Vol. 4, 1521-1526, Ju,u’ 1998 Clinical Cancer Research 1521

CD28, a Marker Associated with Tumoral Expansion in

Multiple Myeloma’

Nelly Robillard,2 Ga#{235}tan Jego,2

Catherine Pellat-Deceunynck, Danielle Pineau,

Denis Puthier, Marie-Paule Mellerin,

Sophie Barill#{233}, Marie-Jos#{233} Rapp,

Jean-Luc Harousseau, Martine Amiot, and

Regis Bataille3

Laboratoire d’H#{233}matologie (N. R.. D. P.. M-P. M.. R. B.l.

Oncogenese Immunoh#{233}matologique. Institut National de Ia Sante et

de Ia Recherche M#{233}dicale Unite 463 1G. J.. C. P-D.. D. P.. S. B..

M. A., R. B.]. and Departement dHematologie Clinique [M-J. R.,J-L. H.]. Institut de Biologic. 44 093 Nantes Cedex 01. France

ABSTRACT

CD28 expression was thoroughly investigated on

plasma cells of monocbonal gammopathy of undetermined

significance, multiple myeloma (MM), and human myeloma

cell lines. CD28� plasma cells were detected in 19% of 31

monoclonal gammopathy of undetermined significance,

41 % of I 16 MM, and 100% of 13 human myeloma cell lines.CD28� myeloma cells were detected in 21 of 79 (26%) MM

cases at diagnosis, 13 of 22 (59%) at medullary relapse (P <

0.009), and 14 of 15 (93%) at extramedullary relapse (P

0.05), including 10 of 10 (100%) secondary plasma cell

leukemias (P = 0.05). Serial studies in individual patients

confirmed the emergence of CD28� myeloma cells with

tumoral expansion and treatment failure. This was signifi-

cantly correlated with the expression of CD28 ligand, i.e.,

CD86 (but not CD8O), and with an increase in the prolifer-ative activity (labeling index) of myeloma cells in bone mar-

row. Whereas the expression of CD56 defines a particular

subset of myeloma patients, CD28 is the only antigen for

which expression correlates with tumor progression. Our

data show that an aggressive compartment of CD28” and

CD86� myeloma cells emerges during the course of MM in

vivo, indicating that CD28 could be aberrantly expressed on

highly malignant (possibly mutated) myeloma cells. Con-

versely, a subset of proliferative plasmablasts coexpressing

CD28 and CD86 could be the normal counterpart of the

Received I 2/2/97: revised 3/16/98: accepted 3/23/98.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked

advertise,nent in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I This work was supported by the Programme Hospitalier de Recherche

Clinique and by La Ligue contre Ic Cancer des Deux S#{232}vres(to G. J.).

2 The first two authors contributed equally to this work.

3 To whom requests for reprints should be addressed. at Laboratoired’H#{233}matologie. Institut de Biologic. 9 Quai Moncousu. 44 093

Nantes Cedex 01. France. Phone: 33 (0)2-40-08-40-49: Fax: 33 (0)2-

40-08-41-14.

clonogenic myeloma stem cell because a subset of CD28�

plasma cells was observed in 6 of 6 cases of reactive plas-

mocytosis.

INTRODUCTION

CD28, a well-documented antigen expressed on human T

lymphocytes. plays an essential role in T-cell activation and

1-cell-B-cell interactions through its specific ligands, especially

CD86 (reviewed in Refs. 1-4). It is now well established that

signaling through the 1-cell receptor is necessary but not suffi-

cient to induce antigen-specific 1-cell activation and cytokine

secretion. A costimulatory signal involving the 1-cell surface

molecule CD28 and its ligands is essential for 1-cell clonal

expansion. lymphokine secretion, and effector function. If this

costimulatory signal is not delivered, I cells enter a state of

long-term unresponsiveness to specific antigen (referred to as

anergy; Refs. 1-4). In 1989, Kozbor et a!. (5) reported the

expression of CD28 on seven of seven HMCLs4 but not on

resting B cells from peripheral blood or lymphoid organs, lym-

phoblastoid cell lines. or any other B-cell tumors. In 1990. Lee

et a!. (6) confirmed the expression of CD28 gene by malignant

plasma cells from RPMI 8226 HMCL as a series of four distinct

CD28 mRNA species: I .3-, 1 .5-, 3.5-, and 3.7-kb transcripts that

were identical to those of I lymphocytes. More recently. we

used flow cytometry and double immunofluorescence to rein-

vestigate the expression of CD28 on normal and malignant

plasma cells and HMCLs (7-9) and found that normal plasma

cells from tonsils and bone marrow did not express CD28 (7).

