Bruno Paiva, PhD Scientific Co-Director of CIMA LAB Diagnostics and Director of the Unit of Cytometry, University of Navarra, Spain

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-
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
CD10- CD19+ CD20++ CD27het CD38het
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. CNAGEP miRNA
2010
MethylationCytogenetics
1995
FISH
2000 2005
NGS
2013
ISS ISS-FISHTC groups
Advancing technology refines PC characterization
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
IGH translocations
t(4;14)
t(14;16)
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. Files 1, 2, 3, 4
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”

11. ≥2 subclones in 35/116 (30%) newly-diagnosed MM patients
Identification of subclonal heterogeneity through
generation of iPEP (immunophenotipyc expression profiling)
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
#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
2N
2N
2N
2N
2N
2N
2N
NT
11% -1p
53% -1p
50% +1p
50% +1p
2N
2N
NT
2N
29%+1p
35%+1p
NT
NT
NT
NT
1q
2N
2N
46% +1q
77% +1q
2N
2N
2N
NT
2N
2N
50% +1q
50% +1q
2N
2N
NT
2N
29%+1p
35%+1p
NT
NT
NT
NT
t(14q32)
neg
neg
80%
91%
neg
neg
61%
56%
neg
neg
67%*
15% *
neg
neg
26%
84%*
neg
neg
24%
neg
neg
neg
RB1 (13q14)
2N
2N
2N
78% del
2N
66% del
2N
2N
2N
2N
70% del
30% del
2N
2N
2N
87% del
2N
2N
2N
15% del
100% del
100% del
TP53 (17p13)
2N
14% del
2N
11% del
2N
2N
2N
2N
2N
2N
60% del
2N
NT
NT
2N
87% del
2N
2N
2N
2N
100% del
100% del

13. FACS-sorted distinct phenotypic subclones are
often 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)

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
Clones with a distinct
pattern of mutations
Bone marrow
MRD

16. 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 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
0,4
0,3
0,2
0,1
0,8
0,7
0,7
0,6
0,6
0,5
1,0
0,9
0,8
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. 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)
TTP
100
80
60
OS
100
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)

18. 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 are
associated 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.

19. 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: 1332-1334
Time 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 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
PCs
Peripheral
blood
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 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

22. Cell competition for potentially overlapping BM niches
% of BM B-cell subsets
Pro-B Pre-B
100%
80%
60%
40%
20%
0%
Smoldering MM
Paiva et al. Leukemia 2011; 25: 697-706
** p ≤.005
vs. HA
* p <.05
vs. HA
Symptomatic MM
100%
80%
60%
40%
20%
0%
% of BM Lymphoid CD34+ HSC
*** p <.001
vs. 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

23. 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.2012.237
6.
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 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
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 #3
Patient #4
Patient #5
Patient #6
Patient #7
Patient #8
Patient #9
Patient #10
…with an unique profile of integrin and adhesion molecules
Paiva B, et al. Blood. 2013;122(22):3591-8.

26. 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.

27. 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 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.

28. % of Annexin-V + ve cells
MM-CTCsBM MM-PCs
100
80
60
40
20
0
Bortezomib
100
80
60
40
20
0
MM-CTCsBM MM-PCs
VRD (BortzLenDex)
100
80
60
40
20
0
MM-CTCsBM MM-PCs
Combined (n=7)
P =.320
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
Paiva B, et al. Blood. 2013;122(22):3591-8.

29. The SDF1/CXCR4 axis
20h
16h8h
4h
24h
20h
16h
12h 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)
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-PCs
C9C
+9q34 (23%)
MM-CTCs
13q14 (2N)
17p13 (2N)
MM-CTCs
C9C
9q34 (2N)
Paiva B, et al. Blood. 2013;122(22):3591-8.

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

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

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

34. 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
A Darwinian view of myeloma treatment
Therapy