JOURNAL OF PATHOLOGY, VOL.

167: 39 1-397 ( 1 992)

TDM35-A NEW MONOCLONAL ANTIBODY TO THE XHl CERVICAL CARCINOMA CELL LINE. CHARACTERIZATION A N D IMMUNOPEROXIDASE LOCALIZATION IN BENIGN A N D MALIGNANT TISSUES TREENA CHANA*, XIN HAN*, BELINDA DAGG*, ANDREW s. L A W R I E ~ROGER , P. GOODING$, DAMIAN L. EUSTACE~,CHRISTOPHER D. M. FLETCHER* AND EADIE HEYDERMAN*

s.

Departments oj Histopathology*, Coagulation Research? and Obstetrics and GynaecologyS, U M D S , St Thomas Hospital, London SEI 7EH, U.K.;$Department oflmmunology, Westminstu Hospital, London S W I P 2AP, U.K. Received 11 October 1991 Accepted 7 January 1992

SUMMARY The murine monoclonal lgGl kappa antibody TDM35 was raised against the cervical carcinoma cell line XH 1 . The antibody recognizes 18.5-66 kDa NCA-like glycoproteins and immunostains a variety of formalin-fixed, paraffinembedded normal, benign, and malignant tissues. It is of value in the diagnosis of carcinoma of the exocrine pancreas and it identifies foci of squamous and glandular differentiation in other tumours. TDM35 should form a useful addition to a panel of antibodies for the evaluation of epithelial lesions. KEY WORDS-TDM~~,monoclonal antibody, NCA-like, immunoperoxidase, tumour localization,XH1 cell line.

INTRODUCTION We have raised a number of murine monoclonal antibodies against X H 1, a new human cervical carcinoma cell line developed in this laboratory from a cervical adenosquamous carcinoma.' Only those antibodies that showed positive immunoperoxidase staining on paraffin-embedded, formalin-fixed tissue sections of an XHI xenograft were selected for further study. We report on the production, characterization, and immunoperoxidase localization of one of them, TDM35, which recognizes an NCA-like antigen in formalin-fixed, paraffinembedded tissue sections. MATERIALS A N D METHODS

Production of TDM35 monoclonal antibody BALb/c mice were immunized with live XHl cervical carcinoma cells.' Immune spleen cells were Addressee for correspondence: Dr X. Han, Department of Histopathology, UMDS, St Thomas Hospital, London SEl7EH, U.K.

0022-341 7/92/080391-07 $08.50 0 I992 by John Wiley & Sons, Ltd.

fused with P3X63Ag8.653 myeloma cells2 in the presence of 50 per cent polyethylene glycol 1500 (Sigma). Hybrids were selected by culture in HAT medium (Sigma).3 Supernatants from wells containing growing hybrids were tested by ELISA on fixed XH 1 cells grown in 96-well plates. Those positive by ELISA were used to immunostain sections of an XH 1 nude mouse xenograft'. A streptavidinlbiotinl complex (ABC) method4 without enzyme predigestion was employed with diaminobenzidine (DAB; Sigma) as chromogen. Endogenous peroxidase was inhibited as previously d e ~ c r i b e d .TDM35 ~ gave particularly strong and discrete staining in the centre of islands of tumour in an XH 1 xenograft and was selected for cloning by limiting dilution. An ELISA hybridoma subtyping kit was used to determine the murine immunoglobulin class (Boehringer Mannheim, BCL, Sussex).

Ascites preparation Ascitic fluid was obtained by intraperitoneal inoculation of TDM35 hybridoma cells into BALb/c

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mice 10 days after priming with an intraperitoneal injection of 0.5ml Pristane (Sigma). An IgG fraction was prepared by affinity chromatography using Sepharose-CL-4B Protein A (Pharmacia Biosystems Ltd., Beds).

George's Hospital, London), cytokeratin (CAM 5.2; Becton Dickinson, Oxford), or with TDM35 supernatant. A colon carcinoma was used as positive control for CEA, DD9, and CAM 5.2, and an omental deposit of an ovarian carcinoma for EMA.

Electrophoresis of X H I cell lysate and Western blotting

Tissue localization Formalin- fixed, para fin-embedded sections were chosen to contain a range of normal tissues and common lesions, and an XHI xenograft was used as a positive control. TDM35 supernatant was used either neat or diluted 1:2 for 1 h at room temperature, with the second biotinylated rabbit antimouse antibody diluted 1:250. Using an automated immunostainer (Histostainer; Leica, Beds) with the incubation chamber set to 37°C and the first antibody incubation carried out overnight, TDM35 supernatant could be used diluted 1:50, the second antibody at 1:5000, and the ABC complex diluted 1:200.