However, this work showed for the first time that CD28 is

expressed on fresh human myeboma cells from some but not all

patients with MM (7). Finally. all HMCLs. unlike nonmalignant

EBV� lymphoblastoid cell lines, express CD28 (7-9). A major

issue is to clarify the clinical significance of this antigen on

human myeloma cells because our initial study showed a po-

tential correlation between CD28 expression and disease sever-

ity (7). This study, which evaluated CD28 expression in a large

number of patients with either benign or malignant plasma cell

disorders, clearly shows that this antigen is a marker associated

with disease progression and treatment failure.

MATERIALS AND METHODS

Patients. The expression of CD28 on plasma cells was

investigated in 31 individuals with MGUS and 1 16 patients with

MM. without (ti 106) or with (ii = 10) a significant leukemic

phase (secondary PCL). For these latter MM patients, the phe-

notype of myeloma cells was determined on bone marrow

4 The abbreviations used are: HMCL, human myeloma cell line: MM.

multiple myeloma: MGUS, monoclonal gammopathy of undetermined

significance; PCL. plasma cell leukemia: mAb, monocbonal antibody:

PE. phycoerythrin: Ab. antibody: BrdUrd. bromodeoxyuridine.

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1522 CD28 Expression on Human Myeloma Cells

myeboma cells or both bone marrow and peripheral blood my-

eboma cells. Low- or high-risk MGUS was classified according

to our previously reported criteria (10). The diagnostic criteria

for MM were those of the Southwest Oncology Group (1 1).

Seventy-nine MM patients (with or without a leukemie phase)

were studied at diagnosis, 22 at medullary relapse, and 15 at

extramedullary relapse (10 secondary PCLs, 3 pleural effusions,

I skin and 1 ascitis) and treatment failure. Six patients with

peripheral blood reactive plasmocytosis during the course of

viral infections (n 4) or B- and 1-cell lymphomas (n 2)

were included. In these patients, peripheral blood plasma cells

were processed as myeloma cells.

HMCLs. The XGs and SBN1 HMCL were established

by ourselves (8, 12). U266 and RPMI 8226 were purchased

from American Type Culture Collection (Manassas, VA), and

OPM2, LP1 , L363, and NCI H929 were from DSM (Germany).

ANBL6 was a generous gift from D. Jelinek (Mayo Clinic,

Rochester, MN).

mAbs. mAbs against the following antigens were used:

Apo 2.7, CD19, CD28, CD38, CD45, CD56, CD8O, CD86, and

CDI38 (i.e., syndeean-l). Apo 2.7-PE, anti-CD]9-PE, anti-

CD28-FITC, anti-CD38-FITC, anti-CD45FITC, control IgG 1-

FITC, and control IgGi-PE were obtained from Immunotech

(Marseilles, France); anti-CD28-PE, anti-CD56-PE, and anti-

CD8O-PE were from Beeton Dickinson (Heidelberg, Germany);

anti-CD86-PE was from Pharmingen (San Diego, CA); rabbit

antihuman K light ehain-PE Ab was from DAKO (Gbostrup,

Denmark); and mouse antihuman X light ehain-PE was from

Caltag laboratories (San Francisco, CA). B-B4 anti-CD138 mAb

was a gift from Dr. J. Wijdenes, Diacbone Research (Besan#{231}on,

France). B-B4 mAb (CD138) is specific for normal and malig-

nant plasma cells in bone marrow and peripheral blood and

recognizes syndeean-l (7, 13). Purified B-B,, mAb was biotiny-

lated as described previously (7).

