IMMUNOPHENOTYPING OF HAEMATOLOGICAL MALIGNANCIES

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1 UTILIDAD DE LA CITOETRIA DE FLUJO EN EL DIAGNÓSTICO, CLASIFICACIÓN Y ONITORIZACIÓN DE HEOPATÍAS ALIGNAS - 953/994: From the development of the instruments & techniques to the WHO classification of haematological malignancies /2006: The ability to specifically identify leukaemic cells: from normal phenotypes to aberrant phenotypic profiles. CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAANCA (SPAIN) Curso Avanzado de Actualización en Oncohematología por citometria de flujo Buenos Aires, de mayo de /-: Recent contributions of immunophenotyping of haematological malignancies: pointing to the future : The Coulter principle and instrument development - 965/68: ultiparameter and multicolour flow cytometry - 953: The Coulter principle and instrument development - 965/68: ultiparameter and multicolour flow cytometry - 970: FACS: fluorescence activated cell sorter. arv van Dilla H. Crissman Wallace Coulter Wofgang Göhde Joe Gray 970 L. Herzenberg & FACS II (976) - 953: The Coulter principle and instrument development - 965/68: ultiparameter and multicolour flow cytometry - 970: FACS: fluorescence activated cell sorter. Fluorescence activated cell sorting Len & Lee Herzenberg Prepared by A.Salvador

2 WHERE CAN I APPLY FLOW CYTOETRY? - 953: The Coulter principle and instrument development - 965/68: ultiparameter and multicolour flow cytometry - 970: FACS: fluorescence activated cell sorter : Production of monoclonal antibodies Kohler G, ilstein C. Continuous cultures of fused cells secreting antibody of pre-defined specificity. Nature 975;256: César ILSTEIN Gunter VALET - 953: The Coulter principle and instrument development - 965/68: ultiparameter and multicolour flow cytometry - 970: FACS: fluorescence activated cell sorter : Production of monoclonal antibodies - 978/80: Immunophenotyping of leukemia cells Immunophenotypic classification of ALL - 98: Definition of aberrant marker expression - 985: Use of Immunophenotyping to classify FAB 7 Tabla 2.- Immunological classification of ALL Phenotype B-ALL Ig+ T-ALL SER+ non-t non-b ALL calla+ calla- CyCD ntdt - 953: The Coulter principle and instrument development - 965/68: ultiparameter and multicolour flow cytometry - 970: FACS: fluorescence activated cell sorter : Production of monoclonal antibodies - 978/80: Immunophenotyping of leukemia cells David ASON Immunophenotypic classification of ALL - 98: Definition of aberrant marker expression - 985: Use of Immunophenotyping to classify FAB 7-985: APAAP technique - 953: The Coulter principle and instrument development - 965/68: ultiparameter and multicolour flow cytometry - 970: FACS: fluorescence activated cell sorter : Production of monoclonal antibodies - 978/80: Immunophenotyping of leukemia cells Immunophenotypic classification of ALL - 98: Definition of aberrant marker expression - 985: Use of Immunophenotyping to classify FAB 7-985: APAAP technique - 986: Benchtop 3-color flow cytometers - 987: Immunophenotypic analysis of normal hematopoiesis Loken et al, Blood, 987 Loken et al, Blood, : The Coulter principle and instrument development - 965/68: ultiparameter and multicolour flow cytometry - 970: FACS: fluorescence activated cell sorter : Production of monoclonal antibodies - 978/80: Immunophenotyping of leukemia cells Immunophenotypic classification of ALL - 98: Definition of aberrant marker expression - 985: Use of Immunophenotyping to classify FAB 7-985: APAAP technique - 986: Benchtop 3-color flow cytometers - 987: Immunophenotypic analysis of normal hematopoiesis - 988: Second IC classification of haematological malignancies - 993: CD45-based blast cell gating/identification - 994: REAL classification of lymphoid neoplasias - 997: WHO classification of lymphoid neoplasias 2