XHI cells were lysed with 10 per cent SDS in 0.01 M HCl and diluted either in a non-reducing buffer (10 mM Tris with 1 mM disodium EDTA, 2.5 per cent SDS, and 0.05 per cent wjv bromophenol blue, pH 8.0) or in a reducing buffer prepared by adding 2.5 per cent dithiothreitol to the non-reducing buffer. Samples were run on a Phast System@ using 12.5 per cent homogenous PhastGels@ together with high and low molecular weight markers (HMW 53-212 kD; LMW 14.4-94 kD) (Pharmacia). One of each pair of gels was stained for protein with Coomassie blue R. The other was electroblotted onto a nitrocellulose membrane (Schleicher and Schuell BA85, Anderman and Co. Ltd., Surrey) using a PhastTransferm system (Pharmacia). The membrane was blocked with a solution of 5 per cent powdered milk (Marvel, Premier Brands U.K. Ltd., Birmingham), washed in distilled water and then in 0.02 M borate-buffered saline (pH 7.2) with 0.1 per cent Tween 20 (BBST), and incubated for 2 h in TDM35 antibody diluted 1:2 in BBST. After washing in BBST, they were incubated for 2 h in alkaline phosphatase conjugated goat anti-mouse antibody diluted 1: 1000 (Dako). Bands were visualized with nitro-blue tetrazolium as chromogen.' Carbohydrate content of antigen Formalin-fixed XH 1 cells grown in 96-well plates were incubated at 37°C for 2 h in either 50 rnU/ml neuraminidase in 0.1 M sodium acetate, 1 per cent periodic acid in distilled water, 0.1 per cent trypsin in PBS (pH 7.2), or in PBS alone as control. An ELISA was used to determine any change in binding of TDM35 supernatant or ascitic fluid after this treatment. Parental myeloma supernatant was used as a negative control. Compurison with C E A , E M A , DD9,und cytokeratin Sections of an XH1 xenograft were immunostained with antibodies to CEA (Amersham, Bucks), EMA (Dako, Bucks), DD9 (Dr Grant, St

Negative control As the antigen recognized by TDM35 has not yet been purified, an absorption specificity control could not be prepared. The negative control employed was parental myeloma supernatant. This control would ensure that staining was due to TDM35 binding and not to endogenous peroxidase, or to binding of the biotinylated anti-species antibody or the streptavidinj biotin/peroxidase complex.' RESULTS TDM35 was shown to be of IgGl kappa immunoglobulin class. Binding of purified ascites to XHl cells was reduced from O D 0.68 & 0.09 to 0.0 1 & 0-01 by periodic acid and of supernatant from OD 0.35 0.05 to 0.02 & 0.02. Neither trypsin nor neuraminidase had any effect on binding, indicating that the determinant recognized was glycosylated but not sialated. SDS polyacrylamide gels of the XHI cell lysate showed multiple bands within the range 1421 2 kD. There was no apparent difference between samples run under reducing or non-reducing conditions, indicating an absence of disulphide bonds between subunits. Tmmunolocalization of bands with TDM35 revealed several bands in the same molecular weight range, with major bands at 18.5, 35, 50, 54, and 66 kD (Fig. I). Antibodies to CEA, EMA, cytokeratin, and DD9 stained the xenograft in a pattern similar to each

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TDM35-IMMUNOPEROXIDASE TISSUE LOCALIZATION 14.4

20.1

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67.0

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94.0

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Fig. 1-12.5 per cent SDS-PAGE of XHI cell lysate stained with Coomassie blue R (lanes 1-3). Western blot using TDM35 (lanes 4 and 5). (1) Molecular weight markers 14.494 kD; (2) XHI cell lysate; (3) XHI cell lysate reduced; (4) Western blot o f X H l cell lysate; ( 5 ) XH1 cell lysate reduced

other and to that seen with TDM35. On the colon carcinoma, however, TDM35 staining was different from CEA and CAM 5.2 staining. CEA staining was seen in necrotic debris within malignant acini, on luminal membranes, and in the cytoplasm of most tumour cells. Very occasional polymorphs were positive. TDM35 staining was also seen in the debris within acini and on luminal membranes, but there was minimal cytoplasmic staining and polymorphs were strongly positive. Staining for CAM 5.2 was pericellular and quite different from CEA or TDM35 staining, and the ovarian serous carcinoma used as a positive control for EMA was negative for TDM35. TDM35 staining was similar to staining of DD9 on the colon carcinoma but staining of luminal membranes was stronger with TDM35, while polymorphs and macrophages were of equal intensity. A variety of benign and malignant tumours were positive with the TDM35 antibody (Figs 2-9). Cervical adenocarcinomas and squamous and adenosquamous carcinomas were all positive. In squamous carcinomas and in the XHl xenograft, staining was maximal at the centre of tumour islands (Fig. 3). In largely undifferentiated cervical carcinomas, the

