1. Genetic profiling of multiple myeloma
Mar Mallo, PhD
Josep Carreras Leukemia Research Institute

2. Disclosures
No conflicts of interest to disclose

3. Diagnosis of hematological neoplasms
Laboratory data
Clinical data
Flow cytometry
IHC
Genetic study
Cytology

4. Heterogeneous disease
Diagnosis of hematological neoplasms
Laboratory data
Clinical data
Flow cytometry
IHC
Genetic study
Cytology
Heterogeneous disease
Different clinical evolution entities
<6 months
Median OS: 3-4 years
>10 years

5. Low infiltration of tumoral cells in the bone marrow
Cytogenetic study limitation of MM
Failure
10-25%
Normal karyotype
40-50%
Low infiltration of tumoral cells in the bone marrow
Abnormal karyotype
30-40%
Agressive?

6. Plasma cells need to be identified/selected
Identification/selection of plasma cells
Identification by FICTION
Combination of FISH and detection of Ig (fluorescent antibody κ or λ)
Selection by immunomagnetic beads
CD138 antibody
Selection by sorter
CD138 antibody

7. (<48 / >75 chromosomes)
Genetic characterization of MM
Cytogenetics
FISH
Microarrays (CGH/SNP)
30%
>90%
90-100%
Low mitotic index
MM hyperdiploid
(48-75 chromosomes) (57%)
MM non-hyperdiploid
(<48 / >75 chromosomes)
Trisomies of odd chromosomes (3, 5, 7, 9, 11, 15, 19 and 21)
Translocations of the immunoglobulin gene (IGH, 14q32) (30-40%)
Deletions
Monosomies (hypodiploid)
WHO classification of Tumours of Haematopoietic and Lymphoid Tissues, 2008

8. Importance of IGH translocations (30-40%)
Cyclin D1 gene – t(11;14)(q13;q32) Cyclin D2 gene – t(12;14)(p13;q32)
Cyclin D3 gene – t(6;14)(p21;q32)
Survival probability
Time from diagnosis (months)
t(4;14) (n=29)
No t(4;14) (n=231)
1.0
0.8
0.6
0.4
0.2
0.0 80 60 40 20
100
P <
FGFR3 gene – t(4;14)(p15;q32)
Patients with t(4;14) respond to treatment but early relapses
C-MAF gene – t(14;16)(q32;q23)
MAFB gene – t(14;20)(q32;q11)
Gutiérrez et al., Leukemia, 2007
Fonseca et al., Blood, 2003
WHO classification of Tumours of Haematopoietic and Lymphoid Tissues, 2008

9. Importance of chromosome 1 alterations
1p deletion
Poor prognosis for both 1p22 and 1p32 deletion
1q gain
Poor prognosis for both OS and PFS
Hebraud et al., Leukemia, 2013
Avet-Loiseau et al., JCO, 2012

10. Importance of 17p deletions
Adverse prognosis
Uncommon in newly diagnosed MM
TP53 mutations are very rare
The lack of P53 may promote the extramedullary disease
Chang et al., Blood 2005; Tiedemann et al., Leukemia 2008; López-Anglada et al. Eur J Haematol 2009; Avet-Loiseau et al., JCO 2013

11. Cytogenetic risk stratification
Cytogenetic analysis is one of the most important prognosis factors in MM: and it is mandatory at diagnosis to define high risk cytogenetic patients
Standard-risk
Intermediate-risk
High-risk*
Trisomies (hyperdiploidy)
t(11;14)
t(6;14)
t(4;14)
17p deletion
t(14;16)
t(14;20)
High risk gene expression profiling signature
* In the presence of concurrent trisomies, patients with high risk cytogenetics should be considered standard-risk
Rajkumar et al., Am J Hematol, 2014

12. Importance of clone size by FISH
Study in 333 newly and 92 relapsed MM patients
IGH related rearrangements observed in majority of purifed plasma cells. Although 13q deletion, 17p deletion and 1q21 amplification were present in different percentages
The optimal cut-off with prognostic value:
10% for 13q deletion % for 17p deletion % for 1q21 amplification
An et al., Clin Cancer Res, 2015

13. Adverse FISH alterations
Strong association among adverse lesions
Adverse FISH alterations
t(4;14); t(14;16); t(14;20); 1q gain; 17p deletion
Within the subset of patients with hyperdiploidy, there is cosegregation of additional adverse cytogenetic lesions as well
Boyd et al., Leukemia, 2012
Pawlyn et al., Blood, 2014

14. Strong association among adverse lesions
Prognostic model in MM based on co-segregating adverse FISH lesions
OS graded by number of adverse lesions
The triple combination of an adverse IGH translocation, +1q and del(17p) was associated with a median OS of 9.1 months
The coexistence of hyperdiploidy did not alter the PFS or OS of patients with the presence of any adverse cytogenetic lesion
61 months
9 months
24 months
42 months
Boyd et al., Leukemia, 2012; Pawlyn et al., Blood, 2014

