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B-CELL NEOPLASMS (PRECURSOR AND MATURE)

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1 B-CELL NEOPLASMS (PRECURSOR AND MATURE)
CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor Immunophenotyping: Standardization and Applications March 9-11, 2012 TMH, Mumbay (India)

2 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) 800s207b Text Molecular and cytogenetic diagnostics in lymphoma patients Prepared by JJM van Dongen 2

3 WHO CLASSIFICATION OF LYMPHOID MALIGNANCIES (2002-2008)
B-cell precursor (immature) derived acute lymphoblastic leukemia/lymphoblastic lymphoma - Mature (peripheral) B-cell neoplasms

4 B-cell precursor acute lymphoblastic leukemia/lymphoma (B-ALL)
B Lymphoblastic Leukemia/Lymphoma, not otherwise specified B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities BCP-ALL/LL with t(9:22) (q34;q11.2); BCR/ABL BCP-ALL/LL with t(v;11q23); MLL rearranged BCP-ALL/LL with t(12;21) (p13;q22); TEL/AML1 (ETV6-RUNX1) BCP-ALL/LL with hyperdiploidy BCP-ALL/LL with hypodiploidy (Hypodiploid ALL) BCP-ALL with t(5;14)(q31;q32)(IL3-IGH) BCP-ALL with t(1;19)(Q23;P13.3); (E2A-PBX1; TCF3/PBX1)

5

6 ALOT 1 tube BCP-ALL T-ALL AML/MDS 4 tubes 4 tubes 4 to 7 tubes

7 ALOT: B-cell precursor ALL Responsible scientist: Ludovic Lhermitte
BM stained with ALOT 8-color tube CyCD3 CD7 sCD3 CD19 CyCD79a CyMPO CD45 CD34 Responsible scientist: Ludovic Lhermitte

8 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) ALOT

9 EuroFlow ALOT: assessment of blast cell lineage
Van Dongen et al: EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia 2012 (under revision)

10 PRECURSOR-B ALL & T-ALL
CLASSIFICATION OF PRECURSOR-B ALL & T-ALL Precursor-B ALL: - B I (CD19+/cCD79a+/CD10-) - B II (CD10+/Ig-) - B III (cIgm+) - B IV (sIg+) T-ALL: - T I (CD7+,cCD3+) - T II (CD2+ &/or CD5+ &/or CD8+ - T III (CD1a+) - T IV (CD1a-,CD3+)

11 BCP-ALL panel BCP-ALL BCP-ALL

12 BCP-ALL panel BCP-ALL

13 BCP-ALL panel ALOT BCP-ALL

14 BCP-ALL panel ALOT BCP-ALL

15 BCP-ALL panel ALOT BCP-ALL

16 BCP-ALL panel ALOT BCP-ALL Positive Diagnosis

17 Differential Diagnosis & Ambiguous lineage acute leukemia
BCP-ALL panel ALOT BCP-ALL Differential Diagnosis & Ambiguous lineage acute leukemia

18 Maturation stage (EGIL)
BCP-ALL panel ALOT BCP-ALL Maturation stage (EGIL)

19 Alternative classification
BCP-ALL panel ALOT BCP-ALL Alternative classification Immunophenotypic features associated with well-defined molecular aberrations

20 BCP-ALL panel ALOT BCP-ALL Prognosis markers

21 BCP-ALL panel ALOT BCP-ALL LAP markers

22 BCP-ALL panel ALOT BCP-ALL

23 BCP-ALL panel 31 parameters ALOT BCP-ALL
LS CD45 CD19 CD34 cyCD3 cyMPO cyCD79a CD7 smCD3 CD20 CD58 CD66C CD10 CD38 smIgK cyIgM CD33 smIgM+CD117 smIgL CD9 TdT CD13 CD22 CD24 CD21 CD15+65 NG2 CD123 CD81 31 parameters

24 BCP-ALL:DETECTION OF ABERRANT PHENOTYPES
Mix of 3 different regenerating B cell populations (Haematogones) BCP-ALL blast cells

25 PRECURSOR B-ALL: DNA ANEUPLOIDY B-CELL PRECURSOR ALL: DNA ANEUPLOIDY
256 512 768 1024 RM FL2-Area -> PRECURSOR B-ALL: DNA ANEUPLOIDY NORMAL B-CELLS NEOPLASTIC RESIDUAL NON-B-CELLS B-CELL PRECURSOR ALL: DNA ANEUPLOIDY

26 IMMUNOPHENOTYPE OF NEOPLASTIC B-CELL PRECURSORS
BCP-ALL Phenotype vs cytogenetics: t(9;22)* Sensitivity 100%, Specificity >90% CD19+ CD10+ CD34++ CD38-/d CD13d 11q23 Sensitivity 95%, Specificity 85% CD19+ CD10- CD20-CD34+CD15+CD t(12;21) Sensitivity 86%, Specificity 100% CD19+ CD10+ CD34d CD45-/d DR++ *Leukemia 15:406  Am J Clin Pathol 111:467  Leukemia 14:1225

