Affy
- 1. Phenotypic identification of subclones in multiple myeloma with different genomic profile, clonogenic potential and drug sensitivity Bruno Paiva University of Navarra, Spain
- 2. The statements in this presentation are those of the author and not of Affymetrix
- 3. Multiple myeloma • Second most common hematological malignancy - Incidence: ~4/100.000 persons/year - Prevalence: 60.000 patients (Europe) - Incidence increases with age: 80% of patients > 60y (rare in <35y) • Clinical Course: Remitting and Relapsing disease - With current treatment • 5-year survival 50% - 70% • Potentially cured ~ 10% Despite the progress in survival with novel agents……. the majority of patients eventually relapses (remains a largely incurable disease)
- 4. B-cell differentiation BM Plasma cells CD10- CD19+ CD20- CD27++ CD38+++ CD138+ PB Plasma cells CD10- CD19+ CD20het CD27++ CD38++ CD138het SLT Plasmablasts CD10het CD19+ CD20+ CD27++ CD38+++ CD138- SLT/PB Memory CD10- CD19+ CD20+ CD27+ CD38+ SLT GC B-cells BM ProB CD10++ CD19+ CD20- CD27- CD38++ BM PreB CD10+ CD19+ CD20het CD27- CD38++ BM/PB Immature CD10het CD19+ CD20+ CD27- CD38het BM/PB/SLT Naive CD10- CD19+ CD20+ CD27- CD38- CD10- CD19+ CD20++ CD27het CD38het
- 5. Plasma cells: terminally differentiated but… … new-born vs. long-lived CD19 CD81 heterogenous heterogenous ( 80% +ve cells) ( 95% +ve cells) CD45 CD56 heterogenous heterogenous ( 80% +ve cells) (95% -ve cells)
- 6. PC characterization Technology Cytogenetics FISH GEP CNA miRNA Methylation NGS 1995 2000 2005 2010 2013 Clinical utility ISS Tx groups TC groups ISS-FISH GEP sig Morgan G. Educational Session ASH 2012
- 7. Substantial baseline clonal heterogeneity and subsequent clonal selection under treatment Keats JJ, et al. Blood. 2012;120:1067-76. Egan JB, et al. Blood. 2012 120: 1060-1066 Bolli N, et al. Nat Commun. 2014;5:2997
- 8. MM: genetic markers with prognostic significance FISH analysis IGH translocations Gene expression t(4;14) t(14;16) t(11;14) Genomic imbalances Non-hyperdiplid 1q gains 1p deletions Monosomy 13 17p deletions SNP-based mapping array 16q deletions 12p deletions 1q gains 5q gains profiling TC classification Molecular classifications (UAMS & Hovon) 70 gene-model (Arkansas group) 15 gene-model (Intergroupe Francophone) Perez-Simon, Blood 1999; Fonseca Blood 2003; Chang Blood 2005; Gutierrez Leukemia 2007; Avet- Loiseau JCO 2010 & Blood 2011; Boyd Leukemia 2011, Kumar Blood 2012; Zhan Blood 2006, Saughnessy Blood 2007; Deacaux Blood 2008; Broyl Blood 2010; Tapper JCO 2011
- 9. Disease models of tumour cell heterogeneity: multiple myeloma Bone marrow Clones with a distinct pattern of mutations
- 10. Identification of subclonal heterogeneity through generation of iPEP (immunophenotipyc expression profiling) • iPEP for all 23 phenotypic markers analysed plus FSC and SSC was generated for every single clonal PC Merging of 4 different tubes using backbone markers Software calculation of “missing values”Files 1, 2, 3, 4
