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29/12/2011
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SNP-based mapping arrays reveal high genomic
complexity in monoclonal gammopathies:
from MGUS to myeloma status
Lucía López Corral, María Eugenia Sarasquete, Sílvia Beà, Ramón García Sanz,
María Victoria Mateos, Luis Antonio Corchete, Joan Bladé, Albert Oriol, Miguel T.
Hernández García, Pilar Giraldo, José Hernández, Marcos González, Jesús María
Hernández Rivas, Jesús F. San Miguel, Norma C. Gutiérrez.
ASH, San Diego. 12/12/2011
1

29/12/2011
Monoclonal Gammopathies: from early to late stages
Introduction
Normal PC
MGUS
SMM
MM
· MM is a malignant and incurable disorder characterized by the
accumulation of clonal plasma cells in the bone marrow.
·MM evolves from a previous premalignant condition in most patients1,2.
Monoclonal Gammopathies: from early to late stages
Introduction
Normal PC
MGUS
SMM
MM
· MM is a malignant and incurable disorder characterized by the
accumulation of clonal plasma cell in the bone marrow.
·MM evolves from a previous premalignant condition in most patients1,2.
1Landgren et al, Leukemia 2009. 2Weiss et al, Expert. Rev. Hematol 2010
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Monoclonal gammopathies: IMWG criteria1
Introduction
MGUS
SMM
MM
· Monoclonal protein
< 30 g/L
30 g/L
YES
and
and/or
and
· BMPC (%)
< 10%
10%
> 10%b
and
and
and
· CRAB symptomsa
NO
NO
YES
a) Myeloma Related Organ or Tissue Impairment (end organ damage) related to Plasma cell proliferative process: anemia with 2 g/dL below the
normal level or <10 g/dL, or serum calcium level >10 mg/L (0.25 mmol/L) above normal or >110 mg/dL (2.75 mmol/L), or lytic bone lesions or
osteoporosis with compressive fractures, or renal insuficiency (creatinine >2 mg/dL or 173 mmol/L),[CRAB: Calcium increase, Renal
impairment, Anemia and Bone lesion] or symptomatic hyperviscosity,, amyloidosis or recurrent bacterial infections (>2 episodes in 12 m).
b) For symptomatic multiple myeloma, a minimum level of M-component or BM plasma cell infiltration (although usually it is >10%, is not
required, provided than this two features coexists with the presence of end organ damage
1International Working Group (BJH 2003; 121:749)
MGUS and SMM: risk of progression to symptomatic MM
Introduction
Smoldering MM
)
10% per year
(%
progression
of
MGUS
1% per year
Probability
Years from diagnosis
Kyle et al, NEJM (2007)
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Multistep transformation model
Introduction
Germinal center
MGUS
Intramedullary MM
Extramedullary MM
HMCL
B cell
Primary IGH translocations
Secondary IGH translocations: CMYC
t(11;14); t(4;14);
Trisomies
del 13/p16/17p
Mutations of P53
t(14;16)
Mutations of N, K-RAS, FGFR3
t(6;14); t(14;20);
The chromosomal regions explored are not involved
in the malignant transformation
This suggests
The progression to MM is associated with subtle
genetic lesions beyond the resolving power of
classical techniques
Hallek (modified), Blood 1998
Multistep transformation model
Introduction
Germinal center
MGUS
Intramedullary MM
Extramedullary MM
HMCL
B cell
Primary IGH translocations
Secondary IGH translocations: CMYC
t(11;14); t(4;14);
Trisomies
del 13/p16/17p
Mutations of P53
t(14;16)
Mutations of N, K-RAS, FGFR3
t(6;14); t(14;20);
The chromosomal regions explored are not involved
in the malignant transformation
This suggests
The progression to MM is associated with subtle
genetic lesions beyond the resolving power of
classical techniques
Hallek (modified), Blood 1998
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29/12/2011
Aims
A comprehensive high-resolution analysis of
