UK NEQAS for Leukocyte Typing Scientific Meeting, Sheffield, UK 24 June 2013

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1 UK NEQAS for Leukocyte Typing Scientific Meeting, Sheffield, UK 24 June 2013 Flow cytometry vs. Molecular techniques for Diagnosis, Classification and Monitoring of Hematological Malignancies Need of innovation, standardization, QC, and continuous education Jacques J.M. van Dongen J.J.M. on behalf van Dongen of UK NEQAS Scientific Meeting, Sheffield, UK, 24 June 2013 Laboratory diagnosis of hematological malignancies 1. Making the diagnosis Normal reactive/regenerating malignant For based on immunophenotyping For example Ig-TCR based clonality diagnostics in suspected lymphoproliferative diseases 2. Classification of hematopoietic malignancies Relation with prognosis: relevance of risk-group definition For example based on immunophenotypic differentiation characteristics For example based on genetic aberrations (fusion genes/fusion proteins) 3. Evaluation of treatment effectiveness (MRD): MRD-based risk-group stratification (treatment reduction or escalation) E.g. based on RQ-PCR of Ig-TCR genes E.g. based on flow cytometric immunophenotyping 1

2 UK NEQAS Scientific Meeting, Sheffield, UK, 24 June 2013 Laboratory diagnosis of hematological malignancies 1. Making the diagnosis Normal reactive/regenerating malignant For based on immunophenotyping: BIOMED-1 and EuroFlow For example Ig-TCR based clonality diagnostics in suspected lymphoproliferative diseases: BIOMED-2/EuroClonality 2. Classification of hematopoietic malignancies Relation with prognosis: relevance of risk-group definition For example based on immunophenotypic differentiation characteristics: EuroFlow For example based on genetic aberrations (fusion genes/fusion proteins): BIOMED-1, EAC and EuroMRD (Ph+ ALL subgroup) 3. Evaluation of treatment effectiveness (MRD): MRD-based risk-group stratification (treatment reduction or escalation) E.g. based on RQ-PCR of Ig-TCR genes: BIOMED-1, EAC and EuroMRD E.g. based on flow cytometric immunophenotyping: BIOMED-1 and EuroFlow UK NEQAS Scientific Meeting, Sheffield, UK, 24 June 2013 Laboratory diagnosis of hematological malignancies 1. Making the diagnosis Normal reactive/regenerating malignant For based on immunophenotyping: BIOMED-1 and EuroFlow For example Ig-TCR based clonality diagnostics in suspected lymphoproliferative diseases: BIOMED-2/EuroClonality 2. Classification of hematopoietic malignancies Relation with prognosis: relevance of risk-group definition For example based on immunophenotypic differentiation characteristics: EuroFlow For example based on genetic aberrations (fusion genes/fusion proteins): BIOMED-1, EAC and EuroMRD (Ph+ ALL subgroup) 3. Evaluation of treatment effectiveness (MRD): MRD-based risk-group stratification (treatment reduction or escalation) E.g. based on RQ-PCR of Ig-TCR genes: BIOMED-1, EAC and EuroMRD E.g. based on flow cytometric immunophenotyping: BIOMED-1 and EuroFlow 2

3 European network for laboratory diagnostics EuroClonality (BIOMED-2) EuroMRD ALL, infant ALL and NHL EuroFlow Since 1996 Since 2001 Since 2006 Participants: based on experience and participation in (inter)national clinical trails Aims: - Innovation and standardization of laboratory diagnostics - Quality control - Continuous education EuroClonality/BIOMED-2 Concerted Action: BMH4-CT Since PCR-based clonality studies for early diagnosis of lymphoproliferative disorders Aberdeen Current chairman: Liz Hodges (2011- till now) Leeds Kiel Nottingham Leicester Cambridge Groningen Norwich Amsterdam Cardiff Arnhem London Rotterdam Münster Berlin Nijmegen Eindhoven Göttingen Southampton Roeselare Frankfurt Würzburg Rouen Paris Heidelberg Créteil Pierre-Benite Founding chairman: J.J.M. van Dongen ( ) 47 laboratories in 8 countries For reprints, contact: b.vanbodegom@erasmusmc.nl Porto Léon Segovia Salamanca Madrid Toledo Lisboa Cadiz Toulouse Availability of BIOMED-2 multiplex tubes: InVivoScribe Technologies, San Diego, CA November

