Proyecto EuroFlow: objetivos, resultados y perspectivas futuras

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1 Curso Avanzado de Actualización en Onco-hematolog hematología por Citometria de flujo, Buenos Aires, 30 de mayo de 0 Proyecto EuroFlow: objetivos, resultados y perspectivas futuras Un nuevo concepto en diagnóstico or citometria de flujo Jacques J.M. van Dongen & Alberto Orfao por el grupo Discrimination between normal malignant immune cells in blood, bone marrow lymphoid tissues Normal days weeks to months Reactive / regeneration Laboratory methods: - Cytomorphology immunophenotyping genetic events treatment Malignant lymphoma multiple myeloma CLL ALL, AML CML - Molecular diagnostics, e.g. PCR-based clonality diagnostics molecular classification via detection of oncogenetic defects Diagnostics for hematological malignancies. Making the diagnosis Normal reactive/regenerating malignant Annually > 300,000 new patients with a hematological malignancy in developed countries. Classification of hematopoietic malignancies - relation with prognosis - relevance of risk-group definition in treatment protocols Based on differentiation characteristics 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) Identification of different B-cell subpopulations in childhood BM Pro-B-cell Pre-B-I cell Pre-B-II cell Immature B-cell Mature B-cell Plasma cell staining TdT///CD0 + ( lymphogate) staining ///CD + ( lymphogate) -PerCP TdT- - - CD- - E.G. van Lochem et al., Cytometry Part B 004; 60B: -3. Flow cytometric immunophenotyping of normal malignant leukocytes Gaps areas for improvement (Status in 005) Management structure of EuroFlow Consortium Erasmus MC USAL DYNOMICS CYTOGNOS IMM UNIKIEL AP-HP UNIVLEEDS PPH/O SAM Rotterdam Salamanca Rotterdam Salamanca Lisbon Kiel Paris Leeds Prague Zabrze no technical stardization in flow cytometry no guidelines for selection of the appropriate antibody clones virtually no new markers introduced over a decade (st-still in development) many oncoproteins (including fusion proteins) not yet included in immunostaining protocols; 3-4-color flow cytometry has many limitations: limited sensitivity limited specificity management of large data files from multiple samples is complex time-consuming; new software needed for: fast easy analysis of data; automated patient reports; introduction of flow data into electronic hospital systems. Advisory Committee - M. Kok - C. Romeo Casabona - A.J.M. de Wild-Chardonnens - J.F. San Miguel - H.J.H.M. Claassen General Assembly (one representative per participating organisation) Coordination Committee (WP) J.J.M. van Dongen, coordinator A. Orfao, vice-coordinator M. Kneba E.A. Macintyre WP WP3 DYNOMICS CYTOGNOS WP4 WP5 A.Orfao M. Kneba WP6 Intellectual Property Committee - E. van Oosterom, lawyer - L.A.C.M. van Wezenbeek, patent attorney - J.J.M. van Dongen, coordinator Management team - A. Steenbergen project () manager - W. Borsje (financial officer) - B. van Bodegom (secretariat) International collaboration between academia industry E.A. Macintyre

2 5 th EuroFlow Educational Workshop, Paris, FR, 9 March 0 Achievements of the EuroFlow Consortium Multicolor flow cytometry ( 8 colors) with full technical stardization inclusion of violet laser selection of appropriate fluorochromes stardization of instrument settings laboratory protocols detailed testing comparison of antibody clones conjugated antibodies (multiple companies) Implementation development of novel software fast easy hling of large data files (including automated pattern recognition) combining multiple tubes: calculation APS view mapping of diagnosis follow-up leukemia samples against templates of Development of 8-color antibody protocols for diagnosis, classification screening tubes (include recognition of normal leukocyte subsets) multi-tube panels for diagnosis classification per disease category special