Clinical question. Screening tube. Diagnostic panel MRD. Clinical question

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1 OW CYTOMETRY UPDATES IN LYMPHOPROLIFERATIVE DISORDERS CANCER RESEARCH CENTER IBSAL UNIVERSITY & UNIVERSITY HOSPITAL, SALAMANCA (SPAIN) DISCLOSURES The EuroFlow Scientific Consortium Iamco-chairof receives royalties from patents I am co-inventor from Cytognos SL and Becton/Dickinson Biosciences. All co-inventors of these patents and their institutions, including myself have declined receiving any compensation or royalty ISLH 2016 Meeting Milano (Italy), May 12 th, 2016 OW CYTOMETRY DIAGNOSTICS IN LYMPHOPROLIFERATIVE DISORDERS The EuroFlow comprehensive approach 1. Making the diagnosis Normal reactive/regenerating malignant Clinical question Atypical lymphocytes Splenomegaly Lymphocytosis cytopenia LN enlargement Sustained monocytosis Eosinophilia non-igm, Bone lesions BM plasmacytosis High monoclonal non-igm Suspicion of lymphoma localization in small cell number samples e.g. CS F, vitreous 2. Classification of hematopoietic malignancies - relation with prognosis - relevance of risk-group definition in treatment protocols 3. Disease staging (e.g. in case of secondary CNS Lymphoma) Screening tube Diagnostic panel Immunophenotyping 4. Identification of therapeutic targets (e.g. for antibody therapy) 5. Evaluation of treatment effectiveness Detection of minimal residual disease (MRD) MRD Mantle cell lymphoma For sure (probability = 100%) Not for sure (probability <100%) Clinical question The EuroFlow comprehensive approach Sustained monocytosis cytopenia Eosinophilia Atypical lymphocytes Splenomegaly Lymphocytosis LN enlargement non-igm, Bone lesions BM plasmacytosis To design optimal Ab combinations To evaluate the selected Ab combinations High monoclonal non-igm Suspicion of lymphoma localization in small cell number samples e.g. CS F, vitreous The EuroFlow comprehensive approach Clinical question Screening tube High suspicion of acute leukemia e.g. blast cells observed ALOT Sustained monocytosis cytopenia Eosinophilia Atypical lymphocytes Splenomegaly Lymphocytosis LN enlargement LST reactive/polyclonal non-igm, Bone lesions BM plasmacytosis PCST first tube of PCD reactive/polyclonal High monoclonal non-igm Suspicion of lymphoma localization in small cell number samples e.g. CS F, vitreous SST Screening tube Diagnostic panel Immunophenotyping To identify normal vs abnormal cells in a sample To classify the abnormal cells into a disease category Diagnostic panel clonal/aberrant B-CLPD limited non- B-CLPD complete FCL other clonal B other? B-CLPD clonal T-CLPD NK-CLPD PCD MRD Mantle cell lymphoma For sure (probability = 100%) Not for sure (probability <100%) MRD Comprehensive network of panels aiming at the diagnosis and characterization of the major WHO entities 1

2 LST Lymphocytosis screening tube LST Lymphocytosis screening tube Atypical lymphocytes High suspicion of Splenomegaly acute leukemia e.g. blast cells observed Lymphocytosis cytopenia LN enlargement Sustained monocytosis Eosinophilia non-igm, Bone lesions BM plasmacytosis High monoclonal non-igm Suspicion of lymphoma localization in small cell number samples e.g. CS F, vitreous Blue CD4 Orange FITC Lambda CD8 PE Kappa CD56 PerCP Cy5.5 CD5 PE Cy7 APC APC H7 TCR CD3 CD38 ALOT LST PCST first tube of PCD SST Able to identify all the sample major populations: Non-hematopoietic cells Blue CD4 Orange FITC Lambda CD8 PE Kappa CD56 PerCP Cy5.5 CD5 PE Cy7 APC APC H7 TCR CD3 CD38 T lymphocytes (T-cell subpopulations) B lymphocytes (B-cell light chain restriction) NK cells Plasma cells B-NHL panel backbone Responsible scientist: J. Flores Montero Responsible scientist: J. Flores Montero GATING B-CELLS IN THE LST TUBE 2

3 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 3

4 Automated gating Classification phase LYMPHOCYTE SCREENING TUBE: DIAGNOSTIC SCREENING OF B-CLPD COMPASS Groups of events to be reclassified into cell populations Reference Database Output for the cluster: Events classified as cell populations Responsible scientists: Rafael Fluxa, Juan Hernandez, Quentin Lecrevisse LYMPHOCYTE SCREENING TUBE: DIAGNOSTIC SCREENING OF B-CLPD LYMPHOCYTE SCREENING TUBE: DIAGNOSTIC SCREENING OF B-CLPD 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% PERCENT TUMOR B-CELLS BY MANUAL vs AUTOMATED GATING (n=113) % of Tumor B-cells identified by Automated software % of Tumor B-cells identified by the Expert 4

