DIAGNÓSTICO Y CLASIFICACIÓN DE LEUCEMIAS AGUDAS CON LOS PANELES EUROFLOW DIAGNOSTICS IN HEMATO-ONCOLOGY. Clinical symptoms Laboratory

Transcription

1 DIAGNÓSTICO Y CLASIFICACIÓN DE LEUCEMIAS AGUDAS CON LOS PANELES EUROFLOW DIAGNOSIS OF CLONAL HAEMATOLOGICAL DISORDERS Clinical symptoms Laboratory and signs findings Morphology + cytochemistry CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Cytogenetics Immunophenotyping Curso Avanzado de Actualización en Oncohematología por Citometria de Flujo, Buenos Aires, 31 de mayo de 2011 Molecular biology/fish DIAGNOSTICS IN HEMATO-ONCOLOGY 1. Making the diagnosis Normal reactive/regenerating malignant Annually > 300,000 new patients with a hematological malignancy in developed countries 2. Classification of hematopoietic malignancies - relation with prognosis - relevance of risk-group definition in treatment protocols Based on differentiation characteristics and particularly on chromosome aberrations, resulting in fusion gene transcripts or aberrantly (over) expressed genes 3. Evaluation of treatment effectiveness Detection of minimal residual disease (MRD): MRD-based risk-group stratification (treatment reduction or treatment intensification) Annually > 400,000 follow-up samples in leukemia patients (ALL, AML, CML) Prepared by JJM van Dongen REQUIRED DEVELOPMENTS IN FLOW CYTOMETRY (status in 2005) Immunobeads introduce combined cellular/immunobead assays special immunobead for leukemias Novel antibodies test new (academic) antibodies for application in intracellular stainings development of new antibodies against oncoproteins and aberrant signalling pathways Multicolor flow cytometry: 8 color comprehensive panels inclusion of solid state violet laser selection of appropriate fluorochromes compare conjugated antibodies (multiple companies) Development of novel software for complex pattern recognition combining multiple tubes: calculate data & multivariate analyses mapping of diagnosis and follow-up leukemia samples against templates of reference normal/control samples THE EUROFLOW APPROACH TO LEUKEMIA/LYMPHOMA IMMUNOPHENOTYPING Clinical question Diagnostic screening tube Diagnostic classification panel MRD monitoring Experience 14 Major groups 154 Nosologic entities Knowledge Evaluation Reference profiles Majority of diseases? Majority of cases? New disease entities? SET UP OF A FCM LABORATORY FOR LEUKEMIA /LYMPHOMA TYPING: CONVENTIONAL PANEL DESIGN Clinical request/need Purchase a flow cytometer Design of MAb panels (Disease category vs cell lineage oriented) Training Immunophenotypic diagnostic activity started New indications Experience Panel optimization Experience 1

2 STANDARDIZATION EFFORTS FOR IMMUNOPHENOTYPIC STUDIES - CLSI (Clinical Laboratory Standards Institute): - Stetler-Stevenson et al.: Clinical flow cytometric analysis of neoplastic hematolymphoid cells; Approved guideline. CLSI document H43-A2. CLSI, CCS (Clinical Cytometry Society): - Davis et al: 2006 Bethesda International Consensus recommendations on the flow cytometric immunophenotypic analysis of hematolymphoid neoplasias. Clin Cytometry, 72B, ESCCA (European Society for Clinical Cell Analysis: - European Leukemia Net ( - Consenso Latinoamericano (Clin Cytometry, 1998 y 2006) LEUKEMIA /LYMPHOMA IMMUNOPHENOTYPING: EVALUATION OF ANTIBODY PANELS Single center panel Single center experience/evaluation Experience-based (subjective) Long time required Limited by new: instruments techniques markers Consensus recommendations Shared experience <subjectivity Multicenter panel Multicenter evaluation possible Prospective evaluation Experimentally supported >objectivity CONSTRUCTION OF EUROFLOW LEUKEMIA/ LYMPHOMA IMMUNOPHENOTYPING ANTIBODY PANEL CONSTRUCTION OF EUROFLOW PANELS: MEDICAL INDICATION ORIENTATION/SCREENING & CLASSIFICATION PANELS Clinical request/need Proposed strategy Acute leukemia Cytopenia Sustained monocytosis Lymphocytosis LN involvement Monoclonal component Monoclonal component non-igm Suspect small cell samples (e.g. CSF, FNA, vitreous) Medical indication Design of MAb panels (Medical indication-oriented) & immunophenotyping strategy Techniques Panel evaluation vs conventional in-use panels Panel optimization (re-design) 2-8 cycles Panel evaluation Panel optimization (re-design) ALOT LST PCD SST T-ALL AML/MDS B-CLPD limited various subtypes of various subtypes of T-ALL various subtypes of AML MDS PNH CML CML-BC other MPD Eosinophilia CLL non-cll reactive/ non-aberrant B-CLPD broad CLL MCL FCL HCL clonal reactive/polyclonal other B-CLPD other clonal B T-CLPD NK-CLPD aberrant γδ+ aberrant αβ+ reactive aberrant NK cells reactive Van Dongen et al: EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. To be published in: Leukemia 2011 ALOT 1 tube T-ALL AML/MDS 4 tubes 4 tubes 4 to 7 tubes 2