tubes for MRD monitoring per disease category Stardization in diagnostic flow cytometry Stardization according to literature generally refers to: lists of CD codes markers per disease category rarely a specific antibody is recommended (almost) never a fluorochrome is proposed HOWEVER: Stardization according to GLP guidelines dems for much higher levels of stardization EuroFlow stardization aims at: usage of comparable flow cytometers (3 lasers 8 colors) full stardization of instrument settings (e.g. based on stard beads) stardized laboratory protocols immunostaining procedures (SOP s) careful selection of optimal antibody clones per marker/cd code selection of optimal 8-color antibody combinations fluorochromes design of combinations of multiple 8-color tubes: estimation APS view new software for fast easy data analysis with automated pattern recognition recognition of normal abnormal leukocyte subsets (complete differentiation pathways) with the same immunostaining protocols mapping of new patient samples against large data base of earlier collected patient samples, analyzed with the same immunostaining protocol Fluorochromes for 8-color flow cytometric immunophenotyping Fluorochromes for 8-color flow cytometric immunophenotyping Fluorochrome Excitation Emission Lasers Peak (nm) Peak (nm) Violet Argon Helium-Neon AmCyan Marina Phycocrythrin () Texas Red PerCP Allophycocyanin () Alexa H Fluorochrome Excitation Emission Lasers Peak (nm) Peak (nm) Violet Argon Helium-Neon /Horizon AmCyan /Horizon Marina Phycoerythrin () Texas Red PerCP Allophycocyanin () Alexa H Construction of EuroFlow panels Fluorochrome conjugates, antibody panels, antibody combinations Synchronized light scatter experiments Local settings EuroFlow settings Fluorochrome s choice CD43 -H CD43 CD03 -H r = 0.77 r = CD03-7 different normal PB samples acquired in 7 different centers Responsible scientist: Sebastian Bottcher Normal PB samples processed according to the stardized EuroFlow sample preparation protocol

3 5th EuroFlow Educational Workshop, Paris, FR, 9 March 0 Immunophenotypic classification & identification of LAIP Achievements of the EuroFlow Consortium Multicolor flow cytometry ( 8 colors) with full technical stardization inclusion of violet laser selection of appropriate fluorochromes stardization of instrument settings laboratory protocols detailed testing comparison of antibody clones conjugated antibodies (multiple companies) Implementation development of novel software fast easy hling of large data files (including automated pattern recognition) combining multiple tubes: calculation APS view mapping of diagnosis follow-up leukemia samples against templates of Development of 8-color antibody protocols for diagnosis, classification screening tubes (include recognition of normal leukocyte subsets) multi-tube panels for diagnosis classification per disease category special tubes for MRD monitoring per disease category TdT+ / + / CD38+ Tube Precursor Analyse 4 -B-ALL Set gate LAIP: Tube CD5 / NG- / + - TdT+ Analyse (D) Set gate Tube 3 CyCD79+ / CyCD3- / MPOAnalyse (D) Set gate JJM van Dongen Department of Immunology, Erasmus MC IMMUNOPHENOTYPIC CHARACTERISTICS OF NORMAL vs LEUKEMIC B-CELLS Lucio et al, Leukemia, 999 MERGED DATA FILES FOR SINGLE STEP GATING Full phenotypic profile 8- COLOR flow cytometry: Bcp-ALL EuroFlow panel (450 bivariate plots) + B-CELLS (D) -PO -Cy7 A single gating step for 5 different data files (tubes) Lucio et al, Leukemia, 999 Merging of data files Multicolor analysis: bar code Simultaneous display of immunophenotype MDS normal A single gating step for five different data files (merged) CD36 CD36 CD33 Pedreira et al, Cytometry 008;73:834 Blast cells (+/+) 3