27 ADULT PRECURSOR B-ALL: IMMUNOPHENOTYPE OF BCR/ABL+ CASES
10 1 2 3 4 HG CD10 -> HG CD34 -> HG HG CD13 -> RFR RFR RFR UVJ CD34 PE -> UVJ CD13 PE -> MO AP ADULT PRECURSOR B-ALL: IMMUNOPHENOTYPE OF BCR/ABL+ CASES Normal BM CD34+ B-cells DNA diploid Bcr/abl+ ALL DNA aneuploid CD CD CD CD34 CD19 CD45 CD38

28 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) vv017 Kindly provided by JJM Van Dongen on behalf of the Euroflow group

29 Weerkamp et al, Leukemia, 2009
BCR-ABL RUO testing by EuroFlow MFI values of the different cell samples (n=217) jd410u Weerkamp et al, Leukemia, 2009 29

30 Results concerning BCR-ABL RUO kit
Weerkamp et al, Leukemia, 2009 High specificity of BCR-ABL RUO  High concordance (100%; 145/145) between BCR-ABL PCR results and the BCR-ABL RUO results;  Results: - 17/78 precursor-B-ALL were BCR-ABL positive in both assays (mainly adults) /19 CML were BCR-ABL positive in both assays (with borderline positivity in one CML case; MFI of 144) /48 of other (acute) leukemias were BCR-ABL positive Mean Fluorescence Intensity (MFI) values  two main groups of positive patient samples were seen: ● high level positivity: MFI values ≥ 1,000 ● lower level positivity: MFI values ≥ 135, but < 1,000  negative samples were defined as MFI values < 135 Different MFI values in precursor-B-ALL and CML Precursor-B-ALL: 88% (15/17) high level positivity CML: 84% (16/19) low level positivity (true low expression or remaining protease activity?) 800s1009 30

31 Immunobead flow cytometry with BD™ CBA Flex beads BCR-ABL t(9;22) fusion protein: Specificity
Black: (t(1;19), neg. control) Blue: TOM-1, BCR-ABL+ (p190) Green: LAMA-84 BCR-ABL+ (p210) Purple: AR230, BCR-ABL+ (p230) 810322Me Catching antibody: anti-BCR (clone 3E2C10) Bead: BD-Flex bead (A7) Detection antibody: biotinylated anti-ABL (clone 8E9) + SA-PE

32 Final Statement about the BCR-ABL RUO testing
The main advantages of the immunobead assay are: Not dependent of the breakpoint position in the fusion gene; No need for special laboratory facilities other then a routine flow cytometer; Providing results within several hours; The possibility to run in parallel to routine immunophenotyping: no extra technician time needed !!; Allowing multiplexing with differently-labeled beads, that can detect different fusion proteins within the same disease category 32

33 PANELS OF IMMUNOBEADS FOR THE CLASSIFICATION OF ACUTE LEUKAEMIAS
Precursor-B-ALL AML ‘MLL’ T-ALL BCR-ABL PML-RARA MLL-AF4 CALM-AF10 TEL-AML1 AML1-ETO MLL-AF9 LMO2 E2A-PBX1 CBFB-MYH11 MLL-AF10 HOX11L2 ( MLL-AF4) MLL-ENL TAL1 MLL-AF6

34 Precursor B-ALL multiplex tube: E2A-PBX1 t(1;19)
Sensitivity for detection on cell line <10% *Exp. Performed on April 27th 2007 Neg. patients Pos patients

35 Precursor B-ALL multiplex tube: TEL-AML1 t(12;21)
Sensitivity for detection REH cell line in: WBC : 10-50% PBMC : 1-10%

36 Precursor B-ALL multiplex tube: MLL-AF4 t(4;11)
Sensitivity for detection MV4;11 line in: 697 cell line: 10% WBC : 10-50% (close to 10%) PBMC: idem 10%

37 SIMULTANEOUS DETECTION OF THE BCR-ABL AND E2A-PBX FUSION PROTEINS
100% K562 100% 697 10% K562/697 10% 697/K562 R2 = green = BCR beads R3 = pink = E2A beads

38 B-CELL NEOPLASMS (PRECURSOR AND MATURE)
CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor Immunophenotyping: Standardization and Applications March 9-11, 2012 TMH, Mumbay (India)