4 Analysis of complete IGH gene rearrangements: VH-JH VH DH JH VH-FR1 primers VH-FR2 primers VH-FR3 primers J H primer V H family primers IGH tube A 5' 3' VH1-FR1 (1-2) (-252) GGCCTCAGTGAAGGTCTCCTGCAAG VH2-FR1 (2-5) (-284) GTCTGGTCCTACGCTGGTGAAACCC VH3-FR1 (3-7) (-256) CTGGGGGGTCCCTGAGACTCTCCTG VH4-FR1 (4-4) (-256) CTTCGGAGACCCTGTCCCTCACCTG VH5-FR1 (5-51)(-255) CGGGGAGTCTCTGAAGATCTCCTGT VH6-FR1 (6) (-263) TCGCAGACCCTCTCACTCACCTGTG IGH tubes A, B, and C 3' 5' CCAGTGGCAGAGGAGTCCATTC (+57) J H consensus IGH tube B VH1-FR2 (1-2) (-192) CTGGGTGCGACAGGCCCCTGGACAA VH2-FR2 (2-5) (-190) TGGATCCGTCAGCCCCCAGGGAAGG VH3-FR2 (3-7) (-189) GGTCCGCCAGGCTCCAGGGAA VH4-FR2 (4-4) (-188) TGGATCCGCCAGCCCCCAGGGAAGG VH5-FR2 (5-51)(-190) GGGTGCGCCAGATGCCCGGGAAAGG VH6-FR2 (6) (-194) TGGATCAGGCAGTCCCCATCGAGAG VH7-FR2 (7) (-192) TTGGGTGCGACAGGCCCCTGGACAA IGH tube C VH1-FR3 (1-2) (-55) VH2-FR3 (2-5) (-54) VH3-FR3 (3-7) (-57) VH4-FR3 (4-4) (-48) VH5-FR3 (5-51)(-69) VH6-FR3 (6) (-63) VH7-FR3 (7) (-69) TGGAGCTGAGCAGCCTGAGATCTGA CAATGACCAACATGGACCCTGTGGA TCTGCAAATGAACAGCCTGAGAGCC GAGCTCTGTGACCGCCGCGGACACG CAGCACCGCCTACCTGCAGTGGAGC GTTCTCCCTGCAGCTGAACTCTGTG CAGCACGGCATATCTGCAGATCAG BIOMED-2 report: Leukemia 2003; 17: BIOMED-2 Concerted Action BMH4-CT : PCR-based clonality studies IGH VH-J H PCR GeneScan analysis IGH tube A: VH-FR1 JH IGH tube B: VH-FR2 JH IGH tube C: VH-FR3 JH 4

5 Analysis of TCRB gene rearrangements Vβ Dβ Jβ V β family primers J β primers TCRB tubes A and B Vβ2 (-204) 5' AACTATGTTTTGGTATCGTCA 3 Vβ4 Vβ5/1 Vβ6a/11 Vβ6b/25 Vβ6c Vβ7 Vβ8a Vβ9 Vβ10 Vβ11 (-201) (-197) (-201) (-201) (-201) (-198) (-201) (-198) (-201) (-198) CACGATGTTCTGGTACCGTCAGCA CAGTGTGTCCTGGTACCAACAG AACCCTTTATTGGTACCGACA ATCCCTTTTTTGGTACCAACAG AACCCTTTATTGGTATCAACAG CGCTATGTATTGGTACAAGCA CTCCCGTTTTCTGGTACAGACAGAC CGCTATGTATTGGTATAAACAG TTATGTTTACTGGTATCGTAAGAAGC CAAAATGTACTGGTATCAACAA Vβ12a/3/13a/15 (-198) ATACATGTACTGGTATCGACAAGAC Vβ13b Vβ13c/12b/14 Vβ16 Vβ17 Vβ18 Vβ19 Vβ20 (-198) (-198) (-201) (-198) (-201) (-201) (-193, inv) GGCCATGTACTGGTATAGACAAG GTATATGTCCTGGTATCGACAAGA TAACCTTTATTGGTATCGACGTGT GGCCATGTACTGGTACCGACA TCATGTTTACTGGTATCGGCAG TTATGTTTATTGGTATCAACAGAATCA CAACCTATACTGGTACCGACA Vβ21 Vβ22 Vβ23/8b Vβ24 (-201) (-201) (-201) (-197) TACCCTTTACTGGTACCGGCAG ATACTTCTATTGGTACAGACAAATCT CACGGTCTACTGGTACCAGCA CGTCATGTACTGGTACCAGCA TCRB tubes A and C: J β A primers 3' 5' GTGGTCTAAGTGTCAACATCCATTC CTGGTCCAATTGGCAACATCCATTC TTCAACCGAGTGACAACATCCATTC CTTGGGTCGAGAGACAGAACCCATAC CTGAGCTGAGAGGTAGGATCCATTC GTCCGAGTGACACTGTCCATAC TCCGACTGGCATGACCCATTC TCCGACTGGCACGACCCGCTC GTCCGAGTGCCAATGTCCATTC 3' 5' AGTGGCACGATCCATTCTTCC ACTGTCACGAGCCATTCGCCC AGAGTCACGACCCATTCGACC CACGAGCCACACGCGC (+53) Jβ1.1 (+53) Jβ1.2 (+55) Jβ1.3 (+56) Jβ1.4 (+55) Jβ1.5 (+58) Jβ1.6 (+56) Jβ2.2 (+58) Jβ2.6 (+52) Jβ2.7 TCRB tubes B and C: J β B primers Dβ (+59) (+58) (+59) (+57) Jβ Jβ2.1 Jβ2.3 Jβ2.4 Jβ2.5 Dβ1 primer Dβ2 primer J β primers TCRB tube C Dβ1 (-252) 5' 3' GCCAAACAGCCTTACAAAGAC 5' 3' Dβ 2 (-137) TTTCCAAGCCCCACACAGTC BIOMED-2 report: Leukemia 2003; 17: BIOMED-2 multiplex TCRB tube B: Vβ -Jβ Vβ Dβ Jβ V β family primers J β B primers bp MwM PB-MNC thymus Cell line PEER ES-9 ES-6 TCRB tube B Vβ-Jβ ss he ho PB-MNC 2100 ES ES nt cell line PEER BIOMED-2 report: Leukemia 2003; 17:

6 UK NEQAS Scientific Meeting, Sheffield, UK, 24 June 2013 Laboratory diagnosis of hematological malignancies 1. Making the diagnosis Normal reactive/regenerating malignant For based on immunophenotyping: BIOMED-1 and EuroFlow For example Ig-TCR based clonality diagnostics in suspected lymphoproliferative diseases: EuroClonality 2. Classification of hematopoietic malignancies Relation with prognosis: relevance of risk-group definition For example based on immunophenotypic differentiation characteristics: EuroFlow For example based on genetic aberrations (fusion genes/fusion proteins): BIOMED-1, EAC and EuroMRD (Ph+ ALL subgroup) 3. Evaluation of treatment effectiveness (MRD): MRD-based risk-group stratification (treatment reduction or escalation) E.g. based on RQ-PCR of Ig-TCR genes: BIOMED-1, EAC and EuroMRD E.g. based on flow cytometric immunophenotyping: BIOMED-1 and EuroFlow 6

7 BIOMED-1 Concerted Action ( ) Van Dongen et al., Leukemia 1999 Europe against Cancer (EAC) Detection of fusion gene transcripts in acute leukemia FG RNA TEL (12p13) AML1 (21q22) ENF ENR bp ENPr341 delta Rn 1.E+01 1.E+00 1.E-01 1.E copies 5 10 copies 3 10 copies 2 10 copies 1 10 copies MNC 1.E-03 Europe Against Cancer 1.E Cycle Gabert et al., Leukemia (2003); 17:2318 7

8 UK NEQAS Scientific Meeting, Sheffield, UK, 24 June 2013 Laboratory diagnosis of hematological malignancies 1. Making the diagnosis Normal reactive/regenerating malignant For based on immunophenotyping: BIOMED-1 and EuroFlow For example Ig-TCR based clonality diagnostics in suspected lymphoproliferative diseases: EuroClonality 2. Classification of hematopoietic malignancies Relation with prognosis: relevance of risk-group definition For example based on immunophenotypic differentiation characteristics: EuroFlow For example based on genetic aberrations (fusion genes/fusion proteins): BIOMED-1, EAC and EuroMRD (Ph+ ALL subgroup) 3. Evaluation of treatment effectiveness (MRD): MRD-based risk-group stratification (treatment reduction or escalation) E.g. based on RQ-PCR of Ig-TCR genes: BIOMED-1, EAC and EuroMRD E.g. based on flow cytometric immunophenotyping: BIOMED-1 and EuroFlow 8

9 Table 1. Advantages and disadvantages of the three main MRD techniques MRD technique Estimated sensitivity Advantages Disadvantages Flow cytometry -4 colors -6-8 colors ?? - Fast - Analysis at cell population level or single cell level - Easy storage of data - Variable sensitivity, because of similarities between normal (regenerating) cells and malignant cells - Limited standardization, no QC rounds RQ-PCR of Ig/TCR genes Applicable in virtually all lymphoid malignancies - Sensitive - Standardized + QC rounds - Time consuming - Expensive - Requires extensive experience and knowledge RQ PCR of fusion transcripts and other aberrancies Relatively easy - Sensitive - Excellent for specific leukemia subgroups, such as BCR-ABL or PML-RARA - Limited standardization (harmonization) - Limited QC rounds (with conversion factors) - Limited applicability in broad patient groups (absence of targets) Differentiation lineage (stage) of hematological malignancies B T NK Myeloid Mϕ Undifferentiated AML >98% <2% CML 100% CML-BC 25-30% <2% 60 to 70% 5% ALL (childhood) 82 to 86% 14-18% <1% <2% ALL (adult) 75 to 80% 20-25% <1% <5% CLL 90 to 95% 5 to 10% 1 to 2% NHL ~90% 5 to 10% <2% 1 to 2% <2% Hodgkin lymphoma +* Multiple myeloma 100% TOTAL 71% (78%) * 5% 1% 14% 1% 1% * It is assumed that in the majority of cases with Hodgkin lymphoma the malignant cells belong to the B- cell lineage 9

10 European Study Group on MRD detection Chairman: J.J.M. van Dongen Since 1994/ laboratories in 19 countries Supported by Leukaemia & Lymphoma Research, LeukemiaNet, and EuroClonality November 2012 PCR analysis of Ig/TCR genes Dept. of Immunology, Erasmus MC, Rotterdam 10

11 RQ-PCR analysis of TCR/Ig gene rearrangements EuroMRD Guideline Report: Van der Velden et al. Leukemia 2007 RQ-PCR analysis of TCRD gene rearrangement Van der Velden et al., Department of Immunology, Erasmus MC, Rotterdam 11

