lmmunocytology in Malignant Pleural Mesothelioma

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1 lmmunocytology in Malignant Pleural Mesothelioma Expression of Tumor Markers and Distribution of Lymphocyte Subsets ]osune Guzman, M.D.;* Klaus]. Bross, M.D.;t Gebhard Wurtemberger, M.D.;t. and Ulrich Costabel, M.D., F.C.C.P. We studied the reactivity of malignant mesothelioma cells with tumor marlters and the phenotypes of lymphocyte subsets in pleural effusions from 14 patients with malignant mesothelioma. For identification of cell surface antigens with monoclonflj antibodies, the adhesive slide assay was used. The reaction pfltlem of mesothelioma cells was found to be CEA neative, Leu M1 negative, EMA positive, BMA- 120 positive, My 4 positive, and BA-2 positive. The surface morphology of mesothelioma cells may be of additional help for diagnosis. By these markers, the distinction between mesotheliomas and car~119mas is facilitated. The differentiation of reactive benign mesothelial hyperplasia from malignant mesothelioma by surface marker staining is not yet possible, however. In many effusions in this study, a concomitant T-lymphocytosis was observed with a nonspecific increase in the CD4/CD8 ratio, as known for other pleural diseases. (Che&t 1989; 95:590-95) CEA = carcinoembryonic antigen; EMA =epithelial membrane antigen; MAbs =monoclonal antibodies; PAP= peroxidase-antiperoxidase; NK =natural killer he cytologic diagnosis of malignant mesothelioma T in serous effusions continues to present difficulties of practical importance. The conventional cytology is often limited by the problems encountered in distinguishing malignant mesothelioma from metastatic adenocarcinoma or from reactive benign mesothelial hyperplasia. Since monoclonal antibodies (MAbs) for the identification of antigens on tumor cells have become available, several reports on the reactivity of mesothelioma cells with tumor markers have been published with different results. Most authors agree that mesothelioma cells are negative for carcinoembryonic antigen (CEA), but show a positive reaction for epithelial membrane antigen (EMA). 1 5 We studied the reaction pattern of malignant mesothelioma cells in pleural effusions with monoclonal antibodies against cell surface antigens using a special method for cell-preparation that allows a clear demonstration of the cell surface configuration. This may be of additional help for the recognition of mesothelioma cells, which in most cases contain profuse, longbranching microvilli, as previously described only in electron microscopic studies.~ The second aim of this study was to determine the phenotypes of pleural lymphocyte subsets in malignant pleural effusions due to mesothelioma, because recently interest has in- From the *Pathologisches lnstitut; the t Abteilung Hiimatologie/ Onkologie; the tabteilung Pneumologie, Universitatsklinik Freiburg i.br.; and the Abteilung Pneumologie/AIIergologie, Ruhrlandklinik, Essen-Heidhausen, West Germany. Manuscript received February 24; revision accepted July 25. Reprint requests: Dr. Guzman, lbthologisches lnstitut der Universitat, Albertstr 19, D-7800 Freiburg, ~st Gennany 590 creased in the local distribution of immune effector cells isolated from tumors or malignant effusions. 