Acta Haematol 1990;83:123-129
Immunophenotypic Analysis of Lymphocytes and Myeloma Cells in Patients with Multiple Myeloma Chihiro Shimazaki, Jerrold Fried, Amaury G. Perez, David A. Scheinberg, Jens Atzpodien, Chang Y. Wang, Rosanne Wisniewolski, Bayard D. Clarkson Laboratories of Hematopoietic Cel! Kinetics and Human Cancer Immunology, Memorial Sloan-Kettering Cancer Center, New York, N.Y.; United Biomedical, Inc., Lake Success, N.Y., USA
Key Words. Immunophénotype • Monoclonal antibodies • Multiple myeloma • Plasma cells Abstract. The immunological phenotypes of lymphocytes and myeloma cells in 48 patients with multiple myeloma (MM) were analyzed using a panel of monoclonal antibodies (mAbs). Myeloma cells were positive for OKTIO, BL3, PCA1 and BA2. In a few cases, they were also positive for the B cell-associated antigens J5, B1 and 12. Eight of 48 cases had more than 15% J5-positive lymphocytes, and some lymphocytes in MM ex pressed plasma cell-associated antigens (PCA1, BL3, OKTIO), suggesting a possible clonal involvement. These observations demonstrate the heterogeneity of surface antigen expression of myeloma cells and suggest that BL3, PCA1, BA2 and J5 may be useful mAbs for purging myeloma cells from bone marrow for autologous transplantation.
Multiple myeloma (MM) is a neoplasm predomi nantly composed of plasma cells in the terminal stage cell of B cell differentiation producing monoclonal immunoglobulins. However, studies using anti-idiotypic antibody have shown that not only myeloma cells but also B and pre-B cells are clonally involved [1-3]. Recently Caligaris-Cappio et al. [4] demon strated that common acute lymphoblastic leukemia antigen (CALLA)-positive pre-B cells are precursors of myeloma cells. However, little is known about the process of in vivo maturation from the precursor cells to differentiated myeloma cells which can be identi fied by morphological examination. In order to further define the immunophénotype of both the morphologically recognizable myeloma cells and the potential progenitor cells appearing morpho logically as small lymphocytes, we examined the sur face antigen expression of lymphocytes and myeloma
cells in the bone marrow of MM and peripheral blood of plasma cell leukemia (PCL) using a panel of mon oclonal antibodies (mAbs) which identify different stages of B cell differentiation and also T cells. We also discuss which mAbs are likely to be most useful for purging myeloma cells and their precursors from the graft ex vivo in preparation for autologous bone marrow transplantation. Materials and Methods Patients
Forty-seven patients with MM and I patient with PCL were in cluded in this study. The untreated patients were studied at the time of diagnosis and the previously treated patients were studied at the time of relapse. Thirty-one cases were IgG myeloma, 8 were IgA, I was IgD and IgM, 5 were Bence Jones myeloma and 2 were non producing myeloma. The patients were classified by the clinical staging system proposed by Durie and Salmon [5]: 4 were in stage I. 18 in stage II, and 26 in stage III. Three cases were diagnosed as plasmablastic subtype by the criteria proposed by Greipp et al. (6).
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Introduction
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Table 1. Monoclonal antibodies used in this study Antibody
Antigen
Antigen expression on normal cells
Reference
J5 BA2 B4 Bl BLI BL3 PCA1 OKTIO 12 Tl 1
CD10a, 95 kD (CALLA) CD9,24 kD CD19,94 kD CD20, 35 kD NAb, glycolipid NA, 105 kD NA, NR1 CD38,45 kD NA, 28-34 kD CD2, 50 kD
pre-B cell, granulocyte pre-B cell pan B cell pan B cell pan B cell plasma cell, mature B cell plasma cell, granulocyte plasma cell, activated T and B cell pan B cell, monocyte T cell
Ritz et al. [9] Kersey et al. [10] Nadler et al. [11] Stashenko et al. [12] Wang et al. [13] Wanget al.[13] Anderson et al. [14] Reinherz et al. [15] Nadler et al. [16] Kamoun et al. [17]
a Clusters of differentiation (CD) defined by the Second International Workshop on Human Leukocyte Differentiation Antigens. b Not applicable. c Not reported.
in RPMI-1640 medium supplemented with 10% fetal calf serum (Hyclone, Logan, Utah), I % penicillin-streptomycin-neomycin (Gibco, Grand Island, N.Y.), and 1% L-glutamine. Cells were either used immediately for cell surface marker anal ysis or were cryopreserved for future use using a mixture contain ing 5% dimethylsulfoxide, 6% hydroxyethyl starch [7], and 45% au tologous plasma or serum. Frozen cells were stored in the vapor phase of liquid nitrogen. Prior to analysis, the cells were thawed in the presence of 60 U of deoxyribonuclease I (Cooper Biomedical, Malvern, Pa.), and then viable cells were separated by sedimenta tion on a Ficoll-Hypaque density cushion. This procedure enriches myeloma cells since freezing destroys mature myeloid cells [8],
icu +-> 4-> ns o co
o
Monoclonal Antibodies
o
CTi
Ten mAbs used in this study are shown in table I together with a summary of their reactivities and references [9-17], J5, B4, Bl, 12, PCA1 and T11 were kindly provided by Coulter Immunology, Hia leah, Fla. BA2 was obtained from Hybritech, San Diego, Calif. OKTIO was obtained from Ortho Immunology, Raritan, N.J. BL1 and BL3 were provided by Dr. C.Y. Wang and R. Wisniewolski.