39 WHO: Mature B-cell Neoplasms

40 B CELL CHRONIC LYMPHOPROLIFERATIVE DISORDERS
Heterogeneous group of diseases typically characterized by a monoclonal expansion of a mature-appearing neoplastic B-lymphocyte Mature/peripheral B cell chronic lymphoid leukemias: Chronic lymphocytic leukemia/Small B cell lymphocytic lymphoma Prolymphocytic leukemia Hairy cell leukemia Mature/pheripheral B-cell lymphomas: Lymphoplasmacytic lymphoma Splenic marginal zone lymphoma Extranodal marginal zone lymphoma (MALT-type) Nodal marginal zone lymphoma Follicular lymphoma Mantle cell lymphoma Diffuse large B-cell lymphoma Burkitt lymphoma Plasma cell neoplasias: Multiple myeloma/plasmacytoma WHO CLASSIFICATION OF B-CLPD

41 DIAGNOSIS OF CLONAL HAEMATOLOGICAL DISORDERS
Clinical symptoms Laboratory and signs findings Morphology + cytochemistry Cytogenetics Immunophenotyping Molecular biology/FISH

42 IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT
TYPES OF B-CLPD (Orfao et al, In: “B-CLL”.Humana Press, 2004) sIg CD5 CD10 CD20 CD11c CD23 CD24 CD25 CD38 CD43 CD79b CD103 FMC7 B-CLL d d -/ / d PLL / /+ -/ /+ -/+ -/ HCL / SMZL / / /+ + LPL / /+ MCL / / /+ FL /+ -/d / LDBCL / / BL / /+ -/

43 MATUTES et al SCORE FOR B-CLL
MARKER PATTERN SCORE CD5 positive CD23 positive CD dim sIg dim FMC7 negative (CD79b) dim Diagnosis of CLL requires a score > 3 (4) Matutes et al, Leukemia 1994

44 WHO: B-cell malignancies
Histology & cytology DLBCL B-PLL Cytogenetics MCL BL Immunophenotype CLL HCL Clinic MALT

45 The EuroFlow comprehensive approach
Clinical question Screening tube Diagnostic panel MRD 45

46 The EuroFlow comprehensive approach
Atypical lymphocytes Monoclonal Suspicion of Clinical question Screening tube Diagnostic panel MRD High suspicion of Splenomegaly component High lymphoma acute leukemia Unexplained L ymphocytosis non-IgM, monoclonal localization in e.g. blast cells observed cytopenia LN enlargement component “small cell number” Bone lesions BM plasmacytosis non-IgM samples e.g. CS F , vitreous Sustained Unexplained Monoclonal monocytosis Eosinophilia component 46

47 The EuroFlow comprehensive approach
Atypical lymphocytes Monoclonal Suspicion of Clinical question Screening tube Diagnostic panel MRD High suspicion of Splenomegaly component High lymphoma acute leukemia Unexplained L ymphocytosis non-IgM, monoclonal localization in e.g. blast cells observed cytopenia LN enlargement component “small cell number” Bone lesions BM plasmacytosis non-IgM samples e.g. CS F , vitreous Sustained Unexplained Monoclonal monocytosis Eosinophilia component PCS T SS T ALO T LS T first tube of PCD reactive/polyclonal reactive/polyclonal clonal/aberrant other ? B-CLPD clonal ALOT – acute leukemia orientation tube LST – lymphocytosis screening tube PCD – Plasma cell discrasia screening tube SST – small sample tube 47

48 The EuroFlow comprehensive approach
Atypical lymphocytes Monoclonal Suspicion of Clinical question Screening tube Diagnostic panel MRD High suspicion of Splenomegaly component High lymphoma acute leukemia Unexplained L ymphocytosis non-IgM, monoclonal localization in e.g. blast cells observed LN enlargement component “small cell number” cytopenia Bone lesions BM plasmacytosis non-IgM samples e.g. CS F , vitreous Sustained Unexplained Monoclonal monocytosis Eosinophilia component PCS T SS T ALO T LS T first tube of PCD first reactive/polyclonal reactive/polyclonal 4 tubes clonal/aberrant other ? B-CLPD first clonal 4 tubes BCP-AL L T -AL L B-CLPD B-CLPD AML/MDS T -CLPD NK-CLPD PCD limited complete Comprehensive network of panels aiming the diagnosis and characterization of the major WHO entities 48

49 The EuroFlow comprehensive approach
Atypical lymphocytes Monoclonal Suspicion of Clinical question Screening tube Diagnostic panel MRD High suspicion of Splenomegaly component High lymphoma acute leukemia Unexplained L ymphocytosis non-IgM, monoclonal localization in e.g. blast cells observed component “small cell number” cytopenia LN enlargement Bone lesions BM plasmacytosis non-IgM samples e.g. CS F , vitreous Sustained Unexplained Monoclonal monocytosis Eosinophilia component PCS T SS T ALO T LS T first tube of PCD first reactive/polyclonal reactive/polyclonal 4 tubes clonal/aberrant other ? B-CLPD first clonal 4 tubes BCP-AL LL T -AL L AML/MDS B-CLPD B-CLPD T -CLPD NK-CLPD PCD limited complete aberrant variou s variou s variou s CL L CL L aberrant ab + subtype s subtype s subtype s NK cells o f BCP-AL L o f T -AL L o f AM L various subtypes non-CL L MC L aberrant gd + reactive of PCD MDS FC L reactive PNH HC L CM L other clonal B CML-BC other MPD 49

