1. Phenotypic identification of subclones in
multiple myeloma with different genomic profile,
clonogenic potential and drug sensitivity
Bruno Paiva
University of Navarra, Spain

2. The statements in this presentation are those of the
author and not of Affymetrix

3. • 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……. the
majority of patients eventually relapses
(remains a largely incurable disease)
Multiple myeloma

4. BM ProB
CD10++ CD19+ CD20- CD27- CD38++
BM PreB
CD10+ CD19+ CD20het CD27- CD38++
BM/PB Immature
CD10het CD19+ CD20+ CD27- CD38het
BM/PB/SLT Naive
CD10- CD19+ CD20+ CD27- CD38-
SLT GC B-cells
CD10- CD19+ CD20++ CD27het CD38het
SLT/PB Memory
CD10- CD19+ CD20+ CD27+ CD38+
PB Plasma cells
CD10- CD19+ CD20het CD27++ CD38++
CD138het
SLT Plasmablasts
CD10het CD19+ CD20+ CD27++ CD38+++
CD138-
BM Plasma cells
CD10- CD19+ CD20- CD27++ CD38+++ CD138+
B-cell differentiation

5. Plasma cells: terminally differentiated but…
… new-born vs. long-lived
CD19
heterogenous
( 80% +ve cells)
CD81
heterogenous
( 95% +ve cells)
CD45
heterogenous
( 80% +ve cells)
CD56
heterogenous
(95% -ve cells)

6. Advancing technology refines PC characterization
1995 2010
CNAGEP miRNA MethylationCytogenetics FISH NGS
2000 2005 2013
ISS ISS-FISHTC groups
Technology
Clinical utility
Tx groups GEP sig
Morgan G. Educational Session ASH 2012

7. Keats JJ, et al. Blood. 2012;120:1067-76. Egan JB, et al. Blood. 2012 120: 1060-1066
Substantial baseline clonal heterogeneity and
subsequent clonal selection under treatment
Bolli N, et al. Nat Commun. 2014;5:2997

8. SNP-based
mapping array
16q deletions
12p deletions
1q gains
5q gains
MM: genetic markers with prognostic significance
FISH analysis
t(14;16)
IGH translocations
t(4;14)
t(11;14)
Genomic imbalances
Non-hyperdiplid
1q gains
1p deletions
Monosomy 13
17p deletions
Gene expression
profiling
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

9. Disease models of tumour cell heterogeneity:
multiple myeloma
Clones with a distinct
pattern of mutations
Bone marrow

10. Identification of subclonal heterogeneity through
generation of iPEP (immunophenotipyc expression profiling)
Files 1, 2, 3, 4
• iPEP for all 23 phenotypic markers analysed plus FSC and SSC was generated for
every single clonal PC
Merging of 4 different tubes using backbone markers
Software calculation
of “missing values”

11. Identification of subclonal heterogeneity through
generation of iPEP (immunophenotipyc expression profiling)
≥2 subclones in 35/116 (30%) newly-diagnosed MM patients
Top-markers for identification of distinct phenotypic subclones
CXCR4, CD44, CD19, HLADR, CD54, CD49e, CD138, β7, CD33, CD20, CD81, CD27, CD56
Paino T, et al. Blood 2013;122(21): abstract 531 (oral presentation)

12. FACS-sorted distinct phenotypic subclones are
often associated with different cytogenetic profiles
Paino T, et al. Blood 2013;122(21): abstract 531 (oral presentation)
Patient Subclones 1p 1q t(14q32) RB1 (13q14) TP53 (17p13)
#1 CD81+ 2N 2N neg 2N 2N
CD81- 2N 2N neg 2N 14% del
#2 Β7+ 2N 46% +1q 80% 2N 2N
Β7- 2N 77% +1q 91% 78% del 11% del
#3 CD45+ 2N 2N neg 2N 2N
CD45- 2N 2N neg 66% del 2N
#4 CD56-, CD81- 2N 2N 61% 2N 2N
CD56+, CD81+ NT NT 56% 2N 2N
#5 CD56+ 11% -1p 2N neg 2N 2N
CD56- 53% -1p 2N neg 2N 2N
#6 CD56+ 50% +1p 50% +1q 67%* 70% del 60% del
CD56- 50% +1p 50% +1q 15% * 30% del 2N
#7 CD19+ 2N 2N neg 2N NT
CD19- 2N 2N neg 2N NT
#8 CD38+, SSC↑ NT NT 26% 2N 2N
CD38low SSC↓ 2N 2N 84%* 87% del 87% del
#9 CD81- 29%+1p 29%+1p neg 2N 2N
CD81+ 35%+1p 35%+1p neg 2N 2N
#10 CD56+ NT NT 24% 2N 2N
CD56- NT NT neg 15% del 2N
#11 CD56+ NT NT neg 100% del 100% del
CD56- NT NT neg 100% del 100% del

