Phenotypic identification of subclones in
multiple myeloma with different genomic profile,
clonogenic potential and drug sensitivity
Bruno PaivaUniversity of Navarra, Spain
• Second most common hematological malignancy– Incidence: ~4/100.000 persons/year– Prevalence: 60.000 patients (Europe)– Incidence increases with age: 80% of patients > 60y (rare in <35y)
• Clinical Course: Remitting and Relapsing disease- With current treatment
• 5-year survival 50% - 70%• Potentially cured ~ 10%
Despite the progress in survival with novel agents……. themajority of patients eventually relapses(remains a largely incurable disease)
Multiple myeloma
BM ProBCD10++ CD19+ CD20- CD27- CD38++
BM PreBCD10+ CD19+ CD20het CD27- CD38++
BM/PB ImmatureCD10het CD19+ CD20+ CD27- CD38het
BM/PB/SLT NaiveCD10- CD19+ CD20+ CD27- CD38-
BM Plasma cellsCD10- CD19+ CD20- CD27++ CD38+++ CD138+
PB Plasma cellsCD10- CD19+ CD20het CD27++ CD38++
CD138het
SLT PlasmablastsCD10het CD19+ CD20+ CD27++ CD38+++
CD138-
SLT/PB MemoryCD10- CD19+ CD20+ CD27+ CD38+
SLT GC B-cellsCD10- CD19+ CD20++ CD27het CD38het
B-cell differentiation
Plasma cells: terminally differentiated but…… new-born vs. long-lived
CD19heterogenous
( 80% +ve cells)
CD81heterogenous
( 95% +ve cells)
CD45heterogenous
( 80% +ve cells)
CD56heterogenous
(95% -ve cells)
CNAGEP miRNA
2010
MethylationCytogenetics
1995
FISH
2000 2005
NGS
2013
ISS ISS-FISHTC groups
Advancing technology refines PC characterizationTechnology
Clinical utility
Tx groups GEP sig
Morgan G. Educational Session ASH 2012
Keats JJ, et al. Blood. 2012;120:1067-76. Egan JB, et al. Blood. 2012 120: 1060-1066
Substantial baseline clonal heterogeneity andsubsequent clonal selection under treatment
Bolli N, et al. Nat Commun. 2014;5:2997
SNP-basedmapping array
16q deletions
12p deletions1q gains
5q gains
MM: genetic markers with prognostic significance
FISH analysis
IGH translocationst(4;14)
t(14;16)
t(11;14)
Genomic imbalances
Non-hyperdiplid
1q gains
1p deletions
Monosomy 13
17p deletions
Gene expressionprofiling
TC classification
Molecular classifications(UAMS & Hovon)
70 gene-model(Arkansas group)
15 gene-model(Intergroupe Francophone)
Perez-Simon, Blood 1999; Fonseca Blood 2003; Chang Blood 2005; Gutierrez Leukemia 2007; Avet- Loiseau JCO 2010 & Blood 2011; Boyd Leukemia 2011, Kumar Blood 2012;Zhan Blood 2006, Saughnessy Blood 2007; Deacaux Blood 2008; Broyl Blood 2010; Tapper JCO 2011
Disease models of tumour cell heterogeneity:multiple myeloma
Clones with a distinctpattern of mutations
Bone marrow
Files 1, 2, 3, 4
Identification of subclonal heterogeneity throughgeneration of iPEP (immunophenotipyc expression profiling)
• iPEP for all 23 phenotypic markers analysed plus FSC and SSC was generated forevery single clonal PC
Merging of 4 different tubes using backbone markers
Software calculationof “missing values”
≥2 subclones in 35/116 (30%) newly-diagnosed MM patients
