A TÉCNICA DO NGF (Next Generation Flow) PARA ESTUDO DE DRM

Transcription

1 A TÉCNICA DO NGF (Next Generation Flow) PARA ESTUDO DE DRM CANCER RESEARCH CENTER IBSAL-UNIVERSITY OF SALAMANCA/CSIC HEMO 2016 Congreso Brasileiro de Hematologia, Hemoterapia y Terapia Celular Florianopolis, 10 de Novembro de 2016

2 Conflict of Interest Disclosure I hereby declare the following potential conflicts of interest concerning my presentation: Membership on an Entity s Advisory Committee: Janssen, Celgene.

3 MRD MONITORING IN HAEMATOLOGICAL MALIGNANCIES In vivo drug kinetics Tumor microenvironment Treatment compliance Therapy Tumor cell features N. of tumor cells Resistance Sensitivity Complete remission Immunological CR Molecular CR Morphology, Cytogenetics Southern-Blot, Imaging FCM DNA aneuploidy F.I.S.H Flow cytometry P.C.R. - Response to therapy impacts on long-term patient outcome. - MRD improves prognostic stratification among patients in CR. - Flow cytometry is well suited for MRD assessment.

4 The MRD Marker Must Be Reliable Specificity: to discriminate malignant vs normal cells Sensitivity: to detect at least 1 malignant cell in a background of 10, ,000 normal cells. Applicability: to be applicable in virtually every patient Reproducibility: standardized methods Clinical utility: available resources & results obtained in a timely manner (around the world) The MRD Technique Must Be Feasible

5 Identification of leukemia-associated immunophenotypes TdT+ / CD19+ / CD38+ Tube 1 Set gate Precursor -B-ALL Analyse (2D) 4 Tube 2 CD15 / NG2- / CD19+ LAIP: CD45 - Tdt lo Analyse (2D) Set gate Tube 3 CyCD79+ / CyCD3- / MPO- Set gate Analyse (2D) JJM van Dongen Department of Immunology, Erasmus MC Lucio et al, Leukemia, 1999

6 Early Flow Cytometry MRD Approaches Investigate a large panel of potentially aberrant markers at diagnosis Identify the most informative (multiple) aberrant phenotypes Construct a patient-specific phenotypic probe Apply the patient-specific probe(s) for follow-up samples.

7 AML: RFS according to LAP+ cells in mcr after Induction Therapy 1 st BM in % 85% ±6% p = LAP+ cells at CR % ±8% < 10-4 (n=8) < 10-3 (n= 37) < 10-2 (n=64) % ±9% 10-2 (n=17) RFS y Months LAP: leukemia associated phenotypes in >75% AML cases San Miguel et al, Blood, 1997 & San Miguel et al, Blood, 2001

8 Early Flow Cytometry MRD Approaches Investigate a large panel of potentially aberrant markers at diagnosis Costly Identify the most informative (multiple) aberrant phenotypes Need for expertise Construct a patient-specific phenotypic probe Need for broad reagent availability Apply the patient-specific probe(s) for follow-up samples. Variable sensitivity and limited reproducibility

9 From: Szczepanski, Orfao et al, Lancet Oncol, 2001; 2: MRD TECHNIQUES FOR HAEMATOPOIETIC MALIGNANCIES Disease category FCM immunophenotyping (sensitivity) PCR/RT-PCR analyses (sensitivity) LAIP sigκ/sigλ Junctional Reg Chromosomal or TCRVβ Ig/TCR genes aberrations ( ) ( ) ( ) ( ) Precursor B-ALL Children 80-90% NA 95% 40-50% Adults 70-80% NA 90% 35-45% T-ALL Children >95% 30-35% >95% 10-25% Adults >95%? 90% 5-10% Chronic B-cell leukemias <5% >95% >95% 10-25% Chronic T-cell leukemias 5-10% 60-65% 95% <5% B-cell lymphomas <5% >95% 70-80% 25-30% T-cell lymphomas 20-25% 50-60% 95% 10-15% AML 70-90% NA 10% 10-30% CML NA NA NA >95%