genomic imbalances from the early to late stages
of monoclonal gammopathies:
DNA: SNP-arrays
1. Copy number abnormalities (CNA)
2. Copy number neutral LOH
(CNN-LOH)
3. Correlation with fragile sites (FRA)
Material and Methods
74 patients with monoclonal gammopathies1
·MGUS2
(n=20)
· BM aspiration
·HR-SMM3
(n=20)
·MM4
(n=34)
Paired peripheral blood
(n=10)
· Cytogenetic Lab
Genome-wide Human
· Positive selection
DNA5:
SNP array 6.0
(CD138+)
Purity >90%
1 IMWG 2003
2 >24 months of stable disease
AutoMACs separation system
3Kyle etl al, NEJM 2007; Perez-persona et al, Blood 2007
4Newly diagnosed untreated patients
5Only high quality DNA was used (ND-1000 spectrophotometer )
The study was approved by the research ethics committees and written informed consent was obtained (Helsinky declaration).
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SNP-arrays methodology
Material and Methods
Genome-Wide Human SNP-Array 6.0 assay protocol (Affymetrix)
· Processing (Fluidics Station 450, GeneChip Scanner 3000 7G and AGCC)
· Filter (Contrast quality control > 0.4 y MAPD < 0.35)
· Normalization (240 hapmap file)
ANALYSIS (Genotyping Console 4.0 Affymetrix-, dCHIP y ChAS Affymetrix-, SPSS 15)
1. > 10 markers per segments
2. > 100 Kb minimun genomic sizes
Criteria
3. <50% overlap with known CNV
4. CNN-LOH >5 Mb
Example of a normal chromosome
Material and Methods
Chromosome 1
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Example of a monosomy
Material and Methods
Chromosome 13
Example of a trisomy
Material and Methods
Chromosome 5
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Example of a trisomy in a minor subclone
Material and Methods
Chromosome 11
Example of a monosomy in a minor subclone
Material and Methods
Chromosome 22
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29/12/2011
Results
A comprehensive high-resolution analysis of
genomic imbalances from early to late stages of
monoclonal gammopathies:
DNA: SNP-arrays
1. Copy number abnormalities
(CNA)
2. Copy number neutral LOH
(CNN-LOH)
3. Correlation with fragile sites
(FRA)
Copy number abnormalities
Results
· CNA1 were identified in 93% (69/74 patients)
· Two MGUS and three SMM patients with no CNA
-
Global CNA
GAINS
LOSSES
Median (range)
Median (range)
Median (range)
MGUS (N=20)
5 (0-12)
1,5 (0-8)
1,5 (0-9)
HR-SMM (N=20)
7,5 (0-23)
3 (0-12)
3,5 (0-14)
MM (N=34)
12 (1-32)
6,5 (1-20)
4 (0-29)
SMM vs MM p=0,025
+
MGUS vs MM P= 0,006
MGUS vs MM P= 0,000
MGUS vs MM P= 0,033
Progressive increase in the incidence of CNA1 from
MGUS to HR-SMM and to MM
1CNA= copy number alterations
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Copy number abnormalities
Results
· CNA1 were identified in 93% (69/74 patients)
· Two MGUS and three SMM patients with no CNA
Global CNA
GAINS
LOSSES
-
Median (range)
Median (range)
Median (range)
MGUS (N=20)
5 (0-12)
1,5 (0-8)
1,5 (0-9)
HR-SMM (N=20)
7,5 (0-23)
3 (0-12)
3,5 (0-14)
MM (N=34)
12 (1-32)
6,5 (1-20)
4 (0-29)
SMM vs MM p=0,025
MGUS vs MM P= 0,006
MGUS vs MM P= 0,000
MGUS vs MM P= 0,033
+
Progressive increase in the incidence of CNA1 from
MGUS to HR-SMM and to MM
1CNA= copy number alterations
Copy number abnormalities
Results
· CNA1 were identified in 93% (69/74 patients)
· Two MGUS and three SMM patients with no CNA
Global CNA
GAINS
LOSSES
-
Median (range)
Median (range)
Median (range)
MGUS (N=20)
5 (0-12)
1,5 (0-8)
1,5 (0-9)
HR-SMM (N=20)
7,5 (0-23)
3 (0-12)
3,5 (0-14)
MM (N=34)
12 (1-32)
6,5 (1-20)
4 (0-29)
SMM vs MM p=0,025
MGUS vs MM P= 0,006
MGUS vs MM P= 0,000
MGUS vs MM P= 0,033
+
Progressive increase in the incidence of CNA1 from
MGUS to HR-SMM and to MM
1CNA= copy number alterations
11

