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CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor Immunophenotyping: Standardization and Applications Multicolor.

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Presentation on theme: "CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor Immunophenotyping: Standardization and Applications Multicolor."— Presentation transcript:

1 CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor Immunophenotyping: Standardization and Applications Multicolor Immunophenotyping: Standardization and Applications March 9-11, 2012 TMH, Mumbay (India) PLASMA CELLS (MORMAL AND NEOPLASTIC)

2 Therapy Treatment compliance In vivo drug kinetics Tumor micro- environment Tumor cell features MRD MONITORING IN HAEMATOLOGICAL MALIGNANCIES N. of tumor cells Resistance Complete remission Immunological CR Molecular CR SensitivitySensitivity - CML - APL - Childhood ALL - … Therapeutic decisions Morphology, Cytogenetics Southern-Blot, FCM DNA aneuploidy FCM DNA aneuploidy F.I.S.H Flow cytometry Flow cytometry P.C.R.

3 MRD TECHNIQUES FOR HAEMATOPOIETIC MALIGNANCIES FCM immunophenotyping PCR/RT-PCR analyses (sensitivity) (sensitivity) Disease category LAIP sIg  /sIg Junctional Reg Chromosomal or TCRV  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 95% >95% 10-25% Chronic T-cell leukemias 5-10% 60-65% 95% <5% B-cell lymphomas 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% From: Szczepanski, Orfao et al, Lancet Oncol, 2001; 2:

4 BACKGROUND IMMUNOPHENOTYPING - Acute Leukemias & Lymphoproliferative disorders: Mandatory for diagnosis & monitoring - Multiple Myeloma: Restricted to research Differential diagnosis of unusual cases - Acute Leukemias & Lymphoproliferative disorders: Mandatory for diagnosis & monitoring - Multiple Myeloma: Restricted to research Differential diagnosis of unusual cases

5 Plasma cell quantification (BM infiltration) Morphological PC count : - area of BM smear - infiltration pattern - area of BM smear - infiltration pattern Variability Immunophenotyping: - precise identification by CD38/CD CD38 FITC -> CD138 PerCP/Cy5 -> Co-expression of CD38/CD CD38 FITC -> TRANSFORMED SSC -> High-intensity CD138 PerCP/Cy5-> TRANSFORMED SSC -> Specific expression + + = = - but…..diluted sample  lower numbers

6 Correlation between Immunophenotyping & Morphology: Prognostic influence of the number of BMPC: Proportion of plasma cell by morphology Proportion of plasma cell by flow cytometry WW W W W W W W W W W W W W W W W W W W W W W W W W W W W W W WW W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W WWW W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W WW W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W WW W W W W W W W W W W W W R 2 = 0, Proportion of plasma cell by morphology Proportion of plasma cell by flow cytometry WW W W W W W W W W W W W W W W W W W W W W W W W W W W W W W WW W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W WWW W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W WW W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W WW W W W W W W W W W W W W R 2 = 0,4

7 High-dose chemotherapy and Novel Drugs Complete remission (CR): 25%-75% Relapse-free survival (RFS) at 5 year: 40%-70% High-dose chemotherapy and Novel Drugs Complete remission (CR): 25%-75% Relapse-free survival (RFS) at 5 year: 40%-70% BACKGROUND However, patients with MM ultimately relapse MINIMAL RESIDUAL DISEASE (MRD) However, patients with MM ultimately relapse MINIMAL RESIDUAL DISEASE (MRD) persistence of residual malignant cells

8 Ocqueteau, Am J Pathol, 1996; San Miguel et al, Blood, 2002

9 MM vs Normal BM plasma cells Abnormal plasma cells Normal plasma cells

10 MM PLASMA CELL CyIg + PC-associated Ags: CD38 ++/ % CD % B-cell-associated Ags: CD % CD % CD % CD % HLA-DR +het ….. 10% CD % FMC % HPC-associated Ags: CD % CD % Adhesion molecules: CD56 +/ %  1/  2 integrins 98% CD54….……….50-70% Co-stimulatory Ags: CD28 +/ % CD % CD81,CD27 -/lo.40-50% CD52……………….10-50% Myeloid-associated Ags: CD % CD33 +/ % Pan-leuc. Ag: CD % Rawstron et al, Haematologica 2008