4 DATA CALCULATION IN THE INFINICYT TM SOFTWARE MERGED AND CALCULATED DATA FILE Tube Exp Low-> Exp Low-> Tube Full phenotypic profile Event X in tube. RCP CY5.5-> FSC->. RCP CY5.5-> Event Y: Nearest neighbour of event X in tube FSC-> Calculated FMC7 value for event X in tube (based on data from event Y in tube ) Impossible phenotypic profile MERGED & CALCULATED LISTMODE DATA FILE Files Merging by Infinicyt program Integration of results from multiple tubes based on 4 to 6 common parameters per tube Parameters 6 Files x 0 parameters x events File x 5 parameters x C R E Parameter coding = Common = Measured = Calculated Colors No. of Total no. of No. of common parameters End result tubes antibodies antibodies Scatter no. of parameters 4-colors (8+) 6-colors (+) 8-colors (0+) 8-colors (3+) events Development of 8-color multi-tube antibody protocols (3 or 4 antibodies in common per tube in each protocol ) Automatic identification of populations Automatic identification of populations Multidimensional analysis: CD8+ T-lymfocytes Monocytes Automated Separation among different cell Populations (APS view) B-cells Neutrophils CD4+ T-lymfocytes NK-cells 4

5 APS Procedure for AUTOMATIC ANALYSIS APS Procedure for groups of patients VISUALIZATION OPTIONS Dots Dots / Mean Mean APS Dots View APS Means View Group of patients with the same panel/protocol applied same disease category APS Procedure for groups of patients Results of synchronized experiments APS view of 30 merged data files from different centers CD4+ Monocytes CD8+ CD3- T cells CD3+ CD4+ T-cells Local normal donors MEAN VALUES REPRESENTATION Each dot = patient/sample CD3+ CD8- CD4- T cells CD3+ CD8+ T cells Group of patients with same panel/protocol applied different disease categories + + B-cells 5 th EuroFlow Educational Workshop, Paris, FR, 9 March 0 Achievements of the EuroFlow Consortium Multicolor flow cytometry ( 8 colors) with full technical stardization inclusion of violet laser selection of appropriate fluorochromes stardization of instrument settings laboratory protocols detailed testing comparison of antibody clones conjugated antibodies (multiple companies) Implementation development of novel software fast easy hling of large data files (including automated pattern recognition) combining multiple tubes: calculation APS view mapping of diagnosis follow-up leukemia samples against templates of Development of 8-color antibody protocols for diagnosis, classification screening tubes (include recognition of normal leukocyte subsets) multi-tube panels for diagnosis classification per disease category special tubes for MRD monitoring per disease category EuroFlow antibody protocols Development of 8-color multi-tube antibody protocols (3 or 4 antibodies in common per tube in each protocol ). Screening tubes (include recognition of normal leukocyte subsets) Acute leukemia orientation tube (): tube (L Lhermitte) Lymphoid screening tube (LST): tube (J Flores Montero) Small sample screening tube (SST): tube (AW Langerak) Plasma cell dyscrasia tubes (PCD): tubes (J Flores Montero). Multi-tube panels for characterization per disease category B-cell precursor ALL (BCP-ALL) protocol: (L Lhermitte) T-cell ALL (T-ALL) protocol: (V Asnafi) AML/MDS protocol: 7 tubes (VHJ van der Velden) B chronic lymphoproliferative diseases (B-CLPD): 5 tubes (S Böttcher) T chronic lymphoproliferative diseases (T-CLPD): 6 tubes (J Almeida) NK chronic lymphoproliferative diseases (NK-CLPD): 3 tubes (J Almeida) 5

