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CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor Immunophenotyping: Standardization and Applications Multicolor.

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Presentation on theme: "CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor Immunophenotyping: Standardization and Applications Multicolor."— Presentation transcript:

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

2 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) DIAGNOSTICS IN HEMATO-ONCOLOGY Prepared by JJM van Dongen

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

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)

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6 ALOT BCP-ALLT-ALLAML/MDS 4 tubes 4 to 7 tubes 1 tube

7 ALOT: B-cell precursor ALL 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) AL OT

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

11 BCP-ALL panel 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 Positive Diagnosis ALOT BCP-ALL

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

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

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

20 BCP-ALL panel Prognosis markers ALOT BCP-ALL

21 BCP-ALL panel LAP markers ALOT BCP-ALL

22 BCP-ALL panel ALOT BCP-ALL

23 BCP-ALL panel 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 ALOT BCP-ALL

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

25 B-CELL PRECURSOR ALL: DNA ANEUPLOIDY

26 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 IMMUNOPHENOTYPE OF NEOPLASTIC B-CELL PRECURSORS

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

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

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

30 1.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) -19/19 CML were BCR-ABL positive in both assays (with borderline positivity in one CML case; MFI of 144) -0/48 of other (acute) leukemias were BCR-ABL positive 2.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 < 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?) Results concerning BCR-ABL RUO kit Weerkamp et al, Leukemia, 2009

31 Immunobead flow cytometry with BD™ CBA Flex beads BCR-ABL t(9;22) fusion protein: Specificity Black: 697 (t(1;19), neg. control) Blue: TOM-1, BCR-ABL + (p190) Green: LAMA-84 BCR-ABL + (p210) Purple: AR230, BCR-ABL + (p230) 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: 1.Not dependent of the breakpoint position in the fusion gene; 2.No need for special laboratory facilities other then a routine flow cytometer; 3.Providing results within several hours; 4.The possibility to run in parallel to routine immunophenotyping: no extra technician time needed !!; 5.Allowing multiplexing with differently-labeled beads, that can detect different fusion proteins within the same disease category

33 PANELS OF IMMUNOBEADS FOR THE CLASSIFICATION OF ACUTE LEUKAEMIAS Precursor-B-ALL AML ‘MLL’ T-ALL BCR-ABLPML-RARA MLL-AF4CALM-AF10 TEL-AML1AML1-ETO MLL-AF9LMO2 E2A-PBX1CBFB-MYH11 MLL-AF10HOX11L2 ( MLL-AF4) MLL-ENLTAL1 MLL-AF6

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

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

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

37 100% K562100% % K562/69710% 697/K562 R2 = green = BCR beads R3 = pink = E2A beads SIMULTANEOUS DETECTION OF THE BCR-ABL AND E2A-PBX FUSION PROTEINS

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

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 symptomsLaboratory and signs findings Morphology + cytochemistry Cytogenetics Immunophenotyping Molecular biology/FISH

42 IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT TYPES OF B-CLPD ( 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 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 MARKER PATTERN SCORE CD5 positive 1 CD5 positive 1 CD23 positive 1 CD22 dim 1 sIg dim 1 FMC7 negative 1 (CD79b) dim 1 Diagnosis of CLL requires a score > 3 (4) 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 Diagnostic panel MRD

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

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

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

49 Clinical question Screening tube Diagnostic panel Diagnostic panel MRD The EuroFlow comprehensive approach Monoclonal component Monoclonal component non-IgM, Bone lesions BM plasmacytosis ALOTLS T PCST first tube of PCD SST BCP-ALLLT-ALL AML/MDS B-CLPD limited T-CLPDNK-CLPD Sustained monocytosis Unexplained Eosinophilia reactive/polyclonal other ? B-CLPD CLL non-CLL CLL MCL FCL HCL other clonal B reactive aberrant ab + reactive aberrant NK cells various subtypes ofBCP-ALL MDS PNH CML CML-BC other MPD High suspicion of acute leukemia e.g. blast cells observed Unexplained cytopenia Atypical lymphocytes Splenomegaly Lymphocytosis LN enlargement clonal reactive/polyclonal clonal/aberrant first 4 tubes PCD various subtypes of PCD High monoclonal component non-IgM Suspicion of lymphoma localization in “small cell number” samples e.g. CSF, vitreous first 4 tubes various subtypes ofT-ALL various subtypes ofAML B-CLPD complete aberrant gd +

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

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

52 Panel construction Selection of: Fluorochromes Antibodies Fluorochrome + antibody combinations 8-color combinations Panels