10 MRD TECHNIQUES FOR HAEMATOPOIETIC MALIGNANCIES Disease category FCM immunophenotyping (sensitivity) PCR/RT-PCR analyses (sensitivity) LAIP sigκ/sigλ Junctional Reg Chromosomal or TCRVβ Ig/TCR genes aberrations ( ) ( ) ( ) ( ) Precursor B-ALL Children >90% NA 95% 40-50% Adults >95% NA 90% 35-45% T-ALL Children >95% 30-35% >95% 10-25% Adults >95%? 90% 5-10% Chronic B-cell leukemias >95% >95% >95% 10-25% Chronic T-cell leukemias 70-80% 60-65% 95% <5% B-cell lymphomas 90% >95% 70-80% 25-30% T-cell lymphomas 75-90% 50-60% 95% 10-15% Multiple myeloma >95% >90% 70-80% NT AML 70-90% NA 10% 30-40%* CML NA NA NA >95%

11 PROGNOSTIC IMPACT OF MRD IN CHILDHOOD ALL

12 Prognostic significance of MRD in CLL trials following current iwcll standard (10-4 ) Reference N Therapy Technique Moreno et al Blood 2006 Bosch et al CCR 2008 Kwok et al 2009 Böttcher et al JCO 2012 Fischer et al 2012 Pettitt et al JCO 2012 Bouvet et al Haematol 2013 Goede et al NEJM 2014 Santacruz et al Haematol 2014 Strati et al Blood 2014 MRD threshold PFS by MRD group 22 auto SCT 3/4-color flow m vs 75 m 44 FCM 4-color flow 10-4 MRD+ CR < MRD- CR (RD) p value < various 4-color flow m vs 91 m < FC/ FCR 4-color flow 45 BR 4-color flow 10-4 & and m 10-4 to < m < m m 10-4 to < m < m 0.2 < < Cam-HDMP 4-color flow m vs. 24 m FCR 4-color flow m vs. NR < Clb-G ASO RQ-PCR m vs. NR NR 96 various 3/4-color flow m vs. 76 m (TFS, CR only) < FCR 4-color flow 10-4 NR < TOTAL 1157 Slide prepared by S. Böttcher

13 CLL8 DCLLSG TRIAL: MRD at interim and final restaging MRD after 3 vs. 6 cycles (FCR) PFS Progression-Free Survival 10-2 < & < 10-2 FC, 18% FCR, 13% FC, 47% FCR, 24% FC, 35% FCR, 63% 10-2 < < 10-4 Low MRD < 10-4 Intermediate 10-4 & < 10-2 High MRD 10-2 Time (months) 3 cycles vs. 6 cycles (FCR) Böttcher S et al. J Clin Oncol 2012;30:

14 Immunophenotypic detection of MRD by multiparameter flow cytometry ADVANTAGES LIMITATIONS* - Highly applicable: >90% - Fast (<2h) - Relatively simple - Quantitative (% cells) - Sensitivity of < Heterogenous tissue (BM) infiltration - Quality of BM sample (PB dilution) - Major tumor cell compartment as the only target - No tumor specific antigens 1 - Fresh (<24h) samples required - Broadly available -Assessment of recovery of normal cell compartments* * Extramedullary relapses 1 Aberrant phenotypes

15 Immunophenotypic detection of MRD by multiparameter flow cytometry ADVANTAGES LIMITATIONS* - Highly applicable: >90% - Fast (<2h) - Relatively simple - Quantitative (% cells) - Sensitivity of < Broadly available -Assessment of recovery of normal cell compartments* - Heterogenous tissue (BM) infiltration - Quality of BM sample (PB dilution) - Major tumor cell compartment as the only target - No tumor specific antigens 1 - Fresh (<24h) samples required - Less sensitive than PCR (& NGS) - Lack of standardization * Extramedullary relapses 1 Aberrant phenotypes

16 CLL MRD: Precision of MRD by flow vs. ASOqPCR Comparative analysis in GCLLSG CLL8 trial FC FCR MRD flow r = 0.95 MRD flow r = < qr < qr ASO IGH RQ-PCR ASO IGH RQ-PCR n = 177 n = 353 Böttcher et al., Leukemia, 2009