5 The EuroFlow comprehensive approach PANEL OF ANTIBODIES FOR FCM ANALYSIS OF CSF SAMPLES FROM B-NHL Clinical question Screening tube Diagnostic panel High suspicion of acute leukemia e.g. blast cells observed ALOT Sustained monocytosis cytopenia Eosinophilia Atypical lymphocytes Splenomegaly Lymphocytosis LN enlargement LST reactive/polyclonal clonal/aberrant B-CLPD limited non- B-CLPD complete FCL non-igm, Bone lesions BM plasmacytosis PCST first tube of PCD reactive/polyclonal other? B-CLPD clonal T-CLPD NK-CLPD High monoclonal non-igm PCD Suspicion of lymphoma localization in small cell number samples e.g. CS F, vitreous SS T Blue CD4 Orange FITC Lambda CD8 PE Kappa CD56 PerCP Cy5.5 CD5 Improved identification of: PE Cy7 APC APC H7 TCR CD3 -Non-hematopoietic cells (e.g. CD38) -Hematopoietic cells: -Plasma cells (CD38++) -Monocytes (CD14+) -Dendritic cells (CD14-) CD38 other clonal B MRD Comprehensive network of panels aiming at the diagnosis and characterization of the major WHO entities Quijano et al, J Clin Oncol, 2009; Stacchini et al, Clin Cytometry 2012; van Dongen et al, Leukemia 2012 OW CYTOMETRIC ANALYSIS OF CSF SAMPLES FROM B-NHL Stabilised CSF samples (Transfix) Add PBS (4/mL) Centrifuge-wash (2x) and concentrate (150 L) Cell staining of 1/3 sample (50 L) Measure in the flow cytometer Restain (if negative) vs panel adapted to B-NHL phenotype Kraan et al, Current Protocols Cytometry, 2008 Cytology Flow Cytometry Cytology P Positive CSF 27/123 (22%) 7/123 (6%) < Susp. - Flow Cytometry *The presence of neoplastic cells was ruled out by further immunocytochemical analyses. Quijano et al, J Clin Oncol, /123 (77%) 17/123 (14%) 1/123 (1%)* 7/123 (6%) - 3/123 (2%) DIAGNOSIS OF LEPTOMENINGEAL DISEASE IN NHL: Cytology vs flow cytometry Schroers* Bommer Craig Stacchini Benevolo Muñiz Wilson Eur J Hematol Cancer Cytopathol J Clin Pathol Cytometry B Blood Blood Haematologica Am No cases No samples 37 70? N % Cytology+ 19% 29%*? 16% 4% 7% 5% % FCM+ 30% 28% 8% 24% 10% 22% 18% % of cells 100% 80% 60% 40% 20% 0% Cytology+/FCM+ Cytology /FCM+ * N. of cells/ L Cytology+/FCM+ Cytology /FCM+ * * 30/37 PCNSL 4% false-positive results by cytomorphology Kiewe et al, Neuro-Oncology, 2010: 11% (7/63) CC+ vs 3% (1/32) FCM+ or ICQ+ in PCNSL *Cut-off: <20% and <1 neoplastic B-cell/ L *p<0.001 Quijano et al, J Clin Oncol,

6 SPANISH SOCIETY OF HEMATOLOGY GUIDELINES FOR DIAGNOSIS OF LEPTOMENINGEAL DISEASE IN LYMPHOCYTE SCREENING TUBE: DIAGNOSTIC SCREENING OF B-CLPD - It is recommended that (standardized) flow cytometry immunophenotyping is incorporated to routine diagnosis of leptomeningeal disease in high-risk in combination with clinical presentation, imaging techniques and conventional cytology. Degree of evidence*: 1+ Recommendation*: A *Scotish Intercollegiate Guidelines Network (SIGN) LYMPHOCYTE SCREENING TUBE: DIAGNOSTIC SCREENING OF B-CLPD Compatible with B-cell chronic lymphocytic leukemia 1= LST R LST + BCLPD classification panel Blue /CD4 Orange FITC sig /CD8 CD23 CD31 CD103 CD62L PE sigk /CD56 CD10 LAIR CD95 CD39 PerCP- Cy5.5 CD5 CD79b CD11c CD22 HLA-DR PECy7 /TCR APC CD3 0 sigm CXCR5 CD27 APC-H7 CD38 CD43 CD81 CD49d /CD4//sIgl/sIgK/CD8/CD56/CD5//CD38/CD23/CD10/CD79b/0/CD43/CD31/LAI R1/CD11c/sIgM/CD81/CD103/CD95/CD22/CXCR5/CD49d/CD62L/CD39/HLA-DR//CD27 30-colors flow cytometry! CONSTRUCTION OF EUROOW LEUKEMIA/ LYMPHOMA IMMUNOPHENOTYPING ANTIBODY PANEL LST + BCLPD classification panel Medical indication Clinical request/need Proposed strategy Panel optimization (re-design) 1= LST 2 3 Blue /CD4 Orange FITC sig /CD8 CD23 CD31 PE sigk /CD56 CD10 LAIR PerCP- Cy5.5 CD5 CD79b CD11c PECy7 /TCR APC CD3 0 sigm APC-H7 CD38 CD43 CD81 Design of MAb panels (Medical indication-oriented) & immunophenotyping strategy 2-8 cycles Panel evaluation 4 5R CD103 CD62L CD95 CD39 CD22 HLA-DR CXCR5 CD27 CD49d /CD4//sIgl/sIgK/CD8/CD56/CD5//CD38/CD23/CD10/CD79b/0/CD43/CD31/LAI R1/CD11c/sIgM/CD81/CD103/CD95/CD22/CXCR5/CD49d/CD62L/CD39/HLA-DR//CD27 Techniques Panel evaluation vs conventional in-use panels Panel optimization (re-design) 30-colors flow cytometry! 6

