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Use of Antibodies to Carcinoembryonic Antigen and Human Milk Fat Globule to Distinguish Carcinoma, Mesothelioma, and Reactive Mesothelium

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Use of Antibodies to Carcinoembryonic Antigen and Human Milk Fat Globule to Distinguish Carcinoma, Mesothelioma, and Reactive Mesothelium J Clin Pathol 1984;37:1215-1221 Use of antibodies to carcinoembryonic antigen and human milk fat globule to distinguish carcinoma, mesothelioma, and reactive mesothelium RJ MARSHALL, A HERBERT, SG BRAYE, DB JONES From the University Department ofHistopathology, Southampton General Hospital, Southampton SUMMARY Antibodies raised against human milk fat globule (HMFG 1 and 2) and carcinoem- bryonic antigen were used in an immunoperoxidase technique to differentiate mesothelioma, carcinoma, and benign, reactive mesothelium. Sixteen mesotheliomas, 27 lung carcinomas, and 13 specimens of reactive mesothelium were examined. Staining for carcinoembryonic antigen was not seen in reactive mesothelium or mesothelioma but was present in 22 of 27 carcinomas. Mesothelioma and carcinoma usually stained with HMFG 1 and 2; reactive mesothelium did not. These three antibodies may help to distinguish carcinoma, mesothelioma, and reactive mesothelium. Distinguishing mesothelioma from carcinoma is a malignant mesothelioma was obtained from well recognised problem.' Histochemistry and elec- pleuro-pneumonectomy and pleurectomy specimens tron microscopy may help to make this distinction or from biopsies taken at thoracoscopy or but do not always give a definitive answer. Anti- thoracotomy (Table 1). Adequate material was bodies to carcinoembryonic antigen (CEA) and available in all cases for definitive diagnosis to be keratin have been assessed with conflicting made of biphasic (seven cases), epithelial (eight results.) It is equally difficult to distinguish benign cases), or mesenchymal (one case) malignant from malignant mesothelium. Morphometry6 and mesothelioma using morphological criteria. All the use of histiocytic markers in an immunoperoxid- cases were stained with periodic acid Schiff after ase technique8 have been advocated for this pur- diastase digestion and were negative. The presence pose. of acid mucopolysaccharide sensitive to hyaluronid- Anti-CEA and HMFG 2 have been used previ- ase digestion was confirmed by staining with Alcian ously to diagnose malignancy in cytological prepara- tions of pleural fluids.9 '0 Their usefulness in this respect depends on a knowledge of the staining Table 1 Malignant mesotheliomas behaviour of reactive mesothelium and of the pat- Case Type ofspecimen Histological Presence of tern of staining of benign and malignant cells in his- no type hyaluronidase tological preparations. sensitive In this study we have evaluated three antibodies, mucopolysaccharide anti-CEA and HMFG 1 and 2, to see if they would 1 Pleuropneumonectomy Epithelial + distinguish between carcinoma, mesothelioma, and 2 Pleuropneumonectomy Epithelial 3 Pleuropneumonectomy Mixed + benign, reactive mesothelium. 4 Pleurectomy Epithelial + 5 Thoracotomy Mixed 6 Thoracoscopy Mixed + Material and methods 7 Thoracoscopy Mixed + 8 Thoracoscopy Mixed 9 Thoracoscopy Mixed + Tissue was examined from 16 mesotheliomas, 27 10 Thoracoscopy Mixed + lung carcinomas, and 13 specimens in which reactive 11 Thoracoscopy Epithelial mesothelium was present. Tissue diagnosed as 12 Thoracoscopy Epithelial + 13 Thoracoscopy Epithelial + 14 Thoracoscopy Epithelial 15 Thoracoscopy Epithelial + 16 Thoracoscopy Mesenchymal- Accepted for publication 24 July 1984 1215 1216 Marshall, Herbert, Braye, Jones blue at pH I or 2-5 in the cytoplasm or acini of 10 of Table 3 Results the 15 cases with an epithelial component. The carcinomas were obtained from lobectomy, No HMFG 1 HMFG 2 CEA pneumonectomy, or lung biopsy specimens. Eight Mesotheliomas Epithelial 6 + + were squamous carcinomas, eight oat cell, four large 2 + _ _ cell, and seven adenocarcinomas, including one Biphasic 6 + + bronchioloalveolar cell carcinoma (Table 2). Mesenchymal 1 Reactive mesothelium was examined in eight Carcinomas pleurae removed after pneumothorax (Table 2). Squamous 7 + + + 1 + + _ Three pericardial and two pleural cases of mesothel- Oat cell 4 + + + ial reaction to tumour infiltration were also 2 + + _ 1 - + _ examined. 