Molcular Bioloy and Cytometry Course, 16 May 2013, Moll Minimal Residual Disease in Acute Myeloid Leukemia Vincent H.J. van der Velden, NL
Survival of AML patients Overall survival 100% 80% 60% 40% 0-14 year 15-44 year 45-54 year 55-64 year 65-74 year >75 year 20% 0% 0 1 2 3 4 5 Years from diagnosis à Clear need for early recognition of good and poor risk patients Dutch Cancer registration: www.ikcnet.nl
Treatment effectiveness in AML Characteristics of tumor cells e.g. - genetic abnormalities - in vitro drug sensitivity - gene expression profile - immunophenotype/morphology In vivo drug distribution e.g. - gastrointestinal absorption - distribution in body (e.g. CNS) - drug metabolism (e.g. polymorphisms in enzymes) - liver excretion - kidney excretion Treatment compliance e.g. - duration of Rx - side effects (e.g. allergy, infections) r e l a t i v e f r e q u e n c y o f l e u k e m i c c e l l s Evaluation of overall treatment effectiveness by detection of MRD 1 10-1 10-2 10-3 10-4 10-5 10-6 10-7 0 cure 0 0.5 1 1.5 2 follow-up in years
Detection of MRD in AML Clinical significance Methods to detect MRD: PCR analysis of genetic abnormalities Flowcytometric analysis of aberrant immunophenotypes
Clinical significance of MRD in AML Does MRD has clinical significance? If so, What time-points are informative? What cut-off levels are informative? Is there any difference between adult and childhood AML?
MRD: prognostic significance - adults Relapse-free survival Relapse-free survival Buccisano et al. Leukemia 2006 After induction 1 Cut-off: 0.035% MRD negative: 35% MRD positive: 65% After consolidation Cut-off: 0.035% MRD negative: 39% MRD positive: 61%
MRD: prognostic significance - adults Relapse-free survival Relapse-free survival Feller et al. Leukemia 2004 After induction 2 Cut-off: 0.14% (median) MRD negative: 50% MRD positive: 50% After consolidation Cut-off: 0.11% (median) MRD negative: 50% MRD positive: 50%
MRD: prognostic significance - childhood Overall survival Event-free survival (3-y) Coustan-smith et al. BJH 2003 Langebrake et al. JCO 2006 After induction 1 After induction 1 Cut-off: 0.1% Cut-off: 0.1% (+0.3%) MRD negative: 66% MRD positive: 34% MRD negative: 60% MRD positive: 40%
MRD: prognostic significance - childhood Relapse-free survival Inaba et al. JCO 2012 Relapse-free survival Van der Velden et al. Leukemia 2010 MRD 0.1% After induction 1 Cut-off: 0.1% MRD negative: 63% MRD positive: 37% After induction 1 Cut-off: 0.1% and 0.5% MRD negative: 34% MRD low positive: 40% MRD high positive: 26%
Clinical significance of MRD in AML MRD is an strong and independent prognostic factor, both in childhood and adult AML Relevant cut-off levels vary between 0.01% and 0.5% Relevant cut-off levels are protocol-dependent
Detection of MRD in AML Clinical significance Methods to detect MRD: PCR analysis of genetic abnormalities Flowcytometric analysis of aberrant immunophenotypes
Molecular MRD analysis in AML Only applicable in limited number of patients Prognostic factor in adult AML Clinical significance still questionable in childhood AML (particularly for AML1- ETO and CBFB-MYH11) Inaba et al, JCO 2012
Detection of MRD in AML Clinical significance Methods to detect MRD: PCR analysis of genetic abnormalities Flowcytometric analysis of aberrant immunophenotypes
Detection of aberrant immunophenotypes 1 Normal bone marrow AML at diagnosis (AML12-GLA-017) asynchronous antigen expression
Detection of aberrant immunophenotypes 2 Normal bone marrow AML at diagnosis (AML12-DUB-005) cross-lineage antigen expression
Detection of aberrant immunophenotypes 3 Detection of leukemia-associated immunophenotypes Type of aberrant immunophenotype childhood AML 1 adult AML 2 Cross-lineage antigen expression 80% 35% (e.g. CD33+CD2+) Antigen over-expression 25% 10% (e.g. CD34++, CD10++) Asynchronous antigen expression 80% 60% (e.g. CD34+HLA-DR-) Ectopic antigen expression <10% <10% (e.g. TdT+CD2+ in blood) one Leukemia-associated immunophenotype >90% >90% 1 Van der Velden et al, Leukemia 2010; 2 Dutch-Belgian working group, submitted