11 CD19 CD38 CD45 CD56 CD28 CD33 CD117 CD20 96% 80% 73% 60% 36% 18% 32% 17% Asynchronous expression Infra-expression Mateo et al. J Clin Oncol; 2008;26:2737 Over-expression 92% Incidence of aberrant phenotypes in PC from MM

12 MOST USEFUL ANTIGENS FOR THE DETECTION OF ABERRANT PC IN MM AntigenExpression % MM with Requirement for NormalAltered altered expression MRD studies CD19+ (>70%) -95% Essential CD56- (>85%) ++75% Essential CD20- (100%) +10% Preferred CD117- (100%) +30% Preferred CD28-/dim (100%) ++15% Recommended CD81 +-/dimN.A. Recommended CD27 ++-/dim40-50% Recommended N.A.: not analyzed/not reported. Rawstron et al, EMN consensus, Haematologica, 2008

13 Immmunophenotype of normal vs clonal PC Clonal PCNormal PC Clonal PC Normal PC Clonal PCNormal PC Clonal PCNormal PC CD38 HLA-I   2-microglobulin CD40 % of positive PC Mean FL Intensity p=0.21p= Clonal PCNormal PCCD95p=0.72 p  Clonal PCNormal PCCD56 p  Clonal PCNormal PCCD86 p  p=0.002 Clonal PCNormal PC CD126 p  Perez-Andres et al, Leukemia, 2005; Perez-Andres et al, Int J Cancer, 2009

14 MGUS vs MM: IMMUNOPHENOTYPIC PANELS N.of PB AMCA FITC PE PerCPCy5.5 APC PE-Cy7 APCH7 colors PO 3 CD38 CD56CD19 4 CD38 CD56CD19 CD45 6 CD38 CD56CD45 cyIgk CD19cyIg 8CD45CD138 CD38 CD56 CD117 cyIgk CD19cyIg

15 Characterization markers CD10, CD20, CD22 CD24, CD27, CD38 CD39, CD43, CD63 CD81, CD95, CD138 Bcl-2, HLA-DR, CyIg Normal B lymphopoiesis CD11a, CD11c, CD31, CD49d, CD62L, CXCR5, CCR6, CD303 B cell homing Known to differentiate CD13, CD15, CD28, CD33, CD56, CD45, CD117,  2 M 7 informative markers

16 NORMAL vs NEOPLASTIC PC: IMMUNOPHENOTYPIC PANELS N.of PB HV500 FITC PE PerCPCy5.5 APC PE-Cy7 Alexa700 colors HV450 PO APC-H7 3 CD38 CD56CD19 4 CD38 CD56CD19 CD45 6 cyIgL cyIgkCD19 CD45 CD56 CD38 CD138 CD117 CD19 CD45 CD56 CD38 8 CD45 CD138cyIgL cyIgk CD138 CD117 CD56 CD38

17 CONSTRUCTION OF EuroFlow MRD PANELS: MM MM #1 MM #2 Normal BM #1 Normal BM #2 SSC CD38-FITC SSC CD38-FITC Identify PCSelect PC Merge PC (n-cases) Principal component analysis (n=12 markers) CD CD CD CD CD CD CD MOST INFORMATIVE MARKERS

18 Most informative markers EuroFlow PANEL: Plasma cell dyscrasias Abnormal PC detection /classification in MGUS & MM (APS view) Normal PCs Abnormal PCs CD19:PE Cy7-A LOGICAL12.16 CD45:PacB-A LOGICAL11.81 Responsible scientist: J.Flores

19 PCD panel: Backbone markers Pac Blue Pac Orange FITCPEPerCP- Cy5.5 PECy7APCAPC-H7 1CD45CD138CD38 CD19 2CD45CD138CD38 CD19 Responsible scientists: Juan Flores

20 PCD panel: Backbone markers Pac Blue Pac Orange FITCPEPerCP- Cy5.5 PECy7APCAPC-H7 1CD45CD138CD38 CD19 2CD45CD138CD38 CD19 Responsible scientists: Juan Flores Pac Blue Pac Orange FITC PEPerCP- Cy5.5 PECy7APCAPC-H7 1CD45CD138CD38CD56CD27 CD19CyIgk CyIg 2CD45CD138CD38CD28  2 M CD19CD117 CD81