29/12/2011
Copy number abnormalities
Results
· CNA1 were identified in 93% (69/74 patients)
· Two MGUS and three SMM patients with no CNA
Global CNA
GAINS
LOSSES
-
Median (range)
Median (range)
Median (range)
MGUS (N=20)
5 (0-12)
1,5 (0-8)
1,5 (0-9)
HR-SMM (N=20)
7,5 (0-23)
3 (0-12)
3,5 (0-14)
MM (N=34)
12 (1-32)
6,5 (1-20)
4 (0-29)
SMM vs MM p=0,025
MGUS vs MM P= 0,006
MGUS vs MM P= 0,000
MGUS vs MM P= 0,033
+
Progressive increase in the incidence of CNA1 from
MGUS to HR-SMM and to MM
1CNA= copy number alterations
Percentages of aberrations per chromosome
Results
LOSSES
GAINS
1p
MGUS vs. MM *
1q
* MGUS vs. MM
2p
2q
3p
* MGUS vs. MM
3q
4p
4q
SMM vs. MM *
5p
5q
6p
6q
* MGUS vs. MM
7p
7q
8p
8q
9p
* MGUS vs. MM
9q
10p
10q
11p
* SMM vs. MM
11q
* MGUS and SMM vs. MM
12p
12q
13q
14q
15q
Chromosomes 16p
16q
MGUS vs. SMM and MM *
17p
17q
18p
18q
19p
* MGUS and SMM vs. MM
19q
* GMSI y MMQ vs. MM
20p
20q
21q
* MGUS and SMM vs. MM
22q
MGUS vs. MM *
Xp
Xq
Yp
Yq
150
100
50
0
50
100
150
% accumulated of cases
MGUS
SMM
MM
*= P values <0.05
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Significantly different frequencies of gains and losses
Results
COPY NUMBER GAINS
MGUS (n=20)
SMM (n=20)
MM (n=34)
P value
patients (%)
patients (%)
patients (%)
1q
20% (4/20)
40% (8/20)
59% (20/34)
P=0.013 (MGUS vs MM)
3p
10% (2/20)
25% (5/20)
38% (13/34)
P=0.05 (MGUS vs MM)
6p
5% (1/20)
15% (3/20)
26% (9/34)
P=0.05 (MGUS vs MM)
9p
25% (5/20)
40% (8/20)
59% (20/34)
P=0.034 (MGUS vs MM)
11p
10% (2/20)
5% (1/20)
35% (12/34)
P=0.019 (MGUS vs MM)
11q
0%
P=0.001 (MGUS vs MM)
(0/20)
10% (2/20)
47% (16/34)
P=0.013 (SMM vs MM)
19p
25%
P=0.011 (MGUS vs MM)
(5/20)
30% (6/20)
65% (22/34)
P=0.029 (SMM vs MM)
19q
25%
P=0.05 (MGUS vs MM)
(5/20)
20% (4/20)
56% (19/34)
P=0.022 (SMM vs MM)
21q
0%
P=0.004 (MGUS vs MM)
(0/20)
5% (1/20)
32% (11/34)
P=0.022 (SMM vs MM)
COPY NUMBER LOSSES
GMSI (n=20)
MMQ (n=20)
MM (n=34)
pacientes (%)
pacientes (%)
pacientes (%)
1p
5% (1/20)
25% (5/20)
44% (15/34)
P=0.006 (MGUS vs MM)
4q
5% (1/20)
0% (0/20)
21% (7/34)
P=0.038 (SMM vs MM)
16q
0%
P=0.02 (MGUS vs SMM)
(0/20)
30% (6/20)
21% (7/34)
P=0.038 (MGUS vs MM)
22q
0% (0/20)
15% (3/20)
23% (8/34)
P=0.020 (MGUS vs MM)
Results
Significantly different frequencies of gains and losses
COPY NUMBER GAINS
MGUS (n=20)
SMM (n=20)
MM (n=34)
P value
patients (%)
patients (%)
patients (%)
1q
20% (4/20)
40% (8/20)
59% (20/34)
P=0.013 (MGUS vs MM)
3p
10% (2/20)
25% (5/20)
38% (13/34)
P=0.05 (MGUS vs MM)
6p
5% (1/20)
15% (3/20)
26% (9/34)
P=0.05 (MGUS vs MM)
9p
25% (5/20)
40% (8/20)
59% (20/34)
P=0.034 (MGUS vs MM)
11p
10% (2/20)
5% (1/20)
35% (12/34)
P=0.019 (MGUS vs MM)
11q
0%
P=0.001 (MGUS vs MM)
(0/20)
10% (2/20)
47% (16/34)
P=0.013 (SMM vs MM)
19p
25%
P=0.011 (MGUS vs MM)
(5/20)
30% (6/20)
65% (22/34)
P=0.029 (SMM vs MM)
19q
25%
P=0.05 (MGUS vs MM)
(5/20)
20% (4/20)
56% (19/34)
P=0.022 (SMM vs MM)
21q
0%
P=0.004 (MGUS vs MM)
(0/20)
5% (1/20)
32% (11/34)
P=0.022 (SMM vs MM)
COPY NUMBER LOSSES
GMSI (n=20)
MMQ (n=20)
MM (n=34)
pacientes (%)
pacientes (%)
pacientes (%)
1p
5% (1/20)
25% (5/20)
44% (15/34)
P=0.006 (MGUS vs MM)
4q
5% (1/20)
0% (0/20)
21% (7/34)
P=0.038 (SMM vs MM)
16q
0%
P=0.02 (MGUS vs SMM)
(0/20)
30% (6/20)
21% (7/34)
P=0.038 (MGUS vs MM)
22q
0% (0/20)
15% (3/20)
23% (8/34)
P=0.020 (MGUS vs MM)
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Percentages of cases carrying specific CNA as
Results
major subpopulations
50%
)
(%
45%
CNA
40%
ith
35%
w
30%
11q gain
11q +
cases
on
25%