9 Lymphocyte subsets in pleural effusions from malignant mesothelioma have previously been described only sporadically. to MATERIAL AND METHODS \\e studied 14 patients with pleural effusions due to malignant mesothelioma of the pleural cavity. Our population under study consisted of 12 men and two women: mean age, 65::!: 10 years; range, 50 to 81 years. The tumors had been classified at the pleuropneumonectomy specimen obtained by surgery. They all were PAS negative. The histology of 12 cases classified as epithelial mesothelioma showed numerous mesothelium-lined clefts and more solid "epithelial" nests of cells. Two cases (nos. 13 and 14) showing a sarcomatous and a solid "epithelial" component were classified as biphasic mesothelioma. The pleural effusions for this study were obtained prior to surgical procedure or any other therapy. Exposure to asbestos was known in only six cases. Cells from 10 ml of pleural effusion stored in EDTA-coated tubes were obtained for immunocytochemical studies. For this purpose the adhesive slide assay was used Mononuclear cells were separated by Ficoll-Hypaque centrifugation." To facilitate the identification of macrophages, an incubation with a solution containing latex beads (1 ml minimum essential medium, 50 ~~ol bovine serum albumin [BSA] 22 percent, 5 ILl latex stock solution/sigma No. LB-8) for 8 min at 37"C was performed. For immunocytochemistry the cells were added to each of the reaction areas of adhesive glass slides (Superior, Bad Mergentheim) and fixed with glutaraldehyde (0.05 percent in 0.1 M phosphate-buffered saline solution, ph 7.8) for 5 min to block Fc-receptors and preserve cell morphology. The adhesive glass slides contain 12 cell-adhesive reaction areas separated by a hydrophobic coating of the remaining area of the slide surface. By this principle, 12 immunoreactions can be performed in parallel on the same glass slide with low amounts of antibodies and cells. 13 Jmmunocytology in Malignant Pleural Mesothelioma (Guzman et a/)

2 co CD3 CD4 CD8 CD14 CD20 CD25 CD38 HLA-DR CDIS Name CEA3-13 EMA BMA-120 OKT3 OKT4 OKT8 My4 Bl anti-tac OKTIO Ia BA-2 Leu M 1 Leu7 OKT9 HLA-1 HLE-1 Table 1-Monoclonol Anitbodiu Uaed in thia Study Supplier Immunbiologie, Freiburg (W. Ger.) Dakopatts Behring Or tho Coulter Coulter Becton-Dickinson Coulter Hybritech Becton-Dickinson Becton-Dickinson Hybritech Hybritech Target Carcinoembryonic antigen 1:50 Dilution Epithelial membrane antigen 1:100 Endothelial cells 1:20 T-lymphocytes 1:100 Helper/inducer T cells 1:50 Suppressor/cytotoxic T cells 1:50 Monocytes, macrophages 1:100 Pan-B-lymphocytes 1:100 Interleukin-2-receptor 1:100 Premature T cells, activated T cells 1:50 B-lymphocytes, activated T-lymphocytes, 1:50 monocytes Cells in early stage of lymphoid 1:50 differentiation 1: Granulocytes (monocytes) 1:100 Natural killer cells 1:100 Transferrin-receptor, proliferating cells 1:100 Human leukocyte antigen 1:100 Human leukocyte antigen 1:100 Cluster designation. The immunostaining was performed according the peroxidaseantiperoxidase (PAP) technique with diaminobenzidine (0.06 percent solution) as substrate followed by postfixation with OsO, 2 percent. The monoclonal antibodies used in this study are listed in Table 1. To evaluate the reaction, the slides were viewed by a light microscope with a magnification 400-fold. Positive-reacting cells were clearly recognized by the dark brown color of the cell membrane (positive cell surface reaction). Two hundred tumor cells or lymphocytes were counted in each reaction area, and the positivereacting cells were calculated as percentage of total tumor cells or lymphocytes, respectively. A differential cell count of mononuclear cells to determine the percentage of tumor cells, macrophages, and lymphocytes was also performed on 200 cells in each case after Ficoll-Hypaque centrifugation. The mean values± SD were calculated for all parameters. RESULTS In 12 of 14 cases, the proportions of tumor cells in the effusions ranged from 2 to 71 percent. In two cases, no malignant cells were present, but an elevated percentage of lymphocytes was observed (95 percent and 96 percent, respectively). The reactivity of the mesothelioma cells with the study markers is shown in Table 2. In all cases EMApositive tumor cells were found, with a mean proportion of 47 ± 33 percent positive-reacting tumor cells (Fig 1). BMA-120-positive tumor cells were aiso present in all cases, with a mean proportion of 80 ± 12 percent of tumor cells being positive (Fig 2). CEA 3-13 positivity of tumor cells was never observed. In addition, the reactivity of the tumor cells with the granulocyte marker Leu M1 was consistently negative. Regarding the monocyte/macrophage marker My4, however, some mesothelioma cells interestingly re- Table 2-Clinical Data of the Study Group with Malignant Meaothelioma and Reactivity of Meaothelioma Cella in the Pleural Fluid* Patient No. Age, yr/sex Cell type CEA3-13 EMA BMA-120 My4 LeuM1 HLA-DR BA-2 OKT9 1 69/M Epithelial /M Epithelial /M Epithelial /F Epithelial IM Epithelial ND /M Epithelial NO /F Epithelial NO /M Epithelial /M Epithelial /M Epithelial /M Epithelial ND /M Epithelial NO /M Biphasic No mesothelioma cells present in the pleural fluid 14 46/M Biphasic No mesothelioma cells present in the pleural fluid Mean±SD 0 47±33 80±12 14± ±17 67±26 30±22 *Monoclonal antibody-positive mesothelioma cells are expressed as percentage of total mesothelioma cells. NO, not determined. CHEST I 95 I 3 I MARCH,

3 which is higher than the normal value of 1.8±0.7 for the peripheral blood ratio in our laboratory. 12 The analysis of activation markers on T cells with the monoclonal antibodies CD 25, CD 38, and anti-hla- DR showed that in all cases, only a few T cells from the pleural fluid expressed these markers (Table 3). The monoclonal antibody Leu 7 directed against natural killer (NK) cells was studied in only five cases and reacted positive with 7 ± 6 percent oflymphocytes (range, one to 16). DISCUSSION This study investigated the reactivity of mesothelioma cells with a panel of MAbs against cell membrane antigens. Our preparation technique making use of the adhesive glass slide assay offers several advantages. It permits the preservation of excellent cell morphology and of the integrity of the cell membrane structure. The cells do not lose their three-dimensional shape during the fixation and staining procedure. The intact structure of the cell membrane allows isolated membrane staining of the cell surface. Because the antibodies cannot penetrate into the cytoplasma, in- FlGVRE 1A( upper). lmmunostaining of mesothelioma cells from the pleural fluid with EMA. The cell surface is partially or completely positive with a hairy reaction pattern (large arrows). Some tumor cells (small arrows), macrophages (m), and lymphocytes (I) are negative. lmmunoperoxidase adhesive slide assay (original magnification x400). FIGVRE 1B (lower). Two groups of mesothelioma cells at higher magnification. Partially positive staining of cell surface with EMA. One tumor cell additionally shows typical roundish intracytoplasmic organella (original magnification x 1,000). acted positive in 11 of 12 cases (mean value 14 ± 15 percent of tumor cells) (Fig 3). A certain percentage of mesothelioma cells also reacted with HLA-DR (mean value 25 ± 17 percent of tumor cells) and with BA-2 (mean value 67 ± 26 percent of tumor cells) in each of the cases studied with these markers. In all cases OKT9 positivity was observed on 30 ± 22 percent of tumor cells. This marker may serve as an index of transferrin-receptor expression, which is connected to cell growth and proliferation. No reactivity of mesothelioma cells with the MAb HLE-1 was found. In all cases mesothelioma cells were HLA-1 positive. The analysis of pleural lymphocyte subpopulations (Table 3) revealed that in all cases the majority of the lymphocytes present were CD3-positive T cells (83 ± 5 percent). The phenotypical characterization of the T-lymphocyte subpopulations demonstrated that 67 ± 9 percent of total lymphocytes were CD4-positive helper/inducer and 20 ± 6 percent were CD8-positive suppressor/cytotoxic T cells. The ratio of helper/inducer to suppressor/cytotoxic T cells was 3. 7 ± 1.4, FlGt:RE 2A (upper). lmmunostaining of mesothelioma cells from pleural fluid with the MAb BMA-120. lmmunoperoxidase adhesive slide assay (original magnification x 400). FlGVRE 2B (lower). Negative control reaction without the first antibody. Mesothelioma cells (arrow), macrophages (m), lymphocytes (l), erythrocytes (e), and one granulocyte (g). No positive-reacting cells present (original magnification X 400). 592 Jmmunocytology in Malignant Pleural Mesothelioma (Guzman at at)