12 UK NEQAS Scientific Meeting, Sheffield, UK, 24 June 2013 Laboratory diagnosis of hematological malignancies 1. Making the diagnosis Normal reactive/regenerating malignant For based on immunophenotyping: BIOMED-1 and EuroFlow For example Ig-TCR based clonality diagnostics in suspected lymphoproliferative diseases: EuroClonality 2. Classification of hematopoietic malignancies Relation with prognosis: relevance of risk-group definition For example based on immunophenotypic differentiation characteristics: EuroFlow For example based on genetic aberrations (fusion genes/fusion proteins): BIOMED-1, EAC and EuroMRD (Ph+ ALL subgroup) 3. Evaluation of treatment effectiveness (MRD): MRD-based risk-group stratification (treatment reduction or escalation) E.g. based on RQ-PCR of Ig-TCR genes: BIOMED-1, EAC and EuroMRD E.g. based on flow cytometric immunophenotyping: BIOMED-1 and EuroFlow 12

13 Comparison between molecular techniques and flow cytometry in hematological malignancies Molecular techniques Flow cytometry Speed 2-3 days (up to weeks) fast: 2-3 hours!! Target DNA or RNA protein/cells (RNA is an instable target) ( end-product ) Applicability depends on disease broad (chromosome aberrations) Multiplexing technically demanding relatively easy (even 25 to 100 tests per tube) Accuracy semi-quantitative quantitative Focus all cells in sample any subpopulation (or: prior purification) Facilities special laboratories needed only standard lab needed (pre-pcr lab, PCR lab, etc) (+ flow cytometer) Since 2006 EuroFlow is an independent scientific consortium, which aims at innovation in flow cytometry for improvement of diagnostic patient care Chairmen: J.J.M. van Dongen & A. Orfao 16 laboratories in 12 countries November

14 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) Multiple variants of BCR-ABL transcripts caused by multiple different BCR breakpoint regions F. Weerkamp, et al. Leukemia 2009; 23:

15 Chromosome aberrations and fusion genes in acute leukemias Chromosome Fusion Relative frequency per type of acute leukemia aberration genes Precursor-B-ALL AML children adults children adults adults <60 y >60y t(1;19)(q23;p13) E2A-PBX1 5-8% 3-4% t(4;11)(q21;q23) MLL-AF4 3-5% a 3-4% <1% <1% <1% t(9;22)(q34;q11) BCR-ABL p % 15-30% <1% <1% <1% BCR-ABL p % 10-15% <1% <1% <1% t(12;21)(p13;q22) TEL-AML % <2% t(8;21)(q22;q22) AML1-ETO % 6-8% 2-3% t(15;17)(q22;q21) PML-RARA % b 5-15% b 2-6% b inv(16)(p13;q22) CBFB-MYH % 5-6% 3-4% TOTAL 40-45% 40-45% 25-30% 20-25% 10-12% a In infant ALL, the frequency of t(4;11) can be as high as 70%. b In southern European regions (ES, FR, and IT) the frequency of t(15;17) with PML-RARA is essentially higher than in northern European regions. L. Dekking et al. Best Pract Res Clin Haematol 2010; 23: UK NEQAS Scientific Meeting, Sheffield, UK, 24 June 2013 Standardization in diagnostic flow cytometry Standardization according to literature generally refers to (status in 2005): lists of CD codes and markers per disease category no guidelines for selection of the appropriate antibody clones (or fluorochromes) no technical standardization in flow cytometry HOWEVER: GLP guidelines demand for much higher levels of standardization EuroFlow standardization aimed at: usage of comparable flow cytometers (3 lasers and 8 colors) full standardization of instrument settings (e.g. based on standard beads) standardized laboratory protocols and immunostaining procedures (SOP s) careful selection of optimal antibody clones per marker/cd code selection of optimal 8-color antibody combinations and fluorochromes design of combinations of multiple 8-color tubes: estimation and APS view new software for fast and easy data analysis with automated pattern recognition recognition of normal and 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 15

16 UK NEQAS Scientific Meeting, Sheffield, UK, 24 June 2013 Achievements in flow cytometry by EuroFlow 1. Full technical standardization of multicolor flow cytometry ( 8 colors) standardization of instrument settings and laboratory protocols selection of fluorochromes and selection of antibody clones per marker EuroFlow protocols work on all tested 8 colors flow cytometers: - DAKO Cyan, LSR-II, FACS Canto-II; - also the late arrivals Navios and MACS Quant can be used!!! 2. Implementation and further development of novel software: Infinicyt fast and easy data handling with automated pattern recognition combining multiple tubes: calculation and APS (principle component analysis) mapping of diagnosis and follow-up leukemia samples against templates of normal/control samples 3. Development of 8-color antibody protocols for diagnosis, classification and monitoring of hematological malignancies 8-color panels are based on recognition of normal cells & differentiation pathways diagnosis and classification tubes are ready; MRD tubes in development flexibility within panels: deletion and inclusion of tubes (and markers) is possible 4. Large EuroFlow data base linked to Infinicyt software (available end 2013/early 2014) Synchronized light scatter experiments Local settings EuroFlow settings 7 different normal PB samples acquired in 7 different centers Normal PB samples processed according to the standardized EuroFlow sample preparation protocol 16