3 CONSTRUCTION OF EUROFLOW ANTIBODY PANELS Step 0: Design strategy Step 1: Selection of fluorochromes Step 2: Selection of markers Step 3: Selection of antibody reagents Step 4: Selection of antibody combinations Step 5: Panel constructed ALOT (Acute Leukemia Orientation Tube) Designed for assessment of the nature of immature blast cell populations in acute leukemia samples Designed to choose appropriate immunophenotypic panel(s) Acute Leukemia Orientation Tube (AL0T) Gating markers Gating Markers Target Antigen Fluorochrome conjugate (first level) (second level) Immaturity markers Lineage markers X My cympo FITC cycd79a PE X B, T CD34 PerCP Cy5.5 - X X CD19 PE CY7 My X B, CD7 APC X X T, My smcd3 APC H7 X T cycd3 Pacific Blue X T PO X - X AL OT ALOT: B-cell precursor ALL BM stained with ALOT 8-color tube CyCD3 CD7 scd3 CD19 CyCD79a CyMPO CD34 Responsible scientist: Ludovic Lhermitte Responsible scientist: Ludovic Lhermitte T-ALL T-ALL AML Responsible scientist: Ludovic Lhermitte Responsible scientist: Ludovic Lhermitte 3

4 T-ALL AML T-ALL AML Responsible scientist: Ludovic Lhermitte Single «virtual» merged tube/data file Responsible scientist: Ludovic Lhermitte T-ALL AML T-ALL AML Responsible scientist: Ludovic Lhermitte Responsible scientist: Ludovic Lhermitte T-ALL AML ALOT (Acute Leukemia Orientation Tube) Responsible scientist: Ludovic Lhermitte Responsible scientist: Ludovic Lhermitte 4

5 ALOT: IMMUNOPHENOTYPIC CLASSIFICATION OF BLASTS Development of immunostainings protocols 8-color combinations T-ALL vs AML vs AML T-ALL vs Acute leukemia Cytopenia Sustained monocytosis Lymphocytosis LN involvement Monoclonal component Monoclonal component non-igm Suspect small cell samples (e.g. CSF, FNA, vitreous) Eosinophilia BLASTS ALOT LST PCD SST BLASTS BLASTS T-ALL vs AML vs AML T-ALL vs PerCP PacBlue PacOrange FITC PE PE Cy7 APC APC H7 Cy5.5 cycd3 cympo cycd79a CD34 CD19 CD7 CD3 Objectives: BLASTS BLASTS BLASTS Assessmentofthenatureofimmatureblastcellpopulationsin acuteleukemiasamples(b, T versus non-lymphoidormixedphenotype); Orientation towards the most appropriate complementary antibody panel(s):, T-ALL, and/oraml/mds Development of immunostainings protocols 8-color combinations panel: Development of immunostainings protocols 8-color combinations panel: PacBlue PacOrange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 PacBlue PacOrange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 cycd3 cympo cycd79a CD34 CD19 CD7 CD3 cycd3 cympo cycd79a CD34 CD19 CD7 CD3 CD20 CD58 CD66c CD34 CD19 CD10 CD38 Kappa Cyµ CD33 CD34 CD19 IgM CD117 Lambda CD9 TdT CD13 CD34 CD19 CD22 CD24 CDw65 NG2 CD34 CD19 CD123 CD81 : : PacBlue PacOrange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 PacBlue PacOrange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 cycd3 cympo cycd79a CD34 CD19 CD7 CD3 cycd3 cympo cycd79a CD34 CD19 CD7 CD3 cycd3 TdT CD99 CD5 CD10 CD1a CD3 cycd3 CD2 CD117 CD4 CD8 CD7 CD3 cycd3 TCRγδ CD33 CD56 cytcrβ CD3 cycd3 CD44 CD13 HLA Dr RA CD123 CD3 Responsible scientist: Ludovic Lhermitte Responsible scientist: Ludovic Lhermitte cycd3 TdT CD99 CD5 CD10 CD1a smcd3 cycd3 TdT CD99 CD5 CD10 CD1a smcd3 cycd3 CD2 CD117 CD4 CD8 CD7 smcd3 cycd3 CD2 CD117 CD4 CD8 CD7 smcd3 cycd3 TCRγδ cycd3 TCRγδ cycd3 CD44 CD13 HLADR RA CD123 smcd3 cycd3 CD44 CD13 HLADR RA CD123 smcd3 5

