Clin. exp. Immunol (1991) 83, 418-422
ADONIS 0009910491000776
Expression of the neural cell adhesion molecule (CD56) by human myeloma cells J. DRACH, C. GATTRINGER & H. HUBER Department of Internal Medicine, Division of Immunohaematology and Oncology, University of Innsbruck, Innsbruck, Austria
(Acceptedfor publication 4 October 1990)
SUMMARY Recent studies in multiple myeloma indicate that molecules associated with different haematopoietic lineages may be expressed aberrantly by myeloma cells. In order to investigate this phenomenon further, we studied the immunophenotype of bone marrow cells from 21 patients with multiple myeloma using a panel of monoclonal antibodies against T,B, myelomonocytic, and natural killer (NK)-cell antigens. Leu-19/NKHl (CD56), a molecule identical to N-CAM, which is normally expressed by neuroectodermal and NK cells, was found in 13 patients (62%). Dual-parameter flow cytometry was used to correlate N-CAM positivity with DNA aneuploidy or cytoplasmic immunoglobulin expression as markers of myeloma cells. When N-CAM was found positive, other haematopoietic antigens were expressed only in three out of 13 cases (23%). In contrast, myeloma cells not expressing N-CAM frequently exhibited pre-B cell markers, myeloid antigen, and HLA-DR, respectively (seven out of eight cases, 88%). Six out of eight N-CAM-negative myelomas were of the IgG lamba isotype, otherwise no clearcut association with basic clinical and laboratory parameters was noted. We conclude that N-CAM expression is a common finding in multiple myeloma. Whether its expression and the observed antigenic heterogeneity is just a manifestation of malignancy or N-CAM may play a role in the biology of multiple myeloma regarding tumour cell spread, remains to be explained.
Keywords multiple myeloma neural cell adhesion molecule immunophenotypic analysis
involvement in the pathogenesis of multiple myeloma (Barlogie et al., 1989). Studying the immunophenotype of bone marrow myeloma cells we found evidence for the expression of the natural killer (NK) cell associated antigen CD56 (Drach, Gattringer & Huber, 1990) which has recently been shown to be identical to the 140-kD isoform of N-CAM (Lanier et al., 1989). Here we studied how frequently N-CAM occurs in plasma cell myeloma and whether N-CAM positive cases represent a distinctive subgroup with regard to immunophenotypic and clinical fea-
INTRODUCTION
Multiple myeloma is a haematological malignancy manifesting itself mainly at the terminal stage of B cell differentiation. This is reflected by the typical plasma cell morphology and the expression of cytoplasmic immunoglobulin (Barlogie et al., 1985b) as well as plasma cell-associated antigens including CD38 (OKTlO, Leu-17), PC-1, and PCA-1 (Anderson et al., 1983). Despite the widespread use of surface marker analysis for classifying and monitoring acute leukaemia and malignant lymphoma, immunophenotyping of myeloma cells has gained importance only recently, since ongoing studies have demonstrated that several lineage associated antigens may be expressed by myeloma cells in an aberrant fashion. The occurrence of preB cell associated features (in particular cALLA/CD10 and CD19) as well as the expression of myelomonocytic, megakaryocytic, and erythroid antigens have been described (Durie & Grogan, 1985; Caligaris-Cappio et al., 1985; Epstein et al., 1988; Grogan et al., 1989; Epstein, Xiao & He, 1990), and these findings lead to the hypothesis of pluripotential stem cell
tures.
MATERIALS AND METHODS Patients
All 21 patients studied had clinical findings consistent with overt multiple myeloma. This included an elevated number of plasma cells in the bone marrow (more than 10%) plus osteoporosis or lytic lesions, together with a monoclonal paraprotein in the serum or urine (or both). Fourteen patients were studied at the time of diagnosis, before the initiation of treatment, and two at relapse. Five patients who had responded to treatment were studied between courses of chemotherapy. One patient pre-
Correspondence: Dr J. Drach, Department of Internal Medicine, University of lnnsbruck, Anichstraf3e 35, A-6020 Innsbruck, Austria.