Identification of subclonal heterogeneity throughgeneration of iPEP (immunophenotipyc expression profiling)
Top-markers for identification of distinct phenotypic subclonesCXCR4, CD44, CD19, HLADR, CD54, CD49e, CD138, β7, CD33, CD20, CD81, CD27, CD56
Paino T, et al. Blood 2013;122(21): abstract 531 (oral presentation)
FACS-sorted distinct phenotypic subclones areoften associated with different cytogenetic profiles
Paino T, et al. Blood 2013;122(21): abstract 531 (oral presentation)
Patient
#1
#2
#3
#4
#5
#6
#7
#8
#9
#10
#11
Subclones
CD81+
CD81-
Β7+
Β7-
CD45+
CD45-
CD56-, CD81-
CD56+, CD81+
CD56+
CD56-
CD56+
CD56-
CD19+CD19-
CD38+, SSC↑CD38low SSC↓
CD81-CD81+CD56+CD56-CD56+CD56-
1p
2N2N2N2N2N2N2NNT
11% -1p53% -1p50% +1p50% +1p
2N2NNT2N
29%+1p35%+1p
NTNTNTNT
1q
2N2N
46% +1q77% +1q
2N2N2NNT2N2N
50% +1q50% +1q
2N2NNT2N
29%+1p35%+1p
NTNTNTNT
t(14q32)
negneg80%91%negneg61%56%negneg
67%*15% *negneg26%
84%*negneg24%negnegneg
RB1 (13q14)
2N2N2N
78% del2N
66% del2N2N2N2N
70% del30% del
2N2N2N
87% del2N2N2N
15% del100% del100% del
TP53 (17p13)
2N14% del
2N11% del
2N2N2N2N2N2N
60% del2NNTNT2N
87% del2N2N2N2N
100% del100% del
FACS-sorted distinct phenotypic subclones areoften associated with different cytogenetic profiles
del(14q32): 67%
del(14q32): 15%
60% del(17p13)
0% del(17p13)
70% del(13q14)
30% del(13q14)
Paino T, et al. Blood 2013;122(21): abstract 531 (oral presentation)
Clonal selection after drug exposure: MRD as areservoir of chemoresistant cells
Baseline Cycle 9 MRD Cycle 18 MRD
PCA in merged files
Paino T, et al. Blood 2013;122(21): abstract 531 (oral presentation)
Disease models of PC heterogeneity: myeloma
Clones with a distinctpattern of mutations
Bone marrow
MRD
Cumulative Proportion Event Free Surviving
Cumulative Proportion Surviving
0 12 72 84 9624 36 48 60
Months from diagnosis
0,1
0,5
0,4
0,3
0,2
1,0
0,9
CR vs nCRCR vs PRnCR vs PR
P=0.01P<10-6
P=0.04
0 12 72 84 9624 36 48 60
Months from diagnosis
0,0
0,4
0,3
0,2
0,1
0,8
0,70,7
0,6
0,6
0,5
1,0
0,9
0,8
CR vs nCR or PRnCR vs PR
P<10-5
P=0.07
CR, n=278 nCR, n=124 PR, n=280 PD, n=25
EFS OS
Lahuerta JJ, et al. J Clin Oncol. 2008;26:5775–82.
The deepest the response, the longer the survivalAchievement of CR as a surrogate marker for extended survival
Median: 61m
Median: 62m
P < 0.001P < 0.001 Median: 36m
Median: 141m
160140120100806040200
40
20
0
140120100806040200
40
20
0
MRD monitoring by 4-color flow: patients <65y
• 125 patients in CR after HDT/ASCT (GEM2000)
TTP100
80
60
OS100
80
60
Flow CR (n=71) MRD positive (n=57)Paiva B et al; Blood. 2008; 15;112(10):4017-23 (f/u updated July 2012)
140120100806040200
80 MRD+ (median 0.02% BM clonal PCs) / High-risk: median PFS 22m
P <0.001
60
40
20
0
MRD myeloma cells with high-risk cytogenetics areassociated with faster relapses
PFS
100
MRD+ (median 0.1% BM clonal PCs) / Standard-risk FISH: median PFS 39m
Paiva B, et al. Blood. 2012;119:687-91.