- 11. Identification of subclonal heterogeneity through generation of iPEP (immunophenotipyc expression profiling) ≥2 subclones in 35/116 (30%) newly-diagnosed MM patients Top-markers for identification of distinct phenotypic subclones CXCR4, CD44, CD19, HLADR, CD54, CD49e, CD138, β7, CD33, CD20, CD81, CD27, CD56 Paino T, et al. Blood 2013;122(21): abstract 531 (oral presentation)
- 12. -sorted distinct phenotypic subclones are often associated with different cytogenetic profiles Patient Subclones 1p 1q t(14q32) RB1 (13q14) TP53 (17p13) #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 CD81+ 2N CD81- 2N Β7+ 2N Β7- 2N CD45+ 2N CD45- 2N CD56-, CD81- 2N CD56+, CD81+ NT CD56+ 11% -1p CD56- 53% -1p CD56+ 50% +1p CD56- 50% +1p CD19+ 2N CD19- 2N CD38+, SSC↑ NT CD38low SSC↓ 2N CD81- 29%+1p CD81+ 35%+1p CD56+ NT CD56- NT CD56+ NT CD56- NT 2N neg 2N neg 46% +1q 80% 77% +1q 91% 2N neg 2N neg 2N 61% NT 56% 2N neg 2N neg 50% +1q 67%* 50% +1q 15% * 2N neg 2N neg NT 26% 2N 84%* 29%+1p neg 35%+1p neg NT 24% NT neg NT neg NT neg 2N 2N 2N 14% del 2N 2N 78% del 11% del 2N 2N 66% del 2N 2N 2N 2N 2N 2N 2N 2N 2N 70% del 60% del 30% del 2N 2N NT 2N NT 2N 2N 87% del 87% del 2N 2N 2N 2N 2N 2N 15% del 2N 100% del 100% del 100% del 100% del Paino T, et al. Blood 2013;122(21): abstract 531 (oral presentation)
- 13. FACS-sorted distinct phenotypic subclones are often associated with different cytogenetic profiles del(14q32): 67% 70% del(13q14) 60% del(17p13) del(14q32): 15% 30% del(13q14) 0% del(17p13) Paino T, et al. Blood 2013;122(21): abstract 531 (oral presentation)
- 14. Clonal selection after drug exposure: MRD as a reservoir of chemoresistant cells Baseline Cycle 9 MRD Cycle 18 MRD PCA in merged files Paino T, et al. Blood 2013;122(21): abstract 531 (oral presentation)
- 15. Disease models of PC heterogeneity: myeloma Bone marrow MRD Clones with a distinct pattern of mutations
- 16. The deepest the response, the longer the survival Achievement of CR as a surrogate marker for extended survival EFS 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 0 12 CR vs nCR or PR nCR vs PR 24 36 48 60 72 Months from diagnosis CR, n=278 OS P<10-5 P=0.07 84 96 nCR, n=124 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 CR vs nCR P=0.01 CR vs PR P<10-6 nCR vs PR P=0.04 12 24 36 48 60 72 84 96 Months from diagnosis PR, n=280 PD, n=25 Lahuerta JJ, et al. J Clin Oncol. 2008;26:5775-82.
- 17. -color flow: patients <65y • 125 patients in CR after HDT/ASCT (GEM2000) TTP OS 100 100 80 80 Median: 141m 60 60 40 Median: 62m 40 20 20 Median: 61m 0 P < 0.001 Median: 36m 0 P < 0.001 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 160 Flow CR (n=71) MRD positive (n=57) Paiva B et al; Blood. 2008; 15;112(10):4017-23 (f/u updated July 2012)
- 18. MRD myeloma cells with high-risk cytogenetics are associated with faster relapses PFS 100 MRD+ (median 0.1% BM clonal PCs) / Standard-risk FISH: median PFS 39m 80 60 40 20 0 0 20 40 MRD+ (median 0.02% BM clonal PCs) / High-risk: median PFS 22m P <0.001 60 80 100 120 140 Paiva B, et al. Blood. 2012;119:687-91.
