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CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN)

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). DIAGNOSTICS IN HEMATO-ONCOLOGY. 1. Making the diagnosis

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CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN)

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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) Prepared by JJM van Dongen

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

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

  7. ALOT 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)

  8. 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)

  9. 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+)

  10. BCP-ALL BCP-ALL panel BCP-ALL

  11. BCP-ALL panel BCP-ALL

  12. BCP-ALL panel ALOT BCP-ALL

  13. BCP-ALL panel ALOT BCP-ALL

  14. BCP-ALL panel ALOT BCP-ALL

  15. BCP-ALL panel ALOT BCP-ALL Positive Diagnosis

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

  17. BCP-ALL panel ALOT BCP-ALL Maturation stage (EGIL)

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

  19. BCP-ALL panel ALOT BCP-ALL Prognosis markers

  20. BCP-ALL panel ALOT BCP-ALL LAP markers

  21. BCP-ALL panel ALOT BCP-ALL

  22. BCP-ALL panel 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

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

  24. PRECURSOR B-ALL: DNA ANEUPLOIDY NEOPLASTIC B-CELLS NORMAL B-CELLS 0 256 512 768 1024 RESIDUAL NON-B-CELLS FL2-Area -> RM30243.100 0 256 512 768 1024 FL2-Area -> RM30243.100 B-CELL PRECURSOR ALL: DNA ANEUPLOIDY

  25. 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+CD65+ 7.1+ • 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

  26. ADULT PRECURSOR B-ALL: IMMUNOPHENOTYPE OF BCR/ABL+ CASES Normal BM CD34+ B-cells CD45 CD19 CD19 CD38 10 0 10 1 10 2 10 3 10 4 0 1 2 3 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 10 10 10 10 CD10 CD13 CD34 CD34 CD13 PE -> CD34 PE -> CD34 -> CD10 -> UVJ39869.004 UVJ39869.002 MO21489004 AP36452.003 DNA diploid Bcr/abl+ ALL CD19 CD19 CD45 CD38 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 CD10 CD13 CD34 CD34 CD10 -> CD13 -> CD34 -> CD34 -> HG30672.002 HG30672.010 HG30672.008 HG30672.006 DNA aneuploid Bcr/abl+ ALL CD19 CD19 CD38 CD45 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 CD10 -> CD13 -> CD34 -> CD34 -> CD10 CD13 CD34 CD34 RFR7690.008 RFR7690.008 RFR7690.007 RFR7690.011

  27. 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

  28. BCR-ABL RUO testing by EuroFlowMFI values of the different cell samples (n=217) Weerkamp et al, Leukemia, 2009

  29. 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) - 19/19 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 • 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 CMLPrecursor-B-ALL: 88% (15/17) high level positivityCML: 84% (16/19) low level positivity (true low expressionor remaining protease activity?)

  30. 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

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

  33. 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

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

  35. 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%

  36. 100% K562 100% 697 R2 = green = BCR beads R3 = pink = E2A beads 10% 697/K562 10% K562/697 SIMULTANEOUS DETECTION OF THE BCR-ABL AND E2A-PBX FUSION PROTEINS

  37. 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)

  38. WHO: Mature B-cell Neoplasms

  39. B CELL CHRONIC LYMPHOPROLIFERATIVE DISORDERS Heterogeneous group of diseases typically characterized by a monoclonal expansion of a mature-appearing neoplastic B-lymphocyte WHO CLASSIFICATION OF B-CLPD 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

  40. DIAGNOSIS OF CLONAL HAEMATOLOGICAL DISORDERS Clinical symptoms Laboratory and signsfindings Morphology + cytochemistry Cytogenetics Immunophenotyping Molecular biology/FISH

  41. 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 -/+ - + + - - + - ++ -/+ -/+ - +

  42. MATUTES et al SCORE FOR B-CLL MARKER PATTERN SCORE 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) Matutes et al, Leukemia 1994

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

  44. Clinical question Screening tube Diagnostic panel MRD The EuroFlow comprehensive approach

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

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

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

  49. 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? “Diagnostic classification” panel 14 Major groups 154 Nosologic entities MRD monitoring

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