50 THE EUROFLOW APPROACH TO LEUKEMIA/LYMPHOMA IMMUNOPHENOTYPING
Clinical question Knowledge Experience Evaluation Reference profiles Diagnostic screening tube Majority of diseases? Majority of cases? New disease entities? S “Diagnostic classification” panel 14 Major groups 154 Nosologic entities MRD monitoring

51 Clinical request/need
CONSTRUCTION OF EUROFLOW LEUKEMIA/ LYMPHOMA IMMUNOPHENOTYPING ANTIBODY PANEL Clinical request/need Proposed strategy Medical indication Panel optimization (re-design) 2-8 cycles Design of MAb panels (Medical indication-oriented) & immuno-phenotyping strategy Panel evaluation S Panel evaluation vs conventional in-use panels Panel optimization (re-design) Techniques

52 Panel construction Selection of: Fluorochromes Antibodies
Fluorochrome + antibody combinations 8-color combinations Panels BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. 52

53 Panel construction Selection of: Fluorochromes Antibodies
Fluorochrome + antibody combinations 8-color combinations Panels Reagent stability Brightness Low fluorescence background levels No spectral overlap between fluorochrome emissions BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. 53

54 FLUOROCHROME SELECTION
Initial selection Further comparisons FL Channel Laser Commonly available fluorochromes 1 Violet Pacific Blue HORIZON V450 2* AmCyan Pacific Orange HORIZON V500 3 Blue FITC Alexa Fluor 488 4 PE 5 PE-TxRed 6 PerCP Cy5.5 PerCP 7 PE Cy7 8 Red APC Alexa Fluor 647 9 APC Cy7 APC H7 Alexa Fluor 700 FL Channel Laser Commonly available fluorochromes 1 Violet Pacific Blue HORIZON V450 2 AmCyan* Pacific Orange HORIZON V500 3 Blue FITC Alexa Fluor 488 4 PE 5 PE-TxRed 6 PerCP Cy5.5 PerCP 7 PE Cy7 8 Red APC Alexa Fluor 647 9 APC Cy7 APC H7 Alexa Fluor 700 *Alternative new additional fluorochromes currently under evaluation Responsible scientists: T.Kalina, J.Flores 54

55 Panel construction – Antibodies
Selection of: Fluorochromes Antibodies Fluorochrome + antibody combinations 8-color combinations Panels BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. 55

56 Panel construction – Antibodies
Backbone antibodies The same antibodies in every tube of the panel Essential for merge-calculation function Characterization antibodies - Lineage assessment, - Differentiation lineage markers, - Maturation stage, - Aberrant markers, - Relation to cytogenetic abnormality, - LAP, - MRD … BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. 56

57 Panel construction – Antibodies
Backbone antibodies The same Ab in every tube of the panel Essential for merge-calculation function Backbone markers: Should identify all cells belonging to the target lineage, either normal or malignant BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. Backbone candidates for B-CLPD: CD19? CD20? CD22? CD37? - Aberrant underexpression of CD19 and/or CD20 frequently observed - κ/CD37/λ/CD19/CD22/CD20 tested in 69 B-NHL cases Responsible scientist: S. Böttcher 57

58 Panel Construction: selection of backbone B cell markers
Conclusion: CD37 & CD22 redundant, as CD20 PacB plus CD19 PE-Cy7 were sufficient to identify all malignant B cells in all cases Responsible scientist: S. Böttcher

59 Panel construction Selection of: Fluorochromes Antibodies
Fluorochrome + antibody combinations 8-color combinations Panels BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. 59

60 Panel construction – Fluorochrome + antibody BB combinations
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 BCP-ALL CD45 CD34 CD19 T-ALL cyCD3 CD3 AML/MDS HLADR CD117 B-CLPD CD20 T-CLPD CD4 CD8 NK-CLPD CD56 PCD CD138 CD38 BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. 60

61 LST – Lymphocytosis screening tube
Monoclonal component non-IgM, Bone lesions BM plasmacytosis ALO T LS PCS first tube of PCD SS Sustained monocytosis Unexplained Eosinophilia High suspicion of acute leukemia e.g. blast cells observed cytopenia Atypical lymphocytes Splenomegaly L ymphocytosis LN enlargement High monoclonal non-IgM Suspicion of lymphoma localization in “small cell number” samples e.g. CS F , vitreous Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR gd CD3 CD38 Responsible scientist: J. Flores Montero 61

62 LST – Lymphocytosis screening tube
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR gδ CD3 CD38 BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. Responsible scientist: J. Flores Montero 62