17 MRD BONE MARROW LEVELS IN MM: FLOW CYTOMETRY vs ASO-PCR (n=102) MRD by flow cytometry MRD by flow cytometry MRD by ASO-PCR PCR - and/or FCM - (n=59) - PCR - FCM - 41 (69%) - PCR + FCM - 11 (19%) - PCR - FCM + 7 (12%) MRD by NGS NGS applicability: 91%; NGS sensitivity :<10-5 ** 2 cases excluded in the final publication Puig et al, Leukemia, 2013 Martinez-Lopez et al, Blood 2014

18 RQ-PCR and 4-color/6-color flow cytometric MRD in childhood ALL percentage of patients 100% 90% 80% 70% 60% 50% 40% 30% 20% negative <0.01% <0.05% % 0.1-1% >1% 10% 0% FCM-4 FCM-6 PCR FCM-4 FCM-6 PCR FCM-4 FCM-6 PCR d15 d33 d78 Denys and Van der Velden et al. Leukemia

19 MRD-based risk groups (day 33 and day 78) in 171 patients of the DCOG-ALL10 protocol RQ-PCR (Ig/TCR) based risk groups HR (6%) MR (62%) LR (30%) NA a FCMbased risk group HR (5%) 6 (4%) 2 (1%) 0 (0%) 0 (0%) MR (38%) 3 (2%) 60 (35%) 1 (1%) 0 (0%) LR (59%) 0 (0%) 44 (26%) 49 (29%) 6 (4%) a a Not applicable: Six patients could not be classified with molecular MRD analysis (no Ig/TCR marker with at least a quantitative range of 10-4 ). Denys and Van der Velden et al. Leukemia

20 Conventional 4-6 color flow MRD requires experience Rawstron et al, Leukemia, 2007 Rawstron et al., Leukemia, 2007

21 FLOW-MRD IN THE BONE MARROW OF MULTIPLE MYELOMA: current practice (Survey of 11/26 responders from 30 major institutions) Variables Median (Range) Number of events: 3-5 x 10 5 (1x10 5-4x10 6 ) No mpcs for MRD+: 25 (20-50)* Sensitivity (Max): 0.005% (0.01% %) No. of markers studied: 8 (6-12) sigk or sigλ: 73% CD19- CD45-: 55% CD19- CD45- CD56+: 55% CD27-/low: 27% Other: 45% *Pathologist dependent Flanders et al Blood 2013

22 Impact of MRD after HDT/ASCT on MM survival: Comparison between different induction regimens MRD value is independent of the induction regimen Spanish GEM2000 and GEM2005<65 Trials Paiva B et al. Blood ; abstr 1910 PFS P < GEM2000: MRD + (n=124) MRD - (n=171) GEM2005<65y:MRD + (n=121) MRD - (n=101) MRD OS MRD + MRD P < MRD - % MRD-negative GEM2000: 42% GEM2005: 45% UK MRC IX Trial Rawstron et al. Haematologica 2015 (published online: DOI ) MRD + MRD + MRD - MRD + MRD - % MRD-negative CTD: 71% CVAD: 54%

23 GEM 2000 vs MRC IX trials: Impact of BM MRD at month +3 after ASCT (n=295 vs 397) Progression-Free Survival % patients Progression-Free 100% 80% 60% 40% 20% 0% p<0.001 p< % 32% 22% 13% Median: 71m Median: 28.6m Median: 37m Median: 15.5m Months from ASCT GEM2000 trial (50% CR) MRD negative 42% (n=125) MRD positive 68% (n=170) MRC IX trial (54% CR) MRD negative 62% (n=247) MRD positive 38% (n=150) Paiva B et al; Blood. 2008, 112: Rawstron AC et al; J Clin Oncol 2013, 31:

24 CONVENTIONAL FLOW-MRD IN MM: CR and MRD positivity rates Series N. % CR % MRD - % CR but MRD+ Rawstron et al 45 73% 53% 27% Blood 2002 San Miguel et al 87 45% 26% (36% & 15%) 50% Blood 2002 Sarrasquete et al 24 58% 54% 37% Haematologica 2005 Paiva et al % 42% 36% Blood 2008 Paiva et al* % 24% 46% J Clin Oncol 2011 Paiva et al % 64% 36% Blood 2012 Rawstron et al % 62% 15% J Clin Oncol 2013 Roussel et al 31 58% 68% NR J Clin Oncol 2014 Paiva et al** % 46% 46% Haematologica 2015 Total 1,271 63% 49% 30% * Only patients >65 years treated with GEM2005; **Only relapsed patients who achieved CR were included