21q gain
21q +
ati
20%
16q del
16q -
22q del
15%
22q -
rsubpopul
10%
joaM 5%
0%
MGUS
SMM
MM
MGUS
SMM
MM
25%
)
(% 20%
CNA
11q +
ith 15%
11q gain
w
21q +
21q gain
cases
16q -
10%
16q del
on
22q -
ati
22q del
5%
subpopul
inorM 0%
MGUS
MGUS
SMM
SMM
MM
MM
Percentages of cases carrying specific CNA as
Results
minor subpopulations
) 50%
(% 45%
CNA 40%
ithw 35%
30%
11q gain
11q +
cases
on 25%
21q gain
21q +
ati
20%
16q del
16q -
22q del
15%
22q -
rsubpopul 10%
joaM 5%
0%
MGUS
MGUS
SMM
SMM
MM
MM
25%
)
(%
20%
CNA
ithw
15%
11q gai
11q n
+
cases
21q gai
21q n
+
on
ati
10%
16q del
16q -
22q del
22q -
subpopul
5%
inorM
0%
MGUS
MGUS
SMM
SMM
MM
MM
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29/12/2011
Percentages of cases carrying specific CNA as
Results
minor and major subpopulations
50%
(%)
45%
CNA
40%
ith
w
35%
30%
11q gain
11q +
cases
ion
25%
21q gain
21q +
20%
16q del
16q -
22q del
15%
22q -
rsubpopulatjo 10%
aM 5%
0%
MGUS
MGUS
SMM
SMM
MM
MM
25%
(%)
20%
CNA
ith
w
15%
11q
11q
11q +
gain
+
cases
21q
21q
21q +
gain
+
ion
16q
16q
16q -
10%
del
22q
22q
22q -
del
5%
subpopulat
inorM
0%
MGUS
SMM
MM
MGUS
SMM
MM
Gained and lost minimal common regions (MCR)
Results
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Gained and lost minimal common regions (MCR)
Results
Losses on the left
Gains on the right
MGUS
SMM
MM
2
1
2
6
8
9
11
12
14
16
17
20
22
X
Homozygous deletions (HZD)
Results
· 12 HZD corresponding to 5 MGUS (25%), 1 SMM (5%) and 3 MM (9%).
· 10 different chromosomal regions involved.
· Median size: 210 Kb. Total of genes: 23.
Patients
Band
Start (Kb)
End (Kb)
Size (Kb)
Genes
15_MGUS
1q25.1
173068.5
173063.2
5.3
RABGAP1L
13_MGUS
1q31,1
195077.6
194981.6
96.0
CFHR1, CFHR3
15_MGUS
2p22.3
34590.3
34546.8
43.5
19_MGUS
2p22.3
34590.6
34549.7
40.9
66_MM
3q26.1
163626.5
163612.0
14.5
11_MGUS
6q14.1
79092.9
79020.7
72.3
9_MGUS
8p11.23-p11.22
39507.6
39350.8
156.8
ADAM3A
19_MGUS
8p11.23-11.22
39506.4
39354.1
152.3
ADAM3A
TRPC6, ANGPTL5, KIAA1377, C11orf70, YAP1, BIRC3, BIRC2,
56_MM
11q22.1-q22.2
102013.9
101523.2
490.7
TMEM123, MMP7, MMP20
40_SMM
13q32.1
94715.6
93912.0
803.6
DCT, TGDS, GPR180, SOX21, ABCC4
74_MM
19q13.31
48434.2
48239.1
195.0
PSG2, PSG5, PSG4, PSG9
40_SMM
22q11.22
21556.1
21110.4
445.7
ZNF280B, ZNF280A, PRAME, GGTL4
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29/12/2011
Results
A comprehensive high-resolution analysis of
genomic imbalances from the early to late stages
of monoclonal gammopathies:
DNA: SNP-arrays
1. Copy number abnormalities
(CNA)
2. Copy number neutral LOH
(CNN-LOH)
3. Correlation with fragile sites
(FRA)
Copy number neutral-LOH and Copy number gain-LOH*
Results
GMSI
MMQ
MM
*
*
*
*
*
1
3
45
67
89
10
11
12
1
2
34
56
7
8
9
10
11
12
*
*
13
14
15
16
17
18
19
20
21
22
13
14
15
16
17
18
19
20
21
22
X
Y
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Copy number neutral-LOH and Copy number gain-LOH*
Results
· 38% of patients (28/74) showed CNN-LOH
· 58 CNN-LOH (52 partial, 6 complete). Median: 2 (1-5) Frequency of CNN-LOH according to entity:
· 7 copy number gain LOH*.
MGUS
25%
GMSI
MMQ
MM
SMM
25%
P= 0.047
MM
52%
*
*
*
*
*
1
3
45
67
89
10
11
12
1
2
34
56
7
8
9
10
11
12
*
*
13
14
15
16
17
18
19
20
21
22
13
14
15
16
17
18
19
20
21
22
X
Y
Results
A comprehensive high-resolution analysis of
genomic imbalances from the early to late stages
of monoclonal gammopathies:
DNA: SNP-arrays
1. Copy number abnormalities
(CNA)
2. Copy number neutral LOH
(CNN-LOH)
3. Correlation with fragile sites
(FRA)
18

