AVANÇOS NO DIAGNÓSTICO E CLASSIFICAÇÃO IMUNOFENOTIPICA DE LEUCEMIAS LINFOCITICAS CRÓNICAS B

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1 AVANÇOS NO DIAGNÓSTICO E CLASSIFICAÇÃO IMUNOFENOTIPICA DE LEUCEMIAS LINFOCITICAS CRÓNICAS B CANCER RESEARCH CENTER IBSAL-UNIVERSITY OF SALAMANCA/CSIC HEMO 2016 Congreso Brasileiro de Hematologia, Hemoterapia y Terapia Celular Florianopolis, 12 de Novembro de 2016

2 FLOW CYTOMETRY DIAGNOSTICS IN HEMATO-ONCOLOGY 1. Making the diagnosis Normal reactive/regenerating malignant 2. Classification of hematopoietic malignancies - relation with prognosis - relevance of risk-group definition in treatment protocols 3. Disease staging (e.g. in case of NHL) 4. Identification of therapeutic targets (e.g. for antibody therapy) 5. Evaluation of treatment effectiveness Detection of minimal residual disease (MRD)

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

4 The EuroFlow comprehensive approach Clinical question High suspicion of acute leukemia e.g. blast cells observed Sustained monocytosis Unexplained cytopenia Unexplained Eosinophilia Atypical lymphocytes Splenomegaly Lymphocytosis LN enlargement Monoclonal component Monoclonal component non-igm, Bone lesions BM plasmacytosis High monoclonal component non-igm Suspicion of lymphoma localization in small cell number samples e.g. CS F, vitreous Screening tube Immunophenotyping Diagnostic panel To identify normal vs abnormal cells in a sample MRD Mantle cell lymphoma For sure (probability = 100%) Not for sure (probability <100%) To classify the abnormal cells into a disease category To design optimal Ab combinations To evaluate the selected Ab combinations

5 The EuroFlow comprehensive approach Clinical question High suspicion of acute leukemia e.g. blast cells observed Sustained monocytosis Unexplained cytopenia Unexplained Eosinophilia Atypical lymphocytes Splenomegaly Lymphocytosis LN enlargement Monoclonal component Monoclonal component non-igm, Bone lesions BM plasmacytosis High monoclonal component non-igm Suspicion of lymphoma localization in small cell number samples e.g. CS F, vitreous Screening tube ALOT LST reactive/polyclonal PCST first tube of PCD reactive/polyclonal SST clonal/aberrant other? B-CLPD clonal Diagnostic panel B-CLPD limited B-CLPD complete T-CLPD NK-CLPD PCD non- MCL FCL HCL other clonal B MRD Comprehensive network of panels aiming at the diagnosis and characterization of the major WHO entities

6 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 γδ CD3 CD38 Able to identify all the sample major populations: Non-hematopoietic cells T lymphocytes B lymphocytes (T-cell subpopulations) (B-cell light chain restriction) NK cells Plasma cells B-NHL panel backbone Responsible scientist: J. Flores Montero

7 GATING B-CELLS IN THE LST TUBE

8 GATING IN THE LST TUBE: 35 different cell populations x mean of 3 gates (105 gates)

9 GATING IN THE LST TUBE: 35 different cell populations x mean of 3 gates (105 gates)

10 GATING IN THE LST TUBE: 35 different cell populations x mean of 3 gates (105 gates)

11 GATING IN THE LST TUBE: 35 different cell populations x mean of 3 gates (105 gates)

12 GATING IN THE LST TUBE: 35 different cell populations x mean of 3 gates (105 gates)

13 GATING IN THE LST TUBE: 35 different cell populations x mean of 3 gates (105 gates)

14 GATING IN THE LST TUBE: 35 different cell populations x mean of 3 gates (105 gates)

15 GATING IN THE LST TUBE: 35 different cell populations x mean of 3 gates (105 gates)

16 Automated gating How does it work? Identifying the pathways that link individual events in an (N)- dimensional space A software tool similar to Compass based on a Reference Database Clustering phase Groups of events Classification phase Cell populations Responsible scientists: Rafael Fluxa, Juan Hernandez, Quentin Lecrevisse

17 LYMPHOCYTE SCREENING TUBE: DIAGNOSTIC SCREENING OF B-CLPD

18 LYMPHOCYTE SCREENING TUBE: DIAGNOSTIC SCREENING OF B-CLPD

19 LYMPHOCYTE SCREENING TUBE: DIAGNOSTIC SCREENING OF B-CLPD

20 LYMPHOCYTE SCREENING TUBE (LST): Automated gating and identification of tumor B-cells Diagnosis N. of Cases (%) % blasts identified median BCLPD 113/113 (100%) 100% OTHER 0/27 (0%) 0%

21 % of Tumor B-cells identified by the Expert PERCENT TUMOR B-CELLS BY MANUAL vs AUTOMATED GATING (n=113) % of Tumor B-cells identified by Automated software