3 CD8 PerCP FC IUNOPHENOTYPING IN THE 80`S: PANELS OF REAGENTS AND TECHNIQUES PANELS OF REAGENTS: - Panels of relevant markers for the diagnostic classification of patients suspected of: DIAGNOSIS OF Clinical symptoms and signs Laboratory findings orphology + cytochemistry TECHNIQUES: - AL, ALL - B-CLPD, T-CLPDT CD DS G Immunophenotyping RD - Isolation of NC - Indirect and direct IF - Single stainings - Difficult to distinguish normal/leukemic leukemic cells IF Few fluorochrome conjugated Ab available Few fluorochrome available Acute leukemias Chronic lymphoid leukemias CLINICAL UTILITY OF IUNOPHENOTYPING OF IUNOPHENOTYPIC CLASSIFICATION OF ACUTE LEUKAEIAS - Acute leukaemias: - Lineage assignment (myeloid vs lymphoid -B or T-) - Diagnosis of biphenotypic leukaemias/mixed lymphoid/myeloid - Phenotypic classification of B-cell precursor ALL - Phenotypic classification of T-ALL - Lineage subclassification of AL (e.g: AL with monocytic maturation) - Chronic lymphoproliferative disorders: - Diagnosis of T & B-cell clonality - Phenotypic classification of T/NK-CLPD - Phenotypic classification of B-cell CLPD EGIL: DEFINITION OF BAL Score B-Lineage T-Lineage yeloid lineage 2 ccd79a c/mcd3 PO cig TCR Lisozyme ccd22 CD2, CD5 CD3, CD33 CD20 CD8, CD0 CD7, CDw Tdt, CD24 Tdt, CD7 CD4, CD5 CDa CD64 Criteria: > 2 points IUNOPHENOTYPIC PATTERNS OF DIFFERENT TYPES OF B-CLPD B (Orfao et al, In: B-CLL.Humana Press, 2004) sig CD5 CD0 CD20 CDc CD23 CD24 CD25 CD38 CD43 CD79b CD03 FC7 TRADITIONAL FC PHENOTYPING B-CLL d + - d -/ /+ + d - - PLL + -/ /+ -/+ + -/+ -/+ -/ HCL / Blood NC SZL + -/ / /+ + Ficoll NC LPL / /+ CL / / /+ FL /+ -/d + -/ TRANSFORED SSC NC 5% LDBCL /+ - -/ BL -/ /+ -/ FSC-Height -> FSC Height CD3 FITC -> EGV CD3 APC CD4 APC -> CD4 APC EGV Criteria for positivity: : >2 3

4 CD3 APC CD8 PerCP ULTICOLOR FLOW CYTOETRY vs SINGLE-STAININGS IN ONE TUBE CD9 PerCPCY5.5 -> CD9 PerCP/Cy CD4 APC APC -> EGV : The Coulter principle and instrument development - 965/68: ultiparameter and multicolour flow cytometry - 970: FACS: fluorescence activated cell sorter : Production of monoclonal antibodies - 978/80: Immunophenotyping of leukemia cells Immunophenotypic classification of ALL - 98: Definition of aberrant marker expression - 985: Use of Immunophenotyping to classify FAB 7-985: APAAP technique - 986: Benchtop 3-color flow cytometers - 987: Immunophenotypic analysis of normal hematopoiesis - 988: Second IC classification of haematological malignancies - 993: CD45-based blast cell gating/identification - 994: REAL classification of lymphoid neoplasias - 997: WHO classification of lymphoid neoplasias - 953/994: From the development of the instruments & techniques to the WHO classification of haematological malignancies /2006: The ability to specifically identify leukaemic cells: from normal phenotypes to aberrant phenotypic profiles /-: Recent contributions of immunophenotyping of haematological malignancies: pointing to the future. FLOW CYTOETRY: TYPE OF INFORATION - Identification of cell populations - Enumeration of cell numbers - Characterization of cell populations Identification of different granulocytic subpopulations in childhood B IUNOPHENOTYPIC IDENTIFICATION OF LINEAGE COITENT OF CD34 + B CELLS yelo/monoblast Promyelocyte yelocyte etamyelocyte Neutrophill Neutrophil precursors staining 5 CD6/CD3/ CD45/CDb SSC FSC SSC CD6-FITC SSC CD3-PE TRANSFORED SSC a CD45 PERCP b b CyPO PE c CDb-APC CD3-PE CD3-PE CD34 APC TRANSFORED SSC ntdt FITC CD6-FITC CDb-APC CD6-FITC E.G. van Lochem et al., Cytometry Part B 2004; 60B: -3. B-cell precursors atarraz S et al. Leukemia