17 UK NEQAS Scientific Meeting, Sheffield, UK, 24 June 2013 Achievements in flow cytometry by EuroFlow 1. Full technical standardization of multicolor flow cytometry ( 8 colors) standardization of instrument settings and laboratory protocols selection of fluorochromes and selection of antibody clones per marker EuroFlow protocols work on all tested 8 colors flow cytometers: - DAKO Cyan, LSR-II, FACS Canto-II; - also the late arrivals Navios and MACS Quant can be used!!! 2. Implementation and further development of novel software: Infinicyt fast and easy data handling with automated pattern recognition combining multiple tubes: calculation and APS (principle component analysis) mapping of diagnosis and follow-up leukemia samples against templates of normal/control samples 3. Development of 8-color antibody protocols for diagnosis, classification and monitoring of hematological malignancies 8-color panels are based on recognition of normal cells & differentiation pathways diagnosis and classification tubes are ready; MRD tubes in development flexibility within panels: deletion and inclusion of tubes (and markers) is possible 4. Large EuroFlow data base linked to Infinicyt software (available end 2013/early 2014) Immunophenotypic classification & identification of LAIP Tube 1 Set gate TdT+ / CD19+ / CD38+ Analyse (2D) 4 Tube 2 CD15 / NG2- / CD19+ Analyse (2D) Set gate Tube 3 CyCD79+ / CyCD3- / MPO- Set gate Analyse (2D) JJM van Dongen Department of Immunology, Erasmus MC Lucio et al, Leukemia,

18 Immunophenotypic characteristics of normal vs. leukemic B-cells CD19+ B-cells 8- COLOR flow cytometry: BCP-ALL EuroFlow panel (450 bivariate plots) CD45-PO SSC SSC CD19-PECy7 Lucio et al, Leukemia, 1999 Automated identification of populations Multidimensional analysis: Automated Separation among different cell Populations (APS view) 18

19 Automated population separation (APS) CD8+ T-lymfocytes Monocytes B-cells Neutrophils CD4+ T-lymfocytes NK-cells APS Procedure for AUTOMATIC ANALYSIS Visualization options Dots Dots / Mean Mean 19

20 APS Procedure for groups of patients APS Dots View APS Means View Group of patients with the same panel/protocol applied and same disease category APS Procedure for groups of patients MEAN VALUES REPRESENTATION Each dot = patient/sample Group of patients with same panel/protocol applied and 2 different disease categories 20

21 UK NEQAS Scientific Meeting, Sheffield, UK, 24 June 2013 Achievements in flow cytometry by EuroFlow 1. Full technical standardization of multicolor flow cytometry ( 8 colors) standardization of instrument settings and laboratory protocols selection of fluorochromes and selection of antibody clones per marker EuroFlow protocols work on all tested 8 colors flow cytometers: - DAKO Cyan, LSR-II, FACS Canto-II; - also the late arrivals Navios and MACS Quant can be used!!! 2. Implementation and further development of novel software: Infinicyt fast and easy data handling with automated pattern recognition combining multiple tubes: calculation and APS (principle component analysis) mapping of diagnosis and follow-up leukemia samples against templates of normal/control samples 3. Development of 8-color antibody protocols for diagnosis, classification and monitoring of hematological malignancies 8-color panels are based on recognition of normal cells & differentiation pathways diagnosis and classification tubes are ready; MRD tubes in development flexibility within panels: deletion and inclusion of tubes (and markers) is possible 4. Large EuroFlow data base linked to Infinicyt software (available end 2013/early 2014) Algorithm for EuroFlow antibody panels in hemato-oncology Screening tubes CSF & vitreous fluid ~100% diagnosis: 4 screening tubes Classification tubes ~98% classification: 33 extra tubes 21

22 Algorithm for EuroFlow antibody panels in hemato-oncology Acute leukemia orientation tube (ALOT) ALOT 1 tube BCP-ALL T-ALL AML/MDS 4 tubes 4 tubes 4 to 7 tubes Pacific Blue Pacific Orange FITC PE PerCP- Cy5.5 PE-Cy7 APC APC- H7 cycd3 CD45 cympo cycd79a CD34 CD19 CD7 smcd3 Responsible scientist: L Lhermitte 22

23 Responsible scientist: L Lhermitte BCP-ALL T-ALL AML Single «virtual» merged tube/data file Responsible scientist: L Lhermitte BCP-ALL T-ALL AML 23

24 Responsible scientist: L Lhermitte BCP-ALL T-ALL AML ALOT (Acute Leukemia Orientation Tube) BCP-ALL (n=254) Responsible scientist: L Lhermitte T-ALL (n=63) AML MPO+ (n=70) AML MPO-/CD7- (n=11) AML MPO-/CD7+ (n=13) 481 overall cases 24

25 ALOT (Acute Leukemia Orientation Tube) BCP-ALL (n=254) T-ALL (n=63) AML MPO+ (n=70) AML MPO-/CD7- (n=11) AML MPO-/CD7+ (n=13) 481 overall cases Algorithm for EuroFlow antibody panels in hemato-oncology 25