6 cycd3 TdT CD99 CD5 CD10 CD1a smcd3 cycd3 TdT CD99 CD5 CD10 CD1a smcd3 cycd3 CD2 CD117 CD4 CD8 CD7 smcd3 cycd3 CD2 CD117 CD4 CD8 CD7 smcd3 cycd3 TCRγδ cycd3 TCRγδ cycd3 CD44 CD13 HLADR RA CD123 smcd3 cycd3 CD44 CD13 HLADR RA CD123 smcd3 Positive Diagnosis cycd3 TdT CD99 CD5 CD10 CD1a smcd3 cycd3 TdT CD99 CD5 CD10 CD1a smcd3 cycd3 CD2 CD117 CD4 CD8 CD7 smcd3 cycd3 CD2 CD117 CD4 CD8 CD7 smcd3 cycd3 TCRγδ cycd3 TCRγδ cycd3 CD44 CD13 HLADR RA CD123 smcd3 cycd3 CD44 CD13 HLADR RA CD123 smcd3 Differential Diagnosis & Ambiguous lineage acute leukemia Classical classification «Maturation stage» (EGIL) cycd3 TdT CD99 CD5 CD10 CD1a smcd3 cycd3 TdT CD99 CD5 CD10 CD1a smcd3 cycd3 CD2 CD117 CD4 CD8 CD7 smcd3 cycd3 CD2 CD117 CD4 CD8 CD7 smcd3 cycd3 TCRγδ cycd3 TCRγδ cycd3 CD44 CD13 HLADR RA CD123 smcd3 cycd3 CD44 CD13 HLADR RA CD123 smcd3 Alternative T-ALL classification Maturation stage Alternative T-ALL classification Maturation stage & Well-defined molecular abberations 6

7 cycd3 TdT CD99 CD5 CD10 CD1a smcd3 cycd3 TdT CD99 CD5 CD10 CD1a smcd3 cycd3 CD2 CD117 CD4 CD8 CD7 smcd3 cycd3 CD2 CD117 CD4 CD8 CD7 smcd3 cycd3 TCRγδ cycd3 TCRγδ cycd3 CD44 CD13 HLADR RA CD123 smcd3 cycd3 CD44 CD13 HLADR RA CD123 smcd3 LAP Markers LS CD19 CD34 cycd3 cympo cycd79a CD7 smcd3 TdT CD99 CD5 C10 CD1a CD2 CD117 CD4 CD8 TCRγδ CD33 CD56 cytcr ΒF1 CD44 CD13 HADR RA CD parameters 13 parameters Colorful dots White dots = normal maturation stage = T-ALL samples 13 parameters panel CD20 CD58 CD66c CD34 CD19 CD10 CD38 smigk cyigm CD33 CD34 CD19 +CD117 smigl CD9 TdT CD13 CD34 CD19 CD22 CD24 Colorful dots White dots = normal maturation stage = T-ALL samples 7

