Microsoft PowerPoint - 18582605_Matsui

Myeloma cancer stem
cells
William Matsui
Division of Hematologic Malignancies
Sidney Kimmel Comprehensive Cancer Center
Johns Hopkins University School of Medicine

Disclosures
William Matsui
Consultant
Pfizer
Bristol Myers-Squibb
Infinity Pharmaceuticals
Research Support
Geron Corporation
Merck

Cancer Stem Cells
Founding observations
Clonogenic tumor cells are rare
In vivo studies
Tumor initiating cells are infrequent in animal models
of cancer (lymphoma, myeloma and sarcoma)
Ectopic growth of human tumors requires many cells
In vitro studies
Few tumor cells can form colonies

Multiple Myeloma
Low in vivo clonogenic potential
Adj. PC5
Spontaneous murine
plasma cell tumor
Splenic MM colony
formation in
syngeneic recipients
CFU-MM 1 in 30,000
Cancer Research, 1968

Multiple Myeloma
Low in vitro clonogenic potential
Primary MM specimens
CFU-MM in 89% of patients
CFU-MM 1 in 1,000 to
100,000
Science, 1977

Multiple Myeloma
Phenotypic heterogeneity
Plasma cells (CD138+CD19neg)
Phenotypically define disease
Constitute the majority of tumor cells
Responsible for disease manifestations
B cells (CD19+CD138neg)
Clonotypic - share identical immunoglobulin
rearrangements
1 in 10,000 to 100,000 tumor cells
Present in circulation and bone marrow
Can differentiate into plasma cells in vitro
Harbor characteristic chromosomal translocations

Myeloma Colony Assay
Clonogenic cells are CD138neg, not CD138+
CD138neg/
CD138+
CD34neg
100
Patient 1
Patient 2
75
Patient 3
#
50
Colony
25
0 0
10
20
30
40
50
Cells plated/ml (x104)
Matsui et al. Blood, 2004

In Vivo Myeloma Growth
CD138neg cells engraft NOD/SCID mice
7.5
CD138+
7.0
(g/ml)
Ig 6.5
CD138+
CD138neg
1.0
CD138neg
human 0.5
CD138
0
Serum

light chain light chain
Matsui et al. Blood, 2004

Circulating MM Precursors
CD19+CD27+ cells engraft NOD/SCID mice
Patient CD138+
Mouse Marrow
CD138
CD19
fluorescence
lambda light chain
Relative
Size (bp)
Matsui et al. Cancer Research, 2008

Multiple Myeloma
Functional heterogeneity
Plasma cells
B cells
CD138+
CD138neg
CD19neg
CD19+CD27+
No Disease
Matsui et al. Blood, 2004; Cancer Research, 2008

Clonogenic Myeloma Cells
Plasma cells in SCID-Hu mice
Yaccoby and Epstein, Blood, 1999

Myeloma Stem Cell Phenotype
Potential confounding factors
Clonogenic assays
Colony formation in vitro
SCID-Hu & SCID-Rab
NOD/SCID & NOD/SCIDIL2cko
Patient specific factors
Disease stage
MGUS vs. MM vs. PCL
Newly diagnosed vs. relapsed
Genetics
Hyperdiploid vs. Ig-translocations
t(11;14) vs t(4;14), del(17p)

Cancer Stem Cells
Relapse requires chemoresistance
and clonogenic potential
Treatment
Burden
Tumor
Detection
Limit
Time

MM Plasma Cells vs Stem Cells
Differential drug sensitivities
RPMI 8226
CD138+
125
100
Recovery
75
Control)(% 50
Clonogenic
25
0
Dex.
Lenalid.
Bortez.
Ritux.+ C' Campath+ C'
(0.1µM)
(1µM)
(10nM)
(10µg/ml)
(10µg/ml)
Matsui et al. Cancer Res, 2008

MM Plasma Cells vs Stem Cells
Differential drug sensitivities
RPMI 8226
Clinical MM progenitors
CD138+
CD138neg (n = 4)
125
CD138neg
100
Recovery
75
Control)(% 50
Clonogenic
25
0
Dex.
Lenalid.
Bortez.
Ritux.+ C' Campath+ C'
(0.1µM)
(1µM)
(10nM)
(10µg/ml)
(10µg/ml)
Matsui et al. Cancer Res, 2008

Normal Stem Cells
Multiple mechanisms of drug resistance
ABC transporters (ABCG2, MDR1)
Active efflux of cytotoxic agents
Intracellular detoxifying enzymes
Xenobiotic neutralization
Quiescence
Resistance to cell cycle-dependent cytotoxics
Low expression of target proteins
All of these processes appear to be active in
myeloma stem cells
Matsui et al. Cancer Research, 2008

Myeloma Stem Cells
Identification of therapeutic targets
Discovery approach
Cellular profiling (GEP, proteomic, kinome)
Requires homogeneous cell populations
Screening (small molecule)
Technical limitations in most clonogenic assays
Educated approach
B cell targeting agents
Already exist for lymphoma and leukemia
Monoclonal Antibodies
TLR9 agonists
Bruton's tyrosine kinase inhibitors

