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Identification of Immature and Mature Myeloma Cells
in the Bone Marrow of Human Myelomas
By Michio M. Kawano, Naihui Huang, Hironori Harada, Yuka Harada, Akira Sakai,
Hideo Tanaka, Kouji Iwato, and Atsushi Kuramoto
With regard to the expression of adhesion molecules, human myeloma cells freshly isolated from bone marrow
were heterogeneous. By two-color analysis with antiVLA-5 antibody (PE staining) and FITC-labeled anti-CD38
antibody, w e found all myeloma cells located at CD38strong positive (CD38+ +) fraction and identified two subpopulationsamong these myeloma cells: CD38+ +VIA-5(VLA-5-) myeloma cells and CD38++VLA-5+ (VLA-5+)
myeloma cells. To clarify the biologic character of these
two subpopulations, the morphology, in vitro proliferative
activity and in vitro M-protein secretion were examined in
each fraction isolated by the purification procedureor a cell
sorter. Morphologicexamination showed that VLA-5- myeloma cells were mostly immature or plasmablastic and
VLA-5+ cells were mature myeloma cells. Furthermore,
VLA-5- myeloma cells proliferated markedly in vitro and
responded to interleukin 6 (IL-6), a growth factor for myeloma cells, while VLA-5+ myeloma cells showed very low
uptakes of 3H-thymidine and no responses to IL-6 but secreted higher amounts of M-protein (immunoglobulin) in
vitro significantly. Therefore, w e could clarify here heterogeneity of human myeloma cells in the bone marrow with
regard to the expressionof VLA-5, one of integrin adhesion
molecules; VLA-5- myeloma cells were proliferative immature cells and VLA-5+ cells were mature myeloma
cells.
0 1993 by The American Society of Hematology.
M
(GMP-140). With regard to the expression of VLA-5, we
present here that myeloma cells could be classified into
VLA-5- and VLA-5’ myeloma cells and clarify the physiologic significance of these subpopulations of human myeloma cells.
YELOMA is considered to be a proliferative disorder
of malignant plasma cells (myeloma cells). But, recently, the presence of precursor or premyeloma cells has
been reported in the peripheral blood from myeloma patients.’,’ In addition, the phenotypic analysis of myeloma
cells showed that some myeloma cells expressed CD 10 antigen3 or myeloid antigen.4 This raised the possibility that
myeloma cells might be derived from immature cells of B
cell lineage or that myeloma cells might consist of heterogeneous subpopulations, that is, immature myeloma cells and
mature cells. On the other hand, we previously showed that
human myeloma cells freshly isolated from bone marrow
could respond to interleukin-6 (IL-6)5and proliferate in vitro.6 However, in approximately one third of the cases of
myeloma, proliferative and IL-6 responsive myeloma cells
were detected significantly. Thus, it also raised the question
whether the restricted subpopulations of myeloma cells
could really respond to IL-6 and proliferate but others could
not. Therefore, in order to clarify the heterogeneity of myeloma cells, we examined their expressions of adhesion molecules by two-color analysis with anti-CD38 antibody and
antibodies to adhesion molecule^^^^ as follows: CD54
(ICAM-I), CD2 (LFA-2), CD1 l a (LFA-la), CDI l b (Macl), CDw49b (VLA-2), CDw49d (VLA-4), CDw49e (VLA5), CDw49f (VLA-6), CD44, CD56, ELAM-I, and CD62
From the Department oflnternal Medicine, Research Institute for
Nuclear Medicine and Biology, Hiroshima University, and National Ohtake Hospital, Hiroshima, Japan.
Submitted December 7, 1992; accepted February 26, 1993.
Supported in part by grants from the Ministry of Education
Science and Culture.
Address reprint requests to Michio M . Kawano, MD, Myeloma
Study Group, Department of Internal Medicine, Research Institute
for Nuclear Medicine and Biology, Hiroshima University, Kasumi
1-2-3, Minami-ku, Hiroshima 734, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-49 71/93/8202-0030%3.00/0
564
MATERIALS AND METHODS
Patients. Thirty-nine myeloma patients were studied: 28 were
IgG type, four IgA type, one IgD type, and six Bence-Jones type;
and four were in the clinical stage’ I, seven stage 11, and 28 stage 111.