Phenotypic Analysis. For immunofluorescence staining,

5 x l0� cells were incubated with different FIIC- or PE-

conjugated or biotinylated mAbs for 30 mm at 4#{176}Cin the

presence of 20% human AB serum, followed by incubation (20

mm at 20#{176}C)with streptavidin coupled to Quantum Red (Sigma

Chemical Co., St. Louis, MO). Cells were fixed in 1% formal-

dehyde and analyzed on a FACSealibur flow cytometer with

Cell Quest Software (Becton Dickinson). Data acquisition was

always performed in two steps: first, 10,000 total cells were

collected; and second, at least 5,000 plasma cells (CDl38�)

were acquired with an activated live gate on SSC versus CD 138

dot plot. Apoptotie plasma cells were recognized by low ex-

pression of CD138 and Apo 2.7 staining and excluded from

analysis.

CDI38 plasma cell identification was controlled by coex-

pression of CD138, CD38, and intracytoplasmie K/X immuno-

fluorescence in a three-color assay. Briefly, cells were first

surface-stained with CD138-biotin-streptavidin-Quantum Red

and CD38-PE as described above, fixed, and permeabilized

overnight in PBS with 1% paraformaldehyde and 0.01% Tween

20. For intracytoplasmie �ciX staining, permeabilized cells were

washed and incubated for 30 mm at 20#{176}Cwith PE-conjugated

Abs in PBS with 0.5% Tween 20. Cells were washed, and at

least 5000 plasma cells (K- or X-positive cells) were collected.

Careful analysis of the three-color staining revealed that >98%

Table 1 Expres sion of CD28, CD8O, and CD 86 by HMCLs

HMCLs CD28 CD8O CD86

U266 + - -

RPMI 8226 + - -

L363 + - + (weak)

ANBL-6 + - + (weak)

NCI-H929 + .- -

LPI + - -

OPM2 + - -

SBNI + - -

XGI + - + (weak)

XG2 + - + (weak)

XG5 + - -

XG6 + - -

XG7 + - -

of either K- or X-positive cells coexpressed CD138 and bright

CD38.

Labeling Index of Myeloma Cells. The labeling index

of myeboma cells was measured according to a modification of

Carayon and Bord (14) using anti-BrdUrd mAb (BUS-]; Cym-

bus Bioscience, Euromedex, Souffelweyersheim, France) and

analyzed by flow cytometry. The percentage of myeloma cells

in S phase was determined by the number of positive cells for

BrdUrd staining within the viable population of intracytoplas-

mie K-/X-positive plasma cells. Briefly, ]06 cells were incubated

with or without (control) 50 p.M BrdUrd for 2 h at 37#{176}Cin 5%

CO2 incubator, washed, and permeabilized overnight in PBS

containing I % paraformaldehyde and 0.01 % Tween 20 at 4#{176}C.

After two washes, cells were incubated for 30 mm at 37#{176}Cwith

50 Kunitz units of DNase I (Sigma). After DNase digestion,

cells were washed in 0.5% Tween 20 PBS and labeled using

PE-coupled anti-light chain Abs for 30 mm at room temperature.

Following two washes, both control cells and the incorporated

BrdUrd cells were stained with 10 p.1 of FITC-BU5-l mAb in

PBS supplemented with 0.5% Tween 20 and 20% AB serum for

45 mm at room temperature. After two washes, the cells were

resuspended in PBS and analyzed immediately on a FACSea1i-

bur flow cytometer. For this purpose, data acquisition was

performed on 50,000 cells and then on 10,000 live gated K-/X-

positive cells when the plasmacytosis represented <20% of

cells.

Statistical Analyses. The Wileoxon rank sum test, the x2test (with Yates correction, if necessary), and the Fisher exact

test were used for statistical studies.

RESULTS

CD28 Is Expressed on All HMCLs. CD28 gene was

expressed on all 13 HMCLs tested (Table 1) as a series of four

distinct transcripts that were identical to those of I lymphocytes

(data not shown). Although CD8O, a CD28 ligand, was not

expressed on HMCLs, a weak but significant expression of

CD86, another CD28 ligand, was found on 4 of 13 (30%)

HMCLs (Table 1).

CD28 Myeboma Cells Emerge during Tumoral Expan-

sion in Patients with MM. To investigate the clinical and

biological significance of CD28 expression in fresh human

myeloma cells, we used the largest pool of patients with plasma

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MGUS MM

Fig. I Changes in CD28 expres-

sion on myeloma cells according

to disease activity in patients with

MM: comparison with MGUSand human myeloma cell lines.

