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1 de DUVE INSTITUTE Hematological malignancies : Aims of the genetic characterization INTERUNIVERSITY CERTIFICATE IN HUMAN GENETICS Université catholique de Louvain Brussels,14/02/2014 Professor Hélène Antoine-Poirel, MD, PhD Center for Human Genetics 1
2 Hematopoiesis
3 Normal hematopoiesis(2) : growth factors
4 Normal hematopoiesis(3) : transcription factors
5 Classification of hematological malignancies (WHO 2008) Clonal proliferation of a bone marrow hematopoietic precursor With block of differentiation = acute Without block of differentiation = chronic Myeloid lymphoid Quantitative abn Qualitative abn Acute Myeloblastic Leukemia Acute Lymphoblastic Leukemia Myeloproliferative neoplasm Myelodysplastic neoplasm
6 Classification of B-cell and T-cell lymphomas (WHO 2008) Anaplastic Large Cell lymphoma
7 Aims of the genetic characterization of cancers 1. Diagnosis Clonality and malignancy Classification (positive and differential diagnosis) 2. Prognosis 3. Minimal residual disease 4. Treatment Molecules targeting the genetic alteration Host polymorphisms triggering the drug response and/or toxicity Pharmacogenetics 5. Cancer genetic predisposition 6. Research 7
8 Clonality as a marker of malignancy All cells derive from the same ancestral cell where occurred the acquired initiating mutation Thrombocytosis, polycythemia, myelofibrosis : Myeloproliferative neoplasm or reactive proliferation? JAK2 V617F mutation = clonal myeloproliferative disorder polycythemia vera / Vaquez (95%) essential thrombocytosis (50-60%) myelofibrosis (50-60%) CalR (ex9) = clonal myeloproliferative disorder essential thrombocytosis (25%) myelofibrosis (35%) 8
9 Myeloproliferative neoplasms CalR CalR
10 Classification of Acute myeloid leukemia (AML) : FAB Classification (1976) Morphology (+ immunophenotype -1991) (AML minimally differentiated M0) AML without maturation M1 AML with maturation M2 Promyelocytic acute leukemia M3 Myelomonocytic leukemia M4 AML-M4 with abn eosinophils M4Eo Monoblastic/monocytic AL M5a/b Erythroid AL M6 Megacaryoblastic AL M7 10
11 Acute myeloid leukemia (AML) WHO classification (2008) Morphology + Immunophenotype + Genetics AML with recurrent genetic abnormalities AML with balanced translocations / inversions t(8;21)(q22;q22) RUNX1-RUNX1T1 inv/t(16;16)(p13;q22) CBFB-MYH11 abnormal eosinophils (M4Eo) t(15;17)(q22;q12) PML-RARa promyelocytic (M3) t(9;11)(p22;q23) MLLT3-MLL t(6;9)(p23;q34) DEK-NUP214 basophilia inv/t(3;3)(q21;q26.2) RPN1-EVI1 t(1;22)(p13;q13) RBM15-MKL1 megacaryoblastic (M7) AML with gene mutations Normal cytogenetics t(8;21), inv/t(16;16) FLT3-ITD, FLT3-TKD MLL-PTD NPM1 mutations (ex12) CEBPA mutations KIT mutations
12 Acute myeloid leukemia (AML) WHO classification (2008) AML with myelodysplasia-related changes following a myelodysplastic syndrome (MDS) or myeloproliferative disorder without prior MDS AML & myelodysplastic syndromes, therapy related Alkylants agents Ionizing radiation therapy Topoisomerase II inhibitors Others AML not otherwise specified AML with minimally differentiation AML without maturation AML with maturation Acute myelomonocytic leukemia Acute monoblastic & monocytic leukemia Acute erythroid leukemia Acute megacaryoblastic leukemia Acute basophilic leukemia Acute panmyelosis with myelofibrosis FAB M0 M1 M2 M4 M5a/b M6 M7 Myeloid sarcoma