418
419
N-CAM in multiple myeloma sented with plasma cell leukaemia. Staging was performed according to the Durie and Salmon staging system (Durie & Salmon, 1975). Bone marrow aspirates were obtained for diagnostic purpose including immunocytochemical investigations. Mononuclear cells were isolated by density centrifugation over FicollIsopaque (Lymphoprep; density 1-077 g/ml) at 400 g for 20 min and washed twice with phosphate-buffered saline (PBS). Immunophenotypic studies Mononuclear cells were examined for ploidy (DNA content) and monoclonal cytoplasmic immunoglobulin by dual-parameter flow cytometry using a dual-staining technique as previously described (Barlogie et al., 1985b) with some modifications. Briefly, cells were incubated overnight at room temperature to shed cytophilic protein and then fixed in 70% ice-cold ethanol for 1 h. After two washes in PBS, aliquots of 1 x 106 cells were incubated with anti-light chain antibodies (fluorescein-conjugated F(ab')2 fragment of rabbit anti-human kappa/lambda light chains; Dakopatts, Copenhagen, Denmark) at a dilution of 1/25. Incubation was carried out at 4°C for 30 min, followed by two washes with PBS. Finally, cells were incubated with 0-5 mg ribonuclease A (Sigma) in I ml of PBS at 37°C for 30 min and DNA-counterstained with propidium iodide (1 ml PBS containing 50 pg of propidium iodide; Sigma). Simultaneous determination of DNA and RNA content was performed after staining with acridine orange (Polysciences, Warrington, PA) as previously described (Andreeffet al., 1980). The expression of surface antigens by myeloma cells was investigated using a panel of commercially available monoclonal antibodies (MoAbs) as listed in Table 1. Each antibody was diluted with PBS supplemented with 1% bovine serum albumin according to titration experiments performed in our laboratory. The FITC-conjugated MoAb was added at saturation concentration to 106 cells; in case of an unlabelled MoAb, FITCconjugated goat anti-mouse IgG (Coulter, Hialeah, FL) was used as a second-step reagent in an indirect immunofluorescence assay. Each incubation step was carried out at 4°C for 30 min. For simultaneous analysis of nuclear DNA content cells were permeabilized by adding 1 ml of cold PBS containing 0-25% saponin (Sigma). After 10 min cells were spun down, treated with ribonuclease A and stained with propidium iodide as described above. To identify co-expression of surface antigens and cytoplasmic light chain, cells were treated with PEconjugated MoAb against the surface marker, fixed with 70% ethanol, and then reacted with FITC-conjugated F(ab')2 fragment of rabbit anti-human kappa/lambda light chains (Dakopatts). Flow cytometry Samples were analysed on a FACStar flow cytometer (Becton Dickinson, Mountain View, CA) equipped with an argon laser emitting at 488 nm. FITC fluorescence was measured using a 530/30 nm band-pass filter, and red fluorescence (PE, propidium iodide) was measured using a 585/40 nm band-pass filter. At least 10 000 events were counted for each sample, and the results were stored in list mode. The final processing of the data was performed using the Consort-30 Data Management system (provided by Becton Dickinson). Cells displaying fluorescence intensities above the upper limit of the negative control distribution which was determined using FITC- and PE-labelled
Table 1. Monoclonal antibodies used for the immunophenotypic analysis
Antibody Leu-4 Leu-12 cALLA
CD3
My-9
CD33 CD13 CD15 CD14 CD38
My-7 Leu-M I Leu-M3 Leu-17
Cellular reactivity*
Source
lymphoid progenitors myeloid progenitors, Mono Mono, G
BD BD BD Coulter Coulter BD BD BD
Antigen
CDl9 CDlO
T B
G Mono Plc, activated T,
thymocytes Leu- 19 Leu-7
Leu-1 1 Mo-I la
N-CAM/CD56 CD57 FcyRIII/CD16 CR3/CD1 lb HLA-DR
NK NK
NK, G Mono, NK, G Mono, activated T, B, progenitor cells
BD BD BD Coulter BD
* Cellular reactivity as defined by the Fourth International Workshop on Human Leucocyte Differentiation Antigens, Vienna, 1989. BD, Becton Dickinson, Mountain View, CA; Coulter, Coulter Immunology, Hialeah, FL; T, T lymphocytes; B, B lymphocytes; Mono, monocytes; G, granulocytes; NK, natural killer cells; Plc, plasma cells.
isotypic control antibodies (Becton Dickinson) were considered
positive. RESULTS Demonstration of N-CAM expression by myeloma cells Dual-parameter flow cytometric analysis was carried out to confirm the expression of N-CAM by myeloma cells. Since multiple myeloma exhibits an abnormal nuclear DNA-content in about 80% of cases (Barlogie et al., 1985b), immunophenotypic features of myeloma cells were examined in relationship to DNA aneuploidy, as depicted in Fig. 1. DNA aneuploid cells were identified as myeloma cells using dual parameter analysis of cytoplasmic immunoglobulin light chains and nuclear DNA content. Only cells with the hyperdiploid DNA stemline contained monoclonal cytoplasmic immunoglobulin (lambda), and thus represented the myeloma tumour cell population (DNA index= 1-20). In addition, the hyperdiploid cells were found to have a markedly increased RNA content which is characteristic of myeloma cells as measured after staining with acridine orange. Testing for N-CAM and nuclear DNA content clearly revealed positivity for N-CAM among the hyperdiploid, i.e. myeloma cells. In cases of multiple myeloma with no DNA aneuploid stemline (five patients in this series), flow cytometric analysis of coexpression of N-CAM and cytoplasmic immunoglobulin was performed.
Immunophenotypic analysis of myeloma cells Using a panel of MoAbs (Table 1) and dual-parameter flow cytometry as described above, bone marrow cells from 21 patients with multiple myeloma were tested for the expression of N-CAM as well as early B, T, myelomonocytic markers, and HLA-DR. As expected of plasma cells, all cases expressed Leu17 (CD38). Positivity for N-CAM was found in 13 patients
420
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J. Drach, C. Gattringer & H. Huber 104
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10 -
102
lo,
0'-lp 100 0
100
0
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300
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101 jVC
10' 0
100
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0
1O00
100
'lU-
z
s 102
(0 10'
0
200
100
200
100
200
z
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Fig. 1. Dual-parameter analysis of myeloma cells. The DNA histogam demonstrates a cell population having a DNA aneuploid stemline. DNA hyperploidy of myeloma cells was defined by simulatneous analysis of DNA content (abscissa) and cytoplasmic immunoglobulin (kappa/lambda; ordinate). Hyperploid myeloid cells, which were further characterized by their high RNA content, reacted positively with the antibody against N-CAM (CD56).
