Lymphomas: M em brane Markers and Cell Flow Cytometric Diagnosis

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1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 17, No. 1 Copyright 1987, Institute for Clinical Science, Inc. Lymphomas: M em brane Markers and Cell Flow Cytometric Diagnosis ARMAND B. GLASSMAN, M.D., SALLY SELF, M.D., and JOYCE CH RISTO PH ER CLA(ASCP)-MT(HEW) Departments of Laboratory Medicine, Basic and Clinical Immunology, and Microbiology Medical University of South Carolina Charleston, SC ABSTRACT Lymphocytes are characterized by m em brane markers which, in part, reflect biological and functional activity. This is particularly true for T lym phocyte subsets identified by m onoclonal antibodies. The B lym phocytes can be identified b u t in a m ore general manner. It has been pro posed that the use of these markers will aid in the differential diagnosis of a variety of lymphomas. The objective of this work was to evaluate the use of m onoclonal antibodies (MAb-s) and surface im m unoglobulin (slg) analysis in a cell flow cytom eter (CFC) as m ethods to identify and classify lymphomas. The cell flow cytometric findings w ere then evaluated in light of th e histopathologic diagnosis (HPD). Fifty-eight (58) patients w ith a variety of lymphomas and benign lymph node disorders were studied. Lymph node tissue samples w ere obtained after surgical rem oval and appropriately prepared for evaluation in a CFC. Results showed that the variety of hyperplasias and reactive follicular lym phadenopathies could not be characterized by the technique or application of CFC alone. Both B cell and T cell lymphomas could be recognized and differentiated by MAb-s and/or light chain monotypism using slg s in a CFC, but morphologic and clinical information were required for diagnostic confirmation. Hodgkin s disease could not be identified by C F C because of the lack of a specific identifiable marker. Cell flow cytom etry provides an easy and rapid adjunct to the diagnosis of a variety of lymphomas. At the present time, m em brane markers in cell suspensions from tissues (lymph nodes) identified by MAb-s and slg s in a C F C cannot be used to provide definitive diagnoses for reactive lym phadenopathies, H odgkin s disease or som e classes of lymphomas. Introduction 1 Lym phocytes are a m orphologically hom ogeneous group of cells with a high nuclear cytoplasmic ratio. They serve as the basic framework for the immune system and are divided into groups on the basis of biological function. Som e are able to differentiate into cells which pro duce antibodies (B-lym phocytes) and others are responsible for cell m ediated im m unity responses including: m ain /87/ $01.20 Institute for Clinical Science, Inc.

2 2 GLASSMAN, SELF, AND CHRISTOPHER tenance of tolerance, delayed h y persensitivity reactions, cytotoxic reactions, suppression, induction and h e lp e r functions, etc. (T-lymphocytes). The developm ent of these functions relies upon a specific recognition of events. Surface receptors on the cell m em brane may relate to the activity that makes a particu la r g ro u p o f ly m p h o c y te s u n iq u e. A lth o u g h som e of th e s e m e m b ra n e m arkers may be shared by other cells (e.g., HLA antigens), others are identified by general characteristics, such as the ability of thym us-dependent (T) lymphocytes to bind sheep erythrocytes (Erosetting). O ther markers are specific for the functional differentiation of smaller groups of cells, such as m arkers for h elp er or suppressor lym phocytes.1 In the course of T and B cell differentiation, lym phocytes express special surface m arkers defining th eir derivation and/or functional stages. Prothymocytes o rig in a te in th e b o n e m a rro w and m ig ra te to th e th y m u s w h e re th e y d evelop know n ch aracteristics and a series of surface m em brane receptors which can be recognized by presently available monoclonal antibodies (table I). Adult T lymphocyte markers that are of particular interest clinically include the T inducer/helper and T cytotoxic/suppressor m arkers.7 The prim itive precursor of the B lymphocyte cell line (Pre-B cell) can be identified in the bone marow. This cell is characterized by the presence of cytoplasm ic im m unoglobulin (clg + ), b u t does not have surface im m unoglobulin (slg ). As B cells m ature, they develop su rfa c e im m u n o g lo b u lin s (slgm +, sigg +, slgd-t- and siga + ); the majority of circulating B cells express slgm + or both slgm + and slgd +. They function as surface rec e p to rs on the cell m em brane and recognize and respond to antigens. Each individual B cell bears e ith e r kappa ( k ) or lam b d a (X.) lig h t chains, but not both. In a clonal prolifer- O K T 3 O K T 4 O K T 8 M A b - s * O K T 11 O K T 6 O K T 10 TABLE I Monoclonal Antibodies (MAB-s) S p e c i f i c