Phenotypic identification of subclones in multiple myeloma with different genomic profile, clonogenic potential and drug sensitivity

Phenotypic identification of subclones in

multiple myeloma with different genomic profile,

clonogenic potential and drug sensitivity

Bruno PaivaUniversity of Navarra, Spain

The statements in this presentation are those of theauthor and not of Affymetrix

• 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


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)


Clonal selection after drug exposure: MRD as areservoir of chemoresistant cells

Baseline Cycle 9 MRD Cycle 18 MRD

PCA in merged files


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?

The pathogenesis of myeloma

Gonzalez, D. et al. Blood. 2007;110(9):3112-21

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


% 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


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)


pattern of mutations EMD

Disease models of PC heterogeneity: myeloma

Bone marrow

MRDPB-CTC

Clones with a distinct

Tumorprogenitor cell

MGUS SMM MM

A Darwinian view of myeloma treatment

Myelomaprogenitor cell

MGUS SMM


Early-treatment

Treatment modifies the balancebetween existing and competingsub-clones, resulting in a reductionof clonal complexity

MGUS SMM MMOriginal clone – Drug X resistant

Myelomaprogenitor cell

Drug X sensitive

Triple-drug combinations to target all different clonesAlways consider retreating with a previous therapy that was functional


Therapy

Phenotypic identification of subclones in multiple myeloma with different genomic profile, clonogenic potential and drug sensitivity

Science