26 Acute leukemia orientation tube (ALOT) ALOT 1 tube BCP-ALL T-ALL AML/MDS 4 tubes 4 tubes 4 to 7 tubes Pacific Blue Pacific Orange FITC PE PerCP- Cy5.5 PE-Cy7 APC APC- H7 cycd3 CD45 cympo cycd79a CD34 CD19 CD7 smcd3 Responsible scientist: L Lhermitte Multi-tube EuroFlow classification panel for B-cell precursor ALL (BCP-ALL)* Responsible scientist: L Lhermitte Tube Pacific Blue Pacific Orange FITC PE PerCP- Cy5.5 PE-Cy7 APC APC-H7 Aim** 1 CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38 Diagnosis and classification of BCP-ALL; Detection of LAP markers; Detection of phenotypes associated with molecular aberrations 2 Smlgκ CD45 Cylgµ CD33 CD34 CD19 Smlgµ and CD117 Smlgλ Diagnosis and classification of BCP-ALL; 3 CD9 CD45 NuTdT CD13 CD34 CD19 CD22 CD24 Diagnosis and classification of BCP-ALL; Detection of phenotypes associated with molecular aberrations; Detection of LAP markers 4 CD21 CD45 CD15 and CDw65 NG2 CD34 CD19 CD123 CD81 Subclassification of BCP-ALL; Detection of LAP markers; Detection of phenotypes associated with molecular aberrations * Backbone markers are indicated in bold; Cy= cytoplasmic; Sm= surface membrane; Nu= nuclear. ** The described marker combinations can also be applied for disease staging and monitoring of treatment effectiveness (MRD diagnostics). 26

27 Multi-tube EuroFlow classification panel for T-ALL.* Responsible scientist: V Asnafi Tube Pacific Blue Pacific Orange FITC PE PerCP- Cy5.5 PE-Cy7 APC APC- H7 Aim** 1 CyCD3 CD45 NuTdT CD99 CD5 CD10 CD1a SmCD3 Diagnosis, classification, and maturation stages of T-ALL; detection of LAP markers; detection of MRD 2 CyCD3 CD45 CD2 CD117 CD4 CD8 CD7 SmCD3 Diagnosis, classification, and maturation stages of T-ALL. 3 CyCD3 CD45 TCRγδ TCRαβ CD33 CD56 CyTCRβ SmCD3 Diagnosis, classification, and maturation stages of T-ALL, according to TCR status; Detection of LAP markers 4 CyCD3 CD45 CD45 CD13 HLADR CD45RA CD123 SmCD3 Classification of T-ALL; Detection of LAP markers * Backbone markers are indicated in bold; Cy= cytoplasmic; Sm= surface membrane; Nu= nuclear. ** The described marker combinations can also be applied for disease staging and monitoring of treatment effectiveness (MRD diagnostics). Multi-tube EuroFlow classification panel for AML/MDS Responsible scientist: VHJ van der Velden Tube Pacific Blue Pacific Orange FITC PE PerCP- Cy5.5 PE-Cy7 APC APC- H7 Aim** AML/ MDS 1 HLADR CD45 CD16 CD13 CD34 CD117 CD11b CD10 Diagnosis and subclassification of AML and PNH especially focused on neutrophilic lineage 2 HLADR CD45 CD35 CD64 CD34 CD117 IREM2 CD14 Diagnosis and subclassification of AML and PNH especially focussed on monocytic lineage 3 HLADR CD45 CD36 CD105 CD34 CD117 CD33 CD71 Diagnosis and subclassification of AML especially focused on erythroid lineage 4 HLADR CD45 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) 27

28 Algorithm for EuroFlow antibody panels in hemato-oncology Panel Construction: Selection of B-cell backbone markers Responsible scientist: S. Böttcher 28

29 B-CLPD panel Pac Blue CD20 CD4 Pac Orange CD45 FITC Lambda CD8 PE Kappa CD56 PerCP Cy5.5 CD5 PE Cy7 APC APC H7 CD19 TCR γδ CD3 CD38 CD20 CD45 CD23 CD10 CD79b CD19 CD200 CD43 CD20 CD45 CD31 LAIR-1 CD11c CD19 IgM CD81 CD20 CD45 CD103 CD95 CD22 CD19 CXCR5 CD49d CD20 CD45 CD62L CD39 HLA-Dr CD19 CD27 CD20/CD4/CD45/Lambda/Kappa/CD8/CD56/CD5/CD19/CD38/CD23/CD10/CD79b/CD200/CD43/CD31/ LAIR/CD11c/IgM/CD81/CD103/CD95/CD22/CXCR5/CD49d/CD62L/CD39/HLA-DR/CD19/CD27 30-color flow cytometry! Responsible scientist: S. Böttcher Designed by: P. Lucio PCA of total immunophenotype Principal component 2 MCL CLL 1 SD 2 SD Principal component 1 Responsible scientist: S. Böttcher 29

30 PCA of total immunophenotype MCL CLL 2 SD separated PC1 1 IgM CD CD79b CD CD Responsible scientist: S. Böttcher PCA of total immunophenotype MCL CLL CD10+ DLBCL BL 2 SD separated 1 SD separated PC1 1 IgM CD CD79b CD CD PC1 1 CD CD IgM CD CD Responsible scientist: S. Böttcher 30

31 PCA of total immunophenotype MCL CLL CD10+ DLBCL BL FL CD10- DLBCL 2 SD separated 1 SD separated Overlap of 1 st SD PC1 1 IgM CD CD79b CD CD PC1 1 CD CD IgM CD CD Responsible scientist: S. Böttcher PCA of total immunophenotype: clearly separated diseases MZL HCL MCL CLL FL CLL Responsible scientist: S. Böttcher 31