21 CD38 FITC -> TRANSFORMED SSC -> CD38 FITC -> CD138 PerCP/Cy5 -> CD38-FITC gated PC T-SSC CD138-PerCP/Cy5.5 MONOCLONAL GAMMOPATHIES: IDENTIFICATION OF CLONAL PLASMA CELLS Clonal PC Normal PC C D 5 6 P E 9 1 D C C P A 5 4 D C CD19-PcpCy5 CD56-PE CD45-APC Perez-Andres, J Biol Reg, 2004

22 MRD TECHNIQUES FOR HAEMATOPOIETIC MALIGNANCIES FCM immunophenotyping PCR/RT-PCR analyses (sensitivity) (sensitivity) Disease category LAIP sIg  /sIg Junctional Reg Chromosomal or TCRV  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 >90% >90% 70-80% NT AML 70-90% NA 10% 30-40%* CML NA NA NA >95% * Increased through the usage of additional molecular markers (e.g.: WT1, NMP1 & FLT3 mutations

23 BM plasma cells in MGUS

24 Differential diagnosis Risk of MGUS transformation 2 Cases with predominantly (>95%) CD19- ve PC.... High risk (26% transformed in 31 months) Risk of MGUS transformation 2 Cases with predominantly (>95%) CD19- ve PC.... High risk (26% transformed in 31 months) 2. Rawstron A, Blood 2003, 102, 36 a (Abstr.116) MGUS MM Only 20% of MM patients showed poly-PC and constantly <5% (median: 0.25%) 1 The most powerful single criteria for differential diagnosis (even in stage I MM) versus >5% poly-PC: 98% MGUS Clonal Poly-Clonal 1. Ocqueteau M, Am J Pathol 1998, 152: 1655

25 Prognostic influence of phenotypic profiles CD56 & CD28 Months from diagnosis p=0.01 CD56+CD28- n= m +/+ or -/- n= m CD56-CD28+ n= m CD56+CD28- n= m +/+ or -/- n= m CD56-CD28+ n= m Months from diagnosis CD56 & CD CD56+CD117+ n= m +/- or +/- n= m CD56-CD117- n= m CD56+CD117+ n= m +/- or +/- n= m CD56-CD117- n= m p=0.001 Months from diagnosis CD28 & CD CD28-CD117+ n= m +/- or +/- n= m CD28+CD117- n= m CD28-CD117+ n= m +/- or +/- n= m CD28+CD117- n= m p= PFS

26 Kyle & Alexanian 1980 a. Estimated incidence: 15% of newly diagnosed MM b. Estimated Risk of progression: 10% per year c vs. 1% on MGUS a Kyle 1980, Alexanian 1980; b Rajkumar 05; c Kyle 05 Introduction Smoldering Multiple Myeloma

27 Proportion of aPC referred to the total-PC (aPC/BMPC) 2 nd step PC compartment 2 nd step PC compartment % aPC/BMPC % nPC/BMPC 1 st step Total cellularity 1 st step Total cellularity PC analysis in BM by FC % PC within BM cellularity % PC within BM cellularity

28 % Total PC in BM*2.8 ( ) % of aPC / BMPC compartment97 (35-100) < 95% aPC / BMPC36 (40%) > 95% aPC / BMPC53 (60%) Flow Cytometry Results * Median (range)

29 Impact of % aPC/BMPC by FC on Progression Free Survival <95% aPC/BMPC n= 36 (4 progressions) >95% aPC/BMPC n= 53 (34 progressions) 92%37% 5 years

30 Multivariate analysis for PFS pHR % a PC /BMPC Immunoparesis

31 Impact of prognostic index on PFS Immunoparesis >95% aPC/BMPCScore (n) - -0 (n=32) + / --/+1 (n=27) ++2 (n=27)

32 Impact of prognostic index on PFS >95% aPC/BMPC or paresis n= 27 (12 progressions) >95% aPC/BMPC + paresis n= 27 (22 progressions) No adverse factors n= 32 (3 progressions) 91% 58% 18% 5 years