7 B-CLPD panel Characterization markers Backbone markers: Should identify all B cells Aberrant underexpression of and/or frequently observed sigκ/cd37/sigλ//cd22/ tested in 69 B-NHL cases Conclusion: CD37 & CD22 redundant, as B plus PE-Cy7 were sufficient to identify all malignant B cells in all cases B PECy7 R=0.46 (n=151) Normal B lymphopoiesis CD10,, 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, 0, sig Panel construction characterization markers vs : PCA of total immunophenotype Tested markers (n=66): Backbone markers (e.g.,, CD22, CD37, ). 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, 0). 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 Principal 2 1 SD 2 SD PC1 1 IgM CD79b CD FINAL: 4 tube 8-color panel (20 antibodies) X univariate analysis X multivariate analysis Principal 1 Responsible scientist: S. Böttcher vs : PCA of total immunophenotype vs : 1 X 1 DIFFERENTIAL DIAGNOSIS vs CD10 - vs CD10 + PC1 1 IgM CD10 70% CD43 13% CD81 10% 8% 0 42% CD11c 39% CD95 19% CD62L 47% CD31 26% CD305 10% 8% CD95 6% CD81 2% 0 + IgM IgM + CD79b CD23 + IgM CD79b CD CD38 87% CD5 13% vs vs vs 0 100% CD5 92% 6% CD81 1% CD39 53% CD5 47% CD103 93% CD11c 7% CD10 99% CD38 1% vs MZL/ CD31 48% CD49d 44% HLADR 3% 3% CXCR5 3% CD23 + CD79b 0 + CD79b 0 + CD23 Responsible scientist: Sebastian Bottcher CD23 71% 3% CD305 44% CD31 28% CD43 26% CD10 2% CD305 74% CD22 15% 8% 4% CD11c 53% CD62L 19% 0 10% CD95 6% CD5 6% 6% 7

8 Separation power of different types of BCLPD CD10 + CD10 - CD10+ CD10- MZL MZL Expert pathologist agreement with the consensus diagnosis Responsible scientist: S. Böttcher 2 SD separated 1 SD separated Overlap of 1st SD 1 x 1comparison n = expert hematopathologists ~1,400 lymphoma cases The NHL Classification Project, Bood 1997;89: Kindly provided by Raul Braylan PCA of B-CLPD panel BCLPD classification panel: modular design vs. vs. vs. vs. CD10- CD10- Full panel MZL CD10+ vs. CD10+ vs. vs. vs. MZL vs.normal Responsible scientist: S. Böttcher Designed by: Q Lecrevisse BCLPD classification panel: modular design CD10- BCLPD classification panel: modular design Full panel Tubes 1 & 2 only MZL MZL Tubes 1 & 2 only CD10+ CD10- CD10+ CD10- MZL CD10+ Tubes 1 (LST) and 2 only: resolve 100% of and 85% cases 8

9 BCLPD classification panel: modular design BCLPD: Diagnostic classification of individual cases vs a reference data base Tubes 1 & 2 only MZL CD10- CD10+ Tubes 1 (LST) and 2 only: resolve 100% of and 85% cases Tubes 1 (LST) only: resolves 48% of and 21% cases BCLPD: Diagnostic classification of individual cases Summary: EuroFlow LST tube and BCLPD Panel for diagnosis and classification of mature B-cell malignancies Overview of registered recipients of EuroFlow protocols (Spring Spring 2016) 5,000 downloads from ~1,500 institutes - 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 diagnosticwho2008subtypes. - 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. - Similarly, the combination of tubes 1 & 3 are sufficient for the diagnosis of. - 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. = number of institutes per country March

10 THE CIC/USAL-IBSAL TEAM EuroFlow consortium aims at innovation in flow cytometry ( THANK YOU 10