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0 1990 The Japanese Society of

Pathology

Expression of Vimentin and Epithelial Membrane Antigen in Human Malignant Lymphomas

Ashit Baran Sarker, Tadaatsu Akagi, Tadashi Yoshino, Yoshihiko Hoshida, Kiyoshi Takahashi, and Yasushi Horie

lmmunoreactivity with monoclonal antibodies against the intermediate filament protein, vimentin, and epithelial membrane antigen (EMA) was examined i n 330 cases of lymphoma (317 non-Hodgkin’s and 13 Hodgkin’s lymphomas), 12 reactive lymph nodes and mononuclear cells of the peripheral blood using either indirect immunoperoxidase staining or the avidin-biotin immunoperoxidase complex technique. The cell origin of each tumor was established using a panel of monoclonal antibodies against lymphocyte differentiation antigens. There were 41 T-cell, 247 B-cell and 29 undetermined lymphomas, and 13 cases of Hodgkin’s disease in the series. Vimentin was expressed in 24 T-cell lymphomas (58.5%) and 60 B-cell lymphomas (24.2%). This difference i n frequency was statistically significant. Vimentin expression i n follicular lymphomas was less frequent than i n diffuse B-cell lymphomas. In diffuse lymphomas, small and medium cell types were more reactive with anti-vimentin than large cell types. Reed-Sternberg cells (R-S cells) in Hodgkin’s disease were positive for vimentin i n 11 cases (84.6%). The frequency of EMA reactivity in lymphomas was low, particularly in T-cell lymphomas. No positive cases were found among follicular lymphomas. I n diffuse non-Hodgkin’s lymphomas, EMA was expressed only i n mixed and large cell types, but never i n smaller ones. In conclusion, monoclonal antibodies against vimentin and EMA appear to be of limited usefulness for the diagnosis of non-Hodgkin’s lymphomas, but anti-vimentin antibody may be used as an adjunct to the diagnosis of R-S cells i n Hodgkin’s disease. Acta Pathol Jpn 4 0 : 581-587, 1990. Key words : Vimentin, Epithelial membrane antigen, Lymphoma, lmmunoperoxidase staining

Received November 29, 1989. Accepted for publication March 26, 1990. Second Department of Pathology, Okayarna University Medical School, Okayama. Second Mailing address: Tadaatsu Akagi Department of Pathology, Okayama University Medical School, Shikata-cho 2-5-1, Okayama 700, Japan.

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INTRODUCTION Vimentin is considered to be a major intermediate filament protein in the cytoplasm of mesenchymal derivatives(1). Abundant vimentin mRNA has also been detected in normal human lymphocytes (2). Immunocytological studies have shown that all mononuclear cells of human peripheral blood express vimentin (3). However, there is still controversy over vimentin expression among lymphoid cells in normal human lymphoid tissues. For example, Giorno (4) reported that very few lymphocytes were immunoreactive for vimentin. Successive studies on human lymphomas have shown that lymphoma cells also retain vimentin, although the extent of immunoreactivity is variable (5-8). Recently, Molter et a/. (9) reported that vimentin expression in a Bcell lineage varied according to the stage of differentiation or maturation. Epithelial membrane antigen (EMA) is a component on the surface membranes of many epithelial cells, and is detected by an antiserum raised against human milk fat globule membranes (10, 11). Anti-EMA antibodies have been used on paraffin-embedded tissue sections employing immunoperoxidase techniques in order to detect metastatic foci of breast cancers in lymph nodes (12, 13) and bone marrow (14), and to differentiate a naplast ic epithelia I tumors from maligna nt lymphomas (15). An early study revealed that normal and neoplastic lymphoid cells were devoid of EMA (1l), but subsequently it was shown by Delsol et a/. (16) that EMA expression is not restricted to epithelial cells, but also appears on normal and neoplastic plasma cells, some non-Hodgkin’s lymphoma cells, and Reed-Sternberg (RS) cells in Hodgkin’s disease. In view of the above discrepancies in findings by different authors, our present study was designed to evaluate vimentin and EMA expression in lymphomas of different cell origin and to assess the value of these

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Vimentin and EMA in Lymphoma (Sarker et a/.)

markers in the diagnosis of lymphomas by comparing their reactivity with that in normal lymphoid tissues and peripheral blood mononuclear cells using immunohistochemical methods.

MATERIALS AND METHODS

H,O,. Intrinsic positive controls for vimentin were fibroblasts, dendritic cells, endothelial cells and macrophages, and those for EMA were plasma cells. Their staining characteristics indicated the reliability of the reaction, and therefore false-negative results were discarded.

Tissue samples

Statistical analysis

A total of 330 cases of lymphoma (317 nonHodgkin's lymphomas and 13 cases of Hodgkin's disease) were examined immunohistochemically. Of the 330 lymphomas, 1 6 1 were nodal and 169 were extranodal. Twelve samples of reactive lymph nodes and normal peripheral blood mononuclear cells were also included in this study. All of these tissue samples were fixed in 10% formalin and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (HE), and examined immuno histochemica Ily. Periphera I blood mononuclear cells were isolated using density gradient centrifugation on Ficoll-Hypaque. Lymphomas were classified according to the criteria of the Japanese Lymphoma Study Group (17). lmmunophenotyping of the lymphomas was carried out with a panel of antibodies against lymphoid cell types.

To verify the significance of differences in expression of vimentin and EMA between T- and B-cell lymphomas, and also between follicular and diffuse lymphomas, respectively, we applied chi-squared test for statistical analysis, the confidence levels being determined using a chi-squared table.