63 LST – Lymphocytosis screening tube
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR gδ CD3 CD38 Able to identify all the sample major populations: Non-hematopoietic cells BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. Responsible scientist: J. Flores Montero 63

64 LST – Lymphocytosis screening tube
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR gδ CD3 CD38 Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. Responsible scientist: J. Flores Montero 64

65 LST – Lymphocytosis screening tube
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR gδ CD3 CD38 Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. B lymphocytes Responsible scientist: J. Flores Montero 65

66 LST – Lymphocytosis screening tube
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR gδ CD3 CD38 Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. B lymphocytes NK cells Responsible scientist: J. Flores Montero 66

67 LST – Lymphocytosis screening tube
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR gδ CD3 CD38 Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. B lymphocytes NK cells Plasma cells Responsible scientist: J. Flores Montero 67

68 LST – Lymphocytosis screening tube
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR gδ CD3 CD38 Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes (T-cell subpopulations) BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. B lymphocytes NK cells Plasma cells Responsible scientist: J. Flores Montero 68

69 LST – Lymphocytosis screening tube
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR gδ CD3 CD38 Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes (T-cell subpopulations) BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. B lymphocytes (B-cell subsets & Ig light chain restriction) NK cells Plasma cells Responsible scientist: J. Flores Montero 69

70 LST – Lymphocytosis screening tube
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR gδ CD3 CD38 Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes (T-cell subpopulations) B lymphocytes (B-cell light chain restriction) BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. NK cells Plasma cells B-NHL panel backbone Responsible scientist: J. Flores Montero 70

71 Panel construction Selection of: Fluorochromes Antibodies
Fluorochrome + antibody combinations 8-color combinations Panels BACKGROUND The development of standardized 8-color flow cytometry (FC) combined with bio-informatic merging of data from multiple tubes allows multiparameter evaluation of surface and intracellular proteins at the single cell level, while maintaining all the advantages of FC diagnostics (rapidity, evaluation of cellular heterogeneity, application to minimal residual disease, etc) AIMS Within the European EuroFlow program, we designed an 8-color acute leukemia screening tube (ALST) for fast and efficient evaluation of patients suspected of acute leukemia in order to choose more detailed immunophenotyping panels. METHODS 157 acute leukemia samples (105 BM, 45 PB, 7 others) were analyzed with the ALST tube by FC in 8 EuroFlow centers, using standard operating procedures which provided intercenter reproducibility. In parallel, conventional procedures for lineage assessment of blast cells were used at each centre. ALST data was collected and analysed centrally using the Infinicyt software (Cytognos®). The blast cell population was identified bio-informatically, extracted from each sample and virtually merged in a common database with the blast cell populations from all other samples. Within this database, each leukemic sample was represented by its median expression for each individual ALST antibody, and the results from all 157 samples underwent unsupervised discrimination by principal component analysis (PCA) using automated software tools. RESULTS Using a pair-wise PCA-based comparison, 152/157 AL could be clearly separated into 3 well defined clusters (89 B-Cell Precursor-ALL, 27 T-ALL and 36 AML), with no mis-classification compared to classical, more extensive FC panels. Five cases (3%) clustered together in between the typical T-ALL and AML clusters, in keeping with an undifferenciated T/Myeloid maturation arrest. These included 3 AML and 2 undifferentiated AL. No intermediate B/Myeloid or T/B clusters were observed. No center dependent effect on clustering was observed, in keeping with the use of highly standard operating flow cytometric procedures. SUMMARY / CONCLUSIONS This ALST FC strategy linked to Infinicyt analysis allows efficient, fast and accurate lineage orientation of acute leukemia samples and multicenter, multiparametric analysis of patient data. In addition, this approach allowed identification of a subgroup of T/Myeloid AL which should be further analysed in both T-ALL and AML panels. Multicenter identification of these relatively rare samples will allow analysis of their clinical and biological characteristics and, if appropriate, their individualization for specific therapy. In conclusion, we propose an easy, efficient and highly sensitive flow-based tool to screen for cell lineage in acute leukemias which contributes to optimise health resources for further FC panels, allow multicenter comparisons and may help in identifying new subgroups of patients. 71

72 Characterization markers
B cell homing Normal B lymphopoiesis CD11a, CD11c, CD31, CD49d, CD62L, CXCR5, CCR6, LAIR1 CD10, CD20, CD22 CD24, CD27, CD38 CD39, CD43, CD63 CD81, CD95, CD138 Bcl-2, HLA-DR, IgM Known to differentiate CD5, CD23, CD25 FMC7, CD79b, CD103, CD200, sIg Responsible scientist: Sebastian Bottcher