32 Separation power of different types of BCLPD CLL DLBCL CD10+ DLBCL CD10- FL HCL LPL MCL MZL BL CLL DLBCL CD10 + DLBCL CD10 - FL HCL LPL MCL Responsible scientist: S. Böttcher 2 SD separated 1 SD separated Overlap of 1st SD 1 x 1comparison n = 150 Expert pathologist agreement with the consensus diagnosis LPL MZL 5 expert hematopathologists ~1,400 lymphoma cases The Non-Hodgkin Lymphoma Classification Project, Bood 1997;89: Kindly provided by Raul Braylan after 25th ICCS meeting 32

33 B-CLPD panel Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 CD20 CD45 Lambda Kappa CD5 CD19 CD3 CD38 CD20 CD45 CD23 CD10 CD79b CD19 CD200 CD43 CD20 CD45 CD31 LAIR-1 CD11c CD19 IgM CD81 CD20 CD45 CD103 CD95 CD22 CD19 CXCR5 CD49d CD20 CD45 CD62L CD39 HLA-Dr CD19 CD27 Responsible scientist: S. Böttcher B-NHL classification panel modular design Full panel CLL MCL CLL HCL CLL MZL CLL FL CLL DLBCL Tubes 1 & 2 only CLL MCL CLL HCL CLL MZL CLL FL CLL DLBCL Responsible scientist: S. Böttcher 33

34 UK NEQAS Scientific Meeting, Sheffield, UK, 24 June 2013 Achievements in flow cytometry by EuroFlow 1. Full technical standardization of multicolor flow cytometry ( 8 colors) standardization of instrument settings and laboratory protocols selection of fluorochromes and selection of antibody clones per marker EuroFlow protocols work on all tested 8 colors flow cytometers: - DAKO Cyan, LSR-II, FACS Canto-II; - also the late arrivals Navios and MACS Quant can be used!!! 2. Implementation and further development of novel software: Infinicyt fast and easy data handling with automated pattern recognition combining multiple tubes: calculation and APS (principle component analysis) mapping of diagnosis and follow-up leukemia samples against templates of normal/control samples 3. Development of 8-color antibody protocols for diagnosis, classification and monitoring of hematological malignancies 8-color panels are based on recognition of normal cells & differentiation pathways diagnosis and classification tubes are ready; MRD tubes in development flexibility within panels: deletion and inclusion of tubes (and markers) is possible 4. Large EuroFlow data base linked to Infinicyt software (available end 2013/early 2014) Reference data files: Normal B-cells vs. B-CLL cells Normal PB CD19+ B-cells CD19+ CLL B-cells CD19-PECy7 CD19-PECy7 Case number Case number CD19-PECy7 Case number CLL cases Normal PB B cells LAIR1 9.3 CD5 8.6 CD79b 8.2 IgM 8.1 Normal PB (n=8) CLL cases (n=6) Designed by: A. Orfao 34

35 New Case plotted against reference data base of normal B-cells and B-CLL cells CD19+ CLL B-cells CD19-PECy7 Case number SSC SSC Normal PB (n=8) CLL cases (n=6) CD19-PECy7 CD19-PECy7 CLL cases Normal PB B cells CD19 PECy7 CLL Normal PB B-cells Case number Designed by: A. Orfao 35

36 Results of synchronized experiments APS view of 30 merged data files from different centers CD14+ Monocytes CD8+ CD3- T cells CD3+ CD4+ T-cells Local normal donors CD3+ CD8- CD4- T cells CD3+ CD8+ T cells CD19+ CD20+ B-cells EuroFlow LST-QC tube (Lymphocyte Screening Tube) B-cells / Kappa vs. Lambda NK-cells CD19 CD5 CD19 Lambda T-cells / CD4+ vs. CD8+ CD3 Kappa CD19 CD8 CD3 CD4 CD3 CD56 Composition of the EuroFlow LST-QC tube(8 color / 11 markers): CD20+CD4 PacB / CD45 PacO CD8+Lambda FITC /CD56+Kappa PE /CD5 PerCP55 /CD19 PC7 CD3 APC/CD81 APC-H7 Responsible scientist: Tomas Kalina 36

37 APS view of QC cell types QC 2010 QC 2011 QC 2012 CD4+ T-celll All QC years Kappa Lambda CD8+ T-cell NK-cell Responsible scientist: Tomas Kalina UK NEQAS Scientific Meeting, Sheffield, UK, 24 June 2013 Current EuroFlow tools for diagnosis and classification The EuroFlow antibody panels were built on an evidence-based strategy, aiming the most informative antibody combinations. Specific antibody clones and fluorochromes been extensively tested in several testing rounds within the EuroFlow laboratories before approval. For maximum information, the EuroFlow antibody panels, the EuroFlow SOPs and the Infinicyt software should be used as one flow-based tool. In the EuroFlow algorithm, clinical relevance, efficiency, accuracy and costeffective aspects were taking in account. Please note: the antibody panels are flexible: not all tubes are needed for inclusion of patients into clinical treatment protocols and own tubes can be added. The combined EuroFlow tools are able to diagnose, characterize and subclassify the major WHO entities (application of reference data base linked to Infinicyt). However, not all classification tubes are needed for assignment to the clinical treatment protocols. EuroFlow reference data base (linked to Infinicyt software) will appear highly valuable for diagnostic patient care: fast and accurate diagnosis! 37