Fig. 1. Forward and 90° light scatter dot plot of myeloma mar row using the FACScan flow cytometer. Bone marrow mononu clear cells from myeloma marrow were divided into five compart ments: red blood cells ( 1), granulocytes (2), monocytes (3), lympho cytes (Lym), and plasma cells (PC). The gates used for surface marker analysis of lymphocytes and plasma cells are indicated.
Cells
Bone marrow cells were obtained from the posterior iliac crest of 4 healthy volunteers and patients with MM after informed writ ten consent. Peripheral blood from 1 patient with PCL was also ex amined. The mononuclear cells were separated through FicollHypaque density centrifugation, washed twice and resuspended
Cell Surface Marker Analysis
Indirect immunofluorescence was performed using methods de scribed previously [18]. Briefly, the cells were suspended in RPMI-1640 medium containing 5% autologous plasma (to mini mize nonspecific binding of mAbs to Fc receptors) and incubated with saturating amounts of mAbs for 30 min at 4°C. Control sam ples were incubated with purified mouse IgGl or IgG2a (Coulter Immunology). After washing twice, the cells were stained with 1:50 dilution of fluorescein isothiocyanate (FITC)-conjugated F(ab')2 fragments of goat antimouse IgG (Cooper Biomedical) for 30 min at 4°C, washed twice and resuspended in phosphate-buffered saline with 2% fetal calf serum. Cells were analyzed using a FACS-IV (Becton Dickinson) or a FACScan flow cytometer (Becton Dickin son). Forward and 90° light scatter signals were used as gates for the selection of lymphocyte and plasma cell populations [19-21], Data were analyzed at the SKI Core Computer Facility using a PDP11/70 computer or by the FACScan Consort 30 software.
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Forward scatter
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Table 2. Immunophénotype of lymphocytes in MM and normal bone marrow
Number of patients3with 15% positive Total % positive cells (mean ± SD)b in MM Normal bone marrowc
BA2
B4
Bl
BL1
BL3
31 9 8
3 3 2
7 4 4
10 4 4
4 0 6
9 4 9
17 6 3
48
8
15
18
10
22
26
9± 8 22 ±6
7 ±9 15 ± 5
13 ± 12 20c
13 ± 17 O'
6± 9 11 ±4
17 ± 26 NT1
5 ±8 0±0
12
Til
3 7 7
2 9 11
0 1 9
17
22
10
18 ± 14 9± 6
17 ± 10 27 ± 5
48 ±25 52 ± 11
OKTIO
Number of patients with less than 5% positive cells, 5-15% positive cells, and greater than 15% positive cells. Percentage of postitve cells for each antigen in MM marrows. Percentage of positive cells in 4 normal bone marrows. Not tested. Data in 1 normal bone marrow.
Results Forward and 90 ° Light Scatter Display o f Myeloma Marrow A representative forward and 90° light scattergram of MM marrow is shown in figure 1. Similar patterns were observed in most patients, including 1 with PCL. Bone marrow mononuclear cells were classified into five compartments: red blood cells, granulocytes, monocytes, lymphocytes and plasma cells. Cells in cluded in each fraction were identified by morpholog ical examination of cells sorted using the FACS-IV (lymphocytes, granulocytes, plasma cells) or by double staining using phycoerythrin-conjugated Leu M3 (monocytes) and FITC-labeled OKTIO (plasma cells). Calligaris-Cappio et al. [4] demonstrated that at least some myeloma precursor cells were CALLApositive small lymphoid cells. Therefore we examined the surface antigen expression on small lymphocytes using the gate shown in figure 1. The purity of the lymphocyte fraction was more than 90% as deter mined by morphological examination of sorted cells in this fraction and by phenotypic analysis using mAbs which demonstrated that RIO (erythroid cell marker) or My8 (mature neutrophil and monocyte) positive cells were below 10% (unpublished data). The gate defining plasma cells is also shown in figure 1. The purity of the plasma cell fraction was as high as that of the lymphocyte fraction (>90%) in cases with greater than 30% myeloma cells in the mar row.
Immunological Phenotype o f Lymphocytes The immunophénotype of lymphocytes in MM marrows including blood from PCL and normal bone marrows is shown in table 2. For each antibody, the patients were divided into three categories depending on the percentages of positive cells. Of 48 patients tested for J5 binding, 6% of lympho cytes were positive as compared to 11 % in normal bone marrow. Only 8 patients had more than 15% CALLApositive cells and no correlation was observed between the percentages of CALLA-positive cells and the clini cal stage of patients (data not shown). The majority of patients exhibited decreased num bers of cells with pan B cell antigens (9, 7 and 17% positive lymphocytes for B4, B1 and 12, respectively) compared to normal marrows. Two of 8 patients were highly positive for BA2 (79 and 38%). Nine of 22 and 3 of 26 patients expressed plasma cell-associated anti gens (BL3 and PCA1 > 15%). OKTIO was expressed in 15% of cells in 7 of 17 as well. The mean percentage of cells positive for these late B and myeloma markers was higher in patients’ marrows than in normal mar row samples. T cells were not reduced in MM (except for 1 case) as compared to normal bone marrow. Immunological Phenotype o f Myeloma Cells Nineteen cases with greater than 30% myeloma cells were studied including 2 cases with fewer mye loma cells initially (18 and 15%), in which myeloma cells were enriched by treatment with freezing and thawing as described above.
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3 b c 11 c
PCA1
J5
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Table 3. Immunological phenotype of myeloma cells
Number of patients'with < 15% positive 15-30% positive 30-60% positive >60% positive Total
J5
BA2
B4
Bl
BLI
BL3
12 4 1 1
1 0 5 2
13 0 0 0
12 0 2 0
6 0 0 0
3 1 3 II
18
8
13
14
6
18
OKTIO
12
7 3 5 4
0 0 4 10
5 2 1 1
19
14
9
PCA1
a Number of patients with less than 15% positive cells, 15-30% positive cells, 30-60% positive cells and greater than 60% positive cells.