(62% of cases), the percentage of positive myeloma cells ranged between 60 and 100% (median 90%). These results along with further phenotypic features are summarized in Table 2. Overall, marked heterogeneity in antigen expression by myeloma cells was observed and, as shown in Table 2, two groupings may be established based on the immunophenotypic results. Group A is comprised of N-CAM-negative cases myeloma cells of which very frequently show expression of other lineage associated antigens (specifically, pre-B cell and/or myeloid markers in six out of eight cases) and HLA-DR (one case), respectively. In contrast, 10 out of 13 cases (77%) with N-CAM positivity, which constitute group B, did not exhibit any additional aberrant antigenic feature tested in this study. In patients 14 and 17, sufficient cells were availble to study further natural killer cell-related antigens (FcyRIII/CD16, CD57, and CR3/CD Ib). Twenty per cent of myeloma cells from patient 17 were found to express CR3/CD lb whereas otherwise no reactivity with any of the antibodies was observed in both patients. Since CR3 also occurs on monocytes and granulocytes, it is important to note that in the patient under discussion no evidence of myelomonocytic differentiation antigens could be demonstrated (Table 2). Table 3 summarizes basic clinical and laboratory parameters in the 21 patients examined in this study. Although the number of patients investigated so far is small, the presence of N-CAM appears to be unrelated to disease stage. The only obvious association is the high frequency of an IgG lambda paraprotein in group A (N-CAM-negative myeloma). Cells from one patient presenting with plasma cell leukaemia (patient I in Table 2) did not exhibit N-CAM expression.
DISCUSSION Cell surface antigens that are involved in cell-cell interactions and cellular adhesion have been identified on several cell types in haematopoietic, muscle, and neural tissues (Edelman, 1986). One of these cellular adhesion molecules is N-CAM, and has been characterized extensively (Cunningham et al., 1987; Barton et al., 1988). Different isoforms of the N-CAM polypeptide are derived by alternative mRNA splicing from a single gene, and the 180-kD and 140-kD transmembrane isoforms differ only by the presence of an additional cytoplasmic domain (Owens, Edelman & Cunningham, 1987; Walsh & Dickson, 1989). Recently, the identity of Leu-19/NKHI, which has previously been characterized on peripheral blood lymphocytes, and the 140-kD isoform of N-CAM have been demonstrated (Lanier et al., 1989), and the molecule has been defined as the CD56 antigen by the 4th International Workshop on Leucocyte Differentiation Antigens, Vienna, 1989. N-CAM is expressed on neuroectodermal cells and represents an important cellular adhesion molecule which mediates homotypic adhesion among neurons and between neurons and muscle (Walsh & Dickson, 1989). In tumours, N-CAM expression has been described in Wilm's tumour, neuroblastoma, Ewing's sarcoma, and small lung cell cancer (Lipinsky et al., 1987; Roth et al., 1988; Patel et al., 1989; Moolenaar et al., 1990). In haematopoietic tissues N-CAM is expressed on NK cells and T lymphocytes mediating MHC-unrestricted cytotoxicity (Lanier et al., 1986). The role of the N-CAM molecule in NK cell function or activation and its possible involvement in lymphocyte adhesion remains to be elucidated. In addition to
421
N-CAM in multiple myeloma Table 2. Immunophenotypic studies of myeloma cells
Patient
N-CAM/CD56
CD3
-
-
Group A I 2 3 4
CDl9
CD13
-
-
-
-
+
CDlO
-
+
+
-
+
-
-
5
6 7 8
CD33
-
-
-
+
-
CD14
HLA-DR
NT
-
+
_ _
_
_
_ _ NT
+ +
-
-
CD15
NT
+
+
+
_
_
NT
Group B 9
+
-
10 11
+ +
-+
12 13 14 15 16 17 18 19 20 21
+ +
NT
+
-
-
-
-
-
-
-
_ _
-
-
-
-
_
-
_
_ -
+
-
-
-
_
_ NT
_
_
_
-
-
-
-
+
-
-
+ + + + + +
+
+
-
_ _
-
+, > 20% of myeloma cells reacted positively with the antibody; ±, positive reaction with 5-20% of myeloma cells; - no reactivity or < 500. NT, not tested.
Table 3. Characteristics of patients iwith N-CAM-negative versus NCAM-positive m!yeloma (MM)
Group A
Group B
N-CAM negative MM
N-CAM positive-MM
8 5 1
13 9 1
2
3
2
6
2 2 9
IgG IgA BJP Kappa Lambda
6 1 1 2
10 2 1 8
6
5
Beta-2 microglobuline > 6 Creatinine > 1*5
1
2
Total number of patients Patients studied at diagnosis Patients studied at relapse Patients studied between courses of chemotherapy stage I stage II stage III
BJP, Bence-Jones protein.