73 Characterization markers
B cell homing Normal B lymphopoiesis CD11a, CD11c, CD31, CD49d, CD62L, CXCR5, CCR6, LAIR1 CD10, CD20, CD22 CD24, CD27, CD38 CD39, CD43, CD63 CD81, CD95, CD138 Bcl-2, HLA-DR, IgM Known to differentiate CD5, CD23, CD25 FMC7, CD79b, CD103, CD200,sIg Responsible scientist: Sebastian Bottcher

74 X X X X X X Panel construction – characterization markers
Tested markers (n=66): Backbone markers (e.g. CD19, CD20, CD22, CD37, CD45). Lineage assignment and maturation stage (e.g. Bcl-2, HLA-DR, IgM, CD10, CD43, CD24, CD27, CD38, CD39, CD63, CD81, CD95, CD138). Disease specific (e.g. CD5, CD23, CD25, CD79b, CD103, CD200). Integrins and chemokine receptors (e.g. CD11a, CD11c, CD31, CD49d CD62L, CXCR5, LAIR1). 150 cases of B-Lymphoproliferative disorders tested; aim: Improve differential classification of B-NHL Avoid markers with redundant information FINAL: 4 tube 8-color panel (20 antibodies) X X X X X X strong correlation to more significant markers X – univariate analysis X – multivariate analysis Responsible scientist: S. Böttcher 74

75 LST + BCLPD classification panel
Pac Blue Pac Orange FITC PE PerCP-Cy5.5 PECy7 APC APC-H7 1= LST CD20 /CD4 CD45 sIgl /CD8 sIgK /CD56 CD5 CD19 /TCRgd CD3 CD38 2 CD20 CD45 CD23 CD10 CD79b CD19 CD200 CD43 3 CD20 CD45 CD31 LAIR CD11c CD19 sIgM CD81 4 CD20 CD45 CD103 CD95 CD22 CD19 CXCR5 CD49d 5R CD20 CD45 CD62L CD39 HLA-DR CD19 CD27 CD20/CD4/CD45/sIgl/sIgK/CD8/CD56/CD5/CD19/CD38/CD23/CD10/CD79b/CD200/CD43/CD31/LAIR1/CD11c/sIgM/CD81/CD103/CD95/CD22/CXCR5/CD49d/CD62L/CD39/HLA-DR/CD19/CD27 30-colors flow cytometry ! Responsible scientist: Sebastian Bottcher

76 LST + BCLPD classification panel
Pac Blue Pac Orange FITC PE PerCP-Cy5.5 PECy7 APC APC-H7 1= LST CD20 /CD4 CD45 sIgl /CD8 sIgK /CD56 CD5 CD19 /TCRgd CD3 CD38 2 CD20 CD45 CD23 CD10 CD79b CD19 CD200 CD43 3 CD20 CD45 CD31 LAIR CD11c CD19 sIgM CD81 4 CD20 CD45 CD103 CD95 CD22 CD19 CXCR5 CD49d 5R CD20 CD45 CD62L CD39 HLA-DR CD19 CD27 CD20/CD4/CD45/sIgl/sIgK/CD8/CD56/CD5/CD19/CD38/CD23/CD10/CD79b/CD200/CD43/CD31/LAIR1/CD11c/sIgM/CD81/CD103/CD95/CD22/CXCR5/CD49d/CD62L/CD39/HLA-DR/CD19/CD27 30-colors flow cytometry ! Responsible scientist: Sebastian Bottcher

77 B-CLPD: 4-COLOUR STAINING PANEL
- FITC PE PerCP/Cy APC - Control Control CD Control - sIgk sIgl CD CD5 - CD CD23 CD CD20 - Fmc7 CD24 CD CD34 - CD43 CD79b CD CD49d - CyBcl2 CD10 CD CD38 - CD103 CD CD CD11c - sIgM CD27 CD CCR6 - CD3 CyZap70 CD CD5

78 REFERENCE DATAFILES FOR CLL B-CELLS Merge Calculated Data files
CLL case 1 Merge Calculated Data files CLL case 2 CD19+ CLL B-cells Case number CD19-PECy7 CLL case 3 CLL case 4 Responsible scientist: Sebastian Bottcher

79 Characterization markers: CD200
MZL MCL HCL LPL FL CLL DLBCL BL Responsible scientist: Sebastian Bottcher

80 Characterization markers: LAIR1(CD305)
MZL MCL HCL LPL FL CLL DLBCL BL Responsible scientist: Sebastian Bottcher

81 Characterization marker: CD5
CLL immunophenotypic diagnosis Characterization marker: CD5 HCL LPL FL MZL MCL CLL BL DLBCL CD10+ CD10- Parameter Significance 1 IgM 14.02 2 CD200 13.76 3 CD79b 11.94 4 CD23 8.57 5 CD38 7.73 Best 5 markers for the DD between CLL and MCL according to EuroFlow analysis Responsible scientist: S. Böttcher