i t y P a n T o r p e r i p h e r a l T cells I n d u c e r / h e l p e r T cells C y t o t o x i c / s u p p r e s s o r T cells E - r o s e t t e r e c e p t o r "Common" thymocytes P r e c u r s o r cells, a c t i v a t e d lymphs, n u l l cells O K M 1 Monocytes, null cells and g r a n u l o c y t e s O K B 2 B cells a n d g r a n u l o c y t e s 0 K B 7 B cells a n d p r e c u r s o r s Leu-M3 M o n o c y t e s, macrophages OKIal, HLA-DR(Ia) C A L L A L eukocyte L e u - 12 T r a n s f e r r i n B cells, m o n o c y t e s, m a c r o phages, a c t i v a t e d T cells C o m m o n a c u t e l y m p h o b l a s t i c l e u k e m i a a n t i g e n A l l leukocytes P a n B o r p e r i p h e r a l B c e l l s T r a n s f e r r i n receptor, a c t i v a t e d a n d / o r p r o l i f e r a t i n g cells, n e o p l a s t i c cells *The O K d e s i g n a t e s T, M, B, a n d lai are O r t h o - m u n e m o n o c l o n a l a n t i b o d i e s a n d are c o m m e r c i a l l y availab l e t h r o u g h O r t h o P h a r m a c e u t i c a l Corporation; the L e u series, H L A - D R ( I a ), CALLA, L e u k o c y t e a n d T r a n s f e r r i n are B e c t o n D i c k i n s o n ' s C o m p a n y MAB-s a n d can be o b t a i n e d f r o m them. ation of B cells, such as a B cell lym phoma, all of the progeny cells will bear only one type of light chain, either kappa or lambda. Thus, light chain monotypism is a very useful m arker for B cell neoplasia. Pre-B and m ature B cells carry receptors for the Fc portion of Igs and for C3b and C4b. These receptors, however, are not specific for the B cell line and can be found on other cells.2 Lymphoma is a general term applied to malignant disorders of the lymphoid tissues. It is hypothesized that the norm al cell m atu ratio n process is in te r rupted at various stages of developm ent w ith the rem aining cells failing to com plete the differentiation process.5 A variety of diagnostic approaches have been used to help identify m alignant from benign proliferating lym phocytes. The clinical findings and histopathologic diagnosis have b e e n th e standard by which the accuracy of in vitro diagnostic techniques are assessed.10 Problem s in

3 LYMPHOMAS AND MARKERS/CELL FLOW CYTOMETRY 3 differential diagnosis of lymphomas are co m p lic a ted by th e d iv ersity of the pathological classifications or formulas that are cu rrently used, and in the subjective in te rp re ta tio n of the m orphology.6,8 Im m unofluorescence and immunoperoxidase m ethods have been used in fixed and frozen tissue sections for the identification of T and B lymphocytes. Monoclonal and monospecific antibodies have been used to identify circulating or separated lymphocytes using a variety of technologies including cell flow cytom e try (CFC), immunofluorescence, immunoenzym atic and solid-phase techniques. The purpose of this paper is to explore th e clin ical a p p licatio n of C F C and MAb-s as an aid for classification of lymphomas. A comparison is made betw een C FC and the m orphologic diagnosis of th e lym phocyte disorders. Methods A spectrum of monoclonal antibodies (MAb-s) w ere used in this study. OKT 3, 4, 6, 8, 10, 11, OKB 2, 7 and OKM1 w ere obtained* as w ere Leu-M3, HLA- DR(Ia), CALLA, Leukocyte, Leu-12 and Transferrin Receptor, t Fluorescein conjugated goat or burro F(ab')2 antisera to hum an kappa, lam bda, m u, delta and gamma chains w ere also obtained. $ Tissue sam ples w ere obtained im m e diately after surgical rem oval and transported in McCoy s modified medium to the laboratory. Sam ples w ere m inced using scissors, and the resulting suspension was w ashed several tim es in phosphate-buffered saline containing 0.2 p e r cent bovine serum album in and 0.01 percent sodium azide (PBS) at 600 X g for three m inutes. After the final wash, cells w ere resuspended in two ml of PBS * Ortho Pharmaceutical Corporation, Raritan, NJ. t B ecton D ickinson M onoclonal C enter Inc., Mountain View, CA. t Kallestad Laboratories Inc., Austin, TX. GIBCO, Grand Island, NY. and filtered through fine nylon m esh to rem ove clum ps. Viability was assessed by trypan blue exclusion. T he cell suspension was adjusted to a concentration of 1 X 107 cells per ml and then stained with the appropriate MAb-s (FITC-conjugated) for surface m arker identification or analyzed for surface im m unoglobulins. A cytocentrifuge preparation of the suspension and a histologic section of the node or tissue were examined to ensure that representative cells were obtained. Cell suspensions w ere analyzed using an O rtho Spectrum I II. 1 The C FC works through the use of laser optics and filters, and a series of electronic devices which converts light im pulses into signals. A com puter processes and displays the generated signals into data and appropriate graphs. The cells (one by one) are rapidly m easured as they