6 Algorithm for EuroFlow antibody panels in hemato-oncology Algorithm for EuroFlow antibody panels in hemato-oncology Atypical lymphocytes High suspicion of Splenomegaly acute leukemia Unexplained Lymphocytosis e.g. blast cells observed cytopenia LN enlargement Sustained Unexplained Monoclonal monocytosis Eosinophilia Monoclonal non-igm, Bone lesions BM plasmacytosis High monoclonal non-igm Suspicion of lymphoma localization in small cell number samples e.g. CSF, vitreous Atypical lymphocytes High suspicion of Splenomegaly acute leukemia Unexplained Lymphocytosis e.g. blast cells observed cytopenia LN enlargement Sustained Unexplained Monoclonal monocytosis Eosinophilia Monoclonal non-igm, Bone lesions BM plasmacytosis High monoclonal non-igm Suspicion of lymphoma localization in small cell number samples e.g. CSF, vitreous LST PCST tube of PCD SST LST PCST tube of PCD SST reactive/polyclonal clonal/aberrant reactive/polyclonal other B-CLPD clonal BCP-ALL T-ALL AML/MDS B-CLPD limited B-CLPD complete T-CLPD NK-CLPD PCD BCP-ALL T-ALL AML/MDS B-CLPD limited B-CLPD T-CLPD NK-CLPD complete PCD of BCP-ALL of T-ALL of AML MDS CLL non-cll CLL MCL FCL aberrant αβ+ aberrant γδ+ reactive aberrant NK cells reactive of PCD immunobead assays for fusion protein detection immunobead assays for fusion protein detection PNH CML CML-BC HCL other clonal B other MPD Acute leukemia orientation tube () tube Single tube EuroFlow screening tube for acute leukemias Acute Leukemia Orientation Tube ()* - H7 BCP-ALL T-ALL AML/MDS 4 to 7 tubes cympo cycd79a CD7 - H7 * Backbone markers are indicated in bold; cy= cytoplasmic; sm= surface membrane. cympo cycd79a CD7 BCP-ALL T-ALL AML (Acute Leukemia Orientation Tube) BCP-ALL cases (n=89) T-ALL cases (n=7) AML cases (n=36) 6

7 (Acute Leukemia Orientation Tube) Multi-tube EuroFlow classification panel for B-cell precursor ALL (BCP-ALL)* Tube -H7 Aim** CD58 CD66c CD0 CD38 Diagnosis classification of BCP-ALL; Detection of LAP markers; Detection of molecular aberrations smlgκ cylgµ CD33 smlgµ CD7 smlgλ Diagnosis classification of BCP-ALL; BCP-ALL (54) T-ALL (63) AML MPO+ (70) AML MPO-7- () AML MPO-7+ (3) 48 overall cases 3 CD9 nutdt CD3 CD CD4 Diagnosis classification of BCP-ALL; Detection of molecular aberrations; Detection of LAP markers 4 CD CD5 NG CD3 CD8 Subclassification of BCP-ALL; Detection of LAP markers; CDw65 Detection of phenotypes associated with molecular aberrations * Backbone markers are indicated in bold; cy= cytoplasmic. ** The described marker combinations can also be applied for disease staging monitoring of treatment effectiveness (MRD diagnostics). BCP-ALL panel BCP-ALL panel Haematogones BCP-ALL TEL-AML BCP-ALL BCR-ABL BCP-ALL Hyperdiploïdes BCP-ALL MLL rearranged Haematogones BCP-ALL TEL-AML BCP-ALL BCR-ABL BCP-ALL Hyperdiploïdes BCP-ALL MLL rearranged Tube Multi-tube EuroFlow classification panel for T-ALL.* Responsible scientist: V Asnafi - H7 Aim** Acute leukemia orientation tube () tube nutdt CD99 CD5 CD0 CDa Diagnosis classification of BCP-ALL; Detection of LAP markers; Detection of molecular aberrations CD CD7 CD4 CD8 CD7 Diagnosis classification of BCP-ALL; BCP-ALL T-ALL AML/MDS 3 TCRγδ TCRαβ CD33 CD56 cytcrβ Diagnosis classification of BCP-ALL; Detection of molecular aberrations; Detection of LAP markers 4 to 7 tubes - H7 4 CD3 RA CD3 Subclassification of BCP- ALL; Detection of LAP markers; Detection of molecular aberrations cympo cycd79a CD7 * Backbone markers are indicated in bold; cy= cytoplasmic; sm= surface membrane; nu= nuclear. ** The described marker combinations can also be applied for disease staging monitoring of treatment effectiveness (MRD diagnostics). 7

8 (Acute Leukemia Orientation Tube) Multi-tube EuroFlow classification panel for B-cell precursor ALL (BCP-ALL)* Tube -H7 Aim** CD58 CD66c CD0 CD38 Diagnosis classification of BCP-ALL; Detection of LAP markers; Detection of molecular aberrations smlgκ cylgµ CD33 smlgµ CD7 smlgλ Diagnosis classification of BCP-ALL; BCP-ALL (54) T-ALL (63) AML MPO+ (70) AML MPO-7- () AML MPO-7+ (3) 3 CD9 nutdt CD3 CD CD4 Diagnosis classification of BCP-ALL; Detection of molecular aberrations; Detection of LAP markers 4 CD CD5 NG CD3 CD8 Subclassification of BCP-ALL; Detection of LAP markers; CDw65 Detection of phenotypes associated with molecular aberrations * Backbone markers are indicated in bold; cy= cytoplasmic. ** The described marker combinations can also be applied for disease staging monitoring of treatment effectiveness (MRD diagnostics). 