25 Standardized EuroFlow NGF-MRD Approaches Development of optimal antibody panel combinations Increased number of cells evaluated ( 10 7 ) Novel data analysis strategies

26 Characterization markers Normal B lymphopoiesis CD10, CD20, CD22 CD24, CD27, CD38 CD39, CD43, CD63 CD81, CD95, CD138 Bcl-2, HLA-DR, CyIg B cell homing CD11a, CD11c, CD31, CD49d, CD62L, CXCR5, CCR6, CD303 Known to differentiate CD13, CD15, CD28, CD33, CD56, CD45, CD117, β 2 M Informative markers

27 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)

28 EuroFlow-IMF NGF-MRD DESIGN 1. IDENTIFICATION OF MOST EFFICIENT MRD DISCRIMINATING MARKERS (n=94) cpc populations from MM patients (n= 63 merged MM patients BM samples) npc reference populations (n= 31 merged normal/reactive BM samples) N. Marker selected Times within top 8 1 CD19:PE Cy CD45:PB 57 3 CD56:PE 56 4 CD81:APC H CyIgλ:APC H CD27:PerCP Cy CD117:APC 38 8 CyIgκ:APC 36 9 B2micro:PerCP Cy CD38:FITC CD138:PO CD28:PE 19

29 EuroFlow-IMF NGF-MRD PANEL OPTIMIZATION OF THE TWO 8-COLOR MM MRD ANTIBODY COMBINATIONS Panel version Tube PerCP PacB PacO FITC PE PE Cy7 APC APC H7 Cy5.5 1 CD45 CD138 CD38 CD56 CD27 CD19 CD117 CD CD45 CD138 CD38 CyIgk CyIgl HV500-C 1 CD45 CD138 CD38 CD56 CD27 CD19 CD117 CD CD45 CD138 CD38 CD56 CD229 CD19 CyIgk CyIgl APC C750 1 CD45 CD138 CD38 CD56 CD27 CD19 CD117 CD CD45 CD138 CD38 CD56 CD229 CD19 CyIgk CyIgl HV450 1 CD138 CD27 CD38 CD56 CD45 CD19 CD117 CD CD138 CD27 CD38 CD56 CD45 CD19 CyIgk CyIgl BV421 BV510 1 CD138 CD27 CD38 (ME) CD56 CD45 CD19 CD117 CD CD138 CD27 CD38 (ME) CD56 CD45 CD19 CyIgk CyIgl ME: multiepitope Flores-Montero, submitted 2016

30 Treatment-independent monoclonal antibody reagents Daratumomab treated MM Isatuximab treated PCs

31 Minimal residual disease MM panel Version 5 final Panel Tube BV421 BV510 BV605 FITC PE 8- color PerCP Cy5.5 PE Cy7 APC APC A700 APC C750 1 CD117 CD81 CD138 CD27 CD38 CD56 CD45 CD19 2 CyIgκ CyIgλ 10- color 1 CD138 CD27 CD45 CD38 CD56 CyIgλ CD19 CD117 CyIgκ CD81 Target clone Conjugate Manufacturer Ref cat CD138 MI15 BV421 BD Biosciences CD27 O323 BV510 BioLegend CD45 HI30 BV605 BD Biosciences CD38 Multiclone FITC Cytognos CYT-38F CD56 C5.9 PE Cytognos CYT-56PE Anti-Lambda JDC-12 PerCPCy5.5 BD Biosciences CD19 J3-119 PECy7 Beckman Coulter IM3628 CD D2 APC BD Biosciences Anti-Kappa Polyclonal APC Dako C0222 Anti-Kappa G APCA700 BD Biosciences CD81 M38 APCC750 Cytognos CYT-81AC750 Anti-Lambda Polyclonal APCC750 Cytognos CYT-LAC750 (goat)