53 Selection of: Fluorochromes Antibodies Fluorochrome + antibody combinations 8-color combinations Panels  Reagent stability  Brightness  Low fluorescence background levels  No spectral overlap between fluorochrome emissions Panel construction

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

55 Panel construction – Antibodies Selection of: Fluorochromes Antibodies Fluorochrome + antibody combinations 8-color combinations Panels

56 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 … Panel construction – Antibodies

57 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 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 Panel construction – Antibodies

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

59 Panel construction Selection of: Fluorochromes Antibodies Fluorochrome + antibody combinations 8-color combinations Panels

60 Panel Pac Blue Pac Orange FITCPE PerCP Cy5.5 PE Cy7 APC APC H7 BCP-ALLCD45CD34CD19 T-ALLcyCD3CD45CD3 AML/MDS HLADR CD45CD34CD117 B-CLPDCD20CD45CD19 T-CLPDCD4CD45CD3CD8 NK-CLPDCD45CD3CD56CD19 PCDCD45CD138CD38CD19 Panel construction – Fluorochrome + antibody BB combinations

61 Monoclonal component Monoclonal component non-IgM, Bone lesions BM plasmacytosis ALOTLST PCST first tube of PCD SST Sustained monocytosis Unexplained Eosinophilia High suspicion of acute leukemia e.g. blast cells observed Unexplained cytopenia Atypical lymphocytes Splenomegaly Lymphocytosis LN enlargement High monoclonal component non-IgM Suspicion of lymphoma localization in “small cell number” samples e.g. CSF, vitreous Pac Blue Pac Orange FITCPE PerCP Cy5.5 PE Cy7APCAPC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR  CD3CD38 LST – Lymphocytosis screening tube Responsible scientist: J. Flores Montero

62 Pac Blue Pac Orange FITCPE PerCP Cy5.5 PE Cy7APCAPC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR  δ CD3CD38 Responsible scientist: J. Flores Montero LST – Lymphocytosis screening tube

63 Pac Blue Pac Orange FITCPE PerCP Cy5.5 PE Cy7APCAPC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR  δ CD3 CD38 Responsible scientist: J. Flores Montero Able to identify all the sample major populations: Non-hematopoietic cells LST – Lymphocytosis screening tube

64 Pac Blue Pac Orange FITCPE PerCP Cy5.5 PE Cy7APCAPC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR  δ CD3 CD38 Responsible scientist: J. Flores Montero Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes LST – Lymphocytosis screening tube

65 Pac Blue Pac Orange FITCPE PerCP Cy5.5 PE Cy7APCAPC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR  δ CD3CD38 Responsible scientist: J. Flores Montero Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes B lymphocytes LST – Lymphocytosis screening tube

66 Pac Blue Pac Orange FITCPE PerCP Cy5.5 PE Cy7APCAPC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR  δ CD3 CD38 Responsible scientist: J. Flores Montero Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes B lymphocytes NK cells LST – Lymphocytosis screening tube

67 Pac Blue Pac Orange FITCPE PerCP Cy5.5 PE Cy7APCAPC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR  δ CD3 CD38 Responsible scientist: J. Flores Montero Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes B lymphocytes NK cells Plasma cells LST – Lymphocytosis screening tube

68 Pac Blue Pac Orange FITCPE PerCP Cy5.5 PE Cy7APCAPC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR  δ CD3 CD38 Responsible scientist: J. Flores Montero Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes B lymphocytes NK cells Plasma cells (T-cell subpopulations) LST – Lymphocytosis screening tube

69 Pac Blue Pac Orange FITCPE PerCP Cy5.5 PE Cy7APCAPC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR  δ CD3 CD38 Responsible scientist: J. Flores Montero Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes B lymphocytes NK cells Plasma cells (T-cell subpopulations) (B-cell subsets & Ig light chain restriction) LST – Lymphocytosis screening tube

70 Pac Blue Pac Orange FITCPE PerCP Cy5.5 PE Cy7APCAPC H7 CD20 CD4 CD45 Lambda CD8 Kappa CD56 CD5 CD19 TCR  δ CD3CD38 Responsible scientist: J. Flores Montero Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes B lymphocytes NK cells Plasma cells (T-cell subpopulations) (B-cell light chain restriction) B-NHL panel backbone LST – Lymphocytosis screening tube

71 Panel construction Selection of: Fluorochromes Antibodies Fluorochrome + antibody combinations 8-color combinations Panels

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

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

74 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 – univariate analysis X – multivariate analysis X X X Panel construction – characterization markers Responsible scientist: S. Böttcher