Two patients with plasma cell leukemia, five patients with polyclonal gammopathy, and five healthy donors were also studied. Informed consent was obtained before bone marrow aspiration procedure in all patients.
Phenotype ofmyeloma cells (plasma cells). Bone marrow mononuclear cells were freshly isolated from bone marrow aspirates in
the myeloma patients by Ficoll-Hypaque centrifugation. The cells
( 5 X IO’) were stained with 2 pg of monoclonal anti-VLA-4, VLA5 , CD44, CD56, CD54, CD2, CD58, CDlla, VLA-2, VLA-6, or
CD62 antibody (Immunotech SA, France) at 4°C for 30 minutes.
After washing, the cells were incubated with phycoerythrin (PE)-labeled goat anti-mouse IgG (Immunotech SA) at 4°C for 30 minutes. The cells were then washed and subsequently incubated with
50 pg of normal mouse IgG to block nonspecific binding at 4°C for
20 minutes. Subsequently, the cells were incubated with FITC-conjugated anti-CD38 antibody (Immunotech SA) at 4°C for 30 minutes. Immunofluorescence of the membrane was measured by flow
cytometer (Cytron; Orhto Diagnostic Systems, Westwood, MA).
Two-color cytograms using fluorescence contour plots of the expression of CD38 antigen (x axis, log scale) and VLA-5 (y axis, log
scale) are presented.
Cell sorting of VL‘4-5- and VLA-5’ myeloma cells. Bone
marrow mononuclear cells were stained in the sterile condition
with FITC-anti-CD38 and PE-anti-VLA-5 as described above.
The cells (1 x IO’) were applied to a cell sorter (FACS-IV, Becton
Dickinson, Mountain View, CA). The cells with CD38 strong positive(++) VLA-5- and the cells with CD38++ VLA-5+ were collected, respectively.
Myeloma cell proliferation assay. The myeloma cells sorted by
the cell sorter (FACS-IV) were cultured at 2 X lo5 cells/mL for 48
hours in 0.2 mL of RPMI-1640 medium (Nissui, Japan) supplemented with 10%fetal calfserum (FCS; MA Bioproducts, Walkersville, MD) and 1 X IO-’ 2-mercaptoethanol(2-ME)in the presence
or absence of recombinant IL-6 (rIL-6) (kindly provided by Drs T.
Blood, VOI 82,NO 2 (July 15).1993:564-570
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565
HETEROGENEITY OF MYELOMA CELLS
Hirano and T. Kishimoto, Osaka University). DNA synthesis was
measured by adding 37 KBq of 3H-thymidine (185 GBq/mmol,
Amersham, UK) during the last 16 hours of culture.
In vitro IgG secretion. Myeloma cells were isolated from bone
marrow aspirates by purification procedure: Percoll (Pharmacia
Fine Chemicals, Uppsala, Sweden) gradient centrifugation, E-rosette formation, and treatment with anti-LeuM 1 antibody (Becton
Dickinson, Sunnyvale, CA) and rabbit complement (Hoechst Behring, Germany) were performed as reported previously.loThus purified myeloma cells (1 X IO6 cells/mL) in IgG myeloma patients
were cultured for 48 hours, and culture supernatants were collected
for measuring the amount of IgG by sandwich method of the enzyme-linked immunosorbent assay (ELISA) as described previ0us1y.l~Data are presented as M-protein (IgG) secretion activity
(mean): in vitro IgG secretion rate (pg/cell/day), concentration of
IgG in the culture supernatant (ng/mL) x I03/culture period (day)
x myeloma cell number (cell/mL).
Statistical significancewas evaluated by the Student’s t-test.
Amplijkation of VDJ segment of rearranged IgH by PCR. High
molecular weight genomic DNA was isolated from myeloma cells
sorted by cell sorter as described elsewhere.” Oligonucleotides as
primers were synthesized by a DNA synthesizer (model 380A, Applied Biosystems, Foster City, CA) and purified using HPLC.