0
this cellular distribution, we report here frequent expression of N-CAM by myeloma cells which has been documented by dualparameter flow cytometry using DNA aneuploidy and/or cytoplasmic immunoglobulin expression as features characteristic myeloma cells. Using this experimental approach, we were able to rule out the possibility that residual T lymphocytes and NK cells might account for N-CAM positivity observed in the bone marrow samples. Moreover, since NK cell activity in the peripheral blood and bone marrow of patients with multiple myeloma has been reported to be increased (Miksche et al., 1989), it was important to correlate N-CAM expression with myeloma associated parameters. Our finding might have important implications with respect to myeloma tumour cell biology. Recent studies in nonHodgkin's lymphoma suggest an association between sion of cellular adhesion molecules and dissemination expresof the disease. In B-cell lymphomas of low-grade malignancy the lack of ICAM- 1 (CD54) and a leukaemic course of the disease were significantly correlated (Stauder et al., 1989). When the lymphocyte homing receptor (H-CAM/CD44) was investigated, lymphocyte homing receptor positive lymphomas were found to disseminate haematogenously; in contrast, lymphomas lacking homing receptor expression usually remained local independent of the histologic subtype and the proliferative activity (Jalkanen, Joensuu & Kiemi, 1990). Thus, it is reasonable to assume that cellular adhesion molecules might also be involved in the spreading characteristics of multiple myeloma resulting in the typical manifestation of the disease. When multiple myeloma is
J. Drach, C. Gattringer & H. Huber
422
detected, it is usually widespread throughout the axial skeleton and bone marrow whereas only few plasma cells are seen in the circulation. Not considering the rare cases presenting with plasma cell leukaemia, dissemination of multiple myeloma only occurs at the terminal stage of the disease. In this connection it is worth noting that not only the expression of N-CAM, but also of ICAM-I on myeloma cells (Stauder et al., 1989) is a common finding. It remains to be investigated whether cases of multiple myeloma lacking these cellular adhesion molecules have a more aggressive course of the disease. Interestingly, one patient with plasma cell leukaemia studied in our series was N-CAM negative. Alternatively, expression of N-CAM/NKH 1 may be interpreted as an NK cell-associated feature, although so far we have no evidence of further NK cell antigens on myeloma cells. Taken together, our immunophenotypic studies demonstrate marked antigenic heterogeneity of myeloma cells, and aberrant antigen expression is more pronounced in cases that are N-CAM negative. As suggested previously, the simultaneous expression of various differentiation antigens by the same tumour cell supports the concept of early stem cell involvement in the pathogenesis of multiple myeloma (Barlogie et al., 1989; Epstein et al., 1990; Drach et al., 1990). Further studies should clarify to what extent other cellular adhesion molecules and integrins occur in multiple myeloma and how the expression correlates with the clinical course. This will help to elucidate a possible role of these molecules in tumour cell spread and extend our knowledge about the biology of multiple myeloma. ACKNOWLEDGMENTS This work was supported by a grant from the Austrian 'Fonds zur Forderung wissenschaftlicher Forschung' (P6066F).
REFERENCES ANDERSON, K.C., PARK, E.K., BATES, M.P., LEONARD, R.C.F., HARDY, R., SCHLOSSMAN, S.F. & NADLER, L.M. (1983) Antigens on human plasma cells identified by monoclonal antibodies. J. Immunol. 130, 1132. ANDREEFF, M., DARZYNKIEWICZ, Z., SHARPLESS, T.K., CLARKSON, B.D. & MELAMED, M.R. (1980) Discrimination of human leukemia subtypes by flow cytometric analysis of cellular DNA and RNA. Blood, 55, 282. BARLOGIE, B., ALEXANIAN, R., DIXON, D., SMITH, L., SMALLWOOD, L. & DELASALLE, K. (1985a) Prognostic implications of tumor cell DNA and RNA content in multiple myeloma. Blood, 66, 338. BARLOGIE, B., ALEXANIAN, R., PERSHOUSE, M., SMALLWOOD, L. & SMITH, L. (1985b) Cytoplasmic immunoglobulin content in multiple myeloma. J. clin. Invest. 76, 765. BARLOGIE, B., EPSTEIN, J., SELVANAYAGAM, P. & ALEXANIAN, R. (1989) Plasma cell myeloma-new biological insights and advances in therapy. Blood, 73, 865. BARTON, C.H., ELSOM, V.L., MOORE, S.E., GORIDIS, C. & WALSH, F.S. (1988) Complete sequence and in vitro expression of a tissue-specific phosphatidylinositol-linked N-CAM isoform from skeletal muscle. Development, 104, 165. CALIGARIS-CAPPIO, F., BERGUI, L., TEsio, L., PIZZOLO, G., MALAVASI, F., CHILOSI, M., CAMPANA, C., VAN CAMP, B. & JANOSSY, G. (1985) Identification of malignant plasma cell precursors in the bone marrow of multiple myeloma. J. Clin Invest. 76, 1243. CUNNINGHAM, B.A., HEMPERLY, J.J., MURRAY, B.A., PREDIGER, E.A., BRACKENBURY, R. & EDELMAN, G.M. (1987) Neural cell adhesion