1 00’

90

80

70

% 60

50

40

30

20

10

0

MGUS Diagnosis Medullary Extramed Cell linesrelapse relapse

Clinical Cancer Research 1523

cell disorders ever studied. CD28 was clearly expressed on

freshly explanted malignant plasma cells in 41 % of patients with

MM. The median percentage of CD28� myeboma cells per

patient was 100% (range = 10-100%). Few patients with

<10% of CD28� myeboma cells were classified as CD28

MM. A more detailed analysis ofCD28 expression on malignant

plasma cells showed a clear correlation with disease progres-

sion. As indicated in Fig. I, CD28� myeboma cells were only

found in 26% of previously untreated patients with MM but in

72% of relapsing patients (P < 0.0001). When relapses were

investigated more carefully, CD28� myeboma cells were found

in 59% of medublary relapses but in 93% of extramedullary

relapses (P = 0.05). For the extramedullary relapses, 10 samples

came from MM patients in leukemie phase (i.e. , secondary

PCL), and 5 came from ascitis, skin lesions, or pleural effusions.

In the former cases, CD28 expression was found in 10 of 10

cases. Of note, in two of these secondary PCLs, CD28 was

expressed on peripheral blood myeboma cells but not on bone

marrow myeboma cells. In the batter eases, CD28 expression was

found in four of five samples. Thus, tumoral expansion was

clearly associated with a growing CD28� myeloma cell com-

partment. It is noteworthy that CD28 expression was observed

in >90% of extramedullaiy tumors, which are usually associ-

ated with treatment failure. Serial evaluations of CD28 were

performed in three patients to clearly show that CD28 expres-

sion emerged during tumoral expansion in individual patients. In

all three eases, medullary relapse was associated with the ap-

pearanee of CD28. In one of these cases, CD28 evaluation was

performed at diagnosis, medublary relapse, and extramedullary

relapse (i.e., secondary PCL). As indicated in Fig. 2, an expand-

ing CD28� myeboma cell compartment was detected in eon-

junction with tumor progression (0, 53, and 94%, respectively,

for the three evaluation times). Interestingly, CD28� plasma

cells were found in 19% of individuals with MGUS. The subset

of CD28� MGUS was too small (ii = 6 of 31) to allow any

conclusions to be drawn. However, the follow-up of these

individuals, as compared to patients with CD28 MGUS, will

MMCell lines

be of particular interest in terms of malignant conversion into

overt MM.

CD28 Myeloma Cells Also Emerge in the Special Subset

of MM Lacking CD56. CD19 and CD56 antigens were thor-

oughly investigated in conjunction with CD28. We and others

have previously reported that malignant plasma cells lack CD19

but overexpress CD56, as compared to normal plasma cells (7.

15-17). As expected, most patients included in the current study

did not express CD19 (98.8% ofeases) but overexpressed CD56

(83% at diagnosis). Interestingly. as previously pointed out by

Kawano and colleagues ( 17), no patient (of 1 16 total patients)

presented the phenotype of normal plasma cells (CDl9�,

CD56). Of note, CD56 was completely lost in malignant

plasma cells of patients with secondary PCL, both in bone

marrow and peripheral blood (ii = 10). To determine whether

this could have been due to late down-regulation of the antigen

in secondary PCL, we reinvestigated CD56 expression in the

original samples at diagnosis from four CD56 secondary

PCLs. In three of these four eases, CD56 was partially or totally

lacking on bone marrow plasma cells at diagnosis. Thus, in

contrast to CD28, CD56 did not correlate with disease progres-

sion but designated a specific subset of myeboma patients de-

tectable at diagnosis. Interestingly, CD28 was more frequently

observed at diagnosis in CD56 (57% of cases) than CD56�

patients (17%, P = 0.002; Table 2). Furthermore, as indicated in

Table 2, CD28 expression increased significantly with disease

progression in both CD56 subsets of patients.

CD28 Expression Correlates. The importance of Ii-

gands in the biology of CD28 led us to investigate the expres-

sion of CD8O and CD86 on fresh human myeboma cells. CD8O

was not found in 20 consecutive patients with MM, regardless of

disease activity, whereas significant CD86 expression was ob-

served in 17 of 25 (68%) consecutive patients with MM. Inter-

estingly, a significant association was found between CD28 and

CD86 expression: all CD28� MM patients (n = 10) expressed

CD86 but only 46% of CD28 patients expressed CD86 (7 of

15: P = 0.018: Fig. 3A).