109
108
107
106
105
104
103
102
101
10
0
Presentation
PR
VGPR
CR
cells
MRD
Immune surveillance of undetectable MRD
(Operational cure)
Modified from Morgan GJ, et al. Blood 2013;122: 1332-1334Time to progression
The paradigm of the myeloma treatment
• To achieve (operational) cure or long-term disease control (through immune surveillance),eradicating the maximum number of tumor cells is a prerequisite
• Maximizing cure rates by personalizing therapy is one of the major aims of modern therapy
Tumor
How is thechemoresistant clone?
CASE ID ISOTYPEPeripheral blood B-cells Peripheral
blood NormalPCs
Peripheralblood
MM-PCsNaive IgM+ Memory IgG+ Memory IgA+ Memory
MGUS 1
MGUS 2
MGUS 3
MM 1
MM 2
MM 3
MM 4
MM 5
MM 6
MM 7
IgG
IgG
IgG
IgG
IgA
IgG
IgA
IgG
IgA
IgG
-NT
NT
-------
----
NT
NT
NT
---
----
NT
--
NT
NT
NT
----
NT
--
NT
NT
NT
----
NT
-----
NT
NT
NT
NT
NT
+NT
+++
Circulating B-cells from patients with MM and MGUSare usually devoided of clonotypic B-cells
FACS of highly purified B-cell maturation subsets (>95%)Sensitivity of ASO-PCR (10-4 - 10-5)N.T.: Not tested
The presence of clonal myeloma PCs in PB of myeloma patients is a frequent findingThiago et al. Haematologica 2013
Cell competition for potentially overlapping BM niches
% of BM B-cell subsets
Pro-B Pre-B100%
80%
60%
40%
20%
0%
Smoldering MM
Paiva et al. Leukemia 2011; 25: 697-706
** p ≤.005vs. HA
* p <.05vs. HA
Symptomatic MM
100%
80%
60%
40%
20%
0%
% of BM Lymphoid CD34+ HSC
*** p <.001vs. HA
1,0%
0,8%
0,6%
0,4%
0,2%
0,0%
% of PB clonal PC
Burger et al. Blood 2006 107: 1761-1767
*** p <.001 vs.MGUS and SMM
1.0%
0.8%
0.6%
0.4%
0.2%
0.1%HA
MGUS
0%
MGUS SMM MM
% of normal BMPC
*** p <.001 vs.MGUS and SMM
1. Billadeau. Blood. 1996 1;88(1):289-96.2.3.4.
Schneider. Br J Haematol. 1997; 97(1):56-64.Kumar. J Clin Oncol. 2005 20;23(24):5668-74.Paiva. Leukemia. 2011; 25(4):697-706.
5. Bianchi. Leukemia. 2012 doi: 10.1038/leu.2012.2376.7.8.
Rawstron. Br J Haematol. 1997 ; 97(1):46-55.Luque. Clin Exp Immunol. 1998 ;112(3):410-8.Nowakowski. Blood. 2005 ;106(7):2276-9.
MM-CTCs are present in every stage and predictdisease transformation/aggressiveness
• MM-CTCs are detected in the PB of MGUS (0% - 81%) 1-4,
smoldering MM (50% - 75%) 1,5, symptomatic MM (35% - 87%) 1,2,4,6-9 and
relapse/refractory MM (52%) 10 patients
• The number of MM-CTCs predicts malignant transformation in
MGUS 3 and smoldering MM 5 and inferior OS in symptomatic 8 and
relapsed/refractory MM 10
9. Chandesris. Br J Haematol 2007; 136: 609–614.10. Peceliunas. Leuk Lymphoma. 2012 ; 53(4):641-7.
• Are all BM MM-PCs capable to egress into PB, or only a specific
sub-clone?
• Do MM-CTCs have stem cell-like features and are enriched by
clonogenic cells?
• Does circadian rhythms also affect MM-CTCs?