32 Minimal residual disease MM panel Version 5 (final) Panel Tube BV421 BV510 BV605 FITC PE 8- color PerCP Cy5.5 PE Cy7 APC APC A700 APC C750 1 CD117 CD81 CD138 CD27 CD38 CD56 CD45 CD19 2 CyIgκ CyIgλ 10- color 1 CD138 CD27 CD45 CD38 CD56 CyIgλ CD19 CD117 CyIgκ CD81 8-color MRD panel (2 tubes) 10-color MRD panel (1 tube) >Time consuming Increased (60%) reagent costs Better reagent performance Increased precision & QC Fast Lower reagent costs Suboptimal reagent performance Single measurement

33 Design of antibody panel phase 4-5: Final results PB PO FITC PE PerCPCy5.5 PE Cy7 APC APCC750 CD20 CD45 CD9 CD66c/ CD123 CD34 CD19 CD10 CD38 CD20 CD45 CD58 CD22 CD34 CD19 CD10 CD81 PB PO FITC PE PerCPCy5.5 PE Cy7 APC APCC750 CD20 CD45 CD81 CD20 CD45 CD81 CD66c/ CD123 CD73/ CD304 CD34 CD19 CD10 CD38 CD34 CD19 CD10 CD38 Currently (phase 5) being evaluated for applicability and sensitivity: - In parallel to RQ-PCR analysis (98% concordance in >300 MRD samples) - Using new EuroFlow bulk lysis protocol (aim: acquire > 5 million cells) - Switch from 8-color to color immunostainings possible

34 CLL MRD Panel: versions 5, 6, 7 TUBE BV421 BV510 FITC PE PerCP- Cy5.5 PECy7 APC APC- C750 v5-1 CD27 CD3 CD79b CD5 CD22 CD19 CD200 CD81 v5-2 CD27 CD3 CD79b CD5 CD200 CD19 ROR1 CD81 V5-3 CD5 CD3 CD79b ROR1 CD27 CD19 CD200 CD81 V5-3 performed very poorly TUBE BV421 BV510 FITC PE PerCP- Cy5.5 PECy7 APC APC- C750 v6-1 CD27 CD3 CD79b CD5 CD22 CD19 CD200 CD81 v6-2 CD27 CD3 CD79b CD5 CD200 CD19 ROR1 CD81 TUBE BV421 BV510 FITC PE PerCP- Cy5.5 PECy7 APC APC- C750 v7-1 CD27 CD3 CD79b CD5 CD22 CD19 CD200 CD81 v7-2 CD27 CD3 CD79b CD5 CD20 CD19 ROR1 CD81 Version 8 currently under construction Responsible scientists: responsible: S. BöttcherAW and Langerak, A.K. Langerak S Böttcher

35 Consensus ERIC recommendations for MRD panels in CLL (Rawstron et al, Leukemia, 2007, 2013 and 2015) ERIC 4-color antibody panel: APC PerCPCy5.5 FITC PE CD5 CD19 CD20 CD38 CD5 CD19 CD22 CD81 CD5 CD19 CD43 CD79b ERIC 6-color antibody panel: CD5 CD19 CD20 CD3 CD38 CD79b CD5 CD19 CD20 CD81 CD22 CD43 + CD3 ERIC 8-color antibody panel*: CD5 CD19 CD20 CD81 CD79b CD22 Two positions free - CD3 - CD38 - CD43 Full concordant with 4-color at 10-4 (linearity between 10-4 and 10-5 ) Easier to perform with less reagents and less sample

36 NEXT GENERATION FLOW (NGF): EuroFlow NGF-MRD Approaches Optimal antibody panel Optimal disease-specific antibody panel applicable to all patients with high sensitivity Bulk lyse sample processing Standardized sample processing systematic acquisition of 10 7 Novel data analysis strategies Infinicyt software (version RC6): PCA, file merge, automated gating Flores-Montero, submitted 2016

37 Highly sensitive (<10-5 ) MM-MRD sample preparation protocol: Bulk lyse -Add lysing buffer to 0.3 to 3mL of sample containing >10 x Incubate >10 x 10 6 cells in 100µL for 10 min (max volume 250 µl) New Sample Preparation SOP -Wash with 2 ml PBS (1x) -Stain with antibody mixture -Add 2mL FACSLyse & incubate for 15 min -Wash with PBS (2x) -Measure >5 x 10 6 cells/tube in the FCM