MRD detection by flow cytometry Diagnosis (AML12-DUB-005) Follow-up 0.03% Sensitivity: 0.1-0.01% (10-3 10-4 )
MRD detection by flow cytometry: pitfalls 1 Heterogeneity of AML blast cells Blast cell subsets observed in ~75% of AML Preferably all subpopulations should be monitored (focus on immature cells) AML at diagnosis (AML12-SHE-014)
MRD detection by flow cytometry: pitfalls 2 Multiple myeloid lineages and differentiation stages Experience and knowledge required for recognition of LAIP (TdT) (MPO) myeloid progenitor cell HLA-DR CD34 CD117 ( CD13 ) ( CD33 ) ( CD7 ) MPO myelomonocytic progenitor cell HLA-DR CD34 CD117 CD13 CD33 ( CD15 ) MPO monoblast HLA-DR CD34 CD13 CD33 CD36 MPO MPO MPO myeloblast (HLA-DR) CD34 CD117 CD13 CD33 (CD15) MPO promonocyte HLA-DR CD13 CD33 CD11b (CD14) CD36 promyelocyte CD117 CD13 CD33 CD15 myelocyte CD13 CD33 CD15 CD11b (CD16) MPO monocyte HLA-DR CD13 CD33 CD11b CD14 CD36 MPO granulocyte CD13 CD33 CD15 CD11b CD16 CyCD68 macrophage HLA-DR ( CD13 ) ( CD33 ) CD11c ( CD14 ) ( CD36 ) ( CD1 ) CD68 RFD9 Monocytic lineage Granulocytic lineage Erythroid lineage immature megakaryoblast HLA-DR CD34 CD33 ( CD36 ) ( CD41/CD61 ) ( CD42 ) proerythroblast CD36 CD71 (CD235a) megakaryoblast CD36 CD41/CD61 CD42 ( CD9 ) erythroblast CD36 CD71 CD235a megakaryocyte CD36 CD41/CD61 CD42 CD9 erythrocytes CD235a platelets CD36 CD41/CD61 CD42 CD9 Megakaryocytic lineage Dendritic cells Eosinophils Basophils Mast cells
Identification of LAIPs: experience is required Electronic data file of diagnostic samples were distributed over 5 labs Identification of LAIP in every center Institute Percentage of missed LAIPs Phase 1 Phase 2 Total period 1 11 (6/55) 3 (3/88) 6 (9/143) 2 39 (18/46) 27 (24/88) 31 (42/134) 3 51 (28/55) 36 (32/88) 42 (60/143) 4 63 (34/54) 40 (34/84) 49 (68/138) 5 61 (20/33) 28 (18/65) 39 (38/98) Missed: 40-66% 25-40% 30-50% Dutch/Belgian MRD working party: Department of Hematology, VUmc, Amsterdam (coordinator: GJ Schuurhuis); Department of Hematology, University Hospital KU, Leuven; Central Hematological Laboratory, University Medical Center, Nijmegen; Department of Clinical and Tumor Immunology, Erasmus MC, Rotterdam; Department of Immunology Erasmus MC, Rotterdam
Non-evaluable patients due to missing of LAIPs Patients (%) in whom none of the consensus LAIPs was identified Samples with no LAIPs (%) 100 80 60 40 20 0 Phase 1 Phase 2 Institute 1 Institute 2 Institute 3 Institute 4 Institute 5 33% 7% Dutch/Belgian MRD working party: Department of Hematology, VUmc, Amsterdam (coordinator: GJ Schuurhuis); Department of Hematology, University Hospital KU, Leuven; Central Hematological Laboratory, University Medical Center, Nijmegen; Department of Clinical and Tumor Immunology, Erasmus MC, Rotterdam; Department of Immunology Erasmus MC, Rotterdam
MRD detection by flow cytometry: pitfalls 3 Sensitivity: 1. How to determine sensitivity Testing LAIP on bone marrow FU samples from other AML patients Testing LAIP on normal bone marrow à not easy! Samples should be matched for age and for time-point in protocol Gated on CD34+CD13- cells Coustan-Smith et al, BJH 2003
Background of LAIP in normal bone marrow Percentage of leukocytes Dutch/Belgian MRD working party: Department of Hematology, VUmc, Amsterdam (coordinator: GJ Schuurhuis); Department of Hematology, University Hospital KU, Leuven; Central Hematological Laboratory, University Medical Center, Nijmegen; Department of Clinical and Tumor Immunology, Erasmus MC, Rotterdam; Department of Immunology Erasmus MC, Rotterdam
MRD detection by flow cytometry: pitfalls 3 Sensitivity: 1. How to determine sensitivity Testing LAIP on bone marrow FU samples from other AML patients Testing LAIP on normal bone marrow à not easy! Samples should be matched for age and for time-point in protocol à not easy! 2. Sensitivity/Specificity Adult AML studies: 0.01% in ~70% of patients Childhood AML studies: 0.01% in ~50% of patients à Due to differences in immunophenotype (related to difference in genotype)? à Due to differences in bone marrow regeneration?