What is the role of MM-CTCs in the pathogenesis ofmultiple myeloma?
The potential to egress into PB is restricted to aminor sub-clone in the BM…
BM MM-PC vs. CTCs: principle component analysis (APS) of 22 antigensPatient #1
Patient #2
Patient #3
Patient #4
Patient #5
Patient #6
Patient #7
Patient #8
Patient #9
Patient #10
…with an unique profile of integrin and adhesion moleculesPaiva B, et al. Blood. 2013;122(22):3591-8.
MM-CTCsBM MM-PCs
MM-CTCs are mostly quiescent
DRAQ5 + 4-color flow cytometry
% of cells in S-phase (n=10)
P=.005
2.5
2.0
1.5
1.0
0.5
0.0
Paiva B, et al. Blood. 2013;122(22):3591-8.
Nº of colonies Nº of clusters
Patient (nº of cells)
#1 (1.200)
#2 (5.300)
#3 (6.500)
#4 (10.000)
#5 (34.900)
#6 (72.000)
#7 (80.000)
#8 (100.000)
BM MM-PCs
0
0
2
0
0
0
0
0
MM-CTCs
0
1
5
0
0
0
0
0
BM MM-PCs
0
0
0
0
0
0
1
0
MM-CTCs
0
0
2
0
0
0
14
0
Clonogenic potential of BM MM-PCs vs. MM-CTCs inco-culture with stromal cells
• Same number of BM MM-PCs and MM-CTCs cells seeded with hTERT stromal cells (10:1 ratio)
All measurements at day 14Colonies: >40 cellsClusters: 10-39 cells
Paiva B, et al. Blood. 2013;122(22):3591-8.
% of Annexin-V + ve cells
MM-CTCsBM MM-PCs
100
80
60
40
20
0
Bortezomib
100
80
60
40
20
0MM-CTCsBM MM-PCs
VRD (BortzLenDex)
100
80
60
40
20
0MM-CTCsBM MM-PCs
Combined (n=7)
P =.320
Paired BM MM-PCs and MM-CTCs show the sameresponse to chemotherapy
• Cytotoxicity measured after 48h• Bortezomib: 2.5nM; Lenalidomide: 1.0 µM; Dexamethasone: 10nM
Paiva B, et al. Blood. 2013;122(22):3591-8.
The SDF1/CXCR4 axis
20h16h8h
4h24h
20h16h
12h 20h16h8h
4h24h
20h16h
12h
CXCR4 (Amount of antigen MFI expression / MM-CTC)
SDF-1α levels (pg/mL)MM-CTCs (median cells/µL)CD34+ HSC (median cells/µL)
MM patients at relapse (n=6)Quantification started at 16:00pm every 4h up to 12:00am next day (when patients' initiated treatment)Time points 16h and 21h have been duplicated to facilitate viewing of the time curve Paiva B, et al. Blood. 2013;122(22):3591-8.
Cytogenetic comparison between paired BM MM-PCs and MM-CTCs: less abnormalities?
• Purity of BM MM-PCs and MM-CTCs FACS sorting ≥95% (n=4)
BM MM-PCs+1q21 (23%)
BM MM-PCs-13q14 (95%)+9q34 (90%)
MM-CTCs+1q21 (28%)
MM-CTCs-13q14 (97%)+9q34 (80%)
BM MM-PCs-13q14 (80%)17p13 (2N)
BM MM-PCsC9C+9q34 (23%)
MM-CTCs13q14 (2N)17p13 (2N)
MM-CTCsC9C9q34 (2N)
Paiva B, et al. Blood. 2013;122(22):3591-8.
pattern of mutations EMD
Disease models of PC heterogeneity: myeloma
Bone marrow
MRDPB-CTC
Clones with a distinct
Myelomaprogenitor cell
MGUS SMM
A Darwinian view of myeloma treatment
Early-treatment
Treatment modifies the balancebetween existing and competingsub-clones, resulting in a reductionof clonal complexity