22 The EuroFlow comprehensive approach Clinical question High suspicion of acute leukemia e.g. blast cells observed Sustained monocytosis Unexplained cytopenia Unexplained Eosinophilia Atypical lymphocytes Splenomegaly Lymphocytosis LN enlargement Monoclonal component Monoclonal component non-igm, Bone lesions BM plasmacytosis High monoclonal component non-igm Suspicion of lymphoma localization in small cell number samples e.g. CS F, vitreous Screening tube ALOT LST reactive/polyclonal PCST first tube of PCD reactive/polyclonal SST clonal/aberrant other? B-CLPD clonal Diagnostic panel B-CLPD limited B-CLPD complete T-CLPD NK-CLPD PCD non- MCL FCL HCL other clonal B MRD Comprehensive network of panels aiming at the diagnosis and characterization of the major WHO entities

23 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) & immunophenotyping strategy Panel evaluation Techniques Panel evaluation vs conventional in-use panels Panel optimization (re-design)

24 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 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

25 B-CLPD panel Backbone markers: Should identify all B cells Aberrant underexpression of CD19 and/or CD20 frequently observed sigκ/cd37/sigλ/cd19/cd22/cd20 tested in 69 B-NHL cases Conclusion: CD37 & CD22 redundant, as CD20 PacB plus CD19 PE-Cy7 were sufficient to identify all malignant B cells in all cases CD20 PacB R=0.46 (n=151) CD19 PECy7 Responsible scientist: Sebastian Bottcher

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

27 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, X HLA-DR, IgM, CD10, CD43, CD24, X CD27, CD38, CD39, CD63, X CD81, CD95, CD138). X Disease specific (e.g. CD5, CD23, CD25, X CD79b, CD103, CD200). Integrins and chemokine receptors (e.g. CD11a, X 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 univariate analysis X multivariate analysis Responsible scientist: S. Böttcher

28 Classification of BCLPD by the EuroFlow panel BL DLBCL FL HCL LPL MCL MZL N

29 Classification of BCLPD by the EuroFlow panel BL DLBCL FL HCL LPL MCL MZL N

30 MCL vs : PCA of total immunophenotype Principal component 2 MCL 1 SD 2 SD PC1 1 IgM CD CD79b CD CD Principal component 1 Responsible scientist: Sebastian Bottcher

31 MCL vs : PCA of total immunophenotype MCL MCL MCL PC1 1 IgM CD CD200 + IgM IgM + CD79b CD23 + IgM 3 CD79b CD MCL MCL MCL 5 CD CD23 + CD79b CD200 + CD79b CD200 + CD23 Responsible scientist: Sebastian Bottcher

32 Diagnostic classification of BCLPD: Individual differential diagnosis CD38 CD39 CD49d CD43 CD95 25% 16% 16% 15% 7%

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

34 LST + BCLPD classification panel Pac Blue Pac Orange FITC PE PerCP- Cy5.5 PECy7 APC APC-H7 1= LST CD20 /CD4 CD45 sigλ /CD8 sigk /CD56 CD5 CD19 /TCRγδ 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/LAI R1/CD11c/sIgM/CD81/CD103/CD95/CD22/CXCR5/CD49d/CD62L/CD39/HLA-DR/CD19/CD27 30-colors flow cytometry! Responsible scientist: Sebastian Bottcher

35 BCLPD classification panel: modular design Tubes 1 & 2 only MCL FL LPL MZL HCL BL CD10- DLBCL CD10+ DLBCL Tubes 1 (LST) and 2 only: resolve 100% of and 85% MCL cases Responsible scientist: Sebastian Bottcher

36 BCLPD classification panel: modular design Tubes 1 & 2 only MCL FL LPL MZL HCL BL CD10- DLBCL CD10+ DLBCL Tubes 1 (LST) and 2 only: resolve 100% of and 85% MCL cases Tubes 1 (LST) only: resolves 48% of and 21% MCL cases Responsible scientist: Sebastian Bottcher

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38 BCLPD classification panel: modular design

39 BCLPD classification panel: modular design

40 BCLPD classification panel: modular design

41 BCLPD classification panel: modular design

42 Summary: Advances in the diagnosis and classification of mature B-cell malignancies - The EuroFlow BCLPD panel consists of a total of 5 tubes containing information about 30 markers, useful for the diagnostic screening and classification of the major BCLPD diagnostic WHO2016 subtypes. - For an optimized efficiency the panel may be applied stepwise. Thus, with only the LST tube, around half cases and a significant percentage of mantle cell lymphoma cases may be unequivocally identified. - Tubes 1 & 2 are typically sufficient for the diagnosis of and the differential diagnosis between and MCL. - Similarly, the combination of tubes 1 & 3 are sufficient for the diagnosis of HCL. - Usage of different multivariate approaches is associated with variable levels of discrimination among the distinct diagnostic categories of BCLPD. - Despite all the above the differential diagnosis between a few entities still remains a challenge.

43 EuroFlow consortium aims at innovation in flow cytometry (

44 THE CIC/USAL-IBSAL TEAM

45 MUITO OBRIGADO