Research. on August 4, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Diagnosis Medullary Extra-Relapse medullary

Relapse

C.)

�:E;::�!�I i�!

�-�;---1)#{176} 10

�. -

C.)

� ‘�

10 10 10 10 10

aC.)

1111 �llIu 1�

� -

C’)

1o� � 1I� 1110 �Jl I;2 �3 �

CD38

C’)�.

c’J�.

�.

0%C,)

�!__

Tho#{176} ;‘�;i �‘�2 � 4

-�.

94%C.)

Q’

C�

�100. � ‘�‘T�2 ;‘�;�� �4

CD28

1524 CD28 Expression on Human Myeloma Cells

5 G. Jego et al. , manuscript in preparation.

Fig. 2 Serial analysis of

CD28 cells in the CD138’

plasma cell population in a pa-

tient with MM at diagnosis,

medullary relapse, and ex-tramedullary relapse (second-

ary leukemic phase).

Given the significant association of a detectable compart-

ment of CD28� myeloma cells with disease progression, we

searched for a correlation between the presence of this compart-

ment and tumor kinetics in the bone marrow of newly diagnosed

patients with MM. It was particularly noteworthy that the me-

dian labeling index of myeloma cells was 4% (range = 0.6-

4.5%) in 7 CD28� patients but only 1.1% (range = 0-4.5%) in

16 CD28 patients (P = 0.02; Fig. 3B). These data indicate that

CD28� MM were more proliferative (and thus more aggressive)

than CD28 MM at diagnosis.

CD28 Is Detected on Reactive Plasma Cells. We pre-

viously found that nonproliferative and highly differentiated

plasma cells within bone marrow did not express CD28 (7). In

view of our current results in MM, it seemed to be of interest to

investigate CD28 expression on reactive plasma cells, which are

highly proliferative but less differentiated (l8).� Six eases of

reactive plasmocytosis were studied using the same method-

ological approach, and the median percentage of reactive poly-

clonal plasma cells in peripheral blood was found to be 9%

(range - 4-70%). In all eases, the phenotype was normal:

CDl38��, CD38���, K�/X�, CD19�, CD1 la�, and CD56.

A significant population of CD28� plasma cells was found in all

eases (range = 1-12%, median = 6%).

DISCUSSION

CD28, a well-documented antigen expressed on human 1

lymphocytes, plays an essential role in 1-cell activation and

1-cell-B-cell interactions through its specific ligands: B7-l , i.e.,

CD8O; and B7-2, i.e., CD86 (]-4). In 1989, Kozbor et a!. (5)

reported the expression of CD28 on 7 of 7 HMCLs, an obser-

vation confirmed by us and others (7-9, 19). Thus, it is clear that

CD28 is a universal marker expressed on 100% of HMCLs but

not on other cells of the B-cell lineage, including EBV� B-cell

lines. At the transcriptional level, its expression on HMCLs is

identical to that of I cells. Although all HMCLs express CD28,

none express CD8O, and only 30% express CD86 weakly,

Research. on August 4, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Bp=0.02 p=0.02

Fig. 3 CD28 expression correlates.A, expression of CD86 on plasma

cells (CD138�) according to CD28expression. B, labeling index of

plasma cells was defined as described

in “Materials and Methods.” Median

values are indicated in the figure (-).

0

(I)U)U)

a.

U)

CDCD0C.)

A

1 00

5 0 ______\ \ \ ‘ \

%‘ ,‘ S “ S

5’’’’’

S S S S S

0� - _____

1� � � 1’ �

‘‘‘5’

‘‘‘‘/,

‘‘‘5’

‘5’’’

‘‘5’’

‘‘‘‘5

‘‘‘‘/,‘‘‘5’

‘‘‘‘5

‘‘5’’

‘5’’’

‘‘5’’

5.

�4.

‘Ca)

�0C

a)

.E 2a)

U)

-I

0

41’ 41’

4�.