Primers used here were: Vsal-5‘-CTGTCGACACGGCCGTGTATTACTG-3’! for the consensussequence of the V region (3’end of
FR3 of V, genes) and Jpst-5’-AACTGCAGAGGAGACGGTGACC-3’ for the consensus sequence of the J region (3‘ end of JH
segments). As for @-actin gene, 5’-TTCTACAATGAGCTGCGTGT-3’(5’ primer) and 5’-GCCAGACAGCACTGTGTTGGJ
(3’ primer). Polymerase chain reaction (PCR) was carried out by
using Thermus aquaticus (Taq) DNA polymerase (Perkin Elmer
Cetus, Nonvalk, CT), and 30 cycles of amplification containing an
annealing step at 55°C for 2 minutes, an elongation step at 72°C for
2 minutes, and a denaturation step at 94°C for 1 minute. The amplified DNA was analyzed by gel electrophoresis in 4% agarose gels
(Nusieve, FMC, Denmark) and then was visualized under ultraviolet light after staining with ethidium bromide.
RESULTS
Heterogeneity of VLA-5 expression on human myeloma
cells. We found the cells located at CD38-strong positive
(CD38++)fraction by two-color analysiswith anti-CD38 antibody were all myeloma cells (plasma cells), and there were
no myeloma cells (plasma cells) at CD38-negative (CD38-)
or CD38-weak positive (CD38+) fraction. This was confirmed by the evidence that we sorted CD38++,CD38+,and
CD38- fractions by the cell sorter, respectively; morphologic examination of these fractions revealed that CD38++
fraction consisted of more than 99% of myeloma cells
(plasma cells), and neither CD38+nor CD38- fraction contained any myeloma cells (plasma cells) (data not shown).
By using two-color staining with anti-CD38 antibody, we
examined the expression of adhesion molecules on myeloma cells (CD38++fraction). Myeloma cells mostly expressed CD44 (Fig lB), CD54, and CD56 on their surface.
As for expression of 01-integnn, most all myeloma cells we
tested expressed VLA-4 (Fig IC) but not VLA-2 or VLA-6,
while the VLA-5 antigen was expressed on the restricted
subpopulations of myeloma cells as shown in Table 1.
There were apparently no correlations between the expressions of VLA-5 antigen and isotypes of M-protein, or clinical stages. Figure 1 clearly shows the presence of two subpopulations of myeloma cells (CD38++fractions): VLA-5- cells
and VLA-5+ cells. In approximately one third ofthe cases of
myeloma patients, myeloma cells consisted of either VLA5- cells (Fig 1E) or VLA-5’ cells (Fig 1 H), and in other cases
ID-FL
CD38
cD38
cD38
LW
F
G
m
v)
5,
81-FL
CD3
LW
BR-FL
C W
CD38
LW
CD38
Fig 1. Two-color analysis of VIA-5 expression on myeloma cells. Human bone marrow mononuclear cells were freshly isolated from
bone marrow aspirates in myeloma patients by Ficoll-Hypaquecentrifugation. Two-color cytograms using FITC-CD38 (x axis, Log scale) and
PE-CD44 (6).-VIA-4 (C), or -VIA-5 (D-H) (y axis, Log scale) (case 1, Table 1) are presented. (A) Forward (y axis) and right (x axis) scattering
profile of bone marrow mononuclear cells in a representative case of myelomas (case 1). A circle represents an analyzing area. All myeloma
cells located at CD38++ (right area of x axis on the cytogram). The expression of VIA-5 on myeloma cells were heterogeneous: VIA-5+
cells are 5.2% (E) (case 13). 36.0% (F) (case 25). 68.3% (G) (case 38). and 89.9% (H) (case 17).
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KAWANO ET AL
566
Table 1. Heterogeneity of VLA-5 Expression on Myeloma Cells
Phenotype (% positive)
Positive in CD38" Cellst
Case
No.
