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Fondazione IRCCS Policlinico- Milano Laboratory
of Molecular Genetics
Bio-Molecular Markers in the Characterization of
Multiple Myeloma Antonino Neri, MD, PhD
XXIII IATMO CONFERENCE Cancer, Cell Death and
Differentiation October 19-21 2007 Jolly Hotel,
Trieste
Department of Medical Sciences Hematology
School University of Milano
2
Pathogenesis of Multiple Myeloma
mechanisms of disease
progression
Normal plasma cells
Karyotype instability IGH translocations 13q
deletion Hyperdiploidy
Myeloma Plasma Cell Leukemia
Karyotype instability IGH translocations 13q
deletion Hyperdiploidy 1q amplification Ras
mutations p53 deletions Secondary
translocations/c-myc FGFR3 mutations
MGUS
Smouldering MM
MGUS
Karyotype instability IGH translocations 13q
deletion Hyperdiploidy 1q amplification
3
Frequency and partners of 14q32 translocations in
MM
(c-MAF) (MAFB)
16q23 20q11

(c-MYC)
4p16.3
8q24

5
(FGFR3 and MMSET)
5
15
(Cyclin D3)
6p21

3
11q13
Others

(Cyclin D1)

20
1q21

(IRTA1/IRTA2)
2p23

(NMYC)
9p13

(PAX5)

?
25
11q23

(MLL1)

?
12p13

(Cyclin D2)

?
1q11 (?)
3q21 (?)
27
None
25
4
t(1114) and Cyclin D1 involvement in MM
12/48 patients (25)
t(1114) 7/39 patients (18)
Clinical symptoms (p0.026) Clinical stage
(p0.044) Tumor burden (p0.022) Patients
survival (p0.346)

Pruneri et al. Am J Pathol, 2000
5
Double-Color FISH Assay for the Detection of the
t(414)(p16.3q32) in MM
Chr 4
PAC 884J17
PAC 1174P18
PCL-1
LB375
LB1017
OPM2
H929
UTMC2
JIM3
KMS11
LB278
4p16.3
cen
tel
5'
3'
FGFR3
WHSC1/ MMSET

Richelda et al. Blood, 1997 Finelli et al. Blood,
1999
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DETECTION OF t(414)(p16.3q32) IN MM BY RT-PCR
OF IGH-MMSET FUSION TRANSCRIPTS
A
KMS-11
LB1017
UTMC2
LB278
LB375
PCL-1
OPM-2
H929
JIM3
LP1
4p16.3
cen
tel
1a
1
2
3
4
5
6 7
8
9
10
23
24
WHSC1/MMSET
FGFR3
B
D
C
KMS-11
H929
OPM-2
?
x
1
2
1
2
1
2
1064 bp
KMS-11
4
5
3
6
MB4-1
1007 bp
438 bp
H929
6
4
5
MB4-2
381 bp
275 bp
OPM-2
271,281
5
6
MB4-3
218 bp
E

Malgeri et al Cancer Res, 2000
542
438
343
239
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IGH TRANSLOCATIONS INVOLVING THE c-MAF LOCUS ON
16q23
LB3782
JJN3
ANBL6
MM.1
KMS11
RPMI
c-maf
cen
tel
D16S518
D16S3125
D16S3516
D16S3324
D16S3213
BAC 70d24
100 kb
Pt. LB3782
c-maf IGH
c-maf
a chr. 16
8
Map of 13q14 region deletion in chronic
lymphocytic leukemia and multiple myeloma
320G21
34F20
cen
tel
normal
terminal deletion
monosomy
9
del(13) is strongly associated with the t(414)
and t(1416) chromosomal translocations
MM patients with 14q32 translocations
MM patients lacking 14q32 translocations
del(13) (43 of all MM patients)
100
100
80
80
60
60
del(13)
del(13)
40
40
20
20
0
0
t(1114)
t(414)
t(1416)
other
14q32
39/176
121/290
42/05
58/68
13/14
Avet-Loiseau et al., Blood 2002
10
Prognostic relevance of major genetic lesions in
Multiple Myeloma
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Chromosome content in Multiple Myeloma (Cytogeneti
cs, DNA index, FISH)
Non- Hyperdiploidy (NHD) lt 48 and/or gt 75
chromosomes Increased prevalence of IGH
translocation
Hyperdiploidy (HD) 48-75 chromosomes Multiple
trisomies involving Chr. 3,5,7,9,11,15,19,21
50
50
Hyperdiploid
normal
Orfao et al., Citometry 1994 Wuilleme et al.,
Leukemia, 2005
12
A FISH-based trisomy index for the detection of
hyperdiploid MM
13

• Chromosome 1 abnormalities in MM occur in 40-60
  of patients
• balanced translocations (rare events)
• deletions of the short arm
• Amplification/polisomy of 1q