Case
1 2 3
% of positive cells J5
BA2
Bl
BL3
PCAI
OKTIO
12
35 17 100
NT NT 90
30 4 0
80 47 0
90 95 0
NT 93 100
NT NT 0
NT = Not tested.
The results of immunophenotypic analysis of mye loma cells are shown in table 3. For the purposes of this discussion, we will consider patients to be posi tive for a given antigen if at least 15% of their mye loma cells are positive for that antigen. OKTIO was positive in all 14 patients tested, and more than 60% of the myeloma cells were positive in 10 of them. BL3 was positive in 15 of 18 patients tested. More than 60% of the myeloma cells were positive in 11 out of 18 patients, and only 3 had fewer than 15% positive cells. PCA1 was positive in 12 of 19 cases and was greater than 60% in 4 of these. J5 was negative in 12 out of 18 cases; however, more than 30% of the cells were pos itive in 2 cases (100% in 1 PCL and 35% in 1 MM, re spectively). Stages of 6 cases with J5-positive mye loma cells were as follows: 1 in stage I, 3 in stage II and 2 in stage III. B4 and BL1 were negative in all cases examined, and B1 was negative except for 2 cases (45 and 30%). BA2 was positive in 7 of 8 cases, and in 2 of them greater than 60% of the myeloma
cells were positive. 12 was negative in 5 of 9 cases; however, more than 60% positive cells were found in 1 case. The immunophenotype of myeloma cells in 3 cases of plasmablastic subtype as proposed by Greipp et al. [6] is shown in table 4. Patient 3 had PCL which had an unusual immunophenotype. OKTIO was highly positive in 2 cases tested, and PCA1 and BL3 were positive in 2 of 3 cases. An interesting Finding was that all cases were positive for J5, in contrast to the obser vation that 12 out of 15 cases in the other type of MM were negative for J5. B1 was positive in 1 case (30%), while 12 out of 13 cases in MM other than this subtype were negative.
Discussion MM is a tumor of the most differentiated B cells and has been considered to be more homogeneous for surface antigen expression than the histologically de fined subgroups at the early and mid-stage of B cell differentiation [22], However, in the present study, we demonstrated that MM might be more heterogeneous than previously considered. We found the major phenotype of MM to be positive for OKTIO, BL3, PCA1 and BA2, and negative for J5, Bl, B4, BL1 and 12. In some cases, however, myeloma cells expressed B cell antigens such as J5, BA2, Bl and 12, recently also reported by others [23-26]. An interesting finding is that more than 30% of the myeloma cells in 7 out of 8 cases expressed antigens detected by BA2, which is ordinarily expressed on early B cells. San Miguel et al. [24] also reported that most myeloma cells (80%) reacted with FMC8, which recognizes CD9 antigen but also reacts with mono-
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Table 4. Immunological phenotype of plasmablastic myeloma
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cytes. Katagiri et al. [23] demonstrated that 3 of 18 pa suggested these cells to be the myeloma stem cell. In tients were positive for BA2 and that the myeloma cell other studies, Lokhorst et al. [30] identified a CALLAline RPM18226 was also positive. It is notable that negative precursor of myeloma cells which was char some myeloma cells express CALLA, especially in the acterized by a small cytoplasmic spot of immunoglob plasmablastic subtype. Durie and Grogan [25] first ulin of the relevant idiotype. This cell was positive for reported CALLA-positive myeloma as a subgroup HB4 and HB6, but did not express other B cell anti with extremely poor prognosis, and San Miguel et al. gens or plasmacytoid antigens. Therefore, definitive [27] demonstrated the phenotype of plasmablastic identification of the myeloma precursor cell remains myeloma to be CD38(OK.T10)+, CD10(J5)+, uncertain. CD9(FMC8) + , CD20(B1)+, HLA-DR + , suggesting Lymphocytes expressing pan B cell antigens (Bl, B4, la) appear to be decreased in MM as compared to that myeloma cells in this type bear a more immature immunophénotype than in the other morphological normal bone marrow. In addition, some lymphocytes types. In our series the stages and the survivals in 6 in MM express late B and plasma cell-associated anti patients with CALLA-positive myeloma cells were gens (PCA1, BL3, OK.T10). The same findings were different, while the patient with massive (100%) seen in the blood of MM, where surface immunoglob CALLA-positive myeloma cells was resistant to ulin-positive B cells were decreased but an abnor chemotherapy and died within 3 months. Considering mally large pool of pre-B cell was present [31]. Some that CALLA is positive on many subtypes of lym of these cells expressed plasma cell antigens [32, 33], phoma [28], the plasmablastic subtype may be closer suggesting that these lymphocytes were involved with to lymphoma than to terminally differentiated mye the neoplastic clone. Studies using anti-idiotype an loma. Our data support this hypothesis; all 3 plasma tibody [1-3] and molecular genetics [34] supported blastic cases which we examined were CALLA-posi this hypothesis. At least part of the heterogeneity of the lymphocyte tive and 1 was also positive for Bl. These findings, taken together with the BA2 expression described compartment with respect to surface antigen expres above, support the close relationship between sion was probably due to the fact that the lymphocyte myeloma cells and B cells of an earlier immuno gate included both normal lymphocytes and an un known percentage of myeloma progenitor cells. Fur phénotype. Caligaris-Cappio et al. [4] reported that half of thermore, most of the cells within the gate were small their myeloma patients had significant numbers of nondividing lymphocytes, as determined by cell sort CALLA-positive lymphocytes (median 30% positive ing; larger proliferating lymphocytes and lymphocells). They demonstrated that these cells can be in plasmacytoid cells were probably intermediate in duced by phorbol ester to differentiate