13 Core Bing Factor : RUNX1/CBFα/AML1 & CBFβ GM-CSF, IL-3, M-CSF-R, TCRα, TCRß p21/waf1, MPO, NE, granzyme B, NP-3. AML-M2 : t(8;21)(q22;q22) AML-M4Eo : t/inv(16)(p13q22) Childhood preb ALL: t(12;21)(p13;q22)
14 Retinoic Acid Receptor - alpha RARα R A AA PML-RARα R R R R AA R AA A
15 MLL (11q23)
16 Molecular heterogeneity of AML with normal karyotype Roboz GJ ASH Education Book December 10, 2011 vol no
17 Cooperating mutations in leucemogenesis Class I mutations Proliferative and / or survival advantage FLT3-ITD FLT3-TKD KIT RAS PTPN11 JAK2 + AML Class II mutations block in hematopoietic differentiation and in resulting apoptosis PML-RARA == RUNX1-RUNX1T1 CBFB-MYH11 MLL fusions CEBPA NPM1? ASH 2005
18 Next Generation Sequencing : new recurring mutations 5 classes of genes involved in leukemogenesis == == == Murati A BMC Cancer 2012
19 Schematic representation of epigenetic regulation of a leukemogenic locus (LL) framed by histone H3 Murati A, et al, BMC Cancer 2012
20 Model of leukemogenesis: slot machine Murati A BMC Cancer 2012
21 Schematic representation of the current knowledge of genotype/phenotype relationships in MDS, MDS/MPN, and a related MPN like CNL SF3B1 & RARS SF3B1/JAK2 or SF3B1/MPL & RARS-T miscellanious? RCUD Various combinations RNA splicing (SRF2,U2F1) DNA methylation (TET2, DNMT3A) RCMD RAEB TET2/SRSF2 & CMML ASXL1 : poor CSF3R & CNL SETBP1 & acml Cazzola M, et al, Blood 2013
22 Acute lymphoblastic leukemia /lymphoma (ALL) FAB (1976) GEIL/EGIL (1994/95) WHO (2001/08) Morphology + Immunophenotype + Genetics ALL-L1 ALL-L2 ALL-L3 Pro-B-ALL Common ALL Pre-B-ALL Mature B-ALL Pro-T-ALL Pre-T-ALL Cortical T-ALL Medullary T-ALL ALL of B-cell precursors t(9;22)(q34;q11.2) 3-4% BCR-ABL t(v;11q23) 2-3% MLL Rgt t(12;21)(p13;q22) 16-29% ETV6-RUNX1 Hyperdiploidy > % Hypodiploidy 5% t(5;14)(q31;q32) IL3-IGH t(1;19)(q23;p13.3) 6% TCF3-PBX1 ALL of T-cell precursors 1p32 25% SIL-TAL1 del(9)(p21) 30% p16 t(10;v)(q24;v) 7-31% TLX1 t(5;14)(q35;q32) 13-20% TLX3 t(10;11)(p13;q14) 9-30% CALM-AF10 t(11;v)(q23;v) 8% MLL-v 9q34 6% NUP214-ABL inv(7)(p15q35) 5% TCRβ-HOXA
23 B-cell Lymphomas
24 B-cell Lymphomas t(14;18)(q32;q21) t(3;v)(q27;v) t(8;14)(q24;q32)
25 Diffuse Large B-Cell Lymphoma : Classification based on gene expression profiling Clinical heterogeneity of DLBCL Non reproducibility of morphology and immunohistochemistry Gene expression profiling : 2 main molecular subtypes of DLBCL : those derived from the lymph node germinal center (GCB) and others derived from an activated B-cell (ABC) (c) NFKB activation REL amplification BCL2 translocation Ash A. Alizadeh, Nature 2000
26 Relativity of the diagnostic value The specificity of genetic alterations is relative BCR-ABL : characteristics of CML (~100%) 25% of adult ALL (children : 2-4%) MYC : characteristics of Burkitt lymphomas (~95%) ~25% of childhood DLBCL (adults : 5-10%) BCL2 : characteristics of follicular lymphomas (~95%) ~20-30% of adult DLBCL (children : ~0%) Majoritary clone Primary genetic event? Sub-clone Secondary event?
