Congreso Nacional del Laboratorio Clínico 2016

Transcription

1 Técnicas de alta sensibilidad en la detección de enfermedad mínima residual en Mieloma Múltiple J. Flores-Montero, MD, PhD Departamento de Medicina, Servicio Central de Citometría, Centro de Investigación del Cáncer, Universidad de Salamanca EuroFlow Consortium X Congreso Nacional del Laboratorio Clínico, Zaragoza, Octubre 2016

2 Quality of response to treatment and progression in MM PR, partial response; VGPR, very good partial response; CR, complete remission; MRD, minimal residual disease; M-c: monoclonal component Paiva et al, Blood 2015

3 Quality of response to treatment and progression in MM Immunofixation (M-c), %BM- PC, sflc ratio or absence of cpc IHC/IMF, Imaging PR, partial response; VGPR, very good partial response; CR, complete remission; MRD, minimal residual disease; M-c: monoclonal component Paiva et al, Blood 2015

4 Quality of response to treatment and progression in MM Impact of treatment response in elderly patients PFS OS Total series (age 54-89) Patients 75 y.o. Guy et al, Blood 2011

5 Quality of response to treatment and progression in MM PR, partial response; VGPR, very good partial response; CR, complete remission; MRD, minimal residual disease; M-c: monoclonal component Paiva et al, Blood 12015

6 Quality of response to treatment and progression in MM MFC, ASO-PCR, NGS, PET/CT PR, partial response; VGPR, very good partial response; CR, complete remission; MRD, minimal residual disease; M-c: monoclonal component Paiva et al, Blood 12015

7 Sensitive techniques for MM MRD evaluation MFC (<8-color) ASO-PCR NGS PET/CT Classic bivariate dot plots representations, normal PC phenotype

8 CD56 CD117 Cy Ig kappa CD38 Sensitive techniques for MM MRD evaluation MFC (<8-color) ASO-PCR NGS PET/CT PC characterization and detection of phenotypically abnormal PC CD138 CD45 CD19 CD27 CD81 Cy Ig lambda

9 Flow cytometry MRD evolution Study Rawstron, Blood 2002 San Miguel, Blood 2002 Paiva, Blood 2008 Paiva, JCO 2011 Paiva, Blood 2012 Rawstron, JCO 2013 Roussel, JCO 2014 Rawstron, Blood 2015 Paiva, Blood 2016 Combination CD38/CD3/CD45 CD38/CD138/CD45 CD38/CD19/CD45 CD38/CD56/CD45 CD38/CD56/CD19/CD45 CD138/CD28/CD33/CD38 CD20/CD117/CD138/CD38 CD38/CD56/CD19/CD45 CD38/CD27/CD45/CD28 b2m/cd81/cd38/cd117 CD138/CD38/CD45/CD19/CD56/CD27 Plus CD81/CD117 CD200/CD52 CD56+CD28/CD138/CD19/CyIgk/CyIg l/cd45/cd38 CD138/CD38/CD45/CD19/CD56/CD27 Plus CD81/CD117 CD45/CD138/CD38/CD56/CD27/CD19 /CD117/CD81 % aberrant N of cells acquired Sensitivity 100% 5x10 4-5x % <2x10 6 (LG) 90% with tube x >2x10 6 /tube (LG) NR x <2x x10-5 5x % 2x

10 Tumor depletion assessed by Flow Cytometry PFS OS Total (n=397) Patients in conventional CR (n=214) Rawstron et al, Blood 2015

11 Sensitive techniques for MM MRD evaluation MFC (<8-color) ASO-PCR NGS PET/CT Courtesy of ERASMUS MC, Rotterdam

12 Sensitive techniques for MM MRD evaluation MFC (<8-color) ASO-PCR NGS PET/CT Courtesy of N. Puig

13 ASO-PCR MRD detection Study Applicability Sensitivity PFS impact OS impact ASO PCR Martinelli, JCO % 10-5 Yes NA Bakkus,Br J Haematol % 10-4 Yes NA Galimberti, Leuk Res 2005* NA NA Yes Sarrasquete, Haematologica % 5x10-5 Yes NA Martinez-Sanchez, Br J Haematol 2008* 91% Yes Yes RQ-ASO Putkonen, Eur J Haematol % Yes No Ladetto, JCO 2010 a 51% 10-6 Yes Yes Korthals, Biol Blood Marrow Transplant % Yes Yes Puig, Leukemia % 10-5 Yes Yes Silvennoinen, Blood Cancer J % 4x10-6 Yes NA *faso; a, Nested Modified from Paiva et al, Cancer Treat Res, 2016