Antibodies and other reagents The antibodies used in this study were monoclonal antibodies (MoAbs) against vimentin and EMA, Pan-B, MB-1, MT-1, UCHL-1, and the anti-kappa and lambda light chains of immunoglobulins. Anti-vimentin, antiEMA and UCHL-1 were purchased from Dakopatts (Copenhagen, Denmark), M B - l and MT-1 were from Bio Science Products AG (Emmenbrucke, Switzerland), anti-kappa and anti-lambda were from Tago, Inc. (California, USA), and Pan-B was from Kyowa Medex Go. (Tokyo, Japan). The antibodies were diluted with phosphate buffered saline supplemented with bovine serum albumin and NaN, to a titer which gave specific staining with minimum nonspecific background. Biotinylated goat anti-mouse IgG and peroxidase-conjugated streptavidin were purchased from Bio Genex Laboratories (California, USA), and diaminobenzidine tetrachloride was from Dojin (Tokyo, Japan).

RESULTS Immunoreactivity of reactive l y m p h nodes and peripheral blood mononuclear cells In every specimen of reactive lymph node examined, follicular center B cells showed no immunoreactivity for vimentin, but many lymphocytes in the mantle zone and paracortical areas did show vimentin expression (Fig. 1). Macrophages, fibroblasts, dendritic and interdigitating reticulum cells, and endothelial cells were also positive for vimentin. On the other hand, only plasma cells were immunoreactive for EMA. All normal peripheral blood mononuclear cells were immunoreactive for vimentin, whereas in contrast, they did not contain any detectable EMA.

Staining procedures Sections were stained by either an indirect two-stage immunoperoxidase procedure or an avidin-biotin-peroxidase complex (ABC) technique (18). lmmunostaining of paraffin sections was preceded by bleaching and destruction of endogenous peroxidase using methanol/

Figure 1. Reactive lymph node. Note many vimentin-positive lymphocytes in the mantle zone and paracortical area. Endothelial cells and tingible-body macrophages in the germinal center are positive for vimentin, but follicular center cells are negative. lmmunostaining for vimentin.

Acta Pathologica Japonica 40 (8): 1990 Table 1. lmmunoreactivity of Vimentin and EMA in Nodal and Extranodal Lymphomas ~

T-cell lymphoma B-cell lymphoma Total FL. medium FL. mixed FL. large DL. small DL. intermediate DL. medium DL. mixed DL. large DL. pleomorphic DL. lymphoblastic Plasmacytoma Hodgkin's disease Total

Vimentin 24/41 (58.5) 601247 (24.2) 84/288 (29.1) 2/16 (12'5) 1/9 (11.1) 0/1 ( 0.0) 3/6 (50.0) 2/7 (28.6) 20/54 (37.0) 36/83 (43.3) 24/132 (18.1) 2/3 (66.6) 1/3 (33.3) 1/3 (33,3) 11/13 (84.6) 103/330 (31.2)

Figures in brackets indicate percentages. EMA : Epithelial membrane antigen.

EMA 1/41 ( 2.4) 121247 ( 4.8) 13/288 ( 4.5) ( O'O) 0/9 ( 0.0) 0/1 ( 0.0) 0/6 ( 0.0) '/' ( O.') 0/54 ( 0.0) 4/83 ( 4.8) 9/132 ( 6.8)

0/3 1/3 1/13 15/330

( ) '.O ( 0.0)

(33.3) ( 7.7) ( 4.5)

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Immunoreactivity of lymphomas (Table 1) Lymphomas expressing monotypically either of the light-chain immunoglobulins were classified as B cell in origin irrespective of expression of T-cell antigens, since B-cell lymphomas often show aberrant expression of Tcell antigens. In cases where the result of immunoglobulin staining was inconclusive, those expressing only B-cell markers, MB-1 and Pan-B, were classified as B cell. Lymphomas were classified as T cell in origin if the cells expressed no B-cell markers and expressed one or more pan T-cell markers, MT-1 and UCHL-1. Those expressing both B- and T-cell antigens or neither of them were classified as undetermined if the immunophenotype could not be definitely predicted on the basis of morphologica I features. Of the 330 lymphomas including 13 cases of Hodgkin's disease, 41 were considered phenotypically to be of T-cell origin, 2 4 7 of B-cell origin, and 29 were

Figure 2. Follicular lymphoma of the lymph node, medium cell type (B-cell origin). a : HE. b : lmmunostaining for vimentin.

Figure 3. Diffuse lymphoma of the lymph node, large cell type ( 6 immunoblastic). a : HE. b: lmmunostaining for vimentin.

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Vimentin and EMA in Lymphoma (Sarker et a/.)

Figure4. Diffuse lymphoma of the lymph node, pleomorphic type (T-cell origin). a : HE. b : lmmunostaining for vimentin.

Figure 5. Diffuse lymphoma of the skin, mixed cell type (T-cell Figure 6. Hodgkin’s disease of the lymph node, mixed cellularorigin). lmmunostaining for vimentin. ity. Arrowhead indicates a vimentin-positive Reed-Sternberg cell. lmmunostaining for vimentin.

Figure 7. Diffuse lymphoma of the stomach, mixed cell type (B-cell origin). a : HE. b : lmmunostaining for vimentin.

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Acta Pathologica Japonica 40 (8) : 1990

Figure 8. Plasmacytoma of the stomach. a : Typical plasrnacytoid cells with Dutcher bodies (arrowheads) show diffuse in filtration. HE. b : lmrnunostaining for EMA.

phenotypically undetermined. Of the 41 T-cell lymphomas, 2 4 were vimentin-positive (58.5%), and of the 247 B-cell lymphomas, 60 were vimentin -positive (24.2%). This discrepancy in proportions was statistically significant by chi-square test (P

Expression of vimentin and epithelial membrane antigen in human malignant lymphomas.

Immunoreactivity with monoclonal antibodies against the intermediate filament protein, vimentin, and epithelial membrane antigen (EMA) was examined in...
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