MM and PC
group
The coexpression of CD11a and CD45bright is the
hallmark of proliferating myeloma cells
Catherine Pellat-Deceunynck, Nelly Robillard, Régis Bataille
Inserm U601, MM and PC group
Nantes Hospital, Nantes, France

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Identification of myeloma cells and bone marrow plasma cells
R1
R2
R3
R1, R2, R3
R4
R1
(log)
CD38PE
R2
CD38PE
CD45APC
R5
SSC-H
SSC-H (log)
CD138PECy5
FSC
SSC-H (log)
R1
R2
R3
R4 and R5
SSC++CD38+++
SSC++CD38+++
excludes occasional
define MM/PC
CD138+++
apoptotic MM/PC
subpopulations
R1 and R2 : identification of MM/PC
R4 and R5 : definition of subpopulations based on CD45 expression and SSC

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BrdU incorporation, staining and analysis
· Overnight BrdU incorporation (+) or not (-) in total blood or bone marrow
· Surface staining: CD38 (PE), CD45 (APC), CD138 (PECy5)
· Fixation and permeabilization
· BrdU (FITC) staining
R1
CD38PE
CD38PE
R2
SSC-H (log)
CD138PECy5
-=
138PECy5
5.5%
138PECy5
0.4%
5.1%
CD
CD
BrdUFITC (+)
BrdUFITC (-)

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BrdU+ myeloma cells are located within the CD45bright SSChigh subset
R1
5.1%
R2
CD38PE
CD38PE
138PECy5CD
SSC-H (log)
CD138PECy5
BrdUFITC
R4
8.5%
R4
50%
138PECy5
R5
138PECy5
CD
CD45APC
R5
CD45APC
SSC-H(log)
CD
1.4%
BrdUFITC

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LI of CD45bright SSChigh and CD45low/neg SSClow myeloma subsets
40
30
Diagnosis (n=25)
(%)
20
Relapse (n=24)
LI
10
0 CD45bright
CD45low/neg

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CD45bright SSChigh and CD45low/neg SSClow myeloma subsets
Myeloma cells (%)
MFI (ratio)
LI (%)
median
range
median
range
median
range
CD45bright SSChigh
12
2-68
34
10-92
6.8
0.5-40
CD45low/neg SSClow
88
32-98
1.2
1-3.8
1
0.2-16.6
N= 49 patients

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Conclusion 1
· Myeloma tumors are always a mix of proliferating CD45bright
SSChigh and non proliferating CD45low/neg SSClow cells.
· All patients have a CD45bright SSChigh compartment (median 12%,
n=49).

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Tonsil and peripheral plasma cells are all CD45bright SSChigh
Tonsil
Blood
Reactive
Plasmacytosis
R1
R1
R1
CD38
CD38
CD38
SSC-H
SSC-H
SSC-H
R2
CD38
CD38
R2
CD38
R2
CD138
CD138
CD138
R4
R4
R4
CD45
R5
CD45
R5
CD45
R5
SSC-H
SSC-H
SSC-H
R4
CD38
CD38
CD38
15%
15%
34%
BrdU
BrdU
BrdU

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BrdU+ BM plasma cells are located within a CD45bright SSChigh subset
R1
11.8%
R2
CD38PE
CD38PE
138PECy5CD
SSC-H (log)
CD138PECy5
BrdUFITC
R4
18.4%
R4
138PECy5
70%
CD45APC
R5
CD
138PECy5
R5
2.6%
CD45APC
SSC-H(log)
CD
BrdUFITC

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Conclusion 2
· PCs from tonsils and blood proliferate and are CD45bright
· PCs from bone marrow are a mix of proliferating CD45bright PCs
and non proliferating CD45low/neg PCs
· CD45low/neg PCs exist only within bone marrow (role of stromal
cells in the down regulation of CD45 and proliferation?)

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Bcl-2 and LI are inversely correlated in PC and MM
Reactive
MM1
BM1
Tonsil
Blood
Plasmacytosis
R4
R4
CD45
R5
CD45
R5
CD45
CD45
CD45
R4
R4
R4
SSC
SSC
SSC
SSC
SSC
R4
9
3
73
3
Bcl2
Bcl2
Bcl2
Bcl2
Bcl2
R5
22
7
Bcl2/LI r=-0.854, p<.001
Bcl2
Bcl2
p<.01
p<.01

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Phenotype of CD45brightSSChigh myeloma cells
MM2
MM3
MM4
MM5
R1
R1
R1
R1
CD38
CD38
CD38
CD38
SSC-H
SSC-H
SSC-H
SSC-H
R2
R2
R2
R2
CD38
CD38
CD38
CD38
CD138
CD138
CD138
CD138
IgG1
IgG1
IgG1
IgG1
CD45
CD45
CD45
CD45
Apo2.7
Apo2.7
Apo2.7
Apo2.7
CD45
CD45
CD45
CD45

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Phenotype of CD45brightSSChigh myeloma cells: coexpression of CD11a
MM2
MM3
MM4
MM5
CD19
CD19
CD19
CD19
CD27
CD27
CD27
CD27
CD28
CD28
CD28
CD28
CD56
CD56
CD56
CD56
CD11a
CD45
CD45
CD45
CD45

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Conclusion
· CD45 delineates two subsets of bone marrow PC and myeloma cells.
CD45bright expression is associated with proliferation.
·· The CD11a+ CD45bright Bcl-2low MM cells could constitute the growth
fraction. This "to be killed" population could be targeted through
CD45 or CD11a targeted therapies.

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MM and PC
group
Philippe Moreau
Nelly Robillard
Steven Le Gouill
Régis Bataille
Jean-Luc Harousseau
Inserm U601, MM and PC group
Clinical Department of Hematology
Nantes Hospital, Nantes, France
Nantes Hospital, Nantes, France

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