14 Sensitive techniques for MM MRD evaluation MFC (<8-color) ASO-PCR NGS PET/CT Normal Clonal MRD Courtesy of ERASMUS MC, Rotterdam

15 Sensitive techniques for MM MRD evaluation MFC (<8-color) ASO-PCR NGS PET/CT Modified from Faham M, et al. Blood. 2012

16 Tumor depletion assessed by NGS TTP OS Total (n=110) Patients in conventional CR (n=32) Martinez-Lopez et al, Blood 2014

17 Sensitive techniques for MM MRD evaluation MFC (<8-color) ASO-PCR NGS PET/CT Sachpekidis et al, Eur J Nucl Med Mol Imagin, 2016

18 Correlation of different techniques MFC (<8-color) ASO-PCR NGS PET/CT PCR vs MFC PCR vs NGS Puig et al, Leukemia 2012; Ladetto et al, Leukemia 2014

19 Sensitive techniques for MM MRD evaluation MFC (<8-color) ASO-PCR NGS PET/CT Applicability >97% 60% to 70% 90% 100% Reproducibility Moderate at Moderate High Not reported (among centers) MRD Availability High Intermediate Limited Intermediate Diagnostic sample Time Important (but not mandatory) 2-3 h Mandatory 5 d (follow-up) 3-4 wk (target identification) Mandatory Important (but not mandatory) >7 d 2 h Sensitivity to High (4 mm) Quantitative Yes (directly; high accuracy) Yes Yes Yes Fresh sample Needed (36 h) Not needed Not needed NA Patchy sample Impacts Impacts Impacts No impact Global cell characterization Yes No No No Standardization Lacking Yes, since 15y (EuroMRD) Not reported No Modified from Paiva et al, Blood 2015

20 Sensitive techniques for MM MRD evaluation MFC (<8-color) ASO-PCR NGS PET/CT Applicability >97% 60% to 70% 90% 100% Reproducibility Moderate at Moderate High Not reported (among centers) MRD Availability High Intermediate Limited Intermediate Diagnostic sample Time Important (but not mandatory) 2-3 h Mandatory 5 d (follow-up) 3-4 wk (target identification) Mandatory Important (but not mandatory) >7 d 2 h Sensitivity to High (4 mm) Quantitative Yes (directly; high accuracy) Yes Yes Yes Fresh sample Needed (36 h) Not needed Not needed NA Patchy sample Impacts Impacts Impacts No impact Global cell characterization Yes No No No Standardization Lacking Yes, since 15y (EuroMRD) Not reported No Modified from Paiva et al, Blood 2015

21 Sensitive techniques for MM MRD evaluation MFC (<8-color) ASO-PCR NGS PET/CT Applicability >97% 60% to 70% 90% 100% Reproducibility Moderate at Moderate High Not reported (among centers) MRD Availability High Intermediate Limited Intermediate Diagnostic sample Time Important (but not mandatory) 2-3 h Mandatory 5 d (follow-up) 3-4 wk (target identification) Mandatory Important (but not mandatory) >7 d 2 h Sensitivity to High (4 mm) Quantitative Yes (directly; high accuracy) Yes Yes Yes Fresh sample Needed (36 h) Not needed Not needed NA Patchy sample Impacts Impacts Impacts No impact Global cell characterization Yes No No No Standardization Lacking Yes, since 15y (EuroMRD) Not reported No Modified from Paiva et al, Blood 2015

22 Sensitive techniques for MM MRD evaluation MFC (<8-color) ASO-PCR NGS PET/CT Applicability >97% 60% to 70% 90% 100% Reproducibility Moderate at Moderate High Not reported (among centers) MRD Availability High Intermediate Limited Intermediate Diagnostic sample Time Important (but not mandatory) 2-3 h Mandatory 5 d (follow-up) 3-4 wk (target identification) Mandatory Important (but not mandatory) >7 d 2 h Sensitivity to High (4 mm) Quantitative Yes (directly; high accuracy) Yes Yes Yes Fresh sample Needed (36 h) Not needed Not needed NA Patchy sample Impacts Impacts Impacts No impact Global cell characterization Yes No No No Standardization Lacking Yes, since 15y (EuroMRD) Not reported No Modified from Paiva et al, Blood 2015