38 UK NEQAS Scientific Meeting, Sheffield, UK, 24 June 2013 New (ongoing) EuroFlow developments 1. Monitoring of treatment effectiveness (MRD tubes)elation with - Acute leukemias (includes recognition of normal precursors) - Chronic lymphoproliferative disorders (includes recognition of normal cells) 2. Antibody panels for application in diagnosis and classification of immunodeficiencies, auto-immune diseases, allergy, and inflammation. 3. New flow cytometric tools for Companion Diagnostics - special focus on antibody-based treatment - monitoring of antibody levels and target saturation levels - etc. 4. Monitoring of other malignancies (solid tumors) Dissection of normal precursor-b-cell differentiation Responsible scientists: V.H.J. van der Velden and E. Mejstrikova 38

39 Dissection of normal precursor-b-cell differentiation Responsible scientists: V.H.J. van der Velden and E. Mejstrikova Precursor B-cell differentiation in normal vs regenerating bone marrow in EuroFlow immunostaining 1 Normal BM Late maturation stages Normal vs Regenerating BM Late maturation stages Early maturation stages Early maturation stages CD19 Gated B-cells B (excluding PC) Responsible scientists: V.H.J. van der Velden and E. Mejstrikova 39

40 Four BCP-ALL cases vs normal precursor B-cells in EuroFlow immunostaining 1 APS view 1 APS view 2 Case 1 Case 2 Case 3 Case 4 Responsible scientists: V.H.J. van der Velden and E. Mejstrikova Designed by: A. Orfao & Q Lecrevisse BCP-ALL are different from normal B-cells (in EuroFlow immunostaining 2) BCP-ALL Case 1 40

41 Case 1: APS view versus normal B-cells (in EuroFlow immunostaining 2) Versus normal B-cells Versus normal B-cells first 6 stages only Relevant parameters for MRD analysis MRD tube design & testing Development of 8-color MRD panels Single-tube antibody EuroFlow MRD protocols under evaluation 1. Acute leukemias (includes recognition of normal precursors) Acute myeloid leukemia panel (AML-MRD): 1 tube per pathway (A. Orfao) B-cell precursor (BCP-ALL-MRD): 1 tube (V. van der Velden, E. Mejstrikova) T-cell ALL (T-ALL-MRD): 1 tube (V.Asnafi) 2. Chronic lymphoproliferative disorders (includes recognition of normal cells) Chronic lymphocytic leukemia (CLL-MRD): 1 tube (A. Langerak) Hairy cell leukemia (HCL-MRD): 1 tube (E. MacIntyre, L. Lhermitte) Mantle cell lymphoma (MCL-MRD): 1 tube (S. Böttcher) Follicular lymphoma (FL-MRD): 1 tube (S. Böttcher) Marginal zone lymphoma (MZL-MRD): 1 tube (P.Lucio) Lymphoplasmacytic lymphoma (LPL-MRD): 1 tube (P. Lucio) Diffuse large B-cell lymphoma (DLBCL-MRD): 1 tube (P. Lucio) Burkitt lymphoma (BL): 1 tube (L.Lhermitte) T-chronic lymphoproliferative diseases (T-CLPD-MRD): 1 tube (J. Almeida) Multiple myeloma (MM): 1 tube (J. Flores) 41

42 UK NEQAS Scientific Meeting, Sheffield, UK, 24 June 2013 New (ongoing) EuroFlow developments 1. Monitoring of treatment effectiveness (MRD tubes)elation with - Acute leukemias (includes recognition of normal precursors) - Chronic lymphoproliferative disorders (includes recognition of normal cells) 2. Antibody panels for application in diagnosis and classification of immunodeficiencies, auto-immune diseases, allergy, and inflammation. 3. New flow cytometric tools for Companion Diagnostics - special focus on antibody-based treatment - monitoring of antibody levels and target saturation levels - etc. 4. Monitoring of other malignancies (solid tumors) Identification of leukocyte subsets in the EuroFlow PID screening tube T-cells Neutrophils DC CD16+ Monocytes Basophils Eosinophil s B-cells Responsible scientist: Martin Perez 42

43 Euroflow is an independent scientific consortium, which aims at innovation in flow cytometry for improvement of diagnostic patient care European network for laboratory diagnostics Scientifically independent consortium Innovation Development of new technologies or technical strategies, using the most suited and advanced equipment. Protection of new intellectual property (IP) by filing of patents on behalf of the consortium (collective IP, collective ownership, collective revenues). Standardization Standardization of methods, including primer sequences, instrument settings, antibodies, fluorochromes, etc. Guidelines for interpretation of results. AIM: fully identical results in all participating laboratories (essential for international treatment protocols) Quality Control Regular quality control rounds (twice per year) + discussion about results. Certificates for participation and performance. Dissemination and continuous education Educational Workshops for dissemination of knowledge and experience Educational lectures on new developments during consortium meetings 43

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