27 Aims of the genetic characterization of cancers 1. Diagnosis Clonality and malignancy Classification (positive and differential diagnosis) 2. Prognosis 3. Minimal residual disease 4. Treatment Molecules targeting the genetic alteration Host polymorphisms triggering the drug response and/or toxicity Pharmacogenetics 5. Cancer genetic predisposition 6. Research 27
28 Childhood ALL : Impact of cytogenetics on survival (Moorman AV Blood 2007)
29 Adult ALL : Impact of cytogenetics on survival Cytogenetics : 1366 (90%) of 1522 patients Successful : 1003 cases (73%) Abnormal clone : 796 cases (79%) (Moorman AV Blood 2007)
30 Adult AML : Impact of cytogenetic entities recognized in 2008 WHO classification on survival Grimwade D et al. Blood 2010;116:
31 Adult AML : Impact of gene mutations on survival 3 prognostic groups using 4 marker combinations: low- risk, biallelic CEBPAm + and/or NPM1m + /DNMT3Am high-risk, DNMT3Am + and/or MLLm + and intermediate, all remaining cases Shen Y et al. Blood 2011;118:
32 Revised risk stratification of AML according to different models Risk profile Dohner H et al. (Blood 2010;115: )( ) Patel JP et al. (NEJM 2012;366: ) Grossmann V et al. (Blood 2012;120: ) Based on cytogenetic analysis mutation analyses of the NPM1, CEBPA, and FLT3 genes excluding cases of acute promyelocytic leukemia integrated cytogenetic and mutational analysis (MLL-PTD, FLT3-ITD, NPM1, CEBPA, TET2, ASXL1, DNMT3A, PHF6, IDH1, and IDH2) only molecular markers (PML-RARA, RUNX1-RUNX1T1, CBFB-MYH11, FLT3-ITD, MLL-PTD, mutations in NPM1, CEPBA, RUNX1, ASXL1, and TP53) Favorable t(8,21)(q22;q22); RUNX1-RUNX1T1 t/inv(16)(p13.1q22); CBFB-MYH11 NPM1 mut/flt3-itd- (*) CEBPA mut (*) Favorable cytogenetics NPM1 mut/flt3-itd-with IDH1 or IDH2 mutations Very favourable: PML-RARA CEBPA double-mut Favorable: RUNX1-RUNX1T1 CBFB-MYH11 NPM1 mut/flt3-itd- Intermediate Unfavorable Intermediate-I: NPM1 mut/flt3-itd + and normal karyotype NPM1 wt/flt3-itd + and normal karyotype NPM1 wt/flt3-itd- and normal karyotype Intermediate-II: t(9,11)(p22;q23); MLLT3-MLL Cytogenetic abnormalities not classified as favorable or adverse inv(3)(q21q26.2) or t(3,3)(q21;q26.2); RPN1-EVI1 t(6,9)(p23;q34); DEK-NUP214 t(v;11)(v;q23); MLL rearranged 5 or del(5q); -7; abnl(17p); complex karyotype CEBPA mut FLT3-ITD- and all these genes wildtype: ASXL1, MLL-PTD, PHF6, and TET2 FLT3-ITD+, trisomy 8-negative and all these genes wild-type: MLL-PTD, TET2, and DNMT3A Unfavorable cytogenetics FLT3-ITD+, CEBPA wt and any of these gene alterations: MLL-PTD, TET2, DNMT3A mutations or trisomy 8 CEBPA single-mut/flt3-itd + NPM1 mut/flt3-itd + Wild-type cases Unfavorable: MLL-PTD and/or RUNX1 and/or ASXL1 mut Very unfavourable: TP53 mut
33 Myelodysplastic syndromes (MDS): recurring chromosomal abnormalities & frequency at diagnosis Unbalanced MDS t-mds Balanced MDS t-mds +8 * 10% t(11;16)(q23;p13.3) 3% -7/del(7q) 10% 50% t(3;21)(q26.2;q22.1) 2% 5/del(5q) 10% 40% t(1.3)(p36.3;q21.2) 1% del(20q) * 5-8% t(2;11)(p21;q23) 1% -Y * 5% t/inv(3)t(q31q26.2) 1% i/t(17q) 3-5% t(6;9)(p23;q34) 1% -13/del(13q) 3% del(11q) 3% del/t(12p) 3% del(9p) 1-2% idic(x)(q13) 1-2% t-mds = therapy related-mds WHO 2008
34 Myelodysplastic syndromes (MDS): International Prognostic Score System IPSS (Greenberg, 1997) Score Prognostic variables % blast cells <5% 5-10% 11-19% 20-30% (AML) Cytopenias Hb<10mg/dL PN<1.8x109/L PQ<100x109/L Karyotype Good Intermediate Poor N -Y del(5q) del(20q) Other Complex ( 3 an) Chr7 abn