into plasma light scatter values between those of lymphocytes and cells which synthesize immunoglobulin of the same plasma cells and may have been included in the mon isotype as the patient’s myeloma cells, implying that ocyte compartment. A possible method for distin these CALLA-positive lymphocytes are myeloma guishing between normal lymphocytes and myeloma progenitor cells. In contrast to their report, we found progenitor cells, at least for hyperdiploid tumors, that only 8 of 48 patients had greater than 15% would be to employ DNA content as an additional CALLA-positive lymphocytes. One possible explana gating parameter (e.g., using propidium iodide). The results obtained in this study suggest possible tion for this discrepancy is that they used immunocytochemical methods which could detect intracytoplas- antibodies for purging myeloma cells ex vivo in pre mic antigens, while we used a flow cytometric method paration for autologous bone marrow transplanta to detect surface antigens. In this context, Caligaris- tion. The important characteristics of mAbs for this Cappio found that some cases have numerous lym purpose is that they react well with myeloma cells phoid cells which do not express CALLA on the sur and/or their precursors but do not react with normal face but are CALLA-positive in the cytoplasm hematopoietic progenitors. In this context, our obser [personal communication]. vations suggest that a cocktail of antibodies which in More recently, Grogan et al. [29] found a novel cludes BL3, PCA1 and BA2 may be useful. Since mye pre-B cell component with coexpression of cytoplas loma cells are heterogeneous for their surface antigen mic p, CALLA, terminal deoxynucléotidyl transfer expression as demonstrated above, a combination of ase and plasma cell antigen (PCA1 and PC-1) and these mAbs may be appropriate. The next logical step
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Immunophénotype of Myeloma Cells
would be to determine the percentage of myeloma cells remaining after incubation with this combina tion. A search for additional antibodies labeling these remaining cells would then be initiated. Because J5-positive cells might include at least some of the myeloma progenitors and some of myeloma cells were positive for J5, this antibody should also be con sidered for use in purging of MM. Experiments in volving the purging of myeloma cells using these mAbs and immunomagnetic beads are currently in progress [35].
Acknowledgements The authors would like to thank Lorraine Horowitz-Fenchel and Susan De Meritt for excellent administrative assistance and Barbara McFlendry for the FACScan analyses. This work was supported in part by grants PO1-CA-20194 and ROl-CA-19117 from the National Institutes of Health, USPHS.
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lymphohemopoietic progenitor cell surface structure identified with monoclonal antibody. J Exp Med 1981;153:726-731. 11 Nadler LM, Anderson KC, Marti G, Bates MP, Park EK, Da ley JF, Schlossman SF: B4, a human B lymphocyte associated antigen expressed on normal, mitogen activated, and malignant B lymphocytes. J Immunol 1983:131:244-250. 12 Stashenko P, Nadler LM, Hardy R, Schlossman SF: Characteri zation of a human B lymphocyte specific antigen. J Immunol 1980;125:1678-1685. 13 Wang CY, Azzo W, Al-katib A, Chiorazzi N, Knowles DM II: Preparation and characterization of monoclonal anti bodies recognizing three distinct differentiation antigens (BL1, BL2, BL3)on human Blymphocytes. J Immunol 1984;133: 684-691. 14 Anderson KC, Pari EK, Bates MP, Leonard RCF, Hardy R, Schlossman SF, Nadler LM: Antigens on human plasma cells identified by monoclonal antibodies. J Immunol 1983:130: 1132-1138. 15 Reinherz EL, Kung PC, Goldstein G, Levy RH, Schlossman SF: Discrete stages of human intrathymic differentiation: Anal ysis of normal thymocytes and leukemic lymphoblasts on T line age. Proc Natl Acad Sci USA 1980;77:1588-1592. 16 Nadler LM, Stashenko P, Hardy R, Pesando JM, Yunis EJ, Schlossman SF: Monoclonal antibodies defining serologically distinct HLA-D/DR related Ia-like antigens in man. Hum Im munol 1981;1:77-90. 17 Kamoun M, Martin PJ, Hanson JA, Brown MA, Siadek AW, Nowinski RC: Identification of human T lymphocyte surface protein associated with the E rosette receptor. J Exp Med 1981;153:207-212. 18 Fried J, Shimazaki C, Perez AG, Wisniewolski R, Wang CY: Application of flow cytometry to the analysis of multiple mye loma cells and their progenitors; in Yen A (ed): Flow Cytome try. Boca Raton, CRC Press, 1989, pp 59-78. 19 Salzman GC, Crowell JM, Martin BA, Trujillo TT, Romero A, Mullaney PF, LaBauve PM: Ceil classification by laser light scattering: Identification and separation of unstained leuko cytes. ActaCytol 1975:19:374-377. 20 Van der Engh G, Visser J: Light scattering properties of pluri potent and committed haemopoietic stem cells. Acta Haematol 1979:62:289-298. 21 Atzpodien J, Gulati S, Kwon J H, Wachter M, Fried J, Clarkson BD: Human bone marrow CFU-GM and BFU-E localized by light scatter cell sorting. Exp Cell Biol 1987;55:265-270. 22 Anderson KC, Bates MP, Slaughenhoupt BL, Pinkus GS, Schlossman SF, Nadler LM: Expression of human B cell-asso ciated antigens on leukemias and lymphomas: A model of hu man B cell differentiation. Blood 1984;63:1424-1433. 23 Katagiri S, Yonezawa T, Tamaki T, Kanayama Y, Kuyama J, Ohnishi M, Tsubakio T, Kurata Y, Tarui S: Surface expression of human myeloma cells: An analysis using a panel of mono clonal antibodies. Acta Haematol 1984;72:372-378. 24 San Miguel JF, Gonzalez CM, Zola H, Borrasca AL: Immuno logical phenotype of neoplasms involving the B cell in the last step of differentiation. BrJ Haematol 1986;62:75-83. 25 Durie BGM, Grogan TM: CALLA-positive myeloma: An ag gressive subtype with poor survival. Blood 1985;66:229-232. 26 Epstein J, Barlogie B, Katzmann J, Alexanian R: Phenotypic heterogeneity in aneuploid multiple myeloma indicates pre-B cell involvement. Blood 1988;71:861-865.