5 ONOCLONAL GAOPATHIES: IDENTIFICATION OF CLONAL PLASA CELLS DIAGNOSIS OF TRANSFORED SSC -> T-SSC CD38-PerCP/Cy5.5 CD38 PerCP/Cy5 -> CD38 FITC -> CD38-FITC CD38 FITC -> Normal PC CD9-PcpCy5 C D 9 CD56-PE C D 5 6 P CD38-FITC gated PC E C D 4 5 A P C CD45-APC Clonal PC Perez-Andres, J Biol Reg, 2004 Clinical symptoms and signs Cytogenetics Laboratory findings orphology + cytochemistry olecular biology/fish Immunophenotyping Immunophenotypic identification of PB B-cells with a CLL-like phenotype Normal B cells Clonal B cells *0.35% of all B-cells & O.O3% of all leucocytes Nieto et al, Blood 2009 INIAL RESIDUAL DISEASE IN B-CLL Brugiatelli et al. (Cancer 989) Robertson LE et al. (Blood 992) Leonormand B et al. (Leukemia 994) Cabezudo E et al. (Leukemia, 997) García-Vela A et al. (Leukemia, 999) Rawstron AC et al. (Blood 200) aloum K et al. (Br J Haematol 2002) Gupta R et al. (Am J Clin Pathol 2004) Bottcher S et al. (Leukemia 2004) oreton P et al. (J Clin Oncol 2005) ontillo et al. (Cancer Invest 2005) Aberrant criteria sigκ + /sigλ + ratio sigκ + /sigλ + ratio CD9 + /CD5 + CD9 + /CD5 + CD9 + /CD79b +d CD9 + /CD20 +d /CD5 + /CD79b +d CD9 + /CD20 +d /CD5 + /CD79b +d CD9 + /CD5 + CD9 + /CD5 + /CD43 + /CD20 +d CD9 + /CD20 +d /CD5 + /CD79b +d CD9 + /CD20 +d /CD5 + /CD79b +d Sensitivity Prognostic value Not analyzed Not analyzed Neoplastic cells Sensitivity ETHODS FOR RD INVESTIGATION P.C.R. F.I.S.H Flow cytometry orphology, Cytogenetics, Southern-Blot Blot, FC DNA aneuploidy FC IUNOPHENOTYPING IN THE 90`S: PANELS OF REAGENTS AND TECHNIQUES PANELS OF REAGENTS: - Panels of informative combinations of reagents for: - AL, ALL, BAL -, W, GUS - B-CLPD, T-CLPDT - DS TECHNIQUES: - Non-NRBC NRBC lysis - Direct IF - ultiple stainings Diagnosis & follow-up of RD in acute leukaemias, CLPD & - Distinct normal vs leukemic phenotypes any fluorochrome conjugated Ab available Increased number of fluorochrome available 5