35 Myelodysplastic syndromes (MDS): Revised IPSS Score IPSS-R (Greenberg, 2012) Prognostic variable 0 0,5 1 1, % Bone marrow blast cells 2 >2-<5% 5-10% >10% Hb 10 8-<10 <8 PQ <100 <50 ANC 0,8 <0,8 Karyotype Very good Good Intermediary Poor Very poor -Y del(11q) +8-7 > 3 abn Normal +19 inv(3)/t(3q)/del(3q) del(5q) i(17q) 2 abn including- 7/del(7q) del(12p) Other simple abn complex = 3 an del(20q) Independant double clones 2 an including del(5q) del(7q-) 5 risk groups Very low Low IntermediaryHigh Very high Score 1,5 <1,5-3 <3-4,5 <4,5-6 >6
36 Multiple Myeloma Mayo Stratification of Myeloma and Risk-Adapted Therapy ( Dispenzieri A et al., Mayo Clin Proc ) High risk (~25% of patients) Al least one criteria : FISH 17p deletion FISH t(4;14) translocation FISH t(14;16) translocation Karyotype 13q deletion Karyotype hypodiploidy Plasma cell labelling index 3% Standard risk (~75% of patients) other FISH or karyotype abn, including : Hyperdiploidy FISH t(11;14) translocation FISH t(6;14) translocation
37 Aims of the genetic characterization of cancers 1. Diagnosis Clonality and malignancy Classification (positive and differential diagnosis) 2. Prognosis 3. Minimal residual disease 4. Treatment Molecules targeting the genetic alteration Host polymorphisms triggering the drug response and/or toxicity Pharmacogenetics 5. Cancer genetic predisposition 6. Research 37
38 Minimal residual disease After induction therapy of leukemias assessment of the complete remission at the different levels : clinical, morphological, immunological, cytogenetic, molecular (real-time PCR)
39 Childhood ALL Evaluation by Real-Time RT-PCR with oligonucleotide probes specific of the patient Immunoglobulin / TCR rearrangement Sensitivity : 10-4 Kaplan-Meier estimates of the Relapse-free Interval according to the - presence or absence and - level of Residual Disease in patients with a first Complete Remission of ALL at the end of induction therapy (Cave H et al. N Engl J Med 1998)
40 Chronic myeloid leukemia (CML) Cytogenetic & molecular response Cytogenetic response : Absent >95% Minimal 66-95% Minor 36-65% Partial 1-35% Complete 0% Assessment on at least 20 mitoses Major molecular response (MMR) : Decreased BCR-ABL transcript level 0.1% of average BCR-ABL transcript level in 30 untreated CML-CP European LeukemiaNet Recommendations (Baccarini, Blood 2013) : definition of the response to TKIs : optimal / warning / failure Based on cytogenetics, FISH and molecular analyses (BCR-ABL quantification & mutation analysis of the ABL TK domain)
41 Aims of the genetic characterization of cancers 1. Diagnosis Clonality and malignancy Classification (positive and differential diagnosis) 2. Prognosis 3. Minimal residual disease 4. Treatment Molecules targeting the genetic alteration Host polymorphisms triggering the drug response and/or toxicity Pharmacogenetics 5. Cancer genetic predisposition 6. Research 41
42 Promyelocytic acute leukemia (AML-M3/APL) faggot cells = bundles of Auer rods
43 Promyelocytic acute leukemia (AML-M3/APL) t(15;17)(q22;q21)
44 Promyelocytic acute leukemia (AML-M3/APL) FISH : fusion PML-RARA
45 Promyelocytic acute leukemia (AML-M3/APL) LMA M3 t(15;17)(q22;q21) = fusion between PML (15q22) and RARA, retinoic acid receptor alpha (17q21) Reduced sensitivity of the nuclear receptor to the ligand, retinoic acid All Trans Retinoic Acid ATRA (Vesanoid ) ( vitamin A)
46 Promyelocytic acute leukemia (AML-M3/APL) 1µM ATRA : differentiation induction with growth inhibition and apoptosis