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Received: August 7, 1989 Accepted: October 10, 1989 Dr. Chihiro Shimazaki Second Department of Medicine Kyoto Prefectural University of Medicine Kawaramachi-hirokoji, Kamikyoku Kyoto 602 (Japan)
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27 San Miguel JF, Moro MJ, Gonzalez M: Plasmablastic multiple myeloma: An immunologically different subtype. Br J Haema tol 1987:66:275. 28 Ritz J, Nadler LM, Bhan AK, Notis-McConarty J, Pesando JM, Schlossman SF: Expression of common acute lymphoblas tic leukemia antigen (CALLA) by lymphomas of B-cell and Tcell lineage. Blood 1981;58:648-652. 29 Grogan TM, Durie BGM, Lomen C, Spier C, Wirt DP, Nagle R, Wilson GS, Richter L, Vela E, Maxey V, McDaniel K. Ran gel C: Delineation of a novel pre-B cell component in plasma cell myeloma: Immunochemical, immunophenotypic, genotyp ic, cytologic, cell culture, and kinetic features. Blood 1988; 70:932-942. 30 Lokhorst HM, Boom SE, Bast BJEG, Peters PJ, Tedder TF, Gerdes J, Petersen E, Ballieux RE: A novel type of proliferating lymphoplasmacytoid cell with a characteristic spotted immu nofluorescence pattern. J Clin Invest 1987;79:1401-1411. 31 Pilarski LM, Mant MJ, Ruether BA: Pre-B cells in peripheral blood of multiple myeloma patients. Blood 1985;66:416-422. 32 Ruiz-Arguelles GJ, Katzmann JA, Greipp PR, Gonchoroff NJ, Garton JP, Kyle RA: Multiple myeloma: Circulating lympho cytes that express plasma cell antigens. Blood 1984;64:352-356. 33 Boccadoro M, Omede P, Massaia M, Dianzani U, Pioppo P, Battaglio S, Meregalli M, Pileri A: Human myeloma: Several subsets of circulating lymphocytes express plasma-cell-asso ciated antigens. Eur J Haematol 1988;40:299-304.
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Immunophenotypic Analysis of Lymphocytes and Myeloma Cells in Patients with Multiple Myeloma Chihiro Shimazaki, Jerrold Fried, Amaury G. Perez, David A. Scheinberg, Jens Atzpodien, Chang Y. Wang, Rosanne Wisniewolski, Bayard D. Clarkson Laboratories of Hematopoietic Cel! Kinetics and Human Cancer Immunology, Memorial Sloan-Kettering Cancer Center, New York, N.Y.; United Biomedical, Inc., Lake Success, N.Y., USA
Key Words. Immunophénotype • Monoclonal antibodies • Multiple myeloma • Plasma cells Abstract. The immunological phenotypes of lymphocytes and myeloma cells in 48 patients with multiple myeloma (MM) were analyzed using a panel of monoclonal antibodies (mAbs). Myeloma cells were positive for OKTIO, BL3, PCA1 and BA2. In a few cases, they were also positive for the B cell-associated antigens J5, B1 and 12. Eight of 48 cases had more than 15% J5-positive lymphocytes, and some lymphocytes in MM ex pressed plasma cell-associated antigens (PCA1, BL3, OKTIO), suggesting a possible clonal involvement. These observations demonstrate the heterogeneity of surface antigen expression of myeloma cells and suggest that BL3, PCA1, BA2 and J5 may be useful mAbs for purging myeloma cells from bone marrow for autologous transplantation.
Multiple myeloma (MM) is a neoplasm predomi nantly composed of plasma cells in the terminal stage cell of B cell differentiation producing monoclonal immunoglobulins. However, studies using anti-idiotypic antibody have shown that not only myeloma cells but also B and pre-B cells are clonally involved [1-3]. Recently Caligaris-Cappio et al. [4] demon strated that common acute lymphoblastic leukemia antigen (CALLA)-positive pre-B cells are precursors of myeloma cells. However, little is known about the process of in vivo maturation from the precursor cells to differentiated myeloma cells which can be identi fied by morphological examination. In order to further define the immunophénotype of both the morphologically recognizable myeloma cells and the potential progenitor cells appearing morpho logically as small lymphocytes, we examined the sur face antigen expression of lymphocytes and myeloma
cells in the bone marrow of MM and peripheral blood of plasma cell leukemia (PCL) using a panel of mon oclonal antibodies (mAbs) which identify different stages of B cell differentiation and also T cells. We also discuss which mAbs are likely to be most useful for purging myeloma cells and their precursors from the graft ex vivo in preparation for autologous bone marrow transplantation. Materials and Methods Patients
Forty-seven patients with MM and I patient with PCL were in cluded in this study. The untreated patients were studied at the time of diagnosis and the previously treated patients were studied at the time of relapse. Thirty-one cases were IgG myeloma, 8 were IgA, I was IgD and IgM, 5 were Bence Jones myeloma and 2 were non producing myeloma. The patients were classified by the clinical staging system proposed by Durie and Salmon [5]: 4 were in stage I. 18 in stage II, and 26 in stage III. Three cases were diagnosed as plasmablastic subtype by the criteria proposed by Greipp et al. (6).