4 F1Gl'RE 3A (upper). lmmunostaining of cells from the pleural fluid with the monocyte/macrophage marker My4. Positive reacting tumor cells I large arrotes ). negative tumor cells I small arrou:s). positive-reacting macrophages (m). negative lymphocytes (l). lm munoperoxidase adhesive slide assay (original magnification X 400). F1Gl'RE 3B llou erj. ~ly4 positive mesothelioma cells at higher magnification. :\ote microvilli on cell surface of two intensively positive tumor cells. Another mesothelioma cell is only weakly and partially positive. ~lacrophages are also positive (m) (original magnification x!,000). tracytoplasmic staining is not observed. This permits the recognition of the typical surface configuration of mesothelioma cells with their long and profuse microvilli in our immunocytochemical preparations, as previously achieved only by electron microscopy. 6 ~ This surface configuration is important for the distinction of mesothelioma cells from adenocarcinoma cells. Regarding the reactivity of mesothelioma cells with MAbs, we never observed an immunostaining with CEA. This is in agreement with most reports from the literature but is in contrast to the weak CEA positivity observed in early studies, 1 s- 18 which were mostly performed on tissue sections and with unabsorbed or polyclonal antibodies to CEA. Thus, nonspecific CEA staining may have occurred in the latter studies. By our opinion, mesothelioma can be excluded when CEA-positive tumor cells are present. The EMA has received a great attention in the literature.u 9 We found strong positivity of mesothelioma cells in all our cases. This is consistent with the results of Cibas et ali but contrary to those of Mason and Bed ross ian. 15 Carcinoma cells (Table 4) and a few activated mesothelioma cells also reacted with this MAb. Our study also demonstrated a strongly positive reaction of mesothelial cells with BMA-120, previously not described for cells from pleural effusions. This finding is not specific, however, for cells of malignant mesothelial origin, because ovarian carcinoma cells in ascites and normal mesothelial cells have also been found to react positive for BMA-120, as detected in our ongoing studies on tumor markers in effusions 21 > (Table 4). As in prior reports, in our study mesothelioma cells did not react with Leu M Since all carcinomas studied in our laboratory did react with Leu M 1 (Table Table 3-Percentage of Tumor Cells, Macrophages, and Immune Effector Cells in the Pleural Fluid of Malignant Mesothelioma* Patient :\o. Tumor Cells ~lacrophages Lymphocytes CD3 CD4 CDS CD4/CD8 CD20 CD25 CD38 HLA-DRt ND ND ND ND ' ND Mean±SD 19±21 34±22 48±30 83±5 67±9 20±6 3.73± ±4 5±2 4±2 16±4 *Differential cell counts are expressed as percentage of total mononucleated cells. Monoclonal antibody positive lymphocytes are expressed as percentage of total lymphocytes. ND, not determined. tonly percentage of positive reacting lymphocytes are shown. CHEST I 95 I 3 I MARCH,