75 LST + BCLPD classification panel CD49dCXCR5CD19CD22CD95CD103CD45CD204 CD27CD19HLA-DRCD39CD62LCD45CD205R CD81sIgMCD19CD11cLAIRCD31CD45CD203 CD43CD200CD19CD79bCD10CD23CD45CD202 CD38CD3CD19 /TCR  CD5sIgK /CD56 sIg /CD8 CD45CD20 /CD4 1= LST APC-H7APCPECy7PerCP- Cy5.5 PEFITCPac Orange Pac Blue CD20/CD4/CD45/sIgl/sIgK/CD8/CD56/CD5/CD19/CD38/CD23/CD10/CD79b/CD200/CD43/CD31/LAI R1/CD11c/sIgM/CD81/CD103/CD95/CD22/CXCR5/CD49d/CD62L/CD39/HLA-DR/CD19/CD27 30-colors flow cytometry ! Responsible scientist: Sebastian Bottcher

76 CD20/CD4/CD45/sIgl/sIgK/CD8/CD56/CD5/CD19/CD38/CD23/CD10/CD79b/CD200/CD43/CD31/LAI R1/CD11c/sIgM/CD81/CD103/CD95/CD22/CXCR5/CD49d/CD62L/CD39/HLA-DR/CD19/CD27 30-colors flow cytometry ! LST + BCLPD classification panel CD49dCXCR5CD19CD22CD95CD103CD45CD204 CD27CD19HLA-DRCD39CD62LCD45CD205R CD81sIgMCD19CD11cLAIRCD31CD45CD203 CD43CD200CD19CD79bCD10CD23CD45CD202 CD38CD3CD19 /TCR  CD5sIgK /CD56 sIg /CD8 CD45CD20 /CD4 1= LST APC-H7APCPECy7PerCP- Cy5.5 PEFITCPac Orange Pac Blue Responsible scientist: Sebastian Bottcher

77 B-CLPD: 4-COLOUR STAINING PANEL - FITCPE PerCP/Cy5.5 APC - ControlControlCD19 Control - sIgksIg CD19 CD5 - CD22 CD23 CD19 CD20 - CD22 CD23 CD19 CD20 - Fmc7CD24CD19 CD34 - CD43CD79bCD19 CD49d - CyBcl2CD10CD19 CD38 - CD103CD25 CD19 CD11c - sIgMCD27CD19 CCR6 - CD3CyZap70CD19 CD5

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

79 Characterization markers: CD200 Responsible scientist: Sebastian Bottcher MZLMCLHCLLPLFLCLLDLBCLBL

80 Characterization markers: LAIR1(CD305) MZLMCLHCLLPLFLCLLDLBCLBL Responsible scientist: Sebastian Bottcher

81 Characterization marker: CD5 MZLMCL HCL LPL FL CLL DLBCL BL CLL immunophenotypic diagnosis CD10+CD10- Best 5 markers for the DD between CLL and MCL according to EuroFlow analysis Responsible scientist: S. Böttcher

82 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 MCLCLL Principal component 1 → Principal component 2 → 1 SD 2 SD Responsible scientist: Sebastian Bottcher

84 PCA of total immunophenotype MCLCLL Principal component 1 → Principal component 2 → 1 SD 2 SD Responsible scientist: Sebastian Bottcher PC1 1IgM CD CD79b CD CD …

85 MZLMCL HCL LPL FLCLL DLBCL BL CD10+ CD10- Best 5 markers for the DD between CLL and MCL according to EuroFlow analysis Responsible scientist: S. Böttcher Characterization marker: smIgM CLL immunophenotypic diagnosis

86 MZL MCL HCLLPL FL CLL DLBCL BL CD10+ CD10- Best 5 markers for the DD between CLL and MCL according to EuroFlow analysis Responsible scientist: S. Böttcher Characterization marker: CD200 CLL immunophenotypic diagnosis