82 V.H.J.van der Velden & J de Vries, unpublished results
Gene expression profiling of ALL cells in BM at diagnosis and purified ALL cells at extramedullary sites BM at Dx CNS Testis PCA study (Principal Component Analysis) V.H.J.van der Velden & J de Vries, unpublished results

83 PCA of total immunophenotype
MCL CLL Principal component 1 → Principal component 2 → 1 SD 2 SD Responsible scientist: Sebastian Bottcher

84 PCA of total immunophenotype
MCL CLL Principal component 1 → Principal component 2 → 1 SD 2 SD PC1 1 IgM 14.09 2 CD200 14.06 3 CD79b 13.39 4 CD23 8.60 5 CD20 6.43 Responsible scientist: Sebastian Bottcher

85 Characterization marker: smIgM
CLL immunophenotypic diagnosis Characterization marker: smIgM HCL LPL FL MZL MCL CLL BL DLBCL CD10+ CD10- Parameter Significance 1 IgM 14.02 2 CD200 13.76 3 CD79b 11.94 4 CD23 8.57 5 CD38 7.73 Best 5 markers for the DD between CLL and MCL according to EuroFlow analysis Responsible scientist: S. Böttcher

86 Characterization marker: CD200
CLL immunophenotypic diagnosis Characterization marker: CD200 HCL LPL FL MZL MCL CLL BL DLBCL CD10+ CD10- Parameter Significance 1 IgM 14.02 2 CD200 13.76 3 CD79b 11.94 4 CD23 8.57 5 CD38 7.73 Best 5 markers for the DD between CLL and MCL according to EuroFlow analysis Responsible scientist: S. Böttcher

87 Major consecutive development steps in the design of the EuroFlow B-CPLD panel
Version Tube Fluorescence channel PacB PacO FITC PE PECy5 PECy7 APC APCCy7 Backbone SmIg CD37 SmIg CD19 CD22 CD20 Backbone 2 1 CD20 SmIg CD37 SmIg CD19 CD22 1 CD20 CD45 Ig Ig PerCPCy5.5 CD19 IgM AF CD5 CD22 2 CD20 CD45 CD103 CD10 CD5 CD19 CD43 CD22 Panel 1 3 CD20 CD45 CD81 CD79b CD5 CD19 CD23 CD22 4 CD20 CD45 CD31 CD63 CD5 CD19 CXCR5 CD22 5 CD20 CD45 CD24 LAIR1 CD5 CD19 CD11a CD22 6 CD20 CD45 CD38 CD25 CD138 CD19 CD11c CD22 1 CD20 CD45 Ig Ig CD22 CD19 CD23 APCH CD81 Panel 2 2 CD20 CD45 CD103 CD25 CD11c CD19 IgM CD24 3 CD20 CD45 CD31 LAIR1 CD5 CD19 CD43 CXCR5 4 CD20 CD45 bcl2 CD10 CD79b CD19 CD38 CD49b 800S108387 PacB: Pacific Blue; FITC: Fluorescein isothiocyanate; PE: Phycoerythrin; Cy5: cyanin5; Cy7: Cyanin7; APC: Allophycocyanin; PerCP: Peridinin-chlorophyll-protein; AF700: Alexa Fluor 700; H7: Hilite7 Responsible scientist: S. Böttcher

88 Major consecutive development steps in the design of the EuroFlow B-CPLD panel (continued)
Version Tube Fluorescence channel PacB PacO FITC PE PECy5 PECy7 APC APCCy7 1 CD20 CD45 Ig Ig CD22 CD19 CD23 CD81 2 CD20 CD45 CD103 CD25 CD11c CD19 IgM Panel 3 3 CD20 CD45 CD31 LAIR1 CD5 CD19 CD43 4 CD20 CD45 bcl2 CD10 CD79b CD19 CD38 CD49d 5 CD20 CD45 CD24 CD95 CD19 CD200 2 CD20 CD45 CD103 CD25 CD11c CD19 IgM CD43 Panel 4 3 CD20 CD45 CD31 LAIR1 CD5 CD19 CD43 CD24 4 CD20 CD45 bcl2 CD10 CD79b CD19 CD83 CD49d 5 CD20 CD45 CD24 CD95 CD19 CD200 CD31 6 CD20 CD45 CD19 CXCR5 CD103 1=LST CD20 CD45 CD8 + CD56 + CD5 CD19 + CD3 CD Ig Ig TCR 2 CD20 CD45 CD23 CD10 CD79b CD19 CD200 CD43 Panel 5 3 CD20 CD45 CD31 LAIR CD11c CD19 IgM CD81 4 CD20 CD45 CD103 CD95 CD22 CD19 CXCR5 CD49d 5 CD20 CD45 CD62L CD39 HLADR CD19 CD27 800S1084