41’41’

41’

I --I-- I

CD28 CD28� CD28 CD28�

(n=15) (n=10) (n=16) (n=7)

Clinical Cancer Research 1525

6 Manuscript in preparation.

T able 2 Expression of C D28 in both CD56� and CD56 subsets of MM

Disease status CD56� subset CD56 subset

Diagnosis

Medullary relapse

Il7%(10/56) �

I56% (9/16) J

P = 0.002

P=0.006

I57%(12/2l)

100% (5/5) P = 0.01

Extramedullary relapse

Extramedullary sites 75% (3/4) 100% (1/1)

Secondary PCL No patient 100% (10/10)

whereas EBV� B-cell lines strongly express both CD8O and

CD86.

More recently, we have found that (a) malignant plasma

cells from some patients with MM express CD28, as do those of

HMCLs (7), and that (b) normal well-differentiated and non-

proliferative plasma cells (in bone marrow) do not express

CD28 (7). Here, we carried out a more thorough assessment of

this “1-cell antigen” on freshly explanted malignant plasma

cells. Our current data clearly show that CD28 appears during

disease progression. A continual increase in the percentages of

MM patients expressing CD28 was found between the initial

and terminal phases of the disease and at treatment failure, when

almost all patients expressed CD28. This pattern was also ob-

served in the important subset of MM lacking CD56 and is

currently being investigated.6 Serial studies have clearly con-

firmed this concept in individual patients. The concept that

CD28 expression is associated with tumoral expansion is also

supported by the fact that the labeling index of newly diagnosed

patients with CD28� MM was 4 times greater than that of

CD28 MM. This point strongly supports the concept that

CD28� MM is more aggressive (because it is more prolifera-

tive) than other forms. From a practical point of view, CD28

expression could be of prognostic value in MM and also in

MGUS because CD28 was found to be expressed in some

patients with these presenting features. Further investigation is

needed to clarify these points. Basically, these in vivo data

suggest that CD28 expression on malignant plasma cells could

reflect intrinsic cell malignancy (in terms of oncogenic events

accumulated in a clone) with higher proliferative capacity. It is

of interest to emphasize that HMCLs generally emerge from

samples obtained during extramedullary relapse or from the

peripheral blood of secondary PCL, for which 100% of samples

and cells express CD28 as HMCLs, with frequent Ras and p53

point mutations (20, 21). Further investigation will be necessary

to clarify the mechanisms leading to the up-regulation of CD28

on these tumors. It would appear that these observations are not

consistent with our finding of CD28 expression in 6 of 6 cases

of reactive plasmocytosis in which plasma cells were not ma-

lignant. However, these cases of reactive plasmocytosis were

highly proliferative.5 Thus, the intrinsic malignancy of myeloma

cells related to the occurrence of Ras and/or p53 mutations

(Refs. 20 and 2 1 ; or other unknown oncogenic mutations) dur-

ing disease progression could make them proliferative enough to

express CD28. Another possibility is that a normal but prolif-

Research. on August 4, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

1526 CD28 Expression on Human Myeloma Cells

erative CD28� plasmablast could be the normal counterpart of

the myeboma stem cell, although it is undetectable in most

patients at diagnosis. It is noteworthy that our observation of

CD28 on reactive plasma cells provides the first evidence of

CD28 expression on the normal B-cell lineage in vivo, a finding

that requires further investigation. Although the expression of

CD28 is clearly associated with tumoral expansion, there is no

clear function for this antigen, not only on myeloma cells but

also on normal (reactive) plasma cells. However, activation of

this antigen through its CD86 ligand (although weakly ex-

pressed) could facilitate myeloma cell survival by up-regulation

of antiapoptotic molecules such as Bel-XL, as described for I

lymphocytes (22, 23). In this context, it is noteworthy that

chemoresistance in MM, which is associated with overexpres-

sion of CD28 (this data), is also related to overexpression of

Bcl-XL (24). Thus, further investigation is needed to delineate

the role of CD28 on reactive plasma cells and human myeboma

cells.

ACKNOWLEDGMENTS

We thank C. Ville for typing this manuscript and M. Etrillard for

technical assistance.

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1998;4:1521-1526. Clin Cancer Res   N Robillard, G Jego, C Pellat-Deceunynck, et al.   myeloma.CD28, a marker associated with tumoral expansion in multiple

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