8 panel panel ALOT ALOT CD20 CD58 CD66c CD34 CD19 CD10 CD38 CD20 CD58 CD66c CD34 CD19 CD10 CD38 smigk cyigm CD33 CD34 CD19 +CD117 smigl smigk cyigm CD33 CD34 CD19 +CD117 smigl CD9 TdT CD13 CD34 CD19 CD22 CD24 CD9 TdT CD13 CD34 CD19 CD22 CD24 Positive Diagnosis panel panel ALOT ALOT CD20 CD58 CD66c CD34 CD19 CD10 CD38 CD20 CD58 CD66c CD34 CD19 CD10 CD38 smigk cyigm CD33 CD34 CD19 +CD117 smigl smigk cyigm CD33 CD34 CD19 +CD117 smigl CD9 TdT CD13 CD34 CD19 CD22 CD24 CD9 TdT CD13 CD34 CD19 CD22 CD24 Differential Diagnosis & Ambiguous lineage acute leukemia Maturation stage (EGIL) panel panel ALOT ALOT CD20 CD58 CD66c CD34 CD19 CD10 CD38 CD20 CD58 CD66c CD34 CD19 CD10 CD38 smigk cyigm CD33 CD34 CD19 +CD117 smigl smigk cyigm CD33 CD34 CD19 +CD117 smigl CD9 TdT CD13 CD34 CD19 CD22 CD24 CD9 TdT CD13 CD34 CD19 CD22 CD24 Alternative classification Prognosis markers Immunophenotypic features associated with welldefined molecular aberrations 8

9 panel panel ALOT ALOT CD20 CD58 CD66c CD34 CD19 CD10 CD38 CD20 CD58 CD66c CD34 CD19 CD10 CD38 smigk cyigm CD33 CD34 CD19 +CD117 smigl smigk cyigm CD33 CD34 CD19 +CD117 smigl CD9 TdT CD13 CD34 CD19 CD22 CD24 CD9 TdT CD13 CD34 CD19 CD22 CD24 LAP markers LS CD19 CD34 cycd3 cympo cycd79a CD7 smcd3 CD20 CD58 CD66C CD10 CD38 smigk cyigm CD33 +CD117 smigl CD9 TdT CD13 CD22 CD NG2 CD123 CD81 31 parameters B-CELL MATURATION IN NORMAL BM NORMAL BM B_CELL MATURATION vs REGENERATION NORMAL BM B_CELL MATURATION vs REGENERATION NORMAL BM B_CELL MATURATION vs REGENERATION 9

10 VS NORMAL BM B-CELL MATURATION (CASE 4) VS NORMAL BM B-CELL MATURATION APS view 1 APS view 2 Case 1 Case 2 Case 3 Case 4 MULTIPLE CASES VS NORMAL BM B-CELL MATURATION panel Haematogones TEL-AML1 BCR-ABL Hyperdiploïdes MLL rearranged Flow cytometry immunophenotyping in AML Definition of which subpopulations to analyse Usually blast cells, Less frequently: maturing neutrophils, monocytes, erythroid cells Define which markers/combinations of markers Define the myeloid lineages involved Define the phenotypic alterations to be considered: Numerical changes Fluorescence intensity Asynchronous expression of maturation-associated markers WHO CLASSIFICATION OF AML* AML with recurrent genetic abnormalities AML with myelodysplasia-related changes AML NOS Therapy-related myeloid neoplasms Myeloid sarcoma Myeloid proliferations related to Down syndrome (DS): - Transient abnormal myelopoiesis - Myeloid leukemia associated with DS Blastic plasmacytoid dendritic cell neoplasm * SM-AHNMD 10

