Tee

Fig. 3 Cell preparations labelled with the Ca I antibody, using the immunoperoxidase method andcounterstained with Mayer's haematoxylin showing positive membrane staining. (a) Normal squamouscells showing positive staining (arrow) x 525. (b) Dyskaryotic squamous cells showing positive staining(arrow) x 525.

epithelia. These workers do, however, report theoccurrence of non-specific binding in the cervicalepithelium. This binding was seen when using con-trol monoclonal IgM antibodies, and could beremoved by incubating the sections with trypsin. Inthe present study there was no non-specific reactionbetween cervical epithelium tissue and control IgMantibody. The binding of the Ca 1 antibody was notremoved with trypsin, and was considered to be dueto the presence of the Ca antigen. The pattern ofstaining found in colon tumour was similar to thatreported by McGee et al.2

If expression of the Ca antigen was predominantlydue to malignant or premalignant changes within thecells of the cervical epithelium the observed stainingmight be expected to occur in abnormal cells only.However, the variety of staining pattern seen in rep-resentatives of all the morphological groups suggeststhat in the cervical epithelium the expression of theCa antigen is not related directly to malignancy.Some link with malignancy is suggested by thosecases in which the occurrence of the antigen corres-ponded to the localised presence of dysplasia.The observation of heterogeneity of staining seen

here corroborates observations made by McGee eta12 who observed variation of staining within singletumours and cited an example of a malignant car-cinoid in which the Ca antigen was detected in onlytwo of three blocks. The distribution of this antigenis clearly not related in a simple way to the presenceor absence of malignancy.

The absence of immunohistochemical staining inthe basal layers of the squamous epithelium isdifficult to explain. The apparent absence of the Caantigen in such a layer of proliferating cells mayhave some significance in a study of the function ofthe Ca antigen, but at present this is not clear.The results reported in this study suggest that the

Ca 1 antibody does not have a significant role in theprimary diagnosis or follow up of dysplastic andneoplastic lesions of the uterine cervix.

This work was supported by the Cancer ResearchCampaign. We are grateful to our clinical colleaguesand in particular to Mr JWW Studd, Dr L Cardozo,Dr S Tuck, Dr D Rosenberg and the nursing staff.We should also like to acknowledge the skilled tech-nical assistance of Mr ET Davies, Miss J Deacon,Mrs G Harris, Mrs S Patel, Mr A Perrin and also thephotographic assistance of Mr KW Davies and MrRJ Senkus. We should also like to thank Dr SChantler and Dr U Beckford of Wellcome Diag-nostics for their advice and support, and Mrs NMBennett for secretarial help.

References

Ashall F, Bramwell ME, Harris H. A new marker for humancancer cells. 1. The Ca antigen and the Ca 1 antibody. Lancet1982;ii: 1-6.

2McGee J O'D, Woods, JC, Ashall F, Bramwell ME, Harris H. Anew marker for human cancer cells. 2. Immunohistochemicaldetection of Ca antigen in human tissues with the Ca antibody.Lancet 1982;ii:7-10.

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i,

...4:%:.""I--.e .:,

Ca antigen in normal, dysplastic and neoplastic squamous epithelia of the human uterine cervix3 Pasca AS, Kummerlander L, Pejtsik B, Krommer K, Pali K.

Herpes simplex virus-specific antigens in exfoliated cervicalcells from women with and without cervical anaplasia. CancerRes 1976;63:319-23.

4Leif RC, Easter HN, Warters RL, Thomas RA, Dunlap LA,Austin MF. Centrifugal cytology 1. A quantitative techniquefor the preparation of glutaraldelyde-fixed cells for the lightand scanning electron microscope. J Histochem Cytochem1971;19:203-15.

I Haines HG, McCoy JP, Hofheinz DE, Ng ABP, Nordqvist SRB,Leif RC. Cervical carcinoma antigens in the diagnosis ofhuman squamous cell carcinoma of the cervix. J Natl CancerInst 1981;66:465-74.

iRequests for reprints to: Dr DEH Tee, Department ofImmunology, King's College Hospital Medical School,Denmark Hill, London SE5 8RX, England.

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