6 Diagnostics in hemato-oncology. aking the diagnosis Normal reactive/regenerating malignant Annually > 0,000 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 (RD): RD-based risk-group stratification (treatment reduction or treatment intensification) Annually > 400,000 follow-up samples in leukemia patients (ALL, AL, CL) JJ van Dongen What problems are we experiencing? - any reagents: costly and complex - Need expertise in normal (& reference) cell populations - Time consuming - Technical limitations - any (my) strategies to reach a similar result but suboptimal - Not standardized: reproducibly harmonized? - Partial and more limited clinical utility than expected STANDARDIZATION EFFORTS FOR IUNOPHENOTYPIC STUDIES - CLSI (Clinical Laboratory Standards Institute): - Stetler-Stevenson Stevenson et al.: Clinical flow cytometric analysis of neoplastic hematolymphoid cells; Approved guideline.. CLSI document H43-A2. CLSI, CCS (Clinical Cytometry Society): - Davis et al: 2006 Bethesda International Consensus recommendations on the flow cytometric immunophenotypic analysis of hematolymphoid neoplasias. Clin Cytometry,, 72B, ESCCA (European Society for Clinical Cell Analysis: - European Leukemia Net ( - Consenso Latinoamericano (Clin Clin Cytometry,, 998 y 2006) Standardization in diagnostic flow cytometry Standardization according to literature generally refers to: lists of CD codes and markers per disease category rarely a specific antibody is recommended and (almost) never a fluorochrome is proposed HOWEVER: Standardization according to GLP guidelines demands for much higher levels of standardization EuroFlow standardization aims at: usage of comparable flow cytometers (3 lasers and 8 colors) full standardization of instrument settings (e.g. based on standard beads) standardized laboratory protocols and immunostaining procedures (SOP s) careful selection of optimal antibody clones per marker/cd code selection of optimal 8-color antibody combinations and fluorochromes design of combinations of multiple 8-color tubes: estimation and APS view new software for fast and easy data analysis with automated pattern recognition recognition of normal and abnormal leukocyte subsets (complete differentiation pathways) with the same immunostaining protocols mapping of new patient samples against large data base of earlier collected patient samples, analyzed with the same immunostaining protocol icroscopy 70s-90s CLINICAL APPLICATIONS OF FLOW CYTOETRY Hybridoma technology onoclonal antibodies Fluorochrome-conjugates Flow cytometry From research laboratories to clinical diagnostics XXI century Digital instruments >4 color flow cytometers Higher analytical speed Exponentially growing amount of complex information/data - 953/994: From the development of the instruments & techniques to the WHO classification of haematological malignancies /2006: The ability to specifically identify leukaemic cells: from normal phenotypes to aberrant phenotypic profiles /-: Recent contributions of immunophenotyping of haematological malignancies: pointing to the future. 6

7 GUEST EDITORIAL: The Continuing Evolution of Hematology BEREND HOUWEN Laboratory Hematology 8: Carden Jennings Publishing Co., Ltd. EXCERPT: The evolution of laboratory hematology is a continual process, as the papers in the following Special Section demonstrate. These papers cover a variety of topics related to the practice of laboratory hematology. Application of extended differential capabilities decreases the need for routine microscopic intervention for samples with nucleated red blood cells. Similarly, as careful examination of flagging performance shows, increased productivity THANK can be achieved by fewer morphology reviews. In most laboratories, rules for review of hematology results are applied by the technologists. any times these rules are quite informal, but when formalized YOU and embedded in a computer-based algorithm, they can form a powerful management tool, as illustrated in one of the papers. RECENT CONTRIBUTIONS OF IUNO- PHENOTYPING IN THE DIAGNOSIS OF - Standardization of immunophenotypic analyses - Diagnosis of clonal multilineage disease - Rapid screening of relevant cytogenetic subgroups of acute leukaemias - Ontogenic characterization of CLPD CONSTRUCTION OF EUROFLOW LEUKEIA/ LYPHOA IUNOPHENOTYPING ANTIBODY PANEL THE EUROFLOW APPROACH TO LEUKEIA/LYPHOA IUNOPHENOTYPING Clinical request/need Proposed strategy Clinical question Experience Knowledge Evaluation edical indication Design of Ab panels (edical indication-oriented) & immunophenotyping strategy Techniques Panel evaluation vs conventional in-use panels Panel optimization (re-design) 2-8 cycles Panel evaluation Panel optimization (re-design) Diagnostic screening tube Diagnostic classification panel RD monitoring 4 ajor groups 54 Nosologic entities Reference profiles ajority of diseases? ajority of cases? New disease entities? CONSTRUCTION OF EUROFLOW PANELS: EDICAL INDICATION ORIENTATION/SCREENING & CLASSIFICATION PANELS Synchronized light scatter experiments Local settings EuroFlow settings 7 different normal PB samples acquired in 7 different centers Van Dongen et al: EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Normal PB samples processed according to the standardized EuroFlow sample preparation protocol 7

