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

Disclosures No conflicts of interest to disclose

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

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

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?

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

(<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

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 < 0.0001 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

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

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

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

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 50% for 17p deletion 20% for 1q21 amplification An et al., Clin Cancer Res, 2015

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

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

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

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

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

Available techniques for genome study Karyotype / FISH Microarrays Sequencing

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

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

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

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

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

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

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

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

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

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