48 overall cases Acute leukemia orientation tube () 5 th EuroFlow Educational Workshop, Paris, FR, 9 March 0 tube Achievements of the EuroFlow Consortium BCP-ALL T-ALL AML/MDS 4 to 7 tubes cympo cycd79a CD7 - H7 Multicolor flow cytometry ( 8 colors) with full technical stardization inclusion of violet laser selection of appropriate fluorochromes stardization of instrument settings laboratory protocols detailed testing comparison of antibody clones conjugated antibodies (multiple companies) Implementation development of novel software fast easy hling of large data files (including automated pattern recognition) combining multiple tubes: calculation APS view mapping of diagnosis follow-up leukemia samples against templates of Development of 8-color antibody protocols for diagnosis, classification screening tubes (include recognition of normal leukocyte subsets) multi-tube panels for diagnosis classification per disease category special tubes for MRD monitoring per disease category Identification of different granulocytic subpopulations in childhood BM Multi-tube EuroFlow classification panel for AML/MDS Responsible scientist: VHJ van der Velden Tube - H7 Aim** Myelo/monoblast Promyelocyte Myelocyte Metamyelocyte Neutrophill AML/ MDS CD6 CD3 CD7 CDb CD0 Diagnosis subclassification of AML PNH especially focused on neutrophilic lineage staining 5 CD6/CD3/ /CDb FSC CD6- CD3- CD35 CD64 CD7 IREM CD4 Diagnosis subclassification of AML PNH especially focussed on monocytic lineage CDb- CD6- CD3- CDb- CD3- CD6-3 4 CD36 nutdt CD05 CD56 CD7 CD7 CD33 CD7 CD7 Diagnosis subclassification of AML especially focused on erythroid lineage Aberrant expression of lymphoid-associated markers abnormal lymphoid maturation E.G. van Lochem et al., Cytometry Part B 004; 60B: -3. * Further information about the markers 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 monitoring of treatment effectiveness (MRD diagnostics) 8

9 Neutrophil maturation Phenotypic changes during normal neutrophil differentiation Stage I Stage II Stage III Stage IV Stage V 0 3 MPO Myeloblast Promyelocyte Myelocyte Metamyelocyte Neutrophil CD5 C P 3- A 3 D C Myeloblast Neutrophil MPO- cmpo -> b/mpo/dr/ Stage I Stage II Stage III Stage IV Stage V CD CD3 CD64 CD33 CDb MPO CD65 CD3 CD CDb -> b/35/45/3. CDb- 0 0 CD6 CD CD5 -> CD5-5/6/45/0.4 Responsible scientist: A. Orfao HLA-DR CD7 CD54 N-DIMENSIONAL NEUTROPHIL MATURATION IN NORMAL BM Maturation stage of neutrophil precursors in normal BM Myeloblasts Promyelocytes Neutrophils Myelocytes Metamyelocytes Bs CD3 PerCP CDb CD6 CD7 CD0 PO CD38 CD5 Maturation stage of neutrophil precursors in normal BM 5 th EuroFlow Educational Workshop, Paris, FR, 9 March 0 Achievements of the EuroFlow Consortium Multicolor flow cytometry ( 8 colors) with full technical stardization inclusion of violet laser selection of appropriate fluorochromes stardization of instrument settings laboratory protocols detailed testing comparison of antibody clones conjugated antibodies (multiple companies) Implementation development of novel software fast easy hling of large data files (including automated pattern recognition) combining multiple tubes: calculation APS view mapping of diagnosis follow-up leukemia samples against templates of Development of 8-color antibody protocols for diagnosis, classification screening tubes (include recognition of normal leukocyte subsets) multi-tube panels for diagnosis classification per disease category special tubes for MRD monitoring per disease category 9