- 19. The paradigm of the myeloma treatment To achieve (operational) cure or long-term disease control (through immune surveillance), eradicating the maximum number of tumor cells is a prerequisite • Maximizing cure rates by personalizing therapy is one of the major aims of modern therapy Tumor cells 109 108 107 106 105 104 103 102 101 Presentation PR VGPR CR MRD How is the chemoresistant clone? Immune surveillance of undetectable MRD 10 (Operational cure) 0 Time to progression Modified from Morgan GJ, et al. Blood 2013;122: 1332-1334
- 20. The pathogenesis of myeloma Gonzalez, D. et al. Blood. 2007;110(9):3112-21
- 21. Circulating B-cells from patients with MM and MGUS are usually devoided of clonotypic B-cells CASE ID ISOTYPE Naive Peripheral blood B-cells IgM+ Memory IgG+ Memory Peripheral Peripheral blood Normal blood IgA+ Memory PCs MM-PCs MGUS 1 IgG - MGUS 2 IgG NT MGUS 3 IgG NT MM 1 IgG - MM 2 IgA - MM 3 IgG - MM 4 IgA - MM 5 IgG - MM 6 IgA - MM 7 IgG - - - - - NT NT NT - - - - - - - - - - - NT NT - - - - NT NT NT NT NT NT - - - - NT - - - - - NT NT NT NT NT + NT + + + FACS of highly purified B-cell maturation subsets (>95%) Sensitivity of ASO-PCR (10-4 - 10-5) N.T.: Not tested The presence of clonal myeloma PCs in PB of myeloma patients is a frequent finding Thiago et al. Haematologica 2013
- 22. Cell competition for potentially overlapping BM niches 1.0% 0.8% 0.6% 0.4% 0.2% 0.1% % of normal BMPC *** p <.001 vs. MGUS and SMM HA MGUS 100% 0% Burger et al. Blood 2006 107: 1761-1767 MGUS SMM % of BM B-cell subsets 100% % of BM Lymphoid CD34+ HSC 1,0% Pro-B Pre-B Smoldering MM MM Symptomatic MM % of PB clonal PC *** p <.001 vs. 80% 60% 40% * p <.05 20% vs. HA 0% 80% 60% ** p ≤.005 vs. HA 40% *** p <.001 vs. HA 20% 0% 0,8% 0,6% 0,4% 0,2% 0,0% MGUS and SMM Paiva et al. Leukemia 2011; 25: 697-706
- 23. -CTCs are present in every stage and predict disease transformation/aggressiveness • MM-CTCs are detected in the PB of MGUS (0% - 81%) 1-4, smoldering MM (50% - 75%) 1,5, symptomatic MM (35% - 87%) 1,2,4,6-9 and relapse/refractory MM (52%) 10 patients • The number of MM-CTCs predicts malignant transformation in MGUS 3 and smoldering MM 5 and inferior OS in symptomatic 8 and relapsed/refractory MM 10 1. Billadeau. Blood. 1996 1;88(1):289-96. 5. Bianchi. Leukemia. 2012 doi: 10.1038/leu.2012.237 2. Schneider. Br J Haematol. 1997; 97(1):56-64. 6. Rawstron. Br J Haematol. 1997 ; 97(1):46-55. 3. Kumar. J Clin Oncol. 2005 20;23(24):5668-74. 7. Luque. Clin Exp Immunol. 1998 ;112(3):410-8. 9. Chandesris. Br J Haematol 2007; 136: 609-614. 4. Paiva. Leukemia. 2011; 25(4):697-706. 8. Nowakowski. Blood. 2005 ;106(7):2276-9. 10. Peceliunas. Leuk Lymphoma. 2012 ; 53(4):641-7.
- 24. What is the role of MM-CTCs in the pathogenesis of multiple myeloma? • Are all BM MM-PCs capable to egress into PB, or only a specific sub-clone? • Do MM-CTCs have stem cell-like features and are enriched by clonogenic cells? • Does circadian rhythms also affect MM-CTCs?