Stage'
1
IA
IA
IA
IA
IIA
IIA
IIA
IIA
IIA
IIA
IIA
MA
IllA
IllA
MA
IllA
IllA
MA
IllA
MA
IllA
MA
IllA
IllA
IllA
IllA
IllA
IllA
IllA
IllA
IllA
IllA
IllA
MA
IllB
IllB
NIB
lllB
IllB
PCL
PCL
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
M-Protein
G-K
G-K
G-K
G-K
G-K
G-K
G-K
G-X
G-X
G-A
A-K
G-K
G-K
G-K
G-K
G-K
G-K
G-K
G-K
G-K
G-K
G-X
G-X
G-X
G-h
G-X
A-K
A-K
A-X
D-X
BJ-K
BJ-X
BJ-X
BJ-X
G-K
G-K
G-X
BJ-K
BJ-X
A-X
A-A
CD38'+
VLA-5+
VLA-4+
CD44+
19.8
27.2
15.6
16.5
18.2
15.0
11.6
18.0
12.2
15.6
16.1
20.6
32.8
11.1
30.7
82.1
21.7
78.7
16.0
97.9
48.2
23.2
25.6
97.8
27.5
36.3
28.6
27.3
66.9
80.5
72.8
40.6
89.8
64.8
18.4
24.1
66.2
45.8
17.2
92.3
80.4
25.7
67.2
52.6
90.2
43.9
6.6
39.6
31.1
60.6
87.8
96.8
4.8
5.2
7.2
26.1
76.2
89.9
30.1
2.1
3.8
12.6
78.4
1.1
96.0
36.0
98.5
72.0
31.9
21.5
2.2
96.4
73.0
99.8
0.9
6.5
52.7
55.1
68.3
77.9
1.1
1.3
99.5
95.6
96.2
98.6
90.8
92.3
98.1
93.2
92.9
93.4
90.4
94.0
90.6
91.2
95.2
95.8
98.7
91.7
90.3
99.2
96.9
91 .o
92.1
97.3
92.8
92.0
97.2
95.6
98.0
89.4
90.8
94.3
99.1
96.2
90.3
92.2
90.5
94.7
91.4
90.2
95.6
93.4
91.8
96.0
94.8
94.0
89.2
86.8
90.3
93.8
90.6
96.2
90.0
82.3
89.3
82.3
90.8
94.6
93.5
91.4
81.7
90.6
88.6
90.0
94.3
90.4
94.7
91.8
97.1
94.2
33.0
92.7
87.0
96.2
84.8
93.8
95.9
90.1
91.3
94.0
86.7
83.9
3.6
1.9
2.6
1.9
1.6
2.0
1.4
1.5
1.2
0.8
91.6
83.1
79.2
78.4
75.0
89.5
84.1
80.7
86.3
87.5
96.0
90.5
89.2
92.2
91.3
90.6
97.5
89.6
90.5
87.5
98.3
93.6
90.7
94.0
98.9
93.0
93.8
90.8
87.9
87.5
Disease
41
42
43
44
45
46
47
48
49
50
Liver cirrhosis
Liver cirrhosis
Collagen disease
Collagen disease
Collagen disease
Healthy
Healthy
Healthy
Healthy
Healthy
* According to criteria of Dune and Salmon?
t Positive percent in CD38++fractions by two-color analysis.
myeloma cells consisted of both VLA-5- and VLA-5' cells
in various proportions (Fig 1 F and G).
In the peripheral blood, only VLA-5- myeloma cells (Fig
2 B and D) not VLA-5+ myeloma cells were detected in
some cases where myeloma cells consisted of VLA-5- cells
alone in the bone marrow (Fig 2A) or myeloma cells contained both VLA-5- and VLA-5' cells (Fig 2C). However,
in approximately one tenth of the cases we tested, circulating myeloma cells were detected in the peripheral blood
significantly (>O. 1% myeloma cells, (CD38++),when we analyzed 30,000 mononuclear cells of peripheral blood. Furthermore, leukemic myeloma cells from two cases of
plasma cell leukemia were isolated from the peripheral
blood by Ficoll-Hypaque centrifugation. Their phenotypes
were also VLA-5- (CD38++VLA-4+)(Table 1 and Fig 2E).
On the other hand, bone marrow mononuclear cells were
isolated from five healthy donors and five patients with
polyclonal gammopathy derived from two cases of liver cirrhosis or three cases of collagen disease, and the phenotypes of their plasma cells were predominantly VLA-5+
(CD38++VLA-4+)(Table 1 and Fig 2F).
Therefore, these results show that in the bone marrow of
myelomas, myeloma cells consist of VLA-5- cells and VLA5' cells, but only VLA-5- myeloma cells are detected in the
peripheral blood, and leukemic plasma cells from plasma
cell leukemia are VLA-5- cells, while normal plasma cells
are predominantly V L A - S cells.