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Introduction
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Table 1. Monoclonal antibodies used in this study Antibody
Antigen
Antigen expression on normal cells
Reference
J5 BA2 B4 Bl BLI BL3 PCA1 OKTIO 12 Tl 1
CD10a, 95 kD (CALLA) CD9,24 kD CD19,94 kD CD20, 35 kD NAb, glycolipid NA, 105 kD NA, NR1 CD38,45 kD NA, 28-34 kD CD2, 50 kD
pre-B cell, granulocyte pre-B cell pan B cell pan B cell pan B cell plasma cell, mature B cell plasma cell, granulocyte plasma cell, activated T and B cell pan B cell, monocyte T cell
Ritz et al. [9] Kersey et al. [10] Nadler et al. [11] Stashenko et al. [12] Wang et al. [13] Wanget al.[13] Anderson et al. [14] Reinherz et al. [15] Nadler et al. [16] Kamoun et al. [17]
a Clusters of differentiation (CD) defined by the Second International Workshop on Human Leukocyte Differentiation Antigens. b Not applicable. c Not reported.
in RPMI-1640 medium supplemented with 10% fetal calf serum (Hyclone, Logan, Utah), I % penicillin-streptomycin-neomycin (Gibco, Grand Island, N.Y.), and 1% L-glutamine. Cells were either used immediately for cell surface marker anal ysis or were cryopreserved for future use using a mixture contain ing 5% dimethylsulfoxide, 6% hydroxyethyl starch [7], and 45% au tologous plasma or serum. Frozen cells were stored in the vapor phase of liquid nitrogen. Prior to analysis, the cells were thawed in the presence of 60 U of deoxyribonuclease I (Cooper Biomedical, Malvern, Pa.), and then viable cells were separated by sedimenta tion on a Ficoll-Hypaque density cushion. This procedure enriches myeloma cells since freezing destroys mature myeloid cells [8],
icu +-> 4-> ns o co
o
Monoclonal Antibodies
o
CTi
Ten mAbs used in this study are shown in table I together with a summary of their reactivities and references [9-17], J5, B4, Bl, 12, PCA1 and T11 were kindly provided by Coulter Immunology, Hia leah, Fla. BA2 was obtained from Hybritech, San Diego, Calif. OKTIO was obtained from Ortho Immunology, Raritan, N.J. BL1 and BL3 were provided by Dr. C.Y. Wang and R. Wisniewolski.
Fig. 1. Forward and 90° light scatter dot plot of myeloma mar row using the FACScan flow cytometer. Bone marrow mononu clear cells from myeloma marrow were divided into five compart ments: red blood cells ( 1), granulocytes (2), monocytes (3), lympho cytes (Lym), and plasma cells (PC). The gates used for surface marker analysis of lymphocytes and plasma cells are indicated.
Cells
Bone marrow cells were obtained from the posterior iliac crest of 4 healthy volunteers and patients with MM after informed writ ten consent. Peripheral blood from 1 patient with PCL was also ex amined. The mononuclear cells were separated through FicollHypaque density centrifugation, washed twice and resuspended
Cell Surface Marker Analysis
Indirect immunofluorescence was performed using methods de scribed previously [18]. Briefly, the cells were suspended in RPMI-1640 medium containing 5% autologous plasma (to mini mize nonspecific binding of mAbs to Fc receptors) and incubated with saturating amounts of mAbs for 30 min at 4°C. Control sam ples were incubated with purified mouse IgGl or IgG2a (Coulter Immunology). After washing twice, the cells were stained with 1:50 dilution of fluorescein isothiocyanate (FITC)-conjugated F(ab')2 fragments of goat antimouse IgG (Cooper Biomedical) for 30 min at 4°C, washed twice and resuspended in phosphate-buffered saline with 2% fetal calf serum. Cells were analyzed using a FACS-IV (Becton Dickinson) or a FACScan flow cytometer (Becton Dickin son). Forward and 90° light scatter signals were used as gates for the selection of lymphocyte and plasma cell populations [19-21], Data were analyzed at the SKI Core Computer Facility using a PDP11/70 computer or by the FACScan Consort 30 software.
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Forward scatter
Immunophénotype of Myeloma Cells
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Table 2. Immunophénotype of lymphocytes in MM and normal bone marrow
Number of patients3with 15% positive Total % positive cells (mean ± SD)b in MM Normal bone marrowc
BA2
B4
Bl
BL1
BL3
31 9 8
3 3 2
7 4 4
10 4 4
4 0 6
9 4 9
17 6 3
48
8
15
18
10
22
26
9± 8 22 ±6
7 ±9 15 ± 5
13 ± 12 20c
13 ± 17 O'
6± 9 11 ±4
17 ± 26 NT1
5 ±8 0±0
12
Til
3 7 7
2 9 11
0 1 9
17
22
10
18 ± 14 9± 6
17 ± 10 27 ± 5
48 ±25 52 ± 11
OKTIO
Number of patients with less than 5% positive cells, 5-15% positive cells, and greater than 15% positive cells. Percentage of postitve cells for each antigen in MM marrows. Percentage of positive cells in 4 normal bone marrows. Not tested. Data in 1 normal bone marrow.