33 MM: IMMUNOPHENOTYPIC IDENTIFICATION OF NEOPLASTIC PLASMA CELLS IN REMISSION BM

34 MM: Diagnostic vs remission BM MRD/remission Diagnosis MM patients show few poly-PC constantly <5% (median: 0.25%) 1 versus Clonal Poly-Clonal

35 FLOW MRD IN MM: Why, when and how? - Does response to therapy impact on long-term patient outcome? - Does flow-based MRD improve prognostic stratification of myeloma patients? -Is flow-based MRD a well suited technique for MRD assessment in MM? - Can flow-based MRD techniques be used in routine diagnostic labs?

36 In the ASCT setting, there is a large body of evidence showing an association between optimal response (CR/VGPR) and long-term outcome (PFS and OS) 10 prospective trials (2991 patients): All showed a positive correlation (statistically significant in 8). Similar findings in 5/8 retrospective trials (Van de Velde, Hematologica 2007, 92, 1399) Impact of CR in the ASCT setting - Significant correlation between maximal response and outcome prospective studies (< ) & rétrospective studies (< ) prospective studies (< ) & rétrospective studies (< ) Is it the same CR & VGPR ??

37 Months from diagnosis 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 Cumulative Proportion Surviving CR vs nCR CR vs PR nCR vs PR P=0.01 P<10 -6 P= Months from diagnosis 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 Cumulative Proportion Event Free Surviving CR vs nCR or PR nCR vs PR P<10 -5 P=0.07 CR, n=278nCR, n=124PR, n=280 PD, n=25 EFS OS Lahuerta et al. JCO 2008;26:5775–5782 CR and nCR are not the same: “depth of response” PETHEMA-GEM 2000: Outcome according to post-transplant response CRnCRPRPD Medians EFS, months Medians OS, monthsNR 6115

38 CR correlates with long-term PFS and OS in elderly patients treated with novel agents Gay et al. Blood 2011 PFS OS P<0.001 CR VGPR PR CR VGPR PR Retrospective analysis: 3 randomized European trials of GIMEMA and HOVON groups (n=1175) First-line treatment MP (n=332), MPT (n=332), VMP (n=257), VMPT-VT (n=254) Significant benefit also seen when analysis is restricted to patients >75 years old

39 Depth of response MR PR VGPR/ nCR CR sCR Molecular/Flow CR Treatment initiation Progression TTP MRD investigation in MM : molecular & Immnunophenotypic tools Which level of response should be measured ? Depth of response is related to TTP

40 GEM 2000 (n=510*) Diagnosis VBMCP/ VBAD GEM 2005<65y (n=369*) Diagnosis Thalidomide/ Dexamethasone (TD) (n=121) VBMCP/ VBAD (x4) Bortezomib (x2) (n=126) ASCT 3m post-ASCT MRD investigation MRD investigation 6 cycles Bortezomib/ Thalidomide/ Dexamethasone (VTD) (n=122) ASCT 3m post-ASCT MRD investigation MRD investigation 6 alterning cycles GEM 2005>65y (n=246*) Diagnosis (n=206) (n=157) * Patients achieving CR or VGPR after treatment without MRD investigation were excluded from the ITT analysis 6 cycles Bortezomib/ Melphalan/ Prednisone (VMP) (n=121) Bortezomib/ Thalidomide/ Prednisone (VTP) (n=125) MRD investigation (n=102) Analysis of immunophenotypic response (IR) by MFC in 619 myeloma patients included in three consecutive Spanish trials (n=295) (n=222)

41 EF + n=108 EF + n=108 # MM-PC # % N-PC/BMPC 0.76 MRD evaluation 86% *MRD+ cases 44 # Results expressed as medians Complete remission Partial Response IFx - n=147 (79%) IFx - n=147 (79%) 0.1 < % 85 <.001 IFx + n=40 (21%) IFx + n=40 (21%) 62% P P *≥0.001% MM-PC Correlation between immunophenotyping & electrophoretic responses at three months post-ASCT (GEM 2000 trial, n=295) Paiva et al; Blood. 2008, 112:

42 GEM2000 & GEM2005: Impact on survival of achieving an Immunophenotypic CR after HDT/ASCT independent of the induction regimen OS PFS P <.001 GEM2000 GEM2005 (<65y) P <.001 P = P =.640 P =.091 P = Paiva et al. Blood ; abstr 1910

43 Paiva et al, JCO, 2011

44 MRD- IFE- 71 m (n= 94) MRD- IFE+ 65 m (n=31) MRD+ IFE- 37 m (n=53) p= At 5 years: 59% 49% 24% 17% MRD+ IFE+ 37 m (n=117) Months GEM2000: Impact on survival of achieving an immunophenotypic CR vs. conventional CR after HDT/ASCT PFS

45 GEM 2000 trial: Multivariate Analysis prisk MRD+ at day High Risk Cytog* ns Age >60y.ns IF+ at dey +100ns ns PFS OS Paiva et al; Blood t(4;14), t(4;16), del (17p)

46 GEM : Immunophenotypic response & FISH for the prediction of early relapse in CR patients after HDT/ASCT (n=241) MRD negative + Standard risk FISH (n=58) MRD positive OR High-risk FISH (n=45) MRD positive + High-risk FISH (n=7) OS PFS P <.001 Months P <.001 Months Medians: NR Median: 97m Median: 43m 80% 93% % Median: 1y after ASCT Median: 64m Median: 35m

47 GEM 2005(>65y): Impact on survival of the depth of response after induction therapy (n=102) Immunophenotypic response (n=31) “Stringent CR” (n=11) CR (n=9) PR (≥70% reduction) (n=51)

48 Updated results from the MRC myeloma IX trial Owen et al. IMW Paris 2011 abstr O intensively treated patients (CVAD or CTD and HDM) at 3 months post-HDM: 66% remained MRD +ve highly predictive of outcome (PFS; p=0.0001) increased MRD -ve rates with consolidation and maintenance  prolongation of PFS 510 non-transplant eligible patients (CTDa or MP) only 8% became MRD- but a significantly improved PFS was demonstrated (p=0.028) Immunophenotypic CR predicted outcome in CR (IFx -) patients and both standard and high-risk cytogenetic groups

49 MM: Flow cytometry immunophenotyping vs. molecular monitoring of MRD ? Molecular techniques Flow cytometry Speed2-3 days (up to weeks) fast: 1-2 hours TargetDNA or RNAprotein/cells (RNA is an instable target)(“end-product”) Applicability70-75%>95% Sensitivity Multiplexingtechnically demandingrelatively easy (even 25 to 100 tests per tube) Accuracysemi-quantitative quantitative Focusall cells in sample any subpopulation (or: prior purification)(FACSorted for further analyses) Facilitiesspecial laboratories neededonly standard lab needed (pre-PCR lab, PCR lab, etc)(+ flow cytometer) Modified from J.J.M. van Dongen

50 HOW TO SIMPLIFY & OPTIMIZE FLOW-BASED MRD STRATEGIES - Improve the design of MRD panels for a greater efficiency and higher reproducibility. - Construct reference data files for normal and neoplastic cells (e.g.: per disease category) - Multi-n-dimensional comparison of normal vs neoplastic cell populations (e.g.: at diagnosis and follow-up) : - Automated PCA-guided approach for homogeneous cell populations(e.g. lymphoid) - Maturation tools for heterogeneous cell populations(e.g. myeloid)

51 CONSTRUCTION OF EuroFlow MRD PANELS: MM MM #1 MM #2 Normal BM #1 Normal BM #2 SSC CD38-FITC SSC CD38-FITC Identify PCSelect PC Merge PC (n-cases) Principal component analysis (n=14 markers/parameters) CD CD CD CD CD CD CD MOST INFORMATIVE MARKERS

52 Automated classification of normal vs aberrant plasma cells in MM Diagnosis Post-transplant Pre-Transplant m-PC (22%) n-PC (0.12%) n-PC (0.03%)+m-PC (0.8%) Comparative View

53 FLOW-BASED MRD IN MM: Conclusions - Response to therapy impacts on long-term patient outcome both in the transplant and non-transplant settings - Flow-based MRD improves prognostic stratification of myeloma patients particualrly among those cases who reach CR - Flow-based MRD is a well suited technique for MRD assessment in MM - Full standardized and automated flow-based MRD approaches for MM have been developped Shall flow-based MRD be introduced in the treatment-decision algorithms of new (randomized) trials ?