1q21.1 and 1q42.13 gain by FISH (63/125 50)
3 copies 51/63 (80)
4 copies 10/63 (15)
gt4 copies 2/63 (5)
Fabris et al., Leukemia 2007
14
Genetic abnormalities and survival in multiple
myeloma (1064 pts.-IFM99 trial)
Avet-Loiseau et al., Blood 109 3489-95, 2007
15
Impact of novel therapies on the prognostic
relevance of molecular lesions in MM Bortezomib
16
Using Oligonucleotide-Based DNA Microarray
Analysis to Address

• definition of gene expression profiles of
  purified plasma cells from MM patients at
  different clinical-pathological stages

• Molecular classification of MM and
  clinical-prognostic implications

17
Unsupervised analysis of plasma cell dyscrasias
4N_12MGUS_132 MM_9PCL
No distinct clusters for MGUS and PCL_ Mattioli
et al. JCO 2005 and unpublished results
18
Supervised multiclass analysis (SAM software)
identifies a probe sets (287) distinguishing
between MGUS and PCL but not MM samples
MGUS
PCL
MM
19
A gene expression signature found in MGUS is
linked to a good prognosis in Multiple
Myeloma Zhan et al. 2006 Blood
20
Gene expression profiling reveals deregulation of
Cyclin D genes in virtually all MM patients a
unifying, early event in pathogenesis
From Hideshima et al. Blood, 2005
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Molecular classification of MM based on IGH
translocations and Cyclin D genes expression
CCND1
CCND3
CCND2
FGFR3
MMSET
C-MAF
MAFB
None
TC5
TC1
TC2
TC3
TC4
t(1416)/t(1420)
t(414)
t(1114)/t(614)
Agnelli at al., JCO 2005 and unpublished results
22
Genetic alterations and pathological features in
stratified TC groups
N/K-RAS mutations
23
Transcriptional Fingerprints (166 probe sets)
Characterizing the TC Groups
Multiclass analysis (PAM software)_ Agnelli et
al., JCO 2005 and unpublished results
24
Molecular classification of multiple myeloma
(Based on the dysregulation of Cyclin D genes
and 5 recurrent IGH translocations)

• Gene expression patterns associated with groups

• Biological and clinical features associated with
  groups

• Prognosis and response to different treatments
  associated with groups

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GENOME-WIDE DNA ANALYSIS BY SINGLE NUCLEOTIDE
POLYMORPHISM (SNPs) ARRAY
SNPs are common variations occuring in the human
genome at a frequency of one in every 300 bases
(10 million positions out of the 3 billion
nucleotide human genome).
These variations can be used to track
inherithance in families, susceptibility to
disease, and allelic imbalances in cancer.
26
Frequency of DNA copy number alterations in MM
(45 pts.)
Human Mapping 50K Xba SNPs Affymetrix
LOSS
GAIN
Agnelli et al., Submitted
27
Transcriptional Fingerprint Characterizing
Chromosome 13 deletion
Pattern of del(13q) SNPs arrays 50K/H
Affymetrix Loss Gain
Agnelli et al. Haematologica, 2007
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Transcriptional fingerprint associated with 1q
gain in MM
Pattern of chromosome 1 alterations SNPs arrays
50K/H Affymetrix Loss Gain
Fabris et al. Leukemia 2007
29
Upregulation of translational machinery
discriminates between HD and NHD myelomas
Agnelli et al., Br.J. Haematol.
2007
30
(No Transcript)
31
Hierarchical clustering generated on SNPs and
gene expression microarrays data (43 pts.)
Agnelli et al., Submitted
Human Mapping 50K Xba SNPs arrays
32
Pathogenetic events and clonal origin in MM
transformation does MM represent different
diseases ?
Precursor cell
Non-hyperdiploid MM
Genomic Instability
del (13) 1q gain
Errors during switch recombination
Hyperdiploid MM
Precursor cell
Genomic Instability
Mechanisms?
33
Acknowledgments

• Centro di Genetica Molecolare- Ematologia 1-
  Fondazione IRCCS Policlinico Milano
• Dipartimento di Scienze Mediche- Università degli
  Studi di Milano
• Luca Agnelli
• Sonia Fabris
• Katia Todoerti
• Laura Mosca
• Michela Mattioli
• Luigia Lombard
• Luca Baldini
• Giorgio Lambertenghi Deliliers
• Divisione di Ematologia Azienda Ospedaliera
  Bianchi- Melacrinò-Morelli, Reggio Calabria,
  Vincenzo Callea
• Divisione di Ematologia Azienda Ospedaliera di
  Cosenza
• Fortunato Morabito
• Myeloma Working Party

Dipartimento di Processi Chimici dellIngegneria,
Università degli Studi di Padova, Padova,
Italy Silvio Bicciato
Supported by Associazione Italiana Ricerca sul
Cancro