10 Algorithm for EuroFlow antibody panels in hemato-oncology Multi-tube EuroFlow classification combinations for B-cell chronic lymphoproliferative diseases (B-CLPD) * Atypical lymphocytes High suspicion of Splenomegaly acute leukemia Unexplained Lymphocytosis e.g. blast cells observed cytopenia LN enlargement Sustained Unexplained Monoclonal monocytosis Eosinophilia Monoclonal non-igm, Bone lesions BM plasmacytosis High monoclonal non-igm Suspicion of lymphoma localization in small cell number samples e.g. CSF, vitreous Tube Responsible scientist: S Böttcher supported by A Rawstron J Flores Montero -H7 Aim** LST reactive/polyclonal clonal/aberrant PCST tube of PCD reactive/polyclonal other B-CLPD SST CD4 CD8 smlgλ CD56 smlgκ CD5 TCRγδ 8 CD38 LST tube; detection of B- CLPD BCP-ALL T-ALL AML/MDS B-CLPD limited clonal B-CLPD T-CLPD NK-CLPD complete PCD CD3 CD0 CD79b 0 CD43 Identification of CLL vs other B-CLPD cases, when combined with LST of BCP-ALL immunobead assays for fusion protein detection of T-ALL of AML MDS PNH CML CML-BC CLL non-cll CLL MCL FCL HCL other clonal B aberrant αβ+ aberrant γδ+ reactive aberrant NK cells reactive of PCD CD3 CD03 CD6L LAIR CD95 CD39 CDc CD smlgµ CXCR5 CD7 CD8 CD49d Further subclassification of non-cll B-CLPD, e.g. HCL, MCL, FL, MZL, LPL, DLBCL other B-CLPD other MPD * Backbone markers are indicated in bold; sm= surface membrane. ** The described marker combinations might also be applied for disease staging monitoring of treatment effectiveness (MRD diagnostics) Multi-tube EuroFlow classification combinations for B-cell chronic lymphoproliferative diseases (B-CLPD) * Algorithm for EuroFlow antibody panels in hemato-oncology Tube Responsible scientist: S Böttcher supported by A Rawstron J Flores Montero -H7 Aim** Atypical lymphocytes High suspicion of Splenomegaly acute leukemia Unexplained Lymphocytosis e.g. blast cells observed cytopenia LN enlargement Sustained Unexplained Monoclonal monocytosis Eosinophilia Monoclonal non-igm, Bone lesions BM plasmacytosis High monoclonal non-igm Suspicion of lymphoma localization in small cell number samples e.g. CSF, vitreous CD4 CD8 smlgλ CD56 smlgκ CD5 TCRγδ 8 CD38 LST tube; detection of B- CLPD LST reactive/polyclonal clonal/aberrant PCST tube of PCD reactive/polyclonal other B-CLPD SST CD3 CD0 CD79b 0 CD43 Identification of CLL vs other B-CLPD cases, when combined with LST BCP-ALL T-ALL AML/MDS B-CLPD limited clonal B-CLPD T-CLPD NK-CLPD complete PCD CD3 CD03 CD6L LAIR CD95 CD39 CDc CD smlgµ CXCR5 CD7 CD8 CD49d Further subclassification of non-cll B-CLPD, e.g. HCL, MCL, FL, MZL, LPL, DLBCL other B-CLPD of BCP-ALL immunobead assays for fusion protein detection of T-ALL of AML MDS PNH CML CML-BC CLL non-cll CLL MCL FCL HCL other clonal B aberrant αβ+ aberrant γδ+ reactive aberrant NK cells reactive of PCD * Backbone markers are indicated in bold; sm= surface membrane. ** The described marker combinations might also be applied for disease staging monitoring of treatment effectiveness (MRD diagnostics) other MPD Technical aspects of EuroFlow protocols: instrument settings, fluorochrome choice, stardization T. Kalina, J. Flores-Montero, Q. Lecrevisse, M. Cullen 3,L. Lhermitte 4, L. Sedek 5, A. Mendonca 6, S. Bötcher 7, J. te Marvelde 8, Mejstříková, O. Hrušák, J.J.M. van Dongen 8, A. Orfao On behalf of the EuroFlow Consortium (EU-FP6, LSHB-CT ), Department of Pediatric Hematology Oncology, Charles University, Prague, Czech Republic;, Department of Medicine, Cancer Research Centre Cytometry Service, University of Salamanca, Salamanca, ES; 3, St. James University Hospital, Leeds, UK; 4, Department of Hematology, Hôpital Necker, Paris, FR 5, Department of Pediatric Hematology Oncology, Medical University of Silesia, Zabrze, PL; 6, Department of Hematology, Instituto Portugues de Oncologia, Lisbon, PT; 7, nd Department of Medicine, University Klinik Schleswig-Holstein, Kiel, DE; 8, Department of Immunology, Erasmus MC, Rotterdam, NL; To be published in: Leukemia 0; 5: xxxx-xxxx EuroFlow antibody panels for stardized n-dimensional flow cytometric immunophenotyping of normal, reactive malignant leukocytes J.J.M. van Dongen, L. Lhermitte, S. Böttcher, J. Almeida, V.H.J. van der