moleculae: structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing. Science, 236, 799. DRACH, J., GATTRINGER, C. & HUBER, H. (1990) Multiple myeloma with coexpression of myeloid and natural killer cell antigens. Blood, 76, 265. DURIE, B.G.M. & SALMON, S.E. (1975) A clinical staging system for multiple myeloma. Correlation of measured myeloma cell mass with presenting clinical features, response to treatment and survival. Cancer, 36, 842. DURIE, B.G.M. & GROGAN, T.M. (1985) CALLA-positive myeloma: an aggressive subtype with poor prognosis. Blood, 66, 229. EDELMAN, G.M. (1986) Cell adhesion molecules in the regulation of animal form and tissue pattern. Annu. Rev. Cell. Biol. 2, 81. EPSTEIN, J., XIAO, H. & HE, X.-Y. (1990) Markers of multiple hematopoietic-cell lineages in multiple myeloma. N. Engl. J. Med. 322, 664. EPSTEIN, J., BARLOGIE, B., KATZMANN, J. & ALEXANIAN, R. (1988) Phenotypic heterogeneity in aneuploid multiple myeloma indicates pre-B cell involvement. Blood, 71, 861. GROGAN, T.M., DURIE, B.G.M., SPIER, C.M., RICHTER, L. & VELA, E. (1989) Myelomonocytic antigen positive multiple myeloma. Blood, 73, 763. JALKANEN, S., JOENSUU, H. & KIEMI, P. (1990) Prognostic value of lymphocyte homing receptor and S-phase fraction in non-Hodgkin's lymphoma. Blood, 75, 1549. LANIER, L.L., LE, A.M., CIVIN, C.I., LOKEN, M.R. & PHILLIPS, J.H. (1986) The relationship of CD16 (Leu-1 1) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J. Immunol. 136, 4480. LANIER, L.L., TESTI, R., BINDL, J. & PHILLIPS, J.H. (1989) Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule. J. exp. Med. 169, 2233. LIPINSKI, M., HIRSCH, M.-R., DEAGoSTINi-BAZIN, H., YAMADA, O., TURSZ, T. & GORIDIS, C. (1987) Characterization of neural cell adhesion molecules (NCAM) expressed by Ewing and neuroblastoma cell lines. Int. J. Cancer, 40, 81. MIKSCHE, M., GISSLINGER, H., SCHEITHAUER, W., LINKESCH, W. & LUDWIG, H. (1989) Natural killer cell activity in peripheral blood and bone marrow of patients with multiple myeloma. In International Conference on Multiple Myeloma, Bologna, Italy p. 159. MOOLENAAR, C.E.C.K., MULLER, E.J., SCHOL, D.J., FIGDOR, C.G., BOCK, E., BITTER-SUERMANN, D. & MICHALIDES, R.J.A.M. (1990) Expression of neural cell adhesion molecule-related sialoglycoprotein in small cell lung cancer and neuroblastoma cell lines H69 and CHP212. Cancer Res. 50, 1102. OWENS, G.C., EDELMANN, G.M. &-CUNNINGHAM, B.A. (1987) Organization of the neural cell adhesion molecule (N-CAM) gene: alternative exon usage as the basis for different membrane-associated domains. Proc. natl Acad. Sci. USA, 84, 294. PATEL, K., MOORE, S.E., DICKSON, G., ROSSELL, R.J., BEVERLEY, P.C., KEMSHEAD, J.T. & WALSH, F.S. (1989) Neural cell adhesion molecule (NCAM) is the antigen recognized by monoclonal antibodies of similar specificity in small-cell lung carcinoma and neuroblastoma. Int. J. Cancer, 44, 573. ROTH, J., ZUBER, C., WAGNER, P., TAATJES, D.J., WEISGERBER, C., HEITZ, P.U., GORIDIS, C. & BITTER-SUERMANN, D. (1988) Reexpression of poly(sialic acid) units of the neural cell adhesion molecule in Wilms tumor. Proc. natl Acad. Sci. USA, 85, 2999. STAUDER, R., GREIL, R., SCHULZ, T.F., THALER, J., GATTRINGER, C., RADASKIEWICZ, T., DIERICH, M.P. & HUBER, H. (1989) Expression of leucocyte function-associated antigen-i and 7F7-antigen, an adhesion molecule related to intercellular adhesion molecule- 1 (ICAM- 1) in non-Hodgkin lymphomas and leukaemias: possible influence on growth pattern and leukaemic behaviour. Clin. exp. Immunol. 77, 234. WALSH, F.S. & DICKSON, G. (1989) Generation of multiple N-CAM polypeptides from a single gene. BioEssays, 11, 83.
ADONIS 0009910491000776
Expression of the neural cell adhesion molecule (CD56) by human myeloma cells J. DRACH, C. GATTRINGER & H. HUBER Department of Internal Medicine, Division of Immunohaematology and Oncology, University of Innsbruck, Innsbruck, Austria
(Acceptedfor publication 4 October 1990)
SUMMARY Recent studies in multiple myeloma indicate that molecules associated with different haematopoietic lineages may be expressed aberrantly by myeloma cells. In order to investigate this phenomenon further, we studied the immunophenotype of bone marrow cells from 21 patients with multiple myeloma using a panel of monoclonal antibodies against T,B, myelomonocytic, and natural killer (NK)-cell antigens. Leu-19/NKHl (CD56), a molecule identical to N-CAM, which is normally expressed by neuroectodermal and NK cells, was found in 13 patients (62%). Dual-parameter flow cytometry was used to correlate N-CAM positivity with DNA aneuploidy or cytoplasmic immunoglobulin expression as markers of myeloma cells. When N-CAM was found positive, other haematopoietic antigens were expressed only in three out of 13 cases (23%). In contrast, myeloma cells not expressing N-CAM frequently exhibited pre-B cell markers, myeloid antigen, and HLA-DR, respectively (seven out of eight cases, 88%). Six out of eight N-CAM-negative myelomas were of the IgG lamba isotype, otherwise no clearcut association with basic clinical and laboratory parameters was noted. We conclude that N-CAM expression is a common finding in multiple myeloma. Whether its expression and the observed antigenic heterogeneity is just a manifestation of malignancy or N-CAM may play a role in the biology of multiple myeloma regarding tumour cell spread, remains to be explained.