- 25. The potential to egress into PB is restricted to a minor sub-clone in the BM… BM MM-PC vs. CTCs: principle component analysis (APS) of 22 antigens Patient #1 Patient #3 Patient #5 Patient #7 Patient #9 Patient #2 Patient #4 Patient #6 Patient #8 Patient #10 …with an unique profile of integrin and adhesion molecules Paiva B, et al. Blood. 2013;122(22):3591-8.
- 26. MM-CTCs are mostly quiescent DRAQ5 + 4-color flow cytometry % of cells in S-phase (n=10) P=.005 2.5 2.0 1.5 1.0 0.5 0.0 BM MM-PCs MM-CTCs Paiva B, et al. Blood. 2013;122(22):3591-8.
- 27. Clonogenic potential of BM MM-PCs vs. MM-CTCs in co-culture with stromal cells • Same number of BM MM-PCs and MM-CTCs cells seeded with hTERT stromal cells (10:1 ratio) Nº of colonies Nº of clusters Patient (nº of cells) BM MM-PCs MM-CTCs BM MM-PCs MM-CTCs #1 (1.200) 0 0 0 0 #2 (5.300) 0 1 0 0 #3 (6.500) 2 5 0 2 #4 (10.000) 0 0 0 0 #5 (34.900) 0 0 0 0 #6 (72.000) 0 0 0 0 #7 (80.000) 0 0 1 14 #8 (100.000) 0 0 0 0 All measurements at day 14 Colonies: >40 cells Clusters: 10-39 cells Paiva B, et al. Blood. 2013;122(22):3591-8.
- 28. Paired BM MM-PCs and MM-CTCs show the same response to chemotherapy • Cytotoxicity measured after 48h • Bortezomib: 2.5nM; Lenalidomide: 1.0 µM; Dexamethasone: 10nM Bortezomib 100 80 60 40 20 0 BM MM-PCs MM-CTCs VRD (BortzLenDex) 100 80 60 40 20 0 BM MM-PCs MM-CTCs Combined (n=7) 100 80 P =.320 60 40 20 0 BM MM-PCs MM-CTCs Paiva B, et al. Blood. 2013;122(22):3591-8.
- 29. MM-CTCs (median cells/µL) CD34+ HSC (median cells/µL) 16h 24h 8h 16h 20h 4h 12h 20h MM patients at relapse (n=6) CXCR4 (Amount of antigen MFI expression / MM-CTC) SDF-1α levels (pg/mL) 16h 24h 8h 16h 20h 4h 12h 20h Quantification started at 16:00pm every 4h up to 12:00am next day (when patients' initiated treatment) Time points 16h and 21h have been duplicated to facilitate viewing of the time curve Paiva B, et al. Blood. 2013;122(22):3591-8.
- 30. Cytogenetic comparison between paired BM MM- PCs and MM-CTCs: less abnormalities? • Purity of BM MM-PCs and MM-CTCs FACS sorting ≥95% (n=4) BM MM-PCs MM-CTCs BM MM-PCs MM-CTCs +1q21 (23%) +1q21 (28%) -13q14 (80%) 13q14 (2N) 17p13 (2N) 17p13 (2N) BM MM-PCs MM-CTCs -13q14 (95%) -13q14 (97%) +9q34 (90%) +9q34 (80%) BM MM-PCs MM-CTCs C9C C9C +9q34 (23%) 9q34 (2N) Paiva B, et al. Blood. 2013;122(22):3591-8.
- 31. Disease models of PC heterogeneity: myeloma Bone marrow MRD PB-CTC Clones with a distinct pattern of mutations EMD
- 32. A Darwinian view of myeloma treatment MGUS SMM MM Tumor progenitor cell
- 33. A Darwinian view of myeloma treatment MGUS SMM Early-treatment Treatment modifies the balance between existing and competing Myeloma progenitor cell sub-clones, resulting in a reduction of clonal complexity
- 34. A Darwinian view of myeloma treatment Therapy MGUS SMM MM Original clone - Drug X resistant Myeloma progenitor cell Drug X sensitive Triple-drug combinations to target all different clones Always consider retreating with a previous therapy that was functional