47 RARα R A AA PML-RARα NPM-RARα NuMA-RARα R R R R AA ATRA sensitivity R R 45 mg/m 2 /jr A A AA AA R AA PLZF-RARα STAT5b-RARα R R R R R R R AA R A Poor ATRA sensitivity R R R R 45 mg/m 2 /jr R AA AA R
48 Chronic myeloid leukemia (CML) LMC t(9;22)(q34;q11) fusion between BCR (22q11) and ABL1 (9q34) Constitutively activated chimeric tyrosine kinase leukemia mouse Tyrosine kinase inhibitor (STI571 / Imatinib / Glivec )
49 Inhibitors of tyrosine kinase activity competitive antagonists of the ATP-binding site of bcr-abl Druker, B. J. Blood 2008;112:
50 Chronic myeloid leukemia (CML) : Glivec resistance LSI BCR/ABL (Vysis) LSI BCR/ABL (Vysis) 006C C BCR-ABL amplification ABL tyrosine kinase domain mutations and overexpression of drug-efflux proteins (ABCB1 and ABCG2) Development of new tyrosine kinase inhibitors (Dasatinib, Nilotinib, )
51 ABL KD Mutations Sensitivity of BCR/ABL Kinase Domain Mutations to TKIs (values are given as IC 50 Redaelli, S. et al. J Clin Oncol; 27:
52 Pharmacogenetics : Host polymorphisms trigger the drug response and/or toxicity Polymorphisms in susceptibility genes for environmental / genotoxic exposures Activation enzymes (phase I) = cytochromes Detoxification enzymes (Phase II) = Glutathion S-Transferase Nucleic acid synthesis : folat metabolism and Methotrexate 6-mercaptopurinethol metabolism : thiopurine methyltransferase (TPMT) deficit
53 Folate metabolism & Methotrexate enzymes subject to common genetic polymorphisms DHFR (dihydrofolate reductase), GGH (gamma glutamyl hydrolase), MTHFR (methylenetetra-hydrofolate reductase), FPGS (folylpoly-glutamate synthetase) Carroll, W. L. et al. Hematology 2003;2003:
54 Thiopurin metabolism : 6-mercaptopurine (Purinethol & ALL) Probability of complete remission vs thiopurine methyltransferase (TPMT) genotype in childhood ALL (SJCRH Total XII trial) Carroll, W. L. et al. Hematology 2003;2003:
55 Perspectives Exhaustive tumor sequencing (exome genome) Genetic «signature» of the tumor : specific & limited repertoire of synergistic «driver» mutations Relapse of acute leukemia : dynamic process with : New mutations Clonal selection : Persistance of a dominant clone and / or Selection & expansion of a minor clone Improved knowledge of the physiopathogeny Towards an integrated & dynamic classification? New therapeutic targets Combined targeted treatments = personalized therapeutic «strategies» (signalling pathways, epigenetic mutations)
56 Aims of the genetic characterization of cancers 1. Diagnosis Clonality and malignancy Classification (positive and differential diagnosis) 2. Prognosis 3. Minimal residual disease 4. Treatment Molecules targeting the genetic alteration Host polymorphisms triggering the drug response and/or toxicity Pharmacogenetics 5. Cancer genetic predisposition 6. Research 56
57 Predisposition to hematological malignancies Very rare familial disorders Bone marrow failures Fanconi anemia (FA complementation groups) Dyskeratosis congenita (TERC / TERT /DKC1) Diamond-Blackfan Anemia (RPS19) Shwachman-Diamond Syndrome (SBDS) Predisposition to AML / MDS CEBPA RUNX1 GATA2 Haematologica 2010 Low penetrance susceptibility Chronic Lymphocytic Leukemia Acute Lymphoblastic Leukemia
58 Thank you! de DUVE INSTITUTE 58
GENETICS OF HEMATOLOGICAL MALIGNANCIES
de DUVE INSTITUTE GENETICS OF HEMATOLOGICAL MALIGNANCIES INTERUNIVERSITY CERTIFICATE IN HUMAN GENETICS Université catholique de Louvain Brussels,19/02/2016 Professor Hélène Antoine-Poirel, MD, PhD Center
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