23 Standardized EuroFlow-IMF NGF-MRD MM Approach

24 Standardized EuroFlow-IMF NGF-MRD MM Approach Development of optimal antibody panel combinations Increased number of cells evaluated ( 10 7 ) Novel data analysis strategies

25 EuroFlow-IMF NGF-MRD DESIGN 1. IDENTIFICATION OF MOST EFFICIENT MRD DISCRIMINATING MARKERS (n=94) cpc populations of 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

26 EuroFlow-IMF NGF-MRD PANEL OPTIMIZATION OF THE TWO 8-COLOR MM MRD ANTIBODY COMBINATIONS Panel version Tube PacB PacO FITC PE PerCP Cy5.5 PE Cy7 APC APC H7 1 CD45 CD138 CD38 CD56 CD27 CD19 CD117 CD81 2 CD45 CD138 CD38 CyIgk CyIgl HV500-C 1 CD45 CD138 CD38 CD56 CD27 CD19 CD117 CD81 2 CD45 CD138 CD38 CD56 CD229 CD19 CyIgk CyIgl APC C750 1 CD45 CD138 CD38 CD56 CD27 CD19 CD117 CD81 2 CD45 CD138 CD38 CD56 CD229 CD19 CyIgk CyIgl HV450 1 CD138 CD27 CD38 CD56 CD45 CD19 CD117 CD81 2 CD138 CD27 CD38 CD56 CD45 CD19 CyIgk CyIgl BV421 BV510 1 CD138 CD27 CD38 (ME) CD56 CD45 CD19 CD117 CD81 2 CD138 CD27 CD38 (ME) CD56 CD45 CD19 CyIgk CyIgl ME: multiepitope Flores-Montero, submitted 2016

27 SSC -A Residual BM PC in MM patient treated with Daratumumab Clon OKT10 Clon AT13 Clon IB4 Clon HB7 Clone LD38 (currently in use) Clon AT1 Clon IB6 Clon SUN4B7 Multi-epitope CD38 FITC Residual plasma cell Unclassified events OKT10, AT13, IB4, AT1, IB6, SUN4B7 clons were kindly supply by Dr. F. Malavasi

28 Material, Methods and Results EuroFlow-IMF NGF-MRD PROTOCOLS DIFFERENT SAMPLE PREPARATION PROTOCOLS Bulk lyse Protocol (A1): 0.5% BSA, with FacsLyse Flores-Montero, submitted 2016

29 AUTOMATED GATING Raw FCS files

30 AUTOMATED GATING Raw FCS files Clusters of events

31 AUTOMATED GATING Raw FCS files Clusters of events Database-matched cell populations

32 NEXT GENERATION FLOW (NGF): EuroFlow-IMF NGF-MRD MM Approach Optimal antibody panel 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

33 VALIDATION OF THE NGF MM MRD METHOD 1. NGF- MRD MM vs 2 nd generation flow (n=103) r= 0.76; p<0.001 MRD NGF levels <LOQ MRD NGF levels >LOQ * 2 cases proven policlonal Follow-up VGPR MM BM samples Flores-Montero, submitted 2016

34 VALIDATION OF THE NGF MM MRD METHOD 1. NGF- MRD MM vs 2 nd generation flow (n=103) r= 0.76; p<0.001 MRD NGF levels <LOQ MRD NGF levels >LOQ * 2 cases proven polyclonal Follow-up VGPR MM BM samples Flores-Montero, submitted 2016

35 VALIDATION OF THE NGF MM MRD METHOD 3. NGF- MRD MM vs NGS (n=27) r= 0.62; p= NGS levels < LOD Follow-up VGPR MM BM samples Flores-Montero, submitted 2016

36 VALIDATION OF THE NGF MM MRD METHOD 2. NGF- MRD MM vs automatic identification of cpc (n=103) r= 0.96; p<0.001 MRD NGF levels <LOQ MRD NGF levels >LOQ Follow-up VGPR MM BM samples Flores-Montero, submitted 2016

37 Concluding remarks: MM MRD assessment with high sensitive techniques helps to improve risk stratification of patients and can potentially be used to guide therapeutic decisions Next Generation Flow assay for high-sensitive, fast and standardized quantification of MRD in MM, overcomes previous limitations of conventional flow-mrd approaches

38 THANK YOU

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