13. FACS-sorted distinct phenotypic subclones are
often associated with different cytogenetic profiles
del(14q32): 67%
0% del(17p13)del(14q32): 15% 30% del(13q14)
70% del(13q14) 60% del(17p13)
Paino T, et al. Blood 2013;122(21): abstract 531 (oral presentation)

14. PCA in merged files
Clonal selection after drug exposure: MRD as a
reservoir of chemoresistant cells
Baseline Cycle 9 MRD Cycle 18 MRD
Paino T, et al. Blood 2013;122(21): abstract 531 (oral presentation)

15. Disease models of PC heterogeneity: myeloma
Clones with a distinct
pattern of mutations
Bone marrow
MRD

16. 0 12 72 84 9624 36 48 60
Months from diagnosis
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
CumulativeProportionSurviving
CR vs nCR
CR vs PR
nCR vs PR
P=0.01
P<10-6
P=0.04
0 12 72 84 9624 36 48 60
Months from diagnosis
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
CumulativeProportionEventFreeSurviving
CR vs nCR or PR
nCR 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 survival
Achievement of CR as a surrogate marker for extended survival

17. MRD monitoring by 4-color flow: patients <65y
Median: 61m
Median: 62m
Median: 141m
P < 0.001
0 20 40 60 80 100 120 140 160
80
60
40
20
0
14040
P < 0.001
0 20
80
60
40
20
0
• 125 patients in CR after HDT/ASCT (GEM2000)
TTP OS
100 100
Median: 36m
60 80 100 120
Flow CR (n=71) MRD positive (n=57)
Paiva B et al; Blood. 2008; 15;112(10):4017-23 (f/u updated July 2012)

18. 120100806040200
80
40
20
0
100
MRD+ (median 0.1% BM clonal PCs) / Standard-risk FISH: median PFS 39m
MRD+ (median 0.02% BM clonal PCs) / High-risk: median PFS 22m
P <0.001
60
MRD myeloma cells with high-risk cytogenetics are
associated with faster relapses
PFS
140
Paiva B, et al. Blood. 2012;119:687-91.

19. • 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
109
108
107
106
105
104
103
102
101
0
Tumor
cells Presentation
PR
VGPR
CR
MRD
Immune surveillance of undetectable MRD
10
(Operational cure)
Modified from Morgan GJ, et al. Blood 2013;122: 1332-1334
Time to progression
The paradigm of the myeloma treatment
How is the
chemoresistant clone?

20. The pathogenesis of myeloma
Gonzalez, D. et al. Blood. 2007;110(9):3112-21

21. CASE ID ISOTYPE
Peripheral blood B-cells Peripheral
blood Normal
Peripheral
blood
MGUS 2 IgG NT NT
MGUS 3 IgG NT NT
MM 1 IgG NT
MM 2 IgA
-
-
-
NT
-
-
-
NT
-
-
-
NT
-
-
-
NT NT
MM 3 IgG NT
MM 4 IgA NT
+
NT
MM 5 IgG
-
-
NT
-
-
NT
MM 6 IgA NT NT
MM 7 IgG
-
-
-
-
-
-
-
-
-
- NT NT
-
-
-
-
-
+
+
+
Circulating B-cells from patients with MM and MGUS
are 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 finding
Thiago et al. Haematologica 2013
Naive IgM+ Memory IgG+ Memory IgA+ Memory PCs MM-PCs
MGUS 1 IgG - - - - - NT

22. Cell competition for potentially overlapping BM niches
% of BM B-cell subsets
Pro-B Pre-B
100%
80%
60%
40%
20%
0%
Paiva et al. Leukemia 2011; 25: 697-706
** p ≤.005
vs. HA
* p <.05
vs. HA
HA
MGUS
Smoldering MM
Symptomatic MM
100%
80%
60%
40%
20%
0%
1,0%
0,8%
0,6%
0,4%
0,2%
0,0%
Burger et al. Blood 2006 107: 1761-1767
% of PB clonal PC
*** p <.001 vs.
MGUS and SMM
1.0%
0.8%
0.6%
0.4%
0.2%
0.1%
0%
MGUS SMM MM
% of normal BMPC
*** p <.001 vs.
MGUS and SMM
% of BM Lymphoid CD34+ HSC
*** p <.001
vs. HA