Stem Cell Self-Renewal
Putative regulatory pathways
Shared regulatory pathways
Similarities between normal and cancer stem cells
Surface phenotypes
Drug resistance mechanisms
Developmental signaling pathways
Hedgehog, Wnt/-catenin, Notch
Telomerase
Epigenetic modifiers

Hedgehog Signaling
Background
Regulates normal stem cell fate decisions
Required during development
Active in tissue repair and regeneration
Active in human cancers
Activating mutations: medulloblastoma, basal cell
carcinoma, rhabdomyosarcoma
Aberrant up regulation: lung, upper GI,
prostatecancers
May regulate cancer stem cells
Hematologic malignancies - multiple myeloma, CML
Solid tumors - breast, pancreatic, glioblastoma

Hedgehog Signaling
Signal transduction
Merchant & Matsui, Clinical Cancer Research, 2010

Hedgehog and Myeloma
Relative over-expression in stem cells
150
6
activity
100
PTCH1
4
Control
SMO
Vector
Expression
reporter
CD138+
50
2
Gli
CD138neg
Relative
0
0
CD138+ CD138+ CD138neg
Relative
NCI-H929
Normal
NCI-H929
Peacock et al. PNAS, 2007

Hedgehog and Myeloma
Inhibition of self-renewal
125
125
100
100
n = 5
Recovery
75
75
Control
50
50
Cyclopamine
5E1
25
25
Clonogenic%
0
0
RPMI 8226
NCI-H929
Clinical CD138neg
Peacock et al. PNAS, 2007

Hedgehog and Myeloma
Impact on cancer stem cells
Control
T = 0
T = 5 days
Cyclopamine
Control
Blue
CD138
CD19
Hoechst
Hoechst Red
Cyclopamine
Peacock et al. PNAS, 2007

Myeloma Stem Cells
Clinical translation
Optimal strategy: sequentially target both
myeloma plasma cells and stem cells
Debulk plasma cells
Target stem cells
High-dose therapy
Rituximab
Rituximab Cytoxan
Rituximab
Day -10
1
4
~20
~45 60 90
180
Correlative studies
Huff et al. AACR, 2008

Cytoxan and Rituximab
Correlative studies
Quantify clonogenic myeloma growth
Colony formation in methylcellulose
Normalize colony numbers to CD34+ cells
Should be unaffected by cytoxan or rituximab
Serial assessment
Pretreatment (baseline), post-cytoxan/pre-rituximab
(~day 25), 2, 3, 6, 12 months
Complete data available on 18/21 patients
Huff et al. AACR, 2008

Correlative Studies
Serial quantification of MM-CFU
(log)
MM-CFU
Relative
Days

Cytoxan and Rituximab
Correlative studies
Baseline
Increased MM colonies associated with shorter time
to progression
HR = 1.34 per additional log unit (95% CI: 0.98, 1.34),
p=0.07
Day 60
Increased risk of progression
HR=1.74 (95% CI: 1.25, 2.43), p=0.001
Day 90
Increased risk of recurrence
HR=1.34 (95% CI: 1.01, 1.78), p=0.043

Correlative Studies
Clonogenic growth and progression
100
free
Declining MM CSC
80
(median = 185 days)
60
Rising MM CSC
(median = 640 days)
progression
40
20
HR = 10.56, 95% CI (2.2, 50.7)
p = 0.003
0
Proportion
0
200 400 600 800 1000 1200
Days

Myeloma Stem Cells
Conclusions
Tumorigenic myeloma cells
Frequency is rare in most studies
Phenotype remains controversial
Technical differences between clonogenic
assays
Role in disease pathophysiology unclear
Initiation, relapse, progression
Proof of clinical relevance is still lacking
May provide novel insights into cancer biology

Cancer Stem Cells
Conclusions
Targeting strategies
B cell based strategies
Several already available for lymphomas and
leukemias
Shared stem cell pathways
Emphasis on self-renewal as a therapeutic target
Clinical translation
Novel clinical trial designs needed to detect
activity against small populations of tumor cells
Stem cell based biomarker strategies may serve as
novel endpoints

Acknowledgements
Akil Merchant
Carol Ann Huff
NIH/NCI
Zeshaan Rasheed
Katy Rogers
Leukemia and Lymphoma
Ana Markovic
Miah Jung
Society
Jasmin Agarwal
International Myeloma
Toshihiko Tanno
Ivan Borrello
Foundation
Sarah Brennan
Kim Noonan
Multiple Myeloma Research
Yiting Lim
Rick Jones
Foundation
Vesselin Penchev
Jamie Barber
American Society of Clinical
Jessical Norberg
Milada Vala
Oncology
Qiuju Wang
Maryland Stem Cell
Giselle Joseph
Neil Watkins
Commission
Angel Gonzales
Craig Peacock
Sidney Kimmel Foundation
Ally Huang
Phil Beachy
William Goodwin Foundation
Chris Murter
Jeanne Kowalski
Pearse Family Foundation
Brianna Mallone
Gabrielle's Angel Foundation