54 CIRCULATING NEOPLASTIC PLASMA CELLS MGUSSMMMMP.-value % of cases with PB M-PC7 (21%)18 (69%)43 (75%)<.001 N. of M-PC/  L <.001 % of PB M-PC Paiva et al, Leukemia (under revision); ASH 2010 (abstract #617)

55 MINIMAL NUMBER OF PC REQUIRED TO BE ANALYZED IN A MRD ASSAY FOR MM N. of tests in the MRD assay N. of total nucleated cells/test N. of events/test to define a PC population 11,000, , , , ,00020 Rawstron, Orfao et al, Haematologica, 2008

56 Grupo Español de Mieloma (GEM) Hospitales Clínico de Barcelona 12 Octubre (Madrid) Clínico de Salamanca Clínico de San Carlos (Madrid) Hospital de Badalona Clínico de Asturias Fr. Peset (Valencia) Universitario de Canarias Rio Ortega (Valladolid) Cínico de Zaragoza Hospital General de Jerez Ramón y Cajal (Madrid) Morales Meseguer (Murcia) La Fe (Valencia) C.U. de Navarra Galdakao (Vizcaya) Clínico de Valladolid Sant Pau (Barcelona) Arnau Vilanova (Lérida) Universitario de Santiago General Universitario de Valencia Universitario de Getafe (Madrid) Insular de las Palmas H. de La Princesa (Madrid) Severo Ochoa (Madrid) Juan XIII (Tarragona) Toledo Gandía (Valencia) Vall D´Hebrón (Barcelona) San Jorge (Huesca) Verge de la Cinta (Tortosa) Alarcos (Ciudad Real) Mataró (Madrid) Juán Canalejo (Coruña) Ferrol Hospitales General de Segovia Cruces (Bilbao) St. Coloma de Gramanet (Barcelona) Gregorio Marañon (Madrid) Carlos Haya (Málaga) H. Tauli (Gerona) Huesca Palencia Alcira (Valencia) H. Del Mar (Barcelona) Mahón (Baleares) Clínico de Málaga Xeral Cies (Vigo) Plasencia Cáceres Algeciras Ávila Jaén S. Pau i Sta Tecla (Tarragona) General de Guadalajara Sagunto (Valencia) Son Dureta (Mallorca) Cuenca Alicante SUS M. Valdecilla (Santander) Albacete H. Del Bierzo Fundación Jiménez Díaz (Madrid) Elda (Alicante) V. Del Rosel (Cartagena) Castellón Mutua Tarrasa Consorcio Tarrasa C. Corachán (Barcelona) G.Mateo, M. Perez-Andres, N.Gutierrez, R.Lopez, M.Mateos, R.Garcia-Sanz, J.Almeida, J.San Miguel Salamanca´Group:

57 Cytometry Service & Department Medicine, CICancer, University Salamanca-CSIC, Salamanca, Spain Martín Perez-Andres Leandro Thiago Alberto Orfao Wilheminenhospital, Wien, Austria Niklas Zojer University Medical Center, Groningen, Netherlands Nico A Bos Serv. Hematology, Hosp. Univ. Salamanca, Salamanca, Spain Bruno Paiva Jesus F San Miguel Vrije Universiteit, Brussel, Belgium Karin Vanderkerken Aalborg Hospital Science & Innovation Center AHSIC Aarhus University Hospital, Denmark Hans E Johnsen University of Heidelberg, Heidelberg, Germany Dirk Hose INSERM, U847, CHU Montpellier, Institute of Research in Biotherapy, Université Montpellier1 Montpellier, France Bernard Klein Anouk Caraux School of Medicine, Southampton, UK Surinder S Sahota Erasmus University Medical Center, Rotterdam, Netherlands Pieter Sonneveld

58 SERVICIO DE CITOMETRIA, DEPARTAMENTO DE MEDICINA Y CENTRO DE INVESTIGACIÓN DEL CÁNCER (IBMCC) UNIVERSIDAD DE SALAMANCA

59 THANKYOU


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