1 - + + Tissue was fixed in 10% neutral buffered formol- Large cell 3 + + + 1 - + + saline, routinely processed, and embedded in Adeno 6 + + + paraffin wax. Sections (5 ,um) were cut and stained 1 + + _ with Mayer's haematoxylin and eosin. Further sec- Reactive mesothelium 10 2 + - _ tions were examined with polyclonal rabbit antihu- 1 - + _ man CEA (Code No Al 15, Dakopatt Ab, Sweden), used at a dilution of 1/500, using the method though no consistent difference in staining pattern described by Mepham et al. " Two monoclonal anti- was seen with these two antibodies. Bronchial bodies, HMFG 1 and 2, (Seward Laboratory, Lon- reserve cells were prominent in seven cases and don) were used at a dilution of 1/3 in an indirect were stained with HMFG 1 and 2 (Fig. 1). immunoperoxidase technique. These antibodies are Normal alveolar epithelium was seen in 10 cases directed against determinants in the membranes of and in nine stained strongly with HMFG 2. HMFG 1 human milk fat globules.'2 They react with a variety stained with varying intensity in five cases. of carcinomas and normal glandular epithelia.'3 14 Reactive alveolar epithelium, present in 10 cases, Sections in which the first stage antibody was stained strongly with HMFG 2 in nine cases and replaced by Tris buffered saline served as negative variably with HMFG 1 in seven cases. controls and showed no staining in any of the cases studied. Sections of breast carcinoma and colonic Anti-CEA carcinoma were used as positive controls for HMFG The anti-CEA antibody stained bronchi and bron- 1 and 2 and CEA respectively. chioles with varying intensity in eight cases. Reserve cells were stained in three of the seven cases in a Results (Table 3) pattern similar to that seen with the HMFG anti- bodies. Normal alveolar epithelium stained strongly NORMAL BRONCHIAL AND ALVEOLAR in nine of 10 cases, as with HMFG 2. The pattern of EPITHELIUM staining of reactive alveolar epithelium was variable HMFG I and 2 and closely paralleled HMFG 1. Bronchial epithelium was present in 14 of the cases examined. HMFG 1 gave focal staining of the cilia REACTIVE MESOTHELIUM and basal plate region of bronchial epithelial cells. HMFG I and 2 HMFG 2 stained the epithelium of respiratory bron- Ten of the 13 specimens of reactive mesothelium chioles with a similar pattern but was generally were negative with HMFG 1 and 2. One case negative in the larger bronchioles and bronchi, stained with HMFG 2 and two cases with HMFG 1. Staining was very focal and seen on the free border Table 2 Carcinomas and reactive mesothelium of the cells. Care was needed to distinguish reactive pneumo- cytes from mesothelium in excised bullous cysts, but identification was made easier by the presence of Carcinoma 27 then stained strongly with Squamous 8 lung tissue. Pneumocytes Oat cell 8 all three antibodies, while mesothelium was gener- Large cell 4 ally negative (Fig. 2). Adeno 7 Reactive 13 Pleurectomy following pneumothorax 8 Anti-CEA Pericardectomy for malignant infiltration 3 This antibody did not stain any reactive mesothelial Pleural biopsies in suspected malignant effusions 2 cells. Use of antibodies to carcinoembryonic antigen and human milk fat globule 1217 ~~~ .....<ae S: ;.x.:e.~~~~~~~~~~- I~~~~~~~~~~~~~ U4~~~~~4 Fig. 1 Bronchus stained for HMFG 1. The cilia and basal plate region are pbsitive. Reserve cells stain on the surface adjacent to the respiratory epithelium (arrow). PAP x 350. o.X:._, 149 * I* *IsoVt I f :...9 *~~~~~~i4. WlJ. A.A:, iI.t 4... %% 41Y 'r, -.. Apo 4 "4:, 4 I.P. %kk. i% 4 * .9. 6 Jp 01. :.. Vp 0i .9I I.0 i 4 ,l5 ^ A . a 0 to1 4 MD . ".. e * j:*4.A Fig. 2 Bullous cyst stained for HMFG 2. The respiratory epithelium on the inner surface is positive. Mesothelial cells do not stain. PAP x 250. 1218 Marshall, Herbert, Braye, Jones I 4 9/? V5 ,; A*Wa. 3p.. s -I.' ).'6 * o .E- 4 .;. } M Fig. 3 Mesothelioma stained for HMFG 1. The solid clump ofcells (top centre) stains weakly. Staining is denser where lumina are formed. PAP x 300. !F^" o.., e ..:-:. ..: .S.: QR .K h.. *3 .0mr Fig. 4 Spindle cell element ofa biphasic mesothelioma stained for HMFG 1. PAP x 400. Use ofantibodies to carcinoembryonic antigen and human milk fat globule 1219 II ,4444-'-t 4444tarw.,f.. 444 'I ; , / W 'J, i t. 4? 1. g ..,sEXs j #t >>8A } %X* tAd ~~~~~~~~~~~~~~~~~~~~~~~t IlR. IV' ?- .4. O-t .q , I -. .. X; w* .s. s v Sst 0 S ... s .sey6: f.h .' v / * lC o *5 v wiPt. t{. .: Fig. 5(a) Squamous carcinoma stained for HMFG z t 4 4s <t e 2. There is dense staining ofthe cell surface in better 4 _S. ) differentiated areas. PAP x 400. (b) Oat cell 8<e e v *%'?-o :.t 2: tW carcinoma stained for HMFG 2. PAP x 400. (c) g Y. 2 *^ ';B: § § * .@ * Poorly differentiated adenocarcinoma stained for ;: e v J 11* s .:5 $ se w a HMFG 1. PAP x 400. *_..eE < vY sSP '8 0.,;. ^4.S ^ Je * Y w * fiiF :e v *^ .Xk§ 9i+ '_/x ..i r s + 'S5s e i $ MESOTHELIOMAS*B!.t. of 27 were positive with HMFG 1. (Table 3). All the 2 'st squamous carcinomas showed focal staining with HMFG I and Twelve of the 16 mesotheliomas were positive with both antibodies. There were individual differences HMFG 1 and 2 (Table 3). Staining was present in in the staining seen with each antibody but no con- the cytoplasm of some cases but was more com- stant pattern emerged. Staining was cytoplasmic monly present at the cell surface. Solid clumps of with surface accentuation in more differentiated epithelial cells did not stain, but where clefts formed areas (Fig. 5a). Four oat cell carcinomas stained within the tumour, the luminal surfaces of the cells strongly with HMFG 1 and 2 (Fig. 5b). became strongly positive (Fig. 3). Three of the seven Two did not stain with HMFG 1 and two others biphasic tumours showed strong, granular, cyto- stained only weakly; these four carcinomas also plasmic staining of the spindle cell element (Fig.
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