38 NGF vs 2 nd GENERATION FLOW-MRD IN MM: NUMBER OF CELLS EVALUATED Total events acquired n = x10 7 1x x10 7 0x nd generation flow EuroFlow-IMF NGF Total events acquired 2 nd generation flow 1 x 10 6 (range: 0,03-6 x 10 6 ) EuroFlow-IMF NGF-MRD 10.4 x 10 6 (range: x 10 6 )

39 Next Generation Flow MRD vs Conventional 2 nd Generation (8-color) Flow-MRD (n=110) > LOQ Sensitivity of routine flow (conventional 8-color flow MRD) Neg < LOQ Neg Sensitivity of Next Generation Flow MRD * 2 samples proven polyclonal by CyIg staining LOQ, limit of quantitation

40 Next Generation Flow MRD vs Conventional 2 nd Generation (8-color) Flow-MRD (n=110) > LOQ Sensitivity of routine flow (conventional 8-color flow MRD) nd generation flow < LOQ MRD-negative cases by the routine approach Next Generation Flow-MRD /73 (75%) 18/73 (25%) Neg Neg Sensitivity of Next Generation Flow MRD * 2 samples proven polyclonal by CyIg staining LOQ, limit of quantitation

41 NEXT GENERATION FLOW (NGF) versus CONVENTIONAL FLOW MRD: Cases with discrepant MRD results Case ID MM Status Conventional 8-color FCM NGF (Tube 1 + Tube 2) % tpc % npc % apc # apc % tpc % npcs % apc # apc (Tube 1+ 2) 1 CR CR CR CR scr CR CR ,866 8 scr CR CR VGPR CR scr scr CR , VGPR CR CR CR CR CR %, percentage; #, number of dots; t, total; n, normal; a, abnormal; PC, plasma cells

42 NEXT GENERATION FLOW (NGF) MM MRD: Cell populations identified in normal vs MM BM N. of cells evaluated/bm sample >5-8 x 10 6 >8-11 x 10 6 >11x 10 6 Total MRD+ 3/13 (23%) 13/23 (56%) 17/29 (60%) 33/65 (51%) MRD- 10/13 (77%) 10/23 (44%) 12/29 (40%) 32/65 (49%)

43 MRD POSITIVITY RATES ACCORDING TO THE SENSITIVITY OF NEXT GENERATION FLOW (NGF): (GEM2012MENOS65 TRIAL) Induction (6 cycles) HDT/ASCT Consolidation MRD-negative 24% 57% 50% MRD-positive 76% 43% 50% MRD-positive % 19% 0% MRD-positive % 28% 33% MRD-positive % 43% 50% Quantitative MRD required!!! Slide prepared by B. Paiva

44 BCP-ALL: IMPACT OF THE NUMBER OF CELLS EVALUATED IN THE SENSITIVITY OF FLOW-MRD Theunissen et al. Blood 2016 (manuscript under review)

45 NEXT GENERATION FLOW (NGF): EuroFlow NGF-MRD Approaches Optimal antibody panel Optimal disease-specific antibody panel applicable to all patients with high sensitivity Bulk lyse sample processing Standardized sample processing systematic acquisition of 10 7 Novel data analysis strategies Infinicyt software (version RC6): PCA, file merge, automated gating Flores-Montero, submitted 2016

46 Automated gating/identification of MRD cells 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

47 Raw FCS files Automated gating

48 Automated gating Raw FCS files Data based matched cell populations

49 Automated gating: gated plasma cells Manual analysis: npc: 0.13% (CyIgk: 0.6% / CyIgλ: 0.5%) apc: 14.04%

50 Automated Gating vs Manual (expert-based) Data Analysis (n=110) > LOQ Sensitivity of Automated gating Neg < LOQ LOQ, limit of quantitation Neg Sensitivity of Expert-based gating TIME OF DATA ANALYSIS (automated vs expert-based analysis): 8h 52m vs. 19h 39m (4m 50s vs. 10m 43s/case)