11 Multi-tube EuroFlow classification panel for AML/MDS Responsible scientist: VHJ van der Velden Multi-tube EuroFlow classification panel for AML/MDS (Part 2) Responsible scientist: VHJ van der Velden Tube Pacific Blue Pacific Orange FITC PE PE-Cy7 APC Aim** Tube Pacific Blue Pacific Orange FITC PE PerCP- Cy5.5 PE-Cy7 APC APC- H7 PerCP- Cy5.5 APC- H7 Aim** AML/ MDS 1 HLADR CD16 CD13 CD34 CD117 CD11b CD10 Diagnosis and subclassification of AML and PNH especially focused on neutrophilic lineage AML/ MDS 5 HLADR NG2 CD34 CD117 CD22 CD38 Aberrant expression of markers; detection of stem cells 2 HLADR CD35 CD64 CD34 CD117 IREM2 CD14 Diagnosis and subclassification of AML and PNH especially focussed on monocytic lineage 6 HLADR CD42a and CD61 CD203c CD34 CD117 CD123 CD4 Diagnosis and subclassification of AML especially focused on megakaryocytic, basophilic, mast cell and plasmacytoid dendritic lineages 3 HLADR CD36 CD105 CD34 CD117 CD33 CD71 Diagnosis and subclassification of AML especially focused on erythroid lineage 4 HLADR nutdt CD56 CD34 CD117 CD7 CD19 Aberrant expression of lymphoid-associated markers and abnormal lymphoid maturation * Further information about the markers and the availability of hybridoma clones is summarized in Appendix A. Backbone markers are indicated in bold; nu= nuclear. ** The described marker combinations might also be applied for disease staging and monitoring of treatment effectiveness (MRD diagnostics) AML- M7 HLADR CD41 CD25 CD34 CD117 CD42b CD9 Characterization of AML- 7 M7, mastocytosis * Further information about the markers and the availability of hybridoma clones is summarized in Appendix A. Backbone markers are indicated in bold; nu= nuclear. ** The described marker combinations might also be applied for disease staging and monitoring of treatment effectiveness (MRD diagnostics) IMMUNOPHENOTYPIC CHARACTERIZATION OF MDS Bone marrow HEMATOPOIESIS T/NK cell precursor Blood - tissues HOW SIMILAR ARE NEOPLASTIC CELLS TO NORMAL CELLS? - Reflect cell lineage and maturation stage. IN WHAT DO NEOPLASTIC CELLS DIFFER FROM NORMAL CELLS? - Reflect derailment of protein expression (underlying genetic abnormalities and/or changes in the BM microenvironment?) Hematopoyetic Stem cell SCF IL3 IL7 Lymphoid precursor Myeloid precursor GM- CSF IL11 TPO IL9 EPO G-CSF CFU-GM CFU-G M-CSF CFU-Eo CFU-M CFU-MC CFU-Bs BFU-E CFU-Mk B-cell precursor pdc precursor Dendritic cells Neutrophil Monocyte/ Macrophage/DC Eosinophil Mast cell Basophil Erythrocytes Platelets WHO CLASSIFICATION OF AML AML with recurrent genetic abnormalities AML with myelodysplasia-related changes AML NOS: - AML with minimal differentiation - AML without maturation - AML with maturation - Acute myelomonocytic leukemia - Acute monoblastic and monocytic leukemia - Acute erythroid leukemia - Acute megakaryoblastic leukemia - Acute basophilic leukemia - Acute panmyelosis with myelofibrosis Therapy-related myeloid neoplasms Myeloid sarcoma Myeloid proliferations related to Down syndrome Blastic plasmacytoid dendritic cell neoplasm IMMUNOPHENOTYPIC IDENTIFICATION OF LINEAGE COMMITMENT OF CD34 + BM CELLS TRANSFORMED SSC CD34 APC a PERCP TRANSFORMED SSC Neutrophil precursors b b 1024 CyMPO PE B-cell precursors c ntdt FITC Matarraz S et al. Leukemia