Clonality of V U - 5 - and VLA-5' myeloma cells. To
confirm whether both VLA-5- and VLA-5+ myeloma cells
show the same clonality, we amplified VDJ segment of IgH
gene of VLA-5- and VLA-5+ myeloma cells sorted with cell
sorter by PCR as described in Materials and Methods. Amplified DNA from VLA-5- myeloma cells had the same
band in length (approximately 1 I O bp) as that from VLA-5+
myeloma cells in the same patients as shown in Fig 3.
VLA-5- myeloma cells contain proliferative and IL-6 responsivefractions, while VLA-5+cells have low proliferative
activity but secrete higher amounts of M-protein (immunoglobulin). Myeloma cells were purified by the procedure
as described in Materials and Methods. Bone marrow mononuclear cells were separated by Ficoll-Hypaque centrifugation, followed by Percoll discontinuous gradient, and then
were subjected to E-rosette formation and treatment with
monoclonal anti-LeuM 1 antibody and rabbit complement.
Thus purified fractions consisted of more than 90% myeloma cells, fewer than 1% monocytes, and 1% myeloid
cells. These purified myeloma cells were cultured for 48
hours in vitro and uptakes of 3H-thymidine (3H-TdR) were
measured as described in the Materials and Methods section. VLA-5- myeloma cells had higher proliferative activity in vitro than VLA-5' cells, and VLA-5- myeloma cells
but not VLA-5+ cells showed good responses to IL-6 (data
not shown).
To confirm that VLA-5- myeloma cells have higher proliferative activity and respond to IL-6 better than VLA-5+
cells, we sorted both VLA-5- and VLA-5+ myeloma cells in
the same patient and examined in vitro proliferative activity
of each fraction. In four cases where myeloma cells con-
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567
HETEROGENEITY OF MYELOMA CELLS
RD-FL
106
"IB
BR-FL
Fig 2. The expression of VLA-5 on myeloma cells in
the peripheral blood and that on normal plasma cells in
the bone marrow. The mononuclear cells from bone
marrow or peripheral blood were separated by FicollHypaque centrifugation. The expression of VIA-5 was
analyzed by two-color staining as described in Materials
and Methods. In case 30 (Table 1) where myeloma cells
consisted of VIA-5- cells ( 4 in the bone marrow (A),
VIA-5- myeloma cells were detected significantly in the
peripheral blood (B). In case 28 where myeloma cells
consisted of both VIA-5- i 4 1 and VLA-5' ( cells in the
bone marrow (C), only VLA-5- myeloma cells were found
in the peripheral blood (D). In a plasma cell leukemia
(case 401,leukemic plasma cells in the peripheral blood
were also VIA-5- cells (E).On the other hand, normal
plasma cells from bone marrow in a patient with liver
ciFrhosis (case 42) showed their phenotype of VIA-5'
( i 1 (Fl.
CD38
CD38
CD38
CD38
CD38
CD38
LOB
n
sisted of both VLA-5- and VLA-5+ cells (case no, 3, 7, 25,
37, Table I), bone marrow mononuclear cells were stained
with anti-VLA-5 antibody and FITC-anti-CD38 antibody,
and applied to cell sorter (FACS-IV). CD38++VLA-5-cells
and CD38++VLA-5+cells were sorted separately as described in Materials and Methods. These sorted cells were
cultured for 48 hours in vitro and uptakes of 3H-TdR of
these cells were measured. VLA-5- myeloma cells showed
higher uptakes of 3H-TdR than VLA-5+ cells (P< .01) and
good responses to IL-6, while VLA-5+ myeloma cells revealed very low proliferative activity in vitro and almost no
responses to IL-6 as shown in Fig 4A. However, it should be
noted that responses of sorted VLA-5- myeloma cells to
IL-6 were weaker than those of purified fractions by Percoll
centrifugation. This may be explained by the fact that it
took a long time to sort the cells and during sorting myeloma cells were somewhat damaged. Therefore, these results suggest that VLA-5- myeloma cells have higher prolif-
erative activity and can respond to IL-6, while VLA-5'
myeloma cells have low proliferative activity without response to IL-6.