51 BCP-ALL: MRD DETECTION BY NGF IN BM

52 Novel multi-color high-throughput flow cytometry for MRD detection in BCP-ALL MRD follow-up at Day +33

53 Novel multi-color high-throughput flow cytometry for MRD detection in BCP-ALL 0.08% MRD MRD follow-up at Day +33 Residual BCP-ALL cells & mature B-cells

54 Novel multi-color high-throughput flow cytometry for MRD detection in BCP-ALL 0.08% MRD MRD follow-up at Day +33 Residual BCP-ALL cells & mature B-cells MRD follow-up at Day +79 Regenerating precursor B-cells

55 Novel multi-color high-throughput flow cytometry for MRD detection in BCP-ALL 0.08% MRD MRD follow-up at Day +33 Residual BCP-ALL cells & mature B-cells MRD follow-up at Day +79 Suspected % MRD Regenerating precursor B-cells

56 Novel multicolor high-throughput NGF for MRD detection in BCP-ALL A Day +15 Day +33 D G Day +78

57 Novel multicolor high-throughput NGF for MRD detection in BCP-ALL A B C Day % Day +33 D E F 0.005% G H I Day +78 <0.0005%

58 NEXT GENERATION FLOW (NGF): EuroFlow-IMF NGF-MRD MM Approach Standardized sample processing systematic acquisition of 10 7 Specific identification of: -Myeloma clonal plasma cells at MRD levels -Other major and minor residual cell subsets Flores-Montero, submitted 2016

59 MM NGF-MRD: Evaluation of the quality of the BM sample (PB contamination) Myeloma PCs Normal PCs Mast cells Myeloid precursors NRBCs B-cell Precursors I B-cell Precursors II Immature B lymphocytes Naive B cells Memory B cells

60 NEXT GENERATION FLOW (NGF): Identified BM cell populations -Myeloma plasma cells -Other major and minor residual cell subsets: -Neutrophils -Eosinophils -Basophils -Dendritic cells -Monocytes -Mast cells -Mesenchymal stem cells -Nucleated red cells -CD117+ myeloid precursors -B-cells: - B-cell precursors (CD27+ and CD27-) - Naive lymphocytes - Memory B-cells - Normal residual PCs: CD19+ (k vs λ) CD19- CD56- (k vs. λ) CD19- CD56+ (k vs. λ)

61 NEXT GENERATION FLOW (NGF): Identified BM cell populations -Myeloma plasma cells -Other major and minor residual cell subsets: -Neutrophils -Eosinophils -Basophils -Dendritic cells -Monocytes -Mast cells -Mesenchymal stem cells -Nucleated red cells -CD117+ myeloid precursors -B-cells: - B-cell precursors (CD27+ and CD27-) - Naive lymphocytes - Memory B-cells - Normal residual PCs: CD19+ (k vs λ) CD19- CD56- (k vs. λ) CD19- CD56+ (k vs. λ)

62 NEXT GENERATION FLOW (NGF): Cell populations identified in normal BM Cell population Normal BM % cutoff MRD+/MRD- samples with counts below Normal BM (n=65) Mast cells <0.002% 5%/12% NRBC <2% 3%/3% CD19 - npc <0.003% 14%/14% CD19 - /CD19 + npc ratio <0.08% 6%/9% CD27 + B-cell precursors <0.004% 14%/14% CD27 - B-cell precursors <0.05% 3%/19% CD27 + /CD27 - B-cell precursor ratio <0.04% 5%/6% Mature B-cells <0.6% 28%/20% B-cell precursor/mature B-cell ratio <0.03% 8%/8% Myeloid precursors <0.2% 8%/6%

63 NEXT GENERATION FLOW: Impact of MRD on Progression-Free Survival (n=79) Patients in VGPR, CR/sCR NGF status NGF and 2 nd generation flow status Progression-free survival (%) NGF- (n=37), 75% PFS: NR* NGF+ (n=42), 75% PFS: 10 months P= NGF- (n=37), 75% PFS: NR* NGF+/2 nd Generation Flow+ (n=26), 75% PFS: 12 months NGF+/2 nd Generation Flow- (n=16), 75% PFS: 10 months P= Time from MRD assessment (months) Relapsed patients in this group corresponded to 1 sample with low acquired cellularity (<5x10 6 ) PB contamination and a second one also showing clear contamination with PB