Results Forward and 90 ° Light Scatter Display o f Myeloma Marrow A representative forward and 90° light scattergram of MM marrow is shown in figure 1. Similar patterns were observed in most patients, including 1 with PCL. Bone marrow mononuclear cells were classified into five compartments: red blood cells, granulocytes, monocytes, lymphocytes and plasma cells. Cells in cluded in each fraction were identified by morpholog ical examination of cells sorted using the FACS-IV (lymphocytes, granulocytes, plasma cells) or by double staining using phycoerythrin-conjugated Leu M3 (monocytes) and FITC-labeled OKTIO (plasma cells). Calligaris-Cappio et al. [4] demonstrated that at least some myeloma precursor cells were CALLApositive small lymphoid cells. Therefore we examined the surface antigen expression on small lymphocytes using the gate shown in figure 1. The purity of the lymphocyte fraction was more than 90% as deter mined by morphological examination of sorted cells in this fraction and by phenotypic analysis using mAbs which demonstrated that RIO (erythroid cell marker) or My8 (mature neutrophil and monocyte) positive cells were below 10% (unpublished data). The gate defining plasma cells is also shown in figure 1. The purity of the plasma cell fraction was as high as that of the lymphocyte fraction (>90%) in cases with greater than 30% myeloma cells in the mar row.
Immunological Phenotype o f Lymphocytes The immunophénotype of lymphocytes in MM marrows including blood from PCL and normal bone marrows is shown in table 2. For each antibody, the patients were divided into three categories depending on the percentages of positive cells. Of 48 patients tested for J5 binding, 6% of lympho cytes were positive as compared to 11 % in normal bone marrow. Only 8 patients had more than 15% CALLApositive cells and no correlation was observed between the percentages of CALLA-positive cells and the clini cal stage of patients (data not shown). The majority of patients exhibited decreased num bers of cells with pan B cell antigens (9, 7 and 17% positive lymphocytes for B4, B1 and 12, respectively) compared to normal marrows. Two of 8 patients were highly positive for BA2 (79 and 38%). Nine of 22 and 3 of 26 patients expressed plasma cell-associated anti gens (BL3 and PCA1 > 15%). OKTIO was expressed in 15% of cells in 7 of 17 as well. The mean percentage of cells positive for these late B and myeloma markers was higher in patients’ marrows than in normal mar row samples. T cells were not reduced in MM (except for 1 case) as compared to normal bone marrow. Immunological Phenotype o f Myeloma Cells Nineteen cases with greater than 30% myeloma cells were studied including 2 cases with fewer mye loma cells initially (18 and 15%), in which myeloma cells were enriched by treatment with freezing and thawing as described above.
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3 b c 11 c
PCA1
J5
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Shimazaki/Fried/Perez/Scheinberg/Atzpodien/Wang/Wisniewolski/Clarkson
Table 3. Immunological phenotype of myeloma cells
Number of patients'with < 15% positive 15-30% positive 30-60% positive >60% positive Total
J5
BA2
B4
Bl
BLI
BL3
12 4 1 1
1 0 5 2
13 0 0 0
12 0 2 0
6 0 0 0
3 1 3 II
18
8
13
14
6
18
OKTIO
12
7 3 5 4
0 0 4 10
5 2 1 1
19
14
9
PCA1
a Number of patients with less than 15% positive cells, 15-30% positive cells, 30-60% positive cells and greater than 60% positive cells.
Case
1 2 3
% of positive cells J5
BA2
Bl
BL3
PCAI
OKTIO
12
35 17 100
NT NT 90
30 4 0
80 47 0
90 95 0
NT 93 100
NT NT 0
NT = Not tested.
The results of immunophenotypic analysis of mye loma cells are shown in table 3. For the purposes of this discussion, we will consider patients to be posi tive for a given antigen if at least 15% of their mye loma cells are positive for that antigen. OKTIO was positive in all 14 patients tested, and more than 60% of the myeloma cells were positive in 10 of them. BL3 was positive in 15 of 18 patients tested. More than 60% of the myeloma cells were positive in 11 out of 18 patients, and only 3 had fewer than 15% positive cells. PCA1 was positive in 12 of 19 cases and was greater than 60% in 4 of these. J5 was negative in 12 out of 18 cases; however, more than 30% of the cells were pos itive in 2 cases (100% in 1 PCL and 35% in 1 MM, re spectively). Stages of 6 cases with J5-positive mye loma cells were as follows: 1 in stage I, 3 in stage II and 2 in stage III. B4 and BL1 were negative in all cases examined, and B1 was negative except for 2 cases (45 and 30%). BA2 was positive in 7 of 8 cases, and in 2 of them greater than 60% of the myeloma
cells were positive. 12 was negative in 5 of 9 cases; however, more than 60% positive cells were found in 1 case. The immunophenotype of myeloma cells in 3 cases of plasmablastic subtype as proposed by Greipp et al. [6] is shown in table 4. Patient 3 had PCL which had an unusual immunophenotype. OKTIO was highly positive in 2 cases tested, and PCA1 and BL3 were positive in 2 of 3 cases. An interesting Finding was that all cases were positive for J5, in contrast to the obser vation that 12 out of 15 cases in the other type of MM were negative for J5. B1 was positive in 1 case (30%), while 12 out of 13 cases in MM other than this subtype were negative.