pass through the sampling area causing a scattering of the light source. N u m e ro u s m e a s u r e m e n ts c a n b e obtained as the light source is scattered. Such m easu rem en ts include forw ard angle light scatter (FALS) which relates to cell size, and 90 light or right angle light scatter (90 LS), which relates to cell g ran u larity or in te rn al stru c tu re complexity. The am ount of fluorescence em itted at various wavelengths provides inform ation on the quantity of cellular constituents being analyzed. The cells are th en displayed in a frequency distribution or histogram and the population of interest is chosen or gated on the principle of FALS versus 90 LS. The com puter w ould then collect and correlate fluorescent data gated on FALS and 90 LS.3-4 Results Fifty-eight patients with a variety of lym phom as and lym ph node disorders w ere studied. The D epartm ent of Laboratory M edicine s Diagnostic Im m unol 1Ortho Diagnostic Systems, Inc., Westwood, MA.

4 4 CLASSMAN, SELF, AND CHRISTOPHER ogy D ivision has d e v e lo p e d its own u n iq u e re p o r t form (fig u re 1). Ten patients with lymphoid hyperplasia were examined by CFC. Analysis of normal nodes was virtually impossible because of th e ir sm all size and d ifficulty in obtaining this tissue. Therefore, a series of reactive nodes w ere examined to provide a standard against which neoplastic nodes could be com pared. In reactive nodes the kappa/lambda ratio of surface immunoglobulin was 1.2 to 2.6, the percent of T cells ranged from 30 to 80 percent, the OKT4/OKT8 ratio varied from 0.6 to 8.5 and the percentage of B cells ranged from 16 to 73 percent. Cell flow cytom eter was not helpful in recognizing different p attern s of hyperplasias, although in nodes with a prominent paracortical hyperplasia there was a tendency for an increased percent of T cells and increased four to eight ratio. In each instance of benign hyperplasia, the histologic diagnosis was required to d etermine the benign response. M aterial from 37 lymphomas was evaluated. For purpose of this discussion they w ere divided into the broad categories o f B cell lym phom a, T cell lym phom a, and H odgkin s disease. This characterization was done on the basis of com bined histopathologic and flow cytom etric data. Given the histopathological diagnosis of a diffuse lym phom a, CFC was definitive in assigning the lymphoma to the B or T cell category. The diagnosis of H odgkin s disease was m ade on m orphology alone. Follicular lym phom as were assigned to the B cell category and in ev ery in stan ce th e C F C data co r roborated the diagnosis. The results are sum m arized in table II. In th e B cell lymphom as, C FC revealed one of two diagnostic p attern s. E ith e r th ere was light chain monotypism among the cells bearing surface immunoglobulin or the preponderance of cells expressed neither slg nor T cell m arkers, but did express B m ark ers (Leu-12, OKB 2, OKB 7, or HLA-DR(Ia).) These patterns were very u s e fu l in so lv in g th e m o rp h o lo g ic dilem m a of follicular hyperplasia versus follicular lym phom a and inflam m atory lym phoid infiltrate versus extranodal lymphoma. In the case of T cell lymphomas, there is no im m unotypic clue of monoclonality, such as light chain monotypism to aid in the diagnosis of lymphom a. Given the histologic diagnosis of diffuse lymphoma, it is possible to establish the cell of origin, either B or T cell by CFC. Also, one is aided by th e p h enom en o n th a t in approxim ately 50 percent of the T cell lym phom as th e cells w ill express an abnormal phenotype. A normal m ature T cell will express the im m unophenotype of OKT 3 +, OKT 11 +, O K T 4 +, OKT 8 -, or OKT 3 +, OKT 11 +, OKT 4 -, OKT 8 +. In this series of lymphomas the following abnorm al phenotypes have been observed: [OKT 11 +, OKT 4 -, OKT 8 - ], [OKT 3 -, OKT 11 +, OKT 4 + ], [OKT 3 -, OKT 11 +, OKT 8 + ]. W ith the exception of an individual who genetically lacks the OKT 4 epitope, a p re p o n d e ra n c e of T cells w ith an abnormal phenotype has only been seen in T cell neoplasms. In those T cell lymphom as w hich express a norm al phenotype C FC is not particularly useful in d iffe re n tia tin g th e lym phom as from hyperplasias or Hodgkin s disease. One m ust rely on histomorphology or molecular biology (clonal T cell antigen-specific receptor gene rearrangem ent).9 The vast m ajority of cells in a node involved by H odgkin s disease are reactive lym phocytes; therefore, it is not surprising that th e C F C profile of a H odgkin s node is identical to that of a reactive node. T he R e e d -S te rb erg cell is not accessible to analysis by C F C in the usual case because they are present in such low num bers and they do not readily go into suspension. Therefore, the diagnosis of Hodgkin s disease is dependent on the morphology.