Velden, J. Flores-Montero, A. Rawstron, V.Asnafi, Q. Lécrevisse, P. Lucio, E. Mejstrikova, T. Szczepański, T. Kalina, R. de Tute, M. Brüggemann, L. Sedek, M. Cullen, A.W. Langerak, A. Mendonça, E. Macintyre, M. Martin-Ayuso,O. Hrusak, M.B. Vidriales, A. Orfao On behalf of the EuroFlow Consortium (EU-FP6, LSHB-CT ), Department of Immunology, Erasmus MC, Rotterdam, NL;, Department of Hematology, Hôpital Necker, University of Paris Descartes, AP-HP, Paris, FR 3, Medical Clinic II, University Medical Center Schleswig-Holstein,Campus Kiel, Kiel, DE; 4, Department of Medicine, Cancer Research Centre (IBMCC-CSIC-USAL) Cytometry Service, University of Salamanca, Salamanca, ES; 5, St. James University Hospital, Leeds, UK; 6, Department of Hematology, Instituto Portugues de Oncologia, Lisbon, PT; 7, Department of Pediatric Hematology Oncology, Charles University, Prague, CZ; 8, Department of Pediatric Hematology Oncology, Medical University of Silesia, Zabrze, PL; 9, Cytognos SL, Salamanca, ES; 0,Department of Hematology, University Hospital, Salamanca, ES. To be published in: Leukemia 0; 5: xxxx-xxxx 0

11 5 th EuroFlow Educational Workshop, Paris, FR, 9 March 0 Detection of minimal residual disease (MRD) Achievements of the EuroFlow Consortium Multicolor flow cytometry ( 8 colors) with full technical stardization inclusion of violet laser selection of appropriate fluorochromes stardization of instrument settings laboratory protocols detailed testing comparison of antibody clones conjugated antibodies (multiple companies) Implementation development of novel software fast easy hling of large data files (including automated pattern recognition) combining multiple tubes: calculation APS view mapping of diagnosis follow-up leukemia samples against templates of relative frequency of leukemic cells detection limit of cytomorphological techniques detection limit of immunophenotyping PCR techniques Development of 8-color antibody protocols for diagnosis, classification screening tubes (include recognition of normal leukocyte subsets) multi-tube panels for diagnosis classification per disease category special tubes for MRD monitoring per disease category 0 cure follow-up in years I C II maintenance Rx Dept. of Immunology, Erasmus MC, Rotterdam Detection of minimal residual disease in ALL PCR analysis of Ig/TCR genes Technique Applicability Detection Remark limit Flow cytometry BCP-ALL: 85% (0-3 -) 0-4 Fast, but variable sensitivity because (4 colors) T-ALL: 90% of similarities between normal (regenerating) cells malignant cells Germline IGH gene VH DH JH s Cµ PCR of Ig/TCR BCP-ALL: 95% Time consuming relatively genes T-ALL: 95% expensive (junctional region sequencing), but applicable in 95% of lymphoid malignancies Rearranged gene V-D-J recombination PCR of fusion BCP-ALL: 40% Limited applicability in ALL, but transcripts T-ALL: 5% potentially useful in specific subgroups, e.g. BCR-ABL cases in specific protocols VH DH JH Dept. of Immunology, Erasmus MC, Rotterdam EuroMRD: European Study Group on MRD detection (ESG-MRD-ALL) European Study Group on MRD detection Chairman: J.J.M. van Dongen AIMS of EuroMRD (focus on PCR ananlysis of Ig/TCR genes): Europe Glasgow Turku Israel Japan. Quality Control Program: times per year: - February / March - August / September. Educational meetings, including evaluation of quality control rounds: times per year: - May / June - October / November 3. Stardization of MRD techniques - stardization techniques within each treatment protocol - guidelines for interpretation of RQ-PCR results 4. Collaborative development clinical evaluation of new MRD strategies new MRD techniques Lisbon Copenhague Sheffield Bristol London Amsterdam Kiel Hamburg Rotterdam Berlin Lille Brussel Frankfurt Rennes Prague Paris Heidelberg Créteil Vienna Zurich Grenoble Monza Bergamo Toulouse Padova Torino Salamanca Rome Petah Tikva Australia Nagakute-cho Singapore Singapore Sydney Rwick Basis for accreditation of MRD diagnostics Palermo 4 laboratories in 7 countries Dept. of Immunology, Erasmus MC, Rotterdam