8 BCLPD panel: classification of an atypical case vs the reference WHO diagnostic groups B-CLPD: Comparative analysis of our case vs multiple reference groups Responsible scientists: Sebastian Bottcher Costa et al, Leukemia, 200 Responsible scientist: Sebastian Bottcher B-CLPD:Comparative analysis of our case vs multiple reference groups REFERENCE DATAFILES: NORAL vs. CLL B-CELLS Normal PB CD9+ B-cells CD9-PECy7 CD9-PECy7 CD9+ CLL B-cells Case number CD9-PECy7 Case number Responsible scientist: Sebastian Bottcher Costa et al, Leukemia, 200 COPARE A CASE VS NORAL & CLL B-CELLS CD9-PECy7 CD9+ CLL B-cells Normal PB (n=8) CLL cases (n=6) CD9-PECy7 CD9-PECy7 Normal PB (n=8) CLL cases (n=6) Normal PB B cells LAIR 9.3 CD5 8.6 CD79b 8.2 Ig 8. RECENT CONTRIBUTIONS OF IUNOPHENOTYPING IN THE DIAGNOSIS OF - Standardization of immunophenotypic analyses SSC SSC Case number CLL cases Case number - Diagnosis of clonal multilineage disease - Rapid screening of relevant cytogenetic subgroups of acute leukaemias - Ontogenic characterization of CLPD CD9 PECy7 CLL cases Normal PB B cells CLL Normal PB B-cells Case number 8

9 PURIFICATION OF B AST CELLS IN ASTOCYTOSIS KIT UTATION IN S: PATTERN OF CELLULAR INVOLVEENT AST CELLS Case ast Cells ONOCYTES Eos Case 2 Neut ast Cells CD34+ HPC CD34+ HPC o NRC NRC Eos Neut CD34+ HPC o Lymphs NEUTROPHILS Lymphs KIT mutation EOSINOPHILS LYPHOCYTES FREQUENCY OF ULTILINEAL KIT UTATION IN ASTOCYTOSIS (n=202) S: PROGNOSTIC FACTORS FOR PROGRESSIONPROGRESSION-FREE SURVIVAL % OF CASES VARIABLE Only C UNIVARIATE ULTIVARIATE ANALYSIS ANALYSIS C & CD34+ HPC yeloid lineages RR (IC 95%) yeloid & lymphoid lineages p-value RR (IC 95%) p-value Age > (.2-50) 0.03 NS Cytopenias 6.6 (.5-79) 0.02 NS High β2-microglobulin.65 (.2-2.3) Germinal KIT mutation 8.0 (.3-49) (.2-2.9) (.4-24) 0.02 SUBTYPE OF ASTOCYTOSIS Escribano et al, J Allergy Clin Immunol, 2009 Teodosio et al, J Alllergy Clin Immunol 200 Frequency of CLLCLL-like BL cells in healthy individuals RECENT CONTRIBUTIONS OF IUNOPHENOTYPING IN THE DIAGNOSIS OF BL: 80/639 (2.5%) Standardization of immunophenotypic analyses 2.5% (session V) (session I) - Rapid screening of relevant cytogenetic subgroups of acute leukaemias (session III) 75 age (years) - Diagnosis of clonal multilineage disease 7.8% % Ontogenic characterization of CLPD (Session III) 5.2% % Percentage of cases 9