5 Table 4-Comparison of the Immunoreactivity of Malignant Mesothelioma, Carcinomas, and Lymphomas in Pleural Effusions and Ascitic Fluid Studied in our Laboratory* Breast Adenocarcinoma, Small Cell Carcinoma Adenocarcinoma, Mesothelioma Cancer Lung Lung Ovaryt Lymphoma (n= 12) (n=30) (n= 10) (n= 13) (n = 17) (n=9) CEA3-13 0/12 21/30 10/10 6/13 5/ EMA 12/12 30/30 10/10 11/13 17/ BMA /12 10(?)/30~ 3(?)/10~ 1(?)/13~ My4 ll/12 0/30 0/10 0/13 0/ Leu M1 0/12 30/30 10/10 ll/11 17/ HLA-DR 12/12 30/30 10/10 ll/11 17/ BA-2 7n 30/ / OKT9 12/12 20/30 8/10 8/13 12/ HLA-1 12/12 14/30 7/10 4/ HLE-1 0/12 0/30 0/10 0/ /9 *The results are sho\\n as number of positive-reacting cases in each group. t Ascitic fluid. ~Weak reaction. Hodgkin cells positive for leu Ml. 4, Fig 4), this MAb may also be of value for the of total cells after Ficoll-Hypaque centrifugation. distinction between mesothelioma and carcinoma. Regarding the distribution of lymphocyte subsets as Activated mesothelial cells are also negative for Leu studied by their reactivity with a panel of MAbs, there Ml. was a distinct increase in the CD4/CD8 ratio, to a In 11 of 12 cases in this study, the mesothelioma mean value of3. 7 ± 1.4, in comparison with the normal cells reacted positive with the marker My 4, whereas value for the peripheral blood of 1.8 ± 0. 7 in our carcinoma cells studied in our laboratory so far never Iaboratory. 14 Elevated CD4/CD8 ratios in pleural effusions are not limited to patients with mesothelioma, reacted with this marker (Table 4). The My 4 and BA- 2 positivity of mesothelioma cells we observed in our however; they have also been observed in other pleural study has not been previously described to our knowledge. Few activated mesothelial cells were also posiing radiotherapy for malignancies, and effusions owing diseases such as tuberculous pleurisy, pleurisy followtive for these MAbs. to congestive heart failure.z.s Contrary to some carcinomas (Table 4), mesothelioma cells do not lose the reactivity with the MAb was found to be CEA negative, Leu M1 negative, The reaction pattern of mesothelioma in this study HLA-1. On the other hand, HLE-1 may be a useful EMA positive, BMA-120 positive, My 4 positive, BAmarker for the identification of lymphomas, since 2 positive, HLA-1 positive, HLE-1 negative, OKT-9 carcinomas and malignant mesothelioma were always positive, Ia positive. Useful markers for the distinction HLE-1 negative, contrary to lymphomas, which were between mesothelioma and carcinomas are CEA, Leu always positive, except for one case with plasmacytoma. staining for CEA, the surface morphology of mesothe- M1, My 4, and BMA-120. In cases with negative This study revealed that most mesotheliomas are lioma cells may allow the differential diagnosis (Fig characterized by a striking concomitant lymphocytosis 1B and 4). The problem is that at present no MAbs in pleural effusions, reaching values of up to 96 percent can conclusively distinguish reactive benign mesothelial hyperplasia from malignant mesothelioma. This differentiation still depends on morphologic criteria of cell number, size, and clustering. Specific markers reacting exclusively with malignant mesothelioma cells and not with reactive mesothelial cells will therefore be required in the future to facilitate the cytologic diagnosis of malignant mesothelioma. FIGt:RE 4. Immunostaining of tumor cells from adenocarcinoma of the lung in the pleural fluid with the granulocyte marker Leu MI. One tumor cell (arrow) and one granulocyte (g) are positive. The macrophages ( ma) are negative. One erythrocyte (e). Original magnification X 1,000. REFERENCES 1 Cibas ES, Corson JM, Pinkus GS. The distinction of adenocarcinoma from malignant mesothelioma in cell blocks of effusions. Hum Pathol 1987; 18: Ghosh AK, Gatter KC, Dunnill MS, Mason DY. Immunohistological staining of reactive mesothelium, mesothelioma, and lung carcinoma with a panel of monoclonal antibodies. J Clin Pathol 1987; 40: Wang NS, Huang SN, Gold P. Absence of carcinoembryonic 594 lmmunocytology in Malignant Pleural Mesothelioma (Guzman eta/)

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