Background
Organization of multiple myeloma parallels CML
· CD138+ plasma cells are terminally
differentiated progeny
· Myeloma stem cells are CD138neg/CD20+
B cells (Matsui et al., Blood 2004)
· Responsible for disease maintenance
· Differentiate into CD138+ plasma cells
Background
Clinical treatment of multiple myeloma
· Standard cytotoxic agents can produce
remissions but unlikely to cure disease
· Myeloma stem cells responsible for relapse and
disease progression are likely chemoresistant
· Long-term remissions likely require
eradication of myeloma stem cells
· Unknown if newer agents (revlimid, bortezomib)
are active against myeloma stem cells
Objectives
· Further characterize myeloma stem
cells
·"Stem cell" properties
·In vitro sensitivity to anti-myeloma
agents
Materials and Methods
· Human myeloma cell lines
· RPMI 8226 and NCI-H929
· CD138+ and CD138neg populations
isolated by FACS
· Clinical myeloma samples
· Clonogenic myeloma cells obtained from
bone marrow mononuclear cells by
immunomagnetic depletion of CD138+
plasma cells and CD34+ normal
hematopoietic progenitors
Materials and Methods
· Stem cell characteristics analyzed by flow
cytometry
· Forward and orthagonal light scatter
· Aldehyde dehydrogenase (ALDH) - Aldefluor
· Side population (SP) - Hoescht 33342
· Memory B cell - CD27
· Relative drug sensitivity
· Cells (CD138+ or CD138neg) treated with drugs for
72 hours
· Clonogenicity evaluated in methylcellulose
Results
Myeloma cell lines contain small cell
populations that lack CD138 expression (*)
RPMI 8226
NCI-H929
number
cell
Relative
*
*
CD138
CD138
Results
RPMI 8226 CD138neg cells are smaller and less
granular than corresponding CD138+ plasma cells
CD138+
CD138neg
SSC
FSC
Results
High ALDH expression enriches for
CD138neg cells
25
RPMI 8662
25
NCI-H929
20
20
neg
15
15
10
10
CD138%
5
5
0
0
ALDHlowALDHhigh
ALDHlowALDHhigh
Results
SP cells from MM cell lines are CD138neg
R1
R2
RPMI 8226
R1
R1
R2
R1
number
R2
NCI-H929
Blue
R1
cell
R2
Hoechst
Relative
Hoechst Red
CD138
Results Clinical Samples
Clonogenic myeloma cells are CD20+ CD27+
and resemble normal memory B cells
150
n = 4
125
100
Formation
75
50
%CFU-MM
25
0 CD138neg CD138neg CD138neg CD138neg
CD20neg CD27neg
CD3neg
Results - Cell Lines
Anti-myeloma agents active against myeloma plasma
cells but not stem cells, while anti-B cell targeting
agents inhibit myeloma stem cells
NCI-H929
125
100
CD138+
ecoveryR
75
CD138neg
Control) 50
(% 25
Clonogenic
0
Dex
Revlimid
Velcade
Rituximab
Campath
(0.1µM)
(1µM)
(10nM)
(10µg/ml)
(10µg/ml)
RPMI 8226
125
100
CD138+
ecoveryR
75
CD138neg
Control) 50
(% 25
Clonogenic
0
Dex
Revlimid
Velcade
Rituximab
Campath
(0.1µM)
(1µM)
(10nM)
(10µg/ml)
(10µg/ml)
Results Patients
Drug sensitivity of myeloma stem cells
from clinical specimens parallels cell lines
125
100
ecoveryR
n = 4
75
50
(CFU-MM) 25
Clonogenic%
0 Control Dex Revlimid Velcade RituximabCampath
Conclusions
· Myeloma Stem Cells
· Malignant memory B cells
· Differentiate into CD138+ plasma cells
· Share characteristics with hematopoietic
stem cells
· Low forward and side scatter quiescent
· Higher expression of Aldh
· Side population phenotype
Conclusions
· Myeloma plasma cells and stem cells
display divergent drug sensitivities
· Typical anti-myeloma agents (decadron,
revlimid, velcade) active against myeloma
plasma cells but not myeloma stem cells
· Anti-B cell agents (rituximab and
Campath) active against myeloma stem
cells rather than plasma cells
Conclusions
· Therapies targeting MM plasma cells
· May produce clinical improvement or even
remissions
· Unlikely to be curative unless targets shared
by MM stem cells
· Targeting MM stem cells
· Stem cell properties (quiescence, ABC
transporters, ALDH, etc) inherent drug
resistance
· Responses likely to be delayed by prolonged
survival of MM plasma cells
Future directions
· Optimal strategy may be to sequentially
target both myeloma plasma cells and
stem cells
Debulk plasma cells - steroids, revlimid,
velcade, high-dose therapy
Target stem cells - rituximab
· Hopkins clinical trial: high-dose
cyclophosphamide rituximab
Document Outline