Evolution Of Genomic Changes In
Myeloma
Masood A. Shammas, PhD
Genomic Instability is a Hallmark of Cancer
Chromosomes in
normal diploid cells
are intact and stable
Chromosomes in
DL
cancer cells show
DP
DT
IN
TL
frequent and ongoing
rearrangements and
MC
abnormalities
Genomic Instability;
A challenge for cancer treatment
Genomic instability seems to appear early and leads
to ongoing accrual of mutational changes, some of
which
underlie
tumor
progression
­
including
acquisition of invasiveness, drug resistance, and
metastasis.
The ability to constantly evolve, not only enables the
cell to acquire new characteristics for disease
progression, but also presents a great challenge for
cancer treatment and diagnosis.
Evolution of Genetic Change in Myeloma
Cultured MM cells for various time points and evaluated for new mutations;
Genotype calls from cells harvested in the beginning of experiment were used as control
to identify LOH in late samples.
ARP
U266
Day 30
Day 365
CHR
CHR2
CHR8
CHR13
16
LOH
Over time
new changes
(Blue Lines)
continue
to appear
17
18
PURPOSE
To evaluate genomic instability in MM patients
over time
To evaluate the significance of newly-acquired
mutations on clinical outcome
To identify the genes implicated in the pathways
leading to genomic instability
Method : Evaluating Genomic Instability In Myeloma
Bone Marrow Samples from Myeloma Patients
at 2 time points, 6 months apart
CD138+ Cells Purified
Genomic DNA
SNP 250K STY Arrays (Affymetrix)
Genomic changes evaluated using dChip
The signal intensities and genotype calls of SNP hybridizations from
early tumor samples were used as baseline to evaluate changes in copy
number and heterozygosity in late tumor samples, respectively
Acquisition of Copy Number Changes Over Time in MM
Relative copy numbers in early (E) and late (L) samples are shown by
different intensities of red colour
12
3
4
5
6
7
8
9
10
11 12
13
14
E L E L E L E L E L E L E L E L E L E L E L E L E L E L
Copy Number Change Events Acquired By
Myeloma Patients In 6 Months
(Event = copy number change in three or more consecutive SNPs)
3
0.1 2.7 events per 100 Inf SNPs
2.5
Events
SNPs
2
1.5
Change
1
Informative
0.5
0
Number
100
Per
1234
567
89
10 11 12 13 14
Copy
Genomic Changes Correlate With Expression
Changes At Certain Loci
Patients 3 and 7;
CHR19, Q-ARM
Copy Number
Expression
Patient 3
Patient 7
Patient 3 Patient 7
E
L
E
L
E
L
E
L
Reduced copy number and expression
Frequent Areas of Acquired Change
Regions With Copy Number Change In
Two or More Patients
Sample ID
Chr Region
Acquired Change
114
1p12
Amps/Losses
2, 7, 10, 14
1q21.1-1q44
Deletion
3, 7
1p21.1-1p31.1
Amplification
3, 11
3p11.1-3p26.3
Deletion
4, 7, 10, 13
4p16.1-4p16.2
Amplification
7, 11, 14
6q13-6q27
Amplification
3, 14
7q
Deletion
3, 13
8p11.21-8p22
Amplification
1, 6
Chr 13 whole
Deletion
3, 11
14q22-14q32.2
Amplification
6, 11
16q12.1-16q24.3
Deletion
3, 4, 11
19q13
Deletion
3, 7, 11
20p11.2-20p13
Amps/Losses
114
xp22.1-xp22.33
Amps/Losses
Prognostic significance of acquired genomic
changes in myeloma patients
Evaluated if copy number changes at 1p12 and xp22.33 correlate with
survival, using our 190 patient SNP data
1.0
1.0
Patients with no loss
Patients with no loss
At Xp22.33 (17 cases)
0.8
at 1p12 (157 cases)
0.8
0.6
0.6
Survival
0.4
Survival
0.4
Patients with loss
0.2
Patients with copy loss
0.2
(Percentage)
at xp22.33 (175 cases)
at 1p12 (33 cases)
(Percentage)
Patient
Patient
P = 0.037
0
P =P0.017
= 0.017
P = 0.037
0
020
40
60
80
020
40
60
80
Patient Surviaval (Months)
Patient Surviaval (Months)
Whole Genome Sequencing of Myeloma Samples Using
Illumina's Solexa Paired-End Technology
Solexa paired-end reads: 400 bp scan experiment
400 bp random fragments of cancer genome
Paired-end sequencing,
37 bps seq
37 bps seq
utilizing 37 bp read-
lengths
80 million reads
Read pairs mapped back to reference genome
Tandem Duplication
Insertion
Rearrangement
Rearrangement
(Inversion)
Deletion
(Translocation)
Wildtype
Reference genome sequence
Whole Genome Sequencing of Myeloma Samples Using
Illumina's Solexa Paired-End Technology
Genome-wide acquired rearrangements
Early Tumor Samples
Late Tumor Samples
The outer ring shows a representation of the normal karyotype (red indicates centromeres). The blue line in the middle ring
indicates copy number as determined by short read data. The inner circle shows the two endpoints of each somatic
rearrangement identified, joined by green lines. Very small rearrangements appear as single lines
Homologous DNA Recombination
Homologous Recombination Activity is
Elevated in Multiple Myeloma (P<0.001)
6
7
/10
NORMAL
PRIMARY
3
CELLS
MYELOMA CELL LINES
CELLS
10 6
8226
ARK
5
ARP
MPS3
MM1S
MPS1
4
ARD
Frequency
3
ARH
MPS2
Cells
Cells
U266
2
1
Fibroblasts
Plasma
Plasma
Recombination
SiRNA Mediated Inhibition of HR Prevents Mutations Whereas
Induction of HR by Nickel Increases Rate of Mutation
CNi
R
CNi
R
1400
CHR
P<0.05
1
Cells) 1200
Induction
Nickel
1000
2
HR
800
3
Control
in 600
4
400
5
LOH
Following
of 200
Control
6
LOH
(%
0
7
8
120
9
Control-RNAi
P<0.05
10
100
Cells)
11
12
Inhibition
rol 80
13
HR
60
14
Cont
15
ing
in
16
40
HsRAD51-RNAi
17
18
19
LOH 20
20
Follow
21
CHR20
H
of
22
%
0
X
LO
(
Elevated HR Leads to Development of Drug
Resistance
A
100
Dex, 10-8M
80
NiCl , 0.3 mg/ml
Cells
2
60
40
Live
20
%
DEX, 10-8M
0
0
7
14
21
Duration of Treatment (Days)
DexR cells (above), grown in Dex 10­6M
100
B
80
Cells
60
40
Live
20
%
Control cells in Dex 10­6M
0
0
37
5
Duration of Treatment (Days)
CONCLUSIONS
· Myeloma patients display a striking genomic
instability
· Some of the evolved molecular changes can
predict clinical outcome
· Recombination events may be responsible for
the genomic instability
·Mechanisms of genomic instability can be
targeted to to prevent development of invasive,
metastatic, or drug-resistant phenotypes.
Herve AVET-LOISEAU
Stéphane MINVIELLE
Philippe MOREAU
DANA-FARBER
CANCER
INSTITUTE
Nikhil MUNSHI
Mariateresa FULCINITI
Samir AMIN
Florence MAGRANGEAS
Scott RODIG
Catherine CHARBONNEL
Steven TREON
Wilfried GOURAUD
Cheng LI
Loïc CAMPION
Kenneth ANDERSON
Michel ATTAL
Thierry FACON
Claire MATHIOT
Gérald MARIT
Jean-Luc HAROUSSEAU