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Correlation with fragile sites (FRA)
Results
· ±110 FRA described*
· Correlation between FRA and cancer
breakpoints in solid tumors.
· For example: FRA16D (16q23.3): WWOX.
Underexpressed in MM cases with 16q LOH or
t(14;16)
·
55% of MCR
·
65% of CNN-LOH and 58% of CNG-LOH
FRA
·
40% of HZD
*http://www.ncbi.nlm.nih.gov/Locuslink/
Correlation with fragile sites (FRA)
Results
· ±110 FRA described*
· Correlation between FRA and cancer
breakpoints in solid tumors.
· For example: FRA16D (16q23.3): WWOX.
Underexpressed in MM cases with 16q LOH or
t(14;16)
·
55% of MCR
·
65% of CNN-LOH and 58% of CNG-LOH
FRA
·
40% of HZD
*http://www.ncbi.nlm.nih.gov/Locuslink/
19

29/12/2011
Summary

The whole genome analysis using SNP-arrays revealed an
increasing genomic complexity from MGUS to SMM and
to MM.

The transition from MGUS to MM was not associated with
a particular chromosomal imbalance, but rather with an
expansion of altered clones that were already present in
MGUS.

More than a half of the genetic lesions were located at
fragile sites.
Summary

The whole genome analysis using SNP-arrays revealed an
increasing genomic complexity from MGUS to SMM and
to MM.

The transition from MGUS to MM was not associated with
a particular chromosomal imbalance, but rather with an
expansion of altered clones that were already present in
MGUS.

More than a half of the genetic lesions were located at
fragile sites.
20

29/12/2011
Summary

The whole genome analysis using SNP-arrays revealed an
increasing genomic complexity from MGUS to SMM and
to MM.

The transition from MGUS to MM was not associated with
a particular chromosomal imbalance, but rather with an
expansion of altered clones that were already present in
MGUS.

More than a half of the genetic lesions were located at
fragile sites.
Acknowledgements
Hospital Universitario de Salamanca (Hematology department):
Norma C. Gutierrez, Maria Eugenia Sarasquete and Jesús F. San Miguel.
Ramón García Sanz, Marcos González, Maria Victoria Mateos and Jesús M. Hernández Rivas.
Luis Antonio Corchete, Isabel Isidro, Teresa Prieto and Sara González.
Hospital Clínic de Barcelona (Institut d'Investigacions Biomediques):
Silvia M. Beà.
Centro de Investigación del Cáncer (Genomic Unit):
Encarna Fermiñán.
Eva García.
Grupo Español del Mieloma (GEM)
21