12 Precursor B-ALL MRD at 6 months of therapy Precursor-B- ALL at diagnosis -PerCP - TdT- Follow-up 6 months -PerCP 0.007% - TdT- Therapy-induced immunophenotypic shifts BCP-ALL panel At diagnosis Day 5 Day 8 TdT- TdT- TdT- -PerCP -PerCP -PerCP Mix of 3 different regenerating B cell populations (Haematogones) BCP-ALL blast cells Van der Sluijs et al, LEUKEMIA 005; 9: Development of 8-color MRD panels Single-tube antibody EuroFlow MRD protocols under evaluation. Acute leukemias (includes recognition of normal precursors) Acute myeloid leukemia panel (AML-MRD): tube per pathway (A. Orfao) B-cell precursor (BCP-ALL-MRD): tube (V. van der Velden, E. Mejstrikova) T-cell ALL (T-ALL-MRD): tube (V.Asnafi). Chronic lymphoproliferative disorders (includes recognition of normal cells) Chronic lymphocytic leukemia (CLL-MRD): tube (A. Langerak) Hairy cell leukemia (HCL-MRD): tube (E. MacIntyre) Mantle cell lymphoma (MCL-MRD): tube (S. Böttcher) Follicular lymphoma (FL-MRD): tube (S. Böttcher) Marginal zone lymphoma (MZL-MRD): tube (P.Lucio) Lymphoplasmacytic lymphoma (LPL-MRD): tube (P. Lucio) Diffuse large B-cell lymphoma (DLBCL-MRD): tube (P. Lucio) Burkitt lymphoma (BL): tube (L.Lhermitte) T-chronic lymphoproliferative diseases (T-CLPD-MRD): tube (J. Almeida) Multiple myeloma (MM): tube (J. Flores)

13 Achievement of the EuroFlow Consortium: New concept in diagnostic flow cytometry. Full technical stardization of multicolor flow cytometry ( 8 colors) stardization of instrument settings laboratory protocols selection of fluorochromes selection of antibody clones per marker EuroFlow protocols work on all tested 8 colors flow cytometers: - DAKO Cyan, LSR-II, FACS Canto-II; - late arrivals (Navios Gallios) still to be tested (new Workpackage). Implementation further development of novel software: Infinicyt fast easy data hling with automated pattern recognition combining multiple tubes: calculation APS (principle analysis mapping of diagnosis follow-up leukemia samples against templates of 3. Development of 8-color antibody protocols for diagnosis, classification 8-color panels are based on recognition of normal cells & differentiation pathways diagnosis classification tubes are ready; MRD tubes in development flexibility within panels: deletion inclusion of markers tubes is possible 4. Large EuroFlow data base linked to Infinicyt software EuroFlow participants University Institutes / Medical Schools Erasmus MC, Rotterdam, NL J.J.M. van Dongen, V.H.J. van der Velden USAL, Salamanca, ES A. Orfao, J. Flores, J. Almeida, Q. Lecrevisse a.o. IMM, Lisbon, PT P. Lucio, A. Mendonça, A. Parreira a.o. UNIKIEL, Kiel, DE M. Kneba, S. Böttcher, M. Ritgen, M. Brüggemann a.o. AP-HP, Paris, FR E. Macintyre, L. Lhermitte, V. Asnafi a.o. UNIVLEEDS, Leeds, GB S. Richards, A.C. Rawstron. P. Evans a.o. DPH/O, Prague, CZ O. Hrusak, T. Kalina, E. Mesjstrikova a.o. SAM, Zabrze, PL T. Szczepanski, L. Sedek a.o. DCOG, The Hague, NL E. Sonneveld, A. van der Sluijs-Gelling a.o. KUL, Leuven, BE N. Boeckx a.o. HGSA, Porto, PT M. Lima, AH Santos UFRJ, Rio de Janeiro, BR C. Pedreira, E.S. Costa Companies (SME s) DYNOMICS, Rotterdam, NL E. Dekking, F. Weerkamp a.o. CYTOGNOS, Salamanca, ES M. Martin, J. Bensadon, J. Hernez, M. Muñoz a.o. Contributors - Acknowledgements Michael Kneba Monika Brüggemann Sebastian Böttcher Stephen Richards Paul Evans Matt Cullens Ruth de Tute Andy Rawstron Elizabeth MacIntyre Vahid Asnafi Ludovic Lhermitte Paulo Lucio Andreia Mendonça Evan Jensen Tomek Szczepanski Lukasz Sedek Ondrej Hrusak Tomas Kalina Ester Mejstrikova MUCHAS GRACIAS Jacques van Dongen Vincent van der Velden Jeroen Te Marvelde Alita van der Sluijs Photo Lukasz Sedek Juan Flores-Montero Julia Almeida Quentin Lecrevisse 3