imniunostain picked out small foci of squamous differentiation not easily detectable on the H&E section. CIN3 lesions of the cervix were positive but so were superficial epithelial cells of the normal ectocervix (Figs 4 and 5). There was very occasional luminal staining in cycling endometrium, and glands showing the Arias Stella reaction showed a marked fine line of positivity on their luminal surface. Serous ovarian carcinomas were negative while 415 mucinous tumours were positive. They showed a ‘smudgy’ pattern of mucin staining in the cytoplasm and in the lumina of malignant acini (Fig. 8). The endometrial carcinomas were difficult to interpret as there was a marked polymorph infiltrate. Occasional cells in 212 were positive. Normal or hyperplastic prostatic glands showed occasional luminal staining. Two of three prostatic carcinomas were positive. Squamous carcinomas in the skin showed much weaker staining than those of cervix or lung, other than in keratinized areas. Malignant eccrine poromas and basal cell and sebaceous carcinomas were negative, except for keratotic foci which were positive. Malignant melanomas and naevi were

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Fig. 2-9.

TDM35-IMMUNOPEROXIDASE TISSUE LOCALIZATION

negative. Sebaceous glands were negative while eccrine sweat ducts and acini were positive. In these and in serous salivary glands, intercellular canaliculi were well shown. Most of the epidermis of the skin was negative but there was occasional weak staining of normal superficial keratinized cells, with strong staining of hyperkeratotic areas. Most lung carcinomas showed some positivity (Fig. 2). All colonic (Fig. 6), gastric, and pancreatic exocrine adenocarcinomas were positive (Fig. 9). There were foci of acinar differentiation in two of the ileal carcinoids. These foci showed luminal staining with TDM35, while the compact sheets of tumour in these and in the other carcinoids were negative. Normal pulmonary alveolar, mesothelial, and gut epithelia were generally negative, except near tumours and in the presence of inflammation. Acini and ductules were positive in foci of chronic pancreatitis, though normal pancreas was negative. TDM35 was negative in the kidney and in renal cell tumours. Normal bladder epithelium and transitional cell carcinomas were negative except in foci of glandular or squamous differentiation (Fig. 7). Adenocarcinomas of the bladder were positive. CNS, pituitary, parathyroid, adrenal, pancreatic islet cell tumours, follicular, papillary, and anaplastic thyroid carcinomas were negative. Medullary carcinomas of the thyroid were either uniformly or focally strongly positive. All normal mesenchymal tissues, brain, endocrine glands, ovary, and testis were negative. Three of twenty-four sarcomas, all synovial sarcomas, were positive with TDM35. One of four biphasic synovial sarcomas showed cytoplasmic positivity of

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the epithelial component comparable to that seen with EMA antibodies; two others had occasional positive cells. Staining was mainly confined to foci showing overt glandular differentiation. This is in contrast to EMA, which also stains solid sheets or nests of epithelial cells and some of the spindle cell component.' TDM35 was negative on epithelioid sarcomas. Polymorphs and macrophages were positive in all tissues in which they were found (Fig. 7). Lymph nodes, lymphoid tissue, and lymphomas were negative. Stratified squamous epithelium on the surface of chronically inflamed tonsils was positive. Sequential sections of all blocks positive for TDM35 were also immunostained with the myeloma supernatant negative control. Polymorphs in one cervical carcinoma were positive, and positive staining still resulted when 1 per cent ovalbumin in PBS was substituted for the myeloma supernatant. None of the other 206 sections treated with the myeloma supernatant was positive. There was no staining when 1 per cent ovalbumin was substituted for both the primary and the secondary biotinylated rabbit anti-mouse antibody, or when DAB alone was used. Thus, the positive polymorph staining in 1/207 (0.48 per cent) of the sections is likely to be due to binding of the secondary antibody to a rare polymorph antigen and this will be investigated further. DISCUSSION Antibodies to XHl cells were raised partly in the hope that they might discriminate qualitatively

immunoperoxidase stains for TDM35

Fig. 2-9-ABC

Fig. 2-Squamous carcinoma of the lung with positivity mainly at the centre of a tumour island and in polymorphs and necrotic debris. Surrounding lymphocytes and plasma cells are negative Fig. 3-XH1

xenograft shows squamous carcinoma morphology and staining mainly in the centre of tumour islands