Keywords multiple myeloma neural cell adhesion molecule immunophenotypic analysis
involvement in the pathogenesis of multiple myeloma (Barlogie et al., 1989). Studying the immunophenotype of bone marrow myeloma cells we found evidence for the expression of the natural killer (NK) cell associated antigen CD56 (Drach, Gattringer & Huber, 1990) which has recently been shown to be identical to the 140-kD isoform of N-CAM (Lanier et al., 1989). Here we studied how frequently N-CAM occurs in plasma cell myeloma and whether N-CAM positive cases represent a distinctive subgroup with regard to immunophenotypic and clinical fea-
INTRODUCTION
Multiple myeloma is a haematological malignancy manifesting itself mainly at the terminal stage of B cell differentiation. This is reflected by the typical plasma cell morphology and the expression of cytoplasmic immunoglobulin (Barlogie et al., 1985b) as well as plasma cell-associated antigens including CD38 (OKTlO, Leu-17), PC-1, and PCA-1 (Anderson et al., 1983). Despite the widespread use of surface marker analysis for classifying and monitoring acute leukaemia and malignant lymphoma, immunophenotyping of myeloma cells has gained importance only recently, since ongoing studies have demonstrated that several lineage associated antigens may be expressed by myeloma cells in an aberrant fashion. The occurrence of preB cell associated features (in particular cALLA/CD10 and CD19) as well as the expression of myelomonocytic, megakaryocytic, and erythroid antigens have been described (Durie & Grogan, 1985; Caligaris-Cappio et al., 1985; Epstein et al., 1988; Grogan et al., 1989; Epstein, Xiao & He, 1990), and these findings lead to the hypothesis of pluripotential stem cell
tures.
MATERIALS AND METHODS Patients
All 21 patients studied had clinical findings consistent with overt multiple myeloma. This included an elevated number of plasma cells in the bone marrow (more than 10%) plus osteoporosis or lytic lesions, together with a monoclonal paraprotein in the serum or urine (or both). Fourteen patients were studied at the time of diagnosis, before the initiation of treatment, and two at relapse. Five patients who had responded to treatment were studied between courses of chemotherapy. One patient pre-
Correspondence: Dr J. Drach, Department of Internal Medicine, University of lnnsbruck, Anichstraf3e 35, A-6020 Innsbruck, Austria.
418
419
N-CAM in multiple myeloma sented with plasma cell leukaemia. Staging was performed according to the Durie and Salmon staging system (Durie & Salmon, 1975). Bone marrow aspirates were obtained for diagnostic purpose including immunocytochemical investigations. Mononuclear cells were isolated by density centrifugation over FicollIsopaque (Lymphoprep; density 1-077 g/ml) at 400 g for 20 min and washed twice with phosphate-buffered saline (PBS). Immunophenotypic studies Mononuclear cells were examined for ploidy (DNA content) and monoclonal cytoplasmic immunoglobulin by dual-parameter flow cytometry using a dual-staining technique as previously described (Barlogie et al., 1985b) with some modifications. Briefly, cells were incubated overnight at room temperature to shed cytophilic protein and then fixed in 70% ice-cold ethanol for 1 h. After two washes in PBS, aliquots of 1 x 106 cells were incubated with anti-light chain antibodies (fluorescein-conjugated F(ab')2 fragment of rabbit anti-human kappa/lambda light chains; Dakopatts, Copenhagen, Denmark) at a dilution of 1/25. Incubation was carried out at 4°C for 30 min, followed by two washes with PBS. Finally, cells were incubated with 0-5 mg ribonuclease A (Sigma) in I ml of PBS at 37°C for 30 min and DNA-counterstained with propidium iodide (1 ml PBS containing 50 pg of propidium iodide; Sigma). Simultaneous determination of DNA and RNA content was performed after staining with acridine orange (Polysciences, Warrington, PA) as previously described (Andreeffet al., 1980). The expression of surface antigens by myeloma cells was investigated using a panel of commercially available monoclonal antibodies (MoAbs) as listed in Table 1. Each antibody was diluted with PBS supplemented with 1% bovine serum albumin according to titration experiments performed in our laboratory. The FITC-conjugated MoAb was added at saturation concentration to 106 cells; in case of an unlabelled MoAb, FITCconjugated goat anti-mouse IgG (Coulter, Hialeah, FL) was used as a second-step reagent in an indirect immunofluorescence assay. Each incubation step was carried out at 4°C for 30 min. For simultaneous analysis of nuclear DNA content cells were permeabilized by adding 1 ml of cold PBS containing 0-25% saponin (Sigma). After 10 min cells were spun down, treated with ribonuclease A and stained with propidium iodide as described above. To identify co-expression of surface antigens and cytoplasmic light chain, cells were treated with PEconjugated MoAb against the surface marker, fixed with 70% ethanol, and then reacted with FITC-conjugated F(ab')2 fragment of rabbit anti-human kappa/lambda light chains (Dakopatts). Flow cytometry Samples were analysed on a FACStar flow cytometer (Becton Dickinson, Mountain View, CA) equipped with an argon laser emitting at 488 nm. FITC fluorescence was measured using a 530/30 nm band-pass filter, and red fluorescence (PE, propidium iodide) was measured using a 585/40 nm band-pass filter. At least 10 000 events were counted for each sample, and the results were stored in list mode. The final processing of the data was performed using the Consort-30 Data Management system (provided by Becton Dickinson). Cells displaying fluorescence intensities above the upper limit of the negative control distribution which was determined using FITC- and PE-labelled
Table 1. Monoclonal antibodies used for the immunophenotypic analysis
Antibody Leu-4 Leu-12 cALLA
CD3
My-9
CD33 CD13 CD15 CD14 CD38
My-7 Leu-M I Leu-M3 Leu-17
Cellular reactivity*
Source
lymphoid progenitors myeloid progenitors, Mono Mono, G
BD BD BD Coulter Coulter BD BD BD
Antigen
CDl9 CDlO
T B
G Mono Plc, activated T,
thymocytes Leu- 19 Leu-7
Leu-1 1 Mo-I la
N-CAM/CD56 CD57 FcyRIII/CD16 CR3/CD1 lb HLA-DR
NK NK
NK, G Mono, NK, G Mono, activated T, B, progenitor cells
BD BD BD Coulter BD
* Cellular reactivity as defined by the Fourth International Workshop on Human Leucocyte Differentiation Antigens, Vienna, 1989. BD, Becton Dickinson, Mountain View, CA; Coulter, Coulter Immunology, Hialeah, FL; T, T lymphocytes; B, B lymphocytes; Mono, monocytes; G, granulocytes; NK, natural killer cells; Plc, plasma cells.