Bone marrow regeneration after course 1 Adults: Myeloblast : 0.8% Lymphoblasts: 0.4% Erythroblasts: 0.4% Children: Myeloblast : 1.8% Lymphoblasts: 3.9% Erythroblasts: 1.0%
MRD detection by flow cytometry: pitfalls 4 10 Center 1 Variability in MRD assessment MRD level (%) 1 0,1 0,01 negative 0 5 10 15 20 25 Samples (ordered according to increasing MRD %) Dutch/Belgian MRD working party: Department of Hematology, VUmc, Amsterdam (coordinator: GJ Schuurhuis); Department of Hematology, University Hospital KU, Leuven; Central Hematological Laboratory, University Medical Center, Nijmegen; Department of Clinical and Tumor Immunology, Erasmus MC, Rotterdam; Department of Immunology Erasmus MC, Rotterdam
Variability in MRD assessment 10 Center 1; Center 2; Center 3; Center 4; 1 MRD level (%) 0,1 0,01 negative 22% 0 5 10 15 20 25 Samples (ordered according to increasing MRD %) Dutch/Belgian MRD working party: Department of Hematology, VUmc, Amsterdam (coordinator: GJ Schuurhuis); Department of Hematology, University Hospital KU, Leuven; Central Hematological Laboratory, University Medical Center, Nijmegen; Department of Clinical and Tumor Immunology, Erasmus MC, Rotterdam; Department of Immunology Erasmus MC, Rotterdam
MRD detection by flow cytometry: pitfalls 5 Immunophenotypic changes between diagnosis and relapse Diagnosis (AML12-GLA-024) Relapse Immunophenotypic shifts occur in >90% of patients Preferably 2 patient-specific labelings should be used per patient In vast majority of patients at least one MRD labelling remains informative
MRD detection by flow cytometry: pitfalls 6 Reporting of data: Percentage per MNC Rubnitz et al. Lancet Oncology 2010
MRD detection by flow cytometry: pitfalls 6 Reporting of data: Percentage per MNC Percentage per leukocytes Including erythroid precursors? Van der Velden et al. Leukemia 2010 Feller et al. Leukemia 2004
MRD detection by flow cytometry: pitfalls 6 Reporting of data: Percentage per MNC Percentage per leukocytes Including erythroid precursors? Correction for %LAIP? Feller et al. Leukemia 2004
Flow cytometric MRD detection Applicable in vast majority of patients Fast But: Sensitivity not yet optimal Not standardized Interpretation/data analysis subjective and experience-based à Improvements are needed
Improving MRD detection by flow cytometry Focus on stem cells (CD34+CD38-) van Rhenen et al. Clin Cancer Res 2005 % CD38-/CD34+ stem cells at diagnosis à High frequency of stem cells associated with poor clinical outcome
CLL1: expressed on AML stem cells van Rhenen et al. Blood 2007 à CLL1 can be used as an AML stem cell specific marker
Clinical significance of stem cell MRD Thesis M. Terwijn, VUmc, Amsterdam Cut off: 3x10-6 (!!)
Improving MRD detection by flow cytometry Additional markers ( 8 color flow) à more specific (?) New software tools? Abnormal? à Erythroid precursors
Conclusions MRD detection has clinical significance in AML Optimal cut-off levels and time-points are protocol dependent At present: very complex, should only be done in limited number of reference laboratories Sensitive, standardized, widely applicable, and reproducible MRD approach is required This may be achieved by improvements in flow cytometry: focus on stem cells new software à more objective and automated analysis possible Standardization
Acknowledgements Department of Immunology Leukemia and Lymphoma Diagnostics Contact: v.h.j.vandervelden@erasmusmc.nl
MRD: prognostic significance overview Study n Cut-off Time point Survival (MRD+ vs MRD-) Adults Al Mawali et al. 09 25 0.15% post-ind RFS 29% vs 56% 22 0.15% post-cons RFS 11% vs 77% Maurillo et al. 08 142 0.035% post-cons 5-y RFS 16% vs 60% Buccisano et al. 06 100 0.035% post-cons RFS 16% vs 75% Kern et al. 04 62 variable post-ind RFS 0% vs 50% post-cons RFS 26% vs 83% San Miguel et al. 01 126 0.01-1% post-ind RFS 0% vs 14% vs 50% vs 84% San Miguel et al. 97 53 0.5% post-ind RFS 33% vs 80% 53 0.2% Post-Cons RFS 31% vs 68% Venditti et al. 04 56 0.035% post-cons RFS 23% vs 83% Children Langebrake et al. 06 150 variable post-ind EFS 50% vs 70% Sievers et al. 03 252 0.5% post-cons 3-y OS 41% vs 69% Coustan-Smith et al. 03 41 0.1% post-ind 2-y OS 30% vs 72%
Improving MRD detection by flow cytometry Additional markers ( 8 color flow)? New software tools?