Discussion MM is a tumor of the most differentiated B cells and has been considered to be more homogeneous for surface antigen expression than the histologically de fined subgroups at the early and mid-stage of B cell differentiation [22], However, in the present study, we demonstrated that MM might be more heterogeneous than previously considered. We found the major phenotype of MM to be positive for OKTIO, BL3, PCA1 and BA2, and negative for J5, Bl, B4, BL1 and 12. In some cases, however, myeloma cells expressed B cell antigens such as J5, BA2, Bl and 12, recently also reported by others [23-26]. An interesting finding is that more than 30% of the myeloma cells in 7 out of 8 cases expressed antigens detected by BA2, which is ordinarily expressed on early B cells. San Miguel et al. [24] also reported that most myeloma cells (80%) reacted with FMC8, which recognizes CD9 antigen but also reacts with mono-
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Table 4. Immunological phenotype of plasmablastic myeloma
127
cytes. Katagiri et al. [23] demonstrated that 3 of 18 pa suggested these cells to be the myeloma stem cell. In tients were positive for BA2 and that the myeloma cell other studies, Lokhorst et al. [30] identified a CALLAline RPM18226 was also positive. It is notable that negative precursor of myeloma cells which was char some myeloma cells express CALLA, especially in the acterized by a small cytoplasmic spot of immunoglob plasmablastic subtype. Durie and Grogan [25] first ulin of the relevant idiotype. This cell was positive for reported CALLA-positive myeloma as a subgroup HB4 and HB6, but did not express other B cell anti with extremely poor prognosis, and San Miguel et al. gens or plasmacytoid antigens. Therefore, definitive [27] demonstrated the phenotype of plasmablastic identification of the myeloma precursor cell remains myeloma to be CD38(OK.T10)+, CD10(J5)+, uncertain. CD9(FMC8) + , CD20(B1)+, HLA-DR + , suggesting Lymphocytes expressing pan B cell antigens (Bl, B4, la) appear to be decreased in MM as compared to that myeloma cells in this type bear a more immature immunophénotype than in the other morphological normal bone marrow. In addition, some lymphocytes types. In our series the stages and the survivals in 6 in MM express late B and plasma cell-associated anti patients with CALLA-positive myeloma cells were gens (PCA1, BL3, OK.T10). The same findings were different, while the patient with massive (100%) seen in the blood of MM, where surface immunoglob CALLA-positive myeloma cells was resistant to ulin-positive B cells were decreased but an abnor chemotherapy and died within 3 months. Considering mally large pool of pre-B cell was present [31]. Some that CALLA is positive on many subtypes of lym of these cells expressed plasma cell antigens [32, 33], phoma [28], the plasmablastic subtype may be closer suggesting that these lymphocytes were involved with to lymphoma than to terminally differentiated mye the neoplastic clone. Studies using anti-idiotype an loma. Our data support this hypothesis; all 3 plasma tibody [1-3] and molecular genetics [34] supported blastic cases which we examined were CALLA-posi this hypothesis. At least part of the heterogeneity of the lymphocyte tive and 1 was also positive for Bl. These findings, taken together with the BA2 expression described compartment with respect to surface antigen expres above, support the close relationship between sion was probably due to the fact that the lymphocyte myeloma cells and B cells of an earlier immuno gate included both normal lymphocytes and an un known percentage of myeloma progenitor cells. Fur phénotype. Caligaris-Cappio et al. [4] reported that half of thermore, most of the cells within the gate were small their myeloma patients had significant numbers of nondividing lymphocytes, as determined by cell sort CALLA-positive lymphocytes (median 30% positive ing; larger proliferating lymphocytes and lymphocells). They demonstrated that these cells can be in plasmacytoid cells were probably intermediate in duced by phorbol ester to differentiate into plasma light scatter values between those of lymphocytes and cells which synthesize immunoglobulin of the same plasma cells and may have been included in the mon isotype as the patient’s myeloma cells, implying that ocyte compartment. A possible method for distin these CALLA-positive lymphocytes are myeloma guishing between normal lymphocytes and myeloma progenitor cells. In contrast to their report, we found progenitor cells, at least for hyperdiploid tumors, that only 8 of 48 patients had greater than 15% would be to employ DNA content as an additional CALLA-positive lymphocytes. One possible explana gating parameter (e.g., using propidium iodide). The results obtained in this study suggest possible tion for this discrepancy is that they used immunocytochemical methods which could detect intracytoplas- antibodies for purging myeloma cells ex vivo in pre mic antigens, while we used a flow cytometric method paration for autologous bone marrow transplanta to detect surface antigens. In this context, Caligaris- tion. The important characteristics of mAbs for this Cappio found that some cases have numerous lym purpose is that they react well with myeloma cells phoid cells which do not express CALLA on the sur and/or their precursors but do not react with normal face but are CALLA-positive in the cytoplasm hematopoietic progenitors. In this context, our obser [personal communication]. vations suggest that a cocktail of antibodies which in More recently, Grogan et al. [29] found a novel cludes BL3, PCA1 and BA2 may be useful. Since mye pre-B cell component with coexpression of cytoplas loma cells are heterogeneous for their surface antigen mic p, CALLA, terminal deoxynucléotidyl transfer expression as demonstrated above, a combination of ase and plasma cell antigen (PCA1 and PC-1) and these mAbs may be appropriate. The next logical step
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Immunophénotype of Myeloma Cells
would be to determine the percentage of myeloma cells remaining after incubation with this combina tion. A search for additional antibodies labeling these remaining cells would then be initiated. Because J5-positive cells might include at least some of the myeloma progenitors and some of myeloma cells were positive for J5, this antibody should also be con sidered for use in purging of MM. Experiments in volving the purging of myeloma cells using these mAbs and immunomagnetic beads are currently in progress [35].
Acknowledgements The authors would like to thank Lorraine Horowitz-Fenchel and Susan De Meritt for excellent administrative assistance and Barbara McFlendry for the FACScan analyses. This work was supported in part by grants PO1-CA-20194 and ROl-CA-19117 from the National Institutes of Health, USPHS.
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Received: August 7, 1989 Accepted: October 10, 1989 Dr. Chihiro Shimazaki Second Department of Medicine Kyoto Prefectural University of Medicine Kawaramachi-hirokoji, Kamikyoku Kyoto 602 (Japan)
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