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. Multiple myeloma
• 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)

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

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

6. PC characterization
Technology
Cytogenetics FISH GEP CNA miRNA Methylation NGS
1995 2000 2005 2010 2013
Clinical utility
ISS Tx groups TC groups ISS-FISH GEP sig
Morgan G. Educational Session ASH 2012

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

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

10. Identification of subclonal heterogeneity through
generation of iPEP (immunophenotipyc expression profiling)
• 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”Files 1, 2, 3, 4

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

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

14. Clonal selection after drug exposure: MRD as a
reservoir 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)

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

16. The deepest the response, the longer the survival
Achievement of CR as a surrogate marker for extended survival
EFS
1,0
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0,0
0 12
CR vs nCR or PR
nCR vs PR
24 36 48 60 72
Months from diagnosis
CR, n=278
OS
P<10-5
P=0.07
84 96
nCR, n=124
1,0
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
CR vs nCR P=0.01
CR vs PR P<10-6
nCR vs PR P=0.04
12 24 36 48 60 72 84 96
Months from diagnosis
PR, n=280 PD, n=25
Lahuerta JJ, et al. J Clin Oncol. 2008;26:5775-82.

17. -color flow: patients <65y
• 125 patients in CR after HDT/ASCT (GEM2000)
TTP OS
100 100
80 80
Median: 141m
60 60
40
Median: 62m 40
20 20 Median: 61m
0
P < 0.001 Median: 36m 0 P < 0.001
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 160
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. MRD myeloma cells with high-risk cytogenetics are
associated with faster relapses
PFS
100
MRD+ (median 0.1% BM clonal PCs) / Standard-risk FISH: median PFS 39m
80
60
40
20
0
0 20 40
MRD+ (median 0.02% BM clonal PCs) / High-risk: median PFS 22m
P <0.001
60 80 100 120 140
Paiva B, et al. Blood. 2012;119:687-91.

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

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

21. Circulating B-cells from patients with MM and MGUS
are usually devoided of clonotypic B-cells
CASE ID ISOTYPE
Naive
Peripheral blood B-cells
IgM+ Memory IgG+ Memory
Peripheral Peripheral
blood Normal blood
IgA+ Memory PCs MM-PCs
MGUS 1 IgG -
MGUS 2 IgG NT
MGUS 3 IgG NT
MM 1 IgG -
MM 2 IgA -
MM 3 IgG -
MM 4 IgA -
MM 5 IgG -
MM 6 IgA -
MM 7 IgG -
-
-
-
-
NT
NT
NT
-
-
-
- -
- -
- -
- -
NT NT
- -
- -
NT NT
NT NT
NT NT
-
-
-
-
NT
-
-
-
-
-
NT
NT
NT
NT
NT
+
NT
+
+
+
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

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

23. -CTCs are present in every stage and predict
disease 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
1. Billadeau. Blood. 1996 1;88(1):289-96. 5. Bianchi. Leukemia. 2012 doi: 10.1038/leu.2012.237
2. Schneider. Br J Haematol. 1997; 97(1):56-64. 6. Rawstron. Br J Haematol. 1997 ; 97(1):46-55.
3. Kumar. J Clin Oncol. 2005 20;23(24):5668-74. 7. Luque. Clin Exp Immunol. 1998 ;112(3):410-8. 9. Chandesris. Br J Haematol 2007; 136: 609-614.
4. Paiva. Leukemia. 2011; 25(4):697-706. 8. Nowakowski. Blood. 2005 ;106(7):2276-9. 10. Peceliunas. Leuk Lymphoma. 2012 ; 53(4):641-7.

24. What is the role of MM-CTCs in the pathogenesis of
multiple myeloma?
• 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?

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 #3 Patient #5 Patient #7 Patient #9
Patient #2 Patient #4 Patient #6 Patient #8 Patient #10
…with an unique profile of integrin and adhesion molecules
Paiva B, et al. Blood. 2013;122(22):3591-8.

26. 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
BM MM-PCs MM-CTCs
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)
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
All measurements at day 14
Colonies: >40 cells
Clusters: 10-39 cells
Paiva B, et al. Blood. 2013;122(22):3591-8.

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

29. MM-CTCs (median cells/µL)
CD34+ HSC (median cells/µL)
16h 24h 8h 16h
20h 4h 12h 20h
MM patients at relapse (n=6)
CXCR4 (Amount of antigen MFI expression / MM-CTC)
SDF-1α levels (pg/mL)
16h 24h 8h 16h
20h 4h 12h 20h
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)
BM MM-PCs MM-CTCs BM MM-PCs MM-CTCs
+1q21 (23%) +1q21 (28%) -13q14 (80%) 13q14 (2N)
17p13 (2N) 17p13 (2N)
BM MM-PCs MM-CTCs
-13q14 (95%) -13q14 (97%)
+9q34 (90%) +9q34 (80%)
BM MM-PCs MM-CTCs
C9C C9C
+9q34 (23%) 9q34 (2N)
Paiva B, et al. Blood. 2013;122(22):3591-8.

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

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

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

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