12 Normal Maturation of CD34+ Neutrophil precursor cells in the BM HEMATOPOIETIC MATURATION IN NORMAL BM STAGE I STAGE II STAGE III Normal BM Gated CD34+ BM cells Monocytic (MPO-/DR+) CD34+ CD34+ CD34 lo CD117+ CD117+ CD117+ HLADR+ HLADR + HLADR lo MPO +/++ MPO++ MPO++ /65- /65+ /65++ T-SSC CD64 PE Erythroid/DC CD64- CD64- CD PERCPCY5,5 -> PerCP CD36 FITC Normal Maturation of Erythroid Cells in the BM IMMUNOPHENOTYPIC IDENTIFICATION OF LINEAGE COMMITMENT OF CD34 + BM CELLS STAGE I STAGE II STAGE III STAGE IV CD34+ CD34+ CD34 lo CD34- CD117+ CD117+ CD117+ CD117 lo HLADR+ HLADR lo HLADR lo HLADR- CD36 lo/+ CD36+ CD36+ CD36+ CD105- CD105+ CD105+ CD105 lo/- CD71 lo CD71 lo CD71 hi CD71 hi lo lo lo - CD64 PE Monocytic CD36 FITC f Erythroid CD123 PE Basophil HLADR FITC pdc e CD117 PE Mast cells h HLA-DR FITC Matarraz S et al. Leukemia 2008 IMMUNOPHENOTYPE OF CD34+ MYELOID-COMMITTED HPC CELL LINEAGE SSC Erythroid Megakaryocytic high Neutrophil Eosinophil Basophil Monocytic Mast cell pdc IMMUNOPHENOTYPE stable CD36+, CD64-, lo, CD105+ high high low CD61+, lo CyMPO+, CD13hi CyMPO-,, /65+, CyEPO+ CD123hi, HLADRlo, CD117lo hi, CD203c+ stable CyMPO-,, CD64+, DR+, CD117lo low CD117hi, HLADRlo, hi stable CD123hi, HLADRhi, CD36+ SSC SSC A HPC CD34 + B-cell precursors NRBC EARLY MYELOID PRECURSORS UNGATED BM EVENTS Maturing Neutrophils Eosinophils Basophils pdc Monocytic cells Mature Lymphocytes CD34 - B-cell precursors -PerCP C UNGATED EVENTS CD117-PE CD117 + / CD34 - precursors CD34 + /CD117 + HPC SSC SSC B CD34-APC D CD117 + / CD34 - Erythroid precursors UNGATED BM EVENTS Gated CD117 + and/or CD34 + events -PerCP CD34 + HPC CD117 + /CD34 - neutrophil precursors CD34 + HPC 12

13 HEMATOPOIETIC MATURATION IN NORMAL BM MONOCYTIC MATURATION IN NORMAL BM T-SSC Normal BM PERCPCY5,5 -> PerCP CD64 PE Gated CD34+ BM cells Monocytic (MPO-/DR+) CD36 FITC Erythroid/DC CD64-PE CD36 FITC -> CD36-FITC FLAER-FITC CD14-APCH7 CD14-APCH7 IREM-2 APC HLADR CD64 CD36 CD14 IREM2 PLASMACYTOID DENDRITIC CELLS PHENOTYPIC CHANGES DURING NORMAL MAST CELL DIFFERENTIATION CD117 Normal BM CD123-PE CD34 APC CD34 cytryptase FcεRI CD203c HLADR FITC -> HLA DR-FITC PerCP/Cy5.5 -> PerCP Cy5.5 ACUTE MAST CELL LEUKEMIA Acute leukemia BM CD123 PE HLA-DR FITC HLADR FITC -> T-SSC PERCPCY5.5 -> PerCPCy5.5 -PerCP Cy5.5 TRANSFORMED SSC PECY5 PC5 -> CD34-PE PECY5 PC5 -> CD34-PE CD117 APC APC -> WHO CLASSIFICATION OF AML* AML with recurrent genetic abnormalities AML with myelodysplasia-related changes AML NOS Therapy-related myeloid neoplasms Myeloid sarcoma Myeloid proliferations related to Down syndrome (DS): - Transient abnormal myelopoiesis - Myeloid leukemia associated with DS Blastic plasmacytoid dendritic cell neoplasm * SM-AHNMD MONOCYTIC LINEAGE IN MDS: ABERRANT PHENOTYPES Normal BM MDS BM T-SSC T-SSC PERCPCY5.5 -> copia -PerCP PERCPCY5.5 -> PerCP MON CD14-PE CD14-PE MAR -> MON IREM2-FITC MAR -> copia IREM2-FITC T-SSC T-SSC CD56-PE CD56 PE -> MON CD56-PE CD56 PE ->