On the other hand, in 12 cases where myeloma cells predominantly consisted of either VLA-5- (7 cases) or VLA-5+
(5 cases) cells, myeloma cells were also purified by the procedure as mentioned above, not by sorting. Thus purified
myeloma cells (in IgG myelomas) were cultured for 48
hours and the secreted M-protein (IgG) in the culture supernatants was measured by the ELISA method as described in
Materials and Methods. As shown in Fig 4B, VLA-5' myeloma cells (5 cases) secreted higher amounts of M-protein
(IgG) in vitro than VLA-5- cells significantly (P < .05):
VLA-5+cells secreted IgG of 40.1 f 17.6 pg/cell/day (n = 5,
mean f SD), and VLA-5- cells 18.5 2 7.7 pg/cell/day (n =
7). Therefore, this data suggest that VLA-5+ myeloma cells
can secrete higher amounts of M-protein than VLA-5- myeloma cells.
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KAWANO ET AL
568
hn
"Y
I
2
(C) VLA-5+ myeloma cells. Therefore, morphologic examination of sorted cells shows that VLA-5- cells are immature
myeloma cells, and VLA-5+ cells are mature myeloma cells.
3 4
310
28 I
271
234
I94
DISCUSSION
VJ
t
I353
1078
872
t-
actin
603
Fig 3. Clonality of VIA-5- and VIA-5+ myeloma cells. Both
VIA-5- and VIA-5+ myeloma cells were sorted by a cell sorter
separately in the same patient as described in Materials and Methods. DNA was extracted from 1 x 1O5 cells of bone marrow mononuclear cells (before sorting) (lane 2). sorted VIA-5- (lane 3) and
VIA-5+ cells (lane 4). respectively, and then PCR amplification using the V. and J, primers of IgH gene or 5' and 3 primers of @-actin
gene was carried out as described in Materials and Methods. Amplified product from VIA-5- myeloma cells showed the same band
in length as that from VIA-5+ myeloma cells. Lane 1 is marker
DNA.
Morphologicallj. immat lire VLA-S- and mature VLA-5'
myeloma c e / k VLA-5- and VLA-5+ myeloma cells were
sorted by the cell sorter (FACS-IV) in 20 cases of myelomas.
Sorted cells were cytospinned and stained with Wright's solution, and observed undera microscope. Accordingtocriteria by Greipp et al," three people examined individual preparations, separately and blindly, in order to confirm the
reproducibility of the data. VLA-5- fractions consisted of
more than 80% of immature and plasmablastic myeloma
cells, and VLA-5+ cells consisted of more than 90% mature
and intermediate myeloma cells. Representative pictures
are shown in Fig 5: (A) bone marrow smear before sorting in
a representative case. (B) sorted VLA-5- myeloma cells. and
In this report. we demonstrated that two-color staining
with FITC-CD38 antibody could clearly distinguish myeloma cells (plasma cells) from other hemotologic cells: myeloid cells, T cells, B cells, NK cells, or erythroid cells. All
myeloma cells (plasma cells) were located at CD38" fraction, and any cells other than myeloma cells were not found
at this CD38" fraction. This was confirmed by morphologic examination of sorted cells. By this two-color analysis,
we analyzed the expression of surface antigens on myeloma
cells and plasma cells in the bone marrow or peripheral
blood, even though the percentage of myeloma cells or
plasma cells was low (not more than 1%).
With regard to the expression of adhesion molecules on
these CD38" myeloma cells, most of the myeloma cells we
tested expressed CD44, CD54, CD56, and VLA-4, but did
not express CD2, CDI 1b, VLA-2, VLA-6, ELAM-I, or
CD62 (data not shown). The expressions of CDI la and
V L A S were observed on restricted subpopulations of myeloma cells. As reported previ~usly,'~
myeloma cells that
coexpressed CD54 and CD 1 I a, formed homotypic cell aggregations in vitro. In order to clarify the heterogeneity of
myeloma cells, we next focused our analysis on the expression of VLA-5. Here, we could clearly demonstrate that
VLA-5- myeloma cells are proliferative and IL-6 responsive
immature cells and predominantly circulate in the peripheral blood. while VLA-5+ myeloma cells are mature myeloma cells having low proliferative activity without response to IL-6 but can secrete higher amounts of M-protein
than VLA-5- cells.