5 CELLULAR IMMUNOLOGY PRO FILE E - r o s e tte s Figure 1. Sample of report form developed by the Diagnostic Immunology Division of the Department of Laboratory Medicine. LYMPHOMAS AND MARKERS/CELL FLOW CYTOM ETRY oi DEPARTMENT OF LABORATORY MEDICINE DIVISION OF DIAGNOSTIC IMMJNCLOGY E PART«Wr ( CELLULAR IMMUNOLOGY PRO FILE DIFFERENTIAL Dx: «3E SEX IDENTIFICATION SAMPLE SOURCE SUrfar Tpimwrlofciulins Polyvalent HEMftgCTiOGY DATA DIFFERENTIAL t SURFACE MARKERS OKT 3 OKT 4 WBC/tnm3. _Lymph_, M eta Myelo Prcrryelo A typ. Lyirph Mono B la s t QEB...2,... t/ni? Idi TdP IgD IoA Lambda CYTOCTBrrgTyY OKT 6 Leu-12 OKT 8 OKT 9 CvtopLaam ic Im m unoglobulins Sudan Black T a rtra te R esistan t Acid Phos. Chi oroacptat-.e -Esterase Non-Soec. E sterase OKT 10 OKT 11 OKM 5 Acid Phoschatase Non-Stjeci f ic R sterase Leu-Ml w ith f lu o r id e in h i b itio n OKT4/t)KT8 Leu-M2 Leu-M3 u n rarae iw rt O fi tefci-w C ell PLT-1 L e u - l la MV 4 Mv 7 Mv 9 Anti-Leukocyte Anfci-fitea Cell H P tt-1 OKT 10 PCA-l Q Q B S IL -2 CKDr

6 6 G LASSM AN, S E L F, A N D C H R IS T O P H E R TABLE I I E v a lu a tio n o f Lymphomas D i a g n o s i s C e l l F l o w C y t o m e t r i c F e a t u r e s * B c e l l l y m p h o m a (22 cases) T c e l l l y m p h o m a (10 cases) H o d g k i n ' s d i s e a s e (5 cases) R a n g e (percent) Non-neoplastic nodes K/ À ratio : (hyperplasia) B cell: (21 cases) T cell: /8 ratio : L i g h t c h a i n m o n o t y p i s m. (17 cases) o r W i t h a b s e n c e o f s u r f a c e i m m u n o g l o b u l i n e x p r e s s i o n a c e l l s e x p r e s s i n g o t h e r B c e l l m a r k e r s (Leu-12, O K B 2, 0 K B 7, (5 cases) A b n o r m a l p h e n o t y p e v e r y s u g g e s t i v e o f l y m p h o m a. (5 cases) h i g h p e r c e n t a g e of If the n e o p l a s t i c c e l l b e a r s a n o r m a l, m a t u r e T c e l l p h e n o t y p e, the d i a g n o s i s o f l y m p h o m a c a n be v e r y d i f f i c u l t. (5 cases) T h e i m m u n o p h e n o t y p i c p a t t e r n is i d e n t i c a l to t h a t of a r e a c t i v e node. etc.). Hodgkin' s disease can be ruled out if the diagnosis of B cell or T cell l y m p h o m a c a n be m a d e b y c r i t e r i a l i s t e d above. *The O K d e s i g n a t e s T, M, B, a n d I a l a r e O r t h o - m u n e m o n o c l o n a l a n t i b o d i e s a n d a r e c o m m e r c i a l l y a v a i l a b l e t h r o u g h O r t h o P h a r m a c e u t i c a l C o r p o r a t i o n ; the L e u series, H L A - D R ( I a ), CALLA, L e u k o c y t e a n d T r a n s f e r r i n a r e B e c t o n D i c k i n s o n ' s C o m p a n y M A b - s a n d c a n be o b t a i n e d f r o m them. Discussion The use of CFC provides an easy and rapid means of identifying T and B lymphocyte subsets in th e clinical laboratory. M ajor benefits include the large n u m b er of cells analyzed, rapidity of assay, the relative ease of preparation and the m ultiparam eter analyses of specimens. The cost, however, of flow cell cytom etry is high. C urrently, the instrum ents that are used in this technique can cost m ore than $100,000. M onoclonal antibodies are expensive and not always readily available. The cost of a one (xl vial of monoclonal antibody can be in excess of $150. W hile many of the available monoclonal antibodies are specific for the identification of subsets of cells and are a valuable tool in the characterization of malignant disorders, they are not always lineage specific. Technical difficulties, although few, are such that a technologist who dedicates him self/herself to the task is essential. Similar