89 B-CLPD: Diagnostic work-flow
Monoclonal component ALOT LST B-CLPD limited broad Eosinophilia reactive/ non-aberrant CLL non-CLL MCL FCL HCL other clonal B Acute leukemia Cytopenia Lymphocytosis LN involvement > 90% pure by BB Responsible scientists: Juan Flores and Sebastian Bottcher

90 BCLPD classification panel: modular design
CLL MCL CLL HCL CLL MZL CLL FL CLL DLBCL Full panel MCL CLL CLL HCL CLL MZL CLL FL CLL DLBCL Tubes 1 & 2 only Responsible scientist: Sebastian Bottcher

91 PCA of total immunophenotype: clearly separated diseases
MZL HCL MCL CLL FL CLL Responsible scientist: S. Böttcher

92 PCA of total immunophenotype
CD10+ DLBCL MCL CLL BL FL CD10- DLBCL 2 SD separated 1 SD separated Overlap of 1st SD PC1 1 IgM 14.09 2 CD200 14.06 3 CD79b 13.39 4 CD23 8.60 5 CD20 6.43 PC1 1 CD38 9.87 2 CD10 8.92 3 IgM 8.48 4 CD200 8.33 5 CD81 7.37 Responsible scientist: Sebastian Bottcher

93 PCA of B-CLPD panel BL vs. DLBCL CD10- BL vs. CLL BL vs. HCL
BL vs. MCL BL vs. DLBCL CD10+ BL vs. FL BL vs. LPL BL vs. MZL BL vs.Normal Responsible scientist: S. Böttcher Designed by: Q Lecrevisse

94 Separation power of different types of BCLPD
CLL DLBCL CD10+ DLBCL CD10- FL HCL LPL MCL MZL BL DLBCL CD10 + DLBCL CD10 - Responsible scientist: S. Böttcher 2 SD separated 1 SD separated Overlap of 1st SD 1 x 1comparison n = 150

95 EuroFlow B-CLPD panel: summary
B-CLPD panel allows unequivocal classification of most mature B-cell malignancies according to WHO Most differential diagnoses achieved (n=32/36) efficiently except for the following 1 vs 1 comparisons: FL vs DLBC MZL vs LPL LPL vs DLBCL MZL vs DLBCL Responsible scientist: Sebastian Bottcher

96 Expert pathologist agreement with the consensus diagnosis
MZL LPL 5 expert hematopathologists ~1,400 lymphoma cases The NHL Classification Project, Bood 1997;89: Kindly provided by Raul Braylan

97 B-CLPD: Comparative analysis of “our case”
vs multiple reference groups Responsible scientists: Sebastian Bottcher Costa et al, Leukemia, 2010

98 B-CLPD:Comparative analysis of “our case” vs multiple reference groups
Responsible scientist: Sebastian Bottcher Costa et al, Leukemia, 2010

99 IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT
TYPES OF B-CLPD CD5 CD19 CD38 CD20 CD23 CD10 CD79b CD43 CD11c IGM CD103 CD22 B-CLL lo hi lo lo lo MCL HCL hi hi / /+ hi hi hi MZL lo /+ LPL lo /+ FL lo LDBCL /+ LDBCL /+ -/+ BL hi lo hi

100 IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT
TYPES OF B-CLPD CD200 CD31 CD305 CD81 CD95 CXCR5 CD49d CD62L CD39 CD27 B-CLL hi hi -/+ lo - MCL lo / HCL hi hi lo MZL lo LPL FL lo lo lo LDBCL lo LDBCL BL lo lo hi

101 UTILITY OF THE NEW SOFTWARE TOOLS
Standardization of FCM immunophenotyping How to get optimal and comparable measurements? Which are the most appropriate fluorochromes? What is the optimal sample preparation protocol? REPRODUCIBLE & OBJECTIVE RESULTS 101

102 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… IMM, Lisbon, PT P. Lucio, A. Mendonça, A. Parreira a.o… UNIKIEL, Kiel, DE M. Kneba, S. Böttcher, M. Ritgen, M. Brüggemann … AP-HP, Paris, FR E. Macintyre, L. Lhermitte, V. Asnafi … UNIVLEEDS, Leeds, GB S. Richards, A.C. Rawstron. P. Evans … DPH/O, Prague, CZ O. Hrusak, T. Kalina, E. Mesjstrikova … SAM, Zabrze, PL T. Szczepanski, L. Sedek … DCOG, The Hague, NL E. Sonneveld, A. van der Sluijs-Gelling ... KUL, Leuven, BE N. Boeckx … 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 … CYTOGNOS, Salamanca, ES M. Martin, J. Bensadon, J. Hernandez, M. Muñoz … S

103 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 Jacques van Dongen Vincent van der Velden Jeroen Te Marvelde Alita van der Sluijs Photo Lukasz Sedek Juan Flores-Montero Julia Almeida Quentin Lecrevisse 103

104 THANK YOU


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