14 N-DIMENSIONAL NEUTROPHIL MATURATION IN NORMAL BM N-DIMENSIONAL NEUTROPHIL MATURATION IN NORMAL BM Myeloblasts Promyelocytes Neutrophils Bands HLADRPE CD117PE PO FITC CD16FITC CD10APC CD38APC CD13PE CD11bAPC Metamyelocytes CD34PerCP Myelocytes Maturation stage of neutrophil precursors in normal BM N-DIMENSIONAL NEUTROPHIL MATURATION IN NORMAL BM VS AML Neutrophil maturation in normal BM: definition of maturation stages based on principal component analysis (PCA) 0f 10 parameters BM 1 Maturation stage of AML blasts BM2 BM3 BM4 BM5 All BM Aberrant phenotype of AML blasts Aberrant markers 26.6 CD SSC 19.2 FSC 19.0 CD11b BM6 BM8 BM7 6.0 Antigen expression patterns during monocytic maturation in normal BM Monocytic maturation in normal BM: definition of maturation stages based on principal component analysis (PCA) of data on 10 parameters BM 1 BM2 BM3 BM5 BM 4 All BM BM 6 BM7 BM8 14

15 Erythroid maturation in normal BM: definition of maturation stages based on principal component analysis (PCA) of data on 10 parameters BM 1 BM2 BM3 AML/MDS vs normal BM: myelomonocytic maturation CD34 + HPC: Immature BM 4 BM5 Normal BM CD117-PE HLADR-FITC Pre-B CyMPO-PE Neutrophil BM 6 All BM BM7 BM8 RAEB-1 CD117-PE Immature Neutrophil HLADR-FITC CyMPO-PE DE NOVO AML: ABERRANT PHENOTYPES & CLONAL HEMATOPOIESIS (n=68) Phenotype Polyclonal AML# Clonal AML* N=12 N=49 Normal phenotype 58% 2% <2 altered lineages 83% 8% N. of altered lineages 0,7 2,7 Total 12/61 (20%) 49/61 (80%) # One ISM-AML case with KIT mutation restricted to mast cells * Two cases showed coexistence of t(8;21) & D816V KIT mutation in all cellular compartments analyzed Fernández et al, 2011 (in preparation) WHO CLASSIFICATION OF AML AML with recurrent genetic abnormalities - AML with t(8;21)(q22;q22); RUNX1-RUNX1T1 - AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 - APL with t(15;17)(q22;q12); PML-RARA - AML with t(9;11)(p22;q23); MLLT3-MLL - AML with t(6;9)(p23;q34); DEK-NUP214 - AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1 - AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1 - AML with mutated NPM1 - AML with mutated CEBPA AML with myelodysplasia-related changes AML NOS Therapy-related myeloid neoplasms Myeloid sarcoma Myeloid proliferations related to Down syndrome Blastic plasmacytoid dendritic cell neoplasm AL: GENOTYPIC-PHENOTYPIC ASSOCIATIONS Diagnosis Genetic Aberrant immunophenotype lesion Bead-based flow cytometric assay for detection of fusion proteins Patents: US 6,610,498 B1 (26 August 2003) US 6,686,165 B2 (3 February 2004) beads coated with anti-rara antibody bead t(9;22)* CD34 hi,cd10+,cd38 lo,cd13 lo 5 PML RARA 3 bead t(12;21) CD34 het,cd10+,cd20 -,CD13 lo 11q23 CD34 +,CD10 -,7.1 +, + AML t(15;17) CD34 -/+, -/lo,cd2 -/lo,cd13 het PML RARA mrna translation transcription PML RARA PML RARA PML RARA fusion proteins cell lysate Inv(16) MPO hi,cd2 -/lo t(8;21) CD19 +,CD q23 CD56 +,7.1 -/+,CD19 -/lo,cd2 -/+ Ortuño F, Orfao A, Cytometry B, 2004 Dept. of Immunology, Erasmus MC, Rotterdam FITC-conjugated anti-pml antibody bead 15

16 Results of PML-RARA fusion protein detection using the immunobead assay At this moment the technical developments for 7 well-defined fusion proteins have (virtually) been completed: MFI value 100,000 10,000 1, BCR1 BCR2 BCR3 negative result of RQ-PCR CML: BCR-ABL : completed RUO kit launched and published precursor-b-all: BCR-ABL TEL-AML E2A-PBX1 MLL-AF4 : completed : completed : completed : completed AML: AML1-ETO : completed CBFB-MYH11: completed Precursor-B-ALL tube Multiplex tube close to completion Core-factor tube Multiplex tube: prototype completed PML-RARA : completed prototype testing completed 10 APL AML CML T-ALL n=46 (non APL) n=1 n=34 n=16 n=66 MUCHAS GRACIAS 16