10 CLL (7) Clinic (ayo) (2,3) Clinic (Leeds) (8) Population (Leeds) (8) Population (Salamanca) (6)* CLL vs CLL-LIKE BL: Genetic features of clonal B-lymphocytes edian CLL count >5,000/uL q4 deletion 54% (78/238) 44% (56/26) 58% (9/33) 39% (5/38) 36% (6/45) Trisomy 2 6% (53/325) 8% (2/26) 2% (7/33) 8% (4/22) 8% (4/45) q deletion 8% (58/235) 2% (2/26) 6% (2/33) (0/2) (0/45) 7p deletion 7% (23/325) 3% (4/26) 3% (/33) (0/0) (0/45) Rawstron et al, Cytometry B, 200 (in press) Source CLL (25) Clinic (Leeds) (8) Clinic (ayo) (2,3) Familial (Duke) (24) Population (Leeds) (8) Population (Italy) (4) Population (Salamanca) (6) CLL vs CLL-LIKE BL: Biological features of clonal B-lymphocytes edian CLL cell count >5, CLL-like B- cells (median %) >95% >95% >95% 25% 8 7% 0.4% Cases with <98% IGHV homology 534/927 (57.6%) 8/20 (9) 84/09 (77%) 2/6 (75%) 8/20 (9) 36/5 (7) 2/7 (29%) Predominant CLL cell IGHV gene 3-07, -69, 4-34, , 3-23, , -69, 4-34, , , 3-23, /6 No CLLassociated Similar to CLL? - No No Rawstron et al, Cytometry B, 200 (in press) FREQUENCY OF CLL-like BL IN HEALTHY ADULTS FREQUENCY OF CLL-like BL IN HEALTHY ADULTS DETECTED FREQUENCY DETECTED FREQUENCY CALCULATED FREQUENCY 0 Whole series Whole series % of cases with a CLL-like clone years 46% 32% 8% years 88% 62% 36% years % of cases with a CLL-like clone years 46% 32% 8% years 88% 62% 36% years PB Volume (ml) PB Volume (ml) Immunophenotypic identification of CLL-like clonal B- cells in healthy adults Volume of PB analyzed: ml FREQUENCY OF CLL-LIKE LIKE BL IN HEALTHY ADULTS: ANALYSIS OF 50 ml OF PB Number of positive cases: 8/9 - Frequency: < 0. - Count: < 0.2 CLL-like B-cells/µL % OF WBC % FRO B- ABSOLUTE CASE ID AGE GENDER sig ONOCLONAL (x0-3 ) CELLS COUNT CASE 78 L+d onoclonal 0,8% 0,04% 0,066 cel/ul CASE 2 77 F L+d Biclonal 0,28% 0,4% 0,002% 0,0 0,008 cel/ul 0,0009 cel/ul Volume of PB analyzed: 50mL CASE 3 72 F L+d Biclonal 0,% 0,08% 0,008% 0,006% 0,009 cel/ul 0,007 cel/ul CASE 4 87 ND CASE 5 73 onoclonal 0,73% 0,05% 0,036 cel/ul CASE 6 77 onoclonal,3% 0,08% 0,2 cel/ul CASE 7 82 F L+d Biclonal 0,8% 0,095% 0,02% 0, 0,009 cel/ul 0,004 cel/ul CASE 8 88 onoclonal 0,4% 0,03% 0,009 cel/ul % of CLL-like cells: 0.02% of the whole B-cell population CASE 9 73 L+d Biclonal 0,49% 0,27% 0,06% 0,009% 0,032 cel/ul 0,08 cel/ul 0

11 AST CELL UNIT, HOSPITAL VIRGEN del VALLE TOLEDO CIC/UNIVERSITY OF SALAANCA L Escribano I Alvarez Twose L Sanchez uñoz I Sánchez S atas A atito EuroFlow consortium aims at innovation in flow cytometry