Fig. &Normal Fig. 5-In

cervix with positivity of superficial cells

CIN3, the staining extends into the intermediate zone

Fig. &Colonic carcinoma positive on luminal membranes, in debris within malignant acini. Unlike with the CEA antibody, there is only minimal cytoplasmic staining Fig. 7-Inflamed

bladder mucosa positive in a focus of squamous metaplasia. Polymorphs in vessels and in the stroma are positive

Fig. 8-Mucinous ovarian cystadenocarcinoma shows thecharacteristic rather smudgy appearance of positively stained mucin within cells and in glandular spaces Fig. 9-Adenocarcinoma

of the exocrine pancreas shows luminal and mainly supranuclear cytoplasmic positivity

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T. CHANA E r AL.

or quantitatively between the various grades of cervical intraepithelial neoplasia (CIN) and normal epithelium in cervical smears. Although staining of CIN3 lesions extended through the intermediate layer, superficial cells of normal ectocervical epithelium were also positive (Figs 4 and 5). TDM35 would therefore be of no value for screening cytological preparations. Expression of the TDM35 antigen appears to be related to terminal differentiation, since staining was maximal on superficial ectocervical cells and in the centre of islands of squamous carcinoma, often where keratinization occurred (Fig. 2). TDM35 recognizes an NCA-like antigen,’.’’ with a tissue distribution similar to that of DD9“ and other NCA antibodies, though there were subtle differences in staining of the colon carcinoma (Fig. 6). The DD9 antibody recognizes a family of protein determinants in 45, 56, and 80-115 kD glycoproteins,” whereas the TDM35 antibody binds to a carbohydrate epitope in glycoproteins of 18.566 kD. We have previously shown that DD9 is of diagnostic value in that all exocrine pancreatic carcinomas studied so far have been positive.I3 TDM35 shows a pattern of staining in these tumours very similar to that of DD9, and 13/13 pancreatic carcinomas were positive. Absence of TDM35 in a metastatic adenocarcinoma would virtually exclude origin from the gastrointestinal tract including the exocrine pancreas. The rather ‘smudgy’ TDM35 staining of niucin in ovarian mucinous adenocarcinomas was not seen in adenocarcinomas from other sites (Fig. 8). Like EMA, TDM35 picked out cells showing squamous metaplasia (Fig. 7), and the common foci of glandular differentiation seen in transitional cell carcinomas of the bladder.14 The very similar pattern of staining for TDM35, CEA, EMA, cytokeratin, and DD9 in the XHl xenograft highlights one of the problems in the interpretation of immunocytochemical results and the need to examine a variety of tissues when screening new antibodies. In this study, differences in the staining pattern of a colon carcinoma and a metastatic ovarian tumour showed that the TDM35 antigen was not the same as CEA, EMA, cytokeratin, or DD9. TDM35 stains polymorphs in decalcified bone marrow and may help to differentiate cells of myeloid lineage from other haematopoietic cells in bone trephines and marrow smears. The antibody may also be of value in the immunolocalization of occult abscesses.15

NCA is a member of the immunoglobulin supergene family and has been shown to be involved in cell adhesi~n.’~.’’ Our XH 1a cervical carcinoma subline grows as compact sheets and is TDM35positive. When injected into nude mice, it forms progressively invasive tumours and colonizes the lungs when injected intravenously. The X H l b subline, in contrast, grows in monolayer culture in a diffuse fibroblast-like fashion and is negative for TDM35. When injected into nude mice, it forms tumours which then regress and it does not form lung colonies on intravenous injection.’ It seems likely that differences in TDM35 gene expression are involved in regulation of the growth pattern of the XH1 cell line in virro, and in invasion, regression, and ‘metastasis’ in vivo. The cell adhesion activity of TDM35 will be investigated in cultures of XH 1 and our other cervical carcinoma cell lines, and studies to identify and clone the TDM35 gene will be carried out. ACKNOWLEDGEMENTS

We should like to thank Mr P. J. Warren for assistance with the early part of this study, the technical staff of the Department of Histopathology for valuable help with the preparation of blocks and H&E sections, and D r Hollowood and Dr Chong for help in the selection of tissues. Mrs V. Pate1 of Universal Fotos, London printed and colourmatched the photomicrographs. This work was supported by the Clothworkers Guild (TC), St Thomas Hospital Research Endowments Fund (XH), the Dunhill Medical Trust (XH), Birthright (DE), the Jean Shanks Foundation (BD), the Cancer Research Campaign (CDMF), and the Gunnar Nilsson Cancer Research Trust Fund (EH). REFERENCES 1. Han X. Lyle R, Eustace DLS, er a/. XH I-a

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