m em brane markers can occur on benign and m alignant cells. In o rd er to classify a m alig n an t lym phom a, c o rre la tio n is needed with the morphologic interpretation of th e tissu e section and u n d e r standing of th e clinical circum stances. It is obvious from the information collected in this study that C FC can provide a useful and suggestive definitive diagnosis in some cases, particularly for B cell lym phom as. T he B cell lym phomas can be recognized either by surface im m unoglobulin light chain m onotypism or absence of slg s on cells with B cell markers. Those cases of T cell lymphom a in which the cells bear an abnormal phenotype can readily be diagnosed as such. H ow ever, in those cases in which the T cells bear a normal m ature im m unophenotype C F C is not diagnostic. H odgkin s disease gives a C FC pattern identical to reactive lymphadenopathy. T herefore, C FC is not generally useful in th e diagnosis of Hodgkin s disease. In sum m ary, m em brane m arkers in cell suspensions from tissues can be identified by using comm ercially available MAb-s in a cell flow cytom eter. This application com plem ents the diagnosis and classification of a malignancy

7 LYMPHOMAS AND MARKERS/CELL FLOW CYTOMETRY 7 w hen the histopathology and clinical characteristics are taken into consideration. Cell flow cytom etry is objective and analytically sensitive, b u t it possesses certain disadvantages, including cost of th e assay and av ailab ility of m arkers. The characterization of lym phomas is aided by C FC, but the standard for identification rem ains the clinical and histopathologic diagnoses. Acknowledgments Grateful acknowledgment is given to the excellent technical assistance of Donna Runey and Gina Ciabattari. The secretarial assistance of Joanne Gibbs and the editorial assistance of Judy Jarecki-Black are appreciated. References 1. AlSENBERG, A. C.: Cell lineage in lymphoproliferative disease. A m. J. M ed. 7 4 : , D o u g l a s, S. D.: D evelopm ent and structure of cells in the im m une system. Basic & Clinical Immunology, 4th ed. Stites, D. P., Stobo, J. D., Fundenburg, H. H., and W ells, J. V., eds. Los Angeles, Lange Medical Publications, 1982, pp H a n s e n, W. P., H o f f m a n, S. H. Ip, and H e a l e y, K. W. : Light scatter as an adjunct to cellular im munofluorescence in flow cytometric systems. J. Clin. Immunol. 2:32S-41S, L o v e t t, E. J., S c h n it z e r, B., K e r e n, D. F., F l in t, A., H u d s o n, J. L., and M c C l a t c h e y, K. D. : Application of flow cytometry to diagnostic pathology. Lab. Invest. 50:2, , LUKES, R. J., et al. : A morphologic and immunologic surface marker study of cases of non- H odgkin lym phom as and related leukem ias. Am. J. Pathol. 9 0 : , M a n n, R. B., Ja f f e, E. S., and B e r a r d, C. W.: Malignant lym phom as A conceptual understanding of morphologic diversity. Am. J. Pathol. 94: , Re in h e r z, E. L. and S c h l o s s m a n, S. F. : The differentiation and function of human T lymphocytes. Cell 9: , The Non-Hodgkin s Lymphoma Pathologic Classification Project: National Cancer Institute Sponsored Study of Classifications of Non-Hodgkin s Lymphomas: Summary and Description of a W orking Form ulation for Clinical Usage. Cancer 49: , WALDMANN, T. A.: Advances in diagnosis of hematologic malignancies. Hosp. Pract. 21:6 9-77, Wa r n k e, R. A. and R o u s e, R. V.: L im itation s e n c o u n te r e d in th e a p p lication o f tis s u e se c tio n im m u n o d ia g n o sis to th e stu d y o f ly m p h o m a s and r ela ted d iso rd ers. H u m. P athol. 16:4, , 1985.