Recently, the phenotypic analysis of myeloma cells
showed that some myeloma cells expressed C D I O antigen,14
and it has been postulated that C D 10' B cells or plasma cells
may be the precursor cells in myelomas and may be derived
from pre-B cellsls or germinal center B cells.16We alsoexamined the expression of CD I O on myeloma cells freshly isolated from bone marrows by two-color analysis with antiCD38 antibody. However. the expressions o f C D 1 0 on their
surfaces were not detected significantly in any of the 41
cases of myelomas we tested (data not shown). Therefore,
the expression of CDlO was not associated with the expression of VLA-5. On the other hand, it has been also postulated that in secondary antibody responses, germinal center
B cells migrated to the bone marrow to terminally differentiate into plasma cells, which produced a specific antibody." It is likely that VLA-5- immature myeloma cells
may be derived from germinal center B cells, which are
CD38+ VLA-4'VLA-5- (our unpublished observation,
May 1992).'* Up to now, no direct evidence supports this
hypothesis. On the other hand, the VLA-5 molecule is considered to be a critical molecule that may be involved in
adhesion of myeloma cells to bone marrow stromal cells
(our unpublished data). The expression of VLA-5 may be
one of the reasons why VLA-5- myeloma cells are circulat-
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HETEROGENEITY OF MYELOMA CELLS
569
Fig 4. In vitro proliferation and M-protein secretion of VIA-5- and VIA-5+ myeloma cells. (A) In
four cases (case 3 , 7 , 2 5 , and 37 in Table 1)where
myeloma cells consisted of VIA-5- and VIA-5+
cells, both VIA-5- (0)and VIA-5+ ( 0 ) myeloma
cells were sorted by a cell sorter, separately, and
cultured for 48 hours in vitro as described in Materials and Methods. (B) In 12 cases (lgG myeloma)
where myeloma cells consisted of either VLA-5(0)
or VIA-5+ (0)cells, myeloma cells were purified by the Percoll discontinuous centrifugation followed by E-rosetting and treatment of anti-LeuM1
antibody, but not by sorting. Thus purified myeloma cells were cultured for 48 hours, and then
culture supematants were harvested to measure
the amounts of lgG by ELSA as described in Materials and Methods.
A
0
I
IO
100
r I L - 6 (U/ml)
-
0
VLA5-
VlA-5’
I
I
-
* A
c
Fig 5 . Morphology of VIA-5- and
VLA-5+ myeloma cells. Myeloma cells
were classified as mature, intermediate,
immature, or plasmablastic accordingto
the criteria of Greipp et ai” VLA-5- and
VIA-5+ myeloma cells were sorted by a
cell sorter, cytospinned, and stained
with Wright‘s staining. (A) A bone
marrow smear before sorting; (B) sorted
VIA-5- myeloma cells; (C) sorted VIA5+ myeloma cells in a representative
case.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
570
KAWANO ET AL
ing and detected in the peripheral blood. However, the adhesion of hematopoietic cells to the stromal cells is considered to be complicated and several adhesion molecules may
be involved in such adhesions. This issue also remains to be
clarified.
Another important finding is that normal plasma cells
from healthy donors and patients with polyclonal gammopathy showed their phenotype of predominant VLA-5'.
These VLA-5' normal plasma cells were mature plasma
cells morphologically. The difference in the biologic and
genetic character between VLA-5' myeloma cells and VLA5+ normal plasma cells remains unclear. It may be possible
that VLA-5+ myeloma cells have no remarkably proliferative activity but can survive for longer than VLA-5' normal
plasma cells.
Finally, we can postulate that in the bone marrow VLA5- immature myeloma cells and VLA-5' mature myeloma
cells are present, and that VLA-5- myeloma cells are proliferative and IL-6 responsive cells, while VLA-5' myeloma
cells are higher producers of M-protein. Therefore, it is
likely that the ratio of these subpopulations may reflect the
clinical features or clinical courses in multiple myelomas.
Further study on biologic characterization of VLA-5- immature myeloma cells will contribute to the understanding
of oncogenesis of myeloma and of the strategy for treatment.
ACKNOWLEDGMENT
We thank Drs T. Hirano and T. Kishimoto (Osaka University,
Japan) for kindly providing rIL-6, Dr K. Okada (Division of Blood
Transfusion, Hiroshima University Hospital, Japan) for flow cytometer (Cytron) and H. Sumida, K. Yamamoto, and Y. Ohto for
their excellent secretarial assistance.
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1993 82: 564-570
Identification of immature and mature myeloma cells in the bone
marrow of human myelomas
MM Kawano, N Huang, H Harada, Y Harada, A Sakai, H Tanaka, K Iwato and A Kuramoto
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