64 NEXT GENERATION FLOW (NGF): Cell populations identified in normal BM Cell population % Normal BM cells (range) Case 1 Case 2 Mast cells 0.006% ( ) 0.002% % Erythroblasts 6.4% (2-11.5) 8.01% 5.1% CD19 - npc 0.05% ( ) 0.004% 0.004% CD19 - /CD19 + npc ratio 0.6% ( ) 0.3% 0.08% CD27 + B-cell precursors 0.08% ( ) % 0.003% CD27 - B-cell precursors 0.4% ( ) 0.001% 0.01% CD27 + /CD27 - B-cell precursor ratio 0.2% (0.04-1) 0.09% 0.3% Mature B-cells 1.6% ( ) 0.2% 0.1% B-cell precursor/mature B-cell ratio 0.2% ( ) 0.02% 0.1% Myeloid precursors 1.8% ( ) 0.3% 0.3%

65 Advantages & disadvantages of high-throughput NGF MRD method Next Generation Flow cytometry (NGF) (EuroFlow 8-12 colors) Advantages Fast (within 3-4 h) Highly standardized with automated gating (e.g. Infinicyt) Efficient data storage / management with easy data comparison and review. Accurate quantitation Increased sensitivity ( ) Information on normal and malignant cells (sample quality and reconstitution of normal compartments) External and internal QC in a per sample basis Ready for IVD development Limitations - Education and training still required - Many cells needed to reach the required sensitivity, e.g. 5.0 x 10 6, if quantitation down to <10-5 is needed - Fresh (<24h) samples Adapted from van Dongen JJM, van der Velden VHJ, Bruggemann M & Orfao A, Blood 2015

66 DEVELOPMENT OF 8-COLOR EUROFLOW MRD ANTIBODY PANELS Single-tube antibody EuroFlow MRD protocols under evaluation 1. Acute leukemias (include recognition of normal precursors) Acute promyelocytic leukemia panel (APL-MRD): 1 tube (A.Orfao) Acute myeloid leukemia panel (AML-MRD): 1-3 tubes (A.Orfao) B-cell precursor (BCP-ALL-MRD): 1-2 tubes (V. van der Velden, E. Mejstrikova) T-cell ALL (T-ALL-MRD): 1-2 tubes (L. Lhermitte) 2. Chronic lymphoproliferative disorders Chronic lymphocytic leukemia (CLL-MRD): 1-2 tubes (S. Böttcher A. Langerak) Hairy cell leukemia (HCL-MRD): 1 tube (L. Lhermitte) Mantle cell lymphoma (MCL-MRD): 1 tube (S. Böttcher) Follicular lymphoma (FL-MRD): 1 tube (S. Böttcher) Marginal zone lymphoma (MZL-MRD): 1 tube (J. Caetano) Lymphoplasmacytic lymphoma (LPL-MRD): 1 tube (J. Caetano) Diffuse large B-cell lymphoma (DLBCL-MRD): 1 tube (J. Caetano) Burkitt lymphoma (BL): 1 tube (L. Lhermitte) T-chronic lymphoproliferative diseases (T-CLPD-MRD): 1 tube (J. Almeida) Multiple myeloma (MM): 1-2 tubes (J. Flores-Montero)

67 AKNOWLEDGEMENTS EuroFlow is a working group of EHA (European Hematology Association) Euroflow is an independent scientific consortium, which aims at innovation in flow cytometry for improvement of diagnostic patient care

68 MUITO OBRIGADO

69 ALL: CELL FREQUENCIES IN BM VS PB

70 NEXT GENERATION FLOW (NGF): Cell populations identified in normal BM Cell population % in Normal BM (range) Mast cells 0.006% ( ) NRBC 6.4% (2-11.5) CD19 - npc 0.05% ( ) Ratio CD19 - /CD19 + npc 0.6% ( ) CD27 + B-cell precursors 0.08% ( ) CD27 - B-cell precursors 0.4% ( ) Ratio CD27 + /CD27 - B-cell precursors 0.2% (0.04-1) Mature B-cells 1.6% ( ) Ratio B-cell precursors / mature B-cells 0.2% ( ) Myeloid precursors 1.8% ( )