isotypic control antibodies (Becton Dickinson) were considered
positive. RESULTS Demonstration of N-CAM expression by myeloma cells Dual-parameter flow cytometric analysis was carried out to confirm the expression of N-CAM by myeloma cells. Since multiple myeloma exhibits an abnormal nuclear DNA-content in about 80% of cases (Barlogie et al., 1985b), immunophenotypic features of myeloma cells were examined in relationship to DNA aneuploidy, as depicted in Fig. 1. DNA aneuploid cells were identified as myeloma cells using dual parameter analysis of cytoplasmic immunoglobulin light chains and nuclear DNA content. Only cells with the hyperdiploid DNA stemline contained monoclonal cytoplasmic immunoglobulin (lambda), and thus represented the myeloma tumour cell population (DNA index= 1-20). In addition, the hyperdiploid cells were found to have a markedly increased RNA content which is characteristic of myeloma cells as measured after staining with acridine orange. Testing for N-CAM and nuclear DNA content clearly revealed positivity for N-CAM among the hyperdiploid, i.e. myeloma cells. In cases of multiple myeloma with no DNA aneuploid stemline (five patients in this series), flow cytometric analysis of coexpression of N-CAM and cytoplasmic immunoglobulin was performed.
Immunophenotypic analysis of myeloma cells Using a panel of MoAbs (Table 1) and dual-parameter flow cytometry as described above, bone marrow cells from 21 patients with multiple myeloma were tested for the expression of N-CAM as well as early B, T, myelomonocytic markers, and HLA-DR. As expected of plasma cells, all cases expressed Leu17 (CD38). Positivity for N-CAM was found in 13 patients
420
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-
10 -
102
lo,
0'-lp 100 0
100
0
200
i04
300
1035 2
101 jVC
10' 0
100
200
0
1O00
100
'lU-
z
s 102
(0 10'
0
200
100
200
100
200
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200
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Fig. 1. Dual-parameter analysis of myeloma cells. The DNA histogam demonstrates a cell population having a DNA aneuploid stemline. DNA hyperploidy of myeloma cells was defined by simulatneous analysis of DNA content (abscissa) and cytoplasmic immunoglobulin (kappa/lambda; ordinate). Hyperploid myeloid cells, which were further characterized by their high RNA content, reacted positively with the antibody against N-CAM (CD56).
(62% of cases), the percentage of positive myeloma cells ranged between 60 and 100% (median 90%). These results along with further phenotypic features are summarized in Table 2. Overall, marked heterogeneity in antigen expression by myeloma cells was observed and, as shown in Table 2, two groupings may be established based on the immunophenotypic results. Group A is comprised of N-CAM-negative cases myeloma cells of which very frequently show expression of other lineage associated antigens (specifically, pre-B cell and/or myeloid markers in six out of eight cases) and HLA-DR (one case), respectively. In contrast, 10 out of 13 cases (77%) with N-CAM positivity, which constitute group B, did not exhibit any additional aberrant antigenic feature tested in this study. In patients 14 and 17, sufficient cells were availble to study further natural killer cell-related antigens (FcyRIII/CD16, CD57, and CR3/CD Ib). Twenty per cent of myeloma cells from patient 17 were found to express CR3/CD lb whereas otherwise no reactivity with any of the antibodies was observed in both patients. Since CR3 also occurs on monocytes and granulocytes, it is important to note that in the patient under discussion no evidence of myelomonocytic differentiation antigens could be demonstrated (Table 2). Table 3 summarizes basic clinical and laboratory parameters in the 21 patients examined in this study. Although the number of patients investigated so far is small, the presence of N-CAM appears to be unrelated to disease stage. The only obvious association is the high frequency of an IgG lambda paraprotein in group A (N-CAM-negative myeloma). Cells from one patient presenting with plasma cell leukaemia (patient I in Table 2) did not exhibit N-CAM expression.