23. MM-CTCs are present in every stage and predict
disease transformation/aggressiveness
1.
2.
3.
4.
Billadeau. Blood. 1996 1;88(1):289-96.
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.2012.237
6. Rawstron. Br J Haematol. 1997 ; 97(1):46-55.
7. Luque. Clin Exp Immunol. 1998 ;112(3):410-8.
8. Nowakowski. Blood. 2005 ;106(7):2276-9.
•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.

24. •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 of
multiple myeloma?

25. The potential to egress into PB is restricted to a
minor sub-clone in the BM…
BM MM-PC vs. CTCs: principle component analysis (APS) of 22 antigens
Patient #1
Patient #2 Patient #4 Patient #6 Patient #8
Patient #3 Patient #5 Patient #7 Patient #9
Patient #10
…with an unique profile of integrin and adhesion molecules
Paiva B, et al. Blood. 2013;122(22):3591-8.

26. 2.5
2.0
1.5
1.0
0.5
0.0
MM-CTCsBM MM-PCs
P=.005
% of cells in S-phase (n=10)
MM-CTCs are mostly quiescent
DRAQ5 + 4-color flow cytometry
Paiva B, et al. Blood. 2013;122(22):3591-8.

27. Clonogenic potential of BM MM-PCs vs. MM-CTCs in
co-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 14
Colonies: >40 cells
Clusters: 10-39 cells
Paiva B, et al. Blood. 2013;122(22):3591-8.
Nº of colonies Nº of clusters
Patient (nº of cells) BM MM-PCs MM-CTCs BM MM-PCs MM-CTCs
#1 (1.200) 0 0 0 0
#2 (5.300) 0 1 0 0
#3 (6.500) 2 5 0 2
#4 (10.000) 0 0 0 0
#5 (34.900) 0 0 0 0
#6 (72.000) 0 0 0 0
#7 (80.000) 0 0 1 14
#8 (100.000) 0 0 0 0

28. %ofAnnexin-V+vecells
MM-CTCsBM MM-PCs
Paired BM MM-PCs and MM-CTCs show the same
response to chemotherapy
P =.320
80
60
40
20
0
100
80
60
40
20
0
MM-CTCsBM MM-PCs
100 100
80
60
40
20
0
MM-CTCsBM MM-PCs
• Cytotoxicity measured after 48h
• Bortezomib: 2.5nM; Lenalidomide: 1.0 µM; Dexamethasone: 10nM
Bortezomib VRD (BortzLenDex) Combined (n=7)
Paiva B, et al. Blood. 2013;122(22):3591-8.

29. The SDF1/CXCR4 axis
16h 24h 8h 16h
20h 4h 12h 20h 20h
16h8h
4h
24h
20h
16h
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.

30. 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)
+1q21 (23%)
MM-CTCs
+1q21 (28%)
BM MM-PCs
-13q14 (95%)
+9q34 (90%)
MM-CTCs
-13q14 (97%)
+9q34 (80%)
BM MM-PCs
C9C
+9q34 (23%)
MM-CTCs
C9C
9q34 (2N)
-13q14 (80%)
17p13 (2N)
BM MM-PCs BM MM-PCs MM-CTCs
13q14 (2N)
17p13 (2N)
Paiva B, et al. Blood. 2013;122(22):3591-8.

31. Disease models of PC heterogeneity: myeloma
Clones with a distinct
pattern of mutations
Bone marrow
EMD
MRD
PB-CTC

32. Tumor
progenitor cell
MGUS SMM MM
A Darwinian view of myeloma treatment

33. Myeloma
progenitor cell
MGUS SMM Early-treatment
A Darwinian view of myeloma treatment
Treatment modifies the balance
between existing and competing
sub-clones, resulting in a reduction
of clonal complexity

34. Myeloma
progenitor cell
MGUS SMM MM
Original clone – Drug X resistant
Drug X sensitive
Triple-drug combinations to target all different clones
Always consider retreating with a previous therapy that was functional
A Darwinian view of myeloma treatment
Therapy