87 Major consecutive development steps in the design of the EuroFlow B-CPLD panel VersionTubeFluorescence channel PacBPacOFITCPEPECy5PECy7APCAPCCy7 Backbone 11SmIg  CD37SmIg CD19CD22CD20 Backbone 21CD20 SmIg  CD37SmIg CD19CD22 1CD20CD45Ig  Ig PerCPCy5.5CD19IgMAF700 CD5CD22 2CD20CD45CD103CD10CD5CD19CD43CD22 Panel 13CD20CD45CD81CD79bCD5CD19CD23CD22 4CD20CD45CD31CD63CD5CD19CXCR5CD22 5CD20CD45CD24LAIR1CD5CD19CD11aCD22 6CD20CD45CD38CD25CD138CD19CD11cCD22 1CD20CD45Ig Ig  CD22CD19CD23APCH7 CD81 Panel 22CD20CD45CD103CD25CD11cCD19IgMCD24 3CD20CD45CD31LAIR1CD5CD19CD43CXCR5 4CD20CD45bcl2CD10CD79bCD19CD38CD49b 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) VersionTubeFluorescence channel PacBPacOFITCPEPECy5PECy7APCAPCCy7 1CD20CD45Ig Ig  CD22CD19CD23CD81 2CD20CD45CD103CD25CD11cCD19IgM Panel 33CD20CD45CD31LAIR1CD5CD19CD43 4CD20CD45bcl2CD10CD79bCD19CD38CD49d 5CD20CD45CD24CD95CD19CD200 1CD20CD45Ig Ig  CD22CD19CD23CD81 2CD20CD45CD103CD25CD11cCD19IgMCD43 Panel 43CD20CD45CD31LAIR1CD5CD19CD43CD24 4CD20CD45bcl2CD10CD79bCD19CD83CD49d 5CD20CD45CD24CD95CD19CD200CD31 6CD20CD45CD19CXCR5CD103 1=LSTCD20CD45CD8 +CD56 +CD5CD19 +CD3CD38 Ig Ig  TCR  2CD20CD45CD23CD10CD79bCD19CD200CD43 Panel 53CD20CD45CD31LAIRCD11cCD19IgMCD81 4CD20CD45CD103CD95CD22CD19CXCR5CD49d 5CD20CD45CD62LCD39HLADRCD19CD27

89 B-CLPD: Diagnostic work-flow Monoclonal component ALOTLST B-CLPD limited B-CLPD broad Eosinophilia reactive/ non-aberrant CLL non-CLL 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 CLLMCL CLLMCL Full panel Tubes 1 & 2 only BCLPD classification panel: modular design CLL HCL CLLHCL CLLMZL CLLMZL CLL FL CLL FL CLLDLBCL CLLDLBCL Responsible scientist: Sebastian Bottcher

91 MZLHCLMCL CLLFLCLL PCA of total immunophenotype: clearly separated diseases Responsible scientist: S. Böttcher

92 PC1 1CD CD IgM8.48 4CD CD … PC1 1IgM CD CD79b CD CD … MCLCLL CD10+ DLBCL BL 2 SD separated 1 SD separated Responsible scientist: Sebastian Bottcher PCA of total immunophenotype FL CD10- DLBCL Overlap of 1 st SD

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

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

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 The NHL Classification Project, Bood 1997;89: Kindly provided by Raul Braylan MZL LPL 5 expert hematopathologists ~1,400 lymphoma cases

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

102 EuroFlow participants University Institutes / Medical Schools Erasmus MC, Rotterdam, NLJ.J.M. van Dongen, V.H.J. van der Velden… USAL, Salamanca, ESA. Orfao, J. Flores, J. Almeida, Q. Lecrevisse… IMM, Lisbon, PTP. Lucio, A. Mendonça, A. Parreira a.o… UNIKIEL, Kiel, DEM. Kneba, S. Böttcher, M. Ritgen, M. Brüggemann … AP-HP, Paris, FRE. Macintyre, L. Lhermitte, V. Asnafi … UNIVLEEDS, Leeds, GBS. Richards, A.C. Rawstron. P. Evans … DPH/O, Prague, CZO. Hrusak, T. Kalina, E. Mesjstrikova … SAM, Zabrze, PL T. Szczepanski, L. Sedek … DCOG, The Hague, NLE. Sonneveld, A. van der Sluijs-Gelling... KUL, Leuven, BEN. Boeckx … HGSA, Porto, PTM. Lima, AH Santos UFRJ, Rio de Janeiro, BRC. Pedreira, E.S. Costa Companies (SME’s) DYNOMICS, Rotterdam, NLE. Dekking, F. Weerkamp … CYTOGNOS, Salamanca, ESM. Martin, J. Bensadon, J. Hernandez, M. Muñoz …

103 Contributors - Acknowledgements Elizabeth MacIntyre Vahid Asnafi Ludovic Lhermitte Juan Flores-Montero Julia Almeida Quentin Lecrevisse Jacques van Dongen Vincent van der Velden Jeroen Te Marvelde Alita van der Sluijs Michael Kneba Monika Brüggemann Sebastian Böttcher Stephen Richards Paul Evans Matt Cullens Ruth de Tute Andy Rawstron Tomek Szczepanski Lukasz Sedek Paulo Lucio Andreia Mendonça Evan Jensen Ondrej Hrusak Tomas Kalina Ester Mejstrikova Photo Lukasz Sedek

104 THANK YOU


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