DISCUSSION Cell surface antigens that are involved in cell-cell interactions and cellular adhesion have been identified on several cell types in haematopoietic, muscle, and neural tissues (Edelman, 1986). One of these cellular adhesion molecules is N-CAM, and has been characterized extensively (Cunningham et al., 1987; Barton et al., 1988). Different isoforms of the N-CAM polypeptide are derived by alternative mRNA splicing from a single gene, and the 180-kD and 140-kD transmembrane isoforms differ only by the presence of an additional cytoplasmic domain (Owens, Edelman & Cunningham, 1987; Walsh & Dickson, 1989). Recently, the identity of Leu-19/NKHI, which has previously been characterized on peripheral blood lymphocytes, and the 140-kD isoform of N-CAM have been demonstrated (Lanier et al., 1989), and the molecule has been defined as the CD56 antigen by the 4th International Workshop on Leucocyte Differentiation Antigens, Vienna, 1989. N-CAM is expressed on neuroectodermal cells and represents an important cellular adhesion molecule which mediates homotypic adhesion among neurons and between neurons and muscle (Walsh & Dickson, 1989). In tumours, N-CAM expression has been described in Wilm's tumour, neuroblastoma, Ewing's sarcoma, and small lung cell cancer (Lipinsky et al., 1987; Roth et al., 1988; Patel et al., 1989; Moolenaar et al., 1990). In haematopoietic tissues N-CAM is expressed on NK cells and T lymphocytes mediating MHC-unrestricted cytotoxicity (Lanier et al., 1986). The role of the N-CAM molecule in NK cell function or activation and its possible involvement in lymphocyte adhesion remains to be elucidated. In addition to
421
N-CAM in multiple myeloma Table 2. Immunophenotypic studies of myeloma cells
Patient
N-CAM/CD56
CD3
-
-
Group A I 2 3 4
CDl9
CD13
-
-
-
-
+
CDlO
-
+
+
-
+
-
-
5
6 7 8
CD33
-
-
-
+
-
CD14
HLA-DR
NT
-
+
_ _
_
_
_ _ NT
+ +
-
-
CD15
NT
+
+
+
_
_
NT
Group B 9
+
-
10 11
+ +
-+
12 13 14 15 16 17 18 19 20 21
+ +
NT
+
-
-
-
-
-
-
-
_ _
-
-
-
-
_
-
_
_ -
+
-
-
-
_
_ NT
_
_
_
-
-
-
-
+
-
-
+ + + + + +
+
+
-
_ _
-
+, > 20% of myeloma cells reacted positively with the antibody; ±, positive reaction with 5-20% of myeloma cells; - no reactivity or < 500. NT, not tested.
Table 3. Characteristics of patients iwith N-CAM-negative versus NCAM-positive m!yeloma (MM)
Group A
Group B
N-CAM negative MM
N-CAM positive-MM
8 5 1
13 9 1
2
3
2
6
2 2 9
IgG IgA BJP Kappa Lambda
6 1 1 2
10 2 1 8
6
5
Beta-2 microglobuline > 6 Creatinine > 1*5
1
2
Total number of patients Patients studied at diagnosis Patients studied at relapse Patients studied between courses of chemotherapy stage I stage II stage III
BJP, Bence-Jones protein.
0
this cellular distribution, we report here frequent expression of N-CAM by myeloma cells which has been documented by dualparameter flow cytometry using DNA aneuploidy and/or cytoplasmic immunoglobulin expression as features characteristic myeloma cells. Using this experimental approach, we were able to rule out the possibility that residual T lymphocytes and NK cells might account for N-CAM positivity observed in the bone marrow samples. Moreover, since NK cell activity in the peripheral blood and bone marrow of patients with multiple myeloma has been reported to be increased (Miksche et al., 1989), it was important to correlate N-CAM expression with myeloma associated parameters. Our finding might have important implications with respect to myeloma tumour cell biology. Recent studies in nonHodgkin's lymphoma suggest an association between sion of cellular adhesion molecules and dissemination expresof the disease. In B-cell lymphomas of low-grade malignancy the lack of ICAM- 1 (CD54) and a leukaemic course of the disease were significantly correlated (Stauder et al., 1989). When the lymphocyte homing receptor (H-CAM/CD44) was investigated, lymphocyte homing receptor positive lymphomas were found to disseminate haematogenously; in contrast, lymphomas lacking homing receptor expression usually remained local independent of the histologic subtype and the proliferative activity (Jalkanen, Joensuu & Kiemi, 1990). Thus, it is reasonable to assume that cellular adhesion molecules might also be involved in the spreading characteristics of multiple myeloma resulting in the typical manifestation of the disease. When multiple myeloma is
J. Drach, C. Gattringer & H. Huber
422
detected, it is usually widespread throughout the axial skeleton and bone marrow whereas only few plasma cells are seen in the circulation. Not considering the rare cases presenting with plasma cell leukaemia, dissemination of multiple myeloma only occurs at the terminal stage of the disease. In this connection it is worth noting that not only the expression of N-CAM, but also of ICAM-I on myeloma cells (Stauder et al., 1989) is a common finding. It remains to be investigated whether cases of multiple myeloma lacking these cellular adhesion molecules have a more aggressive course of the disease. Interestingly, one patient with plasma cell leukaemia studied in our series was N-CAM negative. Alternatively, expression of N-CAM/NKH 1 may be interpreted as an NK cell-associated feature, although so far we have no evidence of further NK cell antigens on myeloma cells. Taken together, our immunophenotypic studies demonstrate marked antigenic heterogeneity of myeloma cells, and aberrant antigen expression is more pronounced in cases that are N-CAM negative. As suggested previously, the simultaneous expression of various differentiation antigens by the same tumour cell supports the concept of early stem cell involvement in the pathogenesis of multiple myeloma (Barlogie et al., 1989; Epstein et al., 1990; Drach et al., 1990). Further studies should clarify to what extent other cellular adhesion molecules and integrins occur in multiple myeloma and how the expression correlates with the clinical course. This will help to elucidate a possible role of these molecules in tumour cell spread and extend our knowledge about the biology of multiple myeloma. ACKNOWLEDGMENTS This work was supported by a grant from the Austrian 'Fonds zur Forderung wissenschaftlicher Forschung' (P6066F).
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