15. Strong association among adverse lesions
Bortezomib regimens improve but not overcome outcome of high-risk cytogenetics
Better than control
Worse than control
Bortezomib regimens
IMiDs regimens
Bergsagel et al., Blood, 2013

16. Recommendations for risk stratification
Investigation recommended for risk stratification
Serum albumin and 2-microglobulin to determine ISS stage
t(4;14), t(14;16), and del(17p) on plasma cells by FISH
LDH
Immunoglobulin type IgA
Histology: plasmablastic disease
Additional investigation for risk stratification
Cytogenetics
Gene expression profiling
Labeling index
MRI/PET scan
DNA copy number alteration by CGH/SNP array
Munshi et al., Blood, 2011

17. Recommendations for risk stratification
Cytological study
plasma cells infiltration?
<30%
>30%
CD138+ isolation
Karyotype
Bad prognosis probes
TP53
IGH split
if IGH positive: (t(4;14), t(14;16), t(11;14))
FISH
Munshi et al., Blood, 2011
Guidelines and Quality Assurance for Acquired Cytogenetics, 2011

18. Available techniques for genome study
Karyotype / FISH
Microarrays
Sequencing

19. Whole-genome and exome sequencing
Massive parallel sequencing in paired samples (tumoral and control) in 203 patients
131 (65%) had evidence of mutations in one or more of the 11 recurrently mutated genes
KRAS
NRAS
FAM46C
TP53
DIS3
MM are highly heterogenous
Mutations are clonal or subclonals, can be initiators or potentiators
Subclones with multiple mutations affecting the same pathway
development of new treatments
Previously identified by Chapman et al. (2011)
BRAF
TRAF3
CYLD
RB1
PRDM1
ACTG1
Pathogenesis of MM
Chapman et al., Nature, 2011
Lohr et al., Cancer Cell, 2014

20. Clonal heterogeneity Stable genomes:
No differences between diagnosis and relapse clones
Low-risk cytogenetics
Linear evolution:
the relapse clone apparently derives from the major subclone at diagnosis, but additionally acquired lesions
High-risk cytogenetics
Branching (nonlinear) model:
the relapse clone clearly derives from a minor subclone, barely present at diagnosis. There is clonal heterogeneity at diagnosis.
High-risk cytogenetics
Keats et al., Blood, 2012
Magrangeas et al., Leukemia, 2012

21. Clonal heterogeneity
The earliest myeloma-initiating clones, some of which only had the initiating t(11;14), were still present at low frequencies at the time of diagnosis
For the first time in myeloma, they demonstrate parallel evolution whereby two independent clones activate the RAS/MAPK pathway through RAS mutations and give rise subsequently to distinct subclonal lineages
Melchor et al., Blood, 2014

22. Impact of intra-clonal heterogeneity for MM treatment
Treating early phases (SMM)
As clonal progression is the key feature of transformation of HR-SMM to MM, to treat predominant clone typical of MM already present at the SMM stage will be the future
Walker et al., Leukemia, 2014

23. Gene expression profiling
Transcriptome studies
Genome
Transcriptome
Proteome
Study of genes differentially expressed
Gene expression profiling
70 genes signature
30% mapping to chr. 1 
(contribution to disease progression)
GEP is useful for risk stratification, it is limited by a lack of a uniform platform and availability, and thus is not easily translated into clinical practice
High risk signature
Shaughnessy et al., Blood, 2007

24. Genetic abnormalities in myeloma cells
It is not all in the genes
Genetic abnormalities in myeloma cells
Microenvironment
Extracellular matrix, stroma cells, osteoblasts/osteoclasts

25. It is not all in the genes
The bone marrow niche in MM
MM cells interact with osteoclasts, osteoblasts, stromal cells, and endothelial cells
Multiple cytokines and chemokines are secreted in response to these cell-cell interactions, leading to enhanced tumor growth, inhibition of osteoblasts, and increased osteoclast activity
Ghobrial et al., Blood, 2012

26. It is not all in the genes
Exosomes and MM
Small membrane vesicles that mediate cell-to-cell communications. They have multiple roles in tumorigenesis and contributes to tumor expansion
Bone marrow mesenchymal stromal cell-derived exosomes facilitate MM progression and can play a role in drug resistance in MM cells
Roccaro et al., Journal of Clinical Investigation, 2013
Wang et al., Blood, 2014

27. Take-home message In terms of diagnosis…
1- Cytogenetic study (FISH) is mandatory, preferentially on selected plasma cells
2- Prognostic implications of chromosomal alterations:
Good prognosis: hyperdiploid, IGH translocations with cycline genes
Poor prognosis: t(4;14), t(14;16), t(14;20), chr 1, 17p deletion
In terms of research…
3- Single cell sequencing techniques defines clonal architecture (clonal heterogeneity)
4- Expression techniques show high risk gene expression signatures
5- Microenvironment plays a role in progression and drug resistance in MM

28. Thanks for your attention
Special thanks to: Norma Gutiérrez (Hospital de Salamanca)
Mª José Calasanz (Universidad de Navarra)
Francesc Solé (IJC)